Comparative cryopreservation of avian spermatozoa: benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival.

PubMed

Blanco, Juan M; Long, Julie A; Gee, George; Wildt, David E; Donoghue, Ann M

2011-02-01

A comparative approach was used to evaluate the cryosurvival of turkey and crane sperm frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants and ATP. A range (6-26%) of DMA concentrations was used alone or in combination with ATP (30, 60 or 118mM) or one of the following osmoprotectants: (1) sucrose (turkey, 8.0%; crane, 5.0%); (2) 5.0% sucrose and 5.0% trehalose; or (3) betaine hydrochloride (0.1, 0.2 or 0.4mM). The viability of thawed sperm was assessed using the nigrosin-eosin stain and sperm motility was determined using the hanging-drop technique. For semen frozen only with DMA, post-thaw sperm motility was greatest (P<0.05) for the 6.0%, 10.0% and 18% concentrations, regardless of species. Turkey sperm frozen with the sucrose/trehalose combination had greater (P<0.05) post-thaw motility for all DMA treatments compared to DMA alone. The lowest concentration of the osmoprotectant betaine hydrochloride substantially improved turkey sperm viability post-thaw in all treatments compared to DMA alone (P<0.05). The post-thaw motility of crane sperm was improved (P<0.05) with a combination of 18.0%, 24.0% or 26.0% DMA and 30mM ATP. Moreover, in the presence of osmoprotectants, crane sperm motility decreased as the osmoprotectant concentration increased. The lowest concentration of ATP also improved crane sperm viability post-thaw, especially for DMA concentrations 18% or greater. The combination of sucrose and trehalose improved (P<0.05) crane sperm viability only with 6% and 10% DMA. These data affirm that there are avian-specific differences in sperm survival after cryopreservation and suggest that post-thaw survival can be enhanced by including species-based osmoprotectant/ATP combinations in a cryodiluent where DMA is the cryoprotectant. Published by Elsevier B.V.

Preservation of tissue microstructure and functionality during freezing by modulation of cytoskeletal structure

PubMed Central

Park, Seungman; Seawright, Angela; Park, Sinwook; Dutton, J Craig; Grinnell, Frederick; Han, Bumsoo

2015-01-01

Cryopreservation is one of the key enabling technologies for tissue engineering and regenerative medicine, which can provide a reliable long-term storage of engineered tissues (ETs) without losing their functionality. However, it is still extremely difficult to design and develop cryopreservation protocols guaranteeing the post-thaw tissue functionality. One of the major challenges in cryopreservation is associated with the difficulty of identifying effective and less toxic cryoprotective agents (CPAs) to guarantee the post-thaw tissue functionality. In this study, thus, a hypothesis was tested that the modulation of the cytoskeletal structure of cells embedded in the extracellular matrix (ECM) can mitigate the freezing-induced changes of the functionality and can reduce the amount of CPA necessary to preserve the functionality of ETs during cryopreservation. In order to test this hypothesis, we prepared dermal equivalents by seeding fibroblasts in type I collagen matrices resulting in three different cytoskeletal structures. These ETs were exposed to various freeze/thaw (F/T) conditions with and without CPAs. The freezing-induced cell-fluid-matrix interactions and subsequent functional properties of the ETs were assessed. The results showed that the cytoskeletal structure and the use of CPA were strongly correlated to the preservation of the post-thaw functional properties. As the cytoskeletal structure became stronger via stress fiber formation, the ETs functionality was preserved better. It also reduced the necessary CPA concentration to preserve the post-thaw functionality. However, if the extent of the freezing-induced cell-fluid-matrix interaction was too excessive, the cytoskeletal structure was completely destroyed and the beneficial effects became minimal. PMID:25679482

Studies on the intracellular Ca2+ concentration of thawed dog spermatozoa: influence of Equex from different sources, two thawing diluents and post-thaw incubation in capacitating conditions.

PubMed

Peña, A I; López-Lugilde, L; Barrio, M; Becerra, J J; Quintela, L A; Herradón, P G

2003-02-01

The addition of 0.5% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA), whose active ingredient is sodium dodecyl sulphate (SDS), to a Tris-egg yolk extender was demonstrated to improve the longevity of frozen-thawed dog spermatozoa during in vitro incubation at 38 degrees C. The aim of the first experiment was to compare the effects of two SDS-containing compounds, Equex STM Paste and Equex Pasta (Minitüb, Tiefenbach, Germany), when added to a Tris-egg yolk based extender, on the post-thaw longevity of dog spermatozoa, as well as on the intracellular Ca2+ concentration of spermatozoa, during post-thaw incubation at 38 degrees C. The post-thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better when using Equex STM Paste. Such prolonged sperm longevity, however, was associated to a higher intracellular Ca2+ concentration in a large subpopulation of the live spermatozoa. A second experiment was aimed to evaluate the effects of sperm dilution immediately post-thaw with a Tris buffer containing glucose or fructose. The two Tris buffers were no different for any of the sperm parameters studied. The aim of a third experiment was to evaluate the sperm longevity, motility patterns and intracellular Ca2+ concentration of cryopreserved dog spermatozoa during post-thaw incubation in capacitating conditions [canine capacitating medium (CCM) with or without 5 microg/ml of heparin]. Heparin had no significant effects on any of the sperm parameters evaluated. During the first 8 h of incubation, the majority of the live spermatozoa had a high intracellular Ca2+ content. However, after 8-10 h of incubation, it had significantly declined. The highest proportion of fast motile sperm, and the highest curvilinear velocity, average path velocity and amplitude of lateral head displacement for the total motile sperm were observed during the 2-4-h incubation period. It was concluded that: (a) the addition of 0.5% (v/v) of Equex STM Paste to a Tris-egg yolk based extender significantly improved the post-thaw longevity of dog spermatozoa, but the same concentration of Equex Pasta had no significant beneficial effects; (b) sperm dilution after thawing with a Tris buffer containing glucose or fructose made no difference in post-thaw sperm longevity; (c) the addition of 5 microg/ml of heparin to CCM had no significant capacitating effects on frozen-thawed dog spermatozoa.

Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol.

PubMed

Chanapiwat, Panida; Kaeoket, Kampon; Tummaruk, Padet

2012-03-01

The present study determined the effect of different types of sugars (lactose, fructose, glucose and sorbitol) used in egg yolk-based extender on the post-thawed boar semen quality. Twenty-two ejaculates from 6 fertility-proven Yorkshire boars were cryopreserved by liquid nitrogen vapor method. Sperm motility, viability, acrosome integrity and intact functional plasma membrane were determined at 0, 2 and 4 hr after thawing. It was found that the lactose-based extender resulted in a higher percentage of post-thawed sperm motility, viability, intact acrosome and functional plasma membrane than sorbitol-based extender (P<0.05) and fructose-based extender yielded a higher post-thawed sperm motility and viability than sorbitol-based extender (P<0.05). It could be concluded that sorbitol was not an effective sugar for the cryopreservation in boar semen.

Genes Upregulated in Winter Wheat (Triticum aestivum L.) during Mild Freezing and Subsequent Thawing Suggest Sequential Activation of Multiple Response Mechanisms.

PubMed

Skinner, Daniel Z

2015-01-01

Exposing fully cold-acclimated wheat plants to a mild freeze-thaw cycle of -3 °C for 24h followed by +3 °C for 24 or 48 h results in dramatically improved tolerance of subsequent exposure to sub-freezing temperatures. Gene enrichment analysis of crown tissue from plants collected before or after the -3 °C freeze or after thawing at +3 °C for 24 or 48 h revealed that many biological processes and molecular functions were activated during the freeze-thaw cycle in an increasing cascade of responses such that over 150 processes or functions were significantly enhanced by the end of the 48 h, post-freeze thaw. Nearly 2,000 individual genes were upregulated more than 2-fold over the 72 h course of freezing and thawing, but more than 70% of these genes were upregulated during only one of the time periods examined, suggesting a series of genes and gene functions were involved in activation of the processes that led to enhanced freezing tolerance. This series of functions appeared to include extensive cell signaling, activation of stress response mechanisms and the phenylpropanoid biosynthetic pathway, extensive modification of secondary metabolites, and physical restructuring of cell membranes. By identifying plant lines that are especially able to activate these multiple mechanisms it may be possible to develop lines with enhanced winterhardiness.

The effects of different levels of vitamin E and vitamin C in modified Beltsville extender on rooster post-thawed sperm quality.

PubMed

Amini, Mahmood Reza; Kohram, Hamid; Zare Shahaneh, Ahmad; Zhandi, Mahdi; Sharideh, Hossein; Nabi, Mohammad Mehdi

2015-12-01

Oxidative damage of sperm by means of reactive oxygen species generated by the cellular components of semen is one of the main reason of declined motility and fertility of sperm during the freeze-thawing process. This study was conducted to determine the influence of vitamin C and vitamin E on rooster post-thawed sperm motility, viability and malondialdehyde (MDA) level. Semen samples from 10 sexually-mature Ross 308 breeder roosters were collected and pooled, divided into nine equal parts and diluted with modified Beltsville extender containing with no antioxidants (control), or containing 100 (C100), 200 (C200), 400 (C400), 800 (C800) µg/mL vitamin C, and 2 (E2), 5 (E5), 10 (E10) and 15 (E15) µg/mL vitamin E. After thawing, total and progressive sperm motility, sperm viability and semen MDA level were assessed. The results shown that C200 and E5 extenders resulted in higher total motility (p < 0.05) compared to other extenders, with exception of E10 extender. Progressive motility was higher in E5 extender (p < 0.05) compared to other extenders, with exception of C200 and E10 extenders. Also, C200 and E5 extenders resulted in higher viability of post-thawed spermatozoa (p < 0.05) compared to other extenders. Finally, the results showed that MDA level was lower in C100 and C200 extenders compared to other extenders (p < 0.05), with exception of E5 extender. In conclusion, the results of the present study demonstrate that C200 and E5 can improve the function of post-thawed rooster spermatozoa.

Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate.

PubMed

Alkmin, Diego V; Perez-Patiño, Cristina; Barranco, Isabel; Parrilla, Inmaculada; Vazquez, Juan M; Martinez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi

2014-10-01

Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24h at 15-17°C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p < 0.01) than BE samples. Control samples showed higher (p < 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p < 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of ebselen supplementation on cryopreservation medium in human semen

PubMed Central

Khodayari Naeini, Zohreh; Hassani Bafrani, Hassan; Nikzad, Hossein

2014-01-01

Background: An effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. Objective: This study examined whether the addition of an antioxidant to cryopreservation medium could improve the post-thaw parameters and evaluation of sperm chromatin quality of cryopreserved human spermatozoa from men with normal semen parameters. Materials and Methods: Semen samples (n=35) were collected by masturbation and assessed following WHO standards. Individual samples were classified as two portions. One portion (n=10) was for elucidate the concentration of ebselen.Then the samples(n=25) were divided in to 5groups.The first aliquot remained fresh.The second aliquots was mixed with cryopreservation medium.The third aliquots were mixed with cryopreservation medium containing solvent of ebselen.The forth and fifth aliquots were mixed with cryopreservation medium containing 1.25 and 2.5 µm of ebselen.Samples were frozen and thawed samples were assessed for sperm parameters.Three-way ANOVA Multivariate measures were used to assess. According to this assesment the differences are observed in existent groups in post-thaw count, motility index, vitality staining, and morphology and DNA fragmentation. Results: After freezing the media containing of ebselen, DNA fragmentation is significantly different in comparison with control group. ebselen with 1.25 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.047). Similarly ebselen with 2.5 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.038). But other parameters were not altered. Conclusion: These results suggest that the addition of ebselen to cryopreservation medium doesnot improve post-thaw parameters and DNA fragmentation of sperm. PMID:24976819

Evaluation of ebselen supplementation on cryopreservation medium in human semen.

PubMed

Khodayari Naeini, Zohreh; Hassani Bafrani, Hassan; Nikzad, Hossein

2014-04-01

An effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. This study examined whether the addition of an antioxidant to cryopreservation medium could improve the post-thaw parameters and evaluation of sperm chromatin quality of cryopreserved human spermatozoa from men with normal semen parameters. Semen samples (n=35) were collected by masturbation and assessed following WHO standards. Individual samples were classified as two portions. One portion (n=10) was for elucidate the concentration of ebselen.Then the samples(n=25) were divided in to 5groups.The first aliquot remained fresh.The second aliquots was mixed with cryopreservation medium.The third aliquots were mixed with cryopreservation medium containing solvent of ebselen.The forth and fifth aliquots were mixed with cryopreservation medium containing 1.25 and 2.5 µm of ebselen.Samples were frozen and thawed samples were assessed for sperm parameters.Three-way ANOVA Multivariate measures were used to assess. According to this assesment the differences are observed in existent groups in post-thaw count, motility index, vitality staining, and morphology and DNA fragmentation. After freezing the media containing of ebselen, DNA fragmentation is significantly different in comparison with control group. ebselen with 1.25 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.047). Similarly ebselen with 2.5 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.038). But other parameters were not altered. These results suggest that the addition of ebselen to cryopreservation medium doesnot improve post-thaw parameters and DNA fragmentation of sperm.

Post-thaw motility of frozen boar sperm does not predict success with in vitro fertilization

USDA-ARS?s Scientific Manuscript database

Using cryopreserved boar sperm rather than liquid semen for in vitro fertilization (IVF) allows improved IVF consistency. However, cryopreservation of boar sperm results in reduced post-thaw motility, fertilization and embryo development. Boars are often screened on an individual basis prior to use ...

Trehalose improves semen antioxidant enzymes activity, post-thaw quality, and fertility in Nili Ravi buffaloes (Bubalus bubalis).

PubMed

Iqbal, Sajid; Andrabi, Syed Murtaza Hassan; Riaz, Amjad; Durrani, Aneela Zameer; Ahmad, Nasim

2016-03-15

Our objectives were to study the effect of trehalose in extender on (1) antioxidant enzymes profile during cryopreservation (after dilution, before freezing, and after thawing), (2) in vitro quality (after thawing), and (3) in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 20) from four buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of trehalose (0.0, 15, 30, 45, and 60 mM) and frozen in French straws. At post dilution, profile of sperm catalase (U/mL) was higher (P < 0.05) in extenders containing 15, 30, and 45 mM of trehalose as compared to control. Although profiles of superoxide dismutase (U/mL) and total glutathione (μM) were higher (P < 0.05) in extenders containing 15 and 30 mM of trehalose as compared to control. At prefreezing, sperm catalase, superoxide dismutase, and total glutathione profiles were higher (P < 0.05) in all the treatment groups as compared to control. At post thawing, the profiles of catalase and total glutathione were higher (P < 0.05) in extender containing 30-mM trehalose as compared to other treatment groups and control. Whereas, profile of superoxide dismutase was higher (P < 0.05) in extenders containing 30, 45, and 60 mM of trehalose as compared to control and 15mM group. Post thaw total sperm motility (%) was higher (P < 0.05) in extender containing 30-mM trehalose as compared to control and 15 and 60-mM groups. Although sperm progressive motility (%), rapid velocity (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), plasma membrane (structural and functional, %), acrosome (%), and DNA (%) integrity were higher (P < 0.05) in extender containing 30 mM trehalose as compared to other treatment groups and control. The fertility rates (61% vs. 43%) were higher (P < 0.05) in buffaloes inseminated with semen doses cryopreserved in extender containing 30 mM of trehalose than the control. It is concluded that addition of 30-mM trehalose in extender improves the semen antioxidant enzymes activity, post thaw quality, and fertility in Nili Ravi buffaloes. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of post-thaw addition of seminal plasma on motility, viability and chromatin integrity of cryopreserved donkey jack (Equus asinus) spermatozoa.

PubMed

Sabatini, C; Mari, G; Mislei, B; Love, Cc; Panzani, D; Camillo, F; Rota, A

2014-12-01

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen-thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post-thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP-αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post-thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post-thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time. © 2014 Blackwell Verlag GmbH.

l-Cysteine improves antioxidant enzyme activity, post-thaw quality and fertility of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa.

PubMed

Iqbal, S; Riaz, A; Andrabi, S M H; Shahzad, Q; Durrani, A Z; Ahmad, N

2016-11-01

The effects of l-cysteine in extender on antioxidant enzymes profile during cryopreservation, post-thaw quality parameters and in vivo fertility of Nili-Ravi buffalo bull spermatozoa were studied. Semen samples from 4 buffalo bulls were diluted in Tris-citric acid-based extender having different concentrations of l-cysteine (0.0, 0.5, 1.0, 2.0 and 3.0 mm) and frozen in 0.5-ml French straws. The antioxidative enzymes [catalase, super oxide dismutase and total glutathione (peroxidase and reductase)] were significantly higher (P < 0.05) at pre-freezing and post-thawing in extender containing 2.0 mm l-cysteine as compared to other groups. Post-thaw total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm s -1 ), straight line velocity (μm s -1 ), curvilinear velocity (μm s -1 ), beat cross frequency (Hz), viable spermatozoa with intact plasmalemma (%), acrosome and DNA integrity (%) were higher with the addition of 2.0 mm l-cysteine as compared to other groups (P < 0.05). The fertility rates (59 versus 43%) were higher (P < 0.05) in buffaloes inseminated with doses containing 2.0 mm of l-cysteine than in the control. In conclusion, the addition of 2.0 mm l-cysteine in extender improved the antioxidant enzymes profile, post-thaw quality and in vivo fertility of Nili-Ravi buffalo bull spermatozoa. © 2016 Blackwell Verlag GmbH.

Freezing-induced deformation of biomaterials in cryomedicine

NASA Astrophysics Data System (ADS)

Ozcelikkale, Altug

Cryomedicine utilizes low temperature treatments of biological proteins, cells and tissues for cryopreservation, materials processing and cryotherapy. Lack of proper understanding of cryodamage that occurs during these applications remains to be the primary bottleneck for development of successful tissue cryopreservation and cryosurgery procedures. An engineering approach based on a view of biological systems as functional biomaterials can help identify, predict and control the primary cryodamage mechanisms by developing an understanding of underlying freezing-induced biophysical processes. In particular, freezing constitutes the main structural/mechanical origin of cryodamage and results in significant deformation of biomaterials at multiple length scales. Understanding of these freezing-induced deformation processes and their effects on post-thaw biomaterial functionality is currently lacking but will be critical to engineer improved cryomedicine procedures. This dissertation addresses this problem by presenting three separate but related studies of freezing-induced deformation at multiple length scales including nanometer-scale protein fibrils, single cells and whole tissues. A combination of rigorous experimentation and computational modeling is used to characterize post-thaw biomaterial structure and properties, predict biomaterial behavior and assess its post-thaw biological functionality. Firstly, freezing-induced damage on hierarchical extracellular matrix structure of collagen is investigated at molecular, fibril and matrix levels. Results indicate to a specific kind of fibril damage due to freezing-induced expansion of intrafibrillar fluid. This is followed by a study of freezing-induced cell and tissue deformation coupled to osmotically driven cellular water transport. Computational and semi empirical modeling of these processes indicate that intracellular deformation of the cell during freezing is heterogeneous and can interfere with cellular water transport, thereby leading to previously unconsidered mechanisms of cell freezing response. In addition, cellular water transport is identified as the critical limiting factor on the amount of freezing-induced tissue deformation, particularly in native tissues with high cell densities. Finally, effects of cryopreservation on post-thaw biological functionality of collagen engineered tissue constructs is investigated where cell-matrix interactions during fibroblast migration are considered as the functional response. Simultaneous cell migration and extracellular matrix deformation are characterized. Results show diminished cell-matrix coupling by freeze/thaw accompanied by a subtle decrease in cell migration. A connection between these results and freezing-induced collagen fibril damage is also suggested. Overall, this dissertation provides new fundamental knowledge on cryodamage mechanisms and a collection of novel multi-purpose engineering tools that will open the way for rational design of cryomedicine technologies.

Impacts of post-mortem ageing prior to freezing on technological and oxidative properties of coarse ground lamb sausage in a model system.

PubMed

Choe, Juhui; Kim, Hyun-Wook; Farouk, Mustafa M; Brad Kim, Yuan H

2017-07-01

The objective of this study was to evaluate the effects of ageing time of lamb loins prior to freezing on technological characteristics and oxidation stability of coarse ground lamb loin sausage using in a model system. Lamb loins ( M. longissimus lumborum , n = 25) were aged at -1.5°C for 0, 1, 2, 3, and 8 wk and then frozen for the remaining days (a total of 30 wk). The aged/frozen/thawed lamb loins were ground, and model sausages were formulated with 75% aged/frozen/thawed lamb loin, 25% water, 1.5% sodium chloride (NaCl) and 0.3% sodium tripolyphosphate. The pH and thaw/purge loss of aged/frozen/thawed lamb loins were evaluated, and protein functionality (protein solubility and emulsifying capacity), water-holding capacity and textural properties of model sausages were determined. Cooked model sausages were vacuum-packaged in a plastic bag and displayed under continuous fluorescent natural white light (3°C±1°C). Colour and lipid oxidation of the cooked model sausages were evaluated on 0 and 21 d of display storage. Ageing prior to freezing had no impact on pH and purge/thaw loss of lamb loins and the colour of cooked sausages (p>0.05) made from the loins. Lamb loins aged for at least 3 wk prior to freezing numerically improved total and myofibrillar protein solubilities (p>0.05) and emulsion activity index (p = 0.009) of meat batter, but decreased cooking loss (p = 0.003) and lipid oxidation (p<0.05) of model sausages. This study suggests that post-mortem ageing of raw meat prior to freezing could improve water-holding capacity and lipid oxidative stability of sausage made from the meat.

Actin Cytoskeletal Disruption following Cryopreservation Alters the Biodistribution of Human Mesenchymal Stromal Cells In Vivo

PubMed Central

Chinnadurai, Raghavan; Garcia, Marco A.; Sakurai, Yumiko; Lam, Wilbur A.; Kirk, Allan D.; Galipeau, Jacques; Copland, Ian B.

2014-01-01

Summary Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and human endothelial cells (80% reduction), respectively. This reduction correlated with a reduced cytoskeletal F-actin content in post-thaw MSCs (60% reduction). In vivo, live human MSCs could be detected in murine lung tissues for up to 24 hr, whereas thawed MSCs were undetectable. Similarly, live MSCs whose actin cytoskeleton was chemically disrupted were undetectable at 24 hr postinfusion. Our data suggest that post-thaw cryopreserved MSCs are distinct from live MSCs. This distinction could significantly affect the utility of MSCs as a cellular therapeutic. PMID:25068122

Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSperm density gradient centrifugation.

PubMed

Maxwell, W M C; Parrilla, I; Caballero, I; Garcia, E; Roca, J; Martinez, E A; Vazquez, J M; Rath, D

2007-10-01

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.

Liquid Storage at 4 deg C of Previously Frozen Red Cells

DTIC Science & Technology

1987-12-01

adenosine tnphosphate (ATP). 2.3- acceptable red cell function. A post-thaw storage ca- diphosphoglycerate (2.3-DPG), glucose, supernatant hemo...and Received for publication September 22. 1986; revision received supernatant hemoglobin levels within the acceptable range, November 29, 1986, and...percent. All units were sterile at the end of the 21-day post- thaw storage period. 6.9 The mean red cell ATP and 2,3-DPG levels are shown in Figure 1

Trehalose improves rabbit sperm quality during cryopreservation.

PubMed

Zhu, Zhendong; Fan, Xiaoteng; Pan, Yang; Lu, Yinghua; Zeng, Wenxian

2017-04-01

High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, acrosome integriy, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry. Copyright © 2017 Elsevier Inc. All rights reserved.

Ascorbic acid, catalase and chlorpromazine reduce cryopreservation-induced damages to crossbred bull spermatozoa.

PubMed

Paudel, K P; Kumar, S; Meur, S K; Kumaresan, A

2010-04-01

The present study evaluated the effectiveness of ascorbic acid, catalase, chlorpromazine and their combinations in reducing the cryodamages to crossbred bull (Bos taurus x Bos indicus) spermatozoa. A total of 32 ejaculates (eight each from four bulls) were diluted in Tris-citric acid-fructose-egg yolk-glycerol extender. Each ejaculate was split into six parts (five treatment and one control). Treatment groups included 10 mm ascorbic acid, 0.1 mm chlorpromazine, 200 IU/ml catalase, 10 mm ascorbic acid + 0.1 mm chlorpromazine or 200 IU/ml catalase + 0.1 mm chlorpromazine in the extender. Fluorescent probes (Fluorescein isothiocyanate--Pisum sativum agglutinin + Propidium iodide) were used for the assessment of spermatozoa viability and acrosomal status. The proportion of acrosome intact live (AIL), acrosome intact dead, acrosome reacted live and acrosome reacted dead sperm was assessed in fresh, equilibrated and frozen-thawed semen. The functional status of the sperm was assessed using hypo-osmotic sperm swelling test (HOSST). Activities of acrosin and hyaluronidase enzyme were also determined. Lipid peroxidation level was assayed based on the melonaldehyde (MDA) production. In cryopreserved semen, the values of AIL spermatozoa, HOSST response, hyaluronidase and acrosin activity were reduced by 53%, 47%, 34% and 54%, respectively from their initial values in fresh semen. However, MDA level was threefold higher in the frozen-thawed sperm compared with fresh sperm. Significant (p < 0.05) improvement in motility, viability, HOSST response, retention of hyaluonidase and acrosin and reduction in MDA was recorded in ascorbic acid, catalase, ascorbic acid + chlorpromazine and catalase + chlorpromazine incorporated groups. The percentage of AIL sperm was significantly (p < 0.05) higher in ascorbic acid, catalase and ascorbic acid + chlorpromazine incorporated groups compared with the control. Chlorpromazine alone did not improve the post-thaw semen quality but when combined with either ascorbic acid or catalase, improvement in semen quality was noticed. It was inferred that incorporation of ascorbic acid, catalase and ascorbic acid + chlorpromazine in semen extender improved the post-thaw semen quality in crossbred bulls.

Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender.

PubMed

Najafi, Abozar; Kia, Hossein Daghigh; Mohammadi, Hossein; Najafi, Mir Hossein; Zanganeh, Zaynab; Sharafi, Mohsen; Martinez-Pastor, Felipe; Adeldust, Hamideh

2014-08-01

The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6mM of either antioxidant improved total motility. Cysteamine at 6mM and ergothioneine at 4 and 6mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6mM and ergothioneine at 4 or 6mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender. Copyright © 2014 Elsevier Inc. All rights reserved.

Assessing the toxic effects of DMSO on cord blood to determine exposure time limits and the optimum concentration for cryopreservation.

PubMed

Fry, L J; Querol, S; Gomez, S G; McArdle, S; Rees, R; Madrigal, J A

2015-08-01

Advantages of using cord blood (CB) over other sources of haematopoietic progenitor cells, such as bone marrow, include the ability to cryopreserve and bank the samples until requested for a transplant. Cryopreservation requires the addition of a cryoprotectant to prevent the formation of intracellular ice during freezing. Dimethyl sulphoxide (DMSO) is commonly used at a concentration of 10% (v/v); however, there is evidence to suggest this chemical is toxic to cells as well as to patients after infusion. The toxic effects of DMSO were assessed through cell viability and in vitro functional assays in fresh and post-thaw CB samples before determining the maximum exposure time and optimal concentration for cryopreservation. A dose-dependent toxicity of DMSO was observed in fresh samples with 40% removing all viable and functional haematopoietic progenitor cells (HPC). In fresh and post-thaw analysis, minimal toxic effect was observed when cryopreservation was delayed for up to 1 h after 10% DMSO addition. After thawing, DMSO washout was superior to dilution or unmanipulated when maintained for long periods (advantage observed 1 h after thawing). Finally, the optimum concentration for cryopreserving CB was found to be 7.5 to 10% with detrimental effects observed outside of this range. These results support the use of 7.5-10% as the optimal DMSO concentration and the maximum exposure time should be limited to <1 h prior to freezing and 30 min post-thaw. © 2015 International Society of Blood Transfusion.

Effect of dilution in sperm maturation media and time of storage on sperm motility and fertilizing capacity of cryopreserved semen of sex-reversed female rainbow trout.

PubMed

Judycka, Sylwia; Ciereszko, Andrzej; Dobosz, Stefan; Zalewski, Tomasz; Dietrich, Grzegorz J

2017-05-01

Masculinized females, also called neomales or sex-reversed females have a male phenotype but retain the female genotype (XX). Therefore, all spermatozoa produced in their functional testes carry an X chromosome, which is desired for the production of all-female rainbow trout populations. Semen of sex-reversed female rainbow trout is of low quality and in vitro maturation is required, which includes dilution of sperm suspensions with specially formulated maturation solutions. The aim of this study was to determine the effect of dilution in different maturation media on sperm quality (sperm motility characteristics and fertilizing capacity) of frozen/thawed sperm of sex-reversed female rainbow trout. The effect of time of post-thaw storage (0, 15, 60 and 120min) on semen quality was also tested. Sperm motility parameters and fertilization rate at the eyed and hatching stages were assessed for post-thaw semen diluted in different media. The cryopreservation procedure resulted in high post-thaw sperm motility of about 57% and did not differ from fresh semen. Unexpectedly, maturation media decreased sperm activation capacity immediately after dilution; however, sperm motility increased over time. Fertilization rates of frozen/thawed semen were high (71-87%) and did not differ significantly between experimental variants at any of tested periods of storage. Our results demonstrated that the effect of the maturation media on frozen/thawed sperm is different from that of fresh sperm. The progressive increase in post-thaw sperm motility in maturation media can potentially be applied to routine hatchery practice. Copyright © 2016 Elsevier Inc. All rights reserved.

Impacts of post-mortem ageing prior to freezing on technological and oxidative properties of coarse ground lamb sausage in a model system

PubMed Central

Choe, Juhui; Kim, Hyun-Wook; Farouk, Mustafa M.; Brad Kim, Yuan H.

2017-01-01

Objective The objective of this study was to evaluate the effects of ageing time of lamb loins prior to freezing on technological characteristics and oxidation stability of coarse ground lamb loin sausage using in a model system. Methods Lamb loins (M. longissimus lumborum, n = 25) were aged at −1.5°C for 0, 1, 2, 3, and 8 wk and then frozen for the remaining days (a total of 30 wk). The aged/frozen/thawed lamb loins were ground, and model sausages were formulated with 75% aged/frozen/thawed lamb loin, 25% water, 1.5% sodium chloride (NaCl) and 0.3% sodium tripolyphosphate. The pH and thaw/purge loss of aged/frozen/thawed lamb loins were evaluated, and protein functionality (protein solubility and emulsifying capacity), water-holding capacity and textural properties of model sausages were determined. Cooked model sausages were vacuum-packaged in a plastic bag and displayed under continuous fluorescent natural white light (3°C±1°C). Colour and lipid oxidation of the cooked model sausages were evaluated on 0 and 21 d of display storage. Results Ageing prior to freezing had no impact on pH and purge/thaw loss of lamb loins and the colour of cooked sausages (p>0.05) made from the loins. Lamb loins aged for at least 3 wk prior to freezing numerically improved total and myofibrillar protein solubilities (p>0.05) and emulsion activity index (p = 0.009) of meat batter, but decreased cooking loss (p = 0.003) and lipid oxidation (p<0.05) of model sausages. Conclusion This study suggests that post-mortem ageing of raw meat prior to freezing could improve water-holding capacity and lipid oxidative stability of sausage made from the meat. PMID:28183171

Negative effect of combined cysteine and glutathione in soy lecithin-based extender on post-thawed ram spermatozoa.

PubMed

Zhandi, Mahdi; Sharafi, Mohsen

2015-09-01

This study was conducted to evaluate the effect of combined cysteine and glutathione in soy lecithin-based semen extender on post-thawed ram sperm quality. A total of 28 ejaculates were collected twice a week (from four rams) during breeding season. In each replicate, semen samples (n = 4, one ejaculate for each ram) were pooled and divided into three equal parts, and each part was diluted with one of following extender: (1) soy lecithin-based extender containing no cysteine and no glutathione (C0-G0), (2) soy lecithin-based extender containing cysteine (5 mM) and glutathione (5 mM) (C5-G5), and (3) soy lecithin-based extender containing cysteine (10 mM) and glutathione (10 mM) (C10-G10). After freeze-thawing process, motility and velocity parameters, plasma membrane integrity and functionality, mitochondrial activity, and apoptosis features of spermatozoa were evaluated. The obtained results showed that total and progressive motility, plasma membrane integrity and functionality, and live post-thawed spermatozoa was lower in C10-G10 extender compared to C0-G0 and C5-G5 extenders (P < 0.05). Also, the percentage of dead spermatozoa was higher in C10-G10 extender compared to C0-G0 extender (P < 0.05). Apoptotic spermatozoa was lower in C10-G10 extender compared to C0-G0 and C5-G5 extenders (P < 0.05). All velocity parameters, exception of BCF, did not different between extenders (P > 0.05). In conclusion, it seems that high concentration of combined cysteine and glutathione in soy lecithin-based semen extender has a detrimental effect of post-thawed ram sperm quality.

Spatial considerations during cryopreservation of a large volume sample.

PubMed

Kilbride, Peter; Lamb, Stephen; Milne, Stuart; Gibbons, Stephanie; Erro, Eloy; Bundy, James; Selden, Clare; Fuller, Barry; Morris, John

2016-08-01

There have been relatively few studies on the implications of the physical conditions experienced by cells during large volume (litres) cryopreservation - most studies have focused on the problem of cryopreservation of smaller volumes, typically up to 2 ml. This study explores the effects of ice growth by progressive solidification, generally seen during larger scale cryopreservation, on encapsulated liver hepatocyte spheroids, and it develops a method to reliably sample different regions across the frozen cores of samples experiencing progressive solidification. These issues are examined in the context of a Bioartificial Liver Device which requires cryopreservation of a 2 L volume in a strict cylindrical geometry for optimal clinical delivery. Progressive solidification cannot be avoided in this arrangement. In such a system optimal cryoprotectant concentrations and cooling rates are known. However, applying these parameters to a large volume is challenging due to the thermal mass and subsequent thermal lag. The specific impact of this to the cryopreservation outcome is required. Under conditions of progressive solidification, the spatial location of Encapsulated Liver Spheroids had a strong impact on post-thaw recovery. Cells in areas first and last to solidify demonstrated significantly impaired post-thaw function, whereas areas solidifying through the majority of the process exhibited higher post-thaw outcome. It was also found that samples where the ice thawed more rapidly had greater post-thaw viability 24 h post-thaw (75.7 ± 3.9% and 62.0 ± 7.2% respectively). These findings have implications for the cryopreservation of large volumes with a rigid shape and for the cryopreservation of a Bioartificial Liver Device. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Association of Vitamin E with Rapid Thawing on Goat Semen

PubMed Central

Penitente-Filho, Jurandy Mauro; Oliveira, Fabrício Albani; Jimenez, Carolina Rodriguez; Dias, Júlio César Oliveira; Oliveira, Gisele Dias; Silveira, Renata Gomes; Silveira, Camila Oliveira; Torres, Ciro Alexandre Alves

2014-01-01

The aim of this study was to evaluate the effects of vitamin E associated with rapid thawing on cryopreserved goat semen. Two bucks were used and eight ejaculates per animal were collected using artificial vagina. Semen was diluted with the following treatments: BIOXCELL (control), BIOXCELL + Equex (sodium lauryl sulphate) and BIOXCELL + vitamin E 100 μM. Semen was packaged into 0.25 mL straws and cooled at 5°C for 1 hour. Freezing was performed in liquid nitrogen vapor (−155°C) during 15 minutes. Then, the straws were immersed in liquid nitrogen (−196°C). Straws were thawed at 38°C/60 seconds or at 60°C/7 seconds with immediate sperm analysis. Hypoosmotic swelling test was performed adding a 20 μL aliquot of thawed semen to 1 mL of hypoosmotic solution (100 mOsm·Kg−1) followed by incubation during 60 minutes in water bath (38°C). Vitamin E did not affect any studied parameters (P > 0.05). Nevertheless, defrosting rate of 60°C/7 seconds improved sperm membrane functional integrity (P < 0.05). Current knowledge about goat semen cryopreservation is not sufficient to ensure high post-thawing recovery rates; thus, this study brings important data about using antioxidants and different thawing rates on cryopreservation process. PMID:24955428

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality.

PubMed

De Mercado, Eduardo; Rodríguez, Ana; Gómez, Emilio; Sanz, Elena

2010-03-01

The aim of this study was to evaluate the cryoprotective effect of different freezing extenders against cryopreservation injuries on Iberian boar sperm. The sperm-rich fraction was collected and pooled from six sexually mature Iberian boars, and was frozen in different extenders containing glucose, lactose or fructose as sugar source and including Orvus ES Paste only in the freezing extender-2 (Glucose; Lactose and Fructose) or in both freezing extenders (Glucose2; Lactose2 and Fructose2). During the cryopreservation process, the supernatant was removed after the centrifugation step, then was extended with freezing extender-1 for the equilibration period and with freezing extender-2 immediately before freezing. Post-thaw sperm characteristics, such as plasma membrane integrity (SYBR-14/PI), mitochondrial function (Rhodamine 123) and acrosome integrity (NAR), were monitored. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in the different experimental treatments. Measurements were taken at 30 and 150 min post-thaw. The state of the acrosome after thawing did not show significant differences between the freezing extenders studied. Freezing-thawing caused a significant decrease (P<0.001) in plasma membrane integrity and in mitochondrial activity in the spermatozoa frozen with Orvus ES Paste in both freezing extenders. Furthermore, spermatozoa frozen with Orvus ES Paste in both freezing extenders exhibited lower (P<0.05) motility and kinematic parameters than those frozen in the absence of Orvus ES Paste in the first freezing extender. The spermatozoa frozen with the Lactose extender and with Orvus ES Paste only in the second freezing extender showed a better evolution of the motility and kinematic characteristics (P<0.05) over time. The deterioration in post-thaw sperm motility and kinematic parameters were concurrent with reduced sperm characteristics. It can be suggested that in the Iberian pig, the beneficial effects of Orvus ES Paste during the freezing process of spermatozoa is time dependent. The analysis of different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, determined that the extenders studied in the present experiment affected the quality of frozen-thawed semen in Iberian boar.

Assessment of gamete quality for the eastern oyster (Crassostrea virginica) by use of fluorescent dyes

USGS Publications Warehouse

Paniagua-Chavez, C. G.; Jenkins, J.; Segovia, M.; Tiersch, T.R.

2006-01-01

Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 (R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity (P = 0.004) and thawing treatments (P = 0.04), and mitochondrial membrane potential (P = 0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P = 0.0258) and with mitochondrial membrane potential (P = 0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol (P = 0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced. ?? 2006 Elsevier Inc. All rights reserved.

Improvement in the Viability of Cryopreserved Cells by Microencapsulation

NASA Astrophysics Data System (ADS)

Matsumoto, Yoshifumi; Morinaga, Yukihiro; Ujihira, Masanobu; Oka, Kotaro; Tanishita, Kazuo

The advantages of microencapsulated cells over those of suspended cells were evaluated for improving viability in cryopreservation. Rat pheochromocytoma (PC12) cells were selected as the test biological cells and then microencapsulated in alginate-polylysine-alginate membranes. These microencapsulated PC12 cells were frozen by differential scanning calorimetry (DSC) at various cooling rates, from 0.5 to 10°C/min. Their latent heat was measured during freezing from 4 to -80°C. The post-thaw viability was evaluated by dopamine-concentration measurement and by trypan blue exclusion assay. Results showed that at cooling rates of 0.5 and 1°C/min, the latent heat of microencapsulated PC12 cells was lower than that of suspended cells. This lower latent heat is caused by the fact that the extra-microcapsule froze and the intra-capsule remained unfrozen due to the formation of ice crystals in the extra-capsule space. The post-thaw viability of microencapsulated PC12 cells was improved when the cooling rate was 0.5 or 1°C/min, compared with that of suspended cells. Therefore, in microencapsulated PC12 cells, maintaining the intra-microcapsules in an unfrozen state during freezing reduces the solution effect and thus improves the post-thaw viability.

Effect of addition of coconut water (Cocos nucifera) to the freezing media on post-thaw viability of boar sperm.

PubMed

Bottini-Luzardo, María; Centurión-Castro, Fernando; Alfaro-Gamboa, Militza; Aké-López, Ricardo; Herrera-Camacho, José

2013-01-01

The aims of this experiment were to evaluate the addition of coconut water in natura to the freezing media, compare the effect of deionized water vs filtered water of coconut over the post-thaw seminal characteristics, and evaluate the effect of the deionized water and in natura coconut water on the seminal characteristics of boar sperm at different post-thaw times. Thirty-four ejaculates were used divided in three aliquots which received one of the following treatments (T): T1, LEY (bidistilled water, lactose, and egg yolk) and LEYGO (LEY + glycerol and Orvus ET paste); T2, LEY(A) (coconut deionized water, lactose, and egg yolk)-LEYGO(A); and T3, LEY(B) (in natura coconut water, lactose, and egg yolk)-LEYGO(B). Samples of boar semen were frozen according to the Westendorf method, thawed at 38°C, and evaluated at three incubation times (0, 30, and 60 min). Seminal characteristics assessed were motility (Mot), acrosomal integrity (AInt), membrane integrity (MInt), and mitochondrial activity (MAct). T1 showed a higher percentage of viable sperm than T3 (Mot 36.5 vs 5.4 %, AInt 61.8 vs 41.2 %, MInt 50.4 vs 41.3 %, and MAct 56.9 vs 50.5 %). T2 kept a higher percentage of viable sperm at all incubation times. In natura coconut water showed a detrimental effect over the viability of the frozen-thawed boar semen. Deionized coconut water improved the boar semen viability post-thaw, outperforming results of in natura coconut water.

Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa.

PubMed

Pontbriand, D; Howard, J G; Schiewe, M C; Stuart, L D; Wildt, D E

1989-08-01

A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.

Use of antioxidants reduce lipid peroxidation and improve quality of crossbred ram sperm during its cryopreservation.

PubMed

Banday, Mohamad Naiem; Lone, Farooz Ahmad; Rasool, Fabiha; Rashid, Muzamil; Shikari, Arif

2017-02-01

Ram sperm are subjected to extreme oxidative stress during their preservation at -196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4-5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance. Copyright © 2016 Elsevier Inc. All rights reserved.

Dietary inclusion of fish oil changes the semen lipid composition but does not improve the post-thaw semen quality of ram spermatozoa.

PubMed

Díaz, Rommy; Torres, Mariana A; Paz, Erwin; Quiñones, John; Bravo, Silvana; Farías, Jorge G; Sepúlveda, Néstor

2017-08-01

The aim of this study was to investigate the effects of dietary fish oil (FO) time-response on the fatty acid profile, cholesterol levels and sperm cryosurvival in ram semen. Criollo Araucano rams were randomly assigned to two groups (n=4) according to the type of supplementation: a control group without FO and a supplemented group fed a diet with 3% FO for 8 weeks. The semen lipid profile and post-thaw sperm quality were analyzed at weeks 0 (pre-supplementation), 4, 8, 12 and 16 (post-supplementation) to evaluate the effects of FO supplementation by time interaction. Post-thaw sperm quality was determined by CASA and flow cytometry. In spermatozoa, the supplemented group increased the linoleic acid (C18:2n6c) and docosahexaenoic acid (DHA; C22:6n3) with levels higher at week 16 (P<0.05). The effect of FO on cholesterol concentration in sperm was significant at the end of the experiment (week 16). In seminal plasma, statistical differences of butyric acid (C4:0), palmitic acid (C16:0), stearic acid (C18:0), eicosatrienoic acid (C20:3n3) and DHA were observed at week 12. The cholesterol concentration was not affected by dietary treatments (P>0.05). However, the post-thaw sperm quality of the FO treatment group decreased. Motility percentage decreased 50% and spermatozoa with permeable plasma membrane and reacted acrosome were higher (63%) at week 16 than the control group. These results showed that DHA was effectively incorporated into semen through dietary supplementation with FO, but evaluations of post-thaw sperm quality confirm alteration specificity related to the structure of the lipid bilayer. Copyright © 2017 Elsevier B.V. All rights reserved.

Cryopreservative effects of the recombinant ice-binding protein from the arctic yeast Leucosporidium sp. on red blood cells.

PubMed

Lee, Sung Gu; Koh, Hye Yeon; Lee, Jun Hyuck; Kang, Sung-Ho; Kim, Hak Jun

2012-06-01

Antifreeze proteins (AFPs) have important functions in many freeze-tolerant organisms. The proteins non-colligatively lower the freezing point and functionally inhibit ice recrystallization in frozen solutions. In our previous studies, we found that the Arctic yeast Leucosporidium sp. produces an AFP (LeIBP), and that the protein could be successfully produced in Pichia expression system. The present study showed that recombinant LeIBP possesses the ability to reduce the damage induced to red blood cells (RBCs) by freeze thawing. In addition to 40 % glycerol, both 0.4 and 0.8 mg/ml LeIBPs significantly reduced freeze-thaw-induced hemolysis at either rapid- (45 °C) or slow-warming (22 °C) temperatures. Post-thaw cell counts of the cryopreserved RBCs were dramatically enhanced, in particular, in 0.8 mg/ml LeIBP. Interestingly, the cryopreserved cells in the presence of LeIBP showed preserved cell size distribution. These results indicate that the ability of LeIBP to inhibit ice recrystallization helps the RBCs avoid critically damaging electrolyte concentrations, which are known as solution effects. Considering all these data, LeIBP can be thought of as a key component in improving RBC cryopreservation efficiency.

A simple, field-friendly technique for cryopreserving semen from Asian elephants (Elephas maximus).

PubMed

Arnold, Danielle M; Gray, Charlie; Roth, Terri L; Mitchell, Sebastian; Graham, Laura H

2017-07-01

The specific objectives of the present study were to investigate the effects of manual seeding, differing freeze and thaw rates as well as storage for 24h at 4°C prior to cryopreservation on post-thaw sperm quality in Asian elephants. Extended semen was cooled in an equitainer to 4°C, frozen in liquid nitrogen vapour at various rates with and without manual seeding or in a dry shipper and thawed at 37, 50 and 75°C. There was a significant effect of freeze rate on post-thaw motility (P<0.0001) and acrosomal integrity (P<0.005). The faster freeze rates in the dry shipper and at 1cm or 2cm above liquid nitrogen consistently provided better cryopreservation than slower freezing rates. Thaw temperature had no effect on post-thaw semen quality but there was an interaction between freeze and thaw rates with higher thaw rates resulting in superior post-thaw semen quality in straws frozen at fast rates. Storage of samples prior to freezing had a detrimental effect on post-thaw semen quality. In summary, our results indicate cooling extended semen in an equitainer and cryopreserving it by placing straws directly in a dry shipper is a simple technique for effectively cryopreserving Asian elephant semen in the field or zoo. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of the holding time at 15 °C prior to cryopreservation, the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation.

PubMed

Tomás, Cristina; Gómez-Fernández, José; Gómez-Izquierdo, Emilio; de Mercado, Eduardo

2014-01-30

The aim of the present study was to evaluate the effect of the holding time at 15 °C prior to cryopreservation (2, 4 and 8h), thawing rate (37 °C for 20s or 70 °C for 8s) and post-thaw incubation temperature (15 °C or 37 °C) on the post-thaw boar sperm quality. These are important time periods in the freezing-thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240 min of incubation. The results show that with the holding time at 15 °C prior to cryopreservation there was not a clear effect until at least 24h of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70 °C for 8s, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37 °C for 20s. In addition after thawing the sperm samples incubated at 15 °C had a sustained sperm quality for longer, up to 4h post-thawing. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of freezing and thawing rates on the post-thaw viability of boar spermatozoa frozen in FlatPacks and Maxi-straws.

PubMed

Eriksson, B M; Rodriguez-Martinez, H

2000-11-01

The effects of different freezing and thawing rates on the post-thaw motility and membrane integrity of boar spermatozoa, processed as split samples in Maxi-straws or flat PET-plastic packages (FlatPack) were studied. A programmable freezing device was used to obtain freezing rates of either 20, 50 or 80 degrees C/min. Thawing of the samples was performed in a bath of circulating water; for 40s at 50 degrees C or 27s at 70 degrees C for Maxi-straws and 23s at 35 degrees C, 13s at 50 degrees C or 8s at 70 degrees C for the FlatPacks. Sperm motility was assessed both visually and with a computer assisted semen analysis (CASA) apparatus, while plasma membrane integrity was assessed using the fluorescent probes Calcein AM and ethidium homodimer-1. Temperature changes during freezing and thawing were monitored in both forms of packaging. Values for motile spermatozoa, sperm velocity and lateral head displacement variables were significantly (p<0.05) higher for samples frozen in FlatPacks than in Maxi-straws, with superior results at higher thawing rates. Freezing at 50 degrees C/min yielded better motility than 20 or 80 degrees C/min, although the effect was rather small. Neither freezing rate nor thawing rate had any effect on membrane integrity (p>0.05). A significant boar effect was seen for several parameters. The most striking difference in temperature courses between containers was a 4-5-fold lowering of the thawing rate, between -20 and 0 degrees C, in the center of the Maxi-straw, compared with the FlatPack. This is apparently due to the insulating effect of the thawed water in the periphery of the Maxi-straw. The improvement in sperm motility seen when using the FlatPack appears to be related to the rapid thawing throughout the sample, which decreases the risk of cell damage due to recrystallization during thawing. Since sperm motility patterns have been reported to be correlated with fertility both in vitro and in vivo it is speculated that the use of the FlatPack might improve the results when using frozen-thawed boar spermatozoa for artificial insemination.

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa.

PubMed

Heise, A; Thompson, P N; Gerber, D

2011-02-01

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa. Copyright © 2010 Elsevier B.V. All rights reserved.

Post-thaw amendment of cryopreserved sperm for use in artificial insemination of a viviparous fish, the green swordtail Xiphophorus helleri

PubMed Central

Dong, Qiaoxiang; Huang, Changjiang; Tiersch, Terrence R.

2017-01-01

Sperm cryopreservation protocols have been developed for live-bearers such as the green swordtail Xiphophorus helleri and the platyfish Xiphophorus couchianus. Despite the high post-thaw motility (~75%) obtained in both species, the requirements of sperm storage within the female reproductive tract coupled with the process of internal fertilization place functional demands upon cryopreserved sperm samples far beyond those of oviparous species. The purpose of this study was to facilitate the artificial insemination process with cryopreserved sperm of X. helleri through evaluation of parameters related to sperm quality after thawing. Specifically, this study evaluated the effects on motility for fresh and thawed sperm samples of centrifugation (for concentration of sperm and washing for removal of cryoprotectant), ionic composition, and additions of glucose and fetal bovine serum (FBS) in extender solutions. Centrifugation at 1000 ×g for 10 min at 4 °C was found to have no adverse effects on sperm motility of fresh samples, and for cryopreserved samples, the removal of glycerol by washing yielded higher and longer post-thaw motility (e.g., 168 h vs. 48 h for the controls). Suspension of fresh sperm samples in magnesium-free Hanks’ balanced salt solution (HBSS) did not affect motility; however, HBSS prepared with the absence of potassium or calcium, and the use of unsupplemented saline (NaCl alone) as extenders significantly reduced sperm motility. The presence of glucose in HBSS yielded higher and longer motility for fresh and thawed samples, but addition of glucose at greater than 2 g/L were unnecessary. Addition of 20% FBS prior to freezing was found to increase the post-thaw motility significantly compared to control treatment with 14% glycerol alone. Also addition of 20% FBS after thawing and centrifugation was found to induce the formation of sperm bundles, which may be beneficial for internal fertilization success. In conclusion, concentration of sperm and the removal of cryoprotectant (through centrifugation), and the addition of 20% FBS in the extender is recommended for future insemination trials with cryopreserved samples. PMID:29269962

Effect of increasing equilibration time of diluted bull semen up to 72 h prior to freezing on sperm quality parameters and calving rate following artificial insemination.

PubMed

Murphy, E M; Eivers, B; O'Meara, C M; Lonergan, P; Fair, S

2018-03-01

An equilibration period of approximately 3-4 h prior to semen cryopreservation is standard practice for maintaining membrane integrity and motility of bull sperm. However, a number of studies indicate that an overnight equilibration period prior to freezing results in improved post-thaw semen quality thus optimising pregnancy rates. The aim of this study was to assess the effect of increasing the equilibration time of bull semen up to 72 h before freezing on sperm quality parameters and calving rate (CR) following artificial insemination (AI) with frozen-thawed semen. The effect of holding semen at 4 °C for 6, 24, 48 or 72 h post dilution before freezing on subsequent post-thaw total and progressive motility (Experiment 1) and field fertility (n = 1640 inseminations, Experiment 2) of frozen-thawed semen was assessed. Equilibration time did not affect post-thaw total and progressive motility (P > 0.05). In addition, there was no effect (P > 0.05) of equilibration time on field fertility with a CR of 53.3, 50.5, 51.3 and 47.3 for the 6, 24, 48 and 72 h treatments, respectively. In conclusion, increasing the equilibration time of diluted bull semen from 6 to 72 h had no significant effect on CR, within the expected range of fertility outcomes, thus providing semen processing centres with flexibility in the time which semen can be held prior to freezing. Copyright © 2017 Elsevier Inc. All rights reserved.

The combinatorial effect of different Equex STM paste concentrations, cryoprotectants and the straw-freezing methods on the post-thaw boar semen quality.

PubMed

Wu, T-W; Cheng, F-P; Chen, I-H; Yang, C-H; Tsai, M-Y; Chang, M-H; Wang, J-H; Wu, J-T

2013-02-01

This study was to evaluate the combinatorial effect (14 treatments, A-N) of different Equex STM paste concentrations, cryoprotectants and the straw-freezing method on the post-thaw boar semen quality. Two ejaculates were collected from each of nine boars (three boars from each of three breeds). Semen was diluted in extenders with different concentrations of Equex STM paste and different cryoprotectants [glycerol or dimethylacetamide (DMA)] before cryopreserving via liquid nitrogen or dry ice. Motility, viability, percentage of spermatozoa with intense acrosomal staining and with normal morphology of post-thaw sperm were evaluated. The qualities of thawed semen were best preserved in treatment H (extender with 0.5% Equex STM paste and 5% glycerol and freezing by dry ice) and were worst in treatment B (extender with 0% Equex STM paste and 5% DMA and freezing by dry ice). Significant difference (p < 0.05) was present in post-thawed sperm motility (63% vs 27%), sperm viability (70% vs 33%) and sperm acrosomal integrity rate (68% vs 29%) between treatments H and B. However, sperm proportion with normal morphology showed no significant difference among treatments (66% vs 66%; p > 0.05). Moreover, statistical analysis suggests that no significant difference was present in semen quality among breed or individual donors (p > 0.05). These findings suggest that Equex STM paste improved the cryosurvival efficiency of boar sperm, and the favourable straw-freezing method changes between glycerol and DMA. © 2012 Blackwell Verlag GmbH.

Protein and solute distribution in drug substance containers during frozen storage and post-thawing: a tool to understand and define freezing-thawing parameters in biotechnology process development.

PubMed

Kolhe, Parag; Badkar, Advait

2011-01-01

Active pharmaceutical ingredient for biotechnology-based drugs, commonly known as drug substance (DS), is often stored frozen for longer shelf-life. Freezing DS enhances stability by slowing down reaction rates that lead to protein instability, minimizes the risk of microbial growth, and eliminates the risk of transport-related stress. High density polyethylene bottles are commonly used for storing monoclonal antibody DS due to good mechanical stress/strain resistant properties even at low temperatures. Despite the aforementioned advantages for frozen storage of DS, this is not devoid of risks. Proteins are known to undergo ice-water surface denaturation, cryoconcentration, and cold denaturation during freezing. A systematic investigation was performed to better understand the protein and solute distribution along with potential of aggregate formation during freeze and thaw process. A significant solute and protein concentration gradient was observed for both frozen and thawed DS bottles. In case of thawed DS, cryoconcentration was localized in the bottom layer and a linear increase in concentration as a function of liquid depth was observed. On the other hand, for frozen DS, a "bell shaped" cryoconcentration distribution was observed between the bottom layers and centre position. A cryoconcentration of almost three-fold was observed for frozen DS in the most concentrated part when freezing was conducted at -20 and -40 °C and 2.5-fold cryoconcentration was observed in the thawed DS before mixing. The information obtained in this study is critical to design freeze thaw experiments, storage condition determination, and process improvement in manufacturing environment. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration, freezing rate and thawing rate on post-thaw semen quality.

PubMed

Iaffaldano, N; Di Iorio, M; Miranda, M; Zaniboni, L; Manchisi, A; Cerolini, S

2016-04-01

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality. 2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN2) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined. 3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found. 4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN2 surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.

Freezability of water buffalo bull (Bubalus bubalis) spermatozoa is improved with the addition of curcumin (diferuoyl methane) in semen extender.

PubMed

Shah, S A H; Andrabi, S M H; Qureshi, I Z

2017-10-01

Effects of curcumin as antioxidant in extender were evaluated on freezability of buffalo spermatozoa. Semen from each of the five bulls (n = 3 replicates, six ejaculates/bull, a total of 30 ejaculates) was diluted in Tris-citric acid extender containing curcumin (0.5, 1.0, 1.5 or 2.0 mM) or control. At pre-freezing and post-thawing, total antioxidant contents (μM/L) and lipid peroxidation levels (μM/ml) were higher (p < .05) and lower (p < .05) respectively, with 1.5 and 2.0 mM compared to 0.5 and 1.0 mM curcumin and control. At post-thawing, progressive motility (PM, %) and rapid velocity (RV, %) were higher (p < .05) with 1.5 mM compared to other doses of curcumin and control (except in case of RV, 1.5 was similar with 1.0 mM). Kinematics (average path velocity, μm/s; straight-line velocity, μm/s; curved-line velocity, μm/s; straightness, %; linearity, %), in vitro longevity (%, PM and RV) and DNA integrity (%) at post-thawing were higher (p < .05) with 1.5 mM compared to control. At post-thawing, supravital plasma membrane integrity (%) and viable spermatozoa with intact acrosome (%) were higher with 1.5 compared to 2.0 mM curcumin and control. We concluded that freezability of water buffalo spermatozoa is improved with the addition of 1.5 mM curcumin in extender. © 2016 Blackwell Verlag GmbH.

Concentration dependence of the cell membrane permeability to cryoprotectant and water and implications for design of methods for post-thaw washing of human erythrocytes.

PubMed

Lahmann, John M; Benson, James D; Higgins, Adam Z

2018-02-01

For more than fifty years the human red blood cell (RBC) has been a widely studied model for transmembrane mass transport. Existing literature spans myriad experimental designs with varying results and physiologic interpretations. In this review, we examine the kinetics and mechanisms of membrane transport in the context of RBC cryopreservation. We include a discussion of the pathways for water and glycerol permeation through the cell membrane and the implications for mathematical modeling of the membrane transport process. In particular, we examine the concentration dependence of water and glycerol transport and provide equations for estimating permeability parameters as a function of concentration based on a synthesis of literature data. This concentration-dependent transport model may allow for design of improved methods for post-thaw removal of glycerol from cryopreserved blood. More broadly, the consideration of the concentration dependence of membrane permeability parameters may be important for other cell types as well, especially for design of methods for equilibration with the highly concentrated solutions used for vitrification. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanism of freeze-thaw injury and recovery: A cool retrospective and warming up to new ideas.

PubMed

Arora, Rajeev

2018-05-01

Understanding cellular mechanism(s) of freeze-thaw injury (FTI) is key to the efforts for improving plant freeze-tolerance by cultural methods or molecular/genetic approaches. However, not much work has been done in the last 25+ years to advance our understanding of the nature and cellular loci of FTI. Currently, two FTI lesions are predominantly implicated: 1) structural and functional perturbations in plasma membrane; 2) ROS-induced oxidative damage. While both have stood the test of time, many questions remain unresolved and other potentially significant lesions need to be investigated. Additionally, molecular mechanism of post-thaw recovery (PTR), a critical component of frost-survival, has not been well investigated. Mechanistic understanding of repair after reversible injury could expand the options for strategies to improve frost-hardiness. In this review, without claiming to be exhaustive, I have attempted to synthesize major discoveries from last several decades on the mechanisms of FTI and the relatively little research conducted thus far on PTR mechanisms. It is followed by proposing of hypotheses for mechanism(s) for irreversible FTI or PTR involving cytosolic calcium and ROS signaling. Perspective is presented on some unresolved questions and research on new ideas to fill the knowledge gaps and advance the field. Copyright © 2018 Elsevier B.V. All rights reserved.

A preliminary study on using autologous and heterologous boar sperm supernatant from freezing processes as post-thawing solution: its effect on sperm motility.

PubMed

Kaeoket, Kampon; Chanapiwat, Panida; Tummaruk, Padet; Techakumphu, Mongkol; Kunavongkrit, Annop

2011-06-01

The aim of this study was to evaluate the effect of post-thawing dilution with autologous and heterologous sperm supernatant on motility of frozen-thawed boar spermatozoa. During the cryopreservation, sperm supernatant (a combination of seminal plasma and semen extender, 50% v/v) or seminal plasma from nine boars (Duroc, Large White, and Landrace; three in each) was collected by centrifugation and stored frozen until use as post-thawing solution. Sperm pellets were further processed and cryopreserved using control-rate freezer and was thawed at 50°C for 12 s. After thawing, frozen thawed semen samples were diluted with seminal plasma (group A), supernatant from Landrace (group B), supernatant from Large White (group C), supernatant from Duroc (group D), and Modena™ semen extender (group E). Post-thawing motility was evaluated using a phase-contrast microscope after thawing at 1, 10, 20, and 30 min. The present results show that at 1 min, a significantly higher percentage (P ≤ 0.001) of progressive motility was found in groups B (53.3%) and C (53.9%) than the other groups. At 10 min, the highest (P ≤ 0.001) progressive motility was found in groups B (65%) and C (61%). At 20 and 30 min, a significantly higher percentage (P ≤ 0.001) of progressive motility was found in groups B (58.9%), C (53.5%), and D (45.6%) than groups A (3.9%) and E (20.6%). It can be stated that supernatant from the freezing processes (consisting of seminal plasma and Modena™, 50% v/v) had a beneficial effect on post-thawing progressive motility of frozen boar semen.

Determination of glutation peroxidase and superoxide dismutase activities in canine seminal plasma and its relation with sperm quality and lipid peroxidation post thaw.

PubMed

Neagu, V R; García, B Macías; Rodríguez, A Morillo; Ferrusola, C Ortega; Bolaños, J M Gallardo; Fernández, L González; Tapia, J A; Peña, F J

2011-01-01

Lipid peroxidation (LPO) of dog spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezeability. Innate levels of LPO were low in fresh spermatozoa but increased after thawing in one of the dogs included in our study. The level of lipid peroxidation in fresh spermatozoa was not correlated with that of thawed spermatozoa. Negative correlations were detected between the activity in seminal plasma of GPx and sperm velocities post thaw (P < 0.01), however SOD activity was positively correlated with the percentage of linear motile sperm post thaw (P < 0.05). Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of thawing methods on frozen semen quality of yak (Poephagus grunniens L.) bulls

PubMed Central

Borah, Binod Kumar Dutta; Deka, Bharat Chandra; Biswas, Ranjan Kumar; Chakravarty, Prithiviraj; Deori, Sourabh; Sinha, Sudip; Ahmed, Kutubuddin

2015-01-01

Aim: To evaluate different thawing temperatures and duration on the post-thaw semen quality of Indian yaks bulls. Materials and Methods: Semen ejaculates from four different yak bulls were collected using artificial vagina method and extended with tris extender containing 6.4% glycerol at 35°C, cooled gradually from 35°C to 5°C at 1°C/3 min and equilibrated at 4-5°C for 4 h and frozen in French mini straws using a programmable bio-freezer and finally stored in liquid nitrogen. Thawing of frozen semen straws was carried out using three methods i.e., 35°C for 60 s (thawing method I), 37°C for 30 s (thawing method II) and 75°C for 9 s (thawing method III). The post-thaw semen quality parameters assessed were sperm motility, percent live sperm, hypo-osmotic swelling test (HOST)-reacted sperm, acrosomal changes, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the extracellular media. Results: The percent sperm motility, total incidence of acrosomal changes, and extracellular release of AST varied significantly (p<0.01) between thawing methods but live sperm and HOST-reacted sperm did not vary significantly between thawing methods. The percent sperm motility of frozen yak semen for thawing method III was significantly (p<0.05) higher than that for thawing methods I and II, the difference between thawing methods I and II being non-significant. The critical difference test revealed that the total incidence of acrosomal changes and extracellular release of AST were significantly (p<0.05) lower when thawing was done using methods I and II than in method III. Conclusion: On the basis of the present experiment, we can conclude that barring the post-thaw sperm motility, thawing of frozen yak semen in water either at 35°C for 60 s or 37°C for 30 s gives better post-thaw semen quality than at 75°C for 09 s. PMID:27047161

The state of T cells before cryopreservation: Effects on post-thaw proliferation and function.

PubMed

Luo, Ying; Wang, Peng; Liu, Hui; Zhu, Zhengyan; Li, Chenglong; Gao, Yingtang

2017-12-01

We aim to assess the effect of the state of T cells before cryopreservation on the post-thaw proliferative capacity, phenotype and functional response. Peripheral blood mononuclear cells (PBMCs) were isolated from a hepatocellular carcinoma (HCC) patient, and the T cells were frozen during cell culture according to our experimental design. After a period of re-culture, the proliferative capacity of the cryopreserved cells, the expression of T cell surface markers and the secretion of IFN-γ and IL-10 were assayed. There was >90% cell viability after thaw in every group. Lymphocytes cryopreserved at day 4, 8 or 12 during the cell culture were allowed to recover for 24 h, whereas lymphocytes cryopreserved while freshly isolated were allowed to recover for 72 h. After the period of re-culture, cryopreservation at day 4, 8 or 12 during T cell culture was not found to alter the T cell subpopulation. The proportions of NKT and Treg cells were unchanged when cells were cryopreserved at day 12 during T cell culture. IFN-γ secretion was not impacted by cryopreservation, and IL-10 secretion was significantly decreased when cells were cryopreserved at day 8 or 12 during T cell culture. The state of T cells before cryopreservation has effects on the post-thaw proliferation capacity, the phenotype and the secretion of IFN-γ and IL-10. Cryopreservation of lymphocytes at day 8 or 12 during the cell culture may be the best choice for T cell immunotherapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Cryopreservation of turkey semen: effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching

USDA-ARS?s Scientific Manuscript database

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw funct...

Cryopreservation of red blood cells.

PubMed

Lagerberg, Johan W

2015-01-01

Cryopreservation of red blood cell concentrates (RBCs) is an important method for maintaining an inventory of rare RBC units and managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect RBCs against freezing damage. The use of thawed RBCs was hampered a 24-h outdating period due to potential bacterial contamination when a functionally open system was used for addition and removal of the glycerol. With the introduction of a functionally closed system for the glycerolization and deglycerolization of RBC units, extended post-thaw storage became possible. Here, we describe the cryopreservation of red blood cells according to the high-glycerol method, using a functionally closed processing system.

Improvement of common carp (Cyprinus carpio) sperm cryopreservation using a programable freezer.

PubMed

Bernáth, Gergely; Żarski, Daniel; Kása, Eszter; Staszny, Ádám; Várkonyi, Levente; Kollár, Tímea; Hegyi, Árpád; Bokor, Zoltán; Urbányi, Béla; Horváth, Ákos

2016-10-01

The applicability of a programmable freezer for the increased-scale cryopreservation of common carp sperm was investigated. The effect of different equilibration times, cryopreservation methods, extenders, dilution ratios, activating solutions on the post-thaw motility of common carp sperm was investigated. The suitable post-thaw storage time-interval as well as fertilizing capacity of cryopreserved sperm was also examined. The motility, curvilinear velocity (VCL) and straightness (STR) values did not decrease significantly during 60min of equilibration neither in equilibrated nor thawed groups. Motility parameters of thawed sperm were similar using a conventional cryopreservation technique using a polystyrene box [motility (33%), VCL (47μm/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54μm/s) and STR (89%)]. The highest motility and VCL was measured with a sugar based extender (grayling extender) at a ratio 1:9 (motility: 52%, VCL: 76μm/s) and 1:20 (motility: 49%, VCL: 76μm/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2min. A storage time of six hours following thawing did not have a significant effect on the motility parameters of thawed carp sperm. Agglutination was observed during cryopreservation of an elevated volume of sperm whereas motility 47%, VCL 62μm/s and STR 91% were measured after thawing. Fertilization rate with thawed sperm (32%) was significantly lower compared to the control group (73%). According to our results, the developed method using a programmable freezer is suitable for the cryopreservation of elevated number of straws. However, carp sperm agglutination during freezing may have a negative effect on the fertilizing capacity. Copyright © 2016 Elsevier Inc. All rights reserved.

Seminal plasma applied post-thawing affects boar sperm physiology: a flow cytometry study.

PubMed

Fernández-Gago, Rocío; Domínguez, Juan Carlos; Martínez-Pastor, Felipe

2013-09-01

Cryopreservation induces extensive biophysical and biochemical changes in the sperm. In the present study, we used flow cytometry to assess the capacitation-like status of frozen-thawed boar spermatozoa and its relationship with intracellular calcium, assessment of membrane fluidity, modification of thiol groups in plasma membrane proteins, reactive oxygen species (ROS) levels, viability, acrosomal status, and mitochondrial activity. This experiment was performed to verify the effect of adding seminal plasma on post-thaw sperm functions. To determine these effects after cryopreservation, frozen-thawed semen from seven boars was examined after supplementation with different concentrations of pooled seminal plasma (0%, 10%, and 50%) at various times of incubation from 0 to 4 hours. Incubation caused a decrease in membrane integrity and an increase in acrosomal damage, with small changes in other parameters (P > 0.05). Although 10% seminal plasma showed few differences with 0% (ROS increase at 4 hours, P < 0.05), 50% seminal plasma caused important changes. Membrane fluidity increased considerably from the beginning of the experiment, and ROS and free thiols in the cell surface increased by 2 hours of incubation. By the end of the experiment, viability decreased and acrosomal damage increased in the 50% seminal plasma samples. The addition of 50% of seminal plasma seems to modify the physiology of thawed boar spermatozoa, possibly through membrane changes and ROS increase. Although some effects were detrimental, the stimulatory effect of 50% seminal plasma could favor the performance of post-thawed boar semen, as showed in the field (García JC, Domínguez JC, Peña FJ, Alegre B, Gonzalez R, Castro MJ, Habing GG, Kirkwood RN. Thawing boar semen in the presence of seminal plasma: effects on sperm quality and fertility. Anim Reprod Sci 2010;119:160-5). Copyright © 2013 Elsevier Inc. All rights reserved.

Hydroxyethyl starch as a substitute for dextran 40 for thawing peripheral blood progenitor cell products.

PubMed

Zhu, Fenlu; Heditke, Sarah; Kurtzberg, Joanne; Waters-Pick, Barbara; Hari, Parameswaran; Margolis, David A; Keever-Taylor, Carolyn A

2015-12-01

Removing DMSO post-thaw results in: reduced infusion reactions, improved recovery and stability of viable CD34+ cells. Validated methods use 5%-8.3% Dextran 40 with 2.5%-4.2% HSA for this purpose. Recent shortages of clinical grade Dextran require identification of suitable alternatives. PBPC were used to compare a standard 2X wash medium of 5 parts 10% Dextran 40 in saline (DEX) with 1 part 25% HSA (8.3% DEX/ 4.2% HSA) with Hydroxyethyl Starch (HES)-based solutions. Cells in replicate bags were diluted with an equal volume of wash solution, equilibrated 5 minutes, the bag filled with wash medium, pelleted and the supernatant expressed. Bags were restored to the frozen volume in wash medium and tested by single platform flow cytometry and CFU. Total viability, viable TNC, MNC, and CD34+ cell recovery, and CD34+ cell viability were compared immediately post-thaw and after 90 minutes. 5.2% HES/4.2% HSA did not differ from our standard in CD34 recovery or viability. Due to concerns that high concentrations of HES could affect renal function we tested 0.6% HES/2.5% HSA resulting in significantly poorer CD34 recovery and viability. Results improved using 2.4% HES/4.2% HSA and when 0.6% HES/4.2%HSA was used no significant differences were seen. CFU assays confirmed no differences between the standard dextran arm and HES at 2.4% or 0.6% so long as HSA was at 4.2%. We conclude that HES from 0.6% to 5.2% with 4.2% HSA is a suitable substitute for Dextran 40 as a reconstitution/washing medium for PBPC products. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Leptin Improves Sperm Cryopreservation via Antioxidant Defense

PubMed Central

Fontoura, Paula; Mello, Mariana Duque; Gallo-Sá, Paulo; Erthal-Martins, Maria Cecília; Cardoso, Maria Cecília Almeida; Ramos, Cristiane

2017-01-01

Background: Leptin and its receptor are present in spermatozoa; however, the role of leptin in sperm function is still controversial. Our present study aimed at demonstrating the effect of cryopreservation on sperm DNA fragmentation (DNAf) and investigating the possible effects of sperm capacitation techniques and leptin in vitro incubation on frozen-thawed sperm DNAf and oxidative stress. Methods: Samples of 45 normospermic men attending for infertility investigation at Vida Centro de Fertilidade, Rio de Janeiro, Brazil, were frozen and thawed with or without capacitation and leptin incubation prior to freezing. Sperm DNA fragmentation was evaluated by Sperm Chromatin Dispersion Assay before and after cryopreservation and oxidative stress parameters were measured by spectrophotometry with and without leptin incubation. Statistical analysis was performed using paired t test to compare DNAf between groups before and after freeze-thaw cycle, to compare groups before and after capacitation and leptin incubation and oxidative measurements before and after leptin incubation. Statistical significance was considered when p≤0.05. Results: Our results revealed a significant post-thaw rise in sperm DNAf compared with fresh samples (p=0.0003). Sperm DNAf was significantly reduced when sperm capacitation was performed before freezing, when compared to those frozen with no previous capacitation (p=0.01). The addition of leptin to capacitated sperm before freezing reduced DNAf (p<0.0001) and enhanced superoxide dismutase (p=0.001) and glutathione peroxidase (p=0.02) antioxidant enzymes activity. Conclusion: The addition of leptin to capacitated sperm can improve sperm DNA quality following cryopreservation, possibly by inducing the activity of certain antioxidant enzymes. PMID:28377896

Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-based extenders in ram sperm cryopreservation.

PubMed

Najafi, Abouzar; Daghigh-Kia, Hossein; Dodaran, Hossein Vaseghi; Mehdipour, Mahdieh; Alvarez-Rodriguez, Manuel

2017-02-01

The aim of this study was to evaluate the effects of glycerol, ethylene glycol or DMSO in a soybean lecithin extender for freezing ram semen. In this study, 20 ejaculates were collected from four Ghezel rams and diluted with soybean lecithin extender with glycerol (7%), ethylene glycol (3%, 5% and 7%) or DMSO (3%, 5% and 7%). Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality, mitochondrial activity (Rhodamine 123) and apoptotic features (Annexin V/Propidium iodide) were assessed after thawing. There was no significant difference between glycerol and ethylene glycol at different concentrations (3% and 5%) regarding sperm total and progressive motility, viability, and membrane integrity. The least percentages of mitochondrial functionality were observed in samples frozen with all different DMSO concentrations tested (P<0.05). Moreover, the percentage of post-thawed dead sperm was the greatest for all the DMSO concentrations compared with other groups (P<0.05). Thus, DMSO had an adverse effect on the post thaw ram sperm parameters. In contrast, ethylene glycol could be a desirable substitute of glycerol in the freezing extender, in view of similar results obtained in post-thaw quality of ram semen cryopreserved in a soybean lecithin extender. We propose that glycerol in a soybean lecithin based extender could be replaced by ethylene glycol at 3% or 5% concentrations. Copyright © 2016 Elsevier B.V. All rights reserved.

Thermal Destabilization of Collagen Matrix Hierarchical Structure by Freeze/Thaw

PubMed Central

Ozcelikkale, Altug; Han, Bumsoo

2016-01-01

This study aims to characterize and understand the effects of freezing on collagen structures and functionality. Specifically, thermodynamic destabilization of collagen at molecular- and fibril-levels by combination of low temperatures and freezing were experimentally characterized using modulated differential scanning calorimetry. In order to delineate the effects of sub-zero temperature and water-ice phase change, we hypothesized that the extent of destabilization can be determined based on post-thaw heat induced thermal denaturation of collagen. It is found that thermal denaturation temperature of collagen in hydrogel decreases by 1.4–1.6°C after freeze/thaw while no such decrease is observed in the case of molecular solution. The destabilization is predominantly due to ice formation. Exposure to low temperatures in the absence of ice has only minimal effect. Calorimetry measurements combined with morphological examination of collagen matrices by scanning electron microscopy suggest that freezing results in destabilization of collagen fibrils due to expansion of intrafibrillar space by ice formation. This fibril-level damage can be alleviated by use of cryoprotectant DMSO at concentrations as low as 0.5 M. A theoretical model explaining the change in collagen post-thaw thermal stability by freezing-induced fibril expansion is also proposed. PMID:26765741

Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm.

PubMed

Murphy, C; English, A M; Holden, S A; Fair, S

2014-03-01

An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples were centrifuged and diluted to 100×10(6)sperm/mL, frozen in 0.5mL straws and stored in liquid nitrogen. Sperm from each treatment was assessed for progressive linear motility (PLM) and acceptable membrane integrity under hypotonic conditions on a phase contrast microscope at 1000× while viability, membrane fluidity and superoxide generation were assessed by flow cytometry. The MβCD1.5 and MβCD0.75 treatments had a greater proportion of viable sperm than the TALP treatment (P<0.01). There was no effect of treatment on PLM or membrane integrity. The MβCD1.5 treatment had a greater proportion of viable sperm positive for membrane fluidity than the TALP treatment (P<0.05). The MβCD1.5 and MβCD0.75 treatments had a lesser proportion of viable sperm positive for superoxide generation than the TALP treatment (P<0.001). This study has demonstrated that adding cholesterol to stallion sperm prior to cryopreservation increases post-thaw viability, with these viable sperm being of better quality in terms of increased membrane fluidity and reduced superoxide generation. Copyright © 2014 Elsevier B.V. All rights reserved.

TEMPORARY STORAGE OF BOVINE SEMEN CRYOPRESERVED IN LIQUID NITROGEN ON DRY ICE AND REFREEZING OF FROZEN-THAWED SEMEN.

PubMed

Abdussamad, A M; Gauly, M; Holtz, W

2015-01-01

Two experiments were conducted. The purpose of Experiment 1 was to investigate whether viability of bovine semen stored in liquid nitrogen (-196°C) will be adversely affected by temporary exposure to dry ice (-79°C). It was convincingly shown that post thaw-motility was not affected, regardless whether semen was thawed immediately or after being returned to liquid nitrogen. Shipping or temporary storage on dry ice, thus, is a viable option. In Experiment 2, refreezing of frozen-thawed semen was attempted. The proportion of motile spermatozoa was reduced by a factor of ten to between 6.0 % and 7.4 %, regardless whether thawing occurred directly after removal from liquid nitrogen or after an interim period on dry ice. When semen was refrozen on dry ice before being returned to liquid nitrogen, motility rates were significantly improved (13.0 % to 17.0 %, P<0.05). In both experiments sperm cells that remained motile displayed vigorous forward movement and normal morphological appearance.

Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants.

PubMed

Wu, Zhuangyuan; Zheng, Xinbiao; Luo, Yongming; Huo, Fei; Dong, Hong; Zhang, Guoting; Yu, Weihao; Tian, Fang; He, Liangjun; Chen, Jingbo

2015-12-01

The present study investigates the effects of five cryoprotectants (CPAs) and cryoprotectant combinations on the post-thaw total motility, progressive motility, viability, mitochondrial membrane potential and acrosome integrity in stallion spermatozoa. In Experiment 1, the objective was to compare the impact of different concentrations (2.5%, 3.5% and 5%) of a single CPA, including glycerol (Gly), ethylene glycol (EG), dimethyl sulphoxide (DMSO), methyl formamide (MF), and dimethylformamide (DMF) for stallion spermatozoa cryopreservation. In Experiment 2, two or more CPAs were used to assess whether this improved post-thaw spermatozoa quality. Gly, MF and DMF, were used to prepare seven combinations of freezing extender with different mixtures of cryoprotectant, and the 3.5% Gly, MF and DMF were used as a control group. The results show that post-thaw total motility, progressive motility, viability, and mitochondrial membrane potential for all concentrations of EG and DMSO were less than the 3.5% and 5% Gly and MF and 2.5% and 3.5% DMF (P<0.05). Use of the 3.5% concentration resulted in the greater post-thaw total motility and progressive motility than the 2.5% and 5% concentrations for all CPAs. The results for the use of different combinations of cryoprotectant indicate there are differences in progressive motility and viability. The viability with the use of Gly(2/3)+MF(1/3) was 44.65% and was greater than the Gly(1/3)+MF(1/3)+DMF(1/3) (30.96%), MF(2/3)+DMF(1/3) (35.05%), Gly (32.21%) and MF(33.76%) (P<0.05). The progressive motility with the use of the MF(2/3)+Gly(1/3) combination was 36.0% and was greater than in the DMF (25.0%) and MF(2/3)+DMF(1/3) (22.7%) (P<0.05). These results suggest that using the appropriate cryoprotectant combination instead of a single cryoprotectant can improve horse spermatozoa cryopreservation. Copyright © 2015 Elsevier B.V. All rights reserved.

Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze-thawing process of ram sperm.

PubMed

Motlagh, Mahdi Khodaei; Sharafi, Mohsen; Zhandi, Mahdi; Mohammadi-Sangcheshmeh, Abdollah; Shakeri, Malak; Soleimani, Masoud; Zeinoaldini, Saeed

2014-10-01

The aim of current study was to evaluate effect of rosemary aqueous extract on post-thawed ram sperm quality in a soybean lecithin-based (SL) extender. Ram semen samples were obtained, extended with SL extender and supplemented with 0% (SL-R0), 2% (SL-R2), 4% (SL-R4), 6% (SL-R6), and 8% (SL-R8) rosemary aqueous extract. Following equilibration, the straws were frozen, and then plunged into the liquid nitrogen. After thawing, sperm motility and velocity parameters, plasma membrane functionality, viability, acrosomal and capacitation status were evaluated. Membrane lipid peroxidation was also analyzed through the malondialdehyde (MDA) concentration. Our results showed that SL-R4 and SL-R6 groups resulted in higher (p < 0.05) percentages of total motility, progressive motility, and plasma membrane functionality, as compared with other groups. Highest (p < 0.05) viable and lowest (p < 0.05) dead spermatozoa were observed in SL-R6 group compared to the other groups. The acrosomal and capacitation status were not affected (p > 0.05) by different levels of rosemary aqueous extract. Lower (p < 0.05) MDA concentration has been observed in SL-R4 and SL-R6 groups. The results of this study demonstrate that supplementation of SL extender with rosemary aqueous extract influences post-thawed ram sperm quality in a dose dependent manner. Copyright © 2014 Elsevier Inc. All rights reserved.

Antioxidant Effect of Xanthan Gum on Ram Sperm after Freezing and Thawing.

PubMed

Gastal, G DA; Silva, E F; Mion, B; Varela Junior, A S; Rosa, C E; Corcini, C D; Mondadori, R G; Vieira, A D; Bianchi, I; Lucia, T

Xanthan gum is used as thickener in media to preserve food products, having cryoprotectant and antioxidant properties that may be relevant for sperm cryopreservation. To evaluate the effects of adding xanthan gum to freezing extenders on post-thawing quality and oxidant activity of ram sperm. Ejaculates from seven rams extended TRIS-egg yolk-glycerol were split in three treatments including xanthan gum (0.15%; 0.20%; and 0.25%) and a control with no xanthan gum. After thawing, motility and production of reactive oxygen species (ROS) with 0.20% and 0.25% xanthan gum were lower than for the control (P < 0.05), but mitochondrial functionality and integrity of membrane, acrosome and DNA did not differ (P > 0.05). Xanthan gum at 0.20% and 0.25% may be an efficient antioxidant for frozen-thawed ram sperm, due to the reduction in ROS production.

The effects of different levels of catalase and superoxide dismutase in modified Beltsville extender on rooster post-thawed sperm quality.

PubMed

Amini, Mahmood Reza; Kohram, Hamid; Zare-Shahaneh, Ahmad; Zhandi, Mahdi; Sharideh, Hossein; Nabi, Mohammad Mehdi

2015-06-01

Oxidative damage of sperm by means of reactive oxygen species generated by the cellular components of semen is one of the main reasons for decreased sperm motility and fertility during the freeze-thawing process. This study was conducted to determine the influence of catalase (CAT) and superoxide dismutase (SOD) on rooster sperm motility, viability and MDA level after freezing and thawing. Semen samples from 10 sexually-mature Ross 308 breeder roosters were collected and pooled, divided into nine equal parts and diluted with modified Beltsville extender containing no antioxidants (control), or supplemented with 50, 100, 200 and 300 μg/mL CAT, or 50, 100, 200 and 300 U/mL SOD. After thawing, sperm motility and motion parameters were assessed using a CASA system. Sperm viability and MDA level were assessed by eosin-nigrosin and MDA test, respectively. The results of this experiment showed that the extender supplemented with 100 and 200 μg CAT, and 50 U SOD had the highest sperm motility (P<0.05) in sperm motility. Also, addition 100, 200 and 300 μg CAT, and 50 U SOD can improve significantly viability after freeze-thaw. Extender supplemented with 100 μg CAT had significantly lower MDA level compared to control and 300 μg CAT. In conclusion, the results of the present study demonstrate that addition of CAT (100 μg/mL) and SOD (50 U/mL) independently have beneficial effect on quality of post-thawed rooster semen. Copyright © 2015 Elsevier Inc. All rights reserved.

High severity experimental burns in Siberian larch forests increase permafrost thaw and larch tree regeneration

NASA Astrophysics Data System (ADS)

Alexander, H. D.; Davydov, S.; Zimov, N.; Mack, M. C.

2013-12-01

Global change models predict increased fire activity in boreal forests as climate warms and dries. We hypothesized that fire-driven decreases in soil organic layer (SOL) depth will (1) increase permafrost thaw by reducing the insulating capacity of the SOL and (2) improve seedbed conditions for tree regeneration. Over time, these changes will lead to altered patterns of above- and belowground carbon (C) accumulation. To test these hypotheses, we conducted plot-level experimental burns in July 2012 in a low-density, mature larch stand near the Northeast Science Station in Cherskii, Siberia. Dried fuels of naturally occurring vegetation were added to plots to achieve four burn severity treatments based on residual SOL depths: control, low (> 8 cm), moderate (5-8 cm), and high severity (2-5 cm). Pre-fire and during two growing seasons post-fire, we measured thaw depth, soil moisture, and soil temperature to determine severity effects on permafrost thaw. We also sowed larch seeds in fall 2012 and quantified germination rates the following growing season. By 1 wk post-fire, thaw depth was 15-25 cm deeper in plots burned at high severity (55 cm) compared to other treatments (30-40 cm). These differences in thaw depth with burn severity were maintained during the subsequent growing season and were associated with increased soil temperature and moisture. Larch regeneration was 10x higher on severely burned plots than those unburned. Our findings highlight the potential for increased fire severity to degrade permafrost and alter successional dynamics and patterns of C accumulation.

Prehospital plasma resuscitation associated with improved neurologic outcomes after traumatic brain injury.

PubMed

Hernandez, Matthew C; Thiels, Cornelius A; Aho, Johnathon M; Habermann, Elizabeth B; Zielinski, Martin D; Stubbs, James A; Jenkins, Donald H; Zietlow, Scott P

2017-09-01

Trauma-related hypotension and coagulopathy worsen secondary brain injury in patients with traumatic brain injuries (TBIs). Early damage control resuscitation with blood products may mitigate hypotension and coagulopathy. Preliminary data suggest resuscitation with plasma in large animals improves neurologic function after TBI; however, data in humans are lacking. We retrospectively identified all patients with multiple injuries age >15 years with head injuries undergoing prehospital resuscitation with blood products at a single Level I trauma center from January 2002 to December 2013. Inclusion criteria were prehospital resuscitation with either packed red blood cells (pRBCs) or thawed plasma as sole colloid resuscitation. Patients who died in hospital and those using anticoagulants were excluded. Primary outcomes were Glasgow Outcomes Score Extended (GOSE) and Disability Rating Score (DRS) at dismissal and during follow-up. Of 76 patients meeting inclusion criteria, 53% (n = 40) received prehospital pRBCs and 47% (n = 36) received thawed plasma. Age, gender, injury severity or TBI severity, arrival laboratory values, and number of prehospital units were similar (all p > 0.05). Patients who received thawed plasma had an improved neurologic outcome compared to those receiving pRBCs (median GOSE 7 [7-8] vs. 5.5 [3-7], p < 0.001). Additionally, patients who received thawed plasma had improved functionality compared to pRBCs (median DRS 2 [1-3.5] vs. 9 [3-13], p < 0.001). Calculated GOSE and DRS scores during follow-up, median 6 [5-7] months, demonstrated increased function in those resuscitated with thawed plasma compared to pRBCs by both median GOSE (8 [7-8] vs. 6 [6-7], p < 0.001) and DRS (0 [0-1] vs. 4 [2-8], p < 0.001). In critically injured trauma patients with TBI, early resuscitation with thawed plasma is associated with improved neurologic and functional outcomes at discharge and during follow-up compared to pRBCs alone. These preliminary data support the further investigation and use of plasma in the resuscitation of critically injured TBI patients. Therapeutic, level V.

Effects of freezing-induced cell-fluid-matrix interactions on the cells and extracellular matrix of engineered tissues.

PubMed

Teo, Ka Yaw; DeHoyos, Tenok O; Dutton, J Craig; Grinnell, Frederick; Han, Bumsoo

2011-08-01

The two most significant challenges for successful cryopreservation of engineered tissues (ETs) are preserving tissue functionality and controlling highly tissue-type dependent preservation outcomes. In order to address these challenges, freezing-induced cell-fluid-matrix interactions should be understood, which determine the post-thaw cell viability and extracellular matrix (ECM) microstructure. However, the current understanding of this tissue-level biophysical interaction is still limited. In this study, freezing-induced cell-fluid-matrix interactions and their impact on the cells and ECM microstructure of ETs were investigated using dermal equivalents as a model ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in type I collagen matrices with varying cell seeding density and collagen concentration. While these dermal equivalents underwent an identical freeze/thaw condition, their spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular level cryoresponse were characterized. The results showed that the extent and characteristics of freezing-induced deformation were significantly different among the experimental groups, and the ETs with denser ECM microstructure experienced a larger deformation. The magnitude of the deformation was well correlated to the post-thaw ECM structure, suggesting that the freezing-induced deformation is a good indicator of post-thaw ECM structure. A significant difference in the extent of cellular injury was also noted among the experimental groups, and it depended on the extent of freezing-induced deformation of the ETs and the initial cytoskeleton organization. These results suggest that the cells have been subjected to mechanical insult due to the freezing-induced deformation as well as thermal insult. These findings provide insight on tissue-type dependent cryopreservation outcomes, and can help to design and modify cryopreservation protocols for new types of tissues from a pre-developed cryopreservation protocol. Copyright © 2011 Elsevier Ltd. All rights reserved.

The benefits of cooling boar semen in long-term extenders prior to cryopreservation on sperm quality characteristics.

PubMed

Wasilewska, K; Zasiadczyk, Ł; Fraser, L; Mogielnicka-Brzozowska, M; Kordan, W

2016-10-01

This study investigated the effects of long-term extenders on post-thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm-rich fractions, collected from five boars, were diluted in Androhep(®) Plus (AHP), Androstar(®) Plus (ASP), Safecell(®) Plus and TRIXcell(®) Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre-freeze and frozen-thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic-like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing-thawing. Differences in the pre-freeze semen were more marked in the sperm motion patterns between the HTs. Pre-freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO-PRO-1(-) /PI(-) ) among the extenders. Post-thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP- and ASP-extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing-thawing. In most of the extenders, the incidence of frozen-thawed spermatozoa with apoptotic-like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long-term preservation extenders modulates post-thaw sperm quality characteristics in an extender-dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen. © 2016 Blackwell Verlag GmbH.

Effect of sex-sorting and cryopreservation on the post-thaw sperm quality of Iberian red deer spermatozoa.

PubMed

Anel-López, L; García-Álvarez, O; Parrilla, I; Del Olmo, D; Maroto-Morales, A; Fernandez-Santos, M R; Ortiz, J A; Soler, A J; Martínez, E M; Vazquez, J M; Garde, J J

2017-02-01

This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitochondrial oxygen consumption is a unique indicator of stallion spermatozoal health and varies with cryopreservation media.

PubMed

Darr, Christa R; Cortopassi, Gino A; Datta, Sandipan; Varner, Dickson D; Meyers, Stuart A

2016-09-15

Mitochondrial oxygen consumption is a sensitive indicator of spermatozoal health in the context of cryopreservation. We investigated oxygen consumption of equine sperm mitochondria during incubation in four commercially available sperm cryopreservation extenders: modified INRA 96, BotuCrio, EZ Freezin-"LE" and "MFR5", in addition to several other parameters including motility, reactive oxygen species (ROS) production and viability. All experimental endpoints, with the exception of average path velocity, were affected significantly by freezing extender type after freezing and thawing. Sperm in INRA 96 had the lowest average progressive motility after thawing (24 ± 4.8%, P < 0.05). Sperm in EZ Freezin-"LE" had the highest post thaw viability (79 ± 3.1%, P < 0.05) and lowest post thaw ROS production (13 ± 2.4%), but sperm in BotuCrio had the highest maximal oxygen consumption levels, while also demonstrating similar ROS production and viability. This difference would not have been detected using conventional sperm analytical methods. In addition, sperm in BotuCrio had the highest average total motility (49 ± 7.4%), progressive motility (41 ± 6.4%), and velocity (VAP, 90 ± 3.6 μm/s) indicating that this medium preserved mitochondrial function optimally after cryopreservation. Mitochondrial oxygen consumption was positively correlated with traditional measures of sperm function including motility and viability (r = 0.62 and r = 0.49, respectively, P < 0.05), thus making it a sensitive method for determining cryopreservation success and mitochondrial function in stallion sperm. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel and traditional traits of frozen-thawed porcine sperm related to in vitro fertilization success

USDA-ARS?s Scientific Manuscript database

Cryopreserved semen allows the use of single ejaculates for repeated analyses, potentially improving in vitro fertilization (IVF) consistency by eliminating inter-ejaculate variability observed with fresh semen. However, the freezing and thawing processes result in compromised sperm function and IVF...

Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

PubMed

Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

2017-03-01

Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and motility parameters. These findings, although preliminary, suggest that resveratrol-induced improvement of cryopreserved sperm functions may be mediated through activation of AMP-activated protein kinase, indicating the importance of AMP-activated protein kinase activity for human spermatozoa functions. Further investigations are required to elucidate the mechanism by which resveratrol ameliorates oxidative stress-mediated damages in an AMP-activated protein kinase-dependent mechanism. © 2016 American Society of Andrology and European Academy of Andrology.

Meat quality of lamb frozen stored up to 21 months: instrumental analyses on thawed meat during display.

PubMed

Muela, E; Monge, P; Sañudo, C; Campo, M M; Beltrán, J A

2015-04-01

The study analysed the effect of frozen storage duration (FSD: 0, 1, 9, 15 or 21 months) and display duration (DD: 0-24 h post-slaughter-, 3 and 6 days) in modified atmosphere packaging (MAP) on lamb quality. pH, colour, lipid oxidation, water holding capacity and instrumental texture were performed on Longissimus muscle in displayed fresh and thawed meat. FSD affected all the variables showing lower differences between fresh and 1 month storage than among them and longer FSD. Only cooking losses were not affected by DD in thawed meats. It was observed a general decrease in quality (lower redness and water holding capacity; higher yellowness and lipid oxidation) as FSD or DD increased and only texture was improved over DD being thawed meat more tender. In conclusion, lamb storage at -18°C should not exceed 1 month if thawed meat would be later displayed in MAP while meat would have an acceptable quality up to 21 months without subsequent display. Copyright © 2015 Elsevier Ltd. All rights reserved.

Colour Changes in Meat of Foals as Affected by Slaughtering Age and Post-thawing Time

PubMed Central

De Palo, P.; Maggiolino, A.; Centoducati, P.; Tateo, A.

2012-01-01

The aim of the present work was to investigate how colour changes of foal meat can vary after thawing out in relation to the slaughtering age of the horses and to the post-thawing time. Eighteen Italian Heavy Draught Horse (IHDH) foals were used for the trial. They were subdivided in three groups according to their slaughtering age (6, 11 and 18 months). Two different surfaces were investigated for each sample: a fresh cut surface (daily renewed cutting surface: DRCS), and not-renewed cutting surface (NRCS). The redness of both investigated surfaces increased with slaughtering age (p<0.01). Moreover, this parameter decreased during post-thawing time (p<0.01) only on the NRCS, probably due to the myoglobin oxidation processes. Colour is an important visual cue denoting perceived quality by consumers. So, by a chromatic perspective the thawed meat of IHDH foals slaughtered at 6 and 11 months proved to be that which best meets the market requirements. PMID:25049544

Effect of various concentrations of butylated hydroxyanisole and butylated hydroxytoluene on freezing capacity of Turkman stallion sperm.

PubMed

Seifi-Jamadi, Afshin; Kohram, Hamid; Zareh-Shahne, Ahmad; Dehghanizadeh, Parvaneh; Ahmad, Ejaz

2016-07-01

The present study aimed to determine the effect of different concentrations of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on post-thaw stallion sperm quality. The ejaculates collected from four healthy mature Turkmen stallions were pooled and divided into eight aliquots. The samples were diluted with extenders containing different concentrations (0.5, 1 or 2mM/mL) of BHA or BHT. The positive control (PC) samples were diluted with extender containing 0.5% ethanol (v/v) whereas; the negative control (NC) samples were diluted with basic extender only. Semen samples were frozen according to a standard protocol. After thawing of samples, sperm motility, viability, membrane integrity, total abnormality and lipid peroxidation were assessed. The greatest (P<0.05) values for total sperm motility, viability and plasma membrane functionality and least values for malonedialdehyde (MDA) concentration were observed in samples supplemented either with 1mM BHT or 2mM BHA. However, the progressive motility was greater (P<0.05) only in samples treated with 2mM BHA. In conclusion, the use of 1mM BHT or 2mM BHA in extender improves the freezing capacity of stallion sperm by reducing oxidative stress during freeze-thaw process. Copyright © 2016 Elsevier B.V. All rights reserved.

Thermomechanical analysis of freezing-induced cell-fluid-matrix interactions in engineered tissues

PubMed Central

Han, Bumsoo; Teo, Ka Yaw; Ghosh, Soham; Dutton, J. Craig; Grinnell, Frederick

2012-01-01

Successful cryopreservation of functional engineered tissues (ETs) is significant to tissue engineering and regenerative medicine, but it is extremely challenging to develop a successful protocol because the effects of cryopreservation parameters on the post-thaw functionality of ETs are not well understood. Particularly, the effects on the microstructure of their extracellular matrix (ECM) have not been well studied, which determines many functional properties of the ETs. In this study, we investigated the effects of two key cryopreservation parameters – i) freezing temperature and corresponding cooling rate; and ii) the concentration of cryoprotective agent (CPA) on the ECM microstructure as well as the cellular viability. Using dermal equivalent as a model ET and DMSO as a model CPA, freezing-induced spatiotemporal deformation and post-thaw ECM microstructure of ETs was characterized while varying the freezing temperature and DMSO concentrations. The spatial distribution of cellular viability and the cellular actin cytoskeleton was also examined. The results showed that the tissue dilatation increased significantly with reduced freezing temperature (i.e., rapid freezing). A maximum limit of tissue deformation was observed for preservation of ECM microstructure, cell viability and cell-matrix adhesion. The dilatation decreased with the use of DMSO, and a freezing temperature dependent threshold concentration of DMSO was observed. The threshold DMSO concentration increased with lowering freezing temperature. In addition, an analysis was performed to delineate thermodynamic and mechanical components of freezing-induced tissue deformation. The results are discussed to establish a mechanistic understanding of freezing-induced cell-fluid-matrix interaction and phase change behavior within ETs in order to improve cryopreservation of ETs. PMID:23246556

Sequestration of PDC-109 protein improves freezability of crossbred bull spermatozoa.

PubMed

Srivastava, N; Srivastava, S K; Ghosh, S K; Singh, L P; Prasad, J K; Kumar, Amit; Perumal, P; Jerome, A; Thamizharasan, A

2012-03-01

A study was carried out to assess the effect of sequestration of PDC-109 protein, a majority constituent of heparin binding proteins (HBP) of seminal plasma, on freezability and in vitro fertilizing ability of crossbred bull spermatozoa after cryopreservation. The study consisted of isolation and characterization of PDC-109 protein to raise anti-sera against it in rabbits. Following which, raised antibodies against PDC-109 protein was quantitated and coated in tubes used for collection of ejaculates. Semen ejaculates thus collected were cryopreserved using EYTG extender. Physico-morphological characteristics, viz. motility, viability, acrosomal integrity and HOS response as an indicator of freezability of cryopreserved spermatozoa were determined at pre freeze as well as post thaw stage. At pre freeze stage, a significant (p<0.05) improvement in viability (83.83 ± 2.18 vs 75.17 ± 2.42) and acrosome integrity (81.33 ± 2.38 vs 72.83 ± 2.39) in antibodies treated group than control was observed. Similarly, increase in HOS responsive spermatozoa was highly significant (p<0.01) than control (78.83 ± 1.69 vs 67.5 ± 1.75). At post thaw stage, significant (p<0.05) improvement in viability (69.50 ± 2.16 vs 60.33 ± 2.19) and HOS responsive spermatozoa (68.67 ± 1.62 vs 58.50 ± 1.32) and highly significant (p<0.01) increase in individual motility (56.17 ± 1.83 vs 47.00 ± 1.86) and acrosome integrity (75.17 ± 2.38 vs 61.83 ± 2.1) was observed in antibodies treated group when compared to control was observed. The results from the study revealed that sequestration of PDC-109 protein from semen samples leads to significant improvement in pre-freeze and post-thaw values of above parameters in cryopreserved spermatozoa. It is thus concluded that sequestration of PDC-109 protein from ejaculates improves freezability of crossbred bull spermatozoa. Copyright © 2012 Elsevier B.V. All rights reserved.

The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA

PubMed Central

2012-01-01

Background Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics). Results Post-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05). Conclusions These results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics. PMID:23217215

Cryopreservation of human insulin expressing cells macro-encapsulated in a durable therapeutic immunoisolating device theracyte.

PubMed

Yakhnenko, Ilya; Wong, Wallace K; Katkov, Igor I; Itkin-Ansari, Pamela

2012-01-01

Encapsulating insulin producing cells (INPCs) in an immunoisolation device have been shown to cure diabetes in rodents without the need for immunosuppression. However, micro-encapsulation in semi-solid gels raises longevity and safety concerns for future use of stem cell derived INPCs. We have focused on a durable and retrievable macro-encapsulation (> 10(6) cells) device (TheraCyte). Cryopreservation (CP) of cells preloaded into the device is highly desirable but may require prolonged exposure to cryoprotectants during loading and post-thaw manipulations. Here, we are reporting survival and function of a human islet cell line frozen as single cells or as islet-like cell clusters. The non-clusterized cells exhibited high cryosurvival after prolonged pre-freeze or post-thaw exposure to 10 percent DMSO. However, both clusterization and especially loading INPCs into the device reduced viable yield even without CP. The survived cryopreserved macro-encapsulated INPCs remained fully functional suggesting that CP of macro-encapsulated cells is a promising tool for cell based therapies.

Display stability of fresh and thawed lamb supplemented with vitamin E or sprayed with an antioxidant borage seed extract.

PubMed

Bellés, Marc; Alonso, Verónica; Roncalés, Pedro; Beltrán, Jose A

2018-06-01

The commercialization of thawed lamb packaged in modified atmosphere and maintained on display could serve as an alternative capable of satisfying the requirements of both customers and distributors. However, previous studies have suggested that lipid oxidation may accelerate post-thawing because peroxidation occurs during frozen storage, thereby leading to rapid and severe secondary lipid oxidation. The addition of an antioxidant compound either in the lamb diet or in the packaged meat could resolve this problem. Therefore, the present study aimed to compare the effect of dietary vitamin E (1000 mg of dl-α-tocopheryl acetate per kg of basal diet) and the spraying of borage seed aqueous extract (10% p/v) on the quality of fresh and thawed lamb leg chops. Both borage extract and vitamin E improved colour (as measured via instrumental and visual assessment of colour) and lipid stability (thiobarbituric acid reactive substances) of fresh and thawed lamb throughout display, although neither of them had any antimicrobial effect. Freezing/thawing accelerated bone marrow darkening and reduced redness but delayed microbial growth. Both of these antioxidant strategies would be very profitable for the preservation of lamb meat, allowing thawed meat packaged in a modified atmosphere to be commercialized. However, additional studies should be carried out to determine how bone darkening in thawed chops can be avoided. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits.

PubMed

Viudes-de-Castro, M P; Lavara, R; Safaa, H M; Marco-Jiménez, F; Mehaisen, G M K; Vicente, J S

2014-05-01

This study was conducted to elucidate the effect of different freezing extenders on two lines selected for hyperprolificacy and longevity (H and LP, respectively). In extender A, dimethyl sulphoxide (Me2SO) and sucrose were used as cryoprotectants. In extenders B and C, the sucrose was replaced by 20% egg yolk, and in extender C the Me2SO was substituted by acetamide. Semen was packaged in 0.25 ml plastic straws and cooled at 5°C for 45 min, and then was frozen in liquid nitrogen vapour for 10 min before being plunged into the liquid nitrogen. Thawing was carried out by immersing the straws in a water bath at 50°C for 10 s. Frozen-thawed semen characteristics and reproductive parameters were affected by freezing. Extender C showed significantly lower post-thawing quality traits than any of the three extenders. Acrosome integrity was significantly improved when Me2SO was used as cryoprotectant. Sucrose replacement by 20% egg yolk had no effect on acrosome integrity but provided significantly lower sperm motility and viability. Freezing extender affected fertility rate, total born, number of implantation sites and gestational losses, obtaining better results when extender A was used. The acrosomal integrity after frozen-thawed process showed a significant correlation with fertility at 12th day and also at birth, indicating that an increase in acrosomal integrity leads to an increase in both fertilities (12th day and at birth). A positive correlation between motility of semen and implantation sites was found. The post-thawing quality traits of semen were not affected by the genetic line, although LP line showed higher total born and lower foetal and gestational losses. The findings of this study suggest that freezing extender composition has a significant effect on the success of rabbit sperm for preservation, and when Me2SO was used as permeable cryoprotectant sucrose provided better protection compared with egg yolk and improved reproductive traits, and, on the other hand, the male genotypes used in the present study had no effect on frozen-thawed sperm parameters but negatively affected some of the reproductive parameters.

A two-step dilution tris-egg yolk extender containing Equex STM significantly improves sperm cryopreservation in the African wild dog (Lycaon pictus).

PubMed

Van den Berghe, Femke; Paris, Monique Christina Johanna; Briggs, Michael Brent; Farstad, Wenche Kristin; Paris, Damien Boyd Bertrand Paul

2018-02-01

Conservation management of endangered African wild dogs (AWD; Lycaon pictus) can benefit greatly from development of sperm freezing and artificial insemination. Previous freezing attempts yielded nearly 0% motile sperm within 2 h of thawing. In this study, two canine freezing protocols were tested: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution. Semen was collected by electroejaculation from n = 24 AWDs, of which eight ejaculates of sufficient quality (four good quality with initial sperm motility of 75.0 ± 4.4% and four poor quality; showing rapid decrease in sperm motility to 3.3 ± 3.3% prior to freezing) were frozen. For good quality samples, motility and sperm motility index persisted for up to 8 h for Protocol 2, and was higher between 2 and 6 h after thawing with a decrease from 4 h of incubation. Motility dropped to nearly 0% after 2 h incubation for Protocol 1. Viability was higher for Protocol 2 throughout the 8 h of incubation, with a decrease after 6 h, compared to 4 h for Protocol 1. Acrosome integrity was higher for Protocol 2 throughout post-thaw incubation, with a decrease after 2 h for both protocols. Protocols did not differ in normal sperm morphology or DNA integrity. Poor quality samples yielded similar results, except for acrosome integrity, which declined for Protocol 2. In conclusion, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it suitable for sperm banking and artificial insemination initiatives. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Different Calcium+2 Intensity Profile and Quality of Oocyte and Goat Sperms after Cryopreservation

NASA Astrophysics Data System (ADS)

Ciptadi, G.; Rahayu, S.; Fatchiyah; Wahyuningsih, S.; Budiarto, A.; Nasich, M.; Putri, A. R. I.; Mudawamah, M.; Ihsan, M. N.

2018-02-01

This research aims were to study the effect of the oocyte and sperms cryopreservation of Indonesian local goat on the post-thawing quality and profile or characters of Calcium+2 intensity in relating with their fertility capacity. A study was conducted to test the freezing method and post-thawing viability both stock cells stored in the deep freezer and liquid nitrogen (-80°C of vs -196°C). A fertility test of sperms has been conducted through in vitro of sperm quality, while the oocytes cryopreserved test was done by in vitro maturation (IVM) rate (%). The profile of Calcium 2+ was performed and analysis by Confocal Laser Scanned Microscope (CLSM). The result showed that IVM rate of goat oocyte is considered lower when cryopreserved in -80°C than in -196°C. Meanwhile, sperm is considered having a good quality in 2 methods of cryopreservation with post-thawing motility > 40 % (SNI 2014). There is an important difference between Calcium intensity of fresh and post-thawing both for oocyte and spermatozoa. Calcium +2 profiles is varied individually on the peak of intensity, but it considered expressed the same profile of each fresh and post-thawing cell. In vitro fertilization test need to be performed to complete the viability and fertility competence of these sperm and oocyte freezing stocks.

Motility and fertilizing ability of cryopreserved Caspian brown trout (Salmo trutta caspius) sperm: Effect of post-thaw storage time and different sperm-to-egg ratios.

PubMed

Golshahi, Karim; Shabani, Nariman; Aramli, Mohammad Sadegh; Noori, Elnaz

2015-10-01

This study was designed to test the effect of post-thaw storage time on sperm motility parameters of Caspian brown trout (n=7). Furthermore, we investigated the effect of sperm-to-egg ratios of 100,000:1, 300,000:1 and 600,000:1 on fertility of cryopreserved Caspian brown semen. Quality was assessed by measuring sperm motility parameters and fertilization rates at the eyed and hatching stages. The percentage of post-thawed sperm motility, curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were not affected by 60 min of storage, whereas a decrease in straight line velocity (VSL), average path velocity (VAP) and linearity (LIN) were found in cryopreserved semen. Thus, the cryopreserved sperm of Caspian brown trout could be stored up to 60 min without loss of the percentage of sperm motility. The fertilization rate was not affected by 60 min of post-thaw storage and was over 70% for sperm-to-egg ratios of both 300,000 and 600,000:1. To our knowledge, this study is the first to report the high post-thaw fertilization ability of Caspian brown trout semen at a sperm-to-egg ratio as low as 300,000:1. This procedure after scaling up can be recommended for routine Caspian brown trout sperm cryopreservation. Copyright © 2015 Elsevier Inc. All rights reserved.

Modification of membrane cholesterol and its impact on frozen-thawed chicken sperm characteristics.

PubMed

Partyka, Agnieszka; Bonarska-Kujawa, Dorota; Sporniak, Marta; Strojecki, Maciej; Niżański, Wojciech

2016-10-01

This study was conducted to determine the changes in chicken sperm plasma membranes fluidity and polarity as lipid packing arrangement induced by cholesterol-loaded cyclodextrin (CLC) and 2-hydroxypropyl-β-cyclodextrin (HBCD) and how sperm cryopreservation outcomes are improved by these changes. Treatment with 2 mg HBCD supported the highest (P < 0.01) percentage of viable spermatozoa compared with the control and CLCs groups after cryopreservation. The percentage of post-thaw progressive and rapid sperm motility was highest in 2 mg HBCD (P < 0.01). After thawing, sperm treated with 1 or 2 mg CLC showed the highest anisotropy at 5, 21, 25 and 40°C (P < 0.01). At 25°C, the lowest anisotropy was observed in the thawed semen from the control group. The highest value (P < 0.01) of generalized polarization (GP) (0.5) at 5°C was observed in the 1 mg CLC treated sample. After 2 h of incubation, the highest percentage of viable spermatozoa was observed in the HBCD group in relation to the other treatments (P < 0.01). Exposure to 1 mg or 2 mg of CLC significantly decreased the percentage of live spermatozoa after thawing (P < 0.01). In conclusion, HBCD appears to play a role in the modification of sperm membranes, increasing their fluidity and preventing them against membrane phase transition to gel, thus minimizing freezing-thaw sperm damage. HBCD treatment enhances chicken sperm viability and motility after cryopreservation and subsequent storage. This novel procedure may be useful for improving the technology for cryopreservation of fowl spermatozoa.

Effects of alginate on frozen-thawed boar spermatozoa quality, lipid peroxidation and antioxidant enzymes activities.

PubMed

Hu, Jinghua; Geng, Guoxia; Li, Qingwang; Sun, Xiuzhu; Cao, Hualin; Liu, Yawei

2014-06-30

Although alginate was reported to play an important role as free radical scavengers in vitro and could be used as sources of natural antioxidants, there was no study about the cryoprotective effects of alginate on boar spermatozoa freezing. The objective of this research was to evaluate the effects of different concentrations of alginate added to the freezing extenders on boar spermatozoa motility, plasma membrane integrity, acrosomal integrity, mitochondrial activities, lipid peroxidation and antioxidative enzymes activities (SOD and GSH-Px) after thawing. Alginate was added to the TCG extender to yield six different final concentrations: 0, 0.2, 0.4, 0.6, 0.8, and 1.0mg/mL. The semen extender supplemented with various doses of alginate increased (P<0.05) total motility. The spermatozoa plasma membrane integrity and mitochondrial activity were improved at four different concentrations: 0.4, 0.6, 0.8, 1.0mg/mL. The addition of alginate also provided significantly positive effect on post-thaw boar spermatozoa acrosomal integrity at concentrations of 0.6, 0.8, 1.0mg/mL, compared with that of the control (P<0.05). The freezing extenders with the presence of alginate led to higher SOD and GSH-Px activities and lower MDA levels, in comparison to the control (P<0.05). In summary, alginate exhibited a dose-related response on frozen-thawed boar spermatozoa motility, functional integrity and antioxidative capacity at appropriate concentrations. Therefore alginate could be employed as an effective cryoprotectant in boar spermatozoa cryopreservation. Copyright © 2014 Elsevier B.V. All rights reserved.

Combinations of glycerol percent, glycerol equilibration time, and thawing rate upon freezability of bull spermatozoa in plastic straws.

PubMed

Wiggin, H B; Almquist, J O

1975-03-01

Twelve ejaculates were used in a central composite experiment to test 15 combinations of glycerol (7, 9, 11, 13, or 15%), glycerol equilibration times (1, 2, 4, 8, or 16 h) and thawing rates (water at 35 C for 15 s, 50 C for 13 s, 65 C for 11 s, 80 C for 9 s, or 95 C for 7 s). Semen was diluted in heated skim milk-glycerol, packaged in .3-ml. Continental U.S. straws and frozen in liquid nitrogen vapor. Based on post-thaw progressive sperm motility after storage at -196 C for 9 to 11 days, estimated optima from multiple regression were 10.7% for glycerol, 2.0 h for glycerol equilibration time, and 76 C for thawing bath temperature. Only the linear effect for each variable was significant. Much faster thawing rates and shorter glycerol equilibration times than those for freezing bull spermatozoa in glass ampules should be used for maximum post-thaw sperm motility in straws.

Liposome encapsulated soy lecithin and cholesterol can efficiently replace chicken egg yolk in human semen cryopreservation medium.

PubMed

Mutalik, Srinivas; Salian, Sujith Raj; Avadhani, Kiran; Menon, Jyothsna; Joshi, Haritima; Hegde, Aswathi Raju; Kumar, Pratap; Kalthur, Guruprasad; Adiga, Satish Kumar

2014-06-01

Cryopreservation of spermatozoa plays a significant role in reproductive medicine and fertility preservation. Chicken egg yolk is used as an extender in cryopreservation of human spermatozoa using glycerol egg yolk citrate (GEYC) buffered medium. Even though 50% survival of spermatozoa is generally achieved with this method, the risk of high levels of endotoxins and transmission pathogens from chicken egg yolk is a matter of concern. In the present study we attempted to establish a chemically defined cryopreservation medium which can replace the chicken egg yolk without affecting sperm survival. Ejaculates from 28 men were cryopreserved with GEYC based freezing medium or liposome encapsulated soy lecithin-cholesterol based freezing medium (LFM). The semen samples were subjected to rapid thawing after 14 days of storage in liquid nitrogen. Post-thaw analysis indicated significantly higher post-thaw motility and sperm survival in spermatozoa cryopreserved with LFM compared to conventional GEYC freezing medium. The soy lecithin and cholesterol at the ratio of 80:20 with sucrose showed the highest percentage of post-thaw motility and survival compared to the other compositions. In conclusion, chemically defined cryopreservation medium with liposome encapsulated soy lecithin and cholesterol can effectively replace the chicken egg yolk from human semen cryopreservation medium without compromising post-thaw outcome.

Effect of alpha-lipoic acid on boar spermatozoa quality during freezing-thawing.

PubMed

Shen, Tao; Jiang, Zhong-Liang; Li, Cong-Jun; Hu, Xiao-Chen; Li, Qing-Wang

2016-04-01

Alpha-lipoic acid (ALA) is known to be a natural antioxidant. The aim of the present study was to evaluate the cryoprotective effect of ALA on the motility of boar spermatozoa and its antioxidant effect on boar spermatozoa during freezing-thawing. Different concentrations (2.0, 4.0, 6.0, 8.0 or 10.0 mg/ml) of ALA were added to the extender used to freeze boar semen, and the effects on the quality and endogenous antioxidant enzyme activities of frozen-thawed spermatozoa were assessed. The results indicated that the addition of ALA to the extender resulted in a higher percentage of motile spermatozoa post-thaw (P < 0.05). The activities of superoxide dismutase, lactate dehydrogenase, glutamic-oxaloacetic transaminase and catalase improved after adding ALA to the extender (P < 0.05). Artificial insemination results showed that pregnancy rate and litter size were significantly higher at 6.0 mg/ml in the ALA group than in the control group (P < 0.05). In conclusion, ALA conferred a cryoprotective capacity to the extender used for boar semen during the process of freezing-thawing, and the optimal concentration of ALA for the frozen extender was 6.0 mg/ml.

Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality.

PubMed

Yang, Chung-Hsun; Wu, Ting-Wen; Cheng, Feng-Pang; Wang, Jiann-Hsiung; Wu, Jui-Te

2016-03-01

The current study aimed to investigate the effects of different concentrations of glycerol (0%, 1%, 2%, 3%, and 5%) and dimethylacetamide (DMA: 0%, 1%, 3%, and 5%) on post-sperm quality characteristics following semen freezing in dry ice (D) or liquid nitrogen (N). Semen was collected from Duroc boars and was allocated to 32 treatment groups for cryopreservation. Analysis of post-thaw semen quality and fertility after artificial insemination (AI) was used to examine the combinatorial effects of different treatments. The best scores for post-thaw sperm motility, sperm viability, and sperm acrosomal integrity were observed in semen frozen in: (a) dry ice in the presence of 5% glycerol and no DMA (16D-treatment); (b) dry ice in the presence of 3% glycerol and no DMA (9D-treatment); and (c) liquid nitrogen in the presence of 3% glycerol and 1% DMA (10N-treatment), with no significant difference observed among these three treatments. The farrowing rates after AI with post-thawed semen after 9D- and 10N-treatments were 33% and 50%, respectively. To summarize, the results of the present study indicated that the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the best post-thaw quality parameters of boar semen. Combinations of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa. Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Physical and kinematic properties of cryopreserved camel sperm after elimination of semen viscosity by different techniques.

PubMed

El-Bahrawy, Khalid; Rateb, Sherif; Khalifa, Marwa; Monaco, Davide; Lacalandra, Giovanni

2017-12-01

This investigation aimed to determine the influence of using different techniques for liquefaction of semen on post-thaw physical and dynamic characteristics of camel spermatozoa. A total of 144 ejaculates were collected from 3 adult camels, Camelus dromedarius, twice-weekly over 3 consecutive breeding seasons. A raw aliquot of each ejaculate was evaluated for physical and morphological properties, whereas the remaining portion was diluted (1:3) with glycerolated Tris lactose egg yolk extender, and was further subjected to one of the following liquefaction treatments: control (untreated), 5μl/ml α-amylase, 0.1mg/ml papain, 5u/ml bromelain, or 40-kHz nominal ultrasound frequency. The post-thaw objective assessment of cryopreserved spermatozoa, in all groups, was performed by a computer-assisted sperm analysis (CASA) system. The results revealed that all liquefaction treatments improved (P<0.05) post-thaw motility, viability and sperm motion criteria. However, an adverse effect (P<0.05) was observed in acrosome integrity, sperm cell membrane integrity and percent of normal sperm in all enzymatically-treated specimens compared to both control and ultrasound-treated semen. These results elucidate the efficiency of utilizing ultrasound technology for viscosity elimination of camel semen. In addition, developing enzymatic semen liquefaction techniques is imperious to benefit from when applying assisted reproductive technologies, particularly AI and IVF, in camels. Copyright © 2017 Elsevier B.V. All rights reserved.

Dimethyleacetamide improves the cryosurvivability of Indian red jungle fowl (Gallus gallus murghi) sperm.

PubMed

Rakha, B A; Ansari, M S; Akhter, S; Zafar, Z; Naseer, A; Hussain, I; Santiago-Moreno, J; Blesbois, E

2017-11-01

It was hypothesized that dimethyleacetamide (DMA) can be used as an alternate to glycerol for cryopreservation of Indian red jungle fowl semen. Four concentrations of DMA (4%, 6%, 8% and 10%) in extender were compared with previously optimized cryopreservation protocol based on 20% glycerol (control) for Indian red jungle fowl. Sperm motility, plasma membrane integrity, viability, and acrosome integrity were assessed at the stage of post-dilution, cooling, equilibration, and freeze-thawing. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) at post-dilution, cooling, equilibration, and freeze-thawing in extender having 6% DMA compared to control and other experimental extenders. The highest (P < 0.05) recovery rates of all aforementioned parameters were also recorded in extender having 6% DMA; thus, 6% DMA was further compared with control (20% glycerol) for fertility after artificial insemination. Eggs were collected for five days after artificial insemination with semen cryopreserved in extender containing 6% DMA and control. The higher no. of fertilized eggs, fertility, no. of hatched eggs, hatch (%) and hatchability were recorded with semen cryopreserved in extender having 6% DMA compared to control. It is concluded that 6% DMA maintained higher post-thaw quality and fertility of Indian red jungle fowl semen and is a better replacement of glycerol. Copyright © 2017 Elsevier Inc. All rights reserved.

Nitrogen availability drives priming effect by altering microbial carbon-use efficiency after permafrost thaw

NASA Astrophysics Data System (ADS)

Chen, L.; Liu, L.; Zhang, Q.; Mao, C.; Liu, F.; Yang, Y.

2017-12-01

Enhanced vegetation growth can potentially aggravate soil C loss by accelerating the decomposition of soil organic matter (SOM) ("priming effect"), thereby reinforcing the positive C-climate feedback in permafrost ecosystems. However, the degree to which priming effect alters permafrost C dynamics is expected to be modified by nitrogen (N) availability after permafrost thaw. Despite this recognition, experimental evidence for the linkage between priming effect and post-thaw N availability is still lacking. Particularly, the microbial mechanisms involved remain unknown. Here, using a thermokarst-induced natural N gradient combined with an isotope-labeled glucose and N addition experiment, we presented a strong linkage between soil N availability and priming effect in Tibetan permafrost. We observed that the magnitude of priming effect along the thaw gradient was negatively associated with soil total dissolved nitrogen (TDN) concentration. This negative effect of post-thaw N availability was further proved by a sharply reduced priming effect following mineral N supply. These two lines of evidence jointly illustrated that the priming effect along the thaw chronosequence was controlled by N availability, supporting the `N mining theory'. In contrast to the prevailing assumption, this N-regulated priming effect was independent from changes in C- or N-acquiring enzyme activities, but positively associated with the change in metabolic quotients (△SOM-qCO2), highlighting that decreased microbial metabolism efficiency rather than increased enzyme activities account for greater priming effect under reduced N availability. Taken together, these findings demonstrate that C dynamics in melting permafrost largely depends on post-thaw N availability due to its effect of retarding SOM mineralization. This C-N interaction and the relevant microbial metabolic efficiency should be considered in Earth System Models for a better understanding of soil C dynamics after permafrost thaw.

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

PubMed Central

Kim, Hak Jun; Lee, Jun Hyuck; Hur, Young Baek; Lee, Chang Woo; Park, Sun-Ha; Koo, Bon-Won

2017-01-01

Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis (TH), ice recrystallization inhibition (IRI), and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type. PMID:28134801

Chicks produced in the Magellanic penguin (Spheniscus magellanicus) after cloacal insemination of frozen-thawed semen.

PubMed

O'Brien, Justine Kellie; Steinman, Karen J; Montano, Gisele A; Dubach, Jean M; Robeck, Todd R

2016-07-01

The in vitro and in vivo functionality of cryopreserved spermatozoa was examined over two breeding seasons in a zoological colony of Magellanic penguins (Spheniscus magellanicus). Frozen-thawed semen was inseminated into five anesthetized females, over a total of eight egg production cycles, with a different male used for each artificial insemination (AI) within each season. Females were maintained within the colony in cordoned nest sites to prevent copulation with their paired male, and were inseminated every 3-10 days until the first oviposition. Semen frozen from seven males using a straw method retained 39.8%, 25.7%, 74.0%, and 52.1% of its initial total motility, progressive motility, average path velocity, and plasma membrane integrity, respectively. Normal morphology of motile cells was reduced (P < 0.05) during freeze-thawing from 76.7% immediately prior to freezing to 65.3% post-thawing. Conceptive females received 1.6 ± 0.2 inseminations before the first oviposition, with 19.2 ± 1.6 × 10(6) motile, morphologically normal spermatozoa per insemination. Overall fertility was 53.3% (8/15 eggs), hatchability was 50.0% (4/8), and genetic analyses confirmed that all embryos and hatchlings were sired by the AI male. Fertile eggs were laid at 4.0-12.1 days after AI, indicating that frozen-thawed spermatozoa resided in the female reproductive tract for up to ∼7.2 days prior to fertilization. Results demonstrate that frozen-thawed Magellanic penguin spermatozoa are fully functional in vivo and support the use of genome banking and AI as tools for managing the sustainability of zoological penguin populations. Zoo Biol. 35:326-338, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Effectiveness of glucose-methanol extender for cryopreservation of Huso huso spermatozoa.

PubMed

Aramli, Mohammad Sadegh; Golshahi, Karim; Nazari, Rajab Mohammad; Aramli, Salim; Banan, Ashkan

2015-11-01

The present approach was designed to evaluate the methanol-glucose extender effects on sperm cryopreservation in beluga sturgeon, Huso huso. Sperm quality was examined by measuring post-thaw sperm motility and fertilizing rate at hatching stage. We first tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30M) in a methanol extender on post-thaw sperm motility. The optimal cryopreservation conditions were found to be 0.2M glucose in the extender. Then, motility and fertilization rates of sperm cryopreserved with 0.2M glucose and 10% methanol (GM) were compared to Tris-sucrose-KCl in 10% methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 15 and 30min equilibration in GM extender before and after cryopreservation were measured. Higher post-thaw sperm motility duration and percentage as well as fertilization rate were obtained with the GM extender when compared to TSKM extender. Equilibration of sperm in extender did not affect the motility quality of either fresh-diluted or frozen/thawed sperm, while fertilization rate showed a significant decline alone after 30min of post-thaw storage. Our results indicated that the use of a simple extender consisting of 0.2M glucose in 10% methanol can be an alternative cryopreservation method to those previously described for sturgeons. Copyright © 2015 Elsevier B.V. All rights reserved.

An investigation into the preservation of microbial cell banks for α-amylase production during 5 l fed-batch Bacillus licheniformis fermentations.

PubMed

Hancocks, Nichola H; Thomas, Colin R; Stocks, Stuart M; Hewitt, Christopher J

2010-10-01

Fluorescent staining techniques were used for a systematic examination of methods used to cryopreserve microbial cell banks. The aim of cryopreservation here is to ensure subsequent reproducible fermentation performance rather than just post thaw viability. Bacillus licheniformis cell physiology post-thaw is dependent on the cryopreservant (either Tween 80, glycerol or dimethyl sulphoxide) and whilst this had a profound effect on the length of the lag phase, during subsequent 5 l fed-batch fermentations, it had little effect on maximum specific growth rate, final biomass concentration or α-amylase activity. Tween 80 not only protected the cells during freezing but also helped them recover post-thaw resulting in shorter process times.

Programmable fast-freezing method improves the post-thaw motion dynamics, integrities of plasmalemma, mitochondrial transmembrane, DNA and, acrosome, and in vivo fertility of water buffalo (Bubalus bubalis) spermatozoa.

PubMed

Khalil Ur Rehman, H; Andrabi, S M H; Ahmed, H; Shah, S A H

2017-10-01

The effects of freezing methods (FR1, nonprogrammable/static, 5 cm above liquid nitrogen [LN 2 ] for 10 min, plunging in LN 2 ; FR2, programmable medium, +4°C to -15°C at 3°C min -1 , from -15 to -80°C at 10°C min -1 and final holding for 1 min at -80°C, plunging in LN 2 ; FR3, programmable fast, from initial holding at +4°C for 2 min, from +4°C to -20°C at 10°C min -1 , from -20°C to -100°C at 30°C min -1 , final holding for 1 min at -100°C and plunging in LN 2 ) were assessed on post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. Mean sperm progressive motility (%), rapid velocity (%), average path velocity (μm s -1 ), straight line velocity (μm s -1 ), curved line velocity (μm s -1 ), integrities (%) of plasmalemma, mitochondrial transmembrane, DNA and acrosome were higher (p < .05) in samples cryopreserved with FR3 compared to FR1 and FR2. Similarly, in vivo fertility (%) of buffalo spermatozoa was higher (p < .05) with FR3 than FR1 (%; 68.0 versus 50.0). We concluded that programmable fast-freezing method (FR3) improves the post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. © 2016 Blackwell Verlag GmbH.

Effect of sucrose and pectin addition on physical, chemical, thermal and rheological properties of frozen/thawed pineapple pulps

NASA Astrophysics Data System (ADS)

Conceição, Márcia Cavalcante; Fernandes, Tatiana Nunes; Prado, Mônica Elisabeth Torres; de Resende, Jaime Vilela

2012-09-01

Pectin (0-1.0 g/100 mL) and sucrose (0-20 g/100 mL) were added to pineapple pulp to improve their rheological properties, thermal properties and stability after freezing and thawing processes. The properties of the mixes were characterized before and after freezing and thawing. Samples were frozen at -20°C, and the freeze concentration was evaluated every 60 min. The thawing rate was evaluated at 19°C and quantified by photographic editing and image analysis software. The thawing rates and values for the freeze concentration were leveled out at pectin concentrations above 0.5 g/100 mL pectin, which indicated that pectin functions to maintain structural homogeneity during freezing. In the thawed samples, the plastic viscosity values were leveled out from pectin concentrations (0.25-0.75 g/100 mL) as the sucrose concentration increased when compared to unfrozen samples. The differences between the rheological parameters of the unfrozen and frozen/thawed pulps, the higher yield stress values after thawing were attributed to the size of suspended particles in the pulp. Applications can specify formulations of frozen products containing pectin, where these properties can be handled after thawing the product.

The Canadian Experiment for Freeze/Thaw in 2012 or 2013 CanEx-FT12 or FT13

NASA Technical Reports Server (NTRS)

Belair, Stephane; Bernier, Monique; Colliander, Andreas; Jackson, Thomas; McDonald, Kyle; Walker, Anne

2011-01-01

General objectives of the experiment are: Pre-launch Calibration/Validation of SMAP Freeze/Thaw products and retrieval algorithms and rehearsal for Soil Moisture Active-Passive (SMAP) post launch validation. The basis of the radar freeze-thaw measurement is the large shift in dielectric constant and backscatter (dB) between predominantly frozen & thawed conditions. The Dielectric constant of liquid water varies with frequency, whereas that of pure ice is constant

Effects of diluents and plasma on honey bee (Apis mellifera L.) drone frozen-thawed semen fertility.

PubMed

Gül, Aziz; Şahinler, Nuray; Onal, Ali G; Hopkins, Brandon K; Sheppard, Walter S

2017-10-01

Cryopreservation is an advanced method used to protect germplasm in liquid nitrogen. Honey bees are of special interest to protect because of their pollination activity and critical role in agriculture. There has been important progress in the cryopreservation of honey bee germplasm in recent years, leading to practical recovery of genetic material for breeding purposes following freezing. However, there remains room for improvement and the goal of the present study was to evaluate the effect of different "extenders" added post-thaw on the fertilization rate of cryopreserved honey bee semen. The purpose of adding extender post-thaw was to dilute the cryoprotectant to remove chemicals after centrifugation because of potential adverse effects. The control consisted of frozen-thawed semen without the addition of an extender; treatment groups included the addition of one of the following extenders: glucose solution, fresh ram semen plasma, fresh honey bee semen plasma, extender solution. All of the above treatments and frozen-thawed control were compared to fresh semen. For each group, 15 virgin queens were instrumentally inseminated with the semen-diluent solution and introduced into nucleus colonies to determine the brood patterns of the queens. Percentages of worker brood produced in the fresh semen, frozen-thawed semen control, glucose, fresh ram semen plasma, fresh honey bee semen plasma, and extender solution supplemented groups were 98.±1.1%, 47.0 ± 0.9%, 3.0 ± 0.8%, 0.3 ± 0.1%, 48.1 ± 4.1% and 40.3 ± 2.4%, respectively. Similiarly, spermatozoa numbers in the spermathecae of the same treatment groups were 3.6 × 10 6 , 1.6 × 10 6 , 7.3 × 10 5 , 4.7 × 10 5 , 8.1 × 10 5 , and 4.6 × 10 5 spermatozoa for the same treatment, respectively. The differences in both worker brood percentage and sperm count in the spermatheca were statistically significant (P < 0.01) among all treatment groups, except the frozen-thawed control group and fresh drone semen plasma group. We found a positive correlation between sperm count in the spermatheca and the percentage of worker brood (r = 0.91). With the exception of fresh honey bee semen plasma, the fertility rate was reduced following the addition of various plasmas and diluents post-freezing. Copyright © 2017 Elsevier Inc. All rights reserved.

Partial deoxygenation of extender improves sperm quality, reduces lipid peroxidation and reactive oxygen species during cryopreservation of buffalo (Bubalus bubalis) semen.

PubMed

Balamurugan, B; Ghosh, S K; Lone, S A; Prasad, J K; Das, G K; Katiyar, R; Mustapha, Abdul Rahman; Kumar, Ajay; Verma, M R

2018-02-01

The present study was designed to investigate the effect of partial deoxygenation of extender on sperm quality, lipid peroxidation (LPO) and reactive oxygen species (ROS) in buffalo (Bubalus bubalis) during cryopreservation of semen. Semen extender was prepared freshly and split into three sub-extenders [Extender I: control (non-deoxygenated), Extender II (partially deoxygenated by using LN 2 flushing) and Extender III (partially deoxygenated mechanically by vacuum pump)]. Amounts of dissolved oxygen (DO) were determined in all the three extenders and also in post-thaw semen. Ejaculates with mass motility of  ≥3+ and individual progressive motility of 70% or greater were collected from Murrah buffalo bulls and utilized in the study. Each semen sample was divided into Groups I (diluted with Extender I), II (diluted with Extender II) and III (diluted Extender III) with a maximum of 60 × 10 6 sperm/mL. French mini straws (0.25 mL) were filled with the extended semen samples, sealed with polyvinyl alcohol powder, kept for 3 h at 5 °C for equilibration and then stored in an automatic programmable freezer until the temperature of straws reached -145 °C followed by plunging the straws into liquid nitrogen (-196 °C). Semen samples were evaluated at pre-freeze and post-thaw stages for various variables [sperm motility, live sperm count, acrosomal integrity, hypo-osmotic swelling (HOS) response, LPO and ROS concentrations]. The mean DO was less (P < 0.05) in Extender II as compared to I and III. The DO was less (P < 0.05) in Group II (semen extended with Extender II) as compared with III (semen extended with Extender III) and I (semen extended with Extender I). The percentages for sperm motility, viability and intact acrosomes (PIA) were greater (P < 0.05) in Groups II and III as compared to the control group at the pre-freeze stage, while at the post-thaw stage, percentages of sperm motility, viability, PIA and HOS response were greater (P < 0.05) in Group II as compared with the control group and Group III. Pre-freeze HOS response (%) was greater (P < 0.05) in Group II as compared with the control and Group III. At the pre-freeze stage, sperm LPO and ROS were less (P < 0.05) in Groups II and III as compared with the control and at post-thaw stage, spermatic LPO and ROS concentrations were less (P < 0.05) in Group II than in the control group and Group III. In conclusion, partial deoxygenation of extender improves sperm quality, reduces sperm LPO and ROS concentrations in buffalo during cryopreservation. Partial deoxygenation of the extender with LN 2 flushing may be one of the ways for improving quality and fertility of frozen-thawed buffalo sperm. Copyright © 2017 Elsevier B.V. All rights reserved.

Cryopreservation has no effect on function of natural killer cells differentiated in vitro from umbilical cord blood CD34(+) cells.

PubMed

Domogala, Anna; Madrigal, J Alejandro; Saudemont, Aurore

2016-06-01

Natural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34(+) cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. We evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro. We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. We found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. We are therefore able to generate large numbers of functional NK cells from CB CD34(+) cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

An Overview of Production and Validation of the SMAP Passive Soil Moisture Product

NASA Technical Reports Server (NTRS)

Chan, S.; O'Neill, P.; Njoku, E.; Jackson, T.; Bindlish, R.

2015-01-01

The Soil Moisture Active Passive (SMAP) mission is an L-band mission scheduled for launch in Jan. 2015. The SMAP instruments consist of a radar and a radiometer to obtain complementary information from space for soil moisture and freeze/thaw state research and applications. By utilizing novel designs in antenna construction, retrieval algorithms, and acquisition hardware, SMAP provides a capability for global mapping of soil moisture and freeze/thaw state with unprecedented accuracy, resolution, and coverage. This improvement in hydrosphere state measurement is expected to advance our understanding of the processes that link the terrestrial water, energy and carbon cycles, improve our capability in flood prediction and drought monitoring, and enhance our skills in weather and climate forecast. For swath-based soil moisture measurement, SMAP generates three operational geophysical data products: (1) the radiometer-only soil moisture product (L2_SM_P) posted at 36-kilometer resolution, (2) the radar-only soil moisture product (L2_SM_A) posted at 3-kilometers resolution, and (3) the radar-radiometer combined soil moisture product (L2_SM_AP) posted at 9-kilometers resolution. Each product draws on the strengths of the underlying sensor(s) and plays a unique role in hydroclimatological and hydrometeorological applications. A full suite of SMAP data products is given in Table 1.

The effect of glycerol-related osmotic changes on post-thaw motility and acrosomal integrity of ram spermatozoa.

PubMed

Fiser, P S; Fairfull, R W

1989-02-01

Ram semen, collected by artificial vagina, was diluted and processed for long-term storage as described by P. S. Fiser, L. Ainsworth, and R. W. Fairfull (Canad. J. Anim. Sci. 62, 425-428, 1982). The concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by a one-step addition at 30 degrees C (Method 1), by cooling the semen to 5 degrees C and addition of the glycerol gradually over 30 min (Method 2), by one-step addition of glycerol prior to equilibration for 2 hr (Method 3), or by cooling to 5 degrees C, followed by a holding period of 2 hr at 5 degrees C, and the one-step addition of glycerol just prior to freezing (Method 4). After thawing, the glycerol concentration of the semen was reduced by stepwise dilution from 4 to 0.4% over 15 or 30 min or by a one-step ten-fold dilution. The average post-thaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.9%) than when the glycerol was added by the other three methods (range, 44.0-46.4% averaged over the glycerol dilution). The average post-thaw percentage of intact acrosomes (61.2%), highest in semen in which the glycerol was added by Method 2, was not significantly different from those in which glycerol was added to semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). However, when averaged over the method of glycerolation, the post-thaw percentage of motile spermatozoa (range, 43.7-44.2%) and the percentage of intact acrosomes (range, 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one-step, 10-fold dilution. These data indicate that post-thaw survival of spermatozoa can be influenced by the way in which glycerol is added prior to freezing. However, post-thaw spermatozoa motility and acrosomal integrity can be maintained even after a rapid decrease in glycerol concentration such as that which accompanies insemination or dilution of semen for assessment of motility.

Interaction of extender composition and freezing method for effective semen cryopreservation in the North American river otter (Lontra canadensis).

PubMed

Bateman, Helen L; Swanson, William F

2017-10-01

Semen cryopreservation and storage in genome resource banks (GRBs), in combination with artificial insemination (AI), could be invaluable for genetic management and conservation of endangered otter species. For any applied conservation benefit, effective methods for otter sperm processing and cryopreservation first must be established. In this study, our objective was to develop an effective semen cryopreservation method for the North American river otter, evaluating the effect of extender composition (i.e., glycerol concentration, Equex STM paste supplementation) and freezing protocol (timing of glycerol addition, pre-freeze cooling rate, freezing/packaging method) on post-thaw sperm motility, longevity and acrosome status. Semen was collected from 14 otters housed at 9 zoos, and following cryopreservation in an egg-yolk based extender, thawed to assess sperm motility and acrosome status immediately post-thaw and during 6 h of in vitro culture. Results indicated that extender containing 4% glycerol was preferable (p < 0.05) to 8% glycerol but the temperature/timing of extender addition containing 4% glycerol did not affect (p > 0.05) post-thaw sperm parameters. Treatments with extender containing Equex and frozen by pelleting on dry ice showed greater (p < 0.05) motility and percentage of intact acrosomes compared to treatments frozen in extender without Equex, regardless of pre-freeze cooling rate. In the absence of Equex, pelleting provided superior post-thaw sperm motility (p < 0.01) and higher (p < 0.001) percentage of sperm with intact acrosomes compared to samples frozen in straws over liquid nitrogen vapor. Results of this study indicate that cryopreservation of otter sperm using an egg-yolk -TEST based extender containing 4% glycerol and 1% Equex, with the pellet freezing method, provided superior post-thaw sperm motility, longevity and acrosomal integrity compared to other combinations. Neither alterations in timing of glycerolated extender addition nor pre-freeze cooling rate had a discernable effect on post-thaw otter sperm parameters. These findings represent the first assessment of semen cryopreservation in any otter species and may be of value as a model for development of semen cryopreservation strategies in other endangered otter species. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of post-thaw incubation on sperm kinematics and acrosomal integrity of ram spermatozoa cryopreserved in medium-sized French straws.

PubMed

Bag, Sadhan; Joshi, Anil; Naqvi, S M K; Mittal, J P

2004-08-01

The objectives were to assess the effect of post-thaw in vitro incubation on motion characteristics and acrosomal integrity of ram spermatozoa of native Malpura and Bharat Merino breeds maintained under a semi-arid tropical environment. Good quality semen samples of both breeds were diluted, packaged in medium-sized straws, and frozen under controlled conditions. Straws were thawed at 60 degrees C for 10s and thawed samples were incubated at 37 degrees C for 4h. Post-thaw motion characteristics and acrosomal integrity of incubated spermatozoa were assessed (by computer-aided semen analysis and Giemsa staining, respectively) just prior to incubation and at hourly intervals thereafter. There was a significant effect of incubation time on motility characteristics and the proportion of spermatozoa with normal acrosomes; 81.4% (arcsin transformed value, 65.2) of spermatozoa were motile at the start of incubation, with 47.9% (arcsin transformed value, 44.4) motile after 4h. At the corresponding times, there were normal acrosomes in 65.8 (arcsin transformed value, 54.8) and 55.7% (arcsin transformed value, 48.9) of spermatozoa, respectively. The percentage straightness of spermatozoa varied during incubation (P < 0.01). However, there was no significant change in percentage linearity, curvilinear velocity, average path velocity, straight line velocity, lateral head displacement, and beat cross frequency of spermatozoa during incubation. There were no breed variations in any motility parameters during incubation, except percentage straightness (P < 0.05), lateral head displacement (P < 0.05) and beat cross frequency (P < 0.01). That sperm motility and acrosomal morphology were very acceptable immediately post-thaw and after 4h of incubation indicated the efficacy of cryopreserving ram spermatozoa under controlled conditions in medium-sized straws.

Liquid nitrogen vapor is comparable to liquid nitrogen for storage of cryopreserved human sperm: evidence from the characteristics of post-thaw human sperm.

PubMed

Hu, Jingmei; Zhao, Shidou; Xu, Chengyan; Zhang, Lin; Lu, Shaoming; Cui, Linlin; Ma, Jinlong; Chen, Zi-Jiang

2015-11-01

To compare the differences in the characteristics of post-thaw human sperm after storage in either liquid nitrogen (LN2; -196 °C) or LN2 vapor (-167 °C). Experimental study. University hospital. Thirty healthy volunteers who agreed to donate their normal semen samples for infertility or research were included in the study. Semen samples (n = 30) were divided into eight aliquots and frozen. Four aliquots of each human semen sample were stored in LN2 (-196 °C), and the other four aliquots were stored in LN2 vapor (-167 °C). After 1, 3, 6, or 12 months, samples were thawed and analyzed. The motility was evaluated by the manual counting method. The viability was estimated by eosin staining. The morphology was analyzed by Diff-Quik staining. The sperm DNA integrity was determined with acridine orange fluorescent staining, and acrosin activity was assayed by the modified Kennedy method. The characteristics of post-thaw human sperm, including motility, viability, morphology, DNA integrity, and acrosin activity, showed no significant difference between LN2 and LN2 vapor storage for the different time periods. LN2 vapor was comparable to LN2 in post-thaw sperm characteristics, suggesting that LN2 vapor may be substituted for LN2 for the long-term storage of human sperm. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric, microscopic and oxidative parameters in ram semen.

PubMed

Mehdipour, Mahdieh; Daghigh Kia, Hossein; Nazari, Maryam; Najafi, Abouzar

2017-10-01

This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l -1 pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l -1 ) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing. Copyright © 2017 Elsevier Inc. All rights reserved.

Cooled semen for fixed-time artificial insemination in beef cattle.

PubMed

Borges-Silva, Juliana C; Silva, Márcio R; Marinho, Daniel B; Nogueira, Eriklis; Sampaio, Deiler C; Oliveira, Luiz Orcírio F; Abreu, Urbano G P; Mourão, Gerson B; Sartori, Roberto

2016-06-01

This study evaluated the use of cooled semen in a fixed-time artificial insemination (FTAI) program compared with frozen-thawed semen to improve pregnancy rates in beef cattle. Ejaculates of three bulls were collected and divided into two treatments: (1) frozen-thawed semen and (2) cooled semen. Egg-yolk extender without glycerol was used for the cooled semen treatment. Straws (25×10 6 spermatozoa) were submitted to cooling for preservation at 5°C for 24h, after which FTAI was performed. Nelore cows (n=838) submitted to FTAI were randomly inseminated using frozen-thawed semen or cooled semen. There was a 20% increase in the pregnancy per AI (P AI -1 ) using cooled semen compared with frozen-thawed semen (59.9±4.7 vs 49.4±5.0%; P<0.005). There was no difference in P AI -1 among the bulls (P=0.40). The frozen-thawed semen had fewer functional spermatozoa than did the cooled semen when evaluated by sperm motility (61.7 vs 81.0%), slow thermoresistance test (41.7 vs 66.7%) and hypoosmotic swelling test (38.3 vs 53.7%; P<0.05). The percentage of sperm abnormalities did not differ between the freeze-thawing and cooling processes (18.6 vs 22.1%; P>0.05). Because there was less damage to spermatozoa and improvement in P AI -1 , the use of cooled semen instead of frozen-thawed semen is an interesting approach to increase reproductive efficiency in cattle submitted to a FTAI protocol.

Rooster Semen Cryopreservation: Effect of Pedigree Line and Male Age on Post-Thaw Sperm Function

USDA-ARS?s Scientific Manuscript database

The fertility rates of cryopreserved poultry semen are highly variable and not reliable for use in preservation of commercial genetic stocks. Our objective was to evaluate the cryosurvival of semen from 8 pedigreed layer lines at the onset and end of production. Semen from 160 roosters (20/line) was...

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

PubMed Central

Braslavsky, Ido

2018-01-01

Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices. PMID:29447224

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate.

PubMed

Bahari, Liat; Bein, Amir; Yashunsky, Victor; Braslavsky, Ido

2018-01-01

Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices.

In vitro evaluation of encapsulated primary rat hepatocytes pre- and post-cryopreservation at -80°C and in liquid nitrogen.

PubMed

Durkut, Serap; Elçin, A Eser; Elçin, Y Murat

2015-02-01

Encapsulation techniques have the potential to protect hepatocytes from cryoinjury. In this study, we comparatively evaluated the viability and metabolic function of primary rat hepatocytes encapsulated in calcium alginate microbeads, in chitosan tripolyphosphate beads, and in three-layered alginate-chitosan-alginate (ACA) microcapsules, before and after cryopreservation at -80°C and in liquid nitrogen (LN2) for 1 and 3 months. Findings demonstrated that LN2 was atop of -80°C in regard to preservation of viability (> 90%) and hepatic functions. LN2-cryopreserved hepatocytes encapsulated in ACA microcapsules retained metabolic function post-thawing, with > 90% of the albumin, total protein and urea syntheses activities, and > 80% of oxidative function.

Evaluation of Cryoprotectant and Cooling Rate for Sperm Cryopreservation in the Euryhaline Fish Medaka Oryzias latipes

PubMed Central

Yang, Huiping; Norris, Michelle; Winn, Richard; Tiersch, Terrence R.

2017-01-01

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: 1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me2SO), N, N- dimethylacetamide (DMA), N, N,-dimethyl formamide (DMF), and glycerol with concentrations of 5, 10, and 15% for 60 min of incubation at 4 °C; 2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and 3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5 and 10%) did not change the sperm motility after 30 min; Me2SO, DMA, and DMF (10 and 15%) and glycerol (5, 10 and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 °C/min to 25 °C/min) and were compared to Me2SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ≤ 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ≤ 10% which was significantly lower than that of methanol and ME. With Me2SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ≤ 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future. PMID:20654608

Characteristics of stallion epididymal spermatozoa at collection and effect of two refrigeration protocols on the quality of the frozen/thawed sperm cells.

PubMed

Guimarães, T; Lopes, G; Ferreira, P; Leal, I; Rocha, A

2012-12-01

Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the extended epididymal sperm cells. Ejaculated sperm samples were obtained from 10 healthy stallions with at least 15 days of sexual rest, before routine orchiectomies. Spermatozoa were recovered from the epididymal tail immediately after castration (EPI), after refrigeration of the epididymis for 24h at 4°C (EPI R) and recovered from epididymal tail immediately after castration and stored for 24h at 4°C (EPI RR). Total motility, straight-line velocity, percentage of rapid cells, viability and morphological defects were similar (p>0.05) among different treatments, and post-thaw viability was higher (p<0.05) in EPI than in the ejaculated sperm. The similarity of post-thaw parameters led us to conclude that immediate collection and refrigeration of the epididymal sperm cells or refrigeration of the whole epididymis are equally efficient as a means of transporting material for 24h before cryopreservation of epididymal spermatozoa. Copyright © 2012 Elsevier B.V. All rights reserved.

Butylated hydroxytoluene can reduce oxidative stress and improve quality of frozen-thawed bull semen processed in lecithin and egg yolk based extenders.

PubMed

Khumran, A M; Yimer, N; Rosnina, Y; Ariff, M O; Wahid, H; Kaka, Asmatullah; Ebrahimi, M; Sarsaifi, K

2015-12-01

The aims of this study were to evaluate the effects of anti-oxidant butylated hydroxytoluene (BHT), when added at different concentrations into lecithin-based Bioxcell(®) (BX) and two egg-yolk-based; Tris (TY) and citrate (CE) semen extenders, on post-thaw bull sperm quality and oxidative stress. A total of 30 ejaculates from three bulls were collected using an electro ejaculator. Ejaculates were extended with one of the BX, TY and CE extenders, which contained different concentrations (0.0 - control, 0.5, 1.0, 1.5, 2.0 and 3.0mM/ml) of BHT. The extended semen samples were chilled to 4 °C, and then frozen slowly to -196 °C in 0.25 ml straws before being stored in liquid nitrogen for 2 weeks. Results showed that supplementation of BHT improved (P<0.05) general motility, progressive motility, morphology, acrosome integrity, DNA integrity and malondialdehyde of sperm at 0.5mM/ml for BX and at 1-1.5mM/ml of BHT for TY and CE when compared with the control. However, greater concentrations of 2.0 and 3.0mM/ml of BHT had a detrimental (P<0.05) effect compared with the control with all extenders evaluated. In conclusion, BHT supplementation at lesser concentrations (0.5-1.5mM/ml) could improve frozen-thawed bull sperm quality by reducing oxidative stress produced during the freezing-thawing procedures in either lecithin or egg-yolk based extenders. Copyright © 2015 Elsevier B.V. All rights reserved.

Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus).

PubMed

Thiangtum, Khongsak; Swanson, William F; Howard, JoGayle; Tunwattana, Wanchai; Tongthainan, Dakara; Wichasilpa, Wisid; Patumrattanathan, Pornchai; Pinyopoommintr, Tanu

2006-01-01

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25 degrees C or after slow cooling to 5 degrees C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (+/- s.e.m.) 43.6 +/- 14.2 x 10(6) motile spermatozoa with 33.5 +/- 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P < 0.05) after thawing. All thawed samples fertilised domestic cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5 degrees C resulted in higher (P < 0.05) post-thaw motility and intact acrosomes than glycerol addition at 25 degrees C. In conclusion, good-quality ejaculates can be obtained from Thai fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports.

PubMed

Chaveiro, A; Machado, L; Frijters, A; Engel, B; Woelders, H

2006-06-01

The aim of this study was to improve the freezing protocol of bull sperm, by investigating the influence on sperm viability after freeze/thawing of different freezing medium components, as well as the effect of cooling rates in the different stages of the cooling protocol, in single factor experiments. The experimental variables were: (1) salt-based versus a sugar-based medium (Tris versus sucrose); (2) glycerol concentration; (3) detergent (Equex) concentration; (4) presence of bicarbonate; (5) rate of cooling from 22 degrees C to holding temperature (CR1); (6) holding temperature (HT); (7) rate of cooling from holding temperature to -6 degrees C (CR2); (8) rate of cooling from -10 to -100 degrees C (CR3). All experiments were performed using five bulls per experiment (three ejaculates per bull). Sperm motility after freezing and thawing was assessed by CASA system, and sperm membrane integrity was assessed by flow cytometry. Sucrose-based medium did not offer a clear significant benefit compared to Tris medium. The concentration of Equex that gave the best results in Tris-based media group and sucrose-based media group was in a range between 2-7 and 4-7 g/l, respectively. In both media groups, a glycerol concentration of 800 mM was the best in any post-thaw viability parameters. In the Tris media group, the presence of bicarbonate had a negative effect on sperm viability. CR1 and CR2 had no significant effect on any of the post-thaw sperm viability parameters, but a CR1=0.2 degrees C/min and CR2=4 degrees C/min appeared to give better results in both media. The holding temperature (HT) that gave the best results was found to be in the range of 5-9 degrees C. There was a significant disadvantage of using a low CR3 of 10 degrees C/min, while 150 degrees C/min appeared to be the best cooling rate for either medium.

Quality of Clotting Factor Activity in Fresh Frozen Plasma at Thaw with a Microwave System and after Storage at 4 degrees C for 48 Hours.

PubMed

Kuta, Piotr; Hauck-Dlimi, Barbara; Strobel, Julian; Zimmermann, Robert; Eckstein, Reinhold

2016-01-01

Uncontrolled hemorrhage in polytrauma patients usually results in rapid need of blood products. Despite the shorter thawing times of microwave devices for heating fresh frozen plasma (FFP), their use has remained controversial, and just a few laboratory analyses have been published on this topic. The aim of this study was to analyse the quality of clotting factors immediately after thawing FFP with a microwave device and after 48-hour post thaw storage at 4 degrees C. 24 FFP units of all four ABO blood groups (six of each blood group) were thawed with a Transfusio-therm 2000 and later stored at 4 degrees C for 48 hours. Samples were drawn aseptically and investigated on various clotting factors and protein proteases (fibrinogen, antithrombin, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, vWF antigen and activity, protein S, and protein C) using standard coagulation and chromogenic assays immediately after thawing and again after a 48-hour storage period at 4 degrees C. All units were tested for both anaerobic and aerobic microbial contamination using standard operating procedures immediately after thawing. After thawing, all coagulation factors and protein protease activities were within normal ranges. Blood group O individuals had approximately 25% lower plasma levels of vWF antigen and activity. After a 48-hour storage period at 4 degrees C, FVIII and FIX activities declined significantly in all blood groups, whereas the remaining clotting factors remained comparably stable. Immediately after rapid thawing using a microwave system, all FFP units contained adequate coagulation factor activities to maintain hemostatic activity at the time of product thaw. The post thaw refrigerated storage caused an anticipated decrease in factor VIII and IX activities, but retained normal coagulation factor levels of many plasma proteins. Therefore we conclude that the Transfusio-therm 2000 has no clinically significant influence on the activity of clotting factors and plasma proteases in FFP units.

Effective freezing rate for semen cryopreservation in endangered Mediterranean brown trout (Salmo trutta macrostigma) inhabiting the Biferno river (South Italy).

PubMed

Iaffaldano, Nicolaia; Di Iorio, Michele; Manchisi, Angelo; Esposito, Stefano; Gibertoni, Pier Paolo

2016-10-01

This study was designed to determine: (i) the in vitro effects of different freezing rates on post-thaw semen quality of Mediterranean brown trout (Salmo trutta macrostigma) from the Biferno river; and (ii) the in vivo fertilization and hatching percentage of freezing rate giving rise to the best post-thaw semen quality. Pooled semen samples were diluted 1:3 (v:v) in a freezing extender composed of 300 mM glucose, 10% egg yolk and 10% dimethyl sulfoxide (DMSO). The extended semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen surface (1, 5 or 10 cm) for 10 min to give three different freezing rates. Semen samples were thawed at 30°C for 10 s. The variables assessed after thawing were sperm motility, duration of motility and viability. Our results clearly indicate a significant effect of freezing rate on post-thaw semen quality. Semen frozen 5 cm above the liquid nitrogen surface showed the best quality after freezing/thawing. Based on these in vitro data, 2 groups of 200 eggs were fertilized with fresh semen or semen frozen 5 cm above the liquid nitrogen surface. Fertilization and hatching rates recorded for eggs fertilized with frozen semen were significantly lower (25.4% and 22.5%, respectively) than the ones obtained using fresh semen (87.8% and 75.5%, respectively). An effective freezing protocol will allow for the creation of a sperm cryobank to recover the original population of Mediterranean brown trout in the Biferno river.

Comparative analysis of cryopreservation methods in Chlamydomonas reinhardtii.

PubMed

Scarbrough, Chasity; Wirschell, Maureen

2016-10-01

Chlamydomonas is a model organism used for studies of many important biological processes. Traditionally, strains have been propagated on solid agar, which requires routine passaging for long-term maintenance. Cryopreservation of Chlamydomonas is possible, yet long-term viability is highly variable. Thus, improved cryopreservation methods for Chlamydomonas are an important requirement for sustained study of genetically defined strains. Here, we tested a commercial cryopreservation kit and directly compared it's effectiveness to a methanol-based method. We also tested thaw-back procedures comparing the growth of cells in liquid culture or on solid agar media. We demonstrated that methanol was the superior cryopreservation method for Chlamydomonas compared to the commercial kit and that post-thaw culture conditions dramatically affect viability. We also demonstrated that cryopreserved cells could be successfully thawed and plated directly onto solid agar plates. Our findings have important implications for the long-term storage of Chlamydomonas that can likely be extended to other algal species. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Cryopreservation of Cynomolgus Macaque (Macaca fascicularis) Sperm by Using a Commercial Egg-YolkFree Freezing Medium.

PubMed

Yan, Yaping; Ao, Lei; Wang, Hong; Duan, Yanchao; Chang, Shaohui; Chen, Bingbing; Zhi, Dalong; Li, Sujuan; Niu, Yuyu; Ji, Weizhi; Si, Wei

2016-11-01

Conventional TRISegg yolk (TEY) freezing medium for the cryopreservation of NHP sperm has the risk of contamination due to widespread zoonotic diseases. This study was aimed at determining the optimal glycerol concentration, freezing rate, and holding time in liquid N2 vapor for the cryopreservation of cynomolgus macaque sperm by using a commercial egg-yolkfree freezing medium (SC medium) designed for human sperm cryopreservation. Sperm motility and acrosomal integrity after freezing were assessed. Sperm in SC medium (dilution ratio, 3:1) frozen at cooling rates of 67 and 183C/min in liquid N2 vapor showed higher post-thaw motility than did samples frozen at 435C/min. At the cooling rate of 183C/min and dilution in SC medium at a 3:1 ratio, post-thaw motility was higher after a holding time of 10 min than after 30 min (recommended by the manufacturer). In addition, post-thaw motility of sperm frozen in SC medium was higher with dilution ratios of 3:1, 4.5:1, and 6:1 compared with 9:1, 10.5:1, and 12:1, and the sample diluted 12:1 showed the lowest percentage of thawed sperm with intact acrosomes. Sperm showed higher post-thaw motility after freezing in TEY than in SC medium; acrosomal integrity did not differ between the 2 media. Our results indicated that cynomolgus macaque sperm can be cryopreserved successfully by using a commercial egg-yolkfree freezing medium, which provides an option for genetic preservation with decreased zoonotic risk in this important NHP species.

Effects of arginine and trehalose on post-thawed bovine sperm quality.

PubMed

Öztürk, Caner; Güngör, Şükrü; Ataman, Mehmet Bozkurt; Bucak, Mustafa Numan; Başpinar, Nuri; Ili, Pınar; Inanç, Muhammed Enes

2017-09-01

The present study was conducted to examine the protective role of arginine and trehalose on post-thaw bull sperm and oxidative stress parameters. Five ejaculates for each bull were used in the study. Each ejaculate, split into three equal aliquots and diluted at 37 °C with base extenders containing 2 mM arginine, 25 mM trehalose and no antioxidant (control) was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. Supplementation of the semen extender with arginine decreased the percentages of post-thawed subjective motility (29 ± 8.21%), CASA motility (12.2 ± 5.69%) and progressive motility (3.52 ± 2.13%), compared with the controls (43 ± 2.73%, 55.4 ± 6.78% and 33.48 ± 4.14%, respectively, P < 0.05). Supplementation of the semen extender with trehalose produced a higher mitochondrial activity and sperm viability (36.3 ± 3.99% and 44.1 ± 2.18%) compared with the control (13 ± 8.15 and 31.7 ± 3.94%, respectively, P < 0.05). It was established that trehalose (95.1%) and arginine (92.8%) protect DNA integrity compared to the control (90.4%) (P < 0.05). Trehalose supplementation in semen extenders provided great benefit in terms of viability, mitochondrial activity, and intact sperm DNA on frozen-thawed bull sperm.

Improvement of cloning efficiency in minipigs using post-thawed donor cells treated with roscovitine.

PubMed

Hwang, Seongsoo; Oh, Keon Bong; Kwon, Dae-Jin; Ock, Sun-A; Lee, Jeong-Woong; Im, Gi-Sun; Lee, Sung-Soo; Lee, Kichoon; Park, Jin-Ki

2013-11-01

Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1 h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells. The GT KO/hCD46 KI donor cells were cultured for either 3 days (control group) or 1 h after thawing with 15 μM roscovitine (experimental group) prior to the nuclear transfer. The relative percentage of the transgenic donor cells that entered into G0/G1 was 93.7 % (±2.54). This was different from the donor cells cultured for 1 h with the roscovitine-treated group (84.6 % ±4.6) (P < 0.05) and without roscovitine (78.6 % ±5.5) (P < 0.01), respectively. The pregnancy rate and delivery rate in the roscovitine group (8/12 and 6/8, respectively) were significantly higher (P < 0.01) than those in the control group (6/19 and 3/6, respectively). In the experimental group, 12 GT KO/hCD46 KI transgenic minipigs were successfully generated, and five minipigs among them survived for more than 6 months so far. The recipient-based individual cloning efficiency ranged from 0.74 to 2.54 %. In conclusion, gene-modified donor cells can be used for cloning of MGH minipigs if the cells are post-thawed and treated with roscovitine for 1 h prior to nuclear transfer.

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing.

PubMed

Fraser, L; Strzezek, J

2007-06-01

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing-thawing caused a significant increase (P<0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P<0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing-thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P<0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P<0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.

Effect of different cryo-protectants on the viability of frozen/thawed semen from boars of the Piau breed.

PubMed

Pinho, R O; Lima, D M A; Shiomi, H H; Siqueira, J B; Silva, H T; Lopes, P S; Guimarães, S E F; Guimarães, J D

2014-05-01

The objective of this study was to evaluate the effect of different cryo-protectants (glycerol, dimethylacetamide and dimethylformamide alone or combined and added to lactose-egg yolk extender) on the viability of frozen/thawed semen from the Piau breed as assessed by in vitro testing. Frozen semen samples (n=20) were used from five male swine. Five different freezing extenders, including 2% glycerol (Group 1 - G), 2% glycerol and 3% dimethylacetamide (Group 2 - GA), 2% glycerol and 3% dimethylformamide (Group 3 - GF), 5% dimethylacetamide (Group 4 - A) and 5% dimethylformamide (group 5 - F), were evaluated. To assess post-thawing sperm quality, sperm motility and morphology were evaluated. Sperm viability was determined using the hypoosmotic swelling test, supravital staining, and a fluorescent assay (carboxyfluorescein diacetate and propidium iodide). The mean total sperm motility of semen immediately after thawing was 46.2±1.3, 57.7±1.5, 53.2±2.1, 51.7±1.2, and 46.5±1.6% for groups 1-5, respectively. Groups 2 (GA) and 3 (GF) had greater motility values (P<0.05). Fluorescent assay values of 22.3±2.3%, 35.2±3.7%, 30.8±3.4%, 36.6±3.7%, and 26.5±3.8% were obtained for Groups 1-5, respectively, showing that Group 4 (A) sperm had greater viability than those from Group 1 (G), although there was no differences between the other treatments (P>0.05). The other complementary tests (hypoosmotic swelling test and supra-vital staining) demonstrated that sperm in Groups 2 (GA), 3 (GF) and 4 (A) had the greatest viability and there were no significant differences among these three groups (P>0.05). The most effective cryo-protectant combinations likely minimized and controlled the deleterious processes that occur in the sperm cell during freezing/thawing, thus improving post-thawing sperm viability. In conclusion, the combination of amides (3%) and glycerol (2%) or dimethylacetamide (5%) alone were more efficient at cryo-protection than glycerol alone for semen freezing in the Piau swine breed. Copyright © 2014 Elsevier B.V. All rights reserved.

High-throughput cryopreservation of spermatozoa of blue catfish (Ictalurus furcatus): Establishment of an approach for commercial-scale processing.

PubMed

Hu, E; Yang, Huiping; Tiersch, Terrence R

2011-02-01

Hybrid catfish created by crossing of female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) are being used increasingly in foodfish aquaculture because of their fast growth and efficient food conversion. However, the availability of blue catfish males is limited, and their peak spawning is at a different time than that of the channel catfish. As such, cryopreservation of sperm of blue catfish could improve production of hybrid catfish, and has been studied in the laboratory and tested for feasibility in a commercial dairy bull cryopreservation facility. However, an approach for commercially relevant production of cryopreserved blue catfish sperm is still needed. The goal of this study was to develop practical approaches for commercial-scale sperm cryopreservation of blue catfish by use of an automated high-throughput system (MAPI, CryoBioSystem Co.). The objectives were to: (1) refine cooling rate and cryoprotectant concentration, and evaluate their interactions; (2) evaluate the effect of sperm concentration on cryopreservation; (3) refine cryoprotectant concentration based on the highest effective sperm concentration; (4) compare the effect of thawing samples at 20 or 40°C; (5) evaluate the fertility of thawed sperm at a research scale by fertilizing with channel catfish eggs; (6) test the post-thaw motility and fertility of sperm from individual males in a commercial setting, and (7) test for correlation of cryopreservation results with biological indices used for male evaluation. The optimal cooling rate was 5°C/min (Micro Digitcool, IMV) for high-throughput cryopreservation using CBS high-biosecurity 0.5-ml straws with 10% methanol, and a concentration of 1×10(9)sperm/ml. There was no difference in post-thaw motility when samples were thawed at 20°C for 40s or 40°C for 20s. After fertilization, the percentage of neurulation (Stage V embryos) was 80±21%, and percentage of embryonic mobility (Stage VI embryo) was 51±22%. There was a significant difference among the neurulation values produced by thawed blue catfish sperm, fresh blue catfish sperm (P=0.010) and channel catfish sperm (P=0.023), but not for Stage VI embryos (P≥0.585). Cryopreserved sperm from ten males did not show significant variation in post-thaw motility or fertility at the neurulation stage. This study demonstrates that the protocol established for high-throughput cryopreservation of blue catfish sperm can provide commercially relevant quantities and quality of sperm with stable fertility for hybrid catfish production and provides a model for establishment of commercial-scale approaches for other aquatic species. Copyright © 2010 Elsevier Inc. All rights reserved.

CHARACTERISTICS AND FERTILITY OF SUMATRAN TIGER SPERMATOZOA CRYOPRESERVED WITH DIFFERENT SUGARS.

PubMed

Wayan Kurniani Karja, Ni; Fahrudin, Mokhamad; Setiadi, Mohamad Agus; Tumbelaka, Ligaya Ita; Sudarwati, Retno; Hastuti, Yohana Tri; Mulia, Bongot Huas; Widianti, Ardyta; Sultan, Keni; Terazono, Tsukasa; Namula, Zhao; Taniguchi, Masayasu; Tanihara, Fuminori; Takemoto, Tatsuya; Kikuchi, Kazuhiro; Sato, Yoko; Otoi, Takeshige

Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.

Influence of freezing and thawing on the hydration characteristics, quality, and consumer acceptance of whole muscle beef injected with solutions of salt and phosphate.

PubMed

Pietrasik, Z; Janz, J A M

2009-03-01

Effects of salt/phosphate injection level (112% or 125% pump), salt level (0.5% or 1.5% salt), and freezing/thawing on hydration characteristics, quality, and consumer acceptance of beef semitendinosus were investigated. All enhancement treatments decreased shear force by 25-35%, but negatively affected colour. Increased salt concentration yielded lower purge and cooking losses, and higher water holding capacity. The higher injection level reduced water binding properties, however, the loss in functionality with higher water addition was overcome with increased salt content. Freezing and subsequent thawing was generally detrimental to colour and water binding properties and tended to increase shear force. Freezing and subsequent thawing did not affect fluid release in steaks held for 1 day before analysis, but resulted in decreased water retention in samples held for 7 days. Holding vacuum packaged steaks for 7 days generally increased package purge and negatively affected colour parameters, although water binding characteristics were improved. Consumer panel results demonstrated a negative effect on juiciness and tenderness where meat subject to low salt/high injection was frozen then thawed - the low salt level was insufficient to maintain any positive effect of injection treatment. In general, salt/phosphate injection improved product acceptability and increased willingness to purchase.

Dose-dependent effects of homologous seminal plasma on motility and kinematic characteristics of post-thaw stallion epididymal spermatozoa.

PubMed

Neuhauser, S; Dörfel, S; Handler, J

2015-05-01

Preservation of epididymal spermatozoa is important to save genetic material of endangered species and breeds, or in case of unexpected injury, which will end the breeding career of valuable sires. Seminal plasma (SP) influences sperm quality in a dose-dependent manner and its addition to preserved semen immediately before insemination may be beneficial for sperm fertility. Increased plasma membrane stability of epididymal spermatozoa reduces freezing injury of cells, and the addition of SP after freezing and thawing might have activating and protecting effects on spermatozoa within the female genital tract. In this study, epididymal spermatozoa were harvested by retrograde flush of the epididymal cauda immediately after routine castration and frozen. Seminal plasma was collected from other six stallions. Homologous SP (SP from the same species, but from a different animal) was added to frozen-thawed epididymal spermatozoa at concentrations of 0, 5, 20, 50 and 80% SP. Addition of SP increased sperm motility and influenced kinematic values in a dose-dependent manner (p < 0.05). Motility improved at concentrations of 20 and 50% SP, but did not further increase at 80% SP. There was no difference in sperm motility among SP from six different donor stallions regardless of the concentrations of SP (p > 0.05). Total and progressive motility of ten frozen-thawed epididymal spermatozoa samples collected from different stallions after dilution with extender and 5, 20, 50 or 80% SP differed significantly (p < 0.05). In conclusion, addition of homologous SP to frozen-thawed stallion epididymal spermatozoa immediately improved motility in a dose-dependent manner regardless of semen quality of SP donor stallions. This might positively influence fertility when SP is added before insemination. Moreover, there seems to be a threshold level of SP concentration for optimal improvement of sperm motility. © 2015 American Society of Andrology and European Academy of Andrology.

Freezing behavior of adherent neuron-like cells and morphological change and viability of post-thaw cells.

PubMed

Uemura, Makoto; Ishiguro, Hiroshi

2015-04-01

Freezing of nerve cells forming a neuronal network has largely been neglected, despite the fact that the cryopreservation of nerve cells benefits the study of cells in the areas of medicine and poison screening. Freezing of nerve cells is also attractive for studying cell morphology because of the characteristic long, thread-like neurites extending from the cell body. In the present study, freezing of neuron-like cells adhering to the substrate (differentiated PC12 cells), in physiological saline, was investigated in order to understand the fundamental freezing and thawing characteristics of nerve cells with neurites. The microscopic freezing behavior of cells under different cooling rates was observed. Next, the post-thaw morphological changes in the cells, including the cytoskeleton, were investigated and post-thaw cell viability was evaluated by dye exclusion using propidium iodide. Two categories of morphological changes, beading and shortening of the neurites, were found and quantified. Also, the morphological changes of neurites due to osmotic stress from sodium chloride were studied to gain a better understanding of causation. The results showed that morphological changes and cell death were promoted with a decrease in end temperature during freezing. Copyright © 2015 Elsevier Inc. All rights reserved.

Emerging technologies in medical applications of minimum volume vitrification

PubMed Central

Zhang, Xiaohui; Catalano, Paolo N; Gurkan, Umut Atakan; Khimji, Imran; Demirci, Utkan

2011-01-01

Cell/tissue biopreservation has broad public health and socio-economic impact affecting millions of lives. Cryopreservation technologies provide an efficient way to preserve cells and tissues targeting the clinic for applications including reproductive medicine and organ transplantation. Among these technologies, vitrification has displayed significant improvement in post-thaw cell viability and function by eliminating harmful effects of ice crystal formation compared to the traditional slow freezing methods. However, high cryoprotectant agent concentrations are required, which induces toxicity and osmotic stress to cells and tissues. It has been shown that vitrification using small sample volumes (i.e., <1 μl) significantly increases cooling rates and hence reduces the required cryoprotectant agent levels. Recently, emerging nano- and micro-scale technologies have shown potential to manipulate picoliter to nanoliter sample sizes. Therefore, the synergistic integration of nanoscale technologies with cryogenics has the potential to improve biopreservation methods. PMID:21955080

Factors affecting storage of Slovak native rabbit semen in the gene bank.

PubMed

Kulíková, Barbora; Oravcová, Marta; Baláži, Andrej; Supuka, Peter; Chrenek, Peter

2017-10-01

In this study, fresh and frozen-thawed semen of Nitra and Zobor rabbit breeds were evaluated for potential inter-breed or inter-male differences in sperm quality traits. Individual male semen from four rabbits of each breed were diluted (v:v; 1:1) in a freezing medium composed of a commercial diluent, 16% of dimethyl sulphoxide (DMSO), 4% of Ficoll 70 and 2% of sucrose and frozen in liquid nitrogen vapours before being plunged into liquid nitrogen. Different motility traits, viability and plasma membrane integrity of fresh and frozen-thawed semen were evaluated in vitro using computer-assisted sperm analysis and flow cytometry. To evaluate the sperm fertilization ability, artificial insemination of fresh and frozen-thawed sperm was performed. Our results showed the effect of breed (P ≤ 0.05) on frozen-thawed sperm viability and plasma membrane integrity. Moreover, individual variability in semen quality among the rabbits was revealed (0.31 to 0.71 among quality traits). Our results thereby confirmed that the cryopreservation procedure could not ensure comparable sperm post-thaw survival for different breeds or males. Nevertheless, correlations between numbers of fresh total motile and progressively moving sperm and several quality parameters measured post thawing were revealed. Therefore, we suggest that the objective assessment of fresh rabbit sperm motility may be an effective indicator of frozen-thawed semen quality. Consequently, regular semen assessment is required in order to preserve good-quality insemination doses from native breeds.

Snowmelt and Surface Freeze/Thaw Timings over Alaska derived from Passive Microwave Observations using a Wavelet Classifier

NASA Astrophysics Data System (ADS)

Steiner, N.; McDonald, K. C.; Dinardo, S. J.; Miller, C. E.

2015-12-01

Arctic permafrost soils contain a vast amount of organic carbon that will be released into the atmosphere as carbon dioxide or methane when thawed. Surface to air greenhouse gas fluxes are largely dependent on such surface controls as the frozen/thawed state of the snow and soil. Satellite remote sensing is an important means to create continuous mapping of surface properties. Advances in the ability to determine soil and snow freeze/thaw timings from microwave frequency observations improves upon our ability to predict the response of carbon gas emission to warming through synthesis with in-situ observation, such as the 2012-2015 Carbon in Arctic Reservoir Vulnerability Experiment (CARVE). Surface freeze/thaw or snowmelt timings are often derived using a constant or spatially/temporally variable threshold applied to time-series observations. Alternately, time-series singularity classifiers aim to detect discontinuous changes, or "edges", in time-series data similar to those that occur from the large contrast in dielectric constant during the freezing or thaw of soil or snow. We use multi-scale analysis of continuous wavelet transform spectral gradient brightness temperatures from various channel combinations of passive microwave radiometers, Advanced Microwave Scanning Radiometer (AMSR-E, AMSR2) and Special Sensor Microwave Imager (SSM/I F17) gridded at a 10 km posting with resolution proportional to the observational footprint. Channel combinations presented here aim to illustrate and differentiate timings of "edges" from transitions in surface water related to various landscape components (e.g. snow-melt, soil-thaw). To support an understanding of the physical basis of observed "edges" we compare satellite measurements with simple radiative transfer microwave-emission modeling of the snow, soil and vegetation using in-situ observations from the SNOw TELemetry (SNOTEL) automated weather stations. Results of freeze/thaw and snow-melt timings and trends are reported for Alaska and the North-West Canadian Arctic for the period 2002 to 2015.

Impact of freeze-thaw on liquefaction potential and dynamic properties of Mabel Creek silt.

DOT National Transportation Integrated Search

2009-02-01

"This study examines the influence of temperature rise and freeze-thaw cycles on the soil liquefaction potential. More specifically, dynamic properties and post-cyclicloading : settlement of fine-grained soils are evaluated in this study. The results...

Modeling conductive heat transfer during high-pressure thawing processes: determination of latent heat as a function of pressure.

PubMed

Denys, S; Van Loey, A M; Hendrickx, M E

2000-01-01

A numerical heat transfer model for predicting product temperature profiles during high-pressure thawing processes was recently proposed by the authors. In the present work, the predictive capacity of the model was considerably improved by taking into account the pressure dependence of the latent heat of the product that was used (Tylose). The effect of pressure on the latent heat of Tylose was experimentally determined by a series of freezing experiments conducted at different pressure levels. By combining a numerical heat transfer model for freezing processes with a least sum of squares optimization procedure, the corresponding latent heat at each pressure level was estimated, and the obtained pressure relation was incorporated in the original high-pressure thawing model. Excellent agreement with the experimental temperature profiles for both high-pressure freezing and thawing was observed.

Microwave Blood Thawing: Biochemical Analysis of Small Samples of Thawed Red Blood Cells.

DTIC Science & Technology

1984-01-01

dissociation curve at three DPG levels . . . 42 7 Changes in DPG concentration over the 6 hours post-wash . . . 43 8 Changes in pH over the 6 hours post...citrate dextrose ATP Adenosine-5’ -triphosphate CPD Citrate phosphate dextrose DPG 2,3- diphosphoglycerate FDA Food and Drug Administration gRBC... diphosphoglycerate (DPG) concentration, and * reduced glutathione (GSH) concentration. Not all parameters were measured on all units at this step of the preparation

Effect of controlled and uncontrolled rate of cooling, prior to controlled rate of freezing, on motion characteristics and acrosomal integrity of cryopreserved ram spermatozoa.

PubMed

Joshi, Anil; Kumar, Davendra; Naqvi, S M K; Maurya, V P

2008-12-01

A programmable cell freezer provides ideal cryobiological conditions for controlled-rate cooling and freezing of ram spermatozoa. The purpose of this study was to investigate the effects of controlled (Group 1) and uncontrolled (Group 2) cooling conditions prior to programmable freezing of ram semen on post-thaw sperm motion characteristics and acrosomal integrity of ram spermatozoa. Semen samples of good initial motility obtained from adult Malpura rams were pooled, diluted to 1 × 10(9) spermatozoa per milliliter with Egg yolk-TEST-glycerol extender, and packaged in 0.25 mL straws. Straws representing Group 1 were cooled in a programmable cell freezer from 25°C to 5°C at the rate of -0.15°C per minute followed by a holding time of 2 h for equilibration, while straws of Group 2 were allowed to cool slowly up to 5°C and equilibrate for 2 h in the cold cabinet. After equilibration, straws of Group 2 were also loaded in the cell freezer for freezing straws of both the treatment groups simultaneously from 5°C to -125°C at the rate of -25°C per minute. Thawing of straws was done at 50°C for 10 s and the quality of frozen-thawed spermatozoa was objectively assessed by using sperm motility analyzer. Thawed samples were also evaluated for acrosomal integrity after staining the dried semen smears with Giemsa stain. The average post-thaw motility of straws was significantly higher (P < 0.05) in samples frozen after controlled cooling, compared with samples frozen after uncontrolled rate of cooling. The percent of spermatozoa with normal acrosome was also significantly (P < 0.05) higher in Group 1, compared to Group 2. The results indicate that controlled-rate cooling has a significant effect on post-thaw motility and acrosomal integrity of frozen-thawed ram spermatozoa, compared to uncontrolled-rate cooling prior to programmable freezing.

Effect of rainbow trout (Oncorhynchus mykiss) seminal plasma on the post-thaw quality of ram semen cryopreserved in a soybean lecithin-based or egg yolk-based extender.

PubMed

Ustuner, Burcu; Alcay, Selim; Toker, M Berk; Nur, Zekariya; Gokce, Elif; Sonat, Fusun Ak; Gul, Zulfiye; Duman, Muhammed; Ceniz, Cafer; Uslu, Aydın; Sagirkaya, Hakan; Soylu, M Kemal

2016-01-01

The aim of the current study was to evaluate the effects of different concentrations of rainbow trout seminal plasma (RTSP) (0.1%, 1% and 10%) in extenders containing either egg yolk or lecithin for use in Awassi ram semen cryopreservation. Pooled sperm were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in egg yolk or lecithin extender containing no RTSP, 0.1%, 1% or 10% RTSP (v/v). Semen samples were assessed for sperm motility, plasma membrane integrity [hypoosmotic swelling test (HOST) and Hoechst 33258] and defective acrosomes [FITC-conjugated Pisum sativum agglutinin (PSA-FITC)] at the following five time points: after dilution with extender A; after equilibration; and post-thaw at 0h, 3h and 5h. Malondialdehyde (MDA) was examined only after thawing. Freezing and thawing procedures (dilution, equilibration and post-thaw incubation at 0h, 3h and 5h) negatively affected the motility (P<0.001) and acrosome integrity (P<0.001). Additionally, freezing and thawing negatively affected the plasma membrane integrity, as determined by the HOST and Hoechst 33258 (P<0.001). The extender group affected the motility (P<0.001) and the HOST results (P<0.001). Levels of MDA in the egg yolk extender with 1% RTSP group were significantly lower than in the lecithin control group (P<0.05). In conclusion, the egg yolk extender groups that were supplemented with 10% and 1% RTSP provided greater cryoprotective effects for semen survivability during 5h incubation than the other extender groups. Copyright © 2015 Elsevier B.V. All rights reserved.

Cryopreservation of Cynomolgus Macaque (Macaca fascicularis) Sperm by Using a Commercial Egg-Yolk–Free Freezing Medium

PubMed Central

Yan, Yaping; Ao, Lei; Wang, Hong; Duan, Yanchao; Chang, Shaohui; Chen, Bingbing; Zhi, Dalong; Li, Sujuan; Niu, Yuyu; Ji, Weizhi; Si, Wei

2016-01-01

Conventional TRIS–egg yolk (TEY) freezing medium for the cryopreservation of NHP sperm has the risk of contamination due to widespread zoonotic diseases. This study was aimed at determining the optimal glycerol concentration, freezing rate, and holding time in liquid N2 vapor for the cryopreservation of cynomolgus macaque sperm by using a commercial egg-yolk–free freezing medium (SC medium) designed for human sperm cryopreservation. Sperm motility and acrosomal integrity after freezing were assessed. Sperm in SC medium (dilution ratio, 3:1) frozen at cooling rates of –67° and –183°C/min in liquid N2 vapor showed higher post-thaw motility than did samples frozen at –435 °C/min. At the cooling rate of –183 °C/min and dilution in SC medium at a 3:1 ratio, post-thaw motility was higher after a holding time of 10 min than after 30 min (recommended by the manufacturer). In addition, post-thaw motility of sperm frozen in SC medium was higher with dilution ratios of 3:1, 4.5:1, and 6:1 compared with 9:1, 10.5:1, and 12:1, and the sample diluted 12:1 showed the lowest percentage of thawed sperm with intact acrosomes. Sperm showed higher post-thaw motility after freezing in TEY than in SC medium; acrosomal integrity did not differ between the 2 media. Our results indicated that cynomolgus macaque sperm can be cryopreserved successfully by using a commercial egg-yolk–free freezing medium, which provides an option for genetic preservation with decreased zoonotic risk in this important NHP species. PMID:27931311

Effect of Camellia sinensis supplementation and increasing holding time on quality of cryopreserved boar semen.

PubMed

Gale, I; Gil, L; Malo, C; González, N; Martínez, F

2015-06-01

Cryopreservation of boar semen is still considered suboptimal due to the low fertility when compared with fresh semen. This study was performed to evaluate the effects of green tea (Camellia sinensis) supplementation of the freezing extender at different concentration (0, 2.5%, 5%, 10%) and also to determine the influence of increasing holding time from 2 to 24 h at 15 °C. Seventeen ejaculates from nine boars were used to make pools of three of them and then cryopreserved. Sperm motility, viability, acrosome integrity, membrane functionality (HOST) and capacitation status were determined before freezing and at 0, 30, 60, 90 and 120 min after thawing. Lipid peroxidation was evaluated just after thawing. The main findings emerging from this study were the following: (i) no improvement in quality of thawed spermatozoa with addition of tea to the freezing extender, (ii) no improvement in quality of thawed spermatozoa with prolonged holding time, (iii) lower peroxidation rate in presence of tea 5% and (iv) a decrease in the number of uncapacited viable spermatozoa with any tea supplementation. We conclude that amplification of holding time in semen cryopreservation process does not vary results, facilitating freezing protocol. Tea supplementation reduces lipoxidation but did not improve quality parameters. © 2014 Blackwell Verlag GmbH.

The post-thaw irradiation of avian spermatozoa with He-Ne laser differently affects chicken, pheasant and turkey sperm quality.

PubMed

Iaffaldano, N; Paventi, G; Pizzuto, R; Passarella, S; Cerolini, S; Zaniboni, L; Marzoni, M; Castillo, A; Rosato, M P

2013-11-30

The effects of post-thaw Helium-Neon (He-Ne) laser irradiation on mobility and functional integrity of frozen/thawed chicken, pheasant and turkey spermatozoa were investigated. Cytochrome C oxidase (COX) activity was also determined as a measure of the effect of irradiation on mitochondrial bioenergetics. Semen samples from each species were collected, processed and frozen according to the pellet procedure. After thawing, each semen sample was divided into two subsamples: the first one was the control; the second one was irradiated with a single mode continuous He-Ne laser wave (wavelength 632.8 nm; 6 mW; 3.96 J/cm(2)). Then the samples were assessed for sperm mobility (Accudenz(®) swim-down test), viability (SYBR-14/PI staining), osmotic-resistance (HOS test) and COX activity. The irradiation was effective P<0.05 increasing sperm motility in the turkey semen (0.228 ± 0.01 compared with 0.294 ± 0.02). The irradiation also caused an increase (P<0.05) of the COX activity in pheasant (+135 ± 4%) and turkey (+116 ± 4%) sperm, without affecting viability and osmotic-resistance. The COX was positively correlated (P<0.05) with the viability of chicken sperm, however no significant interactions were found between mobility and COX activity in the three avian species. Due to the difference in energetic metabolism among avian species used in this study, the He-Ne laser irradiation has a differential action on bio-stimulation of turkey, chicken and pheasant spermatozoa. The present results are the first to elucidate the possibility for restoration of motility of cryopreserved avian spermatozoa by bio-stimulation provided via He-Ne laser irradiation. Copyright © 2013 Elsevier B.V. All rights reserved.

Permafrost Thaw increases Emissions of Nitrous Oxide from Subarctic Peatlands

NASA Astrophysics Data System (ADS)

Voigt, C.; Marushchak, M. E.; Lamprecht, R. E.; Jackowicz-Korczynski, M.; Lindgren, A.; Mastepanov, M.; Christensen, T. R.; Granlund, L.; Tahvanainen, T.; Martikainen, P. J.; Biasi, C.

2017-12-01

Permafrost soils in the Arctic are thawing, exposing not only carbon but also large nitrogen stocks. The decomposition of this vast pool of long-term immobile C and N stocks results in the release of greenhouse gases to the atmosphere. Among these, carbon dioxide (CO2) and methane (CH4) are being studied extensively, and gaseous C release from thawing permafrost is known to be substantial. Most recent studies, however, show that Arctic soils may further be a relevant source of the strong greenhouse gas nitrous oxide (N2O). As N2O is almost 300 times more powerful in warming the climate than CO2 based on a 100-yr time horizon, the release of N2O from thawing permafrost could create a significant non-carbon permafrost-climate feedback. To study the effect of permafrost thaw on N2O fluxes, we collected peat mesocosms from a Subarctic permafrost peatland, and subjected these intact soil-plant systems to sequential thawing from the top of the active layer down to the upper permafrost layer. Measurements of N2O fluxes were coupled with detailed soil analyses and process studies. Since N2O fluxes are highly dependent on moisture conditions and vegetation cover, we applied two distinct moisture treatments (dry vs. wet) and simulated permafrost thaw in vegetated as well as in naturally bare mesocosms. Under dry conditions, permafrost thaw clearly increased N2O emissions. We observed the largest post-thaw emissions from bare peat surfaces, a typical landform in subarctic peatlands previously identified as hot spots for Arctic N2O emissions. There, permafrost thaw caused a five-fold increase in emissions (0.56 vs. 2.81 mg N2O m-2 d-1). While water-logged conditions suppressed N2O emissions, the presence of vegetation lowered, but did not prevent post-thaw N2O release. Based on these findings, we show that one fourth of the Arctic land area could be vulnerable for N2O emissions when permafrost thaws. Our results demonstrate that Arctic N2O emissions may be larger than previously thought, and that the source strength will be crucially governed by moisture conditions at times of thaw, as well as on future changes in vegetation coverage.

Microbial functional diversity covaries with permafrost thaw-induced environmental heterogeneity in tundra soil.

PubMed

Yuan, Mengting M; Zhang, Jin; Xue, Kai; Wu, Liyou; Deng, Ye; Deng, Jie; Hale, Lauren; Zhou, Xishu; He, Zhili; Yang, Yunfeng; Van Nostrand, Joy D; Schuur, Edward A G; Konstantinidis, Konstantinos T; Penton, Christopher R; Cole, James R; Tiedje, James M; Luo, Yiqi; Zhou, Jizhong

2018-01-01

Permafrost soil in high latitude tundra is one of the largest terrestrial carbon (C) stocks and is highly sensitive to climate warming. Understanding microbial responses to warming-induced environmental changes is critical to evaluating their influences on soil biogeochemical cycles. In this study, a functional gene array (i.e., geochip 4.2) was used to analyze the functional capacities of soil microbial communities collected from a naturally degrading permafrost region in Central Alaska. Varied thaw history was reported to be the main driver of soil and plant differences across a gradient of minimally, moderately, and extensively thawed sites. Compared with the minimally thawed site, the number of detected functional gene probes across the 15-65 cm depth profile at the moderately and extensively thawed sites decreased by 25% and 5%, while the community functional gene β-diversity increased by 34% and 45%, respectively, revealing decreased functional gene richness but increased community heterogeneity along the thaw progression. Particularly, the moderately thawed site contained microbial communities with the highest abundances of many genes involved in prokaryotic C degradation, ammonification, and nitrification processes, but lower abundances of fungal C decomposition and anaerobic-related genes. Significant correlations were observed between functional gene abundance and vascular plant primary productivity, suggesting that plant growth and species composition could be co-evolving traits together with microbial community composition. Altogether, this study reveals the complex responses of microbial functional potentials to thaw-related soil and plant changes and provides information on potential microbially mediated biogeochemical cycles in tundra ecosystems. © 2017 John Wiley & Sons Ltd.

Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation.

PubMed

Clulow, J R; Buss, H; Evans, G; Sieme, H; Rath, D; Morris, L H A; Maxwell, W M C

2012-02-01

Sex-sorted, frozen-thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex-sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex-sorted frozen-thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82(®) and a modified lactose-ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex-sorted stallion spermatozoa. No significant differences between the two freezing media could be identified, suggesting that both cryodiluents would be suitable for incorporation into a sex-preselection protocol for stallion spermatozoa. Experiment 2 compared Kenney's modified Tyrode's (KMT) and Sperm TALP (Sp-TALP) as the staining and incubation medium for stallion spermatozoa prior to sex-sorting. A significant increase in the percentage of acrosome-reacted spermatozoa occurred after staining and incubation in the clarified Sp-TALP compared with KMT. As no improvements in sorting rates were achieved using Sp-TALP, it was concluded that stallion sorting protocols could include KMT as the staining and incubation medium while either INRA 82(®) or lactose-EDTA could be employed as a cryodiluents. © 2011 Blackwell Verlag GmbH.

Transformation of Upland Water and Carbon Dynamics by Thawing Permafrost in the Alaskan Interior

NASA Astrophysics Data System (ADS)

Ewing, S. A.; Paces, J. B.; O'Donnell, J. A.; Kanevskiy, M. Z.; Shur, Y.; Jorgenson, M. T.; Harden, J.; Aiken, G. R.; Striegl, R.

2009-05-01

Large arctic rivers can provide an integrated signal of regional permafrost thaw and associated carbon dynamics. A long-term (30-y) decrease in dissolved organic carbon (DOC) and increase in dissolved inorganic carbon in the Yukon River Basin (YRB) suggest increased flow through mineral soils as a result of permafrost thaw. We used U series isotopes to test for the influence of thaw on soil and surface waters in small upland catchments at two sites within the YRB. In natural waters, 234U/238U activity ratios exceed 1.00 (secular equilibrium) as a function of water-rock contact time. Previous work has shown that in major YRB rivers, seasonally and spatially variable 234U/238U ratios could indicate both groundwater inputs and permafrost thaw, with ratios ranging from 1.1 to 2.6. We show that 234U/238U ratios in soil and surface water from these small catchments span the range of values observed in the major rivers, and indicate greater influence of older water where the mineral soil and underlying sediment facilitate drainage and permafrost degradation. Analysis of deep, ice-rich loess permafrost cores (2-10 m) reveals that thaw of Pleistocene ice can release high concentrations of DOC (>1000 ppm) and ammonium in thaw waters. The age and chemical composition of these waters allows for improved prediction of downstream carbon dynamics upon thaw. Field observation of hillslope soil sequences indicates that both topography and mineral substrate influence the effects of thaw on water and carbon dynamics in small catchments.

Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm.

PubMed

Nichi, M; Rijsselaere, T; Losano, Jda; Angrimani, Dsr; Kawai, Gkv; Goovaerts, Igf; Van Soom, A; Barnabe, V H; De Clercq, Jbp; Bols, Pej

2017-04-01

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2-3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures. © 2016 Blackwell Verlag GmbH.

A comparative study on the cryogenic preservation of semen from the sandhill crane and the domestic fowl

USGS Publications Warehouse

Sexton, T.J.; Gee, G.F.; Watson, P.F.

1978-01-01

SYNOPSIS: Recent findings on the cryogenic preservation of semen from the crane, Grus canadensis pratensis and the domestic fowl, Gallus domesticus, are compared. Highest levels of post-thaw motility for crane semen (55%) were obtained when semen was diluted 1:1 with the Beltsville Poultry Semen Extender (BPSE) and held for 30 min at 5 C before it was equilibrated with 4% dimethyl sulfoxide (DMSO) for 15 min. In contrast, post-thaw motility for fowl spermatozoa was highest (80%) when semen was diluted 1:3 with BPSE and held for 60 min at 5 C before it was equilibrated with 4% DMSO for 60 min. Post-thaw motility of spermatozoa of both species was highest when the following freezing rates were used: l C per min from +5 to -20 C, 50 C per min from -20 to -80 C, then plunging into liquid nitrogen which resulted in a rate of 160 C per min from -80 to -196 C. One of four crane eggs resulting from insemination with frozen-thawed semen was fertile, whereas 27 of 55 fowl eggs were fertile, but this difference may have been due largely to fewer spermatozoa being inseminated into the female crane than into the fowl.

Supplementing zinc oxide nanoparticles to cryopreservation medium minimizes the freeze-thaw-induced damage to spermatozoa.

PubMed

Isaac, Ann V; Kumari, Sandhya; Nair, Ramya; Urs, Deepak Raj; Salian, Sujith Raj; Kalthur, Guruprasad; Adiga, Satish Kumar; Manikkath, Jyothsna; Mutalik, Srinivas; Sachdev, Divya; Pasricha, Renu

2017-12-16

The sperm DNA integrity post cryopreservation of human semen samples is one of the serious concerns in human infertility treatment. In the present study, the beneficial effects of zinc oxide nanoparticles in preserving the functional ability of spermatozoa was explored. Ejaculates of normozoospermic men cryopreserved along with Zinc oxide nanoparticles (ZnONPs) exhibited non-significantly higher percentage of total and progressive motility in frozen-thawed samples compared to control. The sperm chromatin damage and malondialdehyde (MDA) level was significantly lower in ZnONPs group (P < 0.01 and P < 0.05 respectively) and the spermatozoa's ability to undergo acrosome reaction was also unaltered. Fluorescence microscopy and High resolution transmission electron microscopy analysis demonstrated that the ZnONPs do not penetrate the membrane of spermatozoa but stay around the spermatozoa. In conclusion, the presence of ZnONPs during cryopreservation appears to be beneficial to the spermatozoa as they withstand freeze-thaw process competently better than control, without any adverse effect shown. Copyright © 2017 Elsevier Inc. All rights reserved.

Characteristics of high-quality Asian elephant (Elephas maximus) ejaculates and in vitro sperm quality after prolonged chilled storage and directional freezing.

PubMed

O'Brien, J K; Steinman, K J; Montano, G A; Love, C C; Saiers, R L; Robeck, T R

2013-01-01

The in vitro quality of spermatozoa from one elephant (Elephas maximus) was examined after chilled storage and directional freezing (DF). High-quality, non-contaminated ejaculates (77.6±6.0% progressive motility, 3.9±1.5 µg creatinine mL(-1) raw semen, 2.7±0.6% detached heads) were cryopreserved after 0 (0hStor), 12 (12hStor) and 24 h (24hStor) of chilled storage. At 0 h and 6h post-thawing, total motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and normal morphology were similar (P>0.05) across treatments. In contrast, progressive motility, rapid velocity and several kinematic parameters were lower (P<0.05) for 24Stor compared with 0hStor at 0 h post-thaw. By 6 h post-thaw, amplitude of lateral head displacement and velocity parameters (average pathway, straight-line and curvilinear velocity) were lower (P<0.05) for 24hStor compared with 0hStor and 12hStor. DNA integrity was high and remained unchanged (P>0.05) across all groups and processing stages (1.6±0.6% of cells contained fragmented DNA). Results indicate that DF after up to 12 h of chilled storage results in a post-thaw sperm population of acceptable quality for artificial insemination. These findings have implications for the cryopreservation of sex-sorted spermatozoa, which typically undergo more than 12 h of chilled storage prior to sorting and preservation.

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: II—Mathematical prediction and experimental validation of optimal cryopreservation protocols☆

PubMed Central

Kashuba, Corinna M.; Benson, James D.; Critser, John K.

2014-01-01

In Part I, we documented differences in cryopreservation success measured by membrane integrity in four mouse embryonic stem cell (mESC) lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1), and we demonstrated a potential biophysical basis for these differences through a comparative study characterizing the membrane permeability characteristics and osmotic tolerance limits of each cell line. Here we use these values to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures. We subsequently verified these predictions experimentally for their effects on post-thaw recovery. From this study, we determined that a cryopreservation protocol utilizing 1 M propylene glycol, a cooling rate of 1 °C/minute, and plunging into liquid nitrogen at −41 °C, combined with subsequent warming in a 22 °C water bath with agitation, significantly improved post-thaw recovery for three of the four mESC lines, and did not diminish post-thaw recovery for our single exception. It is proposed that this protocol can be successfully applied to most mESC lines beyond those included within this study once the effect of propylene glycol on mESC gene expression, growth characteristics, and germ-line transmission has been determined. Mouse ESC lines with poor survival using current standard cryopreservation protocols or our proposed protocol can be optimized on a case-by-case basis using the method we have outlined over two papers. For our single exception, the CBA cell line, a cooling rate of 5 °C/minute in the presence of 1.0 M dimethyl sulfoxide or 1.0 M propylene glycol, combined with plunge temperature of −80 °C was optimal. PMID:24560712

Seminal PDC-109 protein vis-à-vis cholesterol content and freezability of buffalo spermatozoa.

PubMed

Singh, Mahak; Ghosh, S K; Prasad, J K; Kumar, Anuj; Tripathi, R P; Bhure, S K; Srivastava, N

2014-01-10

Advancements in reproductive technologies have shown seminal plasma (SP) as a nutritive-protective medium for spermatozoa metabolism, function and transport. At the same time quality variables and thus freezability of spermatozoa are influenced by SP proteins originating from male reproductive tract. One such protein, viz. PDC-109 is reported to influence freezability of spermatozoa in cattle. Thus the present investigation was designed to evaluate effect of seminal PDC-109 protein concentration on post-thaw cholesterol content and semen quality variables (SQP) as an indicator of membrane integrity and freezability, respectively of buffalo spermatozoa. Ejaculates (n=42) selected on the basis of mass activity and individual motility were divided into three parts, first part for SP proteins isolation, second for cholesterol estimation and third part was cryo-preserved to evaluate freezability based on post-thaw SQP, viz. individual progressive motility, viability and acrosome integrity of spermatozoa. A total of 28 (66.7%) and 14 (33.3%) ejaculates from four bulls were found as freezable or non-freezable, respectively. Though total seminal plasma protein (TSPP) concentration was found similar in freezable and non-freezable ejaculates, the heparin binding proteins (HBP) content in non-freezable semen was greater (P<0.01) than freezable ejaculates. There was a similar trend for the PDC-109 protein content in respective ejaculates. Cholesterol content of spermatozoa and SQP were greater (P<0.05 and 0.01, respectively) in freezable as compared to non-freezable ejaculates of each bull at post-thaw stage. This study showed that concentrations of HBP and PDC-109 in non-freezable semen might be responsible for greater cryo-damage reflecting in poor freezability of buffalo spermatozoa. Copyright © 2013 Elsevier B.V. All rights reserved.

Seasonal Preservation Success of the Marine Dinoflagellate Coral Symbiont, Symbiodinium sp.

PubMed Central

Hagedorn, Mary; Carter, Virginia L.

2015-01-01

Coral reefs are some of the most diverse and productive ecosystems on the planet, but are threatened by global and local stressors, mandating the need for incorporating ex situ conservation practices. One approach that is highly protective is the development of genome resource banks that preserve the species and its genetic diversity. A critical component of the reef are the endosymbiotic algae, Symbiodinium sp., living within most coral that transfer energy-rich sugars to their hosts. Although Symbiodinium are maintained alive in culture collections around the world, the cryopreservation of these algae to prevent loss and genetic drift is not well-defined. This study examined the quantum yield physiology and freezing protocols that resulted in survival of Symbiodinium at 24 h post-thawing. Only the ultra-rapid procedure called vitrification resulted in success whereas conventional slow freezing protocols did not. We determined that success also depended on using a thin film of agar with embedded Symbiodinium on Cryotops, a process that yielded a post-thaw viability of >50% in extracted and vitrified Symbiodinium from Fungia scutaria, Pocillopora damicornis and Porites compressa. Additionally, there also was a seasonal influence on vitrification success as the best post-thaw survival of F. scutaria occurred in winter and spring compared to summer and fall (P < 0.05). These findings lay the foundation for developing a viable genome resource bank for the world’s Symbiodinium that, in turn, will not only protect this critical element of coral functionality but serve as a resource for understanding the complexities of symbiosis, support selective breeding experiments to develop more thermally resilient strains of coral, and provide a ‘gold-standard’ genomics collection, allowing for full genomic sequencing of unique Symbiodinium strains. PMID:26422237

Effects of storage in different semen extenders on the pre-freezing and post-thawing quality of boar spermatozoa.

PubMed

Dziekońska, A; Zasiadczyk, Ł; Lecewicz, M; Strzeżek, R; Koziorowska-Gilun, M; Fraser, L; Mogielnicka-Brzozowska, M; Kordan, W

2015-01-01

The aim of this study was to investigate the effects of storage of semen in different commercial extenders on the pre-freezing and post-thawing quality of boar spermatozoa. Semen was diluted in BTS, Androhep (AH) and Gedil (GD), stored for 24 h at 17°C, and then frozen in accordance with the cryopreservation protocol. Analyses of the quality of spermatozoa included: motility, normal apical ridge (NAR) acrosome, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), measurements of ATP content and activity of superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Prior to the freezing process, no significant effect of the extender on the sperm quality parameters was noted. After thawing the spermatozoa it was demonstrated that the type of extender used influenced PMI, MMP, ATP content and activity of GPx. In the AH extender the percentage of spermatozoa with PMI and ATP content in spermatozoa was significantly higher (P<0.05) as compared to the BTS or GD extenders. In addition, semen stored in the AH was characterised by a statistically higher (P<0.05) percentage of spermatozoa with MMP and increased activity of GPx as compared with the BTS. The results obtained indicate that for the cryopreservation process, boar spermatozoa stored for 24 hours in liquid state can be used. However, the type of extender used prior to freezing may have a significant effect on the post-thawing quality of the spermatozoa. The AH extender better secured the quality of thawed boar spermatozoa as compared with the BTS or GD.

Sage (Salvia officinalis) and fennel (Foeniculum vulgare) improve cryopreserved boar epididymal semen quality study.

PubMed

Monton, A; Gil, L; Malo, C; Olaciregui, M; Gonzalez, N; de Blas, I

2015-01-01

The aim of this study was to evaluate the effect of fennel and sage extracts and the influence of the egg yolk source (fresh or pasteurized) on the success of freezing boar epididymal spermatozoa. In experiment 1, epididymal sperm was recovered by flushing and cryopreserved in a lactose-egg yolk solution supplemented with various concentrations (10, 5 and 2.5 g/L) of sage or fennel. Sperm quality was evaluated (motility, viability, HOST and acrosome integrity) at 0 h and 2 h after thawing. Fennel 10 g/L and sage 5 g/L and control (no extracts) were selected for experiment 2 which also compared fresh or pasteurized egg yolk in the freezing extender and measured DNA integrity of the frozen sperm. Results showed that the interaction between fennel and sage antioxidants with fresh egg yolk significantly improved post thaw sperm quality and protected boar epididymal spermatozoa from cryopreservation damage as a result of oxidative stress.

Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains.

PubMed

Ando, Akira; Nakamura, Toshihide; Murata, Yoshinori; Takagi, Hiroshi; Shima, Jun

2007-03-01

Yeasts used in bread making are exposed to freeze-thaw stress during frozen-dough baking. To clarify the genes required for freeze-thaw tolerance, genome-wide screening was performed using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 58 gene deletions that conferred freeze-thaw sensitivity. These genes were then classified based on their cellular function and on the localization of their products. The results showed that the genes required for freeze-thaw tolerance were frequently involved in vacuole functions and cell wall biogenesis. The highest numbers of gene products were components of vacuolar H(+)-ATPase. Next, the cross-sensitivity of the freeze-thaw-sensitive mutants to oxidative stress and to cell wall stress was studied; both of these are environmental stresses closely related to freeze-thaw stress. The results showed that defects in the functions of vacuolar H(+)-ATPase conferred sensitivity to oxidative stress and to cell wall stress. In contrast, defects in gene products involved in cell wall assembly conferred sensitivity to cell wall stress but not to oxidative stress. Our results suggest the presence of at least two different mechanisms of freeze-thaw injury: oxidative stress generated during the freeze-thaw process, and defects in cell wall assembly.

Trehalose in glycerol-free freezing extender enhances post-thaw survival of boar spermatozoa

PubMed Central

ATHURUPANA, Rukmali; TAKAHASHI, Daisen; IOKI, Sumire; FUNAHASHI, Hiroaki

2015-01-01

Cryopreservation of boar semen is still considered suboptimal due to lower fertility as compared with fresh samples when glycerol, a permeating cryoprotectant, is used. Trehalose is a non-permeable cryoprotectant and nonreducing disaccharide known to stabilize proteins and biologic membranes. The aim of this study was to evaluate the cryosurvival and in vitro penetrability of boar spermatozoa when glycerol was replaced with trehalose in a freezing extender. Ejaculated Berkshire semen samples were diluted in egg yolk-based freezing extender containing glycerol (100 mM) or trehalose (0, 50, 100, 150, 200 and 250 mM) and cryopreserved using a straw freezing procedure. Thawed samples were analyzed for motility, viability, mitochondrial membrane potential (MMP), and acrosome integrity. In experiment 2, penetrability of spermatozoa cryopreserved with 100 mM glycerol or trehalose was examined. Replacement of cryoprotectant glycerol (100 mM) with trehalose had no effect on sperm viability, but replacing it with 100 mM trehalose improved motility, MMP and acrosome integrity significantly. Sperm motility and MMP were considerably higher in 100 mM trehalose, whereas the acrosome integrity was substantially higher in 100–250 mM trehalose. The in vitro penetration rate was also significantly higher in spermatozoa cryopreserved with trehalose (61.3%) than in those cryopreserved with glycerol (43.6%). In conclusion, 100 mM non-permeable trehalose can be used to replace glycerol, a permeating cryoprotectant, for maintenance of better post-thaw quality of boar spermatozoa. PMID:25754239

Season-induced changes in bovine sperm motility following a freeze-thaw procedure.

PubMed

Orgal, Shlomo; Zeron, Yoel; Elior, Nili; Biran, David; Friedman, Eran; Druker, Shaked; Roth, Zvi

2012-01-01

Decreased conception rate of dairy cows in the summer is mainly associated with the deleterious effects of environmental thermal stress on the female reproductive tract. Here, we suggest that decreased reproductive performance might be partially due to inferior-quality semen. Semen from five representative bulls was collected in summer (August to September) and winter (December to January) and evaluated with a computerized sperm-quality analyzer for bulls (SQA-Vb). No seasonal effect was found in fresh ejaculate, but sperm examined post-thawing showed lower velocity, motility and progressive motility (P<0.04) in summer vs. winter samples. Element concentrations in the seminal plasma, determined by inductively coupled plasma-atomic emission spectrometry, differed between seasons, with higher (P<0.01) concentration values of K, Mg, Na and S elements in winter vs. summer samples. Therefore, season-induced alterations in seminal plasma element concentration should be taken into account when using an extender for cryopreservation. Acrosome integrity was assessed by a triple-fluorescence test using Hoechst 33342, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Acrosome reaction was examined by a one-step staining method using FITC-PSA. The proportion of sperm cells with a damaged acrosome post-thawing tended to be higher (P<0.07) in semen collected during the summer vs. winter. Such alterations suggest that seasonal reductions in sperm function might also be involved in the decreased conception rate of dairy cows in summer.

Porcine uterus cryopreservation: an analysis of contractile function using different uterotonics.

PubMed

Schölch, Daniel; Schölch, Sebastian; Strahl, Olga; Hoffmann, Inge; Beckmann, Matthias W; Dittrich, Ralf

2012-10-01

Cryopreservation of whole organs has become increasingly successful in recent years, and establishing reliable methods for confirming the success of specific cryopreservation procedures has therefore become extremely important. On the assumption that methods such as histological evaluation do not provide definitive evidence of long-term cryopreservation and that clear signs of conserved function in an organ are good evidence of its viability, contractile function was analysed in porcine uteri (n=60), either after long-term (group A) or short-term (group B) cryopreservation and post-thaw treatment with three different uterotonics. A slow freezing protocol was used to preserve the organs. Fifteen fresh uteri were analysed similarly for contractile function, which was evaluated by measuring intrauterine pressure after administration of oxytocin, prostaglandin E(1) (PGE(1)), and carbachol. After cryopreservation, all but three uteri (95%) showed rhythmic contractions similar to those in fresh uteri except for differences in the heights of contraction peaks, with lower contractions in PGE(1) subgroup B (P<0.05). With the exception of three nonresponsive uteri in group A, there were no differences in contractility between uteri after long-term cryopreservation and fresh uteri. The results of this study thus contribute to the debate on whether slow freezing or vitrification techniques are best for whole-organ cryopreservation. In summary, (1) preservation of muscular function in porcine uteri is feasible with a slow freezing protocol; (2) measurement of contractile function following administration of uterotonics is a useful method of confirming functionality; and (3) long-term cryopreservation does not significantly impair post-thaw contractibility in comparison with fresh uteri. Copyright © 2012 Elsevier Inc. All rights reserved.

Human oocyte cryopreservation: 5-year experience with a sodium-depleted slow freezing method.

PubMed

Boldt, Jeffrey; Tidswell, Non; Sayers, Amy; Kilani, Rami; Cline, Donald

2006-07-01

A slow freezing/rapid thawing method for the cryopreservation of human oocytes has been employed using a sodium-depleted culture media. In 53 frozen egg-embryo transfer (FEET) cycles, a 60.4% survival rate post-thaw was obtained and a 62.0% fertilization rate following intracytoplasmic sperm injection. Overall pregnancy rates were 26.4% per thaw attempt, 30.4% per patient, and 32.6% per embryo transfer. Pregnancy rates using sodium-depleted phosphate-buffered saline (PBS) as the base medium were 20.0% per thaw, 21.7% per patient, and 26.3% per transfer. With sodium-depleted modified human tubal fluid (mHTF) as the base for the cryopreservation medium, rates were 32.1% per thaw attempt, 39.1% per patient, 37.5% per transfer. The overall implantation rates were 4.2% per thawed oocyte and 13.6% per embryo, (PBS: 3.0% per egg, 10.6% per embryo; mHTF:5.3% per oocyte; 15.9% per embryo). These data indicate that the use of a sodium-depleted media with slow freezing and rapid thawing can yield acceptable pregnancy rates after FEET.

Effect of equilibration times, freezing, and thawing rates on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa.

PubMed

Shah, S A H; Andrabi, S M H; Qureshi, I Z

2016-09-01

The effects of equilibration times (E1, 2 h; E2, 4 h; E3, 6 h), freezing rates (FR1, manual, 5 cm above liquid nitrogen (LN2 ) for 10 min, plunging in LN2 ; FR2, programmable ultra-fast, holding at +4 °C for 2 min, from 4 to -10 °C at -10 °C/min, from -10 to -20 °C at -15 °C/min, from -20 to -120 °C at -60 °C/min, holding at -120 °C for 30 sec, plunging in LN2 ), and thawing rates (T1, 37 °C for 30 sec; T2, 50 °C for 15 sec; T3, 70 °C for 7 sec) were evaluated on quality of buffalo bull spermatozoa. Progressive motility (%), rapid velocity (%), average path velocity (VAP, μm/s), straight line velocity (VSL, μm/s), and mitochondrial transmembrane potential (%) were higher (p < 0.05) with E2, FR2, and T3 compared to other groups. Sperm curved line velocity (VCL, μm/s) was higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm straightness (%) and linearity (LIN, %) were higher (p < 0.05) with E2 compared to other groups. Sperm LIN was affected (p < 0.05) with T3 compared to other groups. Supravital-plasma membrane integrity (%), viability and acrosome integrity (%) of spermatozoa were higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm DNA integrity (%) was higher (p < 0.05) with FR2 and T1 compared to other groups. We concluded that inclusion of 4 h-equilibration time, programmable ultra-fast freezing rate, and rapid thawing at 70 °C for 7 sec in cryopreservation protocol improves the post-thaw quality of buffalo bull spermatozoa. © 2016 American Society of Andrology and European Academy of Andrology.

Recent developments in novel freezing and thawing technologies applied to foods.

PubMed

Wu, Xiao-Fei; Zhang, Min; Adhikari, Benu; Sun, Jincai

2017-11-22

This article reviews the recent developments in novel freezing and thawing technologies applied to foods. These novel technologies improve the quality of frozen and thawed foods and are energy efficient. The novel technologies applied to freezing include pulsed electric field pre-treatment, ultra-low temperature, ultra-rapid freezing, ultra-high pressure and ultrasound. The novel technologies applied to thawing include ultra-high pressure, ultrasound, high voltage electrostatic field (HVEF), and radio frequency. Ultra-low temperature and ultra-rapid freezing promote the formation and uniform distribution of small ice crystals throughout frozen foods. Ultra-high pressure and ultrasound assisted freezing are non-thermal methods and shorten the freezing time and improve product quality. Ultra-high pressure and HVEF thawing generate high heat transfer rates and accelerate the thawing process. Ultrasound and radio frequency thawing can facilitate thawing process by volumetrically generating heat within frozen foods. It is anticipated that these novel technologies will be increasingly used in food industries in the future.

Observations on procedures for thawing and spit-roasting frozen dressed chickens, and post-cooking care and storage: with particular reference to food-poisoning bacteria

PubMed Central

Roberts, Diane

1972-01-01

A comparison was made of four methods of thawing frozen chickens and an average thaw time for each method was determined. Fully and partially thawed chickens, inoculated with salmonellas, Clostridium welchii and Staphylococcus aureus were cooked in a spit-roasting oven at different temperatures for different lengths of time. The chickens were examined freshly cooked and after storage under various conditions. Spit roasting fully thawed chickens until the outer skin was golden brown was sufficient heat-treatment to kill salmonellas and Staph. aureus but Cl. welchii could survive. Salmonellas could also survive if the chickens were not fully thawed before cooking. Incorrect storage after cooking was shown to encourage the growth of pathogens. The incidence of intestinal pathogens in frozen dressed chickens and environmental hazards in spit-roasting establishments were also studied. Of raw chickens examined 35% contained salmonellas (9 serotypes), 63% contained Cl. welchii and 63% Staph. aureus. PMID:4342001

Prognostic value of a pre-freeze hypo-osmotic swelling test on the post-thaw quality of dog semen.

PubMed

Karger, S; Geiser, B; Grau, M; Burfeind, O; Heuwieser, W; Arlt, S P

2016-03-01

Throughout cryopreservation, sperm are exposed to major osmotic challenges. Only intact membranes of sperm cells are able to regulate these volumetric changes, which can be determined by the hypo-osmotic swelling test (HOS test). Correlations between the HOS test and conventional semen variables are inconsistent. Therefore, the objectives of this study were (1) to examine relationships between HOS test results and standard semen variables before freezing and after thawing and (2) to evaluate the prognostic value of the HOS assessments on post-thaw quality of dog semen. Semen of 35 dogs was collected and analyzed before freezing and after thawing following a 7-day freeze-thaw interval. Conventional semen variables such as sperm cell motility, membrane integrity morphology were evaluated and the HOS test was conducted with results from this test being recorded. In fresh semen the HOS test was positively correlated with progressive motility of sperm cells: r=0.52, sperm cell membrane integrity: r=0.50 and normal sperm cell morphology: r=0.46 (P<0.05). In frozen-thawed semen, the data obtained with the HOS test were positively correlated with progressive sperm cell motility: r=0.67 and membrane integrity: r=0.86 (P<0.05). The data obtained with the HOS test in fresh semen were positively correlated with sperm cell membrane integrity: r=0.50 normal sperm cell morphology: r=0.55 and data from the HOS test (r=0.43; P<0.05) with frozen-thawed semen. For the prediction of individual cryopreservation capacity, results from assessment of the fresh semen variables of good and poor semen quality were statistically compared. Based on these results, it is not possible to predict the quality of frozen-thawed dog semen using the HOS test. Copyright © 2016 Elsevier B.V. All rights reserved.

Rooster semen cryopreservation: effect of pedigree line and male age on postthaw sperm function.

PubMed

Long, J A; Bongalhardo, D C; Pelaéz, J; Saxena, S; Settar, P; O'Sullivan, N P; Fulton, J E

2010-05-01

The fertility rates of cryopreserved poultry semen are highly variable and not reliable for use in preservation of commercial genetic stocks. Our objective was to evaluate the cryosurvival of semen from 8 pedigreed layer lines at 2 different ages: the onset and end of commercial production. Semen from 160 roosters (20/line) was frozen individually with 11% glycerol at 6 and 12 mo of age. Glycerol was removed from thawed semen by Accudenz gradient centrifugation. The viability of thawed sperm from each male was determined using fluorescent live-dead staining and flow cytometry; sperm velocity parameters were measured using computerized motion analysis. The fertilizing ability of thawed sperm was evaluated in vitro by assessing hydrolysis of the inner perivitelline membrane. The postthaw function of sperm from the elite lines varied widely, despite the fact that fresh semen from all of these lines typically yielded high fertility rates. The percentage of thawed sperm with intact plasma membranes ranged from 27.8 + or - 2.1 to 49.6 + or - 1.9 and varied among lines and between age groups. Thawed sperm from 2 lines consistently demonstrated the highest and lowest motility parameters, whereas the velocity parameters of the remaining 6 lines varied widely. The mean number of hydrolysis points per square millimeter of inner perivitelline membrane ranged from 12.5 + or - 4.1 (line 2) to 103.3 + or - 30.2 (line 6). Age effects were observed for 4 out of 8 lines; however, improved postthaw sperm function at 12 mo of age was not consistent for all 3 assays. These results demonstrate variability among pedigreed lines in withstanding glycerol-based semen cryopreservation and provide a model for delineating genotypic and phenotypic factors affecting sperm cryosurvival.

Comparison of protocols for cryopreservation of rhesus monkey spermatozoa by post-thaw motility recovery and hyperactivation.

PubMed

Nichols, S M; Bavister, B D

2006-01-01

Cryopreservation of spermatozoa is useful for gene banking and for in vitro fertilisation (IVF). This study compared several published cryopreservation techniques to find the most efficient for rhesus macaques. Effectiveness was assessed by sperm longevity (post-thaw motility % and duration) and ability to hyperactivate in response to chemical activators (caffeine, dibutyryl cyclic AMP). Each ejaculate from three males was treated with four published cryopreservation protocols (Seier et al. 1993; Sanchez-Partida et al. 2000; Si et al. 2000; Isachenko et al. 2005). Upon thawing, each sub-sample was incubated either at 37 degrees C in 5% CO2 in air with or without activators or at approximately 22 degrees C in atmospheric air without activators for 0-24 h. Samples cryopreserved using one method showed zero motility and were not included in the 2 ;2 G-test statistical analysis. The other methods all demonstrated good immediate post-thaw motility rates (68%, 73% and 62% respectively) and underwent capacitation after exposure to activators. Sperm motility in each treatment decreased over time at both temperatures but overall, incubation at 22 degrees C preserved motility better in all three methods. In summary, cryopreservation of rhesus spermatozoa using the method published by Sanchez-Partida et al. or Seier et al. appeared best, potentially supporting gene banking as well as allowing for multiple IVF uses from the same sample.

Improvement of tolerance to freeze-thaw stress of baker's yeast by cultivation with soy peptides.

PubMed

Izawa, Shingo; Ikeda, Kayo; Takahashi, Nobuyuki; Inoue, Yoshiharu

2007-06-01

The tolerance to freeze-thaw stress of yeast cells is critical for frozen-dough technology in the baking industry. In this study, we examined the effects of soy peptides on the freeze-thaw stress tolerance of yeast cells. We found that the cells cultured with soy peptides acquired improved tolerance to freeze-thaw stress and retained high leavening ability in dough after frozen storage for 7 days. The final quality of bread regarding its volume and texture was also improved by using yeast cells cultured with soy peptides. These findings promote the utilization of soy peptides as ingredients of culture media to improve the quality of baker's yeast.

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei: Istiophoridae).

PubMed

van der Straten, K M; Leung, L K-P; Rossini, R; Johnston, S D

2006-01-01

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation, mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

Optimization of conditions for the cryopreservation of yellow catfish (Pelteobagrus fulvidraco) sperm.

PubMed

Yang, Sen; Han, Linqiang; Huang, Rushou; Liufu, Yongzhong; Meng, Zining; Lin, Haoran

2017-06-01

Yellow catfish (Pelteobagrus fulvidraco) is a promising aquaculture species in China with an increasing market demand. To serve the growing demand of male broodstock for artificial fertilization and the preservation of valuable strains for selective breeding, we tried to develop a species-specific cryopreservation protocol for yellow catfish sperm in this study. Important factors such as cryoprotectant, freezing height above the liquid nitrogen (LN) surface, dilution ratio, equilibration time, thawing temperature and cool storage before freezing were standardized. Among the cryoprotectants tested here, 10% Me 2 SO was the most suitable for sperm cryopreservation. Freezing at 7 cm above the LN surface for 10 min yielded the highest post-thaw motility. Further evaluation showed that dilution ratio of 1:3 and 1:5 produced higher post-thaw motility than semen diluted at 2:1, 1:1, 1:9 or 1:19. Equilibration times from 0 to 30 min did not cause significant differences in both equilibrated and post-thaw motility. Also, cool storage up to 24 h did not affect the suitability of sperm for cryopreservation. After thawing, sperm could be stored at 4 °C for 2 h without a reduction in motility parameters. With the combination of optimized freezing conditions, the fertilization and hatching rate of cryopreserved sperm were 87.1 ± 5.2% and 78.5 ± 7.4%, respectively, which were similar to those of fresh sperm (91.8 ± 3.5% and 83.7 ± 2.5%). In general, the cryopreservation protocol optimized here would facilitate breeding practice and hatchery operation in this economically important fish. Copyright © 2017. Published by Elsevier Inc.

A simple and highly effective method for slow-freezing human pluripotent stem cells using dimethyl sulfoxide, hydroxyethyl starch and ethylene glycol.

PubMed

Imaizumi, Keitaro; Nishishita, Naoki; Muramatsu, Marie; Yamamoto, Takako; Takenaka, Chiemi; Kawamata, Shin; Kobayashi, Kenichiro; Nishikawa, Shin-Ichi; Akuta, Teruo

2014-01-01

Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional -80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application.

A Simple and Highly Effective Method for Slow-Freezing Human Pluripotent Stem Cells Using Dimethyl Sulfoxide, Hydroxyethyl Starch and Ethylene Glycol

PubMed Central

Imaizumi, Keitaro; Nishishita, Naoki; Muramatsu, Marie; Yamamoto, Takako; Takenaka, Chiemi; Kawamata, Shin; Kobayashi, Kenichiro; Nishikawa, Shin-ichi; Akuta, Teruo

2014-01-01

Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional −80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application. PMID:24533137

Lutein, Trolox, ascorbic acid and combination of Trolox with ascorbic acid can improve boar semen quality during cryopreservation.

PubMed

Varo-Ghiuru, Florin; Miclea, Ileana; Hettig, Andrea; Ladoşi, Ioan; Miclea, Vasile; Egerszegi, István; Zăhan, Marius

2015-01-01

Due to pour quality of cryopreserved boar semen, artificial innsemination with frozen-thawed semen is quite limited. Developing protocols of boar semen cryopreservation represents a priority but also a challange. The goal of the present study was to evaluate the antioxidant potential of lutein, Trolox, ascorbic acid, and certain combinations of Trolox with ascorbic acid on boar semen cryopreservation procedure. Antioxidants were added to lactose-egg yolk extender, containing a final concentration of 3% glycerol and 0.5% Equex-STM. Semen of six boars was cryopreserved using straw-freezing procedure. After cryopreservation semen was thawed and evaluated for motility, normal apical ridge (NAR), hypo-osmotic swelling test (HOST) and DNA fragmentation index (DFI). Data were analyzed by one-way ANOVA. The results showed better motility after thawing at the concentration of 10 μM lutein, 200 μM Trolox, 200 μM ascorbic acid and 400-200 μM Trolox and ascorbic acid. The supplementation on boar freezing extender with 10 μM lutein increased post-thawed motility, NAR and HOST values (P < 0.01), and decrease DFI (P < 0.05) in comparison with control group. Similar results were obtained using 400-200 μM Trolox and ascorbic acid, with better results in the case of DFI (P < 0.01). In comparison with the control group, a concentration of 200 μM Trolox and 200 μM ascorbic acid provided significant differences (P < 0.01) of motility and NAR. The analysis of sperm characteristics showed that lutein and the mix between Trolox and ascorbic acid used in boar semen cryopreservation can improve the quality of spermatozoa.

Sorted clastic stripes, lobes and associated gullies in high-latitude craters on Mars: Landforms indicative of very recent, polycyclic ground-ice thaw and liquid flows

NASA Astrophysics Data System (ADS)

Gallagher, C.; Balme, M. R.; Conway, S. J.; Grindrod, P. M.

2011-01-01

Self-organised patterns of stone stripes, polygons, circles and clastic solifluction lobes form by the sorting of clasts from fine-grained sediments in freeze-thaw cycles. We present new High Resolution Imaging Science Experiment (HiRISE) images of Mars which demonstrate that the slopes of high-latitude craters, including Heimdal crater - just 25 km east of the Phoenix Landing Site - are patterned by all of these landforms. The order of magnitude improvement in imaging data resolution afforded by HiRISE over previous datasets allows not only the reliable identification of these periglacial landforms but also shows that high-latitude fluviatile gullies both pre- and post-date periglacial patterned ground in several high-latitude settings on Mars. Because thaw is inherent to the sorting processes that create these periglacial landforms, and from the association of this landform assemblage with fluviatile gullies, we infer the action of liquid water in a fluvio-periglacial context. We conclude that these observations are evidence of the protracted, widespread action of thaw liquids on and within the martian regolith. Moreover, the size frequency statistics of superposed impact craters demonstrate that this freeze-thaw environment is, at least in Heimdal crater, less than a few million years old. Although the current martian climate does not favour prolonged thaw of water ice, observations of possible liquid droplets on the strut of the Phoenix Lander may imply significant freezing point depression of liquids sourced in the regolith, probably driven by the presence of perchlorates in the soil. Because perchlorates have eutectic temperatures below 240 K and can remain liquid at temperatures far below the freezing point of water we speculate that freeze-thaw involving perchlorate brines provides an alternative "low-temperature" hypothesis to the freeze-thaw of more pure water ice and might drive significant geomorphological work in some areas of Mars. Considering the proximity of Heimdal crater to the Phoenix Landing Site, the presence of such hydrated minerals might therefore explain the landforms described here. If this is the case then the geographical distribution of martian freeze-thaw landforms might reflect relatively high temperatures (but still below 273 K) and the locally elevated concentration of salts in the regolith.

Intensification of freeze-thaw cycles in the soil on post-fire alpine slopes of Mount Shirouma-dake, northern Japanese Alps central Japan

NASA Astrophysics Data System (ADS)

Sasaki, A.; Suzuki, K.

2016-12-01

This is the continuous study to clarify the geo-environmental changes on the post-fire alpine slopes of Mount Shirouma-dake in the Northern Japanese Alps. The fire occurred at May 9, 2009 on the alpine slopes of Mount Shirouma-dake, and the fire spread to the Pinus pumila communities and grasslands. Although the grass had a little damage by the fire, the P. pumila received nearly impact of the fire. In the P. pumila communities where the leaf burnt, forest floor is exposed and become easy to be affected by atmospheric condition such as rain, wind, snow, and etc. First, we observed condition of the micro-landforms on post-fire slopes repeatedly for seventh years after the fire. As the results of the observation, it is clear that remarkable changes of these micro-landforms have not occurred but some litters on the forest-floor in the fired P. pumila communities are flushed out to surroundings. The litter layer on the forest-floor in the fired P. pumila communities were 3-4 cm thick in August of 2011, but it became 0.5 cm thick in September of 2014. The P. pumila communities established on the slopes consists of angular and sub-angular gravel with openwork texture, which are covered by thin soil layer. On July of 2016, the litter layer almost entirely flushed out and surface of soil layer is exposed to atmosphere. In addition, we observe the ground temperature and soil moisture, under the fired P. pumila communities and the no fired P. pumila communities since October 2009, to find influence of the fire. The ground temperature sensors were installed into at 1 cm, 10 cm, and 40 cm depth. The soil moisture sensors were installed into at 1 cm and 10 cm depth. The 1 cm depth of the soil on the post-fire slopes, several times of diurnal freeze-thaw cycles occurred on October and November since 2011, but it had not occurred in 2009 and 2010. In particular, more than 20 times of diurnal freeze-thaw cycles occurred on freezing period of 2014. The diurnal freeze-thaw cycles continue to be increasing until thawing period of 2016. The period of seasonal frost at 10 cm and 40 cm depth on the post-fire slopes are extended for two weeks. Snowmelt water is especially thought to be act on re-freezing of post-fire slopes on thawing period. These thermal condition changes are triggered by decrease in the thickness of the litter layer on the fired P. pumila communities.

Retained fertilizing capability in cryopreserved feline spermatozoa.

PubMed

Chatdarong, K

2017-04-01

Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization (IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection (ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7 days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7 days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2 hr after a jungle cat (Felis chaus) aged 10 years died during anaesthesia plus another necropsy-to-sperm recovery time of 25 hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation. © 2016 Blackwell Verlag GmbH.

Rapid carbon loss and slow recovery following permafrost thaw in boreal peatlands

USGS Publications Warehouse

Jones, Miriam C.; Harden, Jennifer W.; O'Donnell, Jonathan A.; Manies, Kristen L.; Jorgenson, Torre; Treat, Claire C.; Ewing, Stephanie

2017-01-01

Permafrost peatlands store one-third of the total carbon (C) in the atmosphere and are increasingly vulnerable to thaw as high-latitude temperatures warm. Large uncertainties remain about C dynamics following permafrost thaw in boreal peatlands. We used a chronosequence approach to measure C stocks in forested permafrost plateaus (forest) and thawed permafrost bogs, ranging in thaw age from young (<10 years) to old (>100 years) from two interior Alaska chronosequences. Permafrost originally aggraded simultaneously with peat accumulation (syngenetic permafrost) at both sites. We found that upon thaw, C loss of the forest peat C is equivalent to ~30% of the initial forest C stock and is directly proportional to the prethaw C stocks. Our model results indicate that permafrost thaw turned these peatlands into net C sources to the atmosphere for a decade following thaw, after which post-thaw bog peat accumulation returned sites to net C sinks. It can take multiple centuries to millennia for a site to recover its prethaw C stocks; the amount of time needed for them to regain their prethaw C stocks is governed by the amount of C that accumulated prior to thaw. Consequently, these findings show that older peatlands will take longer to recover prethaw C stocks, whereas younger peatlands will exceed prethaw stocks in a matter of centuries. We conclude that the loss of sporadic and discontinuous permafrost by 2100 could result in a loss of up to 24 Pg of deep C from permafrost peatlands.

Expansion and cryopreservation of porcine and human corneal endothelial cells.

PubMed

Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W

2017-08-01

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing

NASA Astrophysics Data System (ADS)

Briard, Jennie G.; Poisson, Jessica S.; Turner, Tracey R.; Capicciotti, Chantelle J.; Acker, Jason P.; Ben, Robert N.

2016-03-01

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70-80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing

PubMed Central

Briard, Jennie G.; Poisson, Jessica S.; Turner, Tracey R.; Capicciotti, Chantelle J.; Acker, Jason P.; Ben, Robert N.

2016-01-01

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming. PMID:27021850

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing.

PubMed

Briard, Jennie G; Poisson, Jessica S; Turner, Tracey R; Capicciotti, Chantelle J; Acker, Jason P; Ben, Robert N

2016-03-29

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70-80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.

Fertility test of frozen boar semen.

PubMed

Osinowa, O; Salamon, S

1976-10-01

The fertility results of two experiments are presented. In experiment 1, the semen was frozen in tris-fructose-EDTA or BF3 diluents at 0-25 X 10(9)/ml sperm concentration and extended after thawing with either seminal plasma (SP) or the freezing medium (FM) containing no cryoprotective agent. In the second experiment the semen was glycerolated by two methods, frozen at 1-0 X 10(9)/ml sperm concentration, and extended wtih FM before insemination. Fertility after double insemination within one oestrus with semen frozen in tris-fructose-EDTA or BF3 diluents varied depending on the medium used for extension of thawed semen. The farrowing rates for semen frozen in the former diluent with FM and SP post-thawing media were 4/8 and 1/8 respectively, and for semen frozen BF3 diluent with FM and SP post-thawing extenders 1/8 and 5/8. The mean farrowing for the 32 animals inseminasted was 34-4%. Pregnancies for semen frozen in tris-fructose-EDTA and glycerolated at 30 or 5 degrees C were 5/12 and 4/12 respectively, and for single and double inseminations 6/12 and 3/12 respectively. Of 24 animals inseminated 37-5% farrowed.

Does natural honey act as an alternative to antibiotics in the semen extender for cryopreservation of crossbred ram semen?

PubMed Central

Banday, M. N.; Lone, F. A.; Rasool, F.; Rather, H. A.; Rather, M. A.

2017-01-01

Antibiotics are added to semen extenders to take care of heavy microbial load, however, their continuous use poses a constant threat of developing antibiotic resistance by the common microbes present in the semen. Our hypothesis was that natural honey, having antibacterial activity and rich in fructose could replace the use of antibiotics and fructose in the semen extender. Twenty-four ejaculates from six crossbred rams were obtained and extended with tris-based extender without (control) and with honey at 2.5% (T1), 5% (T2) and 7% (T3). Sperm quality was measured in terms of percentage sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa. The semen samples at post-thaw were also evaluated for total viable count (colony forming units/ml). At post-thaw, control exhibited significantly (P<0.05) higher sperm motility in comparison to T2 and T3. The percent of live sperm count, intact acrosome and HOST reacted spermatozoa were significantly higher (P<0.05) for control than all other treatment groups at post-thaw. Among treatment groups, T1 maintained significantly higher (P<0.05) percentage of live sperm count, intact acrosome and HOST reacted spermatozoa than T2 and T3. The total viable count at post-thaw was significantly lower (P<0.05) for control than all the treatment groups. In conclusion, honey cannot be used as an alternative to antibiotics to take care of heavy microbial load in semen, however, levels up to 2.5% may be supplemented to semen as an energy source. PMID:29387098

Soil data from fire and permafrost-thaw chronosequences in upland Picea mariana stands near Hess Creek and Tok, interior Alaska

USGS Publications Warehouse

O'Donnell, Jonathan A.; Harden, Jennifer W.; Manies, Kristen L.; Jorgenson, M. Torre; Kanevskiy, Mikhail; Xu, Xiaomei

2013-01-01

Soils of the Northern Circumpolar Permafrost region harbor 1,672 petagrams (Pg) (1 Pg = 1,000,000,000 kilograms) of organic carbon (OC), nearly 50 percent of the global belowground OC pool (Tarnocai and others, 2009). Of that soil OC, nearly 88 percent is presently stored in perennially frozen ground. Recent climate warming at northern latitudes has resulted in warming and thawing of permafrost in many regions (Osterkamp, 2007), which might mobilize OC stocks from associated soil reservoirs via decomposition, leaching, or erosion. Warming also has increased the magnitude and severity of wildfires in the boreal region (Turetsky and others, 2011), which might exacerbate rates of permafrost degradation relative to warming alone. Given the size and vulnerability of the soil OC pool in permafrost soils, permafrost thaw will likely function as a strong positive feedback to the climate system (Koven and others, 2011; Schaefer and others, 2011). In this report, we report soil OC inventories from two upland fire chronosequences located near Hess Creek and Tok in Interior Alaska. We sampled organic and mineral soils in the top 2 meters (m) across a range of stand ages to evaluate the effects of wildfire and permafrost thaw on soil C dynamics. These data were used to parameterize a simple process-based fire-permafrost-carbon model, which is described in detail by O’Donnell and others (2011a, b). Model simulations examine long-term changes in soil OC storage in response to fire, permafrost thaw, and climate change. These data also have been used in other papers, including Harden and others (2012), which examines C recovery post-fire, and Johnson and others (2011), which synthesizes data within the Alaska Soil Carbon Database. Findings from these studies highlight the importance of climate and disturbance (wildfire, permafrost thaw) on soil C storage, and loss of soil C from high-latitude ecosystems.

Combining reduced glutathione and ascorbic acid has supplementary beneficial effects on boar sperm cryotolerance.

PubMed

Giaretta, Elisa; Estrada, Efrén; Bucci, Diego; Spinaci, Marcella; Rodríguez-Gil, Joan E; Yeste, Marc

2015-02-01

The main aim of this work was to evaluate how supplementing freezing and thawing media with reduced glutathione (GSH) and l-ascorbic acid (AA) affected the quality parameters of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12 ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to which 5 mM of GSH and 100 μM of AA were added separately or together at two different steps of freeze-thawing. Various sperm parameters (levels of free cysteine residues in sperm nucleoproteins, sperm viability, acrosome membrane integrity, intracellular peroxide and superoxide levels [ROS], and total and progressive motility) were evaluated before freezing and at 30 and 240 minutes after thawing. Both GSH and AA significantly improved boar sperm cryotolerance when they were separately added to freezing and thawing media. However, the highest improvement was recorded when both freezing and thawing media were supplemented with 5 mM of GSH plus 100 μM of AA. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels and had no impact on superoxide levels. According to our results, we can conclude that supplementation of freezing and thawing media with both GSH and AA has a combined, beneficial effect on frozen-thawed boar sperm, which is greater than that obtained with the separate addition of either GSH or AA. Copyright © 2015 Elsevier Inc. All rights reserved.

Multi-omics of permafrost, active layer and thermokarst bog soil microbiomes

NASA Astrophysics Data System (ADS)

Hultman, Jenni; Waldrop, Mark P.; Mackelprang, Rachel; David, Maude M.; McFarland, Jack; Blazewicz, Steven J.; Harden, Jennifer; Turetsky, Merritt R.; McGuire, A. David; Shah, Manesh B.; Verberkmoes, Nathan C.; Lee, Lang Ho; Mavrommatis, Kostas; Jansson, Janet K.

2015-05-01

Over 20% of Earth's terrestrial surface is underlain by permafrost with vast stores of carbon that, once thawed, may represent the largest future transfer of carbon from the biosphere to the atmosphere. This process is largely dependent on microbial responses, but we know little about microbial activity in intact, let alone in thawing, permafrost. Molecular approaches have recently revealed the identities and functional gene composition of microorganisms in some permafrost soils and a rapid shift in functional gene composition during short-term thaw experiments. However, the fate of permafrost carbon depends on climatic, hydrological and microbial responses to thaw at decadal scales. Here we use the combination of several molecular `omics' approaches to determine the phylogenetic composition of the microbial communities, including several draft genomes of novel species, their functional potential and activity in soils representing different states of thaw: intact permafrost, seasonally thawed active layer and thermokarst bog. The multi-omics strategy reveals a good correlation of process rates to omics data for dominant processes, such as methanogenesis in the bog, as well as novel survival strategies for potentially active microbes in permafrost.

Multi-omics of permafrost, active layer and thermokarst bog soil microbiomes.

PubMed

Hultman, Jenni; Waldrop, Mark P; Mackelprang, Rachel; David, Maude M; McFarland, Jack; Blazewicz, Steven J; Harden, Jennifer; Turetsky, Merritt R; McGuire, A David; Shah, Manesh B; VerBerkmoes, Nathan C; Lee, Lang Ho; Mavrommatis, Kostas; Jansson, Janet K

2015-05-14

Over 20% of Earth's terrestrial surface is underlain by permafrost with vast stores of carbon that, once thawed, may represent the largest future transfer of carbon from the biosphere to the atmosphere. This process is largely dependent on microbial responses, but we know little about microbial activity in intact, let alone in thawing, permafrost. Molecular approaches have recently revealed the identities and functional gene composition of microorganisms in some permafrost soils and a rapid shift in functional gene composition during short-term thaw experiments. However, the fate of permafrost carbon depends on climatic, hydrological and microbial responses to thaw at decadal scales. Here we use the combination of several molecular 'omics' approaches to determine the phylogenetic composition of the microbial communities, including several draft genomes of novel species, their functional potential and activity in soils representing different states of thaw: intact permafrost, seasonally thawed active layer and thermokarst bog. The multi-omics strategy reveals a good correlation of process rates to omics data for dominant processes, such as methanogenesis in the bog, as well as novel survival strategies for potentially active microbes in permafrost.

Evaluation of combined effects of ageing period and freezing rate on quality attributes of beef loins.

PubMed

Kim, Yuan H Brad; Liesse, Charlotte; Kemp, Robert; Balan, Prabhu

2015-12-01

The objective of our study was to evaluate the combined effects of ageing period and different freezing rates on meat quality attributes of beef loins. Pairs of loins (M. longissimus at 1 day post mortem) from 12 carcasses were divided into four equal portions and randomly assigned to four ageing/freezing treatments (aged only, frozen only, and 3 or 4 weeks ageing at -1.5°C then frozen). Two freezing methods (fast freezing by calcium chloride immersion or slow freezing by air freezer at -18°C) were applied to the loin sections. Fast freezing had no effect on shear force (P>0.05), but significantly improved the water-holding capacity of the aged/frozen loins by reducing purge and drip losses. Ageing-then-freezing significantly improved shear force values of loins compared to both the aged only and frozen only loins. These observations suggest that fast freezing will add more value to the aged/frozen/thawed meat by minimising the amount of water-loss due to the freezing/thawing process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Royal jelly supplementation in semen extender enhances post-thaw quality and fertility of Nili-Ravi buffalo bull sperm.

PubMed

Shahzad, Qaisar; Mehmood, Muhammad Usman; Khan, Hamayun; ul Husna, Asma; Qadeer, Saima; Azam, Asima; Naseer, Zahid; Ahmad, Ejaz; Safdar, Muhammad; Ahmad, Mushtaq

2016-04-01

Two experiments were conducted to evaluate the effect of royal jelly (RJ) on post-thaw sperm quality, in vitro and in vivo fertility rate of cryopreserved buffalo bull sperm. The semen was collected from three mature regular donor buffalo bulls, ejaculates were pooled and semen evaluated initially. In Experiment 1, the ejaculates were extended in tris-citric acid diluter supplemented with different RJ concentrations (0, 0.05, 0.1, 0.2, 0.3 or 0.4%). The diluted semen was cooled to 4°C, packaged into 0.5 mL straws and frozen using standard procedure. The straws were thawed and assessed for sperm progressive motility, viability, plasma membrane, acrosome, and chromatin integrity. The results indicated that sperm progressive motility was significantly greater (P<0.05) in 0.05, 0.1, 0.2 and 0.3% RJ than 0.4% RJ supplemented and control groups. The sperm viability, plasma membrane and acrosome integrity were significantly improved (P<0.05) in 0.1% RJ supplemented group the compared to other treatment groups. In Experiment 2, cryopreserved sperm with 0.1% RJ supplementation and control (without RJ supplementation) were used to observe the in vitro fertilizing potential and in vivo fertility. In vitro fertilization method was applied to assess the cleavage rate; whereas, AI was performed in buffalo during in vivo fertility trial. The buffaloes were inseminated 12h after standing estrus and pregnancy diagnosis was performed through ultrasonography. The results revealed that the cleavage rate was higher (P<0.05) in 0.1% RJ as compared to control group. However, the pregnancy rate was similar (P>0.05) between 0.1% RJ supplemented and control groups. It is concluded that supplementation of RJ in freezing extender can improve the cryosurvival rate and in vitro fertilizing capacity of buffalo bull sperm. Copyright © 2016 Elsevier B.V. All rights reserved.

Incubation of spermatozoa with Anandamide prior to cryopreservation reduces cryocapacitation and improves post-thaw sperm quality in the water buffalo (Bubalus bubalis).

PubMed

Kumar, Puneeth; Mohanty, Tushar Kumar; Kumaresan, Arumugam; Nag, Pradeep; Saraf, Kaustubh Kishor; Kumar, Vimlesh; Lathika, Sreela; Nayak, Samiksha; Bhakat, Mukesh

2018-02-01

Anandamide (AEA), an endocannabinoid, has been shown to reduce capacitation and acrosomal exocytosis in human spermatozoa. Because buffalo spermatozoa are highly susceptible to cryopreservation induced damage, AEA was assessed as to whether it could protect spermatozoa from cryo-damage. Six ejaculates from six Murrah buffalo bulls (total 36 ejaculates) were utilized for the study. Each ejaculate was divided into four aliquots; spermatozoa in Aliquot 1 were extended in Tris-Citrate-Egg Yolk and frozen as per the standard protocol. Spermatozoa in Aliquots 2, 3 and 4 were incubated with AEA at 1 nM, 1 μM and 10 μM, respectively in Tris-Citrate extender for 15 min at 37 °C before cryopreservation. Cryopreserved spermatozoa were thawed at 37 °C for 30 s before assessment of sperm motility, membrane integrity, capacitation, acrosome reaction, mitochondrial membrane potential (MMP) and lipid peroxidation status. The proportion of motile and membrane intact spermatozoa were greater (P < 0.05) with use of 1 μM AEA incorporated group compared with other groups. The proportion of un-capacitated and acrosome intact spermatozoa was greater (P < 0.05) with use of 1 or 10 μM of AEA compared with the other groups. When compared to the control group, use of 1 μM AEA resulted in a greater proportion of spermatozoa with high MMP (P < 0.05). There was no significant difference in the lipid peroxidation status of spermatozoa among any of the four groups. It was inferred that the protective role of AEA during cryopreservation of buffalo spermatozoa was dose dependent and incubation of spermatozoa with AEA at 1 μM concentration prior to cryopreservation reduced cryo-capacitation and improved post-thaw sperm quality in buffalo. Copyright © 2017 Elsevier B.V. All rights reserved.

Rhesus monkey sperm cryopreservation with TEST-yolk extender in the absence of permeable cryoprotectant.

PubMed

Dong, Qiaoxiang; Correa, Liane M; VandeVoort, Catherine A

2009-02-01

Recently, there has been increased interest in ultra-rapid freezing with mammalian spermatozoa, especially for vitrification in the absence of cryoprotectants. Sperm cryopreservation in non-human primates has been successful, but the use of frozen-thawed sperm in standard artificial insemination (AI) remains difficult, and removal of permeable cryoprotectant may offer opportunities for increased AI success. The present study intended to explore the possibility of freezing rhesus monkey sperm in the absence of permeable cryoprotectants. Specifically, we evaluated various factors such as presence or absence of egg yolk, the percentage of egg yolk in the extenders, and the effect of cooling and thawing rate on the success of freezing without permeable cryoprotectants. Findings revealed that freezing with TEST in the absence of egg yolk offers little protection (<15% post-thaw motility). Egg yolk of 40% or more in TEST resulted in decreased motility, while egg yolk in the range of 20-30% yielded the most motile sperm. Cooling at a slow rate (29 degrees C/min) reduced post-thaw motility significantly for samples frozen with TEST-yolk alone, but had no effect for controls in the presence of glycerol. Similarly, slow thawing in room temperature air is detrimental for freezing without permeable cryoprotectant (<2% motility). In addition to motility, the ability of sperm to capacitate based on an increase in intracellular calcium levels upon activation with cAMP and caffeine suggested no difference between fresh and frozen-thawed motile sperm, regardless of treatment. In summary, the present study demonstrates that ejaculated and epididymal sperm from rhesus monkeys can be cryopreserved with TEST-yolk (20%) in the absence of permeable cryoprotectant when samples were loaded in a standard 0.25-mL straw, cooled rapidly in liquid nitrogen vapor at 220 degrees C/min, and thawed rapidly in a 37 degrees C water bath. This study also represents the first success of freezing without permeable cryoprotectant in non-human primates.

Rapid carbon loss and slow recovery following permafrost thaw in boreal peatlands.

PubMed

Jones, Miriam C; Harden, Jennifer; O'Donnell, Jonathan; Manies, Kristen; Jorgenson, Torre; Treat, Claire; Ewing, Stephanie

2017-03-01

Permafrost peatlands store one-third of the total carbon (C) in the atmosphere and are increasingly vulnerable to thaw as high-latitude temperatures warm. Large uncertainties remain about C dynamics following permafrost thaw in boreal peatlands. We used a chronosequence approach to measure C stocks in forested permafrost plateaus (forest) and thawed permafrost bogs, ranging in thaw age from young (<10 years) to old (>100 years) from two interior Alaska chronosequences. Permafrost originally aggraded simultaneously with peat accumulation (syngenetic permafrost) at both sites. We found that upon thaw, C loss of the forest peat C is equivalent to ~30% of the initial forest C stock and is directly proportional to the prethaw C stocks. Our model results indicate that permafrost thaw turned these peatlands into net C sources to the atmosphere for a decade following thaw, after which post-thaw bog peat accumulation returned sites to net C sinks. It can take multiple centuries to millennia for a site to recover its prethaw C stocks; the amount of time needed for them to regain their prethaw C stocks is governed by the amount of C that accumulated prior to thaw. Consequently, these findings show that older peatlands will take longer to recover prethaw C stocks, whereas younger peatlands will exceed prethaw stocks in a matter of centuries. We conclude that the loss of sporadic and discontinuous permafrost by 2100 could result in a loss of up to 24 Pg of deep C from permafrost peatlands. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing.

PubMed

Varghese, Tincy; Divyashree, Bannur C; Roy, Sudhir C; Roy, Kajal S

2016-03-15

The post-thaw fertility of frozen-thawed mammalian spermatozoa is substantially low as compared with that of fresh sperm. Furthermore, the post-thaw fertility of the cryopreserved buffalo sperm has been reported to be poor as compared with that of cattle sperm. Recently, heat shock protein 70 (HSP70) has been found to play a critical role in mammalian fertilization and early embryonic development in boar and cattle. However, the presence of such fertility-related HSP70 in buffalo sperm and its status after cryopreservation has not been reported so far. Thus, a study was conducted to determine the effect of cryopreservation on the level and distribution pattern of HSP70 molecule in buffalo sperm after cryopreservation. Buffalo semen samples, after dilution in semen extender, were aliquoted in straws and divided into two groups. One group was not cryopreserved, and the other group was cryopreserved for 60 days. Sperm proteins were extracted from both non-cryopreserved (NC) and cryopreserved (C) sperm and subjected to Western blot analysis for detection of HSP70 using a monoclonal anti-HSP70 antibody. The distribution pattern of these proteins in buffalo sperm was also monitored before and after cryopreservation using indirect immunofluorescence technique. A prominent 70-kDa protein band of HSP70 protein was detected in protein extracts of both NC and C buffalo sperm. Densitometry analysis revealed that the intensity of 70-kDa HSP70 protein band of cryopreserved sperm decreased significantly (P < 0.05) compared with that of NC sperm. However, the level of HSP70 in cryopreserved extended seminal plasma (ESP) did not change as compared with that of NC samples indicating a possible degradation of HSP70 in the spermatozoa itself rather than leakage of the protein into the ESP. Furthermore, Western blot also confirmed that several HSP70 immunoreactive protein bands detected in the ESP were contributed by the egg yolk that was added to the extender. Immunocytochemistry revealed that HSP70 proteins were distributed over the apical region of sperm head and/or acrosome, post-acrosomal, and middle piece regions of NC buffalo spermatozoa. However, the fluorescence signal of apical region of sperm head was lost significantly (P < 0.05) after a cycle of freezing and thawing. Thus, the present study confirmed that there was loss of HSP70 from buffalo sperm head after freezing and thawing of buffalo spermatozoa, and this may be one of the causes of the reduced post-thaw fertility of sperm in this species. Copyright © 2016 Elsevier Inc. All rights reserved.

The utility of nanowater for ram semen cryopreservation.

PubMed

Murawski, Maciej; Schwarz, Tomasz; Grygier, Joanna; Patkowski, Krzysztof; Oszczęda, Zdzisław; Jelkin, Igor; Kosiek, Anna; Gruszecki, Tomasz M; Szymanowska, Anna; Skrzypek, Tomasz; Zieba, Dorota A; Bartlewski, Pawel M

2015-05-01

Nanowater (NW; water declusterized in the low-temperature plasma reactor) has specific physicochemical properties that could increase semen viability after freezing and hence fertility after artificial insemination (AI) procedures. The main goal of this study was to evaluate ram semen quality after freezing in the media containing NW. Ejaculates from 10 rams were divided into two equal parts, diluted in a commercially available semen extender (Triladyl®; MiniTüb GmbH, Tiefenbach, Germany) prepared with deionized water (DW) or NW, and then frozen in liquid nitrogen. Semen samples were examined for sperm motility and morphology using the sperm class analyzer system and light microscopy. Cryo-scanning electron microscopy (cryo-SEM) was employed to determine the size of extracellular water crystals in frozen semen samples. Survival time at room temperature, aspartate aminotransferase (AspAT) and alkaline phosphatase (ALP) concentrations post-thawing as well as conception/lambing rates after laparoscopic intrauterine AI of 120 ewes were also determined. There were no significant differences between DW and NW groups in sperm progressive motility (26.4 ± 12.2 and 30.8 ± 12.4%) or survival time (266.6 ± 61.3 and 270.9 ± 76.7 min) after thawing and no differences in the percentages of spermatozoa with various morphological defects before or after freezing. There were, however, differences (P < 0.05) in AspAT (DW: 187.1 ± 160.4 vs. NW: 152.7 ± 118.3 U/l) and ALP concentrations (DW: 2198.3 ± 1810.5 vs. NW: 1612.1 ± 1144.8 U/l) in semen samples post-thawing. Extracellular water crystals were larger (P < 0.05) in ejaculates frozen in NW-containing media. Ultrasonographic examinations on day 40 post-AI revealed higher (P < 0.05) conception rates in ewes inseminated with NW (78.3%) compared with DW semen (58.3%), and the percentages of ewes that carried lambs to term were 73.3% and 45.0% in NW and DW groups, respectively (P < 0.01). In summary, the use of a semen extender prepared with NW was associated with a substantial improvement in the fertilizing ability of frozen-thawed ram semen and lamb productivity of inseminated ewes. © 2014 by the Society for Experimental Biology and Medicine.

The utility of nanowater for ram semen cryopreservation

PubMed Central

Murawski, Maciej; Schwarz, Tomasz; Patkowski, Krzysztof; Oszczęda, Zdzisław; Jelkin, Igor; Kosiek, Anna; Gruszecki, Tomasz M; Szymanowska, Anna; Skrzypek, Tomasz; Zieba, Dorota A; Bartlewski, Pawel M

2015-01-01

Nanowater (NW; water declusterized in the low-temperature plasma reactor) has specific physicochemical properties that could increase semen viability after freezing and hence fertility after artificial insemination (AI) procedures. The main goal of this study was to evaluate ram semen quality after freezing in the media containing NW. Ejaculates from 10 rams were divided into two equal parts, diluted in a commercially available semen extender (Triladyl®; MiniTüb GmbH, Tiefenbach, Germany) prepared with deionized water (DW) or NW, and then frozen in liquid nitrogen. Semen samples were examined for sperm motility and morphology using the sperm class analyzer system and light microscopy. Cryo-scanning electron microscopy (cryo-SEM) was employed to determine the size of extracellular water crystals in frozen semen samples. Survival time at room temperature, aspartate aminotransferase (AspAT) and alkaline phosphatase (ALP) concentrations post-thawing as well as conception/lambing rates after laparoscopic intrauterine AI of 120 ewes were also determined. There were no significant differences between DW and NW groups in sperm progressive motility (26.4 ± 12.2 and 30.8 ± 12.4%) or survival time (266.6 ± 61.3 and 270.9 ± 76.7 min) after thawing and no differences in the percentages of spermatozoa with various morphological defects before or after freezing. There were, however, differences (P < 0.05) in AspAT (DW: 187.1 ± 160.4 vs. NW: 152.7 ± 118.3 U/l) and ALP concentrations (DW: 2198.3 ± 1810.5 vs. NW: 1612.1 ± 1144.8 U/l) in semen samples post-thawing. Extracellular water crystals were larger (P < 0.05) in ejaculates frozen in NW-containing media. Ultrasonographic examinations on day 40 post-AI revealed higher (P < 0.05) conception rates in ewes inseminated with NW (78.3%) compared with DW semen (58.3%), and the percentages of ewes that carried lambs to term were 73.3% and 45.0% in NW and DW groups, respectively (P < 0.01). In summary, the use of a semen extender prepared with NW was associated with a substantial improvement in the fertilizing ability of frozen-thawed ram semen and lamb productivity of inseminated ewes. PMID:25491414

Sericin supplementation improves semen freezability of buffalo bulls by minimizing oxidative stress during cryopreservation.

PubMed

Kumar, Pradeep; Kumar, Dharmendra; Sikka, P; Singh, P

2015-01-01

The variety of mammalian cells has been successfully cryopreserved by use of the silk protein sericin due to its strong free-radical-scavenging and potent antioxidant activity. The present study was conducted to examine the protective role of sericin on buffalo spermatozoa during cryopreservation. Semen of four breeding bulls was collected twice a week using artificial vagina technique. The ejaculates of four bulls were pooled, divided into five equal fractions, diluted with the extender supplemented with different concentrations of sericin (0, 0.25, 0.5, 1.5 and 2%) and then cryopreserved. Post-thawed motility was objectively assessed by computer assisted sperm analyzer. Sperm plasma membrane integrity was assessed by hypo-osmotic swelling test (HOST). Malondialdehyde (MDA) concentration, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in frozen-thawed extended seminal plasma by spectrophotometry. The extender supplemented with 0.25, 0.5 and 1% sericin resulted in the higher sperm motility and GPx acivity. Furthermore, plasma membrane integrity and SOD activity were found to be higher (P<0.05) in group supplemented with 0.25 and 0.5% sericin (P<0.05). The MDA concentration was found to be significantly lower (P<0.05) in 0.25 and 0.5% sericin treated groups than control and other treated groups. In conclusion, the supplementation of 0.25-0.5% sericin in semen extender improves frozen-thawed semen quality through protecting sperm from oxidative stress. Copyright © 2014 Elsevier B.V. All rights reserved.

Freezing-thawing and sub-sampling influence the marination performance of chicken breast meat.

PubMed

Bowker, B; Zhuang, H

2017-09-01

Vacuum-tumbling marination is often used to improve the yield and quality of whole or portioned broiler breast fillets. The relationship between the marination performance of whole Pectoralis major muscles and breast fillet sub-samples is not well understood. The objective was to determine the effects of sub-sampling and freezing-thawing on the marination performance and cook loss of broiler breast meat. Paired right and left breast fillets were marinated as whole fillets or sub-samples (cranial and mid-caudal portions). Samples were marinated at 48 h postmortem (fresh) or stored at -20°C and then thawed prior to marination (frozen-thawed). Samples were vacuum-tumbled in 20% wt/wt brine (5% NaCl, 3% STP) and weighed pre-marination, during marination (15, 30, and 45 min), and 24 h post-marination. Samples were then cooked to 75°C for determination of cook loss. Marinade uptake was greater in caudal sub-samples than intact fillets and cranial sub-samples after 15 min of marination (P < 0.0001). After 30 min, marinade uptake was greater in caudal sub-samples and intact fillets than cranial sub-samples (P < 0.05). After 45 min, marinade uptake for fresh samples was greatest in intact fillets and lowest in cranial sub-samples. For frozen-thawed samples, marinade uptake at 45 min was greater in caudal sub-samples and intact fillets than cranial sub-samples (P < 0.0001). Marinade uptake in sub-samples at 30 min was greater in frozen-thawed versus fresh fillets (P < 0.05). Differences in marinade retention were not observed. Cook loss was similar between fresh and frozen-thawed samples but was greater in sub-samples compared to intact fillets (P < 0.0001). Correlations between marinade uptake in intact fillets and cranial sub-samples were greater in fresh (r = 0.64 to 0.78) than frozen-thawed samples (r = 0.39 to 0.59). Correlations between marinade uptake in intact fillets and caudal sub-samples were greater in frozen-thawed (r = 0.79 to 0.82) than fresh samples (r = 0.46 to 0.63). Data suggest that the relationships between marination performance of whole breast fillets and fillet sub-samples are dependent upon prior sample handling and intra-fillet sampling location. Published by Oxford University Press on behalf of Poultry Science Association 2017.

Impact of freezing and thawing on the quality of meat: review.

PubMed

Leygonie, Coleen; Britz, Trevor J; Hoffman, Louwrens C

2012-06-01

This comprehensive review describes the effects of freezing and thawing on the physical quality parameters of meat. The formation of ice crystals during freezing damages the ultrastructure and concentrates the solutes in the meat which, in turn, leads to alterations in the biochemical reactions that occur at the cellular level and influence the physical quality parameters of the meat. The quality parameters that were evaluated are moisture loss, protein denaturation, lipid and protein oxidation, colour, pH, shear force and microbial spoilage. Additionally mechanisms employed to mitigate the effects of freezing and thawing were also reviewed. These include the use of novel methods of freezing and thawing, ante and post mortem antifreeze protein inclusion and vitamin E supplementation, brine injection and modified atmospheric packaging. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparative analysis of boar seminal plasma proteome from different freezability ejaculates and identification of Fibronectin 1 as sperm freezability marker.

PubMed

Vilagran, I; Yeste, M; Sancho, S; Castillo, J; Oliva, R; Bonet, S

2015-03-01

Variation in boar sperm freezability (i.e. capacity to withstand cryopreservation) between ejaculates is a limitation largely reported in the literature. Prediction of sperm freezability and classification of boar ejaculates into good (GFEs) and poor freezability ejaculates (PFEs) before cryopreservation takes place may increase the use of frozen-thawed spermatozoa. While markers of boar sperm freezability have been found from sperm cell extracts, little attention has been paid to seminal plasma. On this basis, the present study compared the fresh seminal plasma proteome of 9 GFEs and 9 PFEs through two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography mass spectrometry (LC-MS/MS). The ejaculates were previously classified as GFE or PFE upon their sperm viability and progressive motility assessments at 30 and 240 min post thawing. From a total of 51 spots, four were found to significantly (p < 0.05) differ between GFEs and PFEs, and two were identified as fibronectin-1 (FN1) and glutathione peroxidase 5 (GPX5). These two potential markers were further studied by western blot and correlation analysis between protein relative abundances in fresh seminal plasma and regression factors from principal component analyses (PCA) run using post-thawing sperm quality parameters. Results confirmed that FN1 is a reliable marker of boar sperm freezability, because GFEs presented significantly (p < 0.05) higher FN1-amounts than PFEs and FN1 was found to be correlated with the first PCA component at 240 min post thawing. In contrast, GPX5 was not validated as a boar sperm freezability marker. We can thus conclude that levels of FN1 in fresh seminal plasma from boar semen may be used as a sperm freezability marker, thereby facilitating the use of frozen-thawed boar spermatozoa. © 2015 American Society of Andrology and European Academy of Andrology.

Effects of holding time during cooling and of type of package on plasma membrane integrity, motility and in vitro oocyte penetration ability of frozen-thawed boar spermatozoa.

PubMed

Eriksson, B M; Vazquez, J M; Martinez, E A; Roca, J; Lucas, X; Rodriguez-Martinez, H

2001-05-01

The effect of a prolonged holding time (HT) during cooling on plasma membrane integrity (PMI), motility and in vitro oocyte penetration ability of boar spermatozoa frozen-thawed in different types of package was investigated. Boar semen was frozen in a split-sample design using 3 different HTs (3, 10 and 20 h) during cooling and three different types of freezing package: Maxi-straws, Medium-straws and FlatPacks. Assessment of PMI (SYBR-14 and propidium iodide, fluorescence microscopy) and sperm motility (visually and with CASA) was done during cooling (at 32 degrees C, 15 degrees C, 5 degrees C) and post-thaw (PT). The in vitro oocyte penetration ability of the spermatozoa was tested only PT, using a homologous in vitro penetration assay (hIVP). During cooling the HTs used had no significant (p<0.05) effect on either PMI or percentage of motile spermatozoa Post-thaw PMI was significantly higher (p<0.05) for 10 h and 20 h HT compared with 3 h, and the percentage of motile spermatozoa decreased significantly with 20 h HT as opposed to 3 h and 10 h. Regarding the freezing packages, the FlatPacks and Maxi-straws yielded significantly more PMI than did the Medium-straws (p<0.05). Post-thaw motility was significantly higher for FlatPacks than for straws, in terms of both percentage motile spermatozoa, and sperm velocity and lateral head displacement (LHD). The hIVP did not show any significant differences among the HTs, although FlatPacks yielded a significantly higher penetration rate and more spermatozoa per penetrated oocyte (p<0.05) than did the straws. Changes in motility patterns, toward a more circular motility during cooling and PT, could be noticed where individual spermatozoa showed a capacitation-like motility pattern. The changes were more obvious with 10-h and 20-h HTs than with 3-h HT.

L-Carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperm.

PubMed

Fattah, A; Sharafi, M; Masoudi, R; Shahverdi, A; Esmaeili, V; Najafi, A

2017-02-01

Rooster semen cryopreservation is not efficient for artificial insemination in breeder flocks. L-Carnitine (LC) has been evaluated for effectiveness in cryopreservation media on the characteristics of rooster sperm after freeze-thawing. Motility characteristics, membrane functionality, abnormal morphology, apoptotic like changes, mitochondria activity and lipid peroxidation of rooster sperms were assessed after freeze-thawing with different concentrations of LC in Beltsville medium. Semen samples were collected from 12 roosters, twice a week, and diluted in the extenders that contained different concentrations of LC. Supplementation of Beltsevile with 1 and 2 mM LC was found to result in higher total motility (68.2± 1.7% and 69.1± 1.7%, respectively), progressive motility (28.4± 1.6%, 29.8± 1.6%), membrane functionality (76.2± 1.9% and 75.9± 1.9%), viability (58.2 ± 1.1%, 59.1 ± 1.1%) and lower significant of lipid peroxidation (2.53 ± 0.08 nmol/ml, 2.49 ± 0.08 nmol/ml) compared to control group containing no LC. Lower motility, progressive motility, and viability were observed in frozen-thawed sperm in extender containing 8 mM LC (35.8± 1.7%, 9.6± 1.2% and 27.1 ± 1.2%, respectively) compared to control. Morphology and mitochondrial activity were not affected by different concentrations of LC. Our results showed that supplementation of Beltsville extender with 1 and 2 mM LC significantly improved the quality of rooster sperm quality after freeze-thawing. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation.

PubMed

Naaldijk, Yahaira; Friedrich-Stöckigt, Annett; Sethe, Sebastian; Stolzing, Alexandra

2016-10-01

Easy, cost-effective and reliable cryopreservation protocols are crucial for the successful and effective application of tissue engineering. Several different protocols are in use, but no comprehensive comparisons across different machine-based and manual methods have been made. Here, we compare the effects of different cooling rates on the post-thaw survival and proliferative capacity of two basic cell lines for skin tissue engineering fibroblasts and keratinocytes, cultured and frozen in suspension or as a monolayer. We demonstrate that effectiveness of cryopreservation cannot be reliably determined immediately after thawing: the results at this stage were not indicative of cell growth in culture 3 days post-thaw. Cryopreservation of fibroblasts in an adherent state greatly diminishes their subsequent growth potential. This was not observed when freezing in suspension. In keratinocytes, however, adherent freezing is as effective as freezing in suspension, which could lead to significant cost and labour savings in a tissue-engineering environment. The 'optimal' cryopreservation protocol depends on cell type and intended use. Where time, ease and cost are dominant factors, the direct freezing into a nitrogen tank (straight freeze) approach remains a viable method. The most effective solution across the board, as measured by viability 3 days post-thaw, was the commonly used, freezing container method. Where machine-controlled cryopreservation is deemed important for tissue-engineering Good Manufacturing Practice, we present results using a portfolio of different cooling rates, identifying the 'optimal' protocol depending on cell type and culture method. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

Effect of cryopreservant combinations on the motility and morphology of curimba (Prochilodus lineatus) sperm.

PubMed

Felizardo, V O; Mello, R A; Murgas, L D S; Andrade, E S; Drumond, M M; Rosa, P V

2010-12-01

This study investigated the application of intra- and extra-cellular cryoprotectant combinations on the quality of curimba Prochilodus lineatus semen subjected to cryopreservation. Semen treatments were tested with 8% DMSO or methanol as intracellular cryoprotectant, 5% egg yolk or lactose as extracellular cryoprotectant and 5% BTS. These cryoprotectant combinations are suitable for curimba but have not been tested at the lesser concentrations proposed or in combination with BTS. Semen samples collected from 19 curimbas were diluted into one of four cryoprotectant combinations: DMSO+yolk; DMSO+lactose; methanol+yolk; and methanol+lactose. After dilution, semen samples were cryopreserved in 0.5 mL straws for 10 days in a liquid nitrogen tank. Semen was thawed in a water bath at 60°C for 8s. We evaluated the quality of fresh, diluted (pre-freezing) and post-freezing semen according to sperm motility rate (%) and duration (s). Sperm morphology was also analyzed in thawed semen. Sperm motility rate decreased progressively after dilution and thawing. The motility rate in post-freezing semen was higher in the treatments using DMSO+lactose and methanol+yolk. Sperm motility duration in post-freezing sperm was greater in the treatments using methanol rather than DMSO as intracellular cryoprotectant, irrespective of the extracellular cryoprotectant used. Abnormality frequency in thawed sperm was less in semen treated with egg yolk than with lactose. Thus the use of methanol intracellular cryoprotectant is recommended along with yolk extracellular cryoprotectant in the cryopreservation process for curimba semen. Copyright © 2010 Elsevier B.V. All rights reserved.

Influence of waxy rice flour substitution for wheat flour on characteristics of batter and freeze-thawed cake.

PubMed

Jongsutjarittam, Nisachon; Charoenrein, Sanguansri

2013-09-12

This study aimed to improve the freeze-thawed cake properties by10-20% waxy rice flour (WRF) substitution for wheat flour (WF). Viscosity of WRF-substituted batters was lower; consequently, trapped air was less uniformly distributed than WF batter. After five freeze-thaw cycles, firmness and enthalpy of melting retrograded amylopectin of WF- and WRF-substituted cakes increased and the matrix surrounding the air pores from SEM images was denser than in fresh-baked cakes. Sensory evaluation showed an increase in firmness and a decrease in firmness acceptability of freeze-thawed cakes. However, freeze-thawed cake with WRF substitution had significantly less firmness, less dense matrix and more acceptability than WF cake. This could have been due to a low amylose content of WRF and the spread of ruptured waxy rice starch granules around swollen wheat starch granules as observed by CLSM. Thus, WRF could be used for WF substitution to improve the firmness in freeze-thawed cake. Copyright © 2013 Elsevier Ltd. All rights reserved.

Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing.

PubMed

Santiago-Moreno, J; Esteso, M C; Pradiee, J; Castaño, C; Toledano-Díaz, A; O'Brien, E; Lopez-Sebastián, A; Martínez-Nevado, E; Delclaux, M; Fernández-Morán, J; Zhihe, Z

2016-05-01

This work examines the effects of subsequent cycles of freezing-thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density-gradient centrifugation (DGC) can increase the number of freezing-thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing-thawing cycles. Although repeated freezing-thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG-selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 μm, the head width 3.6 μm, area 14.3 μm(2) and perimeter length 14.1 μm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing-thawing, even without DGC selection. © 2015 Blackwell Verlag GmbH.

Lateral and subsurface flows impact arctic coastal plain lake water budgets

USGS Publications Warehouse

Koch, Joshua C.

2016-01-01

Arctic thaw lakes are an important source of water for aquatic ecosystems, wildlife, and humans. Many recent studies have observed changes in Arctic surface waters related to climate warming and permafrost thaw; however, explaining the trends and predicting future responses to warming is difficult without a stronger fundamental understanding of Arctic lake water budgets. By measuring and simulating surface and subsurface hydrologic fluxes, this work quantified the water budgets of three lakes with varying levels of seasonal drainage, and tested the hypothesis that lateral and subsurface flows are a major component of the post-snowmelt water budgets. A water budget focused only on post-snowmelt surface water fluxes (stream discharge, precipitation, and evaporation) could not close the budget for two of three lakes, even when uncertainty in input parameters was rigorously considered using a Monte Carlo approach. The water budgets indicated large, positive residuals, consistent with up to 70% of mid-summer inflows entering lakes from lateral fluxes. Lateral inflows and outflows were simulated based on three processes; supra-permafrost subsurface inflows from basin-edge polygonal ground, and exchange between seasonally drained lakes and their drained margins through runoff and evapotranspiration. Measurements and simulations indicate that rapid subsurface flow through highly conductive flowpaths in the polygonal ground can explain the majority of the inflow. Drained lakes were hydrologically connected to marshy areas on the lake margins, receiving water from runoff following precipitation and losing up to 38% of lake efflux to drained margin evapotranspiration. Lateral fluxes can be a major part of Arctic thaw lake water budgets and a major control on summertime lake water levels. Incorporating these dynamics into models will improve our ability to predict lake volume changes, solute fluxes, and habitat availability in the changing Arctic.

Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells.

PubMed

Li, Rui; Yu, Guanglin; Azarin, Samira M; Hubel, Allison

2018-05-01

Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.

Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide

PubMed Central

2012-01-01

Background Mesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed. Results The study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a ‘straight freeze’ protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods. Conclusion A 5% DMSO / 5% HES solution cryopreservation solution using a ‘straight freeze’ approach can be recommended for rat MSC. PMID:22889198

Improvement of stress tolerance and leavening ability under multiple baking-associated stress conditions by overexpression of the SNR84 gene in baker's yeast.

PubMed

Lin, Xue; Zhang, Cui-Ying; Bai, Xiao-Wen; Feng, Bing; Xiao, Dong-Guang

2015-03-16

During the bread-making process, industrial baker's yeast cells are exposed to multiple baking-associated stresses, such as elevated high-temperature, high-sucrose and freeze-thaw stresses. There is a high demand for baker's yeast strains that could withstand these stresses with high leavening ability. The SNR84 gene encodes H/ACA snoRNA (small nucleolar RNA), which is known to be involved in pseudouridylation of the large subunit rRNA. However, the function of the SNR84 gene in baker's yeast coping with baking-associated stresses remains unclear. In this study, we explored the effect of SNR84 overexpression on baker's yeast which was exposed to high-temperature, high-sucrose and freeze-thaw stresses. These results suggest that overexpression of the SNR84 gene conferred tolerance of baker's yeast cells to high-temperature, high-sucrose and freeze-thaw stresses and enhanced their leavening ability in high-sucrose and freeze-thaw dough. These findings could provide a valuable insight for breeding of novel stress-resistant baker's yeast strains that are useful for baking. Copyright © 2015 Elsevier B.V. All rights reserved.

Permafrost thaw in a nested groundwater-flow system

USGS Publications Warehouse

McKenzie, Jeffery M.; Voss, Clifford I.

2013-01-01

Groundwater flow in cold regions containing permafrost accelerates climate-warming-driven thaw and changes thaw patterns. Simulation analyses of groundwater flow and heat transport with freeze/thaw in typical cold-regions terrain with nested flow indicate that early thaw rate is particularly enhanced by flow, the time when adverse environmental impacts of climate-warming-induced permafrost loss may be severest. For the slowest climate-warming rate predicted by the Intergovernmental Panel on Climate Change (IPCC), once significant groundwater flow begins, thick permafrost layers can vanish in several hundred years, but survive over 1,000 years where flow is minimal. Large-scale thaw depends mostly on the balance of heat advection and conduction in the supra-permafrost zone. Surface-water bodies underlain by open taliks allow slow sub-permafrost flow, with lesser influence on regional thaw. Advection dominance over conduction depends on permeability and topography. Groundwater flow around permafrost and flow through permafrost impact thaw differently; the latter enhances early thaw rate. Air-temperature seasonality also increases early thaw. Hydrogeologic heterogeneity and topography strongly affect thaw rates/patterns. Permafrost controls the groundwater/surface-water-geomorphology system; hence, prediction and mitigation of impacts of thaw on ecology, chemical exports and infrastructure require improved hydrogeology/permafrost characterization and understanding

Transmembrane ion distribution during recovery from freezing in the woolly bear caterpillar Pyrrharctia isabella (Lepidoptera: Arctiidae).

PubMed

Boardman, Leigh; Terblanche, John S; Sinclair, Brent J

2011-08-01

During extracellular freezing, solutes in the haemolymph are concentrated, resulting in osmotic dehydration of the cells, which must be reversed upon thawing. Here, we used freeze tolerant Pyrrharctia isabella (Lepidoptera: Arctiidae) larvae to examine the processes of ion redistribution after thawing. To investigate the effect of the intensity of cold exposure on ion redistribution after thawing, we exposed caterpillars to -14°C, -20°C or -30°C for 35h. To investigate the effect of duration of cold exposure on ion redistribution after thawing, we exposed the caterpillars to -14°C for up to 6 weeks while sampling several time points. The concentrations of Na(+), K(+), Mg(2+) and Ca(2+) were measured after thawing in the haemolymph, fat body, muscle, midgut tissue and hindgut tissue. Being frozen for long durations (>3 weeks) or at low temperatures (-30°C) both result in 100% mortality, although different ions and tissues appear to be affected by each treatment. Both water distribution and ion content changes were detected after thawing, with the largest effects seen in the fat body and midgut tissue. Magnesium homeostasis appears to be vital for post-freeze survival in these larvae. The movement of ions during thawing lagged behind the movement of water, and ion homeostasis was not restored within the same time frame as water homeostasis. Failure to regain ion homeostasis after thawing is therefore implicated in mortality of freeze tolerant insects. Copyright © 2011 Elsevier Ltd. All rights reserved.

Influence of freeze-thawing on hyaluronic acid binding of human spermatozoa.

PubMed

Nijs, Martine; Creemers, Eva; Cox, Annemie; Janssen, Mia; Vanheusden, Elke; Castro-Sanchez, Yovanna; Thijs, Herbert; Ombelet, Willem

2009-08-01

Mature human spermatozoa have at least three specific hyaluronic acid (HA) binding proteins present on their sperm membrane. These receptors play a role in the acrosome reaction, hyaluronidase activity, hyaluronan-mediated motility and sperm-zona and sperm-oolemmal binding. Cryopreservation of spermatozoa can cause ultrastructural and even molecular damage. The aim of this study was to investigate if HA binding receptors of human spermatozoa remain functional after freeze-thawing. Forty patients were enrolled in the study. Semen samples were analysed before and after cryopreservation. Parameters analysed included concentration, motility, morphology and hyaluronan binding. Samples were frozen in CBS straws using a glycerol-glucose-based cryoprotectant. HA binding was studied using the sperm-hyaluronan binding assay. Freeze-thawing resulted in a significant decline in motility: the percentage of motile spermatozoa reduced from 50.6 to 30.3% (P < 0.001). HA binding properties of frozen-thawed spermatozoa remained unchanged after the freeze-thawing process: 68.5 +/- 17.1% spermatozoa of the neat sample were bound to HA, as were 71.3 +/- 20.4 of the frozen-thawed sample. This study indicates that freeze-thawing did not alter the functional hyaluronan binding sites of mature motile spermatozoa, and therefore will not alter their fertilizing potential.

Numerical Simulation of Thawing Process of Biological Tissue

NASA Astrophysics Data System (ADS)

Momose, Noboru; Tada, Yukio; Hayashi, Yujiro

Heat transfer and simplified physicochemical model for thawing of the frozen biological cell element consisting of cell and extracellular region was proposed. The melting of intra-and extra-cellular ice, the water transport through cell membrane and other microscale behavior during thawing process were discussed as a function of temperature. Recovery of the cell volume and change of osmotic pressure difference during thawing were clarified theortically in connection with heating velocity, initial cell volume and membrane permeability. Extending this model, the thawing of cellular tissue consisted of numerous cell elements was also simulated. There was a position where osmotic pressure difference became maximum during thawing. Summarizing these results, the thawing damage due to osmotic stress was discussed in relation with the heating operation and the size effect of tissue.

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility.

PubMed

Januskauskas, A; Johannisson, A; Rodriguez-Martinez, H

2003-09-01

This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.

Semen characterization, seasonality of production, and in vitro sperm quality after chilled storage and cryopreservation in the king penguin (Aptenodytes patagonicus).

PubMed

O'Brien, Justine K; Robeck, Todd R

2014-01-01

Research was conducted to examine seasonal seminal traits and to establish short-term and long-term sperm preservation methods in the king penguin (Aptenodytes patagonicus) for use in genome banking and artificial insemination (AI). Spermic ejaculates (n = 87) obtained using a cooperative method were collected across multiple (n = 6, Male 1) and a single (Male 2) breeding season(s). Non-contaminated ejaculates (n = 69) were 0.36 ± 0.32 ml at 56.3 ± 62.7 × 10(7)  sperm/ml with 85.3 ± 10.6% total motility (TMot), 52.5 ± 12.9% progressive motility (PMot), 86.6 ± 24.3 µm/sec average path velocity (VAP) and 92.3 ± 3.7% plasma membrane intact. In vitro quality of chilled semen was best maintained over 48 hr at 5°C than 21°C, with decreased (P < 0.05) motility and morphology parameters observed by 24 and 6 hr, respectively. A comparison of two freezing methods (straw [STR] vs. directional [DF]) demonstrated similar effects on post-thaw quality at 0 and 3 hr, with the exception of plasma membrane integrity which was higher (P < 0.05) at 0 hr for DF (48.7 ± 6.5%) than STR (41.2 ± 7.0%). At 0 hr post-thaw, DF samples retained 46%, 69%, and 52% of their initial PMot, VAP, and plasma membrane integrity, respectively. Normal morphology of motile cells was reduced (P < 0.05) during freeze-thawing from 84% post-collection to 37% and 34% at 0 and 3 hr post-thaw, respectively. Results indicate that chilled and cryopreserved semen from the king penguin has potential for use in AI. © 2014 Wiley Periodicals, Inc.

Impact of Cryopreservation on Caprine Fetal Adnexa Derived Stem Cells and Its Evaluation for Growth Kinetics, Phenotypic Characterization, and Wound Healing Potential in Xenogenic Rat Model.

PubMed

Somal, Anjali; Bhat, Irfan A; B, Indu; Singh, Anuj P; Panda, Bibhudatta S K; Desingu, Perumal A; Pandey, Sriti; Bharti, Mukesh K; Pal, Amar; Saikumar, Guttula; Chandra, Vikash; Sharma, Guttula Taru

2017-08-01

This study was conducted to know the impact of cryopreservation on caprine fetal adnexa derived mesenchymal stem cells (MSCs) on the basic stem cell characteristics. Gravid caprine uteri (2-3 months) were collected from local abattoir to derive (amniotic fluid [cAF], amniotic sac [cAS], Wharton's jelly [cWJ], and cord blood [cCB]) MSCs and expanded in vitro. Cells were cryopreserved at 3rd passage (P3) using 10% DMSO. Post-thaw viability and cellular properties were assessed. Cells were expanded to determine growth kinetics, tri-lineage differentiation, localization, and molecular expression of MSCs and pluripotency markers; thereafter, these cells were transplanted in the full-thickness (2 × 2cm 2 ) rat skin wound to determine their wound healing potential. The post-thaw (pt) growth kinetics study suggested that cWJ MSCs expanded more rapidly with faster population doubling time (PDT) than that of other fetal adnexa MSCs. The relative mRNA expression of surface antigens (CD73, CD90, and CD 105) and pluripotency markers (Oct4, KLF, and cMyc) was higher in cWJ MSCs in comparison to cAS, cAF, and cCB MSCs post-thaw. The percent wound contraction on 7th day was more than 50% for all the MSC-treated groups (pre and post-thaw), against 39.55% in the control group. On day 28th, 99% and more wound contraction was observed in cAF, cAF-pt, cAS-pt, cWJ, cWJ-pt, and cCB, MSCs with better scores for epithelization, neovascularization, and collagen characteristics at a non-significant level. It is concluded that these MSCs could be successfully cryopreserved without altering their stemness and wound healing properties. J. Cell. Physiol. 232: 2186-2200, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cholesterol addition aids the cryopreservation of dromedary camel (Camelus dromedarius) spermatozoa.

PubMed

Crichton, Elizabeth G; Pukazhenthi, Budhan S; Billah, M; Skidmore, Julian A

2015-01-15

The cryopreservation of dromedary camel (Camelus dromedarius) sperm has proved challenging with little success reported. The routine application of artificial insemination with frozen semen would assist the flow of valuable genetic material nationally and internationally. The current study sought to examine the effects of cholesterol (cholesterol-loaded cyclodextrin [CLC]) preloading on camel sperm cryosurvival. Ejaculates (n = 3 males; 3 ejaculates per male) were collected using an artificial vagina during the breeding season and extended in HEPES-buffered Tyrode's albumin lactate pyruvate (TALP) and allowed to liquefy in the presence of papain (0.1 mg/mL) before removal of the seminal plasma by centrifugation. Sperm pellets were resuspended (120 million/mL) in fresh TALP and incubated (15 minutes; 37 °C) with 0, 1.5, or 4.5 mg CLC/mL. Sperm suspensions were then centrifuged and reconstituted in INRA-96 containing 20% (v:v) egg yolk and 2.5% (v:v) methylformamide, loaded in 0.5-mL plastic straws, sealed, and cooled for 20 minutes at 4 °C. Straws were frozen over liquid nitrogen (4 cm above liquid; 15 minutes), plunged, and stored. Sperm motility, forward progressive status, and acrosomal integrity were recorded at 0 and 3 hours after thawing and compared with these same parameters before freezing. Aliquots also were stained with chlortetracycline hydrochloride to assess spontaneous sperm capacitation status before freezing and post-thaw. Pretreatment with CLC (1.5 and 4.5 mg/mL) enhanced cryosurvival. Post-thaw sperm motility was highest (P < 0.05) in 1.5 mg CLC/mL immediately after thawing (44%) and after 3 hours incubation at room temperature (34%). Highest post-thaw sperm progressive status was also achieved in the presence of 1.5 CLC. Greater proportions of spermatozoa retained acrosomal membrane integrity when cryopreserved in the presence of CLC, but there was no difference between 1.5 and 4.5 CLC. Although thawed spermatozoa underwent spontaneous capacitation during in vitro incubation, cryopreservation and CLC treatment exerted no effect. In summary, dromedary camel sperm benefit from exposure to CLC before cryopreservation; this may facilitate the routine collection and storage of sperm from this species. Copyright © 2015 Elsevier Inc. All rights reserved.

Oxygenated thawing and rewarming alleviate rewarming injury of cryopreserved pancreatic islets.

PubMed

Komatsu, Hirotake; Barriga, Alyssa; Medrano, Leonard; Omori, Keiko; Kandeel, Fouad; Mullen, Yoko

2017-05-06

Pancreatic islet transplantation is an effective treatment for Type 1 diabetic patients to eliminate insulin injections; however, a shortage of donor organs hinders the widespread use. Although long-term islet storage, such as cryopreservation, is considered one of the key solutions, transplantation of cryopreserved islets is still not practical due to the extensive loss during the cryopreservation-rewarming process. We have previously reported that culturing islets in a hyperoxic environment is an effective treatment to prevent islet death from the hypoxic injury during culture. In this study, we explored the effectiveness of thawing and rewarming cryopreserved islets in a hyperoxic environment. Following cryopreservation of isolated human islets, the thawing solution and culture media were prepared with or without pre-equilibration to 50% oxygen. Thawing/rewarming and the pursuant two-day culture were performed with or without oxygenation. Short-term recovery rate, defined as the volume change during cryopreservation and thawing/rewarming, was assessed. Ischemia-associated and inflammation-associated gene expressions were examined using qPCR after the initial rewarming period. Long-term recovery rate, defined as the volume change during the two-day culture after the thawing/rewarming, was also examined. Islet metabolism and function were assessed by basal oxygen consumption rate and glucose stimulated insulin secretion after long-term recovery. Oxygenated thawing/rewarming did not alter the short-term recovery rate. Inflammation-associated gene expressions were elevated by the conventional thawing/rewarming method and suppressed by the oxygenated thawing/rewarming, whereas ischemia-associated gene expressions did not change between the thawing/rewarming methods. Long-term recovery rate experiments revealed that only the combination therapy of oxygenated thawing/rewarming and oxygenated culture alleviated islet volume loss. These islets showed higher metabolism and better function among the conditions examined. Oxygenated thawing/rewarming alleviated islet volume loss, with the help of oxygenated culture. Copyright © 2017. Published by Elsevier Inc.

Red wolf (Canis rufus) sperm quality and quantity is affected by semen collection method, extender components, and post-thaw holding temperature.

PubMed

Franklin, Ashley D; Waddell, William T; Goodrowe, Karen L

2018-08-01

Cryopreserving genetic resources is becoming increasingly important for species management. In the zoo-based red wolf (Canis rufus) population, inbreeding continues to increase in the absence of new founders. Through banking sperm, we preserve genetic diversity and create the ability to decrease inbreeding accumulation in the future. The quality and quantity of banked sperm can be affected by cryopreservation media and semen collection methods. This study's objectives were to further optimize semen extender used for red wolf sperm cryopreservation, investigate effects of post-thaw holding temperature, and to determine if urethral catheterization is an effective method for semen collection in this species. Semen collection via electroejaculation (EE) was performed on 39 adult red wolf males (ages 1 to 11) from 15 institutions. Urethral catheterization (UC) was attempted on a subset (n = 14) of those males, prior to EE. Thirteen different semen extenders were used for cryopreservation, which varied in osmolarity (HI or NORM), sugar source (glucose, fructose, or a combination), and cryoprotectant (glycerol or DMSO). Significant decreases in percent motility, forward progressive status (FPS), and acrosomal integrity were observed over time across all extenders (P < 0.0001). Among the extender components examined, post-thaw sperm motility and FPS were lower in DMSO versus glycerol based treatments (P < 0.005). Therefore, DMSO should be considered unsuitable as a cryoprotectant when freezing red wolf sperm. Effects of osmolarity and sugar source were minimal and temporally variable, however notably, a higher percentage of morphologically normal sperm were observed in the fructose-based extenders compared to glucose-based extenders post-thaw (P < 0.05). Additionally, post-thaw sperm motility and FPS declined more rapidly in samples maintained at 37 °C compared to samples held at room temperature (P < 0.05). Greater volumes of semen were collected using EE compared to UC (P = 0.041), and sperm samples collected using EE also had greater motility and FPS (P < 0.05). Additionally, though no gross morphological differences were observed, there were fewer sperm with intact acrosomes in the samples collected via UC (P = 0.0443). Thus, UC should not be considered sufficient for semen collection in red wolves when the desired fate of sperm is cryopreservation and/or AI. However, UC does provide an opportunity for a basic reproductive evaluation of a red wolf male. Copyright © 2018 Elsevier Inc. All rights reserved.

Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach

NASA Astrophysics Data System (ADS)

Sultani, A. Billal; Marquez-Curtis, Leah A.; Elliott, Janet A. W.; McGann, Locksley E.

2016-10-01

Cryopreservation of human umbilical vein endothelial cells (HUVECs) facilitated their commercial availability for use in vascular biology, tissue engineering and drug delivery research; however, the key variables in HUVEC cryopreservation have not been comprehensively studied. HUVECs are typically cryopreserved by cooling at 1 °C/min in the presence of 10% dimethyl sulfoxide (DMSO). We applied interrupted slow cooling (graded freezing) and interrupted rapid cooling with a hold time (two-step freezing) to identify where in the cooling process cryoinjury to HUVECs occurs. We found that linear cooling at 1 °C/min resulted in higher membrane integrities than linear cooling at 0.2 °C/min or nonlinear two-step freezing. DMSO addition procedures and compositions were also investigated. By combining hydroxyethyl starch with DMSO, HUVEC viability after cryopreservation was improved compared to measured viabilities of commercially available cryopreserved HUVECs and viabilities for HUVEC cryopreservation studies reported in the literature. Furthermore, HUVECs cryopreserved using our improved procedure showed high tube forming capability in a post-thaw angiogenesis assay, a standard indicator of endothelial cell function. As well as presenting superior cryopreservation procedures for HUVECs, the methods developed here can serve as a model to optimize the cryopreservation of other cells.

Out-of-season sperm cryopreserved in different media of the Amazonian freshwater fish pirapitinga (Piaractus brachypomus).

PubMed

Nascimento, A F; Maria, A N; Pessoa, N O; Carvalho, M A M; Viveiros, A T M

2010-04-01

The pirapitinga (Piaractus brachypomus) is a freshwater fish that inhabits the Amazon and Orinoco River basins. The use of cryopreserved sperm has been considered to facilitate procedures during the artificial reproduction. The aim of the present study was to develop a freezing protocol for pirapitinga sperm collected outside the spawning season. Sperm samples were diluted in four freezing media prepared by a combination of two extenders (glucose and BTS-Beltsville Thawing Solution) and two cryoprotectant agents (DMSO and methylglycol) loaded into 0.5-mL straws, frozen in a nitrogen-vapor shipping dewar (dry-shipper) and stored in liquid nitrogen at -196 degrees C. Post-thaw sperm motility was evaluated both subjectively using a light microscope and by a computer-assisted sperm analyzer (CASA). Curvilinear, average path and straight-line velocities were also determined. There were no differences (P>0.05) in post-thaw sperm motility between evaluations performed subjectively and using the CASA. Sperm samples cryopreserved in glucose-methylglycol yielded the greatest post-thaw sperm motility (81%) and fastest sperm velocities when compared to the samples frozen in the other three media (P<0.05). Out-of-season sperm cryopreserved in glucose and methylglycol under the conditions described above is of high quality and can therefore be used to facilitate artificial reproduction procedures, as only females will need handling for hormonal induction and gamete collection during the spawning season. Although the CASA system provides precise data on sperm motility, the subjective evaluation is practical and can be conducted by well-trained personnel at commercial fish farms as an acceptable evaluation of sperm quality. Copyright 2009 Elsevier B.V. All rights reserved.

Challenges in cryopreserving endangered mammal spermatozoa: morphology and the value of acrosomal integrity as markers of cryo-survival.

PubMed

Pukazhenthi, Budhan; Santymire, Rachel; Crosier, Adrienne; Howard, JoGayle; Wildt, David E

2007-01-01

The science of cryobiology is essential to the effective, practical use of semen for assisted breeding to help manage small populations of rare wildlife species. In this review, we describe challenges associated with cryopreserving gametes from wild fauna. Based on more than 25 years of experience across a diversity of mammals, it appears that the primary driving force dictating cryo-survival of a spermatozoon is its initial pre-freeze quality and morphology, especially having a morphologically normal, intact acrosome. This assertion is supported through extensive studies of three animal groups that routinely ejaculate semen containing (1) normal sperm/acrosomal quality (examples, Eld's deer, Cervus eldi and giant panda, Ailuropoda melanoleuca), (2) normal acrosomal quality, but from teratospermic donors (>70% pleiomorphic sperm; cheetah, Acinonyx jubatus and black-footed ferret, Mustela nigripes) and (3) abnormal acrosomal quality and general teratospermia (clouded leopard, Neofelis nebulosa). Data revealed that species producing high quality sperm with > 70% normal, intact acrosomes were best able to survive cryopreservation (-80% intact acrosomes post-thaw). Species that were teratospermic, but with high proportions of intact acrosomes (72 to 88%) in ejaculates varied significantly (4 to 55% intact acrosomes post-thaw) in sperm survival to freeze-thawing. Spermatozoa from the clouded leopard (that was both teratospermic while producing only 11% normal acrosomes in fresh semen) failed to survive cryopreservation despite using an array of conventional and unconventional freezing approaches. These observations (combined with zona penetration assays and artificial insemination results) suggest that proportions of malformed sperm and especially initial structural integrity of the acrosome are more important predictors of sperm survivability post-thaw than initial sperm motility scores.

The effect of fire and permafrost interactions on soil carbon accumulation in an upland black spruce ecosystem of interior Alaska: implications for post-thaw carbon loss

Treesearch

Jonathan A. O' Donnell; Jennifer W. Harden; A. David McGuire; Mikhail Z. Kanevskiy; M. Torre Jorgenson; Xiaomei Xu

2010-01-01

High-latitude regions store large amounts of organic carbon (OC) in active-layer soils and permafrost, accounting for nearly half of the global belowground OC pool. In the boreal region, recent warming has promoted changes in the fire regime, which may exacerbate rates of permafrost thaw and alter soil OC dynamics in both organic and mineral soil. We examined how...

Stress preconditioning of semen before cryopreservation improves fertility and increases the number of offspring born: a prospective randomised study using a porcine model.

PubMed

Horváth, A; Szenci, O; Nagy, K; Végh, L; Pribenszky, Cs

2016-03-01

The aim of the present study was to investigate the effect of applying sublethal stress treatment at room temperature, before cryopreservation (hydrostatic pressure (HP): 40MPa, 80min) of 34 boar ejaculate samples, on post-thawed motility and sow fertility. Sows (n=102) were randomly allocated into equal groups inseminated with HP-treated or untreated frozen-thawed semen. Sows were inseminated twice, 10h apart, with 6×10(9) spermatozoa per dose without oestrus synchronisation. Rates of non-return of oestrus and pregnancy, and total numbers of piglets and live piglets were significantly higher (P<0.05) in the HP-treated group. There was also a numerical, albeit non-significant (P>0.05), improvement in the farrowing rate in the HP-treated group. Although the number of live piglets per litter decreased approximately 15% in both groups by 42 days after farrowing, but this remained significantly higher in the HP-treated group. Although total and progressive sperm motility were significantly (P<0.001) higher in the HP-treated group, there were no significant differences (P>0.05) in these parameters between pregnant and non-pregnant sows in either group; thus motility can indicate, but not predict, improved fertility. In conclusion, HP treatment, with sperm cryopreservation, increases in vitro sperm motility and improves reproductive performance without adversely affecting the health of the piglets.

Controls on methane released through ebullition in peatlands affected by permafrost degradation

USGS Publications Warehouse

Klapstein, Sara J.; Turetsky, Merritt R.; McGuire, A. David; Harden, Jennifer W.; Czimczik, C.I.; Xu, Xiaomei; Chanton, J.P.; Waddington, James Michael

2014-01-01

Permafrost thaw in peat plateaus leads to the flooding of surface soils and the formation of collapse scar bogs, which have the potential to be large emitters of methane (CH4) from surface peat as well as deeper, previously frozen, permafrost carbon (C). We used a network of bubble traps, permanently installed 20 cm and 60 cm beneath the moss surface, to examine controls on ebullition from three collapse bogs in interior Alaska. Overall, ebullition was dominated by episodic events that were associated with changes in atmospheric pressure, and ebullition was mainly a surface process regulated by both seasonal ice dynamics and plant phenology. The majority (>90%) of ebullition occurred in surface peat layers, with little bubble production in deeper peat. During periods of peak plant biomass, bubbles contained acetate-derived CH4 dominated (>90%) by modern C fixed from the atmosphere following permafrost thaw. Post-senescence, the contribution of CH4 derived from thawing permafrost C was more variable and accounted for up to 22% (on average 7%), in the most recently thawed site. Thus, the formation of thermokarst features resulting from permafrost thaw in peatlands stimulates ebullition and CH4 release both by creating flooded surface conditions conducive to CH4 production and bubbling as well as by exposing thawing permafrost C to mineralization.

Comparison of Mitochondrial Function in Boar and Bull Spermatozoa Throughout Cryopreservation Based on JC-1 Staining.

PubMed

Hu, C H; Zhuang, X J; Wei, Y M; Zhang, M; Lu, S S; Lu, Y Q; Yang, X G; Lu, K H

Poor reproductivity hampers the commercialization of cryopreserved boar semen. This study was to determine the differences in the sperm mitochondrial function between boar and bull semen at different cryopreservation stages. Boar and bull fresh, equilibrated, and frozen-thawed spermatozoa were evaluated for mitochondrial function using JC-1 under a fluorescent microscope. Bull and boar percentage of spermatozoa staining green (PSSG) showed no difference between fresh and equilibrated semen (P> 0.05). However, frozen-thawed bull and boar semen demonstrated significantly higher PSSG (P < 0.01) than fresh and equilibrated semen. Frozen-thawed boar semen represented a significantly higher PSSG (P < 0.01) than bull semen. Negative cryopreservation influence on boar and bull spermatozoa was not significantly produced by pre-freezing procedures, but rather by freezing and thawing. Cryopreservation has more pronounced negative effects on boar than on bull spermatozoa, which partly explains lagged commercialization of frozen boar semen.

Patterns of nitrogen export from a seasonal freezing agricultural watershed during the thawing period.

PubMed

Zhao, Qiang; Chang, Dan; Wang, Kang; Huang, Jiesheng

2017-12-01

The objectives of this study were to investigate water, ammonium nitrogen (NH 4 + -N), and nitrate nitrogen (NO 3 - -N) export processes during the thawing period in a watershed with heavy agricultural activities and to evaluate contributions of N (i.e., NO 3 - -N and NH 4 + -N) from different source areas under different climate conditions. Experiments were conducted within the 75km 2 agricultural Heidingzi watershed in northeast China. The thawing period was divided into four stages: early-melt, late-melt, rain-on-melt, and post-melt. Drainage regions (DRs) were separated into three types. The processes of water and N discharge from soil into rivers were monitored in these DRs during the thawing periods of 2014, 2015, and 2016. Results show that the processes of water and N discharge were not synchronous during the thawing period. Variations in discharge concentrations of NH 4 + -N and NO 3 - -N during the thawing period were mainly affected by the flushing effect, which was controlled by the physical state of the surface water (snow or ice) and the melt rate of frozen soil. Contributions of N export from the DRs varied under different land uses and climate conditions during the thawing period. NO 3 - -N export was mainly from maize fields. Thawing stages with high NO 3 - -N export were always accompanied by higher discharge rates. NH 4 + -N export mainly occurred during the early-melt and late-melt stages and from riverside rural regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Metagenomic analysis of a permafrost microbial community reveals a rapid response to thaw

USGS Publications Warehouse

MacKelprang, R.; Waldrop, M.P.; Deangelis, K.M.; David, M.M.; Chavarria, K.L.; Blazewicz, S.J.; Rubin, E.M.; Jansson, J.K.

2011-01-01

Permafrost contains an estimated 1672????????Pg carbon (C), an amount roughly equivalent to the total currently contained within land plants and the atmosphere. This reservoir of C is vulnerable to decomposition as rising global temperatures cause the permafrost to thaw. During thaw, trapped organic matter may become more accessible for microbial degradation and result in greenhouse gas emissions. Despite recent advances in the use of molecular tools to study permafrost microbial communities, their response to thaw remains unclear. Here we use deep metagenomic sequencing to determine the impact of thaw on microbial phylogenetic and functional genes, and relate these data to measurements of methane emissions. Metagenomics, the direct sequencing of DNA from the environment, allows the examination of whole biochemical pathways and associated processes, as opposed to individual pieces of the metabolic puzzle. Our metagenome analyses reveal that during transition from a frozen to a thawed state there are rapid shifts in many microbial, phylogenetic and functional gene abundances and pathways. After one week of incubation at 5 ??C, permafrost metagenomes converge to be more similar to each other than while they are frozen. We find that multiple genes involved in cycling of C and nitrogen shift rapidly during thaw. We also construct the first draft genome from a complex soil metagenome, which corresponds to a novel methanogen. Methane previously accumulated in permafrost is released during thaw and subsequently consumed by methanotrophic bacteria. Together these data point towards the importance of rapid cycling of methane and nitrogen in thawing permafrost. ?? 2011 Macmillan Publishers Limited. All rights reserved.

Cryopreservation of Viable Human Lung Tissue for Versatile Post-thaw Analyses and Culture

PubMed Central

Baatz, John E.; Newton, Danforth A.; Riemer, Ellen C.; Denlinger, Chadrick E.; Jones, E. Ellen; Drake, Richard R.; Spyropoulos, Demetri D.

2018-01-01

Clinical trials are currently used to test therapeutic efficacies for lung cancer, infections and diseases. Animal models are also used as surrogates for human disease. Both approaches are expensive and time-consuming. The utility of human biospecimens as models is limited by specialized tissue processing methods that preserve subclasses of analytes (e.g. RNA, protein, morphology) at the expense of others. We present a rapid and reproducible method for the cryopreservation of viable lung tissue from patients undergoing lobectomy or transplant. This method involves the pseudo-diaphragmatic expansion of pieces of fresh lung tissue with cryoprotectant formulation (pseudo-diaphragmatic expansion-cryoprotectant perfusion or PDX-CP) followed by controlled-rate freezing in cryovials. Expansion-perfusion rates, volumes and cryoprotectant formulation were optimized to maintain tissue architecture, decrease crystal formation and increase long-term cell viability. Rates of expansion of 4 cc/min or less and volumes ranging from 0.8–1.2 × tissue volume were well-tolerated by lung tissue obtained from patients with chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis, showing minimal differences compared to standard histopathology. Morphology was greatly improved by the PDX-CP procedure compared to simple fixation. Fresh versus post-thawed lung tissue showed minimal differences in histology, RNA integrity numbers and post-translational modified protein integrity (2-dimensional differential gel electrophoresis). It was possible to derive numerous cell types, including alveolar epithelial cells, fibroblasts and stem cells, from the tissue for at least three months after cryopreservation. This new method should provide a uniform, cost-effective approach to the banking of biospecimens, with versatility to be amenable to any post-acquisition process applicable to fresh tissue samples. PMID:24982205

Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen.

PubMed

Rakha, B A; Ansari, M S; Akhter, S; Hussain, I; Blesbois, E

2016-11-01

The population of red jungle fowl is declining and needs special attention for its conservation with suitable approaches. For ex situ in vitro conservation of Indian red jungle fowl, establishment of semen cryobank is an appropriate option, for which an extender with adequate retrieval capacity for functional spermatozoa is required. Therefore, studies were designed to evaluate a wide range of extenders for cryopreservation of Indian red jungle fowl (Gallus gallus murghi) sperm to achieve maximal post-thawed semen quality and fertility. For this purpose, semen from eight mature cocks were collected, initially evaluated (percent sperm motility, volume and concentration), pooled, assessed for motility, plasma membrane integrity, viability and acrosome integrity, and divided into six aliquots for dilution (1:5; 37°C) in Beltsville poultry, red fowl extender, Lake, EK, Tselutin poultry and chicken semen extenders. Diluted semen was cooled from 37°C to 4°C @ -0.275°C/min. Glycerol (20%) was added to chilled semen, equilibrated for 10min, filled in 0.5mL French straws, kept over LN 2 vapours for 10min and plunged into LN 2 and stored at -196°C. Percentages of motility, plasma membrane integrity, viability and acrosome integrity were higher (P<0.05) in red fowl extender at 0, 2 and 4h of incubation post-thaw. After cryopreservation and post-thawing at 37°C the highest (P<0.05) recovery rates and absolute livability index was also recorded in red fowl extender that was thus used for further artificial insemination of cooled-diluted (Liquid) and cryopreserved sperm. The no. of fertilized eggs (Liquid, 20.6±0.4; Cryopreserved, 12.6±0.5), percent fertility (86.7±2.2; 57.2±3.9), no. of hatched chicks (18.2±0.8; 10.0±0.3), percent hatch (76.5±2.7; 45.3±2.2) and hatchability of fertilized eggs (88.3±3.4; 79.6±3.4) were higher with sperm respectively freshly cooled-diluted or cryopreserved in red fowl extender. However, the rates obtained with frozen-thawed sperm were already successful for cryo-banking purpose and artificial insemination practice. In conclusion, we show the first fertility success obtained with cryopreserved Indian jungle fowl sperm. In addition, the red fowl extender is superior in maintaining the quality of Indian red jungle fowl cryopreserved sperm compared to Beltsville poultry, Lake, EK, Tselutin poultry and chicken semen extender. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm.

PubMed

Brito, Maíra M; Lúcio, Cristina F; Angrimani, Daniel S R; Losano, João Diego A; Dalmazzo, Andressa; Nichi, Marcílio; Vannucchi, Camila I

2017-01-02

In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

The influence of temperature treatment before cryopreservation on the viability and potency of cryopreserved and thawed CD34+ and CD45+ cord blood cells.

PubMed

Schwandt, Svenja; Liedtke, Stefanie; Kogler, Gesine

2017-08-01

Hematopoietic stem cell (HSC) viability and potency is crucial for qualified cord blood (CB) transplants. This study analyzes time and temperature condition before cryopreservation for the viability of CD34 + /CD45 + cells after cryopreservation. Cell viabilities were determined by antibody co-staining with 7-aminoactinomycin D detecting necrotic cells, and subsequent flow cytometric analysis. Additionally, Annexin V staining for determination of apoptotic cells and colony-forming unit (CFU) assays for testing functional potency of HSCs were performed. For all cell types assessed (CD45 + /CD34 + cells, lymphocytes and granulocytes), the highest viabilities were obtained for CB maintained at 4°C or room temperature (RT; 22 ± 4°C) and cryopreserved directly after collection. Starting material were CB units with an age of 24.7 ± 3.5 h after birth. Post-thaw CD34 + cell results were > 90% after temperature treatment of t = 24 h (48 h total age) and > 70% after t = 48 h (72 h total age) at 4°C (48 h, 91.4 ± 5.5%; 72 h, 75.0 ± 12.0%) and RT (48 h, 84.2 ± 9.7%; 72 h, 72.6 ± 0.6%). Viabilities for 30°C samples were < 80% after t = 24 h (48 h total age, 79.8 ± 3.1%) and < 50% after t = 48 h of treatment (72 h total age, 46.8 ± 14.3%). Regarding CFU recovery of pre-freeze (without volume reduction) and thawed CB, a trend toward the highest recoveries was observed at 4°C/RT. The difference between 4°C (77.5 ± 12.0%) and 30°C samples (53.9 ± 4.8%) was shown to be significant in post-thaw samples after t = 24 h treatment (48 h total age; P = 0.0341). Delays between collection and cryopreservation should be minimized because increasing time reduces numbers of viable cells and CFUs before/after cryopreservation. CB units should be maintained at 4°C/RT to retain the highest possible potency of the cells after thawing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Effect of vitamin E and selenium nanoparticles on post-thaw variables and oxidative status of rooster semen.

PubMed

Safa, Soroush; Moghaddam, Gholamali; Jozani, Raziallah Jafari; Daghigh Kia, Hossein; Janmohammadi, Hossein

2016-11-01

This study was conducted to determine the combined effects of adding vitamin E (VitE) and selenium nanoparticle (Nano-Se) as antioxidant supplements to rooster semen extender for freezing. Semen samples were collected from 12 White Leghorn roosters and pooled. Subsequently, the samples were divided into nine equal groups using modified Beltsville extender. Extenders were supplemented with either two amounts of VitE (5 and 10μg/mL) or two amounts of Nano-Se (1% and 2%) or a combination of both VitE and Nano-Se, and no antioxidants extender (control group). Using 5μg/mL VitE and 1% of Nano-Se improved (P<0.05) total sperm motility (79.28±3.86%), progressive sperm motility (18.03±1.02%), sperm viability (81.46±2.16%) and integrity of the sperm membrane (77.21±2.12%) after the freeze-thawing process compared with control group (54.08±3.86%, 10.96±1.02%, 60.53±2.16%, and 54.47±2.12%; respectively). Also, extenders supplemented with 5μg/mL Vit E or 5μg/mL VitE and 1% Nano-Se had a lesser malondialdehyde (MDA) concentration compared to control extender (1.15±0.32, and 1.29±0.32, respectively). Total abnormal morphology of sperm was decreased (P<0.05) by adding 5 or 10μg/mL VitE alone or in combined with 1% or 2% Nano-Se. Moreover, extenders containing Nano-Se demonstrated greater glutathione peroxidase (GPx) activity compared to other extenders. Catalase (CAT) activity was greater in extender supplemented with 10μg/mL VitE and 2% Nano-Se. Moreover, higher TAC was observed in extenders supplemented with VitE and Nano-Se. It can be concluded that addition of 5μg/mL vitamin E in combined with 1% Nano-Se improved the post-thawing quality and oxidative variables of rooster semen. Copyright © 2016 Elsevier B.V. All rights reserved.

A new media without animal component for sperm cryopreservation: motility and various attributes affecting paternal contribution of sperm.

PubMed

Tiwari, Akansha; Tekcan, Merih; Sati, Leyla; Murk, William; Stronk, Jill; Huszar, Gabor

2017-05-01

Our aim was the development of a safe sperm cryopreservation New Media (NM), composed of consistent and reproducible components devoid of any animal origin, and evaluation of NM in terms of its effect on sperm structure and function as compared to regularly used yolk media (TYM) (Irvine Scientific). We evaluated patient semen samples and cryopreserved them in duplicates in either NM or TYM. The samples were cryopreserved for either a short term of 1 week or long term of 1 month prior to thawing. The parameters investigated include sperm motility via computer-assisted semen analysis (CASA), sperm concentration, and sperm biomarkers that promote paternal contribution of spermatozoa to fertilization including hyaluronic acid binding, chromatin maturity, apoptotic markers, cytoplasmic retention, and sperm DNA integrity. As compared to TYM, NM was equally capable of sperm cryopreservation with both short-term and long-term storage in media, and after freeze-thaw and gradient processing of sperm. HA binding of sperm was comparable post thaw in both NM and yolk media. There are also no differences observed between the samples cryopreserved in NM or TYM in terms of their aniline blue staining, CK immunocytochemistry, caspase 3 immunostaining, or DNA nick translation. NM has the advantage of being xeno-free, yet in preservation of sperm motility and other sperm attributes, the NM is as effective as the TYM.

Freeze/thaw stress induces organelle remodeling and membrane recycling in cryopreserved human mature oocytes.

PubMed

Nottola, Stefania Annarita; Albani, Elena; Coticchio, Giovanni; Palmerini, Maria Grazia; Lorenzo, Caterina; Scaravelli, Giulia; Borini, Andrea; Levi-Setti, Paolo Emanuele; Macchiarelli, Guido

2016-12-01

Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration. Samples were studied by light and transmission electron microscopy. We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed. This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism.

Effect of Marine-Derived Ice-Binding Proteins on the Cryopreservation of Marine Microalgae

PubMed Central

Kim, Hak Jun; Koo, Bon-Won; Kim, Doa; Seo, Ye Seul; Nam, Yoon Kwon

2017-01-01

Ice-binding protein (IBPs) protect cells from cryo-injury during cryopreservation by inhibiting ice recrystallization (IR), which is a main cause of cell death. In the present study, we employed two IBPs, one, designated LeIBP from Arctic yeast, and the other, designated FfIBP from Antarctic sea ice bacterium, in the cryopreservation of three economically valuable marine microalgae, Isochrysis galbana, Pavlova viridis, and Chlamydomonas coccoides. Both of the IBPs showed IR inhibition in f/2 medium containing 10% DMSO, indicating that they retain their function in freezing media. Microalgal cells were frozen in 10% DMSO with or without IBP. Post-thaw viability exhibited that the supplementation of IBPs increased the viability of all cryopreserved cells. LeIBP was effective in P. viridis and C. coccoides, while FfIBP was in I. galbana. The cryopreservative effect was more drastic with P. viridis when 0.05 mg/mL LeIBP was used. These results clearly demonstrate that IBPs could improve the viability of cryopreserved microalgal cells. PMID:29194380

Freezing Injury in Onion Bulb Cells

PubMed Central

Palta, Jiwan P.; Levitt, Jacob; Stadelmann, Eduard J.

1977-01-01

Onion (Allium cepa L.) bulbs were subjected for 12 days to either a moderate freeze (−4 C) or a severe freeze (−11 C). They were then thawed slowly over ice. During 7 to 12 days following the thaw, the injury progressed with time in the severely frozen bulbs, but appeared completely repaired in the moderately frozen bulbs. This was shown by the following post-thawing changes. Infiltration of the intercellular spaces increased from 80 to 90% to 100% after the severe freeze, and decreased from 30 to 50% to zero after the moderate freeze. All of the cells were alive immediately after thawing whether the freeze was moderate or severe. Corresponding to the infiltration results 7 to 12 days later, many to most were dead following the severe freeze, all were alive following the moderate freeze. The conductivity of the effusate from pieces of bulb tissue increased after the severe freezing, and decreased after the moderate freezing. The concentration of K+, total solutes, and sugars in the effusate paralleled the conductivity changes. Neither the pH of the effusate nor the permeability of the cells (as long as cells were living) to water was changed following either the severe or the moderate freezes. Some treatments of the thawed tissue following the severe freeze halted the progress of injury. The above results indicate that the semipermeable properties of the cell are uninjured but that the ion and sugar transport mechanism is damaged by freezing. Most likely the primary injury is to the active transport mechanism involved in their transport. It must be concluded that the final injury following freezing and thawing cannot be evaluated from the degree of infiltration or the conductivity of the effusate immediately after thawing, since injury may progress or recede following the thawing. PMID:16660101

The quality of great scallop (Pecten maximus) sperm after thawing.

PubMed

Suquet, Marc; Gourtay, Clémence; Donval, Anne; Le Goïc, Nelly; Quere, Claudie; Malo, Florent; Le Grand, Jaqueline; Ratiskol, Dominique; Mingant, Christian; Fauvel, Christian

2016-04-01

Most publications devoted to the cryopreservation of mollusc sperm have focused on the definition of technical protocols, avoiding the description of sperm quality after thawing. The present study investigated the effects of cryopreservation on sperm quality in the great scallop. Wild scallop were fished during the natural spawning period and conditioned in the hatchery before use. Sperm samples were obtained after intragonadal injection of serotonin and cryopreserved using a previously published protocol. Sperm quality was assessed using a panel of four parameters: sperm motility characteristics, using a computer assisted sperm analysis plugin with Image J, intracellular ATP content using an ATP-Lite kit, sperm integrity, using flow cytometry and sperm morphology, using transmission electron microscopy. For each parameter, fresh (control) and thawed spermatozoa were compared. A significant decrease of both the percentage of motile spermatozoa (reduction: 75%) and sperm swimming speed (86%) were observed for thawed sperm compared with fresh sperm. The percentage of living spermatozoa, as assessed using flow cytometry, was significantly lower for thawed sperm (72.4±2.5%) compared with fresh sperm (86.4±1.1). However, no significant difference of intracellular sperm ATP content was observed between fresh and thawed sperm. Post thawing, while some spermatozoa showed little or no morphological differences compared with fresh sperm, others had undergone drastic changes, including swelling of the plasma membrane, structural alterations of the chromatin and damage to mitochondria. In conclusion, the descriptive parameters studied in the present work showed that the quality of thawed great scallop sperm was lower than that of fresh cells but was still sufficient for use in aquaculture programs and sperm cryobanking for this species. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows.

PubMed

Noguchi, Michiko; Yoshioka, Koji; Hikono, Hirokazu; Suzuki, Chie; Kikuchi, Kazuhiro

2015-12-01

We compared the effects of extenders of frozen-thawed semen on post-thaw sperm characteristics and the distribution of frozen-thawed spermatozoa in the female genital tract after fixed-timed deep intrauterine insemination (DIUI) in sows. Frozen semen samples were thawed and diluted in either modified Modena solution (mMS) or porcine fertilization medium (PFM) containing theophylline, adenosine and cysteine. Sperm quality, assessed in vitro based on motility using a computer-assisted sperm analyzer and the integrity of the plasma and acrosomal membranes using flow cytometry, was evaluated at 0.5, 1.5, 3 and 6h after thawing. Progressive motility and the percentage of spermatozoa with damaged acrosomal membranes in PFM were significantly better than in mMS throughout the 6h. Sows with estrus synchronized using prostaglandin F2 alpha, equine chorionic gonadotropin and human chorionic gonadotropin (hCG) were inseminated once with mMS- or PFM-diluted 5 × 10(8) frozen-thawed spermatozoa by DIUI at 34 h after the hCG injection. At 4h after DIUI, reproductive tracts were recovered from 30 sows. There were significantly fewer polymorphonuclear leukocytes (PMNs) and more spermatozoa outside PMNs in the uterine horn after PFM treatment than with mMS. When 22 sows were administered DIUI with 10 × 10(8) frozen-thawed spermatozoa at 36 h after hCG, the pregnancy rates did not differ significantly between the mMS- (36%) and PFM- (64%) treated groups. Thus, PFM enhanced progressive sperm motility but increased sperm membrane damage compared with mMS; it also suppressed the migration of PMNs into the uterine lumen. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of different antioxidant additives in semen diluent on cryopreservability (-196°C) of buffalo semen.

PubMed

Patel, Hardik A; Siddiquee, G M; Chaudhari, Dinesh V; Suthar, Vishal S

2016-03-01

The aim of this study was to evaluate the effect of different antioxidant additives in standard tris-fructose-egg yolk-glycerol (TFYG) extender on the cryopreservability of buffalo semen. Semen collection using artificial vagina, twice weekly for 5 weeks from three pedigreed health breeding bulls of Mehsani breed, aged between 6 and 8 years. Immediately after initial evaluation all 30 qualifying ejaculates (10/bull) were split into three aliquots and diluted at 34°C keeping the concentration of 100 million spermatozoa/ml with standard TFYG extender as control and TFYG having two antioxidant additives - Cysteine HCl at 1 mg/ml and ascorbic acid at 0.2 mg/ml to study their comparative performance. Semen filled in French Mini straws using IS-4 system and gradually cooled to 4°C and equilibrated for 4 h in cold handing cabinet. After completion of equilibration, straws were cryopreserved in LN2 by Programmable Bio-freezer. Semen was examined at post-dilution, post-equilibration, and post-thaw stages for sperm quality parameters, and at each stage plasma was separated for enzymatic analysis of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP). The mean percentage of sperms in TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage in terms of progressive motility (52.83±0.52, 57.83±0.52, 57.83±0.52), livability (78.70±0.21, 82.33±0.23, 81.73±0.22), and abnormality (5.43±0.21, 5.03±0.17, 5.23±0.18) varied significantly (p<0.05) between control TFYG and TFYG having antioxidant additives. The mean U/L activities of AST (78.70±0.47, 72.80±0.48, 73.30±0.54), LDH (172.70±0.41, 155.78±0.42, 156.33±0.41), and AKP (103.61±0.34, 90.20±0.34, 91.03±0.34) in semen diluted with TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage, respectively, which showed significantly (p<0.05) higher leakage of enzymes in control TFYG than TFYG incorporated with additives. Incorporation of antioxidant additives such as cysteine HCl and ascorbic acid in standard TFYG diluents improves sperm quality parameters, reduces enzyme leakage, and ultimately advances cryopreservability of buffalo semen.

An approach to successful freezing of demi-embryos derived from day-7 bovine embryos.

PubMed

Niemann, H; Brem, G; Sacher, B; Smidt, D; Kräusslich, H

1986-04-01

The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.

Migration of Water in Litopenaeus Vannamei Muscle Following Freezing and Thawing.

PubMed

Deng, Qi; Wang, Yaling; Sun, Lijun; Li, Jianrong; Fang, Zhijia; Gooneratne, Ravi

2018-06-15

Water and protein are major constituents of shrimp, any changes in protein and the state of water influence the quality of shrimp. Therefore, a study to examine the law of moisture migration and protein denaturation under different freezing and thawing conditions is important. The proton density images of thawed frozen-shrimp revealed that the water loss during quick-freezing was much greater than that during slow freezing or microfreezing. At room temperature (25 °C), the water loss from brine-thawing was more than still-water thawing and still-water thawing was more than thawing spontaneously. Freezing-thawing resulted in uniform water redistribution in shrimp muscle. Nuclear magnetic resonance technology (low field magnetic imaging) was used to directly monitor the dynamic processes of fluidity state in shrimp and indirectly monitor protein denaturation and thereby determine the optimal method of freezing-thawing shrimp. Our research showed that microfreezing preservation minimized weight loss, juice leakage and protein denaturation in shrimp muscle during thawing. Water is one of the major components in most organs and is an important factor that influences the shrimp muscle quality. Water migration patterns and subsequent effects on the shrimp muscle under different freezing and thawing conditions were examined using low field nuclear magnetic resonance (NMR) technology. This research provides a theoretical foundation for shrimp processing plants to improve the freezing and thawing process to obtain optimal quality and flavor of shrimp products. © 2018 Institute of Food Technologists®.

Incipient balancing selection through adaptive loss of aquaporins in natural Saccharomyces cerevisiae populations.

PubMed

Will, Jessica L; Kim, Hyun Seok; Clarke, Jessica; Painter, John C; Fay, Justin C; Gasch, Audrey P

2010-04-01

A major goal in evolutionary biology is to understand how adaptive evolution has influenced natural variation, but identifying loci subject to positive selection has been a challenge. Here we present the adaptive loss of a pair of paralogous genes in specific Saccharomyces cerevisiae subpopulations. We mapped natural variation in freeze-thaw tolerance to two water transporters, AQY1 and AQY2, previously implicated in freeze-thaw survival. However, whereas freeze-thaw-tolerant strains harbor functional aquaporin genes, the set of sensitive strains lost aquaporin function at least 6 independent times. Several genomic signatures at AQY1 and/or AQY2 reveal low variation surrounding these loci within strains of the same haplotype, but high variation between strain groups. This is consistent with recent adaptive loss of aquaporins in subgroups of strains, leading to incipient balancing selection. We show that, although aquaporins are critical for surviving freeze-thaw stress, loss of both genes provides a major fitness advantage on high-sugar substrates common to many strains' natural niche. Strikingly, strains with non-functional alleles have also lost the ancestral requirement for aquaporins during spore formation. Thus, the antagonistic effect of aquaporin function-providing an advantage in freeze-thaw tolerance but a fitness defect for growth in high-sugar environments-contributes to the maintenance of both functional and nonfunctional alleles in S. cerevisiae. This work also shows that gene loss through multiple missense and nonsense mutations, hallmarks of pseudogenization presumed to emerge after loss of constraint, can arise through positive selection.

Optimization of ethylene glycol concentrations, freezing rates and holding times in liquid nitrogen vapor for cryopreservation of rhesus macaque (Macaca mulatta) sperm.

PubMed

Yang, Shihua; Ping, Shuhuang; Si, Wei; He, Xiechao; Wang, Xinyi; Lu, Yongqing; Ji, Shaohui; Niu, Yuyu; Ji, Weizhi

2011-06-01

Ethylene glycol (EG) has been speculated to be the most appropriate penetrating cryoprotectant for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient. The present study aimed to determine the optimal EG concentration, freezing rate and holding time in liquid nitrogen (LN(2)) vapor for rhesus sperm cryopreservation. Among six tested EG concentrations (0, 0.18, 0.35, 0.7, 1.4 and 2.1 M), 0.7 M EG showed the most effective cryoprotection (P<0.05). Sperm frozen with 0.7 M EG at -183°C/min showed higher post-thaw motility than sperm frozen at -10, -67 or -435°C/min (P<0.05). Sperm frozen in LN(2) vapor at -183°C/min with 0.7 M EG and a holding time of 10 min showed higher post-thaw motility compared with a holding time of 5 or 15 min (P<0.05). The function of sperm cryopreserved at the optimized EG concentration, freezing rate and holding time was further evaluated by in vitro fertilization. Of the 36 oocytes collected from gonadotropin-stimulated rhesus macaques, 61.1% were fertilized, and 61.1, 44.4 and 36.1% of the oocytes developed to 2 cells, morulae and blastocysts, respectively. Our findings provide an alternative penetrating cryoprotectant and optimal protocol for genetic preservation purposes in this important species.

Plasma IGF-I, INSL3, testosterone, inhibin concentrations and scrotal circumferences surrounding puberty in Japanese Black beef bulls with normal and abnormal semen.

PubMed

Weerakoon, W W P N; Sakase, M; Kawate, N; Hannan, M A; Kohama, N; Tamada, H

2018-07-01

The relationships between semen abnormalities and peripheral concentrations of testicular and metabolic hormones in beef bulls are unclear. Here we compared plasma insulin-like growth factor I (IGF-I), insulin-like peptide 3 (INSL3), testosterone, inhibin concentrations, and scrotal circumferences surrounding puberty in Japanese Black beef bulls (n = 66) with normal or abnormal semen. We collected blood samples and measured scrotal circumferences monthly from 4 to 24 months of age. Semen was collected weekly from 12 months until at least 18 months of age. Fresh semen was evaluated for semen volume, sperm motility, concentrations, and morphological defects. The normal fresh semen was frozen by a standard method and examined for post-thaw sperm motility and fertility. Bulls were classified as having either normal post-thaw semen (n = 45) or abnormal semen (n = 21, when at least one of the above test items was abnormal for 6 months). Abnormal semen was classified into abnormal fresh or low-fertility post-thaw which evaluated for rates of transferable embryos. The abnormal fresh was categorized as having sperm morphological defects, low motility, and morphological defects plus low motility. Scrotal circumferences were smaller for the abnormal-semen group vs. the normal-semen group at 20 and 24 months (p < 0.05). Plasma IGF-I, INSL3, and inhibin concentrations in the abnormal-semen group were lower than those of the normal-semen group (p < 0.05) surrounding puberty (4-6, 8, 18-22, and 24 months for IGF-I; 6, 9, 11-14, 17, and 20-21 months for INSL3; 5, 8-13, 16, 17, 19, and 20 months for inhibin). The plasma testosterone concentrations were lower in the abnormal-semen bulls vs. normal-semen bulls only at 22 months (p < 0.05). Analyses of the classified abnormal semen showed lower plasma INSL3 concentrations for morphological defects plus low motility in fresh semen (p < 0.05) and lower IGF-I and inhibin concentrations for low-fertility post-thaw semen (p < 0.05) compared to the normal semen. Our results suggest that reduced secretions of IGF-I, INSL3, and inhibin surrounding puberty may be associated with semen aberration in beef bulls. Notably, the combined sperm abnormality of morphological defects and low motility in fresh semen could involve lowered INSL3, whereas the low-fertility post-thaw semen might be related to decreases of IGF-I and/or inhibin. Pre-puberty blood IGF-I, INSL3 and inhibin concentrations could be used as indicators to predict aberrant semen in beef bulls. Copyright © 2018 Elsevier Inc. All rights reserved.

Postharvest Ultrasound-Assisted Freeze-Thaw Pretreatment Improves the Drying Efficiency, Physicochemical Properties, and Macamide Biosynthesis of Maca (Lepidium meyenii).

PubMed

Chen, Jin-Jin; Gong, Peng-Fei; Liu, Yi-Lan; Liu, Bo-Yan; Eggert, Dawn; Guo, Yuan-Heng; Zhao, Ming-Xia; Zhao, Qing-Sheng; Zhao, Bing

2018-04-01

A novel technique of ultrasound-assisted freeze-thaw pretreatment (UFP) was developed to improve the drying efficiency of maca and bioactive amide synthesis in maca. The optimal UFP conditions are ultrasonic processing 90 min at 30 °C with 6 freeze-thaw cycles. Samples with freeze-thaw pretreatment (FP), ultrasound pretreatment (UP), and UFP were prepared for further comparative study. A no pretreatment (NP) sample was included as a control. The results showed that UFP improved the drying efficiency of maca slices, showing the highest effective moisture diffusivity (1.75 × 10 -9 m 2 /s). This result was further supported by low-field nuclear magnetic resonance (LF-NMR) analysis and scanning electron microscopy (SEM). The rehydration capacity and protein content of maca slices were improved by UFP. More importantly, contents of bioactive macamides and their biosynthetic precursors were increased in 2.5- and 10-fold, respectively. In conclusion, UFP is an efficient technique to improve drying efficiency, physicochemical properties, and bioactive macamides of maca, which can be applied in the industrial manufacture of maca products. © 2018 Institute of Food Technologists®.

Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: comparison between cysteine and rosemary (Rosmarinus officinalis).

PubMed

Malo, C; Gil, L; Gonzalez, N; Martínez, F; Cano, R; de Blas, I; Espinosa, E

2010-08-01

Anti-oxidants partially ameliorated the detrimental effects of reactive oxidative substances produced during cryopreservation. The objective of the study was to determine the effect of anti-oxidant addition to the freezing extender on boar semen qualities and fertility capacity. Ejaculates were collected from a previously selected boar and semen samples were processed using the straw freezing procedure. In experiment 1, semen samples were cryopreserved in lactose-egg yolk solution supplemented with various concentrations of cysteine (0, 5 and 10mM) to determinate a cysteine concentration capable of producing a protective effect during cryopreservation. Semen quality (total motility, progressive motility, viability, acrosome integrity and hypoosmotic swelling test) was evaluated after freezing and thawing and then every hour for 3h. In experiment 2, ejaculates were cryopreserved with lactose-egg yolk extender with or without the following anti-oxidants: cysteine, rosemary (Rosmarinus officinalis) and cysteine plus rosemary. Semen quality was evaluated. In the experiment 3, fertility capacity of semen frozen in anti-oxidant supplementation extenders was examined in vitro. A total of 2232 oocytes were in vitro matured and inseminated with frozen-thawed sperm. In summary: (i) the effective concentration of cysteine in freezing extender was 10mM; (ii) the addition of exogenous rosemary or cysteine to the freezing extender positively affected post-thawed viability and acrosome integrity. Only rosemary supplementation improved total motility at 3h and progressive motility at any time; (iii) the inclusion of rosemary into the extender was effective in penetration and cleavage rate and also in the efficiency of the fertilization system. (c) 2010 Elsevier Inc. All rights reserved.

Effect of Repeated Freeze-Thaw Cycles on Beef Quality and Safety

PubMed Central

Rahman, Mohammad Hafizur; Hossain, Mohammad Mujaffar; Rahman, Syed Mohammad Ehsanur; Hashem, Mohammad Abul

2014-01-01

The objectives of this study were to know the effect of repeated freeze-thaw cycles of beef on the sensory, physicochemical quality and microbiological assessment. The effects of three successive freeze-thaw cycles on beef forelimb were investigated comparing with unfrozen fresh beef for 75 d by keeping at −20±1℃. The freeze-thaw cycles were subjected to three thawing methods and carried out to know the best one. As the number of freeze-thaw cycles increased color and odor declined significantly before cook within the cycles and tenderness, overall acceptability also declined among the cycles after cook by thawing methods. The thawing loss increased and dripping loss decreased significantly (p<0.05). Water holding capacity (WHC) increased (p<0.05) until two cycles and then decreased. Cooking loss increased in cycle 1 and 3, but decreased in cycle 2. pH decreased significantly (p<0.05) among the cycles. Moreover, drip loss, cooking loss and WHC were affected (p<0.05) by thawing methods within the cycles. 2-Thiobarbituric acid (TBARS) value increased (p<0.05) gradually within the cycles and among the cycles by thawing methods. Total viable bacteria, total coliform and total yeast-mould count decreased significantly (p<0.05) within and among the cycles in comparison to the initial count in repeated freeze-thaw cycles. As a result, repeated freeze-thaw cycles affected the sensory, physicochemical and microbiological qua- lity of beef, causing the deterioration of beef quality, but improved the microbiological quality. Although repeated freeze-thaw cycles did not affect much on beef quality and safety but it may be concluded that repeated freeze and thaw should be minimized in terms of beef color for commercial value and WHC and tenderness/juiciness for eating quality. PMID:26761286

A Loss in the Plasma Membrane ATPase Activity and Its Recovery Coincides with Incipient Freeze-Thaw Injury and Postthaw Recovery in Onion Bulb Scale Tissue 1

PubMed Central

Arora, Rajeev; Palta, Jiwan P.

1991-01-01

Plasma membrane ATPase has been proposed to be functionally altered during early stages of injury caused by a freeze-thaw stress. Complete recovery from freezing injury in onion cells during the postthaw period provided evidence in support of this proposal. During recovery, a simultaneous decrease in ion leakage and disappearance of water soaking (symptoms of freeze-thaw injury) has been noted. Since reabsorption of ions during recovery must be an active process, recovery of plasma membrane ATPase (active transport system) functions has been implicated. In the present study, onion (Allium cepa L. cv Downing Yellow Globe) bulbs were subjected to a freeze-thaw stress which resulted in a reversible (recoverable) injury. Plasma membrane ATPase activity in the microsomes (isolated from the bulb scales) and ion leakage rate (efflux/hour) from the same scale tissue were measured immediately following thawing and after complete recovery. In injured tissue (30-40% water soaking), plasma membrane ATPase activity was reduced by about 30% and this was paralleled by about 25% higher ion leakage rate. As water soaking disappeared during recovery, the plasma membrane ATPase activity and the ion leakage rate returned to about the same level as the respective controls. Treatment of freeze-thaw injured tissue with vanadate, a specific inhibitor of plasma membrane ATPase, during postthaw prevented the recovery process. These results indicate that recovery of freeze-injured tissue depends on the functional activity of plasma membrane ATPase. PMID:16668063

Sperm sex-sorting and preservation for managing the sex ratio and genetic diversity of the southern white rhinoceros (Ceratotherium simum simum).

PubMed

O'Brien, J K; Roth, T L; Stoops, M A; Ball, R L; Steinman, K J; Montano, G A; Love, C C; Robeck, T R

2015-01-01

White rhinoceros ejaculates (n=9) collected by electroejaculation from four males were shipped (10°C, 12h) to develop procedures for the production of chilled and frozen-thawed sex-sorted spermatozoa of adequate quality for artificial insemination (AI). Of all electroejaculate fractions, 39.7% (31/78) exhibited high quality post-collection (≥70% total motility and membrane integrity) and of those, 54.8% (17/31) presented reduced in vitro quality after transport and were retrospectively determined to exhibit urine-contamination (≥21.0μg creatinine/ml). Of fractions analyzed for creatinine concentration, 69% (44/64) were classified as urine-contaminated. For high quality non-contaminated fractions, in vitro parameters (motility, velocity, membrane, acrosome and DNA integrity) of chilled non-sorted and sorted spermatozoa were well-maintained at 5°C up to 54h post-collection, whereby >70% of post-transport (non-sorted) or post-sort (sorted) values were retained. By 54h post-collection, some motility parameters were higher (P<0.05) for non-sorted spermatozoa (total motility, rapid velocity, average path velocity) whereas all remaining motion parameters as well as membrane, acrosome and DNA integrity were similar between sperm types. In comparison with a straw method, directional freezing resulted in enhanced (P<0.05) motility and velocity of non-sorted and sorted spermatozoa, with comparable overall post-thaw quality between sperm types. High purity enrichment of X-bearing (89±6%) or Y-bearing (86±3%) spermatozoa was achieved using moderate sorting rates (2540±498X-spermatozoa/s; 1800±557Y-spermatozoa/s). Collective in vitro characteristics of sorted-chilled or sorted-frozen-thawed spermatozoa derived from high quality electroejaculates indicate acceptable fertility potential for use in AI. Copyright © 2014 Elsevier B.V. All rights reserved.

Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen-thawed semen without affecting seminal kinetics.

PubMed

Guimarães, T; Lopes, G; Pinto, M; Silva, E; Miranda, C; Correia, M J; Damásio, L; Thompson, G; Rocha, A

2015-01-15

Freezability of equine semen may be influenced by microorganism population of semen. The objective of this study was to verify the effect of single-layer density gradient centrifugation (SLC) of fresh semen before cryopreservation on semen's microbial load (ML) and sperm cells kinetics after freezing-thawing. For that, one ejaculate was collected from 20 healthy stallions and split into control (C) samples (cryopreserved without previous SLC) and SLC samples (subjected to SLC). Semen cryopreservation was performed according to the same protocol in both groups. Microbial load of each microorganism species and total microbial load (TML) expressed in colony-forming units (CFU/mL) as well as frozen-thawed sperm kinetics were assessed in both groups. Additional analysis of the TML was performed, subdividing the frozen-thawed samples in "suitable" (total motility ≥ 30%) and "unsuitable" (total motility < 30%) semen for freezing programs, and comparing the C and SLC groups within these subpopulations. After thawing, SLC samples had less (P < 0.05) TML (88.65 × 10(2) ± 83.8 × 10(2) CFU/mL) than C samples (155.69 × 10(2) ± 48.85 × 10(2) CFU/mL), mainly due to a reduction of Enterococcus spp. and Bacillus spp. A relationship between post-thaw motility and SLC effect on ML was noted, as only in samples with more than 30% total motility was ML reduced (P < 0.05) by SLC (from 51.33 × 10(2) ± 33.26 × 10(2) CFU/mL to 26.68 × 10(2) ± 12.39 × 10(2) CFU/mL in "suitable" frozen-thawed semen vs. 240.90 × 10(2) ± 498.20 × 10(2) to 139.30 × 10(2) ± 290.30 × 10(2) CFU/mL in "unsuitable" frozen-thawed semen). The effect of SLC on kinetics of frozen-thawed sperm cells was negligible. Copyright © 2015 Elsevier Inc. All rights reserved.

The effect of cryopreservation on goat semen characteristics related to sperm freezability.

PubMed

Dorado, J; Muñoz-Serrano, A; Hidalgo, M

2010-08-01

Seminal quality parameters were used to evaluate the effect of freeze-thawing procedure on goat sperm characteristics, and to relate possible changes in sperm parameters to cryopreservation success. Semen samples (n=110) were frozen with TRIS and milk-based extenders and thawed. Sperm quality parameters (motility, morphology and acrosome) were compared between fresh and frozen-thawed samples. Sperm freezability was judged by classifying the semen samples as "suitable" or "not suitable" according to the sperm quality parameters assessed before and after thawing. Fertility data was obtained after cervical insemination with frozen semen doses. The ejaculates were grouped into two categories according to their fertility results. In experiment 1, significant differences were found between semen extenders (P<0.001), bucks (P<0.05) and ejaculates within the same male (P<0.05) in terms of sperm quality. There was no seasonal effect (P>0.05) on the majority of the sperm parameters assessed after thawing. Moreover, significant differences (P<0.001) in semen parameters assessed in fresh semen and frozen-thawed samples were found between groups. The effect of the freeze-thawing procedure on sperm quality parameters was also different (P<0.05) between extenders within the same group. The number of sperm quality parameters that had changed after cryopreservation was lower in "suitable" semen samples before and after thawing. In experiment 2, no differences (P>0.05) in semen parameters assessed in fresh semen and frozen-thawed samples were found between groups. The effect of freezing and thawing on sperm quality parameters were different (P<0.05) between extenders within the same group. Only mean beat cross frequency (BCF) values were significantly higher (P<0.05) in TRIS diluted samples that led to successful pregnancies after artificial insemination. In conclusion, CASA-derived motility parameters, together with traditional semen assessment methods, give valuable information on sperm quality before and after freezing. Therefore, the identification of ejaculates as "good" or "bad" based on fresh and post-thaw semen parameters studied in the present experiment were good indicators of goat semen freezability, although the fertilizing capacity of frozen-thawed goat spermatozoa are not revealed by this quality study. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Spermatozoa in the sperm-peak-fraction of the boar ejaculate show a lower flow of Ca(2+) under capacitation conditions post-thaw which might account for their higher membrane stability after cryopreservation.

PubMed

Hossain, Md Sharoare; Johannisson, Anders; Siqueira, Amanda Pimenta; Wallgren, Margareta; Rodriguez-Martinez, Heriberto

2011-10-01

Boar spermatozoa collected in the ejaculate sperm peak-portion (P1, first 10 mL of the sperm-rich fraction, SRF), had shown a higher resilience to freezing and thawing compared to spermatozoa from the rest of the ejaculate (2nd portion of the SRF plus the post-sperm-rich fraction, PSRF), even when using a simplified freezing technique, as long as spermatozoa were incubated in their own seminal plasma (SP). This experiment studied the stability of P1- and SRF-P1 boar spermatozoa frozen in MiniFlatPacks (MFP), post-thaw, using flow cytometry. Since spermatozoa from either portion showed similar cryosurvival and low proportions of unstable membranes (<3%, annexin-V/propidium iodide staining), and only a tendency for SRF-P1 live spermatozoa to depict acrosome exocytosis (FITC-PNA/PI/H33342); they were explored for Ca(2+) contents using a Fluo-4 probe under in vitro capacitating conditions (mBO+ medium), as well they were tested for their ability to sustain a short Ca(2+)-ionophore (A23187) in vitro challenge. The proportions of live spermatozoa depicting high Ca(2+)-levels were initially <2% but increased over incubation time, particularly in SRF-P1(P<0.05), while proportions of live spermatozoa with low Ca(2+)-levels were basically constant over incubation time (~11-14%), for either portion. Incubation in capacitation medium did not modify the proportions of low-Ca(2+) but dramatically increased the proportions of high-Ca(2+) spermatozoa (P<0.001) already after 15 min exposure, highest for SRF-P1 spermatozoa. While the proportion of live spermatozoa with intact acrosome was significantly decreased among SRF-P1 (P<0.001), that of P1-spermatozoa remained unchanged, probably owing to the lowest relative content of cytosolic Ca(2+). The results suggest that spermatozoa in the P1-portion are more resilient to express acrosome exocytosis post-thaw compared to those bathing in the rest of the SRF-fraction when cryopreserved using a simplified technique, in MFPs. Copyright © 2011 Elsevier B.V. All rights reserved.

Nonlinear CO 2 flux response to 7 years of experimentally induced permafrost thaw

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mauritz, Marguerite; Bracho, Rosvel; Celis, Gerardo

Rapid Arctic warming is expected to increase global greenhouse gas concentrations as permafrost thaw exposes immense stores of frozen carbon (C) to microbial decomposition. Permafrost thaw also stimulates plant growth, which could offset C loss. Using data from 7 years of experimental Air and Soil warming in moist acidic tundra, we show that Soil warming had a much stronger effect on CO 2 flux than Air warming. Soil warming caused rapid permafrost thaw and increased ecosystem respiration (R eco), gross primary productivity (GPP), and net summer CO 2 storage (NEE). Over 7 years R eco, GPP, and NEE also increasedmore » in Control (i.e., ambient plots), but this change could be explained by slow thaw in Control areas. In the initial stages of thaw, R eco, GPP, and NEE increased linearly with thaw across all treatments, despite different rates of thaw. As thaw in Soil warming continued to increase linearly, ground surface subsidence created saturated microsites and suppressed R eco, GPP, and NEE. However R eco and GPP remained high in areas with large Eriophorum vaginatum biomass. In general NEE increased with thaw, but was more strongly correlated with plant biomass than thaw, indicating that higher R eco in deeply thawed areas during summer months was balanced by GPP. Summer CO 2 flux across treatments fit a single quadratic relationship that captured the functional response of CO 2 flux to thaw, water table depth, and plant biomass. These results demonstrate the importance of indirect thaw effects on CO 2 flux: plant growth and water table dynamics. Nonsummer R eco models estimated that the area was an annual CO 2 source during all years of observation. As a result, nonsummer CO 2 loss in warmer, more deeply thawed soils exceeded the increases in summer GPP, and thawed tundra was a net annual CO 2 source.« less

Nonlinear CO 2 flux response to 7 years of experimentally induced permafrost thaw

DOE PAGES

Mauritz, Marguerite; Bracho, Rosvel; Celis, Gerardo; ...

2017-02-16

Rapid Arctic warming is expected to increase global greenhouse gas concentrations as permafrost thaw exposes immense stores of frozen carbon (C) to microbial decomposition. Permafrost thaw also stimulates plant growth, which could offset C loss. Using data from 7 years of experimental Air and Soil warming in moist acidic tundra, we show that Soil warming had a much stronger effect on CO 2 flux than Air warming. Soil warming caused rapid permafrost thaw and increased ecosystem respiration (R eco), gross primary productivity (GPP), and net summer CO 2 storage (NEE). Over 7 years R eco, GPP, and NEE also increasedmore » in Control (i.e., ambient plots), but this change could be explained by slow thaw in Control areas. In the initial stages of thaw, R eco, GPP, and NEE increased linearly with thaw across all treatments, despite different rates of thaw. As thaw in Soil warming continued to increase linearly, ground surface subsidence created saturated microsites and suppressed R eco, GPP, and NEE. However R eco and GPP remained high in areas with large Eriophorum vaginatum biomass. In general NEE increased with thaw, but was more strongly correlated with plant biomass than thaw, indicating that higher R eco in deeply thawed areas during summer months was balanced by GPP. Summer CO 2 flux across treatments fit a single quadratic relationship that captured the functional response of CO 2 flux to thaw, water table depth, and plant biomass. These results demonstrate the importance of indirect thaw effects on CO 2 flux: plant growth and water table dynamics. Nonsummer R eco models estimated that the area was an annual CO 2 source during all years of observation. As a result, nonsummer CO 2 loss in warmer, more deeply thawed soils exceeded the increases in summer GPP, and thawed tundra was a net annual CO 2 source.« less

Nonlinear CO2 flux response to 7 years of experimentally induced permafrost thaw.

PubMed

Mauritz, Marguerite; Bracho, Rosvel; Celis, Gerardo; Hutchings, Jack; Natali, Susan M; Pegoraro, Elaine; Salmon, Verity G; Schädel, Christina; Webb, Elizabeth E; Schuur, Edward A G

2017-09-01

Rapid Arctic warming is expected to increase global greenhouse gas concentrations as permafrost thaw exposes immense stores of frozen carbon (C) to microbial decomposition. Permafrost thaw also stimulates plant growth, which could offset C loss. Using data from 7 years of experimental Air and Soil warming in moist acidic tundra, we show that Soil warming had a much stronger effect on CO 2 flux than Air warming. Soil warming caused rapid permafrost thaw and increased ecosystem respiration (R eco ), gross primary productivity (GPP), and net summer CO 2 storage (NEE). Over 7 years R eco , GPP, and NEE also increased in Control (i.e., ambient plots), but this change could be explained by slow thaw in Control areas. In the initial stages of thaw, R eco , GPP, and NEE increased linearly with thaw across all treatments, despite different rates of thaw. As thaw in Soil warming continued to increase linearly, ground surface subsidence created saturated microsites and suppressed R eco , GPP, and NEE. However R eco and GPP remained high in areas with large Eriophorum vaginatum biomass. In general NEE increased with thaw, but was more strongly correlated with plant biomass than thaw, indicating that higher R eco in deeply thawed areas during summer months was balanced by GPP. Summer CO 2 flux across treatments fit a single quadratic relationship that captured the functional response of CO 2 flux to thaw, water table depth, and plant biomass. These results demonstrate the importance of indirect thaw effects on CO 2 flux: plant growth and water table dynamics. Nonsummer R eco models estimated that the area was an annual CO 2 source during all years of observation. Nonsummer CO 2 loss in warmer, more deeply thawed soils exceeded the increases in summer GPP, and thawed tundra was a net annual CO 2 source. © 2017 John Wiley & Sons Ltd.

The improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability.

PubMed

Yeste, Marc; Estrada, Efrén; Pinart, Elisabeth; Bonet, Sergi; Miró, Jordi; Rodríguez-Gil, Joan E

2014-04-01

Reduced glutathione (GSH) improves boar sperm cryosurvival and fertilising ability when added to freezing extenders. Poor freezability ejaculates (PFE) are known to present lower resistance than good freezability ejaculates (GFE) to cryopreservation procedures. So far, no study has evaluated whether the ability of GSH to counteract the cryopreservation-induced injuries depends on ejaculate freezability (i.e. GFE vs. PFE). For this reason, thirty boar ejaculates were divided into three equal volume fractions and cryopreserved with or without GSH at a final concentration of either 2 or 5mM in freezing media. Before and after freeze-thawing, sperm quality was evaluated through analysis of viability, motility, integrity of outer acrosome membrane, ROS levels, integrity of nucleoprotein structure, and DNA fragmentation. Ejaculates were classified into two groups (GFE or PFE) according to their post-thaw sperm motility and viability assessments in negative control (GSH 0mM), after running cluster analyses. Values of each sperm parameter were then compared between treatments (GSH 0mM, GSH 2mM, GSH 5mM) and freezability groups (GFE, PFE). In the case of GFE, GSH significantly improved boar sperm cryotolerance, without differences between 2 and 5mM. In contrast, PFE freezability was significantly increased when supplemented with 5mM GSH, but not when supplemented with 2mM GSH. In conclusion, PFE need a higher concentration of GSH than GFE to improve their cryotolerance. Copyright © 2014 Elsevier Inc. All rights reserved.

Freeze-thaw method improves the detection of volatile compounds in insects using Headspace Solid-Phase Microextraction (HS-SPME)

USDA-ARS?s Scientific Manuscript database

Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC-MS) is commonly used in analyzing insect volatiles. In order to improve the detection of volatiles in insects, a freeze-thaw method was applied to insect samples before the HS-SPME-GC-MS analysis. ...

Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages

PubMed Central

Fan, Xiaoteng; Pan, Yang; Lv, Shan; Pan, Chuanying; Lei, Anmin

2017-01-01

The process of cryopreservation results in over-production of reactive oxygen species, which is extremely detrimental to spermatozoa. The aim of this study was to investigate whether addition of cysteine to freezing extender would facilitate the cryosurvival of rabbit spermatozoa, and if so, how cysteine protects spermatozoa from cryodamages. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of cysteine. The motility, intact acrosomes, membrane integrity, mitochondrial potentials, 8-hydroxyguanosine level and sperm-zona pellucida binding capacity were examined. Furthermore, glutathione peroxidase (GPx) activity, glutathione content (GSH), and level of reactive oxygen species (ROS) and hydrogen peroxide of spermatozoa were analyzed. The values of motility, intact acrosomes, membrane integrity, mitochondrial potentials and sperm-zona pellucida binding capacity of the frozen-thawed spermatozoa in the treatment of cysteine were significantly higher than those of the control. Addition of cysteine to extenders improved the GPx activity and GSH content of spermatozoa, while lowered the ROS, DNA oxidative alterations and lipid peroxidation level, which makes spermatozoa avoid ROS to attack DNA, the plasma membrane and mitochondria. In conclusion, cysteine protects spermatozoa against ROS-induced damages during cryopreservation and post-thaw incubation. Addition of cysteine is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry. PMID:28700739

Effect of extruded wheat flour and pre-gelatinized cassava starch on process and quality parameters of French-type bread elaborated from frozen dough.

PubMed

Ortolan, Fernanda; Brites, Lara Tatiane G; Montenegro, Flávio M; Schmiele, Marcio; Steel, Caroline J; Clerici, Maria Teresa P S; Almeida, Eveline L; Chang, Yoon K

2015-10-01

This study aimed to verify the potential of extruded wheat flour (EWF) or pre-gelatinized cassava starch (PGS) to improve the process and the quality of French bread elaborated from frozen dough. Three formulations were prepared: 100% control wheat flour (CWF) and the other two formulations with 5% substitution of wheat flour by EWF or PGS. Frozen doughs were frozen stored for seven days and after this period they were thawed, fermented, baked and evaluated for physical, chemical and technological characteristics. Available glucose levels found for EWF (12g/100g), and PGS (11.7g/100g) in relation to CWF (7.1g/100g) showed higher sugar availability for yeasts at the initial stage of proofing, and may also have had a cryoprotective effect when freezing bread doughs. The frozen doughs with EWF or PGS, when thawed and fermented, presented higher volume increase, but after baking, they presented lower volume when compared to the control bread. The results of this study are promising for the use of extruded wheat flour or pre-gelatinized cassava starch as sugar providers for doughs' post-freezing proofing process, improving frozen dough process of French-type bread. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cryopreservation of epididymal stallion sperm.

PubMed

Olaciregui, M; Gil, L; Montón, A; Luño, V; Jerez, R A; Martí, J I

2014-02-01

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose-egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters. Copyright © 2014 Elsevier Inc. All rights reserved.

Stabilization of heavy metals in municipal sewage sludge by freeze-thaw treatment with a blend of diatomite, FeSO4, and Ca(OH)2.

PubMed

Wang, Jing; Fu, Rongbing; Xu, Zhen

2017-08-01

In this work, the effects of diatomite with 15% FeSO 4 •7H 2 O and 7.5% Ca(OH) 2 on sludge stabilization were investigated using batch leaching tests. The influence of cell rupture caused by freezing and thawing on stabilization was also evaluated. The results indicated that the optimal diatomite percentage was 2%. Cell rupture by freezing and thawing reduced heavy metal leachability, followed by cell death and decrease of organic groups. The concentration of heavy metals in sludge leachate increased after cell rupture, indicating that the heavy metal leachability was reduced after freezing and thawings. Moreover, the stabilization effects were generally improved after freezing and thawing. As compared with the stabilization of the original sludge, the unstable fractions decreased and the residual fractions of the heavy metals increased in the stabilized sludge after cell rupture. This study developed a method to stabilize heavy metals in municipal sewage sludge. Diatomite combined with FeSO 4 ·7H 2 O and Ca(OH) 2 improved the treatment of sewage sludge contaminated by heavy metals. Cell lysis by freeze-thaw treatment reduced the risk of leaching heavy metals caused by cell death and decreased major organic groups in the sludge.

Effect of basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa.

PubMed

Anel, L; Gomes-Alves, S; Alvarez, M; Borragan, S; Anel, E; Nicolas, M; Martinez-Pastor, F; de Paz, P

2010-09-01

The improvement of freezing extenders is critical when defining sperm cryopreservation protocols for wild species, in order to create germplasm banks. The aim of this study was to evaluate the effect of additives (Equex Paste and EDTA) supplementation, egg-yolk (10 and 20%) and glycerol (4 and 8%) concentrations and extender osmolality (300 and 320 mOsm/kg) on the post-thawing quality of brown bear semen. Semen was obtained from 20 adult males by electroejaculation, and centrifugated individually (600 x g for 6 min). The pellets were diluted 1:1 in the corresponding extender TTF (TES-Tris-Fructose with the aforementioned variants) and cooled to 5 degrees C. Then, it was diluted down to 100 x 10(6) spz/mL, loaded in 0.25 mL straws and frozen at -20 degrees C/min. After thawing (in water at 65 degrees C for 6s), the semen samples were assessed for motility (CASA), viability (SYBR-14 with propidium iodide), acrosomal status (PNA-FITC with propidium iodide) and mitochondrial activity (JC-1). Extender supplementation with additives rendered significantly higher results for these sperm parameters. Comparing the two percentages of egg yolk, 20% egg yolk showed the highest motility results, percentages of viable spermatozoa and viable spermatozoa with intact acrosome. No differences were detected among samples frozen using 4 or 8% glycerol. For extender osmolality, 300 mOsm/kg showed higher values of VAP, VCL, VSL, and ALH than 320 mOsm/kg. Based on the best performance of sperm motility, viability and acrosome status, we conclude that the most suitable extender to cryopreserve brown bear spermatozoa was TTF adjusted to 300 mOsm/kg, supplemented with 20% egg yolk, 4-8% glycerol, and the additives 1% Equex paste and 2% EDTA. Copyright 2010 Elsevier Inc. All rights reserved.

Clove and rosemary essential oils and encapsuled active principles (eugenol, thymol and vanillin blend) on meat quality of feedlot-finished heifers.

PubMed

de Oliveira Monteschio, Jéssica; de Souza, Kennyson Alves; Vital, Ana Carolina Pelaes; Guerrero, Ana; Valero, Maribel Velandia; Kempinski, Emília Maria Barbosa Carvalho; Barcelos, Vinícius Cunha; Nascimento, Karina Favoreto; do Prado, Ivanor Nunes

2017-08-01

Forty Nellore heifers were fed (73days) with different diets: with or without essential oils (clove and/or rosemary essential oil) and/or active principle blend (eugenol, thymol and vanillin). The pH, fat thickness, marbling, muscle area and water losses (thawing and drip) were evaluated 24h post mortem on the Longissimus thoracis, and the effects of aging (14days) was evaluated on the meat cooking losses, color, texture and lipid oxidation. Antioxidant activity was also evaluated. Treatments had no effect (P>0.05) on pH, fat thickness, marbling, muscle area, thawing and drip losses. However, treatments affected (P<0.05) cooking losses, color, texture and lipid oxidation. The diets with essential oil and the active principle blend reduced the lipid oxidation and reduced the color losses in relation to control diet. Aging affected (P<0.05) texture and lipid oxidation. The essential oil and active principles or its blend have potential use in animal feed aiming to maintain/improve meat quality during shelf-life. Copyright © 2017. Published by Elsevier Ltd.

Studies of male reproduction in captive African wild dogs (Lycaon pictus).

PubMed

Johnston, S D; Ward, D; Lemon, J; Gunn, I; MacCallum, C A; Keeley, T; Blyde, D

2007-08-01

Implementation of assisted breeding in the captive African wild dog is restricted by a current lack of knowledge on their reproductive physiology and the apparent difficulty of effectively manipulating the complex social dynamic of the pack in order to conduct reproductive procedures. In this study, we describe protocols for the safe and repeated capture and restraint of the African wild dog (n=7) as well as techniques for assessment of male reproductive function, semen collection and preservation. In a serendipitous finding, captive African wild dogs appeared to display significant seasonal change in male reproduction. Testicular volume and tone, spermatorrhea and the ability to collect semen by electroejaculation all increased significantly during late summer and then subsequently declined by early spring. While there were no detectable seasonal changes in testosterone concentration in the population as whole, the alpha-dominant male in both years of the study, had a highly elevated testosterone concentration compared to subordinate males. Semen collection by electroejaculation during the late summer was most effective in peri-pubertal males (15 months) when all seven electroejaculates were of adequate quality for cryopreservation. In the second breeding season (27 months), there were numerous changes in the pack hierarchy and electroejaculation was not as successful (3/7). The characteristics of electroejaculated semen collected in the breeding season are described for seven animals including the first descriptions and incidence of sperm abnormalities in the species. Semen (n=7) was frozen using a Tris-citrate fructose buffer and final egg yolk and glycerol concentration of 20% and 4%, respectively. Sperm were loaded into 0.25 mL straws, frozen in liquid nitrogen vapor and then thawed at 37 degrees C. Initial post-thaw survival of spermatozoa was encouraging (% motile: 31.8+/-5.8%; rate: 2.8+/-0.3; % intact plasma membranes: 33.4+/-5.3% and the % of damaged acrosomes: 4.4+/-1.5%) but following 2 h incubation at 37 degrees C, post-thaw survival declined markedly.

Biotic responses buffer warming-induced soil organic carbon loss in Arctic tundra.

PubMed

Liang, Junyi; Xia, Jiangyang; Shi, Zheng; Jiang, Lifen; Ma, Shuang; Lu, Xingjie; Mauritz, Marguerite; Natali, Susan M; Pegoraro, Elaine; Penton, C Ryan; Plaza, César; Salmon, Verity G; Celis, Gerardo; Cole, James R; Konstantinidis, Konstantinos T; Tiedje, James M; Zhou, Jizhong; Schuur, Edward A G; Luo, Yiqi

2018-05-26

Climate warming can result in both abiotic (e.g., permafrost thaw) and biotic (e.g., microbial functional genes) changes in Arctic tundra. Recent research has incorporated dynamic permafrost thaw in Earth system models (ESMs) and indicates that Arctic tundra could be a significant future carbon (C) source due to the enhanced decomposition of thawed deep soil C. However, warming-induced biotic changes may influence biologically related parameters and the consequent projections in ESMs. How model parameters associated with biotic responses will change under warming and to what extent these changes affect projected C budgets have not been carefully examined. In this study, we synthesized six data sets over five years from a soil warming experiment at the Eight Mile Lake, Alaska, into the Terrestrial ECOsystem (TECO) model with a probabilistic inversion approach. The TECO model used multiple soil layers to track dynamics of thawed soil under different treatments. Our results show that warming increased light use efficiency of vegetation photosynthesis but decreased baseline (i.e., environment-corrected) turnover rates of SOC in both the fast and slow pools in comparison with those under control. Moreover, the parameter changes generally amplified over time, suggesting processes of gradual physiological acclimation and functional gene shifts of both plants and microbes. The TECO model predicted that field warming from 2009 to 2013 resulted in cumulative C losses of 224 or 87 g m -2 , respectively, without or with changes in those parameters. Thus, warming-induced parameter changes reduced predicted soil C loss by 61%. Our study suggests that it is critical to incorporate biotic changes in ESMs to improve the model performance in predicting C dynamics in permafrost regions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Development of the Soil Moisture Active Passive (SMAP) radiometer derived landscape freeze/thaw product

NASA Astrophysics Data System (ADS)

Colliander, A.; Xu, X.; Dunbar, R. S.; Derksen, C.; Kim, Y.; Kimball, J. S.

2016-12-01

A baseline SMAP mission objective was to determine the land surface binary freeze/thaw (FT) state for northern (>45°N) regions with 80% spatial classification accuracy at 3 km resolution and 2-day average intervals. These requirements were initially achieved from the SMAP radar until the sensor failed in July 2015. The FT algorithm is now transitioning to using SMAP radiometer inputs. The main compromises of this change are a coarse (36 km) radiometer footprint, enhanced noise and potential FT signal degradation from seasonal vegetation biomass, soil moisture and surface inundation changes. The new daily passive FT product (L3_FT_P) is based on the same seasonal threshold algorithm as the radar derived product (L3_FT_A): instantaneous SMAP measurements are compared to reference signatures acquired during seasonal frozen and thawed states. Instead of radar inputs, the normalized polarization ratio (NPR) is calculated from SMAP radiometer measurements. The L3_FT_P algorithm is applied using NPR inputs, whereby NPR decreases and increases are associated with respective landscape freezing and thawing. A lower NPR under frozen conditions is due to smaller V-pol brightness temperature increases and larger H-pol increases. Using in situ measurements from core validation sites, the temporal behavior of backscatter and NPR measurements were evaluated during the spring 2015 radar and radiometer overlap period. The transition from frozen to thawed states produced a NPR response similar in timing and magnitude to the radar response, resulting in similar freeze to thaw seasonal transition dates. While the post-thaw radar backscatter consistently remained at elevated values relative to the frozen state, the NPR drifted downwards following the main thaw transition (due to de-polarization of the scene), which may introduce false freeze classification errors. Both radar and radiometer results tended to lead observed soil thawing due to strong sensitivity of the microwave retrievals to wet snow. Continued analysis of SMAP radiometer measurements will help to identify different landscape components of the SMAP freeze-thaw temporal signal.

On Ideology, Language, and Identity: Language Politics in the Soviet and Post-Soviet Lithuania

ERIC Educational Resources Information Center

Balockaite, Rasa

2014-01-01

The paper illuminates links between state politics and language politics in Lithuania during different historical periods: (a) the thaw period, (b) the stagnation period, (c) the liberalization periods of Soviet socialism, and (d) the two post-Soviet decades characterized by both nationalism and liberalization. Based on analysis of the texts by…

Artificial insemination with frozen-thawed boar sperm.

PubMed

Yeste, Marc; Rodríguez-Gil, Joan E; Bonet, Sergi

2017-09-01

Artificial insemination with frozen-thawed semen in pigs is not a routine technique; its use is restricted to specific cases, such as preservation of valuable genetic material (germplasm banks), safety strategies in case of natural disasters, long-distance transport of sperm, and in combination with sex-sorting. Cryoinjuries resulting from freeze-thawing protocols are a major concern with regard to the fertilization capacity of the treated sperm, which is lower than that of liquid-stored semen. Here, we provide an overview of artificial insemination using cryopreserved sperm, and summarize the factors that influence cryopreservation success before, during, and after freeze-thaw (i.e., sperm selection before starting the cryopreservation process, holding time, use of cryoprotectants, and rates of freezing and thawing) and that are driving the identification of biomarkers to predict sensitivity to cryodamage. Three different artificial insemination techniques (conventional or intracervical; intrauterine; and deep intrauterine) are also discussed with regards to their relevance when using frozen-thawed semen. Finally, we review the use of additives to freezing and thawing media, given reports that they may maintain and improve the quality and fertilizing capacity of frozen-thawed sperm. In sum, artificial insemination with frozen-thawed boar sperm can provide reasonable fertility outcomes, if freezable ejaculates, specific additives, and appropriate insemination techniques are used. © 2017 Wiley Periodicals, Inc.

Improved spring load restriction guidelines using mechanistic analysis

DOT National Transportation Integrated Search

2000-07-01

This project used research to develop more effective criteria for placement and removal of spring load restrictions (SLR). Researchers investigated a method that uses a thawing index equation based on air temperatures to predict thawing events. Resul...

Comparison between mechanical freezer and conventional freezing using liquid nitrogen in normozoospermia.

PubMed

Rahana, A R; Ng, S P; Leong, C F; Rahimah, M D

2011-10-01

This study evaluated the effect of human semen cryopreservation using an ultra-low temperature technique with a mechanical freezer at -85°C as an alternative method to the conventional liquid nitrogen technique at -196°C. This was a prospective experimental study conducted in the Medically Assisted Conception unit, Department of Obstetrics and Gynaecology, National University Hospital, Malaysia from January 1, 2006 to April 30, 2007. All normozoospermic semen samples were included in the study. The concentration, motility and percentage of intact DNA of each semen sample were assessed before and after freezing and thawing on Days 7 and 30 post freezing. Sperm cryopreservation at -85°C was comparable to the conventional liquid nitrogen technique for a period of up to 30 days in a normozoospermic sample. There was no statistical difference in concentration (Day 7 p-value is 0.1, Day 30 p-value is 0.2), motility (Day 7 p-value is 0.9, Day 30 p-value is 0.5) and proportion of intact DNA (Day 7 p-value is 0.1, Day 30 p-value is 0.2) between the ultra-low temperature technique and conventional liquid nitrogen cryopreservation at Days 7 and 30 post thawing. This study clearly demonstrates that short-term storage of sperm at -85°C could be a viable alternative to conventional liquid nitrogen cryopreservation at -196°C due to their comparable post-thaw results.

Sequestration of PDC-109 protein by specific antibodies and egg yolk cryoprotects bull spermatozoa.

PubMed

Srivastava, N; Srivastava, S K; Ghosh, S K; Jerome, A; Das, G K; Mehrotra, S

2013-10-01

PDC-109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time- and concentration-dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC-109 either by anti-PDC-109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY + Ab in minimizing cryoinjury to bull spermatozoa. PDC-109 protein was purified by applying two-step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion-exchange chromatography. After checking the Ab cross-reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris-glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY + TG (group I), Ab + TG (group II) or EY + Ab + TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC-PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein-sequestered-semen (zona-penetration assay) were evaluated. A significant (p < 0.05) improvement in post-thaw SQPs as well as in non-capacitated spermatozoa observed at pre-freeze and post-thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein-mediated cryoinjury. From this study, it can be concluded that sequestration of PDC-109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra-low temperatures. © 2013 Blackwell Verlag GmbH.

Enhanced fertility prediction of cryopreserved boar spermatozoa using novel sperm function assessment.

PubMed

Daigneault, B W; McNamara, K A; Purdy, P H; Krisher, R L; Knox, R V; Rodriguez-Zas, S L; Miller, D J

2015-05-01

Due to reduced fertility, cryopreserved semen is seldom used for commercial porcine artificial insemination (AI). Predicting the fertility of individual frozen ejaculates for selection of higher quality semen prior to AI would increase overall success. Our objective was to test novel and traditional laboratory analyses to identify characteristics of cryopreserved spermatozoa that are related to boar fertility. Traditional post-thaw analyses of motility, viability, and acrosome integrity were performed on each ejaculate. In vitro fertilization, cleavage, and blastocyst development were also determined. Finally, spermatozoa-oviduct binding and competitive zona-binding assays were applied to assess sperm adhesion to these two matrices. Fertility of the same ejaculates subjected to laboratory assays was determined for each boar by multi-sire AI and defined as (i) the mean percentage of the litter sired and (ii) the mean number of piglets sired in each litter. Means of each laboratory evaluation were calculated for each boar and those values were applied to multiple linear regression analyses to determine which sperm traits could collectively estimate fertility in the simplest model. The regression model to predict the percent of litter sired by each boar was highly effective (p < 0.001, r(2) = 0.87) and included five traits; acrosome-compromised spermatozoa, percent live spermatozoa (0 and 60 min post-thaw), percent total motility, and the number of zona-bound spermatozoa. A second model to predict the number of piglets sired by boar was also effective (p < 0.05, r(2) = 0.57). These models indicate that the fertility of cryopreserved boar spermatozoa can be predicted effectively by including traditional and novel laboratory assays that consider functions of spermatozoa. © 2015 American Society of Andrology and European Academy of Andrology.

Paleoecological inferences of recent alluvial damming of a lake basin due to retrogressive permafrost thaw slumping

NASA Astrophysics Data System (ADS)

Quinlan, R.; Delaney, S.; Lamoureux, S. F.; Kokelj, S. V.; Pisaric, M. F.

2014-12-01

Expected climate impacts of future warming in the Arctic include thawing of permafrost landscapes in northern latitudes. Thawing permafrost is expected to have major consequences on hydrological dynamics, which will affect the limnological conditions of Arctic lakes and ponds. In this study we obtained a sediment core from a small lake (informally named "FM1") near Fort McPherson, Northwest Territories, Canada, with a large retrogressive thaw slump (nearly 1 kilometre in diameter) within its catchment. A radiocarbon date from the base of the FM1 sediment core suggests the lake formed between 990-1160 Cal AD. The analysis of aerial photographs indicate the thaw slump initiated between 1970-1990, and sediment geochemistry analysis indicated major changes in sediment content at 54-cm sediment core depth. Analyses of subfossil midge (Chironomidae) fossils inferred that, pre-slump, lake FM1 was shallow with a large bog or wetland environment, with midge assemblages dominated by taxa such as Limnophyes and Parametriocnemus. Post-thaw midge assemblages were dominated by subfamily Chironominae (Tribe Tanytarsini and Tribe Chironomini) taxa, and the appearance of deepwater-associated taxa such as Sergentia suggests that lake FM1 deepened, possibly as a result of alluvial damming from slump materials washing into the lake near its outlet. Most recent stratigraphic intervals infer a reversion back to shallower conditions, with a slight recovery of bog or wetland-associated midge taxa, possibly due to rapid basin infilling from increased deposition rates of catchment-derived materials. Results emphasize that there may be a variety of different outcomes to Arctic lake and pond ecosystems as a result of permafrost thawing, contingent on system-specific characteristics such as slump location relative to the lake basin, and relative inflow and outflow locations within the lake basin.

Extending the Season for Concrete Construction and Repair. Phase II - Defining Engineering Parameters

DTIC Science & Technology

2006-04-01

dosages, seemed to improve the freeze –thaw durability of concrete. Phase II found what appears to be a maximum dosage after which freeze –thaw...durability becomes a concern. That is because cement hydration can only create a finite amount of space to absorb these chemicals. Thus, for freeze ...protection, admixture dosages should be designed according to water content as specified in Phase I, while, for freeze –thaw durability, admixture dosages

Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze-thawing by stabilising disulfide bonds.

PubMed

Yeste, Marc; Flores, Eva; Estrada, Efrén; Bonet, Sergi; Rigau, Teresa; Rodríguez-Gil, Joan E

2013-01-01

One important change the head of boar spermatozoa during freeze-thawing is the destabilisation of its nucleoprotein structure due to a disruption of disulfide bonds. With the aim of better understanding these changes in frozen-thawed spermatozoa, two agents, namely reduced glutathione (GSH) and procaine hydrochloride (ProHCl), were added at different concentrations to the freezing media at different concentrations and combinations over the range 1-2mM. Then, 30 and 240 min after thawing, cysteine-free residue levels of boar sperm nucleoproteins, DNA fragmentation and other sperm functional parameters were evaluated. Both GSH and ProHCl, at final concentrations of 2mM, induced a significant (P<0.05) increase in the number of non-disrupted sperm head disulfide bonds 30 and 240 min after thawing compared with the frozen-thawed control. This effect was accompanied by a significant (P<0.05) decrease in DNA fragmentation 240 min after thawing. Concomitantly, 1 and 2mM GSH, but not ProHCl at any of the concentrations tested, partially counteracted the detrimental effects caused by freeze-thawing on sperm peroxide levels, motility patterns and plasma membrane integrity. In conclusion, the results show that both GSH and ProHCl have a stabilising effect on the nucleoprotein structure of frozen-thawed spermatozoa, although only GSH exerts an appreciable effect on sperm viability.

Inducing Heat Shock Proteins Enhances the Stemness of Frozen-Thawed Adipose Tissue-Derived Stem Cells.

PubMed

Shaik, Shahensha; Hayes, Daniel; Gimble, Jeffrey; Devireddy, Ram

2017-04-15

Extensive research has been performed to determine the effect of freezing protocol and cryopreservation agents on the viability of adipose tissue-derived stromal/stem cells (ASCs) as well as other cells. Unfortunately, the conclusion one may draw after decades of research utilizing fundamentally similar cryopreservation techniques is that a barrier exists, which precludes full recovery. We hypothesize that agents capable of inducing a subset of heat shock proteins (HSPs) and chaperones will reduce the intrinsic barriers to the post-thaw recovery of ASCs. ASCs were exposed to 43°C for 1 h to upregulate HSPs, and the temporal HSP expression profile postheat shock was determined by performing quantitative polymerase chain reaction (PCR) and western blotting assays. The expression levels of HSP70 and HSP32 were found to be maximum at 3 h after the heat shock, whereas HSP90 and HSP27 remain unchanged. The heat shocked ASCs cryopreserved during maximal HSPs expression exhibited increased post-thaw viability than the nonheat shocked samples. Histochemical staining and quantitative reverse transcription-PCR indicated that the ASC differentiation potential was retained. Thus, suggesting that the upregulation of HSPs before a freezing insult is beneficial to ASCs and a potential alternative to the use of harmful cryoprotective agents.

Fatty acids and plasmalogens of the phospholipids of the sperm membranes and their relation with the post-thaw quality of stallion spermatozoa.

PubMed

Macías García, B; González Fernández, L; Ortega Ferrusola, C; Morillo Rodríguez, A; Gallardo Bolaños, J M; Rodríguez Martinez, H; Tapia, J A; Morcuende, D; Peña, F J

2011-03-15

Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parameters studied the ratio of percentage of C16 plasmalogens related to total phospholipids was the one with the better diagnostic value. For potentially bad freezers, the significant area under the ROC-curve was 0.74, with 75% sensitivity and 79.9% specificity for a cut off value of 26.9. Also the percentage of plasmalogens respect to total phospholipids gave good diagnostic value for bad freezers. On the other hand, the percentage of C18 fatty aldehydes related to total phospholipids of the sperm membrane properly forecasted freezeability with an area under the ROC curve of 0.70 with 70% sensitivity and 62.5% specificity for a cut off value of 0.32. Copyright © 2011 Elsevier Inc. All rights reserved.

Mouse Sperm Cryopreservation Using Cryoprotectant Containing l-Glutamine.

PubMed

Takeo, Toru; Nakagata, Naomi

2018-06-01

Efforts to advance sperm cryopreservation are ongoing and include modifications in the cryoprotective agents. The addition of l-glutamine maintains post-thaw motility and reduces plasma membrane damage to sperm. © 2018 Cold Spring Harbor Laboratory Press.

Attempts to Produce Experimental Edema Disease in Swine by Parenterally Injecting Escherichia Coli Serotype 0139:K82:H1*

PubMed Central

Pickrell, J. A.; Link, R. P.; Simon, J.; Rhoades, H. E.; Gossling, J.

1969-01-01

Twenty-two pigs were inoculated parenterally with various E. coli 0139:K82:H1 preparations. Clinical signs of disease in pigs injected with freeze-thaw extract consisted of early listlessness, diarrhea and, later, hyperirritability of varying intensity in some animals. Hemorrhagic gastroenteritis involving the duodenum, spiral colon and the fundic portion of the stomach, and ulceration of the fundic stomach were observed at post-mortem examination of pigs inoculated parenterallly with living culture or freeze-thaw extract. No significant lesions were observed in pigs inoculated with ultrasonic or hypotonic acid-saline extract. In pigs injected with living culture or freeze-thaw extract, the histological alterations consisted of moderate perivascular edema of the brain, marked hepatic parenchymal cell degeneration, hepatic subserosal edema and “toxic” lymph nodes, when compared to the control group. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:4237302

Application of Spaceborne Scatterometer for Mapping Freeze-Thaw State in Northern Landscapes as a Measure of Ecological and Hydrological Processes

NASA Technical Reports Server (NTRS)

McDonald, Kyle; Kimball, John; Zimmermann, Reiner; Way, JoBea; Frolking, Steve; Running, Steve

1994-01-01

Landscape freeze/thaw transitions coincide with marked shifts in albedo, surface energy and mass exchange, and associated snow dynamics. monitoring landscape freeze/thaw dynamics would improve our ability to quantify the interannual variability of boreal hydrology and river runoff/flood dynamics, The annual duration of frost-free period also bounds the period of photosynthetic activity in borel and arctic regions thus affecting the carbon budget and the interannual variability fo regional carbon fluxes.

Successful vitrification of human amnion-derived mesenchymal stem cells.

PubMed

Moon, Jeong Hee; Lee, Jung Ryeol; Jee, Byung Chul; Suh, Chang Suk; Kim, Seok Hyun; Lim, Hyun Jung; Kim, Hae Kwon

2008-08-01

A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs. HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs. The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.

Sperm Flagellum Volume Determines Freezability in Red Deer Spermatozoa

PubMed Central

Ros-Santaella, José Luis; Domínguez-Rebolledo, Álvaro Efrén; Garde, José Julián

2014-01-01

The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 µm3 vs 6.61 µm3, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = −0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success. PMID:25380133

[Cryopreservation of mouse embryos in ethylene glycol-based solutions: a search for the optimal and simple protocols].

PubMed

Luo, Ming-Jiu; Liu, Na; Miao, De-Qiang; Lan, Guo-Cheng; Suo-Feng; Chang, Zhong-Le; Tan, Jing-He

2005-09-01

Although ethylene glycol (EG) has been widely used for embryo cryopreservation in domestic animals, few attempts were made to use this molecule to freeze mouse and human embryos. In the few studies that used EG for slow-freezing of mouse and human embryos, complicated protocols for human embryos were used, and the protocols need to be simplified. Besides, freezing mouse morula with EG as a cryoprotectant has not been reported. In this paper, we studied the effects of embryo stages, EG concentration, duration and procedure of equilibration, sucrose supplementation and EG removal after thawing on the development of thawed mouse embryos, using the simple freezing and thawing procedures for bovine embryos. The blastulation and hatching rates (81.92% +/- 2.24% and 68.56% +/- 2.43%, respectively) of the thawed late compact morulae were significantly (P < 0.05) higher than those of embryos frozen-thawed at other stages. When mouse late compact morulae were frozen with different concentrations of EG, the highest rates of blastocyst formation and hatching were obtained with 1.8mol/L EG. The blastulation rate was significantly higher when late morulae were equilibrated in 1.8 mol/L EG for 10 min prior to freezing than when they were equilibrated for 30 min, and the hatching rate of embryos exposed to EG for 10 min was significantly higher than that of embryos exposed for 20 and 30 min. Both rates of blastocyst formation and hatching obtained with two-step equilibration were higher (P < 0.05) than with one-step equilibration in 1.8 mol/L EG. Addition of sucrose to the EG-based solution had no beneficial effects. On the contrary, an increased sucrose level (0.4 mol/L) in the solution impaired the development of the frozen-thawed embryos. In contrast, addition of 0.1 mol/L sucrose to the propylene glycol (PG)-based solution significantly improved the development of the frozen-thawed embryos. Elimination of the cryoprotectant after thawing did not improve the development of the thawed embryos. The cell numbers were less (P < 0.05) in blastocysts developed from the thawed morulae than in the in vivo derived ones. In summary, embryo stage, EG concentration, duration and procedure of equilibration and sucrose supplementation had marked effects on development of the thawed mouse embryos, and a protocol for cryopreservation of mouse embryos is recommended in which the late morulae are frozen in 1.8 mol/L EG using the simple freezing and thawing procedures of bovine embryos after a two-step equilibration and the embryos can be cultured or transferred without EG removal after thawing.

Semen of spinal cord injured men freezes reliably.

PubMed

Padron, O F; Brackett, N L; Weizman, M S; Lynne, C M

1994-01-01

The objectives of the present study were to: 1) determine the effect of cryopreservation on the percent and the grade of motility of sperm from spinal cord injured (SCI) men and 2) determine which method of freezing yields the best post-thaw motility in sperm from SCI men. Antegrade semen samples were obtained from 9 SCI subjects and 10 age-matched healthy control subjects. Motility in fresh samples was determined and cryopreservative medium was added to each sample. Aliquots of each sample were frozen according to three methods: 1) liquid nitrogen vapor only (V); 2) vapor for 12 minutes followed by submersion into liquid nitrogen (V+N2); and 3) direct submersion into liquid nitrogen (N2). Samples were frozen for 1 week, then thawed. The post-thaw percent and grade of motility was determined. The mean percent motility of fresh samples for SCI subjects (21.0%) was significantly lower than for control subjects (55.7%). After thawing, the mean percent drop in motility for V, V+N2, and N2 for controls was 65.2%, 73.5%, and 79.4%, respectively, and for SCI subjects, it was 64.7%, 74.5%, and 81.6%, respectively. There was no statistically significant difference between control and SCI subjects by method of freezing. Vapor only as a freezing method was superior to all other methods for retention of sperm motility in both control and SCI subjects. We conclude that the semen of SCI men may be frozen reliably and that their sperm retain motility similar to that of normal men. Vapor only, being the most gentle method used, gives the best recovery of sperm motility in either group.

Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

PubMed

Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

2016-05-01

Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells. © 2016 The Protein Society.

The antioxidant effects of soybean lecithin- or low-density lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa.

PubMed

Alvarez-Rodríguez, M; Alvarez, M; Anel-López, L; Martínez-Rodríguez, C; Martínez-Pastor, F; Borragan, S; Anel, L; de Paz, P

2013-01-01

Egg yolk low-density lipoproteins (LDL) and soybean lecithin were evaluated as replacements for egg yolk in extenders used for the cryopreservation of brown-bear spermatozoa. The motility, viability and acrosomal status of post-thawed spermatozoa were analysed, and an egg-yolk extender was used as a control. The total antioxidant capacity of these extenders was tested. Soybean lecithin showed an effect that was dependent on the soybean concentration (2%, 3.5% and 5%) and source (Type A: 24% L-α-phosphatidylcholine, and Type B: 14-23% L-α-phosphatidylcholine). Only semen cryopreserved with 5% Type A soybean exhibited a sperm motility similar to that of semen cryopreserved in egg-yolk-based extender after thawing, although the sperm viability and acrosome status were not as high. Semen frozen in an extender containing LDL (10-15%) exhibited improved sperm viability in comparison with the control, but sperm motility was lower. The LDL-based extender exhibited a higher anti-oxidant activity than the egg-yolk extender and soy lecithin-based extenders. The extenders with higher anti-oxidant activity showed improvements in frozen sperm viability but lower semen motility. These results indicate that soybean lecithin did not have the same protective effect as egg yolk during the freezing of brown-bear spermatozoa but suggest that LDL (10-15%) could be a useful substitute for egg yolk in these extenders.

The effect of cholesterol loaded cyclodextrins on post-thawing quality of buffalo semen in relation to sperm DNA damage and ultrastructure.

PubMed

Ezz, Mohamed Aboul; Montasser, Abd Elmonem; Hussein, Mamdouh; Eldesouky, Ashraf; Badr, Magdy; Hegab, Abd Elraouf; Balboula, Ahmed; Zaabel, Samy M

2017-03-01

The cryopreservation of germ cells is a major tool for the propagation of animals with desired genetic traits. Although cryopreservation of spermatozoa in some animals is effective, its effectiveness is variable. For example, cryopreservation efficiency of buffalo bull spermatozoa remains very poor. In this study, we evaluated sperm DNA damage and ultrastructure in buffalo bull spermatozoa vitrified in the presence or absence of cholesterol-loaded cyclodextrins (CLC). Our results showed that cryopreserved buffalo spermatozoa had elevated levels of deteriorated plasma and mitochondrial membranes, which are the likely causes of DNA damage after vitrification. Accordingly, the levels of the activity of Alanine Aminotransferase (ALT), Alkaline phosphatase (ALP) and Aspartate Aminotransferase (AST) were also elevated following exposure of buffalo bull spermatozoa to a cycle of freezing-thawing. Importantly, supplementation of Tris-Egg Yolk-Glucose (TEYG) extender with (CLC) improved the quality of buffalo spermatozoa following cryopreservation. This protective effect of CLC is likely due to decreasing mitochondrial and plasma membrane deterioration with subsequent inhibition of DNA damage. These results suggest that cholesterol loss is the likely reason for poor semen quality in buffaloes following cryopreservation, and provide evidence that manipulating lipid content during cryopreservation is a promising strategy to improve the quality of buffalo semen. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

Effects of Freeze-Thawing and Intravenous Infusion on Mesenchymal Stromal Cell Gene Expression.

PubMed

Hoogduijn, Martin J; de Witte, Samantha F H; Luk, Franka; van den Hout-van Vroonhoven, Mirjam C G N; Ignatowicz, Lech; Catar, Rusan; Strini, Tanja; Korevaar, Sander S; van IJcken, Wilfred F J; Betjes, Michiel G H; Franquesa, Marcella; Moll, Guido; Baan, Carla C

2016-04-15

Mesenchymal stromal cells (MSC) are increasingly used as an investigative therapeutic product for immune disorders and degenerative disease. Typically, MSC are isolated from human tissue, expanded in culture, and cryopreserved until usage. The safety and efficacy of MSC therapy will depend on the phenotypical and functional characteristics of MSC. The freeze-thawing procedure may change these characteristics. Furthermore, the cells encounter a microenvironment after administration that may impact their properties. It has been demonstrated that the majority of MSC localize to the lungs after intravenous infusion, making this the site to study the effects of the in vivo milieu on administered MSC. In this study, we investigated the effect of freeze-thawing and the mouse lung microenvironment on human adipose tissue-derived MSC. There were effects of freeze-thawing on the whole genome expression profile of MSC, although the effects did not exceed interdonor differences. There were no major changes in the expression of hemostatic regulators on transcriptional level, but significantly increased expression of procoagulant tissue factor on the surface of thawed adipose MSC, correlating with increased procoagulant activity of thawed cells. Exposure for 2 h to the lung microenvironment had a major effect on MSC gene expression and affected several immunological pathways. This indicates that MSC undergo functional changes shortly after infusion and this may influence the efficacy of MSC to modulate inflammatory responses. The results of this study demonstrate that MSC rapidly alter in response to the local milieu and disease-specific conditions may shape MSC after administration.

Increased coagulation and fibrinolytic potential of solvent-detergent plasma: a comparative study between Omniplasma and fresh frozen plasma.

PubMed

van Beers, J J B C; van Egmond, L T; Wetzels, R J H; Verhezen, P W M; Beckers, E A M; van Oerle, R; Spronk, H M H; Straat, R J M H E; Henskens, Y M C

2016-07-01

In this study, differences in levels of proteins involved in coagulation and fibrinolysis were compared between fresh frozen (quarantine plasma) and Omniplasma. Furthermore, thawing conditions and plasma stability after thawing were studied. 10 Omniplasma and 10 quarantine plasma units were used to study different procoagulation, anticoagulation and fibrinolytic parameters. Analysis took place at different time-points during plasma storage at 2-6°C. At baseline, significant reduced levels of factor V, free protein S, α2-antiplasmin and tPA-induced ROTEM lysis time were observed in Omniplasma as compared to quarantine plasma. Moreover, thrombin generation, IXa-AT complex levels and factor XIa were significantly increased in Omniplasma. The majority of the parameters studied remained stable in Omniplasma 48 h after thawing, with the exception of factor VIII (decrease) and IXa-AT (increase). Our results suggest an increased coagulation potential, presumingly as a result of contact activation during the production process and also, an increased fibrinolytic potential in Omniplasma. The stability of Omniplasma, based upon the different parameters studied, is comparable to Q-plasma. A maximum post-thawing time of 48 hfor Omniplasma can be suggested. © 2016 International Society of Blood Transfusion.

Addition of cholesterol-loaded cyclodextrins to the thawing extender: effects on boar sperm quality.

PubMed

Tomás, C; Gómez-Fernández, J; Gómez-Izquierdo, E; Mocé, E; de Mercado, E

2014-06-01

The aim of the present study was to evaluate the effect that the addition of cholesterol-loaded cyclodextrins (CLC) to the thawing extender has on the quality of frozen-thawed boar sperm. Pooled semen (n = 5) from three boars was used for the experiments. The semen was cryopreserved with an egg-yolk-based extender, it was diluted after thawing in Beltsville thawing solution (BTS) supplemented with different concentrations of CLC (0, 12.5, 25, 50 or 100 mg/500 × 10(6) sperm), and these samples were incubated at 37°C for 150 min. The following parameters of sperm quality were evaluated 30 and 150 min after incubation: sperm with intact plasma membrane (SIPM; %), sperm with normal acrosomal ridge (NAR; %), total motile sperm (TMS; %), progressively motile sperm (PMS; %) and kinetic parameters. Both SIPM and NAR increased (p < 0.05) when the thawing extender was supplemented with 12.5, 25 and 50 mg CLC/500 × 10(6) sperm. Nevertheless, motility decreased (p < 0.05) when the concentration of CLC exceeded 12.5 mg CLC/500 × 10(6) sperm. In conclusion, our results suggest that the supplementation of thawing extenders with CLC improves sperm viability and reduces acrosome damage after freezing/thawing. © 2014 Blackwell Verlag GmbH.

Retention of membrane charge attributes by cryopreserved-thawed sperm and zeta selection.

PubMed

Kam, Tricia L; Jacobson, John D; Patton, William C; Corselli, Johannah U; Chan, Philip J

2007-09-01

Mature sperm can be selected based on their negative zeta electrokinetic potential. The zeta selection of cryopreserved sperm is unknown. The objective was to study the effect of zeta processing on the morphology and kinematic parameters of cryopreserved-thawed sperm. Colloid-washed sperm (N = 9 cases) were cryopreserved for 24 h, thawed and diluted in serum-free medium in positive-charged tubes. After centrifugation, the tubes were decanted, serum-supplemented medium was added and the resuspended sperm were analyzed. Untreated sperm and fresh sperm served as the controls. There were improvements in strict normal morphology in fresh (11.8 +/- 0.3 versus control 8.8 +/- 0.3 %, mean +/- SEM) and thawed (8.7 +/- 0.2 versus control 5.4 +/- 0.2%) sperm after zeta processing. Percent sperm necrosis was reduced after zeta processing (66.0 +/- 0.6 versus unprocessed 74.6 +/- 0.3%). Progression decreased by 50% but not total motility after zeta processing of thawed sperm. The results suggested that the cryopreservation process did not impact the sperm membrane net zeta potential and higher percentages of sperm with normal strict morphology, acrosome integrity and reduced necrosis were recovered. The zeta method was simple and improved the selection of quality sperm after cryopreservation but more studies would be needed before routine clinical application.

Application of seminal plasma in sex-sorting and sperm cryopreservation.

PubMed

de Graaf, S P; Leahy, T; Marti, J; Evans, G; Maxwell, W M C

2008-11-01

Substantial dilution of boar semen during processing decreased the concentration of seminal plasma, perhaps contributing to the decline in sperm quality after cryopreservation and sex-sorting. Results of replacing seminal plasma in investigations from many laboratories have been contradictory. Results and discussion here suggest that whereas membrane status can be influenced by seminal plasma, the action of its various components, both positive and negative, is determined in part by the membrane status of the spermatozoa to which it is being exposed. Although progress has been made in identifying components of seminal plasma responsible for its protective effect (notably PSP-I/II spermadhesin for sex-sorted boar spermatozoa), little is known (in any species) regarding how external factors may influence their levels, and their functionality, in seminal plasma. It is noteworthy that seminal plasma is beneficial to post-thaw quality of sex-sorted ram spermatozoa only when added before freezing, not after thawing. Therefore, the action of seminal plasma and its components is dependent on sperm-related factors, in particular the type of processing to which they have been previously exposed. Further research is needed to unravel these biological complexities, and then characterise and synthesise useful proteins within seminal plasma.

Carbon monoxide stunning of Atlantic salmon (Salmo salar L.) modifies rigor mortis and sensory traits as revealed by NIRS and other instruments.

PubMed

Concollato, Anna; Parisi, Giuliana; Masoero, Giorgio; Romvàri, Robert; Olsen, Rolf-Erik; Dalle Zotte, Antonella

2016-08-01

Methods of stunning used in salmon slaughter are still the subject of research. Fish quality can be influenced by pre-, ante- and post-mortem conditions, including handling before slaughter, slaughter methods and storage conditions. Carbon monoxide (CO) is known to improve colour stability in red muscle and to reduce microbial growth and lipid oxidation in live fish exposed to CO. Quality differences in Atlantic salmon, Salmo salar L., stunned by CO or percussion, were evaluated and compared by different techniques [near infrared reflectance spectroscopy (NIRS), electronic nose (EN), electronic tongue (ET)] and sensory analysis. Thawed samples, freeze-dried preparates and NIRS devices proved to be the most efficient combinations for discriminating the treatments applied to salmon, i.e. first the stunning methods adopted, then the back-prediction of the maximum time to reach rigor mortis and finally to correlate some sensory attributes. A trained panel found significant differences between control and CO-stunned salmon: reduced tactile crumbliness, reduced odour and aroma intensities, and reduced tenderness of CO-treated fillets. CO stunning reduced radiation absorbance in spectra of thawed and freeze-dried fillets, but not fillet samples stored in ethanol, where it may have interacted with myoglobin and myosin. The good results in a rapid discrimination of thawed samples detected by NIRS suggest suitable applications in the fish industry. CO treatment could mitigate sensory perception, but consumer tests are needed to confirm our findings. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Application of Spaceborne Scatterometer for Mapping Freeze-Thaw State in Northern Landscapes as a Measure of Ecological and Hydrological Processes

NASA Technical Reports Server (NTRS)

McDonald, Kyle; Kimball, John; Zimmermann, Reiner; Way, JoBea; Frolking, Steve; Running, Steve

1999-01-01

Landscape freeze/thaw transitions coincide with marked shifts in albedo, surface energy and mass exchange, and associated snow dynamics. Monitoring landscape freeze/thaw dynamics would improve our ability to quantify the interannual variability of boreal hydrology and river runoff/flood dynamics. The annual duration of frost-free period also bounds the period of photosynthetic activity in boreal and arctic regions thus affecting the annual carbon budget and the interannual variability of regional carbon fluxes. In this study, we use the NASA scatterometer (NSCAT) to monitor the temporal change in the radar backscatter signature across selected ecoregions of the boreal zone. We have measured vegetation tissue temperatures, soil temperature profiles, and micrometeorological parameters in situ at selected sites along a north-south transect extending across Alaska from Prudhoe Bay to the Kenai Peninsula and in Siberia near the Yenisey River. Data from these stations have been used to quantify the scatterometer's sensitivity to freeze/thaw state under a variety of terrain and landcover conditions. Analysis of the NSCAT temporal response over the 1997 spring thaw cycle shows a 3 to 5 dB change in measured backscatter that is well correlated with the landscape springtime thaw process. Having verified the instrument's capability to monitor freeze/thaw transitions, regional scale mosaicked data are applied to derive temporal series of freeze/thaw transition maps for selected circumpolar high latitude regions. These maps are applied to derive areal extent of frozen and thawed landscape and demonstrate the utility of spaceborne radar for operational monitoring of seasonal freeze-thaw dynamics and associated biophysical processes for the circumpolar high latitudes.

Past permafrost on the Mid-Atlantic coastal plain, eastern United States

USGS Publications Warehouse

French, H.; Demitroff, M.; Newell, Wayne L.

2009-01-01

Sand-wedge casts, soil wedges and other non-diastrophic, post-depositional sedimentary structures suggest that Late-Pleistocene permafrost and deep seasonal frost on the Mid-Atlantic Coastal Plain extended at least as far south as southern Delaware, the Eastern Shore and southern Maryland. Heterogeneous cold-climate slope deposits mantle lower valley-side slopes in central Maryland. A widespread pre-existing fragipan is congruent with the inferred palaeo-permafrost table. The high bulk density of the fragipan was probably enhanced by either thaw consolidation when icy permafrost degraded at the active layer-permafrost interface or by liquefaction and compaction when deep seasonal frost thawed. ?? 2009 John Wiley & Sons, Ltd.

Soy lecithin interferes with mitochondrial function in frozen-thawed ram spermatozoa.

PubMed

Del Valle, I; Gómez-Durán, A; Holt, W V; Muiño-Blanco, T; Cebrián-Pérez, J A

2012-01-01

Egg yolk and milk are the 2 major membrane cryoprotectants commonly used in freezing media for the long-term preservation of semen (alone or in combination with others). However, in recent years, there have been increasing arguments against the use of egg yolk or milk because of the risk of introducing diseases through the use of cryopreserved semen. In this study, we analyzed the protective effect of lecithin as an alternative to egg yolk for the cryopreservation of ram semen, using a range of functional markers for sperm viability, motility, apoptosis, and mitochondrial functionality analyses (mitochondrial inner membrane surface [MIMS], mitochondrial inner membrane potential [MIMP], and cell membrane potential) as methods of assessment in samples diluted in 3 different media: Tris-citrate-glucose as control and 2 media supplemented with soy lecithin or egg yolk. The results showed that lecithin was able to effectively protect certain sperm quality characteristics against freezing-induced damage. However, lecithin induced loss of mitochondrial membrane potential or mitochondrial loss that was not reflected by modifications in sperm motility in fresh semen. MIMS and MIMP values decreased in thawed lecithin-treated samples, concomitant with a lower (P < .05) percentage of total and progressively motile cells, compared with those in egg yolk-containing samples. Further incubation of thawed samples revealed changes in motility and mitochondrial functionality that otherwise would not have been detected. These results indicated that lecithin may have affected the inner mitochondrial membrane in frozenthawed spermatozoa and confirmed that sublethal damages that seriously affect sperm functionality, not detected by classic sperm quality analyses, can be evidenced by changes in the inner mitochondrial membrane surface. These findings strengthen the relationship between mitochondrial membrane potential and motility and show that the mitochondrial alterations induced by the cryopreservation process could be specific targets for the improvement of semen cryopreservation protocols.

Petroleum hydrocarbon biodegradation under seasonal freeze-thaw soil temperature regimes in contaminated soils from a sub-Arctic site.

PubMed

Chang, Wonjae; Klemm, Sara; Beaulieu, Chantale; Hawari, Jalal; Whyte, Lyle; Ghoshal, Subhasis

2011-02-01

Several studies have shown that biostimulation in ex situ systems such as landfarms and biopiles can facilitate remediation of petroleum hydrocarbon contaminated soils at sub-Arctic sites during summers when temperatures are above freezing. In this study, we examine the biodegradation of semivolatile (F2: C10-C16) and nonvolatile (F3: C16-C34) petroleum hydrocarbons and microbial respiration and population dynamics at post- and presummer temperatures ranging from -5 to 14 °C. The studies were conducted in pilot-scale tanks with soils obtained from a historically contaminated sub-Arctic site in Resolution Island (RI), Canada. In aerobic, nutrient-amended, unsaturated soils, the F2 hydrocarbons decreased by 32% during the seasonal freeze-thaw phase where soils were cooled from 2 to -5 °C at a freezing rate of -0.12 °C d(-1) and then thawed from -5 to 4 °C at a thawing rate of +0.16 °C d(-1). In the unamended (control) tank, the F2 fraction only decreased by 14% during the same period. Biodegradation of individual hydrocarbon compounds in the nutrient-amended soils was also confirmed by comparing their abundance over time to that of the conserved diesel biomarker, bicyclic sesquiterpanes (BS). During this period, microbial respiration was observed, even at subzero temperatures when unfrozen liquid water was detected during the freeze-thaw period. An increase in culturable heterotrophs and 16S rDNA copy numbers was noted during the freezing phase, and the (14)C-hexadecane mineralization in soil samples obtained from the nutrient-amended tank steadily increased. Hydrocarbon degrading bacterial populations identified as Corynebacterineae- and Alkanindiges-related strains emerged during the freezing and thawing phases, respectively, indicating there were temperature-based microbial community shifts.

In vitro and in vivo survival of bisected sheep embryos derived from frozen-thawed unsorted, and frozen-thawed sex-sorted and refrozen-thawed ram spermatozoa.

PubMed

Morton, Katherine M; Rowe, Anthony M; Chis Maxwell, W M; Evans, Gareth

2006-04-15

Ovine IVP embryos were derived from frozen-thawed unsorted and frozen-thawed sex-sorted spermatozoa that had been refrozen and thawed. The embryos were bisected and cultured in vitro, or transferred to recipient ewes to determine their survival in vitro and in vivo. Oocyte progression to the blastocyst stage was similar for unsorted (97/232, 41.8%) and sex-sorted spermatozoa (113/286, 39.5%; P > 0.05). Embryo survival in vitro post-bisection was similar for demi-embryos derived from unsorted and sex-sorted sperm, and embryos bisected at the blastocyst and expanded blastocyst stage (P > 0.05). A higher proportion of recipient ewes were pregnant at Day 63 after transfer of two intact embryos derived from unsorted (17/21, 80.9%) than two demi-embryos derived from unsorted (5/15, 33.3%) or sex-sorted spermatozoa (7/17, 41.2%). The number of fetuses per original embryo at Day 63 did not differ among groups (unsorted intact: 23/42, 54.8%; unsorted demi: 7/15, 46.7%; sex-sorted demi: 10/17, 58.8%) and twin pregnancies were observed in all groups. Embryo survival to term was high, and was not significantly different among intact (unsorted: 22/42, 52.4%) and demi-embryos (unsorted: 4/15, 26.7%; sex-sorted spermatozoa: 7/17, 41.2%; P > 0.05). Dizygotic twins (n = 6 sets) were born after the transfer of two intact embryos derived from unsorted spermatozoa, but only singleton lambs resulted from the transfer of demi-embryos. In conclusion, bisected IVP embryos successfully developed into morphologically normal lambs. However, embryo survival to term was neither increased nor decreased by embryo bisection.

Hemostatic profile and safety of pooled cryoprecipitate up to 120 hours after thawing.

PubMed

Lokhandwala, Parvez M; O'Neal, Adrian; Patel, Eshan U; Brunker, Patricia A R; Gehrie, Eric A; Zheng, Gang; Kickler, Thomas S; Ness, Paul M; Tobian, Aaron A R

2018-05-01

AABB standards state that cryoprecipitate should be transfused within 4 to 6 hours after thawing. We evaluated coagulation factor levels and sterility of thawed pooled cryoprecipitate to assess whether shelf life can be safely extended. Donor cryoprecipitate pools (n = 20, 10 group A, 10 group O) were held at ambient temperature and sampled at 0, 4, 8, 24, 48, 72, 96, and 120 hours post-thawing for fibrinogen, Factor (F)VIII, and von Willebrand factor (vWF) levels. Samples were tested at 0 and 120 hours for sterility (BacT/Alert system). Sixty additional cryoprecipitate pools were evaluated after 72 hours. Longitudinal differences in component levels were determined by linear fixed-effects regression. Group O cryoprecipitate had significantly lower FVIII (p = 0.002) and vWF activity (p = 0.006) compared to group A at 0 hours, but were not statistically different in fibrinogen levels (p = 0.33). Fibrinogen levels were stable over 5 days: 501 ± 81 mg/unit (mean ± standard deviation) at 0 hours to 506 ± 102 mg/unit at 120 hours (p = 0.73). Similarly, there was no decline in vWF activity: 200 ± 53 IU/unit at 0 hours to 209 ± 57 IU/unit at 120 hours (p = 0.084). The FVIII activity significantly declined on average by 9.6 IU (95% confidence interval, 5.5-13.8) between 0 hours (111 ± 33 IU/unit) and 120 hours post-thaw (101 ± 33) (p < 0.001). No organisms were detected when cryoprecipitate pools were cultured at 0 hours, but at 120 hours Staphylococcus epidermidis was identified from one pool, potentially a contaminant introduced during repeated sampling. No cultures were positive among the 60 additional cryoprecipitate pools assessed at 72 hours. Extended cryoprecipitate storage at ambient temperature did not affect fibrinogen levels over 120 hours. Sterility of products held at ambient temperature for an extended period of time could be assessed by secondary culture. © 2018 AABB.

The transcriptional response of microbial communities in thawing Alaskan permafrost soils.

PubMed

Coolen, Marco J L; Orsi, William D

2015-01-01

Thawing of permafrost soils is expected to stimulate microbial decomposition and respiration of sequestered carbon. This could, in turn, increase atmospheric concentrations of greenhouse gasses, such as carbon dioxide and methane, and create a positive feedback to climate warming. Recent metagenomic studies suggest that permafrost has a large metabolic potential for carbon processing, including pathways for fermentation and methanogenesis. Here, we performed a pilot study using ultrahigh throughput Illumina HiSeq sequencing of reverse transcribed messenger RNA to obtain a detailed overview of active metabolic pathways and responsible organisms in up to 70 cm deep permafrost soils at a moist acidic tundra location in Arctic Alaska. The transcriptional response of the permafrost microbial community was compared before and after 11 days of thaw. In general, the transcriptional profile under frozen conditions suggests a dominance of stress responses, survival strategies, and maintenance processes, whereas upon thaw a rapid enzymatic response to decomposing soil organic matter (SOM) was observed. Bacteroidetes, Firmicutes, ascomycete fungi, and methanogens were responsible for largest transcriptional response upon thaw. Transcripts indicative of heterotrophic methanogenic pathways utilizing acetate, methanol, and methylamine were found predominantly in the permafrost table after thaw. Furthermore, transcripts involved in acetogenesis were expressed exclusively after thaw suggesting that acetogenic bacteria are a potential source of acetate for acetoclastic methanogenesis in freshly thawed permafrost. Metatranscriptomics is shown here to be a useful approach for inferring the activity of permafrost microbes that has potential to improve our understanding of permafrost SOM bioavailability and biogeochemical mechanisms contributing to greenhouse gas emissions as a result of permafrost thaw.

The transcriptional response of microbial communities in thawing Alaskan permafrost soils

PubMed Central

Coolen, Marco J. L.; Orsi, William D.

2015-01-01

Thawing of permafrost soils is expected to stimulate microbial decomposition and respiration of sequestered carbon. This could, in turn, increase atmospheric concentrations of greenhouse gasses, such as carbon dioxide and methane, and create a positive feedback to climate warming. Recent metagenomic studies suggest that permafrost has a large metabolic potential for carbon processing, including pathways for fermentation and methanogenesis. Here, we performed a pilot study using ultrahigh throughput Illumina HiSeq sequencing of reverse transcribed messenger RNA to obtain a detailed overview of active metabolic pathways and responsible organisms in up to 70 cm deep permafrost soils at a moist acidic tundra location in Arctic Alaska. The transcriptional response of the permafrost microbial community was compared before and after 11 days of thaw. In general, the transcriptional profile under frozen conditions suggests a dominance of stress responses, survival strategies, and maintenance processes, whereas upon thaw a rapid enzymatic response to decomposing soil organic matter (SOM) was observed. Bacteroidetes, Firmicutes, ascomycete fungi, and methanogens were responsible for largest transcriptional response upon thaw. Transcripts indicative of heterotrophic methanogenic pathways utilizing acetate, methanol, and methylamine were found predominantly in the permafrost table after thaw. Furthermore, transcripts involved in acetogenesis were expressed exclusively after thaw suggesting that acetogenic bacteria are a potential source of acetate for acetoclastic methanogenesis in freshly thawed permafrost. Metatranscriptomics is shown here to be a useful approach for inferring the activity of permafrost microbes that has potential to improve our understanding of permafrost SOM bioavailability and biogeochemical mechanisms contributing to greenhouse gas emissions as a result of permafrost thaw. PMID:25852660

Embolism Formation during Freezing in the Wood of Picea abies1

PubMed Central

Mayr, Stefan; Cochard, Hervé; Améglio, Thierry; Kikuta, Silvia B.

2007-01-01

Freeze-thaw events can cause embolism in plant xylem. According to classical theory, gas bubbles are formed during freezing and expand during thawing. Conifers have proved to be very resistant to freeze-thaw induced embolism, because bubbles in tracheids are small and redissolve during thawing. In contrast, increasing embolism rates upon consecutive freeze-thaw events were observed that cannot be explained by the classical mechanism. In this study, embolism formation during freeze-thaw events was analyzed via ultrasonic and Cryo-scanning electron microscope techniques. Twigs of Picea abies L. Karst. were subjected to up to 120 freeze-thaw cycles during which ultrasonic acoustic emissions, xylem temperature, and diameter variations were registered. In addition, the extent and cross-sectional pattern of embolism were analyzed with staining experiments and Cryo-scanning electron microscope observations. Embolism increased with the number of freeze-thaw events in twigs previously dehydrated to a water potential of −2.8 MPa. In these twigs, acoustic emissions were registered, while saturated twigs showed low, and totally dehydrated twigs showed no, acoustic activity. Acoustic emissions were detected only during the freezing process. This means that embolism was formed during freezing, which is in contradiction to the classical theory of freeze-thaw induced embolism. The clustered pattern of embolized tracheids in cross sections indicates that air spread from a dysfunctional tracheid to adjacent functional ones. We hypothesize that the low water potential of the growing ice front led to a decrease of the potential in nearby tracheids. This may result in freezing-induced air seeding. PMID:17041033

Royal jelly supplemented soybean lecithin-based extenders improve post-thaw quality and incubation resilience of goat spermatozoa.

PubMed

Alcay, Selim; Toker, M Berk; Onder, N Tekin; Gokce, Elif

2017-02-01

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thaw quality and incubation resilience of goat spermatozoa. Semen samples were collected from five goats. Pooled semen were diluted with soybean lecithin-based extender without RJ (control) or supplemented with different concentrations (0.25, 0.5 and 0.75%) of RJ (RJ0.25, RJ0.5, RJ0.75 respectively), at a final concentration of 150 × 10 6 spermatozoon/mL. Semen samples were assessed for sperm motility, plasma membrane integrity using hypoosmotic swelling test (HOST) damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The addition of RJ (0.5%, 0.75%) led to higher percentages of subjective motilities (55.33 ± 2.29%, 57.67 ± 2.58%) compared to control and RJ0.25 groups (49.00 ± 2,80%, 51.67 ± 3.09%) (P < 0.05) following the freeze-thawing process. RJ0.5 and RJ0.75 groups had higher plasma membrane functional integrities (66.40 ± 1.34%, 68.20 ± 2.05%) and lower defected acrosome rates (24.60 ± 3.36%, 23.80 ± 2.27%) compared to the other groups (P < 0.05). DNA damaged spermatozoa in all groups were not significant (P > 0.05). In the end of incubation, motility and HOST rates of RJ0.5 (14.00 ± 3.87%, 31.20 ± 3.70%) and RJ0.75 (15.00 ± 3.27%, 29.20 ± 2.59%) groups were higher than control (8.00 ± 2.54%, 18.20 ± 3.11%) and RJ0.25 (9.00 ± 2.07%, 20.60 ± 2.88%) groups (P < 0.05). Also defected acrosome and DNA fragmation rates of RJ0.5 (32.20 ± 1.30%, 5.4 ± 0.55%) and RJ0.75 (29.20 ± 1.30%, 5.80 ± 0.45%) groups were significantly lower than control (38.80 ± 0.84%, 7.40 ± 1.34%) and RJ0.25 (39.80 ± 2.05%, 7.00 ± 1.58) groups. This study shows that RJ supplemented extenders have beneficial effect on goat sperm parameters at 0 h and 6 h of incubation. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells.

PubMed

Cai, Guoping; Lai, Binbin; Hong, Huaxing; Lin, Peng; Chen, Weifu; Zhu, Zhong; Chen, Haixiao

2017-07-01

Cryopreservation is widely used in regenerative medicine for tissue preservation. In the present study, the effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells (HUVECs) were investigated. After 0, 4, 8, 12 or 24 weeks of cryopreservation in liquid nitrogen, the HUVECs were thawed. The excretory functions markers (endothelin‑1, prostaglandin E1, von Willebrand factor and nitric oxide) of HUVECs were measured by ELISA assay. The expression of intercellular adhesion molecule‑1 (ICAM‑1) in HUVECs was analyzed using flow cytometry. An angiogenesis assay was used to determine the angiogeneic capabilities of the thawed HUVECs. The results demonstrated that cryopreserved/thawed and recultivated HUVECs were unsuitable for tissue‑engineered microvascular construction. Specifically, the excretory function of the cells was significantly decreased in the post‑cryopreserved HUVECs at 24 weeks. In addition, the level of ICAM‑1 in HUVECs was significantly upregulated from the fourth week of cryopreservation. Furthermore, the tube‑like structure‑forming potential was weakened with increasing cryopreservation duration, and the numbers of lumen and the length of the pipeline were decreased in the thawed HUVECs, in a time‑dependent manner. In conclusion, the results of the present study revealed that prolonged cryopreservation may lead to HUVEC dysfunction and did not create stable cell lines for tissue‑engineered microvascular construction.

Birth of kids after artificial insemination with sex-sorted, frozen-thawed goat spermatozoa.

PubMed

Bathgate, R; Mace, N; Heasman, K; Evans, G; Maxwell, W M C; de Graaf, S P

2013-12-01

Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX(®) ) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris-citrate-glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids. © 2013 Blackwell Verlag GmbH.

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics.

PubMed

Purdy, P H; Tharp, N; Stewart, T; Spiller, S F; Blackburn, H D

2010-10-15

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility. Copyright © 2010 Elsevier Inc. All rights reserved.

Effect of green tea (Camellia sinensis) extract and pre-freezing equilibration time on the post-thawing quality of ram semen cryopreserved in a soybean lecithin-based extender.

PubMed

Mehdipour, Mahdieh; Daghigh Kia, Hossein; Najafi, Abouzar; Vaseghi Dodaran, Hossein; García-Álvarez, Olga

2016-12-01

The aim of this study was to determine the effect of Camellia sinensis extract as antioxidant supplement and pre-freezing equilibration times in a soybean lecithin extender for freezing ram semen. In this study, a total of 20 ejaculates were collected from four Ghezel rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and Camellia sinensis extract (5, 10, and 15 mg/L) and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 4 h after equilibration. Sperm motility characteristics, membrane integrity, abnormal morphology, mitochondria activity, apoptotic status, MDA and antioxidant activities (GPx, SOD and total antioxidant capacity (TAC)) were evaluated following freeze-thawing. Camellia sinensis extract at level 10 mg/L led to the highest total and progressive motilities percentages, in comparison to other treatments (P < 0.05). Our results showed that Camellia sinensis extract at level of 5 and 10 mg/L led to higher plasma membrane integrity, mitochondria activity and Total antioxidant capacity (TAC) in comparison to the level of 15 mg/L and control group (P < 0.05). Camellia sinensis extract at 10 mg/L level produced the highest percentage of live spermatozoa and the lowest apoptotic spermatozoa in comparison to all treatments (P < 0.05). In addition, level of MDA formation significantly decreased at this concentration, 10 mg/L, compared to all treatments (P < 0.05). No differences (P > 0.05) were observed between equilibration times (0 h vs. 4 h) for sperm samples incubated with or without different concentrations of Camellia sinensis extract. In conclusion, addition of Camellia sinensis extract at level of 10 mg/L can improve post-thawing quality of ram semen cryopreserved in a soybean lecithin extender. However, further research is needed to standardize the process of Camellia sinensis extraction and specially for identifying which compounds are responsible of its beneficial effect on ram sperm cryopreservation. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of holding and equilibration time on post-thaw quality of shipped boar semen.

PubMed

Schäfer, J; Waberski, D; Jung, M; Schulze, M

2017-12-01

Cryopreservation of boar semen is of growing interest for breeding companies. Overnight-shipping of pre-diluted ejaculates to specialized laboratories offers a practicable method, but requires fine-tuned protocols. In this study, the impact of holding post shipping at 17°C for 2 or 24h (n=10 samples) and of equilibration in lactose-egg yolk extender without glycerol at 5°C for 2, 4, 24 or 48h (n=11 samples) before freezing was investigated. Sperm-rich fractions of ejaculates from 21 mature Pietrain boars were collected at a single boar stud. After pre-dilution (1+1, v:v) with Beltsville thawing solution, samples were sent to the laboratory. Temperature profiles during transport and initial equilibration time were recorded. Semen quality post-thaw (PT) was evaluated using CASA and flow cytometry. Holding of 2h after shipping resulted in higher sperm motility (P=0.013) and beat cross frequency (BCF; P=0.047) compared to 24h. Differences between both groups vanished with prolonged incubation at 38°C PT. Equilibration at 5°C for 4h yielded the highest motility and BCF, whereas the equilibration for 48h impaired sperm motility. Membrane integrity, mitochondrial activity and DNA fragmentation index were not affected by any protocol modification. In conclusion, processing of pre-diluted boar semen shipped overnight within 2h after arrival at the laboratory is preferred to 24h of additional holding at 17°C. Extending the equilibration period in lactose-egg yolk extender without glycerol at 5°C from 2h to 4h before freezing is recommended. Copyright © 2017 Elsevier B.V. All rights reserved.

Sperm cryopreservation of African catfish, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio.

PubMed

Viveiros, A T; So, N; Komen, J

2000-12-01

Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clarias gariepinus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2,000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degrees C/min) to various endpoint temperatures (range -25 to -70 degrees C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (P > 0.05) were obtained by spermatozoa frozen at -5 degrees C/min to -45 to -50 degrees C and at -10 degrees C/min to -55 degrees C. In 3-step freezing programs, at -5 degrees C/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degrees C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P > 0.05) were produced by spermatozoa frozen to, and held at, -35 degrees C for 5 min and at -40 degrees C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5 degrees C/min, 5-min hold at -40 degrees C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization conditions. Again, no difference (P > 0.05) in hatching rate was observed between frozen and fresh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish.

Consequences of uncontrolled cooling during sterlet (Acipenser ruthenus) sperm cryopreservation on post-thaw motility and fertilizing ability.

PubMed

Horokhovatskyi, Yevhen; Rodina, Marek; Asyabar, Hadiseh Dadras; Boryshpolets, Sergii; Dzyuba, Borys

2017-06-01

The significant influence of the number and position of fish sperm sample straws in uncontrolled cooling devices on post-thaw spermatozoa parameters, such as motility and fertilizing ability, is presented in this study. The two most popular uncontrolled cooling devices were used in this study: a Styrofoam box setup with a polystyrene floating raft on liquid nitrogen and the dry shipper setup with a straw holder. We tested the effect of different quantities of straws (6 or 60) placed on the polystyrene floating raft and the position of the straws in the holder (on the periphery or in the centre). Using these cooling methods, sperm of 10 male sterlets diluted with methanol containing cryoprotective medium was frozen. All temperature changes were recorded by a thermocouple inside the straw, and the thermogram intervals were analysed. Spermatozoa motility was evaluated by video microscopy with integrated computer-assisted sperm analysis software. Fertilization trials were conducted at a 10 5 spermatozoa/egg ratio. Post-thaw spermatozoa parameters, including the percent of motile spermatozoa, curvilinear velocity, velocity according to the smoothed path, linearity of track, beat-cross frequency and fertilization rate, were significantly decreased in the 60-straw floating raft setup in comparison to all of the other cooling methods. The freezing rate between -10 °C and -30 °C was significantly decreased by up to 18.6 ± 0.61 °C/min for the 60-straw floating raft setup in comparison to the other freezing conditions. Considering the above results, efforts to standardize cryopreservation protocols using uncontrolled cooling devices should take into account the amount of straws subjected to freezing. Copyright © 2017. Published by Elsevier Inc.

Cryoprotective role of organic Zn and Cu supplementation in goats (Capra hircus) diet.

PubMed

Arangasamy, Arunachalam; Krishnaiah, Mayasula Venkata; Manohar, Narasimhaiah; Selvaraju, Sellappan; Rani, Guvvala Pushpa; Soren, Nira Manik; Reddy, Ippala Janardhan; Ravindra, Janivara Parameshwaraiah

2018-04-01

The current study focused on cryopreservation and assessment of characters of post-thaw semen of indigenous Osmanabadi bucks maintained with standard diet, supplemented with different concentrations of organic zinc (Zn), copper (Cu) or in combination, for a period of 180 days. The different doses of organic Zn and Cu were fed per kg DM basis, Zn groups (low: Zn20, medium: Zn40 and high: Zn60), Cu groups: (low: Cu12.5, medium: Cu25 and high: Cu37.5) and combination of Zn + Cu groups (low: Zn20 + Cu12.5, medium: Zn40 + Cu25 and high: Zn60 + Cu37.5) respectively. The control group bucks were maintained mainly on the basal diet without any additional mineral supplementation. Two hundred and forty (240) semen samples were collected from 40 bucks aged 11 months, through electro ejaculator method, processed and analysed for sperm quality parameters both at pre freeze and post-thaw stage. The semen samples were diluted in Tris egg yolk extender, cooled and equilibrated for 4 h at 5 °C, cryopreserved using programmable freezer (PLANER Kryo 360-1.7) and stored at -196 °C. The organic trace minerals (Zn, Cu and Zn + Cu) protected the spermatozoa against the cryoinjury and maintained higher post-thaw semen parameters except in high Zn group. Additional feeding of organic Cu and Zn to bucks had a protective role and resulted in higher sperm liveability, plasma membrane and acrosome integrities, motility and velocity and reduced oxidative stress in supplemented goats (P < 0.05). Copyright © 2018 Elsevier Inc. All rights reserved.

Development of soya milk extender for semen cryopreservation of Karan Fries (crossbreed cattle).

PubMed

Singh, V K; Singh, A K; Kumar, R; Atreja, S K

2013-01-01

Egg yolk based semen extenders are used widely, with the potential risk of xenobiotic contamination. This study was designed to develop a soya milk based extender to substitute egg yolk based extender for bovine semen cryopreservation. In the first experiment soya milk was prepared from fresh soya bean (Glycine max). Concentration of soya milk in tris based extender was standardized based on quality parameters of spermatozoa during liquid preservation at 5°C up to 72 h and compared with egg yolk tris (EYT) extender. Sperm in soya milk tris (SMT) extender with 25 percent soya milk showed no significant (P > 0.05) differences in all the quality parameters like motility, viability, membrane integrity and acrosome integrity, as compared to sperm in EYT extender up to 72h in liquid dilution. In the second experiment the Karan Fries semen was cryopreserved in SMT extender with 25 percent soya milk (selected from the first experiment) using different concentration of glycerol, as cryoprotectant, ranging from 6-7 percent with a difference of 0.2 percent to standardize optimum concentration based on post thaw motility of spermatozoa. Glycerol at a final concentration of 6.4 percent was found to be the best among all. Further, semen samples were split and cryopreserved in newly developed SMT extender containing 6.4 percent glycerol and compared with conventional EYT extender for post thaw sperm quality parameters and degree of cryocapacitation. There were no significant (P > 0.05) differences between sperm in EYT extender and SMT extender for post thaw motility, viability, membrane integrity, acrosome integrity and cryocapacitation. In conclusion, the newly developed SMT extender maintained comparable semen quality as compared to EYT extender hence it can.

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas).

PubMed

Robeck, T R; Steinman, K J; Montano, G A; Katsumata, E; Osborn, S; Dalton, L; Dunn, J L; Schmitt, T; Reidarson, T; O'Brien, J K

2010-10-01

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean +/- SD) 73.0 +/- 12.2, 38.4 +/- 8.8, 88.0 +/- 0.1, and 59.3 +/- 15.7%, respectively. A series of GnRH injections (3 x 250 microg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 +/- 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 +/- 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 +/- 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 +/- 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 +/- 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species. (c) 2010 Elsevier Inc. All rights reserved.

The cryoprotective effect of trehalose supplementation on boar spermatozoa quality.

PubMed

Hu, J-H; Li, Q-W; Jiang, Z-L; Yang, H; Zhang, S-S; Zhao, H-W

2009-08-01

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200 mm), and try to determine the optimum concentration of trehalose. We chose sperm motility, mitochondrial activity, acrosome integrity and membrane integrity as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100 mm trehalose-supplemented extenders, with values of 49.89% for motility, 44.69% for mitochondrial activity, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance diminished significantly for 200 . The synergic effect of trehalose and glycerol resulted in better cryosurvival of boar spermatozoa than that of a single cryoprotectant. In conclusion, when trehalose-supplementation was added up to 100 mm, trehalose confers a greater cryoprotective capacity to the extender, and the sperm motility, mitochondrial activity, membrane integrity and acrosome integrity parameters were significantly improved during frozen-thawed process.

Improvement strategies in ovine artificial insemination.

PubMed

Anel, L; Alvarez, M; Martinez-Pastor, F; Garcia-Macias, V; Anel, E; de Paz, P

2006-10-01

Artificial insemination in ram is scarcely widespread comparing with other domestic species. This has been due not only to fertility results being irregular and low but also because of the difficulty in the application of enhancements such as the use of frozen-thawed sperm. Although there is a lot of information on the use of different options to improve these AI results (such as transcervical application, the use of thawed sperm, etc.) commercial programmes can be classified on two general categories: those using refrigerated semen (15 degrees C) by superficial intracervical deposition (vaginal), and, more restricted, those using thawed sperm by intrauterine deposition (laparoscopy). In the present work, we have summarized our viewpoint on three general research lines for the improvement of AI results in sheep: semen preservation, AI procedures and semen assessment. Briefly, in ram it is necessary to develop a medium term methodology of sperm refrigeration (3-5 days), which would allow the distribution of sperm doses to a widespread area. Nevertheless, it is also necessary to develop an intrauterine transcervical AI technique, which allows thawed semen to be applied by vaginal insemination. Besides, the low predictive value of classic assessment techniques limits the ability to adjust the number of spermatozoa per dose according to its actual fertility.

Influence of Rapid Freeze-Thaw Cycling on the Mechanical Properties of Sustainable Strain-Hardening Cement Composite (2SHCC)

PubMed Central

Jang, Seok-Joon; Rokugo, Keitetsu; Park, Wan-Shin; Yun, Hyun-Do

2014-01-01

This paper provides experimental results to investigate the mechanical properties of sustainable strain-hardening cement composite (2SHCC) for infrastructures after freeze-thaw actions. To improve the sustainability of SHCC materials in this study, high energy-consumptive components—silica sand, cement, and polyvinyl alcohol (PVA) fibers—in the conventional SHCC materials are partially replaced with recycled materials such as recycled sand, fly ash, and polyethylene terephthalate (PET) fibers, respectively. To investigate the mechanical properties of green SHCC that contains recycled materials, the cement, PVA fiber and silica sand were replaced with 10% fly ash, 25% PET fiber, and 10% recycled aggregate based on preliminary experimental results for the development of 2SHCC material, respectively. The dynamic modulus of elasticity and weight for 2SHCC material were measured at every 30 cycles of freeze-thaw. The effects of freeze-thaw cycles on the mechanical properties of sustainable SHCC are evaluated by conducting compressive tests, four-point flexural tests, direct tensile tests and prism splitting tests after 90, 180, and 300 cycles of rapid freeze-thaw. Freeze-thaw testing was conducted according to ASTM C 666 Procedure A. Test results show that after 300 cycles of freezing and thawing actions, the dynamic modulus of elasticity and mass loss of damaged 2SHCC were similar to those of virgin 2SHCC, while the freeze-thaw cycles influence mechanical properties of the 2SHCC material except for compressive behavior. PMID:28788522

Single-layer centrifugation through PureSperm® 80 selects improved quality spermatozoa from frozen-thawed dog semen.

PubMed

Dorado, J; Alcaraz, L; Gálvez, M J; Acha, D; Ortiz, I; Urbano, M; Hidalgo, M

2013-08-01

The aim of this study was to investigate whether single-layer centrifugation (SLC) with PureSperm® 80 could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from 5 dogs was collected and cryopreserved following a standard protocol. After thawing, semen samples were divided into two aliquots: one of them was used as control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining) and viability (triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123), were performed on aliquots of fresh semen, frozen-thawed control and frozen-thawed SLC treated samples. A multivariate clustering procedure separated 26,051 motile spermatozoa into three subpopulations (sP): sP1 consisting of highly active but non-progressive spermatozoa (40.3%), sP2 consisting of spermatozoa with high velocity and progressive motility (30.0%), and sP3 consisting of poorly active and non-progressive spermatozoa (29.7%). SLC with PureSperm® 80 yielded sperm suspensions with improved motility, morphology, viability and acrosome integrity (P<0.001). The frozen-thawed SLC treated samples were enriched in sP2, reaching a proportion of 44.1% of the present spermatozoa. From these results, we concluded that SLC with PureSperm® 80 may be an alternative and successful method for improving the quality of frozen-thawed dog spermatozoa. Moreover, sP2 (high-speed and progressive spermatozoa) was more frequently observed after SLC. Finally, this study also demonstrated that the general motile sperm structure present in dogs remained constant despite the effect caused by either cryopreservation or separation by SLC through PureSperm® 80. Copyright © 2013 Elsevier B.V. All rights reserved.

Nonsurgical Artificial Insemination in the Ewe

USDA-ARS?s Scientific Manuscript database

Artificial insemination (AI) is not readily utilized with sheep because of a lack of knowledge and infrastructure. Therefore, we investigated the effects of the timing of AI with a synchronized estrous on fertility and the impact of cryopreservation diluents on post-thaw sperm quality. The estrous...

Impact of long term cryopreservation on single umbilical cord blood transplant outcomes

PubMed Central

Mitchell, R.; Wagner, J.E.; Brunstein, C.G.; Cao, Q.; McKenna, D.H.; Lund, T.C.; Verneris, M.R.

2015-01-01

Umbilical cord blood (UCB) has the advantage of being collected and cryopreserved for years prior to use. In vitro or in murine models suggest that the duration of storage does not affect UCB progenitor cell performance, however the impact of UCB age on clinical outcomes has not been definitely defined. This study sought to determine the effect of UCB unit cryopreservation time on hematopoietic potency. We analyzed 288 single UCB units used for transplantation from 1992–2013, with unit cryopreservation time ranging from 0.08 to 11.07 years. UCB unit post thaw characteristics were examined, including percent recovery of total nucleated cells (TNC). The number of years the UCB unit spent in cryopreservation had no impact on TNC recovery nor UCB unit post-thaw viability. Duration of cryopreservation also had no impact on neutrophil or platelet engraftment in single UCB transplants. These results show that UCB units can undergo cryopreservation for at least 10 years with no impact on clinical outcomes. PMID:25262882

Review of Thawing Time Prediction Models Depending
on Process Conditions and Product Characteristics

PubMed Central

Kluza, Franciszek; Spiess, Walter E. L.; Kozłowicz, Katarzyna

2016-01-01

Summary Determining thawing times of frozen foods is a challenging problem as the thermophysical properties of the product change during thawing. A number of calculation models and solutions have been developed. The proposed solutions range from relatively simple analytical equations based on a number of assumptions to a group of empirical approaches that sometimes require complex calculations. In this paper analytical, empirical and graphical models are presented and critically reviewed. The conditions of solution, limitations and possible applications of the models are discussed. The graphical and semi--graphical models are derived from numerical methods. Using the numerical methods is not always possible as running calculations takes time, whereas the specialized software and equipment are not always cheap. For these reasons, the application of analytical-empirical models is more useful for engineering. It is demonstrated that there is no simple, accurate and feasible analytical method for thawing time prediction. Consequently, simplified methods are needed for thawing time estimation of agricultural and food products. The review reveals the need for further improvement of the existing solutions or development of new ones that will enable accurate determination of thawing time within a wide range of practical conditions of heat transfer during processing. PMID:27904387

New strategies of boar sperm cryopreservation: development of novel freezing and thawing methods with a focus on the roles of seminal plasma.

PubMed

Okazaki, Tetsuji; Shimada, Masayuki

2012-09-01

Cryopreservation of boar spermatozoa offers an effective means of long-term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70-80%) can be achieved by AI with frozen-thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria-released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll-like receptor-4 (TLR-4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo-capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen-thawed boar sperm. © 2012 Japanese Society of Animal Science.

Post-wildfire landscape change and erosional processes from repeat terrestrial lidar in a steep headwater catchment, Chiricahua Mountains, Arizona, USA

NASA Astrophysics Data System (ADS)

DeLong, Stephen B.; Youberg, Ann M.; DeLong, Whitney M.; Murphy, Brendan P.

2018-01-01

Flooding and erosion after wildfires present increasing hazard as climate warms, semi-arid lands become drier, population increases, and the urban interface encroaches farther into wildlands. We quantify post-wildfire erosion in a steep, initially unchannelized, 7.5 ha headwater catchment following the 2011 Horseshoe 2 Fire in the Chiricahua Mountains of southeastern Arizona. Using time-lapse cameras, rain gauges, and repeat surveys by terrestrial laser scanner, we quantify the response of a burned landscape to subsequent precipitation events. Repeat surveys provide detailed pre-and post-rainfall measurements of landscape form associated with a range of weather events. The first post-fire precipitation led to sediment delivery equivalent to 0.017 m of erosion from hillslopes and 0.12 m of erosion from colluvial hollows. Volumetrically, 69% of sediment yield was generated from hillslope erosion and 31% was generated from gully channel establishment in colluvial hollows. Processes on hillslopes included erosion by extensive shallow overland flow, formation of rills and gullies, and generation of sediment-laden flows and possibly debris flows. Subsequent smaller rain events caused ongoing hillslope erosion and local deposition and erosion in gullies. Winter freeze-thaw led to soil expansion, likely related to frost-heaving, causing a net centimeter-scale elevation increase across soil-mantled slopes. By characterizing landscape form, the properties of near-surface materials, and measuring both precipitation and landscape change, we can improve our empirical understanding of landscape response to environmental forcing. This detailed approach to studying landscape response to wildfires may be useful in the improvement of predictive models of flood, debris flow and sedimentation hazards used in post-wildfire response assessments and land management, and may help improve process-based models of landscape evolution.

Post-wildfire landscape change and erosional processes from repeat terrestrial lidar in a steep headwater catchment, Chiricahua Mountains, Arizona, USA

USGS Publications Warehouse

DeLong, Stephen B.; Youberg, Ann M.; DeLong, Whitney M.; Murphy, Brendan P.

2018-01-01

Flooding and erosion after wildfires present increasing hazard as climate warms, semi-arid lands become drier, population increases, and the urban interface encroaches farther into wildlands. We quantify post-wildfire erosion in a steep, initially unchannelized, 7.5 ha headwater catchment following the 2011 Horseshoe 2 Fire in the Chiricahua Mountains of southeastern Arizona. Using time-lapse cameras, rain gauges, and repeat surveys by terrestrial laser scanner, we quantify the response of a burned landscape to subsequent precipitation events. Repeat surveys provide detailed pre-and post-rainfall measurements of landscape form associated with a range of weather events. The first post-fire precipitation led to sediment delivery equivalent to 0.017 m of erosion from hillslopes and 0.12 m of erosion from colluvial hollows. Volumetrically, 69% of sediment yield was generated from hillslope erosion and 31% was generated from gully channel establishment in colluvial hollows. Processes on hillslopes included erosion by extensive shallow overland flow, formation of rills and gullies, and generation of sediment-laden flows and possibly debris flows. Subsequent smaller rain events caused ongoing hillslope erosion and local deposition and erosion in gullies. Winter freeze-thaw led to soil expansion, likely related to frost-heaving, causing a net centimeter-scale elevation increase across soil-mantled slopes. By characterizing landscape form, the properties of near-surface materials, and measuring both precipitation and landscape change, we can improve our empirical understanding of landscape response to environmental forcing. This detailed approach to studying landscape response to wildfires may be useful in the improvement of predictive models of flood, debris flow and sedimentation hazards used in post-wildfire response assessments and land management, and may help improve process-based models of landscape evolution.

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders.

PubMed

Gadea, Joaquín; Sellés, Elena; Marco, Marco Antonio; Coy, Pilar; Matás, Carmen; Romar, Raquel; Ruiz, Salvador

2004-08-01

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.

ROCK inhibitor Y-27632 increases thaw-survival rates and preserves stemness and differentiation potential of human Wharton's jelly stem cells after cryopreservation.

PubMed

Gauthaman, Kalamegam; Fong, Chui-Yee; Subramanian, Arjunan; Biswas, Arijit; Bongso, Ariff

2010-12-01

The ROCK inhibitor Y-27632 inhibits apoptosis and increases proliferation of frozen-thawed cells. We examined the role of Y-27632 on human umbilical cord Wharton's jelly stem cells (hWJSCs) for (1) thaw-survival (2) proliferation and (3) preservation of stemness and differentiation potential after cryopreservation. hWJSCs were allotted to 4 groups [Gp I: Untreated hWJSC controls; Gp II: Pretreatment with Y-27632 (10 μM) for 24 h before freezing; Gp III: Y-27632 (10 μM) in freezing medium and Gp IV: Pretreatment with Y-27632 (10 μM) for 24 h and inclusion in freezing medium]. All groups were frozen using a rapid freezing method and stored at -196°C in liquid nitrogen for 90 days before evaluation for apoptosis, cell proliferation, stemness and differentiation. After thawing, Groups II, III and IV showed improved cell attachment, increased thaw-survival (live/dead cell counts) and increased cell proliferation (Trypan blue and MTT assay) compared to controls. CD marker stemness profiles, morphology and normal karyotypes were maintained in the treatment groups after thawing and there was no obvious evidence of apoptosis (Annexin V-FITC and TUNEL assays). After thawing, qRT-PCR demonstrated up-regulation of the anti-apoptotic BCL2 gene and down-regulation of the pro-apoptotic BAX gene and cell cycle regulators (P53 and P21) in the treatment groups. Treated frozen-thawed hWJSCs from all groups differentiated into a neuronal phenotype (neuronal morphology and expression of GFAP, β-3 tubulin and SOX2). Increased thaw-survival and retention of stemness and differentiation potential in hWJSCs following cryopreservation is useful for their storage in cord blood banks for future regenerative medicine purposes.

In vitro culture thawed human ovarian tissue: NIV versus slow freezing method.

PubMed

Xiao, Zhun; Wang, Yan; Li, Ling-Ling; Li, Shang-wei

2013-01-01

The aim of this study was to determine if the needle immersed vitrification method (NIV) can improve the growth potential of thawed ovarian tissue in vitro culture. Human ovarian cortical tissues were cryopreserved using NIV and slow freezing method. After 14 days of culture, the preservation outcomes of NIV and slow freezing groups were analyzed histologically using light microscope and apoptosis was assessed by TUNEL assay. The result showed that the percentage of morphologically abnormal primordial follicles was lower in NIV group than in slow freezing group (P < 0.05). The incidence of TUNEL-positive primordial follicles was lower in NIV group than in slow freezing group (P < 0.05). The study showed that cryopreservation of human ovarian tissue with NIV was effective in improving the growth potential of frozen-thawed ovarian tissue in vitro culture.

Hyaluronic acid improves frozen-thawed sperm quality and fertility potential in rooster.

PubMed

Lotfi, Saied; Mehri, Morteza; Sharafi, Mohsen; Masoudi, Reza

2017-09-01

Beneficial effects of Hyaluronic acid (HA) has not been yet assessed for cryopreservation of rooster sperm. This study was conducted to evaluate the effects of different concentrations of HA (0, 1, 2, 4 and 8mM) in Beltsville extender on the cryopreservation of rooster sperm. Semen samples were collected from six Ross broiler breeders (24-week) using abdominal massage, then divided into five equal aliquots and cryopreserved in Beltsville extender that contained different concentrations of HA. Motion characteristics, morphology, membrane functionality, viability, acrosome integrity, lipid peroxidation and fertility potential of sperm were assessed after thawing. HA at concentration of 2mM (HA2) resulted in the highest (P<0.05) total motility (55.3±1.1%) and progressive motility (25.2±0.8%) compared to the other groups. HA8 produced the lowest significant (P<0.05) percentage of total (38.6±1.1%) and progressive (14.7±0.8%) motility. High significant percentage of membrane functionality were observed in HA1 and HA2 (43.2±1.0 and 46.1±1.0%, respectively) compared to HA4 (40.1±1.0%) and HA8 (32.5±1.0%). Moreover, HA1 and HA2 produced the higher percentage of acrosome integrity (54.8±1.2 and 57.5±1.2, respectively) compared to other groups. HA1 and HA2 reduced (P<0.05) malondialdehyde formation (3.66±0.08 and 3.75±0.08 nmol/ml) compared to other groups. Fertility rate and hatching rate obtained from artificial insemination were significantly higher in HA1 (63.7 and 54.7%) and HA2 (67.5 and 57.7%) compared to control group (40 and 37%). Our results showed that supplementation of Beltsville extender with 1 and 2mM HA significantly improved the quality of rooster sperm after freeze thawing. Copyright © 2017 Elsevier B.V. All rights reserved.

The effect of permafrost thaw on short- and long-term carbon accumulation in permafrost mires

NASA Astrophysics Data System (ADS)

Olid, Carolina; Klaminder, Jonatan; Monteux, Sylvain; Johansson, Margareta; Dorrepaal, Ellen

2017-04-01

Permafrost stores twice as much carbon (C) as is currently present in the atmosphere. During recent years, warmer temperatures in the Arctic has caused rapid thawing of permafrost, which have dramatically altered permafrost C storage by increasing both microbial decomposition and plant productivity. Although current research focuses on the effects of climate change on these two processes, there are still no scientific consensus about the magnitude or even the direction of future C feedbacks from permafrost ecosystems. Field manipulation experiments have been widely used during the last decade to improve our knowledge about the net effects of permafrost thaw in the permafrost C storage. However, due to the slow response (decades) of permafrost ecosystems to environmental changes and the short-time nature of these experiments (usually shorter than 5-9 years), there are still concerns when attempting to extrapolate the results to predict long term effects. In addition, measurements are mostly taken exclusively during the summer season, without taking into account inter-annual variability in C fluxes and underestimating microbial activity throughout the cold season. The need to develop a comprehensive understanding of C fluxes over the entire year and at long temporal scales sets the basis of this study. This study aims to quantify the effects of permafrost thawing in permafrost C fluxes using a 12 years permafrost thaw experiment in northern Sweden. Our aims were to quantify the effect of permafrost thaw in both decomposition and primary production in active layer and newly thawed permafrost, and its implications for the C balance. Based on previous observations, we hypothesized that 1) soil decomposition rates were higher in manipulated thaw plots. However, 2) the observed increase in nutrients availability and the higher presence of vascular plants after thawing stimulate primary production, which compensates to some extent the increased C losses by respiration. To validate these hypotheses, an empirically based peat development model was applied to peat cores collected from manipulated thaw (n=6) and control (n=6) plots. Short- (decades) and long-(centuries) term C accumulation rates were estimated using a combined 210Pb and 14C chronology. In contrast to previous studies, our approach is long term and allows applying an empirical mass balance to evaluate depth-explicit changes in C inputs, C losses, and net C accumulation rates in response to permafrost thaw. Comparing shallow versus deep soil C does not only reflect short versus long-term C dynamics but also shows how the responses vary depending on different soil conditions. Our goal is to provide insights to better understand permafrost C dynamics to improve theoretical and predictive climate impact models.

Value of a quality assessment program in optimizing cryopreservation of peripheral blood mononuclear cells in a multicenter study.

PubMed

Aziz, Najib; Margolick, Joseph B; Detels, Roger; Rinaldo, Charles R; Phair, John; Jamieson, Beth D; Butch, Anthony W

2013-04-01

Cryopreservation of peripheral blood mononuclear cells (PBMC) allows assays of cellular function and phenotype to be performed in batches at a later time on PBMC at a central laboratory to minimize assay variability. The Multicenter AIDS Cohort Study (MACS) is an ongoing prospective study of the natural and treated history of human immunodeficiency virus (HIV) infection that stores cryopreserved PBMC from participants two times a year at four study sites. In order to ensure consistent recovery of viable PBMC after cryopreservation, a quality assessment program was implemented and conducted in the MACS over a 6-year period. Every 4 months, recently cryopreserved PBMC from HIV-1-infected and HIV-1-uninfected participants at each MACS site were thawed and evaluated. The median recoveries of viable PBMC for HIV-1-infected and -uninfected participants were 80% and 83%, respectively. Thawed PBMC from both HIV-1-infected and -uninfected participants mounted a strong proliferative response to phytohemagglutinin, with median stimulation indices of 84 and 120, respectively. Expression of the lymphocyte surface markers CD3, CD4, and CD8 by thawed PBMC was virtually identical to what was observed on cells measured in real time using whole blood from the same participants. Furthermore, despite overall excellent performance of the four participating laboratories, problems were identified that intermittently compromised the quality of cryopreserved PBMC, which could be corrected and monitored for improvement over time. Ongoing quality assessment helps laboratories improve protocols and performance on a real-time basis to ensure optimal cryopreservation of PBMC for future studies.

Comparison of two methodologies for the enrichment of mononuclear cells from thawed cord blood products: The automated Sepax system versus the manual Ficoll method.

PubMed

Kaur, Indreshpal; Zulovich, Jane M; Gonzalez, Marissa; McGee, Kara M; Ponweera, Nirmali; Thandi, Daljit; Alvarez, Enrique F; Annandale, Kathy; Flagge, Frank; Rezvani, Katayoun; Shpall, Elizabeth

2017-03-01

Umbilical cord blood (CB) is being used as a source of hematopoietic stem cells (HSCs) and immune cells to treat many disorders. Because these cells are present in low numbers in CB, investigators have developed strategies to expand HSCs and other immune cells such as natural killer (NK) cells. The initial step in this process is to enrich mononuclear cells (MNCs) while depleting unwanted cells. The manual method of MNC enrichment is routinely used by many centers; however, it is an open system, time-consuming and operator dependent. For clinical manufacturing, it is important to have a closed system to avoid microbial contamination. In this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units. Using Sepax, we observed higher recovery of total nucleated cells (TNC), CD34 + cells, NK cells and monocytes when compared to manual enrichment, despite similar TNC and CD34 + viability with the two methods. Even though the depletion of red blood cells, granulocytes and platelets was superior using the manual method, significantly higher CFU-GM were obtained in MNCs enriched using Sepax compared to the manual method. This is likely related to the fact that the automated Sepax significantly shortened the processing time (Sepax: 74 - 175 minutes versus manual method: 180 - 290 minutes). The use of DNAse and MgCl 2 during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery. We optimized enrichment of MNCs from cryopreserved CB products in a closed system using the Sepax which is a walk away and automated processing system. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Effects of straw mulching on soil evaporation during the soil thawing period in a cold region in northeastern China

NASA Astrophysics Data System (ADS)

Fu, Qiang; Yan, Peiru; Li, Tianxiao; Cui, Song; Peng, Li

2018-04-01

To study the effect of straw mulching on soil water evaporation, it is necessary to measure soil water evaporation under different conditions of straw mulching during the soil thawing period. A field experiment was conducted in winter, and soil evaporation was measured using a microlysimeter on bare land (LD) and 4500 (GF4500), 9000 (GF9000) and 13500 kg/hm2 (GF13500) straw mulch. The influence of different quantities of straw mulch on soil water evaporation during the thawing period was analyzed using the Mallat algorithm, statistical analysis and information cost function. The results showed that straw mulching could delay the thawing of the surface soil by 3-6 d, decrease the speed at which the surface soil thaws by 0.40-0.80 cm/d, delay the peak soil liquid water content, increase the soil liquid water content, reduce the cumulative evaporation by 2.70-7.40 mm in the thawing period, increase the range of soil evaporation by 0.04-0.10 mm in the early stage of the thawing period, and reduce the range of soil evaporation by 0.25-0.90 mm in the late stage of the thawing period. Straw mulching could reduce the range of and variation in soil evaporation and can reduce the effect of random factors on soil evaporation. When the amount of straw mulch exceeded 9000 kg/hm2, the effect of increasing the amount of straw mulch on daily soil water evaporation was small.

Developmental consequences of cryopreservation of mammalian oocytes and embryos.

PubMed

Smith, Gary D; Silva E Silva, Cristine Ane

2004-08-01

During the last three decades, significant advances have been made in successful cryopreservation of mammalian preimplantation embryos, and more recently oocytes. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte/embryo cryopreservation success has increased over time, there is still room for improvement. Oocytes and embryos are susceptible to cryo-damage, which collectively entails cellular damage caused by mechanical, chemical, or thermal forces during the freeze-thaw process. Basic studies focused on understanding cellular structures, their composition, and more importantly their functions, in normal cell developments will continue to be critical in assessing, understanding, and correcting oocyte/embryo cryo-damage. This review will delineate many of the oocyte/embryo intracellular and extracellular structures that are or may be compromised during cryopreservation. A global theme presented throughout this review is that many structural components of the oocyte/embryo also have essential functional roles in development. Compromising these cellular structures, and thus their cellular homeostatic functions, can deleteriously influence initial cryo-survival or compromise subsequent normal development through effects on the oocyte and/or early embryo.

Cryopreservation of boar semen in mini- and maxi-straws.

PubMed

Bwanga, C O; de Braganca, M M; Einarsson, S; Rodriguez-Martinez, H

1990-10-01

Split ejaculates from four boars were frozen with a programmable freezing machine, in mini- (0.25 ml) and maxi- (5 ml) plastic straws with an extender at either acidic (6.3) or alkaline (7.4) pH. Glycerol (3%) was used as cryoprotectant. The freezing of the semen was monitored by way of thermocouples placed in the straws. Post-thaw motility and acrosome integrity were evaluated; the latter using phase contrast microscopy, eosin-nigrosin stain and electron microscopy. Post-thaw sperm motility was significantly higher when semen was frozen in mini-straws than in maxi-straws. For the mini-straws, the motility was better when semen was exposed to an acidic environment during freezing, but this beneficial effect of the low extracellular pH was not evident when maxi-straws were thawed. The motility of the spermatozoa diminished significantly during the thermoresistance test (0 h and 2 h time) at 37 degrees C in a similar way for both straws and extracellular pH's. The freezing procedure, no matter the extracellular pH, did not cause major acrosomal damages, but significantly more normal apical ridges were present in the mini-straws than in the maxi-straws. This in vitro evaluation indicated that the freezing method employed was better for mini- than for maxi-straws since the freezing of the 5 ml volumes was not homogeneous, due to the large section area between the surface and the core of the straw.

Current biotechnologies and future possibilities in preservation of animal germplasm

USDA-ARS?s Scientific Manuscript database

Animal germplasm (semen, eggs, embryos, DNA, tissue) can be preserved from all agricultural species and result in the regeneration of animal lines or breeds. However, there are limitations to successful utilization of the material that are attributed to the type, post-thaw quality, and available as...

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

PubMed

Torres, Mariana Andrade; Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell'Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant'Anna; Sepúlveda, Néstor; de Andrade, André Furugen Cesar

2016-01-01

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

PubMed Central

Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell’Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant’Anna; Sepúlveda, Néstor

2016-01-01

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility. PMID:27529819

The Replacement of Monosaccharide by Mannitol or Sorbitol in the Freezing Extender Enhances Cryosurvival of Ram Spermatozoa.

PubMed

Wu, Guo Quan; Lv, Chun Rong; Jiang, Yan Ting; Wang, Si Yu; Shao, Qing Yong; Hong, Qiong Hua; Quan, Guo Bo

2016-10-01

In this study, the protective effects of monosaccharides (glucose and fructose) and sugar alcohols (mannitol, sorbitol, and xylitol) on frozen ram spermatozoa were evaluated and compared. The motility, moving velocity, and hypoosmotic swelling capability of spermatozoa frozen with monosaccharide or sugar alcohol were measured using a computer-assisted spermatozoa analyzer system. The acrosome status, membrane integrity, distribution of phosphatidylserine (PS), and mitochondrial membrane potential (MMP) were analyzed using fluorescence staining and flow cytometry. The results indicated that similar to glucose or fructose, the presence of sugar alcohol in the freezing extender cannot significantly improve the motility and moving velocity of ram spermatozoa equilibrated at 5°C. In terms of motility, pathway velocity, curve velocity, hypoosmotic swelling capability, acrosome and membrane integrity, and MMP, the inclusion of mannitol or sorbitol in the extender can significantly improve the quality of frozen-thawed ram spermatozoa compared to glucose or fructose. However, the effects of mannitol or sorbitol on linear velocity and PS distribution of frozen-thawed spermatozoa were similar to those of the monosaccharides (p > 0.05). In addition, the ability of xylitol to protect acrosome and maintain MMP in frozen-thawed spermatozoa was significantly higher compared with glucose or fructose (p < 0.05), although it could not improve the other evaluated parameters. Finally, there is no significant difference existing between mannitol and sorbitol with respect to the above evaluated parameters. In conclusion, the replacement of glucose or fructose by mannitol or sorbitol in a freezing extender can improve the postthaw quality of ram spermatozoa under specific freezing conditions. Moreover, the protective effects of mannitol and sorbitol on frozen-thawed ram spermatozoa are superior to that of xylitol. However, in the presence of sugar alcohols, the cryoinjury on spermatozoa membrane is still serious. In the future, the question of protecting the membrane of frozen-thawed spermatozoa needs further research.

Urea loading enhances freezing survival and postfreeze recovery in a terrestrially hibernating frog.

PubMed

Costanzo, Jon P; Lee, Richard E

2008-09-01

We tested the hypothesis that urea, an osmolyte accumulated early in hibernation, functions as a cryoprotectant in the freeze-tolerant wood frog, Rana sylvatica. Relative to saline-treated, normouremic (10 micromol ml(-1)) frogs, individuals rendered hyperuremic (70 micromol ml(-1)) by administration of an aqueous urea solution exhibited significantly higher survival (100% versus 64%) following freezing at -4 degrees C, a potentially lethal temperature. Hyperuremic frogs also had lower plasma levels of intracellular proteins (lactate dehydrogenase, creatine kinase, hemoglobin), which presumably escaped from damaged cells, and more quickly recovered neurobehavioral functions following thawing. Experimental freezing-thawing did not alter tissue urea concentrations, but did elevate glucose levels in the blood and organs of all frogs. When measured 24 h after thawing commenced, glucose concentrations were markedly higher in urea-loaded frogs as compared to saline-treated ones, possibly because elevated urea retarded glucose clearance. Like other low-molecular-mass cryoprotectants, urea colligatively reduces both the amount of ice forming within the body and the osmotic dehydration of cells. In addition, by virtue of certain non-colligative properties, it may bestow additional protection from freeze-thaw damage not afforded by glucose.

New aspects of boar semen freezing strategies.

PubMed

Grossfeld, R; Sieg, B; Struckmann, C; Frenzel, A; Maxwell, W M C; Rath, D

2008-11-01

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

PubMed Central

Jitraruch, Suttiruk; Hughes, Robin D.; Filippi, Celine; Lehec, Sharon C.; Glover, Leanne; Mitry, Ragai R.

2017-01-01

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine–tryptophan–ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use. PMID:28901189

Delayed soil thawing affects root and shoot functioning and growth in Scots pine.

PubMed

Repo, Tapani; Lehto, Tarja; Finér, Leena

2008-10-01

In boreal regions, soil can remain frozen after the start of the growing season. We compared relationships between root characteristics and water relations in Scots pine (Pinus sylvestris L.) saplings subjected to soil frost treatments before and during the first week of the growing period in a controlled environment experiment. Delayed soil thawing delayed the onset of sap flow or totally blocked it if soil thawing lagged the start of the growing period by 7 days. This effect was reflected in the electrical impedance of needles and trunks and in the relative electrolyte leakage of needles. Prolonged soil frost reduced or completely inhibited root growth. In unfrozen soil, limited trunk sap flow was observed despite unfavorable aboveground growing conditions (low temperature, low irradiance, short photoperiod). Following the earliest soil thaw, sap flow varied during the growing season, depending on light and temperature conditions, phenological stage of the plant and the amount of live needles in the canopy. The results suggest that delayed soil thawing can reduce tree growth, and if prolonged, it can be lethal.

Inclusion of seminal plasma in sperm cryopreservation of Iberian pig.

PubMed

Gómez-Fernández, José; Gómez-Izquierdo, Emilio; Tomás, Cristina; González-Bulnes, Antonio; Sánchez-Sánchez, Raúl; de Mercado, Eduardo

2012-01-01

The aim of the present study was to evaluate the inclusion of seminal plasma (SP) in the freezing extender, trying to preserve as much as possible of SP with spermatozoa from Iberian pigs, thus improving the conservation of animal genetic resources of this breed. Experiment 1, evaluated the effect of substituting water with SP as diluent in the freezing media in different proportions (0%, 10%, 25%, 50%, 75% and 100%), over pre-freezing (at 10°C and 5°C) and post-thawing sperm quality. The results showed that over 50% of SP in the extender, significantly decreased sperm quality in comparison to the control sample (0% SP) and the samples with 10% and 25% of SP (P<0.05). No significant differences were found between the control sample and the samples with 10% and 25% SP (P>0.05), but treatment with 25% did not show significant differences between the time of incubation at 37°C after thawing (P>0.05), showing greater sperm quality resistance over time. Experiment 2, evaluated the effect of prolonged incubation period, until 480min (simulating the lifespan of sperm in the female genital tract), of sperm samples with 0%, 10% and 25% of SP. Treatment with 25% of SP maintained better sperm quality over time, compared to control sample. Significant differences were observed especially in the parameters of motility analysis (TMS, total motile spermatozoa; PMS: progressive motility spermatozoa. P<0.05). In Experiment 3, the effect of the presence of SP was evaluated during the thawing process. Although some differences were observed between treatments, these differences were not as clear as the previous experiments. In conclusion, replacement of 25% of the water by SP as diluent in the freezing extender could be considered the maximum percentage of inclusion, without harmful effects to the sperm. In addition, this proportion of SP maintained Iberian sperm quality for longer time when it was present during the freezing and thawing process. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of cyclodextrin-loaded cholesterol conjugates on plasma membrane viability of Piau swine breed frozen/thawed spermatozoa.

PubMed

Pinho, R O; Lima, D M A; Shiomi, H H; Siqueira, J B; Silveira, C O; Faria, V R; Lopes, P S; Guimarães, S E F; Guimarães, J D

2016-08-01

The objective of this study was to investigate the effect of cyclodextrin-loaded cholesterol conjugates addition to freezing extenders on plasma membrane viability of frozen-thawed spermatozoa of the Piau swine breed. Twenty semen samples were used from five males. The freezing extender was based on lactose-egg yolk extender, added to 2% glycerol, 3% dimethylacetamide. The addition of cyclodextrin-loaded cholesterol conjugates was performed after centrifugation, when semen was diluted with the cooling extender. Four groups were subjected to the following treatment: without addition (group 1); 1.5 mg of cyclodextrin-loaded cholesterol/120 × 10(6) sperm (group 2); 1.5 mg of cyclodextrin-loaded cholestanol/120 × 10(6) sperm (group 3); 1.5 mg of cyclodextrin-loaded desmosterol/120 × 10(6) sperm (group 4). To check post-thawing sperm quality sperm motility and sperm morphology evaluation were used. Additionally, to check sperm viability the hypoosmotic swelling test, supravital staining, and fluorescent assay were used. The mean values recorded for total sperm motility of semen immediately after thawing were 54.5 ± 5.8, 55.5 ± 5.3, 53.7 ± 6.7, and 52.5 ± 6.6% respectively for groups one to four, without difference between themselves (p > 0.05). Regarding fluorescent assay the results were 28.3 ± 13.2, 26.9 ± 12.2, 22.2 ± 11.4, and 32.0 ± 15.3% respectively for groups one to four, also without difference between groups (p > 0,05). Similarly, complementary tests for evaluating the integrity and functionality of the plasma membrane showed no difference between treatments (p > 0.05). In conclusion, use of cyclodextrin-loaded cholesterol conjugates added to the plasma membrane of sperm did not demonstrate any additive effect on increasing and/or maintaining sperm motility. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of reduced glutathione supplementation in semen extender on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa.

PubMed

Shah, Nadeem; Singh, Vijay; Yadav, Hanuman Prasad; Verma, Meena; Chauhan, Dharmendra Singh; Saxena, Atul; Yadav, Sarvajeet; Swain, Dilip Kumar

2017-07-01

To provide new insights into the mechanisms through which reduced glutathione (GSH) is able to protect spermatozoa, we tested the hypothesis that cryocapacitation and apoptosis like changes can contribute to the negative effect of freezing and thawing on bull spermatozoa, and that GSH prevent this damage. Having known protective effects of GSH in terms of a potent antioxidant, we evaluated capacitation, tyrosine phosphorylation and apoptosis like changes in bull spermatozoa after freezing and thawing in egg yolk tris glycerol extender containing (0.5m M-GSH-T1 & 1mM GSH-T2) and without GSH serving as the control (C). Forty ejaculates were collected from four Hariana bulls and were pooled due to non significant variations among the bull ejaculates for the evaluation of sperm attributes. Capacitation like changes, tyrosine phosphorylation, localization of tyrosine phosphorylated proteins, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA fragmentation after final dilution, 4h of equilibration at 4°C and 24h after freezing and thawing were evaluated. GSH supplementation at 0.5mM showed significant reduction in B- and AR- pattern spermatozoa during all stages of semen freezing and thawing. Immunoblot revealed six proteins which were tyrosine phosphorylated and protein of 30 and 75kDa (p30, p75) were the major tyrosine phosphorylted proteins. On further analysis, the p30 showed differential variation in intensity in all the three groups after freezing and thawing. Positive immune reactivity for tyrosine phosphorylated proteins was found in neck, middle piece and post-acrosomal regions of spermatozoa. Addition of 0.5mM GSH decreased percentage of spermatozoa showing fragmented DNA and increased the percentage of spermatozoa having high transmembrane mitochondrial potential (P<0.05). This study demonstrates that GSH favours survival of bull spermatozoa by interfering with apoptotic and cryocapacitation pathways, and thereby protects the spermatozoa from deleterious effects of cryopreservation. The findings of the study indicated that GSH at 0.5mM can be effectively used as an additive in bull semen extender for freezing and thawing. Copyright © 2017 Elsevier B.V. All rights reserved.

Linking tree demography to climate change feedbacks: fire, larch forests, and carbon pools of the Siberian Arctic

NASA Astrophysics Data System (ADS)

Alexander, H. D.; Loranty, M. M.; Natali, S.; Pena, H., III; Ludwig, S.; Spektor, V.; Davydov, S. P.; Zimov, N.; Mack, M. C.

2017-12-01

Fire severity is increasing in larch forests of the Siberian Arctic as climate warms, and initial fire impacts on tree demographic processes could be an especially important determinant of long-term forest structure and carbon (C) dynamics. We hypothesized that (1) larch forest regrowth post-fire is largely determined by residual soil organic layer (SOL) depth because of the SOL's role as a seedbed and thermal regulator, and (2) changes in post-fire larch recruitment impact C accumulation through stand density impacts on understory microclimate and permafrost thaw. We tested these hypotheses by (1) experimentally creating a soil burn severity gradient in a Cajander larch (Larix cajanderi Mayr.) forest near Cherskiy, Russia and (2) quantifying C pools across a stand density gradient within a 75-year old fire scar. From 2012-2015, we added larch seeds to plots burned at different severities and monitored recruitment along with permafrost and active layer (i.e., subject to annual freeze-thaw) conditions (SOL depth, temperature, moisture, and thaw depth). Across the density gradient, we inventoried larch trees and harvested ground-layer vegetation to estimate aboveground contribution to C pools. We quantified woody debris C pools and sampled belowground C pools (soil, fine roots, and coarse roots) in the organic + upper (0-10 cm) mineral soil. Larch recruits were rare in unburned and low severity plots, but a total of 6 new germinants m-2 were tallied in moderate and high severity plots during the study. Seedling survival for > 1 year was only 40 and 25% on moderate and high severity treatments, respectively, but yielded net larch recruitment of 2 seedlings m-2, compared to 0.3 seedlings m-2 on low severity plots. Density of both total and established recruits increased with decreasing residual SOL depth, which correlated with increased soil temperature, moisture, and thaw depth. At 75-year post-fire, total C pools increased with increased larch density, largely due to increased tree aboveground C pools and decreased ground-layer vegetation C pools, which corresponded to higher canopy cover, cooler soils, and shallower active layer depths. Our findings highlight the potential for a climate-driven increase in fire severity to alter tree recruitment, successional dynamics, and C cycling in Siberian larch forests.

Enhancing the Properties of Conductive Polymer Hydrogels by Freeze-Thaw Cycles for High-Performance Flexible Supercapacitors.

PubMed

Li, Wanwan; Lu, Han; Zhang, Ning; Ma, Mingming

2017-06-14

We report that a postsynthesis physical process (freeze-thaw cycles) can reform the microstructure of conductive polymer hydrogels from clustered nanoparticles to interconnected nanosheets, leading to enhanced mechanical and electrochemical properties. The polyaniline-poly(vinyl alcohol) hydrogel after five freeze-thaw cycles (PPH-5) showed remarkable tensile strength (16.3 MPa), large elongation at break (407%), and high electrochemical capacitance (1053 F·g -1 ). The flexible supercapacitor based on PPH-5 provided a large capacitance (420 mF·cm -2 and 210 F·g -1 ) and high energy density (18.7 W·h·kg -1 ), whose robustness was demonstrated by its 100% capacitance retention after 1000 galvanostatic charge-discharge cycles or after 1000 mechanical folding cycles. The outstanding performance enables PPH-5 based supercapacitor as a promising power device for flexible electronics, which also demonstrates the merit of freeze-thaw cycles for enhancing the performance of functional hydrogels.

Fertility disturbances of dimethylacetamide and glycerol in rooster sperm diluents: Discrimination among effects produced pre and post freezing-thawing process.

PubMed

Abouelezz, F M K; Sayed, M A M; Santiago-Moreno, J

2017-09-01

With avian sperm cryopreservation protocols, the most widely used cryoprotectants (CPAs) are the glycerol (GLY; in gradual freezing: in-straw freezing method), and the dimethylacetamide (DMA; in pellets by plunging into liquid nitrogen: in-pellet rapid freezing method). Use of both methods results in a small portion of thawed live sperm with lesser fertilizing ability compared with the semen samples immediately after collection. This study was conducted to assess the pre-freezing damage occurring to the sperm due to the interaction with the cryoprotectants (CPAs) GLY (8%) and DMA (5%), as well as the post-freezing damage resulting from both freezing methods Data for each treatment, in fresh and frozen-thawed samples, were compared for sperm motility, fertilizing capacity and sperm-egg penetration holes/germinal disc (SP holes/GD). Hens (n=50) were artificially inseminated (10 hens/treatment) six times with 3day intervals between inseminations. The treatment of fresh sperm with DMA led to a reduction (P<0.05) in the count of SP holes/GD (21.4) and the fertility rate (66.7%). The addition and elimination of GLY in fresh samples resulted in a lesser (P<0.05) number of SP holes/GD (11.8) and the fertility rate (i.e., 50.0%). The number of SP-holes/GD was least in frozen-thawed samples using both DMA and GLY (14.2 and 9.2, respectively). The fertility rate when using semen frozen with DMA in- pellets was greater (P<0.05) than with use of semen that had been frozen using GLY in straws (46.4% compared with 31.3%). The reduction in fertility compared with the control when semen was cryopreserved using GLY was 64.1%; the GLY addition and elimination was responsible for two thirds of this reduction. The reduction in fertility when using semen cryopreserved with DMA was 46.7%; half of the reduction was attributed to the treatment with DMA. In conclusion, the mechanical damage attributed to the process for reducing GLY concentrations was more harmful to sperm fertilizing capacity than the toxicity of DMA and freeze/thaw process. For both freezing methods, the amount of sperm cryo-damage was similar, when the damage attributed to the CPA addition and elimination process was excluded. Copyright © 2017 Elsevier B.V. All rights reserved.

Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time.

PubMed

López-Urueña, E; Alvarez, M; Gomes-Alves, S; Martínez-Rodríguez, C; Borragan, S; Anel-López, L; de Paz, P; Anel, L

2014-06-01

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 10(6) spermatozoa/mL in a TES-Tris-Fructose-based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at -196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of omega-3 and omega-6 polyunsaturated fatty acid enriched diet on plasma IGF-1 and testosterone concentration, puberty and semen quality in male buffalo.

PubMed

Tran, L V; Malla, B A; Sharma, A N; Kumar, Sachin; Tyagi, Nitin; Tyagi, A K

2016-10-01

The objective of the present study was to evaluate the effect of omega-3 and omega-6 PUFA enriched diet on plasma IGF-1 and testosterone concentrations, puberty, sperm fatty acid profile and semen quality in male buffalo. Eighteen male buffalo calves were distributed randomly in three different groups and fed concentrate mixture along with green fodder and wheat straw in 50:40:10 ratios as per requirements. Basis ration of animals in group I was supplemented with 4% of prilled fat (PFA), while in group II and group III were added 4.67% of Calcium salt from Soybean (CaSFA) and Linseed oil (CaLFA), respectively. Male buffalo fed omega-3 PUFA high diet significantly increased concentrations of IGF-1 and testosterone in plasma as compared to two other diets (p<0.05). The age of puberty and scrotal circumference significantly increased by dietary fat effect (p<0.05) of which n-3 PUFA enriched diet (CaLFA) had the largest influence as compared to other diets (PFA and CaSFA). Feeding of n-3 PUFA rich diet significantly increased the DHA (C22:6n-3) content in sperm (p<0.05), which contributed to increased fluidity of plasma membrane, elevated quality of sperm (motility, viability) and in vitro fertility (plasma membrane integrity, acrosome integrity) in both fresh and post-thawing semen. These findings indicate that feeding of n-3 PUFA enriched diet increased IGF-1 and testosterone secretion, reduced pubertal age and improved both fresh and post-thawing semen quality in male buffalo. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative studies of mesenchymal stem cells derived from different cord tissue compartments - The influence of cryopreservation and growth media.

PubMed

Dulugiac, Magda; Moldovan, Lucia; Zarnescu, Otilia

2015-10-01

We have identified some critical aspects concerning umbilical cord tissue mesenchymal stem cells: the lack of standards for cell isolation, expansion and cryopreservation, the lack of unanimous opinions upon their multilineage differentiation potential and the existence of very few results related to the functional characterization of the cells isolated from cryopreserved umbilical cord tissue. Umbilical cord tissue cryopreservation appears to be the optimal solution for umbilical cord tissue mesenchymal stem cells storage for future clinical use. Umbilical cord tissue cryopreservation allows mesenchymal stem cells isolation before expected use, according with the specific clinical applications, by different customized isolation and expansion protocols agreed by cell therapy institutions. Using an optimized protocol for umbilical cord tissue cryopreservation in autologous cord blood plasma, isolation explant method and growth media supplemented with FBS or human serum, we performed comparative studies with respect to the characteristics of mesenchymal stem cells (MSC) isolated from different compartments of the same umbilical cord tissue such as Wharton's jelly, vein, arteries, before cryopreservation (pre freeze) and after cryopreservation (post thaw). Expression of histochemical and immunohistochemical markers as well as electron microscopy observations revealed similar adipogenic, chondrogenic and osteogenic differentiation capacity for cells isolated from pre freeze and corresponding post thaw tissue fragments of Wharton's jelly, vein or arteries of the same umbilical cord tissue, regardless growth media used for cells isolation and expansion. Our efficient umbilical cord tissue cryopreservation protocol is reliable for clinical applicability of mesenchymal stem cells that could next be isolated and expanded in compliance with future accepted standards. Copyright © 2015 Elsevier Ltd. All rights reserved.

Carbon and geochemical properties of cryosols on the North Slope of Alaska

USGS Publications Warehouse

Mu, Cuicui; Zhang, Tingjun; Schuster, Paul F.; Schaefer, Kevin; Wickland, Kimberly P.; Repert, Deborah A.; Liu, Lin; Schaefer, Tim; Cheng, Guodong

2014-01-01

Cryosols contain roughly 1700 Gt of Soil organic carbon (SOC) roughly double the carbon content of the atmosphere. As global temperature rises and permafrost thaws, this carbon reservoir becomes vulnerable to microbial decomposition, resulting in greenhouse gas emissions that will amplify anthropogenic warming. Improving our understanding of carbon dynamics in thawing permafrost requires more data on carbon and nitrogen content, soil physical and chemical properties and substrate quality in cryosols. We analyzed five permafrost cores obtained from the North Slope of Alaska during the summer of 2009. The relationship between SOC and soil bulk density can be adequately represented by a logarithmic function. Gas fluxes at − 5 °C and 5 °C were measured to calculate the temperature response quotient (Q10). Q10 and the respiration per unit soil C were higher in permafrost-affected soils than that in the active layer, suggesting that decomposition and heterotrophic respiration in cryosols may contribute more to global warming.

Identifying active methane-oxidizers in thawed Arctic permafrost by proteomics

NASA Astrophysics Data System (ADS)

Lau, C. M.; Stackhouse, B. T.; Chourey, K.; Hettich, R. L.; Vishnivetskaya, T. A.; Pfiffner, S. M.; Layton, A. C.; Mykytczuk, N. C.; Whyte, L.; Onstott, T. C.

2012-12-01

The rate of CH4 release from thawing permafrost in the Arctic has been regarded as one of the determining factors on future global climate. It is uncertain how indigenous microorganisms would interact with such changing environmental conditions and hence their impact on the fate of carbon compounds that are sequestered in the cryosol. Multitudinous studies of pristine surface cryosol (top 5 cm) and microcosm experiments have provided growing evidence of effective methanotrophy. Cryosol samples corresponding to active layer were sampled from a sparsely vegetated, ice-wedge polygon at the McGill Arctic Research Station at Axel Heiberg Island, Nunavut, Canada (N79°24, W90°45) before the onset of annual thaw. Pyrosequencing of 16S rRNA gene indicated the occurrence of methanotroph-containing bacterial families as minor components (~5%) in pristine cryosol including Bradyrhizobiaceae, Methylobacteriaceae and Methylocystaceae within alpha-Proteobacteria, and Methylacidiphilaceae within Verrucomicrobia. The potential of methanotrophy is supported by preliminary analysis of metagenome data, which indicated putative methane monooxygenase gene sequences relating to Bradyrhizobium sp. and Pseudonocardia sp. are present. Proteome profiling in general yielded minute traces of proteins, which likely hints at dormant nature of the soil microbial consortia. The lack of specific protein database for permafrost posted additional challenge to protein identification. Only 35 proteins could be identified in the pristine cryosol and of which 60% belonged to Shewanella sp. Most of the identified proteins are known to be involved in energy metabolism or post-translational modification of proteins. Microcosms amended with sodium acetate exhibited a net methane consumption of ~65 ngC-CH4 per gram (fresh weight) of soil over 16 days of aerobic incubation at room temperature. The pH in microcosm materials remained acidic (decreased from initial 4.7 to 4.5). Protein extraction and characterization identified ~350 proteins, confirmed enhanced microbial activities and significant shift in community structure within the microcosms. Although the activity of Shewanella sp. was suppressed by the incubation conditions, other bacteria were activated. This was shown by at least 3-fold increase in the number of identified proteins, which were primarily players in cellular energy metabolism. Among them, Geobacter sp. and methane-oxidizers, Bradyrhizobium sp., Methylosinus sp. and Methylocystis sp. appear dominant. In order to advance the protein database for better biodiversity and functional identification, we are currently using duo extraction protocols and consolidating metagenome data obtained from the same soil samples. A depth profile (from active to permafrost layer) for methanotrophs is being determined by examining pristine cores, thawed cryosols as well as enrichment cultures. The proteome information from these samples will be presented, which will be complemented by molecular studies.

Incipient Balancing Selection through Adaptive Loss of Aquaporins in Natural Saccharomyces cerevisiae Populations

PubMed Central

Will, Jessica L.; Kim, Hyun Seok; Clarke, Jessica; Painter, John C.; Fay, Justin C.; Gasch, Audrey P.

2010-01-01

A major goal in evolutionary biology is to understand how adaptive evolution has influenced natural variation, but identifying loci subject to positive selection has been a challenge. Here we present the adaptive loss of a pair of paralogous genes in specific Saccharomyces cerevisiae subpopulations. We mapped natural variation in freeze-thaw tolerance to two water transporters, AQY1 and AQY2, previously implicated in freeze-thaw survival. However, whereas freeze-thaw–tolerant strains harbor functional aquaporin genes, the set of sensitive strains lost aquaporin function at least 6 independent times. Several genomic signatures at AQY1 and/or AQY2 reveal low variation surrounding these loci within strains of the same haplotype, but high variation between strain groups. This is consistent with recent adaptive loss of aquaporins in subgroups of strains, leading to incipient balancing selection. We show that, although aquaporins are critical for surviving freeze-thaw stress, loss of both genes provides a major fitness advantage on high-sugar substrates common to many strains' natural niche. Strikingly, strains with non-functional alleles have also lost the ancestral requirement for aquaporins during spore formation. Thus, the antagonistic effect of aquaporin function—providing an advantage in freeze-thaw tolerance but a fitness defect for growth in high-sugar environments—contributes to the maintenance of both functional and nonfunctional alleles in S. cerevisiae. This work also shows that gene loss through multiple missense and nonsense mutations, hallmarks of pseudogenization presumed to emerge after loss of constraint, can arise through positive selection. PMID:20369021

Nitrogen availability increases in a tundra ecosystem during five years of experimental permafrost thaw.

PubMed

Salmon, Verity G; Soucy, Patrick; Mauritz, Marguerite; Celis, Gerardo; Natali, Susan M; Mack, Michelle C; Schuur, Edward A G

2016-05-01

Perennially frozen soil in high latitude ecosystems (permafrost) currently stores 1330-1580 Pg of carbon (C). As these ecosystems warm, the thaw and decomposition of permafrost is expected to release large amounts of C to the atmosphere. Fortunately, losses from the permafrost C pool will be partially offset by increased plant productivity. The degree to which plants are able to sequester C, however, will be determined by changing nitrogen (N) availability in these thawing soil profiles. N availability currently limits plant productivity in tundra ecosystems but plant access to N is expected improve as decomposition increases in speed and extends to deeper soil horizons. To evaluate the relationship between permafrost thaw and N availability, we monitored N cycling during 5 years of experimentally induced permafrost thaw at the Carbon in Permafrost Experimental Heating Research (CiPEHR) project. Inorganic N availability increased significantly in response to deeper thaw and greater soil moisture induced by Soil warming. This treatment also prompted a 23% increase in aboveground biomass and a 49% increase in foliar N pools. The sedge Eriophorum vaginatum responded most strongly to warming: this species explained 91% of the change in aboveground biomass during the 5 year period. Air warming had little impact when applied alone, but when applied in combination with Soil warming, growing season soil inorganic N availability was significantly reduced. These results demonstrate that there is a strong positive relationship between the depth of permafrost thaw and N availability in tundra ecosystems but that this relationship can be diminished by interactions between increased thaw, warmer air temperatures, and higher levels of soil moisture. Within 5 years of permafrost thaw, plants actively incorporate newly available N into biomass but C storage in live vascular plant biomass is unlikely to be greater than losses from deep soil C pools. © 2015 John Wiley & Sons Ltd.

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation.

PubMed

Onofre, J; Baert, Y; Faes, K; Goossens, E

2016-11-01

Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the human is lacking. In addition, few to no data are available on the safety aspects inherent to offspring generation with gametes derived from frozen-thawed TT or TCSs. Moreover, clarification is needed on whether it is better to cryopreserve TT or TCS. Fertility restoration techniques are very promising and expected to be implemented in the clinic in the near future. However, inter-center variability needs to be overcome and the gametes produced for reproduction purposes need to be subjected to safety studies. With the perspective of a future clinical application, there is a dire need to optimize and standardize cryopreservation and safety testing before using frozen-thawed TT of TCSs for fertility restoration. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation

PubMed Central

Onofre, J.; Baert, Y.; Faes, K.; Goossens, E.

2016-01-01

BACKGROUND Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. OBJECTIVE AND RATIONALE This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. SEARCH METHODS An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. OUTCOMES The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the human is lacking. In addition, few to no data are available on the safety aspects inherent to offspring generation with gametes derived from frozen-thawed TT or TCSs. Moreover, clarification is needed on whether it is better to cryopreserve TT or TCS. WIDER IMPLICATIONS Fertility restoration techniques are very promising and expected to be implemented in the clinic in the near future. However, inter-center variability needs to be overcome and the gametes produced for reproduction purposes need to be subjected to safety studies. With the perspective of a future clinical application, there is a dire need to optimize and standardize cryopreservation and safety testing before using frozen-thawed TT of TCSs for fertility restoration. PMID:27566839

Effect of Multiple Freezing/Thawing Cycles on the Structural and Functional Properties of Waxy Rice Starch

PubMed Central

Tao, Han; Yan, Juan; Zhao, Jianwei; Tian, Yaoqi; Jin, Zhengyu; Xu, Xueming

2015-01-01

The structural and functional properties of non-gelatinized waxy rice starch were investigated after 1, 3, 7, and 10 freezing/thawing cycles. Freezing caused an increasing damaged starch from 1.36% in native waxy rice starch to 5.77% in 10 freezing/thawing-treated starch (FTS), as evidenced by the cracking surface on starch granules. More dry matter concentration was leached, which was characterized by high amylopectin concentration (4.34 mg/mL). The leaching was accompanied by a decrease in relative crystallinity from 35.19% in native starch to 31.34% in 10 FTS. Freezing treatment also led to significant deviations in the functional characteristics, for instance decreased gelatinization temperature range, enthalpy, and pasting viscosities. The resistant starch content of 10FTS significantly decreased from 58.9% to 19%, whereas the slowly digested starch content greatly increased from 23.8% in native starch to 50.3%. The increase in susceptibility to enzyme hydrolysis may be attributed to porous granular surface, amylopectin leaching, and the decrease in the relative crystallinity caused by freezing water. PMID:26018506

Effect of multiple freezing/thawing cycles on the structural and functional properties of waxy rice starch.

PubMed

Tao, Han; Yan, Juan; Zhao, Jianwei; Tian, Yaoqi; Jin, Zhengyu; Xu, Xueming

2015-01-01

The structural and functional properties of non-gelatinized waxy rice starch were investigated after 1, 3, 7, and 10 freezing/thawing cycles. Freezing caused an increasing damaged starch from 1.36% in native waxy rice starch to 5.77% in 10 freezing/thawing-treated starch (FTS), as evidenced by the cracking surface on starch granules. More dry matter concentration was leached, which was characterized by high amylopectin concentration (4.34 mg/mL). The leaching was accompanied by a decrease in relative crystallinity from 35.19% in native starch to 31.34% in 10 FTS. Freezing treatment also led to significant deviations in the functional characteristics, for instance decreased gelatinization temperature range, enthalpy, and pasting viscosities. The resistant starch content of 10FTS significantly decreased from 58.9% to 19%, whereas the slowly digested starch content greatly increased from 23.8% in native starch to 50.3%. The increase in susceptibility to enzyme hydrolysis may be attributed to porous granular surface, amylopectin leaching, and the decrease in the relative crystallinity caused by freezing water.

A practical algorithm to estimate soil thawing onset with the soil moisture active passive (SMAP) data

NASA Astrophysics Data System (ADS)

Chen, X.; Liu, L.

2016-12-01

The Soil Moisture Active Passive (SMAP) satellite simultaneously collected active and passive microwave data at L-band from April to July, 2015. The L-band radiometer brightness temperature (TB) data are strongly sensitive to the change of soil moisture, therefore, can be used to estimate freeze/thaw state of soil. We applied an edge detection method to detect the onset of thawing based on the SMAP level-1C TB data. This method convolves the first derivative of the Gaussian function as a kernel with the TB time series. When thawing occurs, soil moisture increases abruptly and leads to a decrease in TB. Therefore, a primary thaw event can be identified when the convolved signal reaches a local minimum. Considering the noise of the radiometer data, not all local minimums correspond to a thaw event. Therefore, we further applied a filter based on a priori or in situ soil temperature observation to eliminate false events. We compared the TB-based estimates with in situ measurements of soil temperature, moisture, and snow depth from April to June from 5 SNOTEL sites in Alaska. Our results show that at 4 out of the 5 sites the estimated thawing onsets and in-situ data agree within 5 to 10 days. However, we found a distinct inconsistency of 41 days at the fifth site. One possible reason is the mismatch in spatial coverage: one pixel of SMAP radiometer data has a size of 36 km, within which different areas may have different freeze/thaw states. The SMAP radar backscatter coefficient (σ0) data are also very sensitive to soil moisture, and has finer spatial resolution of 1 km, making it more directly comparable with the in situ measurements. We applied a seasonal threshold method to estimate thawing onset based on this data. Firstly, we set a thaw onset based on the in situ soil temperature and moisture measurements at 5 cm depth. Then we averaged σ0 observations from April 14th to 7 days before the thaw onset to represent the frozen soil, and used the mean value from 7 days after the thawing onset to June 1st as thawed reference. Next, the σ0-based freeze/thaw distribution within radiometer pixel can be obtained. Assuming TB and have a linear relationship in 36 km scale during a short time, SMAP provide a down scaling method to obtain 9 km resolution TB data. For further work, we plan to apply the edge detection method on this TB data to estimate the soil state in 9 km.

Impact of long-term cryopreservation on single umbilical cord blood transplantation outcomes.

PubMed

Mitchell, Richard; Wagner, John E; Brunstein, Claudio G; Cao, Qing; McKenna, David H; Lund, Troy C; Verneris, Michael R

2015-01-01

Umbilical cord blood (UCB) may be collected and cryopreserved for years before use. In vitro and murine models suggest that the duration of storage does not affect UCB progenitor cell performance; however, the impact of UCB age on clinical outcomes has not been definitely defined. This study sought to determine the effect of UCB unit cryopreservation time on hematopoietic potency. We analyzed 288 single UCB units used for transplantation from 1992 to 2013, with unit cryopreservation time ranging from .08 to 11.07 years. UCB unit post-thaw characteristics were examined, including percent recovery of total nucleated cells (TNC). The number of years the UCB unit spent in cryopreservation had no impact on TNC recovery nor UCB unit post-thaw viability. Duration of cryopreservation also had no impact on neutrophil or platelet engraftment in single UCB transplantations. These results show that UCB units can undergo cryopreservation for at least 10 years with no impact on clinical outcomes. Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Factors impacting the success of post-mortem sperm rescue in the rhinoceros.

PubMed

Roth, T L; Stoops, M A; Robeck, T R; O'Brien, J K

2016-04-01

The goal of this study was to identify factors that influenced the ability to successfully rescue sperm post-mortem from rhinoceroses maintained in North American zoos. Factors considered included procedural technicalities, individual rhinoceros characteristics and timing. Gross testicular pathology was noted in 17.4% of males (4/23) but did not impact sperm recovery except in one case of azoospermia (4.3%). Of the males in which sperm recovery was attempted (n=21), 62% yielded quality samples considered adequate for cryopreservation (≥ 30% motility with ≥ 2.0 forward progressive status). A high percentage of males (70.6%; 12/17) from which reproductive tissue was removed an d cooled ≤ 4 h after death yielded quality sperm samples, whereas only 25% (1/4) of males from which tissue was removed>4h after death yielded quality samples. Quality samples were recovered 1-51 h post-mortem from rhinoceroses 8 to 36 years old. Neither type of illness (prolonged or acute), or method of death (euthanasia or natural) affected the ability to harvest quality samples (P > 0.05). The Indian rhinoceros yielded significantly more sperm on average (40 × 10(9)) than the African black rhinoceros (3.6 × 10(9); P < 0.01) and the African white rhinoceros (3.2 × 10(9); P < 0.05). Across all species and samples assessed (n = 11), mean post-thaw sperm motility (41%), was only 15% less than pre-freeze motility (56%) and only decreased to 22% during the 6h post-thaw assessment period. Rhinoceros sperm rescue post-mortem is relatively successful across a wide range of variables, especially when tissues are removed and cooled promptly after death, and should be considered standard practice among zoos. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimized Standard Operating Procedures for the Analysis of Cerebrospinal Fluid Aβ42 and the Ratios of Aβ Isoforms Using Low Protein Binding Tubes.

PubMed

Vanderstichele, Hugo Marcel Johan; Janelidze, Shorena; Demeyer, Leentje; Coart, Els; Stoops, Erik; Herbst, Victor; Mauroo, Kimberley; Brix, Britta; Hansson, Oskar

2016-05-31

Reduced cerebrospinal fluid (CSF) concentration of amyloid-β1-42 (Aβ1-42) reflects the presence of amyloidopathy in brains of subjects with Alzheimer's disease (AD). To qualify the use of Aβ1-42/Aβ1-40 for improvement of standard operating procedures (SOP) for measurement of CSF Aβ with a focus on CSF collection, storage, and analysis. Euroimmun ELISAs for CSF Aβ isoforms were used to set up a SOP with respect to recipient properties (low binding, polypropylene), volume of tubes, freeze/thaw cycles, addition of detergents (Triton X-100, Tween-20) in collection or storage tubes or during CSF analysis. Data were analyzed with linear repeated measures and mixed effects models. Optimization of CSF analysis included a pre-wash of recipients (e.g., tubes, 96-well plates) before sample analysis. Using the Aβ1-42/Aβ1-40 ratio, in contrast to Aβ1-42, eliminated effects of tube type, additional freeze/thaw cycles, or effect of CSF volumes for polypropylene storage tubes. 'Low binding' tubes reduced the loss of Aβ when aliquoting CSF or in function of additional freeze/thaw cycles. Addition of detergent in CSF collection tubes resulted in an almost complete absence of variation in function of collection procedures, but affected the concentration of Aβ isoforms in the immunoassay. The ratio of Aβ1-42/Aβ1-40 is a more robust biomarker than Aβ1-42 toward (pre-) analytical interfering factors. Further, 'low binding' recipients and addition of detergent in collection tubes are able to remove effects of SOP-related confounding factors. Integration of the Aβ1-42/Aβ1-40 ratio and 'low-binding tubes' into guidance criteria may speed up worldwide standardization of CSF biomarker analysis.

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells 1

PubMed Central

Arora, Rajeev; Palta, Jiwan P.

1988-01-01

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K+, along with small but significant loss of cellular Ca2+. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca2+ from membrane by extracellular K+ and subsequent perturbation of K+ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca2+ compared to control. Loss of membrane-associated Ca2+ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca2+-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl2. Our results suggest that the loss of membrane-associated Ca2+, in part, plays a role in initiation and progression of freezing injury. Images Fig. 1 Fig. 2 PMID:16666196

Mixing of thawed coagulation samples prior to testing: Is any technique better than another?

PubMed

Lima-Oliveira, Gabriel; Adcock, Dorothy M; Salvagno, Gian Luca; Favaloro, Emmanuel J; Lippi, Giuseppe

2016-12-01

Thus study was aimed to investigate whether the mixing technique could influence the results of routine and specialized clotting tests on post-thawed specimens. The sample population consisted of 13 healthy volunteers. Venous blood was collected by evacuated system into three 3.5mL tubes containing 0.109mmol/L buffered sodium citrate. The three blood tubes of each subject were pooled immediately after collection inside a Falcon 15mL tube, then mixed by 6 gentle end-over-end inversions, and centrifuged at 1500g for 15min. Plasma-pool of each subject was then divided in 4 identical aliquots. All aliquots were thawed after 2-day freezing -70°C. Immediately afterwards, the plasma of the four paired aliquots were treated using four different techniques: (a) reference procedure, entailing 6 gentle end-over-end inversions; (b) placing the sample on a blood tube rocker (i.e., rotor mixing) for 5min to induce agitation and mixing; (c) use of a vortex mixer for 20s to induce agitation and mixing; and (d) no mixing. The significance of differences against the reference technique for mixing thawed plasma specimens (i.e., 6 gentle end-over-end inversions) were assessed with paired Student's t-test. The statistical significance was set at p<0.05. As compared to the reference 6-time gentle inversion technique, statistically significant differences were only observed for fibrinogen, and factor VIII in plasma mixed on tube rocker. Some trends were observed in the remaining other cases, but the bias did not achieve statistical significance. We hence suggest that each laboratory should standardize the procedures for mixing of thawed plasma according to a single technique. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use.

PubMed

Gülen, D; Maas, S; Julius, H; Warkentin, P; Britton, H; Younos, I; Senesac, J; Pirruccello, Samuel M; Talmadge, J E

2012-05-01

In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs. Copyright © 2012 Elsevier B.V. All rights reserved.

Balancing risk and benefit: maintenance of a thawed Group A plasma inventory for trauma patients requiring massive transfusion.

PubMed

Mehr, Chelsea R; Gupta, Rajan; von Recklinghausen, Friedrich M; Szczepiorkowski, Zbigniew M; Dunbar, Nancy M

2013-06-01

Transfusion of plasma and red blood cell (RBC) units in a balanced ratio approximating 1:1 has been shown in retrospective studies to be associated with improved outcomes for trauma patients. Our low-volume rural trauma center uses a trauma-activated transfusion algorithm. Plasma is thawed upon activation to avoid wastage. However, the time required for plasma thawing has made achievement of a 1:1 ratio early in resuscitation challenging. In this study, the time required for plasma thawing is characterized, and a potential solution is proposed. A retrospective chart study of 38 moderately and massively transfused (≥6 U in the first 24 hours) trauma patients admitted from January 2008 to March 2012 was performed. We evaluated the time required to dispense plasma and the number of RBCs dispensed before plasma in these patients. The average time between the dispense of RBCs and plasma was 26 minutes (median, 28; range, 0-48 minutes). The average number of RBCs dispensed before plasma was 8 U (median, 7 U; range, 0-24 U). Nearly one third of massively transfused patients had 10 RBCs or greater dispensed before plasma was available. There exists the potential for delayed plasma availability owing to time required for thawing, which may compromise the ability to provide balanced plasma to RBC transfusion to trauma patients. Maintenance of a thawed Group AB plasma inventory may not be operationally feasible for rural centers with low trauma volumes. Use of a thawed Group A plasma inventory is a potential alternative to ensure rapid plasma availability. Therapeutic study, level V.

Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen-thawed, capacitated stallion sperm.

PubMed

Morató, Roser; Soares, Juleide M De Souza; Orero, Guifré; Mogas, Teresa; Miró, Jordi

2013-06-01

The effect of combining double layer density gradient centrifugation (DL-DGC) with different capacitation treatments on the fertilising capacity of frozen-thawed stallion sperm was examined via a heterologous assay involving in vitro-matured, zona pellucida-free bovine oocytes. In a first experiment, aliquots of frozen-thawed stallion sperm were subjected to one of five capacitation treatments without DL-DGC - ionomycin at 1.0μM, 0.1μM, 0.05μM or 0.01μM, or caffeine at 200μg/mL. The fertilising capacity of the semen was then assessed at 18h by staining the above oocytes with 4,6-diamidino-2-phenylindole (DAPI) and examining for sperm penetration, the number of penetrated spermatozoa per oocyte, and male pronucleus formation. In a second experiment, aliquots of frozen-thawed stallion sperm were subjected to DL-DGC selection - or not - and then further subjected to the two best capacitation treatments (0.1μM and 0.05μM ionomycin). The fertilising capacity of the semen was then determined as above. The DL-DGC/capacitated sperm samples showed the highest mean penetration rates: 24.16% following capacitation with 0.1μM ionomycin, and 12.21% following capacitation with 0.05μM ionomycin. The capacitated but non-DL-DGC-selected sperm returned significantly lower values: 6.26% and 7.02% for the same ionomycin treatments respectively. These findings suggest that combining DL-DGC selection with ionomycin capacitation improves the fertilising capacity of frozen-thawed stallion sperm. Copyright © 2013 Elsevier B.V. All rights reserved.

Is sperm freezability related to the post-thaw lipid peroxidation and the formation of reactive oxygen species in boars?

PubMed

Gómez-Fernández, J; Gómez-Izquierdo, E; Tomás, C; Mocé, E; de Mercado, E

2013-04-01

The aim of the present study was to determine whether the levels of reactive oxygen species (ROS) substances production and the levels of lipid peroxidation of the sperm membrane were related to the quality that the ejaculates exhibited after cryopreservation in boars. Ejaculates from 42 healthy boars were used in this study and they were cryopreserved with the lactose-egg yolk extender (LEY). Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37 °C after thawing: the percentage of sperm with intact plasma membrane (SIPM), intracellular reactive oxygen substances production through mean of DCF fluorescence intensity of total sperm (mean-DCF) and the percentage of viable and non-viable sperm containing oxidized BODIPY (VSOB and NVSOB). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. The classification of the ejaculates into good or bad freezers was performed through hierarchical cluster analysis from SIPM and TMS at 150 min post-thawing. The ejaculates of those males classified as good freezers exhibited higher (p < 0.05) SPIM, TMS and PMS than the bad freezers, although both groups presented similar (p > 0.05) VSOB, NVSOB and mean-DCF. Therefore, these results show that lipid peroxidation and the amount of reactive oxygen substances in the sperm after cryopreservation are similar between boars classified as good or bad freezers. © 2012 Blackwell Verlag GmbH.

Long-term cryopreservation of sperm from Mandarin fish Siniperca chuatsi.

PubMed

Ding, Shuyan; Ge, Jiachun; Hao, Chen; Zhang, Mingsheng; Yan, Weihui; Xu, Zhiqiang; Pan, Jianlin; Chen, Songlin; Tian, Yongsheng; Huang, Yahong

2009-07-01

In order to develop cryopreservation techniques for long-term preserving the sperm of Mandarin fish Siniperca chuatsi, we examined the effects of various extender and cryopreservation on post-thaw motility. We found the optimal freezing procedures for the Mandarin fish sperm is diluting the semen in D-15 extender, chilling it to 4 degrees C, adding ME2SO to a final concentration of 10% (v/v), then transferring the semen in cryotubes, holding the cryotubes for 10 min at 6 cm (about -180 degrees C) above the surface of liquid nitrogen, for 5 min on the surface of liquid nitrogen, and finally plunged into liquid nitrogen. After thawed at 37 degrees C for 60s, the sperm had the highest post-thaw motility (96.00+/-1.73%). The optimal fertilization procedures for the frozen sperm is mixing the eggs with sperm, then adding 1 ml of swimming medium (SM=45 mM NaCl+5 mM KCl+20mM Tris-HCl, pH 8.0) immediately. At the sperm/egg ratio of 100,000:1, the fertilization rate and the hatching rate of the frozen sperm cryopreserved for 1 week or 1 year in liquid nitrogen (66.01+/-5.14% and 54.76+/-4.40% & 62.97+/-14.28% and 52.58+/-11.17%) were similar to that of fresh sperm (69.42+/-8.11% and 59.82+/-5.27%) (p>0.05). This is the first report that the Mandarin fish (S. chuatsi) sperm can successfully fertilized eggs after long-term cryopreservation.

Examining the role of shrub expansion and fire in Arctic plant silica cycling

NASA Astrophysics Data System (ADS)

Carey, J.; Fetcher, N.; Parker, T.; Rocha, A. V.; Tang, J.

2017-12-01

All terrestrial plants accumulate silica (SiO2) to some degree, although the amount varies by species type, functional group, and environmental conditions. Silica improves overall plant fitness, providing protection from a variety of biotic and abiotic stressors. Plant silica uptake serves to retain silica in terrestrial landscapes, influencing silica export rates from terrestrial to marine systems. These export rates are important because silica is often the limiting nutrient for primary production by phytoplankton in coastal waters. Understanding how terrestrial plant processes influence silica export rates to oceanic systems is of interest on the global scale, but nowhere is this issue more important than in the Arctic, where marine diatoms rely on silica for production in large numbers and terrestrial runoff largely influences marine biogeochemistry. Moreover, the rapid rate of change occurring in the Arctic makes understanding plant silica dynamics timely, although knowledge of plant silica cycling in the region is in its infancy. This work specifically examines how shrub expansion, permafrost thaw, and fire regimes influence plant silica behavior in the Alaskan Arctic. We quantified silica accumulation in above and belowground portions of three main tundra types found in the Arctic (wet sedge, moist acidic, moist non-acidic tundra) and scaled these values to estimate how shrub expansion alters plant silica accumulation rates. Results indicate that shrub expansion via warming will increase silica storage in Arctic land plants due to the higher biomass associated with shrub tundra, whereas conversion of tussock to wet sedge tundra via permafrost thaw would produce the opposite effect in the terrestrial plant BSi pool. We also examined silica behavior in plants exposed to fire, finding that post-fire growth results in elevated plant silica uptake. Such changes in the size of the terrestrial vegetation silica reservoir could have direct consequences for the rates and timing of silica delivery to coastal receiving waters in the Arctic.

Development and Characterization of UHMWPE Fiber-Reinforced Hydrogels For Meniscal Replacement

NASA Astrophysics Data System (ADS)

Holloway, Julianne Leigh

Meniscal tears are the most common orthopedic injuries to the human body. The current treatment of choice, however, is a partial meniscectomy that leads to osteoarthritis proportional to the amount of tissue removed. As a result, there is a significant clinical need to develop materials capable of restoring the biomechanical contact stress distribution to the knee after meniscectomy and preventing the onset of osteoarthritis. In this work, a fiber-reinforced hydrogel-based synthetic meniscus was developed that allows for tailoring of the mechanical properties and molding of the implant to match the size, shape, and property distribution of the native tissue. Physically cross-linked poly(vinyl alcohol) (PVA) hydrogels were reinforced with ultrahigh molecular weight polyethylene (UHMWPE) fibers and characterized in compression (0.1-0.8 MPa) and tension (0.1-250 MPa) showing fine control over mechanical properties within the range of the human meniscus. Morphology and crystallinity analysis of PVA hydrogels showed increases in crystallinity and PVA densification, or phase separation, with freeze-thaw cycles. A comparison of freeze-thawed and aged, physically cross-linked hydrogels provided insight on both crystallinity and phase separation as mechanisms for PVA gelation. Results indicated both mechanisms independently contributed to hydrogel modulus for freeze-thawed hydrogels. In vitro swelling studies were performed using osmotic solutions to replicate the swelling pressure present in the knee. Minimal swelling was observed for hydrogels with a PVA concentration of 30-35 wt%, independently of hydrogel freeze-thaw cycles. This allows for independent tailoring of hydrogel modulus and pore structure using freeze-thaw cycles and swelling behavior using polymer concentration to match a wide range of properties needed for various soft tissue applications. The UHMWPE-PVA interface was identified as a significant weakness. To improve interfacial adhesion, a novel biocompatible PVA grafting technique was developed to form a direct covalent linkage at the fiber-matrix interface. Chemical grafting was tailored as a function of the number of sites available for covalent bonding and the percentage of sites reacted. PVA grafting resulted in significant improvements to interfacial shear strength from 11 kPa without any treatment to above 220 kPa following grafting. After grafting, failure was observed cohesively within the PVA hydrogel indicating the UHMWPE-PVA interface was successfully optimized. Lastly, in vitro gait simulations and an in vivo sheep study demonstrated the feasibility and biocompatibility of the proposed UHMWPE-PVA composite. The results from this work can be applied to designing materials for other soft tissue applications, including the anterior cruciate ligament (ACL) and the annulus fibrosus.

Criteria to assess human oocyte quality after cryopreservation.

PubMed

Coticchio, G; Bonu, M A; Bianchi, V; Flamigni, C; Borini, A

2005-10-01

Oocyte cryopreservation certainly represents one of the most attractive developments in the field of assisted reproduction, with the aim of preserving female fertility and circumventing the ethical and legal drawbacks associated with embryo freezing. Despite the achievement of the first pregnancy from frozen oocytes dating back as early as 1987, since then fewer than 150 pregnancies have been reported. Over a long period of time, application of oocyte storage on a large scale has been prevented by various factors, namely poor post-thaw survival. Fertilization rates remained low even after the introduction of intracytoplasmic sperm injection. Modifications of slow-freezing protocols, mainly based on the increase of the concentration of sucrose used as non-penetrating cryoprotectant (CPA) and the replacement of sodium with choline, appear to have decisively improved survival rates to over 80%. Investigations at the cellular level on thawed oocytes are largely lacking. Fertilization rates have also benefited from protocol modifications, reaching values indistinguishable from those normally obtained with fresh material. Vitrification protocols have also been tested, giving rise to improvements whose reproducibility is still uncertain. Data on the dynamics of fertilization and preimplantation development of embryos derived from frozen oocytes are extremely scarce. At the moment, clinical efficiency of oocyte cryopreservation cannot be precisely assessed because of the lack of controlled studies, although it appears to be considerably lower than that achieved with embryo freezing. In summary, encouraging advances have been made in the field of oocyte cryopreservation, but presently no protocol can ensure standards of success and safety comparable to those guaranteed by embryo storage.

Seasonal and Downslope Changes in the Pore Water Geochemistry of Tundra Soils Near Nome, Alaska

NASA Astrophysics Data System (ADS)

Philben, M. J.; Zheng, J.; Wullschleger, S. D.; Graham, D. E.; Gu, B.

2017-12-01

Thawing permafrost is exposing vast stores of organic matter to decomposition in previously frozen tundra soils. In low-relief and poorly drained areas, the complexity of microbial metabolism under anaerobic conditions complicates the prediction of resulting CO2 and CH4 emissions. To improve this understanding, we investigated the dissolved gas and major ion concentrations and DOM composition in depth profiles of soil pore water collected from the Teller Road site near Nome, AK, as part of the Next Generation Ecosystem Experiment (NGEE)-Arctic. Pathways of anaerobic organic matter degradation were inferred based on two complementary approaches: first, we compared the composition of soil pore waters of saturated areas in the peat plateau and the base of the hillslope, collected early and late in the thaw season (July and September) to assess seasonal changes in the soil solution chemistry. CH4 and low molecular weight organic acids (e.g., acetate, formate, and propionate) were both near or below the detection limit in July but accumulated later in the season. In contrast, SO42- and Fe(III) concentrations were high in July and low in September, while Fe(II) was higher in September. These results suggest SO42- and Fe(III) reduction were the primary pathways for anaerobic respiration early in the thaw season, while methanogenesis increased in September as labile organic acids accumulated. Second, we assessed the change in DOM composition in a transect of piezometers, capturing the degradation of organic matter during transport down a hillslope. The DOC concentration did not change, but SUVA254 declined and the organic acid concentration increased downslope. In addition, Fourier-transform infrared spectroscopy indicated the ratio of carboxyl to amide and aromatic functional groups increased downslope. These parameters show that although there was no net loss of DOC along the transect, it was transformed to less aromatic and potentially more labile forms. Together, these results suggest that the depletion of electron acceptors in anoxic pore waters over the course of the thaw season leads to accumulation of labile C compounds, which are likely vulnerable to decomposition following thaw or disturbance.

Utilization of a freeze-thaw treatment to enhance phenolic ripening and tannin oxidation of grape seeds in red (Vitis vinifera L.) cultivars.

PubMed

Rustioni, Laura; Cola, Gabriele; VanderWeide, Josh; Murad, Patrick; Failla, Osvaldo; Sabbatini, Paolo

2018-09-01

Phenolic ripening represents a major interest for quality wine producers. Nevertheless, climatic or genotypical limitations can often prevent optimal maturation process. During winemaking seeds can be easily separated and technologically processed to improve their quality. Relying on the key role of oxidation for phenolic ripening, a freeze-thaw treatment was proposed to improve the fruit quality for potential use in challenging growing conditions. The experiment was carried on in two distinctive viticultural areas, Michigan and Italy. Five cultivars (Cabernet Franc, Cabernet Sauvignon, Merlot, Pinot noir and Chambourcin) and six cultivars (Cabernet Sauvignon, Sangiovese, Syrah, Croatina, Barbera and Nebbiolo) were used in Michigan and Italy, respectively. Samples were collected at different phenological stages, to describe the natural ripening process and grape seeds were characterized before and after a freeze-thaw treatment. Colorimetric and spectrophotometric data highlighted similarities among natural and artificial seed ripening promising future applications for the wine industries. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effects of Cryopreservation Duration on the Outcome of Single-Unit Cord Blood Transplantation.

PubMed

Jaing, Tang-Her; Chen, Shih-Hsiang; Wen, Yu-Chuan; Chang, Tsung-Yen; Yang, Ya-Chun; Tsay, Pei-Kwei

2018-01-01

Cryopreservation is widely used in umbilical cord blood (UCB) banking, yet its impact on progenitor cell function remains largely unaddressed. It is unknown whether long-term cryopreservation affects UCB transplantation outcomes. Herein, we evaluated the impact of UCB age on clinical outcomes and investigated the effect of cryopreservation duration of UCB on hematopoietic potency in 91 patients receiving single cord blood transplantations. UCB cryopreservation duration was 0.7 to 13.4 y. The most common indication of transplant was thalassemia (48%). There was no significant association between cryopreservation duration and neutrophil engraftment probability ( P = 0.475). Cryopreservation duration did not affect the post-thaw viability and subsequent neutrophil engraftment rate. Therefore, UCB units can undergo cryopreservation for at least 8 y with no impact on clinical outcomes.

Field information links permafrost carbon to physical vulnerabilities of thawing

USGS Publications Warehouse

Harden, Jennifer W.; Koven, Charles; Ping, Chien-Lu; Hugelius, Gustaf; McGuire, A. David; Camill, P.; Jorgenson, Torre; Kuhry, Peter; Michaelson, Gary; O'Donnell, Jonathan A.; Schuur, Edward A.G.; Tamocai, Charles; Johnson, Kevin; Grosse, G.

2012-01-01

Deep soil profiles containing permafrost (Gelisols) were characterized for organic carbon (C) and total nitrogen (N) stocks to 3m depths. Using the Community Climate System Model (CCSM4) we calculate cumulative probability functions (PDFs) for active layer depths under current and future climates. The difference in PDFs over time was multiplied by C and N contents of soil horizons in Gelisol suborders to calculate newly thawed C and N, Thawing ranged from 147 PgC with 10 PgN by 2050 (representative concentration pathway RCP scenario 4.5) to 436 PgC with 29 PgN by 2100 (RCP 8.5). Organic horizons that thaw are vulnerable to combustion, and all horizon types are vulnerable to shifts in hydrology and decomposition. The rates and extent of such losses are unknown and can be further constrained by linking field and modelling approaches. These changes have the potential for strong additional loading to our atmosphere, water resources, and ecosystems.

Evaluation of cholesterol- treated dromedary camel sperm function by heterologous IVF and AI.

PubMed

Crichton, Elizabeth G; Malo, Clara; Pukazhenthi, Budhan S; Nagy, Peter; Skidmore, Julian A

2016-11-01

Cholesterol (cholesterol-loaded cyclodextrins: CLC) treatment of dromedary camel sperm prior to freezing enhances cryosurvival. The present study first validated the efficacy of a heterologous zona-free goat oocyte assay (n=115 oocytes) to evaluate camel sperm function in vitro (Experiment 1: n=6 bulls), then examined the effects of CLC treatment (1.5mg/mL CLC; CLC+) versus no treatment (0 CLC) of fresh (Experiment 2: n=4 bulls) and frozen-thawed (Experiment 3: n=5 bulls) camel sperm to penetrate, de-condense and form pro-nuclei in in vitro-matured goat oocytes. Finally, the ability of fresh 0 CLC and CLC+ sperm to fertilize in vivo was studied by artificially inseminating super-ovulated females (n=7-9 per treatment) and examining embryo production (Experiment 4: n=4-5 bulls/treatment). Camel spermatozoa penetrated (60%) and formed pro-nuclei (33%) in goat oocytes demonstrating the utility of this heterologous system for assessing sperm function in vitro. For fresh spermatozoa, 0 CLC-treated sperm performed better than their CLC+ counterparts for all parameters measured (P<0.05). In contrast, cryopreservation resulted in a sharp decline in sperm-oocyte interaction in 0 CLC aliquots but remained unaltered in CLC+ aliquots demonstrating a protective effect of cholesterol treatment. There was no difference between treatments in the in vitro fertilizing ability of frozen-thawed sperm or in the numbers of embryos retrieved following AI with fresh 0 CLC or CLC+ sperm. We conclude that although CLC treatment of dromedary camel sperm improves sperm motility it fails to confer an advantage to them in terms of improved in vitro sperm-oocyte interaction or in vivo fertilization under the conditions tested. Copyright © 2016 Elsevier B.V. All rights reserved.

Detrimental Effects of Non-Functional Spermatozoa on the Freezability of Functional Spermatozoa from Boar Ejaculate

PubMed Central

Martinez-Alborcia, Maria J.; Valverde, Anthony; Parrilla, Inmaculada; Vazquez, Juan M.; Martinez, Emilio A.; Roca, Jordi

2012-01-01

In the present study, the impact of non-functional spermatozoa on the cryopreservation success of functional boar spermatozoa was evaluated. Fifteen sperm-rich ejaculate fractions collected from five fertile boars were frozen with different proportions of induced non-functional sperm (0 –native semen sample-, 25, 50 and 75% non-functional spermatozoa). After thawing, the recovery of motile and viable spermatozoa was assessed, and the functional of the spermatozoa was evaluated from plasma membrane fluidity and intracellular reactive oxygen species (ROS) generation upon exposure to capacitation conditions. In addition, the lipid peroxidation of the plasma membrane was assessed by the indirect measurement of malondialdehyde (MDA) generation. The normalized (with respect to a native semen sample) sperm motility (assessed by CASA) and viability (cytometrically assessed after staining with Hoechst 33342, propidium iodide and fluorescein-conjugated peanut agglutinin) decreased (p<0.01) as the proportion of functional spermatozoa in the semen samples before freezing decreased, irrespective of the semen donor. However, the magnitude of the effect differed (p<0.01) among boars. Moreover, semen samples with the largest non-functional sperm subpopulation before freezing showed the highest (p<0.01) levels of MDA after thawing. The thawed viable spermatozoa of semen samples with a high proportion of non-functional spermatozoa before freezing were also functionally different from those of samples with a low proportion of non-functional spermatozoa. These differences consisted of higher (p<0.01) levels of intracellular ROS generation (assessed with 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester; CM-H2DCFDA) and increased (p<0.01) membrane fluidity (assessed with Merocyanine 540). These findings indicate that non-functional spermatozoa in the semen samples before freezing negatively influence the freezability of functional spermatozoa. PMID:22567165

Experimental study on durability improvement of fly ash concrete with durability improving admixture.

PubMed

Quan, Hong-zhu; Kasami, Hideo

2014-01-01

In order to improve the durability of fly ash concrete, a series of experimental studies are carried out, where durability improving admixture is used to reduce drying shrinkage and improve freezing-thawing resistance. The effects of durability improving admixture, air content, water-binder ratio, and fly ash replacement ratio on the performance of fly ash concrete are discussed in this paper. The results show that by using durability improving admixture in nonair-entraining fly ash concrete, the compressive strength of fly ash concrete can be improved by 10%-20%, and the drying shrinkage is reduced by 60%. Carbonation resistance of concrete is roughly proportional to water-cement ratio regardless of water-binder ratio and fly ash replacement ratio. For the specimens cured in air for 2 weeks, the freezing-thawing resistance is improved. In addition, by making use of durability improving admixture, it is easier to control the air content and make fly ash concrete into nonair-entraining one. The quality of fly ash concrete is thereby optimized.

Morphometric changes in boar spermatozoa induced by cryopreservation.

PubMed

García-Herreros, M; Barón, F J; Aparicio, I M; Santos, A J; García-Marín, L J; Gil, M C

2008-09-01

Computer-assisted sperm morphometry analysis was used to determine the effects of cryopreservation on boar sperm head and midpiece morphometry. Sperm-rich fractions were collected from five mature boars. Three microscope slides were prepared from single extended sperm samples prior freezing and post-thawing. All slides were stained with Hemacolor, and 250 sperm images were obtained from each slide. The sperm head dimensions for length, width, area, perimeter and four shape factors and sperm-midpiece dimensions for area, width, angle and distance were determined in each spermatozoa. The effects of sperm freezing on sperm dimensions within and among boars were determined. A previous discriminant analysis of the results was able to correctly classify a 78.3 and 82% of fresh and frozen-thawed spermatozoa respectively. Sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for length, width, area and perimeter. Sperm midpieces were also significantly smaller in cryopreserved spermatozoa for width and area. The highest changes in morphometric dimensions after the freeze-thawing process were found in the midpiece of spermatozoa. The variability of morphometric measurements only was significantly different between fresh and thawed samples for head rugosity and midpiece area. The effects of cryopreservation on morphometric parameters were similar in the boars, which allow us to conclude that cryopreservation process does not have a different effect in each individual boar. In summary, morphometric changes associated with the cryopreservation process on boar spermatozoa do not apparently depends on an effect at individual level.

Long-term comparison of Kuparuk Watershed active layer maps, northern Alaska, USA

NASA Astrophysics Data System (ADS)

Nyland, K. E.; Queen, C.; Nelson, F. E.; Shiklomanov, N. I.; Streletskiy, D. A.; Klene, A. E.

2017-12-01

The active layer, or the uppermost soil horizon that thaws seasonally, is among the most dynamic components of the permafrost system. Evaluation of the thickness and spatial variation of the active layer is critical to many components of Arctic research, including climatology, ecology, environmental monitoring, and engineering. In this study we mapped active-layer thickness (ALT) across the 22,278 sq. km Kuparuk River basin on Alaska's North Slope throughout the summer of 2016. The Kuparuk River extends from the Brooks Range through the Arctic Foothills and across the Arctic Coastal Plain physiographic provinces, and drains into the Beaufort Sea. Methodology followed procedures used to produce an ALT map of the basin in 1995 accounting for the effects of topography, vegetation, topoclimate, and soils, using the same spatial sampling scheme for direct ALT and temperature measurement at representative locations and relating these parameters to vegetation-soil associations. A simple semi-empirical engineering solution was used to estimate thaw rates for the different associations. An improved lapse-rate formulation and a higher-resolution DEM were used to relate temperature to elevation. Three ALT maps were generated for the 2016 summer, combining measured thaw depth, temperature records, the 25 m ArcticDEM, high resolution remote sensed data, empirical laps rates, and a topoclimatic index through the thaw solution. These maps were used to track the spatial progression of thaw through the 2016 summer season and estimate a total volume of thawed soil. Maps produced in this study were compared to the 1995 map to track areas of significant geographic changes in patterns of ALT and total volume of thawed soil.

Effects of adding different levels of Glutamine to modified Beltsville extender on the survival of frozen rooster semen.

PubMed

Khiabani, Aytak Bakhshayesh; Moghaddam, Gholamali; Kia, Hossein Daghigh

2017-09-01

The aim of the present study was to investigate the effects of l-glutamine on the quality of frozen-thawed rooster semen. Semen samples were collected from eight mature roosters (Ross 308). After initial semen assessments, samples of adequate quality were mixed together and diluted with modified Beltsville extender without l-glutamine (control) and supplemented with 2.5, 5, and 7.5mM l-glutamine. Semen straws were subjected to cryopreservation and evaluated twice at 15-day intervals. After thawing, sperm viability, total and progressive sperm motilities were measured by Eosin-Nigrosine and Computer-Aided Sperm Analysis (CASA), respectively. The results showed that sperm functions decreased on day 30 compared to day 15. The extender supplemented with 5mM glutamine improved (p<0.05) sperm viability, total and progressive sperm motilities compared to other treatments and the control group. The best level of glutamine appeared to be 2.5mM, as it provided the highest sperm membrane integrity and the lowest level of abnormalities. The results of this study suggest that the addition of glutamine to the diluent improves semen quality and using glutamine allows rooster sperm to be frozen for longer. Copyright © 2017 Elsevier B.V. All rights reserved.

Investigation of Freeze and Thaw Cycles of a Gas-Charged Heat Pipe

NASA Technical Reports Server (NTRS)

Ku, Jentung; Ottenstein, Laura; Krimchansky, Alexander

2012-01-01

The traditional constant conductance heat pipes (CCHPs) currently used on most spacecraft run the risk of bursting the pipe when the working fluid is frozen and later thawed. One method to avoid pipe bursting is to use a gas-charged heat pipe (GCHP) that can sustain repeated freeze/thaw cycles. The construction of the GCHP is similar to that of the traditional CCHP except that a small amount of non-condensable gas (NCG) is introduced and a small length is added to the CCHP condenser to serve as the NCG reservoir. During the normal operation, the NCG is mostly confined to the reservoir, and the GCHP functions as a passive variable conductance heat pipe (VCHP). When the liquid begins to freeze in the condenser section, the NCG will expand to fill the central core of the heat pipe, and ice will be formed only in the grooves located on the inner surface of the heat pipe in a controlled fashion. The ice will not bridge the diameter of the heat pipe, thus avoiding the risk of pipe bursting during freeze/thaw cycles. A GCHP using ammonia as the working fluid was fabricated and then tested inside a thermal vacuum chamber. The GCHP demonstrated a heat transport capability of more than 200W at 298K as designed. Twenty-seven freeze/thaw cycles were conducted under various conditions where the evaporator temperature ranged from 163K to 253K and the condenser/reservoir temperatures ranged from 123K to 173K. In all tests, the GCHP restarted without any problem with heat loads between 10W and 100W. No performance degradation was noticed after 27 freeze/thaw cycles. The ability of the GCHP to sustain repeated freeze/thaw cycles was thus successfully demonstrated.

Resilience of norovirus GII.4 to freezing and thawing: implications for virus infectivity.

PubMed

Richards, Gary P; Watson, Michael A; Meade, Gloria K; Hovan, Gregory L; Kingsley, David H

2012-12-01

Genogroup II.4 norovirus (NoV) remains the predominant NoV strain in food- and water-borne outbreaks. Capsid integrity as well as viral RNA persistence were determined for GII.4 NoV by real-time RT-PCR after 1-14 freeze/thaw (F/T) cycles (-80 °C/+22 °C) or after -80 °C storage for up to 120 days. In both cases, capsid integrity and viral RNA titers remained stable. RNase was exogenously added after 1-14 F/T cycles, but did not alter the amount of genomic NoV RNA detected, indicating that capsids remained intact. Presumptive NoV infectivity was evaluated in functional studies by a porcine gastric mucin binding assay. Viruses frozen and thawed up to 14× bound similarly to porcine mucin, suggesting no reduction in virus infectivity. Overall, this study shows that a) NoV particles retain their integrity for at least 14 F/T cycles, b) long-term (120 day) frozen storage does not decrease NoV RNA titers, and c) capsid binding to receptor-like glycoprotein moieties remains unaltered after 14 F/T cycles. This work indicates that freezing and thawing of foods or beverages would not be a practical processing intervention to reduce NoV contamination. Likewise, repeated freezing and thawing, as might be encountered during winter months, is not expected to inactivate NoV in the environment. Results do show that laboratory samples destined for molecular biological analyses or for use as positive controls may be repeatedly frozen and thawed without any anticipated reduction in NoV RNA titers. This study documents the cryostability of NoV capsids and RNA to freezing and thawing and to the possible retention of virus infectivity.

Boar spermatozoa cryopreservation in low glycerol/trehalose enriched freezing media improves cellular integrity.

PubMed

Gutiérrez-Pérez, Oscar; Juárez-Mosqueda, María de Lourdes; Carvajal, Salvador Uribe; Ortega, María Elena Trujillo

2009-06-01

The use of glycerol for boar semen cryopreservation results in low fertility, possibly due to toxicity. This has led to recommend the use of solutions with less than 4% glycerol. Trehalose is a disaccharide known to stabilize proteins and biologic membranes during processes such as cryopreservation. Thus, it was decided to evaluate the cryoprotective effect of glycerol/trehalose mixtures. Effects on motility (M), viability (Vb) and acrosomal integrity (nA) were evaluated. Sperm samples were frozen in three different extenders: G4 contained 4% glycerol; T1 contained 1% glycerol plus 250 mM trehalose and T0.5 was constituted by 0.5% glycerol plus 250 mM trehalose. All extenders yielded similar post-freezing/thawing motility rates. Viability was diminished in T0.5 as compared to the others. In regard to acrosome integrity, it was twice as high (P<0.05) in the trehalose enriched media as in G4, the glycerol-only extender. Thus, T1 twice as many spermatozoa were alive, motile and intact, than in either T0.5 or G4, i.e. during freeze/thawing the use of T1 resulted in twice as many fertile cells as when using the other extenders. During our study, we noted that there were wide individual variations both in sperm viability and in motility.

Influence of a static magnetic field on the slow freezing of human erythrocytes.

PubMed

Lin, Chun-Yen; Chang, Wei-Jen; Lee, Sheng-Yang; Feng, Sheng-Wei; Lin, Che-Tong; Fan, Kan-Shin; Huang, Haw-Ming

2013-01-01

The aim of this study was to test whether or not a strong static magnetic field (SMF) had a positive effect on the survival rate of frozen erythrocytes. Human erythrocytes were slow freezing at a rate of -1°C/min, to a final temperature of -20°C. During the freezing process, the cells were simultaneously exposed to an SMF with a magnetic induction of 0.2 or 0.4 T. After the cells were thawed, the survival rate, morphology, and function of the thawed erythrocytes were evaluated. Furthermore, tests of membrane fluidity were performed to assess the effect of the SMF on the cell membrane. The slow freezing process coupled with an SMF increased the survival rate of frozen erythrocytes, without any negative effect on the cell morphology or function. The increases in relative survival rates of frozen erythrocytes were 5.7% and 9.1% when the cells were frozen in 0.2 T and 0.4 T groups, respectively. In addition, the 0.4 T group significantly increased the membrane rigidity of the erythrocytes. Slow freezing coupled with a strong SMF produced positive effects on the survival rate of thawed erythrocytes, without changing their normal function.

Beneficial effect of extracellular adenosine 5'-triphosphate treatment on the Indochinese leopard (Panthera pardus delacouri) sperm quality after cryopreservation.

PubMed

Thuwanut, P; Tipkantha, W; Siriaroonrat, B; Comizzoli, P; Chatdarong, K

2017-04-01

The Indochinese leopard (Panthera pardus delacouri) population, included in CITES Appendix I, has been declining for decades. Proper gamete preservation condition is critical for breeding programme management using artificial insemination or in vitro fertilization (IVF). The present study aimed at investigating the impact of post-thawing treatment of leopard semen with extracellular adenosine 5'-triphosphate (ATPe) on sperm quality (including morphological traits and ability to fertilize an oocyte). Semen from six adult male leopards was collected by electroejaculation (one ejaculation per cat). After the evaluation of the fresh sample quality, the semen was cryopreserved (10 × 10 6 cells per straw; two straws per cat). After thawing, the sperm sample from the first straw of each cat was divided into three aliquots: control (no ATPe), supplemented with 1.0 or 2.5 mM ATPe that were evaluated for sperm quality at 10, 30 min and 3 hr post-thawing. The sperm sample from the second straw, supplemented with 0, 1.0 or 2.5 mM ATPe for 30 min, was assessed for IVF with domestic cat oocytes. Sperm quality (all metrics) was negatively affected by the cryopreservation process (p ≤ .05). However, the percentage of sperm motility, level of progressive motility and percentage of plasma membrane integrity did not differ (p > .05) among post-thawing groups. The sperm mitochondrial membrane potential was enhanced (p ≤ .05) by ATPe treatment (1.0 and 2.5 mM; 10 min to 3 hr of incubation). Furthermore, incubation of ATPe (1.0 and 2.5 mM) for 30 min could promote sperm velocity patterns (curvilinear velocity; VCL and straight line velocity; VSL) (p ≤ .05). The percentage of pronuclear formation and cleaved embryos was increased (p ≤ .05) after 1.0 ATPe treatment (49.8 ± 2.8; 45.9 ± 1.5) compared to 0 mM (41.4 ± 3.3; 38.9 ± 0.5) whereas the number of sperm binding/oocyte did not significantly differ among groups. In summary, we suggest that ATPe activated the velocity of Indochinese leopard sperm motility that may lead to faster sperm/oocyte binding and sperm penetration (factors of successful embryo development). © 2016 Blackwell Verlag GmbH.

The Impact of Reproductive Technologies on Stallion Mitochondrial Function.

PubMed

Peña, F J; Plaza Davila, M; Ball, B A; Squires, E L; Martin Muñoz, P; Ortega Ferrusola, C; Balao da Silva, C

2015-08-01

The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant. Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen-thawed stallion sperm. Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies. © 2015 Blackwell Verlag GmbH.

Theoretical study of heat transfer with moving phase-change interface in thawing of frozen food

NASA Astrophysics Data System (ADS)

Leung, M.; Ching, W. H.; Leung, D. Y. C.; Lam, G. C. K.

2005-02-01

A theoretical solution was obtained for a transient phase-change heat transfer problem in thawing of frozen food. In the physical model, a sphere originally at a uniform temperature below the phase-change temperature is suddenly immersed in a fluid at a temperature above the phase-change temperature. As the body temperature increases, the phase-change interface will be first formed on the surface. Subsequently, the interface will absorb the latent heat and move towards the centre until the whole body undergoes complete phase change. In the mathematical formulation, the nonhomogeneous problem arises from the moving phase-change interface. The solution in terms of the time-dependent temperature field was obtained by use of Green's function. A one-step Newton-Raphson method was specially designed to solve for the position of the moving interface to satisfy the interface condition. The theoretical results were compared with numerical results generated by a finite difference model and experimental measurements collected from a cold water thawing process. As a good agreement was found, the theoretical solution developed in this study was verified numerically and experimentally. Besides thawing of frozen food, there are many other practical applications of the theoretical solution, such as food freezing, soil freezing/thawing, metal casting and bath quenching heat treatment, among others.

Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral

PubMed Central

Hagedorn, Mary; Carter, Virginia; Martorana, Kelly; Paresa, Malia K.; Acker, Jason; Baums, Iliana B.; Borneman, Eric; Brittsan, Michael; Byers, Michael; Henley, Michael; Laterveer, Michael; Leong, Jo-Ann; McCarthy, Megan; Meyers, Stuart; Nelson, Brian D.; Petersen, Dirk; Tiersch, Terrence; Uribe, Rafael Cuevas; Woods, Erik; Wildt, David

2012-01-01

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (−196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems. PMID:22413020

Cryopreservation studies of an artificial co-culture between the cobalamin-requiring green alga Lobomonas rostrata and the bacterium Mesorhizobium loti.

PubMed

Ridley, Christian J A; Day, John G; Smith, Alison G

2018-01-01

Algal-bacterial co-cultures, rather than cultures of algae alone, are regarded as having the potential to enhance productivity and stability in industrial algal cultivation. As with other inocula in biotechnology, to avoid loss of production strains, it is important to develop preservation methods for the long-term storage of these cultures, and one of the most commonly used approaches is cryopreservation. However, whilst there are many reports of cryopreserved xenic algal cultures, little work has been reported on the intentional preservation of both algae and beneficial bacteria in xenic cultures. Instead, studies have focused on the development of methods to conserve the algal strain(s) present, or to avoid overgrowth of bacteria in xenic isolates during the post-thaw recovery phase. Here, we have established a co-cryopreservation method for the long-term storage of both partners in a unialgal-bacterial co-culture. This is an artificial model mutualism between the alga Lobomonas rostrata and the bacterium Mesorhizobium loti , which provides vitamin B 12 (cobalamin) to the alga in return for photosynthate. Using a Planer Kryo 360 controlled-rate cooler, post-thaw viability (PTV) values of 72% were obtained for the co-culture, compared to 91% for the axenic alga. The cultures were successfully revived after 6 months storage in liquid nitrogen, and continued to exhibit mutualism. Furthermore, the alga could be cryopreserved with non-symbiotic bacteria, without bacterial overgrowth occurring. It was also possible to use less controllable passive freezer chambers to cryopreserve the co-cultures, although the PTV was lower. Finally, we demonstrated that an optimised cryopreservation method may be used to prevent the overgrowth potential of non-symbiotic, adventitious bacteria in both axenic and co-cultures of L. rostrata after thawing.

Effects of caffeine supplementation in post-thaw human semen over different incubation periods.

PubMed

Pariz, J R; Hallak, J

2016-11-01

This study aimed to evaluate the effects of caffeine supplementation in post-cryopreservation human semen over different incubation periods. After collection by masturbation, 17 semen samples were analysed according to World Health Organization criteria, processed and cryopreserved with TEST-yolk buffer (1 : 1) in liquid nitrogen. After a thawing protocol, samples were incubated with 2 mm of caffeine for 0, 5, 15, 30 or 60 min, followed by analysis of motility and mitochondrial activity using 3,3'-diaminobenzidine (DAB). Mean variance analysis was performed, and P < 0.05 was the adopted significance threshold. Samples incubated for 15 min showed increased progressive motility compared to other periods of incubation, as well as a reduced percentage of immotile spermatozoa (P < 0.05). In samples incubated for 5 min, increased mitochondrial activity above 50% was observed (DABI and DABII). Although cryosurvival rates were low after the cryopreservation process, incubation with caffeine was associated with an increase in sperm motility, particularly 15-min incubation, suggesting that incubation with caffeine can be an important tool in patients with worsening seminal quality undergoing infertility treatment. © 2016 Blackwell Verlag GmbH.

Sperm quality and cryopreservation of Brazilian freshwater fish species: a review.

PubMed

Viveiros, A T M; Godinho, H P

2009-03-01

The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil.

Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

PubMed

Johnston, S D; Satake, N; Zee, Y; López-Fernández, C; Holt, W V; Gosálvez, J

2012-06-01

This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic injury model appears to explain post-thaw changes in koala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.

Effect of pre- and post-marination aging on meat quality attributes of early deboned (2 h postmortem) broiler breast fillets.

PubMed

Kuttappan, V A; Gunsaulis, V B; Mauromoustakos, A; Meullenet, J F; Owens, C M

2016-11-01

Marination is an effective method that can be used to improve the tenderness of early deboned breast fillets. The present study was designed to evaluate the effect of pre- and post-marination aging of 2 h postmortem (PM) deboned chicken fillets to get optimum meat quality. In this study, a total of 300 broilers (43 to 46 d of age) were processed using an in-line system and deboned at 2 h PM. Fillets were marinated, at either 2.5, 4, 6, 8 or 24 h PM, using vacuum tumbling (20 min) with a 15% marinade (final concentration of 0.5% salt and 0.45% phosphate). A non-marinated control (CON) was included. The left (HOLD) fillets were aged (held at 4°C for 24 h) prior to freezing post-marination while the right (NO HOLD) fillets were frozen immediately after marination to simulate various commercial practices. Marination pickup (MPU), total marinade retained after thawing (TMR), total purge loss after thawing (TPL), cook loss (CL), and Meullenet-Owens Razor Shear energy (MORSE) values were measured. Both in HOLD and NO HOLD fillets, there was an increase (P < 0.05) in MPU and TMR as the time of marination increased from 2.5 to 24 h PM. Furthermore, the HOLD fillets had a higher (P < 0.05) TPL when compared to the NO HOLD group. There was a higher (P < 0.05) CL for the CON fillets when compared to all marinated fillets suggesting that marination resulted in better water holding capacity. However, both in HOLD and NO HOLD groups, the MORSE values for the marinated fillets decreased (P < 0.05) from 4 h PM onwards, with 8 and 24 h PM having lower (P < 0.05) values than all other treatments. The results of this study suggest that pre-marination aging (aging after deboning prior to marination) of early (2 h PM) deboned fillets to 8 h PM can provide better tumble marination pickup and retention as well as tenderness (or lower shear values). © 2016 Poultry Science Association Inc.

In vitro maintenance, cooling and cryopreservation of red wolf (Canis rufus) spermatozoa.

PubMed

Goodrowe, K L; Mastromonaco, G F; Walker, S L; Bateman, H L; Ryckman, D P; Platz, C C; Waddell, W T

2001-01-01

A current priority for the preservation of the endangered red wolf (Canis rufus) is the development of a sperm-based genome resource bank. The aims of this study were to examine the effects of (i) holding temperature on the motility of spermatozoa over time, and (ii) cooling methods on the characteristics of spermatozoa after cooling and cryopreservation. Electroejaculates (n = 11; fresh) were evaluated for the percentage of motile spermatozoa, cell and acrosome morphology (Spermac (Meditech 1st Canada Inc, Montreal, Ontario) and fluorescein isothiocyanate-labelled Pisum sativum agglutinin lectin (PSA/FITC; Sigma Diagnostics, Oakville, Ontario) staining), and zona penetration. Semen samples were then divided into two equal samples and centrifuged to remove seminal plasma. One half of the ejaculate sample was re-suspended in sperm-Tyrode's albumin lactate pyruvate (TALP), divided into three aliquots and maintained either at room temperature (approximately 21-23 degrees C), 0 degree C or 37 degrees C. Sperm motility was examined at 0.5 and 1.0 h, and subsequently every hour for 10 h. Motility of spermatozoa decreased after 2 h, but was consistently greater at room temperature than at 37 degrees C or 0 degree C. The other half of the ejaculate sample was re-suspended in an egg yolk-based extender and divided into two aliquots. One aliquot was cooled in a refrigerator (5 degrees C) for 30 min, whereas the second aliquot was put into a beaker containing water at 37 degrees C, which was then placed into an ice bath until the sample reached 0 degree C (approximately 120 min). Spermatozoa were evaluated after cooling and after freezing and thawing treatments. No differences were observed between cooling treatments either after cooling or freezing and thawing. However, marked decreases in intact acrosomes, post-thaw motility and normal morphology of spermatozoa after treatment demonstrate that further investigations are necessary to improve cryopreservation methods in this species.

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I--Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols.

PubMed

Kashuba, Corinna M; Benson, James D; Critser, John K

2014-04-01

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols. Copyright © 2014 Elsevier Inc. All rights reserved.

Prerequisites for successful human sperm cryobanking: sperm quality and prefreezing holding time.

PubMed

Yavetz, H; Yogev, L; Homonnai, Z; Paz, G

1991-04-01

Frozen-thawed donor semen was used in artificial inseminations and in vitro fertilization programs. Semen accepted for donation was characterized (mean +/- SE) by sperm concentration of 150 +/- 18.6 x 10(6)/mL, normal morphology of 57% +/- 1.4%, good progressive motility at 1 hour of 57% +/- 1.0%, and post-thaw motility of 45% +/- 1.0%. Delay of the freezing process for greater than 1 hour after semen delivery caused a deleterious effect to the freezability of sperm. The average monthly fecundability for the 1st 6 months after inseminations was 13.6%. This value decreased dramatically to 2.6% from the 7th month onward. In 74 IVF/embryo transfer (ET) cycles, the fertilization rate was 55.3% +/- 3.8%, pregnancy rate (PR) per ET was 39.6%, and the PR per woman was 42.8%.

Effect of cryopreservation on the nuclear chromatin decondensation ability of human spermatozoa.

PubMed

Huret, J L

1984-01-01

The possible effect of cryopreservation on human sperm chromatin decondensation ability has been investigated. Comparisons of the actions of the decondensation-inducing agent 1% (w/v) sodium dodecyl sulphate + 6 mM EDTA were made on 30 ejaculates between spermatozoa in seminal plasma, spermatozoa in semen diluted with cryoprotective medium (CPM) and spermatozoa frozen and thawed in the semen-CPM mixture. The results, analyzed as paired series, showed no significant differences between the spermatozoa under the three treatment conditions. Thus, spermatozoa cryopreserved by a method routinely used for semen storage for subsequent artificial insemination showed a nuclear stability equivalent to that of fresh semen. The CPM by itself had no effect upon chromatin instability. No correlation was found between the percentage recovery of post-thaw motility (an usual index for judging semen cryopreservation) and the tests of chromatin decondensation.

Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation.

PubMed

Moraes, E A; Matos, W C G; Graham, J K; Ferrari, W D

2015-07-01

This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the experiments, the treated sperm were incubated for 15min at 22°C to allow for the incorporation of cholesterol or cholestanol. In each experiment, treated sperm incubated for 15min at 22°C to allow for incorporation of cholesterol or cholestanol. The samples were then diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5°C over a 2h period. Loaded into 0.25ml polyvinylchloride straws, frozen in liquid nitrogen vapor for 10min, and then plunged into liquid nitrogen until further use. Higher percentages of motile sperm and viable cells were achieved after thawing for stallion sperm treated with CLC and CnLC compared to control (P<0.05). Addition of CnLC also resulted in more number sperm binding to chicken egg perivitelline membrane (CEPM) after cryopreservation than cholestanol and control sperm (P<0.05). In conclusion, CnLC and CLC improved the percentage of post-thaw motility of equine sperm and CnLC provided greater binding efficiency. Copyright © 2015 Elsevier B.V. All rights reserved.

Observations using Phosphorus-31 nuclear magnetic resonance (31P-NMR) of structural changes in freeze-thawed hen egg yolk.

PubMed

Wakamatsu, Hiroki; Handa, Akihiro; Chiba, Kazuhiro

2018-04-01

Hen egg yolk (EY) has a complicated structure consisting of lipids and proteins, and its structure is deeply related with its functional properties. 31 P-NMR is an efficient technique to non-destructively detect the dynamic behaviour of phospholipids, the main component of bio-membranes. We determined conditions for measuring the 31 P NMR spectra of EY and identified the components. 31 P-NMR was used to detect phosvitin, inorganic phosphate, and lipoprotein as well as structural changes such as granule collapse and freeze-thaw denaturation as signal changes. Freeze-thaw denaturation generated a new denaturation peak. We separated aggregates of LDL from freeze-thawed plasma using centrifugation. TEM and 31 P-NMR observations revealed that the denaturation peak corresponded to LDL aggregates. The 31 P-NMR spectra suggested the formation of multiple forms of LDL aggregates in which the head groups of phospholipid molecules adopt a face-to-face orientation, similar to that observed following the flocculation of lipoproteins or in the lamellar-like structures of phospholipids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Density gradient centrifugation prior to cryopreservation and hypotaurine supplementation improve post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia.

PubMed

Brugnon, F; Ouchchane, L; Pons-Rejraji, H; Artonne, C; Farigoule, M; Janny, L

2013-08-01

Can selection of spermatozoa by density gradient centrifugation prior to cryopreservation and/or hypotaurine supplementation improve the post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia? Sperm selection by density gradient centrifugation before freezing and supplementation of the media by hypotaurine is beneficial for the cryopreservation of semen samples of patients with oligoasthenoteratozoospermia. Sperm from men with oligoasthenoteratozoospermia are more susceptible than normal to cryoinjury. Density gradient centrifugation before sperm freezing may allow the selection of a subpopulation of spermatozoa more resistant to cryopreservation. Hypotaurine is an antioxidant with a protective effect on sperm functions. The experiment was carried out according to a factorial design involving two binary factors resulting in four treatment combinations which were randomly allocated in oligoasthenoteratozoospermia sperm samples from 64 patients recruited between January 2009 and June 2010. Semen was provided by 64 men undergoing evaluation for infertility at the Centre for Reproductive Medicine of the University Hospital in Clermont-Ferrand, France, between January 2009 and June 2010. Four treatment combinations were tested: sperm freezing before selection without (F-S/H-; n = 16) and with hypotaurine supplementation (F-S/H+; n = 16); sperm selection before freezing without (S-F/H-; n = 16) and with hypotaurine supplementation (S-F/H+; n = 16). Measurements of sperm recovery rates and markers of apoptosis (externalization of phosphatidylserine (PS), mitochondrial membrane potential and DNA fragmentation) were compared in recovered spermatozoa after each procedure. Higher recovery rates of progressive and total motile spermatozoa were observed when sperm selection was performed before freezing (P < 0.05). The protective effect of hypotaurine was only observed on the percentage of live spermatozoa with PS externalization among total live spermatozoa (AN+ PI-/((AN+ PI-) + (AN- PI-)) when the sperm selection by density gradient centrifugation was performed before freezing (S-F/H+ versus S-F/H-: 6.8 ± 1.09 versus 11.8 ± 2.03%, P = 0.04). The percentage of mitochondrial membrane potential (DiOC6(3) (high)) spermatozoa was higher (P = 0.001) when sperm selection was done before freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 58.1 ± 3.50 versus 46.7 ± 5.48%) or without (S-F/H- versus F-S/H-: 57.0 ± 5.18 versus 35.4 ± 4.99%) hypotaurine supplementation. The percentages of TUNEL+ spermatozoa were significantly lower (P = 0.001) when sperm selection was done before sperm freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 38.6 ± 9.59 versus 55.7 ± 5.88%) or without hypotaurine supplementation (S-F/H- versus F-S/H-: 37.2 ± 7.91 versus 71.0 ± 5.66%). The ICSI outcomes were not assessed and the fertility of the spermatozoa remains unknown. Sperm selection by density gradient centrifugation before freezing and hypotaurine supplementation could improve the cryopreservation of sperm from oligoasthenoteratozoospermic men and make a larger number of functional spermatozoa available for ICSI. This work was supported by a hospital grant (Projet Hospitalier Recherche Clinique, CHU Clermont Ferrand, France). None of the authors has any conflict of interest to declare.

Comparison of the clinical outcomes between fresh blastocyst and vitrified-thawed blastocyst transfer.

PubMed

Ku, Pei-Yun; Lee, Robert Kuo-Kuang; Lin, Shyr-Yeu; Lin, Ming-Huei; Hwu, Yuh-Ming

2012-12-01

To compare the clinical outcomes between fresh and vitrified-thawed day 5 blastocyst transfers. Retrospective case control study. Tertiary referral center. Patients 38 years of age or less who underwent IVF/ICSI cycles with fresh or frozen-thawed blastocysts transferred from June 1, 2009 to November 30, 2011 Vitrification and thawing of day 5 blastocysts using the Cryotop method. (Kitazato BioPharma Co., Ltd., Fuji city, Shizuoka, Japan) Clinical pregnancy rate, implantation rate, ongoing pregnancy rate, and multiple pregnancy rates. Of the 118 cycles in the fresh transfer group, 234 blastocysts were transferred. The clinical pregnancy rate was 66.1 % and implantation rate was 50.9 %. The ongoing pregnancy rate was 56.8 % and the rates for singleton and twin pregnancies were 53.7 % and 44.8 %. Of the 59 cycles in the vitrified-thawed group, 111 blastocysts were transferred. The clinical pregnancy rate was 59.3 % and implantation rate was 43.2 %. The ongoing pregnancy rate was 47.5 % and the rates for singleton and twin pregnancies were 60.7 % and 39.3 %. The clinical pregnancy rate, implantation rate and ongoing pregnancy rate did not differ significantly between the two groups. The implantation rates were not significantly different between the fresh and the vitrified-thawed groups. Thus, single embryo transfer may be considered in fresh cycles to decrease multiple pregnancy rates. The surplus embryos should be vitrified for the frozen embryo transfer to improve the cumulative pregnancy rate.

Improved fertility in gilts and sows after artificial insemination of frozen-thawed boar semen by supplementation of semen extender with caffeine and CaCl2.

PubMed

Yamaguchi, Shoichiro; Funahashi, Hiroaki; Murakami, Tetsuya

2009-12-01

Supplementation of semen extender with caffeine and CaCl(2) for artificial insemination (AI) of fresh spermatozoa has been demonstrated to reduce recruitment of uterine polymorphonuclear leukocytes (PMNs) and the activity of phagocytosis. Here, we determined if addition of caffeine and CaCl(2) to semen extender improves the fertility of frozen-thawed boar semen. In experiment 1, gilts were cervically inseminated twice with frozen-thawed boar spermatozoa (25 x 10(8) cells per dose) suspended in Modena solution (n=7) or modified Beltsville Thawing Solution supplemented with caffeine and CaCl(2) (BCC, n=7). The gilts were slaughtered 4 h later, and their oviducts and uterine horns plus the body of the uterus were flushed to recover PMNs and non-phagocytosed spermatozoa. There was no difference in the total number of uterine PMNs between gilts inseminated with Modena solution and those inseminated with BCC (3.8 x 10(8) vs. 1.5 x 10(8) cells, respectively); however, the total number of uterine spermatozoa was higher when gilts were inseminated with BCC (40.6 x 10(6) cells) compared with those inseminated with Modena solution (1.4 x 10(6) cells, P<0.05). In experiment 2, gilts and sows were subjected to intrauterine insemination twice with frozen-thawed spermatozoa suspended (25 x 10(8) sperm per dose) in Modena (n=21) or BCC (n=21). The overall pregnancy and farrowing rates were higher in females inseminated with BCC (71.4 and 61.9%, respectively) compared with those inseminated with Modena solution (38.1 and 28.6%, respectively, P<0.05). However, no significant difference in litter size of piglets was observed between treatments (7.2 +/- 1.6 piglets for Modena solution vs. 8.2 +/- 0.9 piglets for BCC solution). In conclusion, we demonstrated that use of BCC solution for frozen-thawed boar semen produced better pregnancy and farrowing rates following AI than Modena solution, probably by reducing the phagocytosis of spermatozoa.

The paleoecology, peat chemistry and carbon storage of a discontinuous permafrost peatland

NASA Astrophysics Data System (ADS)

Talbot, Julie; Pelletier, Nicolas; Olefeldt, David; Turetsky, Merritt; Blodau, Christian; Sonnentag, Oliver; Quinton, William

2017-04-01

Permafrost in peatlands strongly influences ecosystem biogeochemical functioning, vegetation composition and hydrological functions. Permafrost peatlands of northwestern Canada store large amounts of carbon but the peatlands located at the southern margin of the permafrost zone are thawing rapidly. This thaw triggers changes in vegetation, hydrology and peat characteristics, and may affect carbon stocks. We present data from a permafrost plateau to thermokarst bog chronosequence located in the southern portion of the Scotty Creek watershed near Fort Simpson, Northwest Territories, Canada. We assessed changes in plant communities, hydrology, biogeochemistry and permafrost status over 9000 years of peatland development using plant macrofossil, testate amoeba and peat chemical characteristics. Peat accumulation started after the infilling of a lake 8500 cal. yr BP. Minerotrophic peat prevailed at the site until permafrost formed around 5000 cal. yr BP. Permafrost apparently formed three times, although there is spatial variability in the permafrost aggradation - degradation cycles. Permafrost thawed 550 cal. yr BP in the center of the thermokarst bog. Ombrotrophic peat is a fairly recent feature of the peat profiles, only appearing after the most recent permafrost thaw event. Both allogenic (temperature/precipitation/snow cover changes and wildfire) and autogenic (peat accumulation, Sphagnum growth) processes likely influenced permafrost aggradation and thaw. While apparent carbon accumulation rates were lower during present and past permafrost periods than during non-permafrost periods, long term carbon accumulation remained similar between cores with different permafrost period lengths. Deep peat was more decomposed in the thermokarst bog peat profile than in the permafrost plateau profile, highlighting the importance of considering potential deep peat carbon losses to project the fate of thawing permafrost peat carbon stores. Average long-term carbon accumulation derived from the peat cores (n=3, 20.6 ± 1.9 g C m-2 a-1) is in the same range than the contemporary landscape-scale carbon balance measured from eddy covariance at the site ( 15 g C m-2 a-1). While the carbon to nitrogen ratio tends to decrease with peat depth, the carbon to phosphorus ratio tends to increase, perhaps indicating a preferential uptake of phosphorus over nitrogen by plants.

Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population

PubMed Central

Madeddu, Manuela; Berlinguer, Fiammetta; Ledda, Massimo; Leoni, Giovanni G; Satta, Valentina; Succu, Sara; Rotta, Andrea; Pasciu, Valeria; Zinellu, Angelo; Muzzeddu, Marco; Carru, Ciriaco; Naitana, Salvatore

2009-01-01

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December – March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 μl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique. PMID:19228408

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa.

PubMed

Ghallab, AbdelRaouf M; Shahat, Abdallah M; Fadl, Aya M; Ayoub, Mohamed M; Moawad, Adel R

2017-12-01

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5-ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds.

PubMed

Kaeoket, K; Sang-urai, P; Thamniyom, A; Chanapiwat, P; Techakumphu, M

2010-06-01

During the cryopreservation process, the level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), in the sperm plasma membrane decreases significantly because of lipid peroxidation, which may contribute to sperm loss quality (i.e. fertility) of frozen-thawed semen. The aim of this study was to investigate the effect of supplementation of DHA (fish oil) in freezing extender II on frozen-thawed semen quality. Semen from 20 boars of proven motility and morphology, were used in this study. Boar semen was split into four groups, in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various levels of fish oil to reach DHA level of 1X (group I, control, no added fish oil), 6X (group II), 12X (group III) and 18X (group IV). Semen solutions were frozen by using a controlled rate freezer. After cryopreservation, frozen semen was thawed and evaluated for progressive motility, viability by using SYBR-14/Ethidiumhomodimer-1 (EthD-1) staining and acrosome integrity by using FITC-PNA/EthD-1 staining. There was a significantly higher (p < 0.001) percentage of progressive motility, viability and acrosome integrity in DHA (fish oil) supplemented groups than control group. Generally, there seemed to be a dose-dependent effect of DHA, with the highest percentage of progressive motility, viability and acrosome integrity in group-III. In conclusion, supplementation of the LEY extender with DHA by adding fish oil was effective for freezing boar semen as it resulted in higher post-thaw plasma membrane integrity and progressive motility.

Lactose-egg yolk diluent supplemented with N-acetyl-D-glucosamine affect acrosome morphology and motility of frozen-thawed boar sperm.

PubMed

Yi, Y J; Im, G S; Park, C S

2002-12-16

These experiments were carried out to investigate the effect of N-acetyl-D-glucosamine, and to obtain additional information about the effect of orvus es paste (OEP) and egg yolk concentration in the freezing of boar sperm in the maxi-straw. The highest post-thaw acrosomes of normal apical ridge (NAR) and motility were obtained with 0.025 or 0.05% N-acetyl-D-glucosamine concentration in the first diluent. However, there were no effects of N-acetyl-D-glucosamine among the diluents with or without N-acetyl-D-glucosamine at the second dilution. The N-acetyl-D-glucosamine in the first and second diluents was added at room temperatures (20-23 degrees C) and 5 degrees C, respectively. It is suggested that the temperature of N-acetyl-D-glucosamine addition is important for the effect of boar sperm protection during freezing and thawing. When the 0.05% N-acetyl-D-glucosamine was supplemented in the first diluent, the optimum final OEP content was 0.5%. The optimum content of egg yolk in the diluent with 0.05% N-acetyl-D-glucosamine concentration was 20% and egg yolk was one of the main cryoprotective agents. In conclusion, we found out that the diluent with 0.025 or 0.05% soluble N-acetyl-D-glucosamine in the first diluent, 0.5% final orvus es paste concentration and 20% egg yolk concentration significantly enhanced NAR acrosomes and motility of boar sperm after freezing and thawing. Copyright 2002 Elsevier Science B.V.

Studies on the protective efficacy of freeze thawed promastigote antigen of Leishmania donovani along with various adjuvants against visceral leishmaniasis infection in mice.

PubMed

Thakur, Ankita; Kaur, Harpreet; Kaur, Sukhbir

2015-09-01

Visceral leishmaniasis (VL) caused by Leishmania donovani persists as a major public health issue in tropical and subtropical areas of the world. Current treatment of this disease relies on use of drugs. It is doubtful that chemotherapy can alone eradicate the disease, so there is a need for an effective vaccine. Killed antigen candidates remain a good prospect considering their ease of formulation, stability, low cost and safety. To enhance the efficacy of killed vaccines suitable adjuvant and delivery system are needed. Therefore, the current study was conducted to determine the protective efficacy of freeze-thawed L. donovani antigen in combination with different adjuvants against experimental infection of VL. For this, BALB/c mice were immunized thrice at an interval of two weeks. Challenge infection was given two weeks after last immunization. Mice were sacrificed after last immunization and on different post challenge/infection days. Immunized mice showed significant reduction in parasite burden, enhanced DTH responses with increased levels of Th1 cytokines and lower levels of Th2 cytokines, thus indicating the development of a protective Th1 response. Maximum protection was achieved with liposome encapsulated freeze thawed promastigote (FTP) antigen of L. donovani and it was followed by group immunized with FTP+MPL-A, FTP+saponin, FTP+alum and FTP antigen (alone). The present study highlights greater efficacy of freeze thawed promastigote antigen as a potential vaccine candidate along with effective adjuvant formulations against experimental VL infection. Copyright © 2015 Elsevier GmbH. All rights reserved.

Experimental Study on Durability Improvement of Fly Ash Concrete with Durability Improving Admixture

PubMed Central

Quan, Hong-zhu; Kasami, Hideo

2014-01-01

In order to improve the durability of fly ash concrete, a series of experimental studies are carried out, where durability improving admixture is used to reduce drying shrinkage and improve freezing-thawing resistance. The effects of durability improving admixture, air content, water-binder ratio, and fly ash replacement ratio on the performance of fly ash concrete are discussed in this paper. The results show that by using durability improving admixture in nonair-entraining fly ash concrete, the compressive strength of fly ash concrete can be improved by 10%–20%, and the drying shrinkage is reduced by 60%. Carbonation resistance of concrete is roughly proportional to water-cement ratio regardless of water-binder ratio and fly ash replacement ratio. For the specimens cured in air for 2 weeks, the freezing-thawing resistance is improved. In addition, by making use of durability improving admixture, it is easier to control the air content and make fly ash concrete into nonair-entraining one. The quality of fly ash concrete is thereby optimized. PMID:25013870

Resveratrol and Epigallocatechin-3-gallate addition to thawed boar sperm improves in vitro fertilization.

PubMed

Gadani, B; Bucci, D; Spinaci, M; Tamanini, C; Galeati, G

2017-03-01

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2 mM; Experiment 1) or EGCG (25, 50 or 100 μM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and in vitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1 h of incubation at 37 °C. The addition of different doses of Res or EGCG to thawing extender for 1 h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (P < 0.01) while only Res 2 mM induced a significant increase of this parameter (P < 0.01). In addition, EGCG 25 and 50 μM supplementation significantly increased total fertilization efficiency as compared to control (EGCG 25 μM: 40.3 ± 8.2 vs 26.8 ± 9.5, P < 0.05; EGCG 50 μM: 40.4 ± 7.8 vs 26.8 ± 9.5, P < 0.01). The same effect was observed with Res 2 mM (51.0 ± 7.6 vs 29.6 ± 11.3, P < 0.01). In conclusion, our results indicate that the addition of different doses of the two antioxidants to thawed spermatozoa for one hour, even if does not exert any effect on sperm viability and acrosome integrity, efficiently improves in vitro penetration rate. Moreover, both molecules (EGCG 25 and 50 μM and Res 2 mM) significantly increases the total efficiency of fertilization. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of permafrost thaw on nitrogen availability and plant nitrogen acquisition in Interior Alaska

NASA Astrophysics Data System (ADS)

Finger, R.; Euskirchen, E. S.; Turetsky, M.

2013-12-01

The degradation of ice-rich permafrost, which covers a large portion of Interior Alaska, typically leads to thermokarst and increases in soil saturation. As a result, conifer peat plateaus degrade and are often replaced by wet collapse scar bogs. This state change results in profound changes in regional hydrology, biogeochemical cycling, and plant community composition. Preliminary data suggest that permafrost thaw can increase surface soil inorganic nitrogen (IN) concentrations but it is still unknown whether these changes in nutrient availability are short-lived (pulse releases) and whether or not they impact collapse scar vegetation composition or productivity, particularly as collapse scars undergo succession with time-after-thaw. Therefore we are currently examining changes in plant community composition, N availability and plant N acquisition along three thermokarst gradients in Interior Alaska. Each gradient is comprised of a forested permafrost peat plateau, adjacent ecotones experiencing active permafrost degradation (including a collapsing forest canopy and a saturated moat), and a collapse scar bog where permafrost has completely degraded. We predicted that IN concentrations would be highest along the active thaw margin, and lowest in the peat plateau. We also predicted that IN concentrations would be positively related to shifts in vegetation community composition, nutrient use efficiency (NUE) and tissue 15N concentrations. Preliminary results have shown that IN concentrations increase in newer collapse scar features as well as with thaw depth. Our data also show a shift from feather moss and ericaceous shrub-dominate understories in the permafrost plateau to Sphagnum and sedge dominated thaw ecotone and bog communities. Further successional development of the collapse scar bog results in the reintroduction of small evergreen and deciduous shrubs as the peat mat develops. Over time, collapse scar succession and peat accumulation appears to lead to progressive N limitations, resulting in the dominance of plants with higher NUE. This likely has implications for plant litter quality, and could inhibit decomposition processes. We are collecting additional data to compare species-level NUE and nutrient resorption efficiency. We also will measure δ15N of aboveground plant organs, roots, soil, and pore water to explore sources of plant N, which we expect will influenced rooting depth as permafrost thaws as well as differences in mycorrhizal associations along our thaw gradient. Because thawing permafrost soils are anticipated to mobilize large amounts of N from soils, our results will improve our understanding of how permafrost thaw influences vegetation and soil N pools, soil N availability, and plant nutrition.

Freezing and thawing or freezing, thawing, and aging effects on beef tenderness.

PubMed

Grayson, A L; King, D A; Shackelford, S D; Koohmaraie, M; Wheeler, T L

2014-06-01

The objective of this study was to determine the effect of freezing and thawing or freezing and thawing with an additional aging period after frozen storage on the tenderness of longissimus lumborum (LL) and semitendinosus (ST) steaks relative to aged, fresh steaks. Left-side LL and ST (n = 35 each) were obtained from U.S. Select carcasses classified at the grading stand by the U.S. Meat Animal Research Center visible and near-infrared spectroscopy tenderness system to have predicted slice shear force greater than 16.5 kg at 14 d postmortem. At 2 d postmortem, 2.54 cm thick steaks were cut from each muscle and assigned to 1 of the following treatments: 2 d fresh (2FRESH), 2 d freeze + thaw (2FREEZE), 2 d freeze + thaw + 12 d age (2FREEZE+12AGE), 14 d fresh (14FRESH), 14 d freeze + thaw (14FREEZE), 14 d freeze + thaw + 14 d age (14FREEZE+14AGE), and 28 d fresh (28FRESH). Steaks assigned to a freezing treatment were frozen at -26°C for 30 d before thawing/cooking or thawing with an additional aging period at 2°C. Slice shear force for LL and ST was lower (P < 0.01) for 2FREEZE (27.4 and 24.5 kg) and 14FREEZE (22.4 and 22.4 kg) compared to 2FRESH (33.0 and 29.2 kg) and 14FRESH (25.3 and 25.5 kg), respectively. Slice shear force for LL and ST was lower (P < 0.01) for 2FREEZE+12AGE (17.8 and 20.8 kg) and 14FREEZE+14AGE (14.6 and 19.0 kg) compared to 14FRESH (25.3 and 25.5 kg) and 28FRESH (18.7 and 21.7 kg), respectively. Desmin degradation for LL was not different (P > 0.05) between 2FREEZE (21.0%) and 2FRESH (14.6%) or between 14FREEZE (40.4%) and 14FRESH (38.4%); however, desmin degradation was higher (P < 0.06) in 2FREEZE+12AGE (46.7%) and 14FREEZE+14AGE (71.1%) when compared to 14FRESH (38.4%) and 28FRESH (60.5%), respectively. Cooking loss for LL was higher (P < 0.01) in 2FREEZE+12AGE (15.2%) compared to 14FRESH (14.0%) but was not different (P > 0.05) between 14FREEZE+14AGE (15.0%) and 28FRESH (14.3%). Freezing and thawing or a combination of freezing, thawing, and aging resulted in increased tenderness for LL and ST steaks when compared to fresh steaks with the same aging time. These results indicate freezing could be incorporated into normal commercial product distribution processes to improve the consistency of meat tenderness. Researchers who freeze steaks before tenderness assessment should be aware and acknowledge that freezing affects tenderness data.

Some observations on soil freezing in forest and range lands of the Pacific Northwest.

Treesearch

Charles E. Hale

1950-01-01

It is well known that freezing and thawing of the surface soil and humus greatly affect their capacity to absorb water. Post and Dreibelbis (1) in Ohio reported that percolation was materially reduced or ceased entirely when the frost depth was three inches or greater. They also stated that "freezing of the surface soil undoubtedly has considerable influence on...

Comparison of In Vitro- and Chorioallantoic Membrane (CAM)-Culture Systems for Cryopreserved Medulla-Contained Human Ovarian Tissue

PubMed Central

Isachenko, Vladimir; Mallmann, Peter; Petrunkina, Anna M.; Rahimi, Gohar; Nawroth, Frank; Hancke, Katharina; Felberbaum, Ricardo; Genze, Felicitas; Damjanoski, Ilija; Isachenko, Evgenia

2012-01-01

At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5–2.0×1.0–1.2×0.8–1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian medulla-contained strips. PMID:22479331

Alpha-ketoglutarate enhances freeze-thaw tolerance and prevents carbohydrate-induced cell death of the yeast Saccharomyces cerevisiae.

PubMed

Bayliak, Maria M; Hrynkiv, Olha V; Knyhynytska, Roksolana V; Lushchak, Volodymyr I

2018-01-01

Stress resistance and fermentative capability are important quality characteristics of baker's yeast. In the present study, we examined protective effects of exogenous alpha-ketoglutarate (AKG), an intermediate of the tricarboxylic acid cycle and amino acid metabolism, against freeze-thaw and carbohydrate-induced stresses in the yeast Saccharomyces cerevisiae. Growth on AKG-supplemented medium prevented a loss of viability and improved fermentative capacity of yeast cells after freeze-thaw treatment. The cells grown in the presence of AKG had higher levels of amino acids (e.g., proline), higher metabolic activity and total antioxidant capacity, and higher activities of catalase, NADP-dependent glutamate dehydrogenase and glutamine synthase compared to control ones. Both synthesis of amino acids and enhancement of antioxidant system capacity could be involved in AKG-improved freeze-thaw tolerance in S. cerevisiae. Cell viability dramatically decreased under incubation of stationary-phase yeast cells in 2% glucose or fructose solutions (in the absence of the other nutrients) as compared with incubation in distilled water or in 10 mM AKG solution. The decrease in cell viability was accompanied by acidification of the medium, and decrease in cellular respiration, aconitase activity, and levels of total protein and free amino acids. The supplementation with 10 mM AKG effectively prevented carbohydrate-induced yeast death. Protective mechanisms of AKG could be associated with the intensification of respiration and prevention of decreasing protein level as well as with direct antioxidant AKG action.

Effect of Freezing Rate and Microwave Thawing on Texture and Microstructural Properties of Potato (Solanum tuberosum).

PubMed

Phinney, David M; Frelka, John C; Wickramasinghe, Anita; Heldman, Dennis R

2017-04-01

Food freezing is a preservation process that works by lowering temperature while simultaneously decreasing water activity. It is accepted that although freezing preserves foods, it generally has a negative effect on textural quality. This research investigated the texture response of potatoes (Solanum tuberosum) as a function of time to freeze (defined as the time for the center temperature to reach -20 °C) and thawing process. Potatoes slices (6 mm) were blanched then frozen in an ethanol/carbon dioxide bath, a pilot scale high velocity air freezer (HVAF) and a still air freezer to achieve various times to freeze. Slices were stabilized at -20 °C and thawed by 2 methods; room temperature air and microwave. Afterwards, samples were allowed to come to room temperature prior to texture profile analysis (TPA). Results indicate a maximum texture loss of the potato was reached at a time to freeze of approximately 8 min (corresponding to the HVAF). The texture difference between room temperature and microwave thawing methods was not shown to be significant (P = 0.05). SEM images showed the cellular structure of the potato in a HVAF to be similar to that of the still air freezer, validating that the matrix was maximally damaged in both conditions. This work created a continuous quality loss model for the potato as a function of time to freeze and showed no textural benefit to high velocity over still air freezing. © 2017 Institute of Food Technologists®.

Changes to the Carbon and Energy fluxes in a Northern Peatland with Thawing Permafrost

NASA Astrophysics Data System (ADS)

Harder, S. R.; Roulet, N. T.; Crill, P. M.; Strachan, I. B.

2017-12-01

The maintenance of thaw of high carbon density landscapes in the permafrost region ultimately depends of how the energy balance is partitioned as temperatures and precipitation change, yet there are comparatively few energy balance studies, especially in peatlands that contain permafrost. While permafrost peatlands are currently net sinks of carbon, as Arctic temperatures rise and permafrost thaws, the future of these ecosystems and their capacity for carbon uptake is in question. Since 2012 we have been measuring the spatially integrated CO2, energy and water vapour fluxes from the Stordalen peatland (68°22'N, 19°03'E) using eddy covariance (EC). The Stordalen peatland is a heterogeneous peatland in the discontinuous permafrost zone where permafrost thaw is actively occurring, resulting in large changes to the landscape from year to year. Areas where permafrost is present are elevated by up to 1.5 m compared to the areas where permafrost has thawed causing differences in water table depth, peat temperatures, snow distribution, vegetation community and therefore in the carbon and energy fluxes. Our EC tower is located on the edge of a permafrost peat plateau (or palsa) where one fetch measures fluxes from an area underlain by permafrost and the other fetch sees the portion of the peatland where the permafrost has thawed. Within each sector, we have an array of soil temperature and water content sensors to determine the physical characteristics of each fetch. Extensive vegetation surveys (based on plant functional types or PFTs) have also been conducted to run a footprint analysis on the flux data to complete a comparative analysis of the magnitude and variability of the carbon and energy exchanges from PFT. The footprint analysis allows us to explain the difference in energy and carbon fluxes by examining the ecological, biogeochemical and physical characteristics within each footprint. We see distinctly different energy partitioning between the fetches containing intact permafrost and those where the permafrost has thawed: the evaporative efficiency is higher and the Bowen ration lower for the thawed fetches. Our results also show differences in the carbon fluxes depending on the tower footprint.

Micromechanical properties of canine femoral articular cartilage following multiple freeze-thaw cycles.

PubMed

Peters, Abby E; Comerford, Eithne J; Macaulay, Sophie; Bates, Karl T; Akhtar, Riaz

2017-07-01

Tissue material properties are crucial to understanding their mechanical function, both in healthy and diseased states. However, in certain circumstances logistical limitations can prevent testing on fresh samples necessitating one or more freeze-thaw cycles. To date, the nature and extent to which the material properties of articular cartilage are altered by repetitive freezing have not been explored. Therefore, the aim of this study is to quantify how articular cartilage mechanical properties, measured by nanoindentation, are affected by multiple freeze-thaw cycles. Canine cartilage plugs (n = 11) from medial and lateral femoral condyles were submerged in phosphate buffered saline, stored at 3-5°C and tested using nanoindentation within 12h. Samples were then frozen at -20°C and later thawed at 3-5°C for 3h before material properties were re-tested and samples re-frozen under the same conditions. This process was repeated for all 11 samples over three freeze-thaw cycles. Overall mean and standard deviation of shear storage modulus decreased from 1.76 ± 0.78 to 1.21 ± 0.77MPa (p = 0.91), shear loss modulus from 0.42 ± 0.19 to 0.39 ± 0.17MPa (p=0.70) and elastic modulus from 5.13 ± 2.28 to 3.52 ± 2.24MPa (p = 0.20) between fresh and three freeze-thaw cycles respectively. The loss factor increased from 0.31 ± 0.38 to 0.71 ± 1.40 (p = 0.18) between fresh and three freeze-thaw cycles. Inter-sample variability spanned as much as 10.47MPa across freezing cycles and this high-level of biological variability across samples likely explains why overall mean "whole-joint" trends do not reach statistical significance across the storage conditions tested. As a result multiple freeze-thaw cycles cannot be explicitly or statistically linked to mechanical changes within the cartilage. However, the changes in material properties observed herein may be sufficient in magnitude to impact on a variety of clinical and scientific studies of cartilage, and should be considered when planning experimental protocols. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cryopreservation of bull spermatozoa alters the perinuclear theca.

PubMed

Martínez, Carmen Omega; Juárez-Mosqueda, María de Lourdes; Hernández, Jorge; Valencia, Javier

2006-11-01

The perinuclear theca (PT) is involved in several important sperm functions leading to fertilization. The objective of this study was to investigate the effect of cryopreservation of bull spermatozoa on the integrity of the PT and the relationship between PT integrity and semen characteristics. Semen from seven bulls was evaluated before and after cryopreservation, comparing the integrity of the plasma membrane (hypo-osmotic test), percentage of live and dead spermatozoa (triple stain), acrosome integrity (triple stain) and the integrity of the PT (negative stain by electron microscopy). Cryopreservation of bull semen caused substantial damage to the PT; the proportion of spermatozoa with a damaged PT was 15.2% versus 52.5% (P<0.05) in fresh versus frozen-thawed spermatozoa, respectively. Furthermore, on average, 67.4% (range, 64-72%) of fresh spermatozoa were live, compared to 53.1% (range, 49-58%) for frozen-thawed spermatozoa; there was an inverse correlation between the percentage of live spermatozoa and the percentage with damage to the PT. Although 59.1% of frozen-thawed spermatozoa had an intact acrosome, only 43.7% of them still remained alive. In frozen-thawed semen, there was a high correlation (r=0.69) between live spermatozoa with an intact acrosome and spermatozoa that maintained an intact PT. In conclusion, freezing/thawing of bull spermatozoa altered the PT and maintaining PT integrity may be necessary to maintain acrosome integrity.

A finite element analysis of the freeze/thaw behavior of external artery heat pipes

NASA Technical Reports Server (NTRS)

Lu, X. J.; Peterson, G. P.

1993-01-01

A two-dimensional finite element model was used to determine the freeze/thaw characteristics of an external artery heat pipe. During startup, the working fluid, which was located in the liquid channel and the circumferential wall grooves, experienced a phase transformation from a solid to a liquid state. The transient heat conduction equations with moving interfacial conditions were solved using the appropriate initial boundary conditions. The modelling results include the cross-sectional temperature distribution and the interfacial or melt front position as a function of time. A fixed grid approach was adopted in the model for the phase-change process during thawing of frozen working fluid. The interfacial position between the liquid and solid regions was found by balancing the latent heat caused by interfacial movement with the heat addition or extraction at the related grid points.

Sperm kinematics and subpopulational responses during the cryopreservation process in caprine ejaculates.

PubMed

Barbas, J P; Leahy, T; Horta, A E; García-Herreros, M

2018-03-20

Sperm cryopreservation in goats has been a challenge for many years due to the detrimental effects of seminal plasma enzymes produced by the bulbo-urethral glands which catalyse the hydrolysis of lecithins in egg yolk to fatty acids and lysolecithins which are deleterious to spermatozoa. This fact implies to carry out additional processing steps during sperm cryopreservation for seminal plasma removal triggering different sperm responses which may affect sperm functionality. The objective of the present study was to determine specific sperm subpopulation responses in different handling steps during the cryopreservation process by using functional sperm kinematic descriptors in caprine ejaculates. Buck ejaculates (n = 40) were analysed for sperm concentration, viability, morphology and acrosome integrity. Moreover, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system after five different handling steps (fresh sperm, 1st washing, 2nd washing, cooling and frozen-thawed sperm) during a standard cryopreservation protocol for goat semen. The results were analysed using Principal Component Analysis (PCA) and multivariate clustering procedures to establish the relationship between the distribution of the subpopulations found and the functional sperm motility in each step. Except for the 1st and 4th steps, four sperm kinematic subpopulations were observed explaining more than 75% of the variance. Based on velocity and linearity parameters and the subpopulations disclosed, the kinematic response varies among processing steps modifying sperm movement trajectories in a subpopulation-specific and handling step-dependent manner (p < 0.001). The predominant motile subpopulation in freshly ejaculated buck sperm had very fast velocity characteristics and a non-linear trajectory (41.1%). Washing buck sperm twice altered the subpopulation structure as well as cooling which resulted in a dramatic reduction in sperm velocities (p < 0.01). Frozen-thawed spermatozoa showed similar characteristics to cooled sperm except there was a further increase in linearity with a large proportion of sperm attributed to new slow, linear cluster (32.5%). In conclusion, this study confirms the variability and heterogeneity of goat sperm kinematic patterns throughout the cryopreservation process and suggests that the predominant motility pattern (assayed in vitro via CASA) of high quality spermatozoa might be typified by high speed and a non-linear trajectory. The relationships among the number and distribution of sperm subpopulations and the different handling steps were particularlly relevant, specially after the cooling and the post-thawing steps, when effects derived from these critical handling steps were evident and altered drastically the sperm motion patterns. Copyright © 2018 Elsevier Inc. All rights reserved.

Potential Arctic tundra vegetation shifts in response to changing temperature, precipitation and permafrost thaw

NASA Astrophysics Data System (ADS)

van der Kolk, Henk-Jan; Heijmans, Monique M. P. D.; van Huissteden, Jacobus; Pullens, Jeroen W. M.; Berendse, Frank

2016-11-01

Over the past decades, vegetation and climate have changed significantly in the Arctic. Deciduous shrub cover is often assumed to expand in tundra landscapes, but more frequent abrupt permafrost thaw resulting in formation of thaw ponds could lead to vegetation shifts towards graminoid-dominated wetland. Which factors drive vegetation changes in the tundra ecosystem are still not sufficiently clear. In this study, the dynamic tundra vegetation model, NUCOM-tundra (NUtrient and COMpetition), was used to evaluate the consequences of climate change scenarios of warming and increasing precipitation for future tundra vegetation change. The model includes three plant functional types (moss, graminoids and shrubs), carbon and nitrogen cycling, water and permafrost dynamics and a simple thaw pond module. Climate scenario simulations were performed for 16 combinations of temperature and precipitation increases in five vegetation types representing a gradient from dry shrub-dominated to moist mixed and wet graminoid-dominated sites. Vegetation composition dynamics in currently mixed vegetation sites were dependent on both temperature and precipitation changes, with warming favouring shrub dominance and increased precipitation favouring graminoid abundance. Climate change simulations based on greenhouse gas emission scenarios in which temperature and precipitation increases were combined showed increases in biomass of both graminoids and shrubs, with graminoids increasing in abundance. The simulations suggest that shrub growth can be limited by very wet soil conditions and low nutrient supply, whereas graminoids have the advantage of being able to grow in a wide range of soil moisture conditions and have access to nutrients in deeper soil layers. Abrupt permafrost thaw initiating thaw pond formation led to complete domination of graminoids. However, due to increased drainage, shrubs could profit from such changes in adjacent areas. Both climate and thaw pond formation simulations suggest that a wetter tundra can be responsible for local shrub decline instead of shrub expansion.

Functional status of acute stroke patients in University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia.

PubMed

Rameezan, B A R; Zaliha, O

2005-12-01

Stroke is a leading cause of death and disability in most developed countries and developing nations. Majority of the stroke survivors are left with significant physical and cognitive impairments. In addition to the improved acute stroke care, they often benefit from rehabilitation in improving their function. This was the first study done to document function for post stroke patients in Malaysia. It was prospective study conducted to document functional status of acute stroke patients upon admission, discharge and at 3 months post stroke. Assessment of functional status for these patients are based on their activities of daily living and ambulation i.e. self-care, sphincter control, mobility, locomotion, communication and social cognition. It is also aimed to describe their demographic and clinical characteristics. Correlation of functional status at 3 months post stroke with the initial severity of stroke was also explored. A total of fifty-one patients with acute stroke in University Malaya Medical Centre (UMMC) were recruited. The patient's age ranged from 38 to 83 years with a mean of 60.2 years. Thirty-six patients (71%) were first stroke sufferers and fifteen patients (29%) had recurrent stroke. At discharge from acute stay, 13% of patients were able to ambulate with aids and 87% needed assistance for ambulation in varying degrees. Eighty-two percent of patients showed improvement in overall function (both motor and cognition) at 3 months post stroke. Sixty percent of patients were independent in ambulation and 40% required assistance. Significant correlation was seen between the initial severity of stroke and functional status at 3 months post stroke. Functional status of patients with stroke has improved at 3 months post stroke. A comprehensive rehabilitation medicine programme should be incorporated into management of stroke patients to expedite functional recovery and improve patient's independence.

Optimization of viral resuspension methods for carbon-rich soils along a permafrost thaw gradient

DOE PAGES

Trubl, Gareth; Solonenko, Natalie; Chittick, Lauren; ...

2016-05-17

Permafrost stores approximately 50% of global soil carbon (C) in a frozen form; it is thawing rapidly under climate change, and little is known about viral communities in these soils or their roles in C cycling. In permafrost soils, microorganisms contribute significantly to C cycling, and characterizing them has recently been shown to improve prediction of ecosystem function. In other ecosystems, viruses have broad ecosystem and community impacts ranging from host cell mortality and organic matter cycling to horizontal gene transfer and reprogramming of core microbial metabolisms. Here we developed an optimized protocol to extract viruses from three types ofmore » high organic-matter peatland soils across a permafrost thaw gradient (palsa, moss-dominated bog, and sedge-dominated fen). Three separate experiments were used to evaluate the impact of chemical buffers, physical dispersion, storage conditions, and concentration and purification methods on viral yields. The most successful protocol, amended potassium citrate buffer with bead-beating or vortexing and BSA, yielded on average as much as 2-fold more virus-like particles (VLPs) g –1of soil than other methods tested. All method combinations yielded VLPs g –1of soil on the 10 8order of magnitude across all three soil types. The different storage and concentration methods did not yield significantly more VLPs g –1of soil among the soil types. In conclusion, this research provides much-needed guidelines for resuspending viruses from soils, specifically carbon-rich soils, paving the way for incorporating viruses into soil ecology studies.« less

Optimization of viral resuspension methods for carbon-rich soils along a permafrost thaw gradient

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trubl, Gareth; Solonenko, Natalie; Chittick, Lauren

Permafrost stores approximately 50% of global soil carbon (C) in a frozen form; it is thawing rapidly under climate change, and little is known about viral communities in these soils or their roles in C cycling. In permafrost soils, microorganisms contribute significantly to C cycling, and characterizing them has recently been shown to improve prediction of ecosystem function. In other ecosystems, viruses have broad ecosystem and community impacts ranging from host cell mortality and organic matter cycling to horizontal gene transfer and reprogramming of core microbial metabolisms. Here we developed an optimized protocol to extract viruses from three types ofmore » high organic-matter peatland soils across a permafrost thaw gradient (palsa, moss-dominated bog, and sedge-dominated fen). Three separate experiments were used to evaluate the impact of chemical buffers, physical dispersion, storage conditions, and concentration and purification methods on viral yields. The most successful protocol, amended potassium citrate buffer with bead-beating or vortexing and BSA, yielded on average as much as 2-fold more virus-like particles (VLPs) g –1of soil than other methods tested. All method combinations yielded VLPs g –1of soil on the 10 8order of magnitude across all three soil types. The different storage and concentration methods did not yield significantly more VLPs g –1of soil among the soil types. In conclusion, this research provides much-needed guidelines for resuspending viruses from soils, specifically carbon-rich soils, paving the way for incorporating viruses into soil ecology studies.« less

Evaluation of the in vivo and ex vivo optical properties in a mouse ear model

NASA Astrophysics Data System (ADS)

Salomatina, E.; Yaroslavsky, A. N.

2008-06-01

Determination of in vivo optical properties is a challenging problem. Absorption and scattering measured ex vivo are often used for in vivo applications. To investigate the validity of this approach, we have obtained and compared the optical properties of mouse ears in vivo and ex vivo in the spectral range from 370 to 1650 nm. Integrating sphere spectrophotometry in combination with the inverse Monte Carlo technique was employed to determine absorption coefficients, μa, scattering coefficients, μs, and anisotropy factors, g. Two groups of mice were used for the study. The first group was measured in vivo and ex vivo within 5-10 min post mortem. The second group was measured in vivo and ex vivo every 24 h for up to 72 h after sacrifice. Between the measurements the tissues were kept at 4 °C wrapped in a gauze moistened with saline solution. Then the specimens were frozen at -25 °C for 40 min, thawed and measured again. The results indicate that the absorption coefficients determined in vivo and ex vivo within 5-10 min post mortem differed considerably only in the spectral range dominated by hemoglobin. These changes can be attributed to rapid deoxygenation of tissue and blood post mortem. Absorption coefficients determined ex vivo up to 72 h post mortem decreased gradually with time in the spectral regions dominated by hemoglobin and water, which can be explained by the continuing loss of blood. Absorption properties of the frozen-thawed ex vivo tissues showed increase in oxygenation, which is likely caused by the release of hemoglobin from hemolyzed erythrocytes. Scattering of the ex vivo tissues decreased gradually with time in the entire spectral range due to the continuing loss of blood and partial cell damage. Anisotropy factors did not change considerably.

Evaluation of the in vivo and ex vivo optical properties in a mouse ear model.

PubMed

Salomatina, E; Yaroslavsky, A N

2008-06-07

Determination of in vivo optical properties is a challenging problem. Absorption and scattering measured ex vivo are often used for in vivo applications. To investigate the validity of this approach, we have obtained and compared the optical properties of mouse ears in vivo and ex vivo in the spectral range from 370 to 1650 nm. Integrating sphere spectrophotometry in combination with the inverse Monte Carlo technique was employed to determine absorption coefficients, mu(a), scattering coefficients, mu(s), and anisotropy factors, g. Two groups of mice were used for the study. The first group was measured in vivo and ex vivo within 5-10 min post mortem. The second group was measured in vivo and ex vivo every 24 h for up to 72 h after sacrifice. Between the measurements the tissues were kept at 4 degrees C wrapped in a gauze moistened with saline solution. Then the specimens were frozen at -25 degrees C for 40 min, thawed and measured again. The results indicate that the absorption coefficients determined in vivo and ex vivo within 5-10 min post mortem differed considerably only in the spectral range dominated by hemoglobin. These changes can be attributed to rapid deoxygenation of tissue and blood post mortem. Absorption coefficients determined ex vivo up to 72 h post mortem decreased gradually with time in the spectral regions dominated by hemoglobin and water, which can be explained by the continuing loss of blood. Absorption properties of the frozen-thawed ex vivo tissues showed increase in oxygenation, which is likely caused by the release of hemoglobin from hemolyzed erythrocytes. Scattering of the ex vivo tissues decreased gradually with time in the entire spectral range due to the continuing loss of blood and partial cell damage. Anisotropy factors did not change considerably.

Effect of freeze/thaw cycles on several biomarkers in urine from patients with kidney disease.

PubMed

Zhang, Yinan; Luo, Yi; Lu, Huijuan; Wang, Niansong; Shen, Yixie; Chen, Ruihua; Fang, Pingyan; Yu, Hong; Wang, Congrong; Jia, Weiping

2015-04-01

Urine samples were collected from eleven randomly selected patients with kidney disease, including diabetic nephropathy, chronic nephritis, and nephritic syndrome. Urine samples were treated with one of four protocols for freezing and thawing: freeze directly and thaw directly; freeze directly and thaw by temperature gradient; freeze by temperature gradient and thaw directly; and freeze by temperature gradient and thaw by temperature gradient. After one to six freeze/thaw cycles at -20°C or -80°C, different biomarkers showed differential stabilities. The concentrations of total protein, calcium, and potassium did not change significantly after five freeze/thaw cycles at either -20°C or -80°C. Albumin could only sustain three freeze/thaw cycles at -20°C before it started to degrade. We recommend that urine be stored at -80°C as albumin and the organic ions could sustain five and six freeze/thaw cycles, respectively, using the simple "direct freeze and direct thaw" protocol. Furthermore, in most cases, gradient freeze/thaw cycles are not necessary for urine sample storage.

Sperm characteristics and heterologous in vitro fertilisation capacity of Iberian ibex (Capra pyrenaica) epididymal sperm, frozen in the presence of the enzymatic antioxidant catalase.

PubMed

López-Saucedo, J; Paramio, M T; Fierro, R; Izquierdo, D; Catalá, M G; Coloma, M A; Toledano-Díaz, A; López-Sebastián, A; Santiago-Moreno, J

2014-06-01

The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<0.01). The percentage of cleaved embryos at 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates. Copyright © 2014 Elsevier Inc. All rights reserved.

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy.

PubMed

Ford, Tom; Wenden, Claire; Mbekeani, Alison; Dally, Len; Cox, Josephine H; Morin, Merribeth; Winstone, Nicola; Hill, Adrian V S; Gilmour, Jill; Ewer, Katie J

2017-04-04

Ex vivo functional immunoassays such as ELISpot and intracellular cytokine staining (ICS) by flow cytometry are crucial tools in vaccine development both in the identification of novel immunogenic targets and in the immunological assessment of samples from clinical trials. Cryopreservation and subsequent thawing of PBMCs via validated processes has become a mainstay of clinical trials due to processing restrictions inherent in the disparate location and capacity of trial centres, and also in the need to standardize biological assays at central testing facilities. Logistical and financial requirement to batch process samples from multiple study timepoints are also key. We used ELISpot and ICS assays to assess antigen-specific immunogenicity in blood samples taken from subjects enrolled in a phase II malaria heterologous prime-boost vaccine trial and showed that the freeze thaw process can result in a 3-5-fold reduction of malaria antigen-specific IFNγ-producing CD3 + CD4 + effector populations from PBMC samples taken post vaccination. We have also demonstrated that peptide responsive CD8 + T cells are relatively unaffected, as well as CD4 + T cell populations that do not produce IFNγ. These findings contribute to a growing body of data that could be consolidated and synthesised as guidelines for clinical trials with the aim of increasing the efficiency of vaccine development pipelines. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Effect of Liquid Nitrogen on Bone Graft Survival.

PubMed

Sirinoglu, Hakan; Çilingir, Özlem Tuğçe; Çelebiler, Ozhan; Ercan, Feriha; Numanoglu, Ayhan

2015-08-01

Liquid nitrogen is used in medicine for cancer treatment and tissue preservation; however, bone viability after its application is controversial. This study aims to evaluate both the tissue viability and the clinical and histopathologic findings following liquid nitrogen application with different thawing techniques in rats. Mandibular bone grafts were taken from 45 Wistar rats and freezed in liquid nitrogen for 20 minutes. In the rapid-thawing technique (Rapid Thawing-1, Rapid Thawing-2), the grafts were held for 20 minutes in room temperature; in the slow-thawing technique (Slow Thawing-1, Slow Thawing-2), 20 minutes in -20°C, 20 minutes in +4°C, and 20 minutes in room temperature, respectively. In Rapid Thawing-2 and Slow Thawing-2 groups, autografts were implanted to their origin for 3 weeks and bone staining with India ink was performed and samples taken for histologic examination. The amount of cells and blood vessels and the density of bone canaliculi were significantly reduced in Rapid Thawing-1 and Slow Thawing-1 groups comparing to the Control group. However, the reduction rate was more significant in the Slow Thawing-1 group. Histomorphometric evaluation of the healing autografts after 3 weeks revealed that the decreased amounts of canaliculi were not changed in Slow Thawing-2 group. The study results demonstrated that bone tissue survives after liquid nitrogen treatment regardless of the performed thawing technique; however, slow thawing causes more tissue damage and metabolism impairment. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

A practical method to detect the freezing/thawing onsets of seasonal frozen ground in Alaska

NASA Astrophysics Data System (ADS)

Chen, Xiyu; Liu, Lin

2017-04-01

Microwave remote sensing can provide useful information about freeze/thaw state of soil at the Earth surface. An edge detection method is applied in this study to estimate the onsets of soil freeze/thaw state transition using L band space-borne radiometer data. The Soil Moisture Active Passive (SMAP) mission has a L band radiometer and can provide daily brightness temperature (TB) with horizontal/vertical polarizations. We use the normalized polarization ratios (NPR) calculated based on the Level-1C TB product of SMAP (spatial resolution: 36 km) as the indicator for soil freeze/thaw state, to estimate the freezing and thawing onsets in Alaska in the year of 2015 and 2016. NPR is calculated based on the difference between TB at vertical and horizontal polarizations. Therefore, it is strongly sensitive to liquid water content change in the soil and independent with the soil temperature. Onset estimation is based on the detection of abrupt changes of NPR in transition seasons using edge detection method, and the validation is to compare estimated onsets with the onsets derived from in situ measurement. According to the comparison, the estimated onsets were generally 15 days earlier than the measured onsets in 2015. However, in 2016 there were 4 days in average for the estimation earlier than the measured, which may be due to the less snow cover. Moreover, we extended our estimation to the entire state of Alaska. The estimated freeze/thaw onsets showed a reasonable latitude-dependent distribution although there are still some outliers caused by the noisy variation of NPR. At last, we also try to remove these outliers and improve the performance of the method by smoothing the NPR time series.

Methane dynamics regulated by microbial community response to permafrost thaw.

PubMed

McCalley, Carmody K; Woodcroft, Ben J; Hodgkins, Suzanne B; Wehr, Richard A; Kim, Eun-Hae; Mondav, Rhiannon; Crill, Patrick M; Chanton, Jeffrey P; Rich, Virginia I; Tyson, Gene W; Saleska, Scott R

2014-10-23

Permafrost contains about 50% of the global soil carbon. It is thought that the thawing of permafrost can lead to a loss of soil carbon in the form of methane and carbon dioxide emissions. The magnitude of the resulting positive climate feedback of such greenhouse gas emissions is still unknown and may to a large extent depend on the poorly understood role of microbial community composition in regulating the metabolic processes that drive such ecosystem-scale greenhouse gas fluxes. Here we show that changes in vegetation and increasing methane emissions with permafrost thaw are associated with a switch from hydrogenotrophic to partly acetoclastic methanogenesis, resulting in a large shift in the δ(13)C signature (10-15‰) of emitted methane. We used a natural landscape gradient of permafrost thaw in northern Sweden as a model to investigate the role of microbial communities in regulating methane cycling, and to test whether a knowledge of community dynamics could improve predictions of carbon emissions under loss of permafrost. Abundance of the methanogen Candidatus 'Methanoflorens stordalenmirensis' is a key predictor of the shifts in methane isotopes, which in turn predicts the proportions of carbon emitted as methane and as carbon dioxide, an important factor for simulating the climate feedback associated with permafrost thaw in global models. By showing that the abundance of key microbial lineages can be used to predict atmospherically relevant patterns in methane isotopes and the proportion of carbon metabolized to methane during permafrost thaw, we establish a basis for scaling changing microbial communities to ecosystem isotope dynamics. Our findings indicate that microbial ecology may be important in ecosystem-scale responses to global change.

Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry.

PubMed

Kavak, A; Johannisson, A; Lundeheim, N; Rodriguez-Martinez, H; Aidnik, M; Einarsson, S

2003-04-15

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye.

PubMed

Stressmann, Maja; Kitao, Satoshi; Griffith, Marilyn; Moresoli, Christine; Bravo, León A; Marangoni, Alejandro G

2004-05-01

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

PubMed Central

Berlinguer, Fiammetta; Madeddu, Manuela; Pasciu, Valeria; Succu, Sara; Spezzigu, Antonio; Satta, Valentina; Mereu, Paolo; Leoni, Giovanni G; Naitana, Salvatore

2009-01-01

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation. PMID:19900288

Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen

PubMed Central

Tarig, A. A.; Wahid, H.; Rosnina, Y.; Yimer, N.; Goh, Y. M.; Baiee, F. H.; Khumran, A. M.; Salman, H.; Assi, M. A.; Ebrahimi, M.

2017-01-01

Aim: The objective of this study was to evaluate the effects of different concentrations of soybean lecithin (SL) and virgin coconut oil (VCO) in Tris-based extender on chilled and frozen-thawed bull semen quality parameters. Materials and Methods: A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test. Results: The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups. Conclusion: In conclusion, supplementation of 1.5% SL in 2% VCO Tris-based extender enhanced the chilled bull semen. However, there was no marked improvement in the frozen-thawed quality parameters after treatment. PMID:28717321

Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen.

PubMed

Tarig, A A; Wahid, H; Rosnina, Y; Yimer, N; Goh, Y M; Baiee, F H; Khumran, A M; Salman, H; Assi, M A; Ebrahimi, M

2017-06-01

The objective of this study was to evaluate the effects of different concentrations of soybean lecithin (SL) and virgin coconut oil (VCO) in Tris-based extender on chilled and frozen-thawed bull semen quality parameters. A total of 24 ejaculates were collected from four bulls via an electroejaculator. Semen samples were diluted with 2% VCO in Tris-based extender which consists of various concentrations of SL (1, 1.25, 1.5, and 1.75%). A 20% egg yolk in Tris used as a positive control (C+). The diluted semen samples were divided into two fractions; one for chilling which were stored at 4°C for 24, 72, and 144 h before evaluated for semen quality parameters. The second fraction used for freezing was chilled for 3 h at 4°C, packed into 0.25 mL straws and then cryopreserved in liquid nitrogen. The samples were then evaluated after 7 and 14 days. Chilled and frozen semen samples were thawed at 37°C and assessed for general motility using computer-assisted semen analysis, viability, acrosome integrity and morphology (eosin-nigrosin stain), membrane integrity, and lipid peroxidation using thiobarbituric acid reaction test. The results showed that all the quality parameters assessed were significantly (p<0.05) improved at 1.5% SL concentration in chilled semen. Treatment groups of 1, 1.25, 1.5, and 1.75% SL were higher in quality parameters than the control group (C+) in chilled semen. However, all the quality parameters in frozen-thawed semen were significantly higher in the C+ than the treated groups. In conclusion, supplementation of 1.5% SL in 2% VCO Tris-based extender enhanced the chilled bull semen. However, there was no marked improvement in the frozen-thawed quality parameters after treatment.

Influence of cryoprotectants glycerol and amides, combined with antioxidants on quality of frozen-thawed boar sperm.

PubMed

Buranaamnuay, K; Grossfeld, R; Struckmann, C; Rath, D

2011-08-01

The present study was undertaken to examine whether the cooling and freezing extenders containing a mixture of antioxidants (AOs) catalase, Na-pyruvate and mercaptoethanol and one of three types of cryoprotectants (CPs) would be able to improve the quality of frozen-thawed boar sperm. The collected semen, only the sperm-rich fraction, was diluted 1:1 with Androhep plus™ extender, stored at 15°C for 2 h and centrifuged. The centrifuged sperm pellet was re-suspended in lactose-egg yolk extender and divided into four groups for mixing with freezing extenders containing different kinds of CPs at 5°C: (I) glycerol (GLY) as control; (II) GLY with AOs; (III) dimethyl formamide (DMF) with AOs and (IV) dimethyl acetamide (DMA) with AOs. Processed sperm were packaged in 0.25-mL straws and frozen using a controlled rate freezer. After thawing, the diluted thawed sperm were incubated at 38°C for 10 min and was assessed for motility by CASA, membrane/acrosome integrity by FITC-PNA/PI and DNA integrity (DFI) by SCSA. All sperm parameters evaluated, except DFI, were negatively affected (P<0.001) when using DMF (III) or DMA (IV) as CPs instead of GLY (I and II). Total sperm motility was lower (P<0.001) in the samples supplemented with AOs (32.4 ± 1.2, 23.9 ± 1.5, 6.9 ± 0.7, and 10.3 ± 0.9%, for treatments I, II, III and IV, respectively). The quality of sperm frozen in DMF was not different from DMA (P>0.05). There was no difference in DFI among the studied groups (P>0.05). In conclusion, based on the present results, addition of AOs to cooling and freezing extenders and/or replacement of GLY with DMF or DMA could not improve quality of frozen-thawed boar sperm. Copyright © 2011 Elsevier B.V. All rights reserved.

Relative importance of seed drying rate, desiccation tolerance, and cryotolerance for the conservation of Ardisia elliptica, A. brunnescens and A. virens.

PubMed

Yao, X; Goodale, U M; Li, Z L; Huang, Y; Wang, X F; Cheng, F Y; Tan, Y H; Xiao, C F; Lan, Q Y

2014-01-01

The pan-tropical genus Ardisia has more than 400 species and is of high horticultural and medicinal value. Due to overexploitation it is important to conserve the germplasm of this genus. To investigate the feasibility and methods of cryopreservation for long-term seed storage of three Ardisia species: A. elliptica Thunb., A. brunnescens Walker, and A. virens Kurz. We tested whether rapid desiccation can increase desiccation tolerance and cryotolerance, and whether the thawing rate can affect cryopreservation success. Seeds were subjected to three desiccation treatments: 1) activated silica gel at 25 +/- 2 degree C, and 4% relative humidity (RH); 2) saturated NaCl solution in closed jars in 25 +/- 2 degree C and 75% RH; and 3) air-drying at room conditions at 27 +/- 2 degree C and RH 60% for different desiccation durations (12h, 24h, 48h, 96h, and 196h). Seeds were then assessed for desiccation tolerance and cryotolerance after rapid thawing (direct immersion in 36 degree C water bath for 2 min) or slow thawing (at 27°C for 1 h). For all three species, desiccation method and duration significantly affected cryotolerance (P < 0.0001). Fast desiccation did not improve germination compared to slower desiccation (P < 0.01). Whereas A. elliptica germination was unaffected by desiccation duration, drying time significantly (P < 0.0001) affected germination percentage in the other two species especially after 48h. Although slow thawing improved cryotolerance of A. brunnescens seeds (P < 0.05), there was no significant effect of thawing rate on A. elliptica. A. virens seed did not survive cryopreservation. Cryopreservation protocols of Ardisia species may be species-specific and should be established for each species in the genus so that cryopreservation can be used as a successful conservation strategy.

Effects of refrigeration and freezing on the electromechanical and biomechanical properties of articular cartilage.

PubMed

Changoor, Adele; Fereydoonzad, Liah; Yaroshinsky, Alex; Buschmann, Michael D

2010-06-01

In vitro electromechanical and biomechanical testing of articular cartilage provide critical information about the structure and function of this tissue. Difficulties obtaining fresh tissue and lengthy experimental testing procedures often necessitate a storage protocol, which may adversely affect the functional properties of cartilage. The effects of storage at either 4°C for periods of 6 days and 12 days, or during a single freeze-thaw cycle at -20°C were examined in young bovine cartilage. Non-destructive electromechanical measurements and unconfined compression testing on 3 mm diameter disks were used to assess cartilage properties, including the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Cartilage disks were also examined histologically. Compared with controls, significant decreases in SPI (to 32.3±5.5% of control values, p<0.001), Ef (to 31.3±41.3% [corrected] of control values, p=0.046), Em (to 6.4±8.5% of control values, p<0.0001), and an increase in k (to 2676.7±2562.0% of control values, p=0.004) were observed at day 12 of refrigeration at 4°C, but no significant changes were detected at day 6. A trend toward detecting a decrease in SPI (to 94.2±6.2% of control values, p=0.083) was identified following a single freeze-thaw cycle, but no detectable changes were observed for any biomechanical parameters. All numbers are mean±95% confidence interval. These results indicate that fresh cartilage can be stored in a humid chamber at 4°C for a maximum of 6 days with no detrimental effects to cartilage electromechanical and biomechanical properties, while one freeze-thaw cycle produces minimal deterioration of biomechanical and electromechanical properties. A comparison to literature suggested that particular attention should be paid to the manner in which specimens are thawed after freezing, specifically by minimizing thawing time at higher temperatures.

Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa.

PubMed

Akhter, S; Ansari, M S; Andrabi, S M H; Rakha, B A; Ullah, N; Khalid, M

2012-10-01

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen. © 2011 Blackwell Verlag GmbH.

[Cooling of boar spermatozoa prior to freezing and post thaw quality and evaluation of membrane state using chlortetracycline (CTC) staining].

PubMed

Kotzias-Bandeira, E; Waberski, D; Weitze, K F

1997-08-01

The influence of an extended holding time at room temperature (+18 degrees C) before freezing on boar sperm quality was investigated. 17 ejaculates were collected from 5 different boars by separation in sperm rich and sperm poor fraction. The ejaculate were split, diluted 1+1 with Merck I-Medium, and submitted to three different treatments before freezing: 1. Sperm rich fraction, cooling to +20 degrees C for 1.5 h and subsequent cooling to +15 degrees C for 2.5 h; 2. Sperm rich fraction, cooling to +18 degrees C for 4 h and subsequent holding time at +18 degrees C for 16 h; 3. Whole ejaculate (sperm rich fraction plus seminal plasma), cooling to +18 degrees C for 4 h and subsequent holding time at +18 degrees C for 16 h. Subjectively assessed post thaw motility (SMOT), computer-measured motility (CMOT), and acrosome integrity (NAR), assessed by phase contrast microscopy were significantly (p < 0.05) higher after extended holding time (procedure 2 and 3) compared to short holding time (procedure 1). The exposure to seminal plasma during holding had no significant effect. Chlortetracyclin (CTC) staining of sperm membranes gave no reliable information in the presence of an EDTA-containing preservation medium, used routinely in the preservation process.

Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy

PubMed Central

2017-01-01

Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE), a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80%) and yield (>70%). Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies. PMID:28122047

Influence of seminal plasma on the kinematics of boar spermatozoa during freezing.

PubMed

Rodríguez-Martínez, H; Saravia, F; Wallgren, M; Roca, J; Peña, F J

2008-11-01

Sperm motility is, for its relation to cell viability and fertility, a central component of the spermiogram, where consideration of motion patterns allows discrimination of sub-populations among boar spermatozoa. Extension and cryo-preservation imposes changes in these patterns in connection to handling, additives, temperature changes and the removal of boar seminal plasma (BSP) which apparently makes spermatozoa susceptible to oxidative stress, thus affecting survival and motility post-thaw. Detailed kinematic analyses during sperm cooling are sparse, particularly when considering the instrumentation and settings used for analyses, the effect of extenders, and of the BSP the processed spermatozoa are exposed to. Spermatozoa present in the first collectable 10mL of the sperm-rich fraction of the ejaculate (portion 1, P1-BSP), have shown an increased ability to sustain motility during and after cryo-preservation than spermatozoa immersed in the rest of the ejaculate (portion 2, P2). When P2-spermatozoa were cleansed from their BSP and exposed for 60min to pooled P1-BSP, their motility post-thaw increased to similar levels as P1-spermatozoa. This BSP-influence is sire-dependent, presumably related to the protein concentration in the different ejaculate portions, and apparently unrelated to changes in membrane integrity or membrane stability through conventional, controlled cooling.

A combination of solvent extraction and freeze thaw for oil recovery from petroleum refinery wastewater treatment pond sludge.

PubMed

Hu, Guangji; Li, Jianbing; Hou, Haobo

2015-01-01

A combination of solvent extraction and freeze thaw was examined for recovering oil from the high-moisture petroleum refinery wastewater treatment pond sludge. Five solvents including cyclohexane (CHX), dichloromethane (DCM), methyl ethyl ketone (MEK), ethyl acetate (EA), and 2-propanol (2-Pro) were examined. It was found that these solvents except 2-Pro showed a promising oil recovery rate of about 40%, but the recycling of DCM solvent after oil extraction was quite low. Three solvents (CHX, MEK and EA) were then selected for examining the effect of freeze/thaw treatment on improving the quality of recovered oil. This treatment increased the total petroleum hydrocarbon (TPH) content in recovered oil from about 40% to 60% for both MEK and EA extractions, but little effect was observed for CHX extraction. Although the solid residue after oil recovery had a significantly decreased TPH content, a high concentration of heavy metals was observed, indicating that this residue may require proper management. In general, the combination of solvent extraction with freeze/thaw is effective for high-moisture oily hazardous waste treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

Preparation, optimization and property of PVA-HA/PAA composite hydrogel.

PubMed

Chen, Kai; Liu, Jinlong; Yang, Xuehui; Zhang, Dekun

2017-09-01

PVA-HA/PAA composite hydrogel is prepared by freezing-thawing, PEG dehydration and annealing method. Orthogonal design method is used to choose the optimization combination. Results showed that HA and PVA have the maximum effect on water content. PVA and freezing-thawing cycles have the maximum effect on creep resistance and stress relaxation rate of hydrogel. Annealing temperature and freezing-thawing cycles have the maximum effect on compressive elastic modulus of hydrogel. Comparing with the water content and mechanical properties of 16 kinds of combination, PVA-HA/PAA composite hydrogel with freezing-thawing cycles of 3, annealing temperature of 120°C, PVA of 16%, HA of 2%, PAA of 4% has the optimization comprehensive properties. PVA-HA/PAA composite hydrogel has a porous network structure. There are some interactions between PVA, HA and PAA in hydrogel and the properties of hydrogel are strengthened. The annealing treatment improves the crystalline and crosslinking of hydrogel. Therefore, the annealing PVA-HA/PAA composite hydrogel has good thermostability, strength and mechanical properties. It also has good lubrication property and its friction coefficient is relative low. Copyright © 2017 Elsevier B.V. All rights reserved.

Resilient modulus of freeze-thaw affected granular soils for pavement design and evaluation. Part 4: Field validation tests at Albany County Airport

NASA Astrophysics Data System (ADS)

Johnson, T. C.; Crowe, A.; Erickson, M.; Cole, D. M.

1986-10-01

Stress-deformation data for unbound base, subbase, and silty sand subgrade soils in two airfield pavements were obtained from in situ tests and laboratory tests. Surface deflections were measured in the in situ tests, with a falling-weight deflectometer, when the soils were frozen, thawed, and at various stages of recovery from thaw weakening. The measured deflections were used to judge the validity of procedures developed for laboratory triaxial tests to determine nonlinear resilient moduli of specimens in the frozen, thawed and recovering states. The validity of the nonlinear resilient moduli, expressed as functions of externally applied stress and moisture tension, was confirmed by using the expressions to calculate surface deflections that were found to compare well with deflections measured in the in situ tests. The tests on specimens at various stages of recovery are especially significant because they show a strong dependence of the resilient modulus on moisture tension, leading to the conclusion that predictions or in situ measurements of moisture tension can be used to evaluate expected seasonal variation in the resilient modulus of granular soils.

Constraints for the thawing and freezing potentials

NASA Astrophysics Data System (ADS)

Hara, Tetsuya; Suzuki, Anna; Saka, Shogo; Tanigawa, Takuma

2018-01-01

We study the accelerating present universe in terms of the time evolution of the equation of state w(z) (redshift z) due to thawing and freezing scalar potentials in the quintessence model. The values of dw/da and d^2w/da^2 at a scale factor of a = 1 are associated with two parameters of each potential. For five types of scalar potentials, the scalar fields Q and w as functions of time t and/or z are numerically calculated under the fixed boundary condition of w(z=0)=-1+Δ. The observational constraint w_obs (Planck Collaboration, arXiv:1502.01590) is imposed to test whether the numerical w(z) is in w_obs. Some solutions show thawing features in the freezing potentials. Mutually exclusive allowed regions in the dw/da vs. d^2w/da^2 diagram are obtained in order to identify the likely scalar potential and even the potential parameters for future observational tests.

Survival and engraftment of dopaminergic neurons manufactured by a Good Manufacturing Practice-compatible process.

PubMed

Peng, Jun; Liu, Qiuyue; Rao, Mahendra S; Zeng, Xianmin

2014-09-01

We have previously reported a Good Manufacturing Practice (GMP)-compatible process for generating authentic dopaminergic neurons in defined media from human pluripotent stem cells and determined the time point at which dopaminergic precursors/neurons (day 14 after neuronal stem cell [NSC] stage) can be frozen, shipped and thawed without compromising their viability and ability to mature in vitro. One important issue we wished to address is whether dopaminergic precursors/neurons manufactured by our GMP-compatible process can be cryopreserved and engrafted in animal Parkinson disease (PD) models. In this study, we evaluated the efficacy of freshly prepared and cryopreserved dopaminergic neurons in the 6-hydroxydopamine-lesioned rat PD model. We showed functional recovery up to 6 months post-transplantation in rats transplanted with our cells, whether freshly prepared or cryopreserved. In contrast, no motor improvement was observed in two control groups receiving either medium or cells at a slightly earlier stage (day 10 after NSC stage). Histologic analysis at the end point of the study (6 months post-transplantation) showed robust long-term survival of donor-derived tyrosine hydroxylase (TH)(+) dopaminergic neurons in rats transplanted with day 14 dopaminergic neurons. Moreover, TH(+) fibers emanated from the graft core into the surrounding host striatum. Consistent with the behavioral analysis, no or few TH(+) neurons were detected in animals receiving day 10 cells, although human cells were present in the graft. Importantly, no tumors were detected in any grafted rats, but long-term tumorigenic studies will need to determine the safety of our products. Dopaminergic neurons manufactured by a GMP-compatible process from human ESC survived and engrafted efficiently in the 6-OHDA PD rat model. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing.

PubMed

Deller, Robert C; Vatish, Manu; Mitchell, Daniel A; Gibson, Matthew I

2014-01-01

The cryopreservation of cells, tissue and organs is fundamental to modern biotechnology, transplantation medicine and chemical biology. The current state-of-the-art method of cryopreservation is the addition of large amounts of organic solvents such as glycerol or dimethyl sulfoxide, to promote vitrification and prevent ice formation. Here we employ a synthetic, biomimetic, polymer, which is capable of slowing the growth of ice crystals in a manner similar to antifreeze (glyco)proteins to enhance the cryopreservation of sheep and human red blood cells. We find that only 0.1 wt% of the polymer is required to attain significant cell recovery post freezing, compared with over 20 wt% required for solvent-based strategies. These results demonstrate that synthetic antifreeze (glyco)protein mimics could have a crucial role in modern regenerative medicine to improve the storage and distribution of biological material for transplantation.

Cryopreservation of canine sperm using egg yolk and soy bean based extenders.

PubMed

Sánchez-Calabuig, María Jesús; Maillo, Verónica; Beltrán-Breña, Paula; de la Fuente Martínez, Julio; Galera-Carrillo, Silvestre; Pérez-Gutiérrez, José Félix; Pérez-Cerezales, Serafín

2017-09-01

Animal protein-based extenders are widely used despite being a potential source of bacterial or mycoplasma contamination. Its replacement with vegetal protein-based extenders could represent an interesting alternative for dog sperm cryopreservation. This technique could be further improved by the addition of Tris-Glucose-Citric acid (TGC) that could physically protect the spermatozoa and improve its homeostasis. The aim of this study was to evaluate a cryopreservation protocol for dog spermatozoa using a soybean-based extender (LP1 ℗ ) as well as the effects of the addition of (TGC) immediately after the semen collection. Eleven ejaculates from purebred adult dogs were collected, centrifuged in the absence or presence of TGC and processed as fresh or cryopreserved spermatozoa with: egg yolk-based extender (CaniPRO) or LP1 ℗ . Freezing the spermatozoa in LP1 ℗ reduced the amplitude of the lateral head displacement, the percentage of spermatozoa that showed the intact acrosome and the mitochondrial function (P<0.05). These samples also showed a trend towards increased percentage of apoptotic spermatozoa (P<0.05). The addition of TGC before centrifugation did not improve the seminal parameters and adversely affected motility (P<0.05) in the spermatozoa cryopreserved in CaniPRO. However, TGC did not affect motility and increased (P<0.05) the percentage of intact acrosomes in the spermatozoa cryopreserved in LP1 ℗ , reaching similar values than those cryopreserved in CaniPRO. In conclusion, LP1 ® plus TGC provide the same level of protection to dog spermatozoa cryopreservation than the egg yolk based extender CaniPRO when comparing standard post-thaw sperm quality parameters. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

Computer-Assisted Sperm Analysis (CASA) parameters and their evolution during preparation as predictors of pregnancy in intrauterine insemination with frozen-thawed donor semen cycles.

PubMed

Fréour, Thomas; Jean, Miguel; Mirallié, Sophie; Dubourdieu, Sophie; Barrière, Paul

2010-04-01

To study the potential of CASA parameters in frozen-thawed donor semen before and after preparation on silica gradient as predictors of pregnancy in IUI with donor semen cycles. CASA parameters were measured in thawed donor semen before and after preparation on a silica gradient in 132 couples undergoing 168 IUI cycles with donor semen. The evolution of these parameters throughout this process was calculated. The relationship with cycle outcome was then studied. Clinical pregnancy rate was 18.4% per cycle. CASA parameters on donor semen before or after preparation were not significantly different between pregnancy and failure groups. However, amplitude of lateral head displacement (ALH) of spermatozoa improved in all cycles where pregnancy occurred, thus predicting pregnancy with a sensitivity of 100% and a specificity of 20%. Even if CASA parameters do not seem to predict pregnancy in IUI with donor semen cycles, their evolution during the preparation process should be evaluated, especially for ALH. However, the link between ALH improvement during preparation process and pregnancy remains to be explored. Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.

The cryoprotective effect of trehalose supplementation on boar spermatozoa quality.

PubMed

Hu, Jian-Hong; Li, Qing-Wang; Li, Gang; Jiang, Zhong-Liang; Bu, Shu-hai; Yang, Hai; Wang, Li-Qiang

2009-05-01

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200mmol/l), and tried to determine the optimum concentration of trehalose. We chose sperm motility, acrosome integrity, membrane integrity and cryocapacitation as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100mmol/l trehalose-supplemented extenders, with values of 49.89% for motility, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance was diminished significantly for 200mmol/l of trehalose. Before and after capacitation, the CTC score for semen diluted by extender containing 100mmol/l trehalose was 3.68% and 43.82%, respectively. In conclusion, trehalose could confer a greater cryoprotective capacity to boar spermatozoa. Trehalose-supplementation with 100mmol/l concentration in basic extender could significantly improve sperm motility, membrane integrity and acrosome integrity parameters, and reduce boar spermatozoa cryocapacitation during the cryopreservation process.

Environmental Factors Related to a Semiarid Climate Influence the Freezability of Sperm from Collared Peccaries (Pecari tajacu Linnaeus, 1758).

PubMed

Maia, Keilla M; Souza, Ana L P; Praxedes, Erica C G; Bezerra, Luana G P; Silva, Andreia M; Campos, Livia B; Moreira, Samara S J; Apolinário, Carlos A C; Souza, João B F; Silva, Alexandre R

2018-04-30

The influence of environmental factors in a semiarid climate on characteristics of fresh and frozen/thawed sperm collected from collared peccaries (Pecari tajacu) was assessed. Semen from 11 male collared peccaries was collected by electroejaculation during the peaks of the dry and rainy periods while rainfall indices, air temperatures, relative humidity levels, and wind speeds were measured. The number, motility, morphology, osmotic response, and membrane integrity of sperm in the collected ejaculates were assessed. Samples were then frozen in liquid nitrogen, thawed, and reassessed. The rainfall index of the rainy period (73.2 mm) was significantly higher than that of the dry period (13.6 mm) and the relative humidity was significantly higher during the rainy period (74.6%) than it was during the dry period (66.8%). Air temperature and wind speed did not differ between the two periods. Characteristics of sperm in the fresh samples were not affected by environmental parameters. In contrast, computerized analysis revealed that sperm in samples frozen during the rainy period exhibited better post-thaw membrane integrity (28.6 ± 6%), motility (29.5 ± 7.7%), and rapid sperm population (13.7 ± 6.2%) than did sperm in samples frozen during the dry period (23.4 ± 3% membrane integrity, 14.6 ± 4.1% motility, and 4.1 ± 1.2% rapid sperm; p < 0.05). Other characteristics of the frozen/thawed sperm did not differ depending on the period in which they were collected. We demonstrated that environmental parameters did not affect the quality of fresh sperm, but could influence the freezability of sperm collected from collared peccaries raised under a semiarid climate.

Loss of the ability to generate large burst-forming unit-like megakaryocytic colonies from thawed cord blood in semisolid cultures after short term suspension culture.

PubMed

Eskola, M; Bäckman, S; Möttönen, S; Kekomäki, R

2015-04-01

Total colony-forming cells from thawed cord blood units (CBUs) include megakaryocytic colony-forming units (CFU-Mks), which survive the freezing process. The aim of this study was to evaluate whether different megakaryocytic progenitors from unseparated CBUs survive the freezing process and a short-term liquid culture. Thawed samples of CBUs were cultured in liquid medium. During the cultures, serial samples were drawn to assess the growth of different megakaryocytic progenitors in a semisolid collagen medium with identical cytokines as in the liquid medium. Megakaryocytic cells were detected using immunohistochemistry and flow cytometry. In suspension culture, the megakaryocytic progenitors almost completely lost the ability to generate large (burst-forming unit-like, BFU-like) megakaryocytic colonies in semisolid cultures (large colonies, median count per chamber d0: 7.25 vs. d7: 1.5; P < 0.0001), whereas the number of small colonies (median count per chamber d0: 7.25 vs. d7: 16.0; P = 0.0505) peaked at day seven. Further 7-day culture in suspension resulted in the decline of small colonies as well (d7: 16.0 vs. d14: 5.75; P = 0.0088). Total CFU-Mk count declined from 23.3 (range 12.5-34.0) at d0 to 7.25 (range 1.0-13.5) at d14 (P < 0.0001). Immediately post-thaw, CBUs possess an ability to generate large BFU-like megakaryocytic colonies, whereas the colonies were not detectable in most CBUs in semisolid culture after a short suspension culture. Small CFU-Mks were observed throughout the cultures. It may be that the BFU-Mk colonies matured and acquired CFU-Mk behaviour. © 2014 International Society of Blood Transfusion.

Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing.

PubMed

Santiago-Moreno, J; Esteso, M C; Castaño, C; Toledano-Díaz, A; Delgadillo, J A; López-Sebastián, A

2017-06-01

Seminal plasma removal is routine in goat sperm cryopreservation protocols. The classical washing procedure designed to accomplish this usually leaves the pellet resulting from use of this procedure contaminated with dead sperm, debris, and cells other than sperm. This contamination negatively affects viability of sperm after cryopreservation. The present research was conducted to compare the effect on chilled and frozen-thawed goat sperm of the classical washing method to that of a selective washing method involving density gradient centrifugation (DGC). In the first experiment, sperm variables were measured in freshly collected sperm, and again after its washing with both methods and chilling at 5°C for 0, 3, 24, 48, 72 or 96h. The DGC-washed sperm had greater (P<0.01) straight line velocity (VSL), average path velocity (VAP) and progression ratio values at all chilling times. The amplitude of lateral head displacement (ALH) was, however, less (P<0.001) in the DGC-washed sperm at all chilling times. There was a negative correlation (P<0.05) between ALH and VSL. In the second experiment involving the freezing-thawing of sperm washed by using either method, aliquots were post-wash diluted with a Tris-citric acid/glucose/egg yolk/glycerol-based medium and frozen in liquid nitrogen for 5days. After thawing, neither the VCL, VSL nor VAP of the DGC-washed samples were affected, whereas the traditionally washed samples had less motility. In conclusion, the use of DGC was associated with enhanced sperm motility variables after chilling and freezing-thawing. This procedure would, therefore, be a useful means of removing seminal plasma from goat semen and obtaining greater quality sperm for insemination purposes. Copyright © 2017. Published by Elsevier B.V.

Soil-water dynamics and unsaturated storage during snowmelt following wildfire

USGS Publications Warehouse

Ebel, Brian A.; Hinckley, E.S.; Martin, Deborah

2012-01-01

Many forested watersheds with a substantial fraction of precipitation delivered as snow have the potential for landscape disturbance by wildfire. Little is known about the immediate effects of wildfire on snowmelt and near-surface hydrologic responses, including soil-water storage. Montane systems at the rain-snow transition have soil-water dynamics that are further complicated during the snowmelt period by strong aspect controls on snowmelt and soil thawing. Here we present data from field measurements of snow hydrology and subsurface hydrologic and temperature responses during the first winter and spring after the September 2010 Fourmile Canyon Fire in Colorado, USA. Our observations of soil-water content and soil temperature show sharp contrasts in hydrologic and thermal conditions between north- and south-facing slopes. South-facing burned soils were ∼1–2 °C warmer on average than north-facing burned soils and ∼1.5 °C warmer than south-facing unburned soils, which affected soil thawing during the snowmelt period. Soil-water dynamics also differed by aspect: in response to soil thawing, soil-water content increased approximately one month earlier on south-facing burned slopes than on north-facing burned slopes. While aspect and wildfire affect soil-water dynamics during snowmelt, soil-water storage at the end of the snowmelt period reached the value at field capacity for each plot, suggesting that post-snowmelt unsaturated storage was not substantially influenced by aspect in wildfire-affected areas. Our data and analysis indicate that the amount of snowmelt-driven groundwater recharge may be larger in wildfire-impacted areas, especially on south-facing slopes, because of earlier soil thaw and longer durations of soil-water contents above field capacity in those areas.

Effect of Salvia miltiorrhiza polysaccharides on boar spermatozoa during freezing-thawing.

PubMed

Shen, Tao; Jiang, Zhong-Liang; Liu, Hong; Li, Qing-Wang

2015-08-01

Salvia miltiorrhiza polysaccharides (SMPs) were extracted from S. miltiorrhiza in this study. The aim of the present study was to evaluate the effect of SMP on the motility of boar sperm, including the antioxidant effect of SMP on boar sperm and the effect of SMP on the in vivo fertilizing ability of frozen-thawed boar sperm. Fifty ejaculates from 5 Swagger boars were collected and diluted with an extender, which contained 3% glycerol (v/v) with five concentrations of SMP (0.2, 0.4, 0.6, 0.8, and 1.0mg/mL). The semen was frozen in 0.25mL straws at 1.0×10(9) cells/mL. Sixty gilts were inseminated using fresh semen, frozen semen with 0.4mg/mL of SMP and frozen semen without SMP. The results indicate that the addition of SMP to the extender results in a higher percentage of motile sperm post-thaw (P<0.05). The activities of superoxide dismutase, lactate dehydrogenase, glutamic-oxalacetic transaminease and catalase were all determined to be significantly higher than the control group after adding SMP to the extender (P<0.05). The artificial insemination (AI) results demonstrated that the litter size was significantly higher in the 0.4mg/mL of SMP group than in the control group (P<0.05). In conclusion, during the process of freezing, SMP can protect boar sperm from peroxidative damage and increase sperm motility and litter size during the process of freezing-thawing. The optimal concentration of SMP for the frozen extenders in this study was determined to be 0.4mg/mL. Copyright © 2015. Published by Elsevier B.V.

Microparticles variability in fresh frozen plasma: preparation protocol and storage time effects

PubMed Central

Kriebardis, Anastasios G.; Antonelou, Marianna H.; Georgatzakou, Hara T.; Tzounakas, Vassilis L.; Stamoulis, Konstantinos E.; Papassideri, Issidora S.

2016-01-01

Background Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma. Materials and methods Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors’ haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel. Results Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors. Discussion The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles. PMID:27136430

Microparticles variability in fresh frozen plasma: preparation protocol and storage time effects.

PubMed

Kriebardis, Anastasios G; Antonelou, Marianna H; Georgatzakou, Hara T; Tzounakas, Vassilis L; Stamoulis, Konstantinos E; Papassideri, Issidora S

2016-05-01

Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma. Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors' haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel. Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors. The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles.

Pain and Function Recovery Trajectories following Revision Hip Arthroplasty: Short-Term Changes and Comparison with Primary Hip Arthroplasty in the ADAPT Cohort Study.

PubMed

Lenguerrand, Erik; Whitehouse, Michael R; Wylde, Vikki; Gooberman-Hill, Rachael; Blom, Ashley W

2016-01-01

Patients report similar or better pain and function before revision hip arthroplasty than before primary arthroplasty but worse results are reported after revision surgery than after primary surgery. The trajectory of post-operative recovery during the first months and any differences by type of surgery have received little attention. We explored the trajectories of change in pain and function after revision hip arthroplasty to 12-months post-operatively and compare them with those observed after primary hip arthroplasty. This study is a prospective cohort study of patients undergoing primary (n = 80 with 92% for an indication of osteoarthritis) and revision (n = 43) hip arthroplasties. WOMAC pain and function scores and walking speed were collected pre-operatively, at 3 and 12-months post-operatively. Multilevel regression models were used to chart and compare the trajectories of change (0-3 months and 3-12 months) between types of surgery. The improvements in pain and function following revision arthroplasty occurred within the first 3-months with no evidence of further change beyond this initial period. While the pattern of recovery was similar to the one observed after primary arthroplasty, improvements in the first 3-months were smaller after revision compared to primary arthroplasty. Patients listed for revision surgery reported lower pre-operative pain levels but similar post-operative levels compared to those undergoing primary surgery. At 12-months post-operation patients who underwent a revision arthroplasty had not reached the same level of function achieved by those who underwent primary arthroplasty. The post-operative improvements in pain and function are larger following primary hip arthroplasty than following revision hip arthroplasty. Irrespectively of surgery type, most of the improvements occur in the first three post-operative months. More research is required to identify whether the recovery following revision surgery could be improved with specific post-operative interventions.

Freezability of water buffalo spermatozoa is improved with the addition of catalase in cryodiluent.

PubMed

Ali, L; Hassan Andrabi, S M; Ahmed, H; Hussain Shah, A A

Catalase enzyme is usually distributed in mammalian seminal plasma, where it decomposes hydrogen peroxide into water and oxygen and enhances sperm survivability. To evaluate the effect of catalase (0, 100, 200 or 300 IU/ml) added in tris-citric acid (TCA) based extender on motion characteristics, viability and DNA integrity of bubaline spermatozoa at post dilution (PD) and post thawing (PT) stages of cryopreservation. Collection of semen was done in four Nili-Ravi bulls with an artificial vagina (42 degree C). Qualified semen samples from each bull were further subdivided into four aliquots for dilution with the experimental TCA extender containing either 0.0 (T1), 100 IU (T2), 200 IU (T3) or 300 IU (T4) catalase (activity12660 U/mg). At PT, mean computer progressive motility, average path velocity, straight line velocity, curvilinear velocity, visual motility and DNA integrity were higher (P < 0.05) in catalase fortified treatment groups as compared with control. Regarding plasma membrane integrity and supra-vital plasma membrane integrity, at PT the mean values were higher (P < 0.05) in T4 as compared with control. At PD and PT, mean acrosomal integrity of buffalo bull spermatozoa was higher (P < 0.05) in T4 group as compared with control. Addition of catalase at a concentration of 300IU/ml in TCA cryodiluent improved the freezability of water buffalo spermatozoa.

Thawing of Frozen Tuna Meat

NASA Astrophysics Data System (ADS)

Tanaka, Takeo; Nishiwaki, Kôji; Kakuda, Kitonari; Tomimatsu, Takao

Frozen southern bluefin tuna meat discolors easily and sometimes contracts when thawed caused by thaw rigor. These phenomenon often become problematic in the transaction or handling of this kind of frozen tuna. Frozen meat blocks of southern Bluefin tuna were thawed separately by air thawing, running water thawing and microwave thawing. Changes occurring during thawing were checked for meat color by met-myoglobin ratio determination and for contract by microscopic observation. Results are as follows : (1) Discoloration scarcely occurred in the process of running water thawing (at 10°C for 50 min, or at 0°C for 6 hr). (2) No contraction was observed during thawing with running water described above and air thawing (at 18-20°C for 6 hr). (3) Discoloration and contraction seemed to be minimized, as to latently contractile blocks, when meat temperature passed through rapidly between -10°C and -5°C, and slowly (for 5-6 hr) between -5°C and -1°C. When the block was originally not contractile, discloration was minimized by rising meat temperature rapidly from -10°C to -l°C.

Shifting post production patterns: exploring changes in New Zealand's seafood processing industry.

PubMed

Stringer, Christina; Simmons, Glenn; Rees, Eugene

2011-01-01

This paper examines the changing nature of New Zealand's seafood companies' production practices. The past 15 years has seen the offshore outsourcing of post-harvest fish gain unprecedented momentum. The growth in offshore processing is a further stage in an increasingly globalised fisheries value chain. Fish is head and gutted, frozen and then transported to processing sites in China where it is thawed, value-added processed and refrozen for export to the original sourcing country or third country markets. Reasons advanced by the industry for this shift in production practices include quota reductions, increasing production costs and the sale of trawlers.

Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin.

PubMed

Takeo, Toru; Nakagata, Naomi

2011-11-01

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.

Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants.

PubMed

Baumber, Julie; Ball, Barry A; Linfor, Jennifer J

2005-05-01

To evaluate the effect of the addition of enzyme scavengers and antioxidants to the cryopreservation extender on characteristics of equine spermatozoa after freezing and thawing. 2 ejaculates collected from each of 5 stallions. Equine spermatozoa were cryopreserved in freezing extender alone (control samples) or with the addition of catalase (200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione (10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or 500 microM or 1 mM), or the vehicle for alpha-tocopherol (0.5% ethanol). After thawing, spermatozoal motility was assessed via computer-assisted analysis and DNA fragmentation was assessed via the comet assay. Spermatozoal mitochondrial membrane potential, acrosomal integrity, and viability were determined by use of various specific staining techniques and flow cytometry. The addition of enzyme scavengers or antioxidants to cryopreservation extender did not improve spermatozoal motility, DNA fragmentation, acrosomal integrity, viability, or mitochondrial membrane potential after thawing. Superoxide dismutase increased DNA fragmentation, likely because of the additional oxidative stress caused by the generation of hydrogen peroxide by this enzyme. Interestingly, the addition of the vehicle for alpha-tocopherol resulted in a significant decrease in live acrosome-intact spermatozoa. The addition of antioxidants to the cryopreservation extender did not improve the quality of equine spermatozoa after thawing, which suggests that the role of oxidative stress in cryopreservation-induced damage of equine spermatozoa requires further investigation. Our data suggest that solubilizing alpha-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of alpha-tocopherol may be preferred for future investigations.

Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation.

PubMed

Gómez-Torres, María José; Medrano, Llanos; Romero, Alejandro; Fernández-Colom, Pedro José; Aizpurúa, Jon

2017-10-01

Human spermatozoa cryopreservation techniques are used to maintain and protect male fertility in cases such as infertility and malignancy treatments. However, during cryopreservation, the spermatozoa's metabolic rate is reduced and they undergo dramatic functional and structural changes owing to exposure to cryoprotectants and freezing-thawing procedures. While the effects of cryopreservation on cells are documented, to date the induced cryodamage on structural and/or functional sperm biomarkers is not well established at multivariate scale. To address this question, we performed basic sperm analysis, sperm DNA fragmentation assessment, spontaneous acrosome reaction measurement, and cytoskeleton evaluation after thawing samples from subjects with normal and low-quality semen. A cryodamage rate was used to determine the effects of the freeze-thaw process on spermatozoa. In addition, a Principal Component Analysis (PCA) was used for data reduction and to evaluate sperm-specific patterns during the cryopreservation process. We found that the vitality, progressive motility and sperm count from low-quality samples after cryopreservation show higher damage rates (≥40%) than in normal sperm samples. However, cytoskeleton, DNA, tail and mid-piece and acrosome display the highest cryodamage rates (∼50-99%) and are equally susceptible to cryopreservation-induced damage in both low- and normal-quality semen samples. Overall, the evaluation of these parameters provides meaningful information about different aspects of sperm functionality after cryopreservation. Copyright © 2017 Elsevier Inc. All rights reserved.

Consumer Attitudes Toward Storing and Thawing Chicken and Effects of the Common Thawing Practices on Some Quality Characteristics of Frozen Chicken

PubMed Central

Benli, Hakan

2016-01-01

In this study, a survey was conducted to both evaluate the consumers’ general attitudes for purchasing and storing the raw chicken and determine the thawing practices used for defrosting frozen chicken at home. About 75% of the consumers indicated purchasing chicken meat at least once a week or more. Furthermore, the majority (82.16%) of those who stored at least a portion of the raw chicken stated freezing the raw chicken meat at home. Freezing the chicken meat was considered to have no effect on the quality by 43.49% of the consumers while 56.51% thought that freezing had either negative or positive effects on the quality. The survey study indicated that top five most commonly used thawing practices included thawing on the kitchen counter, thawing in the refrigerator, thawing in the warm water, thawing in the microwave, and thawing under tap water. In addition, an experimental study was conducted to determine the effects of these most commonly used thawing practices on some quality characteristics of the chicken meat including pH, drip loss, cooking loss, color analysis and textural profile analysis. Although, L* value for thawing on the kitchen counter was the lowest, after cooking, none of the thawing treatments have a significant effect on the color values. Thawing in the microwave produced the highest drip loss of 3.47% while the lowest drip loss of 0.62% was observed with thawing in the refrigerator. On the other hand, thawing in the microwave and refrigerator caused the lowest cooking loss values of 18.29% and 18.53%, respectively. Nevertheless, there were no significant differences among textural parameter values of the defrosted and then cooked samples using the home based thawing practices, indicating similar quality characteristics among the samples. PMID:26732333

The NASA Soil Moisture Active Passive (SMAP) Mission - Science and Data Product Development Status

NASA Technical Reports Server (NTRS)

Nloku, E.; Entekhabi, D.; O'Neill, P.

2012-01-01

The Soil Moisture Active Passive (SMAP) mission, planned for launch in late 2014, has the objective of frequent, global mapping of near-surface soil moisture and its freeze-thaw state. The SMAP measurement system utilizes an L-band radar and radiometer sharing a rotating 6-meter mesh reflector antenna. The instruments will operate on a spacecraft in a 685 km polar orbit with 6am/6pm nodal crossings, viewing the surface at a constant 40-degree incidence angle with a 1000-km swath width, providing 3-day global coverage. Data from the instruments will yield global maps of soil moisture and freeze/thaw state at 10 km and 3 km resolutions, respectively, every two to three days. The 10-km soil moisture product will be generated using a combined radar and radiometer retrieval algorithm. SMAP will also provide a radiometer-only soil moisture product at 40-km spatial resolution and a radar-only soil moisture product at 3-km resolution. The relative accuracies of these products will vary regionally and will depend on surface characteristics such as vegetation water content, vegetation type, surface roughness, and landscape heterogeneity. The SMAP soil moisture and freeze/thaw measurements will enable significantly improved estimates of the fluxes of water, energy and carbon between the land and atmosphere. Soil moisture and freeze/thaw controls of these fluxes are key factors in the performance of models used for weather and climate predictions and for quantifYing the global carbon balance. Soil moisture measurements are also of importance in modeling and predicting extreme events such as floods and droughts. The algorithms and data products for SMAP are being developed in the SMAP Science Data System (SDS) Testbed. In the Testbed algorithms are developed and evaluated using simulated SMAP observations as well as observational data from current airborne and spaceborne L-band sensors including data from the SMOS and Aquarius missions. We report here on the development status of the SMAP data products. The Testbed simulations are designed to capture various sources of errors in the products including environment effects, instrument effects (nonideal aspects of the measurement system), and retrieval algorithm errors. The SMAP project has developed a Calibration and Validation (Cal/Val) Plan that is designed to support algorithm development (pre-launch) and data product validation (post-launch). A key component of the Cal/Val Plan is the identification, characterization, and instrumentation of sites that can be used to calibrate and validate the sensor data (Level l) and derived geophysical products (Level 2 and higher).

[Effects of an Oral Care Program on the Swallowing Function in Post-Operative Patients With Oral Cancer].

PubMed

Hsiang, Ching-Chi; Hwu, Yueh-Juen

2017-04-01

Oral cancer is the fourth leading cause of death among men in Taiwan. Dysphagia, choking, and aspiration pneumonia are often noted in post-operative patients with oral cancer. Improving patients' swallowing function is an urgent problem that cannot be neglected. To investigate the effects of an oral care program on the swallowing function of post-operative patients with oral cancer. A quasi-experimental research design was conducted and post-operative patients with oral cancer were recruited. The experimental group (n = 20) received 12 weeks of the oral care program intervention, while the control group (n = 20) received standard post-operative care. The modified barium swallow (MBS) study and self-rated degree of dysphagia were compared between the two groups after the intervention period. Post-intervention scores on the MBS test and for the self-rated degree of dysphagia were significantly better in the experimental group than in the control group (p < .001). Performing the oral care program was found to improve the swallowing function of post-operative patients with oral cancer. The results of the present study provide a reference for healthcare providers to improve quality of care.

MAL62 overexpression and NTH1 deletion enhance the freezing tolerance and fermentation capacity of the baker's yeast in lean dough.

PubMed

Sun, Xi; Zhang, Cui-Ying; Wu, Ming-Yue; Fan, Zhi-Hua; Liu, Shan-Na; Zhu, Wen-Bi; Xiao, Dong-Guang

2016-04-04

Trehalose is related to several types of stress responses, especially freezing response in baker's yeast (Saccharomyces cerevisiae). It is desirable to manipulate trehalose-related genes to create yeast strains that better tolerate freezing-thaw stress with improved fermentation capacity, which are in high demand in the baking industry. The strain overexpressing MAL62 gene showed increased trehalose content and cell viability after prefermention-freezing and long-term frozen. Deletion of NTH1 in combination of MAL62 overexpression further strengthens freezing tolerance and improves the leavening ability after freezing-thaw stress. The mutants of the industrial baker's yeast with enhanced freezing tolerance and leavening ability in lean dough were developed by genetic engineering. These strains had excellent potential industrial applications.

Virtual Global Transplant Laboratory Standard Operating Procedures for Blood Collection, PBMC Isolation, and Storage.

PubMed

Higdon, Lauren E; Lee, Karim; Tang, Qizhi; Maltzman, Jonathan S

2016-09-01

Research on human immune responses frequently involves the use of peripheral blood mononuclear cells (PBMC) immediately, or at significantly delayed timepoints, after collection. This requires PBMC isolation from whole blood and cryopreservation for some applications. It is important to standardize protocols for blood collection, PBMC isolation, cryopreservation, and thawing that maximize survival and functionality of PBMC at the time of analysis. This resource includes detailed protocols describing blood collection tubes, isolation of PBMC using a density gradient, cryopreservation of PBMC, and thawing of cells as well as preparation for functional assays. For each protocol, we include important considerations, such as timing, storage temperatures, and freezing rate. In addition, we provide alternatives so that researchers can make informed decisions in determining the optimal protocol for their application.

Salivary levels of phosphorus and urea as indices of their plasma levels in nephropathic patients.

PubMed

Bilancio, Giancarlo; Cavallo, Pierpaolo; Lombardi, Cinzia; Guarino, Ermanno; Cozza, Vincenzo; Giordano, Francesco; Palladino, Giuseppe; Cirillo, Massimo

2018-03-30

Phosphorus and urea are measurable in saliva. Measurements of saliva phosphorus (S-Pho) and saliva urea (S-Urea) could be useful because of low invasivity. Data are limited to saliva tests methodology and to correlations between plasma and saliva compositions. S-Pho and S-Urea were investigated focusing on blind duplicates, differences between collection sites, differences between collection times, freezing-thawing effects, and plasma-saliva correlations. Tests were performed using fresh saliva collected by synthetic swap early morning after overnight fast (standard). Methodology was investigated in fifteen healthy volunteers. Plasma-saliva correlations were investigated in thirty nephropathic outpatients. S-Pho and S-Urea in all measurements ranged above detection limits (0.3 mmol/L). In healthy volunteers, S-Pho and S-Urea were similar in duplicates (results for S-Pho and S-Urea: % difference between samples ≤ 4.85%; R between samples ≥ .976, P < .001), in samples from different mouth sites (≤4.24%; R ≥ .887, P < .001), and in samples of different days (≤5.61%; R ≥ .606, P < .01) but, compared to standard, were substantially lower in after-breakfast samples (-28.0% and -21.3%; R ≥ .786, P < .001) and slightly lower in frozen-thawed samples (-12.4% and -5.92%; R ≥ .742, P < .001). In nephropathic patients, S-Pho was higher than but correlated with plasma phosphorus (saliva/plasma ratio 4.80; R = .686, P < .001), whereas S-Urea and plasma urea were similar and correlated with each other (saliva/plasma ratio 0.96; R = .944, P < .001). Post-dialysis changes in S-Pho and S-Urea paralleled post-dialysis changes in plasma phosphorus and urea. S-Pho and S-Urea reflect plasma phosphorus and plasma urea. Early morning fasting fresh samples are advisable because collection time and freezing-thawing affect saliva tests. © 2018 Wiley Periodicals, Inc.

The thin brown line: The crucial role of peat in protecting permafrost in Arctic Alaska

NASA Astrophysics Data System (ADS)

Gaglioti, B.; Mann, D. H.; Farquharson, L. M.; Baughman, C. A.; Jones, B. M.; Romanovsky, V. E.; Williams, A. P.; Andreu-Hayles, L.

2017-12-01

Ongoing warming threatens to thaw Arctic permafrost and release its stored carbon, which could trigger a permafrost-carbon feedback capable of augmenting global warming. The effects of warming air temperatures on permafrost are complicated by the fact that across much of the Arctic and Subarctic a mat of living plants and decaying litter cover the ground and buffer underlying permafrost from air temperatures. For simplicity here, we refer to this organic mat as "peat". Because this peat modifies heat flow between ground and air, the rate and magnitude of permafrost responses to changing climate - and hence the permafrost-carbon feedback - are partly slaved to the peat layer's slower dynamics. To explore this relationship, we used 14C-age offsets within lake sediments in Alaskan watersheds underlain by yedoma deposits to track the changing responses of permafrost thaw to fluctuating climate as peat accumulated over the last 14,000 years. As the peat layer built up, warming events became less effective at thawing permafrost and releasing ancient carbon. Consistent with this age-offset record, the geological record shows that early in post-glacial times when the peat cover was still thin and limited in extent, warm intervals triggered extensive thermokarst that resulted in rapid aggradation of floodplains. Today in contrast, hillslopes and floodplains remain stable despite rapid warming, probably because of the buffering effects of the extensive peat cover. Another natural experiment is provided by tundra fires like the 2007 Anaktuvuk River fire that removed the peat cover from tundra underlain by continuous permafrost and resulted in widespread thermkarsting. Further support for peat's critical role in protecting permafrost comes from the results of modeling how permafrost temperatures under different peat thicknesses respond to warming air temperature. Although post-industrial warming has not yet surpassed the buffering capacity of 14,000 years of peat buildup in Arctic Alaska, modeling suggests that a threshold is imminent.

Conservation science in a terrorist age: the impact of airport security screening on the viability and DNA integrity of frozen felid spermatozoa.

PubMed

Gloor, Kayleen T; Winget, Doug; Swanson, William F

2006-09-01

In response to growing terrorism concerns, the Transportation Security Administration now requires that all checked baggage at U.S. airports be scanned through a cabinet x-ray system, which may increase risk of radiation damage to transported biologic samples and other sensitive genetic material. The objective of this study was to investigate the effect of these new airport security regulations on the viability and DNA integrity of frozen felid spermatozoa. Semen was collected from two domestic cats (Felis silvestris catus) and one fishing cat (Prionailurus viverrinus), cryopreserved in plastic freezing straws, and transferred into liquid nitrogen dry shippers for security screening. Treatment groups included frozen samples from each male scanned once or three times using a Transportation Security Administration-operated cabinet x-ray system, in addition to non-scanned samples (i.e., negative control) and samples previously scanned three times and exposed to five additional high-intensity x-ray bursts (i.e., positive control). Dosimeters placed in empty dry shippers were used to quantify radiation exposure. Following treatment, straws were thawed and spermatozoa analyzed for post-thaw motility (percentage motile and rate of progressive movement), acrosome status, and DNA integrity using single-cell gel electrophoresis (i.e., the comet assay). Dosimeter measurements determined that each airport screening procedure produced approximately 16 mrem of radiation exposure. Our results indicated that all levels of radiation exposure adversely affected (P < 0.05) post-thaw sperm motility, but the percentage of acrosome-intact spermatozoa did not differ (P > 0.05) among treatment groups. Results also showed that the amount of double-stranded DNA damage was greater (P < 0.05) in sperm samples from both cat species scanned three times compared to samples scanned once or negative controls. Findings suggest that new airport security measures may cause radiation-induced damage to frozen spermatozoa and other valuable biologic samples transported on passenger aircraft and that alternative modes of sample transportation should be used whenever possible.