Assessment of the effect of prolonged forced swimming on CD-1 mice sperm morphology with and without antioxidant supplementation.

PubMed

Rodriguez, I; Diaz, A; Vaamonde, D

2016-04-01

As physical exercise has been shown to negatively affect sperm morphology, this study was undertaken to assess the effect of a 3-min forced swimming protocol during 50 days, with and without administration of antioxidants [N-acetylcysteine (NAC) and trans-resveratrol], on sperm morphology in CD-1 mice. Forty-four 13-week-old CD-1 mice were randomly allocated to four different groups: mice not submitted to exercise, control group (CG), mice submitted to swimming without administration of antioxidants (EX), mice submitted to swimming that received trans-resveratrol supplementation [exercise group (EX)+Resv] and mice submitted to swimming exercise that received NAC supplementation (EX+NAC). The EX showed 30.5% of spermatozoa with normal morphology, showing significant differences with regard to the CG, which showed 58.5%. The groups receiving antioxidant supplements showed significantly higher percentages of spermatozoa with normal morphology in comparison with the EX group (EX+Resv: 64.1%, EX+NAC: 48.2%). The imposed model of forced swimming caused alterations in sperm morphology. The antioxidants employed seem to be suitable antioxidants for avoiding exercise-associated sperm morphology anomalies in prolonged forced swimming exercise. Trans-resveratrol has proven to be more efficient for this purpose. © 2015 Blackwell Verlag GmbH.

Study on the short-term effects of increased alcohol and cigarette consumption in healthy young men’s seminal quality

PubMed Central

Silva, Joana Vieira; Cruz, Daniel; Gomes, Mariana; Correia, Bárbara Regadas; Freitas, Maria João; Sousa, Luís; Silva, Vladimiro; Fardilha, Margarida

2017-01-01

Many studies have reported a negative impact of lifestyle factors on testicular function, spermatozoa parameters and pituitary-gonadal axis. However, conclusions are difficult to draw, since studies in the general population are rare. In this study we intended to address the early and late short-term impact of acute lifestyle alterations on young men’s reproductive function. Thirty-six healthy male students, who attended the Portuguese academic festivities, provided semen samples and answered questionnaires at three time-points. The consumption of alcohol and cigarette increased more than 8 and 2 times, respectively, during the academic festivities and resulted in deleterious effects on semen quality: one week after the festivities, a decrease on semen volume, spermatozoa motility and normal morphology was observed, in parallel with an increase on immotile spermatozoa, head and midpiece defects and spermatozoa oxidative stress. Additionally, three months after the academic festivities, besides the detrimental effect on volume, motility and morphology, a negative impact on spermatozoa concentration was observed, along with a decrease on epididymal, seminal vesicles and prostate function. This study contributed to understanding the pathophysiology underlying semen quality degradation induced by acute lifestyle alterations, suggesting that high alcohol and cigarette consumption are associated with decreased semen quality in healthy young men. PMID:28367956

Cystic fibrosis transmembrane conductance regulator is correlated closely with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters.

PubMed

Jiang, L-Y; Shan, J-J; Tong, X-M; Zhu, H-Y; Yang, L-Y; Zheng, Q; Luo, Y; Shi, Q-X; Zhang, S-Y

2014-10-01

Cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated to be expressed in mature spermatozoa and correlated with sperm quality. Sperm CFTR expression in fertile men is higher than that in infertile men suffering from teratospermia, asthenoteratospermia, asthenospermia and oligospermia, but it is unknown whether CFTR is correlated with sperm parameters when sperm parameters are normal. In this study, 282 healthy and fertile men with normal semen parameters were classified into three age groups, group (I): age group of 20-29 years (98 cases, 27.1 ± 6.2), group (II): age group of 30-39 years (142 cases, 33.7 ± 2.6) and group (III): age group of more than or equal to 40 years (42 cases, 44.1 ± 4.6). Sperm concentration, total count and progressive motility were analysed by computer-assisted sperm analysis. Sperm morphology was analysed by modified Papanicolaou staining. Sperm CFTR expression was conducted by indirect immunofluorescence staining. There was a significant positive correlation (P < 0.001) between CFTR expression and sperm progressive motility (r = 0.221) and normal morphology (r = 0.202), but there were no correlations between sperm CFTR expression and semen volume, sperm concentration, sperm total count as well as male age (P > 0.05). Our findings show that CFTR expression is associated with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters, but not associated with the number of spermatozoa and male age. © 2013 Blackwell Verlag GmbH.

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.

Effect of varicocelectomy on the abnormal retention of residual cytoplasm by human spermatozoa.

PubMed

Zini, A; Buckspan, M; Jamal, M; Jarvi, K

1999-07-01

Abnormal retention of cytoplasmic residues by human spermatozoa is associated with the generation of reactive oxygen species (ROS) in semen and defective sperm function. We have examined the effect of varicocelectomy on the retention of residual cytoplasm by human spermatozoa. Clinical reports of 43 men who underwent microsurgical varicocelectomy at our institution during a 1 year period beginning July 1996 were reviewed. Standard semen parameters (concentration, motility and morphology) and residual cytoplasm retention (monitored by Papanicolaou stain) were assessed before and 6 months after varicocelectomy. The percentage of spermatozoa with residual cytoplasm decreased significantly following varicocelectomy compared to pre-operatively (25.8 versus 18.1% respectively). The percentages of motile spermatozoa and normal forms increased significantly (P = 0.0003, P = 0.005 respectively) following varicocelectomy (22.6 versus 32.9% and 46.4 versus 54.4% respectively). Our data suggest that varicocelectomy can improve the disposal of residual sperm cytoplasm by the testis and/or epididymis in infertile men with varicocele. These data also suggest that varicocelectomy reduces the potential for ROS generation by human spermatozoa in these men.

Stallion spermatozoa selected by single layer centrifugation are capable of fertilization after storage for up to 96 h at 6°C prior to artificial insemination

PubMed Central

2012-01-01

Background One of the challenges faced by equine breeders is ensuring delivery of good quality semen doses for artificial insemination when the mare is due to ovulate. Single Layer Centrifugation (SLC) has been shown to select morphologically normal spermatozoa with intact chromatin and good progressive motility from the rest of the ejaculate, and to prolong the life of these selected spermatozoa in vitro. The objective of the present study was a proof of concept, to determine whether fertilizing ability was retained in SLC-selected spermatozoa during prolonged storage. Findings Sixteen mares were inseminated with SLC-selected sperm doses that had been cooled and stored at 6°C for 48 h, 72 h or 96 h. Embryos were identified in 11 mares by ultrasound examination 16–18 days after presumed ovulation. Conclusion SLC-selected stallion spermatozoa stored for up to 96 h are capable of fertilization. PMID:22788670

Influence of temperature and sperm preparation on the quality of spermatozoa.

PubMed

Thijssen, Annelies; Klerkx, Elke; Huyser, Carin; Bosmans, Eugene; Campo, Rudi; Ombelet, Willem

2014-04-01

This study investigated the effects of long-term (24h) in-vitro sperm incubation at room temperature (RT; 23°C) versus testis temperature (35°C) on various sperm-quality parameters. Semen samples (n=41) were prepared both by density-gradient centrifugation (DGC) and the swim-up technique in order to compare the influence of sperm preparation on sperm quality after incubation. Progressive motility and morphology were significantly higher after incubation at RT compared with 35°C (P<0.001 and P<0.01, respectively). The proportions of acrosome-reacted, apoptotic and dead spermatozoa were significantly lower in samples incubated for 24h at RT compared with 35°C (P<0.001, P=0.01 and P<0.001, respectively). The number of motile, morphologically normal, non-acrosome-reacted and nonapoptotic spermatozoa recovered after sperm preparation was significantly higher in DGC compared with swim-up samples (P<0.001). However, spermatozoa prepared by swim-up showed better survival after incubation compared with DGC-prepared spermatozoa, especially when incubated at 35°C. In conclusion, this study indicates a significantly better and longer preservation of sperm quality when incubation is performed at RT. These findings may convince laboratories to change the routinely used sperm storage conditions in order to maximize the quality of the prepared sperm sample. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa.

PubMed

Terrell, Kimberly A; Wildt, David E; Anthony, Nicola M; Bavister, Barry D; Leibo, S P; Penfold, Linda M; Marker, Laurie L; Crosier, Adrienne E

2012-04-01

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing. We hypothesized that freezing/thawing impairs sperm metabolism and that swim-up, but not density gradient centrifugation, recovers metabolically-normal spermatozoa. Ejaculates were cryopreserved, thawed, and processed by swim-up, Accudenz gradient centrifugation, or conventional washing (representing the 'control'). Sperm glucose and pyruvate uptake, lactate production, motility, and acrosomal integrity were assessed. Mitochondrial membrane potential (MMP) was measured in cat spermatozoa. In both species, lactate production, motility, and acrosomal integrity were reduced in post-thaw, washed samples compared to freshly-collected ejaculates. Glucose uptake was minimal pre- and post-cryopreservation, whereas pyruvate uptake was similar between treatments due to high coefficients of variation. In the cat, swim-up, but not Accudenz processing, recovered spermatozoa with increased lactate production, pyruvate uptake, and motility compared to controls. Although confounded by differences in non-specific fluorescence among processing methods, MMP values within treatments were positively correlated to sperm motility and acrosomal integrity. Cheetah spermatozoa isolated by either selection method exhibited improved motility and/or acrosomal integrity, but remained metabolically compromised. Collectively, findings revealed a metabolically-robust subpopulation of cryopreserved cat, but not cheetah, spermatozoa, recovered by selecting for motility rather than morphology. Published by Elsevier Inc.

Colloid centrifugation of boar semen.

PubMed

Morrell, J M; Wallgren, M

2011-09-01

Colloid centrifugation of boar semen has been reported sporadically for at least the last two decades, beginning with density gradient centrifugation (DGC) and progressing more recently to single layer centrifugation (SLC). Single layer centrifugation through a species-specific colloid has been shown to be effective in selecting the best spermatozoa (spermatozoa with good motility and normal morphology) from boar sperm samples. The method is easier to use and less time-consuming than DGC and has been scaled-up to allow whole ejaculates from other species, e.g. stallions, to be processed in a practical manner. The SLC technique is described, and various scale-up versions are presented. The potential applications for SLC in boar semen preservation are as follows: to improve sperm quality in artificial insemination (AI) doses for 'problem' boars; to increase the shelf-life of normal stored sperm samples, either by processing the fresh semen before preparing AI doses or by processing the stored semen dose to extract the best spermatozoa; to remove pathogens (viruses, bacteria), thus improving biosecurity of semen doses and potentially reducing the use of antibiotics; to improve cryosurvival by removing dead and dying spermatozoa prior to cryopreservation; to select spermatozoa for in vitro fertilization. These applications are discussed and practical examples are provided. Finally, a few thoughts about the economic value of the technique to the boar semen industry are presented. © 2011 Blackwell Verlag GmbH.

Alpaca semen characteristics under free and directed mounts during a mating period.

PubMed

Urquieta, Bessie; Flores, Paloma; Muñoz, Camila; Bustos-Obregón, Eduardo; García-Huidobro, Jorge

2005-12-01

Most studies in alpaca reproductive biology have been focused on female physiology. Only recent research is being conducted in order to increase the knowledge on males. Semen characteristics during breeding periods will contribute to understanding the poor fertility rates in alpaca. Ten adult male alpacas were distributed randomly into two groups and submitted alternatively to two regimens of semen collection of 12 days duration (day 1, initial day of semen collection). Semen samples were collected using an artificial vagina and a receptive, non-pregnant female. With regimen 1, males were maintained with females except for the days of sexual rest (6 and 7). Semen was collected on days 1, 5, 8 and 12. With regimen 2, males were exposed to females for daily semen collection only, before and after sexual rest. Mating duration, color and volume of ejaculates, spermatozoa concentration and morphology were evaluated. No statistical differences for the variables were found between regimens that were used for semen collection. With respect to influence of day, however, the total numbers of spermatozoa ejaculated on days 1 and 5 of semen collection were statistically different (p<0.05). Azoospermic samples increased on days 5 and 12 of semen collection. Partial recovery in spermatozoa concentration and number of spermatozoa ejaculated were observed after sexual rest. Although normal spermatozoa percentage was less on day 1 (p<0.05) as compared with values found in the following ejaculates (days 5 and 12), the total number of normal spermatozoa was greater. These results support the conclusion that when male alpaca have a daily ejaculation during five consecutive days, they might copulate without having enough spermatozoa for fertilization towards the end of the mating period.

The effect of DMA level on morphology and fertilising ability of Japanese quail (Coturnix japonica) spermatozoa.

PubMed

Chełmońska, Bronisława; Łukaszewicz, Ewa; Kowalczyk, Artur; Jerysz, Anna

2006-01-20

The effect of different levels (2, 4 or 6%) of DMA (dimethylacetamide) on the morphology and fertilising ability of unfrozen quail spermatozoa was evaluated. Semen was collected from 72 males kept individually in cages and randomly divided into four groups: Group I--control -- fresh undiluted semen (12 males) and three experimental groups (20 males each) - semen diluted 1:1 with Lake's extender and supplemented with 2% (Group II), 4% (Group III) or 6% (Group IV) of DMA (final concentration). Sperm morphology was evaluated at each step of semen preparation, i.e. in fresh and diluted semen, semen supplemented with DMA and semen that remained after insemination. For fertility tests, 36 females were divided into four groups (nine females each). Females in the control group were inseminated with 10 microl of fresh semen, in the experimental groups with 40 microl of diluted semen. Each stage of quail semen treatment had a deleterious effect on sperm morphology. The highest percentage of morphologically normal cells in semen evaluated after insemination, was observed in samples with 2% DMA, and the lowest--in samples with 6% DMA. Semen dilution and DMA addition significantly affected the fertilising potency of spermatozoa. Fertility of eggs collected from the control group (71.5% on average) was significantly higher (P

Evidence for compromised metabolic function and limited glucose uptake in spermatozoa from the teratospermic domestic cat (Felis catus) and cheetah (Acinonyx jubatus).

PubMed

Terrell, Kimberly A; Wildt, David E; Anthony, Nicola M; Bavister, Barry D; Leibo, Stanley P; Penfold, Linda M; Marker, Laurie L; Crosier, Adrienne E

2010-11-01

Cheetahs and certain other felids consistently ejaculate high proportions (≥ 60%) of malformed spermatozoa, a condition known as teratospermia, which is prevalent in humans. Even seemingly normal spermatozoa from domestic cat teratospermic ejaculates have reduced fertilizing capacity. To understand the role of sperm metabolism in this phenomenon, we conducted a comparative study in the normospermic domestic cat versus the teratospermic cat and cheetah with the general hypothesis that sperm metabolic function is impaired in males producing predominantly pleiomorphic spermatozoa. Washed ejaculates were incubated in chemically defined medium containing glucose and pyruvate. Uptake of glucose and pyruvate and production of lactate were assessed using enzyme-linked fluorescence assays. Spermatozoa from domestic cats and cheetahs exhibited similar metabolic profiles, with minimal glucose metabolism and approximately equimolar rates of pyruvate uptake and lactate production. Compared to normospermic counterparts, pyruvate and lactate metabolism were reduced in teratospermic cat and cheetah ejaculates, even when controlling for sperm motility. Rates of pyruvate and lactate (but not glucose) metabolism were correlated positively with sperm motility, acrosomal integrity, and normal morphology. Collectively, our findings reveal that pyruvate uptake and lactate production are reliable, quantitative indicators of sperm quality in these two felid species and that metabolic function is impaired in teratospermic ejaculates. Furthermore, patterns of substrate utilization are conserved between these species, including the unexpected lack of exogenous glucose metabolism. Because glycolysis is required to support sperm motility and capacitation in certain other mammals (including dogs), the activity of this pathway in felid spermatozoa is a target for future investigation.

Magnetic-activated cell sorting before density gradient centrifugation improves recovery of high-quality spermatozoa.

PubMed

Berteli, T S; Da Broi, M G; Martins, W P; Ferriani, R A; Navarro, P A

2017-07-01

Recent studies have evaluated the use of magnetic-activated cell sorting (MACS) to reduce apoptotic spermatozoa and improve sperm quality. However, the efficiency of using MACS alone, before or after sperm processing by density gradient centrifugation (DGC) has not yet been established. The purpose of this study is to determine the optimal protocol of MACS in assisted reproduction techniques (ART). Thus, we compared sperm quality obtained by DGC alone (DGC), DGC followed by MACS (DGC-MACS), MACS followed by DGC (MACS-DGC), and MACS alone (MACS), and found that the combined methods (MACS-DGC and DGC-MACS) led to retrieval of less spermatozoa with fragmented DNA compared to the single protocols. However, MACS-DGC protocol led to a significantly higher percentage of spermatozoa with progressive motility and normal morphology than DGC-MACS protocol. These findings suggest the potential clinical value of using MACS-DGC to improve sperm quality in seminal preparation for ART. © 2017 American Society of Andrology and European Academy of Andrology.

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.

Spermatozoa quality assessment: a combined holographic and Raman microscopy approach

NASA Astrophysics Data System (ADS)

De Angelis, Annalisa; Ferrara, Maria A.; Di Caprio, Giuseppe; Managò, Stefano; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

2015-05-01

Semen analysis is widely used as diagnostic tool for assessing male fertility, controlling and managing the animal reproduction. The most important parameters measured in a semen analysis are the morphology and biochemical alterations. For obtaining such information, non-invasive, label-free and non-destructive techniques have to be used. Digital Holography (DH) combined with Raman Spectroscopy (RS) could represent the perfect candidate for a rapid, non-destructive and high-sensitive morphological and biochemical sperm cell analysis. In this study, DH-RS combined approach is used for a complete analysis of single bovine spermatozoa. High-resolution images of bovine sperm have been obtained by DH microscopy from the reconstruction of a single acquired hologram, highlighting in some cases morphological alterations. Quantitative 3D reconstructions of sperm head, both normal and anomalous, have been studied and an unexpected structure of the post-acrosomal region of the head has been detected. Such anomalies have been also confirmed by Raman imaging analysis, suggesting the protein vibrations as associated Raman marker of the defect.

Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.

PubMed

Ollero, M; Gil-Guzman, E; Lopez, M C; Sharma, R K; Agarwal, A; Larson, K; Evenson, D; Thomas, A J; Alvarez, J G

2001-09-01

Reactive oxygen species (ROS)-induced damage of membrane phospholipids and DNA in human spermatozoa has been implicated in the pathogenesis of male infertility. In this study, variations in ROS production, DNA structure (as measured by the sperm chromatin structure assay) and lipid composition, were studied in human spermatozoa at different stages of maturation. Sperm subsets were isolated by discontinuous density gradient centrifugation of semen samples obtained from healthy donors and from infertility patients. DNA damage and ROS production were highest in immature spermatozoa with cytoplasmic retention and abnormal head morphology, and lowest in mature spermatozoa. Docosahexaenoic acid and sterol content were highest in immature germ cells and immature spermatozoa, and lowest in mature spermatozoa. The relative proportion of ROS-producing immature spermatozoa in the sample was directly correlated with DNA damage in mature spermatozoa, and inversely correlated with the recovery of motile spermatozoa. There was no correlation between DNA damage and sperm morphology in mature spermatozoa. The high levels of ROS production and DNA damage observed in immature spermatozoa may be indicative of derangements in the regulation of spermiogenesis. DNA damage in mature spermatozoa may be the result of oxidative damage by ROS-producing immature spermatozoa during sperm migration from the seminiferous tubules to the epididymis.

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.

Semen quality during vincristine treatment in dogs with transmissible venereal tumor.

PubMed

Saratsis, P; Ypsilantis, P; Tselkas, K

2000-03-15

The aim of this study was to evaluate the direct effects of vincristine on semen quality in dogs with transmissible venereal tumor (TVT). We examined the semen of 17 dogs suffering from TVT during vincristine treatment. Each animal received 0.6 mg, i.v. vincristine sulphate per square meter of body surface, per week for 4 wk until complete regression of the tumor. The following semen parameters were evaluated: semen volume (second fraction), sperm concentration, total spermatozoa per ejaculate, percentage of progressively motile spermatozoa, percentage of dead spermatozoa, percentage of swollen spermatozoa (hypo-osmotic swelling test) and percentage of morphologically abnormal spermatozoa (primary and secondary defects). Semen was collected and evaluated prior to the beginning of treatment, 3 d after each vincristine injection and 15 d after the last injection. Semen characteristics transiently deteriorated during treatment, but returned to normal 15 d later. These changes were attributed to a direct effect of vincristine on the extragonadal spermatozoal reserves contained in the epididymis and ductus deferens. A GnRH stimulation test was also performed after each semen collection in order to assess the function of the hypothalamic-pituitary-Leydig cell axis. No effect was noted on the above axis.

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.

The Expression Level of Septin12 Is Critical for Spermiogenesis

PubMed Central

Lin, Ying-Hung; Lin, Yung-Ming; Wang, Ya-Yun; Yu, I-Shing; Lin, Yi-Wen; Wang, Yun-Han; Wu, Ching-Ming; Pan, Hsien-An; Chao, Shin-Chih; Yen, Pauline H.; Lin, Shu-Wha; Kuo, Pao-Lin

2009-01-01

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis. PMID:19359518

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.

Cadmium toxicity at low concentration on rabbit spermatozoa motility, morphology and membrane integrity in vitro.

PubMed

Roychoudhury, Shubhadeep; Massanyi, Peter; Bulla, Jozef; Choudhury, Manabendra Dutta; Lukac, Norbert; Filipejova, Terezia; Trandzik, Jozef; Toman, Robert; Almasiova, Viera

2010-09-01

In this study the effect of cadmium on various parameters of spermatozoa motility, morphology as well as on the spermatozoa membrane integrity in rabbits was analyzed in vitro, experimental concentrations ranging from 0.62 to 0.98 micro g CdCl(2)/mL. Pooled rabbit (n = 5) semen was cultured in vitro with cadmium and subsequently diluted to various experimental concentrations apart from control which received no cadmium exposure. Using computer assisted semen analysis method (CASA) we detected decrease of total motility with in the higher concentration range at Time 0. However, with increasing time (after 1 and 2 h of culture), cadmium exerted deleterious effect leading to significant motility reduction in comparison to control. A similar trend was exhibited in case of progressive motility, too. Most of the spermatozoa distance and velocity parameters detected no significant change in comparison to control at the beginning of culture (Time 0), although the toxic effect became significant (P < 0.05) with the passage of culture time (Times 1 and 2 h) in all concentrations. Analysis of spermatozoa morphology detected significant (P < 0.05) alterations at higher concentrations. At higher concentrations acrosomal changes, head without flagellum/separated flagellum, broken flagellum and other abnormalities were significantly higher (P < 0.05), while knob-twisted flagellum and small heads differed significantly (P < 0.05) in comparison to control at all concentrations. In regards to flagellum torso, flagellum ball and retention of cytoplasmic drop statistically higher values (P < 0.05) were noted at the maxium experimental concentration only. Annexin analysis for detection of spermatozoa with disordered membranes revealed higher occurrence of positive spermatozoa in cadmium exposed groups. Annexin-positive reactions suggested alterations in anterior part of head (acrosome) and in flagellum (mitochondrial segment) of spermatozoa. This paper underlines that cadmium is highly toxic for rabbit spermatozoa, as visualized by the toxic effects on parameters of spermatozoa motility, morphology and membrane integrity. The toxic effect is more drastic at higher concentrations. This study also indicates that cadmium requires a minimum one hour incubation time to exert its deletorious effects on various parameters of spermatozoa, particularly at low concentrations.

Effects of advanced selection methods on sperm quality and ART outcome.

PubMed

Yetunde, I; Vasiliki, M

2013-10-01

In assisted reproductive technology (ART), the role of spermatozoa has evolved over the years. In the past, early methods of selecting sperm for ART only focused on selecting motile and morphologically normal appearing sperm. It has become evident that these methods are inefficient in identifying the most suitable sperm for fertilization. Novel methods have thus been created to identify highly motile, morphologically normal, viable non-apoptotic spermatozoa with intact membranes and high DNA integrity for use in ART. These advanced methods of selection utilize our knowledge of unique characteristics of sperm, such as sperm surface charge, the presence of hyaluronic acid binding sites on sperm, sperm ultramorphology, markers of apoptosis and zona pellucida binding on sperm. These methods have shown potential promise in improving ART outcomes. Future developments may include Raman spectroscopy, confocal light absorption and scattering spectroscopic microscopy, and polarization microscopy. While these novel techniques have potential, they come with a cost burden and further studies are required to demonstrate their impact on ART outcomes. Furthermore, clinicians and human reproductive scientists need to continue to gather knowledge about human fertilization and determine the most physiological methods of sperm selection.

Morphology and ultrastructure of Brachymystax lenok tsinlingensis spermatozoa by scanning and transmission electron microscopy.

PubMed

Guo, Wei; Shao, Jian; Li, Ping; Wu, Jinming; Wei, Qiwei

2016-08-01

This study was conducted to investigate Brachymystax lenok tsinlingensis spermatozoa cell morphology and ultrastructure through scanning and transmission electron microscopy. Findings revealed that the spermatozoa can be differentiated into three major parts: a spherical head without an acrosome, a short mid-piece, and a long, cylindrical flagellum. The mean length of the spermatozoa was 36.11±2.84μm, with a spherical head length of 2.78±0.31μm. The mean anterior and posterior head widths were 2.20±0.42μm and 2.55±0.53μm, respectively. The nuclear fossa was positioned at the base of the nucleus that contained the anterior portion of flagellum and a centriolar complex (proximal and distal centrioles). The short mid-piece was located laterally to the nucleus and possessed just one spherical mitochondrion with a mean diameter of 0.65±0.14μm. The spermatozoa flagellum was long and cylindrical, and could be separated into two parts: a long main-piece and a short end-piece. The main piece of the flagellum had short irregular side-fins. The axoneme composed the typical '9+2' microtubular doublet structure and was enclosed by the cell membran e. This study confirmed that B. lenok tsinlingensis spermatozoa can be categorized as teleostean "Type I" spermatozoa; 'primitive' or 'ect-aquasperm type' spermatozoa. To the best of the authers knowledge, this was the first study conducted on the morphology and ultrastructure of B. lenok tsinlingensis spermatozoa. Copyright © 2016 Elsevier Ltd. All rights reserved.

Efficacy of a sperm-selection chamber in terms of morphology, aneuploidy and DNA packaging.

PubMed

Seiringer, M; Maurer, M; Shebl, O; Dreier, K; Tews, G; Ziehr, S; Schappacher-Tilp, G; Petek, E; Ebner, T

2013-07-01

Since most current techniques analysing spermatozoa will inevitably exclude these gametes from further use, attempts have been made to enrich semen samples with physiological spermatozoa with good prognosis using special sperm-processing methods. A particular sperm-selection chamber, called the Zech-selector, was found to be effective in completely eliminating spermatozoa with DNA strand breaks. The aim of this study was to further analyse the subgroup of spermatozoa accumulated using the Zech-selector. In detail, the potential of the chamber to select for proper sperm morphology, DNA status and chromatin condensation was tested. Two samples, native and processed semen, of 53 patients were analysed for sperm morphology (×1000, ×6300), DNA packaging (fragmentation, chromatin condensation) and chromosomal status (X, Y, 18). Migration time (the time needed for proper sperm accumulation) was significantly correlated to fast progressive motility (P=0.002). The present sperm-processing method was highly successful with respect to all parameters analysed (P<0.001). In particular, spermatozoa showing numeric (17.4% of patients without aneuploidy) or structural chromosomal abnormalities (90% of patients without strand-breaks) were separated most effectively. To summarize, further evidence is provided that separating spermatozoa without exposure to centrifugation stress results in a population of highly physiological spermatozoa. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

DNA fragmentation of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome.

PubMed

Avendaño, Conrado; Franchi, Anahí; Duran, Hakan; Oehninger, Sergio

2010-07-01

To evaluate DNA fragmentation in morphologically normal sperm recovered from the same sample used for intracytoplasmic sperm injection (ICSI) and to correlate DNA damage with embryo quality and pregnancy outcome. Prospective study. Academic center. 36 infertile men participating in the ICSI program. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling (TUNEL) assay and morphologic assessment by phase contrast. Simultaneous assessment of sperm morphology and DNA fragmentation by TUNEL assay was performed in the same cell, then the percentage of normal sperm with fragmented DNA (normal SFD) was correlated with embryo quality and pregnancy outcomes. A highly statistically significant negative correlation was found between the percentage of normal SFD and embryo quality. This association was confirmed for the transferred embryos and for the total embryo cohort. The receiver operating characteristics curve analysis demonstrated that the percentage of normal SFD and embryo quality were statistically significant predictors of pregnancy. When the percentage of normal SFD was

Effects on boar semen quality after infection with porcine reproductive and respiratory syndrome virus: a case report

PubMed Central

2013-01-01

The effect of porcine reproductive and respiratory syndrome virus (PRRSV) on semen quality was examined in a group of 11 spontaneously infected boars in a commercial boar stud. Semen samples were collected 4 weeks prior to 4 weeks post-infection (wpi). Infection with PRRSV of the European genotype subtype 1 (EU-1) was verified by specific quantitative real-time polymerase chain reaction (RT-PCR) in 36% of the serum samples. All boars seroconverted before 4 wpi and remained in normal condition throughout the study. Comparison of the percentage of morphologically intact spermatozoa revealed an increase of acrosome-defective spermatozoa (P = 0.012) between −4 and 4 wpi. Significant deleterious effects on semen quality were detected for membrane integrity when semen had been stored for 2 days after sampling. Analysis of sperm subpopulations in a thermoresistance test on day 7 after sampling revealed alterations in the percentage of circular, progressively motile spermatozoa (P = 0.013), in the percentage of non-linear, progressively motile spermatozoa (P = 0.01), and on the amplitude of lateral sperm head displacement (P = 0.047). There was no difference in the incidence of mitochondrially active spermatozoa (P = 0.075). Investigation of routine production data between pre- and post-infection status showed no differences on ejaculate volume (P = 0.417), sperm concentration (P = 0.788), and percentage of motile spermatozoa (P = 0.321). This case report provides insights into a potential control strategy for PRRSV outbreaks in boar studs. PMID:23442207

Correlation between sperm DNA fragmentation index and CMA3 positive spermatozoa in globozoospermic patients.

PubMed

Hosseinifar, H; Yazdanikhah, S; Modarresi, T; Totonchi, M; Sadighi Gilani, M A; Sabbaghian, M

2015-05-01

The absence of the acrosome causes the situation which is called globozoospermia. There are a few studies, mostly as case reports, about correlation between levels of sperm DNA damage in patients with total round-headed spermatozoa. We investigated this correlation as well as CMA3 positive spermatozoa in 20 globozoospermic men (with more than 90% round-headed spermatozoa) attending to Royan Institute. Semen samples divided into three parts to semen analysis, to measure DNA fragmentation index (DFI) using sperm chromatin structure assay (SCSA) and to detect CMA3(+) sperm cells by chromomycin A3 staining and fluorescent microscopy. Our results showed that there were significant differences in sperm concentration, total sperm motility, and normal morphology between patients and controls group (p < 0.001). Moreover, the average of DFI and CMA3 positive spermatozoa in patients group significantly increases compared with control group (p < 0.001). A significant correlation between DFI and CMA3(+) in total population was also detected in patients group (r = 0.45, p = 0.046). To our knowledge, this is the largest study about correlation between DNA damage levels and CMA3 positive spermatozoa with round head sperm cells in total globozoospermic men. It seems that the increase in DNA damage may be because of defective sperm DNA compaction, as we detected CMA3 positive sperm cells in these patients. © 2015 American Society of Andrology and European Academy of Andrology.

Sperm with large nuclear vacuoles and semen quality in the evaluation of male infertility.

PubMed

Komiya, Akira; Watanabe, Akihiko; Kawauchi, Yoko; Fuse, Hideki

2013-02-01

This study compared the sperm nuclear vacuoles and semen quality in the evaluation of male infertility. One hundred and forty-two semen samples were obtained from patients who visited the Male Infertility Clinic at Toyama University Hospital. Semen samples were evaluated by conventional semen analyses and the Sperm Motility Analysis System (SMAS). In addition, spermatozoa were analyzed at 3,700-6,150x magnification on an inverted microscope equipped with DIC/Nomarski differential interference contrast optics. A large nuclear vacuole (LNV) was defined as one or more vacuoles with the maximum diameter showing > 50% width of the sperm head. The percentage of spermatozoa with LNV (% LNV) was calculated for each sample. Correlations between the % LNV and parameters in SMAS and conventional semen analyses were analyzed. Processed motile spermatozoa from each sample were evaluated. The mean age of patients was 35 years old. Semen volume was 2.9 ± 1.6mL (0.1-11.0; mean ± standard deviation, minimum-maximum), sperm count was 39.3 ± 54.9 (x10(6)/mL, 0.01-262.0), sperm motility was 25.1 ± 17.8% (0-76.0), and normal sperm morphology was 10.3 ± 10.1% (0-49.0). After motile spermatozoa selection, we could evaluate % LNV in 125 ejaculates (88.0%) and at least one spermatozoon with LNV was observed in 118 ejaculates (94.4%). The percentage of spermatozoa with LNV was 28.0 ± 22.4% (0-100) and % LNV increased significantly when semen quality decreased. The correlation between the % LNV and the semen parameters was weak to moderate; correlation coefficients were -0.3577 in sperm count (p < 0.0001), -0.2368 in sperm motility (p = 0.0084), -0.2769 in motile sperm count (p = 0.019), -0.2419 in total motile sperm count (p = 0.0070), and -0.1676 in normal sperm morphology (p = 0.0639). The % LNV did not show a significant correlation with the SMAS parameters except for weak correlation to beat/cross frequency (r = -0.2414, p = 0.0071). The percentage of spermatozoa with LNV did not have a strong correlation with parameters in conventional semen analysis and SMAS in the patients with male infertility; however, a certain level of negative influence of LNV to sperm quality cannot be excluded.

Fatty acid composition of spermatozoa is associated with BMI and with semen quality.

PubMed

Andersen, J M; Rønning, P O; Herning, H; Bekken, S D; Haugen, T B; Witczak, O

2016-09-01

High body mass index (BMI) is negatively associated with semen quality. In addition, the composition of fatty acids of spermatozoa has been shown to be important for their function. The aim of the study was to examine the association between BMI and the composition of spermatozoa fatty acids in men spanning a broad BMI range. We also analysed the relation between fatty acid composition of spermatozoa and semen characteristics, and the relationship between serum fatty acids and spermatozoa fatty acids. One hundred forty-four men with unknown fertility status were recruited from the general population, from couples with identified female infertility and from morbid obesity centres. Standard semen analysis (WHO) and sperm DNA integrity (DFI) analysis were performed. Fatty acid compositions were assessed by gas chromatography. When adjusted for possible confounders, BMI was negatively associated with levels of sperm docosahexaenoic acid (DHA) (p < 0.001) and palmitic acid (p < 0.001). The amount of sperm DHA correlated positively with total sperm count (r = 0.482), sperm concentration (r = 0.469), sperm vitality (r = 0.354), progressive sperm motility (r = 0.431) and normal sperm morphology (r = 0.265). A negative association was seen between DHA levels and DNA fragmentation index (r = -0.247). Levels of spermatozoa palmitic acid correlated positively with total sperm count (r = 0.227), while levels of linoleic acid correlated negatively (r = -0.254). When adjusted for possible confounders, only the levels of arachidonic acid showed positive correlation between spermatozoa and serum phospholipids (r = 0.262). Changes in the fatty acid composition of spermatozoa could be one of the mechanisms underlying the negative association between BMI and semen quality. The relationship between fatty acids of spermatozoa and serum phospholipids was minor, which indicates that BMI affects fatty acid composition of spermatozoa through regulation of fatty acid metabolism in the testis. The role of dietary intake of fatty acids on the spermatozoa fatty acid composition remains to be elucidated. © 2016 American Society of Andrology and European Academy of Andrology.

Pregnancy rates after artificial insemination with cooled stallion spermatozoa either with or without single layer centrifugation.

PubMed

Morrell, J M; Richter, J; Martinsson, G; Stuhtmann, G; Hoogewijs, M; Roels, K; Dalin, A-M

2014-11-01

A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from "normal" ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sperm quality seen in SLC-selected sperm samples compared with uncentrifuged controls in laboratory tests are reflected in an increased pregnancy rate after artificial insemination. In a multicentre study, SLC-selected sperm samples and uncentrifuged controls from eight stallions were inseminated into approximately 10 mares per treatment per stallion. Ultrasound examination was carried out approximately 16 days after insemination to detect an embryonic vesicle. The pregnancy rates per cycle were 45% for controls and 69% for SLC-selected sperm samples, which is statistically significant (P < 0.0018). Thus, the improvement in sperm quality reported previously for SLC-selected sperm samples is associated with an increase in pregnancy rate, even for ejaculates from stallions with no known fertility problem. Copyright © 2014 Elsevier Inc. All rights reserved.

Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey mouse lemur, Microcebus murinus.

PubMed

Aslam, H; Schneiders, A; Perret, M; Weinbauer, G F; Hodges, J K

2002-02-01

Germ cell production and organization of the testicular epithelium in a prosimian species, the grey mouse lemur, Microcebus murinus, was investigated to extend knowledge of comparative primate spermatogenesis. In addition, semen samples collected from adult male lemurs (body weight 53-92 g; n = 16) by rectal probe electroejaculation were evaluated using computer-assisted morphometric and kinematic analysis of spermatozoa. Epididymidal spermatozoa were collected from six animals after hemicastration; the testes were weighed and prepared for stereological analysis and flow cytometry. The relative testis mass (as a percentage of body weight) ranged between 1.17 and 5.6%. Twelve stages of testicular seminiferous epithelium as described for macaques were applied and only a single stage was observed in most of the seminiferous tubule cross-sections. On average (mean SD), a single testis contained 1870 +/- 829 x 10(6) germ cells and 35 +/- 12 x 10(6) Sertoli cells. Germ cell ratios (preleptotene:type B spermatogonia = 2, round spermatid:pachytene = 3; elongated spermatid:round spermatids = 1) indicated high spermatogenic efficacy. Sperm head dimensions and tail lengths of the ejaculated and epididymidal spermatozoa were similar. Percentages of defects (neck/mid-piece and tail) were low ( 10%) and similar for ejaculated and epididymidal spermatozoa. Spermatozoa were highly motile, characterized by extensive lateral head displacement, but relatively low progressive motility. In conclusion, the grey mouse lemur has unusually large testes with a highly efficient spermatogenic process and large sperm output. These features, together with the high proportion of morphologically normal and highly motile spermatozoa in the ejaculates, indicate that Microcebus murinus is a species in which sperm competition after ejaculation is likely to occur. The predominantly single spermatogenic stage system seems to be an ancestral feature among primates.

An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet

PubMed Central

2012-01-01

Recent studies have shown cytoplasmic droplets to be normal morphological occurrences in human male spermatozoa. When the cytoplasm around the sperm midpiece is present in large amounts, however, pathological effects may transpire. The cytoplasmic droplet then becomes known as excess residual cytoplasm, which can impair overall sperm function and produce higher levels of reactive oxygen species, potentially leading to male infertility. Though the distinction between cytoplasmic droplets and excess residual cytoplasm has been made, some studies fail to recognize the difference and incorrectly label the latter as a cytoplasmic droplet. This review attempts to clarify excess residual cytoplasm’s effect on fertility, examine the enzymes responsible, and suggest tests and possible treatment options for those affected by this defect. PMID:23159014

Comparative analyses of semen and endocrine characteristics of free-living versus captive jaguars (Panthera onca).

PubMed

Morato, R G; Conforti, V A; Azevedo, F C; Jacomo, A T; Silveira, L; Sana, D; Nunes, A L; Guimarães, M A; Barnabe, R C

2001-11-01

Semen and blood samples were obtained from free-living (n = 6) and captive (n = 8) jaguars (Panthera onca) to compare reproductive characteristics between the two populations. Semen samples were analysed for volume (ml), percentage of motile spermatozoa, rate of forward progression (0-5), concentration (10(6) ml(-1)), total sperm count (10(6)) and sperm morphology. Serum testosterone concentration was determined by radioimmunoassay. Although ejaculate volume was greater in captive jaguars (n = 47 samples) than in free-living jaguars (n = 7 samples) (P < 0.05), the free-living jaguars produced more total spermatozoa (59.3 +/- 12.8 versus 152.0 +/- 88.0 x 10(6), respectively; not significant) with better viability and forward progression (2.8 +/- 0.1 versus 3.5 +/- 0.2, respectively; P < 0.05) and more spermatozoa with normal morphology (73.5 +/- 3.9 versus 5.0 +/- 1.1%, respectively; P < 0.05). Serum testosterone concentrations were similar for captive and free-living male jaguars (3.1 +/- 0.7 and 2.1 +/- 0.8 ng ml(-1), respectively). In summary, the data showed that semen may be collected successfully from free-living jaguars and evaluated under field conditions to establish normative reproductive values in this species. The results also indicate that jaguars maintained in zoos show inferior seminal characteristics compared with free-living animals.

The use of semen evaluation and assisted reproduction in Spix's macaws in terms of species conservation.

PubMed

Fischer, Dominik; Neumann, Daniel; Purchase, Cromwell; Bouts, Tim; Meinecke-Tillmann, Sabine; Wehrend, Axel; Lierz, Michael

2014-01-01

The Spix's macaw (Cyanopsitta spixii) is the rarest parrot on earth. The remaining captive population consists of 79 individuals. Captive propagation is ongoing to increase the number of individuals for future reintroduction back into the wild. Unfortunately, from 2004 to 2012, only 33 chicks hatched from 331 eggs. Semen evaluation and assisted reproduction might help to overcome this problem. Therefore, a recently developed electro-stimulated semen collection technique was used in Spix's macaws. Semen collection was successful in 39 of 78 attempts in 10 out of 17 males. Examination of the semen included evaluation of volume, color, consistency, contaminations and pH, as well as determination of motility, viability, morphology, concentration, and total count of spermatozoa. The median volume of semen samples was 5.6 µl. On average, 34.7 ± 21.9% (median 30%) of the sperm were motile and 23.1 ± 22.1% (median 16.5%) were progressively motile. In addition to spermatozoa, round cells were detected in the samples. Median sperm concentration was 15,500/µl (range 500-97,500/µl) and median viability was 50% (range 5-87%). Morphological examination revealed in 26.5% normal spermatozoa, high numbers of malformations of the head (50.2%) and tail region (20.5%), with 29% of all sperm showing multiple abnormalities. Artificial insemination was performed in three females; two eggs laid after artificial insemination had spermatozoa present on the perivitelline layer, suggesting the possible success of the insemination technique. Although no fertilization could be demonstrated, these preliminary results are promising, as they indicate that assisted reproduction might be a tool for species conservation in the Spix's macaw. © 2014 Wiley Periodicals Inc.

Effects of millimeter-wave electromagnetic exposure on the morphology and function of human cryopreserved spermatozoa.

PubMed

Volkova, N A; Pavlovich, E V; Gapon, A A; Nikolov, O T

2014-09-01

Exposure of human cryopreserved spermatozoa to millimeter-wave electromagnetic radiation of 0.03 mW/cm2 density for 5 min in normozoospermia and for 15 min in asthenozoospermia lead to increase of the fraction of mobile spermatozoa without impairing the membrane integrity and nuclear chromatin status and without apoptosis generation.

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.

Predictive value of sperm morphology and progressively motile sperm count for pregnancy outcomes in intrauterine insemination.

PubMed

Lemmens, Louise; Kos, Snjezana; Beijer, Cornelis; Brinkman, Jacoline W; van der Horst, Frans A L; van den Hoven, Leonie; Kieslinger, Dorit C; van Trooyen-van Vrouwerff, Netty J; Wolthuis, Albert; Hendriks, Jan C M; Wetzels, Alex M M

2016-06-01

To investigate the value of sperm parameters to predict an ongoing pregnancy outcome in couples treated with intrauterine insemination (IUI), during a methodologically stable period of time. Retrospective, observational study with logistic regression analyses. University hospital. A total of 1,166 couples visiting the fertility laboratory for their first IUI episode, including 4,251 IUI cycles. None. Sperm morphology, total progressively motile sperm count (TPMSC), and number of inseminated progressively motile spermatozoa (NIPMS); odds ratios (ORs) of the sperm parameters after the first IUI cycle and the first finished IUI episode; discriminatory accuracy of the multivariable model. None of the sperm parameters was of predictive value for pregnancy after the first IUI cycle. In the first finished IUI episode, a positive relationship was found for ≤4% of morphologically normal spermatozoa (OR 1.39) and a moderate NIPMS (5-10 million; OR 1.73). Low NIPMS showed a negative relation (≤1 million; OR 0.42). The TPMSC had no predictive value. The multivariable model (i.e., sperm morphology, NIPMS, female age, male age, and the number of cycles in the episode) had a moderate discriminatory accuracy (area under the curve 0.73). Intrauterine insemination is especially relevant for couples with moderate male factor infertility (sperm morphology ≤4%, NIPMS 5-10 million). In the multivariable model, however, the predictive power of these sperm parameters is rather low. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Efficacy of evaluation of rooster sperm morphology using different staining methods.

PubMed

Lukaszewicz, E; Jerysz, A; Partyka, A; Siudzińska, A

2008-12-01

This work focused on inexpensive methods of evaluation fowl sperm morphology, based on eosin-nigrosin smears, which can determine disorders in spermatogenesis and can be recommended for evaluating the fertilising potency and selecting males in flocks reproduced by artificial insemination. Four fowl breeds (Black Minorca, Italian Partridge, Forwerk and Greenleg Partridge) were used to determine the efficacy of sperm morphology evaluation using four eosin-nigrosin staining methods (according to Blom, Bakst and Cecil, Morisson, Jaśkowski) and three examiners of different experience (high, medium, novice). There were significant (P< or = 0.01) differences in sperm morphology between Blom's staining method and those of Bakst and Cecil, Morisson or Jaśkowski, irrespective of fowl breed and examiners experience. Blom stain caused sperm head swelling and showed a drastic reduction in the proportion of live spermatozoa with normal morphology. The staining method had a greater influence on sperm morphology evaluation than the experience of the examiners.

Automatic detection of spermatozoa for laser capture microdissection.

PubMed

Vandewoestyne, Mado; Van Hoofstat, David; Van Nieuwerburgh, Filip; Deforce, Dieter

2009-03-01

In sexual assault crimes, differential extraction of spermatozoa from vaginal swab smears is often ineffective, especially when only a few spermatozoa are present in an overwhelming amount of epithelial cells. Laser capture microdissection (LCM) enables the precise separation of spermatozoa and epithelial cells. However, standard sperm-staining techniques are non-specific and rely on sperm morphology for identification. Moreover, manual screening of the microscope slides is time-consuming and labor-intensive. Here, we describe an automated screening method to detect spermatozoa stained with Sperm HY-LITER. Different ratios of spermatozoa and epithelial cells were used to assess the automatic detection method. In addition, real postcoital samples were also screened. Detected spermatozoa were isolated using LCM and DNA analysis was performed. Robust DNA profiles without allelic dropout could be obtained from as little as 30 spermatozoa recovered from postcoital samples, showing that the staining had no significant influence on DNA recovery.

Relationship between phospholipase C-zeta, semen parameters, and chromatin status.

PubMed

Tavalaee, Marziyeh; Kiani-Esfahani, Abbas; Nasr-Esfahani, Mohammad H

2017-08-01

The need for additional tests to complement basic sperm analysis in clinics is well appreciated. In this regard, a number of tests such as sperm DNA integrity test as a tool in diagnosis and treatment of infertility are suggested. But recent studies have focused on main sperm factors involved in oocyte activation such as phospholipase C-zeta (PLCζ) that initiate intracellular Ca 2+ signaling and embryogenesis. Therefore, this study aimed to investigate the relationship between PLCζ, basic semen parameters, sperm DNA fragmentation (SDF), and protamine deficiency in men with normal (n=32) and abnormal (n=23) semen parameters. Unlike SDF and protamine deficiency, as negative factors related to fertility, the mean value of PLCζ as positive factor related to infertility was significantly lower in men with abnormal semen parameters compared to men with normal semen parameters. Significant correlations were also observed between sperm concentration, motility, and abnormal morphology with the percentage of PLCζ positive spermatozoa. In addition, logistic regression analysis revealed that sperm morphology is more predictive than sperm motility and concentration for PLCζ presence. In addition, a statistically significant negative relationship was observed between the percentage of PLCζ positive spermatozoa and SDF. These findings suggested during ICSI, selection of sperm based on morphology has a profound effect on its ability to induce oocyte activation based on the likelihood of PLCζ expression. Therefore, assessment of PLCζ as an index for fertilization potential of a semen sample in men with severe teratozoospermia may define individuals who are candidates for artificial oocyte activation (AOA) and may avoid failed fertilization post ICSI.

Oxidative phosphorylation is essential for felid sperm function, but is substantially lower in cheetah (Acinonyx jubatus) compared to domestic cat (Felis catus) ejaculate.

PubMed

Terrell, Kimberly A; Wildt, David E; Anthony, Nicola M; Bavister, Barry D; Leibo, S P; Penfold, Linda M; Marker, Laurie L; Crosier, Adrienne E

2011-09-01

Compared with the normospermic domestic cat, sperm metabolic function is compromised in the teratospermic cat and cheetah, but the pathway(s) involved in this deficiency are unknown. Glycolysis is essential for sperm motility, yet it appears to function normally in spermatozoa of either species regardless of structural morphology. We conducted a comparative study to further understand the mechanisms of energy production in felid spermatozoa, with the hypothesis that oxidative phosphorylation is required for normal sperm function and is impaired in teratospermic ejaculates. Electroejaculates from both species were stained with MitoTracker to quantify mitochondrial membrane potential (MMP) or were incubated to assess changes in sperm function (motility, acrosomal integrity, and lactate production) after mitochondrial inhibition with myxothiazol. Sperm midpiece dimensions also were quantified. Sperm mitochondrial fluorescence (directly proportional to MMP) was ~95% lower in the cheetah compared with the normospermic and teratospermic cat, despite the cheetah having a 10% longer midpiece. In both species, MMP was increased 5-fold in spermatozoa with retained cytoplasm compared with structurally normal cells. Inhibition of oxidative phosphorylation impaired sperm function in both species, but a 100-fold higher inhibitor concentration was required in the cat compared with the cheetah. Collectively, findings revealed that oxidative phosphorylation was required for sperm function in the domestic cat and cheetah. This pathway of energy production appeared markedly less active in the cheetah, indicating a species-specific vulnerability to mitochondrial dysfunction. The unexpected, cross-species linkage between retained cytoplasmic droplets and elevated MMP may reflect increased concentrations of metabolic enzymes or substrates in these structures.

The influence of macro- and microelements in seminal plasma on diluted boar sperm quality.

PubMed

Pipan, Maja Zakošek; Mrkun, Janko; Strajn, Breda Jakovac; Vrtač, Katarina Pavšič; Kos, Janko; Pišlar, Anja; Zrimšek, Petra

2017-02-10

Growing evidence indicates that macro- and microelements in the seminal plasma of humans and various domestic animals are of great importance due to their roles in sperm metabolism, function, survival and oxidative stress. In the present study, we therefore determined the concentrations of macro- and microelements in fresh boar seminal plasma and their relation to sperm quality parameters after 3 days of liquid storage was assessed. Twenty ejaculates from eight boars were collected, and semen volume, concentration, sperm motility, morphology, tail membrane integrity, plasma membrane permeability, mitochondrial membrane potential and DNA fragmentation were determined on the day of collection (day 0) and day 3 (72 h) of storage at 15-17 °C. Seminal plasma was separated and the concentrations of macroelements (Na, K, Ca, and Mg) and microelements (Cu, Fe, Zn and Se) were determined. After 3 days of storage Se levels correlated significantly with sperm motility, progressive motility and morphology, all of which are routinely used for semen evaluation. On day 3, Se levels also correlated with tail membrane integrity, viability and intact DNA (P < 0.05). The correlation coefficients showed that mitochondrial function was better preserved at higher levels of Zn, while higher levels of Cu decreased mitochondrial function, but led to the better preservation of DNA. It was also evident that higher levels of Fe were associated with higher proportions of live spermatozoa and of spermatozoa with normal morphology after 3 days of storage (P < 0.05), while higher levels of Ca and Mg in fresh seminal plasma were associated with lower percentages of progressive motile spermatozoa and with a decreased proportion of spermatozoa with intact DNA (P < 0.05). Multivariate analysis including microelements showed that Se significantly affected sperm quality parameters, mentioned above, after 3 days of storage. Macro- and microelements were associated with boar sperm quality and may be important biomarkers of boar sperm quality after liquid storage. Our results demonstrate that the evaluation of Se in fresh boar seminal plasma can serve as an additional tool in predicting sperm quality after storage.

Dry Preservation of Spermatozoa: Considerations for Different Species.

PubMed

Patrick, Jennifer; Comizzoli, Pierre; Elliott, Gloria

2017-04-01

The current gold standard for sperm preservation is storage at cryogenic temperatures. Dry preservation is an attractive alternative, eliminating the need for ultralow temperatures, reducing storage maintenance costs, and providing logistical flexibility for shipping. Many seeds and anhydrobiotic organisms are able to survive extended periods in a dry state through the accumulation of intracellular sugars and other osmolytes and are capable of returning to normal physiology postrehydration. Using techniques inspired by nature's adaptations, attempts have been made to dehydrate and dry preserve spermatozoa from a variety of species. Most of the anhydrous preservation research performed to date has focused on mouse spermatozoa, with only a small number of studies in nonrodent mammalian species. There is a significant difference between sperm function in rodent and nonrodent mammalian species with respect to centrosomal inheritance. Studies focused on reproductive technologies have demonstrated that in nonrodent species, the centrosome must be preserved to maintain sperm function as the spermatozoon centrosome contributes the dominant nucleating seed, consisting of the proximal centriole surrounded by pericentriolar components, onto which the oocyte's centrosomal material is assembled. Preservation techniques used for mouse sperm may therefore not necessarily be applicable to nonrodent spermatozoa. The range of technologies used to dehydrate sperm and the effect of processing and storage conditions on fertilization and embryogenesis using dried sperm are reviewed in the context of reproductive physiology and cellular morphology in different species.

Dry Preservation of Spermatozoa: Considerations for Different Species

PubMed Central

Patrick, Jennifer; Comizzoli, Pierre

2017-01-01

The current gold standard for sperm preservation is storage at cryogenic temperatures. Dry preservation is an attractive alternative, eliminating the need for ultralow temperatures, reducing storage maintenance costs, and providing logistical flexibility for shipping. Many seeds and anhydrobiotic organisms are able to survive extended periods in a dry state through the accumulation of intracellular sugars and other osmolytes and are capable of returning to normal physiology postrehydration. Using techniques inspired by nature's adaptations, attempts have been made to dehydrate and dry preserve spermatozoa from a variety of species. Most of the anhydrous preservation research performed to date has focused on mouse spermatozoa, with only a small number of studies in nonrodent mammalian species. There is a significant difference between sperm function in rodent and nonrodent mammalian species with respect to centrosomal inheritance. Studies focused on reproductive technologies have demonstrated that in nonrodent species, the centrosome must be preserved to maintain sperm function as the spermatozoon centrosome contributes the dominant nucleating seed, consisting of the proximal centriole surrounded by pericentriolar components, onto which the oocyte's centrosomal material is assembled. Preservation techniques used for mouse sperm may therefore not necessarily be applicable to nonrodent spermatozoa. The range of technologies used to dehydrate sperm and the effect of processing and storage conditions on fertilization and embryogenesis using dried sperm are reviewed in the context of reproductive physiology and cellular morphology in different species. PMID:28398834

[Effect of spermatozoa from different sources on normal fertilization of oocytes and embryo quality and development in intracytoplasmic sperm injection cycles].

PubMed

Xie, Duo; Qiu, Zhuolin; Luo, Chen; Chu, Qingjun; Quan, Song

2014-06-01

To evaluate the impact of spermatozoa from different sources on normal fertilization of oocytes, embryo quality and embryo developmental potential in intracytoplasmic sperm injection (ICSI) cycles. A retrospective analysis was conducted among 197 patients undergoing ICSI cycles in our center. The patients were classified into 3 groups according to the sources of semen, namely ejaculated spermatozoa group (n=102), percutaneous epididymal sperm aspiration (PESA) group (n=68), and testicular sperm aspiration (TESA) group (n=27). The ejaculated spermatozoa group was further classified into oligoasthenoteratozoospermia (n=67) and cryptozoospermia (n=35) subgroups. The normal fertilization, high-quality embryo, implantation and clinical pregnancy rates were compared among the groups; the rate of high-quality blastocyst formation in in-vitro culture of non-top quality embryos was also observed. The patients with PESA showed significantly higher normal fertilization rate (75.6%) than those in oligoasthenoteratozoospermia (64.8%), cryptozoospermia (62.1%), and TESA (61.6%) groups (P<0.05). No significant differences were found in the high-quality embryo, implantation, and clinical pregnancy rates among the groups (P>0.05). The rate of high-quality blastocyst formation in the in-vitro culture of non-top quality embryos was also comparable among the groups (P>0.05). Although spermatozoa obtained with by PESA is associated with a higher normal fertilization rate, the sources of spermatozoa do not significantly affect the embryonic quality and developmental potential in ICSI cycles.

Influence of sexual stimulation on sperm parameters in semen samples collected via masturbation from normozoospermic men or cryptozoospermic men participating in an assisted reproduction programme.

PubMed

Yamamoto, Y; Sofikitis, N; Mio, Y; Miyagawa, I

2000-05-01

To evaluate the influence of sexual stimulation via sexually stimulating videotaped visual images (VIM) on sperm function, two semen samples were collected from each of 19 normozoospermic men via masturbation with VIM. Two additional samples were collected from each man via masturbation without VIM. The volume of seminal plasma, total sperm count, sperm motility, percentage of morphologically normal spermatozoa, outcome of hypo-osmotic swelling test and zona-free hamster oocyte sperm penetration assay, and markers of the secretory function of prostate were significantly larger in semen samples collected via masturbation with VIM than masturbation without VIM. The improved sperm parameters in the samples collected via masturbation with VIM may reflect an enhanced prostatic secretory function and increased loading of the vas deferens at that time. In a similar protocol, two semen samples were collected via masturbation with VIM from each of 22 non-obstructed azoospermic men. Semen samples from these men had been occasionally positive in the past for a very small number of spermatozoa (cryptozoospermic men). Two additional samples were collected from each cryptozoospermic man via masturbation without VIM. The volume of seminal plasma, total sperm count, sperm motility, and a marker of the secretory function of prostate were significantly larger in semen samples collected via masturbation with VIM. Fourteen out of the 22 men were negative for spermatozoa in both samples collected via masturbation without VIM. These men demonstrated spermatozoa in both samples collected via masturbation with VIM. Six men with immotile spermatozoa in both samples collected via masturbation without VIM exposed motile spermatozoa in both samples collected via masturbation with VIM. High sexual stimulation during masturbation with VIM results in recovery of spermatozoa of greater fertilizing potential both in normozoospermic and cryptozoospermic men. The appearance of spermatozoa after masturbation with VIM in the vast majority of cryptozoospermic men is of clinical significance in programmes applying intracytoplasmic sperm injections for the management of severe male infertility and obviates the need for testicular biopsy.

The effect of Eurycoma longifolia on sperm quality of male rats.

PubMed

Chan, Kit-Lam; Low, Bin-Seng; Teh, Chin-Hoe; Das, Prashanta K

2009-10-01

The present study investigated the effects of a standardized methanol extract of E. longifolia Jack containing the major quassinoid constituents of 13alpha(21)-epoxyeurycomanone (1), eurycomanone (2), 13alpha,21-dihydroeurycomanone (3) and eurycomanol (4) on the epididymal spermatozoa profile of normal and Andrographis paniculata induced infertile rats. The standardized MeOH extract at doses of 50, 100 and 200 mg/kg, the EtOAc fraction (70 mg/kg), and standardized MeOH extract at 200 mg/kg co-administered with the EtOAc fraction of A. paniculata at 70 mg/kg were each given orally to male Sprague-Dawley albino rats for 48 consecutive days. The spermatozoa count, morphology, motility, plasma testosterone level and Leydig cell count of the animals were statistically analyzed by ANOVA with a post-hoc Tukey HSD test. The results showed that the sperm count of rats given the standardized MeOH extract alone at doses of 50, 100 and 200 mg/kg were increased by 78.9, 94.3 and 99.2%, respectively when compared with that of control (p < 0.01). The low count, poor motility and abnormal morphology of the spermatozoa induced by the A. paniculata fraction were significantly reversed by the standardized MeOH extract of E. longifolia (p < 0.001). The plasma testosterone level of the rats treated with the standardized MeOH extract at 200 mg/kg was significantly increased (p < 0.01) when compared with that of the control and infertile animals. The spermatocytes in the seminiferous tubules and the Leydig cells appeared normal. Testosterone level was significantly higher in the testes (p < 0.01) than in the plasma after 30 days of oral treatment with the standardized MeOH extract. Interestingly, eurycomanone (2) alone was detected in the rat testis homogenates by HPLC-UV and confirmed by LC/MS, and may have contributed towards the improvement of sperm quality. Thus, the plant may potentially be suitable for the management of male infertility.

The Usefulness of Selected Physicochemical Indices, Cell Membrane Integrity and Sperm Chromatin Structure in Assessments of Boar Semen Sensitivity

PubMed Central

Wysokińska, A.; Kondracki, S.; Iwanina, M.

2015-01-01

The present work describes experiments undertaken to evaluate the usefulness of selected physicochemical indices of semen, cell membrane integrity and sperm chromatin structure for the assessment of boar semen sensitivity to processes connected with pre-insemination procedures. The experiments were carried out on 30 boars: including 15 regarded as providers of sensitive semen and 15 regarded as providers of semen that is little sensitive to laboratory processing. The selection of boars for both groups was based on sperm morphology analyses, assuming secondary morphological change incidence in spermatozoa as the criterion. Two ejaculates were manually collected from each boar at an interval of 3 to 4 months. The following analyses were carried out for each ejaculate: sperm motility assessment, sperm pH measurement, sperm morphology assessment, sperm chromatin structure evaluation and cell membrane integrity assessment. The analyses were performed three times. Semen storage did not cause an increase in the incidence of secondary morphological changes in the group of boars considered to provide sperm of low sensitivity. On the other hand, with continued storage there was a marked increase in the incidence of spermatozoa with secondary morphological changes in the group of boars regarded as producing more sensitive semen. Ejaculates of group I boars evaluated directly after collection had an approximately 6% smaller share of spermatozoa with undamaged cell membranes than the ejaculates of boars in group II (p≤0.05). In the process of time the percentage of spermatozoa with undamaged cell membranes decreased. The sperm of group I boars was characterised with a lower sperm motility than the semen of group II boars. After 1 hour of storing diluted semen, the sperm motility of boars producing highly sensitive semen was already 4% lower (p≤0.05), and after 24 hours of storage it was 6.33% lower than that of the boars that produced semen with a low sensitivity. Factors that confirm the accuracy of insemination male selection can include a low rate of sperm motility decrease during the storage of diluted semen, low and contained incidence of secondary morphological changes in spermatozoa during semen storage and a high frequency of spermatozoa with undamaged cell membranes. PMID:26580438

Microfluidic separation of motile sperm with millilitre-scale sample capacity

NASA Astrophysics Data System (ADS)

Nosrati, Reza; Vollmer, Marion; Eamer, Lise; Zeidan, Krista; San Gabriel, Maria C.; Zini, Armand; Sinton, David

2012-11-01

Isolating motile from non-motile spermatozoa has been a challenge since the establishment of in vitro fertilization. Microfluidic approaches have been employed for this purpose, but current devices are limited by low sample volume. Here, we present a high-throughput microfluidic device that separates spermatozoa from one millilitre of raw semen sample based on the hydrodynamic characteristics of swimming sperm in a confined geometry. The device consists of two layers: an outer injection ring on top aligned with a network of radial microchannels at the bottom guiding motile sperm into an inner collection chamber. This approach (1) maximizes exposure of the sperm to the fluid channels, (2) maximizes surface area density (3) prevents fluid flow bias, and (4) employs a non-Newtonian viscoelastic medium consistent with the in vivo environment. Tests with human and bull spermatozoa indicate an increase in motile sperm concentration from 62.2% in raw semen to 99.2% in separated sample combined with a higher incidence of normal morphology. DNA integrity testing is currently underway. In conclusion, we present an effective one-step procedure to perform semen purification and separation on a millilitre-scale with clinically relevant numbers.

DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

PubMed

Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

2014-10-15

Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies. Copyright © 2014 Elsevier Inc. All rights reserved.

Sperm preparation for ART

PubMed Central

Henkel, Ralf R; Schill, Wolf-Bernhard

2003-01-01

The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm. PMID:14617368

Effects of artificial illumination on turkey sperm viability.

PubMed

Williams, C J; Siopes, T D

1985-12-01

The effects of direct exposure of turkey semen to artificial lighting on the quality of the spermatozoa were investigated. Undiluted (neat) and diluted semen were exposed to light or dark treatments for 4 or 6 hr while held at 5, 15, or 25 C. The percentage of normal, abnormal and dead sperm, and the percent fertility was determined after various light and dark treatments. Neat semen held in light at 5, 15, and 25 C, and dilute semen held at 5 and 25 C, contained significantly greater numbers of normal spermatozoa than semen exposed to the dark. A significant rise in the number of abnormal and dead spermatozoa was seen in treated samples held in the dark. Both neat and extended semen exposed to blue light (peak 450 nm) contained significantly greater numbers of normal spermatozoa and fewer abnormal spermatozoa than semen treated with red light (peak 650 nm) after both 4 and 6 hr of treatment. The fertilizing capacity of spermatozoa exposed to light was greater than that of spermatozoa exposed to dark. Artificial insemination of hens with semen exposed to light or darkness for 6 hr resulted in an initial percentage of fertile eggs of about 40% and 24% from the light and dark treated semen, respectively. It was concluded that light resulted in improved quality of turkey semen during a short-term holding period.

Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model.

PubMed

Patrick, Jennifer L; Elliott, Gloria D; Comizzoli, Pierre

2017-11-01

Characterizing the resilience of mammalian cells to non-physiological conditions is necessary to develop preservation and long-term storage strategies at low or ambient temperatures. Using the domestic cat model, the objective of the study was to characterize structural integrity (morphology and DNA damage) as well as functional properties (sperm aster formation and embryo formation after sperm injection) of spermatozoa after microwave-assisted drying to a moisture content compatible with storage in a glassy state at supra-zero temperatures. In Experiment 1, cat epididymal spermatozoa were porated with hemolysin and dried (using a commercial microwave oven set to 20% power) in the presence of trehalose for up to 50 min in a low humidity environment (11%) before measuring moisture content and sample temperature. In Experiment 2, morphology and DNA integrity were evaluated in sperm dried for up to 30 min (using the same method as above) versus fresh spermatozoa. In Experiment 3, the functionality of sperm dried for 30 min versus fresh sperm cells was evaluated after injection into oocytes based on sperm aster formation (5 h post-injection) and embryo development in vitro over 7 days. Moisture contents compatible with dry state storage were reached after 30 min of microwave-assisted drying. After rehydration, sperm morphology was not affected and the percentages of cells with damaged DNA (∼6.5%) was similar to the fresh controls. Sperm aster diameters appeared to be generally smaller for dried-rehydrated cells compared to the fresh controls. This observation was consistent with a lower proportion of blastocyst formation after injection with dried spermatozoa (6.5%) compared to fresh spermatozoa (15%). However, the blastocyst quality based on the total blastomere number was not affected by the sperm treatment. This is the first and encouraging report in any species so far demonstrating that spermatozoa can be dried using microwaves without causing irreversible damage to the cellular structure and function. Published by Elsevier Inc.

Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations.

PubMed

Celik-Ozenci, Ciler; Jakab, Attila; Kovacs, Tamas; Catalanotti, Jillian; Demir, Ramazan; Bray-Ward, Patricia; Ward, David; Huszar, Gabor

2004-09-01

We hypothesize that the potential relationship between abnormal sperm morphology and increased frequency of numerical chromosomal aberrations is based on two attributes of diminished sperm maturity: (i) cytoplasmic retention and consequential sperm shape abnormalities; and (ii) meiotic errors caused by low levels of the HspA2 chaperone, a component of the synaptonemal complex. Because sperm morphology and aneuploidies were assessed in semen, but not in the same spermatozoa, previous studies addressing this relationship were inconclusive. We recently demonstrated that sperm shape is preserved following fluorescence in situ hybridization (FISH). Thus, we examined the shape and chromosomal aberrations in the same sperm. We performed phase contrast microscopy and FISH, using centromeric probes for chromosomes X, Y, 10, 11 and 17 in 15 men. The fluorescence and respective phase contrast images were digitized using the Metamorph program. We studied 1286 sperm (256 disomic, 130 diploid and 900 haploid sperm) by three criteria: head and tail dimensions, head shape and Kruger strict morphology. Furthermore, in each analysis, we considered whether disomic or diploid sperm may be distinguished from haploid sperm. There was an overall, but not discriminative, relationship between abnormal sperm dimensions or shape and increased frequencies of numerical chromosomal aberrations. However, approximately 68 of the 256 disomic, and four of 130 diploid sperm showed head and tail dimensions comparable with the most normal, lowest tertile of the 900 haploid spermatozoa. Considering all 1286 sperm, among those with the most regular, symmetrical shape (n = 367), there were 63 and five with disomic and diploid nuclei, respectively. In line with these findings, among the 256 disomic sperm, 10% were Kruger normal. Sperm dimensions or shape are not reliable attributes in selection of haploid sperm for ICSI.

The Trumorph® system: The new universal technique for the observation and analysis of the morphology of living sperm. [corrected].

PubMed

Soler, C; García-Molina, A; Contell, J; Silvestre, M A; Sancho, M

2015-07-01

Evaluation of sperm morphology is a fundamental component of semen analysis, but its real significance has been obscured by a plethora of techniques that involve fixation and staining procedures that induce artefacts. Here we describe Trumorph℗®, a new method for sperm morphology assessment that is based upon examination of wet preparations of living spermatozoa immobilized by a short 60°C shock using negative phase contrast microscopy. We have observed samples from five animals of the following species: bull, boar, goat and rabbit. In every case, all the components of the sperm head and tail were perfectly defined, including the acrosome and midpiece (in all its length, including cytoplasmic droplets). A range of morphological forms was observed, similar to those found by conventional fixed and stained preparations, but other forms were found, distinguishable only by the optics used. The ease of preparation makes it a robust method applicable for analysis of living unmodified spermatozoa in a range of situations. Subsequent studies on well-characterized samples are required to describe the morphology of potentially fertilizing spermatozoa. Copyright © 2015 Elsevier B.V. All rights reserved.

Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia

PubMed Central

Liu, Shan-Wen; Li, Yuan; Zou, Li-Li; Guan, Yu-Tao; Peng, Shuang; Zheng, Li-Xin; Deng, Shun-Mei; Zhu, Lin-Yan; Wang, Li-Wei; Chen, Li-Xin

2017-01-01

Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l−1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer-aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l−1) to a hypotonic solution (290 mOsm l−1), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4′-diisothiocyanatostilbene-2,2′- isulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DIDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC-3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid-piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC-3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia. PMID:27270342

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

PubMed

Morales, P; Vantman, D; Barros, C; Vigil, P

1991-03-01

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 degrees C) on sperm quality.

PubMed

Filliers, M; Rijsselaere, T; Bossaert, P; De Causmaecker, V; Dewulf, J; Pope, C E; Van Soom, A

2008-12-01

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR((R))14-PI) and DNA fragmentation (TUNEL). After addition of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 degrees C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P<0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.

Energy metabolism of spermatozoa of the sand dollar Clypeaster japonicus: the endogenous substrate and ultrastructural correlates.

PubMed

Mita, M; Yasumasu, I; Nakamura, M

1994-07-01

Energy metabolism in spermatozoa of the sand dollar-sea urchin Clypeaster japonicus was examined. The spermatozoa contained triglyceride and cholesterol ester besides several kinds of phospholipids and cholesterol. Glycogen and glucose were present at extremely low levels. Following incubation of spermatozoa in seawater, the triglyceride content decreased rapidly. Other lipids, however, remained at constant levels. High lipase activity was demonstrated in the spermatozoa. Also, [1-14C]oleic acid was oxidized to 14CO2. Ultrastructural study showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and vacuoles of various sizes appeared near the lipid globules. Thus, it is concluded that C. japonicus spermatozoa obtain energy through oxidation of fatty acid from triglyceride stored in the lipid globules at the midpieces.

Preservation of mithun (Bos frontalis) semen at refrigeration temperature.

PubMed

Karunakaran, M; Dhali, A; Mech, A; Khate, K; Rajkhowa, C; Mishra, D P

2007-10-01

The objective of the present study was to investigate the possibility of preserving mithun (Bos frontalis) spermatozoa at refrigeration temperature using tris-egg yolk diluent. Semen samples were collected from four adult mithun bulls through rectal massage method. Good quality semen samples (n=30) were preserved at 4 degrees C using tris-egg yolk diluent for 72 h. Progressive motility, live spermatozoa count and morphological abnormalities were evaluated every 12 h until 72 h of preservation. The colour, consistency and mass activity of fresh semen samples were found to be creamy white, medium and 3+ to 4+ (5+ scale), respectively. The average (mean+/-S.E.) volume (ml), pH and spermatozoa concentration (10(6) ml(-1)) of fresh semen samples were found to be 0.6+/-0.01, 6.8+/-0.03 and 425+/-48, respectively. Progressive motility and live spermatozoa count were found to be less than 30% (P<0.01) after 48 h of storage. Head (P<0.05), midpiece (P<0.05), tail (P<0.01) and total (P<0.01) abnormalities were found to be increased significantly over the time of storage. It was observed that progressive motility and live spermatozoa count remained above 30% and 40%, respectively, until 36 h of storage. Simultaneously the percentage of morphologically abnormal spermatozoa was found to be significantly low until 36 h of storage. The results indicate that it is possible to preserve mithun spermatozoa at refrigeration temperature in tris-egg yolk diluent, which can be further used for artificial insemination within 36 h of storage.

DEFINING THE SPERMATOZOA RNA FINGERPRINT FOR THE NORMAL FERTILE MALE

EPA Science Inventory

Defining the spermatozoa RNA fingerprint for the normal fertile male
G. Charles Ostermeier1, David Dix2, David Miller3, and Stephen A. Krawetz1

1Departments of Ob/Gyn, CMMG & ISC, Wayne State University, USA.
2Reproductive Toxicology Division, U.S. Environmental Pr...

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.

Biophysical studies of spermatozoa

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

Pistenma, David Andrew

1970-12-01

The objectives of this thesis include characterization of spermatozoa according to several physical properties (morphology, size, electrophoretic mobility, sedimentation rate and specific gravity), correlation of these properties with several biological properties (viability, intrinsic motility, fertilizing capacity, antigenicity and genetic composition) and an evaluation of interrelationships among these properties and with selected experimental variables.

The importance of transmission electron microscopy analysis of spermatozoa: Diagnostic applications and basic research.

PubMed

Moretti, Elena; Sutera, Gaetano; Collodel, Giulia

2016-06-01

This review is aimed at discussing the role of ultrastructural studies on human spermatozoa and evaluating transmission electron microscopy as a diagnostic tool that can complete andrology protocols. It is clear that morphological sperm defects may explain decreased fertilizing potential and acquire particular value in the field of male infertility. Electron microscopy is the best method to identify systematic or monomorphic and non-systematic or polymorphic sperm defects. The systematic defects are characterized by a particular anomaly that affects the vast majority of spermatozoa in a semen sample, whereas a heterogeneous combination of head and tail defects found in variable percentages are typically non-systematic or polymorphic sperm defects. A correct diagnosis of these specific sperm alterations is important for choosing the male infertility's therapy and for deciding to turn to assisted reproduction techniques. Transmission electron microscopy (TEM) also represents a valuable method to explore the in vitro effects of different compounds (for example drugs with potential spermicidal activity) on the morphology of human spermatozoa. Finally, TEM used in combination with immunohistochemical techniques, integrates structural and functional aspects that provide a wide horizon in the understanding of sperm physiology and pathology. transmission electron microscopy: TEM; World Health Organization: WHO; light microscopy: LM; motile sperm organelle morphology examination: MSOME; intracytoplasmic morphologically selected sperm injection: IMSI; intracytoplasmic sperm injection: ICSI; dysplasia of fibrous sheath: DFS; primary ciliary dyskinesia: PCD; outer dense fibers: ODF; assisted reproduction technologies: ART; scanning electron microscopy: SEM; polyvinylpirrolidone: PVP; tert-butylhydroperoxide: TBHP.

Evaluating γH2AX in spermatozoa from male infertility patients.

PubMed

Zhong, Hui-zhi; Lv, Fu-tong; Deng, Xue-lian; Hu, Ying; Xie, Dan-ni; Lin, Bin; Mo, Zeng-nan; Lin, Fa-quan

2015-09-01

To investigate whether γH2AX levels were different in the spermatozoa of healthy men compared with infertility patients, and to assess the possible correlations between γH2AX and conventional semen parameters and double-stranded breaks (DSBs) identified with the use of comet assay. Prospective study. Clinical laboratory. Semen from 100 male infertile patients and 100 healthy sperm donors. Human sperm samples were analyzed in terms of World Health Organization parameters. The γH2AX levels were detected by means of flow cytometry. DSBs of sperm were detected by means of comet assay. Morphology slides were made and the sperm morphology assessed according to strict criteria. Conventional semen analyses, γH2AX levels in sperm, DNA DSBs in sperm, and correlations among γH2AX, conventional semen analyses, and DSBs. Concentration, viability, motility, and normal sperm morphology were significantly lower in male infertility patients compared with healthy men. Also, γH2AX levels and the number of DSBs were significantly higher in the sperm of infertile subjects compared with healthy men. γH2AX levels correlated negatively with conventional semen parameters and positively with DSBs. A threshold γH2AX level of 18.55% was identified as a cutoff value to discriminate infertile subjects from fertile control subjects with a specificity of 86.0% and a sensitivity of 83.0%. The positive and negative predictive values of the 18.55% γH2AX threshold were high: 87.7% and 85.5%, respectively. γH2AX levels were higher in the sperm of male infertility patients than in healthy men. γH2AX levels in sperm, as evaluated with the use of flow cytometry, might be a useful biomarker for evaluating DSBs in human spermatozoa. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

The effect of a single sauna exposure on spermatozoa.

PubMed

Brown-Woodman, P D; Post, E J; Gass, G C; White, I G

1984-01-01

Semen was collected at weekly intervals for 3 wk before and 10 wk after the sauna exposure at 85 degrees C for 20 minutes. The numbers, morphology, ultrastructure, motility, viability and metabolism of the sperm was assessed. Sperm numbers fell within one wk and slowly returned to normal in 5 wk. The earliest ultrastructural change was swelling of the plasma membrane, followed by an increase in the number of immature forms and disorganization of the arrangement of the mitochondria. Motility, glucose utilization and lactic acid accumulation of the sperm rose temporarily immediately after sauna.

Early and late effects of Ibuprofen on mouse sperm parameters, chromatin condensation, and DNA integrity in mice.

PubMed

Roodbari, Fatemeh; Abedi, Nahid; Talebi, Ali Reza

2015-11-01

There are few studies indicating the detrimental effects of ibuprofen on sperm fertility potential and DNA integrity. To determine the effects of Ibuprofen on sperm parameters, chromatin condensation and DNA integrity of mice. In this experimental study, 36 adult male mice with average weight 37 gr were divided into three groups, including control (group I, n=12), normal dosage of ibuprofen (group II, n=12) and high dosage (group III, n=12). Ibuprofen with different doses was dissolved in daily water of animals. After 35, 70 and 105 days, the cauda epididymis of mice were cut and incubated in Ham's F10 media. Sperm samples were analyzed for parameters (motility, morphology and count), DNA integrity (SCD test) and chromatin condensation (chromomycin A3 and Aniline blue staining). After 35 days, in addition to above mentioned sperm parameters, all of the treated mice showed statistically significant increase in spermatozoa with immature chromatin (P<0.05). However, after 70 days, the rate of sperm DNA fragmentation assessed by SCD was increased in group II (66.5±0.7) and the percentage of immature spermatozoa (AB(+) and CMA3(+)) was higher in group III (77.5±0.7 and 49.5±6.3 respectively) than other groups. After 105 days, the AB(+) spermatozoa were increased in both normal dose and high dose groups. Ibuprofen may cause a significant reduction in sperm parameters and sperm chromatin/DNA integrity in mice. It should be noted that these deleterious effects are dose-dependent and can be seen in early and late stage of drug treatments.

Ultrastructure of spermatozoa of spider crabs, family Mithracidae (Crustacea, Decapoda, Brachyura): Integrative analyses based on morphological and molecular data.

PubMed

Assugeni, Camila de O; Magalhães, Tatiana; Bolaños, Juan A; Tudge, Christopher C; Mantelatto, Fernando L; Zara, Fernando J

2017-12-01

Recent studies based on morphological and molecular data provide a new perspective concerning taxonomic aspects of the brachyuran family Mithracidae. These studies proposed a series of nominal changes and indicated that the family is actually represented by a different number and representatives of genera than previously thought. Here, we provide a comparative description of the ultrastructure of spermatozoa and spermatophores of some species of Mithracidae in a phylogenetic context. The ultrastructure of the spermatozoa and spermatophore was observed by scanning and transmission electron microscopy. The most informative morphological characters analysed were thickness of the operculum, shape of the perforatorial chamber and shape and thickness of the inner acrosomal zone. As a framework, we used a topology based on a phylogenetic analysis using mitochondrial data obtained here and from previous studies. Our results indicate that closely related species share a series of morphological characteristics of the spermatozoa. A thick operculum, for example, is a feature observed in species of the genera Amphithrax, Teleophrys, and Omalacantha in contrast to the slender operculum observed in Mithraculus and Mithrax. Amphithrax and Teleophrys have a rhomboid perforatorial chamber, while Mithraculus, Mithrax, and Omalacantha show a wider, deltoid morphology. Furthermore, our results are in agreement with recently proposed taxonomic changes including the separation of the genera Mithrax (previously Damithrax), Amphithrax (previously Mithrax) and Mithraculus, and the synonymy of Mithrax caribbaeus with Mithrax hispidus. Overall, the spermiotaxonomy of these species of Mithracidae represent a novel set of data that corroborates the most recent taxonomic revision of the family and can be used in future taxonomic and phylogenetic studies within this family. © 2017 Wiley Periodicals, Inc.

Impact of radio frequency electromagnetic radiation on DNA integrity in the male germline.

PubMed

Aitken, R J; Bennetts, L E; Sawyer, D; Wiklendt, A M; King, B V

2005-06-01

Concern has arisen over human exposures to radio frequency electromagnetic radiation (RFEMR), including a recent report indicating that regular mobile phone use can negatively impact upon human semen quality. These effects would be particularly serious if the biological effects of RFEMR included the induction of DNA damage in male germ cells. In this study, mice were exposed to 900 MHz RFEMR at a specific absorption rate of approximately 90 mW/kg inside a waveguide for 7 days at 12 h per day. Following exposure, DNA damage to caudal epididymal spermatozoa was assessed by quantitative PCR (QPCR) as well as alkaline and pulsed-field gel electrophoresis. The treated mice were overtly normal and all assessment criteria, including sperm number, morphology and vitality were not significantly affected. Gel electrophoresis revealed no gross evidence of increased single- or double-DNA strand breakage in spermatozoa taken from treated animals. However, a detailed analysis of DNA integrity using QPCR revealed statistically significant damage to both the mitochondrial genome (p < 0.05) and the nuclear beta-globin locus (p < 0.01). This study suggests that while RFEMR does not have a dramatic impact on male germ cell development, a significant genotoxic effect on epididymal spermatozoa is evident and deserves further investigation.

Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki).

PubMed

Della Togna, Gina; Trudeau, Vance L; Gratwicke, Brian; Evans, Matthew; Augustine, Lauren; Chia, Han; Bronikowski, Edward J; Murphy, James B; Comizzoli, Pierre

2017-03-15

Knowledge of basic gamete biology is critical to better protect and propagate endangered amphibian species and also to develop reproductive technologies combined with germplasm cryopreservation. The objectives of the study were to test different hormonal stimulations and then characterize the quantity and quality of Panamanian golden frog (Atelopus zeteki) spermatozoa. Following intraperitoneal injection of the gonadotropin-releasing hormone agonist (des-Gly 10 , D-Ala 6 , Pro-NHEt 9- -GnRH 1, 2 or 4 μg/g of body weight), human chorionic gonadotropin (hCG; 5 or 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 29 males were collected at different time points (from 0.5 to 24 h post-injection) to analyze the concentration, motility, and morphology of the spermatozoa. Peak of sperm concentration was observed at 3.5 h post injection for all hormonal treatments. Amphiplex™ led to the highest sperm concentrations (4.45 ± 0.07 × 10 6  cells/mL) followed by 4 μg/gbw GnRH-A (2.65 ± 0.21 × 10 6  cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 10 6  cells/mL). More than 60% of spermatozoa were motile following all treatments but the highest motility (>90%) was obtained from the 10 IU/gbw hCG treatment. Spermic urine samples obtained with all hormone treatments had higher pH (ranging from 7.1 to 7.8) than the urine alone (6.7-6.8). Spermatozoa were filiform and elongated with an apical acrosome, a mitochondrial sheath, a small midpiece and a long tail with an undulating membrane. More than 80% of cells were morphologically normal and 50-70% had intact DNA. These sperm characteristics were not influenced by hormonal treatments. This first comprehensive characterization of sperm samples following optimized hormonal stimulations in A. zeteki lays the foundation for more fundamental studies, reproductive technologies, and future preservation strategies. Copyright © 2016. Published by Elsevier Inc.

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.

Proteomic analysis of mature and immature ejaculated spermatozoa from fertile men

PubMed Central

Cui, Zhihong; Sharma, Rakesh; Agarwal, Ashok

2016-01-01

Dysfunctional spermatozoa maturation is the main reason for the decrease in sperm motility and morphology in infertile men. Ejaculated spermatozoa from healthy fertile men were separated into four fractions using three-layer density gradient. Proteins were extracted and bands were digested on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. Western blotting was performed to verify the expression levels of the proteins of interest. 1469 proteins were identified in four fractions of spermatozoa. The number of detected proteins decreased according to the maturation level of spermatozoa. During spermatozoa maturation, proteins involved in gamete generation, cell motility, energy metabolism and oxidative phosphorylation processes showed increasing expression levels and those involved in protein biosynthesis, protein transport, protein ubiquitination, and response to oxidative stress processes showed decreasing expression levels. We validated four proteins (HSP 70 1A, clusterin, tektin 2 and tektin 3) by Western blotting. The study shows protein markers that may provide insight into the ejaculated spermatozoa proteins in different stages of sperm maturation that may be altered or modified in infertile men. PMID:26510506

Effects of treatment with butylated hydroxytoluene on the susceptibility of boar spermatozoa to cold stress and dilution.

PubMed

Bamba, K; Cran, D G

1992-05-01

Boar spermatozoa acquired resistance to cold shock immediately after exposure to 2.0 mmol butylated hydroxytoluene (BHT) l-1 when Beltsville thawing solution was used as a basic diluent, as judged by motility (the proportion of motile spermatozoa) and acrosomal integrity. The concentration of BHT could be reduced to 0.2 mmol l-1 without decreasing the protective action. However, motility was altered in the presence of greater than 0.15 mmol BHT l-1. Beltsville freezing 5 (BF5) diluent was more effective than Beltsville thawing solution in protecting spermatozoa from cold shock, but addition of BHT to BF5 diluent did not affect the motility and acrosomal morphology of spermatozoa before or after cold shock. Dilution of BHT-treated spermatozoa with BF5 diluent did not restore motility and did not afford further protection against cold shock; it was detrimental to spermatozoa treated with 2 mmol BHT l-1 for greater than 15 min. Egg yolk or lecithin had a detrimental effect. When spermatozoa were treated with 0.05-0.10 mmol BHT l-1 before slow cooling to 5 degrees C, the progressive motility and acrosomal integrity were maintained better after storage for 6 days than in untreated spermatozoa.

Improvement of liquid stored boar semen quality by removing low molecular weight proteins and supplementation with α-tocopherol.

PubMed

Zakošek Pipan, M; Mrkun, J; Nemec Svete, A; Zrimšek, P

2017-11-01

Seminal plasma contains low-molecular weight components that can exert a harmful effect on sperm function. We have evaluated the effects of removing low-molecular weight components from seminal plasma and adding α-tocopherol on boar semen quality after 72h of liquid storage. Semen was evaluated on the basis of motility, morphology, acrosome integrity, plasma membrane modifications, mitochondrial activity, DNA fragmentation and lipid peroxidation. Thiobarbituric acid reactive substances (TBARS), 8-isoprostane, and antioxidant status (total antioxidant capacity (TAC) and superoxide dismutase activity (SOD)) were measured in seminal plasma. Removal of low-molecular weight components from seminal plasma, together with the addition of α-tocopherol, kept the lipid peroxidation and mitochondrial activity and DNA fragmentation at the same level as in native semen samples. Dialysing semen and adding 200μM of α-tocopherol led to higher progressive motility, a higher proportion of morphologically normal spermatozoa and a significantly lower level of acrosomal reacted spermatozoa compared to non-dialyzed semen samples after 72h of storage. In conclusion, liquid stored boar semen was better preserved, and oxidative stress in the semen was reduced when semen was dialyzed and α-tocopherol was added prior to storage. Copyright © 2017 Elsevier B.V. All rights reserved.

Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid.

PubMed

Agha-Rahimi, A; Khalili, M A; Nottola, S A; Miglietta, S; Moradi, A

2016-11-01

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p < 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p < 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF. © 2016 American Society of Andrology and European Academy of Andrology.

Seminal Plasma pH, Inorganic Phosphate, Total and Ionized Calcium Concentrations In The Assessment of Human Spermatozoa Function.

PubMed

Banjoko, S Olatunbosun; Adeseolu, Fasiu O

2013-11-01

Fertilization in humans is dependent on viability of the male spermatozoa among other factors and there have been conflicting reports on the role of pH, calcium and phosphate concentrations in sperm function. This study therefore aimed to investigate seminal plasma pH, inorganic phosphate, total and ionized calcium concentrations relative to spermatozoa function. Seminal plasma concentrations of pH, total calcium, ionized calcium (Ca(++)); inorganic phosphate, motility and spermatozoa count were determined in 80 males by standard methods. Forty-nine of the subjects had normal spermatozoa motility (> 60%) and 31 had hypomotility (< 60%). The hypomotility group exhibited lower calcium ion (Ca(2)+) concentrations; 0.19+0.01mmol/L compared with normal motility group; 0.24+0.01mmol/L (p<0.001) the latter also had significantly higher inorganic phosphate; 7.83+1.27 while the former had 5.64+1.62mmol/L (p= 0.004). The mean spermatozoa counts for hypomotility and normal motility group were 42.0 ± 13 x 106 , 72.35 + 20 x 106 respectively (p< 0.001). No significant differences were observed in pH, volume of ejaculate and total calcium concentration between the hypomotility and normal motility groups The mean concentrations of pH were 7.51 ± 0.02 and 7.54 ± 0.03 respectively (p= 0.21) and total calcium; 3.10 ± 0.12 and 3.36 ± 0.14mmol/L respectively (p= 0.16 ). There was a significant difference in percentage of abnormal forms in both groups with hypomotile group having 36% compared to mormal motility group with 5% (p< 0.05). Correlations were observed between seminal concentrations of calcium ions, inorganic phosphate, spermatozoa count and motility but not with total calcium concentrations and pH and therefore should be considered in understanding male infertility and preparation of media for sperm preservation for in vitro fertilization.

Variation of rabbit sperm physiologic parameters after the X-ray exposure.

PubMed

Klepko, A V; Gorban, L V; Motryna, O A; Kondratova, Y A; Chernyshov, A V; Andreychenko, S V

2013-01-01

To study the impact of X-irradiation on the morpho-functional characteristics of rabbit spermatozoa and the contents of fructose, L-carnitine, α-tocopherol, ascorbate and citrate in the animal sperm. The total body irradiation of rabbits (Soviet Shinchilla) was performed at the RUM-17 device in the dose range of 1.0-7.0 Gy with 2.8·10-3 Gy/sec intensity. The animal sperm was collected by the artificial vagina and then separated on seminal plasma and spermatozoa through centrifugation. Sperm concentration, motility, morphology and linear velocity were identified using the "MBI-6" light microscope. The quantitative determination of ascorbate, α-tocopherol and L-carnitine in seminal plasma was made on the "Agilent 1200" liquid chromatograph, while the concentration of fructose and citrate was measured on a spectrophotometer. The X-ray irradiation was shown to cause a dose-dependent suppressing effect on the prostate, seminal vesicles and epididymices expresed through the decreased content of fructose, citrate, L-carnitine, α-tocopherol and ascorbate in a rabbit sperm. Simultaneously the exacerbation of morphologic anomalies (damage of acrosomes, heads and tails) in spermatozoa along with spermatozoid motility and linear velocity decrease were elucidated. The run-time pattern of morpho-functional changes in spermatozoa together with component content of rabbit sperm in response to the total body irradiation was studied. Consequently the dose dependent raise of morphologic anomalies (damage of acrosomes, heads and tails) in spermatozoa and the slowing of spermatozoid linear velocity was found out. The sperm capacity to fulfill the post-radiation recovery was proven up. The effectiveness of the latter was shown to be dependent upon the radiation dose absorbed and the duration of post-irradiation period. Klepko A. V., Gorban L. V., Motryna O. A., Kondratova Ju. A., Chernyshov A. V., Andrejchenko S. V., 2013.

Effect of cryopreservation on sperm DNA integrity in patients with teratospermia.

PubMed

Kalthur, Guruprasad; Adiga, Satish Kumar; Upadhya, Dinesh; Rao, Satish; Kumar, Pratap

2008-06-01

To test whether sperm with abnormal head morphology are more likely to undergo DNA damage and/or chromatin modification during the process of freeze-thawing. In this prospective study, the semen samples from forty-four men attending the infertility clinic were included. Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. The sperm morphology and the sperm DNA damage were evaluated before and after cryopreservation. The relationship between sperm head abnormalities and freeze-thaw-induced DNA modification was assessed. University hospital fertility center. Men attending infertility clinic for semen analysis. The normospermic and teratospermic semen samples were evaluated for DNA damage before and after cryopreservation by comet assay and acridine orange bindability test. Elucidation of association between sperm morphologic defect and cryodamage. A threefold increase in the amount of DNA damage was observed in teratospermic samples compared with their normospermic counterparts, indicating a higher susceptibility of morphologically abnormal sperm to cryodamage. The susceptibility of morphologically abnormal sperm to DNA damage/chromatin modification during the freeze-thaw process is significantly higher than that of sperm with normal morphology.

Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle.

PubMed

Rahman, Mohammad Bozlur; Kamal, Md Mostofa; Rijsselaere, Tom; Vandaele, Leen; Shamsuddin, Mohammed; Van Soom, Ann

2014-10-01

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n=1239) were collected at three different time points (12, 18 and 24h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P>0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P<0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

Selection of High-Quality Spermatozoa May Be Promoted by Activated Vitamin D in the Woman.

PubMed

Bøllehuus Hansen, Lasse; Rehfeld, Anders; de Neergaard, Rosanna; Nielsen, John Erik; Iversen, Lea Hedegaard; Boisen, Ida Marie; Mortensen, Li Juel; Lanske, Beate; Almstrup, Kristian; Carlsen, Elisabeth; Berg, Anders Hayden; Jørgensen, Niels; Andersen, Anders Nyboe; Juul, Anders; Blomberg Jensen, Martin

2017-03-01

The vitamin D receptor (VDR) and enzymes involved in activation (CYP2R1, CYP27B1) and inactivation (CYP24A1) of vitamin D are expressed in ovary, testes, and spermatozoa. Determine responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in spermatozoa from normal and infertile men, and identify the site of exposure and how 1,25(OH)2D3 influences sperm function. Spermatozoa expressing VDR, CYP2R1, CYP27B1, and CYP24A1 were analyzed in normal and infertile men. 25-Hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D3], and 1,25(OH)2D3 were measured in serum, seminal fluid, cervical secretions, and ovarian follicular fluid. 1,25(OH)2D3 was tested on human spermatozoa. Tertiary center for fertility. Protein expression in spermatozoa and semen quality were assessed in 230 infertile and 114 healthy men. Vitamin D metabolites were measured in fluids from 245 men and 13 women, while 74 oocytes and 17 semen donors were used for sperm-function tests. VDR and CYP24A1 expressions in spermatozoa, fluid concentrations of 25-OHD, 24,25(OH)2D3, and 1,25(OH)2D3, and 1,25(OH)2D3-induced effects on intracellular calcium concentration ([Ca2+]i) and sperm-oocyte binding in vitro. VDR and CYP24A1 were expressed in a >2-fold higher fraction of spermatozoa from normal than infertile men (P < 0.01). Concentrations of 25-OHD, 24,25(OH)2D, and 1,25(OH)2D3 were undetectable in seminal fluid but high in ovarian follicular fluid. Follicular concentrations of 1,25(OH)2D3 induced a modest increase in [Ca2+]i and sperm-oocyte binding in vitro (P < 0.05). Presence of VDR and CYP24A1 mainly in spermatozoa of higher quality supports that 1,25(OH)2D3 available in the female reproductive tract may promote selection of the best gametes for fertilization. Copyright © 2017 by the Endocrine Society

Evolutionary morphology of the male reproductive system, spermatozoa and seminal fluid of spiders (Araneae, Arachnida)--current knowledge and future directions.

PubMed

Michalik, Peter; Ramírez, Martín J

2014-07-01

The male reproductive system and spermatozoa of spiders are known for their high structural diversity. Spider spermatozoa are flagellate and males transfer them to females in a coiled and encapsulated state using their modified pedipalps. Here, we provide a detailed overview of the present state of knowledge of the primary male reproductive system, sperm morphology and the structural diversity of seminal fluids with a focus on functional and evolutionary implications. Secondly, we conceptualized characters for the male genital system, spermiogenesis and spermatozoa for the first time based on published and new data. In total, we scored 40 characters for 129 species from 56 families representing all main spider clades. We obtained synapomorphies for several taxa including Opisthothelae, Araneomorphae, Dysderoidea, Scytodoidea, Telemidae, Linyphioidea, Mimetidae, Synotaxidae and the Divided Cribellum Clade. Furthermore, we recovered synspermia as a synapomorphy for ecribellate Haplogynae and thus propose Synspermiata as new name for this clade. We hope that these data will not only contribute to future phylogenetic studies but will also stimulate much needed evolutionary studies of reproductive systems in spiders. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quality of canine spermatozoa retrieved by percutaneous epididymal sperm aspiration.

PubMed

Varesi, S; Vernocchi, V; Faustini, M; Luvoni, G C

2013-02-01

To investigate the feasibility of percutaneous epididymal sperm aspiration in dogs and whether it might provide a population of epididymal spermatozoa similar to the population that can be obtained by processing isolated epididymis caudae. Concentration and total sperm number, motility, morphology and acrosomal integrity of spermatozoa retrieved by percutaneous epididymal sperm aspiration, in vitro aspiration and mincing of the cauda of the epididymis were compared. Percutaneous epididymal sperm aspiration is a feasible procedure to retrieve a population of spermatozoa in dogs. Quality is similar to that of spermatozoa collected in vitro, although a wide variation amongst animals was observed. In case of ejaculation failure due to pathological conditions in dogs, the collection of spermatozoa from the cauda of the epididymis could be an option for providing gametes for assisted reproductive technologies. Percutaneous epididymal sperm aspiration can be used in dogs with compromised reproductive performance, in which orchiectomy cannot be performed for medical or owner reasons. Further studies aimed to investigate whether the percutaneous epididymal sperm aspiration technique might be feasible for repeated semen collection and to accurately evaluate side effects are required. © 2013 British Small Animal Veterinary Association.

Effects of storage time on the motility, mortality and calcium levels of Atlantic salmon Salmo salar spermatozoa.

PubMed

Parodi, J; Guerra, G; Cuevas, M; Ramírez-Reveco, A; Romero, F

2017-04-01

This study estimates spermatozoa mortality, morphology, motility and intracellular calcium levels in Atlantic salmon Salmo salar milt after prolonged storage. Milt samples were preserved at 4° C for 25 days and then evaluated for mortality. Motility remained high for the first 3 days and the mortality was low during the first 5 days of storage. A decrease of >50% in calcium content was observed after 5 days of storage. When spermatozoa were activated, calcium levels increased >200% in relative fluorescence units (RFU); this rate of increase was lost when the samples were stored for extended periods of time and was only partially manifested in a zero calcium solution. The results suggest that in vitro storage of S. salar spermatozoa at 4° C for a period of 3 days preserves motility and limits mortality to levels similar to those of fresh spermatozoa. This method also maintains intracellular calcium storage critical for spermatozoa performance. © 2017 The Fisheries Society of the British Isles.

Chlamydiae in the ejaculate: their influence on the quality and morphology of sperm.

PubMed

Veznik, Zdenek; Pospisil, Leopold; Svecova, Drahomira; Zajicova, Atanaska; Unzeitig, Vit

2004-07-01

Given the lack of information concerning the role of Chlamydia trachomatis in male fertility, the aim of this study was to ascertain and analyze the quality of Chlamydiae-positive and -negative semen. Sperm count was performed according to the 1999 World Health Organization (WHO) laboratory manual for examination of human semen and sperm-cervical mucus interaction, and sperm survival was assessed by a 120-min test. The evaluation of the morphological examination of ejaculates was carried out using the sasmo (strict morphological analysis of ejaculates) computer program. Chlamydiae were detected by immunofluorescent reaction using the Progen Biotechnik GmbH diagnostic set. Fisher's exact test and the chi-quadrate test were used for statistical analysis. Of the total of 627 sperm samples examined, Chlamydiae were detected in 136 cases (21.7%). Sperm analysis showed significant differences between Chlamydiae-positive and -negative samples. The Chlamydiae-contaminated group showed normal sperm morphology 14.4% lower, volume 6.4% lower, concentration 8.3% lower, motility 7.8% and velocity 9.3% lower than in Chlamydiae-negative samples. The average values for normal spermatozoa and motility in the Chlamydiae-positive group were also significantly reduced. Chlamydia trachomatis was found to be a possible factor in sperm pathology. These results could help to elucidate the role of Chlamydia trachomatis in male infertility.

Ultrastructure variation in the spermatozoa of Pseudopaludicola frogs (Amphibia, Anura, Leptodactylidae), with brief comments on its phylogenetic relevance.

PubMed

dos Santos, Julio Sérgio; Introíni, Gisele Orlandi; Veiga-Menoncello, Ana Cristina Prado; Recco-Pimentel, Shirlei Maria

2015-12-01

The taxonomic history of the small frogs of the genus Pseudopaludicola from South America has been controversial. Phylogenetic inferences based on molecular data have identified four Pseudopaludicola clades, correlating with the known variation in karyotypes (2n = 22, 20, 18, and 16). In this study, the ultrastructure of the spermatozoa was analyzed in 12 species of the Pseudopaludicola, with the aim of describing their morphology and identifying characters that may contribute to a better understanding of the phylogenetic relationships. The spermatozoa presented marked differences in tail structures. The tails of the spermatozoa of the species with 2n = 22 chromosomes (Pseudopaludicola sp. 1 [P. pusilla group], Pseudopaludicola falcipes, P. mineira, and Pseudopaludicola saltica), as well as Pseudopaludicola ameghini and Pseudopaludicola ternetzi (2n=20), have juxta-axonemal fibers, undulating membranes and axial fibers. In contrast, in the species with 2n = 18 (P. facureae, P. giarettai, Pseudopaludicola canga, P. atragula, and Pseudopaludicola sp. 2) and 2n = 16 (Pseudopaludicola mystacalis), there are no evident axial or juxta-axonemal fibers, but a paraxonemal rod with a thick undulating membrane, which is shorter than that found among Pseudopaludicola species. The ultrastructural morphological differences observed in the spermatozoa of these species may be phylogenetically informative, given that they coincide with the consensus phylogeny of the group and appear to represent a progressive simplification of the spermatozoon. © 2015 Wiley Periodicals, Inc.

Sperm quality in men is improved by supplementation with a combination of L-arginine, L-citrullin, roburins and Pycnogenol®.

PubMed

Stanislavov, R; Rohdewald, P

2014-12-01

The aim of this study was to investigate the influence of Prelox®R, a combination of French maritime pine bark extract (Pycnogenol®), L-arginine, L-citrulline and roburins, on male fertility. Sperm quality of 50 subfertile men was tested in monthly intervals in a double-blind, randomized, placebo controlled, crossover study. Patients received 2 tablets Prelox®R or placebo twice daily during test periods. Following a run-in period of 1 month, patients received either Prelox®R or a placebo for 1 month. After a wash-out period of 1 month, patients received Prelox®R or a placebo in a crossover manner for 1 month. Sperm volume, concentration of spermatozoa, total count, motility, vitality and morphology were measured by standard methods of calculation of the Fertility Index (FI) in monthly intervals. Activity of e-NOS in sperm was evaluated in parallel by measuring the quantity of L-citulline produced from L-arginine. Supplementation with Prelox®R enhanced sperm volume and concentration, motility, vitality and morphology significantly versus placebo. The Fertility Index rose to normal values during treatment with Prelox®R. e-NOS activity in sperm was elevated by Prelox®R. No adverse effects were reported. Prelox®R offers a safe method to improve quality of human spermatozoa in subfertile men.

Optimization of IVF pregnancy outcomes with donor spermatozoa.

PubMed

Wang, Jeff G; Douglas, Nataki C; Prosser, Robert; Kort, Daniel; Choi, Janet M; Sauer, Mark V

2009-03-01

To identify risk factors for suboptimal IVF outcomes using insemination with donor spermatozoa and to define a lower threshold that may signal a conversion to fertilization by ICSI rather than insemination. Retrospective, age-matched, case-control study of women undergoing non-donor oocyte IVF cycles using either freshly ejaculated (N=138) or cryopreserved donor spermatozoa (N=69). Associations between method of fertilization, semen sample parameters, and pregnancy rates were analyzed. In vitro fertilization of oocytes with donor spermatozoa by insemination results in equivalent fertilization and pregnancy rates compared to those of freshly ejaculated spermatozoa from men with normal semen analyses when the post-processing motility is greater than or equal to 88%. IVF by insemination with donor spermatozoa when the post-processing motility is less than 88% is associated with a 5-fold reduction in pregnancy rates when compared to those of donor spermatozoa above this motility threshold. When the post-processing donor spermatozoa motility is low, fertilization by ICSI is associated with significantly higher pregnancy rates compared to those of insemination. While ICSI does not need to be categorically instituted when using donor spermatozoa in IVF, patients should be counseled that conversion from insemination to ICSI may be recommended based on low post-processing motility.

In-depth proteomic analysis of carp (Cyprinus carpio L) spermatozoa.

PubMed

Dietrich, Mariola A; Arnold, Georg J; Fröhlich, Thomas; Ciereszko, Andrzej

2014-12-01

Using a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography-electrospray ionization tandem mass spectrometry, we identified 348 proteins in carp spermatozoa, most of which were for the first time identified in fish. Dynein, tubulin, HSP90, HSP70, HSP60, adenosylhomocysteinase, NKEF-B, brain type creatine kinase, mitochondrial ATP synthase, and valosin containing enzyme represent high abundance proteins in carp spermatozoa. These proteins are functionally related to sperm motility and energy production as well as the protection of sperm against oxidative injury and stress. Moreover, carp spermatozoa are equipped with functionally diverse proteins involved in signal transduction, transcription, translation, protein turnover and transport. About 15% of proteins from carp spermatozoa identified here were also detected in seminal plasma which may be a result of leakage from spermatozoa into seminal plasma, adsorption of seminal plasma proteins on spermatozoa surface, and expression in both spermatozoa and cells secreting seminal plasma proteins. The availability of a catalog of carp sperm proteins provides substantial advances for an understanding of sperm function and for future development of molecular diagnostic tests of carp sperm quality, the evaluation of which is currently limited to certain parameters such as sperm count, morphology and motility or viability. The mass spectrometry data are available at ProteomeXchange with the dataset identifier PXD000877 (DOI: http://dx.doi.org/10.6019/PXD000877). Copyright © 2014 Elsevier Inc. All rights reserved.

[Expression of DKKL1 in spermatozoa of men with asthenospermia].

PubMed

Yan, Qiu-Xia; Ma, Yi; Chen, Run-Qiang; Zhou, Xiu-Qin; Qiao, Jing; Xian, Ying-Jie; Feng, Ling; Chen, Cai-Rong

2018-03-20

To compare the expression of DKKL1 in ejaculated spermatozoa of normal fertile men and men with asthenospermia and investigate the role of DKKL1 in the pathogenesis of asthenospermia. The characteristics of semen samples collected from normal fertile men and men with asthenospermia were analyzed using computer-assisted sperm analysis according to WHO criteria. The ejaculated sperms were isolated by Percoll discontinuous density gradients to detect the expression of DKKL1 mRNA and protein using real-time PCR and Western blotting. The expression of DKKL1 mRNA was significantly down-regulated by 11.1 times in asthenospermic men as compared with that in normal fertile men (P<0.01). Western blotting showed that the expression of DKKL1 protein was down-regulated by 2.4 times in asthenospermic men compared to normal fertile men. The expression of DKKL1, which may play an important role in sperm motility,is significantly decreased in ejaculated spermatozoa of men with asthenospermia.

Effects of leptin on sperm count and morphology in Sprague-Dawley rats and their reversibility following a 6-week recovery period.

PubMed

Almabhouh, F A; Osman, K; Siti Fatimah, I; Sergey, G; Gnanou, J; Singh, H J

2015-09-01

Altered epididymal sperm count and morphology following leptin treatment has been reported recently. This study examined the effects of 42 days of leptin treatment on sperm count and morphology and their reversibility during a subsequent 56-day recovery period. Twelve-week-old male Sprague-Dawley rats were randomised into four leptin and four saline-treated control groups (n = 6). Intraperitoneal injections of leptin were given daily (60 μg Kg(-1) body weight) for 42 days. Controls received 0.1 ml of 0.9% saline. Leptin-treated animals and their respective age-matched controls were euthanised on either day 1, 21, 42 or 56 of recovery for collection of epididymal spermatozoa. Sperm concentration was determined using a Makler counting chamber. Spermatozoa were analysed for 8-hydroxy-2-deoxyguanosine and DNA fragmentation (Comet assay). Data were analysed using anova. Sperm concentration was significantly lower but fraction of abnormal spermatozoa, and levels of 8-hydroxy-2-deoxyguanosine were significantly higher in leptin-treated rats on day 1 of recovery. Comet assays revealed significant DNA fragmentation in leptin-treated rats. These differences were reduced by day 56 of recovery. It appears that 42 days of leptin treatment to Sprague-Dawley rats has significant adverse effects on sperm count and morphology that reverse following discontinuation of leptin treatment. © 2014 Blackwell Verlag GmbH.

A new insight into male fertility preservation for patients with completely immotile spermatozoa.

PubMed

Chen, Huanhua; Feng, Guixue; Zhang, Bo; Zhou, Hong; Wang, Caizhu; Shu, Jinhui; Gan, Xianyou; Lin, Ruoyun; Huang, Dongmei; Huang, Yingqin

2017-09-18

Sperm cryopreservation is the most effective method to preserve male fertility but this is normally used for motile spermatozoa. Thus, only motile spermatozoa are used for cryopreservation in most reproductive medicine centers worldwide. The immotile spermatozoa from some problematic patients are usually discarded, resulting in a missed opportunity of sterility cryopreservation for future assisted reproductive treatments. Many studies have shown that successful fertilization can be obtained after selection of viable sperm from the completely immotile spermatozoa before ICSI. Whether the completely immotile spermatozoa are worth of freezing has not been realized The aim of this study is to explore the clinical value of cryopreservation of immotile spermatozoa. Completely immotile spermatozoa were collected and frozen, and subsequently viable but immotile frozen-thawed spermatozoa were selected by laser plus for ICSI. Main outcomes included spermatozoa survival index, fertilization rate and good quality embryo rate. After identification by laser, the fresh samples of spermatozoa presented with a mean survival rate of 54.86% and 26.05%, and this was reduced to 44.13% and 18.13% in frozen-thawed spermatozoa samples, which showed a frozen-thawed spermatozoa survival index of 0.80 and 0.70 in the testicular and ejaculate sperm, respectively. There were no statistically differences in fertilization rate (80% vs80.51%, 75.00% vs 81.48%), cleavage rate (95.45% vs 98.95%, 100.00% vs 95.45%) and good quality embryo rate (40.48% vs 52.13%, 33.33%vs38.10%) between the frozen-thawed immotile spermatozoa group and the routine fresh immotile spermatozoa ICSI group in both testicular and ejaculate sperm, respectively. The results of the study show that completely immotile spermatozoa can be frozen in order to preserve male fertility as long as viable spermatozoa are present. This procedure provides a further possibility for fertility preservation for patients with completely immotile spermatozoa.

Genetic control of gamete quality in the mouse--a tribute to Halina Krzanowska.

PubMed

Styrna, Jozefa

2008-01-01

In this article, we summarise the principal research findings of the distinguished Polish scientist, Professor Halina Krzanowska, related to the genetic control of mammalian gamete quality. During the early stages of her career, Halina Krzanowska conducted experiments on poultry and then she moved on to work on mice. All her research on gamete quality was conducted on the research models, consomic, congenic and recombinant inbred strains, which Krzanowska developed herself. These models differed mostly in their fertility. Krzanowska was one of the first researchers to demonstrate the influence of chromosome Y on the morphology of mice spermatozoa. She also showed that the uterotubal junction is in vivo a selection barrier for the morphologically abnormal spermatozoa, whereas in vitro abnormal spermatozoa are able to participate in fertilization, the function of selective barrier being performed by the granulosa cell layer and the zona pellucida. Another model which Krzanowska produced were chimaeras, which she used to find out if the percentage of abnormal spermatozoa and the efficiency of fertilization are determined by germ cells themselves or by environmental factors and she discovered that sperm head shape, the proportion of abnormal sperm and fertilizing capacity are determined mainly by the genotype of germ cells and only minimally by environmental factors.

Do photoperiod and endocrine disruptor 4-tert-octylphenol effect on spermatozoa of bank vole (Clethrionomys glareolus)?

PubMed

Kotula-Balak, Malgorzata; Grzmil, Pawel; Chojnacka, Katarzyna; Andryka, Katarzyna; Bilinska, Barbara

2014-05-15

Photoperiod is an environmental signal that controls physiology and behavior of all organisms. Bank voles, which are seasonal breeders, are stimulated to reproduce by the long photoperiod associated with spring and summer. To date, physiology of bank vole spermatozoa has not been explored, although they constitute an interesting model for examining the relationship between photoperiod and xenoestrogen on spermatozoa function. In an attempt to evaluate the acute effect of 4-tert-octylphenol (OP) an in vitro system was used. Spermatozoa isolated from the cauda epididymidies of long-day (LD; 18 h light: 6 h darkness) and short-day (SD; 6 h light: 18 h darkness) bank voles were treated with two OP concentrations (10(-4) M and 10(-8)M, respectively). OP-treated spermatozoa were used for the examination of motility parameters (computer-assisted semen analyzer CEROS), acrosome integrity (Commassie blue staining), cAMP production (immunoenzymatic assay) and cell viability (flow-cytometry analysis). The study revealed the photoperiod-dependent effect of short OP-treatment on motility parameters of vole spermatozoa. In LD spermatozoa, an increase of velocities: (curvilinear velocity [VCL], average path velocity [VAP] straight line velocity [VSL]) and head activity (amplitude of the lateral head displacement, [ALH]) was found. Interestingly, in SD spermatozoa opposite effect on VCL, VAP, VSL and ALH was observed, however only after treatment with 10(-4)M OP. The dose-dependent influence of OP upon acrosome integrity, as well as cAMP levels, in relation to the reproductive status of voles was observed. Moreover, OP exposure affected spermatozoa morphology rather than spermatozoa viability. Copyright © 2014 Elsevier Inc. All rights reserved.

Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology.

PubMed

Huszar, G; Vigue, L

1993-03-01

Our previous creatine phosphokinase (CK) activity studies in human sperm revealed differences among men and among sperm populations within the same specimen. Samples with low sperm concentrations, high incidence of abnormal sperm morphology, and diminished fertility had higher per sperm CK activity. In the present work, we demonstrated, with 14C-FDNB covalent CK active site modification and with direct CK immunocytochemistry, that the higher CK activity is related to an increased content of CK and of other proteins in sperm. Also, sperm heads with higher CK content were significantly larger and rounder and showed a higher incidence of amorph configuration. We suggest that these biochemical and morphological irregularities are related and are due to a failure of spermatogenesis, more specifically, to a higher retention of cytoplasm, which in normal sperm development is lost to the Sertoli cells as residual bodies. Thus higher CK activity and larger or irregular head size in human sperm signify cellular immaturity and a failure to complete spermatogenesis.

Deletion of the Slo3 gene abolishes alkalization-activated K+ current in mouse spermatozoa

PubMed Central

Zeng, Xu-Hui; Yang, Chengtao; Kim, Sung Tae; Lingle, Christopher J.; Xia, Xiao-Ming

2011-01-01

Mouse spermatozoa express a pH-dependent K+ current (KSper) thought to be composed of subunits encoded by the Slo3 gene. However, the equivalence of KSper and Slo3-dependent current remains uncertain, because heterologous expression of Slo3 results in currents that are less effectively activated by alkalization than are native KSper currents. Here, we show that genetic deletion of Slo3 abolishes all pH-dependent K+ current at physiological membrane potentials in corpus epididymal sperm. A residual pH-dependent outward current (IKres) is observed in Slo3−/− sperm at potentials of >0 mV. Differential inhibition of KSper/Slo3 and IKres by clofilium reveals that the amplitude of IKres is similar in both wild-type (wt) and Slo3−/− sperm. The properties of IKres suggest that it likely represents outward monovalent cation flux through CatSper channels. Thus, KSper/Slo3 may account for essentially all mouse sperm K+ current and is the sole pH-dependent K+ conductance in these sperm. With physiological ionic gradients, alkalization depolarizes Slo3−/− spermatozoa, presumably from CatSper activation, in contrast to Slo3/KSper-mediated hyperpolarization in wt sperm. Slo3−/− male mice are infertile, but Slo3−/− sperm exhibit some fertility within in vitro fertilization assays. Slo3−/− sperm exhibit a higher incidence of morphological abnormalities accentuated by hypotonic challenge and also exhibit deficits in motility in the absence of bicarbonate, revealing a role of KSper under unstimulated conditions. Together, these results show that KSper/Slo3 is the primary spermatozoan K+ current, that KSper may play a critical role in acquisition of normal morphology and sperm motility when faced with hyperosmotic challenges, and that Slo3 is critical for fertility. PMID:21427226

High throughput, parallel imaging and biomarker quantification of human spermatozoa by ImageStream flow cytometry.

PubMed

Buckman, Clayton; George, Thaddeus C; Friend, Sherree; Sutovsky, Miriam; Miranda-Vizuete, Antonio; Ozanon, Christophe; Morrissey, Phil; Sutovsky, Peter

2009-12-01

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.

Importance of sperm morphology during sperm transport and fertilization in mammals.

PubMed

García-Vázquez, Francisco A; Gadea, Joaquín; Matás, Carmen; Holt, William V

2016-01-01

After natural or artificial insemination, the spermatozoon starts a journey from the site of deposition to the place of fertilization. However, only a small subset of the spermatozoa deposited achieves their goal: to reach and fertilize the egg. Factors involved in controlling sperm transport and fertilization include the female reproductive tract environment, cell-cell interactions, gene expression, and phenotypic sperm traits. Some of the significant determinants of fertilization are known (i.e., motility or DNA status), but many sperm traits are still indecipherable. One example is the influence of sperm dimensions and shape upon transport within the female genital tract towards the oocyte. Biophysical associations between sperm size and motility may influence the progression of spermatozoa through the female reproductive tract, but uncertainties remain concerning how sperm morphology influences the fertilization process, and whether only the sperm dimensions per se are involved. Moreover, such explanations do not allow the possibility that the female tract is capable of distinguishing fertile spermatozoa on the basis of their morphology, as seems to be the case with biochemical, molecular, and genetic properties. This review focuses on the influence of sperm size and shape in evolution and their putative role in sperm transport and selection within the uterus and the ability to fertilize the oocyte.

Importance of sperm morphology during sperm transport and fertilization in mammals

PubMed Central

García-Vázquez, Francisco A; Gadea, Joaquín; Matás, Carmen; Holt, William V

2016-01-01

After natural or artificial insemination, the spermatozoon starts a journey from the site of deposition to the place of fertilization. However, only a small subset of the spermatozoa deposited achieves their goal: to reach and fertilize the egg. Factors involved in controlling sperm transport and fertilization include the female reproductive tract environment, cell-cell interactions, gene expression, and phenotypic sperm traits. Some of the significant determinants of fertilization are known (i.e., motility or DNA status), but many sperm traits are still indecipherable. One example is the influence of sperm dimensions and shape upon transport within the female genital tract towards the oocyte. Biophysical associations between sperm size and motility may influence the progression of spermatozoa through the female reproductive tract, but uncertainties remain concerning how sperm morphology influences the fertilization process, and whether only the sperm dimensions per se are involved. Moreover, such explanations do not allow the possibility that the female tract is capable of distinguishing fertile spermatozoa on the basis of their morphology, as seems to be the case with biochemical, molecular, and genetic properties. This review focuses on the influence of sperm size and shape in evolution and their putative role in sperm transport and selection within the uterus and the ability to fertilize the oocyte. PMID:27624988

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

Morphometric characteristics and chromatin integrity of spermatozoa in three Italian dog breeds.

PubMed

Lange-Consiglio, A; Antonucci, N; Manes, S; Corradetti, B; Cremonesi, F; Bizzaro, D

2010-12-01

Studies in many species indicate that variation of spermatozoan head morphology is a sensitive biomarker for abnormal chromatin structure and resultant clinical fertility. This preliminary study evaluated spermatozoan head morphometry in different dog breeds and assessed whether morphometric parameters could reflect spermatozoan DNA fragmentation in dogs. Spermatozoan morphometry and DNA quality (measured by TUNEL flow cytometry) were assessed in semen from 11 dogs of three Italian breeds (Cirneco dell'Etna, Piccolo Levriero Italiano and Segugio Maremmano). Morphometric data showed that Segugio dogs had significantly larger (33·67%) spermatozoa and that Piccolo Levrieros had a higher incidence of long (46·75%) and elliptical spermatozoan heads (11·5%) when compared with the samples from other breeds. Moreover, the predominance of elliptical spermatozoa in one dog (23%) was significantly related to the percentage of spermatozoa with fragmented DNA (12·6%), whereas in another dog, where no more than 1% of spermatozoa was elliptical, only 0·36% of spermatozoa had damaged DNA. It is noteworthy that the breeding record of the former dog in the previous 12 months showed poor fertility and fecundity. These data suggest that spermatozoan head morphometry could be breed related and that there is a significant correlation between DNA fragmentation and elliptical spermatozoa in individual animals. This finding, albeit limited in our study to a single case, is possibly related to clinical infertility. © 2010 British Small Animal Veterinary Association.

Effect of prostatic fluid on the quality of fresh and frozen-thawed canine epididymal spermatozoa.

PubMed

Korochkina, E; Johannisson, A; Goodla, Lavanya; Morrell, J M; Axner, E

2014-12-01

Canine epididymal spermatozoa have a low freeze-tolerance ability compared with ejaculated spermatozoa, which could arise from the absence of prostatic fluid (PF). Therefore, the purpose of this work was to elucidate the influence of PF on the quality of canine epididymal sperm before and after freezing. Caudae epididymides were retrieved from eight dogs after routine castration. Spermatozoa were released by slicing the tissue and were extended in either Tris solution or PF before freezing. Frozen sperm samples were thawed at 70 °C for 8 seconds in a waterbath. Sperm concentration, motility using computer-assisted sperm analysis, morphology, plasma membrane, acrosome and chromatin integrity were assessed in the fresh sperm samples (after 20 minutes incubation) and at 0 and 4 hours after thawing. Progressive motility, distance straight line, distance average path, average path velocity, curvilinear velocity, straight line velocity, straightness, linearity, wobble, and beat cross frequency were significantly increased after extraction into PF. There was a higher proportion of spermatozoa with DNA damage in the PF treatment group at 4 hours after thawing than in the Tris treatment group (15.8% vs. 6.7%, P < 0.05). These results suggest that the addition of PF to canine spermatozoa activates sperm motility in fresh spermatozoa but has a negative effect on chromatin integrity after freezing-thawing. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization and cooled storage of semen from corn snakes (Elaphe guttata).

PubMed

Fahrig, Brooke M; Mitchell, Mark A; Eilts, Bruce E; Paccamonti, Dale L

2007-03-01

The phylogenetic order Squamata has many representatives that could benefit from the use of semen preservation as a tool for assisting conservation. To date, few studies have been made evaluating the potential for collecting and preserving semen from snakes. The objectives of this study were to characterize semen parameters of the corn snake (Elaphe guttata), including appearance, volume, concentration, sperm motility, and sperm morphology, and to determine the longevity of corn snake sperm motility stored at 4 degrees C. Single semen samples were collected from 22 adult corn snakes. The appearance of the corn snake semen was generally cloudy, and the color was white to tan. Corn snake spermatozoa initially exhibited a median motility of 92.5%. Corn snakes were found to produce small-volume ejaculates (median 0.01 ml). However, the overall concentration of the snake ejaculate was high (chi = 852 x 10(6) +/- 585 x 10(6) spermatozoa/ml). Morphologically, a mean of 75.7 +/- 9.3% of the sperm cells in an ejaculate were normal. Snake ejaculate with a white appearance had significantly higher sperm concentrations (chi = 1,859 x 10(6) +/- 1,008 x 106 sperm cells/ml; F = 15.74, P = 0.001) than tan ejaculates (chi = 601 x 10(6) +/- 439 x 106 sperm cells/ml). Sperm motility decreased significantly in samples that were stored at 4 degrees C for greater than 48 hr in a refrigerator or Equitainer I. This is the first study to characterize semen volume, appearance, and concentration; sperm motility; and sperm morphology in captive corn snakes. The information derived from this study can be used to develop a model for a collection, cooled storage, and shipping program for semen from endangered or threatened captive and wild snakes.

Transmission electron microscopy analysis of the origin and incidence of sperm intranuclear cytoplasmic retention in fertile and teratozoospermia men.

PubMed

Zhu, W-J

2018-03-01

The human sperm nucleus contains cytoplasm. However, the origin and incidence of human sperm intranuclear cytoplasmic retention (INCR) remain unknown. The objectives of this study were to observe the morphological origin of INCR within the seminiferous epithelium and investigate the incidence of INCR in fertile and teratozoospermia men using transmission electron microscopy (TEM). By TEM, INCR initially appeared in elongating round spermatid nuclei and varied in size, number, shape, content, location and distribution within sperm nuclei. The teratozoospermia group (n = 16) demonstrated a higher incidence of INCR than did the fertile group (n = 16) (17.6 ± 5.2% vs. 9.7 ± 3.4%; p = 0.000). In the fertile group, no correlations were found between the incidence of INCR and abnormal sperm morphology, nuclear vacuole, acrosome integrity, motility or concentration (p > 0.05). However, the incidence of INCR exhibited a positive relationship with sperm abnormal morphology in the teratozoospermia group (r = 0.616, p = 0.011). These results demonstrate that INCR occurs in the early process of spermatogenesis and is an alteration found in the nucleus. Spermatozoa from teratozoospermia men contained more INCRs than those from fertile males. More attention should be paid to the possibility of spermatozoa containing INCR when using spermatozoa with abnormal head morphology for clinical or diagnostic purposes. © 2018 American Society of Andrology and European Academy of Andrology.

The osmotic tolerance of boar spermatozoa and its usefulness as sperm quality parameter.

PubMed

Yeste, Marc; Briz, Mailo; Pinart, Elisabeth; Sancho, Sílvia; Bussalleu, Eva; Bonet, Sergi

2010-06-01

Predicting the fertility outcome of ejaculates is very important in the field of porcine reproduction. The aims of this study were to determine the effects of different osmotic treatments on boar spermatozoa and to correlate them with fertility and prolificacy, assessed as non-return rates within 60 days (NRR(60d)) of the first inseminations, and litter size (LS), respectively. Sperm samples (n=100) from one hundred healthy Piétrain boars were used to assess 48 treatments combining different osmolalities (ranged between 100 and 4000 mOsm kg(-1)), different compounds used to prepare anisotonic solutions, and two different modalities: return and non-return to isotonic conditions. Sperm quality was evaluated before and after applying the treatments on the basis of analyses of sperm viability, motility, morphology and percentages of acrosome-intact spermatozoa. Statistical analyses were performed using a one-way ANOVA and post hoc Tukey's test, linear regression analyses (Pearson correlation and multiple regression) and Jackknife cross-validation. Although three conventional parameters: sperm viability, sperm morphology and the percentages of acrosome-intact spermatozoa were significantly correlated with NRR(60d) and with LS, their respective osmotic tolerance parameters (defined for each parameter and treatment regarding with negative control) presented a higher Pearson coefficient with both fertility and prolificacy in three treatments (150 mOsm kg(-1) with non-return to isotonic conditions, 200 mOsm kg(-1) with return and 500 mOsm kg(-1) using sodium citrate and non-return to isotonic conditions). We conclude that osmotic resistance in sperm viability, sperm morphology and acrosome-intactness in the treatments mentioned above could be assessed along with classical parameters to better predict the fertilising ability of a given ejaculate. Copyright (c) 2010 Elsevier B.V. All rights reserved.

[Macrocephalic spermatozoa. What would be the impact on reproduction?].

PubMed

Guichaoua, M-R; Mercier, G; Geoffroy-Siraudin, C; Paulmyer-Lacroix, O; Lanteaume, A; Metzler-Guillemin, C; Perrin, J; Achard, V

2009-09-01

We want to highlight the risk of infertility and failure of Assisted Reproductive Technologies due to the presence of macrocephalic spermatozoa (MS) in the sperm at rate equalling or superior to 20% in at least one semen analysis. We did a retrospective analysis of 19 infertile patients presenting MS at average rate between 14.3 and 49.7%. For each patient, at least one semen analysis showed a MS rate equal or superior to 20%. We did an automated analysis of the spermatozoa surface for 13 patients and a detailed analysis of the MS morphology in 18 patients. Thirteen couples benefited of one or more IVF with or without ICSI. The semen analysis shows an impairment of one or more parameter of the sperm in all patients. Three morphological aspects for MS were highlighted: MS with irregular head, MS with regular head, and MS with multiple heads, with a dominance of irregular heads. The spermatozoa surface analysis shows a significant increase of the average surface and of the standard deviation (p<0.0001). The average rate of pregnancies by transfer is decreased compared to usual rates in our laboratories (13% versus 28%). We want to sensitize biologist and clinical doctors to the existence of partial forms of this syndrome, which could be related to infertility with impaired sperm parameters and low pregnancy rates after FIV or ICSI.

Does genome organization matter in spermatozoa? A refined hypothesis to awaken the silent vessel.

PubMed

Ioannou, Dimitrios; Tempest, Helen G

2018-01-02

The spermatozoon is considered by many to be a silent vessel whose only function is to safely deliver the paternal genome to the maternal oocyte. As a result, the paternal contribution to fertilization and embryogenesis is frequently overlooked. However, the spermatozoon is a highly elaborate and specialized cell that is formed through the process of spermatogenesis. Spermatogenesis is a complex cellular program of differentiation that produces mature spermatozoa, which are essential for reproduction, fertilization, and normal embryonic development. The sperm cell is unique in morphology, chromatin structure, and function. Increasing evidence demonstrates that perturbations in chromatin integrity and organization could have a significant clinical impact on fertilization and embryogenesis. In this article we will review the evidence that demonstrates the paternal genome to be highly packaged and uniquely organized. We will postulate how the integrity and organization of the paternal genome likely has functional consequences that are critical for the establishment and maintenance of a viable pregnancy. In doing so, we hope to dispel the common myth that the sperm cell is a silent vessel; instead we will demonstrate the sperm cell to be a highly segmentally organized, epigenetically primed cell. 2D: two-dimension; 3C: chromosome conformation capture; 3D: three-dimension; 4D: four-dimension; CTs: chromosome territories; FISH: fluorescence in situ hybridization; IMSI: intra cytoplasmic morphologically selected sperm injection; ICSI: intracytoplasmic sperm injection; IVF: in-vitro fertilization; mESCs: mouse embryonic stem cells; NORs: nuclear organizing regions; TADs: topologically associated domain.

Sperm morphology and chromatin integrity in Swedish warmblood stallions and their relationship to pregnancy rates

PubMed Central

Morrell, Jane M; Johannisson, Anders; Dalin, Anne-Marie; Hammar, Linda; Sandebert, Thomas; Rodriguez-Martinez, Heriberto

2008-01-01

Background Artificial insemination is not as widely used in horses as in other domestic species, such as dairy cattle and pigs, partly because of the wide variation in sperm quality between stallion ejaculates and partly due to decreased fertility following the use of cooled transported spermatozoa. Furthermore, predictive tests for sperm fertilising ability are lacking. The objective of the present study was to assess sperm morphology and chromatin integrity in ejaculates obtained from 11 warmblood breeding stallions in Sweden, and to evaluate the relationship of these parameters to pregnancy rates to investigate the possibility of using these tests predictively. Methods Aliquots from fortyone ejaculates, obtained as part of the normal semen collection schedule at the Swedish National Stud, were used for morphological analysis by light microscopy, whereas thirtyseven were used for chromatin analysis (SCSA) by flow cytometry. The outcome of inseminations using these ejaculates was made available later in the same year. Results Ranges for the different parameters were as follows; normal morphology, 27–79.5%; DNA-fragmentation index (DFI), 4.8–19.0%; standard deviation of DNA fragmentation index (SD_DFI) 41.5–98.9, and mean of DNA fragmentation index (mean_DFI), 267.7–319.5. There was considerable variation among stallions, which was statistically significant for all these parameters except for mean_DFI (P < 0.001, P < 0.01, P < 0.001 and P < 0.2 respectively). There was a negative relationship between normal morphology and DFI (P < 0.05), between normal morphology and SD_DFI (P < 0.001), and between normal morphology and mean_DFI (P < 0.05). For specific defects, there was a direct relationship between the incidence of pear-shaped sperm heads and DFI (P < 0.05), and also nuclear pouches and DFI (P < 0.001), indicating that either morphological analysis or chromatin analysis was able to identify abnormalities in spermiogenesis that could compromise DNA-integrity. A positive relationship was found between normal morphology and pregnancy rate following insemination (r = 0.789; P < 0.01) and a negative relationship existed between DFI and pregnancy rate (r = -0.63; P < 0.05). Sperm motility, assessed subjectively, was not related to conception rate. Conclusion Either or both of the parameters, sperm morphology and sperm chromatin integrity, seem to be useful in predicting the fertilising ability of stallion ejaculates, particularly in determining cases of sub-fertility. PMID:18179691

Sperm yield after single layer centrifugation with Androcoll-E is related to the potential fertility of the original ejaculate.

PubMed

Morrell, J M; Stuhtmann, G; Meurling, S; Lundgren, A; Winblad, C; Macias Garcia, B; Johannisson, A

2014-05-01

Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and good chromatin, retrospective analysis was carried out to investigate whether sperm yield after SLC is linked to potential fertility. Commercial semen doses for AI from 24 stallions (five stallions with four ejaculates each, 19 stallions with three ejaculates each; n = 77) obtained during the breeding season were cooled, and sent overnight to the Swedish University of Agricultural Sciences in an insulated box for evaluation, with other doses being sent to studs for commercial AI. On arrival at Swedish University of Agricultural Sciences, the semen was used for SLC and also for evaluation of sperm motility, membrane integrity, chromatin integrity, and morphology. The seasonal pregnancy rates for each stallion were available. The yield of progressively motile spermatozoa after SLC (calculated as a proportion of the initial load) was found to be highly correlated with pregnancy rate (r = 0.75; P < 0.001). Chromatin damage was highly negatively correlated with pregnancy rate (r = -0.69; P < 0.001). Pregnancy rate was also correlated with membrane integrity (r = 0.58; P < 0.01), progressive motility (r = 0.63; P < 0.01), and normal morphology (r = 0.45; P < 0.05). In conclusion, these preliminary results show that sperm yield after SLC is related to the potential fertility of the original ejaculate, and could be an alternative indicator of stallion fertility if breeding data are not available. Single layer centrifugation is fast (30 minutes) and does not require expensive equipment, whereas other assays require a flow cytometer and/or specialist skills. An additional option could be to transport semen doses to a laboratory for SLC if the stud personnel do not want to perform the procedure themselves. Copyright © 2014 Elsevier Inc. All rights reserved.

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

PubMed Central

Prapaiwan, N.; Tharasanit, T.; Punjachaipornpol, S.; Yamtang, D.; Roongsitthichai, A.; Moonarmart, W.; Kaeoket, K.; Manee-in, S.

2016-01-01

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa. PMID:26954170

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa.

PubMed

Prapaiwan, N; Tharasanit, T; Punjachaipornpol, S; Yamtang, D; Roongsitthichai, A; Moonarmart, W; Kaeoket, K; Manee-In, S

2016-05-01

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

SPINK2 deficiency causes infertility by inducing sperm defects in heterozygotes and azoospermia in homozygotes.

PubMed

Kherraf, Zine-Eddine; Christou-Kent, Marie; Karaouzene, Thomas; Amiri-Yekta, Amir; Martinez, Guillaume; Vargas, Alexandra S; Lambert, Emeline; Borel, Christelle; Dorphin, Béatrice; Aknin-Seifer, Isabelle; Mitchell, Michael J; Metzler-Guillemain, Catherine; Escoffier, Jessica; Nef, Serge; Grepillat, Mariane; Thierry-Mieg, Nicolas; Satre, Véronique; Bailly, Marc; Boitrelle, Florence; Pernet-Gallay, Karin; Hennebicq, Sylviane; Fauré, Julien; Bottari, Serge P; Coutton, Charles; Ray, Pierre F; Arnoult, Christophe

2017-08-01

Azoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Varicocele is associated with abnormal retention of cytoplasmic droplets by human spermatozoa.

PubMed

Zini, A; Defreitas, G; Freeman, M; Hechter, S; Jarvi, K

2000-09-01

To determine whether varicocele is associated with retention of sperm cytoplasmic droplets in infertile men. Retrospective study. University infertility clinic. Nonazoospermic men with idiopathic (n = 69) and varicocele-associated infertility (n = 73), and 20 fertile controls presenting for vasectomy. None. Standard semen parameters and percentage of spermatozoa with cytoplasmic droplets on Papanicolaou smears. No statistically significant differences were found between the fertile and infertile groups with respect to semen volume. Fertile controls had significantly greater mean percent sperm motility and normal morphology than infertile men. The mean percentage of sperm with residual cytoplasm was statistically significantly different in all three groups. Infertile men with varicocele had the highest percentage of sperm with cytoplasmic droplets, the next highest level being in men with idiopathic infertility and the lowest level in fertile controls (11.7 +/- 1.0, 8.1 +/- 0.9 and 3.2 +/- 0.4%, respectively, P<.0001). Our data show that idiopathic and even moreso, varicocele-related male infertility are conditions associated with impaired disposal of residual sperm cytoplasm by the testis and/or epididymis. These data provide a possible mechanism for the observed semen abnormalities and reduced fertility potential associated with varicocele and idiopathic male infertility.

Field fertility of sex-sorted and non-sorted frozen-thawed stallion spermatozoa.

PubMed

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

2008-11-01

In the 2004/2005 breeding season, the fertility of sex-sorted (SS) and non-sorted (NS) frozen stallion spermatozoa from two Hannovarian stallions was compared. A hysteroscopic insemination technique [Morris, L.H., Tiplady, C., Allen, W.R., 2003a. Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction. Equine Vet. J. 35, 197-201] was used to deposit low doses (6, 13 or 25 x 10(6) frozen-thawed SS or NS spermatozoa) onto the utero-tubal junction at 32 or 38 h after the administration of Chorulon (2500 IU, Intervet). Fertility was low, with one pregnancy (13 x 10(6) spermatozoa, 500 microL) obtained after artificial insemination with frozen SS spermatozoa (n=29 cycles) which resulted in the birth of a filly. Two pregnancies were obtained in mares inseminated with 6 x 10(6) NS spermatozoa in 250 microL (n=31 cycles). Mares failing to conceive on two experimental cycles were allocated to the conventional insemination group. Insemination with >500 x 10(6) motile NS frozen-thawed spermatozoa, yielded satisfactory per cycle conception rates (35.5%, 22/62) for both stallions combined and was within the values of their normal fertility as quoted by the stud's records. This suggests that the quality of the frozen semen was acceptable and that the freezing processes yielded viable spermatozoa capable of fertilisation. The poor fertility after hysteroscopic insemination with low doses of sex-sorted or non-sorted spermatozoa from the same stallions may be directly attributable to the low dose insemination conditions with frozen-thawed rather than sex-sorted spermatozoa.

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.

Immunological observations following vasectomy.

PubMed

Choi, Y J; Reiner, L; Ney, C

1979-09-15

Lymphocytotoxic antibodies (LCA) against panels of normal lymphocytes and leukemic B-cells were demonstrated in vasectomized men. Since vasectomy is known to induce antibody formation to spermatozoa, the demonstration of these lymphocytotoxic antibodies may be related to antigenic constituents of spermatozoa such as HLA or B-cell alloantigens. Long term follow-up is needed to clarify the clinical significance of these antibodies.

Current perspectives of CASA applications in diverse mammalian spermatozoa.

PubMed

van der Horst, Gerhard; Maree, Liana; du Plessis, Stefan S

2018-03-26

Since the advent of computer-aided sperm analysis (CASA) some four decades ago, advances in computer technology and software algorithms have helped establish it as a research and diagnostic instrument for the analysis of spermatozoa. Despite mammalian spermatozoa being the most diverse cell type known, CASA is a great tool that has the capacity to provide rapid, reliable and objective quantitative assessment of sperm quality. This paper provides contemporary research findings illustrating the scientific and commercial applications of CASA and its ability to evaluate diverse mammalian spermatozoa (human, primates, rodents, domestic mammals, wildlife species) at both structural and functional levels. The potential of CASA to quantitatively measure essential aspects related to sperm subpopulations, hyperactivation, morphology and morphometry is also demonstrated. Furthermore, applications of CASA are provided for improved mammalian sperm quality assessment, evaluation of sperm functionality and the effect of different chemical substances or pathologies on sperm fertilising ability. It is clear that CASA has evolved significantly and is currently superior to many manual techniques in the research and clinical setting.

Ejaculate traits in the Namibian cheetah (Acinonyx jubatus): influence of age, season and captivity.

PubMed

Crosier, Adrienne E; Marker, Laurie; Howard, JoGayle; Pukazhenthi, Budhan S; Henghali, Josephine N; Wildt, David E

2007-01-01

The objective was to examine the influence of animal age, season and captivity status on seminal quality in wild-born cheetahs (Acinonyx jubatus) in Namibia, Africa. Animals were divided into three age categories: juvenile (14-24 months; n = 16 males, 23 ejaculates); adult (25-120 months; n = 76 males, 172 ejaculates); and aged (>120 months; n = 5 males, 5 ejaculates). Seasons were categorised into hot-wet (January-April), cold-dry (May-August) and hot-dry (September-December). A comparison between freshly wild-caught (n = 29 males, 41 ejaculates) and captive-held cheetahs (n = 68 males, 159 ejaculates) was also conducted. Raw ejaculates contained 69.0 +/- 1.1% motile spermatozoa (mean +/- s.e.m.) with 73.6 +/- 1.5% of these cells containing an intact acrosome. Overall, 18.4 +/- 0.9% of spermatozoa were morphologically normal, with midpiece anomalies being the most prevalent (approximately 39%) defect. Juvenile cheetahs produced ejaculates with poorer sperm motility, forward progressive status, lower seminal volume and fewer total motile spermatozoa than adult and aged animals. Spermatogenesis continued unabated throughout the year and was minimally influenced by season. Proportions of sperm malformations were also not affected by season. Ejaculates from captive cheetahs had increased volume and intact acrosomes, but lower sperm density than wild-caught counterparts. In summary, Namibian cheetahs produce an extraordinarily high proportion of pleiomorphic spermatozoa regardless of age, season or living (captive versus free-ranging) status. Young males less than 2 years of age produce poorer ejaculate quality than adult and aged males. Because (1) all study animals were wild born and (2) there was little difference between freshly caught males and those maintained in captivity for protracted periods, our results affirm that teratospermia in the cheetah is mostly genetically derived. It also appears that an ex situ environment for the Namibian cheetah can ensure sperm quality comparable with that for free-living males.

Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis).

PubMed

Ashish, Shende; Bhure, S K; Harikrishna, Pillai; Ramteke, S S; Muhammed Kutty, V H; Shruthi, N; Ravi Kumar, G V P P S; Manish, Mahawar; Ghosh, S K; Mihir, Sarkar

2017-04-01

The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Effect of heat stress on the fertility of male mice in vivo and in vitro.

PubMed

Yaeram, J; Setchell, B P; Maddocks, S

2006-01-01

A study was conducted to determine whether following exposure of male mice to high temperatures, the ability of their spermatozoa to fertilise ova was reduced, especially during the period before the males became completely infertile. Male mice placed in a microclimate chamber at 36 degrees C for two periods, each of 12 h on successive days, were less able to fertilise control females in vivo when mated and, even in those females that became pregnant, litter size was reduced. However, these effects were associated with falls in testis weight and numbers of spermatozoa in the testis and epididymis. To determine whether the effect on fertility was a result of the decreased spermatozoa numbers, spermatozoa were collected from the epididymides of heated and control males. Equal numbers of motile spermatozoa from an unselected sample or those subjected to a swim-up procedure to separate those that were motile from the immotile ones in the sample were then mixed in vitro with oocytes from superovulated normal females. Similar numbers of spermatozoa from both control and heated males bound to the zona pellucida but smaller percentages of the oocytes were fertilised by spermatozoa from the heated males and fewer of these spermatozoa penetrated the ova. The effects were first seen 7 days after the heat exposure and became more obvious after 10 or 14 days.

Tests to measure the quality of spermatozoa at spermiation

PubMed Central

Amann, Rupert P.

2010-01-01

This commentary is to critique the revised World Health Organization (WHO) semen analysis manual as it pertains to characteristics of a spermatozoon at spermiation. The aims of the revised WHO manual include improving the 'quality of semen analysis' without any restriction to clinical use. Furthermore, the manual states that semen analysis may be useful for (a) 'investigating male fertility status' and (b) 'monitoring spermatogenesis during and following male fertility regulation.' However, if the analysis of ejaculated spermatozoa is intended for the purposes described in (b), then cells that are abnormal at spermiation must be identified. This paper takes the position that the manual does not identify methods to estimate the quality of spermatozoa at spermiation. Instead, it uses a 'gold standard' of sperm passing through the cervical mucus or arriving near the site of fertilization. Although this standard is appropriate for drawing conclusions regarding the probability that an individual could impregnate his partner, it is not appropriate for studying illness of the testes per se. Herein, the measures of sperm quality presented in the WHO manual are critiqued with respect to the detection of spermatozoa that were abnormal at spermiation vs. those that became abnormal subsequently. Quality assessments based on the percentage of motile or 'viable' spermatozoa are meaningless. Alternative quality attributes defining spermatozoa at spermiation are presented in this paper. In conclusion, assessment of spermatozoal quality at spermiation, on the basis of quality attributes of individual ejaculated spermatozoa, is best achieved through application of (a) a new paradigm for the morphological evaluation of sperm quality and (b) modern analytical techniques to evaluate, in an adequate sample, several appropriate independent attributes in each spermatozoon in order to more accurately identify the proportion of abnormal spermatozoa. PMID:20111084

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.

Quantity and quality of guinea pig (cavia porcellus) spermatozoa after administration of methanol extract of bitter melon (momordica charantia) seed and depot medroxy progesterone acetate (DMPA)

NASA Astrophysics Data System (ADS)

Ilyas, Syafruddin; Hutahaean, Salomo; Nursal

2018-03-01

The discovery of male contraceptive drugs continues to be pursued, due to the few participation of men associated with the lack of contraceptive options for men. The combination of bitter melon seed methanol extract and DMPA are the options that currently apply to men. Therefore, the use of guinea pigs as experimental animals conducted research using experimental methods with complete randomized design (CRD). There are 4 control groups and 4 treatment groups. The first group, control group of dimethyl sulphoxide (DMSO) for 0 week (K0), The second one, bitter melon seed extract of 50 mg/100g Body Weight/day for 0 week (P0), the third one, control group of dimethyl sulfoxide (DMSO) for 4 weeks (K1), the fourth one, bitter melon seed extract of 50 mg/100g BW/day for 4 weeks + Depot medroxy Progesterone Acetate (P1), the fifth one, control group of dimethyl sulfoxide (DMSO) for 8 weeks (K2), the sixth one, bitter melon seed extract of 50 mg/100g BW/day for 8 weeks + DMPA (P2), the seventh one, control group of dimethyl sulfoxide (DMSO) for 12 weeks (K3), the eighth one, bitter melon seed extract of 50 mg/100g BW/day for 12 weeks + DMPA (P3). Methanol extract of bitter melon seed to decrease the quantity and quality of guinea pig spermatozoa decreased significantly, i.e. viability and normal morphology of spermatozoa (p<0.05).

Current concepts of molecular events during bovine and porcine spermatozoa capacitation.

PubMed

Vadnais, Melissa L; Galantino-Homer, Hannah L; Althouse, Gary C

2007-01-01

Spermatozoa are required to undergo the processes of capacitation before they obtain fertilizing ability. The molecular changes of capacitation are still not fully understood. However, it is accepted that capacitation is a sequential process involving numerous physiological changes including destabilization of the plasma membrane, alterations of intracellular ion concentrations and membrane potential, and protein phosphorylation. There are no known morphological changes that occur to the spermatozoon during capacitation. The purpose of this review is to summarize current evidence on the molecular aspects of capacitation both in vivo and in vitro in bovine and porcine spermatozoa. For the purpose of this review, the process of sperm capacitation will encompass maturational events that occur following ejaculation up to binding to the zona pellucida, that triggers acrosomal exocytosis and initiates fertilization.

Sperm characteristics and teratology in rats following vas deferens occlusion with RISUG and its reversal.

PubMed

Lohiya, N K; Suthar, R; Khandelwal, A; Goyal, S; Ansari, A S; Manivannan, B

2010-02-01

The functional success of the reversal of vas occlusion by styrene maleic anhydride (RISUG), using the solvent vehicle, Dimethyl Sulphoxide (DMSO), has been investigated. Reversal with DMSO was carried out in Wistar albino rats 90 days after bilateral vas occlusion. The body weight, organ weight, sperm characteristics, fertility test and teratology, including skeletal morphology were evaluated in vas occlusion and reversal animals and in F(1) progenies to assess the functional success of the occlusion and reversal. Body weight, organ weight and the cauda epididymal sperm characteristics of vas occlusion and reversal animals and of F(1) progenies were comparable to control. Ejaculated spermatozoa in the vaginal smear showed detached head/tail, acrosomal damage, bent midpiece, bent tail and morphological aberrations in sperm head after vas occlusion, which returned to normal, 90 days after reversal. Monthly fertility test, post-injection showed 0% fertility, which improved gradually and 100% fertility was achieved 90 days after reversal. The fertility/pregnancy/implantation record and skeletal morphology of the offspring were comparable to control. The results suggest functional success and safety of vas occlusion reversal by DMSO.

Expression, Localization of SUMO-1, and Analyses of Potential SUMOylated Proteins in Bubalus bubalis Spermatozoa

PubMed Central

Brohi, Rahim Dad; Wang, Li; Hassine, Najla Ben; Cao, Jing; Talpur, Hira Sajjad; Wu, Di; Huang, Chun-Jie; Rehman, Zia-Ur; Bhattarai, Dinesh; Huo, Li-Jun

2017-01-01

Mature spermatozoa have highly condensed DNA that is essentially silent both transcriptionally and translationally. Therefore, post translational modifications are very important for regulating sperm motility, morphology, and for male fertility in general. Protein sumoylation was recently demonstrated in human and rodent spermatozoa, with potential consequences for sperm motility and DNA integrity. We examined the expression and localization of small ubiquitin-related modifier-1 (SUMO-1) in the sperm of water buffalo (Bubalus bubalis) using immunofluorescence analysis. We confirmed the expression of SUMO-1 in the acrosome. We further found that SUMO-1 was lost if the acrosome reaction was induced by calcium ionophore A23187. Proteins modified or conjugated by SUMO-1 in water buffalo sperm were pulled down and analyzed by mass spectrometry. Sixty proteins were identified, including proteins important for sperm morphology and motility, such as relaxin receptors and cytoskeletal proteins, including tubulin chains, actins, and dyneins. Forty-six proteins were predicted as potential sumoylation targets. The expression of SUMO-1 in the acrosome region of water buffalo sperm and the identification of potentially SUMOylated proteins important for sperm function implicates sumoylation as a crucial PTM related to sperm function. PMID:28659810

Effect of repeated collection on semen characteristics of alpacas.

PubMed

Bravo, P W; Flores, D; Ordoñez, C

1997-09-01

Semen characteristics of alpacas were studied after repeated collections. Twelve adult males were divided into three groups of four each for semen collection once, twice, or three times every other day. The duration of copulation; volume of ejaculate; pH; motility; sperm concentration (number of sperm/milliliter semen); total number of sperm per ejaculate; and percentages of live, normal, and abnormal spermatozoa were analyzed by regression analysis. Semen color and consistency were analyzed by the chi-square test. Between the first, second, and third ejaculations, there were differences (p < 0.05) in sperm concentration; percentages of normal spermatozoa and abnormal spermatozoa; sperm with abnormal heads and abnormal tails; and consistency (viscous, viscous, and semi-viscous). There were no differences (p > 0.05) in ejaculated volume, percentage of live spermatozoa, pH, percentage of cytoplasmic droplets, and duration of copulation. Some males from which semen was collected on the three-mating schedule ejaculated only seminal plasma during the second and third copulation starting on Day 10 of the study. There were differences between males (p < 0.05) for most of the characteristics studied. In sum, frequency of mating affected some semen characteristics that may be important determinants of the fertility of male alpacas.

Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months

PubMed Central

Wakayama, Sayaka; Kamada, Yuko; Yamanaka, Kaori; Kohda, Takashi; Suzuki, Hiromi; Shimazu, Toru; Tada, Motoki N.; Osada, Ikuko; Nagamatsu, Aiko; Kamimura, Satoshi; Nagatomo, Hiroaki; Mizutani, Eiji; Ishino, Fumitoshi; Yano, Sachiko

2017-01-01

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at –95 °C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS. PMID:28533361

Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months.

PubMed

Wakayama, Sayaka; Kamada, Yuko; Yamanaka, Kaori; Kohda, Takashi; Suzuki, Hiromi; Shimazu, Toru; Tada, Motoki N; Osada, Ikuko; Nagamatsu, Aiko; Kamimura, Satoshi; Nagatomo, Hiroaki; Mizutani, Eiji; Ishino, Fumitoshi; Yano, Sachiko; Wakayama, Teruhiko

2017-06-06

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 °C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.

High resolution light microscopic evaluation of boar semen quality sperm cytoplasmic droplet retention in relationship with boar fertility parameters.

PubMed

Lovercamp, K W; Safranski, T J; Fischer, K A; Manandhar, G; Sutovsky, M; Herring, W; Sutovsky, P

2007-01-01

The purpose of this study was to investigate the relationship between fertility and quantitative measures of boar semen quality, including various patterns of sperm cytoplasmic droplet (CD) retention, as determined by high power differential interference contrast (DIC) microscopy. A total of 116 ejaculates were collected from a nucleus herd of 18 Large White boars over an eight month period. Semen quality parameters were analyzed for each ejaculate by calculating the percentage of normal spermatozoa, spermatozoa possessing a CD in the proximal, distal, or distal midpiece reflex position, total spermatozoa with an attached cytoplasmic droplet, spermatozoa with non-CD related aberrations and total spermatozoa with abnormalities. Of the 116 ejaculates received, 71 ejaculates from 13 boars had corresponding fertility data from single-sire inseminations of multiparous sows. The fertility data included farrowing rate (FR) and total number born (TNB). The monthly FR encompassed one month before and one month after the date of semen collection. Detection of differences for fertility and semen quality parameters was performed by separating the boars into either an above-average or below-average group based on the mean FR (74.01 +/- 1.43%) or TNB (12.34 +/- 0.17) for the study. For FR, the boars in the below-average group had a significantly lower percentage of normal spermatozoa and significantly higher percentage of spermatozoa possessing distal CDs, total attached CDs and total abnormalities compared to the boars in the above-average group. Conversely, for TNB there were no significant differences between the above- and below-average groups for the semen quality parameters. These data suggest that the attached CD may negatively affect FR, but not TNB. The detection of relationships between the boar fertility parameters and the retention of the sperm CD after ejaculation, document the advantage of high power DIC microscopy in conventional semen evaluation.

Poor Centrosomal Function of Cat Testicular Spermatozoa Impairs Embryo Development In Vitro after Intracytoplasmic Sperm Injection1

PubMed Central

Comizzoli, Pierre; Wildt, David E.; Pukazhenthi, Budhan S.

2007-01-01

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h post-activation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages. PMID:16687647

Studies on sperm storage in the vas deferens of the spinifex hopping mouse (Notomys alexis).

PubMed

Peirce, E J; Moore, H D M; Leigh, C M; Breed, W G

2003-02-01

The cauda epididymidis, with its relatively cool temperature (32-35 degrees C), is considered to be the main site of sperm storage in male mammals. However, in the adult male spinifex hopping mouse, Notomys alexis, similar numbers of spermatozoa are found in the vas deferens to those in the cauda epididymidis. The present study shows that, unlike in the laboratory mouse in which spermatozoa of the vas deferens are found mainly in the epididymal region of the duct, spermatozoa in the hopping mouse are localized mainly to the middle and urethral regions of the vas deferens which lies in the inguinal and lower abdominal region of the body cavity. After ligation of the vas deferens close to its connection with the epididymis, many spermatozoa in the vas deferens retain the potential for motility for up to 2 weeks, indicating that the viability of spermatozoa is not compromised by being restricted to core body temperature. This urethral region of the vas deferens, in which spermatozoa reside, has a highly divergent structural organization compared with that of common laboratory rodents in which there is an expanded lumen with a network of epithelial folds. Ultrastructural observations of the cells lining the duct indicate that there are not any marked differences in morphology compared with the cells lining the duct in common laboratory murids, but the infoldings of the vas deferens of the hopping mouse are highly vascular which might facilitate supply of oxygen and nutrients to the spermatozoa residing in the lumen.

Adaptation of Motility Analysis Apparatus for Space Science and Microgravity Ground-Based Experiments

NASA Technical Reports Server (NTRS)

Johnson, Jacqueline U.

1996-01-01

Previous space flight studies have described unfavorable effects of microgravity on testicular morphology and spermatogenesis (Cosmos 1887 Biosputnik flight, 9/29/87 - 10/12/87). The flight animals demonstrated small reductions in testicular and epididymal size, the phenomenon explained as resulting water loss. Yet, light microscopic histological preparations revealed few spermatozoa in the rete testis of the flight males compared to control animals. The cause for this finding was subjectively assessed to be due to "the anatomical dislocation of the organs... and a disturbance in testicular blood supply". Unfortunately, the reported effects of microgravity on the reproductive processes (particularly within males) are few and divergent. If habitation in space is a futuristic goal, more objective testing (of male and female gametes) in a microgravity environment will provide insight to the developmental potential of these reproductive cells. As part of the Marshall Space Flight Centers' Summer Faculty Fellowship Program within the Biophysics Branch, a key component of the research investigation was to develop a test to evaluate individual cell motility and orientation in varying gravitational environments, using computerized assessment of sperm cell concentration, morphology and motility to provide objective, quantitative experimental control. In previous work performed jointly by the author and a NASA colleague, it has been shown that macroscopic motile aggregates of spermatozoa were not altered by the absence of microgravity. Variations in the number of normal versus abnormal sperm due to microgravity influences have yet to be established. It is therefore of interest to monitor the cytoskeletal matrix (microtubulin) of these organisms as a possible indicator of cell viability and/or function.

Relationship between sperm quality traits and field-fertility of porcine semen

PubMed Central

Lymberopoulos, A. G.; Khalifa, T. A. A.

2010-01-01

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 × 108 sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 × 106 sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 × 109 spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2~76.5%) and SCI (0.16~4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity. PMID:20458156

Evaluation of the damage in fish spermatozoa cryopreservation

NASA Astrophysics Data System (ADS)

Li, Jun; Liu, Qinghua; Zhang, Shicui

2006-12-01

Cryodamages occur during sperm cryopreservation. Cryopreservation of fish sperm usually results in marked decrease in sperm quality, such as swelling or disruption of the plasma membrane, mitochondrial dysfunction, diminished sperm motility, impaired velocity, shorter motility period, denaturation, and release of some enzymes from spermatozoa. In this paper, damages in morphology, physiology, biochemistry and metabolism, and genetic integrity of fish semen after cryopreservation are discussed. New approaches in assessment of fish thawed sperm quality such as computer assisted sperm analysis, flow cytometic analysis combined with fluorescent probes and single cell gel electrophoresis are also briefly reviewed.

Sperm motility and morphology changes in rats exposed to cadmium and diazinon.

PubMed

Adamkovicova, Maria; Toman, Robert; Martiniakova, Monika; Omelka, Radoslav; Babosova, Ramona; Krajcovicova, Vladimira; Grosskopf, Birgit; Massanyi, Peter

2016-08-08

Humans are ubiquitously exposed to multiple environmental contaminants. Consequences of combined action on the reproductive system remain unknown. This study aimed to assess single and joint effects of cadmium and diazinon exposure on sperm quality parameters. Male adult Wistar rats were randomized into 4 groups of ten animals each. Group A was used as a control, animals from group B were exposed to cadmium (30 mg/L), rats from group C were administered with diazinon (40 mg/L), and rats from group D were exposed simultaneously to cadmium (30 mg/L) and diazinon (40 mg/L) via drinking water for 90 days. Sperm morphology and motility were evaluated using a bright field microscope and a computer-assisted semen analysis. The percentage of motile spermatozoa and morphologically normal sperm was markedly reduced in rats from the group B. Rats from the C group showed an increase in velocity parameters, amplitude of lateral head displacement, decrease in beat-cross frequency, and an increase in abnormal sperm morphology. Simultaneous coexposure to cadmium and diazinon increased distance and velocity parameters, and amplitude of lateral head displacement. Reductions were observed in straightness, linearity, wobble, and beat-cross frequency. The decreased normal sperm morphology rates were related to defects of the sperm tail. Exposure to cadmium and diazinon at relatively low doses impairs sperm quality and can reduce male fertility. Cadmium and diazinon caused significant changes on sperm morphology with varying effects on motility patterns. These parameters were significantly higher in the group D as compared to the group C. The findings have important implications for reproductive risk assessment of combined exposures to multiple chemicals.

FISH assessment of aneuploidy frequencies in mature and immature human spermatozoa classified by the absence or presence of cytoplasmic retention.

PubMed

Kovanci, E; Kovacs, T; Moretti, E; Vigue, L; Bray-Ward, P; Ward, D C; Huszar, G

2001-06-01

Previously, a relationship has been found between diminished cellular maturity of human spermatozoa and low-level expression of the testis-specific chaperone protein, HspA2. Because HspA2 is a component of the synaptonemal complex in rodents, and assuming that this is also the case in men, it was postulated that the frequency of chromosomal aneuploidies would be higher in immature versus mature spermatozoa. This question was examined in spermatozoa from semen and from 80% Percoll pellets (enriched for mature spermatozoa) of the same ejaculate in 10 oligozoospermic men. Immature spermatozoa with retained cytoplasm, which signifies spermiogenetic arrest, were identified by immunocytochemistry. Using fluorescence in-situ hybridization (FISH), approximately 7000 sperm nuclei were evaluated in each of the 20 fractions (142 086 spermatozoa in all) using centromeric probes for the X, Y and 17 chromosomes. The proportions of immature spermatozoa were 45.4 +/- 3.4 versus 26.6 +/- 2.2% in the two semen versus the Percoll groups (medians: 48.2 versus 25%, P < 0.001, n = 300 spermatozoa per fraction, total 6000 spermatozoa). There was also a concomitant decline in total disomy, total diploidy and total aneuploidy frequencies in the 80% Percoll versus semen fractions (0.17 versus 0.54%, 0.14 versus 0.26% and 0.31 versus 0.81% respectively, P < 0.001 in all comparisons). The mean decline of aneuploidies was 2.7-fold. With regard to the hypothesis that aneuploidies are related to sperm immaturity, there was a close correlation between the incidence of immature spermatozoa and disomies (r = 0.7, P < 0.001) but no correlation with diploidies (r = 0.03), indicating that disomies originate primarily in immature spermatozoa. It is suggested that the common factor underlying sperm immaturity and aneuploidies is the diminished expression of HspA2. In addition, the lack of this chaperone may also cause diminished cellular transport of proteins, such as DNA-repair enzymes or of the retention of cytoplasm that is extruded from normally maturing spermatozoa during spermiogenesis.

Smoking is associated with the retention of cytoplasm by human spermatozoa.

PubMed

Mak, V; Jarvi, K; Buckspan, M; Freeman, M; Hechter, S; Zini, A

2000-09-01

To determine whether cigarette smoking is associated with the abnormal retention of residual sperm cytoplasm in infertile men. Semen samples were obtained from 87 consecutive non-azoospermic men with idiopathic infertility (18 smokers and 69 nonsmokers) and from 20 men presenting for vasectomy (fertile controls). Standard semen parameters and the percentage of spermatozoa with residual cytoplasm (on Papanicolaou smears) were recorded. Subject age, semen volume, and sperm density, motility, and morphology were not significantly different between the two groups of infertile men. However, a significant difference was found in the mean +/- SEM percentages of sperm with cytoplasm droplets between smokers and nonsmokers (12.9% +/- 1.7% and 8.1% +/- 0.9%, respectively; P < 0.001). Our data suggest that cigarette smoking is associated with retention of sperm cytoplasmic droplets in infertile men, a morphologic characteristic associated with impaired sperm function.

In vitro fertilization of mouse ova by spermatozoa exposed isothermally to radio-frequency radiation

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

Cleary, S.F.; Liu, L.M.; Graham, R.

Mouse spermatozoa were exposed in vitro for 1 h to 27- or 2,450-MHz CW RF radiation at SARs of 0 to 90 W/kg under isothermal (37 +/- 0.2 degrees C) conditions. Exposure at either frequency to RF radiation at SARs of 50 W/kg or greater resulted in a statistically significant reduction in the ability of irradiated sperm to fertilize mouse ova in vitro (P less than .05). Over the range of SARs there was no apparent difference in the effects of 27- vs. 2,450-MHz RF radiation. There were no readily detectable exposure effects on spermatozoan morphology, ultrastructure, or capacitation. Themore » reduction of in vitro fertilization is attributed to a direct effect of RF radiation on spermatozoa rather than to heating.« less

The epididymis, cytoplasmic droplets and male fertility.

PubMed

Cooper, Trevor G

2011-01-01

The potential of spermatozoa to become motile during post-testicular maturation, and the relationship between the cytoplasmic droplet and fertilizing capacity are reviewed. Post-testicular maturation of spermatozoa involves the autonomous induction of motility, which can occur in vivo in testes with occluded excurrent ducts and in vitro in testicular explants, and artefactual changes in morphology that appear to occur in the testis in vitro. Both modifications may reflect time-dependent oxidation of disulphide bonds of head and tail proteins. Regulatory volume decrease (RVD), which counters sperm swelling at ejaculation, is discussed in relation to loss of cytoplasmic droplets and consequences for fertility. It is postulated that: (i) fertile males possess spermatozoa with sufficient osmolytes to drive RVD at ejaculation, permitting the droplet to round up and pinch off without membrane rupture; and (ii) infertile males possess spermatozoa with insufficient osmolytes so that RVD is inadequate, the droplet swells and the resulting flagellar angulation prevents droplet loss. Droplet retention at ejaculation is a harbinger of infertility caused by failure of the spermatozoon to negotiate the uterotubal junction or mucous and reach the egg. In this hypothesis, the epididymis regulates fertility indirectly by the extent of osmolyte provision to spermatozoa, which influences RVD and therefore droplet loss. Man is an exception, because ejaculated human spermatozoa retain their droplets. This may reflect their short midpiece, approximating head length, permitting a swollen droplet to extend along the entire midpiece; this not only obviates droplet migration and flagellar angulation but also hampers droplet loss.

Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast.

PubMed

Yurchenko, Olga V

2012-02-01

Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9+2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 μm in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 μm in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically. Copyright © 2011 Elsevier Ltd. All rights reserved.

Internalization of silver nanoparticles into mouse spermatozoa results in poor fertilization and compromised embryo development

PubMed Central

Yoisungnern, Ton; Choi, Yun-Jung; Woong Han, Jae; Kang, Min-Hee; Das, Joydeep; Gurunathan, Sangiliyandi; Kwon, Deug-Nam; Cho, Ssang-Goo; Park, Chankyu; Kyung Chang, Won; Chang, Byung-Soo; Parnpai, Rangsun; Kim, Jin-Hoi

2015-01-01

Silver nanoparticles (AgNPs) have many features that make them attractive as medical devices, especially in therapeutic agents and drug delivery systems. Here we have introduced AgNPs into mouse spermatozoa and then determined the cytotoxic effects of AgNPs on sperm function and subsequent embryo development. Scanning electron microscopy and transmission electron microscopy analyses showed that AgNPs could be internalized into sperm cells. Furthermore, exposure to AgNPs inhibited sperm viability and the acrosome reaction in a dose-dependent manner, whereas sperm mitochondrial copy numbers, morphological abnormalities, and mortality due to reactive oxygen species were significantly increased. Likewise, sperm abnormalities due to AgNPs internalization significantly decreased the rate of oocyte fertilization and blastocyst formation. Blastocysts obtained from AgNPs-treated spermatozoa showed lower expression of trophectoderm-associated and pluripotent marker genes. Overall, we propose that AgNPs internalization into spermatozoa may alter sperm physiology, leading to poor fertilization and embryonic development. Such AgNPs-induced reprotoxicity may be a valuable tool as models for testing the safety and applicability of medical devices using AgNPs. PMID:26054035

Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae).

PubMed

Su, Cai Xia; Chen, Jie; Shi, Fu Ming; Guo, Ming Shen; Chang, Yan Lin

2017-07-01

The acrosome complex plays an indispensable role in the normal function of mature spermatozoa. However, the dynamic process of acrosome complex formation in insect remains poorly understood. Gampsocleis gratiosa Brunner von Wattenwyl possesses the typical characteristic of insect sperms, which is tractable in terms of size, and therefore was selected for the acrosome formation study in this report. The results show that acrosome formation can be divided into six phases: round, rotating, rhombic, cylindrical, transforming and mature phase, based on the morphological dynamics of acrosome complex and nucleus. In addition, the cytoskeleton plays a critical role in the process of acrosome formation. The results from this study indicate that: (1) glycoprotein is the major component of the acrosome proper; (2) the microfilament is one element of the acrosome complex, and may mediate the morphologic change of the acrosome complex; (3) the microtubules might also shape the nucleus and acrosome complex during the acrosome formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative analysis of in vitro characteristics of fresh and frozen-thawed urethral and epididymal spermatozoa from cats (Felis domesticus).

PubMed

Prochowska, Sylwia; Niżański, Wojciech; Partyka, Agnieszka

2016-11-01

The first aim of this study was to provide a comprehensive analysis of structural and functional features of spermatozoa in semen collected from the same cat by two methods: urethral catheterization and epididymis slicing. The second aim was to assess if feline urethral (CT) and epididymal (EP) spermatozoa undergo the same changes during cryopreservation and to compare the postthaw characteristics of spermatozoa collected by the two methods. In the first phase, CT and EP semen were collected from 20 cats, and sperm motility, viability, morphology, computer-assisted sperm analysis (CASA) parameters, membrane and acrosome integrity, mitochondrial potential, lipid peroxidation, and chromatin status were assessed. In the second phase, both types of semen collected from 10 cats were cryopreserved, thawed, and the same sperm parameters were assessed as in fresh semen. Fresh CT spermatozoa (phase I) showed higher (P < 0.05) motility (subjective: median 75.0% vs. 62.5%; by CASA: mean ± SD 60.2 ± 10.7% vs. 43.1 ± 16.7%), morphology (mean ± SD, 57.5 ± 9.6% vs. 45.2 ± 15.9%), and membrane integrity (median live: 89.2% vs. 79.8%). Other parameters were not different between CT and EP spermatozoa. After cryopreservation (phase II), spermatozoa from both types of semen did not differ significantly, except for lipid peroxidation of live sperm cells (median CT: 3.5%, EP: 1.7%, P < 0.05). Urethral and EP spermatozoa showed a similar, significant drop in motility (CT to 18.6 ± 10.3% and EP to 21.6 ± 12.1%, P < 0.05), progressive motility (CT to 6.8 ± 5.9% and EP to 8.3 ± 8.8%, P < 0.05), and rapid movement (from 34.3 ± 20.6% to 8.5 ± 7.0% in CT and from 26.0 ± 14.7% to 10.1 ± 10.4% in EP, P < 0.05), whereas other motion characteristics assessed by CASA were not affected (P > 0.05). The strongest change after cryopreservation was noted in high mitochondrial potential (median CT: 1.3%, EP: 2.2%). Although cryopreservation increased acrosome damage and lipid peroxidation, the level of these changes in the population of live sperm cells remained low (median acrosome damage: CT: 3.3%, EP: 4.5%, lipid peroxidation CT: 3.5%, EP: 1.7%). Cryopreservation did not affect chromatin structure (median percent DNA fragmentation index, CT: 3.3%, EP: 2.3%). In this study, we confirmed that urethral catheterization for collection of semen allows the retrieval of spermatozoa with quality equally good as in those obtained by epididymal slicing. Spermatozoa from both types of semen collected showed similar characteristics after freezing/thawing so both types can be used for semen banking. Copyright © 2016 Elsevier Inc. All rights reserved.

Supplementation of soybean lecithin-based semen extender by antioxidants: complementary flowcytometric study on post-thawed ram spermatozoa.

PubMed

Sharafi, Mohsen; Zhandi, Mahdi; Akbari Sharif, Abbas

2015-06-01

The purpose of the current study was to evaluate the effects of cysteine (C) and glutathione (G) on the post-thawed ram sperm quality. Collected semen samples from four mature rams were diluted with five soybean lecithin (SL)-based extenders containing: no antioxidant (SL-0), 5 mM cysteine (SL-C5), 10 mM cysteine (SL-C10), 5 mM glutathione (SL-G5) and 10 mM glutathione (SL-G10). After freeze-thawing process, motion and velocity parameters, plasma membrane integrity and functionality, morphological abnormality, lipid peroxidation, acrosomal status, mitochondria activity, and apoptosis status of post-thawed ram spermatozoa were assessed. The results showed that SL-C10 increased the total motility and plasma membrane integrity (p < 0.05) of post-thawed ram spermatozoa (55.86 ± 1.37 and 60.57 ± 1.34 %) compared to other extenders. Progressive motility was significantly higher in SL-C10 (24.71 ± 1.13 %) compared to SL-0 (20 ± 1.13 %) and SL-G10 (15 ± 1.13 %). Mitochondrial activity was significantly higher in SL-C10 (56.83 ± 2.29 %) compared to SL-G10 (38.75 ± 2.29 %). Capacitation and acrosomal status, lipid peroxidation, and the percentage of dead spermatozoa were not affected by different extenders. The percentage of live spermatozoa was higher in SL-C10 (56.33 ± 1.35 %) compared to other extenders. Also, SL-C10 resulted in a lower percentage of apoptotic spermatozoa (14.17 ± 0.53 %) compared to other extenders. The results of this study showed that supplementation of SL-based ram semen extender with 10 mM cysteine resulted in an improved quality of post-thawed ram spermatozoa.

Body mass index and human sperm quality: neither one extreme nor the other.

PubMed

Luque, E M; Tissera, A; Gaggino, M P; Molina, R I; Mangeaud, A; Vincenti, L M; Beltramone, F; Larcher, J Sad; Estofán, D; Fiol de Cuneo, M; Martini, A C

2017-04-01

The aim of the present study was to investigate the still contentious association between body mass index (BMI) and seminal quality. To this end, 4860 male patients (aged 18-65 years; non-smokers and non-drinkers), were classified according to BMI as either underweight (UW; BMI <20kgm -2 ; n=45), normal weight (NW; BMI 20-24.9kgm -2 ; n=1330), overweight (OW; BMI 25-29.9kgm -2 ; n=2493), obese (OB; BMI 30-39.9kgm -2 ; n=926) or morbidly obese (MOB; BMI ≥40kgm -2 ; n=57). Conventional semen parameters and seminal concentrations of fructose, citric acid and neutral α-glucosidase (NAG) were evaluated. The four parameters that reflect epididymal maturation were significantly lower in the UW and MOB groups compared with NW, OW and OB groups: sperm concentration, total sperm count (103.3±11.4 and 121.5±20.6 and vs 157.9±3.6, 152.4±2.7 or 142.1±4.3 spermatozoa ejaculate -1 respectively, P<0.05), motility (41.8±2.5 and 42.6±2.6 vs 47.8±0.5, 48.0±0.4 or 46.3±0.6 % of motile spermatozoa respectively, P<0.05) and NAG (45.2±6.6 and 60.1±7.9 vs 71.5±1.9, 64.7±1.3 or 63.1±2.1 mU ejaculate-1 respectively, P<0.05). Moreover, the percentage of morphologically normal spermatozoa was decreased in the MOB group compared with the UW, NW, OW and OB groups (4.8±0.6% vs 6.0±0.8%, 6.9±0.1%, 6.8±0.1 and 6.4±0.2%, respectively; P<0.05). In addition, men in the MOB group had an increased risk (2.3- to 4.9-fold greater) of suffering oligospermia and teratospermia (P<0.05). Both morbid obesity and being underweight have a negative effect on sperm quality, particularly epididymal maturation. These results show the importance of an adequate or normal bodyweight as the natural best option for fertility, with both extremes of the BMI scale as negative prognostic factors.

Effects of severe chronic scrotal Dermatophilus congolensis (kirchi) infection on semen characteristics in Zebu/Friesian crossbred bulls and effect of long-acting terramycin chemotherapy.

PubMed

Sekoni, V O

1993-07-01

The semen characteristics of 12 Zebu/Friesian crossbred bulls, aged 2 to 3 years, were studied during a 21-month period. At the 12th month of the study, the commencement of the rainy season, the bulls were infected naturally with Dermatophilus congolensis . Lesions were scattered over the body and limbs, but were particularly pronounced on the scrotum. Monthly treatments with injection of terramycin were begun as soon as lesions were detected and continued until the end of the study. The lesions worsened and became pronounced particularly on the scrotum of all the bulls. Scrotal scab formation caused by infection became prominent at the 14th month of the study. Until that period, the bulls had normal semen characteristics. From the 15th month until the end of the study, there was progressive deterioration of semen characteristics in all the bulls; this was manifested by some or all of the following effects: decreased volume, increased percentage of dead spermatozoa, increased percentage of total sperm morphological abnormalities, decreased percentage of progressively motile spermatozoa, oligospermia and terminal azoospermia. Therefore, severe chronic scrotal dermatophilosis may be a significant cause of infertility or sterility in bulls.

In vitro study of Zika virus infection in boar semen.

PubMed

Luplertlop, Natthanej; Suwanmanee, San; Ampawong, Sumate; Vongpunsawad, Sompong; Poovorawan, Yong

2017-10-01

Zika virus (ZIKV) is an important arbovirus that is capable of directly infecting neuronal cells. Infection can cause microcephaly in fetuses and Guillain-Barré syndrome in adults. Recent epidemiological studies have shown that ZIKV is sexually transmitted, especially from infected males to uninfected females. This study aimed to investigate the transmission pattern of ZIKV in semen using boar semen. Experiments were performed ex vivo using semen from healthy boar. The samples were infected with ZIKV, and viral RNA was detected and cell morphology was examined at different time points postinfection. ZIKV infection was confirmed by transmission electron microscopy. Viral RNA levels were found to markedly decrease as the time postinfection increased, without any evidence of virus replication. The sperm showed no significant changes in morphology. Transmission electron microscopy revealed the presence of virus-free sperm, suggesting that ZIKV cannot replicate in boar semen. We suggest three possible reasons underlying this phenomenon. First, the spermatozoa of boar might not be the target of ZIKV associated with sexual transmission. Second, the effect of the external environment on spermatozoa may affect ZIKV replication. Third, ZIKV may not be tropic for spermatozoa. This ex vivo study might be used as a platform to study the association of sexual transmission with ZIKV in other longer-lasting cells, such as Leydig or Sertoli cells.

Synergetic Effects of K, Ca, Cu and Zn in Human Semen in Relation to Parameters Indicative of Spontaneous Hyperactivation of Spermatozoa

PubMed Central

Bolanca, Ivan; Obhodas, Jasmina; Ljiljak, Dejan; Matjacic, Lidija; Kuna, Krunoslav

2016-01-01

We have observed that sperm quality parameters indicative of spermatozoa hyperactivation such are lower “linearity” and “straightness”, and as showed by this research “elongation”, were more pronounced in patients with normal spermiogram compared to the group of men with reduced sperm motility who were undergoing routine in vitro fertilisation. The research encompassed 97 men diagnosed with normozoospermia (n = 20), asthenozoospermia (n = 54) and oligoasthenozoospermia (n = 23). The findings indicate that sperm quality of patients with normal spermiogram diagnosed according to WHO criteria, may be compromised by showing premature spontaneous hyperactivation which can decrease the chances of natural conception. We assessed synergistic effects of multiple chemical elements in ejaculated semen to find if premature spontaneous hyperactivation of spermatozoa can be a sign of imbalanced semen composition especially of elements K, Ca, Cu and Zn. Human semen samples showing low or high baseline status of chemical elements concentrations were found in samples from all three diagnostic groups. However, correlation of K/Ca and Cu/Zn ratios, taking into account samples from all three groups of men, were negative at statistical significance level p = 0.01. We tested if the negative correlation between K/Ca and Cu/Zn ratio works for greater number of semen samples. We found the negative correlation to be valid for 175 semen samples at statistical significance of p = 0.00002. The ratio of K/Ca and Cu/Zn, i.e. increased concentrations of K and Zn in comparison to concentrations of Ca and Cu, were associated with a decrease of “straightness” in the group of men with normal spermiogram and pronounced spontaneous hyperactivation of spermatozoa, implying that these elements act in synergy and that the balance of elements and not their absolute concentrations plays the major role in premature spermatozoa hyperactivation in ejaculated semen. PMID:27031102

The effect of increased ozone concentrations in the air on selected aspects of rat reproduction.

PubMed

Jedlińska-Krakowska, M; Gizejewski, Z; Dietrich, G J; Jakubowski, K; Glogowski, J; Penkowski, A

2006-01-01

Five-month-old male rates were exposed to 0.5 ppm ozone for 50 days, 5 hours a day. A week before the completion of ozone exposure, a biological test was performed to determine the fertilization rate and the survival rate of newborns in both ozone-exposed and control animals. After 50 days, the rats were sacrificed with an overdose of halotane, and testes were collected to assess the morphology and motility of spermatozoa. Neither the morphology of spermatozoa nor motility parameters determined by the CASA (computer-assisted sperm analysis) system showed statistically significant differences between ozone-exposed and control males. The number of successful matings and the survival rate of newborns per litter within one year postpartum were also similar in both groups. However, sperm concentration was by 17% lower in ozone-exposed rats, compared with the control animals.

An intact acrosome is required for the chemotactic response to progesterone in mouse spermatozoa.

PubMed

Guidobaldi, Hector A; Hirohashi, Noritaka; Cubilla, Marisa; Buffone, Mariano G; Giojalas, Laura C

2017-04-01

Mammalian sperm become fertilization-competent in the oviduct, during a process known as capacitation that involves the acquisition of the ability to exocytose the acrosome but also the chemotactic responses-both of which contribute to successful fertilization. Chemotaxis is used by spermatozoa to orient and to locate the egg; the acrosome reaction facilitates sperm binding to and fusing with the egg membrane. Mammalian spermatozoa are able to sense picomolar concentrations of progesterone, which drives chemotactic behavior. The state of the acrosome during the chemotactic response, however, is unknown. Genetically modified mouse spermatozoa were employed in a chemotaxis assay under fluorescence microscopy to evaluate their acrosome status while swimming, allowing us to elucidate the acrosome integrity of sperm responding to progesterone-induced chemotaxis. We first showed that wild-type mouse spermatozoa chemotactically respond to a gradient of progesterone, and that the genetic modifications employed do not affect the chemotactic behavior of sperm to progesterone. Next, we found that acrosome-intact, but not acrosome-reacted, spermatozoa orient and respond to picomolar concentrations of progesterone and that chemotaxis normally occurs prior to the acrosome reaction. Our results suggest that premature commitment to acrosome exocytosis leads to navigation failure, so proper control and timing of the acrosome reaction is required for fertilization success and male fertility. © 2017 Wiley Periodicals, Inc.

Dimensions of human ejaculated spermatozoa in Papanicolaou-stained seminal and swim-up smears obtained from the Integrated Semen Analysis System (ISAS®)

PubMed Central

Bellastella, Giuseppe; Cooper, Trevor G.; Battaglia, Marina; Ströse, Anda; Torres, Inma; Hellenkemper, Barbara; Soler, Carles; Sinisi, Antonio A.

2010-01-01

Objective measurements are required for computer-aided sperm morphometric analysis (CASMA) machines to distinguish normal from abnormal sperm heads. The morphometric characteristics of spermatozoa in 72 samples of semen and of spermatozoa from 72 other semen samples after swim-up were quantified by the semi-automated Integrated Sperm Analysis System (ISAS) computer-aided system, which measured the sperm head parameters length (L), width (W), area (A), perimeter (P), acrosomal area (Ac), and the derived values L/W and P/A. For each man a homogeneous population of distributions characterized seminal spermatozoa (7 942 cells: median values L 4.4 μm, W 2.8 μm, A 9.8 μm2, P 12.5 μm, Ac 47.5%, L/W 1.57, P/A 1.27), and there was no significant difference in within- and among-individual variation. Different men could have spermatozoa of significantly different dimensions. Head dimensions for swim-up spermatozoa from different men (4 812 cells) were similar to those in semen, differing only by 2%–5%. The values of L, W and L/W fell within the limits given by the World Health Organization (WHO). Although these samples were not biologically matched, linear mixed-effects statistical analyses permitted valid comparison of the groups. A subpopulation of 404 spermatozoa considered to fit the stringent criteria of WHO 'normal' seminal spermatozoa from both semen and swim-up were characterized by median values (and 95% confidence intervals) of L, 4.3 μm (3.8–4.9), W, 2.9 μm (2.6–3.3), A, 10.2 μm2 (8.5–12.2), P, 12.4 μm (11.3–13.9), Ac, 49% (36–60), L/W, 1.49 (1.32–1.67) and P/A, 1.22 (1.11–1.35). These median values fall within the 95th centile confidence limits given by WHO, but the confidence intervals for L and W were larger. Although these differences in head dimensions among men and after swim-up could be detected by CASMA, the small differences make it unlikely that technicians would be able to distinguish them. The values could be used as default sperm head values for the CASMA machine used here. PMID:20852650

Dimensions of human ejaculated spermatozoa in Papanicolaou-stained seminal and swim-up smears obtained from the Integrated Semen Analysis System (ISAS(®)).

PubMed

Bellastella, Giuseppe; Cooper, Trevor G; Battaglia, Marina; Ströse, Anda; Torres, Inma; Hellenkemper, Barbara; Soler, Carles; Sinisi, Antonio A

2010-11-01

Objective measurements are required for computer-aided sperm morphometric analysis (CASMA) machines to distinguish normal from abnormal sperm heads. The morphometric characteristics of spermatozoa in 72 samples of semen and of spermatozoa from 72 other semen samples after swim-up were quantified by the semi-automated Integrated Sperm Analysis System (ISAS) computer-aided system, which measured the sperm head parameters length (L), width (W), area (A), perimeter (P), acrosomal area (Ac), and the derived values L/W and P/A. For each man a homogeneous population of distributions characterized seminal spermatozoa (7 942 cells: median values L 4.4 μm, W 2.8 μm, A 9.8 μm(2), P 12.5 μm, Ac 47.5%, L/W 1.57, P/A 1.27), and there was no significant difference in within- and among-individual variation. Different men could have spermatozoa of significantly different dimensions. Head dimensions for swim-up spermatozoa from different men (4 812 cells) were similar to those in semen, differing only by 2%-5%. The values of L, W and L/W fell within the limits given by the World Health Organization (WHO). Although these samples were not biologically matched, linear mixed-effects statistical analyses permitted valid comparison of the groups. A subpopulation of 404 spermatozoa considered to fit the stringent criteria of WHO 'normal' seminal spermatozoa from both semen and swim-up were characterized by median values (and 95% confidence intervals) of L, 4.3 μm (3.8-4.9), W, 2.9 μm (2.6-3.3), A, 10.2 μm(2) (8.5-12.2), P, 12.4 μm (11.3-13.9), Ac, 49% (36-60), L/W, 1.49 (1.32-1.67) and P/A, 1.22 (1.11-1.35). These median values fall within the 95th centile confidence limits given by WHO, but the confidence intervals for L and W were larger. Although these differences in head dimensions among men and after swim-up could be detected by CASMA, the small differences make it unlikely that technicians would be able to distinguish them. The values could be used as default sperm head values for the CASMA machine used here.

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.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

PubMed

Yoshida, Masa-aki; Yamada, Lixy; Ochi, Hiroe; Iwata, Yoko; Tamura-Nakano, Miwa; Sawada, Hitoshi; Sauer, Warwick H H; Ogura, Atsushi; Hirohashi, Noritaka

2014-08-01

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa. Copyright © 2014 Elsevier Inc. All rights reserved.

Curcumin Inhibits The Adverse Effects of Sodium Arsenite in Mouse Epididymal Sperm

PubMed Central

Momeni, Hamid Reza; Eskandari, Najmeh

2016-01-01

Background The aim of this study was to investigate the effects of curcumin on epididy- mal sperm parameters in adult male Navel Medical Research Institute (NMRI) mice ex- posed to sodium arsenite. Materials and Methods In this experimental study, we divided the animals into four groups: control, sodium arsenite (5 mg/kg), curcumin (100 mg/kg) and curcumin+sodium arsenite. Exposures were performed by intraperitoneal injections for a 5-week period. After the exposure period, we recorded the animals’ body and left testes weights. The left caudal epididymis was used to count the sperm number and analyze motility, viability, morphological abnormalities, acrosome reaction, DNA integrity, and histone-protamine replacement in the spermatozoa. One-way analysis of variance (ANOVA) followed by the Tukey’s test was used to assess the statistical significance of the data with SPSS 16.0. P<0.05 was considered significant. Results Mice exposed to sodium arsenite showed a significant decrease in the num- ber, motility, viability, normal sperm morphology and acrosome integrity of spermato- zoa compared to the control group. In the curcumin+sodium arsenite group, curcumin significantly reversed these adverse effects to the point where they approximated the control. In addition, the application of curcumin alone had no significant difference in these parameters compared to the control and curcumin+sodium arsenite groups. However, we observed no significant differences in the body and the testis weight as well as the DNA integrity and histone-protamine replacement in the spermatozoa of the four groups. Conclusion Curcumin compensated for the toxic effects of sodium arsenite on a number of sperm parameters in adult mice. PMID:27441059

Chronic consumption of Hypericum humifusum leaf extracts impairs epididymis spermatozoa characters in association with oxidative stress in adult male Wistar rats.

PubMed

Hammami, Imen; Ali, Ridha Ben; Nahdi, Afef; Kallech-Ziri, Olfa; Boussada, Marwa; El May, Ahmed; El May, Michèle Véronique

2017-09-01

Recently, there has been increasing interest in Hypericum (Hypericaceae) genus. The first part of the present study focused on the phytochemical analysis of the methanolic and aqueous extracts of Hypericum humifusum leaves. The second part of the study investigated the effect of Hypericum humifusum leaf extracts on male reproductive parameters. 30 male rats were grouped into control (1mL/rat, distilled water), treated by 200mg/kg body weight (bw) aqueous extract (A200), 400mg/kg bw aqueous extract (A400), 10mg/kg bw methanolic extract (M10) and 20mg/kg bw methanolic extract (M20) groups. The phytochemical analysis revealed the presence of tannins, flavonoids, steroids, carbohydrates, and phenolic compounds. After thirty-day treatment, body and reproductive organs were weighed. Testes in all rat groups were processed for biochemical assays and histopathological examinations. Epididymis sperm analyses were also performed. Testicular tissue homogenate samples were used for Malondialdehyde (MDA), catalase and superoxide dismutase (SOD) measurements. We showed that Hh extracts induced a severe seminiferous tubular damage with an increase in the percentage of empty seminiferous tubules. Epididymis sperm analysis revealed a significant reduction in density and viability of sperm with alteration of spermatozoa morphology. Also, we found that Hh leaf extracts decreased plasma total cholesterol, HDL-cholesterol and triglycerides levels. These results were associated with an increase of MDA levels and a decrease of catalase and SOD activities in testis tissues. Our finding revealed that chronic consumption of Hh extracts induces disruption of normal spermatogenesis by alteration of sperm density, viability, and morphology. This action may be due to an inhibition of the antioxidant-defense system. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Progesterone stimulates the long-distance migration of capacitated ram spermatozoa through viscous media under geotactic condition.

PubMed

Martínez-Rodríguez, Carmen; Anel-López, Luis; Alvarez, Mercedes; Ortega-Ferrusola, Cristina; Boixo, Juan Carlos; Peña, Fernando J; Anel, Luis; de Paz, Paulino

2018-05-15

Forward progressive motility of spermatozoa is an essential prerequisite for reproductive success, and sperm navigation is assisted by guidance mechanisms that may depend on micro-environmental factors. In the present study, we performed an integrated analysis of long-distance ram sperm migration in vitro that combined two environmental factors (10 μM progesterone and a geotactic effect) and the physiological status of the cells (capacitation treatment). A penetration assay was used in which spermatozoa had to travel 20 mm in a viscous medium (two media of differing viscosity: acrylamide and hyaluronic acid) through a tube device. The number of migrating spermatozoa, the physiology of the cells (motility analyzed using a CASA system; acrosomal status, viability and active mitochondria evaluated by flow cytometry; DNA fragmentation index calculated by quantitative PCR) and the morphometry of sperm heads (performed using an image analysis system) were evaluated after long-distance sperm migration. Ram sperm capacitation significantly stimulates cell migration through viscous media under geotactic conditions, and this effect is enhanced by progesterone induction. The rheological characteristics of viscous media have a marked impact on ram sperm migration, and acrylamide more favorably facilitates navigation over a large distance. The migrating spermatozoa are morphologically better adapted (high ellipticity) for displacement in viscous media and exhibit remarkably depleted mitochondrial membrane potential. Copyright © 2018 Elsevier Inc. All rights reserved.

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 cooling rate on sperm quality of cryopreserved Andalusian donkey spermatozoa.

PubMed

Demyda-Peyrás, S; Bottrel, M; Acha, D; Ortiz, I; Hidalgo, M; Carrasco, J J; Gómez-Arrones, V; Gósalvez, J; Dorado, J

2018-06-01

The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN 2 ) vapour; P2 (controlled pre-freeze cooling rate): semen pre-cooled at a controlled rate for 73 min and frozen in LN 2 vapour; and P3 (rapid freezing) semen frozen immediately in LN 2 vapour. After thawing at 37 °C for 30 s, semen samples were assessed for motility, morphology, acrosome and plasma membrane integrity; spermatozoa were also tested for DNA integrity. Significant (P < 0.01) differences were found between the cryopreservation protocols for all sperm parameters evaluated, except for DNA integrity. Semen samples frozen using P2 showed significantly (P < 0.01) higher values for sperm motility, morphology, sperm membrane integrity, and acrosome integrity. On the contrary, P3 reduced sperm motility (P < 0.01) and increased the percentage of spermatozoa with damaged plasma membrane (P < 0.001). In our study, we demonstrated that the sperm of Andalusian donkey is particularly sensitive to the cooling rate used before freezing. Furthermore, Andalusian donkey semen can be successfully cryopreserved using controlled cooling rates combined with freezing in LN 2 vapour. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of psychological stress on fertility hormones and seminal quality in male partners of infertile couples.

PubMed

Bhongade, M B; Prasad, S; Jiloha, R C; Ray, P C; Mohapatra, S; Koner, B C

2015-04-01

The present study evaluated the effect of psychological stress on male fertility hormones and seminal quality in male partner of infertile couples. Seventy male partners of infertile couples were evaluated for level of psychological stress using Hospital Anxiety and Depression Score (HADS) questionnaire, serum total testosterone, luteinising hormone (LH) and follicle-stimulating hormone (FSH) by electrochemiluminescence assay and serum GnRH by ELISA. Seminal analysis was performed as per WHO guideline. Nineteen (27%) of them had HADS anxiety and depression score ≥8 (abnormal HADS score). The persons having abnormal HADS had lower serum total testosterone, higher serum FSH and LH than those of persons having normal HADS. Serum total testosterone correlated negatively with HADS, but LH and FSH correlated positively. There was no change in GnRH with the change in stress or testosterone levels. Sperm count, motility and morphologically normal spermatozoa were lower in persons having abnormal HADS. Sperm count correlated positively with total testosterone and negatively with FSH and LH. Abnormal sperm motility and morphology were related to lower testosterone and higher LH and FSH levels. Psychological stress primarily lowers serum total testosterone level with secondary rise in serum LH and FSH levels altering seminal quality. Stress management is warranted for male infertility cases. © 2014 Blackwell Verlag GmbH.

Magnetic-activated cell sorting for sperm preparation reduces spermatozoa with apoptotic markers and improves the acrosome reaction in couples with unexplained infertility.

PubMed

Lee, Tsung-Hsien; Liu, Chung-Hsien; Shih, Yang-Tse; Tsao, Hui-Mei; Huang, Chun-Chia; Chen, Hsiu-Hui; Lee, Maw-Sheng

2010-04-01

Couples with unexplained infertility (UI) tend to have low fertilization rates with current IVF procedures. Here, we attempted to identify spermatozoa with apoptotic markers in couples with UI and unsuccessful intrauterine insemination (IUI) and we investigated the efficiency and benefit of magnetic-activated cell sorting (MACS) for sperm preparation in such patients. Sixty couples with UI and two IUI failures were recruited. The sperm were prepared by conventional density gradient centrifugation (DGC) and divided into two aliquots. One aliquot was used as a control and the other was further processed by MACS (D + M). Apoptotic markers were identified using fluorescence-labeled dye and flow cytometry, including externalization of phosphatidylserine (EPS), disrupted mitochondrial membrane potential (MMP) and DNA fragmentation. The fertilization potential of prepared spermatozoa was analyzed by basic semen analysis, computer-aided sperm analysis and the induced acrosome reaction test (IART). After DGC, spermatozoa showed 18.6% EPS, 28.3% disrupted MMP and 13.5% DNA fragmentation. Numbers of spermatozoa with apoptotic markers were significantly reduced by D + M, versus DGC alone (P < 0.001). Although the motility of spermatozoa was slightly decreased after MACS, most sperm motion characteristics were not impaired. Interestingly, the IART significantly improved after D + M, versus DGC alone, especially for the couples with a normal hemizona assay (P < 0.001). The spermatozoa prepared by D + M showed a reduced level of apoptotic markers. Improvement in the IART suggests a high fertilization potential of the processed spermatozoa. The identification of apoptotic markers and use of MACS may be helpful in directing the management plan for patients with UI and multiple IUI failures.

Down-regulation of CatSper1 channel in epididymal spermatozoa contributes to the pathogenesis of asthenozoospermia, whereas up-regulation of the channel by Sheng-Jing-San treatment improves the sperm motility of asthenozoospermia in rats.

PubMed

Wang, Ya-Nan; Wang, Bo; Liang, Ming; Han, Cai-Yan; Zhang, Bin; Cai, Jie; Sun, Wei; Xing, Guo-Gang

2013-02-01

To determine the expression of CatSper1 channel in epididymal spermatozoa in a rat model of asthenozoospermia, induced by cyclophosphamide (CP), and further examine the effects of soluble granules of Sheng-Jing-San (SJS), a traditional Chinese medicine recipe, on CatSper1 expression and sperm motility in the CP-induced asthenozoospermic rats. Placebo-controlled, randomized trial. Neuroscience Research Institute, Peking University, China. Sexually mature male Sprague-Dawley rats (n = 60). In the CP group, CP at the dose of 35 mg/kg intraperitoneally injected into rats once a day for 7 days; in the normal saline (NS) group, 0.9% saline solution was injected as control. Sperm motility and count were evaluated by computer-assisted sperm assay (CASA); protein and mRNA expression of CatSper1 channel in epididymal spermatozoa was determined by Western blotting and quantitative real-time RT-PCR, respectively. The rats were randomly divided into five groups with 12 rats in each group: CP, normal saline (NS), CP + SJS, CP + NS, and treatment naïve. In the CP + SJS group, after the last injection of CP, SJS at a dose of 30 mg/kg was intragastrically administrated to rats once a day for 14 days; in CP + NS group, saline solution instead of SJS was administrated as control. In the treatment naïve group, rats were normally fed for 21 days as controls. We found a statistically significant reduction of the CatSper1 channel, which is associated with an impairment of sperm motility in the epididymal spermatozoa of CP-induced asthenozoospermic rats. Soluble granules of SJS could dramatically restore the CP-induced down-regulation of CatSper1 in epididymal spermatozoa, which greatly improved the sperm motility in the asthenozoospermic rats. Down-regulation of the CatSper1 channel in epididymal spermatozoa likely contributes to the pathogenesis of asthenozoospermia, whereas up-regulation of the channel by SJS improves sperm motility and thus can be used as an effective therapeutic strategy for the treatment of male infertility diagnosed with asthenozoospermia. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Sodium-Hydrogen-Exchanger expression in human sperm and its relationship with semen parameters.

PubMed

Zhang, Zhe; Yang, Yuzhuo; Wu, Han; Zhang, Hongliang; Zhang, Haitao; Mao, Jiaming; Liu, Defeng; Zhao, Lianming; Lin, Haocheng; Tang, Wenhao; Hong, Kai; Jiang, Hui

2017-06-01

Sperm-specific sodium-hydrogen exchanger (sNHE) is essential to maintain sperm normal function in mice; however, its role in human sperm has not been clarified to date. The aim of this study is to investigate the expression pattern of sNHE in human spermatozoa and its relationship with sperm functional parameters. Semen samples from 68 asthenozoospermic and 61 normozoospermic men were analyzed for sperm concentration, motility, and acrosome reaction, and high motile spermatozoa were collected by swim-up method. The expression of sNHE in spermatozoa was detected by Western blot and immunofluorescence staining. The relationship between sNHE expression and sperm parameters was assessed. We identified sNHE is mainly localized to the principal piece of the human sperm tail. The expression of sNHE was positively correlated with sperm concentration, total number, and progressive motility. Moreover, sNHE expression was upregulated in swim-up sperm and associated with most of sperm motility parameters including straight line velocity and curvilinear velocity. Our results also showed that sNHE expression is decreased in sperm from patients with asthenozoospermia compared with that from normal controls. However, no correlation was found between sNHE expression and acrosome reaction in spermatozoa. The expression pattern of sNHE suggested that this protein may be involved in the regulation of sperm motility, and aberration of its expression in sperm may contribute to the pathogenesis of asthenozoospermia.

Association of seminal plasma motility inhibitors/semenogelins with sperm in asthenozoospermia-infertile men.

PubMed

Terai, K; Yoshida, K; Yoshiike, M; Fujime, M; Iwamoto, T

2010-01-01

Seminal plasma motility inhibitors (SPMIs) are proteinase-resistant fragments of semenogelin I and II (Sgs), which are the major proteins of semen coagulum. SPMIs inhibit the motility of spermatozoa, and Sgs are thought to be natural regulators of human sperm function. The mechanism underlying sperm motility regulation and its association with defective motility in infertile men remain unclear. The purpose of this study was to investigate the association between SPMIs and spermatozoa in infertile men with asthenozoospermia. Fifty-four semen samples from 37 asthenozoospermic patients and 17 samples from 9 normal healthy subjects were analyzed. Spermatozoa, washed by Percoll density gradients, were immunostained with anti-SPMI antibody and subjected to flow cytometric analysis. The proportion of spermatozoa labeled with the antibody and the average intensity of fluorescence labeling per spermatozoa were analyzed in relation to the parameters used for semen analysis. A significant negative correlation was found between sperm motility and the proportion (R = -0.68) and intensity (R = -0.38) of labeling. These results suggest that SPMIs remain on the sperm surface after liquefaction. This might account for some disorders of sperm motility observed in infertile men with asthenozoospermia. Copyright © 2010 S. Karger AG, Basel.

Picomolar gradients of progesterone select functional human sperm even in subfertile samples.

PubMed

Gatica, L V; Guidobaldi, H A; Montesinos, M M; Teves, M E; Moreno, A I; Uñates, D R; Molina, R I; Giojalas, L C

2013-09-01

More than 1 million infertility treatments are practiced around the world per year, but only 30% of the couples succeed in taking a baby home. Reproductive technology depends in part on sperm quality, which influences not only fertilization but also embryo development and implantation. In order to provide a better quality sperm subpopulation, innovative sperm selection techniques based on physiological sperm features are needed. Spermatozoa at an optimum state may be selected by following an increasing concentration gradient of picomolar progesterone, a steroid secreted by the cumulus cells at the time of ovulation. In this study we developed a method to recruit spermatozoa at the best functional state, based on sperm guidance toward progesterone. The sperm selection assay (SSA) consists of a device with two wells connected by a tube. One well was filled with the sperm suspension and the other with picomolar progesterone, which diffused inside the connecting tube as a gradient. The sperm quality after the SSA was analyzed in normal and subfertile semen samples. Several sperm parameters indicative of sperm physiological state were determined before and after the SSA: capacitation, DNA integrity and oxidative stress. After the SSA, the mean level of capacitated spermatozoa increased three times in normal and in subfertile samples. The level of sperm with intact DNA was significantly increased, while sperm oxidative stress was decreased after sperm selection. Interestingly, the exposure to a progesterone gradient stimulated the completion of capacitation in some spermatozoa that could not do it by themselves. Thus, the SSA supplies a sperm population enriched with spermatozoa at an optimum physiological state that may improve the assisted reproductive technology outcome.

Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus.

PubMed

Fraczek, M; Piasecka, M; Gaczarzewicz, D; Szumala-Kakol, A; Kazienko, A; Lenart, S; Laszczynska, M; Kurpisz, M

2012-10-01

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility. © 2012 Blackwell Verlag GmbH.

A gravitational test of wave reinforcement versus fluid density models

NASA Technical Reports Server (NTRS)

Johnson, Jacqueline Umstead

1990-01-01

Spermatozoa, protozoa, and algae form macroscopic patterns somewhat analogous to thermally driven convection cells. These bioconvective patterns have attracted interest in the fluid dynamics community, but whether in all cases these waves were gravity driven was unknown. There are two conflicting theories, one gravity dependent (fluid density model), the other gravity independent (wave reinforcement theory). The primary objectives of the summer faculty fellows were to: (1) assist in sample collection (spermatozoa) and preparation for the KC-135 research airplane experiment; and (2) to collaborate on ground testing of bioconvective variables such as motility, concentration, morphology, etc., in relation to their macroscopic patterns. Results are very briefly given.

Habitual alcohol consumption associated with reduced semen quality and changes in reproductive hormones; a cross-sectional study among 1221 young Danish men.

PubMed

Jensen, Tina Kold; Gottschau, Mads; Madsen, Jens Otto Broby; Andersson, Anne-Maria; Lassen, Tina Harmer; Skakkebæk, Niels E; Swan, Shanna H; Priskorn, Lærke; Juul, Anders; Jørgensen, Niels

2014-10-02

Study associations between three measures of alcohol consumption (recent, typical/habitual, binging), semen quality and serum reproductive hormones. Cross-sectional population based study. 1221 young Danish men, aged 18-28 years were recruited when they attended a compulsory medical examination to determine their fitness for military service from 2008 to 2012. Total alcohol consumption: (1) in the week preceding (habitual/typical) the visit (recent alcohol intake), (2) in a typical week and (3) frequency of 'binge drinking' (consuming more than 5 units/day)) in the past 30 days was estimated. Semen quality (volume, sperm concentration, total sperm count, and percentages of motile and morphologically normal spermatozoa) and serum concentration of reproductive hormones (follicle-stimulating hormone, luteinising hormone, testosterone, sex hormone binding globulin, oestradiol, free testosterone and inhibin B). Sperm concentration, total sperm count and percentage of spermatozoa with normal morphology were negatively associated with increasing habitual alcohol intake. This association was observed in men reporting at least 5 units in a typical week but was most pronounced for men with a typical intake of more than 25 units/week. Men with a typical weekly intake above 40 units had a 33% (95% CI 11% to 59%) reduction in sperm concentration compared to men with an intake of 1-5 units/week. A significant increase in serum free testosterone with increasing alcohol consumption the week preceding the visit was found. Binging was not independently associated with semen quality. Our study suggests that even modest habitual alcohol consumption of more than 5 units per week had adverse effects on semen quality although most pronounced associations were seen in men who consumed more than 25 units per week. Alcohol consumption was also linked to changes in testosterone and SHBG levels. Young men should be advised to avoid habitual alcohol intake. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Histological observation of germ cell development and discovery of spermatophores in ovoviviparous black rockfish ( Sebastes schlegeli Hilgendorf) in reproductive season

NASA Astrophysics Data System (ADS)

Feng, Junrong; Liu, Liming; Jiang, Haibin; Wang, Maojian; Du, Rongbin

2014-10-01

Black rockfish ( Sebastes schlegeli) is an important species for culture; however, its reproductive characteristics have not been fully documented. In this study, we investigated the morphology and developmental process of germ cells in this ovoviviparous rockfish in reproductive season (October 2011-November 2012) with histological methods. We found that the gonad of mature fish showed notable seasonal changes in developmental characteristics and morphological structure. The sperm cells matured during a period lasting from October to December, significantly earlier than the oocytes did. A large number of spermatozoa and other cells occurred in testis at different developmental stages. Vitellogenesis in oocytes began in October, and gestation appeared in April next year. Spermatophores were discovered for the first time in Sebastes, which assembled in testis, main sperm duct, oviduct and genital tract, as well as ovarian cavity in October and April. These organs may serve either as production or hiding places for spermatophores and spermatozoa which were stored and transported in form of spermatophores. Testicular degeneration started from the distal part of testis in April, with spermatophores assembled in degenerating testis and waiting for transportation. The copulation probably lasted for a long period, during which the spermatozoa were discharged in batches as spermatophores. These spermatophores were coated with sticky materials secreted from the interstitial areas of testis and the main sperm duct, then transported into ovary.

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 effects on boar sperm quality of dietary supplementation with omega-3 polyunsaturated fatty acids differ among porcine breeds.

PubMed

Yeste, Marc; Barrera, Xavier; Coll, David; Bonet, Sergi

2011-07-01

The present study was undertaken to shed light on the relationship between boar sperm quality and dietary supplementation with omega-3 polyunsaturated fatty acids, which has been reported inconsistently in the literature. With this aim, such effects were evaluated and compared among three different porcine breeds: Duroc, Large-White, and Pietrain. Animals were randomly separated into two groups and fed either with a control diet or with a diet supplemented with omega-3. Sperm quality of these boar (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) was assessed every week for a 26-week period. Supplementing boar's diet with omega-3 did not affect ejaculate volume, sperm concentration, sperm motility, sperm viability, and acrosome and mitochondrial sheath integrity. In contrast, supplemented diet positively affected both sperm morphology in Large-White and Pietrain breeds and the osmotic resistance of Pietrain spermatozoa. No effects were seen for the same sperm parameters in Duroc breed. These breed-differences in boar fed with the supplemented diet could explain the contradictions in literature and might be related with differences in the composition of plasma membrane among breeds reported by other authors. Because no harmful effects were observed in the three evaluated breeds, but positive effects in Large-White and Pietrain boar, we can conclude that omega-3 fatty acids may be added to boar's diet at the levels used in this study to improve their sperm quality. More research is, however, needed to determine how these fatty acids differently affect the morphology and the osmotic resistance of the spermatozoa in these breeds. Copyright © 2011 Elsevier Inc. 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.

Puma (Puma concolor) epididymal sperm morphometry

PubMed Central

Cucho, Hernán; Alarcón, Virgilio; Ordóñez, César; Ampuero, Enrique; Meza, Aydee; Soler, Carles

2016-01-01

The Andean puma (Puma concolor) has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component (PC) analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species. PMID:27678466

Puma (Puma concolor) epididymal sperm morphometry.

PubMed

Cucho, Hernán; Alarcón, Virgilio; Ordóñez, César; Ampuero, Enrique; Meza, Aydee; Soler, Carles

2016-01-01

The Andean puma (Puma concolor) has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component (PC) analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species.

Evaluation of gram stain as an alternative in the assessment of human spermatozoa quality.

PubMed

Mantas, D; Msaouel, P; Angelopoulou, R

2006-01-01

During spermiogenesis, protaminosis and sperm chromatin condensation are important prerequisites for the preservation of DNA integrity in spermatozoa. The aim of this study is to assess Gram stain as an alternative technique for the evaluation of human sperm chromatin condensation status. Aniline blue and Gram staining were applied to semen samples from 34 donors in order to determine the relationship between sperm chromatin condensation and infertility. In addition, the possible correlation between morphology and vitality (eosin-Y staining) of spermatozoa compared with their nuclear status (aniline blue and Gram staining) was studied. Chromatin condensation and sperm vitality were significantly higher in fertile men compared to the subfertile. A significant correlation was found between chromatin condensation and (a) sperm vitality (p < 0.01), and (b) nuclear protein status (p < 0.01). Gram staining may be used as a routine method in assisted reproduction laboratories and could assist in the evaluation of sperm quality as well as in the selection of the appropriate fertilization technique.

Hydrodynamics of insect spermatozoa

NASA Astrophysics Data System (ADS)

Pak, On Shun; Lauga, Eric

2010-11-01

Microorganism motility plays important roles in many biological processes including reproduction. Many microorganisms propel themselves by propagating traveling waves along their flagella. Depending on the species, propagation of planar waves (e.g. Ceratium) and helical waves (e.g. Trichomonas) were observed in eukaryotic flagellar motion, and hydrodynamic models for both were proposed in the past. However, the motility of insect spermatozoa remains largely unexplored. An interesting morphological feature of such cells, first observed in Tenebrio molitor and Bacillus rossius, is the double helical deformation pattern along the flagella, which is characterized by the presence of two superimposed helical flagellar waves (one with a large amplitude and low frequency, and the other with a small amplitude and high frequency). Here we present the first hydrodynamic investigation of the locomotion of insect spermatozoa. The swimming kinematics, trajectories and hydrodynamic efficiency of the swimmer are computed based on the prescribed double helical deformation pattern. We then compare our theoretical predictions with experimental measurements, and explore the dependence of the swimming performance on the geometric and dynamical parameters.

Modelling of energy expended by free swimming spermatozoa in temperature-dependent viscous semen.

PubMed

Foo, Jong Yong Abdiel

2010-01-01

Derived models of fertilization kinetics have relied upon estimates of the swimming velocity of spermatozoa from the insemination site to a fallopian tube. However, limited derivations are available describing the probability and energy expended when spermatozoa collide with one another. An analytic approach of spermatozoon motion in a linear viscoelastic fluid is adopted to simplify the derivation. The complex kinematics of motion of an inextensible flagellum is modelled as planar flagellar wave of small amplitude. In humans, a temperature difference is expected between the cooler tubal isthmus and the warmer tubal ampulla. Thus, fluidic characteristics of semen such as viscosity can vary along the female reproductive tract. The results suggest that the probability of spermatozoa colliding in relatively lower viscous semen increases by 64.87% for a 0.5 degrees C surge in temperature. Moreover, this increases for a denser concentration of spermatozoa due to the limited semen volume available to manoeuvre. In addition, the propulsive forces and shear stress were 39.35% lower in less viscous semen due to an increase in temperature of only 0.5 degrees C. Hence, the described derivations herein can assist in the understanding of work done by a normal motile spermatozoon in a pool of semen.

Cofilin is correlated with sperm quality and influences sperm fertilizing capacity in humans.

PubMed

Chen, S M; Chen, X M; Lu, Y L; Liu, B; Jiang, M; Ma, Y X

2016-11-01

Spermatozoa should undergo a series of biochemical modifications in female reproduction tract, which is collectively called sperm capacitation. The capacitated spermatozoa can bind to the egg zona pellucida, resulting in the occurrence of acrosome reaction which enabled spermatozoa penetrate into the egg. The formation of actin plays an important role in these processes. Actin polymerized during sperm capacitation, but the polymers dispersed before acrosome reaction. In this study, we take our focus on actin-binding protein, cofilin. Our results showed that the % and intensity of sperm expressing cofilin in normal sperm were significantly higher than in abnormal sperm, and the sperm expressing cofilin was correlated with sperm quality. Furthermore, treatment with anti-cofilin antibody increased the percentage of sperm capacitation and inhibited progesterone- or A23187- induced acrosome reaction in a dose-dependent manner. The presence of 100 ng/mL anti-cofilin antibodies markedly blocked the sperm penetration of zona-free hamster eggs. Besides, immunofluorescence results revealed that cofilin was colocalized with F-actin in the midpiece of spermatozoa; however, phospho-cofilin was expressed in the tail rather than in the midpiece of spermatozoa, which was not colocalized with F-actin in spermatozoa. Moreover, western blot revealed that phospho-cofilin increased in sperm capacitation, and the total cofilin and cofilin in insoluble fraction increased in acrosome reaction; immunofluorescence results showed that the amount of cofilin in acrosome increased in sperm capacitation. In conclusion, our study revealed that cofilin expression in human sperm is correlated with sperm quality and the alterations of cofilin and phospho-cofilin in fertilization affects sperm capacitation, acrosome reaction, and spermatozoa-oocyte fusion. © 2016 American Society of Andrology and European Academy of Andrology.

ATP-activated P2X2 current in mouse spermatozoa

PubMed Central

Navarro, Betsy; Miki, Kiyoshi; Clapham, David E.

2011-01-01

Sperm cells acquire hyperactivated motility as they ascend the female reproductive tract, which enables them to overcome barriers and penetrate the cumulus and zona pellucida surrounding the egg. This enhanced motility requires Ca2+ entry via cation channel of sperm (CatSper) Ca2+-selective ion channels in the sperm tail. Ca2+ entry via CatSper is enhanced by the membrane hyperpolarization mediated by Slo3, a K+ channel also present in the sperm tail. To date, no transmitter-mediated currents have been reported in sperm and no currents have been detected in the head or midpiece of mature spermatozoa. We screened a number of neurotransmitters and biomolecules to examine their ability to induce ion channel currents in the whole spermatozoa. Surprisingly, we find that none of the previously reported neurotransmitter receptors detected by antibodies alone are functional in mouse spermatozoa. Instead, we find that mouse spermatozoa have a cation-nonselective current in the midpiece of spermatozoa that is activated by external ATP, consistent with an ATP-mediated increase in intracellular Ca2+ as previously reported. The ATP-dependent current is not detected in mice lacking the P2X2 receptor gene (P2rx2−/−). Furthermore, the slowly desensitizing and strongly outwardly rectifying ATP-gated current has the biophysical and pharmacological properties that mimic heterologously expressed mouse P2X2. We conclude that the ATP-induced current on mouse spermatozoa is mediated by the P2X2 purinergic receptor/channel. Despite the loss of ATP-gated current, P2rx2−/− spermatozoa have normal progressive motility, hyperactivated motility, and acrosome reactions. However, fertility of P2rx2−/− males declines with frequent mating over days, suggesting that P2X2 receptor adds a selection advantage under these conditions. PMID:21831833

Quality of 4-hourly ejaculates--levels of calcium and magnesium.

PubMed

Valsa, J; Skandhan, K P; Gusani, P H; Sahab Khan, P; Amith, S

2013-02-01

A four-hourly ejaculation study was conducted in which eleven normal healthy subjects participated. Five of them discontinued after submitting three samples. One alone was present for submission at the end of 16 h (fifth ejaculate), which was his last submission. Physical exhaustion was the sole reason for all participants for their discontinuation from the study. The result showed a decrease in semen volume and sperm count from first to last ejaculate. The increase in motility was probably due to reduction in exposure time to sperm motility inhibitory factors. In general, total motile spermatozoa as well as actively motile spermatozoa progressively increased from first to last ejaculate at the cost of sluggish spermatozoa. A significant increase in seminal plasma calcium and magnesium was seen as well as a significant increase in magnesium inside the cell from the first to the fourth ejaculate. Considering the quality of semen, which was good in sperm count and excellent in motility, calcium and magnesium may be helpful in cleaning motility inhibitory factors of spermatozoa. © 2012 Blackwell Verlag GmbH.

Effects of female bovine plasma collected at different days of the estrous cycle on epididymal spermatozoa motility.

PubMed

Nait Mouloud, M; Ouennoughi, F; Yaiche, L; Kaidi, R; Iguer-Ouada, M

2017-03-15

The aim of this study was to assess the effects of female bovine plasma collected at different days of the reproductive cycle on epididymal spermatozoa motility and to test hypothesis that the subpopulations pattern of motile spermatozoa is affected by this treatment. Blood plasma samples were collected from five Holstein Friesian cows at different stages of the estrous cycle (days 0, 5, 10, 12 and 18), one pregnant cow and one adult bull and were diluted 1:9 (V/V) with normal saline. Female charcoal-treated plasma, Bull plasma and saline were used as controls. Semen samples were obtained from cauda epididymidis through retrograde flushing and diluted in saline to approximately 60 × 106 sperm/ml. The extended semen was diluted 1:2 (V/V) with tested media and motility was evaluated at 15 min and then every hour for 6 h using a computer-assisted semen analysis. Multivariate clustering procedure was applied to identify and quantify specific subpopulations within the semen samples. The statistical analysis clustered all the motile spermatozoa into three separate subpopulations with defined patterns of movement: Subpopulation 1 poorly motile and non-progressive spermatozoa (39.3%), subpopulation 2 including the fastest and the most vigorous spermatozoa (46.4%) and subpopulation 3 represented by slow, non-vigorous but linear spermatozoa (14.3%). Initially, sperm samples supplemented with female, male or female charcoal-treated plasma stimulated equally total motility and spermatozoa belonging to subpopulation 2 regardless of the estrous cycle stage. After 1-h incubation, the motility of these both categories of spermatozoa (total motile and those assigned to subpopulation 2) is enhanced and maintained more in day 12, 18 and pregnant cow plasma than in female plasma from earlier stage of the estrous cycle (day 0, 5 and 10), male plasma and female-charcoal treated plasma. In conclusion, the overall results showed that female plasma stimulated significantly sperm motility, especially at the late stage of the estrous cycle. Additionally, to the diverse compounds contained in blood plasma, progesterone may play a key role in such motility activation. Copyright © 2016 Elsevier Inc. All rights reserved.

Mode of action of α-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by α-chlorohydrin and location of block in glycolysis

PubMed Central

Brown-Woodman, Patricia D. C.; Mohri, Hideo; Mohri, Toshiko; Suter, Dai; White, Ian G.

1978-01-01

1. The effect of α-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by α-chlorohydrin (0.1–1.0mm) than lactate or pyruvate. Inhibition of glycolysis by α-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and α-chlorohydrin. A second, much less sensitive site, of α-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-α-chlorohydrin showed a `block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. α-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-α-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of α-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. α-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with α-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of α-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of α-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of α-chlorohydrin in vitro. PMID:629780

Beneficial effects of low dose Musa paradisiaca on the semen quality of male Wistar rats.

PubMed

Alabi, A S; Omotoso, Gabriel O; Enaibe, B U; Akinola, O B; Tagoe, C N B

2013-03-01

This study aimed at determining the effects of administration of mature green fruits of Musa paradisiaca on the semen quality of adult male Wistar rats. THE ANIMALS USED FOR THE STUDY WERE GROUPED INTO THREE: the control group, given 2 ml of double distilled water, a low dose group given 500 mg/kg/day and a high dose group given 1000 mg/kg/day of the plantain fruits, which was made into flour, and dissolved in 2 ml of double distilled water for easy oral administration. Significant increment in the semen parameters was noticed in animals that received a lower dose of the plantain flour, but those animals who received the high dose had marked and very significant reduction in sperm cell concentration and percentage of morphologically normal spermatozoa. Musa paradisiaca should be consumed in moderate quantities in order to derive its beneficial effects of enhancing male reproductive functions.

Anatomy and histology of the male reproductive tract and spermatogenesis fine structure in the lesser anteater (Tamandua tetradactyla, Myrmecophagidae, Xenarthra): morphological evidences of reproductive functions.

PubMed

Rossi, L F; Luaces, J P; Aldana Marcos, H J; Cetica, P D; Perez Jimeno, G; Merani, M S

2013-08-01

The anatomy and histology of the male genital tract of the lesser anteater were studied. Fine details of spermatozoa regarding their genesis and morphology were also studied in six adult specimens. The testes lie in the pelvic cavity. The deferent duct emerges from the epididymis and opens into the ejaculatory duct, which drains into the membranous urethra. Accessory glands (prostate, seminal vesicle and bulbourethral gland) are histologically similar to those described in other mammals. The short penis presents an urethral orifice, while the corpus spongiosum becomes thinner at the end indicating the absence of a histologically defined glans. The seminiferous epithelium shows: (1) Sertoli cells with deep nuclear indentations, (2) spermatogonia with crusty-like chromatin, (3) spermatocytes at different stages of maturation and (4) three morphologically distinct stages of spermatid differentiation according to nuclear shape, acrosome development and chromatin condensation. Sperm heads appear oval. The length of the spermatozoa averages 67.33 ± 1.60 μm. Two specimens with inactive spermatogenesis were azoospermic. Their testes and epididymis presented sizes smaller than those with active spermatogenesis. These studies together with others in anteaters may contribute to successful breeding in conservation programmes. © 2012 Blackwell Verlag GmbH.

Iatrogenic genetic damage of spermatozoa

PubMed Central

O’Flaherty, Cristian

2016-01-01

Different factors can affect sperm morphology and physiology that negatively influence men fertility. Many studies on humans and animals suggest that both radiation and chemotherapy alter sperm chromatin thus promoting significant damage on sperm DNA and, decreasing the level of protamination, thus altering DNA compaction. Spermatozoa from cancer survivors are affected by chemotherapy even years after the end of the treatment. We are exposed to different toxicants present in the environment (products of air pollution, pesticides, plasticizers, etc), which their impact on men reproduction has not been yet established. This chapter aims to update our knowledge on how sperm chromatin structure is modified by external agents and to describe the different strategies available to better study this complex structure in the infertile men. PMID:23955676

Reproductive, cytological and biochemical toxicity of Yohimbe in male Swiss albino mice.

PubMed

Al-Majed, Abdulhakeem A; Al-Yahya, Abdulaziz A; Al-Bekairi, A M; Al-Shabanah, Othman A; Qureshi, Shoeb

2006-07-01

To study the effect of Corynanthe Yohimbe (Yohimbe) on germ cells in Swiss albino mice. Adult male mice were orally (gavage) treated with different doses (188, 375 and 750 mg/[kg x day]) of aqueous suspension of Yohimbe for 90 days. The following parameters were evaluated: (i) reproductive organ weight, (ii) motility and count of sperm, (iii) study on rate of pregnancy and mean implants, (iv) spermatozoa morphology, (v) cytology of the testes chromosomes, and (vi) biochemical study on estimation of proteins, RNA, DNA, malondialdehyde, nonprotein sulfhydryl (NP-SH) and hormones. The treatment caused significant increase in the weight of seminal vesicles, motility and count of spermatozoa, pre- and post-implants. Male fertility was decreased. These results are confirmed by our data on spermatozoa abnormalities and chromosomal aberrations. The data on biochemical parameters showed increase of malondialdehyde and depletion of NP-SH, proteins, RNA and DNA in the testicular cells. Our results elucidated the role of free radical species in cytological and reproductive changes, possibly, under the influence of yohimbine (principal constituent of Yohimbe) on neurotransmitters, including norephinephrine. These data warrant careful use of Yohimbe.

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.

Production, Preservation, and Transfer of South American Camelid Embryos

PubMed Central

Trasorras, Virginia L.; Carretero, María Ignacia; Neild, Deborah M.; Chaves, Maria Graciela; Giuliano, Susana M.; Miragaya, Marcelo H.

2017-01-01

The current review summarizes progress in the field of in vitro and in vivo production of South American Camelid embryos. Both methods require ovarian superstimulation (with FSH and eCG) to obtain multiple ovulations (in vivo embryo production) or to induce follicle growth for oocyte collection (in vitro embryo production). Moreover, superstimulation entails prior administration of hormones that inhibit follicular growth (progesterone, progestagens, and estrogens). Cumulus-oocyte complexes obtained must mature in vivo (buserelin administration) or in vitro to then be subjected to in vitro fertilization or intracytoplasmic sperm injection. All these techniques also require morphologically normal, motile spermatozoa to achieve fertilization. Methods used to decrease semen viscosity and to select the best spermatozoa (Percoll®; Androcoll-ETM) are described. Additionally, nuclear transfer or cloning has been applied in llamas. Up to now, embryo deep-freezing and vitrification have progressed slowly but are at the height of development. Embryos that are obtained by any of these techniques, either in vivo or in vitro, need to be transferred to synchronized recipient females. The best results are achieved after transfer to the left uterine horn with an ipsilateral ovulation. No live offspring have been obtained after the transfer of cryopreserved embryos. Applying reproductive biotechnologies, such as those described, will permit the expansion of genetically selected animals in the population and also that of wild camelid species, vicunas, and guanacos, whose embryos could then be transferred to the uterus of domestic species. PMID:29181380

Use of biochemical markers to evaluate the quality of fresh and cryopreserved semen from the arctic fox (Vulpes lagopus).

PubMed

Stasiak, K; Glogowski, J; Demianowicz, W; Kowalski, R; Nowak-Tkaczyk, A; Janicki, B

2014-01-01

The aim of this study was to use biochemical markers to evaluate the quality of fresh and cryopreserved semen from the arctic fox (Vulpes lagopus). Twenty-three manually collected ejaculates were analysed for the main indicators of semen quality (sperm concentration and ejaculate volume). Sperm motility and percentage of morphologically normal and abnormal spermatozoa were determined according to the stage of cryopreservation (fresh--measurement A; equilibrated--measurement B; frozen/thawed--measurement C). Furthermore, the seminal plasma and supernatants were analysed after equilibration and freeze/thawing for the activity of the enzymes alkaline phosphatase (ALP), acid phosphatase (AcP), lactate dehydrogenase (LDH) and aspartate aminotransferase (AspAT), and for the activity of acrosin inhibitors (AP). The mean concentration of sperm was 625.1 million/cm3, and ejaculate volume averaged 1.6 cm3. Seminal plasma was characterized by the highest activity of alkaline phosphatase (3.43 x 10(3) U/l) and lowest activity of acrosin inhibitors (4.55 x 10(3) U/l). After equilibration, the supernatants showed the highest activity of acid phosphatase (94.9 U/l) and after freeze-thawing, they showed a high activity of lactate dehydrogenase (535.8 U/l) and aspartate aminotransferase (577.1 U/l), which indicates that these proteins had leaked from spermatozoa into the extracellular medium during the biotechnique of semen cryopreservation. In addition, several significant relationships were found between some indicators of semen quality and plasma and/or supernatant enzyme activity.

Prolyl Endopeptidase (PREP) is Associated With Male Reproductive Functions and Gamete Physiology in Mice.

PubMed

Dotolo, Raffaele; Kim, Jung Dae; Pariante, Paolo; Minucci, Sergio; Diano, Sabrina

2016-03-01

Prolyl endopeptidase (PREP) is a serine protease which has been implicated in many biological processes, such as the maturation and degradation of peptide hormones and neuropeptides, learning and memory, cell proliferation and differentiation, and glucose metabolism. A small number of reports have also suggested PREP participation in both male and female reproduction-associated processes. In the present work, we examined PREP distribution in male germ cells and studied the effects of its knockdown (Prep(gt/gt)) on testis and sperm in adult mice. The protein is expressed and localized in elongating spermatids and luminal spermatozoa of wild type (wt) mice, as well as Sertoli, Leydig, and peritubular cells. PREP is also expressed in the head and midpiece of epididymal spermatozoa, whereas the remaining tail region shows a weaker signal. Furthermore, testis weight, histology of seminiferous tubules, and epididymal sperm parameters were assessed in wt and Prep(gt/gt) mice: wild type testes have larger average tubule and lumen diameter; in addition, lumenal composition of seminiferous tubules is dissimilar between wt and Prep(gt/gt), as the percentage of spermiated tubules is much higher in wt. Finally, total sperm count, sperm motility, and normal morphology are also higher in wt than in Prep(gt/gt). These results show for the first time that the expression of PREP could be necessary for a correct reproductive function, and suggest that the enzyme may play a role in mouse spermatogenesis and sperm physiology. © 2015 Wiley Periodicals, Inc.

High-resolution proton nuclear magnetic resonance characterization of seminolipid from bovine spermatozoa.

PubMed

Alvarez, J G; Storey, B T; Hemling, M L; Grob, R L

1990-06-01

The high-resolution one- and two-dimensional proton nuclear magnetic resonance (1H-NMR) characterization of seminolipid from bovine spermatozoa is presented. The 1H-NMR data was confirmed by gas-liquid chromatography-mass spectrometric analysis of the partially methylated alditol acetates of the sugar unit, mild alkaline methanolysis of the glyceryl ester, mobility on normal phase and diphasic thin-layer chromatography (HPTLC), and fast atom bombardment mass spectrometry (FAB-MS). The structure of the molecule corresponds to 1-O-hexadecyl-2-O-hexadecanoyl-3-O-beta-D-(3'-sulfo)-galactopyranosyl- sn-glycerol.

Impact of storage prior to cryopreservation on plasma membrane function and fertility of boar sperm.

PubMed

Guthrie, H D; Welch, G R

2005-01-15

Occasionally, boar semen must be shipped to another location for cryopreservation. We increased the initial holding time for the cooling of extended semen at 15 degrees C from 3 to 24 h to determine the effects on sperm characteristics and fertility. Thirty-one gilts and sows were inseminated once with subsequently cryopreserved and thawed semen. Increasing the holding time from 3 to 24 h had no significant effect on pregnancy rate 23 days after AI with frozen-thawed semen (64.5%) but decreased (P<0.05) embryo number from 15 to 9 and recovered embryos as fraction of CL from 73 to 47%. While the longer holding time at 15 degrees C did decrease potential litter size, the loss incurred was not too great to preclude the incorporation of a longer holding time into the cryopreservation protocol. An experiment was conducted to test the hypothesis that processing and freeze-thawing of boar semen would induce phospholipid scrambling in the plasma membrane similar to that evoked by incubation in bicarbonate-containing media. Merocyanine staining after incubation in the presence and absence of bicarbonate indicated that changes in plasma membrane phospholipid scrambling of processed and cryopreserved sperm differed from those in fresh semen undergoing bicarbonate-induced capacitation. The level of Annexin-V binding in boar spermatozoa increased from 1.6% in live spermatozoa in fresh semen to 18.7% in cryopreserved sperm. Apoptosis is unlikely to operate in mature spermatozoa. Apoptotic morphology in ejaculated spermatozoa is probably a result of incomplete deletion of apoptotic spermatocytes during spermatogenesis. Increased Annexin-V binding in thawed spermatozoa probably results from plasma membrane damage incurred during freezing and thawing.

Intracytoplasmic Sperm Injection (ICSI) in Extreme Cases of Male Infertility

PubMed Central

Palermo, Gianpiero D.; Neri, Queenie V.; Schlegel, Peter N.; Rosenwaks, Zev

2014-01-01

Introduction Severely compromised spermatogenesis typical of men with virtual azoospermia or non-obstructive azoospermia requires an extreme search for spermatozoa. Our goal was to evaluate the usefulness of a meticulous search carried out in ejaculated or surgically retrieved specimens in achieving pre- and post-implantation embryo development. Patients and Methods In a retrospective cohort study carried out in an academic institution, intracytoplasmic sperm injection (ICSI) outcomes were reviewed as a function of length of microscopic sperm search in ejaculated and surgically retrieved specimens. Couples whose male partner presented with either virtual or non-obstructive azoospermia were treated by ICSI and categorized according to the time spent in identifying and retrieving enough spermatozoa to inject all the oocyte cohort. Semen parameter, fertilization, pregnancies, deliveries, and child welfare in relation to increasing search time were analyzed and compared. Result(s) The maternal and paternal ages were comparable in both ejaculated and testicular sperm extraction (TESE) groups along with the oocytes retrieved. The fertilization rates for both ejaculated and TESE progressively decreased with increasing time (P<0.0001). Clinical pregnancies in the ejaculated cohort remained satifactory. In the TESE cohort, there was a decrease in pregnancy rate with increasing time, from 44% to 23%. In a limited number of cases, offspring health was evaluated in both semen sources and appeared reassuring. Conclusion(s) An extensive and at time exhaustive sperm quest yields kinetically and morphologically impaired spermatozoa without apparent impact on embryo developmental competence. Retrieval of spermatozoa from the seminiferous tubules provided more consistent fertilization and pregnancy outcomes than those retrieved from the ejaculate. A trend indicated that pregnancy rate decreased as search time increased in the TESE group. The utilization of the scarce and unselected spermatozoa did not obviously impair embryo development or cause post-implantation errors. PMID:25437298

Sperm motility parameters and spermatozoa morphometric characterization in marine species: a study of swimmer and sessile species.

PubMed

Gallego, V; Pérez, L; Asturiano, J F; Yoshida, M

2014-09-15

The biodiversity of marine ecosystems is diverse and a high number of species coexist side by side. However, despite the fact that most of these species share a common fertilization strategy, a high variability in terms of the size, shape, and motion of spermatozoa can be found. In this study, we have analyzed both the sperm motion parameters and the spermatozoa morphometric features of two swimmer (pufferfish and European eel) and two sessile (sea urchin and ascidian) marine species. The most important differences in the sperm motion parameters were registered in the swimming period. Sessile species sperm displayed notably higher values than swimmer species sperm. In addition, the sperm motilities and velocities of the swimmer species decreased sharply once the sperm was activated, whereas the sessile species were able to maintain their initial values for a long time. These results are linked directly to the species-specific lifestyles. Although sessile organisms, which show limited or no movement, need sperm with a capacity to swim for long distances to find the oocytes, swimmer organisms can move toward the female and release gametes near it, and therefore the spermatozoa does not need to swim for such a long time. At the same time, sperm morphology is related to sperm motion parameters, and in this study an in-depth morphometric analysis of ascidian, sea urchin, and pufferfish spermatozoa, using computer-assisted sperm analysis software, has been carried out for the first time. A huge variability in shapes, sizes, and structures of the studied species was found using electron microscopy. Copyright © 2014 Elsevier Inc. All rights reserved.

Morphology and histology of male and female reproductive systems in the inseminating species Scoloplax distolothrix (Ostariophysi: Siluriformes: Scoloplacidae).

PubMed

Spadella, Maria Angélica; Oliveira, Claudio; Quagio-Grassiotto, Irani

2008-09-01

The morphology and histology of male and female reproductive systems were examined in Scoloplax distolothrix. Internal insemination was documented in this species by the presence of sperm within the ovaries. Mature males and females have elongated genital papillae, exhibiting a tubular shape in males and a plain heart-shape with two median protuberances in females. The testes are two elongated structures that converge ventrally, under the intestine, towards the genital papilla. They are joined at the caudal end, forming an ovoid single chamber for sperm storage. Secretory regions were not observed. In the lumen of the testicular tubules, spermatozoa can be tightly packed along their lengths, but do not constitute a spermatozeugmata. The lumen of the sperm storage chamber and spermatic duct are filled with free spermatozoa without the accompanying secretions. The ovaries are bird-wing shaped, saccular structures that converge ventrally under the intestine, towards the genital papilla. They are joined at the caudal end, forming a tubular chamber possibly destined for oocyte storage. An oviduct with an irregular outline connects the chamber to the tubular region of the genital papilla. No distinct sperm storage structure was found in the ovaries. The unique male and female genital papillae suggest that these structures are associated with the reproductive mode in scoloplacids, representing evidence for insemination. The occurrence of free spermatozoa, without the accompanying secretions and not arranged in a spermatozeugmata can be associated with the presence of a tubular male genital papilla for sperm transfer to the female genital tract. This reinforces the idea that sperm packets are not necessary for all inseminating species. The male reproductive system in scoloplacids is very different from that in auchenipterids, a second catfish family with insemination, which indicates that the occurrence of insemination is not connected to the internal morphology of reproductive organs.

Toxic effects of 2,4-dichlorophenoxyacetic acid on human sperm function in vitro.

PubMed

Tan, Zhengyu; Zhou, Jun; Chen, Houyang; Zou, Qianxing; Weng, Shiqi; Luo, Tao; Tang, Yuxin

2016-01-01

The herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) is globally used in agriculture and has been linked to human sperm abnormalities in vivo. However, its effects on ejaculated human spermatozoa in vitro have not been characterized. Therefore, we examined the effects of 2,4-D on the functions of ejaculated human spermatozoa in vitro, including: sperm motility, the ability to move through a viscous medium, capacitation, and the acrosome reaction. Different doses of 2,4-D (10 nM, 100 nM, 1 µM, 10 µM, 100 µM, and 200 µM) were applied to human spermatozoa prepared from normal fresh semen samples. The results indicated that 2,4-D did not affect the viability, capacitation, or spontaneous acrosome reactions of human spermatozoa, but it dose-dependently inhibited the total motility, progressive motility, ability to penetrate viscous medium, and progesterone-induced capacitation and acrosome reaction rates. These results suggest that exposure to 2,4-D and its accumulation in the seminal plasma and follicular fluid might increase the risk of infertility. Our findings provide new insights for understanding the male reproductive toxicity of 2,4-D.

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.

New preservation method for mouse spermatozoa without freezing.

PubMed

Van Thuan, Nguyen; Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

2005-02-01

The objective of this study was to investigate the preservation of spermatozoa in a simple medium without freezing and to examine the effects of the preserved sperm on fertilization and development after injection into mature mouse oocytes. Mouse spermatozoa were collected from two caudae epididymides of mature B6D2F1 males and stored under various conditions: 1) in KSOMaa medium (potassium simplex optimized medium with amino acids) supplemented with 0, 1, or 4 mg/ml BSA and held at room temperature (RT, 27 degrees C); 2) in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and held at 4 degrees C, RT, or 37 degrees C (CO2 incubator); 3) in KSOM-BSA with osmolarity ranging from 271 to 2000 mOsmol, adjusted by addition of NaCl and held at 4 degrees C; and 4) a two-step preservation system consisting of storage in 800 mOsmol KSOM-BSA for 1 wk at RT followed by storage at -20 degrees C. Preservation of mouse spermatozoa at 4 degrees C in a medium with high osmolarity (700-1000 mOsmol) resulted in the highest frequency of live births after intracytoplasmic sperm injection (ICSI) into mature oocytes. The optimal conditions for preservation of mouse spermatozoa were 800 mOsmol KSOM containing 4 mg/ml BSA and a holding temperature of 4 degrees C. More than 40% of oocytes injected with sperm heads stored under these conditions for 2 mo developed to the morula/blastocyst stage in vitro and 39% of the embryos developed to term after transfer to recipient mice. Our results also indicate that mouse spermatozoa can be stored in 800 mOsmol KSOM-BSA medium at RT for 1 wk and then at -20 degrees C for up to 3 mo and retain their competence for ICSI. These new preservation methods permit extended conservation of viable spermatozoa that are capable of supporting normal embryonic development and the live birth of healthy offspring after ICSI.

Effects of the anabolic steroid, boldenone undecylenate, on certain reproductive parameters in bulls.

PubMed

Wolfe, D F; Carson, R L; Mysinger, P W; Duran, B S; Rahe, H S

1991-05-01

A total of 17 bulls was used to study the effects of boldenone undecylenate on growth and semen characteristics in beef bulls. In trial 1 nine mature mixed-breed beef bulls with satisfactory semen quality were divided into two groups. Group I (n = 5) received boldenone undecylenate (1.1 mg/kg) at 21 d intervals for a total of seven treatments (147 d). Group II (n = 4) served as untreated controls. Semen was collected from each group by electroejaculation on each treatment day and evaluated according to the standards of the Society for Theriogenology. Although neither the percentage of spermatozoa with primary or secondary morphological abnormalities was different, the ejaculates of Group I bulls contained a higher percentage of abnormal spermatozoa than those in Group II. In trial 2, eight mixed-breed bull calves, average weight 140.4 kg, were maintained under drylot conditions in a single paddock. The bulls were divided into two equal groups. Group I (n = 4) received boldenone undecylenate as in Trial 1. Group II (n = 4) served as untreated controls. The bulls were weighed and the scrotal circumference (SC) was measured every 21 d until it reached 30 cm, at which time semen was collected and evaluated as in Trial 1. Group I bulls had a higher percentage of spermatozoa with primary morphological abnormalities than bulls in Group II. Group I bulls had a higher average daily gain (ADG) than Group II bulls and required 21 d longer for the SC to reach 30 cm. Semen quality for all bulls was satisfactory at each sampling day.

Sperm preparation: state-of-the-art—physiological aspects and application of advanced sperm preparation methods

PubMed Central

Henkel, Ralf

2012-01-01

For assisted reproduction technologies (ART), numerous techniques were developed to isolate spermatozoa capable of fertilizing oocytes. While early methodologies only focused on isolating viable, motile spermatozoa, with progress of ART, particularly intracytoplasmic sperm injection (ICSI), it became clear that these parameters are insufficient for the identification of the most suitable spermatozoon for fertilization. Conventional sperm preparation techniques, namely, swim-up, density gradient centrifugation and glass wool filtration, are not efficient enough to produce sperm populations free of DNA damage, because these techniques are not physiological and not modeled on the stringent sperm selection processes taking place in the female genital tract. These processes only allow one male germ cell out of tens of millions to fuse with the oocyte. Sites of sperm selection in the female genital tract are the cervix, uterus, uterotubal junction, oviduct, cumulus oophorus and the zona pellucida. Newer strategies of sperm preparation are founded on: (i) morphological assessment by means of ‘motile sperm organelle morphological examination (MSOME)' (ii) electrical charge; and (iii) molecular binding characteristics of the sperm cell. Whereas separation methods based on electrical charge take advantage of the sperm's adherence to a test tube surface or separate in an electrophoresis, molecular binding techniques use Annexin V or hyaluronic acid (HA) as substrates. Techniques in this category are magnet-activated cell sorting, Annexin V-activated glass wool filtration, flow cytometry and picked spermatozoa for ICSI (PICSI) from HA-coated dishes and HA-containing media. Future developments may include Raman microspectrometry, confocal light absorption and scattering spectroscopic microscopy and polarization microscopy. PMID:22138904

In vitro embryo production in camel (Camelus dromedarius) from in vitro matured oocytes fertilized with epididymal spermatozoa stored at 4 degrees C.

PubMed

Wani, N A

2009-03-01

Experiments were conducted to study the effect of storing epididymal spermatozoa, in tris-tes- and tris-lactose egg yolk extenders, on their fertilizing ability and subsequent in vitro embryo development. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 degrees C and on ice (0-1 degrees C), respectively. Cumulus oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25COCs/well) containing 500 microL of maturation medium and cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in air for 36 h. Spermatozoa were collected from the cauda epididymides in syringes containing 2-3 mL of either tris-tes- or tris-lactose egg yolk extender. They were cooled down slowly and stored at refrigeration (4 degrees C) temperature. The spermatozoa were evaluated for motility and used for IVF of IVM oocytes on the day of collection and after 2, 4, 6 and 8 days of storage. On the day of IVF, spermatozoa were prepared by the swim up technique and both spermatozoa and oocytes were co-incubated at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air for 15-16 h. Presumptive zygotes were either fixed and stained with Hoechst 33342 for evaluation of fertilization or were cultured in 500 microL of the culture medium at 38.5 degrees C in an atmosphere of 5% CO(2), 5% O(2) and 90% N(2) in air. There was no significant difference (P>0.05) in the proportion of oocytes fertilized with spermatozoa stored in either of the two extenders for up to 8 days. The proportion of oocytes that cleaved (43-60%) and those that developed to blastocysts (14-21%) did not show any difference (P>0.05) either, when spermatozoa from different days of storage were used. First cleavage was observed as early as 16 h after IVF, early blastocysts had developed by day 4, expanded blastocysts after day 5 and hatching of blastocysts started after day 6 of culture. It may be concluded that dromedary epididymal spermatozoa survive in storage for at least 8 days in tris-lactose- and tris-tes egg yolk diluents at 4 degrees C. These spermatozoa maintain fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures.

Correlation study between sperm concentration, hyaluronic acid-binding capacity and sperm aneuploidy in Hungarian patients.

PubMed

Mokánszki, Attila; Molnár, Zsuzsanna; Ujfalusi, Anikó; Balogh, Erzsébet; Bazsáné, Zsuzsa Kassai; Varga, Attila; Jakab, Attila; Oláh, Éva

2012-12-01

Infertile men with low sperm concentration and/or less motile spermatozoa have an increased risk of producing aneuploid spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding may reduce genetic risks such as chromosomal rearrangements and numerical aberrations. Fluorescence in-situ hybridization (FISH) has been used to evaluate the presence of aneuploidies. This study examined spermatozoa of 10 oligozoospermic, 9 asthenozoospermic, 9 oligoasthenozoospermic and 17 normozoospermic men by HA binding and FISH. Mean percentage of HA-bound spermatozoa in the normozoospermic group was 81%, which was significantly higher than in the oligozoospermic (P<0.001), asthenozoospermic (P<0.001) and oligoasthenozoospermic (P<0.001) groups. Disomy of sex chromosomes (P=0.014) and chromosome 17 (P=0.0019), diploidy (P=0.03) and estimated numerical chromosome aberrations (P=0.004) were significantly higher in the oligoasthenozoospermic group compared with the other groups. There were statistically significant relationships (P<0.001) between sperm concentration and HA binding (r=0.658), between sperm concentration and estimated numerical chromosome aberrations (r=-0.668) and between HA binding and estimated numerical chromosome aberrations (r=-0.682). HA binding and aneuploidy studies of spermatozoa in individual cases allow prediction of reproductive prognosis and provision of appropriate genetic counselling. Infertile men with normal karyotypes and low sperm concentrations and/or less motile spermatozoa have significantly increased risks of producing aneuploid (diminished mature) spermatozoa. Selecting spermatozoa by hyaluronic acid (HA) binding, based on a binding between sperm receptors for zona pellucida and HA, may reduce the potential genetic risks such as chromosomal rearrangements and numerical aberrations. In the present study we examined sperm samples of 45 men with different sperm parameters by HA-binding assay and fluorescence in-situ hybridization (FISH). Mean percentage of HA-bound spermatozoa in the normozoospermic group was significantly higher than the oligozoospermic, the asthenozoospermic and the oligoasthenozoospermic groups. Using FISH, disomy of sex chromosomes and chromosome 17, diploidy and estimated numerical chromosome aberration frequencies were significantly higher in the oligoasthenozoospermic group compared with the three other groups. A significant positive correlation was found between the sperm concentration and the HA-binding capacity, and significant negative correlations between the sperm concentration and the estimated numerical chromosomes aberrations as well as between the HA-binding ability and the estimated numerical chromosome aberrations were identified. We conclude that HA-binding assay and sperm aneuploidy study using FISH may help to predict the reproductive ability of selected infertile male patients and to provide appropriate genetic counselling. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Improved quality of cryopreserved cheetah (Acinonyx jubatus) spermatozoa after centrifugation through Accudenz.

PubMed

Crosier, Adrienne E; Henghali, Josephine N; Howard, Jogayle; Pukazhenthi, Budhan S; Terrell, Kimberly A; Marker, Laurie L; Wildt, David E

2009-01-01

Sperm cryopreservation, in combination with assisted reproductive techniques, is a valuable tool for the genetic management of endangered felids. However, the acrosome of the cheetah spermatozoon is especially sensitive to cryopreservation, with approximately 40% of spermatozoa experiencing acrosomal damage immediately after thawing and then another approximately 15% loss during the next 4 hours in vitro. Additionally, thawing causes a reduction in sperm motility by approximately 20% with another decrease of approximately 12% during subsequent incubation in vitro. We hypothesized that slow removal of glycerol from cryopreserved cheetah spermatozoa using an Accudenz gradient would improve acrosomal integrity, sperm motility longevity, and structural morphology. Accudenz was compared with traditional cheetah sperm processing methods for glycerol removal that involves washing, multistep resuspension, and swim-up processing. Electroejaculates (n = 21 total from 8 males) were washed in Ham F10 medium, and sperm pellets were resuspended in TEST-yolk buffer with 0% glycerol. Samples were cryopreserved in straws in 4% final glycerol, thawed, and assessed for percent intact acrosomes (% IA), percent motility (% M), and forward progressive status (FPS; scale, 0-5). Sperm motility index (SMI) was calculated as (% M + [FPS x 20]) / 2. In study 1, glycerol removal by centrifugation through an Accudenz gradient (4%, 10%) was compared with traditional sperm washing (control) and multistep resuspension protocols. At each time after centrifugation (hourly for 4 hours), % IA was improved (P < .05) for Accudenz (range, 36%-39%) compared with control (30%-33%) and multistep (29%-33%) treatments. In study 2, a modified Accudenz protocol was compared with traditional washing and was found to improve (P < .05) SMI (range, 52-64) compared with controls (range, 41-52) at each time postthaw after centrifugation. In study 3, swim-up processed sperm were compared with those treated by centrifugation through Accudenz and traditional sperm washing for improving sperm morphology. The percentage of structurally-normal sperm recovered postthawing increased (P < .05) for both the Accudenz (38%) and swim-up (33%) treatments compared with controls (21%). Percent IA and SMI also were improved (P < .05) for Accudenz (range, 39%-47% and 46-59, respectively) compared with controls (range, 26%-33% and 40-53, respectively). Results indicate that using Accudenz for glycerol removal from cryopreserved cheetah sperm mitigates the significant loss in sperm quality that occurs after freeze-thawing. This alleviation of cellular damage resulting from cryopreservation contributes to a more than 10% improvement in overall sperm motility and, more importantly, allows retention of 40% or more of sperm with intact acrosomes.

Equine sperm-bound antisperm antibodies are associated with poor semen quality.

PubMed

Ferrer, M S; Miller, L M J

2018-06-01

Antisperm antibodies (ASAs) have been associated with infertility in stallions. The objectives of this study were to investigate the frequency of ASA-positive semen samples in satisfactory and non-satisfactory breeder stallions, the association between ASA binding and semen quality, and factors that may affect the diagnosis. Breeding soundness examinations were performed in 21 stallions and the percentage of IgG- and IgA-bound spermatozoa was evaluated using flow cytometry. Median IgG and IgA binding did not differ between the first and second ejaculates. The percentage of IgA-bound spermatozoa was higher in non-satisfactory (n = 10) than satisfactory breeder stallions (n = 11). However, IgG binding or frequency of IgG-positive ejaculates did not differ with stallion classification. The IgG-positive stallions had significantly lower total sperm motility, concentration and total numbers than IgG-negative stallions in the first ejaculate, and lower sperm concentration in the second ejaculate. The IgA-positive stallions had lower total sperm motility, normal spermatozoa and total numbers than IgA-negative stallions in the first ejaculate, and lower total sperm motility, normal spermatozoa and total numbers in the second ejaculate. While IgG binding did not differ with season, IgA binding was higher in the non-breeding season (n = 6 stallions) than the breeding season (n = 15 stallions) in the first ejaculate. Stallion age did not differ with ASA classification. In conclusion, IgG binding was highly prevalent in both groups of stallions, while IgA binding was higher and more prevalent in non-satisfactory breeders. Both isotypes were associated with poor semen quality. Season and sexual rest had an effect on IgA but not IgG binding. Copyright © 2018 Elsevier Inc. All rights reserved.

Vitrification of neat semen alters sperm parameters and DNA integrity.

PubMed

Khalili, Mohammad Ali; Adib, Maryam; Halvaei, Iman; Nabi, Ali

2014-05-06

Our aim was to evaluate the effect of neat semen vitrification on human sperm vital parameters and DNA integrity in men with normal and abnormal sperm parameters. Semen samples were 17 normozoospermic samples and 17 specimens with abnormal sperm parameters. Semen analysis was performed according to World Health Organization (WHO) criteria. Then, the smear was provided from each sample and fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Vitrification of neat semen was done by plunging cryoloops directly into liquid nitrogen and preserved for 7 days. The samples were warmed and re-evaluated for sperm parameters as well as DNA integrity. Besides, the correlation between sperm parameters and DNA fragmentation was assessed pre- and post vitrification. Cryopreserved spermatozoa showed significant decrease in sperm motility, viability and normal morphology after thawing in both normal and abnormal semen. Also, the rate of sperm DNA fragmentation was significantly higher after vitrification compared to fresh samples in normal (24.76 ± 5.03 and 16.41 ± 4.53, P = .002) and abnormal (34.29 ± 10.02 and 23.5 ± 8.31, P < .0001), respectively. There was negative correlation between sperm motility and sperm DNA integrity in both groups after vitrification. Vitrification of neat ejaculates has negative impact on sperm parameters as well as DNA integrity, particularly among abnormal semen subjects. It is, therefore, recommend to process semen samples and vitrify the sperm pellets.

Effect of rapid warming of boar semen on sperm morphology and physiology.

PubMed

Bamba, K; Cran, D G

1985-09-01

The effect of rapid dilution (1:8 with BTS or 1:6.5 with KRP) and temperature change on sperm morphology and physiology were studied using boar spermatozoa pre-diluted in BF5 diluent. Rapid dilution of cold semen (5 degrees C) with a warm solution (37 degrees C) caused marked acrosomal changes which were most prominent in the anterior region. The acrosomal damage appeared to be caused mainly by rapid warming. In contrast to rapid cooling, rapid warming had little effect upon motility, glutamic-oxaloacetic transaminase release and respiration.

Sperm storage in the human cervix: a quantitative study.

PubMed

Insler, V; Glezerman, M; Zeidel, L; Bernstein, D; Misgav, N

1980-03-01

Twenty-five women scheduled for hysterectomy for nonmalignant disease participated in the study. Sperm storage in endocervical crypts was examined in three groups of patients: nine women pretreated with estrogen and inseminated with normal semen, nine women pretreated with gestagen and inseminated with normal semen, and seven women pretreated with estrogen and inseminated with abnormal semen. The number of crypts containing spermatozoa (colonized crypts) and the sperm density per crypt were examined in serially sectioned cervices. In estrogen-pretreated cervices both the percentage of colonized crypts and the sperm density were significantly higher than in gestagen-pretreated cervices. Large and giant crypts proved to be the main storage facility for spermatozoa. The localization of crypts along the endocervical canal did not influence sperm storage. The quality of semen appeared to be of critical importance to sperm storage. The percentage of colonized crypts and sperm density were severly reduced in patients inseminated with abnormal semen.

The effects of trypanosomosis on sperm morphology in Zebu x Friesian crossbred bulls.

PubMed

Sekoni, V O; Rekwot, P I; Bawa, E K

2004-01-01

Detailed studies of sperm morphological abnormalities were carried out on 12 Zebu x Friesian crossbred bulls used in a study of the effects of trypanosomosis. Four bulls were infected with T. vivax, another four with T. congolense, while four served as controls. The infected bulls developed chronic trypanosomosis. All the bulls initially had very low sperm morphological abnormalities that were within acceptable limits for fertile animals. After infection there was a rapid and progressive increase in all sperm abnormalities. Spermatozoa of infected bulls were highly deformed with multiple morphological defects. Mean percentage pre-infection baseline values prior to infection for acrosomal, sperm-head, detached heads, proximal cytoplasmic droplets, distal cytoplasmic droplets, sperm-tail, midpiece and total sperm morphological defects ranged between 0.1 +/- 0.1 for acrosomal and 8.3 +/- 3.2 for total morphological abnormalities in the semen of the bulls. All the infected bulls developed sperm morphological abnormalities of more than a mean of 40.0% from the 4th week after infection until the end of the investigation and were considered unfit for breeding. At 7 weeks post-infection (PI) until the end of the study (12 weeks PI), the controls had a mean of less than 5% sperm morphological defects, while the infected bulls had 100%. Mean percentage values of sperm morphological defects throughout the duration of the investigation for control bulls were low and within the normal range for fertile bulls. These values differed significantly (p<0.001) from the elevated values of the infected bulls. The results show that trypanosomosis due to T. vivax or T. congolense infection can render Zebu x Friesian crossbred bulls unfit for breeding within a very short time. The resultant infertility could be of economic importance in trypanosomosis-endemic sub-Saharan Africa where Zebu x Friesian crossbred bulls are kept.

Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature.

PubMed

Ono, Tetsuo; Mizutani, Eiji; Li, Chong; Wakayama, Teruhiko

2010-08-01

The development of preservation techniques for male gametes at room temperature might allow us to store them in a simple and cost-effective manner. In this study, we studied the use of pure salt or sugar to preserve the whole cauda epididymidis, because it is known that food can be preserved in this way at room temperature for long periods. Mouse epididymides were placed directly in powdered salt (NaCl) or sugars (glucose or raffinose) for 1 day to 1 year at room temperature. Spermatozoa were recovered from the preserved organs after being rehydrated with medium and then isolated sperm heads were microinjected into fresh oocytes. Importantly, the oocyte activation capacity of spermatozoa was maintained after epididymal storage in NaCl for 1 year, whereas most untreated spermatozoa failed to activate oocytes within 1 month of storage. Pronuclear morphology, the rate of extrusion of a second polar body and the methylation status of histone H3 lysine 9 (H3K9me3) in those zygotes were similar to those of zygotes fertilized with fresh spermatozoa. However, the developmental ability of the zygotes decreased within 1 day of sperm storage. This effect led to nuclear fragmentation at the 2-cell embryo stage, irrespective of the storage method used. Thus, although the preserved sperm failed to allow embryo development, their oocyte activation factors were maintained by salt storage of the epididymis for up to 1 year at room temperature.

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen.

PubMed

Lee, Hae-Lee; Kim, Sue-Hee; Ji, Dong-Beom; Kim, Yong-Jun

2009-09-01

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/ propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.

Is intracytoplasmic morphologically selected sperm injection (IMSI) beneficial in the first ART cycle? a multicentric randomized controlled trial.

PubMed

Leandri, R D; Gachet, A; Pfeffer, J; Celebi, C; Rives, N; Carre-Pigeon, F; Kulski, O; Mitchell, V; Parinaud, J

2013-09-01

Intracytoplasmic morphologically selected sperm injection (IMSI), by selecting spermatozoa at high magnification improves the outcome of intracytoplasmic sperm injection (ICSI) mainly after several failures. However, only few monocentric randomized studies are available and they do not analyse results as a function of sperm characteristics. In 255 couples attempting their first assisted reproductive technology (ART) attempt for male infertility (motile sperm count <1×10⁶ after sperm selection, but at least 3×10⁶ spermatozoa per ejaculate to allow a detailed analysis of sperm characteristics), a prospective randomized trial was performed to compare the clinical outcomes of IMSI and ICSI and to evaluate the influence of sperm characteristics on these outcomes. IMSI did not provide any significant improvement in the clinical outcomes compared with ICSI neither for implantation (24% vs. 23%), nor clinical pregnancy (31% vs. 33%) nor live birth rates (27% vs. 30%). Moreover, the results of IMSI were similar to the ICSI ones whatever the degree of sperm DNA fragmentation, nuclear immaturity and sperm morphology. These results show that IMSI instead of ICSI has no advantage in the first ART attempts. However, this does not rule out IMSI completely and more randomized trials must be performed especially regarding patients carrying severe teratozoospermia, or high sperm DNA fragmentation levels or having previous ICSI failures. © 2013 American Society of Andrology and European Academy of Andrology.

Assisted reproductive technology outcomes in azoospermic men: 10 years of experience with surgical sperm retrieval.

PubMed

Semião-Francisco, Luciana -; Braga, Daniela Paes De Almeida Ferreira; Figueira, Rita De Cássia Savio; Madaschi, Camila; Pasqualotto, Fábio Firmbach; Iaconelli, Assumpto; Borges, Edson

2010-03-01

An azoospermic man suffers from an absence of sperm in the ejaculate and this condition is present in about 10% of infertile men. Obstructive azoospermia (OA) is characterized by an occlusion or partial absence of the reproductive tract with the presence of normal spermatogenesis. On the other hand, non-obstructive azoospermia (NOA) is characterized by impaired spermatogenesis. In these cases, spermatozoa can be obtained by percutaneous epididymal or testicular sperm aspiration (PESA and TESA, respectively) and used for intracytoplasmic injection (ICSI). To compare ICSI outcomes using spermatozoa that were surgically retrieved by PESA and TESA, azoospermic patients were divided into the following categories: (i) TESA-NOA (n = 102), (ii) TESA-OA (n = 103), and (iii) PESA-OA (n = 171). Fertilization, pregnancy, and implantation rates were compared between the groups. We noted a lower normal fertilization rate (p = 0.0017) and a higher abortion rate (p = 0.0387) among men in the TESA group who had OA when compared with men in the PESA group who had OA. On the other hand, a lower normal fertilization rate (p = 0.05) and a lower rate of non-cleaved embryos (p = 0.034) was found in the TESA group of NOA patients as compared to the TESA group of OA patients. No statistically significant differences were detected between the TESA and PESA groups and the OA and NOA groups, respectively. The clinical outcomes of embryos arising from ICSI cycles using spermatozoa harvested via PESA and TESA were similar, regardless of whether the patient had obstructive or non-obstructive azoospermia.

A plea for a more physiological ICSI.

PubMed

Ebner, T; Filicori, M; Tews, G; Parmegiani, L

2012-05-01

Intracytoplasmic sperm injection (ICSI) can be considered the most 'revolutionary' in vitro insemination technique because it has efficiently allowed the treatment of male factor infertility. Although ICSI has been successfully and safely applied worldwide for almost 20 years, currently, we have no real knowledge regarding the hypothetical long-term side effects on ICSI adults, given the increased likelihood of spermatozoa with defective nuclear content fertilising the oocytes. The aim of this review article is to investigate the most recent advances of performing ICSI in the safest possible manner, thus, minimising the theoretical hazards of this procedure. To allow for substantiated recommendation which male gametes to choose for physiological ICSI an updated search was performed in Medline and Embase, from 1996 to June 2011. Recent technical advances allow operators to more or less simulate physiological conditions in the laboratory, reducing potential damage to the gametes. It seems possible to prevent fertilisation by DNA-damaged and chromosomal-unbalanced spermatozoa by selecting ICSI sperm by motility and/or maturation markers such as hyaluronic acid or other zona pellucida receptors. Furthermore, novel non-invasive imaging techniques can be valid tools for helping in the morphological selection of ICSI spermatozoa. © 2011 Blackwell Verlag GmbH.

Human sperm DNA integrity: correlation with sperm cytoplasmic droplets.

PubMed

Fischer, Marc Anthony; Willis, Jennifer; Zini, Armand

2003-01-01

To examine the retention of sperm cytoplasmic droplets (CD) and DNA denaturation (DD) in semen from fertile and infertile men. Semen samples were obtained from consecutive nonazoospermic men presenting for infertility evaluation (n = 101) and fertile men presenting for vasectomy (n = 13). The standard semen parameters (sperm concentration, motility, and morphology), sperm DD, and sperm CD were monitored. Sperm DD was evaluated by flow cytometry analysis of acridine orange-treated spermatozoa and expressed as the percentage of spermatozoa demonstrating denatured DNA. The mean (+/-SE) percentages of spermatozoa with CD and DD were significantly higher in infertile than in fertile men (sperm CD 15.7% +/- 0.8% versus 4.8% +/- 0.7% and sperm DD 22.0% +/- 1.5% versus 10.8% +/- 1.8%, respectively). Sperm CD and DD were positively correlated (r = 0.59). Also, sperm CD and DD values correlated inversely with the standard semen parameters. Our data demonstrate that the retention of sperm CD correlates positively with sperm DD and that significantly higher sperm DD and CD are found in infertile than in fertile men. These data suggest that the enhanced susceptibility of sperm DNA to denaturation is associated with the abnormal disposal of residual sperm cytoplasm in the testis and/or epididymis.

Hydrogen Peroxide Modifies Human Sperm Peroxiredoxins in a Dose-Dependent Manner1

PubMed Central

O'Flaherty, Cristian; Rico de Souza, Angela

2010-01-01

Low levels of reactive oxygen species (ROS) modulate signaling pathways required for human sperm activation, but high levels impair sperm function, leading to infertility. Peroxiredoxins (PRDXs) are enzymes with a dual role as ROS scavengers and modulators of ROS-dependent signaling. The present study aimed to characterize PRDXs in human spermatozoa and possible modifications resulting from hydrogen peroxide (H2O2). We found PRDX1, PRDX4, PRDX5, and PRDX6 in both seminal plasma and spermatozoa. Using immunocytochemistry, we demonstrated that these PRDXs are differentially localized in the head, acrosome, mitochondrial sheath, and flagellum. These observations were confirmed by immunoblotting using cytosolic, Triton-soluble and -insoluble, and head and flagella sperm fractions. PRDXs are dose-dependently modified by H2O2, as seen by the formation of disulfide bridges and high-molecular-mass complexes. This first study, to our knowledge, on PRDXs in human spermatozoa indicates that PRDX1, PRDX4, PRDX5, and PRDX6 are modified when spermatozoa are challenged with H2O2. This suggests that PRDXs may protect these cells at high levels of H2O2 but could also control H2O2 levels within different cell compartments so that normal sperm activation can occur. PMID:20864641

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry.

PubMed

Nimptsch, Ariane; Fuchs, Beate; Süß, Rosmarie; Zschörnig, Kristin; Jakop, Ulrike; Göritz, Frank; Schiller, Jürgen; Müller, Karin

2013-08-01

Plasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A2 (PLA2) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA2 digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids.

Low physiological levels of prostaglandins E2 and F2α improve human sperm functions.

PubMed

Rios, Mariana; Carreño, Daniela V; Oses, Carolina; Barrera, Nelson; Kerr, Bredford; Villalón, Manuel

2016-03-01

Prostaglandins (PGs) have been reported to be present in the seminal fluid and cervical mucus, affecting different stages of sperm maturation from spermatogenesis to the acrosome reaction. This study assessed the effects of low physiological PGE2 and PGF2α concentrations on human sperm motility and on the ability of the spermatozoa to bind to the zona pellucida (ZP). Human spermatozoa were isolated from seminal samples with normal concentration and motility parameters and incubated with 1μM PGE2, 1μM PGF2α or control solution to determine sperm motility and the ability to bind to human ZP. The effects of both PGs on intracellular calcium levels were determined. Incubation for 2 or 18h with PGE2 or PGF2α resulted in a significant (P<0.05) increase in the percentage of spermatozoa with progressive motility. In contrast with PGF2α, PGE2 alone induced an increase in sperm intracellular calcium levels; however, the percentage of sperm bound to the human ZP was doubled for both PGs. These results indicate that incubation of human spermatozoa with low physiological levels of PGE2 or PGF2α increases sperm functions and could improve conditions for assisted reproduction protocols.

Methodology of aniline blue staining of chromatin and the assessment of the associated nuclear and cytoplasmic attributes in human sperm.

PubMed

Sati, Leyla; Huszar, Gabor

2013-01-01

In this chapter, the laboratory methods for detection of sperm biomarkers that are aimed at identifying arrested sperm development are summarized. These probes include sperm staining with aniline blue for persistent histones, representing a break in the histone-transition protein-protamine sequence, immunocytochemistry with cytoplasmic sperm proteins, highlighting cytoplasmic retention during spermiogenesis, DNA nick translation testing for DNA chain fragmentation due to various reasons, for instance low HspA2 chaperone protein levels, and consequential diminished DNA repair. Finally, we briefly provide references on our work on sperm hyaluronan binding, abnormal Tybergerg sperm morphology, and the increased levels of chromosomal aneuploidies in sperm with developmental arrest. A very interesting aspect of the biomarker field is the discovery (Sati et al, Reprod Biomed Online 16:570-579, 2008) that the various nuclear and cytoplasmic defects detected by the biomarkers are related, and may simultaneously occur within the same spermatozoa as evidenced by a combination of biomarkers, such as aniline blue staining (persistent histones) coupled with cytoplasmic retention, DNA fragmentation, Caspase-3, Tygerberg abnormal morphology, and increased levels of chromosomal aneuploidies. We show examples of this >80% overlap in staining patterns within the same spermatozoa.

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.

Minimum number of spermatozoa required for normal fertility after deep intrauterine insemination in non-sedated sows.

PubMed

Martinez, E A; Vazquez, J M; Roca, J; Lucas, X; Gil, M A; Parrilla, I; Vazquez, J L; Day, B N

2002-01-01

A fibreoptic endoscope procedure for non-surgical deep intrauterine insemination in non-sedated sows has been reported. However, the endoscope is an expensive and fragile instrument, and is unsuitable for use under field conditions. The aim of this study was to determine the minimum number of spermatozoa required to maintain optimal fertility using a flexible catheter (1.8 m in length, 4 mm in diameter) for deep intrauterine insemination in 2-6 parity non-sedated sows. Crossbred sows were treated with eCG 24 h after weaning and with hCG 72 h later to induce oestrus. Deep intrauterine insemination was performed 36 h after hCG treatment in 117, 126, 60 and 69 sows with 15.0, 5.0, 2.5 or 1.0 x 10(7) spermatozoa in 10 ml, respectively. Weaned sows (n = 147) not treated with hormones and used for standard artificial insemination (AI) (two inseminations per oestrus with 3 x 10(9) spermatozoa in 100 ml) served as controls. The flexible catheter was passed successfully through the cervix into one uterine horn in 95.4% of the sows in an average of 3.7 +/- 0.09 min. Farrowing rates after deep intrauterine insemination with 15 or 5 x 10(7) spermatozoa did not differ from those of the control group (82.9, 76.2 and 83.0%, respectively), but a significant decrease (P < 0.001) was observed in sows inseminated with 2.5 or 1.0 x 10(7) spermatozoa (46.7 and 39.1%, respectively). In contrast, the number of spermatozoa inseminated did not affect prolificacy. Laparotomy revealed that the tip of the flexible catheter reached approximately the anterior third of the uterine horn. Although deep intrauterine insemination was performed in only one uterine horn, the percentages of embryos collected from the tip of both uterine horns 2 days after deep insemination were not significantly different. The results show that in comparison with standard AI, a 20-60-fold reduction in the number of spermatozoa inseminated and an 8-10-fold reduction in the dose volume can be achieved without decreasing fertility when semen is deposited non-surgically into the upper first third of one uterine horn.

Beneficial effects of low dose Musa paradisiaca on the semen quality of male Wistar rats

PubMed Central

Alabi, A. S.; Omotoso, Gabriel O.; Enaibe, B. U.; Akinola, O. B.; Tagoe, C. N. B.

2013-01-01

Background: This study aimed at determining the effects of administration of mature green fruits of Musa paradisiaca on the semen quality of adult male Wistar rats. Materials and Methods: The animals used for the study were grouped into three: the control group, given 2 ml of double distilled water, a low dose group given 500 mg/kg/day and a high dose group given 1000 mg/kg/day of the plantain fruits, which was made into flour, and dissolved in 2 ml of double distilled water for easy oral administration. Results: Significant increment in the semen parameters was noticed in animals that received a lower dose of the plantain flour, but those animals who received the high dose had marked and very significant reduction in sperm cell concentration and percentage of morphologically normal spermatozoa. Conclusion: Musa paradisiaca should be consumed in moderate quantities in order to derive its beneficial effects of enhancing male reproductive functions. PMID:23798793

Thallium-induced testicular toxicity in the rat.

PubMed

Formigli, L; Scelsi, R; Poggi, P; Gregotti, C; Di Nucci, A; Sabbioni, E; Gottardi, L; Manzo, L

1986-08-01

Reproductive tract functions were studied in adult male Wistar rats given 10 ppm thallium as thallium sulfate in the drinking water. After 60 days of treatment, spermatozoa isolated from the cauda epididymides and vas deferens showed reduced motility and immature germ cells were found in the tubular lumen. Histological examination of testes in thallium-treated animals revealed disarrangement of the tubular epithelium and ultrastructural changes in the Sertoli cells with cytoplasmic vacuolation and distension of the smooth endoplasmic reticulum. The activity of testicular beta-glucuronidase was significantly reduced whereas acid phosphatase and sorbitol dehydrogenase activities were unchanged. Plasma testosterone levels were within normal limits. No abnormalities in testicular morphology and biochemistry were seen in animals sacrificed at the end of the first month of thallium exposure. These findings indicate that the male reproductive system is a susceptible target site to toxic effects of thallium under chronic exposure. They also suggest a major involvement of Sertoli cells in the mechanism underlying thallium-induced testicular damage.

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.

A quantitative study of the effects of vasectomy on spermatogenesis in rats.

PubMed Central

McDonald, S W; Scothorne, R J

1988-01-01

A large sample of cross sectional profiles of seminiferous tubules from the left testes of five Albino Swiss rats 6 months after left unilateral vasectomy was compared with those of sham-operated controls. Using the classification of Leblond & Clermont (1952), based primarily on the morphology of the spermatids, the frequency of each stage of the seminiferous cycle was recorded. Profiles were also analysed for distension, reduction in epithelial area and changes in spermatocyte numbers. The lack of significant alterations in either the seminiferous cycle, the numbers of pachytene spermatocyte nuclei or epithelial area in the tubular profiles indicated that there was no alteration in spermatogenic rate after vasectomy. The lack of tubular distension, reduction in spermatocytes per unit length of perimeter or of the presence of mature spermatozoa at inappropriate stages of the cycle indicated the absence of sperm retention. The study makes clear that, at least in Albino Swiss rats 6 months after vasectomy, the apparently healthy tubules were indeed normal. PMID:3248969

Relationship between conventional semen characteristics, sperm motility patterns and fertility of Andalusian donkeys (Equus asinus).

PubMed

Dorado, J; Acha, D; Ortiz, I; Gálvez, M J; Carrasco, J J; Díaz, B; Gómez-Arrones, V; Calero-Carretero, R; Hidalgo, M

2013-12-01

Sperm quality has an important role in determining fertility. The aims of this study were to compare the conventional sperm parameters, plus the characteristics of the motility patterns of the different sperm subpopulations, of donkey donors with different fertility level, and to determine their relationships to fertility. Thirty ejaculates from 6 Andalusian donkeys were assessed for gel-free volume, pH, sperm concentration, motility and morphology. The fertility of donkeys was classified on the basis of pregnancy rates per cycle, where donkeys with a per cycle pregnancy rate ≥60% were considered to be "fertile" (n=3) and those with a per cycle pregnancy rate <40% were categorized to be "sub-fertile" (n=3). Significant differences (P<0.001) between the "fertile" and the "sub-fertile" group were found for total and progressive motility, and for straight line velocity. Sperm variables associated (P<0.05) with an increase in percent pregnant per cycle included total motility (r=0.37), progressive motility (r=0.53), curvilinear velocity (r=0.44), straightness (r=0.39), beat cross frequency (r=0.44), and gel-free volume (r=0.53). Four sperm subpopulations (sP) were identified in fresh semen: sP1 (slow and non-progressive spermatozoa, 20%), sP2 (moderately slow but progressive spermatozoa, 71.2%), sP3 (highly active but non-progressive spermatozoa, 2.9%), and sP4 (highly active and progressive spermatozoa, 5.9%). The lowest percentage (3.1%; P<0.001) of sP4 spermatozoa was observed in the "sub-fertile" group. Three of the sperm subpopulations were related (P<0.05) to fertility (sP2, r=0.54; sP3, r=0.45; sP4, r=0.56). In conclusion, we were able to relate the fertility of donkeys with in vitro measures of sperm motility using computer-assisted sperm analysis techniques. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluating the anti-fertility potential of α-chlorohydrin on testis and spermatozoa in the adult male wild Indian house rat (Rattus rattus).

PubMed

Madhu, Nithar Ranjan; Sarkar, Bhanumati; Biswas, Surjyo Jyoti; Behera, Biplab Kumar; Patra, Ashis

2011-01-01

To examine the effects of α-chlorohydrin on testis and cauda epididymis in the male house rat (Rattus rattus), 24 adult male rats were segregated into two groups. Group I rats were force-fed daily by intragastric intubation with α-chlorohydrin at a single dose of 1.0 mg/100 g body weight/d for 5, 15, and 45 days. Another group was fed with distilled water, which served as the control. The treated male rats were paired with 24 adult proestrus female rats for 5 days after the last oral treatment and fertility was tested. At the end of the experiments, all of the male rats were weighed and killed by cervical dislocation. The right testes were removed, weighed, and processed for ultrastructural changes of spermatozoa from the cauda epididymis and testis under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The seminiferous tubular area, nuclear diameter of the Sertoli and Leydig cells, percentage of spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, spermatozoa, and Sertoli cells in each group were compared morphometrically. Our results showed that the percentages of primary spermatocytes steadily increased from 5 to 15 days, but primary and secondary spermatocytes decreased significantly at 45 days. There was a steady decline in the percentages of spermatozoa and spermatids at all fixation intervals in the treated animals, but the percentages of spermatogonia and Sertoli cells increased significantly at 15 and 45 days. Seminiferous tubular areas, nuclear diameter of Leydig and Sertoli cells, and fertility rates were reduced after 45 days of treatment. SEM and TEM studies revealed severe morphological abnormalities in the spermatozoa, including deglutination of the acrosomal part, loss of head capsules, and fragmentation of tail fibrils. There was an enhanced anti-fertility effect and a lower number of implantation sites in the rats treated for 5 days. Our results validate α-chlorohydrin as a successful anti-fertility agent that prevents spermatogenesis.

Ultrastructure of spermatozoa of Onthophagus taurus (Coleoptera, Scarabaeidae) exhibits heritable variation

NASA Astrophysics Data System (ADS)

Werner, Michael; Simmons, Leigh W.

2011-03-01

Sperm competition is thought to be an important selective pressure shaping sperm form and function. However, few studies have moved beyond gross examinations of sperm morphology. Sperm length is subject to sexual selection via sperm competition in the scarab beetle Onthophagus taurus. Here, the structure and ultrastructure of spermatozoa in this species were investigated using light and electron microscopy. Spermatozoa were found to be filiform, measuring about 1,200 mm in length. The sperm head consists of a three-layered acrosome and a nuclear region bearing the anterior extension of the centriole adjunct. Acrosome and nuclear regions are bilaterally symmetric, with their axes of symmetry being orthogonal to each other. Head and flagellar structures are connected by a well-developed centriole adjunct. The sperm heads are asymmetrically surrounded by accessory material and embedded into the cytoplasm of the spermatocyst cell. The accessory material is produced inside the spermatids and then transferred to the outside due to a new membrane formed around the sperm's organelles. The old spermatid membrane separates the accessory material from the cyst cell. The flagellum contains a 9+9+2 axoneme, two accessory bodies, and two mitochondrial derivatives of unequal size. The major mitochondrial derivative is significantly larger than the minor one. The axoneme is arranged in a sinusoidal manner parallel along the major mitochondrial derivative. The spermatozoa show no progressive motility when released in buffer solution which is likely to be the result of the flagellar arrangement and the structure of the major mitochondrial derivative. The cross-sectional area of the minor and the major mitochondrial derivatives show different patterns of genetic variation. The data provide the first estimates of genetic variation in sperm ultrastructure for any species, and give evidence for the persistence of genetic variation in ultrastructure required for the rapid and divergent evolution that characterizes spermatozoa generally.

Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function.

PubMed

Gomez, E; Buckingham, D W; Brindle, J; Lanzafame, F; Irvine, D S; Aitken, R J

1996-01-01

A method has been developed for quantifying the residual cytoplasm present in the midpiece of human spermatozoa, based upon the imaging of NADH oxidoreductase activity. This procedure used NADH and nitroblue tetrazolium as electron donor and acceptor, respectively, and resulted in the discrete staining of the entire midpiece area, including the residual cytoplasm. Image analysis techniques were then used to generate binary images of the midpiece, from which objective measurements of this cellular domain could be undertaken. Such data were found to be highly correlated with biochemical markers of the cytoplasmic space, such as creatine kinase (CK) and glucose-6-phosphate dehydrogenase (G-6-PDH), in sperm populations depleted of detectable leukocyte contamination. Morphometric analysis of the sperm midpiece was also found to reflect semen quality in that it predicted the proportion of the ejaculate that would be recovered from the high-density region of Percoll gradients and was negatively correlated with the movement and morphology of the spermatozoa in semen. Variation in the retention of excess residual cytoplasm was also associated with differences in the functional competence of washed sperm preparations, both within and between ejaculates. Thus, within-ejaculate comparisons of high- and low-density sperm subpopulations revealed a relative disruption of sperm function in the low-density fraction. This disruption was associated with the presence of excess residual cytoplasm in the midpiece, high concentrations of cytoplasmic enzymes, and the enhanced-generation reactive oxygen species (ROS). Functional differences between individual high-density Percoll preparations were also negatively correlated with the area of the midpiece and the corresponding capacity of the spermatozoa to generate ROS. These findings suggest that one of the factors involved in the etiology of defective sperm function is the incomplete extrusion of germ cell cytoplasm during spermiogenesis as a consequence of which the spermatozoa experience a loss of function associated with the induction of oxidative stress.

Contraception with RISUG® and functional reversal through DMSO and NaHCO3 in male rabbits.

PubMed

Ansari, Abdul S; Badar, Ayesha; Balasubramanian, Krithika; Lohiya, Nirmal K

2017-01-01

The study aimed to evaluate reversal of short- and long-term vas occlusion with reversible inhibition of sperm under guidance (RISUG) using dimethyl sulfoxide (DMSO) and sodium bicarbonate (NaHCO3) in male rabbits (Oryctolagus cuniculus). Animals were divided into seven groups containing five animals each. Fortnightly, semen analysis revealed that sperm concentration and output steadily declined after vas occlusion and complete azoospermia was attained at 30-60 days postinjection. Spermatozoa reappeared at 60-75 days of reversal and normozoospermia was noticed between 135 days and 150 days in the reversal groups. All spermatozoa were found nonmotile prior to azoospermia and a gradual recovery in sperm motility was observed between 105 days and 135 days of reversal. A significant decline in viability of sperms was noticed during vas occlusion up to 30-60 days which recovered at 60-75 days postreversal and normalized by 75-105 days in the reversal groups. A significant enhancement in the sperm abnormalities was recorded in all vas occluded animals as well as those in initial periods of reversal. Other parameters, namely, semen volume, ejaculation time, pH, color, and consistency, remained unaltered during all phases of the study. Fertility test, at the intervals of 15 days, demonstrated that animals exhibited complete sterility during the entire period of vas occlusion. A gradual recovery in fertility was observed with the appearance of spermatozoa following vas occlusion reversal and 100% fertility was observed following 135-150 days of reversal. F1 progeny of reversed animals was found normal. The results suggest that reversal with DMSO or NaHCO3 is feasible, with normal progeny, following short- and long-term contraception.

Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

PubMed

Mitchell, V; Sigala, J; Ballot, C; Jumeau, F; Barbotin, A L; Duhamel, A; Rives, N; Rigot, J M; Escalier, D; Peers, M C

2015-03-01

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk. © 2014 Blackwell Verlag GmbH.

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.

Assessment of sperm nuclear quality after in vitro maturation of fresh or frozen/thawed mouse pre-pubertal testes.

PubMed

Oblette, A; Rives, N; Dumont, L; Rives, A; Verhaeghe, F; Jumeau, F; Rondanino, C

2017-10-01

Is nuclear quality of in vitro generated spermatozoa from fresh or frozen/thawed pre-pubertal mouse testes similar to that of their in vivo counterparts? The production of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was not significantly increased in organotypic cultures compared to in vivo controls. Although murine spermatozoa have been produced in vitro from pre-pubertal testes, their nuclear DNA integrity has never been investigated. Fresh and frozen/thawed testicular fragments from 6 to 7 days postpartum (dpp) mice were cultured for 30 days. Testicular tissues were frozen by controlled slow freezing (CSF) or solid surface vitrification (SSV). In total, 30 fresh, 30 CSF, 30 SSV testes were used for in vitro maturation and 6 testes from 36 to 37 dpp mice were used as in vivo controls. Murine spermatozoa were extracted from pooled in vitro cultured testicular fragments and from in vivo controls. Sperm aneuploidy was analyzed by fluorescence in situ hybridization (FISH), DNA fragmentation by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, chromatin condensation by aniline blue staining, telomere length and number by quantitative FISH, DNA oxidation by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Because of the low spermatogenic yield in cultures, a hundred spermatozoa extracted from pooled tissues were examined and compared to their in vivo counterparts. Most of spermatozoa generated in vitro and in vivo were haploid, contained unfragmented DNA and normally condensed chromatin. A similar proportion of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was found in cultures and in vivo. No significant difference in telomere length was found within the nuclei of in vitro and in vivo generated spermatozoa. However, the number of telomere spots was lower in gametes obtained from cultures of fresh, CSF and SSV testes than in their natural counterparts (P < 0.01). Moreover, the proportion of spermatozoa containing 8-OHdG was significantly increased in frozen/thawed tissues in comparison to fresh tissues and in vivo controls (P < 0.05). None. Further studies will be needed to enhance the production of spermatozoa in organotypic cultures while preserving their quality, to investigate epigenetic modifications and embryonic development. This is the first study comparing the nuclear quality of in vitro and in vivo generated murine spermatozoa. The organotypic culture system will have to be adapted for human tissue and extensive analyses of human gamete quality will have to be performed before potential clinical applications can be envisaged. This work was supported by Rouen University Hospital, Ligue contre le Cancer, Agence de la Biomédecine, Association Laurette Fugain, France Lymphome Espoir, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:journals.permissions@oup.com

Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1.

PubMed

Moretti, E; Pascarelli, N A; Belmonte, G; Renieri, T; Collodel, G

2017-09-01

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology. © 2016 Blackwell Verlag GmbH.

A birth in non-mosaic Klinefelter's syndrome after testicular fine needle aspiration, intracytoplasmic sperm injection and preimplantation genetic diagnosis.

PubMed

Reubinoff, B E; Abeliovich, D; Werner, M; Schenker, J G; Safran, A; Lewin, A

1998-07-01

Non-mosaic Klinefelter patients are generally azoospermic due to primary testicular failure. Nevertheless, in some cases, testicular spermatozoa may be recovered and utilized to fertilize oocytes via intracytoplasmic sperm injection (ICSI). As the risk for an increased number of gonosomes in these spermatozoa is unclear, preimplantation genetic diagnosis (PGD) may be attempted in the resulting embryos. In the present study, we report our experience with the combined approach of sperm retrieval by testicular fine needle aspiration (FNA), ICSI and PGD in seven consecutive non-mosaic Klinefelter individuals. In four patients, between one and five spermatozoa were retrieved in five out of nine consecutive attempts. In a fifth patient, only 10 round spermatids could be isolated. Mature spermatozoa were injected into a total of 16 metaphase-II oocytes, of which 11 (69%) remained intact. Two distinct pronuclei (2PN) were observed in four oocytes (36%) while a single pronucleus (1PN) was documented in two oocytes. Five cleavage stage embryos developed from the oocytes of two couples. Upon the request of one couple, their three embryos (two derived from 1PN oocytes) were transferred without PGD but pregnancy was not achieved. PGD by fluorescence in-situ hybridization (FISH) was performed in the two embryos of the other couple which were derived from normal fertilization. PGD results of one embryo were 18,18,X,X,Y, the embryo was not transferred and FISH analysis of the remaining blastomeres identified variable chromosome numbers in the nuclei. The second embryo was diagnosed as normal and was transferred, resulting in a successful pregnancy and birth. In conclusion, the results of this report indicate that a pregnancy and birth may be attained in azoospermic non-mosaic Klinefelter individuals by testicular FNA combined with ICSI. Due to the unknown risk of gonosomes aneuploidy in embryos from Klinefelter patients, PGD or prenatal diagnosis should be recommended.

Meiotic segregation analysis in spermatozoa of pericentric inversion carriers using fluorescence in-situ hybridization.

PubMed

Morel, F; Laudier, B; Guérif, F; Couet, M L; Royère, D; Roux, C; Bresson, J L; Amice, V; De Braekeleer, M; Douet-Guilbert, N

2007-01-01

Pericentric inversions are structural chromosomal abnormalities resulting from two breaks, one on either side of the centromere, within the same chromosome, followed by 180 degrees rotation and reunion of the inverted segment. They can perturb spermatogenesis and lead to the production of unbalanced gametes through the formation of an inversion loop. We report here the analysis of the meiotic segregation in spermatozoa from six pericentric inversion carriers by multicolour fluorescence in-situ hybridization (FISH) and review the literature. The frequencies of the non-recombinant products (inversion or normal chromosomes) were 80% for the inv(20), 91.41% for the inv(12), 99.43% for the inv(2), 68.12% for the inv(1), 97% for the inv(8)(p12q21) and 60.94% for the inv(8)(p12q24.1). The meiotic segregation of 20 pericentric inversions (including ours) is now available. The frequency of unbalanced spermatozoa varies from 0 to 37.85%. The probability of a crossover within the inverted segment is affected by the chromosome and region involved, the length of the inverted segment and the location of the breakpoints. No recombinant chromosomes were produced when the inverted segment involved <30% of the chromosome length (independent of the size of the inverted segment). Between 30 and 50%, few recombinant chromosomes were produced, inducing a slightly increased risk of aneusomy of recombination in the offspring. The risk of aneusomy became very important when the inverted segment was >50% of the chromosome length. Studies on spermatozoa from inversion carriers help in the comprehension of the mechanisms of meiotic segregation. They should be integrated in the genetic exploration of the infertile men to give them a personalized risk assessment of unbalanced spermatozoa.

Effect of commercial long-term extenders on metabolic activity and membrane integrity of boar spermatozoa stored at 17 degrees C.

PubMed

Dziekońska, A; Fraser, L; Majewska, A; Lecewicz, M; Zasiadczyk, Ł; Kordan, W

2013-01-01

This study was aimed to analyze the metabolic activity and membrane integrity of boar spermatozoa following storage in long-term semen extenders. Boar semen was diluted with Androhep EnduraGuard (AeG), DILU-Cell (DC), SafeCell Plus (SCP) and Vitasem LD (VLD) extenders and stored for 10 days at 17 degrees C. Parameters of the analyzed sperm metabolic activity included total motility (TMOT), progressive motility (PMOT), high mitochondrial membrane potential (MMP) and ATP content, whereas those of the membrane integrity included plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome. Extender type was a significant (P < 0.05) source of variation in all the analyzed sperm parameters, except for ATP content. Furthermore, the storage time had a significant effect (P < 0.05) on the sperm metabolic activity and membrane integrity during semen storage. In all extenders the metabolic activity and membrane integrity of the stored spermatozoa decreased continuously over time. Among the four analyzed extenders, AeG and SCP showed the best performance in terms of TMOT and PMI on Days 5, 7 and 10 of storage. Marked differences in the proportions of spermatozoa with high MMP were observed between the extenders, particularly on Day 10 of storage. There were not any marked differences in sperm ATP content between the extenders, regardless of the storage time. Furthermore, the percentage of spermatozoa with NAR acrosomes decreased during prolonged storage, being markedly lower in DC-diluted semen compared with semen diluted with either AeG or SCP extender. The results of this study indicated that components of the long-term extenders have different effects on the sperm functionality and prolonged semen longevity by delaying the processes associated with sperm ageing during liquid storage.

Rosiglitazone Improves Stallion Sperm Motility, ATP Content, and Mitochondrial Function.

PubMed

Swegen, Aleona; Lambourne, Sarah Renay; Aitken, R John; Gibb, Zamira

2016-11-01

Media used for equine sperm storage often contain relatively high concentrations of glucose, even though stallion spermatozoa preferentially utilize oxidative phosphorylation (OXPHOS) over glycolysis to generate ATP and support motility. Rosiglitazone is an antidiabetic compound that enhances metabolic flexibility and glucose utilization in various cell types, but its effects on sperm metabolism are unknown. This study investigated the effects of rosiglitazone on stallion sperm function in vitro, along with the possible role of AMP-activated protein kinase (AMPK) in mediating these effects. Spermatozoa were incubated with or without rosiglitazone, GW9662 (an antagonist of peroxisome proliferator-activating receptor-gamma), and compound C (CC; an AMPK inhibitor). Sperm motility, viability, reactive oxygen species production, mitochondrial membrane potential (mMP), ATP content, and glucose uptake capacity were measured. Samples incubated with rosiglitazone displayed significantly higher motility, percentage of cells with normal mMP, ATP content, and glucose uptake capacity, while sperm viability was unaffected. The percentage of spermatozoa positive for mitochondrial ROS was also significantly lower in rosiglitazone-treated samples. AMPK localized to the sperm midpiece, and its phosphorylation, was increased in rosiglitazone-treated spermatozoa. CC decreased sperm AMPK phosphorylation and reduced sperm motility, and successfully inhibited the effects of rosiglitazone. Inclusion of rosiglitazone in a room temperature sperm storage medium maintained sperm motility above 60% for 6 days, attaining significantly higher motility than sperm stored in control media. The ability of rosiglitazone to substantially alleviate the time-dependent deterioration of stallion spermatozoa by diverting metabolism away from OXPHOS and toward glycolysis has novel implications for the long-term, functional preservation of these cells. © 2016 by the Society for the Study of Reproduction, Inc.

Liner collection cone and pH effects on postthaw motility, staining, and acrosomes of bovine spermatozoa.

PubMed

Smith, J F; Merilan, C P

1991-04-01

Sixteen ejaculates were collected, four each from four bulls, using artificial vaginas with polyethylene or rubber liner collection cones in a crossover design experiment. The ejaculates were diluted with egg yolk-citrate extender at pH 6.4 or 7.2, cooled, glycerolated, equilibrated, packaged in .5-ml French straws, frozen in nitrogen vapor, and stored in liquid nitrogen. Thirty frozen straws from each ejaculate were thawed rapidly (46.5 degrees C for 12 s), pooled, and then incubated at 46.5 degrees C for periodic evaluation of progressive motility, differential staining, and acrosome morphology under thermal stress conditions. The postthaw motility of spermatozoa and percentage of unstained cells were higher both when collected in polyethylene than in rubber and when extended at pH 7.2 vs. 6.4, but no interaction was found between liner collection cone composition and pII for postthaw motility. Retention of spermatozoan motility during incubation under thermal stress was greater for cells collected in polyethylene, but not different due to pH. Neither pH nor composition of liner collection cone had an effect on postthaw acrosomal scores, but the time required for a 50% increase in severely damaged acrosomes was greater for spermatozoa collected in polyethylene than in rubber liner collection cones.

In-vitro effects of Thymus munbyanus essential oil and thymol on human sperm motility and function.

PubMed

Chikhoune, Amirouche; Stouvenel, Laurence; Iguer-Ouada, Mokrane; Hazzit, Mohamed; Schmitt, Alain; Lorès, Patrick; Wolf, Jean Philippe; Aissat, Kamel; Auger, Jacques; Vaiman, Daniel; Touré, Aminata

2015-09-01

Traditional medicine has been used worldwide for centuries to cure or prevent disease and for male or female contraception. Only a few studies have directly investigated the effects of herbal compounds on spermatozoa. In this study, essential oil from Thymus munbyanus was extracted and its effect on human spermatozoa in vitro was analysed. Gas chromatography and Gas chromatography-mass spectrometry analyses identified 64 components, accounting for 98.9% of the composition of the oil. The principal components were thymol (52.0%), γ-terpinene (11.0%), ρ-cymene (8.5%) and carvacrol (5.2%). Freshly ejaculated spermatozoa was exposed from control individuals to various doses of the essential oil for different time periods, and recorded the vitality, the mean motility, the movement characteristics (computer-aided sperm analysis), the morphology and the ability to undergo protein hyperphosphorylation and acrosomal reaction, which constitute two markers of sperm capacitation and fertilizing ability. In vitro, both the essential oil extracted from T. munbyanus and thymol, the principal compound present in this oil, impaired human sperm motility and its capacity to undergo hyperphosphorylation and acrosome reaction. These compounds may, therefore, be of interest in the field of reproductive biology, as potential anti-spermatic agents. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Semen collection, characterisation and artificial insemination in the beluga (Delphinapterus leucas) using liquid-stored spermatozoa.

PubMed

O'Brien, J K; Steinman, K J; Schmitt, T; Robeck, T R

2008-01-01

Ejaculates were collected from a beluga (Delphinapterus leucas) to gain an understanding of sperm biology and develop a short-term sperm preservation method for use in artificial insemination (AI). Ejaculate parameters and biochemistry, semen production and serum testosterone concentrations of an adult male were characterised for 21 months. Sperm viability, acrosome integrity and morphology did not change (P > 0.05) but ejaculate volume, sperm concentration and total spermatozoa per ejaculate were higher (P < 0.05) from January to June than from July to December. Peak testosterone concentrations (P < 0.05) were observed from October to April (8.0 +/- 1.6 ng mL(-1)). The effects of hyaluronic acid (HA), antioxidants, storage temperature and time on in vitro sperm characteristics were examined. Motility parameters and viability were improved (P < 0.05) when semen was stored at 5 degrees C compared with 21 degrees C. During the first 24 h of storage sperm agglutination was absent only at 5 degrees C in the presence of HA. A nulliparous 28-year-old female was inseminated endoscopically with liquid-stored semen. A pregnancy and birth of a calf was achieved following AI for the first time in this species, thereby validating both the AI technique and the fertility of beluga spermatozoa after chilled storage in a specialised diluent.

A data-driven search for semen-related phenotypes in conception delay

PubMed Central

Patel, C. J.; Sundaram, R.; Buck Louis, G. M.

2016-01-01

SUMMARY Sperm count, morphology, and motility have been reported to be predictive of pregnancy, although with equivocal basis prompting some authors to question the prognostic value of semen analysis. To assess the utility of including semen quality data in predicting conception delay or requiring >6 cycles to become pregnant (referred to as conception delay), we utilized novel data-driven analytic techniques in a pre-conception cohort of couples prospectively followed up for time-to-pregnancy. The study cohort comprised 402 (80%) male partners who provided semen samples and had time-to-pregnancy information. Female partners used home pregnancy tests and recorded results in daily journals. Odds ratios (OR), false discovery rates, and 95% confidence intervals (CIs) for conception delay (time-to-pregnancy > 6 cycles) were estimated for 40 semen quality phenotypes comprising 35 semen quality endpoints and 5 closely related fecundity determinants (body mass index, time of contraception, lipids, cotinine and seminal white blood cells). Both traditional and strict sperm phenotype measures were associated with lower odds of conception delay. Specifically, for an increase in percent morphologically normal spermatozoa using traditional methods, we observed a 40% decrease in conception delay (OR = 0.6, 95% CI = 0.50, 0.81; p = 0.0003). Similarly, for an increase in strict criteria, we observed a 30% decrease in odds for conception delay (OR = 0.7, 95% CI = 0.52, 0.83; p = 0.001). On the other hand, an increase in percent coiled tail spermatozoa was associated with a 40% increase in the odds for conception delay (OR = 1.4, 95% CI = 1.12, 1.75; p = 0.003). However, our findings suggest that semen phenotypes have little predictive value of conception delay (area under the curve of 73%). In a multivariate model containing significant semen factors and traditional risk factors (i.e. age, body mass index, cotinine and ever having fathered a pregnancy), there was a modest improvement in prediction of conception delay (16% increase in area under the curve, p < 0.0002). PMID:27792860

Comparison of a direct and indirect ELISA for quantitating antisperm antibody in semen.

PubMed

Lynch, D M; Howe, S E

1987-01-01

A direct and an indirect quantitative ELISA for antisperm antibody were compared using the spermatozoa and cell-free seminal fluid of 66 infertile males. The normal concentration of sperm binding immunoglobulin was less than or equal to 1.5 fg Ig per spermatozoon for the indirect seminal plasma assay and less than or equal to 1.5 fg Ig per spermatozoon by the direct assay. Of the 66 infertile males, 21% (14/66) had elevated levels of antisperm antibody in their seminal plasma and 26% (17/66) had elevated levels bound directly to their spermatozoa. The direct correlation between the results of these assays was 94%. A simple linear regression analysis between the indirect and direct measurements of antisperm antibody resulted in a correlation coefficient of r = 0.907. There was no statistically significant difference between results from the direct and indirect methods of the patients as a group. However, there was evidence of autospecificity in a small percentage of males who had elevated levels of antisperm antibody by the direct assay that was not detected by the indirect assay using pooled donor spermatozoa.

A critical role of solute carrier 22a14 in sperm motility and male fertility in mice

PubMed Central

Maruyama, Shin-ya; Ito, Momoe; Ikami, Yuusuke; Okitsu, Yu; Ito, Chizuru; Toshimori, Kiyotaka; Fujii, Wataru; Yogo, Keiichiro

2016-01-01

We previously identified solute carrier 22a14 (Slc22a14) as a spermatogenesis-associated transmembrane protein in mice. Although Slc22a14 is a member of the organic anion/cation transporter family, its expression profile and physiological role have not been elucidated. Here, we show that Slc22a14 is crucial for sperm motility and male fertility in mice. Slc22a14 is expressed specifically in male germ cells, and mice lacking the Slc22a14 gene show severe male infertility. Although the overall differentiation of sperm was normal, Slc22a14−/− cauda epididymal spermatozoa showed reduced motility with abnormal flagellar bending. Further, the ability to migrate into the female reproductive tract and fertilise the oocyte were also impaired in Slc22a14−/− spermatozoa. The abnormal flagellar bending was thought to be partly caused by osmotic cell swelling since osmotic challenge or membrane permeabilisation treatment alleviated the tail abnormality. In addition, we found structural abnormalities in Slc22a14−/− sperm cells: the annulus, a ring-like structure at the mid-piece–principal piece junction, was disorganised, and expression and localisation of septin 4, an annulus component protein that is essential for the annulus formation, was also impaired. Taken together, our results demonstrated that Slc22a14 plays a pivotal role in normal flagellar structure, motility and fertility in mouse spermatozoa. PMID:27811987

Testicular function during puberty and young adulthood in patients with Klinefelter's syndrome with and without spermatozoa in seminal fluid.

PubMed

Rohayem, J; Nieschlag, E; Zitzmann, M; Kliesch, S

2016-11-01

Patients with Klinefelter's syndrome experience progressive testicular degeneration resulting in impaired endocrine function and azoospermia. What proportion of adolescents develop testosterone deficiency during puberty and how many have spermatozoa in their semen is unclear to date. We aimed to investigate testicular function during puberty and young adulthood in patients with Klinefelter's syndrome and to assess testosterone effects in target tissues. The clinical data of 281 patients with non-mosaic Klinefelter's syndrome aged 10-25 years without previous testosterone replacement were reviewed. In late pubertal adolescents, semen analyses were evaluated, and testicular volumes, hormone and haemoglobin (Hb) levels, the number of CAG repeats and final height data were compared to those of 233 age-matched controls with pubertal gynaecomastia. Spontaneous pubertal virilisation to Tanner stages IV-V occurred. Serum T levels ≥10 nmol/L were reached in 62% of patients with Klinefelter's syndrome and in 85% of controls at ages 15-25 (T KFS : 12.2 ± 5.4 vs. T C : 16.6 ± 7.2 nmol/L). LH KFS levels were elevated >10 U/L in 84%, and normal in all controls (LH KFS : 18.6 ± 12.2 vs. LH C : 3.5 ± 1.6 U/L). In nine of 130 (7%) adolescents with Klinefelter's syndrome, spermatozoa (oligozoospermia) were found in semen; all had T levels >7 nmol/L and eight of nine had LH levels ≤18 U/L, while their hormone levels, number of CAG repeats and testicular volumes were not different from those of adolescents with azoospermia. Controls had normal sperm concentrations in 73% (46/63). Semen volumes KFS were normal in 55% vs. 78% in controls; Hb KFS was normal in 89% (Hb C : 97%). Mean final height KFS was 185 ± 8 cm vs. 181 ± 7 cm in controls. Hypergonadotropic hypogonadism develops during early puberty in adolescents with Klinefelter's syndrome and remains compensated in over 60% during ages 15-25, with sufficient testosterone secretion for spontaneous accomplishment of pubertal development. Spermatozoa in semen are rare and associated with T levels >7 nmol/L. Parameters reflecting androgen deficiency in target tissues may help to optimise timing of testosterone substitution, which should preferably not be initiated before fertility status has been clarified. © 2016 American Society of Andrology and European Academy of Andrology.

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol.

PubMed

Glazar, Amanda I; Mullen, Steven F; Liu, Jun; Benson, James D; Critser, John K; Squires, Edward L; Graham, James K

2009-10-01

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5 degrees and 22 degrees C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 degrees C than 22 degrees C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC's to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.

Spermatozoa with numerical chromosomal abnormalities are more prone to be retained by Annexin V-MACS columns.

PubMed

Esbert, M; Godo, A; Soares, S R; Florensa, M; Amorós, D; Ballesteros, A; Vidal, F

2017-07-01

Colloidal super-paramagnetic microbeads conjugated with annexin V are effective for separating apoptotic spermatozoa by MACS as a result of the high affinity of annexin V for externalized PS molecules. The effectiveness of the procedure in reducing the percentage of sperm with fragmented DNA and abnormal morphology has also been reported. However, it is still unknown if it could decrease the percentage of aneuploid spermatozoa. The objective of our prospective study, performed on 16 males with abnormal FISH on spermatozoa, was to assess if MACS columns were useful tools to retain spermatozoa carrying chromosomal abnormalities in semen samples processed after density gradient centrifugation (DGC). The pellet obtained after DGC was subjected to MACS, and sperm FISH analyses were performed both in the eluded fraction and in the fraction retained in the column. The observed frequencies of disomy and nullisomy 13, 18, and 21, X and Y, as well as the diploidy rates in the MACS eluded fraction and the fraction retained in the MACS column were recorded. We observed that the frequencies of aneuploidies in the eluded fraction were lower than in the fraction retained in the MACS column (0.59% vs. 0.75%; p = 0.010). DGC determined a significant reduction in sperm concentration (z-ratio = 2.83; p = 0.005) and a significant increase in sperm progressive motility (z-ratio = -3.5; p < 0.001). MACS also led to a significant reduction in sperm concentration (z-ratio = 3.14; p = 0.002) and a significant increase in progressive motility (z-ratio = -2.59; p = 0.01) when compared with the post-DGC sample. Sperm concentration was similar in the two fractions generated by MACS (z-ratio = 0.63; p = 0.52), while progressive motility was significantly higher in the MACS eluded fraction (z-ratio = 2.42; p = 0.02). According to our results, MACS columns are able to selectively retain spermatozoa carrying chromosomal abnormalities. Furthermore, the performance of DGC and MACS on semen samples leads to an enrichment of progressive motility. © 2017 American Society of Andrology and European Academy of Andrology.

Fecal endocrine profiles and ejaculate traits in black-footed cats (Felis nigripes) and sand cats (Felis margarita).

PubMed

Herrick, J R; Bond, J B; Campbell, M; Levens, G; Moore, T; Benson, K; D'Agostino, J; West, G; Okeson, D M; Coke, R; Portacio, S C; Leiske, K; Kreider, C; Polumbo, P J; Swanson, W F

2010-01-15

Information regarding the reproductive biology of black-footed cats (BFC) and sand cats (SC) is extremely limited. Our objectives were to: (1) validate fecal hormone analysis (estrogens, E; progestagens, P; androgens, T) for noninvasive monitoring of gonadal activity; (2) characterize estrous cyclicity, ovulatory mechanisms, gestation, and seasonality; and (3) evaluate male reproductive activity via fecal androgen metabolites and ejaculate traits. In both species, the estrous cycle averaged 11-12 days. In BFC (n=8), estrus lasted 2.2+/-0.2 days with peak concentrations of E (2962.8+/-166.3 ng/g feces) increasing 2.7-fold above basal concentrations. In SC (n=6), peak concentrations of E (1669.9+/-83.5 ng/g feces) during estrus (2.9+/-0.2 days) were 4.0-fold higher than basal concentrations. Nonpregnant luteal phases occurred in 26.5% (26 of 98) of BFC estrous cycles, but were not observed in SC (0 of 109 cycles). In both species, P concentrations during pregnancy were elevated (32.3+/-3.0 microg/g feces BFC; 8.5+/-0.7 microg/g feces SC) approximately 10-fold above basal concentrations. Fecal T concentrations in males averaged 3.1+/-0.1 microg/g feces in BFC and 2.3+/-0.0 microg/g feces in SC. Following electroejaculation, 200 to 250 microl of semen was collected containing 29.9 (BFC) to 36.5 (SC)x10(6) spermatozoa with 40.4 (SC) to 46.8 (BFC)% normal morphology. All females exhibited estrous cycles during the study and spermatozoa were recovered from all males on every collection attempt, suggesting poor reproductive success in these species may not be due to physiological infertility.

The Effect of Photobiomodulation on the Sea Urchin Paracentrotus lividus (Echinodermata) Using Higher-Fluence on Fertilization, Embryogenesis, and Larval Development: An In Vitro Study.

PubMed

Amaroli, Andrea; Gambardella, Chiara; Ferrando, Sara; Hanna, Reem; Benedicenti, Alberico; Gallus, Lorenzo; Faimali, Marco; Benedicenti, Stefano

2017-03-01

The aim of this study was to investigate the photobiomodulation (PBM) effect of the 808 nm diode laser irradiation on spermatozoa, eggs, fertilized eggs, embryos, and larvae of Paracentrotus lividus, using two different power settings. Studies have shown the possible use of PBM in artificial insemination. These have shown the potential effect of low-power laser irradiation on spermatozoa, while there are few studies on the effect of laser photonic energy on oocytes and almost no reports on the influence of lasers in embryogenesis. P. lividus gametes, zygotes, embryos, and larvae were irradiated using the 808 nm diode laser (fluence 64 J/cm 2 using 1 W or 192 J/cm 2 with 3 W) with a flat-top hand-piece delivery, compared to a control without laser irradiation (0 J/cm 2 -0 W). The fertilization rate and the early developmental stages were investigated. The fertilization ability was not affected by the sperm/egg irradiation. At the gastrula stage, no significant differences were observed compared with the control samples. In the late pluteus stage, there were no differences in the developmental percentage observed between the control and the treated samples (1 W), with the exception of larvae from gastrulae and larvae, which were irradiated at 3 W. This study has demonstrated that both the 64 J/cm 2 -1 W and the 192 J/cm 2 -3 W do not induce morphological damage on the irradiated P. lividus gametes whose zygotes generate normal embryos and larvae. Our data therefore support the assumption to use higher fluence in preliminary studies on in vitro fertilization.

Feline spermatozoa from fresh and cryopreserved testicular tissues have comparable ability to fertilize matured oocytes and sustain the embryo development after intracytoplasmic sperm injection.

PubMed

Buarpung, S; Tharasanit, T; Comizzoli, P; Techakumphu, M

2013-01-01

Cryopreservation of testicular tissue associated with intracytoplasmic sperm injection (ICSI) is a critical tool that still needs to be explored for preserving the fertility of endangered species. Using the domestic cat as a model for wild felids, the study aimed at determining the effect of different cryoprotectants and freezing techniques (two-step freezing vs. controlled slow freezing) on the sperm quality (membrane and DNA integrity). Then, spermatozoa were extracted from frozen-thawed testicular tissues and used for ICSI to assess early gamete activation or developmental competence in vitro. The percentage of spermatozoa with intact plasma membrane was not different (P > 0.05) among nonfrozen control, glycerol-, and ethylene glycol-frozen tissues (63.2 ± 2%, 58.2 ± 2.6%, 53.3 ± 2.3%, respectively). However, these percentages were significantly lower (P < 0.05) in groups of dimethyl sulfoxide (46.3 ± 3.3%) and 1,2 propanediol (44.3 ± 2.9%) when compared with control. Conventional freezing combined with 5% (vol/vol) glycerol best preserved sperm membrane integrity (55.0 ± 2.7%) when compared with other freezing techniques. The incidence of DNA fragmentation was found to be low (0.2%-1.1%) in all freezing protocols. After ICSI with frozen testicular spermatozoa, male and female gametes were asynchronously activated and the percentages of normal fertilization at 6, 12, and 18 hours were 11.2%, 20.6%, and 22.1%, respectively. Metaphase II-arrested oocytes containing or not a decondensed sperm head were predominantly found after ICSI with frozen testicular spermatozoa. Although two-pronucleus formation could be observed as soon as 6 hours post ICSI (10%), the rate increased dramatically after 12 and 18 hours post ICSI (17.2% and 19.5%, respectively). ICSI using frozen-thawed testicular spermatozoa yielded cleavage (32.7%), morula (6.5%), and blastocyst (4.4%) percentages similar to nonfrozen control (P > 0.05). It is concluded that conventional freezing technique with glycerol as a principle cryoprotectant is simplified and applicable for cat testicular tissue cryopreservation. We also demonstrate for the first time that feline spermatozoa derived from frozen-thawed testicular tissues retain their fertilizing ability and can be used to produce ICSI-derived embryos. Copyright © 2013 Elsevier Inc. All rights reserved.

Sperm chemorepulsion, a supplementary mechanism to regulate fertilization.

PubMed

Guidobaldi, H A; Cubilla, M; Moreno, A; Molino, M V; Bahamondes, L; Giojalas, L C

2017-08-01

Are human spermatozoa able of chemorepulsive behaviour? Capacitated human spermatozoa are able to be chemorepelled by synthetic Progesterone Receptor Ligands (sPRL, known as contraceptives) and zinc (a cation released by the oocyte upon fertilization). Moving cells can be oriented towards or against a molecular gradient, processes called chemoattraction and chemorepulsion, respectively, which have been described in unicellular organisms such as amoebas and bacteria, to organismic cells such macrophages and developmental cells. In the case of spermatozoa, chemoattraction may help the finding of an oocyte and has been widely studied in various invertebrate and mammalian species; however, chemorepulsion has not yet been verified in spermatozoa. This is an in vitro study involving human, rabbit and mouse spermatozoa which were used to perform 3-30 experiments per treatment. Human sperm samples were obtained by masturbation from healthy donors who gave written consent. Only those samples exhibiting normal semen parameters according to current WHO criteria were included in the study. Rabbit spermatozoa were obtained by artificial vagina whereas mice spermatozoa were obtained from epididymis. The sperm selection assay (SSA), originally designed to evaluate sperm chemoattraction towards progesterone (P), and a video-microscopy and computer motion analysis system were used to test sperm chemorepulsion. Additional kinetic parameters were also determined by video-microscopy and computer motion analysis. In some experiments, the level of induced acrosome-reacted spermatozoa was determined. Rabbit mating manipulation was achieved to perform the sperm-oocyte co-incubation assay. Sperm accumulation in the well containing 100 pg/ml of sPRL was lower than the culture medium negative control (P < 0.05). The percentage of sperm persistence against the well containing 100 pg/ml ulipristal acetate (UPA) (P = 0.001), and the percentage of sperm showing a repulsive pattern of movement (a linear trajectory followed by a transitional one after turning against the UPA), were higher than the culture medium negative control (P = 0.049). Sperm accumulation was diminished when spermatozoa where exposed to a homogeneous distribution of 100 pg/ml sPRL combined with a chemotactic gradient of progesterone (P), with respect to the culture medium negative control (P < 0.05). These results were reverted when non-capacitated spermatozoa were used to perform the same experimental settings. The accumulation of spermatozoa against 100 pg/ml sPRL was lower than the culture medium negative control also in rabbits and mice (P < 0.05). The relative number of rabbit spermatozoa arriving to the vicinity of the oocyte was diminished under the presence of 100 pg/ml UPA (P = 0.004). Sperm accumulation in the well containing zinc was decreased compared to the culture medium negative control (P < 0.05). A homogeneous distribution of zinc combined with a gradient of 10 pM P, was lower than the culture medium negative control (P = 0.016). The results were quite reproducible with two different methodologies (accumulation assay and video-microscopy combined with computer motion analysis), in three mammalian species. The experiments were performed in vitro. Even though a quite complete characterization of sperm chemorepulsion was provided, the molecular mechanism that governs sperm repulsion is currently under investigation. Since the chemorepelled spermatozoa are those physiologically ready to fertilize the oocyte, these findings may have both biological and clinical implications, preventing either polyspermy under natural conditions or fertilization under pharmacological treatment with sPRL. The study was financed by the Universidad Nacional de Cordoba (Argentina). The authors declare that they do not have competing financial interests. N/A. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Bovine epididymal spermatozoa: Resistance to cryopreservation and binding ability to oviductal cells.

PubMed

Cunha, A T M; Carvalho, J O; Kussano, N R; Martins, C F; Mourão, G B; Dode, M A N

2016-12-01

In this study we examined quality, longevity and ability of epididymal sperm (EP) to bind to oviduct explants (OE) after cooling and cryopreservation. Ejaculated (EJ) and EP sperm from seven bulls were evaluated before, during and after cryopreservation for total (TM), progressive motility (PM), sperm morphology, plasma membrane integrity (PMI) and acrosome integrity (ACI). For longevity, cryopreserved EP, EJ and a third group of cells in which EP spermatozoa were incubated with seminal plasm (SP) for 10 min after thawing (EPP group), were compared, and the groups were analyzed at 0, 3, 6, and 24 h for all parameters. Sperm from each group were co-incubated with OE for 30 min, 6 h, and 24 h for binding evaluation. Data were analyzed by the generalized linear models SAS 9.1 (P < 0.05). After cooling, EP displayed higher TM, higher PMI, and higher ACI (P < 0.05) than EJ. No differences were noted in the percentage of spermatozoa with PMI and AI between EJ and EP for fresh, cooled or cryopreserved sperm. However, a reduction in motility occurred in the EJ sperm after cooling, while in EP group such reduction occurred only after cryopreservation. At 6 h of incubation EP and EPP had higher PMI and ACI than EJ (P < 0.05). The number of spermatozoa bound to OE was similar P > 0.05) for all groups either at 30 min or 24 h. We conclude that EP are more resistant to cooling than EJ, and can bind to OE similarly to EJ. Copyright © 2016 Elsevier Inc. All rights reserved.

Negative biomarker-based male fertility evaluation: sperm phenotypes associated with molecular-level anomalies

PubMed Central

Sutovsky, Peter; Aarabi, Mahmoud; Miranda-Vizuete, Antonio; Oko, Richard

2015-01-01

Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry (FC). A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies (ART) and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA (Lens culinaris agglutinin; reveals altered sperm surface) and PNA (Arachis hypogaea/peanut agglutinin; reveals acrosomal malformation or damage). The Postacrosomal Sheath WWI Domain Binding Protein (PAWP), implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery. PMID:25999356

Molecular morphology and function of bull spermatozoa linked to histones and associated with fertility.

PubMed

de Oliveira, Rodrigo V; Dogan, Sule; Belser, Lauren E; Kaya, Abdullah; Topper, Einko; Moura, Arlindo; Thibaudeau, Giselle; Memili, Erdogan

2013-09-01

Sub-par fertility in bulls is influenced by alterations in sperm chromatin, and it might not be solved with increased sperm concentration in artificial insemination. Appropriate histone retention during sperm chromatin condensation plays critical roles in male fertility. The objective of this study was to determine failures of sperm chromatin condensation associated with abnormal persistence or accessibility of histones by aniline blue (ANBL) test, expression levels, and cellular localizations of one variant and two core histones (H3.3, H2B, and H4 respectively) in the spermatozoa of low-fertility (LF) vs high-fertility (HF) bulls. The expression levels and cellular localizations of histones in spermatozoa were studied using immunoblotting, immunocytochemistry, and staining methods. The bioinformatics focused on the sequence identity and evolutionary distance of these proteins among three mammalian species: bovine, mouse, and human. We demonstrated that ANBL staining was different within the LF (1.73 (0.55, 0.19)) and HF (0.67 (0.17, 0.06)) groups (P<0.0001), which was also negatively correlated with in vivo bull fertility (r=-0.90, P<0.0001). Although these histones were consistently detectable and specifically localized in bull sperm cells, they were not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and were evolutionarily conserved in bulls, mice and humans. The H2B variants were more conserved between bulls and humans, than in mice. In conclusion, we showed that H2B, H3.3, and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related to bull fertility.

Digital image analysis of testicular and prostatic ultrasonographic echogencity and heterogeneity in dogs and the relation to semen quality.

PubMed

Moxon, Rachel; Bright, Lucy; Pritchard, Beth; Bowen, I Mark; de Souza, Mírley Barbosa; da Silva, Lúcia Daniel Machado; England, Gary C W

2015-09-01

A semi-automated ultrasonographic method was developed to measure echogenicity and heterogeneity of the testes and prostate gland and relationships of these measures with semen quality were assessed in 43 fertile dogs. The relationship between animal age and body weight upon the volume of the testes, epididymal tail volume and prostate volume were also established. Mean testicular echogenicity was negatively correlated with the percentage of morphologically normal live spermatozoa (more echogenic testes were associated with fewer normal sperm) but not with any other semen quality measure. Mean testicular heterogeneity was positively correlated with the total spermatozoal output (more heterogenous testes, being those with anechoic parenchyma and prominent echogenic stippling, were associated with greater sperm output) but not with any other semen quality measure. There was no relationship between either mean prostatic echogenicity or mean prostatic heterogeneity and any semen quality measure. There was no relationship between age and any testicular or prostatic parameter; however bodyweight was significantly correlated with total testicular volume, total epididymal tail volume and total prostatic volume. Testicular and prostatic ultrasonographic echogenicity and heterogeneity can be objectively assessed using digital image analysis and testicular echogenicity and heterogeneity may be useful adjunct measurements in a breeding soundness examination. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

PubMed Central

Ferrara, Maria Antonietta; Di Caprio, Giuseppe; Managò, Stefano; De Angelis, Annalisa; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

2015-01-01

A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols. PMID:25836358

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

PubMed

Piasecka, M; Gaczarzewicz, D; Laszczyńska, M; Starczewski, A; Brodowska, A

2007-01-01

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

A quantitative description of flagellar movement in golden hamster spermatozoa.

PubMed

Ishijima, S; Mohri, H

1985-01-01

Flagellar movement of golden hamster spermatozoa obtained from the testis and the caput and cauda epididymides was observed by a light microscope while holding them at their heads with a micropipette. Flagellar movement of capacitated spermatozoa and of reactivated spermatozoa demembranated with Triton X-100 was also observed. Testicular and caput epididymal spermatozoa showed weak movement in Tyrode's solution, whereas cauda epididymal spermatozoa showed vigorous movement. The flagellar bends of the cauda epididymal spermatozoa were almost planar. Capacitated spermatozoa moved with waves of a large amplitude. Demembranated spermatozoa reactivated with ATP only had a latent period before the initiation of flagellar movement, and beat at low frequency, whereas demembranated spermatozoa reactivated with both ATP and cAMP began to move immediately at high frequency. Thrust and hydrodynamic power output were calculated using the parameters for the typical waveforms of cauda epididymal spermatozoa before and after capacitation. The possible role of the large amplitude beat in capacitated spermatozoa is discussed. A comparison of the 'principal' and 'reverse' bends in golden hamster sperm flagella as defined by Woolley (1977) with those in sea urchin sperm flagella suggests that the so-called 'principal' bend in golden hamster sperm flagella corresponds to the reverse bend in sea urchin sperm flagella and vice versa.

Sources of spermatozoa loss during collection and artificial insemination of horses.

PubMed

Côté, Michelle A; Blum, Katherine M; Burd, Matthew A

2011-07-01

During artificial insemination of horses, it is important to accurately estimate the number of spermatozoa in each insemination dose. However, little research exists regarding sources of spermatozoa loss during collection and artificial insemination. Therefore, spermatozoal losses were quantified in the dismount loss (187.6×10(6)±62.5×10(6)spermatozoa), gel fraction (179.8×10(6)±61.7×10(6)spermatozoa), and the collection receptacle (136.1×10(6)±26.9×10(6)spermatozoa). Spermatozoal losses were examined in the centrifuge tube (25.8×10(6)±2.1×10(6)spermatozoa), AI pipette during the air removal (90.9×10(6)±8.5×10(6)spermatozoa), and spermatozoa remaining in the AI pipette after insemination (342.9×10(6)±21.4×10(6)spermatozoa). The average cumulative loss was 14.2±2.9% of the total spermatozoa ejaculated with approximately half of the loss due to the process of semen collection and half due to the process of artificial insemination. Spermatozoa retained in the AI pipette, after insemination with extended semen, represented the greatest source of loss. Copyright © 2011 Elsevier B.V. All rights reserved.

Breed differences of bull frozen-thawed semen.

PubMed

Ntemka, A; Tsousis, G; Brozos, C; Kiossis, E; Boscos, C M; Tsakmakidis, I A

2016-12-01

The objective of this study was to investigate the quality of frozen-thawed semen from different bull breeds. Commercial frozen-thawed bull semen samples (26 per breed, 130 totally) of five breeds (Holstein [Η], Brown Swiss [BS], Limousin [L], Belgian Blue [BB], Blonde d' Aquitaine [BA]) were used. After thawing, each semen sample was subjected to thermal resistance test (TR) for 0.5 and 1 hr at 38°C and hypo-osmotic swelling test (HOST) for 1 hr at 150 mOsm at 37°C. Additionally, all samples were evaluated at times 0 hr (thawing), 0.5 hr (TR), 1 hr (TR) for kinetics by CASA [progressive, immotile, rapid, medium, slow moving spermatozoa, curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity (LIN), straightness (STR), beat cross-frequency (BCF), amplitude of lateral head displacement (ALH), wobble (WOB)]. Moreover, directly after thawing, all semen samples were evaluated for morphometry, morphology, viability and DNA fragmentation. Statistical analysis was conducted using a mixed model for repeated measures. The results showed (a) higher VCL after thawing in H, L breeds compared to BB and BA, (b) higher VAP after thawing in L compared to BB, BA, (c) higher values of progressive spermatozoa after TR in H, BS compared to BB, BA, (d) higher values of rapid spermatozoa after thawing and 0.5 hr of TR in H, BS, L compared to BB, BA, (e) lower viability in BA after thawing compared to H, BS, BB, (f) lower morphological abnormalities in H compared to L, BB, (g) higher head length in Η compared to BB. No significant differences were observed in the results from HOST and DNA fragmentation between breeds. In conclusion, quality characteristics of frozen-thawed bull semen are dependent on the breed. Frozen semen from BB and BA breeds should be handled more carefully after thawing, as it is more sensitive to stress. © 2016 Blackwell Verlag GmbH.

Protamine mRNA ratio in stallion spermatozoa correlates with mare fecundity.

PubMed

Paradowska-Dogan, A; Fernandez, A; Bergmann, M; Kretzer, K; Mallidis, C; Vieweg, M; Waliszewski, P; Zitzmann, M; Weidner, W; Steger, K; Kliesch, S

2014-07-01

Highly compacted sperm DNA in protamine toroids and a minor fraction of nucleohistones are prerequisites for the efficient transmission of the paternal genome into the oocyte at fertilization. The objective of this study was to evaluate whether protamines might serve as a prognostic factor for stallion fertility. In situ hybridization detected specific expression of P1 mRNA in the cytoplasm of stage I to VII spermatids, whereas comparable immunohistochemical stainings showed that protein expression was delayed till elongating spermatids in differentiation stages III to VIII. No staining was detectable in cryptorchid testis because of the lack of spermatids in the seminiferous tubules. Using quantitative real-time polymerase chain reaction, we identified mRNA transcripts of P1 and 2 variants of protamine- 2 (P2, P3) in ejaculated spermatozoa from 45 thoroughbred stallions. According to the mare fertility descriptor (i.e. the 'none-return-rate 28 percentage' or NRR28%), stallions were divided into three groups (i.e. high, reduced and low fertility). The P2/P1 mRNA ratio was found to be significantly reduced in the group with lower fertility (p = 0.016) and was slightly correlated with sperm concentration (correlation coefficient r = 0.263). Furthermore, morphologically abnormal sperm count negatively correlated with P2/P1 mRNA ratio, indicating that spermatozoa carrying head defects display a diminished protamine ratio (r = -0.348). Conversely, the P2/P1 ratio was positively correlated with mare fertility or NRR28% (r = 0.274). Interestingly, P3/P1 mRNA ratio remained unaltered in the investigated groups indicating that this variant plays a minor role in equine sperm chromatin compaction. Aberrant protamine transcripts content in equine spermatozoa was not associated with DNA defragmentation rate as measured by flow cytometric acridine orange test. On the basis of these results, we suggest that, similar to human, equine protamine expression constitutes a checkpoint of spermatogenesis and as a corollary the level of protamine mRNA may reflect the quality of spermatogenesis and spermatozoa's fertilizing capacity. © 2014 American Society of Andrology and European Academy of Andrology.

The influence of testicular microlithiasis on the outcomes of in vitro fertilisation in a Chinese Han population.

PubMed

Xu, C; Zhang, F-F; Yang, H-L; Ma, G; Zhang, B; Li, K-J; Zhang, T-J; Liu, J-L; Xuan, X-J

2017-10-01

To investigate the influence of testicular microlithiasis (TM) on the outcomes of in vitro fertilisation (IVF), we retrospectively reviewed the ultrasonography results of the TM patients who underwent IVF treatment in our hospital. They were classified as classic-TM (CTM) or limited-TM (LTM) comparing with patients without TM (Non-TM). Semen parameters, rates of fertilisation, normal fertilisation, good embryos and clinical pregnancy were analysed. The Non-TM group showed higher percentage of morphologically normal spermatozoa than the CTM or LTM group (4.08 ± 2.07 versus 3.40 ± 2.00 versus 3.04 ± 1.66, p = .003) while the other general semen parameters were comparable. The LTM group showed higher rate of fertilisation than the CTM group (85.10% versus 81.12%, p = .044). Moreover, the rates of normal fertilisation of Non-TM (62.47%) and LTM (66.32%) group were significantly higher than the CTM (58.02%) group (p = .027 and p = .001 respectively). There were 333 embryo transfer cycles in total (including 222 fresh and 111 frozen). The overall clinical pregnancy rate was 54.95%, 58.33% and 64.12% in the group of CTM, LTM and Non-TM respectively. However, no statistically significant difference was obtained (p = .326). Our results suggest that TM may have influence on the IVF outcomes. The extent of microlithiasis correlates inversely with the rates of fertilisation and normal fertilisation. © 2016 Blackwell Verlag GmbH.

Ejaculate traits and sperm cryopreservation in the endangered Baird's tapir (Tapirus bairdii).

PubMed

Pukazhenthi, Budhan S; Togna, Gina Della; Padilla, Luis; Smith, Diorene; Sanchez, Carlos; Pelican, Katey; Sanjur, Oris I

2011-01-01

There is little information on the reproductive biology of the male Baird's tapir (Tapirus bairdii). In this study, we characterized the ejaculate traits and evaluated the efficacy of 2 cryodiluents on sperm cryosurvival. Ejaculates were assessed for volume, pH, sperm motility, forward progression, osmolality, sperm concentration, sperm morphology, and acrosomal integrity. For cryopreservation, ejaculates with >50% total sperm motility were washed, and sperm pellets were resuspended in either Botu-Crio (CryoVital, Grandau, Germany) or INRA 96 containing 2% egg yolk and 2.5% each of methyl- and dimethylformamide (INRA 96), and they were cryopreserved over liquid nitrogen vapor. Thawed samples were incubated in vitro (25 °C) and evaluated for percent total sperm motility, forward progression, and acrosomal integrity at hourly intervals for 4 hours. Spermic ejaculates were obtained from all males, and the mean seminal volume, sperm concentration per milliliter, percent sperm motility, progressive status, and percent morphologically normal cells were 20.4 ± 4.3 mL, 101.2 ± 24.0 × 10(6)/mL, 46.1% ± 5.0%, 2.9 ± 0.1, and 6.9% ± 1.4%, respectively. There was a positive significant correlation between percent normal sperm and animal age (r = 0.66; P < .004). Cryopreservation in either Botu-Crio or INRA 96 resulted in a decline (P < .05) in percent sperm motility and acrosomal integrity. Sperm forward progression remained unaffected immediately after thawing in INRA 96 but continued to decline over time. These results characterize, for the first time, the ejaculate traits of the tapir; demonstrate that tapir spermatozoa can be cryopreserved in diluents containing amides alone or in combination with glycerol; and provide fundamental information critical for development of assisted reproductive technologies for the Baird's tapir.

Global analysis of differential gene expression related to long-term sperm storage in oviduct of Chinese Soft-Shelled Turtle Pelodiscus sinensis

PubMed Central

Liu, Tengfei; Yang, Ping; Chen, Hong; Huang, Yufei; Liu, Yi; Waqas, Yasir; Ahmed, Nisar; Chu, Xiaoya; Chen, Qiusheng

2016-01-01

Important evolutionary and ecological consequences arise from the ability of female turtles to store viable spermatozoa for an extended period. Although previous morphological studies have observed the localization of spermatozoa in Pelodiscus sinensis oviduct, no systematic study on the identification of genes that are involved in long-term sperm storage has been performed. In this study, the oviduct of P. sinensis at different phases (reproductive and hibernation seasons) was prepared for RNA-Seq and gene expression profiling. In total, 2,662 differentially expressed genes (DEGs) including 1,224 up- and 1,438 down-regulated genes were identified from two cDNA libraries. Functional enrichment analysis indicated that many genes were predominantly involved in the immune response, apoptosis pathway and regulation of autophagy. RT-qPCR, ELISA, western blot and IHC analyses showed that the expression profiles of mRNA and protein in selected DEGs were in consistent with results from RNA-Seq analysis. Remarkably, TUNEL analysis revealed the reduced number of apoptotic cells during sperm storage. IHC and TEM analyses found that autophagy occurred in the oviduct epithelial cells, where the spermatozoa were closely attached. The outcomes of this study provide fundamental insights into the complex sperm storage regulatory process and facilitate elucidating the mechanism of sperm storage in P. sinensis. PMID:27628424

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.

[Comparative studies on the quantity of fructose and citric acid in the sperm from bulls at different seasons of the year].

PubMed

Doĭcheva, M; Stoianov, T; Tunchev, T

1978-01-01

The contents of fructose and citric acid in the semen of seven bulls of different ages were examined over a one-year period. The bulls were used as sires in the Artificial Insemination Station. The semen production was within the normal range. In the course of the investigations the temperature and moisture regimes of the air in the barns was optimal for the semen production of the bulls. No statistically significant changes in the amount of fructose and citric acid were established at different average diurnal temperatures and the relative moisture content in the different annual seasons. A marked positive correlation (r = 0.070 +/- 0.04) was observed between the changes in the contents of fructose and citric acid throughout the year. These changes were in an inverse correlation with the number of spermatozoa per mL of semen (r = 0.26 +/- 0.08) and in a moderate one for the citric acid (r = 0.49 +/- 0.06). In the bulls of the Red Dannish cattle there was a statistically significant decrease in the number of spermatozoa and a statistically significant increase in the number of immobile spermatozoa in the winter season.

Impact of 5'-amp-activated Protein Kinase on Male Gonad and Spermatozoa Functions.

PubMed

Nguyen, Thi Mong Diep

2017-01-01

As we already know, the male reproductive system requires less energetic investment than the female one. Nevertheless, energy balance is an important feature for spermatozoa production in the testis and for spermatozoa properties after ejaculation. The 5'-AMP-activated protein kinase, AMPK, is a sensor of cell energy, that regulates many metabolic pathways and that has been recently shown to control spermatozoa quality and functions. It is indeed involved in the regulation of spermatozoa quality through its action on the proliferation of testicular somatic cells (Sertoli and Leydig), on spermatozoa motility and acrosome reaction. It also favors spermatozoa quality through the management of lipid peroxidation and antioxidant enzymes. I review here the most recent data available on the roles of AMPK in vertebrate spermatozoa functions.

Optimization of the incubation time and temperature for spermatozoa extraction in freshwater crayfish Pontastacus leptodactylus (Eschscholtz, 1823).

PubMed

Farhadi, Ardavan; Harlıoğlu, Muzaffer Mustafa; Gür, Seyfettin; Acısu, Tutku Can

2018-06-01

Determination and control of spermatozoa quality in crustacean aquaculture is an important issue for successful and controlled reproduction. Investigation of spermatozoa number in spermatophores is a basic and common parameter for determining the reproductive quality in farmed decapods. In the present study, spermatozoa extraction from spermatophores located in the ductus deferens was conducted in Pontastacus leptodactylus using different incubation times and temperatures. The results indicate that the duration of incubation and temperature affected (P < 0.05) spermatozoa extraction. Greater temperatures (40 and 75 °C) resulted in a reduction (P < 0.05) in number of extracted spermatozoa. In contrast, more spermatozoa were extracted when the 4 and 23 °C temperatures were imposed. After 4 h of incubation, the number of extracted spermatozoa were greatest in number at 23 °C. In conclusion, the greater numbers of crayfish spermatozoa can be obtained when the ductus deferens containing spermatophores is incubated at 23 °C for 4 h as compared with other temperatures and incubation durations. The results of present study are useful for assessing spermatozoa quality in aquaculture as well as the extraction of spermatozoa for research purposes. Copyright © 2018 Elsevier B.V. All rights reserved.

Guaifenesin and increased sperm motility: a preliminary case report.

PubMed

Means, Gary; Berry-Cabán, Cristóbal S; Hammermeuller, Kurt

2010-12-20

A review of the literature and an extensive Medline search revealed that this is the first case report of the use of guaifenesin to increase sperm motility. A 32-year-old male presented for an infertility evaluation. He reported an inability to conceive with his wife after 18 months of unprotected intercourse. A semen analysis was performed that included spermatozoa count, liquefaction, morphology, motility, viscosity and volume. Initial results of the semen analysis demonstrated low sperm count and motility. The provider offered treatment with guaifenesin 600 mg extended release tablets twice daily. Two months after guaifenesin therapy the semen analysis was repeated that demonstrated marked improvement in both total sperm count and motility. Evidence for the effectiveness of guaifenesin is almost entirely anecdotal. Given the mechanism of action of guaifenesin, it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility. Additional studies of the effects of guaifenesin on male fertility could yield information of the medication's effect on men with normal or decreased total sperm counts.

Guaifenesin and increased sperm motility: a preliminary case report

PubMed Central

Means, Gary; Berry-Cabán, Cristóbal S; Hammermeuller, Kurt

2011-01-01

Background A review of the literature and an extensive Medline search revealed that this is the first case report of the use of guaifenesin to increase sperm motility. Case A 32-year-old male presented for an infertility evaluation. He reported an inability to conceive with his wife after 18 months of unprotected intercourse. A semen analysis was performed that included spermatozoa count, liquefaction, morphology, motility, viscosity and volume. Initial results of the semen analysis demonstrated low sperm count and motility. The provider offered treatment with guaifenesin 600 mg extended release tablets twice daily. Two months after guaifenesin therapy the semen analysis was repeated that demonstrated marked improvement in both total sperm count and motility. Conclusion Evidence for the effectiveness of guaifenesin is almost entirely anecdotal. Given the mechanism of action of guaifenesin, it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility. Additional studies of the effects of guaifenesin on male fertility could yield information of the medication’s effect on men with normal or decreased total sperm counts. PMID:21403786

Semen characteristics, extension, and cryopreservation of Rusa deer (Rusa timorensis)

PubMed Central

Fitri, Wan-Nor; Wahid, Haron; Rosnina, Yusoff; Jesse, Faez Firdaus Abdullah; Aimi-Sarah, Zainal Abidin; Mohd-Azmi, Mohd Lila; Azlan, Che’ Amat; Azrolharith, Muhammad Rashid; Peter, Innocent Damudu; Ali Baiee, Falah Hasan

2017-01-01

Aim: The objective of this research is to report parameters for breeding soundness evaluation, semen extension, and cryopreservation in Rusa timorensis. Materials and Methods: Seven healthy stags were chosen for semen collection using an electroejaculator. The collections were performed twice in a breeding season between February and June 2016. Samples were collected between 2 and 3 weeks interval, collected twice for each animal. Semen was evaluated, extended, and cryopreserved using four different extenders; Andromed®, BioXcell®, Triladyl®, and a modified Tris-egg yolk combined with Eurycoma longifolia Jack. Results: R. timorensis semen characteristics according to volume (ml), color, sperm concentration (106/ml), general motility (%), progressive motility (%), and % morphology of normal spermatozoa are 0.86±0.18 ml, thin milky to milky, 1194.2±346.1 106/ml, 82.9±2.8%, 76.1±4.8%, and 83.9±4.8%, respectively. Conclusion: Semen characteristics of R. timorensis collected by electroejaculation is good allowing for cryopreservation and future artificial insemination work. The most suitable extender for Rusa deer semen is Andromed®. PMID:28831222

Ultrastructural and hormonal changes in rat cauda epididymal spermatozoa induced by Boswellia papyrifera and Boswellia carterii.

PubMed

Ahmed, Mukhtar; Ali, Daoud; Harrath, Abdel Halim; Hussain, Tajamul; Al-Daghri, Nasser; Alokail, Majed S; Aladakatti, Ravindranath H; Ghodesawar, Mukhtar Ahmed G

2014-04-01

Boswellia papyrifera and Boswellia carterii diffuses smoke polluting air that adversely affects indoor environment that certainly harm human health. Therefore, this study aims at ascertaining the effect of these plants on gonadal hormones and molecular changes in rat spermatozoa. The animals were exposed to 4 g/kg body weight of B. papyrifera and B. carterii daily for 120 days along with suitable controls. Significant decreases in FSH, LH and testosterone levels were evidenced, along with a reduction of protein, sialic acid, and carnitine levels. In sperm physiology, sperm count, motility, speed decrease, whereas sperm anomalies increase. TEM observation indicates morphological changes in plasma and acrosomal membranes, cytoplasmic droplet in the tail region, vacuolated, and disorganization of the mitochondrial sheath. These findings demonstrate that B. papyrifera and B. carterii smoke affects the process of sperm formation and maturation, which indicates the detrimental effects of these plants on the reproductive system. Copyright © 2014 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Nuclear size as estrogen-responsive chromatin quality parameter of mouse spermatozoa.

PubMed

Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Viggiano, Andrea; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

2013-11-01

Recently, we have investigated the endocannabinoid involvement in chromatin remodeling events occurring in male spermatids. Indeed, we have demonstrated that genetic inactivation of the cannabinoid receptor type 1 (Cnr1) negatively influences chromatin remodeling mechanisms, by reducing histone displacement and indices of sperm chromatin quality (chromatin condensation and DNA integrity). Conversely, Cnr1 knock-out (Cnr1(-/-)) male mice, treated with estrogens, replaced histones and rescued chromatin condensation as well as DNA integrity. In the present study, by exploiting Cnr1(+/+), Cnr(+/-) and Cnr1(-/-) epididymal sperm samples, we show that histone retention directly correlates with low values of sperm chromatin quality indices determining sperm nuclear size elongation. Moreover, we demonstrate that estrogens, by promoting histone displacement and chromatin condensation rescue, are able to efficiently reduce the greater nuclear length observed in Cnr1(-/-) sperm. As a consequence of our results, we suggest that nucleus length may be used as a morphological parameter useful to screen out spermatozoa with low chromatin quality. Copyright © 2013 Elsevier Inc. All rights reserved.

[Analysis of morphologic classifications of testicular lesions in male sterility].

PubMed

Lysenko, A I; Kirpatovskiĭ, I D

1991-01-01

An analysis of the authors' own data based on the examination of 697 testis biopsies is given. The material is subdivided into two groups depending on the presence or absence of germ cells: 1) hypospermatogenesis and 2) aspermatogenesis. Hypospermatogenesis is related to any tubule changes resulting in the under-production of spermatozoa. Criteria of tubule changes and a method for determination of the hypospermatogenesis degree are given. Insolvency of some existing histologic classifications is shown.

Diagnostic evaluation of oxidoreductive capability of sperm mitochondria.

PubMed

Piasecka, M; Gaczarzewicz, D; Kurzawa, R; Laszczyńska, M; Kram, A

2004-01-01

In the present paper, morphological and functional features of human sperm midpiece, contributing to the assessment of sperm fertility potential, have been described. The NADH-dependent NBT screening assay was used to identify and visualise: 1/ morphological defects of sperm midpiece, 2/ immature sperm forms with extensive cytoplasmic retention, reflecting developmental failure in spermatogenic remodelling process, 3/ cytoplasmic sperm conglomerates, related to apoptotic bodies and 4/ sperm NADH-dependent oxidoreductase system at the mitochondrial level, related to the reaction intensity. The used assay is an adequate marker of sperm mitochondrial activity and sperm maturity. It can also help discover sperm defects that result in asthenozoospermia and can be used as an additional indicator in the evaluation of the sperm midpiece, as well as in routine morphological examination of spermatozoa, having a considerable predictive value for in vivo and in vitro fertilization.

Study of the role of epididymal alpha-glucosidase in the fertility of male rats by the administration of the enzyme inhibitor castanospermine.

PubMed

Yeung, C H; Cooper, T G

1994-11-01

The activity of epididymal alpha-glucosidase in adult rats was rapidly suppressed to histochemically undetectable levels within 2 days by the continuous release of the enzyme inhibitor castanospermine via a peritoneal osmotic pump at a rate of 100-200 nmol h-1. It was established that mating activities overnight depleted 72% of the spermatozoa in the distal cauda, which was replenished in 2 days, and that fertility began to decline 3 weeks after efferent duct ligation. Male rats of proven mating proficiency and fertility were treated with castanospermine, or buffered saline as control, for up to 30 days and enzyme inhibition was confirmed at the end of treatment by histochemistry. Fertility was normal at the first mating test on day 7, significantly decreased at the second mating on day 9, but recovered in a stepwise manner at subsequent matings on days 12 and 14. Delaying the third mating until day 25 did not sustain the transient subfertility. However, prolonging sperm storage in the distal cauda epididymides and preventing replenishment with freshly matured spermatozoa, by efferent duct ligation for 14 days performed on day 15 during castanospermine administration, caused a decrease in fertility and a change in the kinematics of epididymal spermatozoa of the castanospermine-treated group. In control rats, binding of epididymal spermatozoa to Vicia faba, a lectin specific for glucose and glucosamine, and mannose and mannosamine residues, decreased from the proximal caput to the distal corpus coincident with the increase in alpha-glucosidase activity on the epithelial brush border. Lectin binding then increased in the cauda where enzyme activity was absent. However, castanospermine treatment did not significantly alter this binding profile. The findings suggest that epididymal alpha-glucosidase does not play a crucial role in the development of sperm fertilizing capacity, but may be involved in the preparation of spermatozoa for storage.

The copper transporter (SLC31A1/CTR1) is expressed in bovine spermatozoa and oocytes: Copper in IVF medium improves sperm quality.

PubMed

Anchordoquy, J P; Anchordoquy, J M; Pascua, A M; Nikoloff, N; Peral-García, P; Furnus, C C

2017-07-15

Adequate dietary intake of copper (Cu) is required for normal reproductive performance in cattle. The objective of this study was to investigate the pregnancy rates from cattle with deficient, marginal and adequate Cu plasma concentration at the beginning of artificial insemination protocol. Moreover, we determined Cu concentrations present in bovine oviductal fluid (OF), and the effects of Cu on fertilizing ability of bovine spermatozoa. Also, the presence of Cu transporter, SLC31A1 (also known as CTR1), in spermatozoa and in vitro matured oocyte were investigated. We found no differences in pregnancy rates among animals with adequate, marginal, and deficient Cu concentrations measured in plasma at the beginning of fixed-time artificial insemination (FTAI) protocol. Copper concentrations in OF were 38.3 ± 2.17 μg/dL (mean ± SEM) regardless of cupremia levels. The addition of 40 μg/dL Cu to IVF medium enhanced total and progressive motility, sperm viability, functional sperm membrane integrity (HOST), sperm-zona binding, and pronuclear formation. On the other hand, the presence of Cu in IVF medium did not modify acrosome integrity and cleavage rates after IVF, but impaired blastocyst rates. Cu transporter SLC31A1 was detected in bovine spermatozoa in the apical segment of acrosome, and in the oocyte matured in vitro. In conclusion, the results obtained in the present study determined that cupremia levels at the beginning of FTAI protocol did not influence the pregnancy rates at 60 d after insemination. The presence of CTR1 in bovine mature oocyte and spermatozoa, as well as the beneficial effect of Cu on sperm quality would suggest an important role of this mineral during the fertilization process. Copyright © 2017 Elsevier Inc. 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.

Oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa.

PubMed

Leemans, Bart; Gadella, Bart M; Sostaric, Edita; Nelis, Hilde; Stout, Tom A E; Hoogewijs, Maarten; Van Soom, Ann

2014-07-01

Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca(2+), and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 10(6) spermatozoa/ml), during which it transpired that the highest density (per mm(2)) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pHi) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pHi, presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pHi changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm. © 2014 by the Society for the Study of Reproduction, Inc.

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.

The tolerance of feline corpus and cauda spermatozoa to cryostress.

PubMed

Kunkitti, Panisara; Bergqvist, Ann-Sofi; Sjunnesson, Ylva; Johannisson, Anders; Axnér, Eva

2016-02-01

Epididymal sperm preservation can be used to avoid the total loss of genetic material in threatened species. Spermatozoa from the corpus, as from the cauda, are motile and can undergo capacitation. Thus, they can potentially be preserved for assisted reproductive technologies. However, cryopreservation of spermatozoa has a direct detrimental effect on sperm quality. The aim of this study was to compare the chromatin stability and the survival rate of spermatozoa from the corpus and cauda epididymis after cryopreservation. Epididymal spermatozoa were collected and cryopreserved from the corpus and cauda of 12 domestic cats. Sperm motility, progressive motility, membrane integrity, acrosome integrity, and DNA integrity were evaluated before and after freezing thawing. The average total number of spermatozoa collected from the corpus was lower (10.2 × 10(6) ± 7.4) than that from the cauda epididymis (24.9 × 10(6) ± 14.4; P = 0.005). The percentage of spermatozoa with intact DNA did not differ significantly whether it was collected from the corpus or cauda regions and did not decrease after freezing thawing in either region. However, motility of spermatozoa from both regions was affected by the freezing thawing process with a significant decline in motility after thaw compared with fresh spermatozoa. A significant difference in the percentage of motile sperm between the corpus and cauda was observed after the freezing thawing process (P < 0.001). Although sperm motility was lower in postthaw spermatozoa from the corpus epididymidis than from the cauda, the rate of the reduction did not differ between regions. This study indicates that the cryopreservation process does not have a negative effect on chromatin stability of feline epididymal spermatozoa. Spermatozoa from the corpus region have a similar freezability as spermatozoa from the cauda region. Therefore, preservation of spermatozoa from the corpus and the cauda epididymidis might be of value in preserving genetic material from endangered or valuable felids. Copyright © 2016 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.

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen.

PubMed

Glogowski, J; Demianowicz, W; Piros, B; Ciereszko, A

1998-10-15

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.

Dietary supplementation of antioxidants improves semen quality of IVF patients in terms of motility, sperm count, and nuclear vacuolization.

PubMed

Wirleitner, Barbara; Vanderzwalmen, Pierre; Stecher, Astrid; Spitzer, Dietmar; Schuff, Maximilian; Schwerda, Delf; Bach, Magnus; Schechinger, Birgit; Herbert Zech, Nicolas

2012-12-01

This study aimed to investigate the influence of an oral antioxidative supplementation on sperm quality of in vitro fertilization (IVF) patients, as analyzed by sperm motility according to the WHO criteria and motile sperm organelle morphology examination (MSOME). Semen samples were collected from 147 patients before undergoing an IVF/intracytoplasmic morphologically-selected sperm injection (IMSI) cycle and 2 - 12 months after an antioxidative supplementation. Semen analysis was evaluated according to WHO and MSOME criteria. Spermatozoa were grouped according to the size of nuclear vacuoles within the sperm's heads. Patients were divided into oligoasthenoteratozoospermic (OAT) and non-OAT men. Between first and second semen analysis, patients were supplemented orally with an antioxidative preparation. After the antioxidative therapy we observed a significant reduction in the percentage of immotile sperm cells in the patients. Additionally, the percentage of class I spermatozoa according to MSOME criteria was significantly higher after antioxidative supplementation. In OAT patients the percentage of class I sperm was found to be increased, although not significantly. However, we observed a drastic improvement in sperm motility as well as in total sperm count in this group. The results demonstrated a considerable improvement in semen quality, notably in OAT patients. Considering the putative relationship between semen quality on the one hand and reactive oxygen species on the other, the observed changes in the sperm parameters indicate that a decline in semen quality, and even subtle morphological changes, might be associated with oxidative stress. Our findings suggest that an antioxidative and micronutrient supplementation has a remarkable benefit for IVF patients having restricted sperm parameters, in particular.

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

NASA Astrophysics Data System (ADS)

Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

2016-03-01

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome?

PubMed

Van Saen, D; Vloeberghs, V; Gies, I; Mateizel, I; Sermon, K; De Schepper, Jean; Tournaye, H; Goossens, E

2018-06-01

When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome (KS)? In KS, germ cell loss is not observed in testicular tissue from fetuses in the second semester of pregnancy but present at a prepubertal age when the testicular architecture is still normal, while fibrosis is highly present at an adolescent age. Most KS patients are azoospermic at adult age because of a massive germ cell loss. However, the timing when this germ cell loss starts is not known. It is assumed that germ cell loss increases at puberty. Therefore, testicular sperm extraction (TESE) at an adolescent age has been suggested to increase the chances of sperm retrieval at onset of spermatogenesis. However, recent data indicate that testicular biopsies from peripubertal KS patients contain only a few germ cells. In this study, we give an update on fertility preservation in adolescent KS patients and evaluate whether fertility preservation would be beneficial at prepubertal age. The possibility of retrieving testicular spermatozoa by TESE was evaluated in adolescent and adult KS men. The presence of spermatogonia and the degree of fibrosis were also analysed in testicular biopsies from KS patients at different ages. The patients were divided into four age groups: foetal (n = 5), prepubertal (aged 4-7 years; n = 4), peripubertal (aged 12-16 years; n = 20) and adult (aged 18-41 years; n = 27) KS patients. In peripubertal and adult KS patients, retrieval of spermatozoa was attempted by semen analysis after masturbation, vibrostimulation, electroejaculation or by TESE. MAGE-A4 immunohistochemistry was performed to evaluate the presence of germ cells in testicular biopsies from foetal, prepubertal, peripubertal and adult KS patients. Tissue morphology was evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Testicular spermatozoa were collected by TESE in 48.1% of the adult KS patients, while spermatozoa were recovered after TESE in only one peripubertal patient (5.0%). Germ cells were detectable in testicular biopsies from 21% of adult men for whom no spermatozoa could be retrieved by TESE and in 31.5% of peripubertal KS boys. Very small numbers of spermatogonia (0.03-0.06 spermatogonia/tubule) were detected in three out of four (75%) prepubertal patients. At a foetal age, the number of germ cells was similar for KS and control samples. Increased signs of fibrosis were not present at foetal and prepubertal ages, but peripubertal and adult KS patients showed high levels of fibrosis. N/A. Only four prepubertal biopsies were included in this study, but they all showed a very low germ cell number. A high variability in the number of spermatogonia per mm2 was observed in the limited (n = 5) number of foetal biopsies. However, testicular biopsies from prepubertal and foetal Klinefelter patients are difficult to obtain. Testicular tissue banking at a prepubertal age has been suggested as a potential method for fertility preservation in early diagnosed KS boys. However, our results show that a reduction in germ cell number has already taken place in childhood. Therefore, offering testicular tissue banking in young KS boys to prevent subsequent sterility might be a questionable strategy. However, this should be confirmed in a larger study population. This project was funded by the scientific Fund Willy Gepts from the UZ Brussel (D.V.S., J.D.S.), grants from the Vrije Universiteit Brussel (E.G.) and a Methusalem grant (K.S.). D.V.S is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

Spermatozoa with high mitochondrial membrane potential and low tyrosine phosphorylation preferentially bind to oviduct explants in the water buffalo (Bubalus bubalis).

PubMed

Saraf, Kaustubh Kishor; Kumaresan, Arumugam; Chhillar, Shivani; Nayak, Samiksha; Lathika, Sreela; Datta, Tirtha Kumar; Gahlot, Subhash Chand; Karan, Prabha; Verma, Kiran; Mohanty, Tushar Kumar

2017-05-01

Although it is understood that spermatozoa are subjected to selection processes to form a functional sperm reservoir in the oviduct, the mechanism remains obscure. With the aim to understand the sperm selection process in the oviduct, in the present in vitro study, we analyzed mitochondrial membrane potential and tyrosine phosphorylation status in oviduct-explants bound and unbound spermatozoa. Frozen semen from Murrah buffalo bulls (n=10) used under progeny testing programme were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 4h. Mitochondrial membrane potential (MMP) and tyrosine phosphorylation status of bound and unbound spermatozoa were assessed at 1h and 4h of incubation. The proportion of spermatozoa with high MMP was significantly higher (P<0.001) among the bound spermatozoa (range 84.67-96.56%) compared to unbound (range 8.70-21.03%) spermatozoa. The proportion of tyrosine phosphorylated spermatozoa was significantly higher (P<0.001) among unbound population as compared to bound population. The proportion of spermatozoa displaying tyrosine phosphorylation at acrosomal area was significantly (P<0.05) lower in bound sperm population compared to unbound population. It was inferred that spermatozoa with high MMP and low tyrosine phosphorylation were preferred for oviduct-explants binding in the buffalo. Copyright © 2017 Elsevier B.V. All rights reserved.

Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential.

PubMed

Ball, B A; Vo, A

2001-01-01

Osmotic stress attributed to differences in the relative permeability of cryoprotectants, such as glycerol and water, appears to be an important factor in cryodamage. The objective of this study was to characterize the osmotic tolerance of equine spermatozoa, and to evaluate the effects of addition and removal of cryoprotectants from equine spermatozoa on their motility, and membrane and acrosomal integrity, as well as their mitochondrial membrane potential. Equine spermatozoa had a limited osmotic tolerance to anisosmotic conditions. Although the addition of increasing concentrations of glycerol decreased the motility and viability of equine spermatozoa, the rapid removal of glycerol by dilution in isosmotic media resulted in an even greater decline in motility and viability compared with spermatozoa maintained under anisosmotic conditions. Likewise, the addition and rapid removal of 1.0 M glycerol, ethylene glycol, dimethylsulfoxide, or propylene glycol resulted in a significant decline in sperm motility and viability. Among these cryoprotectants, ethylene glycol had the least detrimental effect on either viability or motility of spermatozoa following the rapid addition and removal of these cryoprotectants. These data demonstrate that equine spermatozoa have a limited osmotic tolerance compared with published reports for mouse or human spermatozoa, and appear to be more similar to boar spermatozoa in their osmotic tolerance. Of the 4 cryoprotectants evaluated in equine spermatozoa, the addition and removal of glycerol resulted in a more marked osmotic stress as indicated by alterations in motility, viability, and acrosomal integrity. These data suggest that alternative cryoprotectants should be considered for cryopreservation of equine spermatozoa in order to reduce osmotic stress associated with the addition of these agents during semen freezing.

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.

Incorporation of nanoparticles within mammalian spermatozoa using in vitro capacitation

USDA-ARS?s Scientific Manuscript database

There is still much unknown about the journey of spermatozoa within the female genital tract. Recent studies have investigated mammalian spermatozoa labeling with fluorescent quantum dot nanoparticles (QD) for non-invasive imaging. Furthermore, the incorporation of these QD within the spermatozoa ma...

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.

The effect of the breeding season, cryopreservation and physiological extender on selected sperm and semen parameters of four ferret species: implications for captive breeding in the endangered black-footed ferret.

PubMed

van der Horst, G; Kitchin, R M; van der Horst, M; Atherton, R W

2009-01-01

In the present investigation, comparative baseline information on selected sperm characteristics of ejaculate spermatozoa of the domestic (Mustela putorius furo), fitch (Mustela sp.) and black-footed ferrets (Mustela nigripes) and the Siberian polecat (Mustela eversmanni) are presented. The main emphasis was to establish differences and similarities among these species in relation to semen and sperm quality during the breeding season, in cryopreservation success and in supporting sperm motility in different extenders or physiological media. The results confirm that most sperm morphology abnormalities were evident during the beginning of the breeding cycle in all four species. No significant interspecies differences were apparent in the sperm attributes examined, for all sampling months during the breeding season. Moreover, all species exhibited comparable patterns of reproductive seasonality. Cryopreservation suppressed sperm characteristics equally in all species studied. Ejaculate spermatozoa of closely related ferret species shared many similar motion characteristics using computer-aided sperm motility analysis. These results suggest that the basic sperm physiology of the ferret species under examination is very similar. Disparate to the interspecies comparisons, there were significant differences for most sperm motion parameters when spermatozoa of any of the ferrets were compared in different extenders. Assisted reproductive technologies developed for use in domestic ferret, fitch ferret or Siberian polecat may be successfully applied to captive breeding of the black-footed ferret using semen during any of the functional breeding months.

Seminal Plasma Characteristics and Expression of ATP-binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability.

PubMed

Schäfer-Somi, S; Palme, N

2016-04-01

The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as < 50% progressive motility and/or > 40% morphologically abnormal sperm and/or < 50% viability. Bad freezers were older than good freezers (5.3 vs 3.4 years, p < 0.05). In bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p < 0.01) and the percentage of sperm with complete loss of ABCA1 signal higher (46.7% vs 30%, p < 0.01); the percentage of dead spermatozoa was higher (36.1% vs 25.5%, p < 0.05), and the concentration of cholesterol and sodium in seminal plasma was lower than in good freezers (p < 0.05). We conclude that in thawed bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings. © 2016 Blackwell Verlag GmbH.

Epididymal C4b-binding protein is processed and degraded during transit through the duct and is not essential for fertility.

PubMed

Nonaka, Mayumi I; Zsigmond, Eva; Kudo, Akihiko; Kawakami, Hayato; Yoshida, Kaoru; Yoshida, Manabu; Kawano, Natsuko; Miyado, Kenji; Nonaka, Masaru; Wetsel, Rick A

2015-04-01

C4b-binding protein (C4BP) is known as one of the circulating complement regulators that prevents excessive activation of the host-defense complement system. We have reported previously that C4BP is expressed abundantly in the rodent epididymis, one of the male reproductive organs connecting the testis and vas deferens, where immature spermatozoa acquire their motility and fertilizing ability during their transit through the duct. Epididymal C4BP (EpC4BP) is synthesized androgen-dependently by the epithelial cells, secreted into the lumen, and bound to the outer membrane of the passing spermatozoa. In this study, we found that EpC4BP is secreted as a large oligomer, similar to the serum C4BP, but is digested during the epididymal transit and is almost lost from both the luminal fluid and the sperm surface in the vas deferens. Such a processing pattern is not known in serum C4BP, suggesting that EpC4BP and serum C4BP might have different functional mechanisms, and that there is a novel function of EpC4BP in reproduction. In addition, the disappearance of EpC4BP from the sperm surface prior to ejaculation suggests that EpC4BP works only in the epididymis and would not work in the female reproductive tract to protect spermatozoa from complement attack. Next, we generated C4BP-deficient (C4BP-/-) mice to examine the possible role of EpC4BP in reproduction. However, the C4BP-/- mice were fertile and no significant differences were observed between the C4BP-/- and wild-type mouse spermatozoa in terms of morphology, motility, and rate of the spontaneous acrosome reaction. These results suggest that EpC4BP is involved in male reproduction, but not essential for sperm maturation. Copyright © 2014 Elsevier GmbH. All rights reserved.

Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status

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

Cummins, J.M.; Fleming, A.D.; Crozet, N.

1986-03-01

Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the headmore » alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.« less

Freeze-dried spermatozoa: An alternative biobanking option for endangered species.

PubMed

Anzalone, Debora Agata; Palazzese, Luca; Iuso, Domenico; Martino, Giuseppe; Loi, Pasqualino

2018-03-01

In addition to the iconic wild species, such as the pandas and Siberian tigers, an ever-increasing number of domestic species are also threatened with extinction. Biobanking of spermatozoa could preserve genetic heritages of extinct species, and maintain biodiversity of existing species. Because lyophilized spermatozoa retain fertilizing capacity, the aim was to assess whether freeze-dried spermatozoa are an alternative option to save endangered sheep breeds. To achieve this objective, semen was collected from an Italian endangered sheep breed (Pagliarola), and a biobank of cryopreserved and freeze-dried spermatozoa was established, and evaluated using IVF (for frozen spermatozoa) and ICSI procedures (for frozen and freeze-dried spermatozoa). As expected, the fertilizing capacity of cryopreserved Pagliarola's spermatozoa was comparable to commercial semen stocks. To evaluate the activating capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI, and allocated to two groups: 56 oocytes were activated by incubation with ionomycin (ICSI-FDSa) and 52 were not activated (ICSI-FDSna). Pronuclear formation (2PN) was investigated at 14-16 h after ICSI in fixed presumptive zygotes. Only artificially activated oocytes developed into blastocysts after ICSI. In the present study, freeze-dried ram spermatozoa induced blastocyst development following ICSI at a relatively high proportion, providing evidence that sperm lyophilization is an alternative, low cost storage option for biodiversity preservation of domestic species. Copyright © 2018 Elsevier B.V. All rights reserved.

Ability of spermatozoa to bind to the zona pellucida during oligozoospermia induced with testosterone during a male contraceptive trial.

PubMed

Liu, D Y; Johnston, R; Baker, H W

1995-06-01

To determine the ability of spermatozoa to bind to the zona pellucida (ZP) in testosterone-induced oligozoospermia, previously fertile men participating in the World Heath Organization (WHO) male contraceptive trial in Melbourne were studied while oligozoospermic to various degrees. Semen analysis were performed according to WHO methods. One or two ejaculates from each subject were cryopreserved before commencing weekly intramuscular injections of 200 mg testosterone enanthate. The frozen spermatozoa were used as controls for ZP-binding tests of spermatozoa obtained during testosterone-induced oligozoospermia (< 10 x 10(6)/ml) in either the suppression or efficacy (n = 6) and recovery (n = 3) phases. Two other subjects in the recovery phase with normozoospermia were also tested. Human oocytes that failed to fertilized in vitro from infertile patients were used for the sperm-ZP binding test. Control (frozen) spermatozoa were labelled with fluorescein isothiocyanate and test (oligozoospermic semen) spermatozoa were labelled with tetramethylrhodamine B isothiocyanate. A mixture of equal numbers of labelled motile control and test spermatozoa were incubated with 4-6 ZP. There was a significantly (p < 0.01) lower number of spermatozoa bound per ZP in oligozoospermic samples (65 +/- 7, mean +/- SEM) than in controls (80 +/- 7). However, there were still large numbers of spermatozoa bound to the ZP for all the oligozoospermic samples. Five subjects had similar numbers of spermatozoa bound to the ZP for both control and oligozoospermic samples. Overall, the ZP-binding ratio of test and control spermatozoa averaged 0.82 (range 0.51-1.13).(ABSTRACT TRUNCATED AT 250 WORDS)

Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma.

PubMed

Maxwell, W M; Welch, G R; Johnson, L A

1996-01-01

Boar, bull and ram spermatozoa were examined after staining with the DNA-permeant Hoechst 33342 fluorochrome and flow cytometric sorting in the presence or absence of seminal plasma. Spermatozoa were assessed for viability with flow cytometry using the live cell nucleic acid stain SYBR-14 and propidium iodide (PI), and for membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum (FITC-PSA) and PI; motility and acrosome integrity were estimated by microscopy. Flow cytometric sorting was compared with pipette dilution of boar and bull spermatozoa into: (1) medium [boar: Test buffer containing 2% yolk (TY) or Beltsville thawing solution (BTS); bull: TY or HEPES buffer containing 0.1% bovine serum albumin (HEPES-BSA)] with or without 10% (v/v) seminal plasma; or (2) an empty tube containing no medium. Sorted spermatozoa were either not centrifuged or centrifuged before assessment during a 4-h holding period. The viability, motility and membrane integrity of boar, bull and ram spermatozoa centrifuged after sorting were also examined when seminal plasma was present or absent from the staining extender and/or the TY collection medium. The results indicate that the viability and membrane integrity of spermatozoa in vitro would be improved if: (1) seminal plasma (10%) was routinely included in the BTS and HEPES-BSA staining extenders for boar spermatozoa and ram spermatozoa, respectively, when used in preparation for flow cytometric sorting; and (2) 10% and 50% seminal plasma were included in the TY collection medium for boar or bull spermatozoa and ram spermatozoa respectively.

The Behavior and Acrosomal Status of Mouse Spermatozoa In Vitro, and Within the Oviduct During Fertilization after Natural Mating.

PubMed

Hino, Toshiaki; Muro, Yuko; Tamura-Nakano, Miwa; Okabe, Masaru; Tateno, Hiroyuki; Yanagimachi, Ryuzo

2016-09-01

Although 90%-100% of mouse oocytes can be fertilized in vitro with capacitated spermatozoa within 1 h after insemination, oocytes within the oviduct are fertilized one by one over a period of several hours. In vitro experiments showed that both acrosome-intact and acrosome-reacted spermatozoa entered the cumulus oophorus, but that acrosome-reacted spermatozoa reached the surface of oocytes more readily than acrosome-intact spermatozoa. During the period of fertilization within the oviduct, acrosome-reacted spermatozoa were seen throughout the isthmus, but with higher incidence in the upper than in the mid- and lower segments of the isthmus. Very few spermatozoa were present in the ampulla, and almost all were acrosome reacted. Although the cumulus oophorus and zona pellucida are known to be able to induce or facilitate the acrosome reaction of spermatozoa, this picture makes it likely that almost all fertilizing mouse spermatozoa within the oviduct begin to react before ascending from the isthmus to the ampulla. We witnessed a reacted spermatozoon that stayed on the zona pellucida of a fertilized oocyte for a while; it then moved out of the cumulus before reaching the zona pellucida of the nearby unfertilized oocyte. We noted that only a few spermatozoa migrate from the isthmus to the ampulla during the progression of fertilization, and this must be one of the reasons why we do not see many spermatozoa swarming around a single oocyte during in vivo fertilization. © 2016 by the Society for the Study of Reproduction, Inc.

Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

PubMed Central

HASEGAWA, Ayumi; MOCHIDA, Keiji; TOMISHIMA, Toshiko; INOUE, Kimiko; OGURA, Atsuo

2014-01-01

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons. PMID:24583808

Sexual maturation and fertility of male Nigerian Dwarf goat (Capra hircus) clones produced by somatic cell nuclear transfer.

PubMed

Gauthier, M; Pierson, J; Drolet, M; Bhatia, B; Baldassarre, H; Keefer, C L

2001-01-01

Three, genetically identical, Nigerian Dwarf bucks produced by somatic cell nuclear transfer (NT) of fetal fibroblasts were monitored for sexual maturation and fertility. Starting at four months of age, these male clones were trained to serve an artificial vagina (AV). Average age of the NT-derived bucks at first semen collection was 20 weeks, which was not different from that of other young bucks of this breed (average age at first collection = 20 weeks). Average sperm production at 5 months of age for the NT-derived bucks was 5.0 x 10(8) spermatozoa, which was comparable to that of dwarf bucks of similar age (3.4 x 10(8) spermatozoa). At seven months of age, semen collected from two NT-derived bucks was used to artificially inseminate six females (three does per buck). Five does were confirmed pregnant by ultrasound at day 42. Nine healthy kids, four males and five females, were born in March and April 2000. Viable spermatozoa were collected from one of the F1 males at 28 weeks of age. These results demonstrated that NT-derived bucks and one of their male offspring developed sexually within the normal timeframe for their breed and that the clones were fertile.

Group management influences reproductive function of the male cheetah (Acinonyx jubatus).

PubMed

Koester, Diana C; Freeman, Elizabeth W; Wildt, David E; Terrell, Kimberly A; Franklin, Ashley D; Meeks, Karen; Crosier, Adrienne E

2017-03-01

Although the free-ranging cheetah is generally socially solitary, as many as 60% of males live in same-sex (usually sibling) coalitions. Under ex situ conditions, the cheetah experiences low reproductive success with only ~18% of males having ever produced young. Most male cheetahs (85%) are managed in captivity in coalitions, but with no data on the influence of social grouping on reproductive parameters. We examined the influence of singleton versus coalition management on various male cheetah physiological traits, including ejaculate quality and gonadal and adrenal hormone metabolite concentrations. We also assessed behaviour within coalitions for evidence of social hierarchy through initiation of interactions with group mates and relatedness to physiological traits. Ejaculate quality (including total motile and structurally normal spermatozoa per ejaculate) and androgen concentration profiles were higher (P<0.05) in coalition compared with singleton males. These results support the conclusion that testis function in the cheetah, specifically related to the development of normal, motile spermatozoa and androgen production, is influenced by management with same-sex conspecifics. The findings have implications for ex situ conservation breeding programs by suggesting that reproductive quality can be enhanced through group maintenance of cheetah males.

Microencapsulation of bovine spermatozoa for use in artificial insemination: a review.

PubMed

Nebel, R L; Vishwanath, R; McMillan, W H; Saacke, R G

1993-01-01

A technique for microencapsulation of bovine spermatozoa has been developed with minimal spermatozoal injury and thus of potential use in artificial insemination. The polymers poly-l-lysine, polyvinylamine and protamine sulfate have proven best for membranes. Encapsulation has been successful with capsules ranging in size from 0.75 to 1.5 mm, and with sperm concentrations from 45 to 180 x 10(6) cells mL-1. Successful extenders include CUE, CAPROGEN, and egg yolk-citrate-glycerol (maximum 10% v/v egg yolk for normal capsular shape). Capsule fragility (ability to rupture under ageing and physical stress) is negatively related to membrane thickness which ranges from 1.92 to 5.32 microns (depending on the concentration of polymer used) and positively related to concentration of sperm encapsulated. Heterospermic studies have shown that encapsulated sperm are capable of fertilization in vivo, but are at a disadvantage to unencapsulated sperm when cows are inseminated at conventional times. Uterine retention of inseminates is favoured by capsules having a 'sticky' membrane. Using current procedures, preliminary homospermic fertility studies indicate that sperm encapsulated with poly-l-lysine or protamine sulfate may achieve normal fertility.

Mfsd14a (Hiat1) gene disruption causes globozoospermia and infertility in male mice.

PubMed

Doran, Joanne; Walters, Cara; Kyle, Victoria; Wooding, Peter; Hammett-Burke, Rebecca; Colledge, William Henry

2016-07-01

The Mfsd14a gene, previously called Hiat1, encodes a transmembrane protein of unknown function with homology to the solute carrier protein family. To study the function of the MFSD14A protein, mutant mice (Mus musculus, strain 129S6Sv/Ev) were generated with the Mfsd14a gene disrupted with a LacZ reporter gene. Homozygous mutant mice are viable and healthy, but males are sterile due to a 100-fold reduction in the number of spermatozoa in the vas deferens. Male mice have adequate levels of testosterone and show normal copulatory behaviour. The few spermatozoa that are formed show rounded head defects similar to those found in humans with globozoospermia. Spermatogenesis proceeds normally up to the round spermatid stage, but the subsequent structural changes associated with spermiogenesis are severely disrupted with failure of acrosome formation, sperm head condensation and mitochondrial localization to the mid-piece of the sperm. Staining for β-galactosidase activity as a surrogate for Mfsd14a expression indicates expression in Sertoli cells, suggesting that MFSD14A may transport a solute from the bloodstream that is required for spermiogenesis. © 2016 Society for Reproduction and Fertility.

Effects of Cryoprotectants and Diluents on the Cryopreservation of Spermatozoa from Far Eastern Catfish, Silurus asotus

PubMed Central

Gil, Hyun Woo; Lee, Tae Ho; Park, In-Seok

2017-01-01

ABSTRACT The aim of this study was to compare the efficacy of cryopreservation methods for ex situ conservation of spermatozoa from far eastern catfish, Silurus asotus. The spermatozoa activity index (SAI) and hatching rates were higher in spermatozoa stored in Alserver’s solution than those of spermatozoa stored in glucose solution. The SAI and hatching rates in all experimental groups gradually decreased with increasing duration of storage. Additionally, the SAI and hatching rates gradually decreased with increasing thawing temperatures at all storage durations (P<0.05). Based on the SAI and hatching rates, our results suggest that the optimal cryopreservation conditions of catfish spermatozoa involve storage in Alserver’s solution with 15% ethylene glycol, and thawing at 25℃. Cryopreservation of spermatozoa is a useful and reliable technique for conserving gene resources and for artificial propagation of far eastern catfish. PMID:28484747

The Challenge of Human Spermatozoa Proteome: A Systematic Review.

PubMed

Gilany, Kambiz; Minai-Tehrani, Arash; Amini, Mehdi; Agharezaee, Niloofar; Arjmand, Babak

2017-01-01

Currently, there are 20,197 human protein-coding genes in the most expertly curated database (UniProtKB/Swiss-Pro). Big efforts have been made by the international consortium, the Chromosome-Centric Human Proteome Project (C-HPP) and independent researchers, to map human proteome. In brief, anno 2017 the human proteome was outlined. The male factor contributes to 50% of infertility in couples. However, there are limited human spermatozoa proteomic studies. Firstly, the development of the mapping of the human spermatozoa was analyzed. The human spermatozoa have been used as a model for missing proteins. It has been shown that human spermatozoa are excellent sources for finding missing proteins. Y chromosome proteome mapping is led by Iran. However, it seems that it is extremely challenging to map the human spermatozoa Y chromosome proteins based on current mass spectrometry-based proteomics technology. Post-translation modifications (PTMs) of human spermatozoa proteome are the most unexplored area and currently the exact role of PTMs in male infertility is unknown. Additionally, the clinical human spermatozoa proteomic analysis, anno 2017 was done in this study.

Influence of mitochondrial membrane potential of spermatozoa on in vitro fertilisation outcome.

PubMed

Marchetti, P; Ballot, C; Jouy, N; Thomas, P; Marchetti, C

2012-04-01

To determine whether the outcome of in vitro fertilisation (IVF) is influenced by the percentage of spermatozoa with functional mitochondria, a total of 91 random couples undergoing IVF were included. Mitochondrial function was determined by flow cytometry and expressed as percentage of spermatozoa. Conventional sperm parameters were studied by light microscopy. Reproductive outcome parameters were fertilisation rate, embryo quality and clinical pregnancy. It was found that the fertilisation rate was correlated with the percentage of spermatozoa (r = 0.24, P = 0.01) as well as with the percentage of highly motile spermatozoa. However, we did not find any relationship between the percentage of spermatozoa and embryo quality. Nevertheless, no patient who exhibited less than 64% of spermatozoa achieved pregnancy. It is concluded that determination of Δψ(m) provides accurate information to guide physicians to identify male patients for whom IVF will be unlikely to result in pregnancy. Therefore, we suggest that the percentage of spermatozoa may contribute to identify the most appropriate treatment for an individual patient. © 2011 Blackwell Verlag GmbH.

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.

Effect of boar ejaculate fraction, extender type and time of storage on quality of spermatozoa.

PubMed

Dziekońska, A; Świąder, K; Koziorowska-Gilun, M; Mietelska, K; Zasiadczyk, Ł; Kordan, W

2017-03-28

The aim of this study was to investigate the effect the sperm-rich fraction (F1) and the post-F1 fraction (F2) on the quality of boar spermatozoa stored in a liquid state. Ejaculates were collected from three Polish Landrace boars. Each ejaculate fraction was diluted with BTS short-term extender and Safe-Cell Plus (SCP) long-term extender and stored for seven days (D1-D7) at 17°C. Analyses included sperm motility parameters, normal apical ridge (NAR) acrosomes and plasma membrane integrity (PMI). Prior to the dilution of fractions, marked changes (p<0.05) were noted between F1 and F2 in progressive motility (PMOT), velocity average pathway (VAP) and velocity straight line (VCL). After the ejaculate was diluted, the type of fraction and type of extender significantly affected (p<0.05) PMOT, being markedly higher (p<0.05) for F1 extended in BTS. No marked changes (p<0.05) were observed between F1 and F2 extended in SCP for any of the analyzed sperm quality parameters during seven days of storage. Significantly higher (p<0.05) values of sperm quality parameters were noted in F1 compared with F2 for BTS on D7 of storage. The results of the four-way ANOVA analysis indicate that boar, fraction of ejaculate, extender type and day of storage had significant effects on the quality of boar stored spermatozoa. The F1 was characterised by higher quality of spermatozoa during storage in comparison with F2 in the short-term extender. Using the long-term extender containing the proteins allowed for a better application of F2, which could be important for the pig industry.

Fertilization capacity of cryopreserved Iberian ibex epididymal sperm in a heterologous in vitro fertilization assay.

PubMed

López-Saucedo, J; Santiago-Moreno, J; Fierro, R; Izquierdo, D; Coloma, M A; Catalá, M G; Jiménez, I; Paramio, M T

2015-02-01

In vitro fertilization (IVF) can be used to assess the fertilization capacity of sperm. Heterologous IVF may be useful when assessing that of wild animals as it is often difficult to obtain adequate numbers of naturally corresponding oocytes. The aim of the present study was to assess the fertilization capacity of frozen-thawed ibex epididymal spermatozoa via heterologous IVF involving the oocytes of prepubertal domestic goats. The effect on fertilization and embryo development of adding oestrous sheep serum (ESS) to the fertilization medium was also examined. Cumulus-oocyte complexes (COCs) were matured in TCM-199 for 24-27 h at 38.5°C in a 5% CO2 in air atmosphere. Frozen-thawed epididymal spermatozoa were selected by density gradient centrifugation. After maturation, the oocytes were co-incubated with spermatozoa in synthetic oviductal fluid (SOF) with different concentrations of ESS: SOF-C (0%), SOF-2 (2%) and SOF-20 (20%). At 17 h post-insemination (hpi), zygotes with one female and one male pronucleus (2PN) were categorised as normal; zygotes with 3PN were recorded as polyspermic, and oocytes with 1PN as asynchronous. Cleavage and blastocyst development were assessed at 48 and 168 hpi respectively. The percentage of zygotes with 2PN was higher in the SOF-2 than in the SOF-20 treatment group (27.7% versus 2.9% P < 0.05). The percentage of blastocysts formed with the SOF-C, SOF-2 and SOF-20 treatments were 1.1%, 7.5% and 0% respectively. These results show that the presence of 2% ESS achieves better results than the use of no serum or the standard 20% concentration. Heterologous IVF may be an effective method for predicting the fertilization capacity of ibex spermatozoa, and therefore perhaps that of other wild mountain ungulates.

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

Spermatozoa isolated from cat testes retain their structural integrity as well as a developmental potential after refrigeration for up to 7 days.

PubMed

Buarpung, Sirirak; Tharasanit, Theerawat; Thongkittidilok, Chommanart; Comizzoli, Pierre; Techakumphu, Mongkol

2015-10-01

The objective of this study was to compare the efficiency of preservation media for isolated feline testicular spermatozoa as well as the concentrations of bovine serum albumin (BSA) on: (1) the membrane (sperm membrane integrity (SMI)) and DNA integrity of spermatozoa; and (2) the developmental potential of spermatozoa after intracytoplasmic sperm injection (ICSI). Isolated cat spermatozoa were stored in HEPES-M199 medium (HM) or Dulbecco's phosphate-buffered saline (DPBS) at 4°C for up to 7 days. Results indicated that HM maintained a better SMI than DPBS throughout the storage periods (P > 0.05). When spermatozoa were stored in HM supplemented with BSA at different concentrations (4, 8 or 16 mg/ml), SMI obtained from HM containing 8 and 16 mg/ml BSA was higher than with 4 mg/ml BSA (P 0.05). In summary, cat spermatozoa immediately isolated from testicular tissue can be stored as a suspension in basic buffered medium at 4°C for up to 7 days. BSA supplementation into the medium improves membrane integrity of the spermatozoa during cold storage. Testicular spermatozoa stored in HM containing 16 mg/ml BSA retained full in vitro developmental potential after ICSI, similar to that of fresh controls even though DNA integrity had slightly declined.

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice

PubMed Central

Horta, Fabrizzio; Alzobi, Hamida; Jitanantawittaya, Sutthipat; Catt, Sally; Chen, Penny; Pangestu, Mulyoto; Temple-Smith, Peter

2017-01-01

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection. PMID:27427551

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

PubMed

Gallo, Alessandra; Boni, Raffaele; Tosti, Elisabetta

2018-01-01

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Abnormal retention of nuclear lamina and disorganization of chromatin-related proteins in spermatozoa from DPY19L2-deleted globozoospermic patients.

PubMed

Paci, Marine; Elkhatib, Razan; Longepied, Guy; Hennebicq, Sylviane; Bessonat, Julien; Courbière, Blandine; Bourgeois, Patrice; Levy, Nicolas; Mitchell, Michael J; Metzler-Guillemain, Catherine

2017-11-01

The aim of this study was to characterize the nuclear lamina (NL) and lamin chromatin-partners in spermatozoa from four DPY19L2-deleted globozoospermic patients. We tested for spermatid transcripts encoding lamins and their chromatin-partners emerin, LAP2α, BAF and BAF-L, by reverse transcriptase-PCR using spermatozoa RNA. We also determined the localization of lamin B1, BAF and BAF-L by immunofluorescent analysis of spermatozoa from all patients. In RNA from globozoospermic and control spermatozoa we detected transcripts encoding lamin B1, lamin B3, emerin, LAP2α and BAF-L, but not A-type lamins. In contrast, BAF transcripts were detected in globozoospermic but not control spermatozoa. The NL was immature in human globozoospermic spermatozoa: lamin B1 signal was detected in the nuclei of globozoospermic spermatozoa in significantly higher proportions than the control (P < 0.05; 56-91% versus 40%) and was predominantly observed at the whole nuclear periphery, not polarized as in control spermatozoa. Conversely, BAF and BAF-L were detected in control, but not globozoospermic spermatozoa. Our results strongly emphasize the importance of the NL and associated proteins during human spermiogenesis. In globozoospermia, the lack of maturation of the NL, and the modifications in expression and location of chromatin-partners, could explain the chromatin defects observed in this rare phenotype. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Cell-mediated immunity to homologous spermatozoa following vasectomy in the human male.

PubMed Central

Nagarkatti, P S; Rao, S S

1976-01-01

Cell-mediated immunity (CMI) to homologous spermatozoal antigens was studied in sixty-two males following vasectomy of a duration of 1-10 years. A group of twenty-two normal, fertile non-vasectomized males was also included in the study. The inhibition in the leucocyte migration test (LMT), in the presence of spermatozoal antigen, was taken as an index of CMI. Twenty of the sixty-two vasectomized males (32.2 percent) showed a positive LMT reaction. When the results were analysed with reference to the duration of vasectomy, it was noted that four cases each (22.2 percent), showed a positive LMT reaction in the groups 0-2 years, and 3.5 years. On the other hand twelve cases gave a positive reaction in the group 6-10 years (46.1 percent). It appears that the incidence of CMI to spermatozoa increases with the duration in vasectomy. PMID:991454

Evaluation of motility, membrane status and DNA integrity of frozen-thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation.

PubMed

Montano, G A; Kraemer, D C; Love, C C; Robeck, T R; O'Brien, J K

2012-06-01

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success.

PubMed

Miller, R R; Sheffer, C J; Cornett, C L; McClean, R; MacCallum, C; Johnston, S D

2004-10-01

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p < or = 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.

The effects of hoechst 33342 staining and the male sample donor on the sorting efficiency of canine spermatozoa.

PubMed

Rodenas, C; Lucas, X; Tarantini, T; Del Olmo, D; Roca, J; Vazquez, J M; Martinez, E A; Parrilla, I

2014-02-01

The aim of this study was to evaluate the influence of Hoechst 33342 (H-42) concentration and of the male donor on the efficiency of sex-sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10(6) sperm/ml, split into four aliquots, stained with increasing H-42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non-viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X- and Y-chromosome-bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H-42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H-42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex-sorting of canine spermatozoa by flow cytometry can be performed successfully using H-42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives. © 2013 Blackwell Verlag GmbH.

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.

Cryopreservation of mouse spermatozoa. I. Effect of seeding on fertilizing ability of cryopreserved spermatozoa.

PubMed

Songsasen, N; Leibo, S P

1997-11-01

To examine the effect of seeding to induce ice formation during cryopreservation on their survival, spermatozoa from B6D2F1 mice were cooled to and held at -4 degrees C for 30 min in phosphate-buffered saline (PBS) alone, in egg yolk-supplemented PBS, or in PBS with raffinose + glycerol as cryoprotective additives (CPAs). Seeding and holding spermatozoa at -4 degrees C did not affect their viability as judged by vital staining. Egg yolk protected spermatozoa against chilling injury, as cooling them to -4 degrees C in the presence of egg yolk yielded higher survivals than those cooled without egg yolk (34.4 +/- 3.4 v 9.0 +/- 1.3% in three replicates of >400 spermatozoa/replicate). To study effects of seeding on their fertilizing ability, spermatozoa in the raffinose-glycerol-egg yolk solution were frozen to -196 degrees C either without seeding or after seeding at -4 degrees C. Development of 222 oocytes into two-cell embryos after in vitro fertilization (IVF) with spermatozoa frozen without seeding was 43%; development rates of 186, 186, and 207 oocytes after IVF with spermatozoa frozen after seeding and being held at -4 degrees C for 5, 10, or 30 min were 46, 44, and 9%, respectively. In a direct comparison, after IVF with seeded or unseeded spermatozoa the respective cleavage rates into two-cell embryos were 83% of 275 oocytes and 69% of 304 oocytes, a difference that was small but significant by chi2 analysis. An additional 925 oocytes were fertilized with spermatozoa after being seeded and frozen to -196 degrees C in four separate batches of CPA solutions. Overall, after IVF with frozen sperm, 68% of those oocytes cleaved into two-cell embryos and 59% developed into 544 blastocysts. Based on these results, we concluded that embryo production by IVF seemed to be improved using spermatozoa frozen after being seeded. Mouse spermatozoa cryopreserved by the method described here are capable of fertilizing oocytes at a rather high rate. Copyright 1997 Academic Press.

Molecular characterization of voltage-gated potassium channel (Kv) and its importance in functional dynamics in bull spermatozoa.

PubMed

Gupta, Rishi Kumar; Swain, Dilip Kumar; Singh, Vijay; Anand, Mukul; Choudhury, Soumen; Yadav, Sarvajeet; Saxena, Atul; Garg, Satish Kumar

2018-07-01

Present study was undertaken to characterize the voltage gated potassium channel (K v 1.1) in bull spermatozoa using sixty four ejaculates collected from four Hariana bulls. Functional characterization was undertaken using a selective blocker of Kv channel, 4-Aminopyridine (4-AP) while molecular presence of Kv on bull spermatozoa by immunoblotting and indirect immunofluorescence. Three sets of 100 μL diluted sperm samples namely-negative control (100 μL of sperm dilution medium (SDM) containing 10 × 10 6  cells), vehicle control (99 μL of SDM containing 10 × 10 6  cells, and DMSO- 1  μL) and 4-AP treatment group (99 μL of SDM containing 10 × 10 6  cells, and drug 1 μL 4-AP) were used in the study. Immunoblotting identified a single band of 56 kDa corresponding to Kv1.1 in Hariana bull spermatozoa. Immunolocalization showed the positive immunoreactivity at head, middle piece and principal piece of the spermatozoa for Kv 1.1. Blocking of Kv using 4-AP resulted in significant (p < 0.05) reduction in sperm progressive motility, per cent capacitated spermatozoa (B-pattern) and acrosome reacted (AR-pattern) spermatozoa, while significant (P < 0.05) increase in per cent swollen spermatozoa. Blocking of Kv channels resulted in significantly (P < 0.05) increased percentage of spermatozoa with lower mitochondrial transmembrane potential. Computer assisted semen analysis (CASA) of motion and kinematic parameters in 4-AP treated spermatozoa indicated reduction in sperm motion parameters like LIN, STR, VSL and VAP and higher ALH, VCL, and BCF indicating hyperactivity of spermatozoa. Based on our findings, it may be concluded that voltage-gated potassium channel (Kv) are present on bull spermatozoa and these are associated with functional dynamics of spermatozoa. However, based on our limited study, it is not possible to deduce that how these channels are associated with induction of hyperactivity. Therefore, further studies are warranted to unravel the mechanistic signaling pathways involved in Kv-mediated alterations in functional dynamics of spermatozoa. Copyright © 2018 Elsevier Inc. All rights reserved.

Ultrastructure of the spermatozoa from a Florida manatee (Trichechus manatus latirostris).

PubMed

Miller, D L; Dougherty, M M; Decker, S J; Bossart, G D

2001-08-01

Semen was opportunistically collected from a free-ranging, 10-year-old, 275 cm (total length) Florida manatee (Trichechus manatus latirostris) during rehabilitation treatments. Ultrastructure of the spermatozoa was examined by scanning and transmission electron microscopy and differed slightly from that described for other mammals. Comparisons to the manatee's closest phylogenetic relatives, the elephant and hyrax, were made. The manatee spermatozoa had a similar acrosome but a distinct annulus and lacked the dense bodies observed in the neck of the elephant spermatozoa. Additionally, manatee spermatozoa lacked the lateral vacuoles observed in the nuclear chromatin from of the hyrax spermatozoa. These data add to our understanding of manatees and allow for comparative studies with other species that may be useful in phylogenetic and reproductive studies.

Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive.

PubMed

Miyata, Haruhiko; Satouh, Yuhkoh; Mashiko, Daisuke; Muto, Masanaga; Nozawa, Kaori; Shiba, Kogiku; Fujihara, Yoshitaka; Isotani, Ayako; Inaba, Kazuo; Ikawa, Masahito

2015-10-23

Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mice lacking Ppp3cc or Ppp3r2 genes (knockout mice) are infertile, with reduced sperm motility owing to an inflexible midpiece. Treatment of mice with cyclosporine A or FK506 creates phenocopies of the sperm motility and morphological defects. These defects appear within 4 to 5 days of treatment, which indicates that sperm-specific calcineurin confers midpiece flexibility during epididymal transit. Male mouse fertility recovered a week after we discontinued treatment. Because human spermatozoa contain PPP3CC and PPP3R2 as a form of calcineurin, inhibition of this sperm-specific calcineurin may lead to the development of a reversible male contraceptive that would target spermatozoa in the epididymis. Copyright © 2015, American Association for the Advancement of Science.

The seminiferous epithelium cycle and daily spermatic production in the adult maned wolf (Chrysocyon brachyurus, Illiger, 1811).

PubMed

Bitencourt, Viviane Lewicki; de Paula, Tarcízio Antônio Rego; da Matta, Sérgio Luis Pinto; Fonseca, Cláudio César; dos Anjos Benjamin, Laércio; Costa, Deiler Sampaio

2007-01-01

The duration of the seminiferous epithelium cycle was estimated in adult maned wolves (Chrysocyon brachyurus, Illiger, 1811), by applying intratesticular injections with tritiated thymidine. The total duration of the seminiferous epithelium cycle in this species was calculated in 8.99 days. So, taking into account that approximately 4.5 cycles of the seminiferous epithelium are necessary for the whole spermatogenesis process to complete, the production of spermatozoa from one spermatogonia will take about 40.45 days. The duration of the spermiogenesis was calculated to be 12.3 days. The eight stages of the seminiferous epithelium cycle were described by the tubular morphology method, which is based either on the form and position of the spermatid nuclei and the occurrence of meiotic divisions. The values of the relative frequency for the pre-meiotic, meiotic and post-meiotic phases in this species were 3.5, 0.78 and 4.8 days, respectively. The maned wolf produces about 29 million spermatozoa a day for each testis gram, therefore being classified among the species provided with a high spermatogenetic efficiency.

A comparative ultrastructural analysis of spermatozoa in Pleurodema (Anura, Leptodactylidae, Leiuperinae).

PubMed

Cruz, Julio C; Ferraro, Daiana P; Farías, Alejandro; Santos, Julio S; Recco-Pimentel, Shirlei M; Faivovich, Julián; Hermida, Gladys N

2016-07-01

This study describes the spermatozoa of 10 of the 15 species of the Neotropical frog genus Pleurodema through transmission electron microscopy. The diversity of oviposition modes coupled with a recent phylogenetic hypothesis of Pleurodema makes it an interesting group for the study of ultrastructural sperm evolution in relation to fertilization environment and egg-clutch structure. We found that Pleurodema has an unusual variability in sperm morphology. The more variable structures were the acrosomal complex, the midpiece, and the tail. The acrosomal complex has all the structures commonly reported in hyloid frogs but with different degree of development of the subacrosomal cone. Regarding the midpiece, the variability is given by the presence or absence of the mitochondrial collar. Finally, the tail is the most variable structure, ranging from single (only axoneme) to more complex (presence of paraxonemal rod, cytoplasmic sheath, and undulating membrane), with the absence of the typical axial fiber present in hyloid frogs, also shared with some other genera of Leiuperinae. J. Morphol. 277:957-977, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Differences in the ability of spermatozoa from individual boar ejaculates to withstand different semen-processing techniques.

PubMed

Parrilla, Inma; del Olmo, David; Sijses, Laurien; Martinez-Alborcia, María J; Cuello, Cristina; Vazquez, Juan M; Martinez, Emilio A; Roca, Jordi

2012-05-01

The present study aimed to evaluate the ability of spermatozoa from individual boar ejaculates to withstand different semen-processing techniques. Eighteen sperm-rich ejaculate samples from six boars (three per boar) were diluted in Beltsville Thawing Solution and split into three aliquots. The aliquots were (1) further diluted to 3×10(7) sperm/mL and stored as a liquid at 17°C for 72 h, (2) frozen-thawed (FT) at 1×10(9) sperm/mL using standard 0.5-mL straw protocols, or (3) sex-sorted with subsequent liquid storage (at 17°C for 6 h) or FT (2×10(7) sperm/mL using a standard 0.25-mL straw protocol). The sperm quality was evaluated based on total sperm motility (the CASA system), viability (plasma membrane integrity assessed using flow cytometry and the LIVE/DEAD Sperm Viability Kit), lipid peroxidation (assessed via indirect measurement of the generation of malondialdehyde (MDA) using the BIOXYTECH MDA-586 Assay Kit) and DNA fragmentation (sperm chromatin dispersion assessed using the Sperm-Sus-Halomax(®) test). Data were normalized to the values assessed for the fresh (for liquid-stored and FT samples) or the sorted semen samples (for liquid stored and the FT sorted spermatozoa). All of the four sperm-processing techniques affected sperm quality (P<0.01), regardless of the semen donor, with reduced percentages of motile and viable sperm and increased MDA generation and percentages of sperm with fragmented DNA. Significant (P<0.05) inter-boar (effect of boars within each semen-processing technique) and intra-boar (effect of semen-processing techniques within each boar) differences were evident for all of the sperm quality parameters assessed, indicating differences in the ability of spermatozoa from individual boars to withstand the semen-processing techniques. These results are the first evidence that ejaculate spermatozoa from individual boars can respond in a boar-dependent manner to different semen-processing techniques. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Izquierdo-Vega, Jeannett A.; FES-Cuautitlan. UNAM. Cuautitlan Izcalli, Estado de Mexico; Sanchez-Gutierrez, Manuel

Fluorosis, caused by drinking water contamination with inorganic fluoride, is a public health problem in many areas around the world. The aim of the study was to evaluate the effect of environmentally relevant doses of fluoride on in vitro fertilization (IVF) capacity of spermatozoa, and its relationship to spermatozoa mitochondrial transmembrane potential ({delta}{psi}{sub m}). Male Wistar rats were administered at 5 mg fluoride/kg body mass/24 h, or deionized water orally for 8 weeks. We evaluated several spermatozoa parameters in treated and untreated rats: i) standard quality analysis, ii) superoxide dismutase (SOD) activity, iii) the generation of superoxide anion (O{sub 2}{supmore » {center_dot}}{sup -}), iv) lipid peroxidation concentration, v) ultrastructural analyses of spermatozoa using transmission electron microscopy, vi) {delta}{psi}{sub m}, vii) acrosome reaction, and viii) IVF capability. Spermatozoa from fluoride-treated rats exhibited a significant decrease in SOD activity ({approx} 33%), accompanied with a significant increase in the generation of O{sub 2}{sup {center_dot}} ({approx} 40%), a significant decrease in {delta}{psi}{sub m} ({approx} 33%), and a significant increase in lipid peroxidation concentration ({approx} 50%), relative to spermatozoa from the control group. Consistent with this finding, spermatozoa from fluoride-treated rats exhibited altered plasmatic membrane. In addition, the percentage of fluoride-treated spermatozoa capable of undergoing the acrosome reaction was decreased relative to control spermatozoa (34 vs. 55%), while the percentage fluoride-treated spermatozoa capable of oocyte fertilization was also significantly lower than the control group (13 vs. 71%). These observations suggest that subchronic exposure to fluoride causes oxidative stress damage and loss of mitochondrial transmembrane potential, resulting in reduced fertility.« less

Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss.

PubMed

Izquierdo-Vega, Jeannett A; Sánchez-Gutiérrez, Manuel; Del Razo, Luz María

2008-08-01

Fluorosis, caused by drinking water contamination with inorganic fluoride, is a public health problem in many areas around the world. The aim of the study was to evaluate the effect of environmentally relevant doses of fluoride on in vitro fertilization (IVF) capacity of spermatozoa, and its relationship to spermatozoa mitochondrial transmembrane potential (DeltaPsi(m)). Male Wistar rats were administered at 5 mg fluoride/kg body mass/24 h, or deionized water orally for 8 weeks. We evaluated several spermatozoa parameters in treated and untreated rats: i) standard quality analysis, ii) superoxide dismutase (SOD) activity, iii) the generation of superoxide anion (O(2)(-)), iv) lipid peroxidation concentration, v) ultrastructural analyses of spermatozoa using transmission electron microscopy, vi) DeltaPsi(m), vii) acrosome reaction, and viii) IVF capability. Spermatozoa from fluoride-treated rats exhibited a significant decrease in SOD activity (~33%), accompanied with a significant increase in the generation of O(2)() (~40%), a significant decrease in DeltaPsi(m) (~33%), and a significant increase in lipid peroxidation concentration (~50%), relative to spermatozoa from the control group. Consistent with this finding, spermatozoa from fluoride-treated rats exhibited altered plasmatic membrane. In addition, the percentage of fluoride-treated spermatozoa capable of undergoing the acrosome reaction was decreased relative to control spermatozoa (34 vs. 55%), while the percentage fluoride-treated spermatozoa capable of oocyte fertilization was also significantly lower than the control group (13 vs. 71%). These observations suggest that subchronic exposure to fluoride causes oxidative stress damage and loss of mitochondrial transmembrane potential, resulting in reduced fertility.

Acrosin activity in turkey spermatozoa: assay by clinical method and effect of zinc and benzamidine on the activity.

PubMed

Glogowski, J; Jankowski, J; Faruga, A; Ottobre, J S; Ciereszko, A

2001-09-15

We optimized a clinical assay developed for measuring total acrosin activity for mammalian and fish semen for use in turkey spermatozoa. The main modifications included dilution of semen to a final concentration of 25 to 1000 x 10(3) spermatozoa, an increase of Triton X-100 concentration to 0.05% and 1 hr preincubation without substrate, Acrosin activity in turkey spermatozoa was much higher than in human spermatozoa (about 100-times) but similar to that of boar sperm. To optimize this assay for turkey spermatozoa, it was necessary to use higher Triton X-100 concentrations in the reaction mixture. There was a better catalytic efficiency at higher temperatures and a special requirement for a preincubation period for proacrosin activation. We observed high inhibition of acrosin activity by zinc added during preincubation (90% at 0.01 mM of zinc chloride). Benzamidine also inhibited turkey acrosin, and the extent of inhibition was similar for the incubation or preincubation period. When zinc ions were added during incubation, this inhibition was lower (24%). The results suggest that zinc influences proacrosin activation of turkey spermatozoa. This influence may be important for successful long-term storage of spermatozoa in the hen's oviduct.

Application of a new convenience gender sorting method for mouse spermatozoa to mouse reproductive engineering technology.

PubMed

Hashimoto, Haruo; Eto, Tomoo; Suemizu, Hiroshi; Ito, Mamoru

2013-02-01

In this study, we attempted to apply new convenience gender sorting methods using sex-determining region Y (SRY) gene expression on Y spermatozoa to mice. Mouse spermatozoa labeled with Cy3-SRY antibody conjugate were used for intracytoplasmic sperm injection (ICSI). In addition, spermatozoa conjugated with SRY antibody were conjugated with magnetic beads (Mag) and were pulled to the bottom of the medium. The supernatant of the medium was used for in vitro fertilization (IVF). The rate of males reproduced by ICSI using the spermatozoa conjugated with Cy3-SRY antibody was 86.1%. The female proportion reproduced by IVF using the spermatozoa separated in the supernatant after Mag-SRY antibody conjugation was 67.3%. These gender sorting methods are effective for the reproduction of transgenic mice.

Sex chromosome-dependent differential viability of human spermatozoa during prolonged incubation.

PubMed

You, Young-Ah; Kwon, Woo-Sung; Saidur Rahman, Md; Park, Yoo-Jin; Kim, Young-Ju; Pang, Myung-Geol

2017-06-01

Are there significant differences in the ability of X chromosome-bearing (X) spermatozoa and Y chromosome-bearing (Y) spermatozoa to survive incubation under stressful conditions? Y spermatozoa are more vulnerable to stress than their X counterparts depending on culture period and temperature, and show higher expression of apoptotic proteins. The primary sex ratio is determined by there being an equal number of spermatozoa carrying X and Y chromosomes. This balance can be skewed by exposure to stressful environmental conditions such as changes in pH, pollutants or endocrine disruptors. However, less is known about the ability of sperm carrying either sex chromosome to withstand environmental stress. The difference in survival between X and Y spermatozoa was evaluated by measuring motility, viability and Y:X chromosome ratio during incubation for 5 days, at three temperatures (4, 22 and 37°C), and three pH conditions (6.5, 7.5 and 8.5). To identify the critical factors that determine the survival of X and Y bearing spermatozoa, we analysed the expression levels of apoptosis-related proteins (Bcl, Bax and Caspase-3), as well as the extent of DNA damage under a subset of conditions. Semen samples were obtained by masturbation from normozoospermic donors after 3 days of sexual abstinence. Four samples with >60% motility from different donors were mixed to obtain sufficient semen and eliminate sampling-related bias. Data are presented as mean ± SD of three independent experiments. Mean age of donors was 28.7 ± 3.2 years. In total, 58 489 spermatozoa were scored. The viability of Y spermatozoa was lower after exposure to different temperatures and culture periods than that of X spermatozoa (P < 0.05). Increased expression of apoptotic proteins in live Y spermatozoa was observed, despite the addition of tocopherol to the culture medium (P < 0.05). Spermatozoa were cultured in vitro during the treatment period. It is difficult to extrapolate the observed lifespan differences to spermatozoa survival in vivo. The experiments were replicated only three times. The prolonged survival of X spermatozoa under stressful conditions might lead to shifts in the ratio of male-to-female births. This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. NRF-2014R1A2A2A01002706). The authors declare no competing financial interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Patients with acephalic spermatozoa syndrome linked to SUN5 mutations have a favorable pregnancy outcome from ICSI.

PubMed

Fang, Jianzheng; Zhang, Jingjing; Zhu, Fuxi; Yang, Xiaoyu; Cui, Yugui; Liu, Jiayin

2018-01-10

Are Sad1 and UNC84 domain containing 5 (SUN5) mutations associated with the outcomes of ICSI in patients with acephalic spermatozoa syndrome (ASS)? Despite highly abnormal sperm morphology, ASS patients with SUN5 mutations have a favorable pregnancy outcome following ICSI. ASS is a rare cause of infertility characterized by the production of a majority of headless spermatozoa, along with a small proportion of intact spermatozoa with an abnormal head-tail junction. Previous studies have demonstrated that SUN5 mutations may cause ASS. Several studies showed that ICSI could help patients with ASS father children. This retrospective cohort study included 11 infertile ASS males with SUN5 mutations. Five of them underwent five ICSI cycles. Their ICSI results were compared to men with ASS without SUN5 mutations (n = 3) and to men with multiple morphological abnormalities of the sperm flagella (MMAF) (n = 9). All ICSI treatments were completed between Jan 2011 and May 2017. Sanger DNA sequencing was used to detect mutations in SUN5. Clinical and biological data were collected from patients at the fertility center. Sanger sequencing validated 11 patients with SUN5 mutations. Three novel mutations in SUN5 (c.829C>T [p.Q277*]; c.1067G>A [p.R356H]; c.211+1 insGT [p.S71Cfs11*]) were identified in three patients. The rates of fertilization, good-quality embryos and pregnancy for five patients with SUN5 mutations following ICSI were 81.5%, 81.8% and 100%, respectively. The rates of fertilization and good-quality embryos in patients with MMAF were significantly lower compared with ASS patients (65.6 versus 82.4%, P = 0.039 and 53.6 versus 85.2%, P = 0.031, respectively). There were no differences in ICSI results between ASS patients with and without SUN5 mutations. Only a small number patients with SUN5 mutations was available because of its rare incidence. Patients with ASS can be effectively treated with ICSI. SUN5 mutations may be one of the genetic causes of ASS. This study was supported by the National Natural Science Foundation of China (81401251, 81370754, and 81170559), the Jiangsu Province Special Program of Medical Science (BL2012009, ZX201110, FXK201221) and a project funded by PAPD of the Priority Academic Program Development of Jiangsu High Education Institutions (JX10231802). None of the authors have any competing interests. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa.

PubMed

Rui, Bruno R; Angrimani, Daniel S R; Losano, João Diego A; Bicudo, Luana de Cássia; Nichi, Marcílio; Pereira, Ricardo J G

2017-12-01

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of Macrophages on the Rooster Spermatozoa Quality.

PubMed

Kuzelova, L; Vasicek, J; Chrenek, P

2015-08-01

The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low-density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer-assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2-3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10-15% of macrophages. Males from macrophage group had lower (p < 0.05) motility parameters (total and progressive movement, velocity curved line) and increased concentration of dead spermatozoa detected by flow cytometry and fluorescence microscopy (p < 0.001 and p ˂ 0.05, respectively). Differences (p < 0.05) between fluorescent microscopy and flow cytometry in results on spermatozoa apoptosis and viability were observed. No significant difference was found between groups in fertility of spermatozoa. In conclusion, the higher presence of macrophages in rooster semen may have a negative effect on some parameters of rooster spermatozoa evaluated in vitro. Furthermore, our study suggests that flow cytometry allows more precise examination of spermatozoa viability and apoptosis in a very short time compared with the fluorescent microscopy. © 2015 Blackwell Verlag GmbH.

Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C.

PubMed

Kikuchi, K; Nagai, T; Kashiwazaki, N; Ikeda, H; Noguchi, J; Shimada, A; Soloy, E; Kaneko, H

1998-09-01

The influence of prolonged storage of boar epididymides on post-thaw sperm motility, and in vitro fertilization was evaluated. Twenty pairs of epididymides were obtained from Large White boars, and spermatozoa from one of each of the pairs were immediately collected and frozen (control group). The remaining epididymides were cooled to 4 degrees C and stored for 1, 2 or 3 d, after which spermatozoa were collected and frozen (experimental groups Day 1, 2 and 3, respectively). Sperm motility was maintained throughout the dilution procedure and then dropped (P < 0.01) after freezing and thawing. During storage the motility of nonfrozen spermatozoa decreased significantly (P < 0.01), reaching a value equal to that of frozen-thawed spermatozoa on Day 3. In vitro fertilization experiments revealed significantly (P < 0.05) lower penetration rates using Day 1, 2 and 3 stored spermatozoa (12, 13 and 2%, respectively) than that of the control group (40%). Oocyte penetration ability seemed to be reflected by acrosome integrity. However, the motility of spermatozoa with the ability to penetrate oocytes in Day 1 and Day 2 groups did not differ from that of the controls. The motility of spermatozoa lacking penetration ability, on the other hand, gradually decreased as the storage period was prolonged. This suggests that the sperm motility and penetration ability are affected by different mechanisms during the cold storage of epididymides. Finally, control and experimental groups exhibited high incidences of monospermic penetration (64 to 90%) and of male pronuclear formation (67 to 71%). These data suggest that cryopreservation of spermatozoa from boar epididymides stored at 4 degrees C for 1 to 2 d can be used for conserving male germ cells when epididymal spermatozoa can not be collected immediately and cryopreserved.

Proteomic Markers of Functional Sperm Population in Bovines: Comparison of Low- and High-Density Spermatozoa Following Cryopreservation.

PubMed

D'Amours, Olivier; Frenette, Gilles; Bourassa, Sylvie; Calvo, Ézéchiel; Blondin, Patrick; Sullivan, Robert

2018-01-05

Mammalian semen contains a heterogeneous population of sperm cells. This heterogeneity results from variability in the complex processes of cell differentiation in the testis, biochemical modifications undergone by spermatozoa during transit along the male reproductive tract, interactions with secretions from accessory sex glands at ejaculation, and, in the context of reproductive technologies, in the ability of ejaculated spermatozoa to resist damage associated with freeze-thaw procedures. When submitted to density gradient centrifugation, ejaculated spermatozoa distribute themselves into two distinct populations: a low-density population characterized by low motility parameters, and a high-density population with high motility characteristics. To understand the origin of ejaculated spermatozoa heterogeneity, cryopreserved semen samples from bulls used by the artificial insemination (A.I.) industry were submitted to Percoll gradient centrifugation. Proteins from low and high density spermatozoa were then extracted with sodium deoxycholate and submitted to proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) methodologies. Quantification of selected sperm proteins was confirmed by multiple reaction monitoring (MRM). Overall, 31 different proteins were more abundant in low-density spermatozoa, while 80 different proteins were more abundant in the high-density subpopulation. Proteins enriched in high-density spermatozoa were markers of sperm functionality such as the glycolytic process, binding to the egg zona pellucida, and motility. Low-density spermatozoa were not solely characterized by loss of proteins and their associated functions. Chaperonin-containing TCP1s and chaperones are hallmarks of the low-density subpopulation. iTRAQ analysis revealed that other proteins such as binder of sperm proteins, histone, GPX5, ELSPBP1, and clusterin are overexpressed in low-density spermatozoa suggesting that these proteins represent defects occurring at different steps during the sperm journey. These differences contribute to the sperm cell heterogeneity present in mammalian semen.

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Ile de France rams.

PubMed

Bravo, J A; Montanero, J; Calero, R; Roy, T J

2011-11-01

The aims of this study were to identify different motile sperm subpopulations in fresh ejaculates from six Ile de France rams, by using a computer-assisted sperm motility analysis (CASA) system, and to evaluate the effects of individual ram and season on population distribution. Overall sperm motility and individual kinematic parameters of motile spermatozoa were evaluated for 125,312 spermatozoa, defined by curvilinear velocity (VCL), linear velocity (VSL), average path velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR), wobble coefficient (WOB), mean amplitude of lateral head displacement (ALH) and frequency of head displacement (BCF). A multivariate cluster analysis was carried out to classify these spermatozoa into a reduced number of subpopulations according to their movement patterns. The statistical analysis clustered the whole motile sperm population into five separate groups: subpopulation 1, constituted by rapid, progressive and non sinuous spermatozoa (VCL=126.41 μm/s, STR=92.87% and LIN=86.47%); subpopulation 2, characterized by progressive spermatozoa with moderate velocity (VCL=74.74 μm/s and STR=84.03%); subpopulation 3, represented by rapid, progressive and sinuous spermatozoa (VCL=130.45 μm/s, STR=76.02% and LIN=47.68%); subpopulation 4 represents rapid nonprogressive spermatozoa (VCL=128.69 μm/s and STR=44.09%); subpopulation 5 includes poorly motile, nonprogressive spermatozoa with a very irregular trajectory (VCL=36.81 μm/s and STR=47.04%). Our results show the existence of five subpopulations of motile spermatozoa in ram ejaculates. The frequency distribution of spermatozoa within subpopulations was quite similar for the six rams, and the five subpopulations turned out to be very stable along seasons. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Membrane status of boar spermatozoa after cooling or cryopreservation.

PubMed

Maxwell, W M; Johnson, L A

1997-07-15

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.

Multi-object tracking of human spermatozoa

NASA Astrophysics Data System (ADS)

Sørensen, Lauge; Østergaard, Jakob; Johansen, Peter; de Bruijne, Marleen

2008-03-01

We propose a system for tracking of human spermatozoa in phase-contrast microscopy image sequences. One of the main aims of a computer-aided sperm analysis (CASA) system is to automatically assess sperm quality based on spermatozoa motility variables. In our case, the problem of assessing sperm quality is cast as a multi-object tracking problem, where the objects being tracked are the spermatozoa. The system combines a particle filter and Kalman filters for robust motion estimation of the spermatozoa tracks. Further, the combinatorial aspect of assigning observations to labels in the particle filter is formulated as a linear assignment problem solved using the Hungarian algorithm on a rectangular cost matrix, making the algorithm capable of handling missing or spurious observations. The costs are calculated using hidden Markov models that express the plausibility of an observation being the next position in the track history of the particle labels. Observations are extracted using a scale-space blob detector utilizing the fact that the spermatozoa appear as bright blobs in a phase-contrast microscope. The output of the system is the complete motion track of each of the spermatozoa. Based on these tracks, different CASA motility variables can be computed, for example curvilinear velocity or straight-line velocity. The performance of the system is tested on three different phase-contrast image sequences of varying complexity, both by visual inspection of the estimated spermatozoa tracks and by measuring the mean squared error (MSE) between the estimated spermatozoa tracks and manually annotated tracks, showing good agreement.

Can Helicobacter pylori infection influence human reproduction?

PubMed

Moretti, Elena; Figura, Natale; Collodel, Giulia; Ponzetto, Antonio

2014-05-21

Helicobacter pylori (H. pylori) infection could be associated with extra-digestive diseases. Here, we report the evidences concerning the decrease in reproductive potential occurring in individuals infected by H. pylori, especially by strains expressing CagA. This infection is more prevalent in individuals with fertility disorders. Infected women have anti-H. pylori antibodies in cervical mucus and follicular fluid that may decrease sperm motility and cross react immunologically with spermatozoa, conceivably hampering the oocyte/sperm fusion. Infection by CagA positive organisms enhances the risk of preeclampsia, which is a main cause of foetus death. These findings are supported by the results of experimental infections of pregnant mice, which may cause reabsorption of a high number of foetuses and alter the balance between Th1 and Th2 cell response. Infected men have decreased sperm motility, viability and numbers of normally shaped sperm and augmented systemic levels of inflammatory cytokines, such as tumor necrosis factor-α, which may damage spermatozoa. In countries where parasitic infestation is endemic, detrimental effects of infection upon spermatozoa may not occur, because the immune response to parasites could determine a switch from a predominant Th1 type to Th2 type lymphocytes, with production of anti-inflammatory cytokines. In conclusion, the evidences gathered until now should be taken into consideration for future studies aiming to explore the possible role of H. pylori infection on human reproduction.

The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

PubMed Central

Redgrove, Kate A.; Nixon, Brett; Baker, Mark A.; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R. John

2012-01-01

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility. PMID:23209833

Oxidation flux change on spermatozoa membrane in important pathologic conditions leading to male infertility.

PubMed

Wiwanitkit, V

2008-06-01

Free radicals or reactive oxygen species mediate their action through proinflammatory cytokines and this mechanism has been proposed as a common underlying factor for male infertility. There is extensive literature on oxidative stress and its role in male infertility and sperm DNA damage and its effects on assisted reproductive techniques. However, there has never been a report on the oxidation flux change in spermatozoa. Here, the author determined the oxidation flux change in such hypoxic cases, using the simulation test based on nanomedicine technique is used. Of interest, change of flux can be detected. The main pathogenesis should be the direct injury of membrane structure of spermatozoa by free radicals which can lead to sperm defect. Therefore, this work can support the finding that the oxidation flux change corresponding to oxygen pressure change in spermatozoa does not exist. However, the flux change can be seen if the membrane thickness of spermatozoa is varied. Thin membrane spermatozoa are more prone to oxidative stress than thick membrane ones. The defect in the enzymatic system within the spermatozoa should be a better explanation for vulnerability of spermatozoa to oxidative stress. The use of enzymatic modification technique by antioxidants can be useful alternative in management of male infertility.

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.

G protein-coupled estrogen receptor (GPER) in adult boar testes, epididymis and spermatozoa during epididymal maturation.

PubMed

Krejčířová, Romana; Maňasová, Marie; Sommerová, Veronika; Langhamerová, Eva; Rajmon, Radko; Maňásková-Postlerová, Pavla

2018-05-04

The G protein-coupled estrogen receptor (GPER) is a transmembrane receptor considered as a mediator of rapid non-genomic responses. GPER has been found in the male reproductive tract of many mammalian species. However, in adult boars, GPER has been reported only in ejaculated spermatozoa. Therefore, we focused on GPER detection in testicular and epididymal tissues and sperm cells in adult boars. We found GPER in Leydig cells and seminiferous tubules of boar testes and in the secretory epithelium of epididymis. A weaker signal was visible in smooth muscle cells and spermatozoa in the epididymal tubule. In spermatozoa isolated from epididymal parts, GPER was found to localize mainly in the sperm acrosome and flagellum. We immunodetected several protein bands in the extracts of the tissues and epididymal spermatozoa. A significantly higher amount of GPER mRNA was detected in the spermatozoa from caput epididymis, whereas the mRNA expression was lower in tissues of testes and caput epididymal. Our results showed the first evidence of GPER in boar epididymal spermatozoa. Moreover, the GPER localization in adult boar testes, epididymis, and mature spermatozoa suggests the involvement of estrogens via transmembrane receptor and rapid non-genomic signaling in both the sperm development and post-testicular maturation. Copyright © 2018 Elsevier B.V. All rights reserved.

Sperm Hy-Liter™: an effective tool for the detection of spermatozoa in sexual assault exhibits.

PubMed

De Moors, Anick; Georgalis, Tina; Armstrong, Gail; Modler, Jeff; Frégeau, Chantal J

2013-05-01

A fluorescence-based assay specifically targeting human spermatozoa was tested and optimized for best staining results using a variety of mock sexual assault samples. Swab clippings versus whole swabs were evaluated for best sample preparation and to simplify workflow (direct application versus swab extraction). The practicality and sensitivity of Sperm Hy-Liter™ was compared to our current phase contrast microscopy protocol for searching for the presence of spermatozoa. Sperm Hy-Liter™ was more sensitive than phase contrast microscopy and was able to detect spermatozoa more effectively in actual sexual assault samples (recent [N=240] or 24 years old [N=4]) containing few spermatozoa. Correlations were drawn between the Sperm Hy-Liter™ spermatozoa counts and the AmpFlSTR(®) Profiler(®) Plus male profiles generated from the sperm cell DNA fractions of semen containing swabs and swab clippings. In addition, recovered spermatozoa from Sperm Hy-Liter™-stained slides with greater than 40 spermatozoa produced full STR male profiles in 20.3% of slides tested and partial STR male profiles in 52.8% of slides tested. The adoption of Sperm Hy-Liter™ offers a means to standardize and improve the efficiency of the microscopic screening of sexual assault evidence. Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.

Diadenosine tetraphosphate (Ap4A) and triphosphate (Ap3A) signaling of human sperm motility.

PubMed

Chan, P J; Su, B C; Tredway, D R

1991-01-01

The ubiquitous dinucleotide polyphosphate, diadenosine tetraphosphate (Ap4A), has been shown to be a signal molecule for DNA replication in mammalian cells. In this study, Ap4A and a related compound, diadenosine triphosphate (Ap3A), were tested for possible signaling functions in human spermatozoa. A computerized automated semen analyzer was used to detect changes in spermatozoa motility parameters. Cryopreserved-thawed donor spermatozoa were washed and incubated in 0.1 mM Ap4A, 0.1 mM Ap3A, or control medium. The data indicated that both Ap4A and Ap3A decreased the percentage of motile spermatozoa after 4 or more hours of incubation in vitro. The two dinucleotide polyphosphates caused an increase in the amplitude of lateral spermatozoa head displacement parameter only at the start of incubation. The other spermatozoa kinematic parameters were unaffected. No opposing ying-yang dual actions of Ap4A to Ap3A were seen. From the results, Ap4A and Ap3A were observed to be potential inhibitory signals of spermatozoa motility after prolonged exposure.

EXTENDING THE VIABILITY OF SPERMATOZOA AND EGGS OF THE SEA URCHIN LYTECHINUS VARIEGATUS.

PubMed

Malgarin, Jéssica; Resgalla, Charrid

2015-01-01

The storage of spermatozoa and eggs of the sea urchin Lytecninus variegatus can meet the demand of different human activities. To develop a protocol easy to reproduce for spermatozoa cryopreservation and cooling of the eggs of the sea urchin. Different formulations of artificial sea water were tested for their effectiveness in the freezing of sea urchin spermatozoa and storage of the eggs. Protocol for freezing of spermatozoa in liquid nitrogen presented the positive results when the cryoprotectant solution was diluted in artificial seawater free of calcium and magnesium. For the conservation of the eggs by cooling, the calcium-free artificial sea water, the calcium- and magnesium-free sea water, and the low-sodium water proved more efficient in preserving the integrity of the eggs. The results showed success in the freezing protocol of spermatozoa and cooling of the eggs mainly in artificial calcium- and magnesium-free sea water.

Natriuretic peptide type C induces sperm attraction for fertilization in mouse

PubMed Central

Kong, Nana; Xu, Xiaoting; Zhang, Yu; Wang, Yakun; Hao, Xiaoqiong; Zhao, Yu; Qiao, Jie; Xia, Guoliang; Zhang, Meijia

2017-01-01

Mammalian spermatozoa undergo selective movement along the isthmus of the oviduct to the ampulla during ovulation, which is a prerequisite for fertilization. The factor(s) that involves in selective spermatozoa movement is still unknown. In this study, we found that the oviductal epithelium in mouse ampulla expressed high levels of natriuretic peptide type C (NPPC) in the presence of ovulated oocyte-cumulus complexes (OCCs). Spermatozoa expressed NPPC receptor natriuretic peptide receptor 2 (NPR2, a guanylyl cyclase) on the midpiece of flagellum. NPPC increased intracellular levels of cGMP and Ca2+ of spermatozoa, and induced sperm accumulation in the capillary by attraction. Importantly, spermatozoa from Npr2 mutant mice were not attracted by NPPC, preventing fertilization in vivo. Oocyte-derived paracrine factors promoted the expression of Nppc mRNA in the ampulla. Therefore, NPPC secreted by oviductal ampulla attracts spermatozoa towards oocytes, which is essential for fertilization. PMID:28054671

Distinct effects of boar seminal plasma fractions exhibiting different protein profiles on the functionality of highly diluted boar spermatozoa.

PubMed

García, E M; Calvete, J J; Sanz, L; Roca, J; Martínez, E A; Vázquez, J M

2009-04-01

The aim of this study was to evaluate how different protein profiles of seminal plasma (SP) fractions affect sperm functionality in vitro. Ejaculates from three boars were separated into six fractions. The fractions differed from each other in their sperm content, in their total SP protein content, and their spermadhesin PSP-I/PSP-II and heparin-binding protein (HBP) concentrations. Spermatozoa were mainly recovered in fraction 2 (sperm-rich fraction, >1800 x 10(6) spermatozoa/ml), whereas the pre-sperm fraction 1 and the post-sperm fractions 4-6 contained low numbers of spermatozoa (<500 x 10(6)/ml). Except in fraction 2, the total SP protein concentration and the concentration of both, spermadhesin PSP-I/PSP-II and the HBPs increased with fraction order. Distinct time-dependent effects were observed on motility characteristics and membrane integrity of highly diluted boar spermatozoa upon incubation with a 10% dilution of the SP from each fraction. The highest sperm viability was recorded after exposure for 5 h to fraction 2, followed by fractions 1 and 3. The percentages of motile spermatozoa also differed significantly among fractions after 5 h of incubation. Spermatozoa incubated with SP of fractions 1-3 showed the highest percentage motility. We conclude that different SP fractions exert distinct effects on the functionality of highly diluted boar spermatozoa. Fractions 1-3 appear to promote sperm survival, whereas fractions 4-6 seem to be harmful for preserving the physiological functions of highly diluted boar spermatozoa.

Develop to Term Rat Oocytes Injected with Heat-Dried Sperm Heads

PubMed Central

Lee, Kyung-Bon; Park, Ki-Eun; Kwon, In-Kiu; Tripurani, Swamy K.; Kim, Keun Jung; Lee, Ji Hye; Niwa, Koji; Kim, Min Kyu

2013-01-01

This study investigated the development of rat oocytes in vitro and in vivo following intracytoplasmic injection of heads from spermatozoa heat-dried at 50°C for 8 h and stored at 4°C in different gas phases. Sperm membrane and chromosome are damaged by the process of heat-drying. Oocyte activation and cleavage of oocytes were worse in oocytes injected with spermatozoa heat-dried and stored for 1 week than unheated, fresh spermatozoa, but in heat-dried spermatozoa, there were no differences in these abilities of oocytes between the samples stored in nitrogen gas and in air. The oocytes injected with heat-dried spermatozoa stored for 1 week could develop to the morula and blastocyst stages without difference between the samples stored in nitrogen gas and in air after artificial stimulation. Cleavage of oocytes and development of cleaved embryos were higher when heat-dried spermatozoa were stored for 3 and 6 months in nitrogen gas than in air. However, the ability of injected oocytes to develop to the morula and blastocyst stages was not inhibited even when heat-dried spermatozoa stored in both atmosphere conditions for as long as 6 months were used. When 2-cell embryos derived from oocytes injected with heads from spermatozoa heat-dried and stored for 1 week and 1 month were transferred, each 1 of 4 recipients was conceived, and the conceived recipients delivered 1 live young each. These results demonstrate that rat oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop to term. PMID:24223784

Hormonal induction of spermatozoa from amphibians with Rana temporaria and Bufo bufo as anuran models.

PubMed

Uteshev, V K; Shishova, N V; Kaurova, S A; Browne, R K; Gakhova, E N

2012-01-01

The use of hormonally induced spermatozoa expressed in urine (HISu) is a valuable component of reproduction technologies for amphibians. Five protocols for sampling HISu from the European common frog (Rana temporaria) were compared: (1) pituitary extracts, (2) 0.12 µg g⁻¹ luteinising hormone-releasing hormone analogue (LHRHa), (3) 1.20 µg g⁻¹ LHRHa, (4) 11.7 IU g⁻¹ human chorionic gonadotrophin (hCG) and (5) 23.4 IU g⁻¹ hCG (g⁻¹ = per gram bodyweight). From 1 to 24h after administration we assessed the number and concentration of spermatozoa in spermic urine and in holding water, and in urine the percentage of motile spermatozoa and their progressive motility. The protocol using 1.20 µg g⁻¹ LHRHa gave the highest total sperm numbers (650 × 10⁶) and the highest percentage (40%) of samples with sperm concentrations above 200 × 10⁶ mL⁻¹. The percentage motility and progressive motility was similar from all protocols. Considerable amounts of spermatozoa were expressed by R. temporaria into their holding water. We tested hormonal priming and spermiation in the common toad (Bufo bufo) using 0.13 µg g⁻¹ LHRHa administered 24h before a final spermiating dose of 12.8 IU g⁻¹ hCG. No spermatozoa were expressed in holding water. Priming resulted in 35% more spermatozoa than without; however, there were no differences in sperm concentrations. Primed B. bufo produced spermatozoa with significantly higher percentage motility, but not progressive motility, membrane integrity, or abnormal spermatozoa than unprimed males.

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).

Oxidoreductive capability of boar sperm mitochondria in fresh semen and during their preservation in BTS extender.

PubMed

Gaczarzewicz, Dariusz; Piasecka, Małgorzata; Udała, Jan; Błaszczyk, Barbara; Laszczyńska, Maria; Kram, Andrzej

2003-07-01

The purpose of our study was to determine the effect of dilution and liquid-preservation of boar sperm on oxidoreductive capability of their mitochondria. The semen was diluted with BTS extender produced from water purified by destillation or by reverse osmosis. The spermatozoa were stored over a four-day period at 16-18 degrees C. The function of sperm mitochondria was assessed using the screening cytochemical test for NADH-dependent oxidoreductases (diaphorase/NADH, related to flavoprotein). Morphological assessment of cytochemical reaction was carried out using a light microscope. The intensity of the reaction was evaluated by means of a computer image analysing system (Quantimet 600S), measuring the integrated optical density (IOD) and mean optical density (MOD) of the reaction product (formazans) occurring in the sperm midpieces. In the non-diluted semen, intensive cytochemical reaction throughout the length of the sperm midpiece was observed. Furthermore, spermatozoa with the intensive reaction displayed the high optical density values. After dilution the semen with two variants of experimental extender, and as the conservation time expired, the cytochemical reaction was less intensive. Moreover, the absence of formazan deposits in various parts of the sperm midpiece was also noted. These morphological features corresponded to low values of optical density. These findings suggest that the dilution of semen and the time of sperm preservation may be critical factors that handicap energy metabolism of sperm mitochondria. The type of water used in preparing BTS extender does not have any significant effect on the oxidoreductive capability of sperm boar mitochondria.

Sperm Morphological Features Associated with Chronic Chagas Disease in the Semen of Experimentally Infected Dogs

PubMed Central

Rodríguez-Morales, Olivia; Pedro-Martínez, Elvia; Hernández-Pichardo, José Ernesto; Alejandre-Aguilar, Ricardo; Aranda-Fraustro, Alberto; Graullera-Rivera, Verónica; Arce-Fonseca, Minerva

2014-01-01

The presence of trypanosomatids in the reproductive systems of different mammals (causing genital lesions in the acute stage of the disease) may predispose the animals to low semen quality. However, there are no studies examining the alterations in the sperm morphological features in the chronic stage of Trypanosoma cruzi infection. Knowledge of these aspects is important to understand the other ways of transmission of the Chagas disease. Progressive motility, mass motility, concentration, and sperm morphology of 84 ejaculates of dogs that were chronically infected with T. cruzi were evaluated. Most of the findings were consistent with the reference values and with those obtained from healthy control dogs. The scrotal circumference was not correlated with spermatozoa concentration in the infected animals. In conclusion, the T. cruzi Ninoa (MHOM/MX/1994/Ninoa) strain does not cause significant alterations in the semen quality of dogs experiencing chronic Chagas disease (at concentrations of 5 × 104 to 1 × 106 parasites per animal). PMID:25114010

Reverse Transcriptase Activity in Mature Spermatozoa of Mouse

PubMed Central

Giordano, Roberto; Magnano, Anna Rosa; Zaccagnini, Germana; Pittoggi, Carmine; Moscufo, Nicola; Lorenzini, Rodolfo; Spadafora, Corrado

2000-01-01

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development. PMID:10725323

Influence of damaged spermatozoa on the fertility of turkey semen stored 24 hours at 5 C.

PubMed

Sexton, T J

1988-10-01

Turkey semen was stored for 24 h at 5 C in the presence or absence of added spermatozoa that were damaged by emersion in liquid nitrogen. The fertility of semen stored in the presence of damaged spermatozoa was significantly lower than semen stored in their absence. Unstored semen was not affected by the presence of damaged spermatozoa. Fertility was affected by a significant semen treatment x holding time interaction.

NOX5 in Human Spermatozoa

PubMed Central

Musset, Boris; Clark, Robert A.; DeCoursey, Thomas E.; Petheo, Gabor L.; Geiszt, Miklos; Chen, Yumin; Cornell, John E.; Eddy, Carlton A.; Brzyski, Robert G.; El Jamali, Amina

2012-01-01

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H2O2 exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca2+ chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H2O2-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the HV1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and HV1. H2O2 treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility. PMID:22291013

A method for tracing exogenous DNA uptake in live spermatozoa and embryos.

PubMed

Mu, Y; Jiao, M; Zhao, Y; Lv, J; Wang, J; Hao, J; Zhang, X; Kong, Q; Liu, Z

2018-03-01

Sperm-mediated gene transfer(SMGT) is a simple method for producing transgenic animals. Due to the lack of repeatability in spermatozoa binding and internalization of exogenous DNA, the efficiency of SMGT is still low. Considering this point, the present work aims to develop a method for evaluating the spermatozoa capacity of binding exogenous DNA after co-incubation with DNA. The main approach is using a Cy5-labelled DNA to trace the exogenous DNA and assess the ability of spermatozoa to take up exogenous DNA. Using this technique, we found that the percentage of spermatozoa that are binding and uptaking DNA is higher at concentration of 10 μg/mL and 100 μg/mL than 5 μg/mL, 1 μg/mL and 0 μg/mL after incubation with Cy5-DNA for 30min at 37oC. After fertilization, the DNA fluorescence signal was also detected in zygotes in groups where spermatozoa were incubated with 10 μg/mL and 100 μg/mL of Cy5-DNA. These results showed a simple and convenient method to trace the exogenous DNA in spermatozoa and zygote when compared to conventional methods of labeling DNA during fertilization, resulting in a real-time observation of the exogenous DNA in spermatozoa and zygote. Copyright© by the Polish Academy of Sciences.

Successful use of the Cryolock device for cryopreservation of scarce human ejaculate and testicular spermatozoa.

PubMed

Stein, A; Shufaro, Y; Hadar, S; Fisch, B; Pinkas, H

2015-03-01

The existing methods for cryopreservation of very low count sperm samples are complex and sub-optimal for individual spermatozoa. Our purpose is to establish an effective simple method for cryoprotecting individual spermatozoa. Samples from patients with OTA were mixed with TYB or HEPES-buffered salt solution with glycerol + glucose and placed on a Cryolock that was plunged directly into liquid nitrogen or exposed to its vapors. Thawing was performed by direct immersion into a drop of warmed medium. The favorable method was tested on diluted samples (10-50 cells) and leftover TESE specimens from patients with azoospermia. Cryopreservation was considered successful if >30 spermatozoa, (>3 motile), or >5 spermatozoa (>1 motile) in diluted and TESE samples, were detected post-thawing. A significantly higher survival rate of seminal spermatozoa was obtained when using the Cryolock with TYB and freezing with liquid nitrogen vapor, compared to HEPES glycerol-glucose (95 vs. 35% respectively). Plunging the Cryolock into liquid nitrogen was detrimental. Cryolock combined with TYB cryoprotection and liquid nitrogen vapor freezing was highly effective for cryopreservation of individual spermatozoa in diluted and TESE samples. The Cryolock may serve for freezing very low-count sperm samples and individual spermatozoa. This method offers simplicity, efficacy, use of available materials, without requiring micromanipulation equipment or skills. © 2015 American Society of Andrology and European Academy of Andrology.

Effects of spermatozoa-oviductal cell coincubation time and oviductal cell age on spermatozoa-oviduct interactions.

PubMed

Aldarmahi, Ahmed; Elliott, Sarah; Russell, Jean; Fazeli, Alireza

2014-01-01

The oviduct plays a crucial role in sperm storage, maintenance of sperm viability and sperm transport to the site of fertilisation. The aim of the present study was to investigate the effects of oviductal cell culture passage number, oviductal cell age and spermatozoa-oviduct coincubation times on gene expression in oviductal cells. Immortalised oviductal epithelial cells (OPEC) obtained from two different cell passages (36 and 57) were subcultured three times with and without spermatozoa for 24 h (control group). In a second study, OPEC were cocultured with spermatozoa for different time intervals (0, 4, 12 and 24 h). Expression of adrenomedullin (ADM), heat shock 70 kDa protein 8 (HSPA8) and prostaglandin E synthase (PGES) in OPEC was measured by quantitative polymerase chain reaction. The expression of ADM and HSPA8 was decreased significantly in OPEC cells from Passage 57, particularly in the later subculture group. These effects on HSPA8, but not ADM, expression in OPEC were further altered after coculture with spermatozoa for 24 h. We also demonstrated that spermatozoa-oviduct coculture for 12 and 24 h resulted in significantly higher expression of ADM, HSPA8 and PGES in OPEC. Overall, the data suggest that the OPEC lose some of their properties as a result of oviductal cell aging and that there are spermatozoa-oviduct interactions leading to increased oviductal cell gene expression.

Improved conception rates in sows inseminated with cryopreserved boar spermatozoa prepared with a more optimal combination of osmolality and glycerol in the freezing extender.

PubMed

Okazaki, Tetsuji; Abe, Shouhachiro; Shimada, Masayuki

2009-04-01

Cryoprotectant agents (CPAs) are added in freezing extenders to prevent intracellular ice crystal formation. However, it has been reported that high dose of CPAs confer toxicity on spermatozoa. Recently, the reduction of intracellular water by a high osmolality solution has also resulted in the suppression of ice crystal formation in spermatozoa, suggesting that the optimal combination of glycerol concentration and freezing extender osmolality could contribute to the development of effective sperm cryopreservation techniques. In this study, we investigated the motility, membrane and acrosomal integrity of frozen-thawed boar spermatozoa treated with freezing extender (NSF) of varying osmolalities (300, 400, 500 mOsm/kg) and final concentrations of glycerol (0.5, 1, 2, 3%). The spermatozoa that were treated at 400 mOsm/kg and 2% glycerol showed significantly higher rates of motility and membrane integrity compared with those in other treatment groups. In addition, the conception and implantation rates of swine artificially inseminated with spermatozoa frozen by the novel freezing extender (conception; 79%, implantation; 57.5%) were significantly higher than those of frozen-thawed spermatozoa treated in the conventional NSF (300 mOsm/kg, 3% glycerol) (conception; 29%, implantation; 33.8%). From these results, we concluded that the novel hyperosmotic (400 mOsm/kg) and low-glycerol (final concentration 2%) freezing extender is beneficial for the cryopreservation of boar spermatozoa.

Effect of D-ribose-L-cysteine on aluminum induced testicular damage in male Sprague-Dawley rats.

PubMed

Falana, Benedict; Adeleke, Opeyemi; Orenolu, Mulikat; Osinubi, Abraham; Oyewopo, Adeoye

2017-06-01

This study investigated the effects of D-ribose and L-cysteine on aluminum-induced testicular damage in male Sprague-Dawley rats. A total number of thirty-five (35) adult male Sprague-Dawley rats were divided into four groups (AD). Group A (comprised five (5) rats) was designated the Control Group that received Physiological Saline; while groups B, C, and D (comprised ten (10) rats) were given 75 mg/kg, 150 mg/kg and 300 mg/kg of body weight of aluminum chloride respectively for 39 days. At day 40, the aluminum-treated groups were subdivided into sub-groups (B1, C1, D1) comprising of five (5) rats each, and 30 mg/kg body weight of Riboceine were administered for twenty (20) days. Groups B, C and D remained on the normal dosage of aluminum chloride for three more weeks (59 days). Andrological parameters (Sperm count, motility, morphology and testosterone) in the aluminum-treated Groups B and C showed no significant difference in their mean values when compared with their control counterparts, whereas there was a significant reduction in the andrological parameters in Group D rats when compared with the Control animals. Histoarchitecture of the testes "stain with H&E" of Group A, B and C rats appeared normal while Group D rats showed testicular damages with several abnormal seminiferous tubules with incomplete maturation of germinal cell layers and absence of spermatozoa in their lumen; Leydig cells appear hyperplastic. Group B1, C1 and D1 andrological and histological parameters appeared normal. Riboceine treatment significantly attenuates aluminum-induced testicular toxicity in male Sprague-Dawley in rats.

Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.

PubMed

Lazaros, Leandros; Kitsou, Chrysoula; Kostoulas, Charilaos; Bellou, Sofia; Hatzi, Elissavet; Ladias, Paris; Stefos, Theodoros; Markoula, Sofia; Galani, Vasiliki; Vartholomatos, Georgios; Tzavaras, Theodore; Georgiou, Ioannis

2017-03-01

To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome. Laboratory study. University research laboratories and academic hospital. Normozoospermic and oligozoospermic white men. RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy. Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa. RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa. Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human spermatozoa; and 4) de novo retrotransposition events occur in human spermatozoa. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Interaction of intact porcine spermatozoa with epithelial cells and neutrophilic granulocytes during uterine passage.

PubMed

Taylor, U; Rath, D; Zerbe, H; Schuberth, H J

2008-04-01

New insemination techniques allow a tremendous sperm reduction for successful artificial insemination (AI) if highly diluted semen is deposited in the tip of the uterine horn and close to the utero-tubal junction. High sperm losses are known to occur during uterine passage and it was the general question whether specific binding mechanisms are involved. Upon arrival in the uterus, spermatozoa are confronted with mainly two different cell types: uterine epithelial cells (UEC) and neutrophilic granulocytes (polymorphonuclear neutrophil, PMN). As cell-sperm interactions can hardly be observed in vivo, an ex vivo system was established to study the interaction between spermatozoa and the UEC. Uterine segments (10 cm) from freshly slaughtered synchronized juvenile gilts were inseminated for 60 min at 38 degrees C. Thereafter spermatozoa were recovered, counted flow cytometrically and examined for changes in viability and mitochondrial membrane potential (MMP). Significantly less spermatozoa with a functioning MMP and intact plasma membranes could be retrieved (55 +/- 7%), while the number of damaged spermatozoa hardly changed (93 +/- 12%), indicating retention of viable sperm cells in the uterine lumen. The interactions between porcine PMN and spermatozoa (motile, immotile, membrane-damaged) were studied in coincubation assays in vitro. The binding of membrane-damaged sperm cells to PMN was virtually non-existent (3 +/- 2%). Viable and motile spermatozoa attached to PMN without being phagocytosed within 60 min (45 +/- 3%), whereas binding to sodium fluoride (NaF)-immobilized spermatozoa was reduced to 20 +/- 2%. The binding of viable sperm to PMN is most likely not lectin-dependent; although both viable cell types were shown to express a broad range of different lectin-binding sugar residues, none of the lectins tested was able to selectively block PMN-sperm binding significantly. The results of the study suggest that viable spermatozoa are already subject to selective processes within the uterus before further selection is initiated at the utero-tubal junction and in the oviductal isthmus.

Aquaporins 7 and 11 in boar spermatozoa: detection, localisation and relationship with sperm quality.

PubMed

Prieto-Martínez, Noelia; Vilagran, Ingrid; Morató, Roser; Rodríguez-Gil, Joan E; Yeste, Marc; Bonet, Sergi

2016-04-01

Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (P<0.05) with sperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.

The test about blood serum capabilities in maintaining the quality of bull spermatozoa during storage in cep diluent at refrigerator temperature

NASA Astrophysics Data System (ADS)

Ducha, Nur

2018-03-01

The storage of spermatozoa requires a protective material from cold shock events and the presence of free radicals.In CEP diluent contain BSA, that was used as spermatozoa protection. This study aim was to examine the ability of cow blood serum in replacing BSA as spermatozoa protective in CEP diluent. Fresh semen from Limousin bull was diluted with CEP diluent + BSA as control, in the treatment group were CEP without BSA, but replaced with 3%, 5%, and 7% serum from fresh blood. Spermatozoa quality tests included motility and viability. The motility of spermatozoa was observed by two people using a light microscope with 200 X magnification at temperature of 37°C. The method of viability observation was eosin nigrosin staining, and observed under a light microscope with 400 X magnification. The results showed that the replacement of cow blood serum with various concentrations gave different effects on the quality of spermatozoa. The best motility and viability of the treatment group was at serum concentrations of 5% after eight days storage and was not significantly different from the controls. The conclusion in this study was cow blood serum can replace BSA in CEP diluents.

Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation.

PubMed

Sati, Leyla; Ovari, Laszlo; Bennett, David; Simon, Stephen D; Demir, Ramazan; Huszar, Gabor

2008-04-01

Individual spermatozoa were assessed with pairs of probes for persistent histones and cytoplasmic retention, persistent histones and DNA fragmentation, and persistent histones and apoptotic markers. The individual spermatozoa were treated sequentially with combinations of probes for these cytoplasmic and nuclear biochemical markers. Sperm fields were recorded with computer-assisted imaging, and staining patterns with the two probes in the same spermatozoa were examined and scored as light, intermediate or dark (mature to arrested-maturity spermatozoa). The effects of arrested sperm maturation were similar with respect to the cytoplasmic and nuclear characteristics of spermatozoa in 84% of cells, indicating that cytoplasmic and nuclear attributes of arrested sperm maturation are related. However, there were moderate (intermediate-dark or intermediate-light patterns, 14.5% of cells) or major (light-dark patterns, 1.6% of cells) discrepancies in the intensity of the double staining patterns. Thus, testing with single maturity markers may not be fully reliable. These findings are important with respect to: (i) arrested sperm maturation; (ii) potential efficacy of antioxidant and similar therapeutic strategies in subfertile men, as spermatozoa with infrastructure defects due to mismaturation or maturation arrest are unlikely to respond to interventions; and (iii) detection of adverse male environmental exposures.

Ultrastructural characters of the spermatozoa in Digeneans of the genus Lecithochirium Lühe, 1901 (Digenea, Hemiuridae), parasites of fishes: comparative study of L. microstomum and L. musculus

PubMed Central

Ndiaye, Papa Ibnou; Quilichini, Yann; Sène, Aminata; Tkach, Vasyl V.; Bâ, Cheikh Tidiane; Marchand, Bernard

2014-01-01

This study provides the first ultrastructural data of spermatozoa in the genus Lecithochirium. The spermatozoa of L. microstomum (from Trichiurus lepturus in Senegal) and L. musculus (from Anguilla anguilla in Corsica) exhibit the general pattern described in the great majority of the Digenea, namely two axonemes with the 9 + “1” pattern typical of the Trepaxonemata, one mitochondrion, a nucleus, parallel cortical microtubules and external ornamentation of the plasma membrane. Spermatozoa of L. microstomum and L. musculus have some specific features such as the presence of a reduced number of cortical microtubules arranged on only one side of the spermatozoon, the lack of spine-like bodies and expansion of the plasma membrane. The external ornamentation of the plasma membrane entirely covers the anterior extremity of the spermatozoa. The ultrastructure of the posterior extremity of the spermatozoa corresponds to the pattern previously described in the Hemiuridae, characterized by only singlets of the second axoneme. A particularity of these spermatozoa is the organization of the microtubule doublets of the second axoneme around the nucleus in the posterior part of the spermatozoon. PMID:25275216

Mitochondrial respiratory efficiency is positively correlated with human sperm motility.

PubMed

Ferramosca, Alessandra; Provenzano, Sara Pinto; Coppola, Lamberto; Zara, Vincenzo

2012-04-01

To correlate sperm mitochondrial respiratory efficiency with variations in sperm motility and with sperm morphologic anomalies. Sperm mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically-treated sperm cells. A possible relationship among sperm mitochondrial respiratory efficiency, sperm motility, and morphologic anomalies was investigated. Mitochondrial respiratory efficiency was positively correlated with sperm motility and negatively correlated with the percentage of immotile spermatozoa. Moreover, midpiece defects impaired mitochondrial functionality. Our data indicate that an increase in sperm motility requires a parallel increase in mitochondrial respiratory capacity, thereby supporting the fundamental role played by mitochondrial oxidative phosphorylation in sperm motility of normozoospermic subjects. These results are of physiopathological relevance because they suggest that disturbances of sperm mitochondrial function and of energy production could be responsible for asthenozoospermia. Copyright © 2012 Elsevier Inc. All rights reserved.

Birth of healthy twins after intracytoplasmic sperm injection using ejaculated immotile spermatozoa from a patient with Kartagener's syndrome.

PubMed

Geber, S; Lemgruber, M; Taitson, P F; Valle, M; Sampaio, M

2012-05-01

This case report demonstrates a successful pregnancy after ICSI combined with hypo-osmotic swelling test in a couple with Kartagener's syndrome with complete immotile ejaculated spermatozoa. Our result suggests that even for complete immotile spermatozoa, the use of hypo-osmotic swelling test is a good alternative to identify viable spermatozoa. When associated with ICSI, it can be a valuable tool to get fertilisation and pregnancy. © 2011 Blackwell Verlag GmbH.

AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility.

PubMed

Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

2017-01-01

AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

PubMed Central

Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

2017-01-01

AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%–80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied. PMID:27678462

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.

Protective effect of Urtica dioica L against nicotine-induced damage on sperm parameters, testosterone and testis tissue in mice.

PubMed

Jalili, Cyrus; Salahshoor, Mohammad Reza; Naseri, Ali

2014-06-01

Nicotine consumption can decrease fertility drive in males by inducing oxidative stress and DNA damage. Urtica dioica L (U.dioica) is a multipurpose herb in traditional medicine for which some anti-oxidative and anti-inflammatory properties have been identified. The main goal is to investigate whether the U.dioica could inhibit nicotine adverse effects on sperm cells viability, count, motility, and testis histology and testosterone hormone. In this study, hydro-alcoholic extract of U.dioica was prepared and various doses of U.dioica (0, 10, 20, and 50 mg/kg) and U.dioica plus nicotine (0, 10, 20, and 50 mg/kg) were administered intraperitoneally to 56 male mice for 28 consequent days. These mice were randomly assigned to 8 groups (n=7) and sperm parameters (sperm cells viability, count, motility, and morphology), testis and prostate weight, testis histology and testosterone hormone were analyzed and compared. The results indicated that nicotine administration (0.5 mg/kg) significantly decreased testosterone level, count and motility of sperm cells, and testis weight compared to control group (p=0.00). However, increasing the dose of U.dioica significantly boosted motility, count, normal morphology of sperm cells, seminiferous tubules diameter, and testosterone in all groups compared to control (p=0.00) and testis weight in 20 and 50 mg/kg doses in comparison with control group (p=0.00). It seems that U.dioica hydro-alcoholic extract administration could increase the quality of spermatozoa and inhibits nicotine-induced adverse effects on sperm parameters.

Protective effect of Urtica dioica L against nicotine-induced damage on sperm parameters, testosterone and testis tissue in mice

PubMed Central

Jalili, Cyrus; Salahshoor, Mohammad Reza; Naseri, Ali

2014-01-01

Background: Nicotine consumption can decrease fertility drive in males by inducing oxidative stress and DNA damage. Urtica dioica L (U.dioica) is a multipurpose herb in traditional medicine for which some anti-oxidative and anti-inflammatory properties have been identified. Objective: The main goal is to investigate whether the U.dioica could inhibit nicotine adverse effects on sperm cells viability, count, motility, and testis histology and testosterone hormone. Materials and Methods: In this study, hydro-alcoholic extract of U.dioica was prepared and various doses of U.dioica (0, 10, 20, and 50 mg/kg) and U.dioica plus nicotine (0, 10, 20, and 50 mg/kg) were administered intraperitoneally to 56 male mice for 28 consequent days. These mice were randomly assigned to 8 groups (n=7) and sperm parameters (sperm cells viability, count, motility, and morphology), testis and prostate weight, testis histology and testosterone hormone were analyzed and compared. Results: The results indicated that nicotine administration (0.5 mg/kg) significantly decreased testosterone level, count and motility of sperm cells, and testis weight compared to control group (p=0.00). However, increasing the dose of U.dioica significantly boosted motility, count, normal morphology of sperm cells, seminiferous tubules diameter, and testosterone in all groups compared to control (p=0.00) and testis weight in 20 and 50 mg/kg doses in comparison with control group (p=0.00). Conclusion: It seems that U.dioica hydro-alcoholic extract administration could increase the quality of spermatozoa and inhibits nicotine-induced adverse effects on sperm parameters. PMID:25071848

Therapeutic and fertility restoration effects of Ionidium suffruticosum on sub-fertile male albino Wistar rats: effects on testis and caudal spermatozoa.

PubMed

Chenniappan, Kuppusamy; Murugan, Kadarkari

2017-12-01

Ionidium suffruticosum (L.) Ging (Violaceae) is an important medicinal plant widely used as a herbal traditional medicine in Ayurveda for the treatment of infertility. Currently, little pharmacological information is available on its male fertility properties following prolonged use. To investigate I. suffruticosum leaf extracts for male fertility parameters. The ethanol lyophilized fraction was administered orally on carbendazim-induced sub-fertility rats (250 mg/kg body weight for 28 days). The effects of fractions on rat's fertility parameters i.e., body and testes weight, sperm motility, sperm vitality, epididymal sperm counts, its morphology, enzyme and antioxidant stress and histopathology were studied and compared with clomiphene citrate. The sub-fertile male rats treated with I. suffruticosum leaf extract increased the body weight of 7 g, testis weight of 97 mg, increased cauda epididymal sperm counts of 34.2 × 10 6 sperm/mL, motility of sperm 46% and vitality 28% also increased and normal sperm morphology also improved up to 32%. The carbendazim-treated group showed loss in body weight of 33 g, testis weight of 851 mg, decreased epididymal sperm counts of 15 × 10 6 sperm/mL, with sluggish motility and a highly significant fall in the live sperms of about 57%. The leaf fraction of I. suffructicosum increased the testicular weight, spermatogenesis, sperm counts, lessened sperm agglutination, and increased testicular oxidative biomarkers, SOD, and CAT. This study therefore supports the usage of I. suffructicosum in traditional medicine for infertility.

Response of spermatozoa to hyposmotic stress reflects cryopreservation success

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

Watson, P.F.; Curry, M.R.; Noiles, E.E.

1992-01-01

Spermatozoa of several species were washed and then subjected to dilution in hyposmotic Tyrode's based solutions. The cells were stained with fluorescent viability stains, carboxyfluorescein diacetate and propidium iodide, and proportions with intact plasma membranes determined by flow cytometry or fluorescence microscopy. Fowl spermatozoa remained almost 100% intact until very low osmolality, and then ruptured. Human spermatozoa showed a similar response with only a small decrease in intact cells before the precipitous decline at low osmolality. Bull spermatozoa were more readily disrupted at higher osmolality, some 40% being damaged before the sudden decline at low osmolality. Ram and boar spermatozoamore » were progressively disrupted even at mild hyposmotic stress, showing approximately 50% of cells ruptured at 150 mOsm.« less

Response of spermatozoa to hyposmotic stress reflects cryopreservation success

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

Watson, P.F.; Curry, M.R.; Noiles, E.E.

1992-06-01

Spermatozoa of several species were washed and then subjected to dilution in hyposmotic Tyrode`s based solutions. The cells were stained with fluorescent viability stains, carboxyfluorescein diacetate and propidium iodide, and proportions with intact plasma membranes determined by flow cytometry or fluorescence microscopy. Fowl spermatozoa remained almost 100% intact until very low osmolality, and then ruptured. Human spermatozoa showed a similar response with only a small decrease in intact cells before the precipitous decline at low osmolality. Bull spermatozoa were more readily disrupted at higher osmolality, some 40% being damaged before the sudden decline at low osmolality. Ram and boar spermatozoamore » were progressively disrupted even at mild hyposmotic stress, showing approximately 50% of cells ruptured at 150 mOsm.« less

Sneaker Male Squid Produce Long-lived Spermatozoa by Modulating Their Energy Metabolism *

PubMed Central

Hirohashi, Noritaka; Tamura-Nakano, Miwa; Nakaya, Fumio; Iida, Tomohiro; Iwata, Yoko

2016-01-01

Spermatozoa released by males should remain viable until fertilization. Hence, sperm longevity is governed by intrinsic and environmental factors in accordance with the male mating strategy. However, whether intraspecific variation of insemination modes can impact sperm longevity remains to be elucidated. In the squid Heterololigo bleekeri, male dimorphism (consort and sneaker) is linked to two discontinuous insemination modes that differ in place and time. Notably, only sneaker male spermatozoa inseminated long before egg spawning can be stored in the seminal receptacle. We found that sneaker spermatozoa exhibited greater persistence in fertilization competence and flagellar motility than consort ones because of a larger amount of flagellar glycogen. Sneaker spermatozoa also showed higher capacities in glucose uptake and lactate efflux. Lactic acidosis was considered to stabilize CO2-triggered self-clustering of sneaker spermatozoa, thus establishing hypoxia-induced metabolic changes and sperm survival. These results, together with comparative omics analyses, suggest that postcopulatory reproductive contexts define sperm longevity by modulating the inherent energy levels and metabolic pathways. PMID:27385589

The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

PubMed

Wong, Chi-Wai; Lam, Kevin K W; Lee, Cheuk-Lun; Yeung, William S B; Zhao, Wei E; Ho, Pak-Chung; Ou, Jian-Ping; Chiu, Philip C N

2017-04-01

Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction? ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content. A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins. The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed. Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays. Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa. N/A. The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted. Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction. This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Ultrastructure of spermiogenesis in Vampyroteuthis infernalis Chun — a relict cephalopod mollusc

NASA Astrophysics Data System (ADS)

Healy, John M.

1990-03-01

Spermiogenesis in the relict deep-sea cephalopod Vampyroteuthis infernalis Chun is examined using transmission electron microscopy (TEM), and the results compared with available data on other cephalopods. Early spermatids of Vampyroteuthis exhibit an ovoid nucleus (with dense irregular patches), numerous mitochondria and a pair of triplet substructure centrioles (arranged parallel to each other). Subsequently, the following morphological changes take place: (1) nuclear contents condense into a fibrous reticulum, then into thick fibres; (2) the acrosomal vesicle (presumably Golgi-derived) positions itself in a shallow depression at the nuclear apex; (3) the flagellum forms from one of the two centrioles; (4) mitochondria cluster around the flagellum at the base of the nucleus; (5) a dense, fibrous plug forms within the basal invagination of the nucleus. Microtubules surround the acrosome and condensing nucleus of spermatids. The dense plug is of special systematic importance since it also occurs in spermatids and spermatozoa of Octopus spp., but not in any investigated species of the Sepiida, Sepiolida or Teuthida. Late spermatids and mature spermatozoa of Vampyroteuthis strongly resemble developing spermatids of Octopus, suggesting a close phylogenetic relationship between Vampyroteuthis (and the Vampyromorpha) and octopods.

Isolation of Toxigenic Nocardiopsis Strains from Indoor Environments and Description of Two New Nocardiopsis Species, N. exhalans sp. nov. and N. umidischolae sp. nov.

PubMed Central

Peltola, Joanna S. P.; Andersson, Maria A.; Kämpfer, Peter; Auling, Georg; Kroppenstedt, Reiner M.; Busse, Hans-Jürgen; Salkinoja-Salonen, Mirja S.; Rainey, Frederick A.

2001-01-01

Nocardiopsis strains were isolated from water-damaged indoor environments. Two strains (N. alba subsp. alba 704a and a strain representing a novel species, ES10.1) as well as strains of N. prasina, N. lucentensis, and N. tropica produced methanol-soluble toxins that paralyzed the motility of boar spermatozoa at <30 μg of crude extract (dry weight) ml−1. N. prasina, N. lucentensis, N. tropica, and strain ES10.1 caused cessation of motility by dissipating the mitochondrial membrane potential, Δψ, of the boar spermatozoa. Indoor strain 704a produced a substance that destroyed cell membrane barrier function and depleted the sperm cells of ATP. Indoor strain 64/93 was antagonistic towards Corynebacterium renale. Two indoor Nocardiopsis strains were xerotolerant, and all five utilized a wide range of substrates. This combined with the production of toxic substances suggests good survival and potential hazard to human health in water-damaged indoor environments. Two new species, Nocardiopsis exhalans sp. nov. (ES10.1T) and Nocardiopsis umidischolae sp. nov. (66/93T), are proposed based on morphology, chemotaxonomic and physiological characters, phylogenetic analysis, and DNA-DNA reassociations. PMID:11526036

In Vitro Measures for Assessing Boar Semen Fertility.

PubMed

Jung, M; Rüdiger, K; Schulze, M

2015-07-01

Optimization of artificial insemination (AI) for pig production and evaluation of the fertilizing capacity of boar semen are highly related. Field studies have demonstrated significant variation in semen quality and fertility. The semen quality of boars is primarily affected by breed and season. AI centres routinely examine boar semen to predict male fertility. Overall, the evaluation of classical parameters, such as sperm morphology, sperm motility, sperm concentration and ejaculate volume, allows the identification of ejaculates corresponding to poor fertility but not high-efficiency prediction of field fertility. The development of new sperm tests for measuring certain sperm functions has attempted to solve this problem. Fluorescence staining can categorize live and dead spermatozoa in the ejaculate and identify spermatozoa with active mitochondria. Computer-assisted semen analysis (CASA) provides an objective assessment of multiple kinetic sperm parameters. However, sperm tests usually assess only single factors involved in the fertilization process. Thus, basing prediction of fertilizing capacity on a selective collection of sperm tests leads to greater accuracy than using single tests. In the present brief review, recent diagnostic laboratory methods that directly relate to AI performance as well as the development of a new boar fertility in vitro index are discussed. © 2015 Blackwell Verlag GmbH.

Segmentation of white rat sperm image

NASA Astrophysics Data System (ADS)

Bai, Weiguo; Liu, Jianguo; Chen, Guoyuan

2011-11-01

The segmentation of sperm image exerts a profound influence in the analysis of sperm morphology, which plays a significant role in the research of animals' infertility and reproduction. To overcome the microscope image's properties of low contrast and highly polluted noise, and to get better segmentation results of sperm image, this paper presents a multi-scale gradient operator combined with a multi-structuring element for the micro-spermatozoa image of white rat, as the multi-scale gradient operator can smooth the noise of an image, while the multi-structuring element can retain more shape details of the sperms. Then, we use the Otsu method to segment the modified gradient image whose gray scale processed is strong in sperms and weak in the background, converting it into a binary sperm image. As the obtained binary image owns impurities that are not similar with sperms in the shape, we choose a form factor to filter those objects whose form factor value is larger than the select critical value, and retain those objects whose not. And then, we can get the final binary image of the segmented sperms. The experiment shows this method's great advantage in the segmentation of the micro-spermatozoa image.

Expression of a Catalytically Inactive Mutant Form of Glutathione Peroxidase 4 (Gpx4) Confers a Dominant-negative Effect in Male Fertility*

PubMed Central

Ingold, Irina; Aichler, Michaela; Yefremova, Elena; Roveri, Antonella; Buday, Katalin; Doll, Sebastian; Tasdemir, Adrianne; Hoffard, Nils; Wurst, Wolfgang; Walch, Axel; Ursini, Fulvio; Friedmann Angeli, José Pedro; Conrad, Marcus

2015-01-01

The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with a targeted mutation of the active site selenocysteine of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4−/− embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breeding and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoa midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mitochondrial Gpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared with selenocysteine at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Because the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and as a structural protein, tightly controlled expression of functional Gpx4 emerges as a key for full male fertility. PMID:25922076

Puberty in the male chimpanzee: progressive maturation of semen characteristics.

PubMed

Marson, J; Meuris, S; Cooper, R W; Jouannet, P

1991-03-01

Cellular and biochemical characteristics of semen obtained by masturbation were studied in 5 pubertal chimpanzees during 1.5 yr. The dental age corresponding to the beginning of the pubertal testicular growth (Po) ranged from 5.0 to 8.3 years (mean = 6.7 +/- 1.2 yr). Time-related variations of all studied parameters were analyzed according to Po and, therefore, independently of the dental age. The emission of first ejaculates, and therefore the onset of activity of the accessory sex gland, was estimated to occur 4 mo after Po. The ejaculate volume (n = 132, r = 0.704, p less than 0.001), as well as the total amounts of l-carnitine (n = 65, r = 0.649, p less than 0.001), fructose (r = 0.522, p less than 0.001), citrate (r = 0.748, p less than 0.001), and acid phosphatase (r = 0.756, p less than 0.001) increased with time. First-obtained ejaculates contained no spermatozoa. Spermarche was estimated to occur 9.4 mo after Po. Total sperm count increased with time (r = 0.595, p less than 0.001). The motility and viability of spermatozoa increased with time (r = 0.474, p less than 0.01 and r = 0.632, p less than 0.001, respectively) while their morphology did not vary. The volume of the liquefied fraction and its content in free spermatozoa considered as available for fecundation remained low until the end of the study, most likely because of a delayed maturation of the prostate. This study shows that the maturation of the male genital tract is a progressive process. (ABSTRACT TRUNCATED AT 250 WORDS)

Effect of semen dilution to low-sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low-density lipoproteins (LDL) extender: comparison to Triladyl and Bioxcell.

PubMed

Vera-Munoz, O; Amirat-Briand, L; Diaz, T; Vásquez, L; Schmidt, E; Desherces, S; Anton, M; Bencharif, D; Tainturier, D

2009-04-01

Artificial insemination with doses containing low-sperm numbers has been utilized to optimize the use of elite bulls. Hen egg yolk is widely used as a cryoprotective agent in semen freezing extender protecting the spermatozoa. Its action is due to the presence of low-density lipoproteins (LDL) in the hen egg yolk. The objectives of the present study were to evaluate the effects of the semen dilution to low-sperm number/dose on sperm motility and integrity of sperm plasma membrane in the cryopreservation process, using two commercial extenders (Triladyl, Bioxcell and LDL extender prepared in our laboratory, 97% purity. Fifteen ejaculates were collected from five fertile crossbred bulls (Bos taurusxBos indicus). After collection, sperm motility was examined by Computer-Assisted Semen Analysis (Hamilton Thorne), morphological sperm characteristics were evaluated by differential interference microscopy and the integrity of plasma membranes was determined using the hypo-osmotic swelling test. The semen was subsequently divided into three aliquots and diluted with the three extenders into 120 x 10(6), 60 x 10(6) and 20 x 10(6)sperm/mL, corresponding to 30 x 10(6), 15 x 10(6) and 5 x10(6) sperm/dose, respectively. This study revealed that LDL extender was more effective in preservation of motility and integrity of the plasma membrane of spermatozoa than Bioxcell and Triladyl (p<0.05), but no significant difference was observed between Triladyland Bioxcell. Therefore we can conclude that LDL extender could be used instead of Triladyl or Bioxcellat low semen concentration per dose for elite bulls, it also could be envisaged for the industry of sex-stored semen.

Thiols of flagellar proteins are essential for progressive motility in human spermatozoa.

PubMed

Cabrillana, María Eugenia; Monclus, María de Los Ángeles; Lancellotti, Tania Estefania Sáez; Boarelli, Paola Vanina; Vincenti, Amanda Edith; Fornés, Miguel Matias; Sanabria, Eduardo Alfredo; Fornés, Miguel Walter

2017-07-01

Male infertility is a disorder of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse. The presence of low-motile or immotile spermatozoa is one of many causes of infertility; however, this observation provides little or no information regarding the pathogenesis of the malfunction. Good sperm motility depends on correct assembly of the sperm tail in the testis and efficient maturation during epididymal transit. Thiols of flagellar proteins, such as outer dense fibre protein 1 (ODF1), are oxidised to form disulfides during epididymal transit and the spermatozoa become motile. This study was designed to determine how oxidative changes in protein thiol status affect progressive motility in human spermatozoa. Monobromobimane (mBBr) was used as a specific thiol marker and disruptor of sperm progressive motility. When mBBr was blocked by dithiothreitol it did not promote motility changes. The analysis of mBBr-treated spermatozoa revealed a reduction of progressive motility and an increased number of spermatozoa with non-progressive motility without affecting ATP production. Laser confocal microscopy and western blot analysis showed that one of the mBBr-positive proteins reacted with an antibody to ODF1. Monobromobimane fluorescence intensity of the sperm tail was lower in normozoospermic than asthenozoospermic men, suggesting that thiol oxidation in spermatozoa of asthenozoospermic men is incomplete. Our findings indicate that mBBr affects the thiol status of ODF1 in human spermatozoa and interferes with progressive motility.

Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks.

PubMed

Namula, Zhao; Sato, Yoko; Kodama, Risa; Morinaga, Kouta; Luu, Vien Viet; Taniguchi, Masayasu; Nakai, Michiko; Tanihara, Fuminori; Kikuchi, Kazuhiro; Nagai, Takashi; Otoi, Takeshige

2013-08-01

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long-term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5-SM and 15-SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5-SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5-SM and 15-SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5-SM group showed similar rates of fertilization and blastocyst formation in the fresh non-stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5-SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks. © 2013 Japanese Society of Animal Science.

Single layer centrifugation-selected boar spermatozoa are capable of fertilization in vitro

PubMed Central

2013-01-01

Background Good quality spermatozoa are important to achieve fertilization, viable embryos and offspring. Single Layer Centrifugation (SLC) through a colloid (Androcoll-P) selects good quality spermatozoa. However, it has not been established previously whether porcine spermatozoa selected by this method maintain their fertility. Methods The semen was prepared either by SLC or by standard centrifugation (control) and used for in vitro fertilization (IVF) at oocyte:spermatozoa ratios of 1:50; 1:100 and 1:300 (or 4 x 103, 8 x 103 and 24 x 103 spermatozoa/ml) to evaluate their subsequent ability to generate blastocysts. In addition, sperm motility was assessed by computer assisted sperm motility analysis. Results Total and progressive motility were significantly higher in sperm samples prepared by SLC compared to uncentrifuged samples. Sperm binding ability, polyspermy, cleavage and blastocyst rates were affected by the oocyte:sperm ratio, but not by sperm treatment. Conclusion The use of SLC does not adversely affect the in vitro fertilizing and embryo-generating ability of the selected spermatozoa compared to their unselected counterparts, but further modifications in the IVF conditions would be needed to improve the monospermy in IVF systems. Since SLC did not appear to have a negative effect on sperm fertilizing ability, and may in fact select for spermatozoa with a greater potential for fertilization, an in vivo trial to determine the usefulness of this sperm preparation technique prior to artificial insemination is warranted. PMID:23497680

Function and culture requirements of snow leopard (Panthera uncia) spermatozoa in vitro.

PubMed

Roth, T L; Howard, J G; Donoghue, A M; Swanson, W F; Wildt, D E

1994-08-01

Electroejaculates from eight snow leopards were used to determine how the motility of spermatozoa was influenced by (i) type of media (Ham's F10, PBS, human tubal fluid or RPMI-1640); (ii) holding temperature (23 degrees C versus 37 degrees C); (iii) washing of spermatozoa and (iv) a sperm metabolic enhancer, pentoxifylline. The duration of sperm motility was assessed by evaluating samples in each treatment every hour for 6 h and a sperm motility index (a value combining percentage sperm motility and rate of forward progression) calculated. Spermatozoa from the Ham's F10, PBS and PBS plus pentoxifylline treatments were also co-incubated with zona-intact, domestic cat eggs that were fixed and evaluated for spermatozoa bound to the zona pellucida, penetrating the outer and inner layers of the zona pellucida and within the perivitelline space. During the 6 h co-incubation, the sperm motility index in PBS with pentoxifylline was greater (P < 0.05) than in PBS alone which, in turn, was greater (P < 0.05) than in the other three test media. Washing the spermatozoa enhanced (P < 0.05) motility in both PBS and PBS plus pentoxifylline relative to unwashed samples, but there was no effect (P > 0.05) of holding temperature. Pentoxifylline supplementation enhanced (P < 0.05) the proportion of cat eggs with bound, but not penetrated, snow leopard spermatozoa in the inner layer of the zona pellucida, and there were no spermatozoa in the perivitelline space.(ABSTRACT TRUNCATED AT 250 WORDS)

Species variation in osmotic, cryoprotectant, and cooling rate tolerance in poultry, eagle, and Peregrine Falcon spermatozoa

USGS Publications Warehouse

Blanco, J.M.; Gee, G.; Wildt, D.E.; Donoghue, A.M.

2000-01-01

Potential factors influencing spermatozoa survival to cryopreservation and thawing were analyzed across a range of the following avian species: domestic chicken (Gallus domesticus), domestic turkey (Meleagris gallopavo), golden eagle (Aquila chrysaetos), Bonelli's eagle (Hieraaetus fasciatus), imperial eagle (Aquila adalberti), and peregrine falcon (Falco peregrinus). Studies focused on spermatozoa tolerance to the following: 1) osmotic stress, 2) different extracellular concentrations of the cryoprotectant dimethylacetamide (DMA), 3) equilibration times of 1 versus 4 h, 4) equilibration temperature of 4 versus 21 degrees C, and 5) rapid versus slow cooling before cryopreservation and standard thawing. Sperm viability was assessed with the live/dead stain (SYBR14/ propidium iodine). Sperm viability at osmolalities >/=800 mOsm was higher (P: /=2.06 M), experienced decreased (P: < 0.05) spermatozoa survival in all species, except the golden eagle and peregrine falcon. Number of surviving spermatozoa diminished progressively with increasing DMA concentrations in all species. Increased equilibration temperature (from 4 to 21 degrees C) markedly reduced (P: < 0.05) spermatozoa survival in all species except the Bonelli's eagle and turkey. Rapid cooling was detrimental (P: < 0.05) to spermatozoa from all species except the imperial eagle and the chicken. These results demonstrate that avian spermatozoa differ remarkably in response to osmotic changes, DMA concentrations, equilibration time, temperature, and survival after fast or slow freezing. These differences emphasize the need for species-specific studies in the development and enhancement of assisted breeding for poultry and endangered species.

Intraperitoneal insemination and retrograde sperm transport in dairy cows.

PubMed

López-Gatius, F; Yániz, J

2000-03-01

To examine the efficiency of retrograde sperm transport following intraperitoneal insemination, live and dead spermatozoa were used at different concentrations, and sperm recovery from cervical mucus (0.5 ml) 2, 6, 12 and 24 h following insemination was evaluated. Forty lactating Friesian cows, in their second to fourth lactation period, were used in this experiment. Thirty-six cows received intraperitoneally either live or dead spermatozoa. Each group of six cows received one of three total sperm numbers of 30, 45 and 90 million. Four cows were inseminated with 90 million spermatozoa into the uterus and served as a control group. All cows were inseminated towards the end of oestrus. After intrauterine insemination sperm recovery declined, but motile and/or immotile spermatozoa were recovered from all cows at any time. In cows inseminated intraperitoneally, sperm was recovered from the cervix at 6-24 h when 90 million were inseminated. A greater number of spermatozoa was recovered after dead rather than after live sperm inseminations. Only immotile, intact or broken spermatozoa and tail-less heads were recovered after intraperitioneal insemination using either live or dead spermatozoa. No sperm was recovered for 30 and 45 million inseminations. Our results show that, following intraperitoneal insemination, there is passive sperm transport from the peritoneal cavity to the genital tract close to the time of ovulation, and suggest a higher sperm retention in the genital tract when live as opposed to dead spermatozoa are used.

RNA PROFILES IN RAT AND MOUSE EPIDIDYMAL SPERMATOZOA

EPA Science Inventory

RNA PROFILES IN RAT AND MOUSE EPIDIDYMAL SPERMATOZOA
Kary E. Thompson1, Hongzu Ren1, Judith E. Schmid1 and David J. Dix1
1Reproductive Toxicology Division, NHEERL, ORD, U.S. Environmental Protection Agency, RTP, NC.

Mature spermatozoa are transcriptionally inactive...

Effects of radiocobalt irradiation of unfertilized or fertilized rabbit OVA in vitro on subsequent fertilization and development in vivo

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

Chang, M C; Hunt, D M; Romanoff, E B

Freshly shed rabbit ova recovered from the Fallopian tubes were irradiated with a radiocobalt source at 45 r to 32,000 r and then transplanted into the left tube of mated rabbits. The ova were recovered and examined microscopically 10 hours to 24 hours, two days and 6 days after transplantation for the determination of fertilization, cleavage, and blastocyst formation. The fetus and uterine contents were examined macroscopically 22 to 25 days after transplantation. The proportion of fertilized ova decreased from 71% at a very low dosage, 45 r, to 46% at a very high dosage, 32,000 r. The proportion ofmore » normally cleaved ova and normal lilastocysts decreased from about 95% at 45 r, to about 3% at 500 r, to 0% at 6,500 r. The proportion of embryonic development decreased from 49% at 45 r, to 21% at 90 r, to 0% at 800 r. A chromosomal bridge was observed in an ovum irradiated at 6,500 r. Failure of second polar body division in one out of 23 ova irradiated at 6,500 r and polyspermy in one out of 32 ova irradiated at 32,000 r before fertilization was observed. When fertilized rabbit ova at the two cell stage were irradiated at 45 r to 6,500 r and examined at various times after transplantation, it was found that the proportion of normal cleavage decreased from 78% at 45 r to 33% at 800 r, to 0% at 6,500 r. The proportion of normal blastocysts decreased from 61% at 45 r, to 20% at 800 r, to 0% at 6,500 r. The proportion of normal embryonic development decreased from 46% at 45 r, to 12% at 500 r, to 0% at 6,500 r. In combination with data from a previous study of the irraiation of rabbit sper matozoa in vitro the following points are revealed: No abnormal fetus, no high proportion of degeneration after implantation, and no disturbance of the sex ratio were observed whether spermatozoa, or ova, unfertilized, or fertilized, were irradiated from 45 r to 800 r. Although there may be a differential sensitivity to various dosages for the subsequent cleavage and blastocyst formation following the irradiation of spermatozoa, unfertilized or fertilized ova at 45 r to 6,500 r, as far as subsequent embryonic development is concerned, the spermatozoa are more radioresistant than either unfertilized or fertilized ova and the unfertilized ova are more radiosensitive than fertilized ova. The chemical constituents of gametes necessary for the future development of the zygotes are more radiosensitive than are those for their fertilization and other activities.« less

Sperm chromatin stability in frozen-thawed semen is maintained over age in AI bulls.

PubMed

Hallap, Triin; Nagy, Szabolcs; Håård, Margareta; Jaakma, Ulle; Johannisson, Anders; Rodriguez-Martinez, Heriberto

2005-04-01

The aim of the present study was to investigate the effect of age of the sire on the in vitro quality of frozen-thawed (FT) bull spermatozoa, both when tested immediately postthaw (PT) and when assessed after cleansing and selection through a swim-up (SU) procedure. Semen samples from six Swedish Red and White Breed (SRB) artificial insemination (AI) bulls at age 1 and again, at 4 years were collected and frozen in 0.25 ml plastic straws. Also, semen was collected from six Estonian Holstein (EHF) bulls at the ages of 3, 5, and 7 years and likewise processed. The FT semen was tested for the susceptibility of sperm nuclear deoxyribonucleic acid (DNA) to undergo acid-induced denaturation in situ, as quantified by flow cytometry (FCM). The DNA denaturability was expressed as function alpha t, i.e., as the ratio of red (denaturated DNA) to red + green (total cellular DNA) fluorescence intensity. The results were expressed as the percentage of cells with high alpha t values, i.e., cells outside the main population (% COMP alpha t). Morphological evaluation of the same samples was performed to detect general and sperm head abnormalities and differences between ages. Fertility results were available as non-return rates (NRRs) for the semen of the sires when they were 1 year (SRB) and 3 years (EHF) old, varying from 62.2 to 70.7% in SRB and from 52.2 to 76.0% in EHF animals. The COMP alpha t values ranged from 0.5-3.6% (PT) to 0.2-1.7% (SU) for SRB bulls and from 0.4-1.8% (PT) to 0.2-1.5% (SU) for EHF bulls. Both breeds lacked differences between ages, either PT or after SU. However, the SU procedure yielded a significantly higher population of spermatozoa with stable DNA following acid-induced denaturation, than PT samples (p < 0.001). No correlation was detected between field fertility and chromatin stability. The results indicate that for these bull populations, the SU procedure was able to select spermatozoa with stable chromatin from the bulk samples. However, the use of DNA denaturation as a challenge to assess sperm chromatin stability did not offer a more accurate tool to evaluate sperm quality than the conventional, light microscopical evaluation of morphology.

Cloned cows with short telomeres deliver healthy offspring with normal-length telomeres.

PubMed

Miyashita, Norikazu; Kubo, Yasuaki; Yonai, Miharu; Kaneyama, Kanako; Saito, Norio; Sawai, Ken; Minamihashi, Akira; Suzuki, Toshiyuki; Kojima, Toshiyuki; Nagai, Takashi

2011-10-01

Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres.

The hOGG1 Ser326Cys polymorphism and male subfertility in Taiwanese patients with varicocele.

PubMed

Chen, S S-S; Chiu, L-P

2018-03-26

To investigate the association between the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene Ser326Cys polymorphism and male subfertility in Taiwanese patients with varicocele, we made a prospective study. Ninety young male patients with varicocele (group 1), 50 young male patients with subclinical varicocele (group 2) and 30 normal young male patients without varicocele (group 3) were recruited in this study. The hOGG1 null homozygous genotype (Cys/Cys) and the occurrence of a 4,977-bp deletion in mitochondrial DNA and mitochondrial copy number in spermatozoa were determined by polymerase chain reaction. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) content of DNA in the spermatozoa was measured using high-performance liquid chromatography, and total antioxidant capacity (TAC) of seminal plasma was detected electrochemically. The rates of male subfertility were 31.1% (28/90) in group 1 and 22% (11/50) in group 2. Of 39 subfertile men, 74.4% (29/39) had the hOGG1 Cys/Cys genotype. Patients in groups 1 and 2 with hOGG1 Cys/Cys genotype had significantly higher 8-OHdG content in sperm DNA, lower mitochondrial copy number in spermatozoa and lower TAC in seminal plasma than those with Ser/Ser or Ser/Cys genotype. Clinicians should pay more attention to patients with varicocele with the hOGG1 Cys/Cys genotype. © 2018 Blackwell Verlag GmbH.

Spermicidal efficacy of VRP, a synthetic cationic antimicrobial peptide, inducing apoptosis and membrane disruption.

PubMed

Ghosh, Prasanta; Bhoumik, Arpita; Saha, Sudipta; Mukherjee, Sandipan; Azmi, Sarfuddin; Ghosh, Jimut K; Dungdung, Sandhya R

2018-02-01

Presently available contraceptives are mostly hormonal or detergent in nature with numerous side effects like irritation, lesion, inflammation in vagina, alteration of body homeostasis, etc. Antimicrobial peptides with spermicidal activity but without adverse effects may be suitable alternatives. In the present study, spermicidal activity of a cationic antimicrobial peptide VRP on human spermatozoa has been elucidated. Progressive forward motility of human spermatozoa was instantly stopped after 100 μM VRP treatment and at 350 μM, all kinds of sperm motility ceased within 20 s as assessed by the Sander-Cramer assay. The spermicidal effect was confirmed by eosin-nigrosin assay and HOS test. VRP treatment (100 μM) in human spermatozoa induced both the intrinsic and extrinsic pathways of apoptosis. TUNEL assay showed VRP treatment significantly disrupted the DNA integrity and changed the mitochondrial membrane permeability as evident from MPTP assay. AFM and SEM results depicted ultra structural changes including disruption of the acrosomal cap and plasma membrane of the head and midpiece region after treatment with 350 μM VRP. MTT assay showed after treatments with 100 and 350 μM of VRP for 24 hr, a substantial amount of Lactobacillus acidophilus (about 90% and 75%, respectively) remained viable. Hence, VRP being a small synthetic peptide with antimicrobial and spermicidal activity but tolerable to normal vaginal microflora, may be a suitable target for elucidating its contraceptive potentiality. © 2017 Wiley Periodicals, Inc.

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding.

PubMed

Plaza Dávila, M; Bucci, D; Galeati, G; Peña, F J; Mari, G; Giaretta, E; Tamanini, C; Spinaci, M

2015-12-01

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 μm) and EGCG (10, 20 and 60 μm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 μm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone. © 2015 Blackwell Verlag GmbH.

Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa

USDA-ARS?s Scientific Manuscript database

Various obstacles are encountered by mammalian spermatozoa during their journey through the female genital tract, and only few or none will reach the site of fertilization. Currently, there are limited technical approaches for non-invasive investigation of spermatozoa migration after insemination. A...

Assessment of reproductive tissues of gilts born from magnetic nanoparticles-treated boar spermatozoa

USDA-ARS?s Scientific Manuscript database

Semen ejaculates contain a heterogeneous population of spermatozoa that may interfere with male fertility. Indeed, poor quality semen generally translates into low fertility rates that are attributed to higher proportions of damaged or abnormal spermatozoa in the ejaculates. It is likely that their ...

Improved exogenous DNA uptake in bovine spermatozoa and gene expression in embryos using membrane destabilizing agents in ICSI-SMGT.

PubMed

Sánchez-Villalba, Esther; Arias, María Elena; Zambrano, Fabiola; Loren, Pía; Felmer, Ricardo

2018-02-01

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

COMPARATIVE ANALYSIS OF MAMMALIAN SPERM MOTILITY

PubMed Central

Phillips, David M.

1972-01-01

Spermatozoa of several mammalian species were studied by means of high-speed cinematography and electron microscopy. Three types of motile patterns were observed in mouse spermatozoa. The first type involved an asymmetrical beat which seemed to propel the sperm in circular paths. The second type involved rotation of the sperm and appeared to allow them to maintain straight paths. In the third type of pattern, the sperm appeared to move by crawling on surfaces in a snakelike manner. Spermatozoa of rabbit and Chinese hamster also had an asymmetrical beat which sometimes caused them to swim in circles. In spite of the asymmetry of the beat, these spermatozoa were also able to swim in straight paths by rotating around a central axis as they swam. Spermatozoa of some species appeared very flexible; their flagella formed arcs with a very small radius of curvature as they beat. Spermatozoa of other species appeared very stiff, and their flagella formed arcs with a very large radius of curvature. The stiffness of the spermatozoan appeared to correlate positively with the cross-sectional area of the dense fibers. This suggests that the dense fibers may be stiff elastic elements. Opossum sperm become paired as they pass through the epididymis. Pairs of opossum spermatozoa beat in a coordinated, alternating manner. PMID:5025110

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.

Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm(®) gradient.

PubMed

Dorado, J; Alcaráz, L; Duarte, N; Portero, J M; Acha, D; Hidalgo, M

2011-05-01

The aims of the present study were to: (1) determine if discrete motile sperm subpopulations exist and their incidence in fresh dog ejaculates, (2) evaluate the effects of cryopreservation on the distribution of spermatozoa within the different subpopulations, and (3) determine the effect of the discontinuous PureSperm(®) gradient on the sperm subpopulation structure of frozen-thawed dog spermatozoa. Semen from 5 dogs were collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm(®). Sperm motility (assessed by computerized-assisted semen analysis, CASA) was assessed before freezing, just after thawing and after preparation on the PureSperm(®) gradients. Cryopreservation had a significant (P<0.001) effect on CASA-derived parameters. PureSperm(®) centrifugation yielded sperm suspensions with improved motility (P<0.01). A multivariate clustering procedure separated 19414 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (20.97%), Subpopulation 2 consisting of slow and low-linear spermatozoa (18.24%), Subpopulation 3 consisting of highly active but non-progressive spermatozoa (20.75%), and Subpopulation 4 consisting of high speed and progressive spermatozoa (40.03%). Although, cryopreservation had a significant (P<0.001) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after freezing and thawing. The selected sperm samples was enrich in Subpopulation 4, reaching a proportion of 31.9% of the present spermatozoa, in contrast with the unselected sperm samples, where this sperm subpopulation accounted for 24.9% of the total. From these results, we concluded that four well-defined motile sperm subpopulations were present either in fresh semen, in unselected sperm samples or in selected preparations from dogs. The discontinuous PureSperm(®) gradient is a simple method to improve the quality of canine frozen-thawed semen samples, since Subpopulation 4 (high-speed and progressive spermatozoa) was more frequently observed after preparation on the gradient. Finally, this study also demonstrated that the general motile sperm structure present in dog remains constant despite the effect caused by either cryopreservation or separation on PureSperm(®) gradient. Copyright © 2011 Elsevier B.V. All rights reserved.

Number of spermatozoa in the crypts of the sperm reservoir at about 24 h after a low-dose intrauterine and deep intrauterine insemination in sows.

PubMed

Tummaruk, P; Tienthai, P

2010-04-01

The aim of this study was to investigate the number of spermatozoa in the crypts of the utero-tubal junction (UTJ) and the oviduct of sows approximately 24 h after intrauterine insemination (IUI) and deep intrauterine insemination (DIUI) and compared with that of conventional artificial insemination (AI). Fifteen crossbred Landrace x Yorkshire (LY) multiparous sows were used in the experiment. Transrectal ultrasonography was performed every 4 h to examine the time of ovulation in relation to oestrous behaviour. The sows were inseminated with a single dose of diluted fresh semen by the AI (n = 5), IUI (n = 5) and DIUI (n = 5) at approximately 6-8 h prior to the expected time of ovulation, during the second oestrus after weaning. The sperm dose contained 3000 x 10(6) spermatozoa in 100 ml for AI, 1,000 x 10(6) spermatozoa in 50 ml for IUI and 150 x 10(6) spermatozoa in 5 ml for DIUI. The sows were anaesthetized and ovario-hysterectomized approximately 24 h after insemination. The oviducts and the proximal part of the uterine horns (1 cm) on each side of the reproductive tracts were collected. The section was divided into four parts, i.e. UTJ, caudal isthmus, cranial isthmus and ampulla. The spermatozoa in the lumen in each part were flushed several times with phosphate buffer solution. After flushing, the UTJ and all parts of the oviducts were immersed in a 10% neutral buffered formalin solution. The UTJ and each part of the oviducts were cut into four equal parts and embedded in a paraffin block. The tissue sections were transversely sectioned to a thickness of 5 mum. Every fifth serial section was mounted and stained with haematoxylin and eosin. The total number of spermatozoa from 32 sections in each parts of the tissue (16 sections from the left side and 16 sections from the right side) was determined under light microscope. The results reveal that most of the spermatozoa in the histological section were located in groups in the epithelial crypts. The means of the total number of spermatozoa in the sperm reservoir (UTJ and caudal isthmus) were 2296, 729 and 22 cells in AI, IUI and DIUI groups, respectively (p < 0.01). The spermatozoa were found on both sides of the sperm reservoir in all sows in the AI and the IUI groups. For the DIUI group, spermatozoa were not found on any side of the sperm reservoir in three out of five sows, found in unilateral side of the sperm reservoir in one sow and found in both sides of the sperm reservoir in one sow. No spermatozoa were found in the cranial isthmus, while only one spermatozoon was found in the ampulla part of a sow in the IUI group. In conclusion, DIUI resulted in a significantly lower number of spermatozoa in the sperm reservoir approximately 24 h after insemination compared with AI and IUI. Spermatozoa could be obtained from both sides of the sperm reservoir after AI and IUI but in one out of five sows inseminated by DIUI.

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.

Osmotic characteristics and fertility of murine spermatozoa collected in different solutions.

PubMed

Si, Wei; Men, Hongsheng; Benson, James D; Critser, John K

2009-02-01

Osmotic stress is an important factor that can result in cell damage during cryopreservation. Before ejaculation or collection for cryopreservation, murine spermatozoa are stored in epididymal fluid, a physiologically hyperosmotic environment (approximately 415 mmol/kg). The objectives of this study were to determine the osmotic tolerance limits of sperm motion parameters of ICR and C57BL/6 mouse spermatozoa collected in isosmotic (290 mmol/kg) and hyperosmotic (415 mmol/kg) media, and the effect of the osmolality of sperm collection media on sperm fertility after cryopreservation. Our results indicate that murine spermatozoa collected in media with different osmolalities (290 and 415 mmol/kg Dulbecco's phosphate buffered saline (DPBS)) appeared to have different osmotic tolerances for the maintenance of sperm motility and other motion parameters in both mouse strains. The hypo- and hyperosmotic treatments decreased motility and affected other motion parameters of spermatozoa collected in 290 mmol/kg DPBS. The extent of the change of motion parameters after treatments corresponded with the levels of osmotic stress. However, for spermatozoa collected in 415 mmol/kg DPBS, exposure to 290 mmol/kg DPBS tended to increase sperm motility and the quality of their motion parameters. The osmolality of sperm collection medium can affect murine sperm fertility. Spermatozoa collected in 415 mmol/kg medium showed higher fertility compared with spermatozoa collected in 290 mmol/kg as assessed by IVF. Results characterizing murine sperm osmotic tolerance collected in media with different osmolalities from different strains and the effect of collection media osmolality on sperm fertility after cryopreservation will be useful in designing cryopreservation protocols.

Live births from artificial insemination of microfluidic-sorted bovine spermatozoa characterized by trajectories correlated with fertility

PubMed Central

Nagata, Maria Portia B.; Endo, Kenji; Ogata, Kazuko; Yamanaka, Kenichi; Egashira, Junki; Katafuchi, Naoto; Yamanouchi, Tadayuki; Matsuda, Hideo; Goto, Yuki; Sakatani, Miki; Hojo, Takuo; Nishizono, Hirofumi; Yotsushima, Kenji; Takenouchi, Naoki; Hashiyada, Yutaka; Yamashita, Kenichi

2018-01-01

Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans. PMID:29555773

Dietary exposure to aflatoxin in human male infertility in Benin City, Nigeria.

PubMed

Ibeh, I N; Uraih, N; Ogonar, J I

1994-01-01

To discover the relationship between aflatoxin levels, if any, in serum of infertile men in comparison with random controls from the community. In a parallel experiment, adult male rats were given an aflatoxin-contaminated diet. 100 adult males, yielding 50 semen samples, from men attending Infertility Clinics at a university teaching hospital and 50 normal men in the same community. The staple foods of the men were assayed for aflatoxin content. The rats were given the aflatoxin-rich diet, and their spermatozoa were examined and their ability to reproduce assessed. A random sampling of semen from 100 adult males comprising 50 samples drawn from infertile men and 50 drawn from normal individuals within the same community revealed the presence of aflatoxins in 20 semen samples from the infertile group (40.0%) and four samples from the fertile group (8.0%). The mean aflatoxin concentrations were 1.660 +/- 0.04 micrograms/mL (infertile men) and 1.041 +/- 0.01 micrograms/mL (fertile men). Infertile men with aflatoxin in their semen showed a higher percentage of spermatozoal abnormality (50.0%) than the fertile men (10.0-15.0%). Dietary exposure of adult male Albino rats to aflatoxin (8.5 micrograms AF1/g of Guinea growers feed for 14 days) produced deleterious effects on the spermatozoa of the affected rats, producing features that resemble those seen in semen of infertile men exposed to aflatoxin.

Physiology of spermatozoa at high dilution rates: the influence of seminal plasma.

PubMed

Maxwell, W M; Johnson, L A

1999-12-01

Extensive dilution of spermatozoa, as occurs during flow-cytometric sperm sorting, can reduce their motility and viability. These effects may be minimized by the use of appropriate dilution and collection media, containing balanced salts, energy sources, egg yolk and some protein. Dilution and flow-cytometric sorting of spermatozoa, which involves the removal of seminal plasma, also destabilizes sperm membranes leading to functional capacitation. This membrane destabilization renders the spermatozoa immediately capable of fertilization in vitro, or in vivo after deposition close to the site of fertilization, but shortens their lifespan, resulting in premature death if the cells are deposited in the female tract distant from the site of fertilization or are held in vitro at standard storage temperatures. This functional capacitation can be reversed in boar spermatozoa by inclusion of seminal plasma in the medium used to collect the cells from the cell sorter and, consequently, reduces their in vitro fertility. It has yet to be determined whether seminal plasma would have similar effects on flow cytometrically sorted spermatozoa of other species, and what its effects might be on the in vivo fertility of flow sorted boar.

Influence of environmentally relevant concentrations of vinclozolin on quality, DNA integrity, and antioxidant responses of sterlet Acipenser ruthenus spermatozoa.

PubMed

Gazo, Ievgeniia; Linhartova, Pavla; Shaliutina, Anna; Hulak, Martin

2013-04-25

The effects of vinclozolin (VIN), an anti-androgenic fungicide, on quality, oxidative stress, DNA integrity, and ATP level of sterlet (Acipenser ruthenus) spermatozoa were investigated in vitro. Fish spermatozoa were incubated with different concentrations of vinclozolin (0.5, 2, 10, 15, 20 and 50 μg/l) for 2 h. A dose-dependent reduction in spermatozoa motility and velocity was observed at concentrations of 2-50 μg/l. A dramatic increase in DNA fragmentation was recorded at concentrations 10 μg/l and above. After 2 h exposure at higher test concentrations (10-50 μg/l), oxidative stress was apparent, as reflected by significantly higher levels of protein and lipid oxidation and significantly greater superoxide dismutase activity. Intracellular ATP content of spermatozoa decreased with increasing concentrations of VIN. The results demonstrated that VIN can induce reactive oxygen species stress in fish spermatozoa, which could impair the sperm quality, DNA integrity, ATP content, and the antioxidant defense system. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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.

Sperm transport and retention at the fertilization site is orchestrated by a chemical guidance and oviduct movement.

PubMed

Guidobaldi, H A; Teves, M E; Uñates, D R; Giojalas, L C

2012-05-01

In mammals, only a few spermatozoa arrive at the fertilization site. During the last step in the journey to the egg, apart from their self-propulsion, spermatozoa may be assisted by oviduct movement and/or a guidance mechanism. The proportion of rabbit spermatozoa that arrive at the fertilization site was determined under in vivo conditions, in which either the ovulation products (secreting chemoattractants) and/or the oviduct movement (causing the displacement of the oviductal fluid) was inhibited. When only one of these components was inhibited, sperm transport to the fertilization site was partially reduced. However, when both the ovulation products and the oviduct movement were inhibited, almost no spermatozoa arrived at the fertilization site. The results suggest that spermatozoa are transported to and retained at the fertilization site by the combined action of a chemical guidance and the oviduct movement. A working model is proposed to explain how these two mechanisms may operate to transport spermatozoa to the fertilization site, probably as an evolutionary adaptation to maximize the chance of fertilizing an egg.

Sneaker Male Squid Produce Long-lived Spermatozoa by Modulating Their Energy Metabolism.

PubMed

Hirohashi, Noritaka; Tamura-Nakano, Miwa; Nakaya, Fumio; Iida, Tomohiro; Iwata, Yoko

2016-09-09

Spermatozoa released by males should remain viable until fertilization. Hence, sperm longevity is governed by intrinsic and environmental factors in accordance with the male mating strategy. However, whether intraspecific variation of insemination modes can impact sperm longevity remains to be elucidated. In the squid Heterololigo bleekeri, male dimorphism (consort and sneaker) is linked to two discontinuous insemination modes that differ in place and time. Notably, only sneaker male spermatozoa inseminated long before egg spawning can be stored in the seminal receptacle. We found that sneaker spermatozoa exhibited greater persistence in fertilization competence and flagellar motility than consort ones because of a larger amount of flagellar glycogen. Sneaker spermatozoa also showed higher capacities in glucose uptake and lactate efflux. Lactic acidosis was considered to stabilize CO2-triggered self-clustering of sneaker spermatozoa, thus establishing hypoxia-induced metabolic changes and sperm survival. These results, together with comparative omics analyses, suggest that postcopulatory reproductive contexts define sperm longevity by modulating the inherent energy levels and metabolic pathways. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Spectral interferometry for morphological imaging in in vitro fertilization (IVF) (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhu, Yizheng; Li, Chengshuai

2016-03-01

Morphological assessment of spermatozoa is of critical importance for in vitro fertilization (IVF), especially intracytoplasmic sperm injection (ICSI)-based IVF. In ICSI, a single sperm cell is selected and injected into an egg to achieve fertilization. The quality of the sperm cell is found to be highly correlated to IVF success. Sperm morphology, such as shape, head birefringence and motility, among others, are typically evaluated under a microscope. Current observation relies on conventional techniques such as differential interference contrast microscopy and polarized light microscopy. Their qualitative nature, however, limits the ability to provide accurate quantitative analysis. Here, we demonstrate quantitative morphological measurement of sperm cells using two types of spectral interferometric techniques, namely spectral modulation interferometry and spectral multiplexing interferometry. Both are based on spectral-domain low coherence interferometry, which is known for its exquisite phase determination ability. While spectral modulation interferometry encodes sample phase in a single spectrum, spectral multiplexing interferometry does so for sample birefringence. Therefore they are capable of highly sensitive phase and birefringence imaging. These features suit well in the imaging of live sperm cells, which are small, dynamic objects with only low to moderate levels of phase and birefringence contrast. We will introduce the operation of both techniques and demonstrate their application to measuring the phase and birefringence morphology of sperm cells.

Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution.

PubMed

Centurion, Fernando; Vazquez, Juan M; Calvete, Juan J; Roca, Jordi; Sanz, Libia; Parrilla, Inmaculada; Garcia, Eva M; Martinez, Emilio A

2003-08-01

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300,000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38 degrees C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.

Sexual behavior, reproductive physiology and sperm competition in male mammals.

PubMed

Dixson, Alan F; Anderson, Matthew J

2004-11-15

Sperm competition involves competition between the gametes of two or more males of a species for fertilization of a given set of ova. Sperm competition is widespread among mammals, as in many other groups of vertebrates. Effects of sexual selection, via sperm competition, upon the evolution of reproductive physiology and behavior are much better understood in invertebrates (and especially in insects) than is the case for mammals. However, if the reproductive organs of male mammals are viewed as an integrated system for production and delivery of spermatozoa (and accessory glandular secretions) to females, then it is logical to assume that sperm competition might influence the evolution of all parts of the system, as well as associated physiological mechanisms (e.g., testicular endocrinology) and behavior (e.g., copulatory patterns). Here we analyze and review relationships between mating systems, relative testes sizes and sperm morphology, phallic morphology, circulating testosterone levels and sexual behavior in male mammals.

Murine sperm capacitation, oocyte penetration and decondensation following moderate alcohol intake.

PubMed

Sánchez, Melisa C; Fontana, Vanina A; Galotto, Camila; Cambiasso, Maite Y; Sobarzo, Cristian M A; Calvo, Lucrecia; Calvo, Juan C; Cebral, Elisa

2018-06-01

Male chronic alcohol abuse causes testicular failure and infertility. We analyzed the effects of moderate sub-chronic alcohol intake on sperm morphology, capacitation, fertilization and sperm head decondensation. CF-1 male mice were administered 15% ethanol in drinking water for 15 days; control mice received ethanol-free water. Similar patterns of tyrosine phosphorylation were observed in capacitated spermatozoa of control and treated males. Percentage of hyperactivation (H) and spontaneous (SAR) and progesterone-induced (IAR) acrosome reaction significantly decreased at 120 and 150 min of capacitation in treated males compared to controls (H: 14.1 ± 2.5 vs 23.7 ± 2.6, P  < 0.05; SAR-T120 min: 17.9 ± 2.5 vs 32.9 ± 4.1, P  < 0.01; IAR-150 min: 43.3 ± 3.5 vs 73.1 ± 1.1, P  < 0.001, n  = 6). During in vitro fertilization (2.5, 3.5 and 4.5 h post-insemination), there was an increased percentage of fertilized oocytes (with a decondensed sperm head and one or two pronuclei) in treated males ( P  < 0.001, n  = 7). After 60 min of in vitro decondensation with glutathione plus heparin, the percentage of decondensed sperm heads was significantly higher in treated males than in controls (mean ± s.d.: 57.1 ± 5.6 vs 48.3 ± 4.5, P  < 0.05, n  = 5). The percentage of morphologically normal sperm heads was significantly decreased in treated males with respect to controls ( P  < 0.001, n  = 9). These results show that short-term moderate alcohol consumption in outbred mice affect sperm morphology, hyperactivation, acrosomal exocytosis, and the dynamics of in vitro fertilization and in vitro sperm nuclear decondensation. © 2018 Society for Reproduction and Fertility.

Distribution profiles of transient receptor potential melastatin- and vanilloid-related channels in rat spermatogenic cells and sperm.

PubMed

Li, Shilin; Wang, Xinghuan; Ye, Haixia; Gao, Weicheng; Pu, Xiaoyong; Yang, Zhonghua

2010-03-01

In the present study, we aimed to investigate the expression and distribution of transient receptor potential melastatin (TRPM)- and vanilloid (TRPV)- related channels in rat spermatogenic cells and spermatozoa. Spermatogenic cells and spermatozoa were obtained from male Sprague-Dawley rats. Reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of all TRPM and TRPV channel members with specific primers. Western blot analysis was applied for detecting the expression of TRPM and TRPV channel proteins. Immunohistochemistry staining for TRPM4, TRPM7 and TRPV5 was also performed in rat testis. The mRNAs of TRPM3, TRPM4, TRPM7 and TRPV5 were detected in the spermatogenic cells and spermatozoa in rat. Western blot analysis verified the expression of TRPM4, TRPM7 and TRPV5 in the rat spermatogenic cells and spermatozoa. Immunocytochemistry staining for TRPM and TRPV channel families indicated that TRPM4 and TRPM7 proteins were highly expressed in different stages of spermatogenic cells and spermatozoa, while TRPV5 protein was lowly expressed in these cells. Our results demonstrate that mRNAs or proteins for TRPM3, TRPM4, TRPM7 and TRPV5 exist in rat spermatogenic cells and spermatozoa. These data presented here may assist in elucidating the possible physiological function of TRPM and TRPV channels in spermatogenic cells and spermatozoa.

pH controls spermatozoa motility in the Pacific oyster (Crassostrea gigas)

PubMed Central

Suquet, Marc; Malo, Florent; Queau, Isabelle; Pignet, Patricia; Ratiskol, Dominique; Le Grand, Jacqueline; Huber, Matthias; Cosson, Jacky

2018-01-01

ABSTRACT Investigating the roles of chemical factors stimulating and inhibiting sperm motility is required to understand the mechanisms of spermatozoa movement. In this study, we described the composition of the seminal fluid (osmotic pressure, pH, and ions) and investigated the roles of these factors and salinity in initiating spermatozoa movement in the Pacific oyster, Crassostrea gigas. The acidic pH of the gonad (5.82±0.22) maintained sperm in the quiescent stage and initiation of flagellar movement was triggered by a sudden increase of spermatozoa external pH (pHe) when released in seawater (SW). At pH 6.4, percentage of motile spermatozoa was three times higher when they were activated in SW containing 30 mM NH4Cl, which alkalinizes internal pH (pHi) of spermatozoa, compared to NH4Cl-free SW, revealing the role of pHi in triggering sperm movement. Percentage of motile spermatozoa activated in Na+-free artificial seawater (ASW) was highly reduced compared to ASW, suggesting that change of pHi triggering sperm motility was mediated by a Na+/H+ exchanger. Motility and swimming speed were highest in salinities between 33.8 and 42.7‰ (within a range of 0 to 50 ‰), and pH values above 7.5 (within a range of 4.5 to 9.5). PMID:29483075

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification.

PubMed

Pradiee, J; Esteso, M C; Castaño, C; Toledano-Díaz, A; Lopez-Sebastián, A; Guerra, R; Santiago-Moreno, J

2017-04-01

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre-freezing equilibration times (2-3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60-85°C min -1 ), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified-warmed sperm variables were at their best when the spermatozoa was diluted in TCG-6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing. © 2016 Blackwell Verlag GmbH.

Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa.

PubMed

Ebner, T; Shebl, O; Moser, M; Mayer, R B; Arzt, W; Tews, G

2011-01-01

Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0–42.1%). Density gradient did not significantly improve the quality of spermatozoa selected(14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and,with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report ona sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.

PubMed

Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar; Henning, Heiko

2016-11-15

Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. © 2016. Published by The Company of Biologists Ltd.

Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation.

PubMed

García-Álvarez, Olga; Maroto-Morales, Alejandro; Ramón, Manuel; del Olmo, Enrique; Jiménez-Rabadán, Pilar; Fernández-Santos, M Rocio; Anel-López, Luis; Garde, J Julián; Soler, Ana J

2014-06-01

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm samples were incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. A significantly greater increase (P≤0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as 'hyperactivated like', with major changes occurring after 1h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place.

Effect of a pre-freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics.

PubMed

Tomás, C; Blanch, E; Fazeli, A; Mocé, E

2013-01-01

The aim of this work was to examine how a pre-freezing treatment with cholesterol-loaded cyclodextrins (CLC) affects boar sperm longevity, capacitation dynamics, ability to bind to a porcine telomerase-immortalised oviductal epithelial cell line (TERT-OPEC) in vitro and DNA integrity dynamics after freeze-thawing. Although the samples treated with CLC exhibited lower sperm quality than the control samples (P<0.05) immediately after thawing, these differences disappeared (P>0.05) after long-term incubation (26h at 37 or 16°C). Additionally, the CLC-treated spermatozoa underwent similar capacitation and DNA fragmentation dynamics as the control spermatozoa (P>0.05). However, CLC-treated spermatozoa were better able to bind to TERT-OPEC in vitro (P<0.0001). In conclusion, the pre-freezing treatment of boar spermatozoa with CLC enhanced the ability of the spermatozoa to bind to TERT-OPEC in vitro, which could have an effect on the establishment of the sperm reservoir in the ampullary--isthmic junction in vivo. Additionally, frozen-thawed spermatozoa can be stored at 16°C for at least 6h without a significant observable decline in sperm quality, which could be beneficial for the transport of thawed diluted doses of spermatozoa from the laboratory to the farm.

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

PubMed Central

Nguyen, Quynh Thu; Wallner, Ulrike; Schmicke, Marion; Waberski, Dagmar

2016-01-01

ABSTRACT Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa. PMID:27612509

Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action

PubMed Central

Zhu, Zhendong; Fan, Xiaoteng; Lv, Yinghua; Zhang, Nan; Fan, Chuning; Zhang, Pengfei; Zeng, Wenxian

2015-01-01

The process of cryopreservation results in high concentration of reactive oxygen species which is detrimental to spermatozoa. The aim of this study was to investigate whether addition of vitamin E analogue to freezing extender can facilitate the cryosurvival of spermatozoa in rabbits, and how vitamin E protects spermatozoa against damages during the process of preservation. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of Trolox (a vitamin E analogue). The level of radical oxygen species (ROS) in spermatozoa that was exposed to Trolox was significantly lower than that of the control during each step of the process of preservation. The percentage of frozen-thawed spermatozoa with lipid peroxidation in the Trolox treatments was significantly lower than that of the control. The motility, intact acrosome, membrane integrity and mitochondrial potentials of the frozen-thawed spermatozoa in the treatment of 200 μM Trolox were significantly higher than those of the control. These observations suggest that addition of vitamin E to a freezing extender leads to higher integrity of acrosome, plasma membrane and mitochondrial membrane potential as well as higher motility. Vitamin E protects spermatozoa through its capacity to quench ROS accumulation and lipid peroxidation during the process of preservation. Addition of Trolox is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry. PMID:26700473

Sequelae in male rabbits following developmental exposure to p,p'-DDT or a mixture of p,p'-DDT and vinclozolin: cryptorchidism, germ cell atypia, and sexual dysfunction.

PubMed

Veeramachaneni, D N R; Palmer, J S; Amann, R P; Pau, K-Y F

2007-01-01

Rabbit does (7-9 per group) were treated daily per orum from gestation day 15 through post-natal week 4 to provide per kg body wt 25 micaromol (low) or 250 micromol (high) p,p'-DDT or a mixture of DDT and vinclozolin (12.5 and 125 micromol each). Developmental as well as post-pubertal reproductive sequelae of male progeny were studied. Testicular descent in some pups was impaired by DDT. Serum LH or testosterone was not affected. FSH was lower in mixture- but not in DDT-exposed rabbits. Lack of sexual interest, penile erection and ejaculation were observed in some mixture rabbits. Sperm counts were unaffected, but morphologically normal spermatozoa were fewer; nuclear and acrosomal morphogenesis was disrupted. Atypical germ cells resembling carcinoma in situ were found. Also considering data for vinclozolin [Veeramachaneni DNR, Palmer JS, Amann RP, Kane CM, Higuchi TT, Pau K-YF. Disruption of sexual function, FSH secretion, and spermiogenesis in rabbits following developmental exposure to vinclozolin, a fungicide. Reproduction 2006;131:805-16], we concluded that DDT causes cryptorchidism and germ cell atypia, vinclozolin permanently disrupts FSH secretion and sexual function, and the mixture causes the full spectrum of dysgenesis.

Spermatozoa Production by Triploid Males in the New Zealand Freshwater Snail Potamopyrgus antipodarum

PubMed Central

Soper, D.M.; Neiman, M.; Savytskyy, O.P.; Zolan, M.E.; Lively, C.M.

2013-01-01

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum 1) have testes that produce morphologically normal sperm, 2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and 3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I likely accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. PMID:24307744

Semen parameters and level of microsatellite heterozygosity in Noriker draught horse stallions.

PubMed

Aurich, Christine; Achmann, Roland; Aurich, Jörg E

2003-07-01

It was the aim of the present study to determine physiological values for different semen parameters in an endangered draught horse breed, the Austrian Noriker. Because small population size is often believed to cause a decrease in fertility and/or semen quality through inbreeding and a reduction in genetic variation, the general genomic heterogeneity of the breed was estimated on the basis of microsatellite variation and correlated to semen parameters. Semen could be collected from 104 of 139 stallions with semen collection being more often successful in younger stallions. Mean volume of ejaculates was 90.8+/-55.1 ml, density 243+/-114 x 10(6)ml(-1), total sperm count 21.0+/-23.7 x 10(9), percentage of morphologically normal spermatozoa 38+/-18% and total motility 50+/-23%. Total sperm count and semen motility were significantly affected by age. Blood samples of 134 stallions were analysed for 12 microsatellite DNA markers. Genotypes of 110 stallions with at least 11 successfully typed markers were used for calculation of heterozygosity. A total of 82 alleles was identified with a mean of 6.8 alleles per marker. Heterozygosity varied between 35 and 76% for the different markers, mean heterozygosity was calculated to 63%. No correlation between heterozygosity and semen parameters was found.

The Impact of Intense Exercise on Semen Quality

PubMed Central

Jóźków, Paweł; Rossato, Marco

2016-01-01

With expanding knowledge on the health benefits of exercise, there is an increasing demand for information on the andrological consequences of participating in sports. These consequences are especially important in the context of infertility problems worldwide. The so-called “male factor” is reported in up to 50% of couples having trouble with conception. The answer to the question, “Is physical activity good for male reproductive health?” is not straightforward. A number of studies have suggested that significant changes in semen parameters may occur due to sports training of certain types, intensities, and durations. The changes to these parameters vary in scope, direction, and magnitude. Findings in recreational athletes have also differed from those in professional athletes. This review of the current literature suggests that intense physical activity may affect the semen concentration, as well as the number of motile and morphologically normal spermatozoa. Training at higher intensities and with increased loads seems to be associated with more profound changes in semen quality. In recreational athletes, exercise has either a positive or neutral effect on semen parameters. Due to many limitations (e.g., global sperm count trends, concerns about the quality control of sperm evaluations, and new standards for semen analysis), comparisons among historical data and their interpretation are difficult. PMID:27645515

Uterosome-like vesicles prompt human sperm fertilizing capability.

PubMed

Franchi, A; Cubilla, M; Guidobaldi, H A; Bravo, A A; Giojalas, L C

2016-12-01

Does the rapid transit through the uterine environment modulate the sperm physiological state? The uterosome-like vesicles (ULVs) secreted by endometrial epithelial cells (EECs) in vitro are able to fuse with human spermatozoa, prompting their fertilizing capacity. Early studies suggest that sperm capacitation begins in the uterus and ends in the oviduct, and that a synergistic effect of both female organs may accelerate this process. Although it has been reported that co-incubation of human spermatozoa with endometrial cell-conditioned medium (CM) stimulates sperm capacitation, the mechanism mediating this communication is unknown. Human ULVs secreted by EECs were characterized and their effect on human sperm physiology was analysed. Spermatozoa were incubated with EEC-derived CM or ULV, after which sperm capacitation was evaluated at different time points. In addition, the interaction of spermatozoa with ULV was analysed. ULVs were isolated by ultracentrifugation and identified using electron microscopy and Western blotting to assess the presence of specific protein markers. Following seminal plasma removal, human spermatozoa were incubated CM or ULV, after which sperm capacitation was evaluated as the ability of the sperm to undergo the induced acrosome reaction and the level of protein tyrosine phosphorylation (PY) determined by Western blot and immunocytochemistry. The interaction of spermatozoa with labelled ULV was analysed by fluorescence microscopy. In all cases, at least three biological replicates from different sperm donors were performed for each set of experiments. Significant differences between mean values were determined by one-way ANOVA followed by Tukey's post hoc test. Differences between treatments were considered statistically significant at P ≤ 0.05. The level of capacitated spermatozoa and those recruited by chemotaxis increased 3- to 4-fold when spermatozoa were incubated in the presence of CM for 4 h. Even a 15 min incubation of spermatozoa with CM was also enough to increase the level of capacitated cells 3- to 4-fold (P < 0.05). Furthermore, a short co-incubation of spermatozoa with ULV stimulates sperm capacitation, as determined by the increase in the level of induced acrosome reaction and the induction of PY. In addition, after the co-incubation of spermatozoa with fluorescent labelled ULV, the sperm cells acquired the fluorescent staining which indicates that ULV might be transferred to the sperm surface by a fusion mechanism. This is an in vitro study performed with human biological material, spermatozoa and endometrial derived cells; the latter being a cell line originally isolated from a uterine adenocarcinoma. The capability of spermatozoa to briefly interact with ULVs supports the hypothesis that any step of sperm transport may have physiological consequences, despite the interaction lasting for only a limited period of time. This way of communication of spermatozoa with cell products of uterine origin opens new frontiers of investigation (e.g. the signalling molecules involved), shedding light on the sperm processes that prepare the male gamete for fertilization, which might have implications for human infertility treatment. N/A. The project was financially supported by SECyT-UNC. The authors declare no conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Home-based treadmill training improved seminal quality in adults with type 2 diabetes.

PubMed

Rosety-Rodriguez, M; Rosety, J M; Fornieles, G; Rosety, M A; Diaz, A J; Rosety, I; Rodríguez-Pareja, A; Rosety, M; Ordonez, F J; Elosegui, S

2014-11-01

This was the first study conducted to determine the influence of home-based treadmill training on seminal quality in adults with type 2 diabetes. Sixty sedentary adults with type 2 diabetes volunteered for the current study. Thirty were randomly allocated to the intervention group and performed a a 14-week, home-based, treadmill training program, 3 sessions per week, consisting of a warm-up (10-15min), 40min treadmill exercise at a work intensity of 55-70% of peak heart rate (increasing by 2.5% each two weeks) measured during a maximal treadmill test, and cooling-down (5-10min). The control group included 30, age and BMI matched adults with type 2 diabetes who did not take part in any training program. Seminal quality analysis included semen volume, sperm concentration, motility and normal morphologic features. Furthermore, total antioxidant status (TAS) as well as glutathione peroxidase (GPX) activity were assessed in seminal plasma. This protocol was approved by an Institutional Ethics Committee. The home-based treadmill training significantly increased sperm concentration as well as percentages of total sperm motility and normal spermatozoa. Furthermore, TAS and GPX activity were increased after the completion of the training program. No significant changes in any of the measured variables were found in the control group. Home-based treadmill training improved seminal quality in adults with type 2 diabetes. A secondary finding was that seminal antioxidant defense system was significantly increased after being exercised. Copyright © 2013 AEU. Published by Elsevier Espana. All rights reserved.

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.

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.

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.

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.

Morphology of ovary and spermathecae of the parasitoid Eibesfeldtphora tonhascai Brown (Diptera: Phoridae).

PubMed

Farder-Gomes, Cliver Fernandes; Santos, Helen Cristina Pinto; Oliveira, Marco Antonio; Zanuncio, José Cola; Serrão, José Eduardo

2018-06-16

Eibesfeldtphora tonhascai (Diptera: Phoridae) is a parasitoid of leaf-cutting ants and a potential biological control agent against these insect pests. This study describes the morphology of the ovary and spermatheca of E. tonhascai. The female reproductive tract of this parasitoid has a pair of meroistic polytrophic ovaries, two lateral oviducts that open into a common oviduct, an elongated accessory gland, and two spermathecae. Young oocytes are small and spherical, and their size increases as yolk is stored in the cytoplasm. This process is followed by chorion production by follicular cells. Mature oocytes are elliptical or torpedo-shaped. The reservoir wall of the spermatheca has type III glandular cells with cytoplasm rich in free ribosomes, rough endoplasmic reticulum, and secretory vesicles. The apical surface of these cells has microvilli associated with mitochondria. The reservoir lumen is lined by a cuticle and filled with spermatozoa. This is the first report of the ovary and spermatheca morphology of E. tonhascai and contributes to the comprehension of the reproductive biology of this parasitoid of leaf-cutting ants.

Seasonal changes in steroid hormone profiles, body weight, semen quality, and the reproductive tract in captive African wild dogs (Lycaon pictus) in South Africa.

PubMed

Newell-Fugate, Annie E; O Nöthling, Johan; J Bertschinger, Henk

2012-09-01

Characterization of reproductive seasonality in the African wild dog (Lycaon pictus) could assist reintroduction programs. Male wild dogs (n=14) were assessed quarterly (January, mid-summer; April, late summer; August, late winter; November, early summer) for serum testosterone, body weight, testicular and prostatic volume, preputial gland measurement, and ejaculate characteristics. Bi-monthly fecal samples were collected from male (n=11) and female (n=4) wild dogs for analysis of fecal androgens and progestagens. Fecal androgens were higher in early summer (246.4 ± 14.5 ng/g) than in early winter (218.6 ± 13.4 ng/g). Serum testosterone was higher in mid-summer (1.4 ± 0.3 ng/ml) than in late winter (0.7 ± 0.1 ng/ml). Number of spermatozoa per ejaculate was greatest in late summer (301.4 ± 39.3 × 10(6)). Other semen parameters peaked in mid-summer (pH: 7.4; progressive motility: 85.0 ± 0.1%; live spermatozoa: 81.0 ± 16%; normal morphology: 71.5 ± 8.2%). Total testicular and prostatic volume were greater during summer (testicular: 36.7 ± 4.2 cm(3); prostatic: 12.0 ± 1.9 cm(3)) than winter (testicular: 25.2 ± 1.9 cm(3); prostatic: 5.8 ± 0.8 cm(3)). Preputial pendulance also was greater in summer (7.1 ± 0.5 cm; n=9) than winter (5.9 ± 0.2 cm). Baseline fecal progestagen metabolites were 6.2 ± 2.5 μg/g and peak fecal progestagen metabolites were 14.7 ± 2.8 μg/g. Copulations resulting in pregnancies (n=2) occurred in late summer and gestation was 71 days. Female wild dogs were seasonally monoestrous with mating in summer and winter. In conclusion, wild dogs are reproductively seasonal with improvement in male reproductive variables during summer and a bi-phasic seasonal pattern to female receptivity. Copyright © 2012 Elsevier Inc. All rights reserved.

Phthalate exposure and semen quality in fertile US men.

PubMed

Thurston, S W; Mendiola, J; Bellamy, A R; Levine, H; Wang, C; Sparks, A; Redmon, J B; Drobnis, E Z; Swan, S H

2016-07-01

Several experimental and observational studies have demonstrated the antiandrogenicity of several phthalates. However, there is limited evidence of an association between phthalate exposure in adult life and semen quality. The aim of this study was to examine phthalate exposure during adulthood in relation to semen quality in fertile US men. This multi-center cross-sectional study included 420 partners of pregnant women who attended a prenatal clinic in one of five US cities during 1999-2001. Nine phthalate metabolites [mono (2-ethylhexyl) phthalate (MEHP), mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono (2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono (2-ethyl-5-carboxypentyl) phthalate (MECPP)], as well as mono-n-butyl phthalate (MBP) and mono-isobutyl phthalate (MiBP), mono (three carboxypropyl) phthalate (MCPP), monobenzyl phthalate (MBzP), and monoethyl phthalate (MEP)] were measured in urine collected at the same time as the semen sample. We regressed natural log-transformed (ln) sperm concentration, ln(total sperm count), ln(total motile sperm count), percent motile spermatozoa, and percent spermatozoa with normal morphology on each of the nine natural log-transformed metabolite concentrations and on the molar-weighted sum of DEHP metabolites in separate models. We fit unadjusted models and models that adjusted for confounders determined a priori. In unadjusted models, ln(MiBP) was significantly and positively associated with motility and ln(MBzP) significantly negatively associated with ln(total sperm count). In adjusted linear models, urinary metabolite concentrations of DEHP, DBP, DEP, and DOP were not associated with any semen parameter. We found an inverse association between ln(MBzP) concentrations and sperm motility (β = -1.47, 95% CI: -2.61, -0.33), adjusted for ln(creatinine concentration), geographic location, age, race, smoking status, stress, recent fever, time from sample collection and time to complete analysis. Several sensitivity analyses confirmed the robustness of these associations. This study and the available literature suggest that impacts of adult exposure to phthalates at environmental levels on classical sperm parameters are likely to be small. © 2015 American Society of Andrology and European Academy of Andrology.

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

PubMed Central

Jin, Mayuko; Fujiwara, Eiji; Kakiuchi, Yasutaka; Okabe, Masaru; Satouh, Yuhkoh; Baba, Shoji A.; Chiba, Kazuyoshi; Hirohashi, Noritaka

2011-01-01

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm–oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm–zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocyte's plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization. PMID:21383182

A pilot study: The effects of repeat washing and fabric type on the detection of seminal fluid and spermatozoa.

PubMed

Nolan, A; Speers, Samuel J; Murakami, Julie; Chapman, Brendan

2018-05-19

In sexual assault cases and more specifically those involving childhood sexual abuse (CSA), victims may have had their potentially semen-stained clothing washed multiple times before a criminal investigation commences. Although it has been previously demonstrated that spermatozoa persist on cotton clothing following a single wash cycle, items of clothing washed multiple times are not routinely examined in these cases because of the assumption that the laundering process would have removed all seminal fluid and spermatozoa. The aim of this study was to examine the persistence of seminal fluid and spermatozoa on a range of fabric types including cotton, nylon, terry towel (100% cotton), polyester fleece, satin and lace which were laundered up to six times. Three techniques were used for the detection of seminal fluid and spermatozoa: an alternative light source, acid phosphatase test and microscopy. The study demonstrated that spermatozoa persisted on cotton and terry towel following six wash cycles. This data emphasises the need to recover and examine items of clothing and bedding of victims for semen, even if the item has been washed multiple times. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative determination of creatine kinase release from herring (Clupea harengus) spermatozoa induced by tributyltin.

PubMed

Grzyb, Katarzyna; Rychłowski, Michał; Biegniewska, Anna; Skorkowski, Edward F

2003-02-01

Creatine kinase (CK, ATP creatine phosphotransferase, EC 2.7.3.2) is an enzyme participating in ATP regeneration, which is the primary source of energy in living organisms. We demonstrated that CK from herring spermatozoa has high activity ( approximately 452 micromol/min/g of fresh semen) and has a different electrophoretic mobility from isoenzymes present in skeletal muscle. In our study, we investigated toxic effect of tributyltin (TBT) on herring spermatozoa using a specific sperm viability kit to observe live and dead sperm cells with a confocal microscope. Treatment of herring spermatozoa with TBT caused a time-dependent decrease of viability: 35% nonviable cells with 5 microM TBT and more than 90% nonviable cells with 10 microM TBT after 6 h exposure. We also monitored CK release from damaged spermatozoa into surrounding medium containing different concentrations of TBT. The higher concentration of TBT was used the more CK release from spermatozoa was observed. We suggest that CK could be a good biomarker of sperm cell membranes degradation in the case when lactate dehydrogenase release from permeabilized cells is not possible for rapid determination of the effect of TBT.

Fluorometric assessments of viability and mitochondrial status of boar spermatozoa following liquid storage.

PubMed

Fraser, L; Lecewicz, M; Strzezek, J

2002-01-01

A study was conducted to assess viability and mitochondrial status of boar spermatozoa stored at 5 degrees C and 16 degrees C. Gel-free ejaculates, collected from 3 mature boars, were extended in a standard diluent (K3) supplemented with a low-density lipoprotein fraction (LDF) isolated from egg yolk, and stored for 96 h at 5 degrees C and 16 degrees C. Motility analysis was conducted after semen dilution (D0) and on D1-D4 of storage. A double staining method, rhodamine 123 (R123) and propidium iodide (PI), was used to assess sperm viability and mitochondrial status. Sperm viability was also assessed using Hoechst 33,258 (H33258) stain. In fresh semen samples, the percentage of motility was significantly correlated with the percentage of viable spermatozoa with functional mitochondria (R123-PI), viable spermatozoa determined by H33258 staining and ATP content (r = 0.88, p < or = 0.01; r = 0.69, p < or = 0.05; r = 0.77, p < or = 0.01, respectively). The ATP content was also positively correlated with the percentage of viable spermatozoa with functional mitochondria (r = 0.76, p < or = 0.01). Sperm cells progressively lost motility, viability and mitochondrial capacity when stored in the supportive media at 5 degrees C and 16 degrees C. Motility estimates were lower (p < or = 0.05) than the percentage of viable spermatozoa with functional mitochondria during storage in K3 and LDF-based diluents on D4 and D3-D4, respectively. Deterioration in motility and membrane integrity was less marked in spermatozoa stored in LDF-based diluents. Spermatozoa doubly-stained with R123-PI appeared to possess some functional mitochondria, particularly in LDF-based diluent semen. Estimates of sperm viability, as determined by R123-PI staining, were equivalent (p > or = 0.05) to estimates made using H33258 staining. A decrease in mitochondrial activity, as measured by R123 uptake, was accompanied by lower ATP content in spermatozoa stored in K3 and LDF-based diluents after 48 h and 72 h of storage, respectively. Fluorometric measurements of viability and mitochondrial status of boar spermatozoa during liquid storage seem to provide reliable information about the sperm functional membranes.

The effect of cryoprotectant on kangaroo sperm ultrastructure and mitochondrial function.

PubMed

McClean, Rhett; Holt, William V; Zee, Yeng Peng; Lisle, Allan; Johnston, Stephen D

2008-12-01

This study examined the effect of cryoprotectants (20% DMSO, a 10% DMSO/10% glycerol mixture, 20% glycerol and 1M sucrose solution) on kangaroo sperm structure and function, along with the effect of varying concentrations of glycerol on sperm mitochondrial function. Eastern grey kangaroo cauda epididymidal spermatozoa were incubated for 10 min at 35 degrees C in each cryoprotectant and the plasma membrane integrity (PMI) and motility assessed using light microscopy. The same samples were fixed for TEM and the ultrastructural integrity of the spermatozoa examined. To investigate the effect of glycerol on the kangaroo sperm mitochondrial function, epididymidal spermatozoa were incubated with JC-1 in Tris-citrate media at 35 degrees C for 20 min in a range of glycerol concentrations (0%, 5%, 10%, 15% and 20%) and the mitochondrial membrane potential (MMP) and plasma membrane integrity determined. As expected, incubation of spermatozoa in 20% glycerol for 10 min resulted in a significant reduction in motility, PMI and ultrastructural integrity. Interestingly, incubation in 20% DMSO resulted in no significant reduction in motility or PMI but a significant loss of structural integrity when compared to the control spermatozoa (0% cryoprotectant). However, 20% DMSO was overall less damaging to sperm ultrastructure than glycerol, a combination of 10% glycerol and 10% DMSO, and sucrose. While all glycerol concentrations had an adverse effect on mitochondrial function, the statistical models presented for the relationship between MMP and glycerol predicted that spermatozoa, when added to 20% glycerol, would lose half of their initial MMP immediately at 35 degrees C and MMP would halve after 19.4 min at 4 degrees C. Models for the relationship between PMI and glycerol predicted that spermatozoa would lose half of their initial PMI after 1.8 min at 35 degrees C and PMI would halve after 21.1 min at 4 degrees C. These results suggest that if glycerol is to be used as a cryoprotectant for kangaroo spermatozoa then it is best administered at 4 degrees C and that mitochondrial function is more sensitive to glycerol than PMI. Future research should be directed at investigating strategies that reduce exposure of spermatozoa to glycerol during processing and that test the cryoprotective properties of 20% DMSO for kangaroo spermatozoa.

Factors controlling sperm migration through the oviduct revealed by gene-modified mouse models

PubMed Central

Fujihara, Yoshitaka; Miyata, Haruhiko; Ikawa, Masahito

2018-01-01

Mammalian fertilization is comprised of many steps including sperm survival in the uterus, sperm migration in the female reproductive tract, physiological and morphological changes to the spermatozoa, and sperm-egg interaction in the oviduct. In vitro studies have revealed essential factors for these fertilization steps for over half a century. However, the molecular mechanism of fertilization has recently been revised by the emergence of genetically modified animals. Here, we focus on essential factors for sperm fertilizing ability and describe recent advances in our knowledge of the mechanisms of mammalian fertilization, especially of sperm migration from the uterus into the oviduct. PMID:29353867

Self-reported onset of puberty and subsequent semen quality and reproductive hormones in healthy young men.

PubMed

Jensen, Tina Kold; Finne, Katrine Folmann; Skakkebæk, Niels E; Andersson, Anna-Maria; Olesen, Inge Ahlmann; Joensen, Ulla Nordström; Bang, Anne Kirstine; Nordkap, Loa; Priskorn, Lærke; Krause, Marianna; Jørgensen, Niels; Juul, Anders

2016-08-01

Is there an association between pubertal onset and subsequent reproductive health in young men? Self-reported later onset of puberty was associated with reduced semen quality and altered serum levels of reproductive hormones among 1068 healthy, young Danish men. The long-term effects of variations in the onset of male puberty on subsequent reproduction remain largely unstudied. In a cross-sectional study, young healthy Danish men were approached when they attended a compulsory medical examination to determine their fitness for military service from 2008 to 2012. A total of 1068 healthy, young Danish men (mean age 19 years) participated. They were asked to assess whether onset of penile and testicular growth, development of pubic hair and voice break occurred earlier, at the same time as or later than their peers. Their semen quality (semen volume, sperm concentration, total sperm count and percentages of motile and morphologically normal spermatozoa) and serum concentrations of sex hormones (LH, FSH, total testosterone, SHBG, inhibin B) and testicular size were determined. The response rate was 29%. Of the 1068 men who then participated, 652 answered the questions about penile growth and pubic hair development and were therefore included in the analysis. Self-reported later onset of puberty was associated with a 25% reduction in sperm concentration (95% CI -41%; -4%), a 40% reduction in total sperm count (-55%; -21%), a 1.6% age point reduction in morphological normal spermatozoa (-2.9; -0.3) and a 1.6 ml reduction in testicular size (-2.4 and -0.8 ml), after adjustment for confounders. Self-reported later onset of puberty was also associated with a 9% (3%; 15%) reduction in free testosterone and a 16% (2%; 31%) increase in FSH, after adjustment for confounders. Our study was cross-sectional and reverse causality cannot be ruled out. In addition, we cannot rule out the possibility that the men with late puberty onset had not yet fully matured although most were in Tanner stage 5. Approximately 15% of young Danish men have self-reported later onset of puberty than their peers. We found poorer testicular function in young men with a history of later pubertal development, suggesting that timing of pubertal onset may be a fundamental marker of male reproductive health. However, we cannot exclude the possibility that these men had not fully matured at the time of examination and therefore their semen quality may yet improve, which makes follow-up important. This work was supported by the Danish Council for Strategic Research, Program Commission on Health, Food and Welfare (project number 2101-08-0058), Rigshospitalet (grants 961506336 and R42-A1326), European Union, DEER (grant agreement no 212844), the Danish Ministry of Health and the Danish Environmental Protection Agency and Kirsten and Freddy Johansens Foundation (grant 95-103-72087). There are no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex.

PubMed

Santolaria, Pilar; Pauciullo, Alfredo; Silvestre, Miguel A; Vicente-Fiel, Sandra; Villanova, Leyre; Pinton, Alain; Viruel, Juan; Sales, Ester; Yániz, Jesús L

2016-01-01

This study was designed to determine the ability of computer-assisted sperm morphometry analysis (CASA-Morph) with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa (SX and SY, respectively). Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average (P < 0.001) although with important differences between bulls. A simultaneous evaluation of all the measured features by discriminant analysis revealed that nuclear area and average fluorescence intensity were the variables selected by stepwise discriminant function analysis as the best discriminators between SX and SY. In the second experiment, the sperm nuclear morphometric results from CASA-Morph in nonsexed (mixed SX and SY) and sexed (SX) semen samples from four bulls were compared. FISH allowed a successful classification of spermatozoa according to their sex chromosome content. X-sexed spermatozoa displayed a larger size and fluorescence intensity than nonsexed spermatozoa (P < 0.05). We conclude that the CASA-Morph fluorescence-based method has the potential to find differences between X- and Y-chromosome-bearing spermatozoa in bovine species although more studies are needed to increase the precision of sex determination by this technique.

Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa.

PubMed

Nicolas, M; Alvarez, M; Borragán, S; Martinez-Pastor, F; Chamorro, C A; Alvarez-Rodriguez, M; de Paz, P; Anel, L

2012-04-01

Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample. Copyright © 2012 Elsevier Inc. All rights reserved.

Adenosine monophosphate-activated kinase, AMPK, is involved in the maintenance of the quality of extended boar semen during long-term storage.

PubMed

Martin-Hidalgo, David; Hurtado de Llera, Ana; Yeste, Marc; Cruz Gil, M; Bragado, M Julia; Garcia-Marin, Luis J

2013-09-01

Boar semen preservation for later use in artificial insemination is performed by diluting semen in an appropriate medium and then lowering the temperature to decrease spermatozoa metabolism. The adenosine monophosphate-activated kinase, AMPK, is a key cell energy sensor that controls cell metabolism and recently has been identified in boar spermatozoa. Our aim was to investigate the role of AMPK in spermatozoa functional parameters including motility, mitochondrial membrane potential, plasma membrane integrity, acrosome integrity, and cell viability during long-term boar semen storage at 17 °C in Beltsville thawing solution. Boar seminal doses were diluted in Beltsville thawing solution in the presence or absence of different concentrations of AMPK inhibitor, compound C (1, 10, and 30 μM) and evaluations were performed at 1, 2, 4, 7, or 10 days. Data demonstrate that AMPK becomes phosphorylated at threonine(172) (active) during storage of boar semen reaching maximum levels at Day 7. Moreover, AMPK inhibition during boar semen storage causes: (1) a potent inhibition of spermatozoa motility; (2) a reduction in the percentage of spermatozoa showing high mitochondria membrane potential; (3) a rise in the percentage of spermatozoa displaying high plasma membrane scrambling; and (4) a loss of acrosomal membrane integrity. Our study suggests that AMPK activity plays an important role in the maintenance of the spermatozoa quality during long-term storage of boar semen. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

The relationship of physical and chemical conditions of CEP diluent with egg yolk addition to bull spermatozoa quality before and after storage at temperaturof 4-5°C

NASA Astrophysics Data System (ADS)

Ducha, N.; Hariani, D.; Budijastuti, W.

2018-01-01

Storage of semen requires diluent to dilute semen and maintain sperm quality. One of the diluent for bull semen was CEP. The purpose of this study was to assess the association of bull spermatozoa quality with the physical and chemical conditions of CEP diluents with the addition of egg yolk before and after the storage process. The study used Limousin bull with 5 replications. The quality of spermatozoa included motility and viability. Physical and chemical conditions included the pH and osmolarity of the diluent. The motility of spermatozoa was observed under a light microscope with 200 X magnification at 37°C by two people. The viability of spermatozoa was observed under a light microscope with 400 X magnification with nigrosine eosin staining. Data were analyzed with ANOVA and continued Duncan’s test. Dilution pH was measured using pH indicator paper ranging from 6-8. The osmolarity of the diluent was measured by electrical osmolarity. The results showed that the addition of egg yolk in the CEP diluent decreased the pH and increased osmolartitas, but the quality of spermatozoa can be kept up to 8 days of storage. The conclusion in this study was the addition of egg yolk in the CEP diluent provided physical and chemical conditions that can maintain the quality of spermatozoa during storage at a temperature of 4-5 ° C.

The influence of direct mobile phone radiation on sperm quality.

PubMed

Gorpinchenko, Igor; Nikitin, Oleg; Banyra, Oleg; Shulyak, Alexander

2014-01-01

It is impossible to imagine a modern socially-active man who does not use mobile devices and/or computers with Wi-Fi function. The effect of mobile phone radiation on male fertility is the subject of recent interest and investigations. The aim of this study was to investigate the direct in vitro influence of mobile phone radiation on sperm DNA fragmentation and motility parameters in healthy subjects with normozoospermia. 32 healthy men with normal semen parameters were selected for the study. Each sperm sample was divided into two equal portions (A and B). Portions A of all involved men were placed for 5 hours in a thermostat, and portions B were placed into a second thermostat for the same period of time, where a mobile phone in standby/talk mode was placed. After 5 hours of incubation the sperm samples from both thermostats were re-evaluated regarding basic motility parameters. The presence of DNA fragmentation in both A and B portions of each sample was determined each hour using a standard sperm chromatin dispersion test. The number of spermatozoa with progressive movement in the group, influenced by electromagnetic radiation, is statistically lower than the number of spermatozoa with progressive movement in the group under no effect of the mobile phone. The number of non-progressive movement spermatozoa was significantly higher in the group, which was influenced by cell phone radiation. The DNA fragmentation was also significantly higher in this group. A correlation exists between mobile phone radiation exposure, DNA-fragmentation level and decreased sperm motility.

[New diagnostic procedures in assessing male fertility].

PubMed

Schütte, B

1989-04-15

In 160 patients with normal and pathological semen samples, we studied the penetration of human spermatozoa in standardized bovine cervical mucus (assay Penetrak). Our results indicate that Penetrak can detect those dysfunctions of sperm motility which cannot be diagnosed by conventional semen analysis - The toluidine blue-pyronine staining is an easy way to differentiate between spermatozoa within an hour. The reliability of this technique is comparable to that of conventional methods and therefore appropriate for routine diagnostics. - As case studies on 140 patients proved, the level of carnitine in the seminal plasm can be regarded as a parameter of epididymal function. In combination with the examination of FSH serum levels, this method may be helpful in the differential diagnosis of azoospermia due to obstruction, Sertoli-cell-only syndrome, or spermatogenic arrest. - In severe oligozoospermia, testicular biopsy using semithin sections may be of high diagnostic and prognostic value. In contrast to paraffin sections, this technique is particularly appropriate for the detection of cytological characteristics of germ cells. The type and number of pathological germ cells are decisive regarding the prognosis. Furthermore, a carcinoma in situ (CIS) can be definitely identified by means of semithin sections. - In a study on 2047 patients, we found seminoma cells in 15 cases (0.73%); 5 of these patients already had a solid seminoma in the testicular rete. Risk-patients are those showing oligozoospermia of less than 10 million spermatozoa per ml or azoospermia associated with unilaterally or bilaterally subnormal testicular volume. In these cases, the testicles are inconspicuous, both palpatorically and sonographifically.

Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

PubMed Central

Sato, Hiroyasu; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Yamamoto, Kei; Masuda, Seiko; Hosono, Tomohiko; Arata, Satoru; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Nakanishi, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Kudo, Ichiro; Murakami, Makoto

2010-01-01

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction. PMID:20424323

The influence of direct mobile phone radiation on sperm quality

PubMed Central

Gorpinchenko, Igor; Nikitin, Oleg; Shulyak, Alexander

2014-01-01

Introduction It is impossible to imagine a modern socially–active man who does not use mobile devices and/or computers with Wi–Fi function. The effect of mobile phone radiation on male fertility is the subject of recent interest and investigations. The aim of this study was to investigate the direct in vitro influence of mobile phone radiation on sperm DNA fragmentation and motility parameters in healthy subjects with normozoospermia. Material and methods 32 healthy men with normal semen parameters were selected for the study. Each sperm sample was divided into two equal portions (A and B). Portions A of all involved men were placed for 5 hours in a thermostat, and portions B were placed into a second thermostat for the same period of time, where a mobile phone in standby/talk mode was placed. After 5 hours of incubation the sperm samples from both thermostats were re–evaluated regarding basic motility parameters. The presence of DNA fragmentation in both A and B portions of each sample was determined each hour using a standard sperm chromatin dispersion test. Results The number of spermatozoa with progressive movement in the group, influenced by electromagnetic radiation, is statistically lower than the number of spermatozoa with progressive movement in the group under no effect of the mobile phone. The number of non–progressive movement spermatozoa was significantly higher in the group, which was influenced by cell phone radiation. The DNA fragmentation was also significantly higher in this group. Conclusions A correlation exists between mobile phone radiation exposure, DNA–fragmentation level and decreased sperm motility. PMID:24982785

Short-Term Storage of Human Spermatozoa in Electrolyte-Free Medium Without Freezing Maintains Sperm Chromatin Integrity Better Than Cryopreservation1

PubMed Central

Riel, Jonathan M.; Yamauchi, Yasuhiro; Huang, Thomas T.F.; Grove, John; Ward, Monika A.

2011-01-01

Previous attempts to maintain human spermatozoa without freezing were based on short-term storage in component-rich medium and led to fast decline in motility and increased incidence of chromosome breaks. Here we report a new method in which sperm are maintained without freezing in an electrolyte-free medium (EFM) composed of glucose and bovine serum albumin. Human sperm were stored in EFM or human tubal fluid medium (HTFM) or were cryopreserved, and their motility, viability, and DNA integrity were examined at different intervals. Cryopreservation led to significant decline in sperm motility and viability and induced DNA fragmentation. Sperm stored in EFM maintained motility and viability for up to 4 and 7 wk, respectively, much longer than sperm stored in HTFM (<2 and <4 wk, respectively). DNA integrity, assessed with comet assay, was also maintained significantly better in EFM than in HTFM. One-week storage in EFM yielded motility and viability similar to that of cryopreserved sperm, but DNA integrity was significantly higher, resembling that of fresh sperm. After several weeks of storage in EFM, sperm were able to activate oocytes, undergo chromatin remodeling, and form normal zygotic chromosomes after intracytoplasmic sperm injection. This study demonstrated that human spermatozoa can be stored in EFM without freezing for several weeks while maintaining motility, viability, and chromatin integrity and that 1-wk storage in EFM offers better protection of sperm DNA integrity than cryopreservation. Sperm storage in EFM may become a viable option for the physicians working in assisted reproduction technology clinics, which would avoid cryodamage. PMID:21593474

Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios.

PubMed

Huszar, G; Sbracia, M; Vigue, L; Miller, D J; Shur, B D

1997-04-01

Sperm creatine phosphokinase (CK) concentrations and the synthesis of the CK-M isoform reflect normal spermiogenesis and predict maturity and fertilizing potential of ejaculated human spermatozoa. Immature spermatozoa, characterized by cytoplasmic retention and low CK-M to CK-B isoform ratios, are deficient in zona binding and fail to cause pregnancies. Because these sperm lack zona-binding ability, we examined in this study whether beta 1,4-galactosyltransferase (GalTase), a key element of sperm-zona interactions in mice, is diminished in immature human sperm. Unexpectedly, GalTase was overexpressed in immature sperm relative to mature sperm: the levels of cytoplasmic CK and plasma membrane GalTase were positively correlated (r = 0.78, p < 0.001, n = 88). Sperm populations with various levels of cellular maturity, prepared by Percoll gradients, had different CK and GalTase concentrations, but within each subpopulation the relationship between CK and GalTase was maintained (p < 0.01-0.001). GalTase activities in intact and vortex-disrupted sperm fractions were similar, showing that GalTase is present on the surface membrane of human sperm--similar to the situation in all other species assayed. The changes previously reported by our laboratory in zona-binding ability and lipid peroxidation rates (which occur simultaneously with cytoplasmic extrusion), decline in CK activity, and increased expression of the CK-M isoform are suggestive of a remodeling of the sperm surface concomitant with cytoplasmic maturation. The changes reported here in GalTase expression on the surface of maturing spermatozoa prove this hypothesis.

DNA fragmentation status in patients with necrozoospermia.

PubMed

Brahem, Sonia; Jellad, Sonia; Ibala, Samira; Saad, Ali; Mehdi, Meriem

2012-12-01

The aim of this study was to determine if a relationship exists between the levels of sperm DNA fragmentation and necrospermia in infertile men. Semen samples obtained from 70 men consulting for infertility evaluation were analyzed according to World Health Organization (WHO) guidelines. Patients were subdivided into three groups according to the percentage of necrotic spermatozoa: normozoospermia (<30%; n = 20), moderate necrozoospermia (50-80%; n = 30), and severe necrozoospermia (>80%; n = 20). DNA fragmentation was detected by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay. The sperm DNA fragmentation index (DFI) was 9.28 ± 2.98% in patients with a normal level of necrotic spermatozoa, 20.25 ± 3.21% in patients with moderate necrozoospermia, and 35.31 ± 5.25% in patients with severe necrozoospermia. There was a statistically significant increase of DNA fragmentation in the necrozoospermic group (P < 0.01). A strong correlation was found between the degree of necrozoospermia and sperm DNA fragmentation. We concluded that patients with necrozoospermia showed a high level of DNA fragmentation compared to normozoospermic men. Severe necrozoospermia (>80%) is a predictive factor for increased sperm DNA damage.

Semen quality parameters as fertility predictors of water buffalo bull spermatozoa during low-breeding season.

PubMed

Ahmed, Hussain; Andrabi, S Murtaza Hassan; Jahan, Sarwat

2016-10-01

The present study was carried out to assess various postthaw semen quality parameters for the prediction of fertility in buffalo bull during low-breeding season. Semen (30 ejaculates) was collected from five adult buffalo bulls with artificial vagina (42 °C). Sperm motility parameters, velocity distribution, motion kinematics, and subpopulations were analyzed by computer-aided sperm motion analyzer (CASA). Moreover, sperm visual motility, supravital plasma membrane integrity, viability/acrosome integrity, viability/mitochondrial transmembrane potential, DNA fragmentation/integrity, and morphology were analyzed by phase-contrast microscope, supravital hypoosmotic swelling test, Trypan blue/Giemsa staining, propidium iodide/"5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide" (JC-1) fluorochromes, neutral comet assay/acridine orange assay and wet mount technique, respectively. Outcome of 528 inseminations was analyzed for in vivo fertility. Pearson's correlation coefficients revealed that sperm progressive motility (%), rapid velocity (%), average path velocity (μm/s), straight line velocity (μm/s), subpopulation one (most rapid, and progressive) of motile spermatozoa (%), supravital plasma membrane integrity (%), and viable spermatozoa with intact acrosome (%) were significantly correlated with in vivo fertility (r = 0.64, P < 0.01; r = 0.57, P < 0.01; r = 0.52, P < 0.01; r = 0.56, P < 0.01; r = 0.73, P < 0.001; r = 0.74, P < 0.001; r = 0.88, P < 0.001); whereas nonviable spermatozoa with damaged acrosome or low-mitochondrial transmembrane potential and comet length (μm) of neutral comet assay were negatively associated with in vivo fertility (r = -0.79, r = -0.75, P < 0.001, and r = -0.60, P < 0.05, respectively). Multiple regression analysis reported that combination of semen quality parameters as predictor of fertility were better (R(2) adjusted = 81.30%, P < 0.001) as compared with single parameter (R(2) adjusted = 50.20%, P < 0.007). It is concluded that assessment of CASA parameters and some other sperm structural and functional parameters, that is, integrity of plasma membrane and acrosome, and transmembrane potential of mitochondria were able to predict the in vivo fertility of water buffalo bull during low-breeding season. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of vitamin E on sperm parameters and DNA integrity in sodium arsenite-treated rats.

PubMed

Momeni, Hamid Reza; Eskandari, Najmeh

2012-05-01

Arsenic as an environmental toxicant is able to exert malformations in male reproductive system by inducing oxidative stress. Vitamin E (Vit.E) is known as antioxidant vitamin. The aim of this study was to investigate the harmful effects of sodium arsenite on sperm parameters and the antioxidant effects of Vit.E on sperm anomalies in sodium arsenite treated rats. Adult male rats were divided into 4 groups: control, sodium arsenite (8 mg/kg/day), Vit.E (100 mg/kg/day) and sodium arsenite+Vit.E. Oral treatments were performed till 8 weeks. Body and left testis weight were recorded and then left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze number, motility, viability and abnormalities of the sperm. Sperm chromatin quality was assessed by nuclear staining using acridine orange and aniline blue. Body and testis weight showed no significant change in 4 groups (p>0.05). A significant decrease in the number, motility, viability and normal sperm morphology was found in sodium arsenite-treated rats compared to the control (p<0.001). Sodium arsenite had no effect on sperm DNA integrity and histon-protamine replacement (p>0.05). In sodium arsenite+Vit.E group, Vit.E could significantly compensate the harmful effects of sodium arsenite on sperm number, motility, viability and morphology compared to sodium arsenite group. In addition, sperm viability and motility was significantly increased in rats treated with Vit.E alone compared to the control and sodium arsenite+Vit.E group. Vitamin E could compensate the adverse effects of sodium arsenite on sperm parameters in adult rats.

Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa.

PubMed

Hereng, T H; Elgstøen, K B P; Cederkvist, F H; Eide, L; Jahnsen, T; Skålhegg, B S; Rosendal, K R

2011-12-01

There has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility. Purified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of (13)C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry. The treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed (13)C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle. Human spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD(+) through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Birth of mice after in vitro fertilization using C57BL/6 sperm transported within epididymides at refrigerated temperatures.

PubMed

Mochida, K; Ohkawa, M; Inoue, K; Valdez, D M; Kasai, M; Ogura, A

2005-07-01

The transportation of cryopreserved spermatozoa is an economical, efficient, and safe method for the distribution of mouse strains from one facility to another. However, spermatozoa from some strains, including C57BL/6 (B6), are very sensitive to freezing and thawing and frequently fail to fertilize eggs by conventional in vitro fertilization methods at the recipient mouse facility. Since many genetically engineered mice have the B6 genetic background, this sensitivity poses a major obstacle to studies of mouse genetics. We investigated the feasibility of transporting spermatozoa within epididymides under non-freezing conditions. First, we examined the interval that B6 and B6D2F1 (BDF1) spermatozoa retained their ability to fertilize when stored within epididymides at low temperatures (5 degrees C or 7 degrees C). Fertilization rates were >50%, irrespective of the spermatozoa used, when epididymides were stored for 3d at 7 degrees C. B6 spermatozoa, but not BDF1 sperm, had better retention of fertilizing ability at 7 degrees C versus 5 degrees C. We then transported freshly collected B6 and BDF1 epididymides from a sender colony to a recipient colony using a common package delivery service, during which the temperature was maintained at 5 degrees C or 7 degrees C for 2d. Sufficiently high fertilization rates (68.0-77.5%) were obtained for all experimental groups, except for B6 spermatozoa transported at 5 degrees C. These spermatozoa were successfully cryopreserved at the recipient facility and, yielded post-thaw fertilization rates of 27.6-66.4%. When embryos derived from the B6 spermatozoa that were transported at 7 degrees C were transferred into recipient females, 52.7% (38/72) developed to term. In conclusion, transportation of epididymides at refrigerated temperatures is a practical method for the exchange of mouse genetic resources between facilities, especially when these facilities do not specialize in sperm cryopreservation. For the B6 mouse strain, the transportation of epididymides at 7 degrees C rather than 5 degrees C, is recommended.

A genetic method for sex determination in Ovis spp. by interruption of the zinc finger protein, Y-linked (ZFY) gene on the Y chromosome.

PubMed

Zhang, Yong Sheng; Du, Ying Chun; Sun, Li Rong; Wang, Xu Hai; Liu, Shuai Bing; Xi, Ji Feng; Li, Chao Cheng; Ying, Rui Wen; Jiang, Song; Wang, Xiang Zu; Shen, Hong; Jia, Bin

2018-03-06

The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene on the Y chromosome have not been completely elucidated, due, in part, to difficulties in gene targeting analysis of the Y chromosome. The zinc finger protein, Y-linked (ZFY) gene was first proposed to be a sex determination factor, although its function in spermatogenesis has recently been elucidated. Nevertheless, ZFY gene targeting analysis has not been performed to date. In the present study, RNA interference (RNAi) was used to generate ZFY-interrupted Hu sheep by injecting short hairpin RNA (shRNA) into round spermatids. The resulting spermatozoa exhibited abnormal sperm morphology, including spermatozoa without tails and others with head and tail abnormalities. Quantitative real-time polymerase chain reaction analysis showed that ZFY mRNA expression was decreased significantly in Hu sheep with interrupted ZFY compared with wild-type Hu sheep. The sex ratio of lambs also exhibited a bias towards females. Together, the experimental strategy and findings of the present study reveal that ZFY also functions in spermatogenesis in Hu sheep and facilitate the use of RNAi in the control of sex in Hu sheep.

Toward an integrative and predictive sperm quality analysis in Bos taurus.

PubMed

Yániz, J L; Soler, C; Alquézar-Baeta, C; Santolaria, P

2017-06-01

There is a need to develop more integrative sperm quality analysis methods, enabling researchers to evaluate different parameters simultaneously cell by cell. In this work, we present a new multi-parametric fluorescent test able to discriminate different sperm subpopulations based on their labeling pattern and motility characteristics. Cryopreserved semen samples from 20 Holstein bulls were used in the study. Analyses of sperm motility using computer-assisted sperm analysis (CASA-mot), membrane integrity by acridine orange-propidium iodide combination and multi-parametric by the ISAS ® 3Fun kit, were performed. The new method allows a clear discrimination of sperm subpopulations based on membrane and acrosomal integrity, motility and morphology. It was also possible to observe live spermatozoa showing signs of capacitation such as hyperactivated motility and changes in acrosomal structure. Sperm subpopulation with intact plasma membrane and acrosome showed a higher proportion of motile sperm than those with damaged acrosome or increased fluorescence intensity. Spermatozoa with intact plasmalemma and damaged acrosome were static or exhibit weak movement. Significant correlations among the different sperm quality parameters evaluated were also described. We concluded that the ISAS ® 3Fun is an integrated method that represents an advance in sperm quality analysis with the potential to improve fertility predictions. Copyright © 2017 Elsevier B.V. All rights reserved.

Variation of sperm head shape and tail length in a species of Australian hydromyine rodent: the spinifex hopping mouse, Notomys alexis.

PubMed

Bauer, M; Breed, W G

2006-01-01

In Australia, there are around 60 species of murid rodents that occur in the subfamily Hydromyinae, most of which produce highly complex, monomorphic, spermatozoa in which the head has an apical hook together with two ventral processes containing filamentous actin and a long tail of species-specific length. One of the few exceptions to this is the spinifex hopping mouse, Notomys alexis, whose spermatozoa have previously been shown to have pleiomorphic heads. In this study, the structural organisation of the sperm head has been investigated in more detail and the variability in length of the midpiece and total length of the sperm tail has been determined for this species. The findings confirm that pleiomorphic sperm heads are invariably present in these animals and that this variability is associated with that of the nucleus, although nuclear vacuoles were not evident. The total length of the sperm tail, as well as that of the midpiece, was also highly variable both within, as well as between, individual animals. The reason(s) for this high degree of variability in sperm morphology is not known but it may relate to a relaxation of the genetic control of sperm form owing to depressed levels of inter-male sperm competition.

TDRP deficiency contributes to low sperm motility and is a potential risk factor for male infertility.

PubMed

Mao, Shanhua; Wu, Fei; Cao, Xinyi; He, Min; Liu, Naijia; Wu, Huihui; Yang, Zhihong; Ding, Qiang; Wang, Xuanchun

2016-01-01

TDRP (Testis Development-Related Protein), a nuclear factor, might play an important role in spermatogenesis. However, the molecular mechanisms of TDRP underlying these fundamental processes remain elusive. In this study, a Tdrp-deficient mouse model was generated. Fertility tests and semen analysis were performed. Tdrp-deficient mice were not significantly different from wild-type littermates in development of testes, genitourinary tract, or sperm count. Morphologically, spermatozoa of the Tdrp-deficient mice was not significantly different from the wild type. Several sperm motility indexes, i.e. the average path velocity (VAP), the straight line velocity (VSL) and the curvilinear velocity (VCL) were significantly decreased in Tdrp-deficient mice (p<0.05). The proportion of slow velocity sperm also increased significantly in the mutant mice (p<0.05). However, fertility tests showed that no significant difference inaverage offspring amount (AOA), frequency of copulatory plug (FCP), and frequency of conception (FC). Furthermore, TDRP1 could interact with PRM2, which might be the molecular mechanism of its nuclear function in spermatozoa. In conclusion, these data collectively demonstrated that Tdrp deficiency impaired the sperm motility, but Tdrp deficiency alone was not sufficient to cause male infertility in mice. Additionally, TDRP1 might participate in spermatogenes is through interaction with PRM2.

Effect of dietary selenium and vitamin E on the ultrastructure and ATP concentration of boar spermatozoa, and the efficacy of added sodium selenite in extended semen on sperm motility.

PubMed

Marin-Guzman, J; Mahan, D C; Whitmoyer, R

2000-06-01

Three experiments evaluated the effects of dietary Se and vitamin E on the ultrastructure of spermatozoa, ATP concentration of spermatozoa, and the effects of adding sodium selenite to semen extenders on subsequent sperm motility. The experiment was a 2 x 2 arrangement of treatments in a randomized complete block design. A total of 10 mature boars were fed from weaning to 18 mo of age diets fortified with two levels of supplemental Se (0 or .5 ppm) or vitamin E (0 or 220 IU/kg diet). The nonfortified diets contained .06 ppm Se and 4.4 IU vitamin E/kg. In Exp. 1, the spermatozoa from all boars were examined by electron microscopy. Vitamin E had no effect on structural abnormalities in the spermatozoa. When the low-Se diet was fed the acrosome or nuclei of the spermatozoa was unaffected, but the mitochondria in the tail midpiece were more oval with wider gaps between organelles. The plasma membrane connection to the tail midpiece was not tightly bound as when boars were fed Se. Immature spermatozoa with cytoplasmic droplets were more numerous when boars were fed the low-Se diet, but the occurrence of midpiece abnormalities occurred in boars fed diets with or without Se or vitamin E. Our results suggest that Se may enhance spermatozoa maturation in the epididymis and may reduce the number of sperm with cytoplasmic droplets. In Exp. 2, the concentration of ATP in the spermatozoa was evaluated in the semen of all treatment boars. When the low-Se diet was fed, ATP concentration was lower (P < .01), whereas vitamin E had no effect on ATP concentration. Experiment 3 investigated the effect of diluting boar semen with a semen extender with sodium selenite added at 0, .3, .6, or .9 ppm Se. Three ejaculates from each boar were used to evaluate these effects on sperm motility to 48 h after dilution. Sperm motility declined (P < .01) when Se was added to the extender, and this decline was exacerbated as the concentration of added Se increased (P < .01). The added Se was demonstrated to be tightly adhered to the spermatozoa. Overall, these results suggest that low Se-diets fed to boars resulted in abnormal spermatozoal mitochondria, a lower ATP concentration in the spermatozoa, and a loose apposition of the plasma membrane to the helical coil of the tail midpiece, but no effect from inadequate vitamin E was demonstrated. Adding sodium selenite to the semen extender reduced sperm cell motility.

Methods for Improving In Vitro and In Vivo Boar Sperm Fertility.

PubMed

Funahashi, H

2015-07-01

Fertility of boar spermatozoa is changed after ejaculation in vivo and in vitro. During processing for in vitro fertilization (IVF), although spermatozoa are induced capacitation, resulting in a high penetration rate, persistent obstacle of polyspermic penetration is still observed with a high incidence. For artificial insemination (AI), we still need a large number of spermatozoa and lose a majority of those in the female reproductive tract. Fertility of cryopreserved boar spermatozoa is still injured through freezing and thawing process. In the present brief review, factors affecting fertility of boar sperm during IVF, AI and cryopreservation are discussed in the context of discovering methodologies to improve it. © 2015 Blackwell Verlag GmbH.

STUDIES ON THE ANTIGENIC STRUCTURE OF SOME MAMMALIAN SPERMATOZOA

PubMed Central

Henle, Werner; Henle, Gertrude; Chambers, Leslie A.

1938-01-01

1. A method has been described for separation of heads and tails of mammalian spermatozoa. 2. By means of absorption technique applied to homologous spermatozoal sera, head-specific and tail-specific antigens could be demonstrated. Both are heat-labile. 3. A heat-stable antigen was found to be common to both heads and tails. This substance is species-specific. 4. Antibodies against the head- and tail-specific antigens led to two different types of agglutination as shown by the slide method. 5. Using heterologous antisera against spermatozoa three different cross-reacting antigens could be observed, two in the heads, one in the tails. 6. One of the head-antigens is not active in the native cell; it comes to action only after breaking the cell. Antibodies against this substance were not found in antisera against native bull spermatozoa but were formed when vibrated spermatozoa or heads were injected into rabbits. 7. The cross-reactions can be removed from an antiserum leaving the head- as well as the tail-specific reaction intact. PMID:19870792

Boar semen controlled delivery system: storage and in vitro spermatozoa release.

PubMed

Torre, M L; Faustini, M; Norberti, R; Stacchezzini, S; Maggi, L; Maffeo, G; Conte, U; Vigo, D

2002-12-13

Swine spermatozoa were encapsulated in barium alginate and protamine-barium alginate membranes to lengthen their preservation time and to provide a means of controlling their release. Precocious acrosome reactions and secondary anomalies were measured as indices of semen quality. These characteristics were observed for two forms of encapsulated spermatozoa when stored at 18 and 38 degrees C for 24 h and for semen diluted in a classical extender at both temperatures. The results indicate that encapsulation enhances semen preservation, providing protection against membrane damage upon dilution. The effect is even more evident at the higher temperature (38 degrees C), where cell metabolism is higher. An in vitro release test of spermatozoa showed a massive cell delivery from barium alginate capsules within 6 h, and a slow release from protamine-barium alginate capsules. The properties of spermatozoa 24 h after release did not differ from the semen stored at the same temperature in capsules, indicating that the release process does not impair semen quality.

Effects of egg yolk, glycerol and the freezing rate on the viability and acrosomal structures of frozen ram spermatozoa.

PubMed

Watson, P F; Martin, I C

1975-04-01

The influence of egg yolk, glycerol and the freezing rate on the survival of ram spermatozoa and on the structure of their acrosomes after freezing was investigated. Egg yolk was shown to be beneficial not only during chilling but also during freezing; of the levels examined, 1-5% gave the greatest protection. Although the presence of glycerol in the diluent improved the survival of spermatozoa, increasing concentrations produced significant deterioration of the acrosomes. With closely controlled linear cooling rates, no overall difference was detected in the survival of spermatozoa frozen at rates between 6 and 24 degrees C per min. However, a significant interaction between freezing rate and the inclusion of glycerol in the diluent showed that glycerol was less important at the highest freezing rate. A sudden cooling phase near to the freezing point following the release of the latent heat of fusion was not detrimental to spermatozoa.

Intracytoplasmic sperm injection outcomes with cryopreserved testicular sperm aspiration samples.

PubMed

Roque, M; Valle, M; Marques, F; Sampaio, M; Geber, S

2016-04-01

Intracytoplasmic sperm injection (ICSI) may be performed with testicular frozen-thawed spermatozoa in patients with nonobstructive azoospermia (NOA). Sperm retrieval can be performed in advance of oocyte aspiration, as it may avoid the possibility of no recovery of spermatozoa on the day of oocyte pickup. There are few studies available in the literature concerning the use of frozen-thawed spermatozoa obtained from testicular sperm aspiration (TESA). To evaluate the effects and the outcomes of ICSI with frozen-thawed spermatozoa obtained by TESA, we performed a retrospective analysis of 43 ICSI cycles using frozen-thawed TESA. We obtained acceptable results with a fertilisation rate of 67.9%, an implantation rate (IR) of 17.1%, and clinical and ongoing pregnancy rates of 41.9% and 37.2% respectively. The results of this study suggest that performing ICSI using cryopreserved frozen-thawed testicular spermatozoa with TESA as a first option is a viable, safe, economic and effective method for patients with NOA. © 2015 Blackwell Verlag GmbH.

Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility.

PubMed

Peddinti, Divyaswetha; Nanduri, Bindu; Kaya, Abdullah; Feugang, Jean M; Burgess, Shane C; Memili, Erdogan

2008-02-22

Male infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. Using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G2/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa. This is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype.

Is vitronectin the velcro that binds the gametes together?

PubMed

Fusi, F M; Bernocchi, N; Ferrari, A; Bronson, R A

1996-11-01

Evidence has been presented that the adhesion of human spermatozoa to the oolemma is mediated by integrins recognizing the Arg-Gly-Asp sequence (RGD). Fibronectin and vitronectin, glycoproteins that contain functional RGD sequences, are both present on human spermatozoa, and integrins that recognize these ligands have been detected on spermatozoa and eggs. In this work, we studied the effects of oligopeptides specifically designed to block fibronectin or vitronectin receptors on the interaction of human spermatozoa with zona-free hamster oocytes. GRGDdSP, a peptide blocking cell attachment to fibronectin, was without effect, while GdRGDSP, which blocks both fibronectin and vitronectin receptors, significantly inhibited the binding of human spermatozoa to the oolemma of zona-free hamster eggs, in a concentration-dependent manner, over a range 1-100 microM. As these experiments suggested that a vitronectin receptor plays a role in sperm-oolemmal adhesion, we performed a series of experiments studying the effects of exogenous vitronectin, when added to spermatozoa and oocytes, on gamete interactions. Sperm-oolemmal adherence, as well as sperm aggregation, was promoted by vitronectin, over range of 2.2 nM to 1 microM, but only in the presence of calcium ions. We propose that vitronectin released during the sperm acrosome reaction is recognized by both gametes and plays a role in their adhesion.