Objective assessment of sperm motion characteristics of Malpura ram lambs raised under intensive management system in semiarid tropical environment.

PubMed

Kumar, Davendra; Joshi, Anil; Naqvi, S M K

2010-04-01

A study was conducted to evaluate the semen production and sperm motion characteristics of ram lambs by computer-aided semen analysis technique. Eight Malpura rams were raised under intensive management system and were trained for semen collection at a weekly interval from the age of 6 months. Rams were scheduled for semen collection at a weekly interval up to 1 year of age to assess their potential for semen production and objective evaluation of semen quality. The average age of ram lambs at the time of first ejaculation was 219 days ranging from 186 to 245 days. The age of ram lambs significantly (p < 0.05) influenced sperm concentration, sperm velocities, and beat frequency of spermatozoa, which were higher in 9-12-month-old compared to 6-9-month-old ram lambs. However, the effect of age was not significant on semen volume, percent motility, percent rapid, medium or slow motile spermatozoa, percent linearity, percent straightness, amplitude of lateral head displacement, percent elongation, and area of sperm head. The body weight of ram lambs was significantly (p < 0.01) and positively correlated (r = 0.46) with age. The results indicate that Malpura ram lambs of 9-12 months of age raised under the intensive management system in a semiarid tropical environment can produce good quality of semen.

Effects of chilled storage and cryopreservation on sperm characteristics, antioxidant enzyme activities, and lipid peroxidation in Pacific cod Gadus microcephalus

NASA Astrophysics Data System (ADS)

Wang, Xueying; Shi, Xuehui; Liu, Yifan; Yu, Daode; Guan, Shuguang; Liu, Qinghua; Li, Jun

2016-07-01

The present study evaluated the effects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (Gr), and lipid peroxidation (measured via malondialdehyde (MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol (PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation (only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could significantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had significant effects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.

An autonomous sperm-like propulsor in a quiescent flow

NASA Astrophysics Data System (ADS)

Kim, Boyoung; Park, Sung Goon; Sung, Hyung Jin

2016-11-01

Flapping motions of flexible fins are widespread in nature. Birds, fish, and insects use their wings, fins, or bodies to stay afloat and to advance forward in the surrounding fluids. It is important to understand the physics of the flapping motions to utilize them for the biomimetic machines. In the present study, we introduce a sperm-like propulsor that consists of a rigid head containing genetic information and a flapping flexible tail for propulsion. The head gives a sinusoidal torque to the leading edge of the tail, and the flexible tail flaps along the leading edge. In other words, the sperm-like propulsor is moved by an oscillating relative angle between the head and the leading edge of the tail. Unlike self-propelled heaving and pitching fins, the 'autonomous' sperm-like propulsor has no prescribed motion or constraint referenced from outside coordinates. The penalty method and the immersed boundary method are used to solve the autonomous sperm-like propulsor in a quiescent flow. The cruising speed and the propulsive efficiency of the propulsor are explored as a function of the head size (D/ L) , the pitching angle (θ0) , the pitching frequency (f) , and the distance from the wall (G/ L) .

Flagellum synchronization inhibits large-scale hydrodynamic instabilities in sperm suspensions

NASA Astrophysics Data System (ADS)

Schöller, Simon F.; Keaveny, Eric E.

2016-11-01

Sperm in suspension can exhibit large-scale collective motion and form coherent structures. Our picture of such coherent motion is largely based on reduced models that treat the swimmers as self-locomoting rigid bodies that interact via steady dipolar flow fields. Swimming sperm, however, have many more degrees of freedom due to elasticity, have a more exotic shape, and generate spatially-complex, time-dependent flow fields. While these complexities are known to lead to phenomena such as flagellum synchronization and attraction, how these effects impact the overall suspension behaviour and coherent structure formation is largely unknown. Using a computational model that captures both flagellum beating and elasticity, we simulate suspensions on the order of 103 individual swimming sperm cells whose motion is coupled through the surrounding Stokesian fluid. We find that the tendency for flagella to synchronize and sperm to aggregate inhibits the emergence of the large-scale hydrodynamic instabilities often associated with active suspensions. However, when synchronization is repressed by adding noise in the flagellum actuation mechanism, the picture changes and the structures that resemble large-scale vortices appear to re-emerge. Supported by an Imperial College PhD scholarship.

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

Boar variability in sperm cryo-tolerance after cooling of semen in different long-term extenders at various temperatures.

PubMed

Wasilewska, K; Fraser, L

2017-10-01

This study investigated individual boar variability in the quality of pre-freeze (PF) and post-thaw (PT) semen cooled in different long-term (LT) extenders and for different holding times (HT). Sperm rich fractions were diluted with Androhep ® Plus (AHP), Androstar ® Plus (ASP), Safecell ® Plus (SCP) and TRIXcell ® Plus (TCP) extenders, stored for 2h at 17°C (HT 1) and additionally for 24h at 10°C (HT 2) and the samples were subsequently evaluated and frozen. Besides the analysis of CASA sperm variables, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), normal apical ridge (NAR) acrosome integrity, and viability (YO-PRO-1 - /PI - ) of sperm were assessed in the PF and PT semen. Results indicated that boar, extender and HT group affected the sperm quality characteristics. There were great variations in PMOT and the sperm motion patterns of the PF semen among the boars. Differences in the HT groups of the PF semen, with respect to the sperm membrane integrity, were less marked among the boars. Consistent variations in TMOT and PMOT in the PT semen were observed among the boars, being greater in the HT 2 group. Most of the CASA-analyzed sperm motion patterns were greater in the HT 2 group of the PT semen. Furthermore, sperm MMP, PMI and viability were greater in the HT 2 group of the PT semen in most of the boars, while consistent differences were observed among the boars for sperm NAR acrosome integrity in either HT group. The significant effect of the cryopreservation process on the sperm membrane proteome was evident from the number of protein bands, detected in the electrophoretic profiles of sperm of the HT 1 and HT 2 groups. The electrophoretic profiles of the PF and PT semen among boars with poor and good semen freezability, however, differed with respect to the abundance and types of sperm membrane-associated proteins. The overall results of this study provided evidence that there are differences among boars in response to the different cooling regimens, and that cooling of extended semen for a 24-h period at 10°C modulated the functions of sperm in an extender-dependent manner, rendering the cells less susceptible to cryo-induced damage. It is suggested that the findings of this study have the potential to improve the technology of boar semen cryopreservation. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of method of euthanasia on sperm motility of mature Sprague-Dawley rats.

PubMed

Stutler, Shannon A; Johnson, Eric W; Still, Kenneth R; Schaeffer, David J; Hess, Rex A; Arfsten, Darryl P

2007-03-01

Euthanasia is one of the most commonly performed procedures in laboratory animal settings. The method of euthanasia may affect experimental results in studies using animals and must be compatible with research objectives including subsequent tissue analyses. Our present study was performed to evaluate the effects of 7 euthanasia methods on sperm motility in mature rats. Rats were euthanized using CO2, 2 commercially available euthanasia solutions (Beuthanasia-D and Sleepaway), and 4 volatile anesthetics (enflurane, halothane, isoflurane, and sevoflurane). Rats euthanized by rapid decapitation alone served as negative controls, and a-chlorohydrin-treated rats euthanized by rapid decapitation were positive controls for sperm impairment. For 5 of these methods, we also measured time to ataxia, recumbency, respiratory arrest, and no auscultable heartbeat. Immediately after euthanasia of each rat, distal caudal epididymides were removed; 1 was processed for automated sperm motility analysis, and the other was frozen for subsequent concentration analysis. Time to all measured parameters was less for volatile anesthetics than for Beuthanasia-D. Times to last respiration and no heartbeat were less for halothane and isoflurane than for enflurane and sevoflurane. Percentage motile sperm did not differ significantly between methods. Percentage progressively motile sperm did not vary significantly between methods except for Beuthanasia-D, for which it was significantly less than the negative control value. Specific sperm motion parameters for each euthanasia method except CO2 and Sleepaway varied significantly from the negative control. Our results indicate that the method of euthanasia is an important consideration when rat sperm motility parameters must be evaluated.

Rheotaxis guides mammalian sperm

PubMed Central

Miki, Kiyoshi; Clapham, David E

2013-01-01

Background In sea urchins, spermatozoan motility is altered by chemotactic peptides, giving rise to the assumption that mammalian eggs also emit chemotactic agents that guide spermatozoa through the female reproductive tract to the mature oocyte. Mammalian spermatozoa indeed undergo complex adaptations within the female (the process of capacitation) that are initiated by agents ranging from pH to progesterone, but these factors are not necessarily taxic. Currently, chemotaxis, thermotaxis, and rheotaxis have not been definitively established in mammals. Results Here, we show that positive rheotaxis, the ability of organisms to orient and swim against the flow of surrounding fluid, is a major taxic factor for mouse and human sperm. This flow is generated within 4 hours of sexual stimulation and coitus in female mice; prolactin-triggered oviductal fluid secretion clears the oviduct of debris, lowers viscosity, and generates the stream that guides sperm migration in the oviduct. Rheotaxic movement is demonstrated in capacitated and uncapacitated spermatozoa in low and high viscosity medium. Finally, we show that a unique sperm motion we quantify using the sperm head's rolling rate reflects sperm rotation that generates essential force for positioning the sperm in the stream. Rotation requires CatSper channels, presumably by enabling Ca2+ influx. Conclusions We propose that rheotaxis is a major determinant of sperm guidance over long distances in the mammalian female reproductive tract. Coitus induces fluid flow to guide sperm in the oviduct. Sperm rheotaxis requires rotational motion during CatSper channel-dependent hyperactivated motility. PMID:23453951

Sperm kinematics and subpopulational responses during the cryopreservation process in caprine ejaculates.

PubMed

Barbas, J P; Leahy, T; Horta, A E; García-Herreros, M

2018-03-20

Sperm cryopreservation in goats has been a challenge for many years due to the detrimental effects of seminal plasma enzymes produced by the bulbo-urethral glands which catalyse the hydrolysis of lecithins in egg yolk to fatty acids and lysolecithins which are deleterious to spermatozoa. This fact implies to carry out additional processing steps during sperm cryopreservation for seminal plasma removal triggering different sperm responses which may affect sperm functionality. The objective of the present study was to determine specific sperm subpopulation responses in different handling steps during the cryopreservation process by using functional sperm kinematic descriptors in caprine ejaculates. Buck ejaculates (n = 40) were analysed for sperm concentration, viability, morphology and acrosome integrity. Moreover, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system after five different handling steps (fresh sperm, 1st washing, 2nd washing, cooling and frozen-thawed sperm) during a standard cryopreservation protocol for goat semen. The results were analysed using Principal Component Analysis (PCA) and multivariate clustering procedures to establish the relationship between the distribution of the subpopulations found and the functional sperm motility in each step. Except for the 1st and 4th steps, four sperm kinematic subpopulations were observed explaining more than 75% of the variance. Based on velocity and linearity parameters and the subpopulations disclosed, the kinematic response varies among processing steps modifying sperm movement trajectories in a subpopulation-specific and handling step-dependent manner (p < 0.001). The predominant motile subpopulation in freshly ejaculated buck sperm had very fast velocity characteristics and a non-linear trajectory (41.1%). Washing buck sperm twice altered the subpopulation structure as well as cooling which resulted in a dramatic reduction in sperm velocities (p < 0.01). Frozen-thawed spermatozoa showed similar characteristics to cooled sperm except there was a further increase in linearity with a large proportion of sperm attributed to new slow, linear cluster (32.5%). In conclusion, this study confirms the variability and heterogeneity of goat sperm kinematic patterns throughout the cryopreservation process and suggests that the predominant motility pattern (assayed in vitro via CASA) of high quality spermatozoa might be typified by high speed and a non-linear trajectory. The relationships among the number and distribution of sperm subpopulations and the different handling steps were particularlly relevant, specially after the cooling and the post-thawing steps, when effects derived from these critical handling steps were evident and altered drastically the sperm motion patterns. Copyright © 2018 Elsevier Inc. All rights reserved.

Clinical Factors Associated with Sperm DNA Fragmentation in Male Patients with Infertility

PubMed Central

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

2014-01-01

Objective. The clinical factors associated with sperm DNA fragmentation (SDF) were investigated in male patients with infertility. Materials and Methods. Fifty-four ejaculates from infertile Japanese males were used. Thirty-three and twenty-one were from the patients with varicoceles and idiopathic causes of infertility, respectively. We performed blood tests, including the serum sex hormone levels, and conventional and computer-assisted semen analyses. The sperm nuclear vacuolization (SNV) was evaluated using a high-magnification microscope. The SDF was evaluated using the sperm chromatin dispersion test (SCDt) to determine the SDF index (SDFI). The SDFI was compared with semen parameters and other clinical variables, including lifestyle factors. Results. The SDFI was 41.3 ± 22.2% (mean ± standard deviation) and did not depend on the cause of infertility. Chronic alcohol use increased the SDFI to 49.6 ± 23.3% compared with 33.9 ± 18.0% in nondrinkers. The SDFI was related to adverse conventional semen parameters and sperm motion characteristics and correlated with the serum FSH level. The SNV showed a tendency to increase with the SDFI. The multivariate analysis revealed that the sperm progressive motility and chronic alcohol use were significant predictors of the SDF. Conclusion. The SCDt should be offered to chronic alcohol users and those with decreased sperm progressive motility. PMID:25165747

Evaluation of the effect of implanted depleted uranium on male reproductive success, sperm concentration, and sperm velocity

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

Arfsten, Darryl P.; Schaeffer, David J.; Johnson, Eric W.

Depleted uranium (DU) projectiles have been used in battle in Iraq and the Balkans and will continue to be a significant armor-penetrating munition for the US military. As demonstrated in the Persian Gulf War, battle injury from DU projectiles and shrapnel is a possibility, and removal of embedded DU fragments from the body is not always practical because of their location in the body or their small size. Previous studies in rodents have demonstrated that implanted DU mobilizes and translocates to the gonads, and natural uranium may be toxic to spermatazoa and the male reproductive tract. In this study, themore » effects of implanted DU pellets on sperm concentration, motility, and male reproductive success were evaluated in adult (P1) Sprague-Dawley rats implanted with 0, 12, or 20, DU pellets of 1x2 mm or 12 or 20 tantalum (Ta) steel pellets of 1x2 mm. Twenty DU pellets of 1x2 mm (760 mg) implanted in a 500-g rat are equal to approximately 0.2 pound of DU in a 154-lb (70-kg) person. Urinary analysis found that male rats implanted with DU were excreting uranium at postimplantation days 27 and 117 with the amount dependent on dose. No deaths or evidence of toxicity occurred in P1 males over the 150-day postimplantation study period. When assessed at postimplantation day 150, the concentration, motion, and velocity of sperm isolated from DU-implanted animals were not significantly different from those of sham surgery controls. Velocity and motion of sperm isolated from rats treated with the positive control compound {alpha}-chlorohydrin were significantly reduced compared with sham surgery controls. There was no evidence of a detrimental effect of DU implantation on mating success at 30-45 days and 120-145 days postimplantation. The results of this study suggest that implantation of up to 20 DU pellets of 1x2 mm in rats for approximately 21% of their adult lifespan does not have an adverse impact on male reproductive success, sperm concentration, or sperm velocity.« less

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.

The addition of ticarcillin-clavulanic acid to INRA 96 extender for stallion semen cooling.

PubMed

Dean, C J; Hobgood, A M; Blodgett, G P; Love, C C; Blanchard, T L; Varner, D D

2012-12-01

A commonly used commercial extender (i.e. INRA 96) contains antimicrobials that may have limited effectiveness. Therefore, addition of ticarcillin-clavulanic acid to this extender is a widespread procedure in the equine breeding industry in the United States. However, such practice has not been critically evaluated. To evaluate the addition of ticarcillin-clavulanic acid to INRA 96 and different extender and antimicrobial storage conditions on sperm function and antimicrobial effectiveness. Gel-free semen (42 ejaculates from 14 mature Quarter Horse stallions) was extended with INRA 96 and stored for 24 h in an Equitainer II. The effects of added ticarcillin-clavulanic acid and different extender storage procedures on sperm motion characteristics (by computer-assisted analysis), sperm membrane integrity (by fluorescence-based measurement) and suppression of bacterial growth (by aerobic and anaerobic culture methods) were evaluated using analysis-of-variance and Chi-square statistical methods. The P value for significance was set at < 0.05. Freezing and thawing of modified or unmodified extender prior to use for stallion semen resulted in reduced sperm quality post cooling for 24 h, as evidenced by a significant reduction in sperm motility (i.e. total and progressive) and sperm membrane integrity. Addition of ticarcillin-clavulanic acid to extender resulted in higher sperm velocity when the reconstituted antimicrobial was subjected to cooled storage, as compared with frozen storage, prior to use. Only 28 of 42 ejaculates (67%) yielded presence of bacteria in neat semen but addition of ticarcillin-clavulanic acid to INRA 96 was not different than INRA 96 alone for inhibiting growth of bacteria (98 vs. 94%, respectively). Addition of ticarcillin-clavulanic acid (1 mg/ml) to INRA 96 did not adversely affect sperm quality in extended semen after cooled storage. Extender freezing and thawing prior to use had detrimental effects on sperm quality. These data suggest that INRA 96 should not be frozen and thawed prior to use. Addition of ticarcillin-clavulanic acid to INRA 96 did not impair sperm quality. All extender treatments effectively controlled the bacterial growth compared with neat semen.

The relationship between mitochondrial DNA copy number and stallion sperm function.

PubMed

Darr, Christa R; Moraes, Luis E; Connon, Richard E; Love, Charles C; Teague, Sheila; Varner, Dickson D; Meyers, Stuart A

2017-05-01

Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, and humans, and has been suggested as a potential biomarker of fertility in stallion sperm. The results of the present study extend this recent discovery using sperm samples from American Quarter Horse stallions of varying age. By determining copy number of three mitochondrial genes, cytochrome b (CYTB), NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 4 (ND4), instead of a single gene, we demonstrate an improved understanding of mtDNA fate in stallion sperm mitochondria following spermatogenesis. Sperm samples from 37 stallions ranging from 3 to 24 years old were collected at four breeding ranches in north and central Texas during the 2015 breeding season. Samples were analyzed for sperm motion characteristics, nuclear DNA denaturability and mtDNA copy number. Mitochondrial DNA content in individual sperm was determined by real-time qPCR and normalized with a single copy nuclear gene, Beta actin. Exploratory correlation analysis revealed that total motility was negatively correlated with CYTB copy number and sperm chromatin structure. Stallion age did not have a significant effect on copy number for any of the genes. Copy number differences existed between the three genes with CYTB having the greatest number of copies (20.6 ± 1.2 copies, range: 6.0 to 41.1) followed by ND4 (15.5 ± 0.8 copies, range: 6.7 to 27.8) and finally ND1 (12.0 ± 1.0 copies, range: 0.4 to 26.6) (P < 0.05). Varying copy number across mitochondrial genes is likely to be a result of mtDNA fragmentation and degradation since downregulation of sperm mtDNA occurs during spermatogenesis and may be important for normal sperm function. Beta regression analysis suggested that for every unit increase in mtDNA copy number of CYTB, there was a 4% decrease in the odds of sperm movement (P = 0.001). Influential analysis suggested that results are robust and not highly influenced by data from individual stallions despite the low number of stallions sampled with low sperm motility. Further genome sequencing is necessary to investigate if mutations or deletions are the underlying causes of inconsistent copy numbers across mitochondrial genes. In conclusion, we show, for the first time, that increased mtDNA copy number is associated with decreased total sperm motility in stallions. We therefore suggest that mtDNA copy number may be an indicator of defective spermatogenesis in stallions. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2010-05-01

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

Assessing reproductive and endocrine parameters in male largescale suckers (Catostomus macrocheilus) along a contaminant gradient in the lower Columbia River, USA

USGS Publications Warehouse

Jenkins, Jill A.; Olivier, H.M.; Draugelis-Dale, R. O.; Eilts, B.E.; Torres, L.; Patiño, R.; Nilsen, Elena B.; Goodbred, Steven L.

2014-01-01

Persistent organochlorine pollutants such as polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (p,p′-DDE), and polybrominated diphenyl ethers (PBDEs) are stable, bioaccumulative, and widely found in the environment, wildlife, and the human population. To explore the hypothesis that reproduction in male fish is associated with environmental exposures in the lower Columbia River (LCR), reproductive and endocrine parameters were studied in male resident, non-anadromous largescale sucker (Catostomus macrocheilus) (LSS) in the same habitats as anadromous salmonids having conservation status. Testes, thyroid tissue and plasma collected in 2010 from Longview (LV), Columbia City (CC), and Skamania (SK; reference) were studied. Sperm morphologies and thyrocyte heights were measured by light microscopy, sperm motilities by computer-assisted sperm motion analysis, sperm adenosine triphosphate (ATP) with luciferase, and plasma vitellogenin (VTG), thyroxine (T4), and triiodothyronine (T3) by immunoassay. Sperm apoptosis, viability, mitochondrial membrane potential, nuclear DNA fragmentation, and reproductive stage were measured by flow cytometry. Sperm quality parameters (except counts) and VTG were significantly different among sites, with correlations between VTG and 7 sperm parameters. Thyrocyte heights, T4, T3, gonadosomatic index and Fulton's condition factor differed among sites, but not significantly. Sperm quality was significantly lower and VTG higher where liver contaminants and water estrogen equivalents were highest (LV site). Total PCBs (specifically PCB-138, -146, -151, -170, -174, -177, -180, -183, -187, -194, and -206) and total PBDEs (specifically BDE-47, -100, -153, and -154) were negatively correlated with sperm motility. PCB-206 and BDE-154 were positively correlated with DNA fragmentation, and pentachloroanisole and VTG were positively correlated with sperm apoptosis and negatively correlated with ATP. BDE-99 was positively correlated with sperm counts and motility; T4 was negatively correlated with counts and positively correlated with motility, thus indicating possible androgenic mechanisms and thyroid endocrine disruption. Male LSS proved to be an informative model for studying reproductive and endocrine biomarkers in the LCR.

In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen.

PubMed

Salmani, Hossein; Towhidi, Armin; Zhandi, Mahdi; Bahreini, Majid; Sharafi, Mohsen

2014-04-01

Soybean lecithin is a suitable plant-based cryoprotectant for freezing ruminant sperm. Optimum level of lecithin was not clear for goat semen cryopreservation. The objective of this study was to investigate the effects of different levels of soybean lecithin in semen extender on post-thaw sperm quality including CASA-motion parameters, viability, plasma membrane integrity and lipid peroxidation. Semen samples were collected from 4 Mahabadi bucks using an artificial vagina. Different concentrations of soy lecithin (SL, 0.5%, 1%, 1.5%, 2% and 2.5% w/v) were compared to 15% (v/v) egg yolk-based extender (TR-EY). No significant difference was observed for sperm progressive motility, viability or plasma membrane integrity in 1.5% SL media (33.8%, 66%, and 62.7%, respectively) and TR-EY medium (35.4%, 67.2%, and 64.9%, respectively). Sperm motion characteristics (VAP, VSL, VCL, ALH and LIN) and rapid spermatozoa were improved with extender containing 1% and 1.5% SL, compared to TR-EY extender. Furthermore, egg yolk produced significantly higher malondialdehyde (4.02±0.21) than other groups. Results suggest that the optimal lecithin concentration in the semen extender was 1.5% and also soy lecithin can substitute for egg yolk during cryopreservation for caprine sperm. Copyright © 2014 Elsevier Inc. All rights reserved.

Spermatogenetic disorders in adult rats exposed to tributyltin chloride during puberty.

PubMed

Yu, Wook Joon; Lee, Beom Jun; Nam, Sang Yoon; Kim, Young Chul; Lee, Yong Soon; Yun, Young Won

2003-12-01

Adverse effects of tributyltin (TBT) chloride were investigated on the reproductive system in male adult rats as exposed during puberty. Fifty Sprague-Dawley rats at the age of 35 days were assigned to five different groups: negative control receiving vehicle, methyltestosterone (10 mg/kg B.W.), and TBT chloride treatments (5, 10, and 20 mg/kg B.W.). Animals were treated by oral gavage for ten consecutive days and sacrificed at 5 weeks after final treatment. The treatment of TBT chloride at the high dose of 20 mg/kg B.W. significantly decreased homogenization-resistant testicular sperm counts (p<0.05). The TBT chloride treatment at the doses of 10 and 20 mg/kg B.W. also significantly decreased caudal epididymal sperm counts (p<0.01). Some of motion kinematic parameters (motility, mean angular displacement, lateral head displacement, and dance) of sperms retrieved from vasa deference were significantly decreased in rats treated with the TBT chloride at the dose of 20 mg/kg B.W. (p<0.05). These results provide a further evidence that an exposure to TBT chloride during pubertal period in male rats produces spermatogenic disorders characterized by decreasing testicular and epididymal sperm counts and some motion parameters of sperms in the vasa deference.

Rhodiola sacra aqueous extract (RSAE) improves biochemical and sperm characteristics in cryopreserved boar semen.

PubMed

Zhao, H-W; Li, Q-W; Ning, G-Z; Han, Z-S; Jiang, Z-L; Duan, Y-F

2009-03-15

Although Rhodiola sacra aqueous extract (RSAE) has been used in many studies as an antioxidant, its effects on semen characteristics and its antioxidant properties during cryopreservation of boar sperm have never been evaluated. Semen was collected from five Duroc boars (2-4-year-old) twice weekly and frozen-thawed in extender with RSEA. Motion characteristics were assessed with a computer-aided semen analysis (CASA) system, whereas other sperm quality end points were assessed by routine methods. The effective concentration of RSEA in extender ranged from 4 to 8mg/L and the effect of RSEA on sperm quality was better in glycerol-free extender than extender containing glycerol (P<0.05). In frozen-thawed boar semen, there was a direct correlation (P<0.05) between RSEA concentration and glutathione (GSH) concentrations, mitochondrial activity, and hypoosmotic swelling test (HOST), and an inverse correlation (r=-0.982, P<0.05) between RSEA concentration and malondialdehyde (all end points were significantly higher at 6mg/L than in the control group). In summary: (i) the effective concentration of RSEA in extender ranged from 4 to 8mg/L; (ii) the effect of RSEA on sperm quality was better in extender without glycerol; and (iii) there was a significant correlation between RSEA concentrations and concentrations of GSH and MAD in frozen-thawed boar semen (antioxidant effects of RSEA were concentration-dependent). Further studies are needed to define the active ingredient in RSEA that protects boar sperm against ROS.

Effect of Sleep Deprivation on the Male Reproductive System in Rats.

PubMed

Choi, Ji Ho; Lee, Seung Hoon; Bae, Jae Hyun; Shim, Ji Sung; Park, Hong Seok; Kim, Young Sik; Shin, Chol

2016-10-01

There has been no study reporting on the influence of sleep deprivation on the male reproductive system including sperm quality. In this study, we hypothesized that sleep deprivation could lead to adverse effect on the male reproductive system. The rats were divided into three groups: 1) control (home-cage, n = 10); 2) SD4 (sleep deprivation for 4 days, n = 10); and 3) SD7 (sleep deprivation for 7 days, n = 10). Sleep deprivation was performed by a modified multiple platform method. Sperm quality (sperm motion parameters and counts), hormone levels (corticosterone and testosterone), and the histopathology of testis were evaluated and compared between the three groups. A statistically significant reduction (P = 0.018) was observed in sperm motility in the SD7 group compared to those of the control group. However, there were no significant differences in other sperm motion parameters, or in sperm counts of the testis and cauda epididymis between three groups. Compared with the control group, the SD4 (P = 0.033) and SD7 (P = 0.002) groups exhibited significant increases of corticosterone levels, but significant decreases of testosterone levels were found in the SD4 (P = 0.001) and SD7 (P < 0.001) groups. Seminiferous tubular atrophy and/or spermatid retention was partially observed in the SD4 and SD7 groups, compared with the normal histopathology of the control group. Sleep deprivation may have an adverse effect on the male reproductive system in rats.

Shape and shear guide sperm cells spiraling upstream

NASA Astrophysics Data System (ADS)

Kantsler, Vasily; Dunkel, Jorn; Goldstein, Raymond E.

2014-11-01

A major puzzle in biology is how mammalian sperm determine and maintain the correct swimming direction during the various phases of the sexual reproduction process. Currently debated mechanisms for sperm long range travel vary from peristaltic pumping to temperature sensing (thermotaxis) and direct response to fluid flow (rheotaxis), but little is known quantitatively about their relative importance. Here, we report the first quantitative experimental study of mammalian sperm rheotaxis. Using microfluidic devices, we investigate systematically the swimming behavior of human and bull sperm over a wide range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions and chirality of the flagellar beat leads to a stable upstream spiraling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilization. To rationalize these findings, we identify a minimal mathematical model that is capable of describing quantitatively the experimental observations.

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.

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.

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

PubMed

Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

2015-03-15

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa. Published by Elsevier Inc.

Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol.

PubMed

Silva, M A; Peixoto, G C X; Lima, G L; Bezerra, J A B; Campos, L B; Paiva, A L C; Paula, V V; Silva, A R

2012-08-01

The objective was to determine the effectiveness of a powdered coconut water-based extender (ACP-116c), plus various concentrations of egg-yolk and glycerol, as an alternative for cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were apportioned into aliquots that were diluted in Tris plus 10% egg yolk and 3% glycerol, or in ACP-116c plus 10 or 20% egg yolk and 1.5 or 3% glycerol. Samples were frozen in liquid nitrogen and, after 1 mo, thawed at 37 °C for 1 min. After thawing, samples were evaluated as reported for fresh semen, and also for sperm membrane integrity (fluorescent probes) and kinematic parameters (computerized analysis). Results were presented as means ± SEM. Freezing and thawing decreased sperm characteristics relative to fresh semen. Overall, ACP-116c plus 20% egg yolk and 3% glycerol provided better (P < 0.05) sperm motility and kinetic rating (48 ± 6.1% and 2.8 ± 0.2, respectively) after thawing than Tris extender (30.4 ± 5.7% and 2.4 ± 0.2). However, there were no differences (P > 0.05) among treatments with regard to the other sperm characteristics. Based on computerized motion analysis, total (26.5 ± 5.9%) and progressive (8.1 ± 2.2%) motility were best preserved (P < 0.05) with the above-mentioned treatment. In conclusion, a coconut water-based extender, ACP-116c, plus 20% egg yolk and 3% glycerol, was effective for cryopreservation of semen from collared peccaries. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Sperm kinematic, head morphometric and kinetic-morphometric subpopulations in the blue fox (Alopex lagopus).

PubMed

Soler, Carles; Contell, Jesús; Bori, Lorena; Sancho, María; García-Molina, Almudena; Valverde, Anthony; Segarvall, Jan

2017-01-01

This work provides information on the blue fox ejaculated sperm quality needed for seminal dose calculations. Twenty semen samples, obtained by masturbation, were analyzed for kinematic and morphometric parameters by using CASA-Mot and CASA-Morph system and principal component (PC) analysis. For motility, eight kinematic parameters were evaluated, which were reduced to PC1, related to linear variables, and PC2, related to oscillatory movement. The whole population was divided into three independent subpopulations: SP1, fast cells with linear movement; SP2, slow cells and nonoscillatory motility; and SP3, medium speed cells and oscillatory movement. In almost all cases, the subpopulation distribution by animal was significantly different. Head morphology analysis generated four size and four shape parameters, which were reduced to PC1, related to size, and PC2, related to shape of the cells. Three morphometric subpopulations existed: SP1: large oval cells; SP2: medium size elongated cells; and SP3: small and short cells. The subpopulation distribution differed between animals. Combining the kinematic and morphometric datasets produced PC1, related to morphometric parameters, and PC2, related to kinematics, which generated four sperm subpopulations - SP1: high oscillatory motility, large and short heads; SP2: medium velocity with small and short heads; SP3: slow motion small and elongated cells; and SP4: high linear speed and large elongated cells. Subpopulation distribution was different in all animals. The establishment of sperm subpopulations from kinematic, morphometric, and combined variables not only improves the well-defined fox semen characteristics and offers a good conceptual basis for fertility and sperm preservation techniques in this species, but also opens the door to use this approach in other species, included humans.

Sperm kinematic, head morphometric and kinetic-morphometric subpopulations in the blue fox (Alopex lagopus)

PubMed Central

Soler, Carles; Contell, Jesús; Bori, Lorena; Sancho, María; García-Molina, Almudena; Valverde, Anthony; Segarvall, Jan

2017-01-01

This work provides information on the blue fox ejaculated sperm quality needed for seminal dose calculations. Twenty semen samples, obtained by masturbation, were analyzed for kinematic and morphometric parameters by using CASA-Mot and CASA-Morph system and principal component (PC) analysis. For motility, eight kinematic parameters were evaluated, which were reduced to PC1, related to linear variables, and PC2, related to oscillatory movement. The whole population was divided into three independent subpopulations: SP1, fast cells with linear movement; SP2, slow cells and nonoscillatory motility; and SP3, medium speed cells and oscillatory movement. In almost all cases, the subpopulation distribution by animal was significantly different. Head morphology analysis generated four size and four shape parameters, which were reduced to PC1, related to size, and PC2, related to shape of the cells. Three morphometric subpopulations existed: SP1: large oval cells; SP2: medium size elongated cells; and SP3: small and short cells. The subpopulation distribution differed between animals. Combining the kinematic and morphometric datasets produced PC1, related to morphometric parameters, and PC2, related to kinematics, which generated four sperm subpopulations – SP1: high oscillatory motility, large and short heads; SP2: medium velocity with small and short heads; SP3: slow motion small and elongated cells; and SP4: high linear speed and large elongated cells. Subpopulation distribution was different in all animals. The establishment of sperm subpopulations from kinematic, morphometric, and combined variables not only improves the well-defined fox semen characteristics and offers a good conceptual basis for fertility and sperm preservation techniques in this species, but also opens the door to use this approach in other species, included humans. PMID:27751987

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

USGS Publications Warehouse

Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki

2015-01-01

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

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

PubMed

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

2016-10-01

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

Robotic ICSI (intracytoplasmic sperm injection).

PubMed

Lu, Zhe; Zhang, Xuping; Leung, Clement; Esfandiari, Navid; Casper, Robert F; Sun, Yu

2011-07-01

This paper is the first report of robotic intracytoplasmic sperm injection (ICSI). ICSI is a clinical procedure performed worldwide in fertility clinics, requiring pick-up of a single sperm and insertion of it into an oocyte (i.e., egg cell). Since its invention 20 years ago, ICSI has been conducted manually by a handful of highly skilled embryologists; however, success rates vary significantly among clinics due to poor reproducibility and inconsistency across operators. We leverage our work in robotic cell injection to realize robotic ICSI and aim ultimately, to standardize how clinical ICSI is performed. This paper presents some of the technical aspects of our robotic ICSI system, including a cell holding device, motion control, and computer vision algorithms. The system performs visual tracking of single sperm, robotic immobilization of sperm, aspiration of sperm with picoliter volume, and insertion of sperm into an oocyte with a high degree of reproducibility. The system requires minimal human involvement (requiring only a few computer mouse clicks), and is human operator skill independent. Using the hamster oocyte-human sperm model in preliminary trials, the robotic system demonstrated a high success rate of 90.0% and survival rate of 90.7% (n=120). © 2011 IEEE

Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome

PubMed Central

Yoon, Sung-Jae; Rahman, Md Saidur; Kwon, Woo-Sung; Park, Yoo-Jin; Pang, Myung-Geol

2016-01-01

Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation. PMID:27031703

Predominance of sperm motion in corners.

PubMed

Nosrati, Reza; Graham, Percival J; Liu, Qiaozhi; Sinton, David

2016-05-23

Sperm migration through the female tract is crucial to fertilization, but the role of the complex and confined structure of the fallopian tube in sperm guidance remains unknown. Here, by confocal imaging microchannels head-on, we distinguish corner- vs. wall- vs. bulk-swimming bull sperm in confined geometries. Corner-swimming dominates with local areal concentrations as high as 200-fold that of the bulk. The relative degree of corner-swimming is strongest in small channels, decreases with increasing channel size, and plateaus for channels above 200 μm. Corner-swimming remains predominant across the physiologically-relevant range of viscosity and pH. Together, boundary-following sperm account for over 95% of the sperm distribution in small rectangular channels, which is similar to the percentage of wall swimmers in circular channels of similar size. We also demonstrate that wall-swimming sperm travel closer to walls in smaller channels (~100 μm), where the opposite wall is within the hydrodynamic interaction length-scale. The corner accumulation effect is more than the superposition of the influence of two walls, and over 5-fold stronger than that of a single wall. These findings suggest that folds and corners are dominant in sperm migration in the narrow (sub-mm) lumen of the fallopian tube and microchannel-based sperm selection devices.

Predominance of sperm motion in corners

PubMed Central

Nosrati, Reza; Graham, Percival J.; Liu, Qiaozhi; Sinton, David

2016-01-01

Sperm migration through the female tract is crucial to fertilization, but the role of the complex and confined structure of the fallopian tube in sperm guidance remains unknown. Here, by confocal imaging microchannels head-on, we distinguish corner- vs. wall- vs. bulk-swimming bull sperm in confined geometries. Corner-swimming dominates with local areal concentrations as high as 200-fold that of the bulk. The relative degree of corner-swimming is strongest in small channels, decreases with increasing channel size, and plateaus for channels above 200 μm. Corner-swimming remains predominant across the physiologically-relevant range of viscosity and pH. Together, boundary-following sperm account for over 95% of the sperm distribution in small rectangular channels, which is similar to the percentage of wall swimmers in circular channels of similar size. We also demonstrate that wall-swimming sperm travel closer to walls in smaller channels (~100 μm), where the opposite wall is within the hydrodynamic interaction length-scale. The corner accumulation effect is more than the superposition of the influence of two walls, and over 5-fold stronger than that of a single wall. These findings suggest that folds and corners are dominant in sperm migration in the narrow (sub-mm) lumen of the fallopian tube and microchannel-based sperm selection devices. PMID:27211846

The fine structure of spermatozoa of Hydrolagus colliei (Chondrichthyes, Holocephali).

PubMed

Stanley, H P

1983-05-01

The ultrastructure of spermatozoa in Hydrolagus colliei is described. Basic similarities of structure to the sperm of the related elasmobranch fish are noted. The most significant features of sperm structure in Hydrolagus that differ from those of elasmobranch fish occur in the tail. The axoneme is eccentrically located and forms a double helix with a single longitudinal column. A second longitudinal column is reduced to a short remnant at the base of the tail. Microtubules within the axoneme are also helically disposed, a feature that is consistent with the rotating motion of the sperm. Abundant glycogen reserves are stored along the length of the tail.

Sperm Motility in Flow

NASA Astrophysics Data System (ADS)

Guasto, Jeffrey; Juarez, Gabriel; Stocker, Roman

2012-11-01

A wide variety of plants and animals reproduce sexually by releasing motile sperm that seek out a conspecific egg, for example in the reproductive tract for mammals or in the water column for externally fertilizing organisms. Sperm are aided in their quest by chemical cues, but must also contend with hydrodynamic forces, resulting from laminar flows in reproductive tracts or turbulence in aquatic habitats. To understand how velocity gradients affect motility, we subjected swimming sperm to a range of highly-controlled straining flows using a cross-flow microfluidic device. The motion of the cell body and flagellum were captured through high-speed video microscopy. The effects of flow on swimming are twofold. For moderate velocity gradients, flow simply advects and reorients cells, quenching their ability to cross streamlines. For high velocity gradients, fluid stresses hinder the internal bending of the flagellum, directly inhibiting motility. The transition between the two regimes is governed by the Sperm number, which compares the external viscous stresses with the internal elastic stresses. Ultimately, unraveling the role of flow in sperm motility will lead to a better understanding of population dynamics among aquatic organisms and infertility problems in humans.

Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments.

PubMed

Tilney, L G

1975-02-01

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.

Rheotaxis facilitates upstream navigation of mammalian sperm cells

PubMed Central

Kantsler, Vasily; Dunkel, Jörn; Blayney, Martyn; Goldstein, Raymond E

2014-01-01

A major puzzle in biology is how mammalian sperm maintain the correct swimming direction during various phases of the sexual reproduction process. Whilst chemotaxis may dominate near the ovum, it is unclear which cues guide spermatozoa on their long journey towards the egg. Hypothesized mechanisms range from peristaltic pumping to temperature sensing and response to fluid flow variations (rheotaxis), but little is known quantitatively about them. We report the first quantitative study of mammalian sperm rheotaxis, using microfluidic devices to investigate systematically swimming of human and bull sperm over a range of physiologically relevant shear rates and viscosities. Our measurements show that the interplay of fluid shear, steric surface-interactions, and chirality of the flagellar beat leads to stable upstream spiralling motion of sperm cells, thus providing a generic and robust rectification mechanism to support mammalian fertilisation. A minimal mathematical model is presented that accounts quantitatively for the experimental observations. DOI: http://dx.doi.org/10.7554/eLife.02403.001 PMID:24867640

Measuring sperm movement within the female reproductive tract using Fourier analysis.

PubMed

Nicovich, Philip R; Macartney, Erin L; Whan, Renee M; Crean, Angela J

2015-02-01

The adaptive significance of variation in sperm phenotype is still largely unknown, in part due to the difficulties of observing and measuring sperm movement in its natural, selective environment (i.e., within the female reproductive tract). Computer-assisted sperm analysis systems allow objective and accurate measurement of sperm velocity, but rely on being able to track individual sperm, and are therefore unable to measure sperm movement in species where sperm move in trains or bundles. Here we describe a newly developed computational method for measuring sperm movement using Fourier analysis to estimate sperm tail beat frequency. High-speed time-lapse videos of sperm movement within the female tract of the neriid fly Telostylinus angusticollis were recorded, and a map of beat frequencies generated by converting the periodic signal of an intensity versus time trace at each pixel to the frequency domain using the Fourier transform. We were able to detect small decreases in sperm tail beat frequency over time, indicating the method is sensitive enough to identify consistent differences in sperm movement. Fourier analysis can be applied to a wide range of species and contexts, and should therefore facilitate novel exploration of the causes and consequences of variation in sperm movement.

Spermiogram and sperm head morphometry assessed by multivariate cluster analysis results during adolescence (12-18 years) and the effect of varicocele

PubMed Central

Vásquez, Fernando; Soler, Carles; Camps, Patricia; Valverde, Anthony; García-Molina, Almudena

2016-01-01

This work evaluates sperm head morphometric characteristics in adolescents from 12 to 18 years of age, and the effect of varicocele. Volunteers between 150 and 224 months of age (mean 191, n = 87), who had reached oigarche by 12 years old, were recruited in the area of Barranquilla, Colombia. Morphometric analysis of sperm heads was performed with principal component (PC) and discriminant analysis. Combining seminal fluid and sperm parameters provided five PCs: two related to sperm morphometry, one to sperm motility, and two to seminal fluid components. Discriminant analysis on the morphometric results of varicocele and nonvaricocele groups did not provide a useful classification matrix. Of the semen-related PCs, the most explanatory (40%) was related to sperm motility. Two PCs, including sperm head elongation and size, were sufficient to evaluate sperm morphometric characteristics. Most of the morphometric variables were correlated with age, with an increase in size and decrease in the elongation of the sperm head. For head size, the entire sperm population could be divided into two morphometric subpopulations, SP1 and SP2, which did not change during adolescence. In general, for varicocele individuals, SP1 had larger and more elongated sperm heads than SP2, which had smaller and more elongated heads than in nonvaricocele men. In summary, sperm head morphometry assessed by CASA-Morph and multivariate cluster analysis provides a better comprehension of the ejaculate structure and possibly sperm function. Morphometric analysis provides much more information than data obtained from conventional semen analysis. PMID:27751986

Automated Analysis of Human Sperm Number and Concentration (Oligospermia) Using Otsu Threshold Method and Labelling

NASA Astrophysics Data System (ADS)

Susrama, I. G.; Purnama, K. E.; Purnomo, M. H.

2016-01-01

Oligospermia is a male fertility issue defined as a low sperm concentration in the ejaculate. Normally the sperm concentration is 20-120 million/ml, while Oligospermia patients has sperm concentration less than 20 million/ml. Sperm test done in the fertility laboratory to determine oligospermia by checking fresh sperm according to WHO standards in 2010 [9]. The sperm seen in a microscope using a Neubauer improved counting chamber and manually count the number of sperm. In order to be counted automatically, this research made an automation system to analyse and count the sperm concentration called Automated Analysis of Sperm Concentration Counters (A2SC2) using Otsu threshold segmentation process and morphology. Data sperm used is the fresh sperm directly in the analysis in the laboratory from 10 people. The test results using A2SC2 method obtained an accuracy of 91%. Thus in this study, A2SC2 can be used to calculate the amount and concentration of sperm automatically

The effects of dietary vitamin B12 deficiency on sperm maturation in developing and growing male rats.

PubMed

Watanabe, Toshiaki; Ohkawa, Keiko; Kasai, Shigenobu; Ebara, Shuhei; Nakano, Yoshihisa; Watanabe, Yoshitaka

2003-03-01

To evaluate the role of vitamin B12 on spermatogenesis, the effects of dietary vitamin B12 deficiency on sperm maturation in developing rat fetuses and young growing rats were examined. The vitamin B12-deficient diet was given to all the animals for three different periods: whole period (gestation to mature), gestation period (gestation to weaning), or immature period (3-12 weeks postnatal). Sperm examination revealed that the sperm count was markedly lower in male progeny (F1) that were vitamin B12-deficient during the whole period. In addition, a significantly higher number of abnormal sperm, such as tailless and amorphous sperm, was observed. In male rats that were vitamin B12-deficient during the immature period, the incidence of abnormal sperms was 14.4% and 4.8% for tailless and short tail, respectively. The motion rates, such as path velocity and straight line velocity, were decreased to 20-40% of the control value in rats that were vitamin B12-deficient both during the whole and gestation periods. However, no effects of vitamin B12 deficiency on sperm motility were observed during the immature and mature periods. From these findings, we suggest that dietary vitamin B12 deficiency during pregnancy may induce irreversible damage in the germ cells of embryos and affect the maturation of spermatozoa.

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.

Comparative study of Sperm Motility Analysis System and conventional microscopic semen analysis

PubMed Central

KOMORI, KAZUHIKO; ISHIJIMA, SUMIO; TANJAPATKUL, PHANU; FUJITA, KAZUTOSHI; MATSUOKA, YASUHIRO; TAKAO, TETSUYA; MIYAGAWA, YASUSHI; TAKADA, SHINGO; OKUYAMA, AKIHIKO

2006-01-01

Background and Aim:  Conventional manual sperm analysis still shows variations in structure, process and outcome although World Health Organization (WHO) guidelines present an appropriate method for sperm analysis. In the present study a new system for sperm analysis, Sperm Motility Analysis System (SMAS), was compared with manual semen analysis based on WHO guidelines. Materials and methods:  Samples from 30 infertility patients and 21 healthy volunteers were subjected to manual microscopic analysis and SMAS analysis, simultaneously. We compared these two methods with respect to sperm concentration and percent motility. Results:  Sperm concentrations obtained by SMAS (Csmas) and manual microscopic analyses on WHO guidelines (Cwho) were strongly correlated (Cwho = 1.325 × Csmas; r = 0.95, P < 0.001). If we excluded subjects with Csmas values >30 × 106 sperm/mL, the results were more similar (Cwho = 1.022 × Csmas; r = 0.81, P < 0.001). Percent motility obtained by SMAS (Msmas) and manual analysis on WHO guidelines (Mwho) were strongly correlated (Mwho = 1.214 × Msmas; r = 0.89, P < 0.001). Conclusions:  The data indicate that the results of SMAS and those of manual microscopic sperm analyses based on WHO guidelines are strongly correlated. SMAS is therefore a promising system for sperm analysis. (Reprod Med Biol 2006; 5: 195–200) PMID:29662398

Comparison of sperm subpopulation structures in first and second ejaculated semen from Japanese black bulls by a cluster analysis of sperm motility evaluated by a CASA system.

PubMed

Kanno, Chihiro; Sakamoto, Kentaro Q; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Katagiri, Seiji; Nagano, Masashi

2017-08-04

In the present study, bull sperm in the first and second ejaculates were divided into subpopulations based on their motility characteristics using a cluster analysis of data from computer-assisted sperm motility analysis (CASA). Semen samples were collected from 4 Japanese black bulls. Data from 9,228 motile sperm were classified into 4 clusters; 1) very rapid and progressively motile sperm, 2) rapid and circularly motile sperm with widely moving heads, 3) moderately motile sperm with heads moving frequently in a short length, and 4) poorly motile sperm. The percentage of cluster 1 varied between bulls. The first ejaculates had a higher proportion of cluster 2 and lower proportion of cluster 3 than the second ejaculates.

Pentoxifylline effects on capacitation and fertility of stallion epididymal sperm.

PubMed

Guasti, P N; Monteiro, G A; Maziero, R R D; Carmo, M T; Dell'Aqua, J A; Crespilho, A M; Rifai, E A; Papa, F O

2017-04-01

The aims of this study were to determinate whether pentoxifylline (PTX) increases the motion parameters of fresh and frozen-thawed equine epididymal spermatozoa, to evaluate the tyrosine phosphorylation of frozen-thawed epididymal sperm in the presence of PTX and to determine whether the PTX-treatment of stallion epididymal sperm prior to freezing improves the fertility response of mares to a reduced number of spermatozoa per insemination dose. Fifty epididymis were flushed with a skim milk based extender with or without PTX. The pre-treatment with PTX enhanced the sperm motility after being harvested (P<0.05); however the freeze-thaw process did not alter the sperm kinematics between control and treated samples (P>0.05). Plasma membrane integrity did not differ between control and PTX group after recovery and after thawing (P>0.05), as observed in tyrosine phosphorylation, which the PTX treatment did not alter the percentage of tail-associated immunofluorescence of cryopreserved epididymal sperm (P>0.05). For the fertility trial, different insemination groups were tested: 800×10 6 epididymal sperm (C800); 100×10 6 epididymal sperm (C100); 100×10 6 epididymal sperm recovered in an extender containing PTX (PTX100). The conception rates for C800; C100 and PTX100 were 68.7% (11/16); 31.5% (5/16) and 50% (8/16), respectively. The conception rate did not differ among groups (P>0.05), however, a low number of animals was used in this study. A trend toward significance (P=0.07) was observed between C800 and C100 groups. In conclusion, PTX has no deleterious effect on sperm motility, viability and capacitation of cryopreserved stallion epididymal sperm. The conventional artificial insemination with 100×10 6 sperm recovered with PTX ensures acceptable conception rates and maximize the limited number of doses of cryopreserved stallion epididymal sperm. Copyright © 2017 Elsevier B.V. All rights reserved.

Premises for fowl sperm preservation based on applied bioenergetics.

PubMed

Froman, D P

2014-02-01

The primary goal of this work was to test whether the sperm mobility assay could be used to derive mathematical relationships from which predictions could be made about sperm cell function. A precondition was random sampling from a pool of sperm. This precondition was met by centrifuging mobile sperm through 12% (wt/vol) Accudenz containing the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and then holding washed sperm at 20°C within buffered potassium chloride. These 2 conditions rendered washed sperm immobile at 20°C. Resumption of sperm mobility was independent of time (P > 0.8558) when sperm were reactivated at body temperature with 2 mM Ca(2+) in isotonic sodium chloride at pH 7.4. Reactivated sperm mobility was 93% of the prewash control. Subsequent experiments served to define a dose response, predict optimal conditions for in vitro sperm mobility, and show how sperm can recover from an imposed non-physiological condition. Thus, functions were derived from which predictions were made. Whereas the utility of BAPTA treatment was confirmed in a new context, such utility did not address the question of whole-cell Ca(2+) flux during sperm cell manipulation. This issue is pivotal for the application of bioenergetics to fowl sperm preservation. Therefore, the secondary goal of this research was to investigate sperm cell Ca(2+) flux using a simulation of conditions encountered by sperm during centrifugation through 12% (wt/vol) Accudenz. These conditions included a temperature of 30°C, a Ca(2+) sink, and no exogenous substrate. Sperm motion was measured with a Hobson SpermTracker. Data points conformed to parabolic functions when motile concentration and velocity were plotted as functions of time. In each case, maximums were observed, e.g., 26 min for motile concentration. The upswing was attributed to a redistribution of intracellular Ca(2+) whereas the downswing was attributed to sperm cell Ca(2+) depletion. A pronounced isothermal increase was observed for each variable when the Ca(2+) sink was overcome with exogenous Ca(2+). Experimental outcomes supported four testable premises applicable to fowl sperm preservation research: 1) the importance of sperm mobility phenotype, 2) the relationship between mitochondrial Ca(2+) cycling and sperm mobility, 3) the utility of the sperm mobility assay for predicting experimental outcomes, and 4) understanding mitochondrial Ca(2+) cycling in terms of whole-cell Ca(2+) flux.

Conformational dynamics and ligand binding in the multi-domain protein PDC109.

PubMed

Kim, Hyun Jin; Choi, Moo Young; Kim, Hyung J; Llinás, Miguel

2010-02-18

PDC109 is a modular multi-domain protein with two fibronectin type II (Fn2) repeats joined by a linker. It plays a major role in bull sperm binding to the oviductal epithelium through its interactions with phosphorylcholines (PhCs), a head group of sperm cell membrane lipids. The crystal structure of the PDC109-PhC complex shows that each PhC binds to the corresponding Fn2 domain, while the two domains are on the same face of the protein. Long timescale explicit solvent molecular dynamics (MD) simulations of PDC109, in the presence and absence of PhC, suggest that PhC binding strongly correlates with the relative orientation of choline-phospholipid binding sites of the two Fn2 domains; unless the two domains tightly bind PhCs, they tend to change their relative orientation by deforming the flexible linker. The effective PDC109-PhC association constant of 28 M(-1), estimated from their potential of mean force is consistent with the experimental result. Principal component analysis of the long timescale MD simulations was compared to the significantly less expensive normal mode analysis of minimized structures. The comparison indicates that difference between relative domain motions of PDC109 with bound and unbound PhC is captured by the first principal component in the principal component analysis as well as the three lowest normal modes in the normal mode analysis. The present study illustrates the use of detailed MD simulations to clarify the energetics of specific ligand-domain interactions revealed by a static crystallographic model, as well as their influence on relative domain motions in a multi-domain protein.

The impact of BMI on sperm parameters and the metabolite changes of seminal plasma concomitantly.

PubMed

Guo, Dan; Wu, Wei; Tang, Qiuqin; Qiao, Shanlei; Chen, Yiqiu; Chen, Minjian; Teng, Mengying; Lu, Chuncheng; Ding, Hongjuan; Xia, Yankai; Hu, Lingqing; Chen, Daozhen; Sha, Jiahao; Wang, Xinru

2017-07-25

The development of male infertility increased rapidly worldwide, which coinciding with the epidemic of obesity. However, the impact of weight abnormalities on sperm quality is still contestable. To assess the correlation between BMI and sperm parameters, we searched relevant articles in PubMed, Embase, Web of science, and Wanfang database published until June 2015 without language restriction. Otherwise, we also recruited some participants who attended fertility clinic as well as some general populations in this report. We performed a systematic review and meta-analysis about BMI and sperm parameters containing total sperm count, concentration, semen volume and sperm motility (overall and progressive). Metabolomic analysis of seminal plasma was performed to explore the mechanism from a new perspective. This study found standardized weighted mean differences (SMD) in sperm parameters (total sperm count, sperm concentration, and semen volume) of abnormal weight groups decreased to different degree compared to normal weight. Dose-response analysis found SMD of sperm count, sperm concentration and semen volume respectively fell 2.4%, 1.3% and 2.0% compared with normal weight for every 5-unit increase in BMI. Metabolomic analysis of seminal plasma showed that spermidine and spermine were likely to play a vital role in the spermatogenesis progress. This systematic review with meta-analysis has confirmed there was a relationship between BMI and sperm quality, suggesting obesity may be a detrimental factor of male infertility.

Sperm quality assessments for endangered razorback suckers Xyrauchen Texanus

USGS Publications Warehouse

Jenkins, Jill A.; Eilts, Bruce E.; Guitreau, Amy M.; Figiel, Chester R.; Draugelis-Dale, Rassa O.; Tiersch, Terrence R.

2011-01-01

Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckers Xyrauchen texanus collected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167–343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (S.E.M.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2; P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2; P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23–82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985; Pr=-0.83; P=0.0116) and mitochondrial function (r=-0.91; P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=-0.77; Pr=-0.66; P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.

Computer-aided sperm analysis: a useful tool to evaluate patient's response to varicocelectomy.

PubMed

Ariagno, Julia I; Mendeluk, Gabriela R; Furlan, María J; Sardi, M; Chenlo, P; Curi, Susana M; Pugliese, Mercedes N; Repetto, Herberto E; Cohen, Mariano

2017-01-01

Preoperative and postoperative sperm parameter values from infertile men with varicocele were analyzed by computer-aided sperm analysis (CASA) to assess if sperm characteristics improved after varicocelectomy. Semen samples of men with proven fertility (n = 38) and men with varicocele-related infertility (n = 61) were also analyzed. Conventional semen analysis was performed according to WHO (2010) criteria and a CASA system was employed to assess kinetic parameters and sperm concentration. Seminal parameters values in the fertile group were very far above from those of the patients, either before or after surgery. No significant improvement in the percentage normal sperm morphology (P = 0.10), sperm concentration (P = 0.52), total sperm count (P = 0.76), subjective motility (%) (P = 0.97) nor kinematics (P = 0.30) was observed after varicocelectomy when all groups were compared. Neither was significant improvement found in percentage normal sperm morphology (P = 0.91), sperm concentration (P = 0.10), total sperm count (P = 0.89) or percentage motility (P = 0.77) after varicocelectomy in paired comparisons of preoperative and postoperative data. Analysis of paired samples revealed that the total sperm count (P = 0.01) and most sperm kinetic parameters: curvilinear velocity (P = 0.002), straight-line velocity (P = 0.0004), average path velocity (P = 0.0005), linearity (P = 0.02), and wobble (P = 0.006) improved after surgery. CASA offers the potential for accurate quantitative assessment of each patient's response to varicocelectomy.

An efficient method for automatic morphological abnormality detection from human sperm images.

PubMed

Ghasemian, Fatemeh; Mirroshandel, Seyed Abolghasem; Monji-Azad, Sara; Azarnia, Mahnaz; Zahiri, Ziba

2015-12-01

Sperm morphology analysis (SMA) is an important factor in the diagnosis of human male infertility. This study presents an automatic algorithm for sperm morphology analysis (to detect malformation) using images of human sperm cells. The SMA method was used to detect and analyze different parts of the human sperm. First of all, SMA removes the image noises and enhances the contrast of the image to a great extent. Then it recognizes the different parts of sperm (e.g., head, tail) and analyzes the size and shape of each part. Finally, the algorithm classifies each sperm as normal or abnormal. Malformations in the head, midpiece, and tail of a sperm, can be detected by the SMA method. In contrast to other similar methods, the SMA method can work with low resolution and non-stained images. Furthermore, an image collection created for the SMA, has also been described in this study. This benchmark consists of 1457 sperm images from 235 patients, and is known as human sperm morphology analysis dataset (HSMA-DS). The proposed algorithm was tested on HSMA-DS. The experimental results show the high ability of SMA to detect morphological deformities from sperm images. In this study, the SMA algorithm produced above 90% accuracy in sperm abnormality detection task. Another advantage of the proposed method is its low computation time (that is, less than 9s), as such, the expert can quickly decide to choose the analyzed sperm or select another one. Automatic and fast analysis of human sperm morphology can be useful during intracytoplasmic sperm injection for helping embryologists to select the best sperm in real time. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Air Pollution and Quality of Sperm: A Meta-Analysis

PubMed Central

Fathi Najafi, Tahereh; Latifnejad Roudsari, Robab; Namvar, Farideh; Ghavami Ghanbarabadi, Vahid; Hadizadeh Talasaz, Zahra; Esmaeli, Mahin

2015-01-01

Context: Air pollution is common in all countries and affects reproductive functions in men and women. It particularly impacts sperm parameters in men. This meta-analysis aimed to examine the impact of air pollution on the quality of sperm. Evidence Acquisition: The scientific databases of Medline, PubMed, Scopus, Google scholar, Cochrane Library, and Elsevier were searched to identify relevant articles published between 1978 to 2013. In the first step, 76 articles were selected. These studies were ecological correlation, cohort, retrospective, cross-sectional, and case control ones that were found through electronic and hand search of references about air pollution and male infertility. The outcome measurement was the change in sperm parameters. A total of 11 articles were ultimately included in a meta-analysis to examine the impact of air pollution on sperm parameters. The authors applied meta-analysis sheets from Cochrane library, then data extraction, including mean and standard deviation of sperm parameters were calculated and finally their confidence interval (CI) were compared to CI of standard parameters. Results: The CI for pooled means were as follows: 2.68 ± 0.32 for ejaculation volume (mL), 62.1 ± 15.88 for sperm concentration (million per milliliter), 39.4 ± 5.52 for sperm motility (%), 23.91 ± 13.43 for sperm morphology (%) and 49.53 ± 11.08 for sperm count. Conclusions: The results of this meta-analysis showed that air pollution reduces sperm motility, but has no impact on the other sperm parameters of spermogram. PMID:26023349

Air pollution and quality of sperm: a meta-analysis.

PubMed

Fathi Najafi, Tahereh; Latifnejad Roudsari, Robab; Namvar, Farideh; Ghavami Ghanbarabadi, Vahid; Hadizadeh Talasaz, Zahra; Esmaeli, Mahin

2015-04-01

Air pollution is common in all countries and affects reproductive functions in men and women. It particularly impacts sperm parameters in men. This meta-analysis aimed to examine the impact of air pollution on the quality of sperm. The scientific databases of Medline, PubMed, Scopus, Google scholar, Cochrane Library, and Elsevier were searched to identify relevant articles published between 1978 to 2013. In the first step, 76 articles were selected. These studies were ecological correlation, cohort, retrospective, cross-sectional, and case control ones that were found through electronic and hand search of references about air pollution and male infertility. The outcome measurement was the change in sperm parameters. A total of 11 articles were ultimately included in a meta-analysis to examine the impact of air pollution on sperm parameters. The authors applied meta-analysis sheets from Cochrane library, then data extraction, including mean and standard deviation of sperm parameters were calculated and finally their confidence interval (CI) were compared to CI of standard parameters. The CI for pooled means were as follows: 2.68 ± 0.32 for ejaculation volume (mL), 62.1 ± 15.88 for sperm concentration (million per milliliter), 39.4 ± 5.52 for sperm motility (%), 23.91 ± 13.43 for sperm morphology (%) and 49.53 ± 11.08 for sperm count. The results of this meta-analysis showed that air pollution reduces sperm motility, but has no impact on the other sperm parameters of spermogram.

Predictive capacity of sperm quality parameters and sperm subpopulations on field fertility after artificial insemination in sheep.

PubMed

Santolaria, P; Vicente-Fiel, S; Palacín, I; Fantova, E; Blasco, M E; Silvestre, M A; Yániz, J L

2015-12-01

This study was designed to evaluate the relevance of several sperm quality parameters and sperm population structure on the reproductive performance after cervical artificial insemination (AI) in sheep. One hundred and thirty-nine ejaculates from 56 adult rams were collected using an artificial vagina, processed for sperm quality assessment and used to perform 1319 AI. Analyses of sperm motility by computer-assisted sperm analysis (CASA), sperm nuclear morphometry by computer-assisted sperm morphometry analysis (CASMA), membrane integrity by acridine orange-propidium iodide combination and sperm DNA fragmentation using the sperm chromatin dispersion test (SCD) were performed. Clustering procedures using the sperm kinematic and morphometric data resulted in the classification of spermatozoa into three kinematic and three morphometric sperm subpopulations. Logistic regression procedures were used, including fertility at AI as the dependent variable (measured by lambing, 0 or 1) and farm, year, month of AI, female parity, female lambing-treatment interval, ram, AI technician and sperm quality parameters (including sperm subpopulations) as independent factors. Sperm quality variables remaining in the logistic regression model were viability and VCL. Fertility increased for each one-unit increase in viability (by a factor of 1.01) and in VCL (by a factor of 1.02). Multiple linear regression analyses were also performed to analyze the factors possibly influencing ejaculate fertility (N=139). The analysis yielded a significant (P<0.05) relationship between sperm viability and ejaculate fertility. The discriminant ability of the different semen variables to predict field fertility was analyzed using receiver operating characteristic (ROC) curve analysis. Sperm viability and VCL showed significant, albeit limited, predictive capacity on field fertility (0.57 and 0.54 Area Under Curve, respectively). The distribution of spermatozoa in the different subpopulations was not related to fertility. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-06-01

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

Cluster analysis reveals seasonal variation of sperm subpopulations in extended boar semen

PubMed Central

IBĂNESCU, Iulian; LEIDING, Claus; BOLLWEIN, Heinrich

2017-01-01

This study aimed to identify motile sperm subpopulations in extended boar semen and to observe the presumptive seasonal variation in their distribution. Data from 4837 boar ejaculates collected over a two-year period were analyzed in terms of kinematic parameters by Computer Assisted Sperm Analysis (CASA). Individual sperm data were used to determine subgroups of motile sperm within the ejaculates using cluster analysis. Four motile sperm subpopulations (SP) were identified, with distinct movement patterns: SP1 sperm with high velocity and high linearity; SP2 sperm with high velocity but low linearity; SP3 sperm with low velocity but high linearity; and SP4 sperm with low velocity and low linearity. SP1 constituted the least overall proportion within the ejaculates (P < 0.05). Season of semen collection significantly influenced the different proportions of sperm subpopulations. Spring was characterized by similar proportions of SP1 and SP4 (NS) and higher proportions of SP3. Summer brought a decrease in both subgroups containing fast sperm (SP1 and SP2) (P < 0.05). During autumn, increases in SP2 and SP4 were recorded. Winter substantially affected the proportions of all sperm subpopulations (P < 0.05) and SP2 became the most represented subgroup, while SP1 (fast and linear) reached its highest proportion compared to other seasons. In conclusion, extended boar semen is structured in distinct motile sperm subpopulations whose proportions vary according to the season of collection. Summer and autumn seem to have a negative impact on the fast and linear subpopulation. Cluster analysis can be useful in revealing differences in semen quality that are not normally detected by classical evaluation based on mean values. PMID:29081440

Recent advances in bird sperm morphometric analysis and its role in male gamete characterization and reproduction technologies

PubMed Central

Santiago-Moreno, Julian; Esteso, Milagros Cristina; Villaverde-Morcillo, Silvia; Toledano-Díaz, Adolfo; Castaño, Cristina; Velázquez, Rosario; López-Sebastián, Antonio; Goya, Agustín López; Martínez, Javier Gimeno

2016-01-01

Postcopulatory sexual selection through sperm competition may be an important evolutionary force affecting many reproductive traits, including sperm morphometrics. Environmental factors such as pollutants, pesticides, and climate change may affect different sperm traits, and thus reproduction, in sensitive bird species. Many sperm-handling processes used in assisted reproductive techniques may also affect the size of sperm cells. The accurately measured dimensions of sperm cell structures (especially the head) can thus be used as indicators of environmental influences, in improving our understanding of reproductive and evolutionary strategies, and for optimizing assisted reproductive techniques (e.g., sperm cryopreservation) for use with birds. Computer-assisted sperm morphometry analysis (CASA-Morph) provides an accurate and reliable method for assessing sperm morphometry, reducing the problem of subjectivity associated with human visual assessment. Computerized systems have been standardized for use with semen from different mammalian species. Avian spermatozoa, however, are filiform, limiting their analysis with such systems, which were developed to examine the approximately spherical heads of mammalian sperm cells. To help overcome this, the standardization of staining techniques to be used in computer-assessed light microscopical methods is a priority. The present review discusses these points and describes the sperm morphometric characteristics of several wild and domestic bird species. PMID:27678467

MONTHLY VARIATION IN SPERM MOTILITY IN COMMON CARP ASSESSED USING COMPUTER-ASSISTED SPERM ANALYSIS (CASA)

EPA Science Inventory

Sperm motility variables from the milt of the common carp Cyprinus carpio were assessed using a computer-assisted sperm analysis (CASA) system across several months (March-August 1992) known to encompass the natural spawning period. Two-year-old pond-raised males obtained each mo...

A coordinated sequence of distinct flagellar waveforms enables a sharp flagellar turn mediated by squid sperm pH-taxis.

PubMed

Iida, Tomohiro; Iwata, Yoko; Mohri, Tatsuma; Baba, Shoji A; Hirohashi, Noritaka

2017-10-11

Animal spermatozoa navigate by sensing ambient chemicals to reach the site of fertilization. Generally, such chemicals derive from the female reproductive organs or cells. Exceptionally, squid spermatozoa mutually release and perceive carbon dioxide to form clusters after ejaculation. We previously identified the pH-taxis by which each spermatozoon can execute a sharp turn, but how flagellar dynamics enable this movement remains unknown. Here, we show that initiation of the turn motion requires a swim down a steep proton gradient (a theoretical estimation of ≥0.025 pH/s), crossing a threshold pH value of ~5.5. Time-resolved kinematic analysis revealed that the turn sequence results from the rhythmic exercise of two flagellar motions: a stereotypical flagellar 'bent-cane' shape followed by asymmetric wave propagation, which enables a sharp turn in the realm of low Reynolds numbers. This turning episode is terminated by an 'overshoot' trajectory that differs from either straight-line motility or turning. As with bidirectional pH-taxes in some bacteria, squid spermatozoa also showed repulsion from strong acid conditions with similar flagellar kinematics as in positive pH-taxis. These findings indicate that squid spermatozoa might have a unique reorientation mechanism, which could be dissimilar to that of classical egg-guided sperm chemotaxis in other marine invertebrates.

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.

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.

Modeling of Fluid-Membrane Interaction in Cellular Microinjection Process

NASA Astrophysics Data System (ADS)

Karzar-Jeddi, Mehdi; Diaz, Jhon; Olgac, Nejat; Fan, Tai-Hsi

2009-11-01

Cellular microinjection is a well-accepted method to deliver matters such as sperm, nucleus, or macromolecules into biological cells. To improve the success rate of in vitro fertilization and to establish the ideal operating conditions for a novel computer controlled rotationally oscillating intracytoplasmic sperm injection (ICSI) technology, we investigate the fluid-membrane interactions in the ICSI procedure. The procedure consists of anchoring the oocyte (a developing egg) using a holding pipette, penetrating oocyte's zona pellucida (the outer membrane) and the oolemma (the plasma or inner membrane) using an injection micropipette, and finally to deliver sperm into the oocyte for fertilization. To predict the large deformation of the oocyte membranes up to the piercing of the oolemma and the motion of fluids across both membranes, the dynamic fluid-pipette-membrane interactions are formulated by the coupled Stokes' equations and the continuum membrane model based on Helfrich's energy theory. A boundary integral model is developed to simulate the transient membrane deformation and the local membrane stress induced by the longitudinal motion of the injection pipette. The model captures the essential features of the membranes shown on optical images of ICSI experiments, and is capable of suggesting the optimal deformation level of the oolemma to start the rotational oscillations for piercing into the oolemma.

Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

NASA Astrophysics Data System (ADS)

Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

2017-04-01

Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa.

PubMed

Contri, Alberto; Valorz, Claudio; Faustini, Massimo; Wegher, Laura; Carluccio, Augusto

2010-08-01

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 x 10(6) sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 x 10(6) sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 x 10(6) sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used. Copyright 2010 Elsevier Inc. All rights reserved.

Gold-standard for computer-assisted morphological sperm analysis.

PubMed

Chang, Violeta; Garcia, Alejandra; Hitschfeld, Nancy; Härtel, Steffen

2017-04-01

Published algorithms for classification of human sperm heads are based on relatively small image databases that are not open to the public, and thus no direct comparison is available for competing methods. We describe a gold-standard for morphological sperm analysis (SCIAN-MorphoSpermGS), a dataset of sperm head images with expert-classification labels in one of the following classes: normal, tapered, pyriform, small or amorphous. This gold-standard is for evaluating and comparing known techniques and future improvements to present approaches for classification of human sperm heads for semen analysis. Although this paper does not provide a computational tool for morphological sperm analysis, we present a set of experiments for comparing sperm head description and classification common techniques. This classification base-line is aimed to be used as a reference for future improvements to present approaches for human sperm head classification. The gold-standard provides a label for each sperm head, which is achieved by majority voting among experts. The classification base-line compares four supervised learning methods (1- Nearest Neighbor, naive Bayes, decision trees and Support Vector Machine (SVM)) and three shape-based descriptors (Hu moments, Zernike moments and Fourier descriptors), reporting the accuracy and the true positive rate for each experiment. We used Fleiss' Kappa Coefficient to evaluate the inter-expert agreement and Fisher's exact test for inter-expert variability and statistical significant differences between descriptors and learning techniques. Our results confirm the high degree of inter-expert variability in the morphological sperm analysis. Regarding the classification base line, we show that none of the standard descriptors or classification approaches is best suitable for tackling the problem of sperm head classification. We discovered that the correct classification rate was highly variable when trying to discriminate among non-normal sperm heads. By using the Fourier descriptor and SVM, we achieved the best mean correct classification: only 49%. We conclude that the SCIAN-MorphoSpermGS will provide a standard tool for evaluation of characterization and classification approaches for human sperm heads. Indeed, there is a clear need for a specific shape-based descriptor for human sperm heads and a specific classification approach to tackle the problem of high variability within subcategories of abnormal sperm cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

PubMed

Margaryan, Hasmik; Dorosh, Andriy; Capkova, Jana; Manaskova-Postlerova, Pavla; Philimonenko, Anatoly; Hozak, Pavel; Peknicova, Jana

2015-03-08

Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

Quantitation of sperm bindable IgA and IgG in seminal fluid.

PubMed

Howe, S E; Lynch, D M

1986-05-01

Seminal fluid and serum from 95 infertile males were assayed for sperm bindable immunoglobulins using an indirect ELISA with whole target sperm. The ELISA method was compared to seminal fluid and serum immobilization and agglutination assays (functional assays). In this infertile group, the ELISA assay was positive in 22% of seminal fluids (greater than 1.2 fg IgA/sperm and greater than 0.3 fg IgG/sperm). The seminal fluid antibodies were IgA and had an accompanying elevated IgG component in 78% of patients. There was a 96% correlation between negative seminal fluid functional assays and negative ELISA, and a 95% correlation between positive seminal fluid functional assays and positive ELISA. Positive serum sperm antibody tests were found in 71% of the infertile males with positive seminal fluid sperm antibodies, but 29% of the infertile males with strongly positive IgA seminal fluid sperm antibodies showed normal levels of serum sperm antibodies by either ELISA or functional assays. The ELISA method gives reproducible quantitation of sperm antibodies in seminal fluid and correlates well with accepted functional assays. Comparisons with serum sperm antibody assays suggests that seminal fluid sperm antibody analysis complements the serum analysis of sperm antibodies.

Morphometric classification of Spanish thoroughbred stallion sperm heads.

PubMed

Hidalgo, Manuel; Rodríguez, Inmaculada; Dorado, Jesús; Soler, Carles

2008-01-30

This work used semen samples collected from 12 stallions and assessed for sperm morphometry by the Sperm Class Analyzer (SCA) computer-assisted system. A discriminant analysis was performed on the morphometric data from that sperm to obtain a classification matrix for sperm head shape. Thereafter, we defined six types of sperm head shape. Classification of sperm head by this method obtained a globally correct assignment of 90.1%. Moreover, significant differences (p<0.05) were found between animals for all the sperm head morphometric parameters assessed.

Exposure to levonorgestrel increases nest acquisition success and decreases sperm motility in the male fathead minnow (Pimephales promelas).

PubMed

Frankel, Tyler; Yonkos, Lance; Ampy, Franklin; Frankel, Jack

2018-04-01

Progestins are utilized as a component of human contraceptives, and commonly enter the environment via wastewater treatment plant effluent. Certain progestins activate fish androgen receptors and cause decreases in fecundity and masculinization of females. We used a nest acquisition assay and computer-assisted sperm analysis to examine the effects of levonorgestrel on male fathead minnow (Pimephales promelas) reproductive fitness. Males were exposed to 0, 10, or 100 ng/L levonorgestrel for 14 d. Combinations of a control male and a male from one of the treatments were placed into a competitive nesting assay, and the time each male spent holding the nest and time spent exhibiting aggressive behaviors were analyzed at 48 h postexposure. Semen samples were analyzed for total motility, straight-line velocity, curvilinear velocity, average path velocity, linearity, beat cross frequency, and wobble at 0, 30, 60, 90, and 120 s postactivation. Males exposed to either 10 or 100 ng/L of levonorgestrel exhibited increased nest acquisition success and lower levels of aggression compared with control-control pairings, as well as decreases in multiple sperm motion characteristics. Our results suggest that further research is required to ascertain the effects of levonorgestrel on male gamete quality and reproductive behaviors. Environ Toxicol Chem 2018;37:1131-1137. © 2017 SETAC. © 2017 SETAC.

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.

Methotrexate Reduces DNA Integrity in Sperm From Men With Inflammatory Bowel Disease.

PubMed

Ley, Dana; Jones, Jeffrey; Parrish, John; Salih, Sana; Caldera, Freddy; Tirado, Edna; Leader, Benjamin; Saha, Sumona

2018-06-01

There are few data on the effects of methotrexate on reproductive capacity in men with inflammatory bowel diseases (IBDs). We performed a case-control study to determine the effects of methotrexate on sperm quality and genetic integrity. We compared sperm samples from 7 men with IBD who had been exposed to methotrexate for at least 3 months with sperm samples collected from 1912 age-matched men at fertility centers (controls) where sperm parameters would be expected to be worse than those of the general population. Sperm were evaluated by basic semen analysis and advanced sperm integrity testing. In samples from men with IBD, all basic semen analysis parameters were within normal limits. However, these samples had reduced sperm integrity, based on significant increases in levels of DNA fragmentation and damage from oxidative stress compared with controls. Our findings indicate that methotrexate can reduce DNA integrity in sperm and cause damage via oxidative stress. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Comparison of effects of cryopreservation diluents on the ability of Ram sperm to reduce resazurin dye

USDA-ARS?s Scientific Manuscript database

Resazurin dye is an effective way to test the metabolism of sperm. As sperm move, they create metabolic waste which is detected by the dye. Another way sperm are evaluated is by Computer-Assisted Sperm Analysis (CASA). CASA detects motility, progression, curvilinear velocity, lateral head amplitude,...

Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis.

PubMed

Zhao, Xing-Chun; Wang, Le; Sun, Jing; Jiang, Bo-Wei; Zhang, Er-Li; Ye, Jian

2016-01-01

Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB-sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases.

Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis

PubMed Central

Zhao, Xing-Chun; Wang, Le; Sun, Jing; Jiang, Bo-Wei; Zhang, Er-Li; Ye, Jian

2016-01-01

Vaginal swabs taken in rape cases usually contain epithelial cells from the victim and sperm from the assailant and forensic DNA analysis requires separation of sperm from these cell mixtures. PH-20, which is a glycosylphosphatidylinositol-anchored hyaluronidase located on the head of sperm, has important functions in fertilization. Here we describe a newly developed method for sperm isolation using anti-PH-20 antibody-coupled immunomagnetic beads (anti-PH-20 IMBs). Optical microscopy and scanning electron microscopy showed the IMBs recognized the head of sperm specifically and exhibited a great capacity to capture sperm cells. However, we found it necessary to incubate the IMB–sperm complex with DNase I before sperm lysis in order to remove any female DNA completely. We compared the sensitivity of anti-PH-20 IMBs in sperm and epithelial cell discrimination to those coated with a different anti-sperm antibody (anti-SP-10, anti-ADAM2 or anti-JLP). Only the anti-PH-20 IMBs succeeded in isolating sperm from cell mixtures at a sperm/epithelial cell ratio of 103:105. Further, our method exhibited greater power and better stability for sperm isolation compared to the traditional differential lysis strategy. Taken together, the anti-PH-20 IMB method described here could be effective for the isolation of sperm needed to obtain a single-sourced DNA profile as an aid to identifying the perpetrator in sexual assault cases. PMID:27442128

Human sperm chromosome analysis after subzonal sperm insemination of hamster oocytes

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

Cozzi, J.

1994-09-01

Sperm microinjection techniques, subzonal sperm insemination (SUZI) and intracytoplasmic sperm injection (ICSI), have achieved a wide spread clinical application for the treatment of male infertility. To date, only one study has focused on sperm karyotypes after microinjection. Martin et al. reported a very high incidence of abnormal human sperm complements after ICSI into hamster oocytes. In the present study, are reported the first human sperm karyotypes after SUZI of hamster oocytes. Spermatozoa from two control donors were treated by calcium ionophore A23187 and injected under the zona of hamster eggs. The microinjected eggs were then cultured for cytogenetic analysis ofmore » the pronuclei. Out of 47 analyzed sperm chromosome metaphases, 5 (10.6%) were abnormal, 4 (8.5%) were hypohaploid and 1 (2.1%) had a structural abnormality. The sex ratio was not significantly different from the expected 1:1 ratio. Rates of chromosomal abnormalities in microinjected spermatozoa were similar to those observed in spermatozoa inseminated with zona free eggs, suggesting that SUZI procedure per se does not increase sperm chromosomal abnormalities.« less

Variance in total levels of phospholipase C zeta (PLC-ζ) in human sperm may limit the applicability of quantitative immunofluorescent analysis as a diagnostic indicator of oocyte activation capability.

PubMed

Kashir, Junaid; Jones, Celine; Mounce, Ginny; Ramadan, Walaa M; Lemmon, Bernadette; Heindryckx, Bjorn; de Sutter, Petra; Parrington, John; Turner, Karen; Child, Tim; McVeigh, Enda; Coward, Kevin

2013-01-01

To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. Laboratory study. University laboratory. Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Hybrid sterility and evolution in Hawaiian Drosophila: differential gene and allele-specific expression analysis of backcross males.

PubMed

Brill, E; Kang, L; Michalak, K; Michalak, P; Price, D K

2016-08-01

The Hawaiian Drosophila are an iconic example of sequential colonization, adaptive radiation and speciation on islands. Genetic and phenotypic analysis of closely related species pairs that exhibit incomplete reproductive isolation can provide insights into the mechanisms of speciation. Drosophila silvestris from Hawai'i Island and Drosophila planitibia from Maui are two closely related allopatric Hawaiian picture-winged Drosophila that produce sterile F1 males but fertile F1 females, a pattern consistent with Haldane's rule. Backcrossing F1 hybrid females between these two species to parental species gives rise to recombinant males with three distinct sperm phenotypes despite a similar genomic background: motile sperm, no sperm (sterile), and immotile sperm. We found that these three reproductive morphologies of backcross hybrid males produce divergent gene expression profiles in testes, as measured with RNA sequencing. There were a total of 71 genes significantly differentially expressed between backcross males with no sperm compared with those backcross males with motile sperm and immotile sperm, but no significant differential gene expression between backcross males with motile sperm and backcross males with immotile sperm. All of these genes were underexpressed in males with no sperm, including a number of genes with previously known activities in adult testis. An allele-specific expression analysis showed overwhelmingly more cis-divergent than trans-divergent genes, with no significant difference in the ratio of cis- and trans-divergent genes among the sperm phenotypes. Overall, the results indicate that the regulation of gene expression involved in sperm production likely diverged relatively rapidly between these two closely related species.

Development of a quantitative validation method for forensic investigation of human spermatozoa using a commercial fluorescence staining kit (SPERM HY-LITER™ Express).

PubMed

Takamura, Ayari; Watanabe, Ken; Akutsu, Tomoko

2016-11-01

In investigations of sexual assaults, as well as in identifying a suspect, the detection of human sperm is important. Recently, a kit for fluorescent staining of human spermatozoa, SPERM HY-LITER™, has become available. This kit allows for microscopic observation of the heads of human sperm using an antibody tagged with a fluorescent dye. This kit is specific to human sperm and provides easy detection by luminescence. However, criteria need to be established to objectively evaluate the fluorescent signals and to evaluate the staining efficiency of this kit. These criteria will be indispensable for investigation of forensic samples. In the present study, the SPERM HY-LITER™ Express kit, which is an improved version of SPERM HY-LITER™, was evaluated using an image analysis procedure using Laplacian and Gaussian methods. This method could be used to automatically select important regions of fluorescence produced by sperm. The fluorescence staining performance was evaluated and compared under various experimental conditions, such as for aged traces and in combination with other chemical staining methods. The morphological characteristics of human sperm were incorporated into the criteria for objective identification of sperm, based on quantified features of the fluorescent spots. Using the criteria, non-specific or insignificant fluorescent spots were excluded, and the specificity of the kit for human sperm was confirmed. The image analysis method and criteria established in this study are universal and could be applied under any experimental conditions. These criteria will increase the reliability of operator judgment in the analysis of human sperm samples in forensics.

The influence of benign prostatic hyperplasia on sperm morphological features and sperm DNA integrity in dogs.

PubMed

Flores, R B; Angrimani, Dsr; Rui, B R; Brito, M M; Abreu, R A; Vannucchi, C I

2017-04-01

Benign prostatic hyperplasia (BPH) has a high incidence in older intact dogs. Due to the increased prostatic oxidative stress and hormonal imbalance of BPH, sperm damage can arise, such as sperm morphological alterations and DNA fragmentation. This study aimed to compare the reproductive potential of healthy dogs and those affected by benign prostatic hyperplasia. Ten dogs were assigned to two experimental groups: dogs without BPH (control; n = 5) and dogs diagnosed with BPH (n = 5), based on clinical signs and ultrasonographic findings. Three semen collections were performed from each dog within one month and analysed using computer-assisted sperm analysis (CASA) and functional tests. Control group showed higher percentage of sperm DNA integrity (95 ± 1.8%) compared to the BPH group (79.2 ± 6.4%). On the other hand, the percentage of minor sperm defects, amplitude of lateral sperm head displacement of the spermatozoa and medium sperm mitochondrial activity were higher in the BPH group. In conclusion, BPH decreases sperm DNA integrity, increases mitochondrial activity, as well as modifies sperm movement pattern. Therefore, a careful sperm analysis of aged dogs with BPH is required before a reproductive programme can be established for such patients. © 2016 Blackwell Verlag GmbH.

Automated analysis of individual sperm cells using stain-free interferometric phase microscopy and machine learning.

PubMed

Mirsky, Simcha K; Barnea, Itay; Levi, Mattan; Greenspan, Hayit; Shaked, Natan T

2017-09-01

Currently, the delicate process of selecting sperm cells to be used for in vitro fertilization (IVF) is still based on the subjective, qualitative analysis of experienced clinicians using non-quantitative optical microscopy techniques. In this work, a method was developed for the automated analysis of sperm cells based on the quantitative phase maps acquired through use of interferometric phase microscopy (IPM). Over 1,400 human sperm cells from 8 donors were imaged using IPM, and an algorithm was designed to digitally isolate sperm cell heads from the quantitative phase maps while taking into consideration both the cell 3D morphology and contents, as well as acquire features describing sperm head morphology. A subset of these features was used to train a support vector machine (SVM) classifier to automatically classify sperm of good and bad morphology. The SVM achieves an area under the receiver operating characteristic curve of 88.59% and an area under the precision-recall curve of 88.67%, as well as precisions of 90% or higher. We believe that our automatic analysis can become the basis for objective and automatic sperm cell selection in IVF. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish

PubMed Central

Park, Daniel S.; Egnatchik, Robert A.; Bordelon, Hali; Tiersch, Terrence R.; Monroe, W. Todd

2013-01-01

Sperm viability in aquatic species is increasingly being evaluated by motility analysis via computer-assisted sperm analysis (CASA) following activation of sperm with manual dilution and mixing by hand. User variation can limit the speed and control over the activation process, preventing consistent motility analysis. This is further complicated by the short interval (i.e., less than 15 s) of burst motility in these species. The objectives of this study were to develop a staggered herringbone microfluidic mixer to: 1) activate small volumes of Danio pearl zebrafish (Danio albolineatus) sperm by rapid mixing with diluent, and 2) position sperm in a viewing chamber for motility evaluation using a standard CASA system. A herringbone micromixer was fabricated in polydimethylsiloxane (PDMS) to yield high quality smooth surfaces. Based on fluorescence microscopy, mixing efficiency exceeding 90% was achieved within 5 s for a range of flow rates (from 50 to 250 μL/h), with a correlation of mixing distances and mixing efficiency. For example, at the nominal flow rate of 100 μL/h, there was a significant difference in mixing efficiency between 3.5 mm (75 ± 4%; mean ± SD) and 7 mm (92 ± 2%; P = 0.002). The PDMS micromixer, integrated with standard volumetric slides, demonstrated activation of fresh zebrafish sperm with reduced user variation, greater control, and without morphologic damage to sperm. Analysis of zebrafish sperm viability by CASA revealed a statistically higher motility rate for activation by micromixing (56 ± 4%) than manual activation (45 ± 7%; n = 5, P = 0.011). This micromixer represented a first step in streamlining methods for consistent, rapid assessment of sperm quality for zebrafish and other aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the PDMS micromixer described herein will improve studies of germplasm physiology and cryopreservation. PMID:22494680

Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish.

PubMed

Park, Daniel S; Egnatchik, Robert A; Bordelon, Hali; Tiersch, Terrence R; Monroe, W Todd

2012-07-15

Sperm viability in aquatic species is increasingly being evaluated by motility analysis via computer-assisted sperm analysis (CASA) following activation of sperm with manual dilution and mixing by hand. User variation can limit the speed and control over the activation process, preventing consistent motility analysis. This is further complicated by the short interval (i.e., less than 15 s) of burst motility in these species. The objectives of this study were to develop a staggered herringbone microfluidic mixer to: 1) activate small volumes of Danio pearl zebrafish (Danio albolineatus) sperm by rapid mixing with diluent, and 2) position sperm in a viewing chamber for motility evaluation using a standard CASA system. A herringbone micromixer was fabricated in polydimethylsiloxane (PDMS) to yield high quality smooth surfaces. Based on fluorescence microscopy, mixing efficiency exceeding 90% was achieved within 5 s for a range of flow rates (from 50 to 250 μL/h), with a correlation of mixing distances and mixing efficiency. For example, at the nominal flow rate of 100 μL/h, there was a significant difference in mixing efficiency between 3.5 mm (75±4%; mean±SD) and 7 mm (92±2%; P=0.002). The PDMS micromixer, integrated with standard volumetric slides, demonstrated activation of fresh zebrafish sperm with reduced user variation, greater control, and without morphologic damage to sperm. Analysis of zebrafish sperm viability by CASA revealed a statistically higher motility rate for activation by micromixing (56±4%) than manual activation (45±7%; n=5, P=0.011). This micromixer represented a first step in streamlining methods for consistent, rapid assessment of sperm quality for zebrafish and other aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the PDMS micromixer described herein will improve studies of germplasm physiology and cryopreservation. Copyright © 2012 Elsevier Inc. All rights reserved.

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)*

PubMed Central

Yu, Heguo; Diao, Hua; Wang, Chunmei; Lin, Yan; Yu, Fudong; Lu, Hui; Xu, Wei; Li, Zheng; Shi, Huijuan; Zhao, Shimin; Zhou, Yuchuan; Zhang, Yonglian

2015-01-01

Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed associations between functional protein acetylation and sperm functions. PMID:25680958

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.

Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters.

PubMed

Boryshpolets, S; Kowalski, R K; Dietrich, G J; Dzyuba, B; Ciereszko, A

2013-10-15

In this study, we examined different computer-assisted sperm analysis (CASA) systems (CRISMAS, Hobson Sperm Tracker, and Image J CASA) on the exact same video recordings to evaluate the differences in sperm motility parameters related to the specific CASA used. To cover a wide range of sperm motility parameters, we chose 12-second video recordings at 25 and 50 Hz frame rates after sperm motility activation using three taxonomically distinct fish species (sterlet: Acipenser ruthenus L.; common carp: Cyprinus carpio L.; and rainbow trout: Oncorhynchus mykiss Walbaum) that are characterized by essential differences in sperm behavior during motility. Systematically higher values of velocity and beat cross frequency (BCF) were observed in video recordings obtained at 50 Hz frame frequency compared with 25 Hz for all three systems. Motility parameters were affected by the CASA and species used for analyses. Image J and CRISMAS calculated higher curvilinear velocity (VCL) values for rainbow trout and common carp at 25 Hz frequency compared with the Hobson Sperm Tracker, whereas at 50 Hz, a significant difference was observed only for rainbow trout sperm recordings. No significant difference was observed between the CASA systems for sterlet sperm motility at 25 and 50 Hz. Additional analysis of 1-second segments taken at three time points (1, 6, and 12 seconds of the recording) revealed a dramatic decrease in common carp and rainbow trout sperm speed. The motility parameters of sterlet spermatozoa did not change significantly during the 12-second motility period and should be considered as a suitable model for longer motility analyses. Our results indicated that the CASA used can affect motility results even when the same motility recordings are used. These results could be critically altered by the recording quality, time of analysis, and frame rate of camera, and could result in erroneous conclusions. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification Male Fertility Through Abnormalities Sperm Based Morphology (Teratospermia) using Invariant Moment Method

NASA Astrophysics Data System (ADS)

Syahputra, M. F.; Chairani, R.; Seniman; Rahmat, R. F.; Abdullah, D.; Napitupulu, D.; Setiawan, M. I.; Albra, W.; Erliana, C. I.; Andayani, U.

2018-03-01

Sperm morphology is still a standard laboratory analysis in diagnosing infertility in men. Manually identification of sperm form is still not accurate, the difficulty in seeing the form of the invisible sperm from the digital microscope image is often a weakness in the process of identification and takes a long time. Therefore, male fertility identification application system is needed Through sperm abnormalities based on sperm morphology (teratospermia). The method used is invariant moment method. This study uses 15 data testing and 20 data training sperm image. That the process of male fertility identification through sperm abnormalities based on sperm morphology (teratospermia) has an accuracy rate of 80.77%. Use of time to process Identification of male fertility through sperm abnormalities Based on sperm morphology (teratospermia) during 0.4369 seconds.

Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate.

PubMed

Reed, Michael L; Ezeh, Peace C; Hamic, Amanda; Thompson, Douglas J; Caperton, Charles L

2009-11-01

Semen specimens (one ejaculate from each of 20 consenting study participants) were subjected to routine semen analysis, an in vitro sperm binding assay (HBA), and a sperm chromatin dispersion assay (HaloSperm), both before and after cryopreservation using cryoprotectant media supplemented with either egg yolk or soy lecithin. Comparing the equivalency of the two phospholipid cryopreservation supplements with regard to postthaw functional parameters demonstrated that there were no statistically significant differences between the two supplements for [1] recovery of motile sperm, [2] maintenance of sperm cell morphology, [3] maintenance of the ability of sperm to bind to hyaluronate in vitro, or [4] maintenance of sperm DNA integrity.

Comparison of Cryopreserved Human Sperm between Ultra Rapid Freezing and Slow Programmable Freezing: Effect on Motility, Morphology and DNA Integrity.

PubMed

Tongdee, Pattama; Sukprasert, Matchuporn; Satirapod, Chonticha; Wongkularb, Anna; Choktanasiri, Wicham

2015-05-01

Cryopreservation of sperm is common methods to preserve male fertility. Sperm freezing, suggest slow programmable freezing caused lower change of sperm morphology than sperm freezing in vapor of liquid nitrogen. Ultra rapid freezing is easy to be worked on, less time, low cost and does not need high experience. To compare the effect on sperm motility, morphology and DNA integrity of post-thawed sperm after ultra rapid freezing and slow programmable freezing methods. Experimental study at laboratory of infertility unit, Department of Obstetrics and Gynecology, Faculty of Medicine Ramathibodi Hospital. Thirty-seven semen samples with normal semen analysis according to World Health Organization (WHO) 1999 [normal sperm volume ( 2 ml) and normal sperm concentration (≥ 20 x10(6)/ml) and sperm motility (≥ 50%)]. Semen samples were washed. Then each semen sample was divided into six cryovials. Two cryovials, 0.5 ml each, were cryopreserved by slow programmable freezing. Four 0.25 ml containing cryovials, were cryopreserved by ultra rapidfreezing method. After cryopreservationfor 1 month, thawedprocess was carried out at room temperature. Main outcomes are sperm motility was determined by Computer-Assisted Semen Analysis (CASA), sperm morphology was determined by eosin-methylene blue staining and sperm DNA integrity was assessed by TUNEL assay. Sperm motility was reduced significantly by both methods, from 70.4 (9.0)% to 29.1 (12.3)% in slowprogrammable freezing and to 19.7 (9.8)% in ultra rapid freezing (p < 0.05). Sperm motility decreased significantly more by ultra rapid freezing (p < 0.001). The percentage of normal sperm morphology and DNA integrity were also reduced significantly by both methods. However, no significant difference between the two methods was found (p > 0.05). Cryopreservation of human sperm for 1 month significantly decreased sperm motility, morphology and DNA integrity in both methods. However sperm motility was decreased more by ultra rapid freezing.

Appraisal and standardization of curvilinear velocity (VCL) cut-off values for CASA analysis of Japanese quail (Coturnix japonica) sperm.

PubMed

Farooq, U; Malecki, I A; Mahmood, M; Martin, G B

2017-06-01

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut-off values of "velocity curvilinear" (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA ® -CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut-off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut-off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut-off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the "25%" setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two-step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA ® -CASA categorization of quail sperm, sperm should be classed as "rapid" at VCL ≥160 μm/s and "slow" at VCL ≤45 μm/s. © 2017 Blackwell Verlag GmbH.

Effect of chamber characteristics, loading and analysis time on motility and kinetic variables analysed with the CASA-mot system in goat sperm.

PubMed

Del Gallego, R; Sadeghi, S; Blasco, E; Soler, C; Yániz, J L; Silvestre, M A

2017-02-01

Several factors unrelated to the semen samples could be influencing in the sperm motility analysis. The aim of the present research was to study the effect of four chambers with different characteristics, namely; slide-coverslip, Spermtrack, ISAS D4C10, and ISAS D4C20 on the sperm motility. The filling procedure (drop or capillarity) and analysis time (0, 120 and 240s), depth of chamber (10 or 20μm) and field on motility variables were analysed by use of the CASA-mot system in goat sperm. Use of the drop-filling chambers resulted in greater values than capillarity-filling chambers for all sperm motility and kinetic variables, except for LIN (64.5% compared with 56.3% of motility for drop- and capillarity-filling chambers respectively, P<0.05). There were no significant differences in total sperm motility between different chamber depths, however, use of the 20μm-chambers resulted in greater sperm progressive motility rate, VSL and LIN, and less VCL and VAP than chambers with a lesser depth. There was less sperm motility and lesser values for kinetic variables as time that elapsed increased between sample loading and sperm evaluation. For sperm motility, use of droplet-loaded chambers resulted in similar values of MOT in all microscopic fields, but sperm motility assessed in capillarity-loaded chambers was less in the central fields than in the outermost microscopic fields. For goats, it is recommended that sperm motility be analysed using the CASA-mot system with a drop-loaded chamber within 2min after filling the chamber. Copyright © 2016 Elsevier B.V. All rights reserved.

Is the quality of donated semen deteriorating? Findings from a 15 year longitudinal analysis of weekly sperm samples.

PubMed

Haimov-Kochman, Ronit; Har-Nir, Ruth; Ein-Mor, Eliana; Ben-Shoshan, Vered; Greenfield, Caryn; Eldar, Ido; Bdolah, Yuval; Hurwitz, Arye

2012-06-01

Studies suggest that global semen quality is declining, but the debate remains open owing to geographic variation. To evaluate temporal trends of sperm parameters - namely concentration, motility and total motile sperm count - in sperm donated during the period 1995-2009. In a retrospective longitudinal cohort study we analyzed the sperm count and motility of 2182 semen samples provided on a weekly basis by 58 young, healthy, fertile, university-educated, paid donors. Despite the lowering of criteria for sperm parameters satisfactory for donation that were implemented in 2004, 38% of applicants for sperm donation are now rejected based on semen quality as compared to a third of applicants 10-15 years ago (P < 0.001). If the old strict criteria were in place 88% of candidates would be rejected today (P < 0.0001). Over the study period, the average sperm parameters dropped from a concentration of 106 +/- 25 million spermatozoa/ml with 79% +/- 4.3% motility to 68 +/- 14 million/ ml with 66% +/- 4.5% motile sperm (P < 0.0001, P < 0.0001, respectively). The total motile sperm count per ejaculate also decreased, from 66.4 +/- 18.2 million to 48.7 +/- 12 million (P < 0.005). When the previous criteria were implemented for the analysis of the latest group of sperm donors, only 18% of donors had an acceptable sperm quality, with an average concentration of 87 +/- 12 million spermatozoa/ml, 73% +/- 2.6% motile sperm and total motile sperm count of 53.1 +/- 3.8 million per ejaculate - still significantly lower than 15 years ago (P= 0.01, P= 0.003, P= 0.058 respectively). The rapid deterioration of sperm quality among fertile semen donors is alarming and may lead to cessation of sperm donation programs.

CASA-Mot in mammals: an update.

PubMed

Yániz, J L; Silvestre, M A; Santolaria, P; Soler, C

2018-03-08

Sperm motility is one of the most widely used parameters of sperm quality. Computer-aided sperm motility analysis (CASA-Mot) systems were developed to reduce the subjectivity of sperm motility assessment, and have had broad scientific and practical acceptance. In this review, the sources of variation and current applications of this technology and its relationships with other sperm quality tests are described in detail. Despite remarkable advances in the technique, there is still great need for standardisation in many species, and the numerous factors that affect the results make it difficult to provide universally accepted criteria for classifying semen samples based on sperm motility characteristics. The main fields for CASA-Mot include the study of male fertility and pathologies, evaluation of the effects of physical and chemical agents, improvement of epidemiological survey studies, more precise calculation of seminal doses for farm animals, realisation of basic studies about sperm function, improvement of sperm technologies such as cryopreservation and quality control analysis. Numerous relationships have been established between CASA-Mot and other sperm quality tests, although most of these parameters are complementary. Future CASA-Mot systems will probably be able to integrate several sperm quality parameters with motility.

Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane.

PubMed

Du, Jian; Shen, Jian; Wang, Yuanxian; Pan, Chuanying; Pang, Weijun; Diao, Hua; Dong, Wuzi

2016-09-13

Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity.

Boar seminal plasma exosomes maintain sperm function by infiltrating into the sperm membrane

PubMed Central

Du, Jian; Shen, Jian; Wang, Yuanxian; Pan, Chuanying; Pang, Weijun; Diao, Hua; Dong, Wuzi

2016-01-01

Seminal plasma ingredients are important for maintenance of sperm viability. This study focuses on the effect of boar seminal plasma exosomes on sperm function during long-term liquid storage. Boar seminal plasma exosomes had typical nano-structure morphology as measured by scanning electron microscopy (SEM) and molecular markers such as AWN, CD9 and CD63 by western blot analysis. The effect on sperm parameters of adding different ratio of boar seminal plasma exosomes to boar sperm preparations was analyzed. Compared to the diluent without exosomes, the diluent with four times or sixteen times exosomes compared to original semen had higher sperm motility, prolonged effective survival time, improved sperm plasma membrane integrity (p < 0.05), increased total antioxidant capacity (T-AOC) activity and decreased malondialdehyde (MDA) content. The diluent containing four times concentration of exosomes compared to original semen was determined to inhibit premature capacitation, but not to influence capacitation induced in vitro. Inhibition of premature capacitation is likely related to the concentration of exosomes which had been demonstrated to transfer proteins including AWN and PSP-1 into sperm. In addition, using fluorescence microscopy and scanning electron microscopy analysis, it was demonstrated that exosomes in diluent were directly binding to the membrane of sperm head which could improve sperm plasma membrane integrity. PMID:27542209

Sperm chromatin structure assay results in Nigerian men with unexplained infertility

PubMed Central

Kolade, Charles Oluwabukunmi

2015-01-01

Objective Several publications have established a relationship between sperm DNA damage and male factor infertility, based on data from America, Europe, and Asia. This study aimed to compare the extent of sperm DNA damage in sperm samples from Nigerian men with unexplained infertility and in sperm samples from a fertile group composed of sperm donors who had successfully impregnated a female partner naturally or through assisted conception. Methods A total of 404 men underwent male fertility evaluation at Androcare Laboratories and Cryobank participated in this study. Semen analysis and a sperm chromatin structure assay (SCSA) were performed on all subjects. Results The men in the unexplained infertility group were slightly older than the men in the fertile sperm group (36±10 years vs. 32±6 years, p=0.051). No significant difference was observed between the two groups in semen analysis parameters (p≥0.05). Men in the unexplained infertility group with normal semen parameters had a significantly higher DNA fragmentation index (DFI) than men in the fertile sperm group (27.5%±7.0% vs. 14.1%±5.3%, p<0.05). In the unexplained infertility group, 63% of the men had a DFI greater than 20%, compared to 4% in the fertile sperm group. In the unexplained infertility group, 15.2% of the subjects had a DFI greater than 30%, compared to 1% in the fertile sperm group. Conclusion Our study showed that the SCSA may be a more reliable predictor of fertility potential than traditional semen analysis in cases of unexplained infertility. PMID:26473109

The Persistence of Sperm and the Development of Time Since Intercourse (TSI) Guidelines in Sexual Assault Cases at Forensic Science Ireland, Dublin, Ireland.

PubMed

Casey, David G; Domijan, Katarina; MacNeill, Sarah; Rizet, Damien; O'Connell, Declan; Ryan, Jennifer

2017-05-01

The persistence of sperm using confirmatory microscopic analysis, the persistence of sperm with tails, time since intercourse (TSI) analysis, and results from the acid phosphatase (AP) reaction from approximately 5581 swabs taken from circa 1450 sexual assault cases are presented. The observed proportions of sperm in the vagina and anus declines significantly after 48 h TSI, and sperm on oral swabs were observed up to 15 h TSI. The AP reaction as a predictor of sperm on intimate swabs is questioned. All AP reaction times gave a low true positive rate; 23% of sperm-positive swabs gave a negative AP reaction time. We show the AP reaction is an unsafe and an unreliable predictor of sperm on intimate swabs. We propose that TSI not AP informs precase assessment and the evaluative approach for sexual assault cases. To help inform an evaluative approach, TSI guidelines are presented. © 2016 American Academy of Forensic Sciences.

Effect of sesame on sperm quality of infertile men.

PubMed

Khani, Behnaz; Bidgoli, Soroor Rabbani; Moattar, Fariborz; Hassani, Hassan

2013-03-01

High level of semen reactive oxygen species is considered as an important factor in male infertility. Sesame has antioxidant properties, which could be effective on improvement of semen parameters. This study was designed to determine the effects of sesame on sperm quality. Twenty-five infertile men entered this clinical trial. They were treated with a 3-months course of taking 0.5 mg/kg sesame. The pre intervention sperm analysis (sperm count, motile sperm percentage and normal morphology sperm percentage) was compared with post treatment sperm analysis. Based on the post intervention seamen analysis, patients were advised to undergo either IVF or ICSI to assess their fertility status. There was significant improvement in the sperm count (10.56 ± 5.25 vs. 22.71 ± 30.14 million per ml) and motility (15.32 ± 13.58 vs. 23.32 ± 20.61 percent) after treatment with sesame (P value: 0.04 and <0.0001 respectively), but there was no significant improvement in sperm morphology after the treatment (10.72 ± 6.66 vs. 13.20 ± 11.14 percent, P value: 0.10). Three patients (12%) underwent IUI, which resulted in 1 successful pregnancy. Two patients (8%) underwent ICSI, which was not successful; however 2 (8%) patients had spontaneous pregnancy. Fortunately, all pregnancies led to live birth. Except 1 case of diarrhea, no other major side effect was reported. Sesame improved sperm count and motility, and can be prescribed as an effective and safe method for male factor infertility.

Sperm whale long-range echolocation sounds revealed by ANTARES, a deep-sea neutrino telescope.

PubMed

André, M; Caballé, A; van der Schaar, M; Solsona, A; Houégnigan, L; Zaugg, S; Sánchez, A M; Castell, J V; Solé, M; Vila, F; Djokic, D; Adrián-Martínez, S; Albert, A; Anghinolfi, M; Anton, G; Ardid, M; Aubert, J-J; Avgitas, T; Baret, B; Barrios-Martí, J; Basa, S; Bertin, V; Biagi, S; Bormuth, R; Bouwhuis, M C; Bruijn, R; Brunner, J; Busto, J; Capone, A; Caramete, L; Carr, J; Celli, S; Chiarusi, T; Circella, M; Coleiro, A; Coniglione, R; Costantini, H; Coyle, P; Creusot, A; Deschamps, A; De Bonis, G; Distefano, C; Di Palma, I; Donzaud, C; Dornic, D; Drouhin, D; Eberl, T; El Bojaddaini, I; Elsässer, D; Enzenhöfer, A; Fehn, K; Felis, I; Fusco, L A; Galatà, S; Gay, P; Geißelsöder, S; Geyer, K; Giordano, V; Gleixner, A; Glotin, H; Gracia-Ruiz, R; Graf, K; Hallmann, S; van Haren, H; Heijboer, A J; Hello, Y; Hernandez-Rey, J J; Hößl, J; Hofestädt, J; Hugon, C; Illuminati, G; James, C W; de Jong, M; Jongen, M; Kadler, M; Kalekin, O; Katz, U; Kießling, D; Kouchner, A; Kreter, M; Kreykenbohm, I; Kulikovskiy, V; Lachaud, C; Lahmann, R; Lefèvre, D; Leonora, E; Loucatos, S; Marcelin, M; Margiotta, A; Marinelli, A; Martínez-Mora, J A; Mathieu, A; Melis, K; Michael, T; Migliozzi, P; Moussa, A; Mueller, C; Nezri, E; Păvălaş, G E; Pellegrino, C; Perrina, C; Piattelli, P; Popa, V; Pradier, T; Racca, C; Riccobene, G; Roensch, K; Saldaña, M; Samtleben, D F E; Sanguineti, M; Sapienza, P; Schnabel, J; Schüssler, F; Seitz, T; Sieger, C; Spurio, M; Stolarczyk, Th; Sánchez-Losa, A; Taiuti, M; Trovato, A; Tselengidou, M; Turpin, D; Tönnis, C; Vallage, B; Vallée, C; Van Elewyck, V; Vivolo, D; Wagner, S; Wilms, J; Zornoza, J D; Zuñiga, J

2017-04-12

Despite dedicated research has been carried out to adequately map the distribution of the sperm whale in the Mediterranean Sea, unlike other regions of the world, the species population status is still presently uncertain. The analysis of two years of continuous acoustic data provided by the ANTARES neutrino telescope revealed the year-round presence of sperm whales in the Ligurian Sea, probably associated with the availability of cephalopods in the region. The presence of the Ligurian Sea sperm whales was demonstrated through the real-time analysis of audio data streamed from a cabled-to-shore deep-sea observatory that allowed the hourly tracking of their long-range echolocation behaviour on the Internet. Interestingly, the same acoustic analysis indicated that the occurrence of surface shipping noise would apparently not condition the foraging behaviour of the sperm whale in the area, since shipping noise was almost always present when sperm whales were acoustically detected. The continuous presence of the sperm whale in the region confirms the ecological value of the Ligurian sea and the importance of ANTARES to help monitoring its ecosystems.

Sperm whale long-range echolocation sounds revealed by ANTARES, a deep-sea neutrino telescope

NASA Astrophysics Data System (ADS)

André, M.; Caballé, A.; van der Schaar, M.; Solsona, A.; Houégnigan, L.; Zaugg, S.; Sánchez, A. M.; Castell, J. V.; Solé, M.; Vila, F.; Djokic, D.; Adrián-Martínez, S.; Albert, A.; Anghinolfi, M.; Anton, G.; Ardid, M.; Aubert, J.-J.; Avgitas, T.; Baret, B.; Barrios-Martí, J.; Basa, S.; Bertin, V.; Biagi, S.; Bormuth, R.; Bouwhuis, M. C.; Bruijn, R.; Brunner, J.; Busto, J.; Capone, A.; Caramete, L.; Carr, J.; Celli, S.; Chiarusi, T.; Circella, M.; Coleiro, A.; Coniglione, R.; Costantini, H.; Coyle, P.; Creusot, A.; Deschamps, A.; de Bonis, G.; Distefano, C.; di Palma, I.; Donzaud, C.; Dornic, D.; Drouhin, D.; Eberl, T.; El Bojaddaini, I.; Elsässer, D.; Enzenhöfer, A.; Fehn, K.; Felis, I.; Fusco, L. A.; Galatà, S.; Gay, P.; Geißelsöder, S.; Geyer, K.; Giordano, V.; Gleixner, A.; Glotin, H.; Gracia-Ruiz, R.; Graf, K.; Hallmann, S.; van Haren, H.; Heijboer, A. J.; Hello, Y.; Hernandez-Rey, J. J.; Hößl, J.; Hofestädt, J.; Hugon, C.; Illuminati, G.; James, C. W.; de Jong, M.; Jongen, M.; Kadler, M.; Kalekin, O.; Katz, U.; Kießling, D.; Kouchner, A.; Kreter, M.; Kreykenbohm, I.; Kulikovskiy, V.; Lachaud, C.; Lahmann, R.; Lefèvre, D.; Leonora, E.; Loucatos, S.; Marcelin, M.; Margiotta, A.; Marinelli, A.; Martínez-Mora, J. A.; Mathieu, A.; Melis, K.; Michael, T.; Migliozzi, P.; Moussa, A.; Mueller, C.; Nezri, E.; Păvălaş, G. E.; Pellegrino, C.; Perrina, C.; Piattelli, P.; Popa, V.; Pradier, T.; Racca, C.; Riccobene, G.; Roensch, K.; Saldaña, M.; Samtleben, D. F. E.; Sanguineti, M.; Sapienza, P.; Schnabel, J.; Schüssler, F.; Seitz, T.; Sieger, C.; Spurio, M.; Stolarczyk, Th.; Sánchez-Losa, A.; Taiuti, M.; Trovato, A.; Tselengidou, M.; Turpin, D.; Tönnis, C.; Vallage, B.; Vallée, C.; van Elewyck, V.; Vivolo, D.; Wagner, S.; Wilms, J.; Zornoza, J. D.; Zuñiga, J.

2017-04-01

Despite dedicated research has been carried out to adequately map the distribution of the sperm whale in the Mediterranean Sea, unlike other regions of the world, the species population status is still presently uncertain. The analysis of two years of continuous acoustic data provided by the ANTARES neutrino telescope revealed the year-round presence of sperm whales in the Ligurian Sea, probably associated with the availability of cephalopods in the region. The presence of the Ligurian Sea sperm whales was demonstrated through the real-time analysis of audio data streamed from a cabled-to-shore deep-sea observatory that allowed the hourly tracking of their long-range echolocation behaviour on the Internet. Interestingly, the same acoustic analysis indicated that the occurrence of surface shipping noise would apparently not condition the foraging behaviour of the sperm whale in the area, since shipping noise was almost always present when sperm whales were acoustically detected. The continuous presence of the sperm whale in the region confirms the ecological value of the Ligurian sea and the importance of ANTARES to help monitoring its ecosystems.

Sperm whale long-range echolocation sounds revealed by ANTARES, a deep-sea neutrino telescope

PubMed Central

André, M.; Caballé, A.; van der Schaar, M.; Solsona, A.; Houégnigan, L.; Zaugg, S.; Sánchez, A. M.; Castell, J. V.; Solé, M.; Vila, F.; Djokic, D.; Adrián-Martínez, S.; Albert, A.; Anghinolfi, M.; Anton, G.; Ardid, M.; Aubert, J.-J.; Avgitas, T.; Baret, B.; Barrios-Martí, J.; Basa, S.; Bertin, V.; Biagi, S.; Bormuth, R.; Bouwhuis, M. C.; Bruijn, R.; Brunner, J.; Busto, J.; Capone, A.; Caramete, L.; Carr, J.; Celli, S.; Chiarusi, T.; Circella, M.; Coleiro, A.; Coniglione, R.; Costantini, H.; Coyle, P.; Creusot, A.; Deschamps, A.; De Bonis, G.; Distefano, C.; Di Palma, I.; Donzaud, C.; Dornic, D.; Drouhin, D.; Eberl, T.; El Bojaddaini, I.; Elsässer, D.; Enzenhöfer, A.; Fehn, K.; Felis, I.; Fusco, L. A.; Galatà, S.; Gay, P.; Geißelsöder, S.; Geyer, K.; Giordano, V.; Gleixner, A.; Glotin, H.; Gracia-Ruiz, R.; Graf, K.; Hallmann, S.; van Haren, H.; Heijboer, A. J.; Hello, Y.; Hernandez-Rey, J. J.; Hößl, J.; Hofestädt, J.; Hugon, C.; Illuminati, G.; James, C. W.; de Jong, M.; Jongen, M.; Kadler, M.; Kalekin, O.; Katz, U.; Kießling, D.; Kouchner, A.; Kreter, M.; Kreykenbohm, I.; Kulikovskiy, V.; Lachaud, C.; Lahmann, R.; Lefèvre, D.; Leonora, E.; Loucatos, S.; Marcelin, M.; Margiotta, A.; Marinelli, A.; Martínez-Mora, J. A.; Mathieu, A.; Melis, K.; Michael, T.; Migliozzi, P.; Moussa, A.; Mueller, C.; Nezri, E.; Păvălaş, G. E.; Pellegrino, C.; Perrina, C.; Piattelli, P.; Popa, V.; Pradier, T.; Racca, C.; Riccobene, G.; Roensch, K.; Saldaña, M.; Samtleben, D. F. E.; Sanguineti, M.; Sapienza, P.; Schnabel, J.; Schüssler, F.; Seitz, T.; Sieger, C.; Spurio, M.; Stolarczyk, Th.; Sánchez-Losa, A.; Taiuti, M.; Trovato, A.; Tselengidou, M.; Turpin, D.; Tönnis, C.; Vallage, B.; Vallée, C.; Van Elewyck, V.; Vivolo, D.; Wagner, S.; Wilms, J.; Zornoza, J. D.; Zuñiga, J.

2017-01-01

Despite dedicated research has been carried out to adequately map the distribution of the sperm whale in the Mediterranean Sea, unlike other regions of the world, the species population status is still presently uncertain. The analysis of two years of continuous acoustic data provided by the ANTARES neutrino telescope revealed the year-round presence of sperm whales in the Ligurian Sea, probably associated with the availability of cephalopods in the region. The presence of the Ligurian Sea sperm whales was demonstrated through the real-time analysis of audio data streamed from a cabled-to-shore deep-sea observatory that allowed the hourly tracking of their long-range echolocation behaviour on the Internet. Interestingly, the same acoustic analysis indicated that the occurrence of surface shipping noise would apparently not condition the foraging behaviour of the sperm whale in the area, since shipping noise was almost always present when sperm whales were acoustically detected. The continuous presence of the sperm whale in the region confirms the ecological value of the Ligurian sea and the importance of ANTARES to help monitoring its ecosystems. PMID:28401960

In vitro alachlor effects on reactive oxygen species generation, motility patterns and apoptosis markers in human spermatozoa.

PubMed

Grizard, Geneviève; Ouchchane, Lemlih; Roddier, Héléne; Artonne, Christine; Sion, Benoît; Vasson, Marie-Paule; Janny, Laurent

2007-01-01

Due to its extensive production and application, the toxicity of chloracetanilide herbicide alachlor[2-chloro-2',6'-diethyl-N-(methoxymethyl)-acetanilide] should be evaluated to establish minimum effects. In this study, we have examined the in vitro effects of alachlor on human sperm motion using a computer-assisted sperm analyser (CASA). An evaluation of both reactive oxygen species (ROS) and markers of apoptosis was also performed to investigate the mechanism by which alachlor modifies the sperm movement. After exposure up to 2 h to alachlor (0, 0.18, 0.37, 0.90 and 1.85 mM), the percentage of viable, motile spermatozoa and sperm velocities were concentration and/or time dependently decreased. The most sensitive parameters were progressive motility, mean average path velocity and mean straight velocity. Alachlor (1.85 mM) induced an increase in ROS production. A decrease of mitochondrial membrane potential (DeltaPsi(m)), an increase of both phosphatidylserine (PS) externalization and DNA fragmentation, which were concentration and/or time dependent, were also observed. It is possible that toxic effects of alachlor result in an oxidative stress which could act as a mediator of apoptosis. Alachlor could also contribute to some hypofertility cases.

Selected sperm traits are simultaneously altered after scrotal heat stress and play specific roles in in vitro fertilization and embryonic development.

PubMed

Lucio, Aline C; Alves, Benner G; Alves, Kele A; Martins, Muller C; Braga, Lucas S; Miglio, Luisa; Alves, Bruna G; Silva, Thiago H; Jacomini, José O; Beletti, Marcelo E

2016-09-01

Improvements in the estimation of male fertility indicators require advances in laboratory tests for sperm assessment. The aims of the present work were (1) to apply a multivariate analysis to examine sperm set of alterations and interactions and (2) to evaluate the importance of sperm parameters on the outcome of standard IVF and embryonic development. Bulls (n = 3) were subjected to scrotal insulation, and ejaculates were collected before (preinsulation = Day 0) and through 56 days (Days 7, 14, 21, 28, 35, 42, 49, and 56) of the experimental period. Sperm head morphometry and chromatin variables were assessed by a computational image analysis, and IVF was performed. Scrotal heat stress induced alterations in all evaluated sperm head features, as well as cleavage and blastocyst rates. A principal component analysis revealed three main components (factors) that represented almost 89% of the cumulative variance. In addition, an association of factor scores with cleavage (factor 1) and blastocyst (factor 3) rates was observed. In conclusion, several sperm traits were simultaneously altered as a result of a thermal insult. These sperm traits likely play specific roles in IVF and embryonic development. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

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.

Detecting coevolution in mammalian sperm-egg fusion proteins.

PubMed

Claw, Katrina G; George, Renee D; Swanson, Willie J

2014-06-01

Interactions between sperm and egg proteins can occur physically between gamete surface-binding proteins, and genetically between gamete proteins that work in complementary pathways in which they may not physically interact. Physically interacting sperm-egg proteins have been functionally identified in only a few species, and none have been verified within mammals. Candidate genes on both the sperm and egg surfaces exist, but gene deletion studies do not support functional interactions between these sperm-egg proteins; interacting sperm-egg proteins thus remain elusive. Cooperative gamete proteins undergo rapid evolution, and it is predicted that these sperm-egg proteins will also have correlated evolutionary rates due to compensatory changes on both the sperm and egg. To explore potential physical and genetic interactions in sperm-egg proteins, we sequenced four candidate genes from diverse primate species, and used regression and likelihood methods to test for signatures of coevolution between sperm-egg gene pairs. With both methods, we found that the egg protein CD9 coevolves with the sperm protein IZUMO1, suggesting a physical or genetic interaction occurs between them. With regression analysis, we found that CD9 and CRISP2 have correlated rates of evolution, and with likelihood analysis, that CD9 and CRISP1 have correlated rates. This suggests that the different tests may reflect different levels of interaction, be it physical or genetic. Coevolution tests thus provide an exploratory method for detecting potentially interacting sperm-egg protein pairs. © 2014 Wiley Periodicals, Inc.

Sperm and Spermatids Contain Different Proteins and Bind Distinct Egg Factors

PubMed Central

Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J.; Gurdon, John B.; Jullien, Jerome

2014-01-01

Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development. PMID:25244019

Analysis of limited fertility in intracytoplasmic sperm injection of sperm obtained by electroejaculation

PubMed Central

Nakamura, Yoshihiro; Kitamura, Masaya; Nishimura, Kenji; Tsujimura, Akira; Takeyama, Masami; Kondoh, Nobuyuki; Miyazaki, Kazunori; Okuyama, Akihiko

2004-01-01

Background and Aims:  We correlated findings in semen from patients with ejaculatory dysfunction with results of in vitro fertilization using their electroejaculated sperm. Methods and Results:  Electroejaculation was carried out in six patients with the above‐mentioned criteria for a total of eight times. Sperm was obtained in six attempts. Intracytoplasmic injection of these sperm was performed in 156 eggs. Sixty‐seven eggs were fertilized; most of these were injected with motile sperm. Two women became pregnant, both after injection with motile sperm. As previously reported, electroejaculated sperm showed low motility and a low fertilization rate, but even motile sperm had a low fertilization rate. Conclusion:  The results of the present study suggest the importance in fertilization of undetermined factors in addition to sperm motility. (Reprod Med Biol 2004; 3: 9–12) PMID:29662380

Computer-aided sperm analysis (CASA) in the medical laboratory: CASA in diagnostic andrology and assisted conception.

PubMed

Tomlinson, Mathew J; Naeem, Asad

2018-03-21

CASA has been used in reproductive medicine and pathology laboratories for over 25 years, yet the 'fertility industry' generally remains sceptical and has avoided automation, despite clear weaknesses in manual semen analysis. Early implementers had difficulty in validating CASA-Mot instruments against recommended manual methods (haemocytometer) due to the interference of seminal debris and non-sperm cells, which also affects the accuracy of grading motility. Both the inability to provide accurate sperm counts and a lack of consensus as to the value of sperm kinematic parameters appear to have continued to have a negative effect on CASA-Mot's reputation. One positive interpretation from earlier work is that at least one or more measures of sperm velocity adds clinical value to the semen analysis, and these are clearly more objective than any manual motility analysis. Moreover, recent CASA-Mot systems offer simple solutions to earlier problems in eliminating artefacts and have been successfully validated for sperm concentration; as a result, they should be viewed with more confidence in relation to motility grading. Sperm morphology and DNA testing both require an evidence-based consensus and a well-validated (reliable, reproducible) assay to be developed before automation of either can be of real clinical benefit.

Validation of the sperm class analyser CASA system for sperm counting in a busy diagnostic semen analysis laboratory.

PubMed

Dearing, Chey G; Kilburn, Sally; Lindsay, Kevin S

2014-03-01

Sperm counts have been linked to several fertility outcomes making them an essential parameter of semen analysis. It has become increasingly recognised that Computer-Assisted Semen Analysis (CASA) provides improved precision over manual methods but that systems are seldom validated robustly for use. The objective of this study was to gather the evidence to validate or reject the Sperm Class Analyser (SCA) as a tool for routine sperm counting in a busy laboratory setting. The criteria examined were comparison with the Improved Neubauer and Leja 20-μm chambers, within and between field precision, sperm concentration linearity from a stock diluted in semen and media, accuracy against internal and external quality material, assessment of uneven flow effects and a receiver operating characteristic (ROC) analysis to predict fertility in comparison with the Neubauer method. This work demonstrates that SCA CASA technology is not a standalone 'black box', but rather a tool for well-trained staff that allows rapid, high-number sperm counting providing errors are identified and corrected. The system will produce accurate, linear, precise results, with less analytical variance than manual methods that correlate well against the Improved Neubauer chamber. The system provides superior predictive potential for diagnosing fertility problems.

A Novel Approach to Identifying Physical Markers of Cryo-Damage in Bull Spermatozoa

PubMed Central

Yoon, Sung-Jae; Kwon, Woo-Sung; Rahman, Md Saidur; Lee, June-Sub; Pang, Myung-Geol

2015-01-01

Cryopreservation is an efficient way to store spermatozoa and plays a critical role in the livestock industry as well as in clinical practice. During cryopreservation, cryo-stress causes substantial damage to spermatozoa. In present study, the effects of cryo-stress at various cryopreservation steps, such as dilution / cooling, adding cryoprtectant, and freezing were studied in spermatozoa collected from 9 individual bull testes. The motility (%), motion kinematics, capacitation status, mitochondrial activity, and viability of bovine spermatozoa at each step of the cryopreservation process were assessed using computer-assisted sperm analysis, Hoechst 33258/chlortetracycline fluorescence, rhodamine 123 staining, and hypo-osmotic swelling test, respectively. The results demonstrate that the cryopreservation steps reduced motility (%), rapid speed (%), and mitochondrial activity, whereas medium/slow speed (%), and the acrosome reaction were increased (P < 0.05). Differences (Δ) of the acrosome reaction were higher in dilution/cooling step (P < 0.05), whereas differences (Δ) of motility, rapid speed, and non-progressive motility were higher in cryoprotectant and freezing as compared to dilution/cooling (P < 0.05). On the other hand, differences (Δ) of mitochondrial activity, viability, and progressive motility were higher in freezing step (P < 0.05) while the difference (Δ) of the acrosome reaction was higher in dilution/cooling (P < 0.05). Based on these results, we propose that freezing / thawing steps are the most critical in cryopreservation and may provide a logical ground of understanding on the cryo-damage. Moreover, these sperm parameters might be used as physical markers of sperm cryo-damage. PMID:25938413

The correlation of sperm morphology with unexplained recurrent spontaneous abortion: A systematic review and meta-analysis

PubMed Central

Cao, Xiaodan; Cui, Yun; Zhang, Xiaoxia; Lou, Jiangtao; Zhou, Jun; Wei, Renxiong

2017-01-01

Sperm morphology displays a potential impact on sperm function and may ultimately impact reproductive function. Current studies have investigated the correlation between sperm morphology with unexplained recurrent spontaneous abortion (RSA) but have shown inconsistent results. Hence, we systematically searched MEDLINE, EMBASE, CNKI databases, as well as the Cochrane Library for studies that examined the association between sperm morphology and unexplained RSA. Fifteen studies were identified, including 883 cases and 530 controls. Our meta-analysis results indicated that the percentage of normal sperm morphology from men with RSA partners was significantly lower than those from normal controls(SMD [95% CI]: − 0.60 [−0.81, −0.40]; P<0.00001) and the percentage of sperm morphologic alterations was significantly higher in patients with RSA compared with the control group (SMD [95% CI]: 0.92 [0.42, 1.43]; P=0.0004). The present study suggested that the percentage of normal sperm morphology may indeed decrease in men from RSA group compared with controls. However, there were some limitations in the study such as the differences in stain techniques and classification criteria. Further evidences are needed to better elucidate the relationship between sperm morphology and unexplained RSA. PMID:28903451

The impact of varicocelectomy on sperm parameters: a meta-analysis.

PubMed

Schauer, Ingrid; Madersbacher, Stephan; Jost, Romy; Hübner, Wilhelm Alexander; Imhof, Martin

2012-05-01

We determined the impact of 3 surgical techniques (high ligation, inguinal varicocelectomy and the subinguinal approach) for varicocelectomy on sperm parameters (count and motility) and pregnancy rates. By searching the literature using MEDLINE and the Cochrane Library with the last search performed in February 2011, focusing on the last 20 years, a total of 94 articles published between 1975 and 2011 reporting on sperm parameters before and after varicocelectomy were identified. Inclusion criteria for this meta-analysis were at least 2 semen analyses (before and 3 or more months after the procedure), patient age older than 19 years, clinical subfertility and/or abnormal semen parameters, and a clinically palpable varicocele. To rule out skewing factors a bias analysis was performed, and statistical analysis was done with RevMan5(®) and SPSS 15.0(®). A total of 14 articles were included in the statistical analysis. All 3 surgical approaches led to significant or highly significant postoperative improvement of both parameters with only slight numeric differences among the techniques. This difference did not reach statistical significance for sperm count (p = 0.973) or sperm motility (p = 0.372). After high ligation surgery sperm count increased by 10.85 million per ml (p = 0.006) and motility by 6.80% (p <0.00001) on the average. Inguinal varicocelectomy led to an improvement in sperm count of 7.17 million per ml (p <0.0001) while motility changed by 9.44% (p = 0.001). Subinguinal varicocelectomy provided an increase in sperm count of 9.75 million per ml (p = 0.002) and sperm motility by 12.25% (p = 0.001). Inguinal varicocelectomy showed the highest pregnancy rate of 41.48% compared to 26.90% and 26.56% after high ligation and subinguinal varicocelectomy, respectively, and the difference was statistically significant (p = 0.035). This meta-analysis suggests that varicocelectomy leads to significant improvements in sperm count and motility regardless of surgical technique, with the inguinal approach offering the highest pregnancy rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Media and dilution procedures tested to minimize handling effects on human, rabbit, and bull sperm for computer-assisted sperm analysis (CASA).

PubMed

Farrell, P B; Foote, R H; McArdle, M M; Trouern-Trend, V L; Tardif, A L

1996-01-01

Proper handling of semen prior to computer-assisted sperm analysis (CASA) is critical if the analysis is to be representative of the fresh sample. The effects of diluting medium or dilution and holding time before CASA on multiple sperm characteristics were studied. Four replicates of unselected semen samples from each of eight human donors were diluted with phosphate-buffered saline (PBS)-glucose plus bovine serum albumin (BSA), with Tyrode's albumen lactate pyruvate (TALP), and with high-potassium TALP (K-TALP) to a concentration of approximately 25 x 10(6) sperm/ml. The diluted semen was held for 0, 1, and 2 hours at approximately 30 degrees C before CASA, with little difference between the three diluents in all 12 variables measured. There was a decline of 3-6% in the proportion of motile sperm over a 2-hour period (P < 0.05). Donors were the largest source of differences (P < 0.05). Rabbit sperm (five bucks, four ejaculates per buck) were processed in a manner similar to that of the human sperm. There was a major effect of media. The average percentages of motile sperm over 2 hours in TALP, K-TALP, and PBS were 76, 42, and 29%, respectively (P < 0.05), with a decline of only 3% in TALP during the 2 hours. Hyperactivity and other characteristics were affected by treatment. Donors were a large source of variation. Bull semen (10 bulls, two ejaculates per bull) either was not diluted or diluted with TALP 2x or 4x and held for 0, 1, and 2 hours at 30 degrees C. It was then diluted to 25 x 10(6) sperm/ml with TALP. There was little change in most sperm characteristics in any treatment during the first hour, although many of the changes were statistically significant. The percentage of motile sperm in undiluted semen declined from 87% to 82% over 2 hours. Modified TALP was a suitable medium for sperm from all three species, and a simple PBS-glucose-BSA medium can be used for human sperm.

The Effect of Omega-3 Fatty Acids, EPA, and/or DHA on Male Infertility: A Systematic Review and Meta-analysis.

PubMed

Hosseini, Banafshe; Nourmohamadi, Mahdieh; Hajipour, Shima; Taghizadeh, Mohsen; Asemi, Zatollah; Keshavarz, Seyed Ali; Jafarnejad, Sadegh

2018-02-16

The objective was to evaluate the effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on sperm parameters including total sperm concentration, sperm motility, sperm DHA, and seminal plasma DHA concentration in infertile men. The literature search was conducted in PubMed, Google Scholar, and Scopus from January 1, 1990 to December 20, 2017. The systematic review and meta-analysis were based on randomized controlled trials in infertile men with DHA or EPA treatments, either alone or in combination with other micronutrients. Three studies met the inclusion criteria: 147 patients in the intervention group and 143 patients in the control group. The analysis showed that omega-3 treatments significantly increased the sperm motility (RR 5.82, 95% CI [2.91, 8.72], p <. 0001, I 2 = 76%) and seminal DHA concentration (RR 1.61, 95% CI [0.15, 3.07], p =. 03, I 2 = 98%). Compared with the controls, the interventions did not affect the sperm concentration (RR 0.31, 95% CI [-8.13, 8.76], p =. 94, I 2 = 95%) or sperm DHA (RR 0.50, 95% CI [-4.17, 5.16], p =. 83, I 2 = 99%). The observed heterogeneity may be due to administration period and dosage of omega-3 fatty acids across the studies. Funnel plot shows no evidence of publication bias. This meta-analysis indicates that supplementing infertile men with omega-3 fatty acids resulted in a significant improvement in sperm motility and concentration of DHA in seminal plasma.

Identification of phosphoproteins coupled to initiation of motility in live epididymal mouse sperm

NASA Technical Reports Server (NTRS)

Tash, J. S.; Bracho, G. E.

1998-01-01

A method for collecting live immotile cauda epididymal mouse sperm that initiate motility by dilution into an activation buffer is described. Sperm in collection buffer showed low percent motility (MOT) and population progression (PRG) that increased 10-fold and 9-fold, respectively, during the first 2 min after dilution into activation buffer. Western phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) analysis revealed a 120 kDa protein that markedly increased in pT content during initiation of motility and may be related to FP130, the motility-coupled axonemal protein of sea urchin sperm. A prominent 82 kDa protein that was pS and pT-phosphorylated in immotile and motile sperm is likely the fibrous sheath component AKAP82 that is phosphorylated during spermatogenesis. Analysis of live human sperm also identified a prominent 120 kDa pT protein. Thus it appears that phosphorylation of FP130 and related 120 kDa proteins in mouse, and perhaps human sperm, represent common targets during motility initiation in sperm. Copyright 1998 Academic Press.

Optimization of the sperm:oocyte ratio and sperm economy in the artificial reproduction of Rhamdia quelen using fructose as a sperm motility modulator.

PubMed

Adames, Maurício Spagnolo; de Toledo, Cesar Pereira Rebechi; Neumann, Giovano; Buzzi, Alexandre Henrique; Buratto, Cíntia Nara; Piana, Pitágoras Augusto; Bombardelli, Robie Allan

2015-10-01

This research was conducted to evaluate the effects of fructose as a modulator of sperm motility and its effects on the reduction in number of sperm cells in IVF using cryopreserved Rhamdia quelen semen. Sperm activation occurred in solutions containing fructose (0.0, 0.9, 1.8, 2.7, 3.6 and 4.5%). The sperm motility rate, velocity and duration of sperm motility were assessed by polynomial regression analysis and grouped by the principal component analysis (PCA). Then, the oocytes were mixed with semen at proportions of 1×10(4), 3×10(4), 5×10(4), 7×10(4) and 9×10(4) for the sperm:oocyte ratio and fertilization was induced by the activation of gametes with the fructose-containing solutions. The fertilization, hatching and larval normality rate were evaluated by response surface protocol and were further grouped by PCA. All sperm variables were affected by the activating solutions, and the most desirable theoretical results for the rate of sperm motility were obtained when using a solution containing 2.85% fructose. In the IVF and incubation assays, there was an interactive effect between the motile sperm:oocyte ratio and the fructose concentration on the rates of oocyte fertilization, hatching and on the clustered index for reproductive success. The results suggest the possibility of reducing the sperm cells on IVF by 17.77% when using a solution containing 2.28% fructose. In conclusion, the use of solutions containing fructose at concentrations that maximize sperm movement allow the reduction of the motile sperm:oocyte ratio, thus promoting sperm metabolic efficiencies and contributing to the feasibility of using cryopreserved semen at a large-scale in IVF. Copyright © 2015 Elsevier B.V. All rights reserved.

MagnetoSperm: A microrobot that navigates using weak magnetic fields

NASA Astrophysics Data System (ADS)

Khalil, Islam S. M.; Dijkslag, Herman C.; Abelmann, Leon; Misra, Sarthak

2014-06-01

In this work, a propulsion system similar in motion to a sperm-cell is investigated. This system consists of a structure resembling a sperm-cell with a magnetic head and a flexible tail of 42 μm and 280 μm in length, respectively. The thickness, length, and width of this structure are 5.2 μm, 322 μm, and 42 μm, respectively. The magnetic head includes a 200 nm-thick cobalt-nickel layer. The cobalt-nickel layer provides a dipole moment and allows the flexible structure to align along oscillating weak (less than 5 mT) magnetic field lines, and hence generates a propulsion thrust force that overcomes the drag force. The frequency response of this system shows that the propulsion mechanism allows for swimming at an average speed of 158 ± 32 μm/s at alternating weak magnetic field of 45 Hz. In addition, we experimentally demonstrate controlled steering of the flexible structure towards reference positions.

New Approaches to Boar Semen Evaluation, Processing and Improvement.

PubMed

Sutovsky, P

2015-07-01

The improvement of boar reproductive performance may be the next frontier in reproductive management of swine herd in Unites States, facilitated by better understanding of boar sperm function and by the introduction of new advanced instrumentation in the andrology field. Objective single ejaculate evaluation and individual boar fertility prediction may be possible by introducing automated flow cytometric semen analysis with vital stains (e.g. acrosomal integrity and mito-potential), DNA fragmentation analysis and biomarkers (ubiquitin, PAWP, ALOX15, aggresome) associated with normal or defective sperm phenotypes. Measurement of sperm-produced reactive oxygen species (ROS) is a helpful indicator of normal semen sample. Semen ROS levels could be managed by the addition of ROS-scavenging antioxidants. Alternative energy regeneration substrates and sperm stimulants such as inorganic pyrophosphate and caffeine could increase sperm lifespan in extended semen and within the female reproductive system. Such technology could be combined with timed sperm release in the female reproductive system after artificial insemination. Sperm phenotype analysis by the image-based flow cytometry will go hand in hand with the advancement of swine genomics, linking aberrant sperm phenotype to the fertility influencing gene polymorphisms. Finally, poor-quality ejaculates could be rescued and acceptable ejaculates improved by semen purification methods such as the nanoparticle-based semen purification and magnetic-activated sperm sorting. Altogether, these scientific and technological advances could benefit swine industry, provided that the challenges of new technology adoption, dissemination and cost reduction are met. © 2015 Blackwell Verlag GmbH.

[Application of a trans-membrane migration method in the study of human sperm motility: a review].

PubMed

Hong, C Y

1991-09-01

Transmembrane migration method is a bioassay specifically designed to study drug effect on human sperm motility. It was first used in the study of sperm immobilizing agents which have a membrane stabilizing effect. Then it was used to investigate the relationship between calcium ion and sperm motility. Recently, this method has been used to screen drugs that stimulate sperm motility. It has also been modified for the study of porcine sperm motility. Computer assisted semen analysis showed that the transmembrane migration method is most suitable for studying drug effect on rapid and straight-forward motility of sperm.

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo).

PubMed

Słowińska, M; Liszewska, E; Dietrich, G J; Ciereszko, A

2012-09-15

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation. Copyright © 2012 Elsevier Inc. All rights reserved.

A Systematic Analysis of a Deep Mouse Epididymal Sperm Proteome

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

Chauvin, Theodore; Xie, Fang; Liu, Tao

Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It is therefore theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end we have performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and havemore » confidently identified 2,850 proteins, which is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, β-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.« less

Sperm dynamics in spiders (Araneae): ultrastructural analysis of the sperm activation process in the garden spider Argiope bruennichi (Scopoli, 1772).

PubMed

Vöcking, Oliver; Uhl, Gabriele; Michalik, Peter

2013-01-01

Storage of sperm inside the female genital tract is an integral phase of reproduction in many animal species. The sperm storage site constitutes the arena for sperm activation, sperm competition and female sperm choice. Consequently, to understand animal mating systems information on the processes that occur from sperm transfer to fertilization is required. Here, we focus on sperm activation in spiders. Male spiders produce sperm whose cell components are coiled within the sperm cell and that are surrounded by a proteinaceous sheath. These inactive and encapsulated sperm are transferred to the female spermathecae where they are stored for later fertilization. We analyzed the ultrastructural changes of sperm cells during residency time in the female genital system of the orb-web spider Argiope bruennichi. We found three clearly distinguishable sperm conditions: encapsulated sperm (secretion sheath present), decapsulated (secretion sheath absent) and uncoiled sperm (cell components uncoiled, presumably activated). After insemination, sperm remain in the encapsulated condition for several days and become decapsulated after variable periods of time. A variable portion of the decapsulated sperm transforms rapidly to the uncoiled condition resulting in a simultaneous occurrence of decapsulated and uncoiled sperm. After oviposition, only decapsulated and uncoiled sperm are left in the spermathecae, strongly suggesting that the activation process is not reversible. Furthermore, we found four different types of secretion in the spermathecae which might play a role in the decapsulation and activation process.

Sperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus).

PubMed

Gennotte, V; François, E; Rougeot, C; Ponthier, J; Deleuze, S; Mélard, C

2012-07-01

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offspring), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology, such as growth, behavior and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm traits measured significantly differed between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males, respectively ranged from 0.92 to 1.33%, from 1.69 to 2.22 ×10(9) cells mL(-1) and from 18'04″ to 27'32″. Mean values of total motility and curvilinear velocity 1 min after sperm activation, respectively ranged from 53 to 58% and from 71 to 76 μm s(-1) for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13% of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype. Copyright © 2012 Elsevier Inc. All rights reserved.

Season of collection and sperm head shape impacts expression of CARHSP and FTL from motile-rich boar sperm

USDA-ARS?s Scientific Manuscript database

The objective of the current study was to evaluate transcript activity of motile-rich sperm collected from June (spring) or August (summer), stored as cooled-extended (ExT) or cryopreserved (FrZ), and selected for least or most sperm head shape change, using Fourier harmonic analysis techniques, bet...

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.

Morphometric and kinematic sperm subpopulations in split ejaculates of normozoospermic men

PubMed Central

Santolaria, Pilar; Soler, Carles; Recreo, Pilar; Carretero, Teresa; Bono, Araceli; Berné, José M; Yániz, Jesús L

2016-01-01

This study was designed to analyze the sperm kinematic and morphometric subpopulations in the different fractions of the ejaculate in normozoospermic men. Ejaculates from eight normozoospermic men were collected by masturbation in three fractions after 3–5 days of sexual abstinence. Analyses of sperm motility by computer-assisted sperm analysis (CASA-Mot), and of sperm morphometry by computer-assisted sperm morphometry analysis (CASA-Morph) using fluorescence were performed. Clustering and discriminant procedures were performed to identify sperm subpopulations in the kinematic and morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three kinematic subpopulations (slow with low ALH [35.6% of all motile spermatozoa], with circular trajectories [32.0%], and rapid with high ALH [32.4%]), and three morphometric subpopulations (large-round [33.9% of all spermatozoa], elongated [32.0%], and small [34.10%]). The distribution of kinematic sperm subpopulations was different among ejaculate fractions (P < 0.001), with higher percentages of spermatozoa exhibiting slow movements with low ALH in the second and third portions, and with a more homogeneous distribution of kinematic sperm subpopulations in the first portion. The distribution of morphometric sperm subpopulations was also different among ejaculate fractions (P < 0.001), with more elongated spermatozoa in the first, and of small spermatozoa in the third, portion. It is concluded that important variations in the distribution of kinematic and morphometric sperm subpopulations exist between ejaculate fractions, with possible functional implications. PMID:27624985

Sperm motility in fish: technical applications and perspectives through CASA-Mot systems.

PubMed

Gallego, V; Asturiano, J F

2018-03-09

Although a relatively high number of sperm quality biomarkers have been reported over the years in several fish species, sperm motility is nowadays considered the best biomarker for fish spermatozoa. The first scientific reports focusing on fish sperm motility date from a century ago, but the objective assessment allowed by computer-aided sperm analysis (CASA-Mot) systems was not applied to fish species until the mid-1980s. Since then, a high number of sperm kinetic parameters from more than 170 fish species have been reported in more than 700 scientific articles, covering a wide range of topics, such as sperm physiology, sperm storage, broodstock management, the phenomenon of sperm competition, ecotoxicology and understanding the life cycle of the species. The sperm kinetic parameters provided by CASA-Mot systems can serve as powerful and useful tools for aquaculture and ecological purposes, and this review provides an overview of the major research areas in which fish sperm motility assessment by a CASA-Mot system has been used successfully.

Human sperm NADH and NADPH diaphorase cytochemistry: correlation with sperm motility.

PubMed

Zini, A; O'Bryan, M K; Israel, L; Schlegel, P N

1998-03-01

We have examined the correlation between the retention of residual sperm cytoplasm and sperm motility in semen from men presenting for infertility evaluation. Semen samples (n = 12) were obtained from nonazoospermic men presenting for infertility evaluation at our institution. Samples were fractionated into high-, intermediate-, and low-density subpopulations by Percoll gradients in order to examine the correlation between the retention of residual sperm cytoplasm and sperm motility. Residual sperm cytoplasm retention was detected by cytochemical staining of sperm for nicotinamide adenine dinucleotide (NADH)- or nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity. The different sperm subpopulations (low, intermediate, and high density) had significantly different percentages of sperm with droplet retention (analysis of variance, P < 0.05). Using either NADH or NADPH diaphorase staining as a marker of the cytoplasmic space, a significant negative correlation was observed between the percentage of sperm with residual cytoplasmic droplets and the percentage of motile sperm (r = -0.58 and -0.61, respectively, P < 0.05). Assessment of residual sperm cytoplasm retention is a simple diagnostic test. Although this test is of unproven value in the management of infertile men, this and other studies suggest that it may provide useful data on sperm function.

The Effect of Folate and Folate Plus Zinc Supplementation on Endocrine Parameters and Sperm Characteristics in Sub-Fertile Men: A Systematic Review and Meta-Analysis.

PubMed

Irani, Morvarid; Amirian, Malihe; Sadeghi, Ramin; Lez, Justine Le; Latifnejad Roudsari, Robab

2017-08-29

To evaluate the effect of folate and folate plus zinc supplementation on endocrine parameters and sperm characteristics in sub fertile men. We conducted a systematic review and meta-analysis. Electronic databases of Medline, Scopus , Google scholar and Persian databases (SID, Iran medex, Magiran, Medlib, Iran doc) were searched from 1966 to December 2016 using a set of relevant keywords including "folate or folic acid AND (infertility, infertile, sterility)".All available randomized controlled trials (RCTs), conducted on a sample of sub fertile men with semen analyses, who took oral folic acid or folate plus zinc, were included. Data collected included endocrine parameters and sperm characteristics. Statistical analyses were done by Comprehensive Meta-analysis Version 2. In total, seven studies were included. Six studies had sufficient data for meta-analysis. "Sperm concentration was statistically higher in men supplemented with folate than with placebo (P < .001)". However, folate supplementation alone did not seem to be more effective than the placebo on the morphology (P = .056) and motility of the sperms (P = .652). Folate plus zinc supplementation did not show any statistically different effect on serum testosterone (P = .86), inhibin B (P = .84), FSH (P = .054), and sperm motility (P = .169) as compared to the placebo. Yet, folate plus zinc showed statistically higher effect on the sperm concentration (P < .001), morphology (P < .001), and serum folate level (P < .001) as compared to placebo. Folate plus zinc supplementation has a positive effect on sperm characteristics in sub fertile men. However, these results should be interpreted with caution due to the important heterogeneity of the studies included in this meta-analysis. Further trials are still needed to confirm the current findings.

Stallion sperm transcriptome comprises functionally coherent coding and regulatory RNAs as revealed by microarray analysis and RNA-seq.

PubMed

Das, Pranab J; McCarthy, Fiona; Vishnoi, Monika; Paria, Nandina; Gresham, Cathy; Li, Gang; Kachroo, Priyanka; Sudderth, A Kendrick; Teague, Sheila; Love, Charles C; Varner, Dickson D; Chowdhary, Bhanu P; Raudsepp, Terje

2013-01-01

Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analyzing sperm and testes RNA on a 21,000-element equine whole-genome oligoarray and by RNA-seq. Microarray analysis revealed 6,761 transcripts in the sperm, of which 165 were sperm-enriched, and 155 were differentially expressed between the sperm and testes. Next, 70 million raw reads were generated by RNA-seq of which 50% could be aligned with the horse reference genome. A total of 19,257 sequence tags were mapped to all horse chromosomes and the mitochondrial genome. The highest density of mapped transcripts was in gene-rich ECA11, 12 and 13, and the lowest in gene-poor ECA9 and X; 7 gene transcripts originated from ECAY. Structural annotation aligned sperm transcripts with 4,504 known horse and/or human genes, rRNAs and 82 miRNAs, whereas 13,354 sequence tags remained anonymous. The data were aligned with selected equine gene models to identify additional exons and splice variants. Gene Ontology annotations showed that sperm transcripts were associated with molecular processes (chemoattractant-activated signal transduction, ion transport) and cellular components (membranes and vesicles) related to known sperm functions at fertilization, while some messenger and micro RNAs might be critical for early development. The findings suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs.

Stallion Sperm Transcriptome Comprises Functionally Coherent Coding and Regulatory RNAs as Revealed by Microarray Analysis and RNA-seq

PubMed Central

Das, Pranab J.; McCarthy, Fiona; Vishnoi, Monika; Paria, Nandina; Gresham, Cathy; Li, Gang; Kachroo, Priyanka; Sudderth, A. Kendrick; Teague, Sheila; Love, Charles C.; Varner, Dickson D.; Chowdhary, Bhanu P.; Raudsepp, Terje

2013-01-01

Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analyzing sperm and testes RNA on a 21,000-element equine whole-genome oligoarray and by RNA-seq. Microarray analysis revealed 6,761 transcripts in the sperm, of which 165 were sperm-enriched, and 155 were differentially expressed between the sperm and testes. Next, 70 million raw reads were generated by RNA-seq of which 50% could be aligned with the horse reference genome. A total of 19,257 sequence tags were mapped to all horse chromosomes and the mitochondrial genome. The highest density of mapped transcripts was in gene-rich ECA11, 12 and 13, and the lowest in gene-poor ECA9 and X; 7 gene transcripts originated from ECAY. Structural annotation aligned sperm transcripts with 4,504 known horse and/or human genes, rRNAs and 82 miRNAs, whereas 13,354 sequence tags remained anonymous. The data were aligned with selected equine gene models to identify additional exons and splice variants. Gene Ontology annotations showed that sperm transcripts were associated with molecular processes (chemoattractant-activated signal transduction, ion transport) and cellular components (membranes and vesicles) related to known sperm functions at fertilization, while some messenger and micro RNAs might be critical for early development. The findings suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs. PMID:23409192

Validation of a novel computer-assisted sperm analysis (CASA) system using multitarget-tracking algorithms.

PubMed

Tomlinson, Mathew James; Pooley, Karen; Simpson, Tracey; Newton, Thomas; Hopkisson, James; Jayaprakasan, Kannamanadias; Jayaprakasan, Rajisha; Naeem, Asad; Pridmore, Tony

2010-04-01

To determine the accuracy and precision of a novel computer-assisted sperm analysis (CASA) system by comparison with existing recommended manual methods. Prospective study using comparative measurements of sperm concentration and motility on latex beads and immotile and motile sperm. Tertiary referral fertility center with strong academic links. Sperm donors and male partners of couples attending for fertility investigations. None. Achievement of Accubead target value for high and low concentration suspensions. Repeatability as demonstrated by coefficients of variation and intraclass correlation coefficients. Correlation and limits of agreement between CASA and manual methods. The CASA measurements of latex beads and sperm concentrations demonstrated a high level of accuracy and repeatability. Repeated Accubead measurements attained the required target value (mean difference from target of 2.61% and 3.71% for high- and low-concentration suspensions, respectively) and were highly reproducible. Limits of agreement analysis suggested that manual and CASA counts compared directly could be deemed to be interchangeable. Manual and CASA motility measurements were highly correlated for grades a, b, and d but could not be deemed to be interchangeable, and manual motility estimates were consistently higher for motile sperm. The novel CASA system was able to provide semen quality measurements for sperm concentration and motility measurements which were at least as reliable as current manual methods. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Advanced forensic validation for human spermatozoa identification using SPERM HY-LITER™ Express with quantitative image analysis.

PubMed

Takamura, Ayari; Watanabe, Ken; Akutsu, Tomoko

2017-07-01

Identification of human semen is indispensable for the investigation of sexual assaults. Fluorescence staining methods using commercial kits, such as the series of SPERM HY-LITER™ kits, have been useful to detect human sperm via strong fluorescence. These kits have been examined from various forensic aspects. However, because of a lack of evaluation methods, these studies did not provide objective, or quantitative, descriptions of the results nor clear criteria for the decisions reached. In addition, the variety of validations was considerably limited. In this study, we conducted more advanced validations of SPERM HY-LITER™ Express using our established image analysis method. Use of this method enabled objective and specific identification of fluorescent sperm's spots and quantitative comparisons of the sperm detection performance under complex experimental conditions. For body fluid mixtures, we examined interference with the fluorescence staining from other body fluid components. Effects of sample decomposition were simulated in high humidity and high temperature conditions. Semen with quite low sperm concentrations, such as azoospermia and oligospermia samples, represented the most challenging cases in application of the kit. Finally, the tolerance of the kit against various acidic and basic environments was analyzed. The validations herein provide useful information for the practical applications of the SPERM HY-LITER™ Express kit, which were previously unobtainable. Moreover, the versatility of our image analysis method toward various complex cases was demonstrated.

Potential Changes in Rat Spermatogenesis and Sperm Parameters after Inhalation of Boswellia papyrifera and Boswellia carterii Incense

PubMed Central

Ahmed, Mukhtar; Al-Daghri, Nasser; Alokail, Majed S.; Hussain, Tajamul

2013-01-01

In this study the effect of Boswellia papyrifera (B. papyrifera) and Boswellia carterii (B. carterii) smoke exposure on spermatogenesis and sperm parameters in male albino rats was investigated. Rats (n = 11) were exposed daily in smoking chambers to smoke emanated by burning 4 g each of either B. papyrifera or B. carterii for 48 days. At the end of exposure duration rats were killed, and the testes were excised and analysed for histopathological and ultrastructural changes. Sperm analysis including total sperm count, motility, velocity and relative percentage of abnormal sperms were recorded. Rats exposed to B. papyrifera and B. carterii showed significant disturbances in spermatogenetic patterns and changes in sperm kinetics compared to unexposed rats. Atrophied seminiferous tubules with dynamic changes were also noticed. The boundaries of intercellular and intracellular vacuoles were seen in the Sertoli cells. Furthermore, in spermatids acrosomal vesicles were not fully formed. Degenerating spermatids were devoid of their nuclear membrane with electron dense matrix and vacuolization. Structural changes in Leydig cells were observed. Sperm analysis in exposed rats exhibited significant decrease in the sperm count, motility, speed and an increase in sperm anomalies when compare to controls. These findings demonstrate that the B. papyrifera and B. carterii smoke affects the process of spermatogenesis and sperm parameters and indicate the detrimental effects of these incense materials on human reproductive system. PMID:23449005

1H Magnetic Resonance Spectroscopy of live human sperm

PubMed Central

Calvert, S J; Paley, M N; Pacey, A A

2017-01-01

Abstract STUDY QUESTION Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa? SUMMARY ANSWER Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface. WHAT IS KNOWN ALREADY Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet (‘80%’ sperm) and the 40/80 interface (‘40%’ sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of ‘40%’ and ‘80%’ sperm. MAIN RESULTS AND THE ROLE OF CHANCE DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65–0.67, lactate/lipid ROC AUC = 0.86–0.87. LIMITATIONS, REASONS FOR CAUTION Only 3–4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the ‘40%’ and ‘80%’ sperm. WIDER IMPLICATIONS OF THE FINDINGS 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest. PMID:28431025

1H Magnetic Resonance Spectroscopy of live human sperm.

PubMed

Reynolds, S; Calvert, S J; Paley, M N; Pacey, A A

2017-07-01

Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa? Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface. Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm. Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet ('80%' sperm) and the 40/80 interface ('40%' sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of '40%' and '80%' sperm. DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65-0.67, lactate/lipid ROC AUC = 0.86-0.87. Only 3-4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the '40%' and '80%' sperm. 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility. This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Testicular sperm is superior to ejaculated sperm for ICSI in cryptozoospermia: An update systematic review and meta-analysis.

PubMed

Kang, Yi-No; Hsiao, Ya-Wen; Chen, Chien-Yu; Wu, Chien-Chih

2018-05-18

Intracytoplasmic sperm injection (ICSI) is well established and provides patients with severely impaired sperm quality with an opportunity to father a child. However, previous studies do not clearly indicate whether male with cryptozoospermia should use testicular sperm or ejaculated sperm for ICSI. The newest systematic review of this topic also gave a controversial conclusion that was based on incorrect pooling result. Moreover, two clinical studies published after the systematic review. In the present update systematic review and meta-analysis, a comprehensive citation search for relevant studies was performed using the Cochrane library databases, Embase, Ovid MEDLINE, PubMed, ScienceDirect, Scopus, and Web of Science up to September 2017. The search returned 313 records, in which six studies were included in quantitative synthesis. These studies involved 578 male infertility patients who had undergone 761 ICSI cycles. The risk ratios favour fresh testicular sperm for good quality embryo rate (1.17, 95% CI 1.05-1.30, P = 0.005), implantation rate (95% CI 1.02-2.26, P = 0.04), and pregnancy rate (RR = 1.74, 95% CI 1.20-2.52, P = 0.004). In conclusion, the existing evidence suggests that testicular sperm is better than ejaculated sperm for ICSI in male with cryptozoospermia.

Comparison of sperm motility subpopulation structure among wild anadromous and farmed male Atlantic salmon (Salmo salar) parr using a CASA system.

PubMed

Caldeira, Carina; García-Molina, Almudena; Valverde, Anthony; Bompart, Daznia; Hassane, Megan; Martin, Patrick; Soler, Carles

2018-04-13

Atlantic salmon (Salmo salar) is an endangered freshwater species that needs help to recover its wild stocks. However, the priority in aquaculture is to obtain successful fertilisation and genetic variability to secure the revival of the species. The aims of the present work were to study sperm subpopulation structure and motility patterns in wild anadromous males and farmed male Atlantic salmon parr. Salmon sperm samples were collected from wild anadromous salmon (WS) and two generations of farmed parr males. Sperm samples were collected from sexually mature males and sperm motility was analysed at different times after activation (5 and 35s). Differences among the three groups were analysed using statistical techniques based on Cluster analysis the Bayesian method. Atlantic salmon were found to have three sperm subpopulations, and the spermatozoa in ejaculates of mature farmed parr males had a higher velocity and larger size than those of WS males. This could be an adaptation to high sperm competition because salmonid species are naturally adapted to this process. Motility analysis enables us to identify sperm subpopulations, and it may be useful to correlate these sperm subpopulations with fertilisation ability to test whether faster-swimming spermatozoa have a higher probability of success.

A taxonomy of possible reasons for and against sperm donation.

PubMed

Bossema, Ercolie R; Janssens, Pim M W; Landwehr, Frieda; Treucker, Roswitha G L; van Duinen, Kor; Nap, Annemiek W; Geenen, Rinie

2013-06-01

Various reasons may guide the decision of men to become a sperm donor. Our aim was to identify a comprehensive set of possible reasons for and against sperm donation. Concept mapping. Assisted reproduction clinics. Nine sperm donors and seven non-sperm donors. Interviews to obtain statements for and against sperm donation, card-sorting tasks to categorize these statements according to similarity, and hierarchical cluster analysis to structure these categorizations. Hierarchical structure with reasons for and against sperm donation. The hierarchical structure with 91 reasons comprised selfishness (including narcissism and procreation), psychosocial drives (including altruism, detached procreation, and sexual/financial satisfaction), and psychosocial barriers (including normative and moral barriers related to oneself, one's spouse, the donor child, and society). The identified hierarchical overview of reasons for and against sperm donation may help potential sperm donors when considering becoming a sperm donor, enable more systematic counseling of potential sperm donors, and guide further research on reasons for and against sperm donation. © 2013 The Authors Acta Obstetricia et Gynecologica Scandinavica © 2013 Nordic Federation of Societies of Obstetrics and Gynecology.

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

Characteristics of testis-specific phosphoglycerate kinase 2 and its association with human sperm quality.

PubMed

Liu, Xue-Xia; Zhang, Hua; Shen, Xiao-Fang; Liu, Fu-Jun; Liu, Juan; Wang, Wen-Juan

2016-02-01

Is there an association between the expression of phosphoglycerate kinase (PGK) 2 in spermatozoa and sperm quality in both elderly men and young asthenozoospermia patients? Spermatozoa from elderly men and young asthenozoospermia patients show decreased expression of PGK2, which has a close positive relationship with sperm quality. PGK1 and PGK2 are involved in spermatogenesis and thought to be related to sperm motility. However, limited information is known about their temporal-spatial expression in human spermatogenesis and their relationship with sperm quality. This was a case-control study including 30 healthy young males (aged 28-31 years), 30 elderly men (aged 68-70 years), and 30 asthenozoospermic patients (aged 25-40 years, progressive motility <32%) who donated semen samples. Furthermore, young testes samples were obtained from five fathers (27-33 years old) who had died in car accidents, while aged testes samples were obtained from five elderly fathers (78-82 years old) who were prostate cancer patients. Semen samples from young adults, elderly men and asthenozoospermic patients were prepared, and their parameters were assessed by Computer-Aided Sperm Analysis (CASA). Sperm proteins were extracted for western blot analysis. Immunohistochemistry was used to characterize the cellular localization of PGK1 and PGK2 in testes samples. Sperm immunofluorescence quantification experiments identified the differential expression of PGK1 and PGK2 in sperm from young adults, elderly men and asthenozoospermic patients. Antibodies against PGK1 and PGK2 were used to test their influence on sperm motility and penetration into viscous media. A modified Kremer test using methyl cellulose was adopted to assess sperm function via penetration into viscous media. Cellular localization analysis showed that PGK1 was mainly expressed in spermatogonia whereas PGK2 was mainly expressed in round spermatids. Expression levels of both PGKs were significantly decreased in the testis with ageing (P < 0.05). Western blot and immunofluorescence quantification showed markedly lower expression of PGK2 (P < 0.05) in sperm from elderly men or asthenozoospermic patients compared sperm from with healthy young men. Sperm functional analysis validated the close relationship between expression of PGK2 and sperm motility (staining percentage, r = 0.60, P < 0.05; intensity, r = 0.59, P < 0.05). Use of an anti-PGK2 antibody on sperm significantly decreased their ability to penetrate into a cervical mucus substitute (P < 0.05). Before any clinical applications using PGK2 to assess sperm quality can be developed, more cases should be used to evaluate this approach. The study provides new insights into the role of PGKs in male reproduction. The results also indicate that PGK2 is a promising molecular candidate for the assessment of sperm quality and the screening of male contraceptive targets. This work was supported by grants from the National Natural Science Foundation of China (no. 81300533, 81370013 and 81000277) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002). The authors declare no competing financial interests. © The Author 2015. 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.

Bovine sperm separation by Swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression.

PubMed

Arias, María Elena; Andara, Katherine; Briones, Evelyn; Felmer, Ricardo

2017-06-01

This study assesses the effect of bovine sperm (obtained from three bulls) separation using density gradients (Percoll and BoviPure) and Swim-up on sperm function and gene expression. Sperm evaluations included the plasma membrane integrity (SYBR14/PI), acrosomal integrity (PNA-FITC/PI), oxidative stress (ROS; CH2FDDA), DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (ΔYm; TMRM) using flow cytometry. Sperm motility was evaluated by computer-assisted sperm analysis (CASA) and gene expression using RT-qPCR. The results showed that separation by Percoll achieves a higher proportion of sperm with intact plasma and acrosomal membranes (89.8 and 87.5%, respectively) than the unseparated control (70.3 and 62.4%, respectively), as well as by Swim-up (74.9 and 63.3%, respectively) and BoviPure (83.3 and 80.4%, respectively). No differences were observed in the proportion of spermatozoa with high ΔΨm between Percoll and BoviPure (84.3% and 83.5%, respectively), which were higher than Swim-up and the unseparated control (72.8% and 43.8%, respectively). The ROS levels were higher in the spermatozoa separated by Percoll and no differences were observed in the sperm DNA integrity between all groups. The motility analysis showed that the separation methods improve (p<0.05) total and progressive motility compared to the control, with Percoll proving the most efficient in this regard. Finally, the gene expression analysis of leptin (LEP), aromatase cytochrome P450 (CYP19) and protamine I (PRM1), after validation of 6 reference genes, showed no differences between groups. In conclusion, bovine sperm separation using density gradient improves the parameters of motility and sperm function without affecting the gene expression. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Semen analysis (image)

MedlinePlus

The sperm count test is performed if a man's fertility is in question. It is helpful in determining if there is a problem in sperm production or quality of the sperm as a cause of infertility. The test may also be used after ...

Effects of whole-body, ionizing radiation on the semen in beagle dogs

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

Day, P.A.

1988-01-01

Six beagle dogs were exposed to a total dose of 183 R of gamma radiation at a dose rate of 1 R/day, while three other dogs were exposed to a single dose of 100 R. Weekly semen analysis was performed on all irradiated dogs plus four nonirradiated dogs. Semen volume, sperm concentration, total sperm count, sperm motility and sperm head morphometry were examined. Dogs exposed to chronic radiation showed a severe decline in sperm numbers, detected after seven weeks of exposure. Sperm concentration and total sperm count were the first parameters affected and were the only parameters consistently affected. Themore » dogs exposed to 100 R as a single dose, did not show a significant decline in sperm numbers. During a 36 week recovery period, the chronically irradiated dogs did show a slight increase in sperm numbers, but they never approached pre-exposure levels.« less

Implementing an open-access CASA software for the assessment of stallion sperm motility: Relationship with other sperm quality parameters.

PubMed

Giaretta, Elisa; Munerato, Mauro; Yeste, Marc; Galeati, Giovanna; Spinaci, Marcella; Tamanini, Carlo; Mari, Gaetano; Bucci, Diego

2017-01-01

Setting an open-access computer assisted sperm analysis (CASA) may benefit the evaluation of motility in mammalian sperm, especially when economic constraints do not allow the use of a commercial system. There have been successful attempts to develop such a device in Zebra fish sperm and the system has been used in very few studies on mammalian spermatozoa. Against this background, the present study aimed at developing an open-access CASA system for mammalian sperm using the horse as a model and based upon the Image J software previously established for Zebra fish sperm. Along with determining the sperm progressive motility and other kinetic parameters (such as amplitude of lateral head displacement), the "results" window was adjusted to simplify subsequent statistical analyses. The path window was enriched with colored sperm trajectories on the basis of the subpopulation they belong to and a number that allowed the sperm track to be associated to the sperm motility data shown in the "results" window. Data obtained from the novel plugin (named as CASA_bgm) were compared with those of the commercial CASA Hamilton-Thorn IVOS Vers.12, through Bland Altman's plots. While the percentage of total and progressive motile sperm, VCL, VAP, VSL, LIN and STR and ALH were in agreement with those obtained with the commercial system, BCF significantly differed between the two systems probably due to their settings. Interestingly, a positive and significant correlation between the percentages of total motile sperm evaluated through CASA_bgm and those showing high mitochondrial membrane potential evaluated by JC-1 staining was found. In conclusion, CASA_bgm ImageJ plugin could be useful and reliable for stallion sperm motility analysis and it is our aim to apply this system to other mammalian species. Copyright © 2016 Elsevier B.V. All rights reserved.

Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin.

PubMed

Masoudi, R; Sharafi, M; Zareh Shahneh, A; Towhidi, A; Kohram, H; Esmaeili, V; Shahverdi, A; Davachi, N Dadashpour

2016-08-01

Semen cryopreservation can provide genetic resources for a large number of females from a small number of superior males. Optimization of cryopreservation media to achieve the highest quality of post-thaw semen is crucial. Soybean lecithin has evaluated as a plant-based cryoprotectant for substitution of egg yolk in ram semen extender. Flow cytometric and fertility assessments were applied following cryopreservation procedure in two experimental groups (SL group: extender containing 1% w/v soybean lecithin and EY group: extender containing 20% v/v egg yolk). The higher percentage of live sperm and the lower percentage of dead sperm were obtained in SL (47.66 ± 1.38, 52.33 ± 1.69, respectively) extender compared to EY (41.16 ± 1.38, 58.83 ± 1.69). For motion characteristics, plasma membrane integrity, acrosome integrity and mitochondria activity, no significant difference was observed between SL and EY extenders. In artificial insemination experiment, there was no significant difference in pregnancy rate, lambing rate and twining rate between SL and EY extenders. It can be concluded that SL extender can be an efficient alternative extender to preserve ram sperm during cryopreservation procedure without adverse effects. Copyright © 2016 Elsevier Inc. All rights reserved.

Supramolecular organization of the sperm plasma membrane during maturation and capacitation.

PubMed

Jones, Roy; James, Peter S; Howes, Liz; Bruckbauer, Andreas; Klenerman, David

2007-07-01

In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm's plasma membrane. We have addressed questions such as the presence of diffusion barriers, confinement of molecules to specific surface domains, polarized diffusion and the role of cholesterol in regulating lipid rafts and signal transduction during capacitation. Atomic force microscopy identified a novel region (EqSS) within the equatorial segment of bovine, porcine and ovine spermatozoa that was enriched in constitutively phosphorylated proteins. The EqSS was assembled during epididymal maturation. Fluorescence imaging techniques were then used to follow molecular diffusion on the sperm head. Single lipid molecules were freely exchangeable throughout the plasma membrane and showed no evidence for confinement within domains. Large lipid aggregates, however, did not cross over the boundary between the post-acrosome and equatorial segment suggesting the presence of a molecular filter between these two domains. A small reduction in membrane cholesterol enlarges or increases lipid rafts concomitant with phosphorylation of intracellular proteins. Excessive removal of cholesterol, however, disorganizes rafts with a cessation of phosphorylation. These techniques are forcing a revision of long-held views on how lipids and proteins in sperm membranes are assembled into larger complexes that mediate recognition and fusion with the egg.

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.

The toxic effect of opioid analgesics on human sperm motility in vitro.

PubMed

Xu, Bo; Wang, Zhi-Ping; Wang, Yan-Juan; Lu, Pei-Hua; Wang, Li-Jun; Wang, Xiao-Hai

2013-04-01

Opioid analgesics are the most common therapeutic analgesic for acute pain. In this study, the toxicological and pharmacological features of a group of opioid analgesics were characterized by the motility of human sperm. Aliquots of sperm were incubated with various concentrations of opioid analgesics in vitro. Computer-assisted sperm analysis was used to assess sperm motility at 15 minutes, 2 hours, and 4 hours after drug addition to the medium. Butorphanol and dezocine showed marked reduction of motility after incubation with sperm for 15 minutes. Butorphanol was more effective than dezocine in immobilizing sperm. Other opioids studied, such as fentanyl, alfentanil, and sufentanil, showed only partial inhibitory activity. Based on the data reported herein, we have found that butorphanol and dezocine exert a sperm-immobilizing effect. However, fentanyl, alfentanil, and sufentanil exhibit only partial inhibition of sperm motility. Given the increasing use of opioids and their potential effect on sperm motility, these findings are greatly relevant to male reproductive health.

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis

PubMed Central

Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

2018-01-01

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval. PMID:28440264

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis.

PubMed

Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

2018-01-01

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval.

TRPM8, a Versatile Channel in Human Sperm

PubMed Central

Ocampo, Ana Y.; Serrano, Carmen J.; Castellano, Laura E.; Hernández-González, Enrique O.; Chirinos, Mayel; Larrea, Fernando; Beltrán, Carmen; Treviño, Claudia L.

2009-01-01

Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis. PMID:19582168

Hemiclonal analysis reveals significant genetic, environmental and genotype x environment effects on sperm size in Drosophila melanogaster.

PubMed

Morrow, E H; Leijon, A; Meerupati, A

2008-11-01

Spermatozoa are the most diverse of all animal cells. Variation in size alone is enormous and yet there are still no clear evolutionary explanations that can account for such diversity. The basic genetics of sperm form is also poorly understood, although sperm size is known to have a strong genetic component. Here, using hemiclonal analysis of Drosophila melanogaster, we demonstrate that there is not only a significant additive genetic component contributing to phenotypic variation in sperm length but also a significant environmental effect. Furthermore, the plasticity of sperm size has a significant genetic component to it (a genotype x environment interaction). A genotype x environment interaction could contribute to the maintenance of the substantial genetic variation in this trait and thereby explain the persistent inter-male differences in sperm size seen in numerous taxa. We suggest that the low conditional dependence and high heritability but low evolvability (the coefficient of additive genetic variation) of sperm length is more consistent with a history of stabilizing selection rather than either sexual selection or strong directional selection.

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.

Are the optimum levels of the catalase and vitamin E in rooster semen extender after freezing-thawing influenced by sperm concentration?

PubMed

Moghbeli, Morteza; Kohram, Hamid; Zare-Shahaneh, Ahmad; Zhandi, Mahdi; Sharafi, Mohsen; Nabi, Mohammad Mehdi; Zahedi, Vahid; Sharideh, Hossein

2016-06-01

To date, there has no report to evaluate the interaction effects of antioxidant and sperm concentration in rooster semen cryopreservation. This study was aimed to investigate the effects of vitamin E (VitE) and catalase (CAT) at different sperm concentrations on the rooster post-thawed sperm quality. Semen samples were collected twice a week from ten roosters (ROS 308) and diluted according to experimental treatments. The treatments consist of different sperm concentrations (200, 400 and 600 × 106 sperm/mL) with supplementation VitE (5 μg/mL; VitE200, VitE400, and VitE600, respectively) or CAT (100 IU/mL; CAT200, CAT400, CAT600, respectively) and without antioxidants [Control (Con); Con200, Con400, Con600, respectively]. After thawing, motion characteristics were assessed using a CASA system. Plasma membrane integrity and malondialdehyde (MDA) level were evaluated with Hypoosmotic swelling test (HOST) and Thiobarbituric acid (TBA), respectively. The higher percentage of total motility, progressive motility, viability and membrane integrity were obtained in VitE400 (81.16 ± 1.21, 18.44 ± 1.19, 85.47 ± 1.07, 86.91 ± 1.16, respectively) and CAT400 (79.38 ± 1.21, 17.19 ± 1.19, 83.42 ± 1.07, 85.73 ± 1.16, respectively) compared to control groups. Moreover, the lowest percentage of MDA was measured in VitE400, VitE600 and CAT400 rather than other groups (1.489, 1.500, 1.510 ± 0.06, respectively). In conclusion, the results of the present study demonstrate that VitE (5 μg/mL) and CAT (100 IU/mL) independently at sperm concentration, 400 million sperm/mL could beneficial effect for preservation of rooster semen during cryopreservation. Copyright © 2016 Elsevier Inc. All rights reserved.

Cytometric analysis of shape and DNA content in mammalian sperm

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

Gledhill, B.L.

1983-10-10

Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, ofmore » accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.« less

Methods for evaluating the effects of environmental chemicals on human sperm production

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

Wyrobek, A.J.

1982-04-20

Sperm tests provide a direct and effective way of identifying chemical agents that induce spermatogenic damage in man. Four human sperm tests are available: sperm count, motility, morphology (seminal cytology), and the Y-body test. These sperm tests have numerous advantages over other approaches for assessing spermatogenic damage, and they have already been used to assess the effects of at least 85 different occupational, envionmental, and drug-related chemical exposures. When carefully controlled, seminal cytology appears to be statistically more sensitive than the other human sperm tests and should be considered an integral part of semen analysis when assessing induced spermatogenic damage.

Comparative Study in Laboratory Rats to Validate Sperm Quality Methods and Endpoints

NASA Technical Reports Server (NTRS)

Price, W. A.; Briggs, G. B.; Alexander, W. K.; Still, K. R.; Grasman, K. A.

2000-01-01

Abstract The Naval Health Research Center, Detachment (Toxicology) performs toxicity studies in laboratory animals to characterize the risk of exposure to chemicals of Navy interest. Research was conducted at the Toxicology Detachment at WPAFB, OH in collaboration with Wright State University, Department of Biological Sciences for the validation of new bioassay methods for evaluating reproductive toxicity. The Hamilton Thorne sperm analyzer was used to evaluate sperm damage produced by exposure to a known testicular toxic agent, methoxyacetic acid and by inhalation exposure to JP-8 and JP-5 in laboratory rats. Sperm quality parameters were evaluated (sperm concentration, motility, and morphology) to provide evidence of sperm damage. The Hamilton Thorne sperm analyzer utilizes a DNA specific fluorescent stain (similar to flow cytometry) and digitized optical computer analysis to detect sperm cell damage. The computer assisted sperm analysis (CASA) is a more rapid, robust, predictive and sensitive method for characterizing reproductive toxicity. The results presented in this poster report validation information showing exposure to methoxyacetic acid causes reproductive toxicity and inhalation exposure to JP-8 and JP-5 had no significant effects. The CASA method detects early changes that result in reproductive deficits and these data will be used in a continuing program to characterize the toxicity of chemicals, and combinations of chemicals, of military interest to formulate permissible exposure limits.

Broad DNA methylation changes of spermatogenesis, inflammation and immune response-related genes in a subgroup of sperm samples for assisted reproduction.

PubMed

Schütte, B; El Hajj, N; Kuhtz, J; Nanda, I; Gromoll, J; Hahn, T; Dittrich, M; Schorsch, M; Müller, T; Haaf, T

2013-11-01

Aberrant sperm DNA methylation patterns, mainly in imprinted genes, have been associated with male subfertility and oligospermia. Here, we performed a genome-wide methylation analysis in sperm samples representing a wide range of semen parameters. Sperm DNA samples of 38 males attending a fertility centre were analysed with Illumina HumanMethylation27 BeadChips, which quantify methylation of >27 000 CpG sites in cis-regulatory regions of almost 15 000 genes. In an unsupervised analysis of methylation of all analysed sites, the patient samples clustered into a major and a minor group. The major group clustered with samples from normozoospermic healthy volunteers and, thus, may more closely resemble the normal situation. When correlating the clusters with semen and clinical parameters, the sperm counts were significantly different between groups with the minor group exhibiting sperm counts in the low normal range. A linear model identified almost 3000 CpGs with significant methylation differences between groups. Functional analysis revealed a broad gain of methylation in spermatogenesis-related genes and a loss of methylation in inflammation- and immune response-related genes. Quantitative bisulfite pyrosequencing validated differential methylation in three of five significant candidate genes on the array. Collectively, we identified a subgroup of sperm samples for assisted reproduction with sperm counts in the low normal range and broad methylation changes (affecting approximately 10% of analysed CpG sites) in specific pathways, most importantly spermatogenesis-related genes. We propose that epigenetic analysis can supplement traditional semen parameters and has the potential to provide new insights into the aetiology of male subfertility. © 2013 American Society of Andrology and European Academy of Andrology.

Symmetry-breaking phase transitions in highly concentrated semen

PubMed Central

Creppy, Adama; Plouraboué, Franck; Praud, Olivier; Druart, Xavier; Cazin, Sébastien; Yu, Hui

2016-01-01

New experimental evidence of self-motion of a confined active suspension is presented. Depositing fresh semen sample in an annular shaped microfluidic chip leads to a spontaneous vortex state of the fluid at sufficiently large sperm concentration. The rotation occurs unpredictably clockwise or counterclockwise and is robust and stable. Furthermore, for highly active and concentrated semen, richer dynamics can occur such as self-sustained or damped rotation oscillations. Experimental results obtained with systematic dilution provide a clear evidence of a phase transition towards collective motion associated with local alignment of spermatozoa akin to the Vicsek model. A macroscopic theory based on previously derived self-organized hydrodynamics models is adapted to this context and provides predictions consistent with the observed stationary motion. PMID:27733694

Sperm freezing to address the risk of azoospermia on the day of ICSI.

PubMed

Montagut, M; Gatimel, N; Bourdet-Loubère, S; Daudin, M; Bujan, L; Mieusset, R; Isus, F; Parinaud, J; Leandri, R

2015-11-01

In which cases is freezing of ejaculated sperm indicated before ICSI? Sperm freezing should be performed only when out of two analyses at least one total sperm count in the ejaculate is lower than 10(6). Due to variations in individual sperm parameters, in cases of severe oligozoospermia there is a risk of absence of spermatozoa on the day of ICSI, leading to cancellation of the attempt. Sperm freezing can avoid this problem but little is known of the parameters governing the decision to freeze sperm or not. This retrospective study included 247 men who underwent sperm cryopreservation to prevent the risk of azoospermia on the day of ICSI, from 2000 to 2012. Receiver operating characteristic curve analysis was used to define the threshold value. The lowest total sperm count per ejaculate was studied as a predictive factor for the use of frozen sperm in a total of 593 ICSI attempts. Moreover, 2003 patients who had at least 4 semen analyses for andrological diagnosis have been studied to evaluate the reproducibility of sperm count. To evaluate the psychological impact of sperm freezing, a questionnaire was administered to 84 men who attended for sperm cryopreservation between June and December 2014. The cost of sperm freezing was analysed according to the French prices. When at least one total sperm count was <10(5) the risk of azoospermia in at least one ICSI attempt was 52% (34/66) versus 3% (5/181) when all counts were ≥10(5) (P < 0.0001). However, the study of the reproducibility of pre-ICSI semen analyses has shown wide variations among ejaculates, and therefore sperm freezing is recommended when one analysis from at least two, showed a sperm count <10(6). Such a policy could allow a saving of about €70 000 by avoiding unnecessary sperm freezings. The psychological impact of sperm freezing was good since >70% of men had positive feelings about this technique. This was a fairly short-term study and preservation of future fertility was not assessed. It appeared impossible to find a threshold that would predict the risk of azoospermia with 100% accuracy. Therefore there is still a risk of absence of spermatozoa on the day of ICSI despite a good negative predictive value when no total sperm count was lower than 10(5). These data suggest that sperm freezing should be proposed when total sperm count is lower than 10(6) to avoid cancellation of the ICSI attempt due to azoospermia. © The Author 2015. 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.

Optimization of ram semen cryopreservation using a chemically defined soybean lecithin-based extender.

PubMed

Emamverdi, M; Zhandi, M; Zare Shahneh, A; Sharafi, M; Akbari-Sharif, A

2013-12-01

The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin-based semen extender as a substitute for egg yolk-based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris-based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post-thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early-apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post-thawed necrotic and late-apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters. © 2013 Blackwell Verlag GmbH.

Tamoxifen is a potent antioxidant modulator for sperm quality in patients with idiopathic oligoasthenospermia.

PubMed

Guo, Li; Jing, Jun; Feng, Yu-Ming; Yao, Bing

2015-09-01

To explore the new mechanisms of tamoxifen (TAM) in the treatment for patients with idiopathic oligoasthenospermia-antioxidation. In a prospective, randomized, controlled clinical trial, 120 cases of idiopathic oligoasthenospermia were enrolled and randomly assigned to the indomethacin group (n = 60) treated with indomethacin (25 mg, bid) and TAM group (n = 60) treated with TAM (10 mg, bid) for 3 months. Before and after treatment, we evaluated semen parameters, serum malondialdehyde (MDA) and total antioxidant capacity (TAC), seminal plasma MDA and TAC, spermatozoa intracellular reactive oxygen species (ROS), sperm succinate dehydrogenase (SDH) activity, sperm mitochondrial membrane potential (MMP), and sperm adenosine triphosphate (ATP) content. The independent t test and one-way repeated measures analysis of variance were used to compare the variables between and within two groups. In the indomethacin group, the percentage of progressive motile sperms, total motility, sperm MMP, and ATP content were increased significantly after 3-month treatment (P < 0.05). In the TAM group, total sperm count, sperm concentration, the percentage of progressive motile sperms, total motility, serum and seminal plasma TAC, sperm MMP, and ATP content were significantly improved or increased (P < 0.05), while spermatozoa intracellular ROS was significantly decreased (P < 0.05). Compared to the indomethacin group, TAM treatment showed better improvement in total sperm count, sperm concentration, serum TAC, seminal plasma TAC, spermatozoa intracellular ROS, and sperm SDH activity. TAM treatment can significantly improve sperm quality, which is achieved through alleviating oxidative stress, improving sperm mitochondrial functionality, and subsequently increasing sperm motility.

Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity.

PubMed

Vijayaraghavan, S; Stephens, D T; Trautman, K; Smith, G D; Khatra, B; da Cruz e Silva, E F; Greengard, P

1996-03-01

Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.

The effect of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) on semen parameters in human males: a systematic review and meta-analysis.

PubMed

Fu, Weihua; Zhou, Zhansong; Liu, Shijian; Li, Qianwei; Yao, Jiwei; Li, Weibing; Yan, Junan

2014-01-01

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is one of the risk factors of impaired male fertility potential. Studies have investigated the effect of CP/CPPS on several semen parameters but have shown inconsistent results. Hence, we performed a systematic literature review and meta-analysis to assess the association between CP/CPPS and basic semen parameters in adult men. Systematic literature searches were conducted with PubMed, EMBASE and the Cochrane Library up to August 2013 for case-control studies that involved the impact of CP/CPSS on semen parameters. Meta-analysis was performed with Review Manager and Stata software. Standard mean differences (SMD) of semen parameters were identified with 95% confidence intervals (95% CI) in a random effects model. Twelve studies were identified, including 999 cases of CP/CPPS and 455 controls. Our results illustrated that the sperm concentration and the percentage of progressively motile sperm and morphologically normal sperm from patients with CP/CPPS were significantly lower than controls (SMD (95% CI) -14.12 (-21.69, -6.63), -5.94 (-8.63, -3.25) and -8.26 (-11.83, -4.66), respectively). However, semen volume in the CP/CPPS group was higher than in the control group (SMD (95% CI) 0.50 (0.11, 0.89)). There was no significant effect of CP/CPPS on the total sperm count, sperm total motility, and sperm vitality. The present study illustrates that there was a significant negative effect of CP/CPPS on sperm concentration, sperm progressive motility, and normal sperm morphology. Further studies with larger sample sizes are needed to better illuminate the negative impact of CP/CPPS on semen parameters.

Monitoring sperm mitochondrial respiration response in a laser trap using ratiometric fluorescence

NASA Astrophysics Data System (ADS)

Mei, Adrian; Botvinick, Elliot; Berns, Michael

2005-08-01

Sperm motility is an important area in understanding male infertility. Various techniques, such as the Computer Assisted Sperm Analysis (CASA), have been used to understand sperm motility. Sperm motility is related to the energy (ATP) production of sperm. ATP is produced by the depolarization of the membrane potential of the inner membrane of the mitochondria. In this study, a mitochondrial dye, JC-1, has been used to monitor the energetics of the mitochondria. This fluorescent dye can emit at two different wavelengths, depending on the membrane potential of the mitochondria. It can fluoresce green at low membrane potential and red at high membrane potential. The ratio of the two colors (red/green) allows for an accurate measurement of the change of membrane potential. Various experiments were conducted to quantify the behavior of the dye within the sperm and the reaction of the sperm to trap. Sperm were trapped using laser tweezers. Results have shown that the ratio drops dramatically when sperm are trapped, indicating a depolarization of the membrane. The physiological response to this depolarization is yet to be determined, but the studies indicate that the sperm could have been slightly damaged by the laser. However, knowing that sperm depolarizes their membrane when trapped can help understand how sperm react to their environment and consequently help treat male infertility.

Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa.

PubMed

Soleilhavoup, C; Tsikis, G; Labas, V; Harichaux, G; Kohnke, P L; Dacheux, J L; Guérin, Y; Gatti, J L; de Graaf, S P; Druart, X

2014-09-23

Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative phosphoproteomics analysis reveals a key role of insulin growth factor 1 receptor (IGF1R) tyrosine kinase in human sperm capacitation.

PubMed

Wang, Jing; Qi, Lin; Huang, Shaoping; Zhou, Tao; Guo, Yueshuai; Wang, Gaigai; Guo, Xuejiang; Zhou, Zuomin; Sha, Jiahao

2015-04-01

One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased frequencies of aberrant sperm as indicators of mutagenic damage in mice.

PubMed

Soares, E R; Sheridan, W; Haseman, J K; Segall, M

1979-02-01

We have tested the effects of TEM in 3 strains of mice using the sperm morphology assay. In addition, we have made an attempt to evaluate this test system with respect to experimental design, statistical problems and possible interlaboratory differences. Treatment with TEM results in significant increases in the percent of abnormally shaped sperm. These increases are readily detectable in sperm treated as spermatocytes and spermatogonial stages. Our data indicate possible problems associated with inter-laboratory variation in slide analysis. We have found that despite the introduction of such sources of variation, our data were consistent with respect to the effects of TEM. Another area of concern in the sperm morphology test is the presence of "outlier" animals. In our study, such animals comprised 4% of the total number of animals considered. Statistical analysis of the slides from these animals have shown that this problem can be dealt with and that when recognized as such, "outliers" do not effect the outcome of the sperm morphology assay.

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.

Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes.

PubMed

Lambrot, R; Xu, C; Saint-Phar, S; Chountalos, G; Cohen, T; Paquet, M; Suderman, M; Hallett, M; Kimmins, S

2013-01-01

Epidemiological studies suggest that a father's diet can influence offspring health. A proposed mechanism for paternal transmission of environmental information is via the sperm epigenome. The epigenome includes heritable information such as DNA methylation. We hypothesize that the dietary supply of methyl donors will alter epigenetic reprogramming in sperm. Here we feed male mice either a folate-deficient or folate-sufficient diet throughout life. Paternal folate deficiency is associated with increased birth defects in the offspring, which include craniofacial and musculoskeletal malformations. Genome-wide DNA methylation analysis and the subsequent functional analysis identify differential methylation in sperm of genes implicated in development, chronic diseases such as cancer, diabetes, autism and schizophrenia. While >300 genes are differentially expressed in offspring placenta, only two correspond to genes with differential methylation in sperm. This model suggests epigenetic transmission may involve sperm histone H3 methylation or DNA methylation and that adequate paternal dietary folate is essential for offspring health.

Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes

PubMed Central

Lambrot, R.; Xu, C.; Saint-Phar, S.; Chountalos, G.; Cohen, T.; Paquet, M.; Suderman, M.; Hallett, M.; Kimmins, S.

2013-01-01

Epidemiological studies suggest that a father’s diet can influence offspring health. A proposed mechanism for paternal transmission of environmental information is via the sperm epigenome. The epigenome includes heritable information such as DNA methylation. We hypothesize that the dietary supply of methyl donors will alter epigenetic reprogramming in sperm. Here we feed male mice either a folate-deficient or folate-sufficient diet throughout life. Paternal folate deficiency is associated with increased birth defects in the offspring, which include craniofacial and musculoskeletal malformations. Genome-wide DNA methylation analysis and the subsequent functional analysis identify differential methylation in sperm of genes implicated in development, chronic diseases such as cancer, diabetes, autism and schizophrenia. While >300 genes are differentially expressed in offspring placenta, only two correspond to genes with differential methylation in sperm. This model suggests epigenetic transmission may involve sperm histone H3 methylation or DNA methylation and that adequate paternal dietary folate is essential for offspring health. PMID:24326934

Sperm competition, sperm numbers and sperm quality in muroid rodents.

PubMed

Gómez Montoto, Laura; Magaña, Concepción; Tourmente, Maximiliano; Martín-Coello, Juan; Crespo, Cristina; Luque-Larena, Juan José; Gomendio, Montserrat; Roldan, Eduardo R S

2011-03-25

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An "overall sperm quality" parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert.

Female promiscuity promotes the evolution of faster sperm in cichlid fishes

PubMed Central

Fitzpatrick, John L.; Montgomerie, Robert; Desjardins, Julie K.; Stiver, Kelly A.; Kolm, Niclas; Balshine, Sigal

2009-01-01

Sperm competition, the contest among ejaculates from rival males to fertilize ova of a female, is a common and powerful evolutionary force influencing ejaculate traits. During competitive interactions between ejaculates, longer and faster spermatozoa are expected to have an edge; however, to date, there has been mixed support for this key prediction from sperm competition theory. Here, we use the spectacular radiation of cichlid fishes from Lake Tanganyika to examine sperm characteristics in 29 closely related species. We provide phylogenetically robust evidence that species experiencing greater levels of sperm competition have faster-swimming sperm. We also show that sperm competition selects for increases in the number, size, and longevity of spermatozoa in the ejaculate of a male, and, contrary to expectations from theory, we find no evidence of trade-offs among sperm traits in an interspecific analysis. Also, sperm swimming speed is positively correlated with sperm length among, but not within, species. These different responses to sperm competition at intra- and interspecific levels provide a simple, powerful explanation for equivocal results from previous studies. Using phylogenetic analyses, we also reconstructed the probable evolutionary route of trait evolution in this taxon, and show that, in response to increases in the magnitude of sperm competition, the evolution of sperm traits in this clade began with the evolution of faster (thus, more competitive) sperm. PMID:19164576

The future of computer-aided sperm analysis

PubMed Central

Mortimer, Sharon T; van der Horst, Gerhard; Mortimer, David

2015-01-01

Computer-aided sperm analysis (CASA) technology was developed in the late 1980s for analyzing sperm movement characteristics or kinematics and has been highly successful in enabling this field of research. CASA has also been used with great success for measuring semen characteristics such as sperm concentration and proportions of progressive motility in many animal species, including wide application in domesticated animal production laboratories and reproductive toxicology. However, attempts to use CASA for human clinical semen analysis have largely met with poor success due to the inherent difficulties presented by many human semen samples caused by sperm clumping and heavy background debris that, until now, have precluded accurate digital image analysis. The authors review the improved capabilities of two modern CASA platforms (Hamilton Thorne CASA-II and Microptic SCA6) and consider their current and future applications with particular reference to directing our focus towards using this technology to assess functional rather than simple descriptive characteristics of spermatozoa. Specific requirements for validating CASA technology as a semi-automated system for human semen analysis are also provided, with particular reference to the accuracy and uncertainty of measurement expected of a robust medical laboratory test for implementation in clinical laboratories operating according to modern accreditation standards. PMID:25926614

Seasonal functional relevance of sperm characteristics in equine spermatozoa.

PubMed

Gamboa, S; Rodrigues, A S; Henriques, L; Batista, C; Ramalho-Santos, J

2010-04-15

A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and lastly by fertility, stressing the importance of individual variations in out-bred animal models. The analysis of multiple ejaculates from the same animals showed clear seasonal-based differences (P<0.05) with poor semen quality in winter and a noticeable improvement in sperm quality with increasing photoperiod. Better semen quality was observed between late March and May. Interactions between month period, animal, and fertility were evident (P<0.05) for sperm concentration, head and tail sperm anomalies, and acrosomal integrity. Thus, it may be advisable to adjust the use of stallion semen according to seasonal variations. Copyright 2010 Elsevier Inc. All rights reserved.

The relationship of bull fertility to sperm nuclear shape

USGS Publications Warehouse

Ostermeier, G.C.; Sargeant, G.A.; Yandell, B.S.; Parrish, J.J.

2001-01-01

group had a linear relationship (r .89, P .05) with fertility. To construct a plot of mean sperm shapes, a novel technique to automatically orient and identify the anterior tip of the sperm head was developed. The mean nuclear shape of high-fertility sperm was more elongated and tapered than those of lower fertility. A discriminant function (P .05) was also constructed that separated the 6 bulls into 2 groups based only on the harmonic amplitudes or sperm nuclear shape. The bulls were correctly classified into the 2 fertility groups. A comparison of sperm chromatin structure analysis (SCSA) and harmonic amplitudes found that overall size variance, anterior roundness, and posterior taperedness of sperm nuclei were related to chromatin stability (P .05). Some of the differences observed in sperm nuclear shape between the high- and lower-fertility bulls may be explained by varying levels of chromatin stability. However, sperm nuclear shape appears to contain additional information from chromatin stability alone. In this particular study, with 6 bulls, all with good chromatin quality, sperm nuclear shape was a better predictor of bull fertility.

Sperm quality after swim up and density gradient centrifugation sperm preparation with supplementation of alpha lipoic acid (ALA): A preliminary study

NASA Astrophysics Data System (ADS)

Lestari, Silvia W.; Lestari, Sarah H.; Pujianto, Dwi A.

2018-02-01

Intra uterine insemination (IUI) as one of the treatment for infertility, persists low success rate. A factor that contributes to the unsuccessful of IUI is sperm preparation, performed through Swim-up (SU) and Density Gradient Centrifugation (DGC) methods. Furthermore, studies have shown that Alpha Lipoic Acid (ALA) is a potent antioxidant that could enhance the sperm motility and protect the DNA integrity of the sperm [1]. This study is aimed to re-evaluate the efficiency of the DGC and SU methods in selecting sperm before being transferred for IUI by the supplementation of ALA based on the sperm DNA integrity. Semen samples were obtained from 13 men from partners of women who are infertile (normozoospermia) and underwent IUI. Semen analysis based on the guideline of World Health Organization (WHO) 2010 was performed to measure the sperm motility and velocity, before and after sperm preparation. Then, samples were incubated with Alpha Lipoic Acid (ALA) in 0.625 mg (ALA 1), 1.25 mg (ALA 2) and 2.5 mg (ALA 3). The Sperm Chromatin Dispersion (SCD) test was performed to evaluate the sperm DNA Fragmentation Index (DFI). The percentage of motile sperm was higher in prepared sperm (post-DGC and post-SU) than in whole semen. Furthermore, the percentage of motile sperm was higher in post-DGC compared to post-SU. The level of DFI after the supplementation of ALA was decreased in prepared sperm compared to the whole semen. ALA was proved capable to select the better sperm quality with decreased sperm DNA fragmentation of prepared sperm in the all of DFI category.

Sperm subpopulations in avian species: a comparative study between the rooster (Gallus domesticus) and Guinea fowl (Numida meleagris).

PubMed

García-Herreros, Manuel

2016-01-01

The main aims of this research were to study possible differences in objective morphometric sperm characteristics, establish normative sperm morphometry standards, and evaluate the presumed different subpopulation distribution of avian spermatozoa from the rooster (Gallus domesticus ) and Guinea fowl (Numida meleagris ) as model avian species. Seventy-two ejaculates (36 per species studied) were obtained manually, following a training period involving gently combined dorso-abdominal and lumbo-sacral massage of the birds. Ejaculates were processed for volume, sperm concentration, viability, motility, and morphology. Moreover, samples were submitted for sperm morphometric assessment using objective Computer-Assisted Semen Analysis for Morphometry (CASA-Morph) methods, with sperm morphometric descriptors evaluated by Principal Component Analysis (PCA) and multivariate clustering analyses. There were several differences observed between the avian species in values obtained for ejaculate volume and sperm concentration (P < 0.001). Irrespective of species, PCA revealed two Principal Components (PCs) explaining more than 80% of the variance. In addition, the number of subpopulations differed with species (three and five subpopulations for rooster and Guinea fowl, respectively). Moreover, the distribution of the sperm subpopulations was found to be structurally different between species. In conclusion, our findings from using CASA-Morph methods indicate pronounced sperm morphometric variation between these two avian species. Because of the strong differences observed in morphometric parameter values and their subpopulation distribution, these results suggest that application of objective analytical methods such as CASA-Morph could substantially improve the reliability of comparative studies and help establish valid normative sperm morphological values for avian species.

Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis.

PubMed

Sakkas, Denny; Alvarez, Juan G

2010-03-01

To review the mechanisms responsible for DNA fragmentation in human sperm, including those occurring during spermatogenesis and transport through the reproductive tract. The mechanisms examined include: apoptosis in the seminiferous tubule epithelium, defects in chromatin remodeling during the process of spermiogenesis, oxygen radical-induced DNA damage during sperm migration from the seminiferous tubules to the epididymis, the activation of sperm caspases and endonucleases, damage induced by chemotherapy and radiotherapy, and the effect of environmental toxicants. The different tests currently used for sperm DNA fragmentation analysis and the factors that determine the predictive value of sperm DNA fragmentation testing and their implications in the diagnosis and treatment of infertility are also discussed. Finally, we also scrutinize how the presence in the embryonic genome of DNA strand breaks or modifications of DNA nucleotides inherited from the paternal genome could impact the embryo and offspring. In particular we discuss how abnormal sperm could be dealt with by the oocyte and how sperm DNA abnormalities, which have not been satisfactorily repaired by the oocyte after fertilization, may interfere with normal embryo and fetal development. Sperm DNA can be modified through various mechanisms. The integrity of the paternal genome is therefore of paramount importance in the initiation and maintenance of a viable pregnancy both in a natural conception and in assisted reproduction. The need to diagnose sperm at a nuclear level is an area that needs further understanding so that we can improve treatment of the infertile couple. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Sperm subpopulations in avian species: a comparative study between the rooster (Gallus domesticus) and Guinea fowl (Numida meleagris)

PubMed Central

García-Herreros, Manuel

2016-01-01

The main aims of this research were to study possible differences in objective morphometric sperm characteristics, establish normative sperm morphometry standards, and evaluate the presumed different subpopulation distribution of avian spermatozoa from the rooster (Gallus domesticus) and Guinea fowl (Numida meleagris) as model avian species. Seventy-two ejaculates (36 per species studied) were obtained manually, following a training period involving gently combined dorso-abdominal and lumbo-sacral massage of the birds. Ejaculates were processed for volume, sperm concentration, viability, motility, and morphology. Moreover, samples were submitted for sperm morphometric assessment using objective Computer-Assisted Semen Analysis for Morphometry (CASA-Morph) methods, with sperm morphometric descriptors evaluated by Principal Component Analysis (PCA) and multivariate clustering analyses. There were several differences observed between the avian species in values obtained for ejaculate volume and sperm concentration (P < 0.001). Irrespective of species, PCA revealed two Principal Components (PCs) explaining more than 80% of the variance. In addition, the number of subpopulations differed with species (three and five subpopulations for rooster and Guinea fowl, respectively). Moreover, the distribution of the sperm subpopulations was found to be structurally different between species. In conclusion, our findings from using CASA-Morph methods indicate pronounced sperm morphometric variation between these two avian species. Because of the strong differences observed in morphometric parameter values and their subpopulation distribution, these results suggest that application of objective analytical methods such as CASA-Morph could substantially improve the reliability of comparative studies and help establish valid normative sperm morphological values for avian species. PMID:27751988

Sperm count. Do we need a new reference value?

PubMed

Cardona Maya, Walter

2010-03-01

To evaluate the sperm count in fertile men, general population, and infertile men in different regions of the world. Sperm counts were recorded according to their fertility status, proven fertility, men recruited from an andrology/infertility clinic, or healthy men. The average of sperm count in the different studies is lower in infertile men that in fertile men (p>0.001) and in the general population (p>0.001). Based on this analysis the normal sperm count is about 65 million per mL. Using these reference value, only the 25% of the studies in infertile men are above this value, and the 75% studies with fertile men (>65 x 106 sperm/mL).

Raman micro-spectroscopy analysis of different sperm regions: a species comparison.

PubMed

Amaral, S; Da Costa, R; Wübbeling, F; Redmann, K; Schlatt, S

2018-04-01

Is Raman micro-spectroscopy a valid approach to assess the biochemical hallmarks of sperm regions (head, midpiece and tail) in four different species? Non-invasive Raman micro-spectroscopy provides spectral patterns enabling the biochemical characterization of the three sperm regions in the four species, revealing however high similarities for each region among species. Raman micro-spectroscopy has been described as an innovative method to assess sperm features having the potential to be used as a non-invasive selection tool. However, except for nuclear DNA, the identification and assignment of spectral bands in Raman-profiles to the different sperm regions is scarce and controversial. Raman spectra from head, midpiece and tail of four different species were obtained. Sperm samples were collected and smeared on microscope slides. Air dried samples were subjected to Raman analysis using previously standardized procedures. Sperm samples from (i) two donors attending the infertility clinic at the Centre of Reproductive Medicine and Andrology; (ii) two C57BL/6 -TgN (ACTbEGFP) 1Osb adult mice; (iii) two adult Cynomolgus monkeys (Macaca fascicularis) and (iv) two sea urchins (Arbacia punctulata) were used to characterize and compare their spectral profiles. Differences and similarities were confirmed by principal component analysis (PCA). Several novel region-specific peaks were identified. The three regions could be differentiated by distinctive Raman patterns irrespective of the species. However, regardless of the specie, their main spectral pattern remains mostly unchanged. These results were corroborated by the PCA analysis and suggest that the basic constituents of spermatozoa are biochemically similar among species. Further research should be performed in live sperm to validate the detected spectral bands and their use as markers of distinctive regions. Raman peaks that have never been described in the sperm cell were detected. Particularly important are those that are unique to the midpiece as they might be a reference to the identification of sperm mitochondria, whose function is highly correlated with that of sperm. In the future, Raman micro-spectroscopy has the potential to be applied in assessment of male fertility. N/A. This work was supported by BMBF project 'Sperm Ident' (FKZ:13N13024) and the DAAD-CRUP bilateral exchange program (AI A06/16-57213087). S.A. is a recipient of a fellowship from the Portuguese foundation for science and technology (FCT-SFRH/BPD/110160/2015) and R.DC. is a recipient of a DAAD PhD stipend (91590556). There is no competing interest.

An inventory of reasons for sperm donation in formal versus informal settings.

PubMed

Bossema, Ercolie R; Janssens, Pim M W; Treucker, Roswitha G L; Landwehr, Frieda; van Duinen, Kor; Nap, Annemiek W; Geenen, Rinie

2014-03-01

The shortage of sperm donors in formal settings (i.e., assisted reproduction clinics) and the availability of sperm donors in informal settings (such as through contacts on the internet) motivated us to investigate why men may prefer either a formal or an informal setting for sperm donation. Interviews with ten sperm donors and non-sperm donors yielded 55 reasons for sperm donation in the two settings. These reasons were categorized according to similarity by 14 sperm donors and non-sperm donors. These categorizations were then structured by means of hierarchical cluster analysis. Reasons favouring formal settings included being legally and physically protected, evading paternal feelings or social consequences, and having a simple, standardized procedure in terms of effort and finances. Reasons favouring informal settings related to engagement, the possibility to choose a recipient, lack of rules and regulations, having contact with the donor child, and having an (intimate) bond with the recipient. The overview of reasons identified may help potential sperm donors decide on whether to donate in a formal or informal setting, and may fuel discussions by professionals about the most appropriate conditions and legislation for sperm donation in formal settings.

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.

A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific features.

PubMed

Perrier, Jean-Philippe; Sellem, Eli; Prézelin, Audrey; Gasselin, Maxime; Jouneau, Luc; Piumi, François; Al Adhami, Hala; Weber, Michaël; Fritz, Sébastien; Boichard, Didier; Le Danvic, Chrystelle; Schibler, Laurent; Jammes, Hélène; Kiefer, Hélène

2018-05-29

Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis. The quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats. These results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention.

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.

Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

PubMed

Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

2016-01-01

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.

Genome amplification of single sperm using multiple displacement amplification.

PubMed

Jiang, Zhengwen; Zhang, Xingqi; Deka, Ranjan; Jin, Li

2005-06-07

Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 mul reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.

Single sperm analysis of the trinucleotide repeat in the Huntington`s disease gene

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

Leeflang, E.P.; Zhang, L.; Hubert, R.

1994-09-01

Huntington`s disease (HD) is one of several genetic diseases caused by trinucleotide repeat expansion. The CAG repeat is very unstable, with size changes occurring in more than 80% of transmissions. The degree of instability of this repeat in the male germline can be determined by analysis of individual sperm cells. An easy and sensitive PCR assay has been developed to amplify this trinucleotide repeat region from single sperm using two rounds of PCR. As many as 90% of the single sperm show amplification for the HD repeat. The PCR product can be easily detected on an ethidium bromide-stained agarose gel.more » Single sperm samples from an HD patient with 18 and 49 repeats were studied. We observed size variations for the expanded alleles while the size of the normal allele in sperm is very consistent. We did not detect any significant bias in the amplification of normal alleles over the larger HD alleles. Our preliminary study supports the observation made by PCR of total sperm that instability of the HD trinucleotide repeat occurs in the germline. HD preimplantation diagnosis on single embryo blastomeres may also possible.« less

Sperm Parameters: Paradigmatic Index of Good Health and Longevity

PubMed Central

Omu, Alexander E.

2013-01-01

Since the discovery of spermatozoon by Anton van Leeuwenhoek in 1677, there has been an ever increasing understanding of its role in reproduction. Many factors adversely affect sperm quality, including varicocele, accessory gland infection, immunological factors, congenital abnormalities, and iatrogenic systemic and endocrine causes, such as diabetes mellitus, obesity, metabolic syndrome, and smoking. The mechanisms responsible for the association between poor sperm parameters and ill health may include oxidative stress, low-grade inflammation, low testosterone, and low sex-hormone-binding globulin. Oxidative stress in the testicular microenvironment may result in decreased spermatogenesis and sperm DNA damage, loss of sperm motility, and abnormal sperm morphology. Low testosterone caused by advanced age, visceral obesity, and inflammation is associated with the development of cardiovascular disease. Hence, semen analysis has an important role in the routine evaluation of idiopathic male infertility, usually manifested as low sperm counts, impaired sperm motility, or absence of sperm, and remains the most common single diagnostic tool. Several studies have shown an inverse relationship between semen quality and medical disorders. This review elucidates the effect of medical disorders and social habits on sperm quality, the mechanisms that are involved in the impairment of sperm quality, and whether or not sperm quality can be used as an index of good health and longevity in a man. PMID:24051979

Effects of glyphosate exposure on sperm concentration in rodents: A systematic review and meta-analysis.

PubMed

Cai, Wenyan; Ji, Ying; Song, Xianping; Guo, Haoran; Han, Lei; Zhang, Feng; Liu, Xin; Zhang, Hengdong; Zhu, Baoli; Xu, Ming

2017-10-01

Correlation between exposure to glyphosate and sperm concentrations is important in reproductive toxicity risk assessment for male reproductive functions. Many studies have focused on reproductive toxicity on glyphosate, however, results are still controversial. We conducted a systematic review of epidemiological studies on the association between glyphosate exposure and sperm concentrations of rodents. The aim of this study is to explore the potential adverse effects of glyphosate on reproductive function of male rodents. Systematic and comprehensive literature search was performed in MEDLINE, TOXLINE, Embase, WANFANG and CNKI databases with different combinations of glyphosate exposure and sperm concentration. 8 studies were eventually identified and random-effect model was conducted. Heterogeneity among study results was calculated via chi-square tests. Ten independent experimental datasets from these eight studies were acquired to synthesize the random-effect model. A decrease in sperm concentrations was found with mean difference of sperm concentrations(MDsperm)=-2.774×10 6 /sperm/g/testis(95%CI=-0.969 to -4.579) in random-effect model after glyphosate exposure. There was also a significant decrease after fitting the random-effect model: MDsperm=-1.632×10 6 /sperm/g/testis (95%CI=-0.662 to -2.601). The results of meta-analysis support the hypothesis that glyphosate exposure decreased sperm concentration in rodents. Therefore, we conclude that glyphosate is toxic to male rodent's reproductive system. Copyright © 2017. Published by Elsevier B.V.

RAT SPERM MOTILITY ANALYSIS: METHODOLOGICAL CONSIDERATIONS

EPA Science Inventory

The objective of these studies was to optimize conditions for computer assisted sperm analysis (CASA) of rat epididymal spermatozoa. ethodological issues addressed include sample collection technique, sampling region within the epididymis, type of diluent medium used, and sample ...

Rat sperm motility analysis: methodologic considerations

EPA Science Inventory

The objective of these studies was to optimize conditions for computer-assisted sperm analysis (CASA) of rat epididymal spermatozoa. Methodologic issues addressed include sample collection technique, sampling region within the epididymis, type of diluent medium used, and sample c...

Identification, RNAi Knockdown and Functional Analysis of an Ejaculate Protein that Mediates a Postmating, Prezgotic Phenotype in a cricket

USDA-ARS?s Scientific Manuscript database

Male ejaculate proteins, including both sperm and seminal fluid proteins, play an important role in mediating reproductive biology. The function of ejaculate proteins can include enabling sperm-egg interactions, enhancing sperm storage, mediating female attractiveness, and even regulating female lif...

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

PubMed

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

2017-05-01

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

Influence of Post-Mortem Sperm Recovery Method and Extender on Unstored and Refrigerated Rooster Sperm Variables.

PubMed

Villaverde-Morcillo, S; Esteso, M C; Castaño, C; Santiago-Moreno, J

2016-02-01

Many post-mortem sperm collection techniques have been described for mammalian species, but their use in birds is scarce. This paper compares the efficacy of two post-mortem sperm retrieval techniques - the flushing and float-out methods - in the collection of rooster sperm, in conjunction with the use of two extenders, i.e., L&R-84 medium and Lake 7.1 medium. To determine whether the protective effects of these extenders against refrigeration are different for post-mortem and ejaculated sperm, pooled ejaculated samples (procured via the massage technique) were also diluted in the above extenders. Post-mortem and ejaculated sperm variables were assessed immediately at room temperature (0 h), and after refrigeration at 5°C for 24 and 48 h. The flushing method retrieved more sperm than the float-out method (596.5 ± 75.4 million sperm vs 341.0 ± 87.6 million sperm; p < 0.05); indeed, the number retrieved by the former method was similar to that obtained by massage-induced ejaculation (630.3 ± 78.2 million sperm). For sperm collected by all methods, the L&R-84 medium provided an advantage in terms of sperm motility variables at 0 h. In the refrigerated sperm samples, however, the Lake 7.1 medium was associated with higher percentages of viable sperm, and had a greater protective effect (p < 0.05) with respect to most motility variables. In conclusion, the flushing method is recommended for collecting sperm from dead birds. If this sperm needs to be refrigerated at 5°C until analysis, Lake 7.1 medium is recommended as an extender. © 2015 Blackwell Verlag GmbH.

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.

Assisted reproductive technology with donor sperm: national trends and perinatal outcomes.

PubMed

Gerkowicz, Sabrina A; Crawford, Sara B; Hipp, Heather S; Boulet, Sheree L; Kissin, Dmitry M; Kawwass, Jennifer F

2018-04-01

Information regarding the use of donor sperm in assisted reproductive technology, as well as subsequent treatment and perinatal outcomes, remains limited. Outcome data would aid patient counseling and clinical decision making. The objectives of the study were to report national trends in donor sperm utilization and live birth rates of donor sperm-assisted reproductive technology cycles in the United States and to compare assisted reproductive technology treatment and perinatal outcomes between cycles using donor and nondonor sperm. We hypothesize these outcomes to be comparable between donor and nondonor sperm cycles. This was a retrospective cohort study using data from all US fertility centers reporting to the Centers for Disease Control and Prevention's National Assisted Reproductive Technology Surveillance System, accounting for ∼98% of assisted reproductive technology cycles (definition excludes intrauterine insemination). The number and percentage of assisted reproductive technology cycles using donor sperm and rates of pregnancy, live birth, preterm birth (<37 weeks), and low birthweight (<2500 g) were the primary outcomes measured. Treatments assessed include use of donor vs nondonor sperm. The trends analysis included all banking and fresh assisted reproductive technology cycles using donor and autologous oocytes performed between 1996 and 2014 (n = 1,710,034). The outcomes analysis was restricted to include only fresh autologous cycles performed between 2010 and 2014 (n = 437,569) to focus on cycles with a potential outcome and cycles reflective of current practice, thereby improving the clinical relevance. Cycles canceled prior to retrieval were excluded. Statistical analysis included linear regression to explore polynomial trends and log-binomial regression to estimate relative risk for outcomes among cycles using donor and nondonor sperm. Of all banking and fresh donor and autologous oocyte assisted reproductive technology cycles performed between 1996 and 2014, 74,892 (4.4%) used donor sperm. In 2014, 7351 assisted reproductive technology cycles using donor sperm were performed, as compared with 1763 in 1996 (6.2% vs 3.8% of all cycles). Among all autologous oocyte cycles performed between 2010 and 2014, the live birth rate was lower for donor sperm (27.9%) than nondonor sperm cycles (32.5%); however, after adjustment for maternal age, donor sperm use was associated with an increased likelihood of live birth (adjusted relative risk, 1.06, 95% confidence interval, 1.01-1.10). Per transfer, there was no significant difference in live birth rates for donor vs nondonor sperm (31.9% vs 36.8%; adjusted relative risk, 1.04, 95% confidence interval, 0.998-1.09). Per singleton live birth, there was no significant difference in preterm birth (11.5% vs 11.8%; adjusted relative risk, 0.98, 95% confidence interval, 0.90-1.06); however, low birthweight delivery was slightly lower in donor sperm cycles (8.8% vs 9.4%; adjusted relative risk, 0.91, 95% confidence interval, 0.83-0.99). Donor sperm use in assisted reproductive technology has increased in the United States, accounting for approximately 6% of all assisted reproductive technology cycles in 2014. Assisted reproductive technology treatment and perinatal outcomes were clinically similar in donor and nondonor sperm cycles. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparison of cryopreserved human sperm in vapor and liquid phases of liquid nitrogen: effect on motility parameters, morphology, and sperm function.

PubMed

Punyatanasakchai, Piyaphan; Sophonsritsuk, Areephan; Weerakiet, Sawaek; Wansumrit, Surapee; Chompurat, Deonthip

2008-11-01

To compare the effects of cryopreserved sperm in vapor and liquid phases of liquid nitrogen on sperm motility, morphology, and sperm function. Experimental study. Andrology laboratory at Ramathibodi Hospital, Thailand. Thirty-eight semen samples with normal motility and sperm count were collected from 38 men who were either patients of an infertility clinic or had donated sperm for research. Each semen sample was divided into two aliquots. Samples were frozen with static-phase vapor cooling. One aliquot was plunged into liquid nitrogen (-196 degrees C), and the other was stored in vapor-phase nitrogen (-179 degrees C) for 3 days. Thawing was performed at room temperature. Motility was determined by using computer-assisted semen analysis, sperm morphology was determined by using eosin-methylene blue staining, and sperm function was determined by using a hemizona binding test. Most of the motility parameters of sperm stored in the vapor phase were not significantly different from those stored in the liquid phase of liquid nitrogen, except in amplitude of lateral head displacement. The percentages of normal sperm morphology in both vapor and liquid phases also were not significantly different. There was no significant difference in the number of bound sperm in hemizona between sperm cryopreserved in both vapor and liquid phases of liquid nitrogen. Cryopreservation of human sperm in a vapor phase of liquid nitrogen was comparable to cryopreservation in a liquid phase of liquid nitrogen.

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.

Queue up, please! Spermathecal filling in the rove beetle Drusilla canaliculata (Coleoptera, Staphylinidae)

NASA Astrophysics Data System (ADS)

Werner, Michael; Gack, Claudia; Speck, Thomas; Peschke, Klaus

2007-10-01

Most studies on insect sperm motility have been conducted in vitro using artificial environments outside the animal’s body. Only little is known about the function of motile insect sperm at different sites within the male or female genital tracts. We dissected genital tracts of female rove beetles ( Drusilla canaliculata) to show that spermatozoa use their own motility to migrate from the spermatophore into the spermatheca. Our dissection method allowed direct observation and filming of the spermathecal filling process inside the female’s genital tract. Spermatozoa were found to enter the spermatheca individually, sometimes in groups of two or three. Although exhibiting only weak motility and no progressive motion in buffer solution, the spermatozoa inside the female show vigorous lashing and reach an average velocity of 47.5 μm s-1. To gain mobility and speed, the spermatozoa likely utilize the relatively small diameter of the spermathecal duct to push themselves off the duct walls, rather than swimming freely in seminal fluid. The spermatozoa (approximately 1,250 μm) are considerably longer than the distance they have to travel along the spermathecal duct (approximately 800 μm). Our study provides the first direct observation of active sperm migration within the female of an insect stressing the importance of the genital tract as a prerequisite for functional sperm motility.

Chromatin-unstable boar spermatozoa have little chance of reaching oocytes in vivo.

PubMed

Ardón, Florencia; Helms, Dietmar; Sahin, Evrim; Bollwein, Heinrich; Töpfer-Petersen, Edda; Waberski, Dagmar

2008-04-01

In the present study, the prevalence of chromatin instability in the fertilizing-competent sperm population in the porcine oviduct in vivo was examined through qualitative analysis of the chromatin structure status of accessory boar sperm found in in vivo-derived embryos. The binding of chromatin-unstable sperm to oviductal epithelium in vitro was also studied. To examine the sperm chromatin state, a modified fluorescence microscopic sperm chromatin structure assay was used. Among a population of 173 fertile boars, individuals were selected for according to their chromatin status: 25 animals showed more than 5% of chromatin-unstable sperm in their ejaculates, and 7 showed consistently elevated percentages of chromatin-unstable sperm in three successively collected semen samples. A positive correlation was found between incidence of chromatin instability and attached cytoplasmic droplets (r=0.44, P<0.01). Analyses of accessory spermatozoa from in vivo-derived embryos demonstrated that the proportion of chromatin-unstable sperm was significantly (P<0.05) reduced in the population of fertilizing-competent sperm in the oviduct compared with the inseminated sperm. Populations of sperm bound to the oviduct in vitro had significantly (P<0.05) lower percentages of chromatin instability than in the original diluted semen sample. In conclusion, numbers of sperm with unstable chromatin are reduced in the oviductal sperm reservoir, possibly because of associated changes in the plasma membrane that prevent sperm from binding to the oviductal epithelium. We conclude that in vivo the likelihood that sperm with unstable chromatin will reach the egg and fertilize it is low.

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

Sperm quality assessment via separation and sedimentation in a microfluidic device.

PubMed

Chen, Chang-Yu; Chiang, Tsun-Chao; Lin, Cheng-Ming; Lin, Shu-Sheng; Jong, De-Shien; Tsai, Vincent F-S; Hsieh, Ju-Ton; Wo, Andrew M

2013-09-07

A major reason for infertility is due to male factors, including the quality of spermatozoa, which is a primary factor and often difficult to assess, particularly the total sperm concentration and its motile percentage. This work presents a simple microfluidic device to assess sperm quality by quantifying both total and motile sperm counts. The key design feature of the microfluidic device is two channels separated by a permeative phase-guide structure, where one channel is filled with raw semen and the other with pure buffer. The semen sample was allowed to reach equilibrium in both chambers, whereas non-motile sperms remained in the original channel, and roughly half of the motile sperms would swim across the phase-guide barrier into the buffer channel. Sperms in each channel agglomerated into pellets after centrifugation, with the corresponding area representing total and motile sperm concentrations. Total sperm concentration up to 10(8) sperms per ml and motile percentage in the range of 10-70% were tested, encompassing the cutoff value of 40% stated by World Health Organization standards. Results from patient samples show compact and robust pellets after centrifugation. Comparison of total sperm concentration between the microfluidic device and the Makler chamber reveal they agree within 5% and show strong correlation, with a coefficient of determination of R(2) = 0.97. Motile sperm count between the microfluidic device and the Makler chamber agrees within 5%, with a coefficient of determination of R(2) = 0.84. Comparison of results from the Makler Chamber, sperm quality analyzer, and the microfluidic device revealed that results from the microfluidic device agree well with the Makler chamber. The sperm microfluidic chip analyzes both total and motile sperm concentrations in one spin, is accurate and easy to use, and should enable sperm quality analysis with ease.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

PubMed

Arias, María Elena; Sánchez-Villalba, Esther; Delgado, Andrea; Felmer, Ricardo

2017-02-01

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics.

PubMed

Purdy, P H; Tharp, N; Stewart, T; Spiller, S F; Blackburn, H D

2010-10-15

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility. Copyright © 2010 Elsevier Inc. All rights reserved.

Effect of 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.

Association between air pollution and sperm quality: A systematic review and meta-analysis.

PubMed

Deng, Zibing; Chen, Fei; Zhang, Meixia; Lan, Lan; Qiao, Zhijiao; Cui, Yan; An, Jinghuan; Wang, Nan; Fan, Zhiwei; Zhao, Xing; Li, Xiaosong

2016-01-01

Exposure to ambient air pollution has been clearly linked to adverse reproductive outcome and fecundation index, but its effects on male semen quality are still uncertain. In this study, we reviewed information from ten studies to get the qualitative evidence of the influence of the ambient air pollution on sperm quality and collected data from six of the ten studies to conduct meta-analysis. The original studies classified participants into different exposure levels and the highest and lowest expose levels were chosen as high expose and low expose groups, respectively. The random-effect model was used in the meta-analysis with the weight mean difference (WMD) as the measure indicator. The WMDs (95% confidence intervals, CIs) of sperm volume, sperm count, semen concentration, sperm progressive motility, total motility, and normal morphology were 0.09 (-0.04, 0.23), 0.46 (-4.47, 5.39), -8.21 (-20.38, 3.96), -7.76 (-16.26, 0.74), -7.61 (-16.97, 1.74) and -3.40 (-7.42, 0.62), respectively. In conclusion, although the differences are not statistically significant between the two groups, the overall trends and evidence from this review indicate the chronic exposure to ambient pollutants at high level may alter men sperm quality. Copyright © 2015 Elsevier Ltd. All rights reserved.

Implementation of novel statistical procedures and other advanced approaches to improve analysis of CASA data.

PubMed

Ramón, M; Martínez-Pastor, F

2018-04-23

Computer-aided sperm analysis (CASA) produces a wealth of data that is frequently ignored. The use of multiparametric statistical methods can help explore these datasets, unveiling the subpopulation structure of sperm samples. In this review we analyse the significance of the internal heterogeneity of sperm samples and its relevance. We also provide a brief description of the statistical tools used for extracting sperm subpopulations from the datasets, namely unsupervised clustering (with non-hierarchical, hierarchical and two-step methods) and the most advanced supervised methods, based on machine learning. The former method has allowed exploration of subpopulation patterns in many species, whereas the latter offering further possibilities, especially considering functional studies and the practical use of subpopulation analysis. We also consider novel approaches, such as the use of geometric morphometrics or imaging flow cytometry. Finally, although the data provided by CASA systems provides valuable information on sperm samples by applying clustering analyses, there are several caveats. Protocols for capturing and analysing motility or morphometry should be standardised and adapted to each experiment, and the algorithms should be open in order to allow comparison of results between laboratories. Moreover, we must be aware of new technology that could change the paradigm for studying sperm motility and morphology.

Assessment of sperm DNA in patients submitted the assisted reproduction technology procedures.

PubMed

Tsuribe, Patrícia Miyuki; Lima Neto, João Ferreira; Golim, Marjorie de Assis; Dell'Aqua, Camila de Paula Freitas; Issa, João Paulo; Gobbo, Carlos Alberto Monte

2016-03-01

This study aimed to produce data on sperm quality while maintaining the integrity of sperm DNA samples taken from patients submitted to in vitro fertilization (IVF) procedures at our center, and determine whether increased levels of histones were associated with sperm DNA damage and decreased fertilization, cleavage, and pregnancy rates. Such findings might shed light on the physiology and outcomes of pregnancy. Semen samples from 27 patients divided into two groups were analyzed. The case group included individuals offered IVF; the control group had subjects with normal spermograms. Sperm DNA structure was assessed through phosphorylated histone H2AX analysis by flow cytometry. The patients with altered sperm parameters had more histones in sperm chromatin than the individuals with normal sperm parameters. Results indicated that increased levels of histone in sperm chromatin do not affect embryo production, but affect the cleavage rate, embryo quality, and might thus reduce pregnancy rates. The integrity of the paternal genome is of paramount importance in the initiation and maintenance of a viable pregnancy in patients treated with assisted reproduction technology procedures. Further studies on sperm diagnostic tests at a nuclear level might improve the treatment offered to infertile couples.

Automated motile cell capture and analysis with optical traps.

PubMed

Shao, Bing; Nascimento, Jaclyn M; Shi, Linda Z; Botvinick, Elliot L

2007-01-01

Laser trapping in the near infrared regime is a noninvasive and microfluidic-compatible biomedical tool. This chapter examines the use of optical trapping as a quantitative measure of sperm motility. The single point gradient trap is used to directly measure the swimming forces of sperm from several different species. These forces could provide useful information about the overall sperm motility and semen quality. The swimming force is measured by trapping sperm and subsequently decreasing laser power until the sperm is capable of escaping the trap. Swimming trajectories were calculated by custom built software, an automatic sperm tracking algorithm called the single sperm tracking algorithm or SSTA. A real-time automated tracking and trapping system, or RATTS, which operates at video rate, was developed to perform experiments with minimal human involvement. After the experimenter initially identifies and clicks the computer mouse on the sperm-of-interest, RATTS performs all further tracking and trapping functions without human intervention. Additionally, an annular laser trap which is potentially useful for high-throughput sperm sorting based on motility and chemotaxis was developed. This low power trap offers a more gentle way for studying the effects of laser radiation, optical force, and external obstacles on sperm swimming pattern.

Effect of sexual steroids on boar kinematic sperm subpopulations.

PubMed

Ayala, E M E; Aragón, M A

2017-11-01

Here, we show the effects of sexual steroids, progesterone, testosterone, or estradiol on motility parameters of boar sperm. Sixteen commercial seminal doses, four each of four adult boars, were analyzed using computer assisted sperm analysis (CASA). Mean values of motility parameters were analyzed by bivariate and multivariate statistics. Principal component analysis (PCA), followed by hierarchical clustering, was applied on data of motility parameters, provided automatically as intervals by the CASA system. Effects of sexual steroids were described in the kinematic subpopulations identified from multivariate statistics. Mean values of motility parameters were not significantly changed after addition of sexual steroids. Multivariate graphics showed that sperm subpopulations were not sensitive to the addition of either testosterone or estradiol, but sperm subpopulations responsive to progesterone were found. Distribution of motility parameters were wide in controls but sharpened at distinct concentrations of progesterone. We conclude that kinematic sperm subpopulations responsive to progesterone are present in boar semen, and these subpopulations are masked in evaluations of mean values of motility parameters. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Testing the limits of Rodent Sperm Analysis: azoospermia in an otherwise healthy wild rodent population.

PubMed

Tannenbaum, Lawrence V; Thran, Brandolyn H; Willams, Keith J

2009-01-01

By comparing the sperm parameters of small rodents trapped at contaminated terrestrial sites and nearby habitat-matched noncontaminated locations, the patent-pending Rodent Sperm Analysis (RSA) method provides a direct health status appraisal for the maximally chemical-exposed mammalian ecological receptor in the wild. RSA outcomes have consistently allowed for as definitive determinations of receptor health as are possible at the present time, thereby streamlining the ecological risk assessment (ERA) process. Here, we describe the unanticipated discovery, at a contaminated US EPA Superfund National Priorities List site, of a population of Hispid cotton rats (Sigmodon hispidus), with a high percentage of adult males lacking sperm entirely (azoospermia). In light of the RSA method's role in streamlining ERAs and in bringing contaminated Superfund-type site investigations to closure, we consider the consequences of the discovery. The two matters specifically discussed are (1) the computation of a population's average sperm count where azoospermia is present and (2) the merits of the RSA method and its sperm parameter thresholds-for-effect when azoospermia is masked in an otherwise apparently healthy rodent population.

Quantification of leptin in seminal plasma of buffalo bulls and its correlation with antioxidant status, conventional and computer-assisted sperm analysis (CASA) semen variables.

PubMed

Kumar, Pradeep; Saini, Monika; Kumar, Dharmendra; Jan, M H; Swami, Dheer Singh; Sharma, R K

2016-03-01

The present study is the first to quantify leptin in seminal plasma of buffalo and investigate its relationship with seminal attributes. Ten ejaculates each from 10 Murrah buffalo bulls were collected. Semen quality variables such as semen volume, sperm concentration, sperm abnormalities, membrane integrity, antioxidant enzyme activities (superoxide dismutase, catalase and total antioxidant capacity), malondialdehyde (MDA) concentration, as well as sperm kinetics and motility variables were evaluated. The leptin concentration in serum and seminal plasma were estimated by the ELISA method. Bulls were classified in two groups on the basis of sperm concentration with Group I having >800 million sperm/mL and Group II <500 million sperm/mL. Greater (P<0.05) mean sperm abnormalities, seminal leptin concentrations and MDA concentrations were recorded in Group II than Group I. The seminal leptin was positively correlated with sperm abnormalities and MDA concentration while being negatively correlated with sperm concentration, but there was no correlation with sperm kinetic and motility variables, sperm membrane integrity and seminal plasma antioxidant enzyme activity. Thus, the data suggest that seminal leptin has a role in spermatogenesis and can be used as a marker for spermatogenesis to predict the capacity of buffalo bulls for semen production. Copyright © 2016 Elsevier B.V. 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.

Scattering of spermatozoa off cylindrical pillars

NASA Astrophysics Data System (ADS)

Bukatin, Anton; Lushi, Enkeleida; Kantsler, Vasily

2017-11-01

The motion of micro-swimmers in structured environments, even though crucial in processes such as in vivo and in vitro egg fertilization, is still not completely understood. We combine microfluidic experiments with mathematical modeling of 3D swimming near convex surfaces to quantify the dynamics of individual sperm cells in the proximity of cylindrical pillars. Our results show that the hydrodynamic and contact forces that account for the shape asymmetry and flagellar motion, are crucial in correctly describing the dynamics observed in the experiments. Last, we discuss how the size of the cylindrical obstacles determines whether the swimmers scatter off or get trapped circling the pillar.

The coevolution of ova defensiveness with sperm competitiveness in house mice.

PubMed

Firman, Renée C; Gomendio, Montserrat; Roldan, Eduardo R S; Simmons, Leigh W

2014-04-01

Theoretical models have suggested that sperm competition can lead to increased ova resistance to fertilization. While there is some comparative evidence that this might be true, there is no experimental evidence to show that ova defensiveness evolves in response to sperm competition. We performed a series of in vitro fertilization assays to gauge the fertilizability of ova produced by female house mice from experimental populations that evolved either with or without sperm competition. Our analysis revealed that after 24 generations of experimental evolution, females that evolved under a polygamous regime produced more defensive ova than females that evolved under a monogamous regime. We therefore provide the first direct line of evidence that sperm competition can generate sexual conflict at the gametic level and lead to asymmetries in fertilization rates among populations. Our results show that females respond to sperm competition via fertilization barriers that have the potential to mediate sperm entry.

Is sperm freezability related to the post-thaw lipid peroxidation and the formation of reactive oxygen species in boars?

PubMed

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

2013-04-01

The aim of the present study was to determine whether the levels of reactive oxygen species (ROS) substances production and the levels of lipid peroxidation of the sperm membrane were related to the quality that the ejaculates exhibited after cryopreservation in boars. Ejaculates from 42 healthy boars were used in this study and they were cryopreserved with the lactose-egg yolk extender (LEY). Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37 °C after thawing: the percentage of sperm with intact plasma membrane (SIPM), intracellular reactive oxygen substances production through mean of DCF fluorescence intensity of total sperm (mean-DCF) and the percentage of viable and non-viable sperm containing oxidized BODIPY (VSOB and NVSOB). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. The classification of the ejaculates into good or bad freezers was performed through hierarchical cluster analysis from SIPM and TMS at 150 min post-thawing. The ejaculates of those males classified as good freezers exhibited higher (p < 0.05) SPIM, TMS and PMS than the bad freezers, although both groups presented similar (p > 0.05) VSOB, NVSOB and mean-DCF. Therefore, these results show that lipid peroxidation and the amount of reactive oxygen substances in the sperm after cryopreservation are similar between boars classified as good or bad freezers. © 2012 Blackwell Verlag GmbH.

Assessment of chromosomal abnormalities in sperm of infertile men using sperm karyotyping and multicolour fluorescence in situ hybridization (FISH)

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

Moosani, N.; Martin, R.H.

1994-09-01

Individuals with male factor infertility resulting from idiopathic oligo-, astheno- or teratozoospermia are frequently offered IVF in an attempt to increase their chances of having a child. A concern remains whether these infertile males have an elevated risk of transmitting chromosomal abnormalities to their offspring. Sperm chromosomal complements from these men were assayed using the human sperm/hamster oocyte fusion system and fluorescence in situ hybridization (FISH) on sperm nuclei. For each of 5 infertile patients, 100 sperm karyotypes were analyzed and multicolour FISH analysis was performed on a minimum of 10,000 sperm nuclei for each chromosome-specific DNA probe for chromosomesmore » 1 (pUC1.77), 12 (D12Z3), X (XC) and Y (DYZ3). As a group, the infertile patients showed increased frequencies of both numerical ({chi}{sup 2}=17.26, {proportional_to} <0.001) and total abnormalities ({chi}{sup 2}=7.78, {proportional_to} <0.01) relative to control donors when assessed by sperm karyotypes. Analysis of sperm nuclei by FISH indicated a significant increase in the frequency of disomy for chromosome 1 in three of the five patients as compared to control donors ({chi}{sup 2}>8.35, {proportional_to} <0.005). In addition, the frequency of XY disomy was significantly higher in four of the five patients studied by FISH ({chi}{sup 2}>10.58, {proportional_to}<0.005), suggesting that mis-segregation caused by the failure of the XY bivalent to pair may play a role in idiopathic male infertility.« less

Environmental osmolality influences sperm motility activation in an anuran amphibian.

PubMed

Byrne, P G; Dunne, C; Munn, A J; Silla, A J

2015-03-01

Evolutionary theory predicts that selection will favour sperm traits that maximize fertilization success in local fertilization environments. In externally fertilizing species, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but there remains limited evidence for adaptive responses to local osmotic environments. In this study, we used a split-sample experimental design and computer-assisted sperm analysis to (i) determine the optimal medium osmolality for sperm activation (% sperm motility and sperm velocity) in male common eastern froglets (Crinia signifera), (ii) test for among-population variation in percentage sperm motility and sperm velocity at various activation-medium osmolalities and (iii) test for among-population covariation between sperm performance and environmental osmolality. Frogs were obtained from nine populations that differed in environmental osmolality, and sperm samples of males from different populations were subjected to a range of activation-medium osmolalities. Percentage sperm motility was optimal between 10 and 50 mOsm kg(-1) , and sperm velocity was optimal between 10 and 100 mOsm kg(-1) , indicating that C. signifera has evolved sperm that can function across a broad range of osmolalities. As predicted, there was significant among-population variation in sperm performance. Furthermore, there was a significant interaction between activation-medium osmolality and environmental osmolality, indicating that frogs from populations with higher environmental osmolality produced sperm that performed better at higher osmolalities in vitro. This finding may reflect phenotypic plasticity in sperm functioning, or genetic divergence resulting from spatial variation in the strength of directional selection. Both of these explanations are consistent with evolutionary theory, providing some of the first empirical evidence that local osmotic environments can favour adaptive sperm motility responses in species that use an external mode of fertilization. © 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.

Sperm cryopreservation affects postthaw motility, but not embryogenesis or larval growth in the Brazilian fish Brycon insignis (Characiformes).

PubMed

Viveiros, A T M; Isaú, Z A; Caneppele, D; Leal, M C

2012-09-01

Sperm cryopreservation is an important method for preserving genetic information and facilitating artificial reproduction. The objective was to investigate whether the cryopreservation process affects postthaw sperm motility, embryogenesis, and larval growth in the fish Brycon insignis. Sperm was diluted in methyl glycol and Beltsville Thawing solution, frozen in a nitrogen vapor vessel (dry shipper) and stored in liquid nitrogen. Half of the samples were evaluated both subjectively (% of motile sperm and motility quality score-arbitrary grading system from 0 [no movement] to 5 [rapidly swimming sperm]) and in a computer-assisted sperm analyzer (CASA; percentage of motile sperm and velocity). The other half was used for fertilization and the evaluation of embryogenesis (cleavage and gastrula stages), hatching rate, percentage of larvae with normal development and larval growth up to 112 days posthatching (dph). Fresh sperm was analyzed subjectively (percentage of motile sperm and motility quality score) and used as the control. In the subjective analysis, sperm motility significantly decreased from 100% motile sperm and quality score of 5 in fresh sperm to 54% motile sperm and quality score of 3 after thawing. Under computer-assisted sperm analyzer evaluation, postthaw sperm had 67% motile sperm, 122 μm/sec of curvilinear velocity, 87 μm/sec of straight-line velocity and 103 μm/sec of average path velocity. There were no significant differences between progenies (pooled data) for the percentage of viable embryos in cleavage (62%) or gastrula stages (24%) or in the hatching rate (24%), percentage of normal hatched larvae (93%), larval body weight (39.8 g), or standard length (12.7 cm) at 112 days posthatching. Based on these findings, cryopreserved sperm can be used as a tool to restore the population of endangered species, such as B. insignis, as well as for aquaculture purposes, without any concern regarding quality of the offspring. Copyright © 2012 Elsevier Inc. All rights reserved.

Sperm Na+, K+-ATPase and Ca2+-ATPase activity: A preliminary study of comparison of swim up and density gradient centrifugation methods for sperm preparation

NASA Astrophysics Data System (ADS)

Lestari, Silvia W.; Larasati, Manggiasih D.; Asmarinah, Mansur, Indra G.

2018-02-01

As one of the treatment for infertility, the success rate of Intrauterine Insemination (IUI) is still relatively low. Several sperm preparation methods, swim-up (SU) and the density-gradient centrifugation (DGC) are frequently used to select for better sperm quality which also contribute to IUI failure. Sperm selection methods mainly separate the motile from the immotile sperm, eliminating the seminal plasma. The sperm motility involves the structure and function of sperm membrane in maintaining the balance of ion transport system which is regulated by the Na+, K+-ATPase, and Ca2+-ATPase enzymes. This study aims to re-evaluate the efficiency of these methods in selecting for sperm before being used for IUI and based the evaluation on sperm Na+,K+-ATPase and Ca2+-ATPase activities. Fourteen infertile men from couples who underwent IUI were involved in this study. The SU and DGC methods were used for the sperm preparation. Semen analysis was performed based on the reference value of World Health Organization (WHO) 2010. After isolating the membrane fraction of sperms, the Na+, K+-ATPase activity was defined as the difference in the released inorganic phosphate (Pi) with and without the existence of 10 mM ouabain in the reaction, while the Ca2+-ATPase was determined as the difference in Pi contents with and without the existence of 55 µm CaCl2. The prepared sperm demonstrated a higher percentage of motile sperm compared to sperm from the whole semen. Additionally, the percentage of motile sperm of post-DGC showed higher result than the sperm from post-SU. The velocity of sperm showed similar pattern with the percentage of motile sperm, in which the velocity of prepared sperm was higher than the sperm from whole semen. Furthermore, the sperm velocity of post-DGC was higher compared to the sperm from post-SU. The Na+, K+-ATPase activity of prepared sperm was higher compared to whole semen, whereas Na+, K+-ATPase activity in the post DGC was higher than post SU. The Ca2+-ATPase activity of prepared sperm was higher compared to whole semen, whereas Ca2+-ATPase activity in the post DGC was higher than post SU. The SU and the DGC methods were able to perform sperm selection by showing a high result of Na+, K+-ATPase and Ca2+-ATPase activities, moreover DGC method selected the sperm with high activities of both the Na+, K+-ATPase and Ca2+-ATPase better compared to SU method.

Toward pre-conceptual genetic analysis of human spermatozoa.

PubMed

Dozortsev, Dmitri; Serafim, Rui; Cardoso, J Jakson; Abdelmassih, Soraya; Nagy, Peter; Diamond, Michael P; Abdelmassih, Roger

2003-01-01

Nuclei of mature mammalian spermatozoa are extraordinarily resistant to chemical and thermal injury. Additionally, decondensation of spermatozoa DNA can be accompanied by little or no visual changes of the sperm head. This study tested whether human spermatozoa could be recovered following several cycles of primer extension preamplification (PEP) and used to achieve fertilization and subsequent development of human oocytes. An attempt was also made to amplify PEP buffer after spermatozoon removal. The results demonstrate that the sperm head can be successfully recovered following treatment with KOH or proteinase K followed by one to four cycles of PEP. It is also shown that following this treatment, the spermatozoa can be injected into the oocytes and will transform into a pronucleus if the oocyte is activated by sperm cytosolic fraction. In some cases, it was also possible to obtain polymerase chain reaction signals using a buffer after sperm cells were removed following several cycles of PEP. Although sperm participation in development was confirmed by fluorescence in-situ hybridization, light microscopy revealed some degree of damage to spermatozoal chromosomes. It is concluded that pre-conceptual analysis of sperm cells may be possible, but more research is necessary to determine the optimal conditions that would preserve sperm DNA integrity while allowing accurate diagnoses.

Epigenetic Transgenerational Inheritance of Altered Sperm Histone Retention Sites.

PubMed

Ben Maamar, Millissia; Sadler-Riggleman, Ingrid; Beck, Daniel; Skinner, Michael K

2018-03-28

A variety of environmental toxicants and factors have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Epigenetic alterations in the germline (sperm or egg) are required to transmit transgenerational phenotypes. The current study was designed to investigate the potential role of histones in sperm to help mediate the epigenetic transgenerational inheritance. The agricultural fungicide vinclozolin and the pesticide DDT (dichlorodiphenyltrichloroethane) were independently used to promote the epigenetic transgenerational inheritance of disease. Purified cauda epididymal sperm were collected from the transgenerational F3 generation control and exposure lineage male rats for histone analysis. A reproducible core of histone H3 retention sites was observed using an H3 chromatin immunoprecipitation (ChIP-Seq) analysis in control lineage sperm. Interestingly, the same core group of H3 retention sites plus additional differential histone retention sites (DHRs) were observed in the F3 generation exposure lineage sperm. Although new histone H3 retention sites were observed, negligible change in histone modification (methylation of H3K27me3) was observed between the control and exposure lineages. Observations demonstrate that in addition to alterations in sperm DNA methylation and ncRNA previously identified, the induction of differential histone retention sites (DHRs) also appear to be involved in environmentally induced epigenetic transgenerational inheritance.

Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation.

PubMed

Matamoros-Volante, Arturo; Moreno-Irusta, Ayelen; Torres-Rodriguez, Paulina; Giojalas, Laura; Gervasi, María G; Visconti, Pablo E; Treviño, Claudia L

2018-02-01

Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. None. This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology. This work was supported DGAPA (IN203116 to C. Treviño), Fronteras-CONACyT No. 71 and Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH (RO1 HD38082) to P.E. Visconti and by a Lalor Foundation fellowship to M.G. Gervasi. A. Matamoros is a student of the Maestría en Ciencias Bioquímicas-UNAM program supported by CONACyT (416400) and DGAPA-UNAM. A. Moreno obtained a scholarship from Red MacroUniversidades and L. Giojalas obtained a schloarhip from CONICET and Universidad Nacional de Cordoba. The authors declare there are not conflicts 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

Seminal plasma and sperm proteome of ring-tailed coatis (Nasua nasua, Linnaeus, 1766).

PubMed

Silva, Herlon Victor Rodrigues; Rodriguez-Villamil, Paula; Magalhães, Francisco Felipe de; Nunes, Thalles Gothardo Pereira; Freitas, Luana Azevedo de; Ribeiro, Leandro Rodrigues; Silva, Alexandre Rodrigues; Moura, Arlindo A; Silva, Lúcia Daniel Machado da

2018-04-15

Ring-tailed coati is listed as a species of least concern in the International Union for Conservation of Nature (IUCN) Red List, however, there has been a sharp decline in their population. The present study was conducted to evaluate the major proteins of both seminal plasma and sperm in ring-tailed coatis. Semen sample was collected from three adult coatis and evaluated for their morphological characteristics. Further, the sample was centrifuged to separate spermatozoa from seminal plasma, and then stored in liquid nitrogen. The seminal plasma and sperm proteins were subjected to one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by mass spectrometry. Gene ontology and protein networks were analyzed using bioinformatics tools. Based on sperm concentration and average protein content of the semen, the concentration of protein/spermatozoon was found to be 104.69 ± 44.43 μg. The analysis of SDS-PAGE gels showed 20.3 ± 3.1 and 17 ± 2 protein bands/lane for seminal plasma and sperm, respectively. In-gel protein digestion and peptide analysis by mass spectrometry revealed 238 and 246 proteins in the seminal plasma and sperm, respectively. The gene ontology analysis revealed that the proteins of seminal plasma mainly participated in cellular (35%) and regulatory (21%) processes. According to their cellular localization, seminal plasma proteins were categorized as structural (18%), extracellular (17%), and nuclear (14%) proteins with molecular functions, such as catalytic activity (43%) and binding (43%). The sperm proteins were also involved in cellular (38%) and regulatory (23%) processes, and mainly categorized as extracellular (17%), nuclear (13%), and cytoplasmic (10%) proteins. The major molecular functions of the sperm proteins were catalytic activity (44%) and binding (42%). These results indicated that the seminal plasma of ring-tailed coati has an array of proteins that can potentially modulate several sperm functions, from sperm protection to oocyte binding. However, further studies are necessary to interpret the roles of these major seminal plasma proteins in coatis. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation.

PubMed

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

2012-07-01

The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose-egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (P<0.05). However, from the three monosaccharides tested, glucose provided the best sperm quality after freezing-thawing. With respect to the disaccharides studied, samples frozen with the extender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (P<0.05) in the samples cryopreserved with the trehalose extender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed

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

2017-04-01

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

Effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility.

PubMed

Soler, Carles; Picazo-Bueno, José Á; Micó, Vicente; Valverde, Anthony; Bompart, Daznia; Blasco, Francisco J; Álvarez, Juan G; García-Molina, Almudena

2018-05-04

Sperm motility is one of the most significant parameters in the prediction of male fertility. Until now, both motility analysis using an optical microscope and computer-aided sperm analysis (CASA-Mot) entailed the use of counting chambers with a depth to 20µm. Chamber depth significantly affects the intrinsic sperm movement, leading to an artificial motility pattern. For the first time, laser microscopy offers the possibility of avoiding this interference with sperm movement. The aims of the present study were to determine the different motility patterns observed in chambers with depths of 10, 20 and 100µm using a new holographic approach and to compare the results obtained in the 20-µm chamber with those of the laser and optical CASA-Mot systems. The ISAS®3D-Track results showed that values for curvilinear velocity (VCL), straight line velocity, wobble and beat cross frequency were higher for the 100-µm chambers than for the 10- and 20-µm chambers. Only VCL showed a positive correlation between chambers. In addition, Bayesian analysis confirmed that the kinematic parameters observed with the 100-µm chamber were significantly different to those obtained using chambers with depths of 10 and 20µm. When an optical analyser CASA-Mot system was used, all kinematic parameters, except VCL, were higher with ISAS®3D-Track, but were not relevant after Bayesian analysis. Finally, almost three different three-dimensional motility patterns were recognised. In conclusion, the use of the ISAS®3D-Track allows for the analysis of the natural three-dimensional pattern of sperm movement.

A novel cost-effective approach to post-vasectomy semen analysis.

PubMed

Senanayake, Eshan; Pacey, Allan A; Maddireddy, Vinod; Shariff, Umar; Hastie, Ken; Rosario, Derek J

2011-05-01

• To examine compliance, clearance rates and cost-effectiveness of a novel approach to managing men following vasectomy based on the testing of sperm viability. • Between January 2003 and March 2005, 832 men undergoing vasectomy were followed prospectively for a minimum of 12 months. • Post-vasectomy semen analysis (PVSA) was carried out at 16 weeks with repeat at 20 weeks only if sperm were detected on initial PVSA i.e. a single clear PVSA on simple microscopy was deemed sufficient for declaring vasectomy successful. • In men with persistent non-motile sperm (PNMS) in the second specimen, comprehensive analysis of number and viability of sperm using a fluorescent probe was carried out on a fresh semen specimen taken in accordance with British Andrology Society (BAS) guidelines. • Overall compliance with the PVSA protocol was 81.3% (95% CI 78.5 to 83.8). No sperm were seen in 540 (78.8%) and 70 (10.3%) at the initial and 2(nd) PVSA respectively. • Persistent spermatozoa at 20 weeks were present in 66 (9.8%, 7.8 to 12.2) cases with 58 (8.6%, 6.7 to 11.0) having PNMS and 8 (1.2%, 0.6 to 2.3) having motile sperm. • Fluorescent viability testing in 53 of the 58 with PNMS showed viable sperm in 2 (3.8%, 1.0 to 12.8). The failure rate of vasectomy defined by PVSA (8 with motile sperm on 2(nd) PVSA and 2 with viable non-motile sperm on fluorescent testing) was 1.2% (0.7 to 2.2). • Average cost per vasectomy of PVSA using this protocol was £10.77 (US$ 16.67) compared with a minimum likely average cost using BAS guidelines of £18.10 (US$ 28). • Demonstrating absence of sperm on simple light microscopy in a single specimen of semen at 16 or 20 weeks post-vasectomy and reserving comprehensive testing of sperm viability for only the higher risk group with PNMS improves compliance and represents a cost-effective strategy for declaring surgical success. This reduces the costs of PVSA by least 40% compared with adherence with BAS guidelines without compromising success in determining outcome after vasectomy. © 2010 THE AUTHORS. BJU INTERNATIONAL © 2010 BJU INTERNATIONAL.

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

PubMed

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

2017-02-01

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

Sperm quality and oxidative status as affected by homogenization of liquid-stored boar semen diluted in short- and long-term extenders.

PubMed

Menegat, Mariana B; Mellagi, Ana Paula G; Bortolin, Rafael C; Menezes, Tila A; Vargas, Amanda R; Bernardi, Mari Lourdes; Wentz, Ivo; Gelain, Daniel P; Moreira, José Cláudio F; Bortolozzo, Fernando P

2017-04-01

Homogenization of diluted boar semen during storage has for a long time been regarded as beneficial. Recent studies indicated an adverse effect of homogenization on sperm quality for yet unknown reasons. This study aimed to verify the effect of homogenization on sperm parameters and to elucidate the impact of oxidative stress. Twenty-one normospermic ejaculates (21 boars) were diluted with Androstar ® Plus (AND) and Beltsville Thawing Solution (BTS). Semen doses were submitted to no-homogenization (NoHom) or twice-a-day manual homogenization (2xHom) during storage at 17°C for 168h. NoHom and 2xHom were similar (P>0.05) for both short- and long-term extenders with respect to motility and kinematics parameters (CASA system), membrane viability (SYBR-14/PI), acrosome integrity, lipid peroxidation, protein oxidation, intracellular reactive oxygen species, sulfhydryl content, and total radical-trapping antioxidant potential. 2xHom reduced sperm motility and motion kinematics (VCL, VSL, VAP, BCF, and ALH) following the thermoresistance test and presented with a slight increase in pH along the storage (P=0.05) as compared to NoHom. Furthermore, 2xHom semen doses presented with a constant SOD and GSH-Px activity during storage whereas enzymatic activity increased for NoHom at the end of the storage. These findings confirm that homogenization of semen doses is detrimental to sperm quality. Moreover, it is shown that the effect of homogenization is unlikely to be primarily related to oxidative stress. Homogenization is not recommended for storage of liquid boar semen for up to 168h in both short- and long-term extenders. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of diluting medium and holding time on sperm motility analysis by CASA in ram.

PubMed

Mostafapor, Somayeh; Farrokhi Ardebili, Farhad

2014-01-01

The aim of this study was to evaluate the effects of dilution rate and holding time on various motility parameters using computer-assisted sperm analysis (CASA). The semen samples were collected from three Ghezel rams. Samples were diluted in seminal plasma (SP), phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and Bioexcell. The motility parameters that computed and recorded by CASA include curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), straightness (STR), linearity (LIN), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF). In all diluters, there was a decrease in the average of all three parameters of sperms movement velocity as the time passed, but density of this decrease was more intensive in SP. The average of ALH between diluters indicated a significant difference, as it was more in Bioexcell in comparison with the similar amount in SP and PBS. The average of LIN in the diluted sperms in Bioexcell was less than two other diluters in all three times. The motility parameters of the diluted sperms in Bioexcell and PBS indicated an important and considerable difference with the diluted sperms in SP. According to the gained results, the Bioexcell has greater ability in preserving motility of sperm in comparison with the other diluters but as SP is considered as physiological environment for sperm. It seems that the evaluation of the motility parameters in Bioexcell and PBS cannot be an accurate and comparable evaluation with SP.

Seasonal variation of human sperm cells among 4,422 semen samples: A retrospective study in Turkey.

PubMed

Ozelci, Runa; Yılmaz, Saynur; Dilbaz, Berna; Akpınar, Funda; Akdag Cırık, Derya; Dilbaz, Serdar; Ocal, Aslı

2016-12-01

We aimed to assess the possible presence of a seasonal pattern in three parameters of semen analysis: sperm concentration, morphology, and motility as a function of the time of ejaculation and sperm production (spermatogenesis) in normal and oligozoospermic men. This retrospective study included a consecutive series of 4,422 semen samples that were collected from patients as a part of the basic evaluation of the infertile couples attending the Reproductive Endocrine Outpatient Clinic of a tertiary women's hospital in Ankara, Turkey, between January 1, 2012 and December 31, 2013. The samples were classified according to sperm concentration: ≥15 x10 6 /mL as normozoospermic samples and 4 -14.99 x10 6 /mL as oligozoospermic samples and seasonal analysis of the semen samples were carried out separately. When the data was analyzed according to the season of semen production, there was no seasonal effect on the sperm concentration. A gradual and consistent decrease in the rate of sperm with fast forward motility was observed from spring to fall with a recovery noticed during the winter. The percentage of sperms with normal morphology was found to be statistically significantly higher in the spring samples compared with the summer samples (p=0.001). Both normozoospermic and oligozoospermic semen samples appeared to have better sperm parameters in spring and winter. The circannual variation of semen parameters may be important in diagnosis and treatment desicions. WHO: World Health Organization; mRNA:messenger ribonucleic acid.

Immature germ cells in semen - correlation with total sperm count and sperm motility.

PubMed

Patil, Priya S; Humbarwadi, Rajendra S; Patil, Ashalata D; Gune, Anita R

2013-07-01

Current data regarding infertility suggests that male factor contributes up to 30% of the total cases of infertility. Semen analysis reveals the presence of spermatozoa as well as a number of non-sperm cells, presently being mentioned in routine semen report as "round cells" without further differentiating them into leucocytes or immature germ cells. The aim of this work was to study a simple, cost-effective, and convenient method for differentiating the round cells in semen into immature germ cells and leucocytes and correlating them with total sperm counts and motility. Semen samples from 120 males, who had come for investigation for infertility, were collected, semen parameters recorded, and stained smears studied for different round cells. Statistical analysis of the data was done to correlate total sperm counts and sperm motility with the occurrence of immature germ cells and leucocytes. The average shedding of immature germ cells in different groups with normal and low sperm counts was compared. The clinical significance of "round cells" in semen and their differentiation into leucocytes and immature germ cells are discussed. Round cells in semen can be differentiated into immature germ cells and leucocytes using simple staining methods. The differential counts mentioned in a semen report give valuable and clinically relevant information. In this study, we observed a negative correlation between total count and immature germ cells, as well as sperm motility and shedding of immature germ cells. The latter was statistically significant with a P value 0.000.

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.

Does being overweight affect seminal variables in fertile men?

PubMed

Taha, Emad A; Sayed, Sohair K; Gaber, Hisham D; Abdel Hafez, Hatem K; Ghandour, Nagwa; Zahran, Asmaa; Mostafa, Taymour

2016-12-01

The effect of being overweight on seminal variables was assesed in 165 fertile men. Participants were divided into three groups: fertile men with normal body mass index (BMI) (18.5-24.9 kg/m 2 ), fertile overweight men (BMI 25-29.9 kg/m 2 ) and fertile obese men (BMI >30 kg/m 2 ). Medical history was taken, a clinical examination conducted. Semen analysis was undertaken and BMI measured. Seminal reactive oxygen species (ROS) was estimated by chemiluminescent assay, sperm vitality by the hypo-osmotic swelling test and sperm DNA fragmentation by propidium iodide staining with flowcytometry. Fertile obese men had significantly lower sperm concentration, progressive sperm motility and sperm normal morphology, with significantly higher seminal ROS and sperm DNA fragmentation compared with fertile normal-weight men and overweight men (all P < 0.05). BMI was negatively correlated with sperm concentration (r = -0.091; P = 0.014), progressive sperm motility (r = -0.697; P = 0.001), normal sperm morphology (r = -0.510; P = 0.001), sperm vitality (r = -0.586; P = 0.001), but positively correlated with sperm DNA fragmentation percentage (r = 0.799; P = 0.001) and seminal ROS (r = 0.673; P = 0.001). Increased BMI was found to affect semen parameters negatively even in fertile men. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

[Prognostic value on recovery rates for the application of sperm preparation techniques and their evaluation in sperm function].

PubMed

Barroso, Gerardo; Chaya, Miguel; Bolaños, Rubén; Rosado, Yadira; García León, Fernando; Ibarrola, Eduardo

2005-05-01

To evaluate sperm recovery and total sperm motility in three different sperm preparation techniques (density gradient, simple washing and swim-up). A total of 290 subjects were randomly evaluated from November 2001 to March 2003. The density gradient method required Isolate (upper and lower layers). Centrifugation was performed at 400 g for 10 minutes and evaluation was done using the Makler counting chamber. The simple washing method included the use of HTF-M complemented with 7.5% of SSS, with centrifugation at 250 g, obtaining at the end 0.5 mL of the sperm sample. The swim-up method required HTF-M complemented with 7.5% of SSS, with an incubation period of 60 minutes at 37 degrees C. The demographic characteristics evaluated through their standard error, 95% ICC, and 50th percentile were similar. The application of multiple comparison tests and analysis of variance showed significant differences between the sperm preparations before and after capacitation. It was observed a superior recovery rate with the density gradient and swim-up methods; nevertheless, the samples used for the simple washing method showed a diminished sperm recovery from the original sample. Sperm preparation techniques have become very useful in male infertility treatments allowing higher sperm recovery and motility rates. The seminal parameters evaluated from the original sperm sample will determine the best sperm preparation technique in those patients who require it.

Pregnancy prediction by free sperm DNA and sperm DNA fragmentation in semen specimens of IVF/ICSI-ET patients.

PubMed

Bounartzi, Theofania; Dafopoulos, Konstantinos; Anifandis, George; Messini, Christina I; Koutsonikou, Chrysoula; Kouris, Spyros; Satra, Maria; Sotiriou, Sotirios; Vamvakopoulos, Nicholas; Messinis, Ioannis E

2016-04-01

The purpose of this study was to evaluate the predictive value of free sperm plasma DNA (f-spDNA) and sperm DNA fragmentation (SDF), in semen specimens from men undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments. Fifty-five semen samples were evaluated during 55 consecutive IVF/ICSI-ET cycles. F-spDNA was determined by conventional quantitative real-time PCR-Sybr green detection approach, while evaluation of sperm DNA damage was performed using the sperm chromatin dispersion (SCD) assay. While f-spDNA only correlated with total sperm count, SDF correlated with many semen parameters (including sperm concentration, total sperm count and the per cent of non-progressive sperm). Neither SDF nor the proportion of sperm with small or no halos correlated with f-spDNA. Interestingly, smoking status correlated with f-spDNA but not with SDF. Although these two factors seem to interact for the prediction of pregnancy, receiver-operating characteristics (ROC) analysis revealed that SDF had a stronger predictive value (AUC = 0.7, p < 0.05) than f-spDNA (AUC = 0.6, p > 0.05). SDF and f-spDNA may not be associated together but they interact at a significant level in order to exert their actions on pregnancy outcome. Among the two markers, SDF appears to have stronger and significantly predictive value for pregnancy success.

A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome

PubMed Central

Simon, Luke; Zini, Armand; Dyachenko, Alina; Ciampi, Antonio; Carrell, Douglas T

2017-01-01

Sperm DNA damage is prevalent among infertile men and is known to influence natural reproduction. However, the impact of sperm DNA damage on assisted reproduction outcomes remains controversial. Here, we conducted a meta-analysis of studies on sperm DNA damage (assessed by SCSA, TUNEL, SCD, or Comet assay) and clinical pregnancy after IVF and/or ICSI treatment from MEDLINE, EMBASE, and PUBMED database searches for this analysis. We identified 41 articles (with a total of 56 studies) including 16 IVF studies, 24 ICSI studies, and 16 mixed (IVF + ICSI) studies. These studies measured DNA damage (by one of four assays: 23 SCSA, 18 TUNEL, 8 SCD, and 7 Comet) and included a total of 8068 treatment cycles (3734 IVF, 2282 ICSI, and 2052 mixed IVF + ICSI). The combined OR of 1.68 (95% CI: 1.49–1.89; P < 0.0001) indicates that sperm DNA damage affects clinical pregnancy following IVF and/or ICSI treatment. In addition, the combined OR estimates of IVF (16 estimates, OR = 1.65; 95% CI: 1.34–2.04; P < 0.0001), ICSI (24 estimates, OR = 1.31; 95% CI: 1.08–1.59; P = 0.0068), and mixed IVF + ICSI studies (16 estimates, OR = 2.37; 95% CI: 1.89–2.97; P < 0.0001) were also statistically significant. There is sufficient evidence in the existing literature suggesting that sperm DNA damage has a negative effect on clinical pregnancy following IVF and/or ICSI treatment. PMID:27345006

Characterisation of the Manduca sexta sperm proteome: Genetic novelty underlying sperm composition in Lepidoptera.

PubMed

Whittington, Emma; Zhao, Qian; Borziak, Kirill; Walters, James R; Dorus, Steve

2015-07-01

The application of mass spectrometry based proteomics to sperm biology has greatly accelerated progress in understanding the molecular composition and function of spermatozoa. To date, these approaches have been largely restricted to model organisms, all of which produce a single sperm morph capable of oocyte fertilisation. Here we apply high-throughput mass spectrometry proteomic analysis to characterise sperm composition in Manduca sexta, the tobacco hornworm moth, which produce heteromorphic sperm, including one fertilisation competent (eupyrene) and one incompetent (apyrene) sperm type. This resulted in the high confidence identification of 896 proteins from a co-mixed sample of both sperm types, of which 167 are encoded by genes with strict one-to-one orthology in Drosophila melanogaster. Importantly, over half (55.1%) of these orthologous proteins have previously been identified in the D. melanogaster sperm proteome and exhibit significant conservation in quantitative protein abundance in sperm between the two species. Despite the complex nature of gene expression across spermatogenic stages, a significant correlation was also observed between sperm protein abundance and testis gene expression. Lepidopteran-specific sperm proteins (e.g., proteins with no homology to proteins in non-Lepidopteran taxa) were present in significantly greater abundance on average than those with homology outside the Lepidoptera. Given the disproportionate production of apyrene sperm (96% of all mature sperm in Manduca) relative to eupyrene sperm, these evolutionarily novel and highly abundant proteins are candidates for possessing apyrene-specific functions. Lastly, comparative genomic analyses of testis-expressed, ovary-expressed and sperm genes identified a concentration of novel sperm proteins shared amongst Lepidoptera of potential relevance to the evolutionary origin of heteromorphic spermatogenesis. As the first published Lepidopteran sperm proteome, this whole-cell proteomic characterisation will facilitate future evolutionary genetic and developmental studies of heteromorphic sperm production and parasperm function. Furthermore, the analyses presented here provide useful annotation information regarding sex-biased gene expression, novel Lepidopteran genes and gene function in the male gamete to complement the newly sequenced and annotated Manduca genome. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hydrodynamic context for considering turbulence impacts on external fertilization.

PubMed

Gaylord, Brian

2008-06-01

Wave-swept shores in the marine environment are characterized by turbulent water motion. This turbulence influences external fertilization in benthic organisms by diluting gametes and producing hydrodynamic shear that is believed to have the capacity to disrupt egg-sperm interaction. However, although turbulence levels associated with decreases in fertilization due to this latter process (shear) have been identified in the laboratory, estimates of the intensities of turbulence in intertidal habitats have been based primarily on scaling arguments of limited precision and unknown accuracy. In the present study, values of energy dissipation rate (a standard measure of the strength of turbulence) were determined for three locations in the surf zone of a rocky shore. These measurements suggest a potential correspondence between threshold levels of turbulence that impair the ability of sperm to fertilize eggs, and actual intensities of turbulence arising in nature.

Effect of semen quality on human sex ratio in in vitro fertilization and intracytoplasmic sperm injection: an analysis of 27,158 singleton infants born after fresh single-embryo transfer.

PubMed

Arikawa, Mikiko; Jwa, Seung Chik; Kuwahara, Akira; Irahara, Minoru; Saito, Hidekazu

2016-04-01

To evaluate the effect of semen quality on human sex ratio in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Retrospective cohort study. Not applicable. A total of 27,158 singleton infants born between 2007 and 2012 after fresh single-embryo transfer. None. Proportion of male infants among liveborn infants. There were 14,996 infants born after IVF, 12,164 infants born after ICSI with ejaculated sperm, and 646 infants born after ICSI with nonejaculated sperm. The sex ratio of IVF was 53.1% (95% confidence interval [CI], 52.3-53.9); the sex ratio of ICSI with ejaculated and nonejaculated sperm demonstrated as statistically significant reduction (48.2%; 95% CI, 47.3-49.1 and 47.7%; 95% CI, 43.8-51.6, respectively). In IVF, lower sperm motility, including asthenozoospermia (sperm motility <40%), was associated with a statistically significantly lower sex ratio compared with normal sperm (51.0%; 95% CI, 48.6-53.3 vs. 53.4%; 95% CI, 52.5-54.3). In ICSI with ejaculated sperm, there was no association between sperm motility and sex ratio. Sperm concentration was not associated with sex ratio in both IVF and ICSI. In IVF, lower sperm motility was associated with a statistically significant reduction in sex ratio; ICSI with either ejaculated or nonejaculated sperm was associated with a statistically significant reduction in sex ratio regardless of semen quality. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

[Relationship of abnormal sperm DNA methylation with early spontaneous abortion].

PubMed

Pan, Lian-Jun; Ma, Jie-Hua; Zhang, Feng-Lei; Zhao, Dan; Pan, Feng; Zhang, Xing-Yuan

2016-10-01

To investigate the relationship between the abnormal sperm DNA methylation level and early spontaneous abortion. We randomly selected 98 males who met the inclusion criteria and whose wives suffered from unexplained abortion or embryo abortion, and included another 46 normal healthy men present for pre-pregnancy check-up as controls. We examined the semen quality and sperm morphology, obtained the sperm DNA fragmentation index (DFI) by modified sperm chromatin dispersion, and measured the sperm DNA methylation level using the methylated DNA quantification kit and the colorimetric method. Compared with the normal controls, the men in the unexplained abortion group showed a significantly lower rate of big-halo sperm (［45.50 ± 26.27］ vs ［36.49 ± 23.06］%, P = 0.038), a higher rate of abnormal-head sperm (［77.08± 12.21］ vs ［81.09± 10.89］%, P = 0.049), and a lower level of sperm DNA methylation (［0.47 ± 0.33］ vs ［0.36 ± 0.26］ ng/μl, P = 0.035). The sperm DNA methylation level was positively correlated with the percentage of big-halo sperm (OR=0.546, P<0.01). Multivariate regression analysis manifested that sperm head abnormality was an independent risk factor of early spontaneous abortion or embryo abortion (OR=1.032, P = 0.049), while the high methylation level was protective factor against early spontaneous abortion or embryo abortion (OR=0.244, P = 0.03). The abnormal level of sperm DNA methylation may be one of the important reasons for early spontaneous abortion or embryo abortion.

Leptin Improves Sperm Cryopreservation via Antioxidant Defense

PubMed Central

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

2017-01-01

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

Review: Diagnosis and impact of sperm DNA alterations in assisted reproduction.

PubMed

Simon, Luke; Emery, Benjamin R; Carrell, Douglas T

2017-10-01

Sperm nuclear and chromatin abnormalities are common among infertile men and are known to influence natural reproduction. These abnormalities are also considered detrimental to normal fertilization, embryo development, and successful implantation and pregnancies following assisted reproductive treatment (ART). Abnormalities in the sperm nucleus can be broadly classified into sperm chromosomal abnormalities (aneuploidies) and sperm DNA abnormalities such as abnormal packing, DNA integrity, or DNA fragmentation. For the past 30 years, numerous tests have been developed to quantify these abnormalities in sperm. In this chapter, we review the causes of sperm DNA and chromosomal abnormalities, describe the commonly used tests to evaluate these abnormalities, and finally review the impact of these abnormalities on male fertility and ART outcomes. We also performed a comprehensive meta-analysis and systematic review from the existing literature to summarize the effect of sperm DNA fragmentation on ART outcomes such as fertilization rate, embryo quality, and clinical pregnancies. A review of the literature presented in this chapter suggests that sperm nuclear and chromatin abnormalities are associated with male infertility, and they reduce the probability of a successful pregnancy following ART. Copyright © 2017. Published by Elsevier Ltd.

[Eosin Y-water test for sperm function examination].

PubMed

Zha, Shu-wei; Lü, Nian-qing; Xu, Hao-qin

2015-06-01

Based on the principles of the in vitro staining technique, hypotonic swelling test, and water test, the Eosin Y-water test method was developed to simultaneously detect the integrity of the sperm head and tail and sperm membrane structure and function. As a widely used method in clinical laboratories in China, the Eosin Y-water test is methodologically characterized by three advantages. Firstly, both the sperm head and tail can be detected at the same time, which allows easy and comprehensive assessment of membrane damage in different parts of sperm. Secondly, distilled water is used instead of the usual formula solution to simplify and standardize the test by eliminating any potential effects on the water molecules through the sperm membrane due to different osmotic pressure or different sugar proportions and electrolyte solutions. Thirdly, the test takes less time and thus can be repeated before and after treatment. This article focuses on the fundamental principles and modification of the Eosin Y-water test and its application in sperm function examination and routine semen analysis for male infertility, assessment of the quality of sperm retrieved by testicular fine needle aspiration, semen cryopreservation program development, and evaluation of sperm membrane integrity after microwave radiation.

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.

The Semen pH Affects Sperm Motility and Capacitation.

PubMed

Zhou, Ji; Chen, Li; Li, Jie; Li, Hongjun; Hong, Zhiwei; Xie, Min; Chen, Shengrong; Yao, Bing

2015-01-01

As the chemical environment of semen can have a profound effect on sperm quality, we examined the effect of pH on the motility, viability and capacitation of human sperm. The sperm in this study was collected from healthy males to avoid interference from other factors. The spermatozoa cultured in sperm nutrition solution at pH 5.2, 6.2, 7.2 and 8.2 were analyzed for sperm total motility, progressive motility (PR), hypo-osmotic swelling (HOS) rate, and sperm penetration. Our results showed that these parameters were similar in pH 7.2 and 8.2 sperm nutrition solutions, but decreased in pH 5.2 and 6.2 solutions. The HOS rate exhibited positive correlation with the sperm total motility and PR. In addition, the sperm Na(+)/K(+)-ATPase activity at different pHs was measured, and the enzyme activity was significantly lower in pH 5.2 and 6.2 media, comparing with that in pH 8.2 and pH 7.2 solutions. Using flow cytometry (FCM) and laser confocal scanning microscopy (LCSM) analysis, the intracellular Ca2(+ )concentrations of sperm cultured in sperm capacitation solution at pH 5.2, 6.2, 7.2 and 8.2 were determined. Compared with that at pH 7.2, the mean fluorescence intensity of sperm in pH 5.2 and 6.2 media decreased significantly, while that of pH 8.2 group showed no difference. Our results suggested that the declined Na(+)/K(+)-ATPase activity at acidic pHs result in decreased sperm movement and capacitation, which could be one of the mechanisms of male infertility.

CASAnova: a multiclass support vector machine model for the classification of human sperm motility patterns.

PubMed

Goodson, Summer G; White, Sarah; Stevans, Alicia M; Bhat, Sanjana; Kao, Chia-Yu; Jaworski, Scott; Marlowe, Tamara R; Kohlmeier, Martin; McMillan, Leonard; Zeisel, Steven H; O'Brien, Deborah A

2017-11-01

The ability to accurately monitor alterations in sperm motility is paramount to understanding multiple genetic and biochemical perturbations impacting normal fertilization. Computer-aided sperm analysis (CASA) of human sperm typically reports motile percentage and kinematic parameters at the population level, and uses kinematic gating methods to identify subpopulations such as progressive or hyperactivated sperm. The goal of this study was to develop an automated method that classifies all patterns of human sperm motility during in vitro capacitation following the removal of seminal plasma. We visually classified CASA tracks of 2817 sperm from 18 individuals and used a support vector machine-based decision tree to compute four hyperplanes that separate five classes based on their kinematic parameters. We then developed a web-based program, CASAnova, which applies these equations sequentially to assign a single classification to each motile sperm. Vigorous sperm are classified as progressive, intermediate, or hyperactivated, and nonvigorous sperm as slow or weakly motile. This program correctly classifies sperm motility into one of five classes with an overall accuracy of 89.9%. Application of CASAnova to capacitating sperm populations showed a shift from predominantly linear patterns of motility at initial time points to more vigorous patterns, including hyperactivated motility, as capacitation proceeds. Both intermediate and hyperactivated motility patterns were largely eliminated when sperm were incubated in noncapacitating medium, demonstrating the sensitivity of this method. The five CASAnova classifications are distinctive and reflect kinetic parameters of washed human sperm, providing an accurate, quantitative, and high-throughput method for monitoring alterations in motility. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The classic EDCs, phthalate esters and organochlorines, in relation to abnormal sperm quality: a systematic review with meta-analysis.

PubMed

Wang, Chao; Yang, Lu; Wang, Shu; Zhang, Zhan; Yu, Yongquan; Wang, Meilin; Cromie, Meghan; Gao, Weimin; Wang, Shou-Lin

2016-01-25

The association between endocrine disrupting chemicals (EDCs) and human sperm quality is controversial due to the inconsistent literature findings, therefore, a systematic review with meta-analysis was performed. Through the literature search and selection based on inclusion criteria, a total of 9 studies (7 cross-sectional, 1 case-control, and 1 pilot study) were analyzed for classic EDCs (5 studies for phthalate esters and 4 studies for organochlorines). Funnel plots revealed a symmetrical distribution with no evidence of publication bias (Begg's test: intercept = 0.40; p = 0.692). The summary odds ratios (OR) of human sperm quality associated with the classic EDCs was 1.67 (95% CI: 1.31-2.02). After stratification by specific chemical class, consistent increases in the risk of abnormal sperm quality were found in phthalate ester group (OR = 1.52; 95% CI: 1.09-1.95) and organochlorine group (OR = 1.98; 95% CI: 1.34-2.62). Additionally, identification of official data, and a comprehensive review of the mechanisms were performed, and better elucidated the increased risk of these classic EDCs on abnormal sperm quality. The present systematic review and meta-analysis helps to identify the impact of classic EDCs on human sperm quality. However, it still highlights the need for additional epidemiological studies in a larger variety of geographic locations.

The classic EDCs, phthalate esters and organochlorines, in relation to abnormal sperm quality: a systematic review with meta-analysis

NASA Astrophysics Data System (ADS)

Wang, Chao; Yang, Lu; Wang, Shu; Zhang, Zhan; Yu, Yongquan; Wang, Meilin; Cromie, Meghan; Gao, Weimin; Wang, Shou-Lin

2016-01-01

The association between endocrine disrupting chemicals (EDCs) and human sperm quality is controversial due to the inconsistent literature findings, therefore, a systematic review with meta-analysis was performed. Through the literature search and selection based on inclusion criteria, a total of 9 studies (7 cross-sectional, 1 case-control, and 1 pilot study) were analyzed for classic EDCs (5 studies for phthalate esters and 4 studies for organochlorines). Funnel plots revealed a symmetrical distribution with no evidence of publication bias (Begg’s test: intercept = 0.40 p = 0.692). The summary odds ratios (OR) of human sperm quality associated with the classic EDCs was 1.67 (95% CI: 1.31-2.02). After stratification by specific chemical class, consistent increases in the risk of abnormal sperm quality were found in phthalate ester group (OR = 1.52 95% CI: 1.09-1.95) and organochlorine group (OR = 1.98 95% CI: 1.34-2.62). Additionally, identification of official data, and a comprehensive review of the mechanisms were performed, and better elucidated the increased risk of these classic EDCs on abnormal sperm quality. The present systematic review and meta-analysis helps to identify the impact of classic EDCs on human sperm quality. However, it still highlights the need for additional epidemiological studies in a larger variety of geographic locations.

Canonical correlation analysis to identify the semen characteristics used to forecast the freeze survival of Curimba (Prochilodus lineatus) spermatozoa.

PubMed

Carvalho, A F S; Murgas, L D S; Ferreira-Machad, M R; Andrade, E S; Felizardo, V O; Allaman, I B; de Paula, F G

　　OBJECTIVE: To identify which sperm characteristics were able to predict more accurately the quality of curimba (Prochilodus lineatus) semen upon freezing using canonical correlation analysis. Eleven fish breeders with initial mean weight of 705.21 ± 111 g were used. For cryopreservation, 200 µL of semen were taken from each animal and diluted in the cryoprotectant solution (10% dimethyl sulfoxide and 5% Beltsville Thawing Solution Minitub) in a 1:4 ratio and placed into 0.5-mL straws. Sperm characteristics (motility, sperm abnormalities, total antioxidant activity and lipid peroxidation) were evaluated. A randomized block design with duplicate samples per treatment (fresh and frozen semen) was used. The block factor was the animals, and the experimental unit the ejaculates. Canonical correlation was used to evaluate the association between sperm characteristics of fresh semen and thawed semen. There was a significant association (P = 0.10) among the variables measured in fresh semen with the variables measured in thawed semen, and 78.6% of the difference observed in the thawed semen can be attributed to variation of variables measured in fresh semen. Sperm motility, motility duration and antioxidant activity of the thawed semen showed an inverse relationship with those of the fresh semen; whereas the minor sperm abnormalities, major sperm abnormalities and lipid peroxidation showed a direct relationship with those of the fresh semen. Only the rate and motility duration of the thawed semen presented high correlation (-0.63 and -0.73, respectively) with the canonical variable represented by the sperm characteristics of fresh semen. The rate and motility duration of fresh semen may be used to predict the quality of the thawed sperm in Prochilodus lineatus.

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.

Hierarchical radial and polar organisation of chromosomes in human sperm.

PubMed

Millan, N M; Lau, P; Hann, M; Ioannou, D; Hoffman, D; Barrionuevo, M; Maxson, W; Ory, S; Tempest, H G

2012-10-01

It is well established that chromosomes occupy distinct positions within the interphase nuclei, conferring a potential functional implication to the genome. In addition, alterations in the nuclear organisation patterns have been associated with disease phenotypes (e.g. cancer or laminopathies). The human sperm is the smallest cell in the body with specific DNA packaging and the mission of delivering the paternal genome to the oocyte during fertilisation. Studies of nuclear organisation in the sperm have postulated nonrandom chromosome position and have proposed a chromocentre model with the centromeres facing toward the interior and the telomeres toward the periphery of the nucleus. Most studies have assessed the nuclear address in the sperm longitudinally predominantly using centromeric or telomeric probes and to a lesser extent with whole chromosome paints. To date, studies investigating the radial organisation of human sperm have been limited. The purpose of this study was to utilise whole chromosome paints for six clinically important chromosomes (18, 19, 21, 22, X, and Y) to investigate nuclear address by assessing their radial and longitudinal nuclear organisation. A total of 10,800 sperm were analysed in nine normozoospermic individuals. The results have shown nonrandom chromosome position for all chromosomes using both methods of analysis. We present novel radial and polar analysis of chromosome territory localization within the human sperm nucleus. Specifically, a hierarchical organisation was observed radially with chromosomes organised from the interior to the periphery (chromosomes 22, 21, Y, X, 19, and 18 respectively) and polar organisation from the sperm head to tail (chromosomes X, 19, Y, 22, 21, and 18, respectively). We provide evidence of defined nuclear organisation in the human sperm and discuss the function of organisation and potential possible clinical ramifications of these results in regards to male infertility and early human development.

Predictive value of sperm-FISH analysis on the outcome of preimplantation genetic diagnosis (PGD) for a pericentric inversion inv5(p15.3q11.2) carrier.

PubMed

Bernicot, I; Dechanet, C; Mace, A; Hedon, B; Hamamah, S; Pellestor, F; Anahory, T

2010-07-01

Pericentric inversions (PIs) are structural chromosomal abnormalities, potentially associated with infertility or multiple miscarriages. More rarely, at meiosis, odd numbers of genetic recombinations within the inversion loop produce recombinant gametes which may lead to aneusomy of recombination in the offspring. We report a FISH segregation analysis of an inv5(p15.3q11.2) carrier, both in sperm and blastomeres. In sperm, we directly evaluated the proportion of recombinant gametes and compared the results with chromosomal abnormalities found in blastomeres collected from embryos obtained following a preimplantation genetic diagnosis (PGD) procedure. A total of 7006 sperm nuclei were analyzed. The size of the inverted segment represented 27% of the total length of chromosome 5. The frequencies of balanced chromosomes (normal or inverted), recombinant chromosomes and unbalanced combinations were 97.1, 0.17 and 2.73%, respectively. Of six embryos, PGD FISH analysis revealed that one was a balanced embryo, whereas five were unbalanced and there were no recombinants. This study demonstrated the value of sperm-FISH analysis in providing reproductive genetic counseling for PI carriers. Our study also highlights the clinical relevance of performing PGD instead of prenatal diagnosis.

Measurement and significance of sperm morphology

PubMed Central

Menkveld, Roelof; Holleboom, Cas AG; Rhemrev, Johann PT

2011-01-01

The measurement or evaluation and clinical significance of human sperm morphology has always been and still is a controversial aspect of the semen analysis for the determination of a male's fertility potential. In this review the background of the development of the evaluation criteria for sperm morphology will be discussed. Aspects of criticism on the strict criteria definition and use of the criteria for sperm morphology evaluation will be discussed as well as possible reasons for the decline in normal sperm morphology values and how we can compromise for this phenomenon resulting in the very low normal reference value as published in the 2010 WHO manual for the Examination and Processing of Human Semen. One of the possible solutions may be to give more attention to a limited number of abnormal sperm morphology categories and the inclusion of sperm morphology patterns. It is concluded in this review that if done correctly and with care and with strict application of existing guidelines as outlined in the 2010 WHO manual, sperm morphology measurement still has a very important role to play in the clinical evaluation of male fertility potential. PMID:21076438

Evaluating the efficacy of DNA differential extraction methods for sexual assault evidence.

PubMed

Klein, Sonja B; Buoncristiani, Martin R

2017-07-01

Analysis of sexual assault evidence, often a mixture of spermatozoa and victim epithelial cells, represents a significant portion of a forensic DNA laboratory's case load. Successful genotyping of sperm DNA from these mixed cell samples, particularly with low amounts of sperm, depends on maximizing sperm DNA recovery and minimizing non-sperm DNA carryover. For evaluating the efficacy of the differential extraction, we present a method which uses a Separation Potential Ratio (SPRED) to consider both sperm DNA recovery and non-sperm DNA removal as variables for determining separation efficiency. In addition, we describe how the ratio of male-to-female DNA in the sperm fraction may be estimated by using the SPRED of the differential extraction method in conjunction with the estimated ratio of male-to-female DNA initially present on the mixed swab. This approach may be useful for evaluating or modifying differential extraction methods, as we demonstrate by comparing experimental results obtained from the traditional differential extraction and the Erase Sperm Isolation Kit (PTC © ) procedures. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of TRPC5 is decreased in the sperm of patients with varicocele-associated asthenozoospermia

PubMed Central

Zhu, Guangbin; Xie, Changying; Yang, Zhonghua; Wang, Yongzhi; Chen, Dong; Wang, Xinghuan

2018-01-01

The present study aimed to determine whether the expression of transient receptor potential channel 5 (TRPC5) protein is altered in spermatozoa of patients with varicocele-associated asthenozoospermia. TRPC5 expression in spermatozoa was determined by polymerase chain reaction and western blotting analyses, and indirect immunofluorescence was used for identification and immunolocalization of the TRPC5 channel in human sperm. Sperm motility and superoxide dismutase (SOD) activity were also determined with a computer-assisted semen analysis system and assay kit, respectively. Compared with levels in control subjects, it was identified that TRPC5 protein expression, SOD activity and cellular motility in the sperm of patients with varicocele-associated asthenozoospermia were reduced (P<0.001). Furthermore, the expression of TRPC5 was positively correlated with sperm motility (r=0.781, P<0.001) and SOD activity (r=0.933, P<0.001), indicated by partial correlation analysis. The present study may provide a novel target for the study and treatment of varicocele-associated asthenozoospermia.

Effect of ram semen extenders and supplements on computer assisted sperm analysis parameters

USDA-ARS?s Scientific Manuscript database

A study evaluated the effects of ram semen extender and extender supplementation on computer assisted sperm analysis (CASA) parameters positively correlated with progressive motility. Semen collected from 5 rams was distributed across treatment combinations consisting of either TRIS citrate (T) or ...

Clinical assessment of the male fertility

PubMed Central

Khatun, Amena; Rahman, Md Saidur

2018-01-01

The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed. PMID:29564308

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.

A technical assessment of the porcine ejaculated spermatozoa for a sperm-specific RNA-seq analysis.

PubMed

Gòdia, Marta; Mayer, Fabiana Quoos; Nafissi, Julieta; Castelló, Anna; Rodríguez-Gil, Joan Enric; Sánchez, Armand; Clop, Alex

2018-04-26

The study of the boar sperm transcriptome by RNA-seq can provide relevant information on sperm quality and fertility and might contribute to animal breeding strategies. However, the analysis of the spermatozoa RNA is challenging as these cells harbor very low amounts of highly fragmented RNA, and the ejaculates also contain other cell types with larger amounts of non-fragmented RNA. Here, we describe a strategy for a successful boar sperm purification, RNA extraction and RNA-seq library preparation. Using these approaches our objectives were: (i) to evaluate the sperm recovery rate (SRR) after boar spermatozoa purification by density centrifugation using the non-porcine-specific commercial reagent BoviPure TM ; (ii) to assess the correlation between SRR and sperm quality characteristics; (iii) to evaluate the relationship between sperm cell RNA load and sperm quality traits and (iv) to compare different library preparation kits for both total RNA-seq (SMARTer Universal Low Input RNA and TruSeq RNA Library Prep kit) and small RNA-seq (NEBNext Small RNA and TailorMix miRNA Sample Prep v2) for high-throughput sequencing. Our results show that pig SRR (~22%) is lower than in other mammalian species and that it is not significantly dependent of the sperm quality parameters analyzed in our study. Moreover, no relationship between the RNA yield per sperm cell and sperm phenotypes was found. We compared a RNA-seq library preparation kit optimized for low amounts of fragmented RNA with a standard kit designed for high amount and quality of input RNA and found that for sperm, a protocol designed to work on low-quality RNA is essential. We also compared two small RNA-seq kits and did not find substantial differences in their performance. We propose the methodological workflow described for the RNA-seq screening of the boar spermatozoa transcriptome. FPKM: fragments per kilobase of transcript per million mapped reads; KRT1: keratin 1; miRNA: micro-RNA; miscRNA: miscellaneous RNA; Mt rRNA: mitochondrial ribosomal RNA; Mt tRNA: mitochondrial transference RNA; OAZ3: ornithine decarboxylase antizyme 3; ORT: osmotic resistance test; piRNA: Piwi-interacting RNA; PRM1: protamine 1; PTPRC: protein tyrosine phosphatase receptor type C; rRNA: ribosomal RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; SRR: sperm recovery rate; tRNA: transfer RNA.

Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera.

PubMed

Whittington, Emma; Forsythe, Desiree; Borziak, Kirill; Karr, Timothy L; Walters, James R; Dorus, Steve

2017-12-02

Rapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology. Comparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects. Our results identify a burst of genetic novelty amongst sperm proteins that may be associated with the origin of heteromorphic spermatogenesis in ancestral Lepidoptera and/or the subsequent evolution of this system. This pattern of genomic diversification is distinct from the remainder of the genome and thus suggests that this transition has had a marked impact on lepidopteran genome evolution. The identification of abundant sperm proteins unique to Lepidoptera, including proteins distinct between specific lineages, will accelerate future functional studies aiming to understand the developmental origin of dichotomous spermatogenesis and the functional diversification of the fertilization incompetent apyrene sperm morph.

Testicular failure

MedlinePlus

... Testicular failure occurs when the testicles cannot produce sperm or male hormones, such as testosterone. Causes Testicular ... semen analysis to examine the number of healthy sperm you are producing. Sometimes, an ultrasound of the ...

The relative nature of fertilization success: Implications for the study of post-copulatory sexual selection

PubMed Central

2008-01-01

Background The determination of genetic variation in sperm competitive ability is fundamental to distinguish between post-copulatory sexual selection models based on good-genes vs compatible genes. The sexy-sperm and the good-sperm hypotheses for the evolution of polyandry require additive (intrinsic) effects of genes influencing sperm competitiveness, whereas the genetic incompatibility hypothesis invokes non-additive genetic effects. A male's sperm competitive ability is typically estimated from his fertilization success, a measure that is dependent on the ability of rival sperm competitors to fertilize the ova. It is well known that fertilization success may be conditional to genotypic interactions among males as well as between males and females. However, the consequences of effects arising from the random sampling of sperm competitors upon the estimation of genetic variance in sperm competitiveness have been overlooked. Here I perform simulations of mating trials performed in the context of sibling analysis to investigate whether the ability to detect additive genetic variance underlying the sperm competitiveness phenotype is hindered by the relative nature of fertilization success measurements. Results Fertilization success values render biased sperm competitive ability values. Furthermore, asymmetries among males in the errors committed when estimating sperm competitive abilities are likely to exist as long as males exhibit variation in sperm competitiveness. Critically, random effects arising from the relative nature of fertilization success lead to an underestimation of underlying additive genetic variance in sperm competitive ability. Conclusion The results show that, regardless of the existence of genotypic interactions affecting the output of sperm competition, fertilization success is not a perfect predictor of sperm competitive ability because of the stochasticity of the background used to obtain fertilization success measures. Random effects need to be considered in the debate over the maintenance of genetic variation in sperm competitiveness, and when testing good-genes and compatible-genes processes as explanations of polyandrous behaviour using repeatability/heritability data in sperm competitive ability. These findings support the notion that the genetic incompatibility hypothesis needs to be treated as an alternative hypothesis, rather than a null hypothesis, in studies that fail to detect intrinsic sire effects on the sperm competitiveness phenotype. PMID:18474087

New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze-thawing.

PubMed

Brouwers, Jos F; Silva, Patricia F N; Gadella, Barend M

2005-01-15

Reactive oxygen species have been implicated in sperm aberrations causing multiple pathologies including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries for semen storage prior to artificial insemination but unusual in porcine breeding industries as semen dilution and storage at 17 degrees C is sufficient for artificial insemination within 2-3 days. However, longer semen storage requires cryopreservation of boar semen. Freeze/thawing procedures induce sperm damage and induce reactive oxygen species in mammalian sperm and boar sperm seems to be more vulnerable for this than bull sperm. We developed a new method to detect reactive oxygen species induced damage at the level of the sperm plasma membrane in bull sperm. Lipid peroxidation in freshly stored and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous lipid classes, phosphatidylcholine and cholesterol and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in the living sperm cells. Cytoplasmatic droplets in incompletely matured sperm cells were intensely peroxidized. Furthermore, lipid peroxidation was particularly strong in the mid-piece and tail of frozen/thawed spermatozoa and significantly less intense in the sperm head. Induction of peroxidation in fresh sperm cells with the lipid soluble reactive oxygen species tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show using the flow cytometer that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze-thawing. Although the method was established on bovine sperm, we discuss the importance of these assays for detecting lipid peroxidation in boar sperm cells.

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.

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

PubMed

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

2016-03-15

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

A Method for Preparation, Storage and Activation of Large Populations of Immotile Sea Urchin Sperm

NASA Technical Reports Server (NTRS)

Bracho, Geracimo E.; Fritch, Jennifer J.; Tash, Joseph S.

1997-01-01

Reversible protein phosphorylation is associated with initiation and modulation of sperm flagellar motility. Many studies aimed at examining the signal transduction mechanisms underlying the expression of motility have relied on detergent-permeabilized sperm reactivated with exogenous 32 P-ATP. However, the reactivation conditions allow variable levels of motility to be expressed and phosphorylation of many proteins that appear to be unrelated to sperm motility. Thus, identification of the few relevant proteins is difficult. We have developed a method to collect and keep sperm immotile until reactivated for analysis to normal motility levels. Artificial sea water (ASW) buffered with 5 mM 2-[N-morpholino]ethanesulfonic acid at pH 6.0 and containing 50 mM KCI, allows collection and storage of immotile sea urchin sperm for up to 96 h at 4-5 C. Motility under these conditions is essentially zero, but sperm is rapidly reactivated to normal motility by diluting with ASW to standard pH (8.0) and KCI concentration (10 mM).

Thiol oxidation by nitrosative stress: Cellular localization in human spermatozoa.

PubMed

Cabrillana, María E; Uribe, Pamela; Villegas, Juana V; Álvarez, Juan; Sánchez, Raúl; Fornés, Miguel W

2016-10-01

Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS(®) system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility. DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO(-): peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis PARP: poli ADP ribose polimerasa VCL: curvilinear velocity VSL: straight-line velocity VAP: average path velocity PRDXs: peroxiredoxins ODF: outer dense fiber ODF1: outer dense fiber 1 PI: propidium iodide DMSO: dimethyl sulfoxide SD: standard deviation analysis of variance.

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

PubMed

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

2015-03-01

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

Defining the "normal" postejaculate urinalysis.

PubMed

Mehta, Akanksha; Jarow, Jonathan P; Maples, Pat; Sigman, Mark

2012-01-01

Although sperm have been shown to be present in the postejaculate urinalysis (PEU) of both fertile and infertile men, the number of sperm present in the PEU of the general population has never been well defined. The objective of this study was to describe the semen and PEU findings in both the general and infertile population, in order to develop a better appreciation for "normal." Infertile men (n = 77) and control subjects (n = 71) were prospectively recruited. Exclusion criteria included azoospermia and medications known to affect ejaculation. All men underwent a history, physical examination, semen analysis, and PEU. The urine was split into 2 containers: PEU1, the initial voided urine, and PEU2, the remaining voided urine. Parametric statistical methods were applied for data analysis to compare sperm concentrations in each sample of semen and urine between the 2 groups of men. Controls had higher average semen volume (3.3 ± 1.6 vs 2.0 ± 1.4 mL, P < .001) and sperm concentrations (112 million vs 56.2 million, P = .011), compared with infertile men. The presence of sperm in urine was common in both groups, but more prevalent among infertile men (98.7% vs 88.7%, P = .012), in whom it comprised a greater proportion of the total sperm count (46% vs 24%, P = .022). The majority of sperm present in PEU were seen in PEU1 of both controls (69%) and infertile men (88%). An association was noted between severe oligospermia (<5 million/mL) with low semen volume (<0.5 mL), and significant sperm counts in PEU (<5 million). Although infertile men tend to have a higher proportion of their total sperm in the urine compared with control, there is a large degree of overlap between the 2 populations, making it difficult to identify a specific threshold to define a positive test. Interpretation of a PEU should be directed by whether the number of sperm in the urine could affect subsequent management.

OBJECTIVE EVALUATION OF HYPERACTIVATED MOTILITY IN RAT SPERMATOZA USING COMPUTER-ASSISTED SPERM ANALYSIS (CASA)

EPA Science Inventory

Objective evaluation of hyperactivated motility in rat spermatozoa using computer-assisted sperm analysis.

Cancel AM, Lobdell D, Mendola P, Perreault SD.

Toxicology Program, University of North Carolina, Chapel Hill, NC 27599, USA.

The aim of this study was t...

Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm.

PubMed

Anel-Lopez, L; Ortega-Ferrusola, C; Álvarez, M; Borragán, S; Chamorro, C; Peña, F J; Morrell, J; Anel, L; de Paz, P

2017-06-26

Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear -80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphometry on sperm yield and its hydrodynamic relationship were studied. The samples selected with Androcoll-Bear 65% showed a very good yield (53.1 ± 2.9) although the yield from Androcoll-Bear 80% was lower (19.3 ± 3.3). The latter showed higher values of motility than the control immediately after post-thawing selection. However, both concentrations of colloid (65 and 80%) showed higher values of viable sperm and viable sperm with intact acrosome than the control. After an incubation of 2 h at 37 °C, the samples from Androcoll-Bear 80% had higher kinematics and proportion of viable sperm with intact acrosome. In the morphometric analysis, the sperm selected by the Androcoll-Bear 80% showed a head with a bigger area which was more elongated than the sperm from other treatments. We conclude that sperm selection with Androcoll-Bear at either 65% or 80% is a suitable technique that allows a sperm population with better quality than the initial sample to be obtained. We recommend the use of Androcoll-Bear 65% since the yield is better than Androcoll-Bear 80%. Our findings pave the way for further research on application of sperm selection techniques to sperm banking in the brown bear.

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations

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

Inawaka, Kunifumi; Kawabe, Mayumi; DIMS Institute of Medical Science, Inc., Ichinomiya

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0 = day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followedmore » by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.« less

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations.

PubMed

Inawaka, Kunifumi; Kawabe, Mayumi; Takahashi, Satoru; Doi, Yuko; Tomigahara, Yoshitaka; Tarui, Hirokazu; Abe, Jun; Kawamura, Satoshi; Shirai, Tomoyuki

2009-06-01

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0=day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.

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.

Molecular andrology as related to sperm DNA fragmentation/sperm chromatin biotechnology.

PubMed

Shafik, A; Shafik, A A; Shafik, I; El Sibai, O

2006-01-01

Genetic male infertility occurs throughout the life cycle from genetic traits carried by the sperm, to fertilization and post-fertilization genome alterations, and subsequent developmental changes in the blastocyst and fetus as well as errors in meiosis and abnormalities in spermatogenesis/spermatogenesis. Genes encoding proteins for normal development include SRY, SOX9, INSL3 and LGR8. Genetic abnormalities affect spermatogenesis whereas polymorphisms affect receptor affinity and hormone bioactivity. Transgenic animal models, the human genome project, and other techniques have identified numerous genes related to male fertility. Several techniques have been developed to measure the amount of sperm DNA damage in an effort to identify more objective parameters for evaluation of infertile men. The integrity of sperm DNA influences a couple's fertility and helps predict the chances of pregnancy and its successful outcome. The available tests of sperm DNA damage require additional large-scale clinical trials before their integration into routine clinical practice. The physiological/molecular integrity of sperm DNA is a novel parameter of semen quality and a potential fertility predictor. Although DNA integrity assessment appears to be a logical biomarker of sperm quality, it is not being assessed as a routine part of semen analysis by clinical andrologists. Extensive investigation has been conducted for the comparative evaluation of these techniques. However, some of these techniques require expensive instrumentation for optimal and unbiased analysis, are labor intensive, or require the use of enzymes whose activity and accessibility to DNA breaks may be irregular. Thus, these techniques are recommended for basic research rather than for routine andrology laboratories.

Semen analysis in fertile patients undergoing vasectomy: reference values and variations according to age, length of sexual abstinence, seasonality, smoking habits and caffeine intake.

PubMed

Sobreiro, Bernardo Passos; Lucon, Antonio Marmo; Pasqualotto, Fábio Firmbach; Hallak, Jorge; Athayde, Kelly Silveira; Arap, Sami

2005-07-07

Recent studies have shown regional and population differences in semen characteristics. The objective was to establish reference values for semen analysis and to verify the effect that age, length of sexual abstinence, seasonality, smoking habits and coffee consumption have on fertile individuals' semen characteristics. Prospective study in the Urology Division, Hospital das Clínicas, Universidade de São Paulo. Between September 1999 and August 2002, 500 fertile men requesting a vasectomy for sterilization purposes were asked to provide a semen sample before the vasectomy. We evaluated the effects of age, sexual abstinence, seasonality, smoking and coffee consumption on semen characteristics. Compared with World Health Organization values, 87.2% of the patients presented sperm morphology below the normal level. A significant decline in semen volume, sperm motility and sperm morphology in patients over 45 years of age was observed. In patients with 5 days or more of abstinence, there was reduced sperm motility. The lowest values for sperm concentration, motility and morphology were observed in summer and the highest in winter. No differences in semen parameters relating to smoking were detected. Patients who drank six or more cups of coffee per day presented higher sperm motility. Our sample had a very low percentage of normal sperm morphology. Only sperm morphology showed a high abnormality rate. Differences in semen parameters with regard to age, length of sexual abstinence, seasonality and coffee consumption were identified. No differences relating to smoking were detected.

Comparison of the frequency of defective sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction between subfertile men with normal and abnormal semen.

PubMed

Liu, De Yi; Liu, Ming Li; Garrett, Claire; Baker, H W Gordon

2007-07-01

The aim of this study was to compare the frequency of defective sperm-zona pellucida (ZP) binding (DSZPB) and defective ZP-induced acrosome reaction (DZPIAR) in subfertile men (i.e. male partners of infertile couples) with normal and abnormal semen analyses. A total of 1030 subfertile men with normal semen analysis (n=255), oligozoospermia (count<20x10(6)/ml, n=136), severe teratozoospermia (strict normal morphology

A novel cross-species inhibitor to study the function of CatSper Ca2+ channels in sperm.

PubMed

Rennhack, Andreas; Schiffer, Christian; Brenker, Christoph; Fridman, Dmitry; Nitao, Elis T; Cheng, Yi-Min; Tamburrino, Lara; Balbach, Melanie; Stölting, Gabriel; Berger, Thomas K; Kierzek, Michelina; Alvarez, Luis; Wachten, Dagmar; Zeng, Xu-Hui; Baldi, Elisabetta; Publicover, Stephen; Kaupp, U Benjamin; Strünker, Timo

2018-05-03

Sperm from many species share the sperm-specific Ca 2+ channel CatSper (cation channel of sperm) that controls the intracellular Ca 2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling CatSper activity and the role of the channel during fertilization differ among species. However, a lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known CatSper inhibitors exhibit considerable side effects and inhibit also Slo3, the K + channel in mammalian sperm. The drug RU1968 was reported to suppress Ca 2+ signaling in human sperm by an unknown mechanism. We resynthesized the drug and revisited its mechanism of action in sperm form humans, mice, and sea urchins. We show by Ca 2+ fluorimetry, single-cell Ca 2+ imaging, electrophysiology, opto-chemistry, and motility analysis that RU1968 inhibits CatSper in sperm from invertebrates and mammals. The drug lacks toxic side effects in human sperm, does not affect mouse Slo3, and inhibits human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, the inhibitor mimics CatSper dysfunction and suppresses motility responses evoked by progesterone, an oviductal steroid that activates CatSper. Finally, we show that the drug abolishes CatSper-mediated chemotactic navigation in sea urchin sperm. We propose RU1968 as a novel tool to elucidate the function of CatSper in sperm across species. This article is protected by copyright. All rights reserved.

Dynamics of sperm transfer in the ant Leptothorax gredleri

NASA Astrophysics Data System (ADS)

Oppelt, Angelika; Heinze, Jürgen

2007-09-01

Mating tactics differ remarkably between and within species of social Hymenoptera (bees, wasps, ants) concerning, e.g., mating frequencies, sperm competition, and the degree of male sperm limitation. Although social Hymenoptera might, therefore, potentially be ideal model systems for testing sexual selection theory, the dynamics of mating and sperm transfer have rarely been studied in species other than social bees, and basic information needed to draw conclusions about possible sperm competition and female choice is lacking. We investigated sperm transfer in the ant Leptothorax gredleri, a species in which female sexuals attract males by “female calling.” The analysis of 38 female sexuals fixed immediately or up to 7 days after copulation with a single male each revealed that the sperm is transferred into the female bursa copulatrix embedded in a gelatinous mass, presumably a spermatophore. Sperm cells rapidly start to migrate from the tip of the spermatophore towards the spermatheca, but transfer is drastically slowed down by an extreme constriction of the spermathecal duct, through which sperm cells have to pass virtually one by one. This results in the spermatheca being filled only between one and several hours after mating. During this time, the posterior part of the spermatophore seals the junction between bursa copulatrix and spermathecal duct and prevents sperm loss. The prolonged duration of sperm transfer might allow female sexuals to chose between ejaculates and explain previously reported patterns of single paternity of the offspring of multiply mated queens.

Semen characteristics after overnight shipping: preservation of sperm concentrations, HspA2 ratios, CK activity, cytoplasmic retention, chromatin maturity, DNA integrity, and sperm shape.

PubMed

Huszar, Gabor; Celik-Ozenci, Ciler; Cayli, Sevil; Kovacs, Tamas; Vigue, Lynne; Kovanci, Ertug

2004-01-01

We tested several approaches that can be used to preserve sperm attributes and the objective biochemical markers of sperm maturity and function for assessment in a remote centralized laboratory after overnight shipping of semen samples. Addition of phenyl-methyl-sulfonyl-fluoride (PMSF) to a final concentration of 20 microg/mL semen at 4 degrees C has preserved sperm concentrations and HspA2 isoform ratios, even at room temperature, simulating a shipping delay in moderate ambient temperatures. Regarding the attributes of individual spermatozoa, the patterns of CK-immunocytochemistry (demonstrates cytoplasmic retention in diminished-maturity spermatozoa); aniline blue staining pattern (tests chromatin maturity); sperm shape assessed by both Kruger strict morphology and computer assisted morphometry; and sperm DNA integrity, as tested by DNA nick translation, all remained unchanged. Thus, the PMSF-4 degrees C conditions preserved sperm concentrations and the cytoplasmic and nuclear biomarkers of sperm cellular maturity and function for next-day analysis. This shipping method will facilitate the early detection of subtle changes in semen quality that can affect sperm function, even when there has been no decline in sperm concentrations to signal possible toxic effects. Furthermore, sample preservation will enable investigators to evaluate semen for toxicology studies and for diagnosis of male infertility from remote locations. Home collection of semen should enhance study participation, and semen assessment in centralized laboratories will address concerns regarding interlaboratory variations and quality control.

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

PubMed

Torres, Mariana Andrade; Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell'Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant'Anna; Sepúlveda, Néstor; de Andrade, André Furugen Cesar

2016-01-01

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility.

Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

PubMed Central

Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell’Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant’Anna; Sepúlveda, Néstor

2016-01-01

Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility. PMID:27529819

Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification

PubMed Central

Xu, Yan; Xie, Jianhui; Chen, Ronghua; Cao, Yu; Ping, Yuan; Xu, Qingwen; Hu, Wei; Wu, Dan; Gu, Lihua; Zhou, Huaigu; Chen, Xin; Zhao, Ziqin; Zhong, Jiang; Li, Rui

2016-01-01

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. Here we combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Our results showed that complete single donor STR profiles could be successfully obtained from sperm/epithelial cell and sperm mixtures from two contributors. For unbalanced sperm/epithelial cells and sperm cells mixtures, sensitivity results revealed that target cells could be detected at as low as 1:32 and 1:8 mixed ratios, respectively. Although highly relies on cell number and blood types or secretor status of the individuals, this procedure would still be useful tools for forensic DNA analysis of multi-suspect sexual assault cases by the combined use of FACS and MACS based on sperm-specific AKAP3 antigen and human blood type antigen. PMID:27857155

Environmental factors contributed to circannual rhythm of semen quality.

PubMed

Mao, Huan; Feng, Lei; Yang, Wan-Xi

2017-01-01

We investigated whether human semen parameters present circannual rhythm or not, and whether environmental factors exert on semen quality. This retrospective study used data of patients mainly from Reproductive Medicine Center and Urology and Andrology Clinic of a general hospital in China. Sperm concentration and motility were measured by computer aided sperm analysis (CASA). Sperm morphology was scored based on the strict criteria (WHO, 2010). The Kruskal-Wallis rank test was used to investigate the relationship between semen parameters and season/month. Partial correlation coefficients were used to analyze the relationship between semen parameters and environmental factors. In this study, we found that sperm concentration and total amount per ejaculate were significantly lower in summer and higher in winter. But, sperm progressive motility and motility were significantly higher in spring and summer (from March to June), lower in autumn and winter (September and October). Unexpectedly, normal sperm morphology and mixed agglutination reaction (MAR) positive rate didn't vary along with season or month. Furthermore, temperature was negatively related to sperm concentration and total amount per ejaculate. Precipitation was positively associated with progressive motility and normal sperm morphology, but negatively related to sperm head defect percentage. The length of sunlight was positively related to progressive motility. The Air Quality Index (AQI) was positively associated with semen volume and sperm total amount per ejaculate. These suggest seasonal and monthly variation underlying some semen parameters.

Impact of seasonal variation, age and smoking status on human semen parameters: The Massachusetts General Hospital experience

PubMed Central

Chen, Zuying; Godfrey-Bailey, Linda; Schiff, Isaac; Hauser, Russ

2004-01-01

Background To investigate the relationship of human semen parameters with season, age and smoking status. Methods The present study used data from subjects recruited into an ongoing cross-sectional study on the relationship between environmental agents and semen characteristics. Our population consisted of 306 patients who presented to the Vincent Memorial Andrology Laboratory of Massachusetts General Hospital for semen evaluation. Sperm concentration and motility were measured with computer aided sperm analysis (CASA). Sperm morphology was scored using Tygerberg Kruger strict criteria. Regression analyses were used to investigate the relationships between semen parameters and season, age and smoking status, adjusting for abstinence interval. Results Sperm concentration in the spring was significantly higher than in winter, fall and summer (p < 0.05). There was suggestive evidence of higher sperm motility and percent of sperm with normal morphology in the spring than in the other seasons. There were no statistically significant relationships between semen parameters and smoking status, though current smokers tended to have lower sperm concentration. We also did not find a statistically significant relationship between age and semen parameters. Conclusions We found seasonal variations in sperm concentration and suggestive evidence of seasonal variation in sperm motility and percent sperm with normal morphology. Although smoking status was not a significant predictor of semen parameters, this may have been due to the small number of current smokers in the study. PMID:15507127

The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa.

PubMed

Anbari, Fatemeh; Halvaei, Iman; Nabi, Ali; Ghazali, Shahin; Khalili, Mohammad Ali; Johansson, Lars

2016-01-01

The goal was to compare the effects of three different sperm preparation media on sperm motility, viability, and DNA integrity of semen samples from normozoospermic men. A total of 15 normozoospermic males were included in the study. The semen analysis (SA) was performed in accordance with the WHO guidelines (2010). After SA, each sample was divided into three aliquots, and swim-up was performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark; Ham's F10, Biochrome, Berlin, Germany; and VitaSperm™, Innovative Biotech, Iran). Sperm motility, viability, and DNA fragmentation were evaluated at 0, 1, 2, and 24 h after swim-up. There were no significant differences, at any time intervals, in the total sperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared using the media from Origio in comparison with VitaSperm™ ( P = 0.03), whereas no significant difference was found against Ham's F10 medium. No significant differences in sperm viability were seen between the media products. However, 1 h after swim-up, the extent of sperm DNA fragmentation was lower in the medium from Origio versus VitaSperm™ ( P = 0.02). The data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs.

The Effect of Glyphosate on Human Sperm Motility and Sperm DNA Fragmentation.

PubMed

Anifandis, George; Katsanaki, Katerina; Lagodonti, Georgia; Messini, Christina; Simopoulou, Mara; Dafopoulos, Konstantinos; Daponte, Alexandros

2018-05-30

Glyphosate is the active ingredient of Roundup ® , which is one of the most popular herbicides worldwide. Although many studies have focused on the reproductive toxicity of glyphosate or glyphosate-based herbicides, the majority of them have concluded that the effect of the specific herbicide is negligible, while only a few studies indicate the male reproductive toxicity of glyphosate alone. The aim of the present study was to investigate the effect of 0.36 mg/L glyphosate on sperm motility and sperm DNA fragmentation (SDF). Thirty healthy men volunteered to undergo semen analysis for the purpose of the study. Sperm motility was calculated according to WHO 2010 guidelines at collection time (zero time) and 1 h post-treatment with glyphosate. Sperm DNA fragmentation was evaluated with Halosperm ® G2 kit for both the control and glyphosate-treated sperm samples. Sperm progressive motility of glyphosate-treated samples was significantly reduced after 1 h post-treatment in comparison to the respective controls, in contrast to the SDF of glyphosate-treated samples, which was comparable to the respective controls. Conclusively, under these in vitro conditions, at high concentrations that greatly exceed environmental exposures, glyphosate exerts toxic effects on sperm progressive motility but not on sperm DNA integrity, meaning that the toxic effect is limited only to motility, at least in the first hour.

Sperm proteins in teleostean and chondrostean (sturgeon) fishes.

PubMed

Li, Ping; Hulak, Martin; Linhart, Otomar

2009-11-01

Sperm proteins in the seminal plasma and spermatozoa of teleostean and chondrostean have evolved adaptations due to the changes in the reproductive environment. Analysis of the composition and functions of these proteins provides new insights into sperm motility and fertilising abilities, thereby creating possibilities for improving artificial reproduction and germplasm resource conservation technologies (e.g. cryopreservation). Seminal plasma proteins are involved in the protection of spermatozoa during storage in the reproductive system, whereas all spermatozoa proteins contribute to the swimming and fertilising abilities of sperm. Compared to mammalian species, little data are available on fish sperm proteins and their functions. We review here the current state of the art in this field and focus on relevant subjects that require attention. Future research should concentrate on protein functions and their mode of action in fish species, especially on the role of spermatozoa surface proteins during fertilisation and on a description of sturgeon sperm proteins.

Challenges in Development of Sperm Repositories for Biomedical Fishes: Quality Control in Small-Bodied Species.

PubMed

Torres, Leticia; Liu, Yue; Guitreau, Amy; Yang, Huiping; Tiersch, Terrence R

2017-12-01

Quality control (QC) is essential for reproducible and efficient functioning of germplasm repositories. However, many biomedical fish models present significant QC challenges due to small body sizes (<5 cm) and miniscule sperm volumes (<5 μL). Using minimal volumes of sperm, we used Zebrafish to evaluate common QC endpoints as surrogates for fertilization success along sequential steps of cryopreservation. First, concentrations of calibration bead suspensions were evaluated with a Makler ® counting chamber by using different sample volumes and mixing methods. For sperm analysis, samples were initially diluted at a 1:30 ratio with Hanks' balanced salt solution (HBSS). Motility was evaluated by using different ratios of sperm and activation medium, and membrane integrity was analyzed with flow cytometry at different concentrations. Concentration and sperm motility could be confidently estimated by using volumes as small as 1 μL, whereas membrane integrity required a minimum of 2 μL (at 1 × 10 6 cells/mL). Thus, <5 μL of sperm suspension (after dilution to 30-150 μL with HBSS) was required to evaluate sperm quality by using three endpoints. Sperm quality assessment using a combination of complementary endpoints enhances QC efforts during cryopreservation, increasing reliability and reproducibility, and reducing waste of time and resources.

Effect of an isotonic lubricant on sperm collection and sperm quality.

PubMed

Agarwal, Ashok; Malvezzi, Helena; Sharma, Rakesh

2013-05-01

To assess the influence of an isotonic lubricant used during sperm sample collection on [1] ease of collection and [2] resultant sperm quality. Paired randomized cross-over design. Tertiary hospital. Healthy men over 18 years old with normal semen analysis as per World Health Organization 2010 guidelines. Collection of semen sample from 22 subjects by masturbation with or without the use of Pre-Seed personal lubricant. Qualitative survey results and quantitative sperm function outcomes were measured to determine resultant sperm quality and collection experience with and without Pre-Seed lubricant. The qualitative questionnaire results showed that 73% of donors prefer the semen collection process with the isotonic lubricant and 55% recommended the use of lubricant in their everyday collection. The motility, viability, membrane integrity, levels of reactive oxygen species, total antioxidant capacity, and percentage of DNA damage in collected semen samples were not affected by the use of the lubricant. More donors prefer, and find it easier, to collect semen samples with the use of the lubricant. The isotonic lubricant Pre-Seed did not compromise sperm quality as evaluated in an array of sperm assays, suggesting its safe use in fertility patients as required during sperm collection. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Selling blood and gametes during tough economic times: insights from Google search.

PubMed

Wu, Jonathan A; Ngo, Tin C; Rothman, Cappy; Breyer, Benjamin N; Eisenberg, Michael L

2015-10-01

To use Google Insights search volume and publicly available economic indicators to test the hypothesis that sperm, egg, and blood donations increase during economic downturns and to demonstrate the feasibility of using Google search volume data to predict national trends in actual sperm, egg, and blood donations rates. Cross-correlation statistical analysis comparing Google search data for terms relating to blood, egg, and sperm donations with various economic indicators including the S&P 500 closing values, gross domestic product (GDP), the U.S. Index of Leading Indicators (U.S. Leading Index), gross savings rate, mortgage interest rates, unemployment rate, and consumer price index (CPI) from 2004-2011. A secondary analysis determined the Pearson correlation coefficient between Google search data with actual sperm, egg, and blood donation volume in the U.S. as measured by California Cryobank, the National Assisted Reproductive Technology Surveillance System, and the National Blood Collection and Utilization Survey, respectively. Significance of cross-correlation and Pearson correlation analysis as indicated by p value. There were several highly significant cross-correlation relationships between search volume and various economic indicators. Correlation between Google search volume for the term 'sperm donation,' 'egg donation,' and 'blood donation' with actual number of sperm, egg and blood donations in the United States demonstrated Pearson correlation coefficients of 0.2 (p > 0.10), -0.1 (p > 0.10), and 0.07 (p > 0.10), respectively. Temporal analysis showed an improved correlation coefficient of 0.9 (p < 0.05) for blood donation when shifted 12 months later relative to Google search volume. Google search volume data for search terms relating to sperm, egg, and blood donation increase during economic downturns. This finding suggests gamete and bodily fluid donations are influenced by market forces like other commodities. Google search may be useful for predicting blood donation trends but is more limited in predicting actual semen and oocyte donation patterns.

BMI in relation to sperm count: an updated systematic review and collaborative meta-analysis.

PubMed

Sermondade, N; Faure, C; Fezeu, L; Shayeb, A G; Bonde, J P; Jensen, T K; Van Wely, M; Cao, J; Martini, A C; Eskandar, M; Chavarro, J E; Koloszar, S; Twigt, J M; Ramlau-Hansen, C H; Borges, E; Lotti, F; Steegers-Theunissen, R P M; Zorn, B; Polotsky, A J; La Vignera, S; Eskenazi, B; Tremellen, K; Magnusdottir, E V; Fejes, I; Hercberg, S; Lévy, R; Czernichow, S

2013-01-01

BACKGROUND The global obesity epidemic has paralleled a decrease in semen quality. Yet, the association between obesity and sperm parameters remains controversial. The purpose of this report was to update the evidence on the association between BMI and sperm count through a systematic review with meta-analysis. METHODS A systematic review of available literature (with no language restriction) was performed to investigate the impact of BMI on sperm count. Relevant studies published until June 2012 were identified from a Pubmed and EMBASE search. We also included unpublished data (n = 717 men) obtained from the Infertility Center of Bondy, France. Abstracts of relevant articles were examined and studies that could be included in this review were retrieved. Authors of relevant studies for the meta-analysis were contacted by email and asked to provide standardized data. RESULTS A total of 21 studies were included in the meta-analysis, resulting in a sample of 13 077 men from the general population and attending fertility clinics. Data were stratified according to the total sperm count as normozoospermia, oligozoospermia and azoospermia. Standardized weighted mean differences in sperm concentration did not differ significantly across BMI categories. There was a J-shaped relationship between BMI categories and risk of oligozoospermia or azoospermia. Compared with men of normal weight, the odds ratio (95% confidence interval) for oligozoospermia or azoospermia was 1.15 (0.93-1.43) for underweight, 1.11 (1.01-1.21) for overweight, 1.28 (1.06-1.55) for obese and 2.04 (1.59-2.62) for morbidly obese men. CONCLUSIONS Overweight and obesity were associated with an increased prevalence of azoospermia or oligozoospermia. The main limitation of this report is that studied populations varied, with men recruited from both the general population and infertile couples. Whether weight normalization could improve sperm parameters should be evaluated further.

BMI in relation to sperm count: an updated systematic review and collaborative meta-analysis

PubMed Central

Sermondade, N.; Faure, C.; Fezeu, L.; Shayeb, A.G.; Bonde, J.P.; Jensen, T.K.; Van Wely, M.; Cao, J.; Martini, A.C.; Eskandar, M.; Chavarro, J.E.; Koloszar, S.; Twigt, J.M.; Ramlau-Hansen, C.H.; Borges, E.; Lotti, F.; Steegers-Theunissen, R.P.M.; Zorn, B.; Polotsky, A.J.; La Vignera, S.; Eskenazi, B.; Tremellen, K.; Magnusdottir, E.V.; Fejes, I.; Hercberg, S.; Lévy, R.; Czernichow, S.

2013-01-01

BACKGROUND The global obesity epidemic has paralleled a decrease in semen quality. Yet, the association between obesity and sperm parameters remains controversial. The purpose of this report was to update the evidence on the association between BMI and sperm count through a systematic review with meta-analysis. METHODS A systematic review of available literature (with no language restriction) was performed to investigate the impact of BMI on sperm count. Relevant studies published until June 2012 were identified from a Pubmed and EMBASE search. We also included unpublished data (n = 717 men) obtained from the Infertility Center of Bondy, France. Abstracts of relevant articles were examined and studies that could be included in this review were retrieved. Authors of relevant studies for the meta-analysis were contacted by email and asked to provide standardized data. RESULTS A total of 21 studies were included in the meta-analysis, resulting in a sample of 13 077 men from the general population and attending fertility clinics. Data were stratified according to the total sperm count as normozoospermia, oligozoospermia and azoospermia. Standardized weighted mean differences in sperm concentration did not differ significantly across BMI categories. There was a J-shaped relationship between BMI categories and risk of oligozoospermia or azoospermia. Compared with men of normal weight, the odds ratio (95% confidence interval) for oligozoospermia or azoospermia was 1.15 (0.93–1.43) for underweight, 1.11 (1.01–1.21) for overweight, 1.28 (1.06–1.55) for obese and 2.04 (1.59–2.62) for morbidly obese men. CONCLUSIONS Overweight and obesity were associated with an increased prevalence of azoospermia or oligozoospermia. The main limitation of this report is that studied populations varied, with men recruited from both the general population and infertile couples. Whether weight normalization could improve sperm parameters should be evaluated further. PMID:23242914

Implication of sperm chromosomal abnormalities in recurrent abortion and multiple implantation failure.

PubMed

Caseiro, Ana Lara; Regalo, Ana; Pereira, Elisa; Esteves, Telma; Fernandes, Fernando; Carvalho, Joaquim

2015-10-01

Currently, some infertility treatment centres provide sperm karyotype analysis, although the impact of sperm chromosomal abnormalities on fertility is not yet fully understood. Several studies using fluorescence in-situ hybridization (FISH) to analyse sperm chromosomal constitution discovered that the incidence of aneuploidy is increased in individuals with a history of repeated abortion or implantation failure and is even higher in cases of oligoasthenoteratozoospermia (OAT), abnormal somatic karyotype or in spermatozoa retrieved directly from the testis or epididymis, showing that the application of FISH in these cases may be of some benefit for improving the reproductive outcome. This article presents the results of clinical trials of FISH analysis on spermatozoa, the medical indications for performing this examination, its results in infertile patients and the advantages when performing genetic counselling prior to treatment. Also discussed is the possibility of applying the latest techniques of genetic analysis in these cases and the potential benefits for improving the prognosis of male infertility. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. 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.

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.

Detection of sperm-reactive antibodies in wild sika deer and identification of the sperm antigens.

PubMed

Kawase, Osamu; Jimbo, Mitsuru

2018-03-16

Antisperm antibodies potentially inhibit sperm functions causing the sterility in humans and experimentally treated animals. However, there is no information about antisperm antibodies emerging spontaneously in wildlife. In this study, we searched for the sperm-reactive antibodies, spontaneously produced in wild sika deer (Cervus nippon), and identified the sperm antigens. We collected 529 fecal masses of sika deer in Japanese cities, from which we extracted the mucosal antibodies to test them for reactivities to deer sperm proteins by ELISA. Two of the extracts contained IgAs that were highly reactive to the sperm proteins. The molecular weights of the active IgAs, partially purified by DEAE-sephadex A-50, were estimated at more than 100 kDa, suggesting that the IgAs evaded drastic digestion in the gastrointestinal tract. Two-dimensional electrophoresis and immunoblotting detected three major antigens, and the following LC-MS/MS analysis identified them as alpha-enolase, phosphoglycerate kinase 2 and acrosin-binding protein. The antibodies were cross-reactive to a recombinant human acrosin-binding protein. To our knowledge, this is the first research to find that the sperm-reactive antibodies are produced spontaneously in wildlife and they recognize a common antigen found in humans.

Panax ginseng induces the expression of CatSper genes and sperm hyperactivation

PubMed Central

Park, Eun Hwa; Kim, Do Rim; Kim, Ha Young; Park, Seong Kyu; Chang, Mun Seog

2014-01-01

The cation channel of sperm (CatSper) protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca2+ influx, real-time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca2+ levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression. PMID:24969054

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

Structural and Functional Studies of the Protamine 2-Zinc Complex from Syrian Gold Hamster (Mesocricetus Auratus) Spermatids and Sperm

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

Dolan, Cheryl E.

The research described in this dissertation consists of four major areas: (1) sequence analysis of protamine 2 from Muroid rodents to identify potential zinc-binding domain(s) of protamine 2; (2) structural studies of the protamine 2-zinc complex from Syrian Gold hamster sperm and spermatids to elucidate the role of zinc during spermiogenesis; (3) structural studies of an unique protamine 2-zinc complex from chinchilla sperm; and (4) Nuclear Magnetic Resonance (NMR) studies of soluble complexes of hairpin oligonucleotides with synthetic arginine-rich peptides or protamine 1 isolated from bull sperm. First, zinc was quantitated in spermatids and sperm by Proton-Induced X-ray Emission (PIXE)more » to determine whether zinc is present in the early stages of spermiogenesis. The PIXE results revealed the zinc content varies proportionately with the amount of protamine 2 in both spermatid and sperm nuclei. An exception was chinchilla sperm containing twice the amount of protamine 2 than zinc. Further analyses by PIXE and X-ray Absorption Spectroscopy (XAS) of zinc bound to protamines isolated from hamster sperm confirmed the majority of the zinc is bound to protamine and identified the zinc ligands of protamine 2 in hamster spermatids and sperm in vivo. These studies established that zinc is bound to the protamine 2 precursor in hamster spermatids and the coordination of zinc by protamine 2 changes during spermiogenesis. Finally, the sequence analysis combined with the XAS results suggest that the zinc-binding domain in protamine 2 resides in the amino-terminus. Similar analyses of chinchilla sperm by XAS were performed to clarify the unusual PIXE results and revealed that chinchilla has an atypical protamine 2-zinc structure. Two protamine 2 molecules coordinate one zinc atom, forming homodimers that facilitate the binding of protamine 2 to DNA and provide an organizational scheme that would accommodate the observed species-specific protamine stoichiometry in mammalian sperm. Based on these results, we propose the binding of zinc to protamine 2 molecules stabilizes a dimerization domain in other mammalian sperm. Future experiments will use the knowledge we gained of the interactions between protamine 1 and DNA from the NMR studies to obtain structural data for the DNA-protamine 2-zinc complex.« less

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

Relevance of testicular histopathology on prediction of sperm retrieval rates in case of non-obstructive and obstructive azoospermia.

PubMed

Cito, Gianmartin; Coccia, Maria E; Dabizzi, Sara; Morselli, Simone; Della Camera, Pier A; Cocci, Andrea; Criscuoli, Luciana; Picone, Rita; De Carlo, Candida; Nesi, Gabriella; Micelli, Elisabetta; Serni, Sergio; Carini, Marco; Natali, Alessandro

2018-03-01

The aim of our research was to establish the relevance of testicular histopathology on sperm retrieval after testicular sperm extraction in patients with non-obstructive azoospermia and in patients with obstructive azoospermia, who already underwent a previous failure testicular fine needle aspiration. We evaluated a total of 82 azoospermic men, underwent testicular sperm extraction, referring to the Assisted Reproductive Technology Centre of the University of Florence, Italy between January 2008 and March 2017. A general and genital physical examination, scrotal and trans-rectal ultrasound, semen analysis, hormone measurements, including follicle-stimulating hormone, luteinizing hormone and total testosterone, were collected. Successful sperm retrieval was obtained in 36 men of total (43.9%). Successful sperm retrieval was 29.5% in non-obstructive azoospermia patients, while men with obstructive azoospermia, who, underwent a previous failure testicular fine needle aspiration, had sperm retrieval in 86% of cases. Mean luteinizing hormone was 6.55 IU/L, total testosterone 4.70 ng/mL, right testicular volume 13.7 mL and left testicular volume 13.6 mL. Mean Follicle-stimulating hormone was 13.45 IU/L in patients with negative sperm retrieval and 8.18 IU/L in men with successful sperm retrieval. According to histology, 20.7% had normal spermatogenesis, 35.3% hypospermatogenesis, 35.3% maturation arrest and 8.5% Sertoli cell-only syndrome. Successful sperm retrieval was 88.2% in patients with normal spermatogenesis, 24.1% in the maturation arrest group and 48.27% in patients with hypospermatogenesis, while negative sperm retrieval was reported in Sertoli cell-only syndrome patients. Seven cases with maturation arrest showed a successful sperm retrieval. Testicular histopathology after testicular sperm extraction offers important information on prediction of sperm retrieval and can guide the surgeon in choosing the more suitable therapeutic practice.

The effect of flash-freezing temperature on stallion sperm DNA structure.

PubMed

Serafini, R; Varner, D D; Bissett, W; Blanchard, T L; Teague, S R; Love, C C

2017-06-01

The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabilities were determined. All comet tail measures were higher following any flash-freezing method, as compared to fresh sperm DNA (P < 0.05), with no difference among flash-frozen treatments (P > 0.05). For most comet variables, intra- and inter-assay variabilities were <10%. Intra- and inter-stallion variabilities revealed that comet head length (HL) and width (CW) were less variable as compared to comet tail values, i.e., % comet tail DNA (T-DNA), tail length (TL), tail moment (OTM), and tail migration (TM). Certain comet tail values in fresh (% T-DNA, and OTM) and flash-frozen sperm (OTM, % T-DNA, TL, and TM) were correlated to the Sperm Chromatin Structure Assay (SCSA) variable, COMP-α t . The comet tail measures were negatively correlated to % morphologically normal sperm (P < 0.05) and positively correlated to % abnormal heads and premature germ cells (P < 0.05). Variables COMP-α t and % total sperm motility were not correlated to any morphologic sperm feature in this group of stallions (P > 0.05). While significant differences in the structure of the sperm DNA were identified in the flash-frozen as compared to the fresh sperm DNA with the neutral comet assay, it cannot be assumed that these changes are fertility limiting. Copyright © 2017. Published by Elsevier Inc.

Effects of hepatitis B virus infection on human sperm chromosomes.

PubMed

Huang, Jian-Min; Huang, Tian-Hua; Qiu, Huan-Ying; Fang, Xiao-Wu; Zhuang, Tian-Gang; Liu, Hong-Xi; Wang, Yong-Hua; Deng, Li-Zhi; Qiu, Jie-Wen

2003-04-01

To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients. Sperm chromosomes of 14 tested subjects (5 healthy controls, 9 patients with HBV infection, including 1 with acute hepatitis B, 2 with chronic active hepatitis B, 4 with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. The total frequency of sperm chromosome aberrations in HBV infection group (14.8 %, 33/223) was significantly higher than that in the control group (4.3 %, 5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4 signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random. HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Effect of nonylphenol on male reproduction: Analysis of rat epididymal biochemical markers and antioxidant defense enzymes

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

Aly, Hamdy A.A., E-mail: hamdyaali@yahoo.com; Domènech, Òscar; Banjar, Zainy M.

The mechanism by which nonylphenol (NP) interferes with male reproduction is not fully elucidated. Therefore, the present study was conducted to evaluate the effect of NP on male reproductive organ's weight, sperm characteristics, and to elucidate the nature and mechanism of action of NP on the epididymis. Adult male Wistar rats were gavaged with NP, dissolved in corn oil, at 0, 100, 200 or 300 mg/kg/day for 30 consecutive days. Control rats were gavaged with vehicle (corn oil) alone. Body weight did not show any significant change while, absolute testes and epididymides weights were significantly decreased. Sperm count in caudamore » and caput/corpus epididymides, and sperm motility was significantly decreased. Daily sperm production was significantly decreased in a dose-related manner. Sperm transit time in cauda epididymis was significantly decreased by 300 mg/kg, while in the caput/corpus epididymis it was significantly decreased by 200 and 300 mg/kg of NP. Plasma LDH was significantly increased while; plasma testosterone was significantly decreased in a dose-related pattern. In the epididymal sperm, NP decreased acrosome integrity, Δψm and 5′-nucleotidase activity. Hydrogen peroxide (H{sub 2}O{sub 2}) production and LPO were significantly increased in a dose-related pattern. The activities of SOD, CAT and GPx were significantly decreased in the epididymal sperm. In conclusion, this study revealed that NP treatment impairs spermatogenesis and has a cytotoxic effect on epididymal sperm. It disrupts the prooxidant and antioxidant balance. This leads oxidative stress in epididymal sperms of rat. Moreover, the reduction in sperm transit time may affect sperm quality and fertility potential. -- Highlights: ► The nature and mechanism of action of NP on rat epididymis were elucidated. ► NP decreased sperm count, motility, daily sperm production and sperm transit time. ► NP decreased sperm acrosome integrity, Δψm and 5′-nucleotidase activity. ► Plasma LDH was significantly increased and testosterone was significantly decreased. ► NP induced oxidative stress in epididymal sperm.« less

The immediate impact of semen diluent and rate of dilution on the sperm quality index, ATP utilization, gas exchange, and ionic balance of broiler breeder sperm.

PubMed

Parker, H M; McDaniel, C D

2006-01-01

The sperm quality index (SQI) is a tool used to predict overall rooster semen quality, fertility, and hatchability. However, semen must be diluted before SQI analysis, and research has shown that the SQI is most predictive of fertility at lower semen dilutions. Therefore, the present study was undertaken to determine why the SQI is not as predictive of fertility at higher semen dilutions and whether semen diluent type alters the SQI, adenosine triphosphate (ATP) utilization, gas exchange, and ionic balance of broiler breeder sperm. Semen was diluted with saline, seminal plasma, or minimum essential medium (MEM) from 2- to 200-fold. The following parameters were measured for each diluent type at each dilution: SQI, ATP, Na+, Ca2+, K+, Cl-, CO2, and O2. To examine the rate of sperm motility, the SQI was expressed as SQI/million sperm per mL (SQI/sperm). There was an interaction between diluent type and dilution for the SQI, SQI/sperm, CO2 generated, O2 used, as well as Na+, Ca2+, and K+ internalization. For sperm diluted with saline, the SQI declined more rapidly with increasing dilution. However, SQI/sperm increased rapidly when semen was diluted with MEM or SP. Sperm diluted in SP used ATP with increasing dilution whereas sperm diluted with saline and MEM generated ATP. Neat semen contained no free O2; however, each diluent type contained abundant O2 resulting in more O2 available as semen was diluted. Sperm diluted in SP produced more CO2 and used more O2 than semen diluted in saline or MEM. For SQI/sperm, ATP and CO2 generated, as well as Na+ and Ca2+ internalization, differences between diluent types occurred when semen was diluted 50-fold and greater. In conclusion, it appears that sperm motility, ATP utilization, gas exchange, and ionic balance are altered by diluent type and rate of dilution. These alterations in semen quality are exacerbated at semen dilutions of 50-fold and greater yielding an SQI that is not indicative of sperm motility or fertility.

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

PubMed

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

2011-03-15

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

Separation of sperm and epithelial cells based on the hydrodynamic effect for forensic analysis

PubMed Central

Liu, Weiran; Chen, Weixing; Liu, Ran; Ou, Yuan; Liu, Haoran; Xie, Lan; Lu, Ying; Li, Caixia; Li, Bin; Cheng, Jing

2015-01-01

In sexual assault cases, forensic samples are a mixture of sperm from the perpetrator and epithelial cells from the victim. To obtain an independent short tandem repeat (STR) profile of the perpetrator, sperm cells must be separated from the mixture of cells. However, the current method used in crime laboratories, namely, differential extraction, is a time-consuming and labor-intensive process. To achieve a rapid and automated sample pretreatment process, we fabricated a microdevice for hydrodynamic and size-based separation of sperm and epithelial cells. When cells in suspension were introduced into the device's microfluidic channels, they were forced to flow along different streamlines and into different outlets due to their different diameters. With the proposed microdevice, sperm can be separated within a short period of time (0.5 h for a 50-μl mock sample). The STR profiles of the products in the sperm outlet reservoir demonstrated that a highly purified male DNA fraction could be obtained (94.0% male fraction). This microdevice is of low-cost and can be easily integrated with other subsequent analysis units, providing great potential in the process of analyzing sexual assault evidence as well as in other areas requiring cell sorting. PMID:26392829

Elliptical Acoustic Particle Motion in Underwater Waveguides

DTIC Science & Technology

2013-03-27

Folkert, ”Tracking sperm whales with a towed acoustic vector sensor,” J. Acoust. Soc. Am. Volume 128, Issue 5, pp. 2681-2694 (2010). 2 Santos, P...modal amplitudes Bm and Cm are weak functions of frequency and range independent. This holds for any normal mode description of the acoustic field in a...wavelengths. Error in measurement aside, the frequency range relation- ship described by the waveguide invariant holds for any directional component of I

Effect of Pseudomonas aeruginosa on sperm capacitation and protein phosphorylation of boar spermatozoa.

PubMed

Sepúlveda, Lilian; Bussalleu, Eva; Yeste, Marc; Bonet, Sergi

2016-05-01

Several studies have reported the detrimental effects that bacteriospermia causes on boar sperm quality, but little is known about its effects on IVC. Considering that, the present study sought to evaluate the effects of different concentrations of Pseudomonas aeruginosa on different indicators of capacitation status (sperm viability, membrane lipid disorder, sperm motility kinematics, and protein phosphorylation of boar spermatozoa) after IVC. Flow cytometry and computer assisted sperm analysis (CASA) revealed that the presence of P aeruginosa in boar sperm samples, mostly at concentrations greater than 10(6) CFU/mL, is associated with a significant (P < 0.05) decrease in the percentages of both sperm membrane integrity and sperm with low membrane lipid disorder, and also with a reduction in sperm motility kinetic parameters when compared with results obtained from the control sample, which presented the typical motility pattern of capacitated-like boar spermatozoa. Moreover, Western blot results also showed significant (P < 0.05) changes in the levels of tyrosine, serine, and threonine protein phosphorylation because of bacterial contamination, the decrease in phosphotyrosine levels of p32, a well-known marker of IVC achievement in boar sperm, being the most relevant. Indeed, after 3 hours of IVC, phosphotyrosine levels of p32 in the control sample were 3.13 ± 0.81, whereas in the tubes with 10(6) and 10(8) CFU/mL were 1.05 ± 0.20 and 0.36 ± 0.07, respectively. Therefore, the present study provides novel data regarding the effects of bacterial contamination on boar sperm, suggesting that the presence of P aeruginosa affects the fertilizing ability of boar sperm by altering its ability to accomplish IVC. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality.

PubMed

Losano, Jda; Angrimani, Dsr; Dalmazzo, A; Rui, B R; Brito, M M; Mendes, C M; Kawai, Gkv; Vannucchi, C I; Assumpção, Meoa; Barnabe, V H; Nichi, M

2017-04-01

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2-deoxy-d-glucose, DOG). Furthermore, treatment with DOG also led to a dose-dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance. © 2017 Blackwell Verlag GmbH.

Semen quality before cryopreservation and after thawing in 543 patients with testicular cancer.

PubMed

MacKenna, Antonio; Crosby, Javier; Huidobro, Cristián; Correa, Eduardo; Duque, Gonzalo

2017-02-01

The main objective of this study was to assess semen characteristics of patients with testicular cancer before cryopreservation and after thawing, to evaluate the consequences of this technique on sperm quality in patients with testicular cancer. Five hundred eighty-nine samples from 543 patients with testicular cancer were cryopreserved between 1995 and 2015, one aliquot per patient was used for a thawing test to assess the impact of cryopreservation on sperm motility; semen analysis was performed before cryo preservation and after thawing, the result interpretation was carried out using the 2010 World Health Organization (WHO) Laboratory Manual, and consent forms were signed by the patients for freezing and when sperm was used for reproductive purposes. Hypospermia was observed in 28.7% of samples, the median sperm concentration was 18 million/mL with 35% oligozoospermia; twenty-two patients (4.1%) had azoospermia and 12.7% had severe oligozoospermia, the median sperm count was 31.3 million and 261 semen samples (44.3%) were normal in all parameters according to the WHO; total motile sperm count before cryopreservation and after thawing was 12 (0-412.2) and 7 (0-303.9) million sperm, respectively (p < 0.00001, 95% CI 5.48-14.91), which represents a 32% reduction; concerning the utilization of cryopreserved semen samples, only twelve patients (2.2%) used their frozen sperm for reproductive purposes. An impairment in semen quality was found in almost half of the samples from patients with testicular cancer, only few patients had azoospermia or severe oligozoospermia; sperm cryopreservation significantly reduces sperm motility and total motile sperm count and very few patients use their frozen sperm for reproductive purposes.

Semen quality before cryopreservation and after thawing in 543 patients with testicular cancer

PubMed Central

MacKenna, Antonio; Crosby, Javier; Huidobro, Cristián; Correa, Eduardo; Duque, Gonzalo

2017-01-01

Objective The main objective of this study was to assess semen characteristics of patients with testicular cancer before cryopreservation and after thawing, to evaluate the consequences of this technique on sperm quality in patients with testicular cancer. Methods Five hundred eighty-nine samples from 543 patients with testicular cancer were cryopreserved between 1995 and 2015, one aliquot per patient was used for a thawing test to assess the impact of cryopreservation on sperm motility; semen analysis was performed before cryo preservation and after thawing, the result interpretation was carried out using the 2010 World Health Organization (WHO) Laboratory Manual, and consent forms were signed by the patients for freezing and when sperm was used for reproductive purposes. Results Hypospermia was observed in 28.7% of samples, the median sperm concentration was 18 million/mL with 35% oligozoospermia; twenty-two patients (4.1%) had azoospermia and 12.7% had severe oligozoospermia, the median sperm count was 31.3 million and 261 semen samples (44.3%) were normal in all parameters according to the WHO; total motile sperm count before cryopreservation and after thawing was 12 (0-412.2) and 7 (0-303.9) million sperm, respectively (p < 0.00001, 95% CI 5.48-14.91), which represents a 32% reduction; concerning the utilization of cryopreserved semen samples, only twelve patients (2.2%) used their frozen sperm for reproductive purposes. Conclusions An impairment in semen quality was found in almost half of the samples from patients with testicular cancer, only few patients had azoospermia or severe oligozoospermia; sperm cryopreservation significantly reduces sperm motility and total motile sperm count and very few patients use their frozen sperm for reproductive purposes. PMID:28333030

Testes Mass, but Not Sperm Length, Increases with Higher Levels of Polyandry in an Ancient Sex Model

PubMed Central

Vrech, David E.; Olivero, Paola A.; Mattoni, Camilo I.; Peretti, Alfredo V.

2014-01-01

There is strong evidence that polyandrous taxa have evolved relatively larger testes than monogamous relatives. Sperm size may either increase or decrease across species with the risk or intensity of sperm competition. Scorpions represent an ancient direct mode with spermatophore-mediated sperm transfer and are particularly well suited for studies in sperm competition. This work aims to analyze for the first time the variables affecting testes mass, ejaculate volume and sperm length, according with their levels of polyandry, in species belonging to the Neotropical family Bothriuridae. Variables influencing testes mass and sperm length were obtained by model selection analysis using corrected Akaike Information Criterion. Testes mass varied greatly among the seven species analyzed, ranging from 1.6±1.1 mg in Timogenes dorbignyi to 16.3±4.5 mg in Brachistosternus pentheri with an average of 8.4±5.0 mg in all the species. The relationship between testes mass and body mass was not significant. Body allocation in testes mass, taken as Gonadosomatic Index, was high in Bothriurus cordubensis and Brachistosternus ferrugineus and low in Timogenes species. The best-fitting model for testes mass considered only polyandry as predictor with a positive influence. Model selection showed that body mass influenced sperm length negatively but after correcting for body mass, none of the variables analyzed explained sperm length. Both body mass and testes mass influenced spermatophore volume positively. There was a strong phylogenetic effect on the model containing testes mass. As predicted by the sperm competition theory and according to what happens in other arthropods, testes mass increased in species with higher levels of sperm competition, and influenced positively spermatophore volume, but data was not conclusive for sperm length. PMID:24736525

Grape juice concentrate alleviates epididymis and sperm damage in cadmium-intoxicated rats.

PubMed

Lamas, Celina de A; Cuquetto-Leite, Livia; do Nascimento da Silva, Emanueli; Thomazini, Bruna F; Cordeiro, Gabriel da S; Predes, Fabrícia de S; Gollücke, Andrea P B; Dolder, Heidi

2017-04-01

The possibility of long-term grape juice concentrate (GJC) consumption conferring a protective effect against cadmium (Cd)-induced damage to the epididymis, completely preserving sperm profile, was evaluated here for the first time in the scientific literature. Male Wistar rats (n = 6/per group) received an intraperitoneal Cd injection (1.2 mg/Kg) at age 80 days and GJC (2 g/Kg) by gavage from 50 days until 136 days old. Groups receiving either Cd or GJC were added. An intraperitoneal injection of saline (0.9%) and water by gavage was administered in the absence of treatment with Cd or GJC. Animals were anaesthetized and exsanguinated at 136 days; the vas deferens, left testis and epididymis were removed; and perfusion continued with fixative. The right epididymis was collected for morphological analysis. Cd had a devastating effect demonstrated by reduced sperm count in testes and epididymis, sperm production and normal sperm count, besides increased epididymis sperm transit time and completely disorganized morphology. These alterations were attributed to higher Cd levels in the testes and a lipid peroxidation (LP) process. Consumption of GJC plus Cd intoxication was effective, reducing metal accumulation and LP. Consequently, we could identify a preserved sperm profile, with improvement in testis and epididymis sperm count, normal sperm structure and sperm transit time. Moreover, GJC extends its protective effect to the epididymis, allowing complete re-establishment of its morphology, ensuring successful sperm maturation process. In conclusion, our study indicates long-term GJC as a promising therapy against reproductive chemical intoxication injury damage, preserving sperm prior to ejaculation. © 2017 The Authors. International Journal of Experimental Pathology © 2017 International Journal of Experimental Pathology.

Sperm quality and DNA integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons.

PubMed

Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Chiu, Chien-Chih; Zhou, Guodong; Chou, Chon-Kit; Lin, Wen-Yi

2016-11-18

The objective of this study was to assess sperm quality and deoxyribonucleic acid (DNA) integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs) as compared to control subjects. The coke oven workers (N = 52) and administrative staff (N = 35) of a steel plant served as the exposed and control groups, respectively. Exposure to PAHs was assessed by measuring 1-hydroxypyren. Analysis of sperm quality (concentration, motility, vitality, and morphology) was performed simultaneously with sperm DNA integrity analysis, including DNA fragmentation, denaturation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). A questionnaire was conducted to collect demographic and potential confounding data. The coke oven workers had lower percentages of sperm motility, vitality and normal morphology than the control group, but the difference was not significant. For DNA integrity, the coke oven workers had significantly higher concentrations of bulky DNA adducts and 8-oxo-dGuo than the control subjects (p = 0.009 and p = 0.048, respectively). However, DNA fragmentation percentages did not significantly increase as compared to those in the subjects from the control group (p = 0.232). There was no correlation between sperm quality parameters and DNA integrity indicators. Occupational exposure of the coke oven workers to PAHs was associated with decreased sperm DNA integrity. Int J Occup Med Environ Health 2016;29(6):915-926. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

Chymotrypsin effects on the determination of sperm parameters and seminal biochemistry markers.

PubMed

Chen, Fang; Lu, Jin-Chun; Xu, Hui-Ru; Huang, Yu-Feng; Lu, Nian-Qing

2006-01-01

Few reports of the effects of treatment with chymotrypsin on the determination of sperm parameters and seminal biochemistry markers are documented. Sperm parameters of 63 liquefied and 27 non-liquefied samples, untreated or treated with chymotrypsin, were evaluated using computer-assisted semen analysis. In addition, biochemistry markers such as gamma-glutamyltranspeptidase, alpha-glucosidase and fructose in 50 liquefied and 39 non-liquefied samples, untreated or treated with chymotrypsin, were determined. Treatment with chymotrypsin had no effect on sperm concentration, motility, motility a and b, straightness, curvilinear velocity, straight line velocity, average path velocity and beat cross frequency in both liquefied and non-liquefied semen. However, linearity (p=0.025) decreased and the amplitude of the lateral head (p=0.029) increased significantly in non-liquefied semen after treatment with chymotrypsin. The levels of gamma-glutamyltranspeptidase, alpha-glucosidase and fructose in seminal plasma were unaffected by chymotrypsin, regardless of liquefaction status. Chymotrypsin had no effects on the detection of sperm parameters and biochemistry markers, and could be used to treat non-liquefied samples before semen analysis in the andrology laboratory.

Association of the VDAC3 gene polymorphism with sperm count in Han-Chinese population with idiopathic male infertility.

PubMed

Pan, Lianjun; Liu, Qingzhen; Li, Jingyun; Wu, Wei; Wang, Xinru; Zhao, Dan; Ma, Jiehua

2017-07-11

Voltage-dependent anion channel (VDAC) is a multifunctional channel protein across the outer mitochondrial membrane of somatic cells and participates in many physiological and pathophysiological processes. Up to now, only a few studies, including our previous studies, showed that VDAC exists in mammalian spermatozoa and is involved in spermatogenesis and sperm functions. There is no report about VDAC genetic variants in germinal tissues or cells. To investigate the possible association between VDAC genetic variants and human sperm quality, we performed semen analysis and variant Genotyping of VDAC3 subtype (rs7004637, rs16891278 and rs6773) of 523 Han-Chinese males with idiopathic infertility respectively by computer assisted semen analysis (CASA) and single nucleotide polymorphism (SNP) Genotyping assay. No significant association was found between rs7004637 and rs6773 genotypes and semen quality. However, the AG genotype of rs16891278 showed a significantly lower sperm concentration compared with the AA genotype (P = 0.044). Our findings suggest that VDAC3 genetic variants may be associated with human sperm count.

Fins improve the swimming performance of fish sperm: a hydrodynamic analysis of the Siberian sturgeon Acipenser baerii.

PubMed

Gillies, Eric A; Bondarenko, Volodymyr; Cosson, Jacky; Pacey, Allan A

2013-02-01

The flagella of sturgeon sperm have an ultrastructure comprising paddle-like fins extending along most of their length. These fins are seen in several other marine and freshwater fish. The sperm of these fish are fast swimmers and are relatively short lived: it is therefore tempting to think of these fins as having evolved for hydrodynamic advantage, but the actual advantage they impart, at such a small length scale and slow speed, is unclear. The phrase "the fins improve hydrodynamic efficiency" is commonly found in biological literature, yet little hydrodynamic analysis has previously been used to support such conjectures. In this paper, we examine various hydrodynamic models of sturgeon sperm and investigate both swimming velocity and energy expenditure. All of the models indicate a modest hydrodynamic advantage of finned sperm, in both straight line swimming speed and a hydrodynamic efficiency measure. We find a hydrodynamic advantage for a flagellum with fins, over one without fins, of the order of 15-20% in straight line propulsive velocity and 10-15% in a hydrodynamic efficiency measure. Copyright © 2012 Wiley Periodicals, Inc.

Development of the NBT assay as a marker of sperm oxidative stress.

PubMed

Tunc, Ozlem; Thompson, Jeremy; Tremellen, Kelton

2010-02-01

Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen samples from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 microg formazan/10(7) sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an individual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.

Cadmium effects on sperm morphology and semenogelin with relates to increased ROS in infertile smokers: An in vitro and in silico approach.

PubMed

Ranganathan, Parameswari; Rao, Kamini A; Sudan, Jesu Jaya; Balasundaram, Sridharan

2018-06-01

Smoking releases cadmium (Cd), the metal toxicant which causes an imbalance in reactive oxygen species level in seminal plasma. This imbalance is envisaged to impair the sperm DNA morphology and thereby result in male infertility. In order to correlate this association, we performed in vitro and in silico studies and evaluated the influence of reactive oxygen species imbalance on sperm morphology impairments due to smoking. The study included 76 infertile smokers, 72 infertile non-smokers, 68 fertile smokers and 74 fertile non-smokers (control). Semen samples were collected at regular intervals from all the subjects. Semen parameters were examined by computer assisted semen analysis, quantification of metal toxicant by atomic absorption spectrophotometer, assessment of antioxidants through enzymatic and non-enzymatic methods, diagnosis of reactive oxygen species by nitro blue tetrazolium method and Cd influence on sperm protein by in vitro and in silico methods. Our analysis revealed that the levels of cigarette toxicants in semen were high, accompanied by low levels of antioxidants in seminal plasma of infertile smoker subjects. In addition the investigation of Cd treated sperm cells through scanning electronic microscope showed the mid piece damage of spermatozoa. The dispersive X-ray analysis to identify the elemental composition further confirmed the presence of Cd. Finally, the in-silico analysis on semenogelin sequences revealed the D-H-D motif which represents a favourable binding site for Cd coordination. Our findings clearly indicated the influence of Cd on reactive oxygen species leading to impaired sperm morphology leading to male infertility. Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.

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.

Sperm quality variables as indicators of bull fertility may be breed dependent.

PubMed

Morrell, Jane M; Nongbua, Thanapol; Valeanu, Sabina; Lima Verde, Isabel; Lundstedt-Enkel, Katrin; Edman, Anders; Johannisson, Anders

2017-10-01

A means of discriminating among bulls of high fertility based on sperm quality is needed by breeding centers. The objective of the study was to examine parameters of sperm quality in bulls of known fertility to identify useful indicators of fertility. Frozen semen was available from bulls of known fertility (Viking Genetics, Skara, Sweden): Swedish Red (n=31), Holstein (n=25) and Others (one each of Charolais, Limousin, Blonde, SKB). After thawing, the sperm samples were analyzed for motility (computer assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity and reactive oxygen species. A fertility index score based on the adjusted 56-day non-return rate for >1000 inseminations was available for each bull. Multivariate data analysis (Partial Least Squares Regression and Orthogonal Partial Least Squares Regression) was performed to identify variables related to fertility; Pearson univariate correlations were made on the parameters of interest. Breed of bull affected the relationship of sperm quality variables and fertility index score, as follows: Swedish Red: %DNA Fragmentation Index, r=-0.56, P<0.01; intact plasma membrane, r=0.40, P<0.05; membrane damaged, not acrosome reacted, r=-0.6, P<0.01; Linearity, r=0.37, P<0.05; there was a trend towards significance for Wobble, r=0.34, P=0.08. Holstein: Linearity was significant r=0.46, P<0.05; there was a trend towards significance for Wobble, r=0.45, P=0.08. In conclusion, breed has a greater effect on sperm quality than previously realized; different parameters of sperm quality are needed to indicate potential fertility in different breeds. Copyright © 2017 Elsevier B.V. All rights reserved.

Simple optical method of qualitative assessment of sperm motility: preliminary results

NASA Astrophysics Data System (ADS)

Sozanska, Agnieszka; Kolwas, Krystyna; Galas, Jacek; Blocki, Narcyz; Czyzewski, Adam

2005-09-01

The examination of quality of the sperm ejaculate is one of the most important steps in artificial fertilization procedure. The main aim of semen storage centres is to characterise the best semen quality for fertilization. Reliable information about sperm motility is also one the most important parameters for in vitro laboratory procedures. There exist very expensive automated methods for semen analysis but they are unachievable for most of laboratories and semen storage centres. Motivation for this study is to elaborate a simple, cheap, objective and repeatable method for semen motility assessment. The method enables to detect even small changes in motility introduced by medical, physical or chemical factors. To test the reliability of the method we used cryopreserved bull semen from Lowicz Semen Storage Centre. The examined sperm specimen was warmed in water bath and then centrifuged. The best semen was collected by the swim-up technique and diluted to a proper concentration. Several semen concentrations and dilutions were tested in order to find the best probe parameters giving repeatable results. For semen visualization we used the phase-contrast microscope with a CCD camera. A PC computer was used to acquire and to analyse the data. The microscope table equipped with a microscope glass pool 0.7mm deep instead of some conventional plane microscope slides was stabilised at the temperature of 37°C. The main idea of our method is based on a numerical processing of the optical contrast of the sperm images which illustrates the dynamics of the sperm cells movement and on appropriate analysis of a grey scale level of the superimposed images. An elaborated numerical algorithm allows us to find the relative amount of motile sperm cells. The proposed method of sperm motility assessment seems to be objective and repeatable.

High-dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism

PubMed Central

Aarabi, Mahmoud; San Gabriel, Maria C.; Chan, Donovan; Behan, Nathalie A.; Caron, Maxime; Pastinen, Tomi; Bourque, Guillaume; MacFarlane, Amanda J.; Zini, Armand; Trasler, Jacquetta

2015-01-01

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR. PMID:26307085

Automated Sperm Head Detection Using Intersecting Cortical Model Optimised by Particle Swarm Optimization.

PubMed

Tan, Weng Chun; Mat Isa, Nor Ashidi

2016-01-01

In human sperm motility analysis, sperm segmentation plays an important role to determine the location of multiple sperms. To ensure an improved segmentation result, the Laplacian of Gaussian filter is implemented as a kernel in a pre-processing step before applying the image segmentation process to automatically segment and detect human spermatozoa. This study proposes an intersecting cortical model (ICM), which was derived from several visual cortex models, to segment the sperm head region. However, the proposed method suffered from parameter selection; thus, the ICM network is optimised using particle swarm optimization where feature mutual information is introduced as the new fitness function. The final results showed that the proposed method is more accurate and robust than four state-of-the-art segmentation methods. The proposed method resulted in rates of 98.14%, 98.82%, 86.46% and 99.81% in accuracy, sensitivity, specificity and precision, respectively, after testing with 1200 sperms. The proposed algorithm is expected to be implemented in analysing sperm motility because of the robustness and capability of this algorithm.

Resolving a genetic paradox throughout preimplantation genetic diagnosis for autosomal dominant severe congenital neutropenia.

PubMed

Malcov, Mira; Reches, Adi; Ben-Yosef, Dalit; Cohen, Tania; Amit, Ami; Dgany, Orly; Tamary, Hannah; Yaron, Yuval

2010-03-01

Severe congenital neutropenia is an inherited disease characterized by low peripheral blood neutrophils, amenable to bone marrow transplantation. Genetic analysis in the family here described detected a ELA2 splice-site mutation in the affected child and also in his asymptomatic father. The parents requested preimplantation genetic diagnosis (PGD), coupled with HLA matching, to obtain a suitable bone marrow donor for the affected child. A PGD protocol was developed, based on multiplex nested PCR for direct analysis of the ELA2 mutation, flanking polymorphic markers and HLA typing. The amplification efficiency of the mutation was > 90% in single leukocytes from the affected child but only 67% in the father. Analysis of single haploid sperm cells from the father demonstrated three different sperm-cell populations: (1) sperm cells harboring the ELA2 mutation on the 'affected' haplotype, (2) sperm cells without the ELA2 mutation on the 'normal' haplotype, and (3) sperm cells without the ELA2 mutation on the 'affected' haplotype. These data demonstrate that the ELA2 mutation in the father occurred de novo during his embryonic development, resulting in somatic as well as germ-line mosaicism. This conclusion was also taken into consideration when PGD was performed. Copyright (c) 2010 John Wiley & Sons, Ltd.

Effect of Palm Pollen on Sperm Parameters of Infertile Man.

PubMed

Rasekh, Athar; Jashni, Hojjatollah Karimi; Rahmanian, Karamatollah; Jahromi, Abdolreza Sotoodeh

2015-04-01

There is a rapidly growing trend in the consumption of herbal remedies in the developing countries. The aim of this study was to determine the effects of orally administered Date Palm Pollen (DPP) on the results of semen analysis in adult infertile men. Forty infertile men participated in our study. They were treated by Pollen powder 120 mg kg(-1) in gelatinous capsules every other day, for two months. Before and at the end of therapy, the semen was collected after masturbation and sperm numbers, motility and morphology were determined. Our findings revealed that consumption of DPP improved the sperm count. The treatment was significantly increased sperm motility, morphology and forward progressive motility. Date palm pollen seems to cure male infertility by improving the quality of sperm parameters.

Plasma membrane changes during the liquid storage of boar spermatozoa: a comparison of methods.

PubMed

Gaczarzewicz, Dariusz; Piasecka, Małgorzata; Udała, Jan; Błaszczyk, Barbara; Stankiewicz, Tomasz; Laszczyńska, Maria

2010-03-01

Studies were performed on boar semen routinely used at the local artificial insemination (AI) centre. The semen was stored in a Safe Cell Plus commercial extender at 17 degrees C for nine days. The aim of our research was focused on changes in sperm plasma membrane integrity. The integrity of the sperm plasma membrane and acrosome as well as sperm motility decreased after dilution and during storage of the semen. The highest percentage of live sperm was identified by the eosin-nigrosin method, a lower percentage by the SYBR-14/PI test, and the lowest percentage of live cells was discovered by the hypoosmotic swelling (HOS) test (P < 0.01). There were significant differences between the results of staining methods and sperm motility (P < 0.01). No significant differences were found between the HOS test results and sperm motility. The plasma membrane integrity parameters positively correlated (P < 0.001) with each other and with sperm motility but negatively with aspartate aminotransferase activity. Our findings confirmed that the boar sperm aging changes, which increased during liquid semen preservation, were connected with the loss of function and integrity of the sperm plasma membrane. The employed complementary tests are comprehensive indicators of sperm membrane integrity during long-term semen preservation, and they can help establish the actual number of 'healthy' cells. The assays may be used in AI laboratories and should be incorporated into the routine of semen analysis.

Senescent sperm performance in old male birds.

PubMed

Møller, A P; Mousseau, T A; Rudolfsen, G; Balbontín, J; Marzal, A; Hermosell, I; De Lope, F

2009-02-01

Senescence is the deterioration of the phenotype with age caused by negative effects of mutations acting late in life or the physiological deterioration of vital processes. Birds have traditionally been assumed to senescence slowly despite their high metabolic rates, high blood sugar levels and high body temperature. Here we investigate the patterns of age-related performance of sperm of a long distance migrant, the barn swallow Hirundo rustica, varying in age from 1 to 6 years, analysed by the computer-assisted sperm analysis equipment. Sperm showed deteriorating performance in terms of linear movement, track velocity, straight line velocity and reduced proportions of rapidly moving, progressive and motile sperm with age. In a second series of experiments, we assessed performance of sperm from the same males in neutral medium and in medium derived from the reproductive tract of females in an attempt to test if sperm of old males performed relatively better in female medium, as expected from extra-pair paternity being negatively related to male age, but not to female age. Older males showed consistently better performance in female medium than in neutral medium in terms of track velocity, straight line velocity and reduced proportions of rapidly moving, progressive and motile sperm, whereas young males showed better performance in neutral medium. These results provide evidence of declining sperm performance for important reproductive variables not only with age, but also with the sperm of old males performing differentially better in female medium than young males.

Role of Trace Elements for Oxidative Status and Quality of Human Sperm.

PubMed

Nenkova, Galina; Petrov, Lubomir; Alexandrova, Albena

2017-08-04

Oxidative stress affects sperm quality negatively. To maintain the pro/antioxidant balance, some metal ions (e.g. copper, zink, iron, selenium), which are co-factors of the antioxidant enzymes, are essential. However, iron and copper could act as prooxidants inducing oxidative damage of spermatozoa. To reveal a possible correlation between the concentrations of some metal ions (iron, copper, zinc, and selenium) in human seminal plasma, oxidative stress, assessed by malondialdehyde and total glutathione levels, and semen quality, assessed by the parameters count, motility, and morphology. Descriptive study. The semen analysis for volume, count, and motility was performed according to World Health Organization (2010) guidelines, using computer-assisted semen analysis. For the determination of spermatozoa morphology, a SpermBlue staining method was applied. Depending on their parameters, the sperm samples were categorized into normozoospermic, teratozoospermic, asthenoteratozoospermic, and oligoteratozoospermic. The seminal plasma content of iron, copper, zinc, and selenium was estimated by atomic absorption spectroscopy. The malondialdehyde and total glutathione levels were quantified spectrophotometrically. In the groups with poor sperm quality, the levels of Fe were higher, whereas those of Zn and Se were significantly lower than in the normozoospermic group. In all groups with poor sperm quality, increased levels of malondialdehyde and decreased glutathione levels were detected as evidence of oxidative stress occurrence. All these differences are most pronounced in the asthenoteratozoospermic group where values differ nearly twice as much compared to the normozoospermic group. The Fe concentration correlated positively with the malondialdehyde (r=0.666, p=0.018), whereas it showed a negative correlation with the level of total glutathione (r=-0.689, p=0.013). The total glutathione level correlated positively with the sperm motility (r=0.589, p=0.044). The elevated levels of Fe and the reduced Se levels are associated with sperm damage. The changes in the concentrations of the trace elements in human seminal plasma may be related to sperm quality since they are involved in the maintenance of the pro-/antioxidative balance in ejaculate.

A Comparative Analysis of the Morphology and Evolution of Permanent Sperm Depletion in Spiders

PubMed Central

2011-01-01

Once thought to be energetically cheap and easy to produce, empirical work has shown that sperm is a costly and limited resource for males. In some spider species, there is behavioral evidence that sperm are permanently depleted after a single mating. This extreme degree of mating investment appears to co-occur with other reproductive strategies common to spiders, e.g. genital mutilation and sexual cannibalism. Here we corroborate that sperm depletion in the golden orb-web spider Nephila clavipes is permanent by uncovering its mechanistic basis using light and electron microscopy. In addition, we use a phylogeny-based statistical analysis to test the evolutionary relationships between permanent sperm depletion (PSD) and other reproductive strategies in spiders. Male testes do not produce sperm during adulthood, which is unusual in spiders. Instead, spermatogenesis is nearly synchronous and ends before the maturation molt. Testis size decreases as males approach their maturation molt and reaches its lowest point after sperm is transferred into the male copulatory organs (pedipalps). As a consequence, the amount of sperm available to males for mating is limited to the sperm contained in the pedipalps, and once it is used, males lose their ability to fertilize eggs. Our data suggest that PSD has evolved independently at least three times within web-building spiders and is significantly correlated with the evolution of other mating strategies that limit males to monogamy, including genital mutilation and sexual cannibalism. We conclude that PSD may be an energy-saving adaptation in species where males are limited to monogamy. This could be particularly important in web-building spiders where extreme sexual size dimorphism results in large, sedentary females and small, searching males who rarely feed as adults and are vulnerable to starvation. Future work will explore possible energetic benefits and the evolutionary lability of PSD relative to other mate-limiting reproductive behaviors. PMID:21264312

[Varicocele and coincidental abacterial prostato-vesiculitis: negative role about the sperm output].

PubMed

Vicari, Enzo; La Vignera, Sandro; Tracia, Angelo; Cardì, Francesco; Donati, Angelo

2003-03-01

To evaluate the frequency and the role of a coincidentally expressed abacterial prostato-vesiculitis (PV) on sperm output in patients with left varicocele (Vr). We evaluated 143 selected infertile patients (mean age 27 years, range 21-43), with oligo- and/or astheno- and/or teratozoospermia (OAT) subdivided in two groups. Group A included 76 patients with previous varicocelectomy and persistent OAT. Group B included 67 infertile patients (mean age 26 years, range 21-37) with OAT and not varicocelectomized. Patients with Vr and coincidental didymo-epididymal ultrasound (US) abnormalities were excluded from the study. Following rectal prostato-vesicular ultrasonography, each group was subdivided in two subsets on the basis of the absence (group A: subset Vr-/PV-; and group B: subset Vr+/PV-) or the presence of an abacterial PV (group A: subset Vr-/PV+; group B: subset Vr+/PV+). Particularly, PV was present in 47.4% and 41.8% patients of groups A and B, respectively. This coincidental pathology was ipsilateral with Vr in the 61% of the cases. Semen analysis was performed in all patients. Patients of group A showed a total sperm number significantly higher than those found in group B. In presence of PV, sperm parameters were not significantly different between matched--subsets (Vr-/PV+ vs. Vr+/PV+). In absence of PV, the sperm density, the total sperm number and the percentage of forward motility from subset with previous varicocelectomy (Vr-/PV) exhibited values significantly higher than those found in the matched--subset (Vr+/PV-). Sperm analysis alone performed in patients with left Vr is not a useful prognostic post-varicocelectomy marker. Since following varicocelectomy a lack of sperm response could mask another coincidental pathology, the identification through US scans of a possible PV may be mandatory. On the other hand, an integrated uro-andrological approach, including US scans, allows to enucleate subsets of patients with Vr alone, who will have an expected better sperm response following Vr repair.

Semen quality analysis of military personnel from six geographical areas of the People's Republic of China.

PubMed

Zou, Zhikang; Hu, Haixiang; Song, Manshu; Shen, Yanling; Guo, Xiuhua; McElreavey, Kenneth; Bittles, Alan H; Wang, Wei

2011-05-01

To examine the determinants of semen quality in a large sample of military personnel from different geographical areas of the People's Republic of China. Cross-sectional study. Six representative geographical regions in China: Beihai, Lhasa, Germu, Xinzhou, Huhehaote, and Mohe. 1,194 army personnel aged 18 to 35 years at the time of their inclusion in the study, sampled between 2007 and 2009. None. Semen volume (in milliliters), sperm concentration (in millions per milliliter), percentage of motile spermatozoa, total sperm count (in millions), and relative risk of subfertility. The median values were 3.0 mL for semen volume, 39.4×10(6) per mL for sperm concentration, 120.1×10(6) for total sperm count, 15.8% for sperm rapid progressive motility, 30.1% for sperm progressive motility, and 43.9% for total motility. We found that 88.3% of the servicemen had at least one semen parameter below normal values according to World Health Organization (WHO) recommendations (1999), and 62.5% according to WHO recommendations (2010). Season, average altitude, and duration of sexual abstinence all were statistically significantly associated with semen quality. The men had markedly lower mean sperm concentrations, sperm counts, and sperm motility compared with WHO recommendations. Possible contributory factors included diet, lifestyle, climate, and altitude. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Meiotic recombination errors, the origin of sperm aneuploidy and clinical recommendations.

PubMed

Tempest, Helen G

2011-02-01

Since the early 1990s male infertility has successfully been treated by intracytoplasmic sperm injection (ICSI), nevertheless concerns have been raised regarding the genetic risk of ICSI. Chromosome aneuploidy (the presence of extra or missing chromosomes) is the leading cause of pregnancy loss and mental retardation in humans. While the majority of chromosome aneuploidies are maternal in origin, the paternal contribution to aneuploidy is clinically relevant particularly for the sex chromosomes. Given that it is difficult to study female gametes investigations are predominantly conducted in male meiotic recombination and sperm aneuploidy. Research suggests that infertile men have increased levels of sperm aneuploidy and that this is likely due to increased errors in meiotic recombination and chromosome synapsis within these individuals. It is perhaps counterintuitive but there appears to be no selection against chromosomally aneuploid sperm at fertilization. In fact the frequency of aneuploidy in sperm appears to be mirrored in conceptions. Given this information this review will cover our current understanding of errors in meiotic recombination and chromosome synapsis and how these may contribute to increased sperm aneuploidy. Frequencies of sperm aneuploidy in infertile men and individuals with constitutional karyotypic abnormalities are reviewed, and based on these findings, indications for clinical testing of sperm aneuploidy are discussed. In addition, the application of single nucleotide arrays for the analysis of meiotic recombination and identification of parental origin of aneuploidy are considered.

Deduction of a calcium ion circuit affecting rooster sperm in vitro.

PubMed

Froman, D P

2016-08-01

Four premises for rooster sperm preservation were outlined previously. Understanding mitochondrial Ca cycling in terms of whole-cell Ca flux was one premise. The present work tested the hypothesis that sperm mitochondria can be damaged by intracellular as well as extracellular Ca. Sperm were washed by centrifugation through 12% (wt/vol) Sperm were washed by centrifugation through 12%(at/vol) Accudenz to procure sperm at a physiological concentration within a chemically-defined suspension. Five solutions were tested. Each solution contained 30 m glucose, and had an osmolality of 320 mmol/kg and a pH of 7.4. Washed sperm were diluted to 2.0 × 10 sperm/mL. Each replicate sperm suspension was cooled to 10°C. Sperm mobility was measured after 1, 2, 4, 8, 12, and 24 h. Data were plotted as a function of time in each experiment. Function type was confirmed by lack of fit analysis. A parabola with a maximum at 3.7 h was observed when sperm were suspended in 205 m taurine buffered with 50 m-tris[hydroxyl-methyl]methyl-2-amino-ethanesulfonic acid (TES). This effect was attributed to a Ca flux from the nuclear envelope into mitochondria. An exponential decay was observed when TES-buffered taurine contained 2 m Ca. This effect was attributed to mitochondrial Ca overload induced by uptake of extracellular Ca. Exponential decay also was observed when TES-buffered taurine contained a Ca chelator. This effect was attributed to a Ca flux from the nuclear envelope through mitochondria and then into an extracellular Ca sink. This possibility was supported by the response of sperm to thapsigargin. Specifically, inhibition of sarcoendoplasmic reticulum Ca-ATPase compromised sperm mobility relative to a buffer control. Finally, a 60 m phosphate buffer containing 2 m citrate yielded a linear relationship in contrast to the TES-buffered solutions tested. Sperm mobility after 24 h of storage in the phosphate buffer was 92% of that observed for prewashed sperm. The linear response was attributed to weak chelators providing resistance within a Ca circuit and thereby preventing mitochondrial Ca overload. Fertility, however, was compromised when hens were inseminated with mobile sperm recovered after either 8 or 24 h of storage at 10°C. In conclusion, sperm cell Ca homeostasis was proven to be critical for maintaining sperm mobility in vitro, but mitochondrial Ca uptake is not the sole phenomenon that compromises sperm function during in vitro storage.

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

PubMed

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

2010-03-01

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

Profiling of sperm proteins and association of sperm PDC-109 with bull fertility.

PubMed

Somashekar, Lakshminarayana; Selvaraju, Sellappan; Parthipan, Sivashanmugam; Ravindra, Janivara Parameswaraiah

2015-01-01

The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (n = 12) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (n = 3) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1-5.6, 37%) and basic (pI; 7.9-10.0, 27%) proteins and their pattern of expression varied significantly (p < 0.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4 kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (p < 0.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility.

Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs.

PubMed

Lucio, C F; Regazzi, F M; Silva, L C G; Angrimani, D S R; Nichi, M; Vannucchi, C I

2016-06-01

Sperm cryopreservation generates sperm damage and reduced fertilization capacity as a consequence of reactive oxygen species formation. Identifying the critical points of the process will contribute to the development of strategies for oxidative stress prevention. Therefore, the aim of this experiment was to verify the occurrence of oxidative stress during the two-step cryopreservation process in dogs. Six healthy mature dogs were used and submitted to the two-step sperm cryopreservation protocol. The sperm analysis was done at three time points: after refrigeration, after glycerolization, and after thawing by sperm motility, measurement of spontaneous and induced oxidative stress, sperm mitochondrial activity, plasma membrane integrity, flow cytometric evaluation of plasma and acrosome membrane integrity, mitochondrial membrane potential, and sperm chromatin structure assay. There was an increase in free radical production after glycerolization (87.4 ± 15.5 ng/mL of spontaneous thiobarbituric acid reactive substances (TBARS) after refrigeration and 1226.3 ± 256.0 ng/mL after glycerolization; P < 0.05), in association with loss of sperm mitochondrial activity. However, frozen-thawed samples had lower sperm motility, lower resistance to oxidative stress (448.7 ± 23.6 ng/mL of induced TBARS after glycerolization and 609.4 ± 35.9 ng/mL after thawing; P < 0.05) and increased lipid peroxidation (4815.2 ± 335.4 ng/mL of spontaneous TBARS after thawing; P < 0.05) as well as increased damage to plasma and acrosomal membranes, compared with refrigeration and glycerolization. In conclusion, the production of free radicals by sperm cells begins during glycerolization. However, sperm oxidative damage intensifies after thawing. Despite intracellular ice formation during cryopreservation, the increased production of reactive oxygen species can be the explanation of the decrease in sperm motility, reduced mitochondrial activity, and sperm plasma membrane and acrosomal damage. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of seminal plasma and flash-freezing on DNA structure of stallion epididymal sperm exposed to different potentiators of DNA damage.

PubMed

Serafini, R; Varner, D D; Blanchard, T L; Teague, S R; LaCaze, K; Love, C C

2018-05-24

The tolerance of sperm DNA structure to seminal plasma and freezing conditions has both clinical and basic biologic relevance. In this study, fresh (FS) or flash-frozen (FZ) stallion epididymal sperm were exposed (SP + ) or unexposed (SP - ) to seminal plasma. Sperm were then evaluated to monitor the degree of change in DNA structure following challenge with chemical (dithiothreitol-DTT), oxidative (iron sulfate; FeSO 4 ) or enzymatic (DNase I) potentiators of DNA damage. For sperm not treated with potentiators (controls), there was no effect of SP treatment (SP - vs. SP + ) or freezing treatment (FS vs. FZ; non-significant) on measures of any DNA assays (i.e., 8-hydroxy, 2'deoxyguanosine [8OHdG], TUNEL, or sperm chromatin structure [SCSA] assays). Group FZ was more susceptible than Group FS to potentiators of DNA damage. Percent 8OHdG-positive sperm was higher in Group FZ/SP - treated with FeSO 4 than all other groups (P < 0.05). Percent TUNEL-positive sperm was similar among FZ/SP - groups treated with DTT, FeSO 4 , or DNase (non-significant) and was higher in these groups than all other treatments (P < 0.05). Percent COMP-α t was higher following treatment with DNase or DTT, as compared to their respective controls, regardless of prior exposure to SP (P < 0.05). Overall, sperm DNA structure was unaffected by seminal plasma or freezing treatment when samples were not exposed to potentiators of sperm DNA damage; however, marked differences were identified in DNA structure when sperm were challenged with chemical, oxidative or enzymatic treatments. These results highlight the importance of challenging DNA structure prior to analysis. The use of potentiators of DNA damage provided a model to evaluate sperm DNA structure following exposure of sperm to various experimental treatments. Copyright © 2018 Elsevier Inc. All rights reserved.

A dictionary learning approach for human sperm heads classification.

PubMed

Shaker, Fariba; Monadjemi, S Amirhassan; Alirezaie, Javad; Naghsh-Nilchi, Ahmad Reza

2017-12-01

To diagnose infertility in men, semen analysis is conducted in which sperm morphology is one of the factors that are evaluated. Since manual assessment of sperm morphology is time-consuming and subjective, automatic classification methods are being developed. Automatic classification of sperm heads is a complicated task due to the intra-class differences and inter-class similarities of class objects. In this research, a Dictionary Learning (DL) technique is utilized to construct a dictionary of sperm head shapes. This dictionary is used to classify the sperm heads into four different classes. Square patches are extracted from the sperm head images. Columnized patches from each class of sperm are used to learn class-specific dictionaries. The patches from a test image are reconstructed using each class-specific dictionary and the overall reconstruction error for each class is used to select the best matching class. Average accuracy, precision, recall, and F-score are used to evaluate the classification method. The method is evaluated using two publicly available datasets of human sperm head shapes. The proposed DL based method achieved an average accuracy of 92.2% on the HuSHeM dataset, and an average recall of 62% on the SCIAN-MorphoSpermGS dataset. The results show a significant improvement compared to a previously published shape-feature-based method. We have achieved high-performance results. In addition, our proposed approach offers a more balanced classifier in which all four classes are recognized with high precision and recall. In this paper, we use a Dictionary Learning approach in classifying human sperm heads. It is shown that the Dictionary Learning method is far more effective in classifying human sperm heads than classifiers using shape-based features. Also, a dataset of human sperm head shapes is introduced to facilitate future research. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen.

PubMed

Seifi-Jamadi, Afshin; Ahmad, Ejaz; Ansari, Mahdi; Kohram, Hamid

2017-04-01

This study was performed to evaluate the effectiveness of quercetin as a non-enzymatic antioxidant in combination with glycerol or Dimethylacetamide (DMA), on freezability of goat semen. Ejaculates from four healthy mature Mahabadi goats were collected using an artificial vagina. After primary processing, semen was pooled and extended by egg yolk based extender supplemented with different concentrations of quercetin (10 or 20 μM) along with 5% glycerol or DMA. The extended semen was frozen and sperm motility parameters, viability, abnormality, membrane integrity and lipid peroxidation were assessed after thawing. Results showed that sperm viability, total motility, progressive motility, straightness (STR) and linearity (LIN) were higher (P < 0.05), and abnormality percentage and MDA concentration were lower (P < 0.05) in extender containing DMA. Similarly, higher (P < 0.05) total motility, progressive motility, viability and membrane integrity along with lower (P < 0.05) MDA level were noted in Q10 group. The lowest (P < 0.05) MDA level was observed in DMA extender containing moderate level of quercetin (Q10D). Also the STR was higher (P < 0.05) in Q10D compared to Q10G and Q20G groups. In conclusion, supplementation of extender with 10 μM quercetin in combination with DMA improves the goat sperm motion kinetics and suppresses lipid peroxidation after freezing and thawing. Furthermore, DMA is more effective cryoprotectant for the freezing of goat sperm. Copyright © 2017 Elsevier Inc. All rights reserved.

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

PubMed

Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

2018-02-01

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

Metal concentrations, sperm motility, and RNA/DNA ratio in two echinoderm species from a highly contaminated fjord (the Sørfjord, Norway).

PubMed

Catarino, Ana I; Cabral, Henrique N; Peeters, Kris; Pernet, Philippe; Punjabi, Usha; Dubois, Philippe

2008-07-01

The present study evaluated the effects of field metal contamination on sperm motility and the RNA/DNA ratio in echinoderms. Populations of Asterias rubens and Echinus acutus that occur naturally along a contamination gradient of sediments by cadmium, copper, lead, and zinc in a Norwegian fjord (the Sørfjord) were studied. Sperm motility, a measure of sperm quality, was quantified using a computer-assisted sperm analysis system. The RNA/DNA ratio, a measure of protein synthesis, was assessed by a one-dye (ethidium bromide)/one-enzyme (RNase), 96-well microplate fluorometric assay. Although both species accumulate metals at high concentrations, neither sperm motility parameters in A. rubens nor the RNA/DNA ratio in both species were affected. The Sørfjord is still one of the most metal-contaminated marine sites in Europe, but even so, populations of A. rubens and E. acutus are able to endure under these conditions.

Acute toxicity effects of perfluorooctane sulfonate on sperm vitality, kinematics and fertilization success in zebrafish

NASA Astrophysics Data System (ADS)

Xia, Jigang; Niu, Cuijuan

2017-07-01

Perfluorooctane sulfonate (PFOS) has emerged as one of the most concerning contaminants in recent years. This study aimed to investigate the acute toxicity effect of PFOS on sperm viability, kinematics and fertilization success in zebrafish ( Danio rerio). Sperm were activated in aqueous media containing a range of PFOS concentrations (0, 0.09, 0.9 and 9 mg/L). Viabilities and kinematics of the sperm exposed to different PFOS treatments were assessed via computer-assisted sperm analysis (CASA) at 20, 40, 60, and 80 s after activation. PFOS exposure decreased the percentage of motile sperm, the curvilinear velocity (VCL), and the mean angular displacement (MAD) of spermatozoa, but showed no influence on the straight-line velocity (VSL) or the angular path velocity (VAP). Furthermore, a significant decrease in fertilization success was observed in spermatozoa that were exposed to 0.9 mg/L PFOS or more. These findings indicate that PFOS pollution in natural aquatic environment may be a potential threaten to successful reproduction of fish.

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm.

PubMed

Coux, Gabriela; Cabada, Marcelo O

2006-04-28

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.

Effects of adding different levels of Glutamine to modified Beltsville extender on the survival of frozen rooster semen.

PubMed

Khiabani, Aytak Bakhshayesh; Moghaddam, Gholamali; Kia, Hossein Daghigh

2017-09-01

The aim of the present study was to investigate the effects of l-glutamine on the quality of frozen-thawed rooster semen. Semen samples were collected from eight mature roosters (Ross 308). After initial semen assessments, samples of adequate quality were mixed together and diluted with modified Beltsville extender without l-glutamine (control) and supplemented with 2.5, 5, and 7.5mM l-glutamine. Semen straws were subjected to cryopreservation and evaluated twice at 15-day intervals. After thawing, sperm viability, total and progressive sperm motilities were measured by Eosin-Nigrosine and Computer-Aided Sperm Analysis (CASA), respectively. The results showed that sperm functions decreased on day 30 compared to day 15. The extender supplemented with 5mM glutamine improved (p<0.05) sperm viability, total and progressive sperm motilities compared to other treatments and the control group. The best level of glutamine appeared to be 2.5mM, as it provided the highest sperm membrane integrity and the lowest level of abnormalities. The results of this study suggest that the addition of glutamine to the diluent improves semen quality and using glutamine allows rooster sperm to be frozen for longer. Copyright © 2017 Elsevier B.V. All rights reserved.

Four-dimensional analysis by high-speed holographic imaging reveals a chiral memory of sperm flagella.

PubMed

Muschol, Michael; Wenders, Caroline; Wennemuth, Gunther

2018-01-01

Here high-speed Digital Holographic Microscopy (DHM) records sperm flagellar waveforms and swimming paths in 4 dimensions (X, Z, and t). We find flagellar excursions into the Z-plane nearly as large as the envelope of the flagellar waveform projected onto the XY-plane. These Z-plane excursions travel as waves down the flagellum each beat cycle. DHM also tracks the heads of free-swimming sperm and the dynamics and chirality of rolling of sperm around their long axis. We find that mouse sperm roll CW at the maximum positive Z-plane excursion of the head, then roll CCW at the subsequent maximum negative Z-plane excursion. This alternating chirality of rolling indicates sperm have a chiral memory. Procrustes alignments of path trajectories for sequences of roll-counterroll cycles show that path chirality is always CW for the cells analyzed in this study. Human and bull sperm lack distinguishable left and right surfaces, but DHM still indicates coordination of Z-plane excursions and rolling events. We propose that sperm have a chiral memory that resides in a hypothetical elastic linkage within the flagellar machinery, which stores some of the torque required for a CW or CCW roll to reuse in the following counter-roll. Separate mechanisms control path chirality.

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

PubMed

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

2017-01-02

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

Artificial fertilisation in a terrestrial toadlet (Pseudophryne guentheri): effect of medium osmolality, sperm concentration and gamete storage.

PubMed

Silla, Aimee J

2013-01-01

Anurans exhibit a greater reproductive diversity than any other vertebrate order. However, studies investigating the effects of the external fertilisation environment on fertilisation success are limited to aquatic-breeding species. This study investigated the effects of fertilisation medium osmolality, sperm concentration and short-term oocyte storage on fertilisation success in a terrestrial-breeding anuran, Pseudophryne guentheri. Split-clutch experimental designs were used to determine optimal fertilisation conditions. To determine the effect of short-term sperm storage, sperm viability was assessed using fluorescence microscopy and percentage sperm motility and velocity quantified with a computer-assisted sperm analysis system. Fertilisation success was highest in media ranging in osmolality from 25 mOsm kg⁻¹ to 100 mOsm kg⁻¹, representing a broader range and higher optimal osmolality than previously reported for aquatic breeders. High rates of fertilisation (>75%) were achieved in relatively low sperm concentrations (2.5×10⁴ mL⁻¹). Oocytes stored in isotonic solutions (200 mOsm kg⁻¹) retained fertilisation capacity (32%) after 8h of storage, while sperm suspensions maintained motility (≥26%) for 13 days. Additional studies on terrestrial-breeding anurans will be required to ascertain whether the optimal fertilisation conditions reported reflect adaptations to achieve fertilisation in a terrestrial environment.

Detection of alterations in human sperm using magnetic orientation techniques

NASA Astrophysics Data System (ADS)

Sakhnini, Lama; Dairi, Maheen; Manaa, Hacene

2007-09-01

In this study we report on magnetic orientation of human sperms. Samples were taken from 17 donors. Normal human sperms became oriented with their long axis perpendicular to the magnetic field ( 1 Tesla maximum). Total orientation was achieved with magnetic field at about one Tesla, while for abnormal sperms the magnetic behavior was different. The dependence of the measured degree of orientation on the intensity of the magnetic field was in good agreement with the theoretical equation for the magnetic orientation of diamagnetic substances. As a result for a numerical analysis based on the equation, the anisotropic diamagnetic susceptibility of normal sperm was found to be ▵ χ= 8×10 -20 J/T2. The degree of orientation was influenced by the alterations in the shape of the head, body or the tail. It has been suggested that the DNA in the sperm head retain the strong magnetic anisotropy to counter balance the magnetic anisotropy retained by flagellum microtubules. Recent studies demonstrated a well-defined nuclear architecture in human sperm nucleus, where the head morphology has significant correlation with sperm chromatin structure assay SCSA. Then as the methods to evaluate SCSA can be difficult and expensive our simple magnetic orientation technique can be an alternative to diagnose alteration in DNA.

Quantitative light and scanning electron microscopy of ferret sperm.

PubMed

Van der Horst, G; Curry, P T; Kitchin, R M; Burgess, W; Thorne, E T; Kwiatkowski, D; Parker, M; Atherton, R W

1991-11-01

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.

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.

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.

Environmental Factors Related to a Semiarid Climate Influence the Freezability of Sperm from Collared Peccaries (Pecari tajacu Linnaeus, 1758).

PubMed

Maia, Keilla M; Souza, Ana L P; Praxedes, Erica C G; Bezerra, Luana G P; Silva, Andreia M; Campos, Livia B; Moreira, Samara S J; Apolinário, Carlos A C; Souza, João B F; Silva, Alexandre R

2018-04-30

The influence of environmental factors in a semiarid climate on characteristics of fresh and frozen/thawed sperm collected from collared peccaries (Pecari tajacu) was assessed. Semen from 11 male collared peccaries was collected by electroejaculation during the peaks of the dry and rainy periods while rainfall indices, air temperatures, relative humidity levels, and wind speeds were measured. The number, motility, morphology, osmotic response, and membrane integrity of sperm in the collected ejaculates were assessed. Samples were then frozen in liquid nitrogen, thawed, and reassessed. The rainfall index of the rainy period (73.2 mm) was significantly higher than that of the dry period (13.6 mm) and the relative humidity was significantly higher during the rainy period (74.6%) than it was during the dry period (66.8%). Air temperature and wind speed did not differ between the two periods. Characteristics of sperm in the fresh samples were not affected by environmental parameters. In contrast, computerized analysis revealed that sperm in samples frozen during the rainy period exhibited better post-thaw membrane integrity (28.6 ± 6%), motility (29.5 ± 7.7%), and rapid sperm population (13.7 ± 6.2%) than did sperm in samples frozen during the dry period (23.4 ± 3% membrane integrity, 14.6 ± 4.1% motility, and 4.1 ± 1.2% rapid sperm; p < 0.05). Other characteristics of the frozen/thawed sperm did not differ depending on the period in which they were collected. We demonstrated that environmental parameters did not affect the quality of fresh sperm, but could influence the freezability of sperm collected from collared peccaries raised under a semiarid climate.

TNF-α inhibitors do not impair sperm quality in males with ankylosing spondylitis after short-term or long-term treatment.

PubMed

Micu, Mihaela C; Micu, Romeo; Surd, Stela; Gîrlovanu, Marinela; Bolboacă, Sorana D; Ostensen, Monika

2014-07-01

The aim of this study was to study the influence of active disease status and TNF-α antagonists on sperm quality in a group of AS patients. Twenty-three active AS patients and 42 controls were recruited. Patients' sperm samples were analysed at baseline (previous to) and at 3-6 months after TNF-α therapy (adalimumab, infliximab, etanercept) administration. Baseline assessment was made for only 20 patients, 2 of them proving to have normal fertility, 2 having a pregnant stable partner and the third having a 9-month-old child. Six patients were retested after 12 months of biologic therapy. Each patient acted as his own comparator. Results were further compared with sperm samples from age-matched controls. Sperm analysis was performed according to the World Health Organization (WHO) 1999 guidelines. Patients' baseline assessment showed normozoospermia in 91% and oligozoospermia in 9% of patients. No significant differences in sperm quality were noticed at follow-up visits compared with baseline. Comparison to controls showed no statistically significant differences in semen quality, with some exceptions: the control group presented a higher percentage of non-progressive and immobile sperm cells and higher numbers of head and tail atypias. Sperm quality in patients with active AS and after receiving short- and long-term TNF-α blocker therapy is comparable to sperm quality in healthy controls. Our study confirms that the disease process of AS does not have a major impact on sperm quality and that treatment with anti-TNF has no negative impact on sperm quality even under long-term treatment. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The Association Between Calcium, Magnesium, and Ratio of Calcium/Magnesium in Seminal Plasma and Sperm Quality.

PubMed

Liang, Hong; Miao, Maohua; Chen, Jianping; Chen, Kanglian; Wu, Bin; Dai, Qi; Wang, Jian; Sun, Fei; Shi, Huijuan; Yuan, Wei

2016-11-01

The study aimed to examine the relationships between calcium, magnesium, and calcium/magnesium ratio in semen plasma and sperm quality. It was a cross-sectional study based on a program aiming at promoting the reproductive health in less-developed areas. A total of 515 men aged between 18 and 55 years provided semen specimens at family planning clinics in Sandu County, Guizhou Province, China. Total calcium and magnesium concentrations in semen plasma were measured with flame atomic absorption spectrometry. Sperm quality, including sperm motility and concentration, was evaluated by using a computer-assisted sperm analysis method. The medians of seminal plasma calcium, magnesium, and zinc concentrations were 9.61, 4.41, and 2.23 mmol/l, respectively. Calcium concentration and calcium/magnesium ratio were negatively associated with sperm concentrations (β = -0.47, P = 0.0123 for calcium; β = -0.25, P = 0.0393 for calcium/magnesium ratio) after adjusting for zinc and other covariates. In stratified analyses, the association between calcium and sperm concentrations only persisted among subjects with a calcium/magnesium ratio of ≤2.5 (β = -0.71, P = 0.0268). In the same stratum, magnesium was associated with increased sperm concentration (β = 0.73, P = 0.0386). Among subjects with a calcium/magnesium ratio of >2.5, neither calcium nor magnesium was associated with sperm concentration. In conclusion, total calcium and magnesium concentrations were associated with sperm concentration among subjects with a lower calcium/magnesium ratio. The calcium and magnesium ratio had a modifying effect on the associations of calcium and magnesium with sperm concentration.

Oxidative Damage to Rhesus Macaque Spermatozoa Results in Mitotic Arrest and Transcript Abundance Changes in Early Embryos1

PubMed Central

Burruel, Victoria; Klooster, Katie L.; Chitwood, James; Ross, Pablo J.; Meyers, Stuart A.

2013-01-01

ABSTRACT Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development. PMID:23904511

Impaired Sperm Maturation in Rnase9 Knockout Mice1

PubMed Central

Westmuckett, Andrew D.; Nguyen, Edward B.; Herlea-Pana, Oana M.; Alvau, Antonio; Salicioni, Ana M.; Moore, Kevin L.

2014-01-01

ABSTRACT Ribonuclease, RNase A family, 9 (RNASE9) is a ribonuclease A superfamily member that is expressed only in the epididymis. It is a small, secreted polypeptide, it lacks ribonuclease activity, and its function(s) is unknown. However, epididymis-specific expression suggests a role in sperm maturation. We generated Rnase9−/− mice to study RNASE9 function in vivo. We confirm that RNASE9 expression is restricted to the epididymis. Within the epididymis, RNASE9 is first detected in midcaput, persists through the distal caput and corpus, and wanes in the cauda. Rnase9−/− mice are born at the expected Mendelian ratio, have normal postnatal growth and development, and have no outwardly apparent phenotype. Spermatogenesis is normal, and Rnase9-null sperm are morphologically normal. Rnase9−/− males have normal fertility in unrestricted mating trials, and fertilization rates in in vitro fertilization assays are indistinguishable from wild-type mice. Visual observations coupled with analyses of sperm velocities shortly after swim out from the corpus shows that motility of Rnase9-null sperm is significantly impaired. However, no differences between wild-type and Rnase9-null sperm are detected by computer-assisted sperm analysis 10–90 min after sperm isolation from the corpus or cauda. Assessment of capacitation-dependent signaling pathways in Rnase9-null sperm showed that, while levels of tyrosine phosphorylation of sperm proteins were normal, there was decreased phosphorylation of protein kinase A substrates upon capacitation compared to wild-type mice. In conclusion, RNASE9 is dispensable for fertility, but the absence of RNASE9 during epididymal transit results in impaired sperm maturation. PMID:24719258

In Vitro System for the Assessment of the Potential Antifertility Affect of Chemicals.

DTIC Science & Technology

1992-07-01

inactivated rabbit, calf, or horse serum; but cell viability was poor. Cell viability was improved in DN alone or in Biggers, Vhittinghaa, and Vhitten...constitute an official endorsement of any commer- cial products. This report may not be cited for purposes of advertisement. Reproduction of this...heat inactivated rabbit, horse , or calf serum and incubated in an atmosphere of 5% C02, 95% air at 37 OC, and sperm motion was recorded on videotape

Cytogenetic and molecular aspects of absolute teratozoospermia: comparison between polymorphic and monomorphic forms.

PubMed

Brahem, Sonia; Elghezal, Hatem; Ghédir, Houda; Landolsi, Hanène; Amara, Abdelbacett; Ibala, Samira; Gribaa, Moez; Saad, Ali; Mehdi, Meriem

2011-12-01

To compare the results of cytogenetic and molecular analysis between absolute polymorphic and monomorphic teratozoospermia. The semen samples from patients with polymorphic teratozoospermia (n = 20), globozoospermia (n = 8), or macrocephalic sperm head syndrome (n = 12), and healthy fertile men (n = 20) were analyzed according to the World Health Organization criteria. The constitutional blood karyotype of the patients was performed on cultured lymphocytes, according to standard techniques. Microdeletion analysis of the Y chromosomes used a sequence tagged site-polymerase chain reaction technique. Triple-color fluorescent in situ hybridization for chromosomes X, Y, and 18 were used to analyze the meiotic segregation. DNA fragmentation was detected using the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay. Whatever the type of teratozoospermia, a normal karyotype and an absence of Y chromosome microdeletion were shown for all patients. A significant increase in the sperm aneuploidy rate and DNA fragmentation were shown, regardless of the type of teratozoospermia. Spermatozoa of the patients with globozoospermia carry an abnormal chromosomal constitution and DNA damage rate with the same frequency as that found in the sperm of patients with absolute polymorphic teratozoospermia. However, a greater sperm aneuploidy rate and DNA fragmentation were found in patients whose teratozoospermia was mainly characterized by increased rates of spermatozoa with macrocephalic head and multiple flagella. Our data have demonstrated that DNA fragmentation and sperm aneuploidy are critical tests in teratozoospermic men, because the results could negatively affect the intracytoplasmic sperm injection outcomes and might play an important role in the counseling of couples considering intracytoplasmic sperm injection. Copyright © 2011 Elsevier Inc. All rights reserved.

Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report.

PubMed

Chithiwala, Zahabiya H; Lee, Hoi Chang; Hill, David L; Jellerette-Nolan, Teru; Fissore, Rafael; Grow, Daniel; Dumesic, Daniel A

2015-09-01

The purpose of this study is to describe impaired oocyte fertilization from phospholipase C-zeta (PLC-ζ) deficiency in normal-appearing sperm that was successfully treated using calcium (Ca(2+)) ionophore with intracytoplasmic sperm injection (ICSI) of oocytes matured in vitro. An infertile couple undergoing in vitro fertilization (IVF) experienced failed oocyte fertilization following ICSI with normal-appearing sperm. A semen sample collected from the patient was used to assess the expression of sperm PLC- ζ protein by Western blot analysis and immunofluorescence and PLC-ζ bioactivity by an in vitro model of Ca(2+) release. A second IVF cycle was performed using Ca(2+) ionophore with ICSI to enhance Ca(2+)-induced oocyte activation of oocytes matured in vitro. Sperm PLC-ζ protein deficiency was demonstrated by Western blot analysis and immunofluorescence and confirmed by reduced PLC-ζ bioactivity using an in vitro model of Ca(2+) release. Nevertheless, with this sperm and supplementation of Ca(2+) ionophore following ICSI, fertilization of four of six oocytes matured in vitro was obtained. In addition, four embryos underwent cleavage and two of them reached the blastocyst stage. Transfer of these blastocysts into the uterus led to a single pregnancy and live birth. Deficiency of PLC-ζ in normal-appearing human sperm is associated with impaired Ca(2+)-dependent oocyte activation during ICSI. Under this condition, use of Ca(2+) ionophore following ICSI of oocytes matured in vitro improves embryo developmental competence, possibly through the activation of Ca(2+)-dependent mechanisms governing fertilization and preimplantation embryogenesis.

Assessment of butorphanol-azaperone-medetomidine combination as anesthesia for semen collection and evaluation of semen quality in white-tailed deer (Odocoileus virginianus).

PubMed

Kirschner, S M; Rodenkirch, R

2017-09-01

The aim of this current study was to evaluate the level of anesthesia produced by a combination of butorphanol-azaperone-medetomidine (BAM) for semen collection by electroejaculation on captive white-tailed bucks (Odocoileus virginianus). Ten male white-tailed deer, weighing 68.2-115.9kg, ranging in age from one to four years were randomly selected from housing pens and anesthetized with the BAM drug combination at a dose volume of 2.0mL each. Semen was collected from each animal using a standard cervid electroejaculation protocol while under BAM anesthesia. Physiological data was recorded following induction of anesthesia and during semen collection. Collected ejaculates were prepared for analysis using a standard extender protocol for cryopreservation. Eleven sperm viability parameters were quantified for each sample using a Computerized Assisted Sperm Analysis system, including total seminal volume; sperm concentration and total sperm number. kinematic parameters of motile spermatozoa were also assessed. Results demonstrated that BAM provided an effective plane of anesthesia for successful collection of viable sperm. Measured physiological variables of heart rate, respiration and body temperature all remained within safe, normal limits. Data recorded on semen characteristics from all collected ejaculates correlated well with key traits determined to be important for successful fertilization through measurement of total semen volume; sperm concentration; total sperm number; and kinematic parameters of motile spermatozoa. There were no serious adverse events. This field study indicates that BAM anesthesia is suitable for semen collection in white-tailed deer. Copyright © 2017 Elsevier B.V. All rights reserved.

Statistical analysis of sperm sorting

NASA Astrophysics Data System (ADS)

Koh, James; Marcos, Marcos

2017-11-01

The success rate of assisted reproduction depends on the proportion of morphologically normal sperm. It is possible to use an external field for manipulation and sorting. Depending on their morphology, the extent of response varies. Due to the wide distribution in sperm morphology even among individuals, the resulting distribution of kinematic behaviour, and consequently the feasibility of sorting, should be analysed statistically. In this theoretical work, Resistive Force Theory and Slender Body Theory will be applied and compared. Full name is Marcos.

Effect of Broadband Nature of Marine Mammal Echolocation Clicks on Click-Based Population Density Estimates

DTIC Science & Technology

2015-09-30

sonar sound and therefore of high relevance to the US Navy, and sperm whale (Physeter macrocephalus) clicks, whose high source level assures long...beaked whale, and Zimmer (2011) for the sperm whale. 5 For background noise we used measurements obtained with the Delphinus towed array at the US...Atlantic Undersea Test and Evaluation Center (AUTEC), where both Blainville’s beaked whales and sperm whales are commonly present. Analysis of the

ANALYSIS OR THE POTENTIAL SPERM BIOMARKER, SP22, IN HUMAN SEMEN

EPA Science Inventory

ANALYSIS OF THE POTENTIAL SPERM BIOMARKER SP22 IN HUMAN SEMEN
Rebecca A. Morris Ph.D.1, Gary R. Klinefelter Ph.D.1, Naomi L. Roberts 1, Juan D. Suarez 1,
Lillian F. Strader 1, Susan C. Jeffay 1 and Sally D. Perreault Ph.D.1

1 U.S. EPA / ORD / National Health a...

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 effect of human sperm chromatin maturity on ICSI outcomes.

PubMed

Gill, Kamil; Rosiak, Aleksandra; Gaczarzewicz, Dariusz; Jakubik, Joanna; Kurzawa, Rafal; Kazienko, Anna; Rymaszewska, Anna; Laszczynska, Maria; Grochans, Elzbieta; Piasecka, Malgorzata

2018-03-29

Because sperm chromatin may play a key role in reproductive success, we verify the associations between sperm chromatin abnormalities, embryo development and the ability to achieve pregnancy. The evaluation of sperm chromatin maturity using aniline blue (AB), chromomycin A3 (CMA3) and toluidine blue (TB) staining were carried out in group of males from infertile couples that underwent ICSI. Low levels of sperm chromatin abnormalities (< 16%) were found in most subjects (> 50%). A higher percentage of TB-positive sperm cells were discovered in the men from couples who achieved ≤ 50% fertilized oocytes compared to men who achieved > 50%. No significant differences were discovered by the applied tests between the men from couples who achieved ≤ 50% and those who achieved > 50% high-quality embryos on the 3rd or 5th day after fertilization, nor between the men from couples who achieved pregnancy and those who failed. The sperm chromatin maturity did not correlate with the ICSI results. However, the ROC analysis revealed a significant predictive value of TB-positive spermatozoa only for fertilization. Therefore, the TB assay can be considered as a useful test for the prediction of fertilization. Our findings suggest that the level of sperm chromatin abnormalities of the examined men was not clinically significant. No found associations between sperm chromatin maturity and embryo development and the ability to achieve pregnancy. We could not exclude the effects of the repairing processes in the fertilized oocyte. The use of complementary tests that verify the status of the sperm chromatin seems justified.

Low levels of PRSS37 protein in sperm are associated with many cases of unexplained male infertility.

PubMed

Liu, Jianbing; Shen, Chunling; Fan, Weimin; Chen, Yan; Zhang, Aijun; Feng, Yun; Li, Zheng; Kuang, Ying; Wang, Zhugang

2016-11-01

PRSS37, a putative trypsin-like serine protease, is highly conserved during mammalian evolution as revealed by multiple sequence alignment. Mice deficient for Prss37 gene exhibit male infertility, but their mating behavior, spermatogenesis, sperm morphology, and motility remain unaffected, similar to a situation called unexplained male infertility (UMI) in men (human being). Here, we demonstrated that PRSS37 is restrictively expressed in human testis, where it is mainly located in the elongating and elongated spermatids during spermiogenesis as shown by immunohistochemical analysis of normal human testicular sections. In mature sperm, PRSS37 appears in the acrosome region and diminishes during acrosome reaction. Further examination reveals that PRSS37 contents in sperm from patients with UMI are dramatically lower than those in sperm from men with proven fertility or from sperm donors. Sperm with low PRSS37 contents exhibit abnormal activation of the proacrosin/acrosin system and premature proteolysis of ADAM2, which may impair the functional competence of human sperm in vivo However, the in vitro fertilization outcomes of sperm with low PRSS37 contents are not affected. Together, these data implicate an important role of PRSS37 for male fertility. PRSS37 can be used as a potential molecular biomarker for evaluating sperm fertilization capability in vivo but not in vitro. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Hyaluronic acid improves frozen-thawed sperm quality and fertility potential in rooster.

PubMed

Lotfi, Saied; Mehri, Morteza; Sharafi, Mohsen; Masoudi, Reza

2017-09-01

Beneficial effects of Hyaluronic acid (HA) has not been yet assessed for cryopreservation of rooster sperm. This study was conducted to evaluate the effects of different concentrations of HA (0, 1, 2, 4 and 8mM) in Beltsville extender on the cryopreservation of rooster sperm. Semen samples were collected from six Ross broiler breeders (24-week) using abdominal massage, then divided into five equal aliquots and cryopreserved in Beltsville extender that contained different concentrations of HA. Motion characteristics, morphology, membrane functionality, viability, acrosome integrity, lipid peroxidation and fertility potential of sperm were assessed after thawing. HA at concentration of 2mM (HA2) resulted in the highest (P<0.05) total motility (55.3±1.1%) and progressive motility (25.2±0.8%) compared to the other groups. HA8 produced the lowest significant (P<0.05) percentage of total (38.6±1.1%) and progressive (14.7±0.8%) motility. High significant percentage of membrane functionality were observed in HA1 and HA2 (43.2±1.0 and 46.1±1.0%, respectively) compared to HA4 (40.1±1.0%) and HA8 (32.5±1.0%). Moreover, HA1 and HA2 produced the higher percentage of acrosome integrity (54.8±1.2 and 57.5±1.2, respectively) compared to other groups. HA1 and HA2 reduced (P<0.05) malondialdehyde formation (3.66±0.08 and 3.75±0.08 nmol/ml) compared to other groups. Fertility rate and hatching rate obtained from artificial insemination were significantly higher in HA1 (63.7 and 54.7%) and HA2 (67.5 and 57.7%) compared to control group (40 and 37%). Our results showed that supplementation of Beltsville extender with 1 and 2mM HA significantly improved the quality of rooster sperm after freeze thawing. Copyright © 2017 Elsevier B.V. All rights reserved.

Trace elements in seminal plasma of men from infertile couples.

PubMed

Guzikowski, Wojciech; Szynkowska, Małgorzata I; Motak-Pochrzęst, Hanna; Pawlaczyk, Aleksandra; Sypniewski, Stanisław

2015-06-19

An analysis of lead, zinc, cadmium and other trace elements in semen of men from infertile couples was performed to determine the association between abnormal semen parameters and enviromental or occupational exposure to some trace metals. Presence of manganese, cobalt, nickel, copper, zinc, molybdenum, cadmium, tin and lead was measured in seminal plasma of 34 men from infertile couples using spectrometry with time-of-flight analysis. Correlations among sperm parameters and trace metals were determined using cluster analysis and Pearson's correlation coefficient. Abnormally high concentrations of lead, cadmium, zinc and cobalt were found in 23 seminal plasma of men from infertile couples. The most consistent evidence was determined for an association between high cadmium concentration in seminal plasma and sperm count, motility and morphology below reference limits (p < 0.01). A correlation of significantly increased tin level and reduced sperm count in semen of men with limited fertility potential was observed (p = 0.04). In our study we observed a correlation of tin level with sperm count in semen of men with limited fertility potential.

Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

PubMed

Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

2014-01-01

Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to the procedures of fish sperm cryopreservation.

Sperm Retrieval in Patients with Klinefelter Syndrome: A Skewed Regression Model Analysis.

PubMed

Chehrazi, Mohammad; Rahimiforoushani, Abbas; Sabbaghian, Marjan; Nourijelyani, Keramat; Sadighi Gilani, Mohammad Ali; Hoseini, Mostafa; Vesali, Samira; Yaseri, Mehdi; Alizadeh, Ahad; Mohammad, Kazem; Samani, Reza Omani

2017-01-01

The most common chromosomal abnormality due to non-obstructive azoospermia (NOA) is Klinefelter syndrome (KS) which occurs in 1-1.72 out of 500-1000 male infants. The probability of retrieving sperm as the outcome could be asymmetrically different between patients with and without KS, therefore logistic regression analysis is not a well-qualified test for this type of data. This study has been designed to evaluate skewed regression model analysis for data collected from microsurgical testicular sperm extraction (micro-TESE) among azoospermic patients with and without non-mosaic KS syndrome. This cohort study compared the micro-TESE outcome between 134 men with classic KS and 537 men with NOA and normal karyotype who were referred to Royan Institute between 2009 and 2011. In addition to our main outcome, which was sperm retrieval, we also used logistic and skewed regression analyses to compare the following demographic and hormonal factors: age, level of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone between the two groups. A comparison of the micro-TESE between the KS and control groups showed a success rate of 28.4% (38/134) for the KS group and 22.2% (119/537) for the control group. In the KS group, a significantly difference (P<0.001) existed between testosterone levels for the successful sperm retrieval group (3.4 ± 0.48 mg/mL) compared to the unsuccessful sperm retrieval group (2.33 ± 0.23 mg/mL). The index for quasi Akaike information criterion (QAIC) had a goodness of fit of 74 for the skewed model which was lower than logistic regression (QAIC=85). According to the results, skewed regression is more efficient in estimating sperm retrieval success when the data from patients with KS are analyzed. This finding should be investigated by conducting additional studies with different data structures.

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.

Effect of hepatitis B virus infection on sperm quality and oxidative stress state of the semen of infertile males.

PubMed

Qian, Li; Li, Qiong; Li, Haibo

2016-09-01

The effects of hepatitis B virus (HBV) infection on sperm quality and oxidative stress state of the semen of infertile males remain undetermined. Normal males and 60 semen samples from infertile males (with or without HBV infection) were subjected to semen analysis. Semen volume, semen pH, sperm density, percentage of forward, movement of sperm, sperm activation rate, sperm survival rate, rate of normal sperm morphology of infertile males with HBV infection were significantly lower than those of infertile males without genital infection and of normal males (P<.05), while interleukin (IL)-17, IL-18, and malondialdehyde (MDA) levels in subjects with HBV infection were significantly higher than those of infertile males without genital infection and of normal males (P<.05). In patients with HBV infection, MDA level was found to be negatively correlated with semen quality, but positively correlated with semen IL-17 and IL-18 concentrations. HBV infection increased MDA level, induced abnormal expression of IL-17 and IL-18, and negatively affected male reproductive capacity, resulting in male infertility. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Extraction of DNA from forensic-type sexual assault specimens using simple, rapid sonication procedures.

PubMed

Crouse, C A; Ban, J D; D'Alessio, J K

1993-10-01

Sonication procedures for the extraction of DNA from forensic-type semen specimens have been developed, which, when compared to currently utilized sperm DNA extraction techniques, are simple, rapid and result in comparable DNA yields. Sperm DNA extraction by sonication was performed on whole semen, seminal stains, buccal swabs and post-coital specimens. Ultrasound disruption of sperm cells and their ultimate release of cellular DNA has been conducted in the presence of sperm wash buffers followed by organic extraction or Chelex 100 with little or no compromise to DNA quality, quantity or amplifiability. Two advantages of sonication over currently used forensic techniques to extract sperm DNA include 1) sperm DNA extraction that occurs within five minutes of sonication compared with an hour or greater for water bath incubations in classic enzyme digestion DNA extractions and 2) one less preparatory step with the Chelex/sonication protocol and three less steps with the sonication/organic protocol compared with other procedures thus eliminating potential sample-to-sample cross-contamination. Sperm DNA extracted by optimum sonication procedures was used for forensic HLA DQ alpha typing and restriction fragment length polymorphisms analysis without any adverse effects on typing results.

Comparison of different extenders on the recovery and longevity of epididymal sperm from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831).

PubMed

da Silva, Andréia Maria; Sousa, Patrícia Cunha; Campos, Lívia Batista; Bezerra, José Artur Brilhante; de Araújo Lago, Arthur Emannuel; de Oliveira, Moacir Franco; Silva, Alexandre Rodrigues

2017-04-01

The aim of this study was to evaluate the performance of cavy (Galea spixii) epididymal sperm following addition to TES or TRIS extenders and using a thermal resistance test (TRT), as well as fluorescence analysis as a complementary method to predict the viability of these gametes. Nine testicle-epididymis complexes were used for sperm collection using a flotation method. Epididymis tails were sliced and one was immersed in 3 ml of TRIS buffer, and the other in 3 ml of TES, for 5 min. After sperm recovery, the samples were subjected to a TRT which involved incubation in a water bath at 37°C for 3 h. During incubation, sample parameters were assessed at 0, 15, 30, 60, 90, 120, 150 or 180 min intervals. Results indicated that the TRIS diluent was more efficient than TES (P < 0.05) for the maintenance of sperm parameters in Spix's yellow-toothed cavies over the whole TRT, maintaining sperm longevity for an extended time. In conclusion, we indicate the use of TRIS diluent for recovery and maintenance of longevity of epididymal sperm from cavies (G. spixii).

Spermaurin, an La1-like peptide from the venom of the scorpion Scorpio maurus palmatus, improves sperm motility and fertilization in different mammalian species.

PubMed

Martinez, Guillaume; Hograindleur, Jean-Pascal; Voisin, Sébastien; Abi Nahed, Roland; Abd El Aziz, Tarek M; Escoffier, Jessica; Bessonnat, Julien; Fovet, Claire-Maëlle; De Waard, Michel; Hennebicq, Sylviane; Aucagne, Vincent; Ray, Pierre F; Schmitt, Eric; Bulet, Philippe; Arnoult, Christophe

2017-02-10

Is it possible to identify original compounds that are able to enhance sperm motility from the venom of the scorpion Scorpio maurus palmatus? We identified a potent disulfide-rich peptide (DRP) of 73 amino acids that significantly improved the motility of fresh and frozen-thawed sperm in different mammalian species, including human, and improved fertilization outcome in mouse IVF experiments. Any disturbance of sperm motility has a strong impact on fertilization and can lead to subfertility or infertility. Significant efforts have, therefore,  been made to identify pharmacological drugs that might improve sperm motility. Such compounds are particularly useful in azoospermia to improve testicular sperm extraction and in the domain of cryopreservation because the motility of frozen-thawed sperm is reduced. This was a basic science/medical research study aimed at identifying original compounds from a library of venoms able to enhance mammalian sperm motility, including human. We first identified in the venom of a scorpion S. m. palmatus a fraction able to potently activate sperm motility. We next purified and characterized the compound by liquid chromatography, mass spectrometry and peptide synthesis. Finally, the potency and toxicity of both purified and synthetic versions of the identified compound on sperm motility were assessed using different in vitro tests in different mammalian species. For human sperm, biological samples were collected from normozoospermic donors and subfertile patients attending a reproduction department for diagnostic semen analysis. Testicular sperm was collected from cynomolgus monkeys (Macaca fascicularis) euthanized for the needs of specific authorized research projects. The peptide was also tested on bovine and mouse epidydimal sperm. We measured different sperm motility parameters with a computer-assisted sperm analysis system in the presence or absence of the peptide. Size exclusion chromatography enabled us to isolate a fraction of the venom of S. m. palmatus able to increase sperm motility. By liquid chromatography and mass spectrometry, a peptide comprising 73 amino acids with 4 disulfide bridges was identified as responsible for the biological activity and called 'spermaurin'. The identity of spermaurin was confirmed by chemical synthesis. We showed that the peptide increased the motility of fresh and frozen-thawed human sperm. We observed that the potency of the peptide was higher on fresh ejaculated spermatozoa with a low motility, achieving a 100% increase of curvilinear velocity in poorly performing sperm. We also demonstrated that peptide is effective on bovine and mouse fresh epididymal, bovine frozen-thawed ejaculated and fresh non-human primate testicular sperm. Finally, in mouse IVF, the production of 2-cell embryos was increased by 24% when sperm were treated with the peptide. This work is an in vitro evaluation of the ability of spermaurin to improve sperm motility parameters. Another limitation of this study is the small number of human sperm samples tested with the natural (n = 36) and synthetic (n = 12) peptides. Moreover, the effect of the peptide on IVF outcome was only tested in mouse and further tests with human and bovine gametes are required to confirm and extend this result in other mammalian species. This work confirms our initial study showing that venoms represent an interesting source of molecules that are able to modify sperm physiology. Moreover, this work presents the first demonstrated biological action of a venom peptide from the scorpion S. m. palmatus with sequence similarities to La1 peptide from Liocheles australasiae (Wood scorpion), a widespread family of DRPs. Not applicable. This work is part of the project 'LAB COM-14 LAB7 0004 01-LIPAV', funded by the program LabCom 2014 from the French Research Agency (ANR). Dr Arnoult reports grants from IMV Technologies during the conduct of the study. In addition, Drs Arnoult, Martinez, Ray and Schmitt have a patent EP16305642.7 pending containing some of the information presented in this manuscript. © 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

Freeze-dried dog sperm: Dynamics of DNA integrity.

PubMed

Olaciregui, M; Luño, V; Gonzalez, N; De Blas, I; Gil, L

2015-10-01

Freeze-drying (FD) has been proposed as an alternative method to preserve spermatozoa. During the FD procedure, sperm DNA might become damaged by both freezing and drying stresses caused by the endonucleases, the oxidative stress and the storage conditions. We examined the DNA integrity of dog sperm freeze-dried with two kinds of chelating agents in FD buffers and storage at two different temperatures. Ejaculated sperm from four dogs were suspended in basic medium (10 mM Tris-HCl buffer+50 mM NaCl) supplemented with 50 mM EGTA or with 50 mM EDTA and then freeze-dried. Sperm samples were stored at 4°C as room temperature, and the analysis of DNA damage was performed after a month and 5 months of storage using a Sperm Chromatin Dispersion test. We found four different sperm populations according to the size of the halos around the sperm head: (1) absent halo, (2) <6 μm, (3) 6-10 μm, (4) >10 μm. All of them coexisted in each freeze-dried dog semen samples and differed significantly among different treatments. The highest percentage of spermatozoa with halo >10 μm was obtained when the semen samples were freeze-dried in EDTA medium and stored at room temperature for five months. Results suggested that both, the kind of chelating agent as well as storage temperature and period, influenced DNA integrity of freeze-dried dog sperm. Copyright © 2015 Elsevier Inc. All rights reserved.

In vivo test of bitter (andrographis paniculata nees.) extract to ejaculated sperm quality

NASA Astrophysics Data System (ADS)

Sumarmin, R.; Huda, NK; Yuniarti, E.; Violita

2018-03-01

Sambiloto or Bitter (Andrographis paniculata Nees.), are often used to treat various diseases, such as influenza, cancer, anti-inflammation, anti-HIV, anti-mitotic and anti-fertility. This study aimed to determine the effects of the bitter (Andrographis paniculata Nees.) extract to ejaculated sperm mice quality (Mus musculus L. Swiss Webster). This research was conducted using Completely Randomized Design with 4 treatments, which are 0.0 g/b.w., (P0), 0.2 g/b.w., (P1), 0,4 g/b.w., (P3), or 0.6 g/b.w., (P4) bitter extract orally for 36 days. After treatment, the mice decapitated, dissected and collected the sperm from vas deferens. Then, the number of sperm counted by used the improved Neubauer and then stained by Eosin to count the abnormal sperm. Data analyzed by ANOVA (Analysis of Variance) then DNMRT. The results showed that the average numbers of sperm are 28.80 x 105 (P0), 19.50 x 105 (P1), 12.50 x105 (P2) and 9.50 x 105 (P3). The average abnormal sperm numbers are 18.33 x 105 (P0), 22.50 x 105 (P1), 31.50 x105 (P2) and 39.33 x 105 (P3). It showed that the effective treatment to decrease sperm number was 0.2 g/b.w., of bitter extract. It can conclude that the bitter (Andrographis paniculata Nees.) extract decreases the quality of the ejaculated sperm of mice (Mus musculus L.)

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.

The combination matters - distinct impact of lifestyle factors on sperm quality: a study on semen analysis of 1683 patients according to MSOME criteria

PubMed Central

2012-01-01

Background Poor sperm quality can negatively affect embryonic development and IVF outcome. This study is aimed at investigating the influence of various lifestyle factors on semen quality according to MSOME (motile sperm organelle morphology examination) criteria. Methods 1683 male patients undergoing assisted reproductive technologies (ART) in our clinic were surveyed about their age, BMI (body mass index), ejaculation frequency, nutrition, sports, sleeping habits and social behavior. Semen samples were collected and evaluation of semen parameters according to MSOME and WHO criteria was performed. Results were grouped and statistically analyzed. Results Although single parameters had minor effects on sperm parameter, the combination of age, BMI, coffee intake, ejaculatory frequency and duration of sexual abstinence were identified as factors having a negative effect on sperm motility. Additionally, we could demonstrate that MSOME quality was reduced. The negative impact of age, BMI and coffee intake on sperm quality could be compensated if patients had a high ejaculation frequency and shorter periods of sexual abstinence. Conclusions Combinations of adverse lifestyle factors could have a detrimental impact on sperm, not only in terms of motility and sperm count but also in terms of sperm head vacuolization. This negative impact was shown to be compensated by higher ejaculation frequency and a shorter period of sexual abstinence. The compensation is most likely due to a shorter storage time in the male gonads, thus reducing the duration of sperms’ exposure to reactive oxygen species (ROS). PMID:23265183

Paper-Based Quantification of Male Fertility Potential.

PubMed

Nosrati, Reza; Gong, Max M; San Gabriel, Maria C; Pedraza, Claudio E; Zini, Armand; Sinton, David

2016-03-01

More than 70 million couples worldwide are affected by infertility, with male-factor infertility accounting for about half of the cases. Semen analysis is critical for determining male fertility potential, but conventional testing is costly and complex. Here, we demonstrate a paper-based microfluidic approach to quantify male fertility potential, simultaneously measuring 3 critical semen parameters in 10 min: live and motile sperm concentrations and sperm motility. The device measures the colorimetric change of yellow tetrazolium dye to purple formazan by the diaphorase flavoprotein enzyme present in metabolically active human sperm to quantify live and motile sperm concentration. Sperm motility was determined as the ratio of motile to live sperm. We assessed the performance of the device by use of clinical semen samples, in parallel with standard clinical approaches. Detection limits of 8.46 and 15.18 million/mL were achieved for live and motile sperm concentrations, respectively. The live and motile sperm concentrations and motility values from our device correlated with those of the standard clinical approaches (R(2) ≥ 0.84). In all cases, our device provided 100% agreement in terms of clinical outcome. The device was also robust and could tolerate conditions of high absolute humidity (22.8 g/m(3)) up to 16 weeks when packaged with desiccant. Our device outperforms existing commercial paper-based assays by quantitatively measuring live and motile sperm concentrations and motility, in only 10 min. This approach is applicable to current clinical practices as well as self-diagnostic applications. © 2015 American Association for Clinical Chemistry.

Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3.

PubMed

Li, Xue-Bo; Wang, Qing-Shan; Feng, Yu; Ning, Shu-Hua; Miao, Yuan-Ying; Wang, Ye-Quan; Li, Hong-Wei

2014-11-01

Forensic DNA analysis of sexual assault evidence requires unambiguous differentiation of DNA profiles in mixed samples. To investigate the feasibility of magnetic bead-based separation of sperm from cell mixtures using a monoclonal antibody against MOSPD3 (motile sperm domain-containing protein 3), 30 cell samples were prepared by mixing 10(4) female buccal epithelial cells with sperm cells of varying densities (10(3), 10(4), or 10(5) cells/mL). Western blot and immunofluorescence assays showed that MOSPD3 was detectable on the membrane of sperm cells, but not in buccal epithelial cells. After biotinylated MOSPD3 antibody was incubated successively with the prepared cell mixtures and avidin-coated magnetic beads, microscopic observation revealed that each sperm cell was bound by two or more magnetic beads, in the head, neck, mid-piece, or flagellum. A full single-source short tandem repeat profile could be obtained in 80% of mixed samples containing 10(3) sperm cells/mL and in all samples containing ≥10(4) sperm cells/mL. For dried vaginal swab specimens, the rate of successful detection was 100% in both flocked and cotton swabs preserved for 1 day, 87.5% in flocked swabs and 40% in cotton swabs preserved for 3 days, and 40% in flocked swabs and 16.67% in cotton swabs preserved for 10 days. Our findings suggest that immunomagnetic bead-based separation is potentially a promising alternative to conventional methods for isolating sperm cells from mixed forensic samples.

Pulmonary exposure to carbonaceous nanomaterials and sperm quality.

PubMed

Skovmand, Astrid; Jacobsen Lauvås, Anna; Christensen, Preben; Vogel, Ulla; Sørig Hougaard, Karin; Goericke-Pesch, Sandra

2018-01-31

Semen quality parameters are potentially affected by nanomaterials in several ways: Inhaled nanosized particles are potent inducers of pulmonary inflammation, leading to the release of inflammatory mediators. Small amounts of particles may translocate from the lungs into the lung capillaries, enter the systemic circulation and ultimately reach the testes. Both the inflammatory response and the particles may induce oxidative stress which can directly affect spermatogenesis. Furthermore, spermatogenesis may be indirectly affected by changes in the hormonal milieu as systemic inflammation is a potential modulator of endocrine function. The aim of this study was to investigate the effects of pulmonary exposure to carbonaceous nanomaterials on sperm quality parameters in an experimental mouse model. Effects on sperm quality after pulmonary inflammation induced by carbonaceous nanomaterials were investigated by intratracheally instilling sexually mature male NMRI mice with four different carbonaceous nanomaterials dispersed in nanopure water: graphene oxide (18 μg/mouse/i.t.), Flammruss 101, Printex 90 and SRM1650b (0.1 mg/mouse/i.t. each) weekly for seven consecutive weeks. Pulmonary inflammation was determined by differential cell count in bronchoalveolar lavage fluid. Epididymal sperm concentration and motility were measured by computer-assisted sperm analysis. Epididymal sperm viability and morphological abnormalities were assessed manually using Hoechst 33,342/PI flourescent and Spermac staining, respectively. Epididymal sperm were assessed with regard to sperm DNA integrity (damage). Daily sperm production was measured in the testis, and testosterone levels were measured in blood plasma by ELISA. Neutrophil numbers in the bronchoalveolar fluid showed sustained inflammatory response in the nanoparticle-exposed groups one week after the last instillation. No significant changes in epididymal sperm parameters, daily sperm production or plasma testosterone levels were found. Despite the sustained pulmonary inflammatory response, an eight week exposure to graphene oxide, Flammruss 101, Printex 90 and the diesel particle SRM1650b in the present study did not appear to affect semen parameters, daily sperm production or testosterone concentration in male NMRI mice.

Human sperm degradation of zona pellucida proteins contributes to fertilization.

PubMed

Saldívar-Hernández, Analilia; González-González, María E; Sánchez-Tusié, Ana; Maldonado-Rosas, Israel; López, Pablo; Treviño, Claudia L; Larrea, Fernando; Chirinos, Mayel

2015-09-02

The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process. Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay. The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP. This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

Physical activity and television watching in relation to semen quality in young men.

PubMed

Gaskins, Audrey Jane; Mendiola, Jaime; Afeiche, Myriam; Jørgensen, Niels; Swan, Shanna H; Chavarro, Jorge E

2015-02-01

Semen quality appears to have declined over the past decades but reasons for this decline are unresolved. The concurrent increase in sedentary behaviour may be a contributing factor. The objective of this study was to evaluate the relationship of physical activity and television (TV) watching with sperm parameters in a population of young, healthy men. Men aged 18-22 years (n=189) from the Rochester Young Men's Study (2009-2010) participated in this analysis. Physical activity (h/week of moderate and vigorous exercise) and TV watching (h/week of TV, video or DVD watching) over the past 3 months were assessed via questionnaire. Semen quality was assessed by sperm concentration, motility, morphology and total sperm count. Sperm concentration and total sperm count were directly related to physical activity after multivariable adjustment (p-trend=0.01 and 0.04); men in the highest quartile of moderate-to-vigorous activity (≥15 h/week) had 73% (95% CI 15% to 160%) higher sperm concentration than men in the lowest quartile (<5 h/week). TV watching was inversely associated with sperm concentration and total sperm count in multivariable analyses (p-trend=0.05 and 0.06); men in the highest quartile of TV watching (>20 h/week) had 44% (95% CI 15 to 63%) lower sperm concentration than men in the lowest quartile (0 h/week). These measures of physical and leisure time activities were not significantly associated with sperm motility or morphology. In this population of healthy men, higher moderate-to-vigorous activity and less TV watching were significantly associated with higher total sperm count and sperm concentration. 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.

Epididymal sperm from Spix's yellow-toothed cavies sperm successfully cryopreserved in Tris extender with 6% glycerol and 20% egg yolk.

PubMed

Silva, Andréia M; Praxedes, Erica C G; Campos, Lívia B; Bezerra, Luana G P; Moreira, Samara S J; Maia, Keilla M; Souza, Ana L P; Silva, Alexandre R

2018-04-01

As a non-threatened hystricognath rodent species, Spix's yellow-toothed cavies can be used as a model for the development of assisted reproductive techniques for the conservation of closely related species. The objective was to establish a functional protocol for cryopreservation of epididymal sperm from these cavies. Twelve sexually mature males, ∼2 y old and weighing ∼300 g, were euthanized. Sperm were recovered by retrograde flushing of the vas deferens and cauda epididymis with Tris extender. Thereafter, sperm were extended in Tris plus 20% egg yolk, with 3%, 6% or 9% glycerol or dimethyl sulfoxide (DMSO), placed in 0.25 mL straws and cryopreserved in liquid nitrogen. Sperm concentration, motility (using computer-assisted sperm analysis; CASA), plasma membrane integrity, osmotic response, morphology and sperm binding-ability were determined in fresh and frozen-thawed sperm. For most sperm endpoints, glycerol was a more desirable cryoprotectant than DMSO. Data (mean ± SEM) were similar with use of 3%, 6%, and 9% glycerol (P > 0.05) in osmotic response (40.66 ± 6.3%, 42.5 ± 7.1%, and 39.5 ± 5.0% respectably), and membrane integrity (55.17 ± 5.5%, 68.4 ± 4.1%, and 59.1 ± 4.9% respectably). Among concentrations assessed, the use of 6% glycerol resulted in the greatest (P < 0.05) post-thaw values for total motility (60.9 ± 4.4%), rapid subpopulation motility (27.7 ± 3.1%) and sperm-binding capability (227.0 ± 20.2). In conclusion, epididymal sperm from the Spix's yellow-toothed cavies (G. spixii) are optimally cryopreserved in Tris extender with 6% glycerol and 20% egg yolk. Copyright © 2018 Elsevier B.V. All rights reserved.

Intermediate scattering function of an anisotropic active Brownian particle

PubMed Central

Kurzthaler, Christina; Leitmann, Sebastian; Franosch, Thomas

2016-01-01

Various challenges are faced when animalcules such as bacteria, protozoa, algae, or sperms move autonomously in aqueous media at low Reynolds number. These active agents are subject to strong stochastic fluctuations, that compete with the directed motion. So far most studies consider the lowest order moments of the displacements only, while more general spatio-temporal information on the stochastic motion is provided in scattering experiments. Here we derive analytically exact expressions for the directly measurable intermediate scattering function for a mesoscopic model of a single, anisotropic active Brownian particle in three dimensions. The mean-square displacement and the non-Gaussian parameter of the stochastic process are obtained as derivatives of the intermediate scattering function. These display different temporal regimes dominated by effective diffusion and directed motion due to the interplay of translational and rotational diffusion which is rationalized within the theory. The most prominent feature of the intermediate scattering function is an oscillatory behavior at intermediate wavenumbers reflecting the persistent swimming motion, whereas at small length scales bare translational and at large length scales an enhanced effective diffusion emerges. We anticipate that our characterization of the motion of active agents will serve as a reference for more realistic models and experimental observations. PMID:27830719

Intermediate scattering function of an anisotropic active Brownian particle.

PubMed

Kurzthaler, Christina; Leitmann, Sebastian; Franosch, Thomas

2016-10-10

Various challenges are faced when animalcules such as bacteria, protozoa, algae, or sperms move autonomously in aqueous media at low Reynolds number. These active agents are subject to strong stochastic fluctuations, that compete with the directed motion. So far most studies consider the lowest order moments of the displacements only, while more general spatio-temporal information on the stochastic motion is provided in scattering experiments. Here we derive analytically exact expressions for the directly measurable intermediate scattering function for a mesoscopic model of a single, anisotropic active Brownian particle in three dimensions. The mean-square displacement and the non-Gaussian parameter of the stochastic process are obtained as derivatives of the intermediate scattering function. These display different temporal regimes dominated by effective diffusion and directed motion due to the interplay of translational and rotational diffusion which is rationalized within the theory. The most prominent feature of the intermediate scattering function is an oscillatory behavior at intermediate wavenumbers reflecting the persistent swimming motion, whereas at small length scales bare translational and at large length scales an enhanced effective diffusion emerges. We anticipate that our characterization of the motion of active agents will serve as a reference for more realistic models and experimental observations.

Intermediate scattering function of an anisotropic active Brownian particle

NASA Astrophysics Data System (ADS)

Kurzthaler, Christina; Leitmann, Sebastian; Franosch, Thomas

2016-10-01

Various challenges are faced when animalcules such as bacteria, protozoa, algae, or sperms move autonomously in aqueous media at low Reynolds number. These active agents are subject to strong stochastic fluctuations, that compete with the directed motion. So far most studies consider the lowest order moments of the displacements only, while more general spatio-temporal information on the stochastic motion is provided in scattering experiments. Here we derive analytically exact expressions for the directly measurable intermediate scattering function for a mesoscopic model of a single, anisotropic active Brownian particle in three dimensions. The mean-square displacement and the non-Gaussian parameter of the stochastic process are obtained as derivatives of the intermediate scattering function. These display different temporal regimes dominated by effective diffusion and directed motion due to the interplay of translational and rotational diffusion which is rationalized within the theory. The most prominent feature of the intermediate scattering function is an oscillatory behavior at intermediate wavenumbers reflecting the persistent swimming motion, whereas at small length scales bare translational and at large length scales an enhanced effective diffusion emerges. We anticipate that our characterization of the motion of active agents will serve as a reference for more realistic models and experimental observations.

[ATP content in cryopreserved sperm of Siberian white cranes Grus leucogeranus (Aves: Gruiformes)].

PubMed

Maksudov, G Iu; Erokhin, A S; Nesterenko, O N; Panchenko, V G

2002-01-01

ATP contents were studied in the native and cryoconserved sperm of Siberian white cranes Grus leucogeranus using bioluminescence analysis. The ATP content in freshly obtained spermatozoa was 12.7 nmol/10(8) cells. No ATP was found in the seminal plasma. In the process of freezing-thawing, the ATP concentration in the spermatozoa decreased by 30%. The differences in the dynamics of ATP content during cryoconservation of sperm of white cranes and other birds and mammals are discussed.

Effect of Broadband Nature of Marine Mammal Echolocation Clicks on Click-Based Population Density Estimates

DTIC Science & Technology

2015-09-30

Blainville’s beaked whales (Mesoplodon densirostris), known to be sensitive to sonar sound and therefore of high relevance to the US Navy, and sperm whale...this study were obtained from von Benda-Beckmann et al. (2010) for the beaked whale, and Zimmer (2011) for the sperm whale. 4 For background...Blainville’s beaked whales and sperm whales are commonly present. Analysis of the noise levels on the system indicated that the background noise was system

Are men talking their reproductive health away?

PubMed

Agarwal, Ashok; Durairajanayagam, Damayanthi

2015-01-01

The advent of mobile phones has revolutionized communication trends across the globe. As the popularity of mobile phone usage continues to escalate, there is now growing concern about the effects of radiofrequency electromagnetic waves (RF-EMW) exposure on biological tissues, such as the brain and testes. Researchers have sought to link the much debated decline in human sperm quality in the last decade, with increased exposure to RF-EMW, particularly through mobile phone usage. In a recent systematic review and meta-analysis on the effect of mobile phone RF-EMW radiation on sperm quality, Adams et al. [1] demonstrated an association between mobile phone exposure and reduced sperm motility and viability, with inconsistent effects on sperm concentration. [1] Results from 10 pooled experimental (in vitro) and observational (in vivo)human studies (n = 1492) led these researchers to suggest that exposure to RF-EMW radiation from carrying a mobile phone in the trouser pocket negatively impacts sperm quality.

Ocean acidification hampers sperm-egg collisions, gamete fusion, and generation of Ca2+ oscillations of a broadcast spawning bivalve, Tegillarca granosa.

PubMed

Shi, Wei; Han, Yu; Guo, Cheng; Zhao, Xinguo; Liu, Saixi; Su, Wenhao; Wang, Yichen; Zha, Shanjie; Chai, Xueliang; Liu, Guangxu

2017-09-01

Although the effect of ocean acidification on fertilization success of marine organisms is increasingly well documented, the underlying mechanisms are not completely understood. The fertilization success of broadcast spawning invertebrates depends on successful sperm-egg collisions, gamete fusion, and standard generation of Ca 2+ oscillations. Therefore, the realistic effects of future ocean pCO 2 levels on these specific aspects of fertilization of Tegillarca granosa were investigated in the present study through sperm velocity trials, fertilization kinetics model analysis, and intracellular Ca 2+ assays, respectively. Results obtained indicated that ocean acidification significantly reduced the fertilization success of T. granosa, which could be accountable by (i) decreased sperm velocity hence reducing the probability for sperm-egg collisions; (ii) lowered probability of gamete fusion for each gamete collision event; and (iii) disrupted intracellular Ca 2+ oscillations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray

PubMed Central

2010-01-01

Background As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals. Methods Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray. Results Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data. Conclusions In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment. PMID:20409345

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray.

PubMed

Xie, Shuwu; Zhu, Yan; Ma, Li; Lu, Yingying; Zhou, Jieyun; Gui, Youlun; Cao, Lin

2010-04-22

As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals. Rats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray. Among all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data. In this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment.

The Disappearing Sperms: Analysis of Reports Published Between 1980 and 2015.

PubMed

Sengupta, Pallav; Dutta, Sulagna; Krajewska-Kulak, Elzbieta

2017-07-01

Reports regarding the changes in sperm concentration in different counties of the world are inconsistent. Furthermore, the reports that sprung up from specific epidemiological and experimental examinations did not include data of prior studies or geographical variations. The current study, following a previous report of massive fall in semen volume over the past 33 years, attempts to delineate the trend of altering sperm concentrations and factors responsible for this by reviewing article published from 1980 to July 2015 with geographic differences. The current study identified an overall 57% diminution in mean sperm concentration over the past 35 years ( r = -.313, p = .0002), which, when analyzed for each geographical region, identified a significant decline in North America, Europe, Asia, and Africa. An increasing trend of sperm concentration was identified only in Australia. The association of male age with such a trend ( R 2 = .979) is reported. The authors also correlated male fertility with sperm concentration. Thus, this comprehensive, evidence-based literature review aims to concisely and systematically present the available data on sperm concentration from 1980 to 2015, as well as to statistically analyze the same and correlate male health with the declining pattern of sperm count in a single scientific review to serve the scientific research zone related to reproductive health. It points to the threat of male infertility in times ahead.

The Disappearing Sperms: Analysis of Reports Published Between 1980 and 2015

PubMed Central

Sengupta, Pallav; Dutta, Sulagna; Krajewska-Kulak, Elzbieta

2016-01-01

Reports regarding the changes in sperm concentration in different counties of the world are inconsistent. Furthermore, the reports that sprung up from specific epidemiological and experimental examinations did not include data of prior studies or geographical variations. The current study, following a previous report of massive fall in semen volume over the past 33 years, attempts to delineate the trend of altering sperm concentrations and factors responsible for this by reviewing article published from 1980 to July 2015 with geographic differences. The current study identified an overall 57% diminution in mean sperm concentration over the past 35 years (r = −.313, p = .0002), which, when analyzed for each geographical region, identified a significant decline in North America, Europe, Asia, and Africa. An increasing trend of sperm concentration was identified only in Australia. The association of male age with such a trend (R2 = .979) is reported. The authors also correlated male fertility with sperm concentration. Thus, this comprehensive, evidence-based literature review aims to concisely and systematically present the available data on sperm concentration from 1980 to 2015, as well as to statistically analyze the same and correlate male health with the declining pattern of sperm count in a single scientific review to serve the scientific research zone related to reproductive health. It points to the threat of male infertility in times ahead. PMID:27099345

A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

PubMed

Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

1997-06-01

Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways.

Autocrine regulation of human sperm motility by tachykinins

PubMed Central

2010-01-01

Background We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. Methods Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). Results The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). Conclusion These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins. PMID:20796280

Autocrine regulation of human sperm motility by tachykinins.

PubMed

Pinto, Francisco M; Ravina, Cristina G; Subiran, Nerea; Cejudo-Román, Antonio; Fernández-Sánchez, Manuel; Irazusta, Jon; Garrido, Nicolas; Candenas, Luz

2010-08-26

We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

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

Distinct sperm nucleus behaviors between genotypic and temperature-dependent sex determination males are associated with replication and expression-related pathways in a gynogenetic fish.

PubMed

Zhu, Yao-Jun; Li, Xi-Yin; Zhang, Jun; Li, Zhi; Ding, Miao; Zhang, Xiao-Juan; Zhou, Li; Gui, Jian-Fang

2018-06-05

Coexistence and transition of diverse sex determination strategies have been revealed in some ectothermic species, but the variation between males caused by different sex determination strategies and the underlying mechanism remain unclear. Here, we used the gynogenetic gibel carp (Carassius gibelio) with both genotypic sex determination (GSD) and temperature-dependent sex determination (TSD) strategies to illustrate this issue. We found out that males of GSD and TSD in gibel carp had similar morphology, testicular histology, sperm structure and sperm vitality. However, when maternal individuals were mated with males of GSD, sperm nucleus swelling and fusing with the female pronucleus were observed in the fertilized eggs. On the contrary, when maternal individuals were mated with males of TSD, sperm nucleus remained in the condensed status throughout the whole process. Subsequently, semen proteomics analysis unveiled that DNA replication and gene expression-related pathways were inhibited in the sperm from males of TSD compared to males of GSD, and most differentially expressed proteins associated with DNA replication, transcription and translation were down-regulated. Moreover, via BrdU incorporation and immunofluorescence detection, male nucleus replication was revealed to be present in the fertilized eggs by the sperm from males of GSD, but absent in the fertilized eggs by the sperm from males of TSD. These findings indicate that DNA replication and gene expression-related pathways are associated with the distinct sperm nucleus development behaviors in fertilized eggs in response to the sperm from males of GSD and TSD. And this study is the first attempt to screen the differences between males determined via GSD and TSD in gynogenetic species, which might give a hint for understanding evolutionary adaption of diverse sex determination mechanisms in unisexual vertebrates.

Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility1

PubMed Central

Nakamura, Noriko; Dai, Qunsheng; Williams, Jason; Goulding, Eugenia H.; Willis, William D.; Brown, Paula R.; Eddy, Edward M.

2013-01-01

ABSTRACT Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4+/Gt). Expression of beta-gal occurred in the testis, and male mice homozygous for the gene trap allele (Eno4Gt/Gt) were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially in Eno4Gt/Gt mice compared to wild-type mice. Sperm from Eno4Gt/Gt mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm. PMID:23446454

Involvement of Fibroblast Growth Factor 2 (FGF2) and its receptors in the regulation of mouse sperm physiology.

PubMed

Saucedo, Lucia; Sobarzo, Cristian; Brukman, Nicolás; Guidobaldi, Hector Alejandro; Lustig, Livia; Giojalas, Laura Cecilia; Buffone, Mariano Gabriel; Vazquez-Levin, Monica Hebe; Marín-Briggiler, Clara

2018-06-04

Fibroblast Growth Factor 2 (FGF2) and its receptors (FGFRs) have been described in several tissues, where they regulate cellular proliferation, differentiation, motility and apoptosis. Although FGF2/FGFRs expression in the male reproductive tract has been reported, there is scarce evidence on their presence in the female reproductive tract and their involvement in the modulation of sperm function. Therefore, the objective of this study was to determine the expression of FGF2 in the female reproductive tract and to assess the role of the FGF2/FGFRs system in the regulation of sperm physiology using the murine model. FGF2 was detected in uterus and oviduct protein extracts, and it was immunolocalized in epithelial cells of the uterus, isthmus and ampulla, as well as in the cumulus oophorus-oocyte complex. The receptors FGFR1, FGFR2, FGFR3 and FGFR4 were immunodetected in the flagellum and acrosomal region of sperm recovered from the cauda epididymis. Analysis of testis sections showed the expression of FGFRs in germ cells at different stages of the spermatogenesis, suggesting the testicular origin of the sperm FGFRs. Sperm incubation with recombinant FGF2 (rFGF2) led to increased sperm motility and velocity, and to enhanced intracellular Ca2+ levels and acrosomal loss compared to the control. In conclusion, this study shows that FGF2 is expressed in tissues of the female reproductive tract. Also, the fact that functional FGFRs are present in mouse sperm and that rFGF2 affects sperm motility and acrosomal exocytosis, suggests the involvement of this system in the in vivo regulation of sperm function.

Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis.

PubMed

Cabrillana, María E; Monclus, María A; Sáez Lancellotti, Tania E; Boarelli, Paola V; Clementi, Marisa A; Vincenti, Amanda E; Yunes, Roberto F M; Fornés, Miguel W

2011-09-01

Mammalian sperm proteins undergo thiol group (SH) oxidation to form disulfides bonds (SS) as they travel through the epididymis during cell maturation. Disulfide bonds are involved in chromatin condensation and tail organelle stabilization. In this work, we used a fluorescent thiol-selective labeling agent, monobromobimane (mBBr), to study the protein thiol status of rat sperm during maturation. Fluorescence signal decrease along the epididymal trip, more evidently in the head, but also in the tail, indicates that both sub cellular regions participate in the thiol changes. The sources of the fluorescence signal are sulfhydryls sperm proteins labeled by mBBr (mBBr-spp). Initial attempts to identify the mBBr-spp labeled were detected in the initial-caput, but not in the distal cauda-segment of the epididymis in sodium dodecyl sulfate (SDS)-PAGE analysis. This phenomenon could be due to protein resistance to solubilization. For this reason, disulfide bond reduction was accomplished by sodium dodecyl sulfate plus dithiothreitol treatment to recover the mBBr signal in SDS-PAGE. Under this protocol, a major 27 kDa protein band displays a strong signal. Protein identification by mass spectrometry and sequence database searching correlated this protein with the outer dense fiber 1 (ODF1). The mBBr specifically bound to N-terminal domain cysteine of ODF1. The mBBr reduces rat sperm motility, quantitatively and qualitatively, and the effects are dose dependent, without significantly increasing the percentage of dead sperm. Thus, we found that ODF1 is highly responsible for mBBr fluorescence detection in the sperm tail, and the motility inhibition by the fluorescence marker indicates that ODF1 N-terminal domain are related to sperm motility. © 2011 Wiley-Liss, Inc. Copyright © 2011 Wiley-Liss, Inc.

High-dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism.

PubMed

Aarabi, Mahmoud; San Gabriel, Maria C; Chan, Donovan; Behan, Nathalie A; Caron, Maxime; Pastinen, Tomi; Bourque, Guillaume; MacFarlane, Amanda J; Zini, Armand; Trasler, Jacquetta

2015-11-15

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Computer assisted sperm analysis of motility patterns of postthawed epididymal spermatozoa of springbok (Antidorcas marsupialis), impala (Aepyceros melampus), and blesbok (Damaliscus dorcus phillipsi) incubated under conditions supporting domestic cattle in vitro fertilization.

PubMed

Chatiza, F P; Bartels, P; Nedambale, T L; Wagenaar, G M

2012-07-15

The need for information on the reproductive physiology of different wildlife species is important for ex situ conservation using such methods as in vitro fertilization (IVF). Information on species reproductive physiology and evaluation of sperm quality using accurate, objective, repeatable methods, such as computer-assisted sperm analysis (CASA) for ex situ conservation has become a priority. The aim of this study was to evaluate motility patterns of antelope epididymal spermatozoa incubated for 4 h under conditions that support bovine IVF using CASA. Cauda epididymal spermatozoa were collected postmortem from testicles of springbok (N=38), impala (N=26), and blesbok (N=42), and cryopreserved in biladyl containing 7% glycerol. Spermatozoa were thawed and incubated in Capacitation media and modified Tyrode lactate (m-TL) IVF media using a protocol developed for domestic cattle IVF. The study evaluates 14 motility characteristics of the antelope epididymal sperm at six time points using CASA. Species differences in CASA parameters evaluated under similar conditions were observed. Several differences in individual motility parameters at the time points were reported for each species. Epididymal sperm of the different antelope species responded differently to capacitation agents exhibiting variations in hyperactivity. Motility parameters that describe the vigor of sperm decreased over time. Spermatozoa from the different antelope species have different physiological and optimal capacitation and in vitro culture requirements. The interspecies comparison of kinematic parameters of spermatozoa between the antelopes over several end points contributes to comparative sperm physiology which forms an important step in the development of species specific assisted reproductive techniques (ARTs) for ex situ conservation of these species. Copyright © 2012 Elsevier Inc. All rights reserved.

Alkylation of sperm DNA is associated with male factor infertility and a reduction in the proportion of oocytes fertilised during assisted reproduction.

PubMed

Stocks, S J; Agius, R M; Cooley, N; Harrison, K L; Brison, D R; Horne, G; Gibbs, A; Povey, A C

2010-04-30

Approximately one-third of IVF cases in the UK are attributed to male factor infertility and in the majority of cases the origin of male infertility is unknown. The integrity of sperm DNA is important both for the success of assisted reproduction and the implications for the off-spring. One type of DNA damage that has not been investigated with respect to fertility outcomes is the adduct N7-methyldeoxyguanosine (N7-MedG), a biomarker for exposure to alkylating agents. A prospective cohort of couples attending for IVF had their N7-MedG levels in sperm measured using an immunoslot blot technique to examine whether sperm N7-MedG levels are associated with male factor infertility, semen quality measures or assisted reproduction outcomes. Sufficient DNA for analysis was obtained from 67/97 couples and N7-MedG was detected in 94% of sperm samples analysed. Men diagnosed with male factor infertility had significantly higher mean levels of N7-MedG in their sperm DNA (P=0.03). Logistic regression analysis showed that N7-MedG levels were significantly negatively associated with the proportion of oocytes successfully fertilised irrespective of the method of fertilisation used (IVF or intra-cytoplasmic sperm injection; ICSI, P<0.001). Therefore exposure to DNA alkylating agents is significantly associated with male infertility and the proportion of oocytes fertilised during assisted reproduction. Reducing such exposure may improve male fertility but further work is required to determine the relative importance of exogenous and endogenous sources of exposure. Copyright 2010 Elsevier B.V. All rights reserved.

Sperm dispersal distances estimated by parentage analysis in a brooding scleractinian coral.

PubMed

Warner, Patricia A; Willis, Bette L; van Oppen, Madeleine J H

2016-03-01

Within populations of brooding sessile corals, sperm dispersal constitutes the mechanism by which gametes interact and mating occurs, and forms the first link in the network of processes that determine specieswide connectivity patterns. However, almost nothing is known about sperm dispersal for any internally fertilizing coral. In this study, we conducted a parentage analysis on coral larvae collected from an area of mapped colonies, to measure the distance sperm disperses for the first time in a reef-building coral and estimated the mating system characteristics of a recently identified putative cryptic species within the Seriatopora hystrix complex (ShA; Warner et al. 2015). We defined consensus criteria among several replicated methods (COLONY 2.0, CERVUS 3.0, MLTR v3.2) to maximize accuracy in paternity assignments. Thirteen progeny arrays indicated that this putative species produces exclusively sexually derived, primarily outcrossed larvae (mean t(m) = 0.999) in multiple paternity broods (mean r(p) = 0.119). Self-fertilization was directly detected at low frequency for all broods combined (2.8%), but comprised 23% of matings in one brood. Although over 82% of mating occurred between colonies within 10 m of each other (mean sperm dispersal = 5.5 m ± 4.37 SD), we found no evidence of inbreeding in the established population. Restricted dispersal of sperm compared to slightly greater larval dispersal appears to limit inbreeding among close relatives in this cryptic species. Our findings establish a good basis for further work on sperm dispersal in brooding corals and provide the first information about the mating system of a newly identified and abundant cryptic species. © 2016 John Wiley & Sons Ltd.

Metabolites involved in cellular communication among human cumulus-oocyte-complex and sperm during in vitro fertilization.

PubMed

Gómez-Torres, María José; García, Eva María; Guerrero, Jaime; Medina, Sonia; Izquierdo-Rico, María José; Gil-Izquierdo, Ángel; Orduna, Jesús; Savirón, María; González-Brusi, Leopoldo; Ten, Jorge; Bernabeu, Rafael; Avilés, Manuel

2015-11-09

Fertilization is a key physiological process for the preservation of the species. Consequently, different mechanisms affecting the sperm and the oocyte have been developed to ensure a successful fertilization. Thus, sperm acrosome reaction is necessary for the egg coat penetration and sperm-oolema fusion. Several molecules are able to induce the sperm acrosome reaction; however, this process should be produced coordinately in time and in the space to allow the success of fertilization between gametes. The goal of this study was to analyze the metabolites secreted by cumulus-oocyte-complex (COC) to find out new components that could contribute to the induction of the human sperm acrosome reaction and other physiological processes at the time of gamete interaction and fertilization. For the metabolomic analysis, eighteen aliquots of medium were used in each group, containing: a) only COC before insemination and after 3 h of incubation; b) COC and capacitated spermatozoa after insemination and incubated for 16-20 hours; c) only capacitated sperm after 16-20 h in culture and d) only fertilization medium as control. Six patients undergoing assisted reproduction whose male partners provided normozoospermic samples were included in the study. Seventy-two COC were inseminated. The metabolites identified were monoacylglycerol (MAG), lysophosphatidylcholine (LPC) and phytosphingosine (PHS). Analysis by PCR and in silico of the gene expression strongly suggests that the cumulus cells contribute to the formation of the PHS and LPC. LPC and PHS are secreted by cumulus cells during in vitro fertilization and they could be involved in the induction of human acrosome reaction (AR). The identification of new molecules with a paracrine effect on oocytes, cumulus cells and spermatozoa will provide a better understanding of gamete interaction.

Evaluation of sperm DNA quality in men presenting with testicular cancer and lymphoma using alkaline and neutral Comet assays.

PubMed

Kumar, K; Lewis, S; Vinci, S; Riera-Escamilla, A; Fino, M-G; Tamburrino, L; Muratori, M; Larsen, P; Krausz, C

2018-01-01

Despite more cancers in young men over the past two decades, improvements in therapies give a greater chance to live full lives following treatment. Sperm genomic quality is variable following cancer diagnosis, so its assessment is important if sperm cryopreservation is being considered. Here, we evaluated DNA damage using two DNA damage assays: an alkaline and for the first time, a neutral Comet assays in men presenting with testicular cancer (n = 19 for alkaline and 13 for neutral group) and lymphoma (n = 13 for alkaline and 09 for neutral group) compared with fertile donors (n = 20 for alkaline and 14 for neutral group). No significant differences were observed in any semen analysis parameters. In contrast, sperm DNA damage was higher in men with testicular cancer than in donors as assessed by both the alkaline (12.4% vs. 37.4%, p < 0.001) and neutral (7.5% vs. 13.4%; p < 0.05) Comet assays. Similar trends were observed in men with lymphoma. Here, sperm DNA damage was higher using both the alkaline (35.0% vs. 12.4%) and neutral (10.7% against 7.5% (p < 0.05) Comet assays. Moreover, the DNA strand breaks (particularly double-strand breaks) were significantly more prominent in men with cancer having abnormal seminal parameters than normozoospermic ones. This study showed that sperm DNA testing using alkaline and neutral Comet assays is more sensitive than semen analysis in detecting impaired sperm quality in men presenting with cancer. It may provide a useful adjunct when considering storage prior to cancer investigations and assisted reproductive techniques (ART)-based treatment. © 2017 American Society of Andrology and European Academy of Andrology.

Differential distribution of sperm subpopulations and incidence of pleiomorphisms in ejaculates of captive howling monkeys ( Alouatta caraya)

NASA Astrophysics Data System (ADS)

Valle, R. R.; Carvalho, F. M.; Muniz, J. A. P. C.; Leal, C. L. V.; García-Herreros, M.

2013-10-01

The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys ( Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [ A, (square micrometers)], Perimeter [ P, (micrometers)], Length [ L, (micrometers)], and Width [ W, (micrometers)]) and shape-derived parameters (Ellipticity [( L/ W)], Elongation [( L - W)/( L + W)], and Rugosity [(4л A/ P 2)]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences ( P < 0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P < 0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species.

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.

Assessment of semen function and lipid peroxidation among lead exposed men

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

Kasperczyk, Aleksandra; Kasperczyk, Slawomir; Horak, Stanislaw

The study population included healthy, fertile men, employees of Zinc and Lead Metalworks (n = 63). Workers exposed to lead were divided into two groups: a group with moderate exposure to lead (ME) - blood lead level (PbB) 25-40 {mu}g/dl and a group with high exposure to lead (HE) PbB = 40-81 {mu}g/dl. The control group consisted of office workers with no history of occupational exposure to lead. Evaluation of lead, cadmium and zinc level in blood and seminal plasma, zinc protoporphyrin in blood (ZPP), 5-aminolevulinic acid in urine (ALA), malondialdehyde (MDA) in seminal plasma and sperm analysis were performed.more » No differences were noted in the concentration of cadmium and zinc in blood and seminal plasma in the study population. Lipid peroxidation in seminal plasma, represented as MDA concentration, significantly increased by about 56% in the HE group and the percentage of motile sperm cells after 1 h decreased by about 34% in comparison to the control group. No statistically significant correlation between other parameters of sperm analysis and lead exposure parameters nor between lead, cadmium and zinc concentration in blood and seminal plasma were found. A positive association between lead intoxication parameters (PbB, ZPP, lead seminal plasma) and MDA concentration in sperm plasma and inverse correlation with sperm cells motility (PbB, ZPP) was found. An increased concentration of MDA was accompanied by a drop in sperm cells motility. In conclusion, we report that high exposure to lead causes a decrease of sperm motility in men most likely as a result of increased lipid peroxidation, especially if the level in the blood surpasses the concentration of 40 {mu}g/dl.« less

Specialized sperm function tests in varicocele and the future of andrology laboratory.

PubMed

Majzoub, Ahmad; Esteves, Sandro C; Gosálvez, Jaime; Agarwal, Ashok

2016-01-01

Varicocele is a common medical condition entangled with many controversies. Though it is highly prevalent in men with infertility, still it marks its presence in males who do have normal fertility. Determining which patients are negatively affected by varicocele would enable clinicians to better select those men who benefitted the most from surgery. Since conventional semen analysis has been limited in its ability to evaluate the negative effects of varicocele on fertility, a multitude of specialized laboratory tests have emerged. In this review, we examine the role and significance of specialized sperm function tests with regards to varicocele. Among the various tests, analysis of sperm DNA fragmentation and measurements of oxidative stress markers provide an independent measure of fertility in men with varicocele. These diagnostic modalities have both diagnostic and prognostic information complementary to, but distinct from conventional sperm parameters. Test results can guide management and aid in monitoring intervention outcomes. Proteomics, metabolomics, and genomics are areas; though still developing, holding promise to revolutionize our understanding of reproductive physiology, including varicocele.

Genetic stability of progeny from an artificial allotetraploid carp using sperm from five fish species.

PubMed

Ye, Yuzhen; Wang, Zhongwei; Zhou, Jianfeng; Wu, Qingjiang

2009-08-01

Microsatellite markers and D-loop sequences of mtDNA from a female allotetraploid parent carp and her progenies of generations 1 and 2 induced by sperm of five distant fish species were analyzed. Eleven microsatellite markers were used to identify 48 alleles from the allotetraploid female. The same number of alleles (48) appeared in the first and second generations of the gynogenetic offspring, regardless of the source of the sperm used as an activator. The mtDNA D-loop analysis was performed on the female tetraploid parent, 25 gynogenetic offspring, and 5 sperm-donor species. Fourteen variable sites from the 1,018 bp sequences were observed in the offspring as compared to the female tetraploid parent. Results from D-loop sequence and microsatellite marker analysis showed exclusive maternal transmission, and no genetic information was derived from the father. Our study suggests that progenies of artificial tetraploid carp are genetically stable, which is important for genetic breeding of this tetraploid fish.

Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios

PubMed Central

Majzoub, Ahmad; Esteves, Sandro C.; Ko, Edmund; Ramasamy, Ranjith; Zini, Armand

2016-01-01

Sperm DNA fragmentation (SDF) has been generally acknowledged as a valuable tool for male fertility evaluation. While its detrimental implications on sperm function were extensively investigated, little is known about the actual indications for performing SDF analysis. This review delivers practice based recommendations on commonly encountered scenarios in the clinic. An illustrative description of the different SDF measurement techniques is presented. SDF testing is recommended in patients with clinical varicocele and borderline to normal semen parameters as it can better select varicocelectomy candidates. High SDF is also linked with recurrent spontaneous abortion (RSA) and can influence outcomes of different assisted reproductive techniques. Several studies have shown some benefit in using testicular sperm rather than ejaculated sperm in men with high SDF, oligozoospermia or recurrent in vitro fertilization (IVF) failure. Infertile men with evidence of exposure to pollutants can benefit from sperm DNA testing as it can help reinforce the importance of lifestyle modification (e.g., cessation of cigarette smoking, antioxidant therapy), predict fertility and monitor the patient’s response to intervention. PMID:28078226

Effects of exposure to 17-alpha-ethynylestradiol on sperm quality of tench (Tinca tinca).

PubMed

Oropesa, A L; Martín-Hidalgo, D; Fallola, C; Gil, M C

2015-10-01

Alterations of sperm quality were studied in tench (Tinca tinca) exposed to sub-lethal doses of 17-alpha-ethynylestradiol-EE2-(50, 100 and 500μg/kg t.w) under semi-static conditions for 30 days. Thus, different biomarkers of sperm quality were assessed: concentration and volume of ejaculate, total number of spermatozoa, percentage of motile spermatozoa, sperm motility and percentage of live and dead spermatozoa. Sperm motility was examined by computer-assisted image analysis and the viability of spermatozoa was assessed through flow cytometry. The most relevant alterations observed were significant reductions in the reproductive parameters such as testicular somatic index, spermatozoa concentration, straight line velocity, curvilinear velocity, average path velocity and wobble in tench exposed to 50μg/kg t.w of EE2. Our study about the effects of EE2 on the sperm quality in tench provides new evidences which strengthen the fact that this synthetic estrogen is included in the list of non-monotonic dose response compounds in animal studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Age-dependent changes in metabolic profile of turkey spermatozoa as assessed by NMR analysis

PubMed Central

Di Iorio, Michele; Mannina, Luisa; Paventi, Gianluca; Rosato, Maria Pina; Cerolini, Silvia; Sobolev, Anatoly P.

2018-01-01

Metabolic profile of fresh turkey spermatozoa at three different reproductive period ages, namely 32, 44 and 56 weeks, was monitored by Nuclear Magnetic Resonance (NMR) spectroscopy and correlated to sperm quality parameters. The age-related decrease in sperm quality as indicated by reduction of sperm concentration, sperm mobility and osmotic tolerance was associated to variation in the level of specific water-soluble and liposoluble metabolites. In particular, the highest levels of isoleucine, phenylalanine, leucine, tyrosine and valine were found at 32 weeks of age, whereas aspartate, lactate, creatine, carnitine, acetylcarnitine levels increased during the ageing. Lipid composition also changed during the ageing: diunsaturated fatty acids level increased from 32 to 56 weeks of age, whereas a reduction of polyunsaturated fatty acids content was observed at 56 weeks. The untargeted approach attempts to give a wider picture of metabolic changes occurring in ageing suggesting that the reduction of sperm quality could be due to a progressive deficiency in mitochondrial energy producing systems, as also prompted by the negative correlation found between sperm mobility and the increase in certain mitochondrial metabolites. PMID:29534088

Absence of effects on the rat sperm quality after subacute exposure to low doses of fungicide prochloraz.

PubMed

Sanabria, Marciana; Pessin, Alessandra; Zanutto, Mirella Rossitto; Perobelli, Juliana Elaine; Guerra, Marina Trevizan; Banzato, Thais Petrochelli; Borges, Cibele dos Santos; Kempinas, Wilma De Grava

2015-01-01

Prochloraz (PCZ) is a fungicide and androgen-receptor antagonist used worldwide in horticulture and agriculture. Pre- and perinatal exposure to this pesticide during sexual differentiation is deleterious for male offspring. Since data on the effects of PCZ on epididymal functions are scarce, and because sperm maturation occurs in this organ, the present investigation aimed to determine whether low PCZ doses administered to rats during the phase of sperm transit through the epididymis might affect the morphophysiology of this organ and sperm quality. Adult male Wistar rats were assigned to 4 different groups: 0 (control, vehicle) or 10, 15, or 30 mg/kg bw/d PCZ diluted in corn oil administered orally for 4 consecutive days. Morphofunctional parameters of the male reproductive tract, hormone concentrations, sperm evaluations, and fertility and histopathologic analysis of testis and epididymis were assessed. There were no statistically significant differences between treated and control groups in relation to all evaluated parameters. Data demonstrated show that PCZ exposure for a brief 4-d exposure and low doses did not produce reproductive toxicity or compromise sperm quality in adult rats.

Male and Couple Fertility Impairment due to HPV-DNA Sperm Infection: Update on Molecular Mechanism and Clinical Impact—Systematic Review

PubMed Central

Gizzo, Salvatore; Ferrari, Bruno; Noventa, Marco; Ferrari, Emanuele; Patrelli, Tito Silvio; Gangemi, Michele; Nardelli, Giovanni Battista

2014-01-01

Recent evidences identify Human Papillomavirus (HPV) sperm infection as a possible cause of male and couple infertility. It acts through different mechanisms at various steps of human conception and early gestational development. We performed a systematic review to assess the role of HPV semen infection on male and couple infertility. Analysis of available and eligible data does not permit us to fund clear evidences about clinical impact of HPV infection on fertility, although sperm parameters impairment is the most widely recognized effect. Regarding biomolecular implications, the available data are often conflicting. More studies are required to define the role of HPV sperm infection in clinical practice. The great majority of evidences are obtained by in vitro studies and this fact represents a limitation for the clinical management of HPVDNA sperm infection. Understanding the biological significance of HPV-DNA semen infection could permit us to explain most of the idiopathic male and couple infertility, leading to a better management of infertile men and a better timing for sperm banking storage before ART cycles. PMID:24783196

Human genetic mapping studies using single sperm typing

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

Hubert, R.S.

1993-01-01

Sperm typing is a powerful technique that uses the polymerase chain reaction (PCR) to analyze DNA sequences within single sperm cells in order to construct genetic maps. This methodology was used to estimate the recombination fraction between D3S2 and D3S2 which was found to be 0.28 (95% CI = 0.20-0.36). Pedigree analysis was unable to determine genetic distance between these two markers due to their low informativeness. We also showed that dinucleotide and tetranucleotide repeat polymorphisms can be analyzed in single cells without using radioactivity or denaturing gels. This provides a rich new source of DANA polymorphisms for genetic mappingmore » by sperm typing. In addition, an approach that uses the sperm typing methodology is described that can define the physical boundaries of meiotic recombination hotspots. The hotspot at 4p16.3 near the Huntington disease gene was localized to an interval between D4S10 and D4S126. These studies demonstrated the usefulness of sperm typing as a tool for the study of human genetic.« less

Selective Loss of Sperm Bearing a Compound Chromosome in the Drosophila Female

PubMed Central

Dernburg, A. F.; Daily, D. R.; Yook, K. J.; Corbin, J. A.; Sedat, J. W.; Sullivan, W.

1996-01-01

The Drosophila compound entire second chromosome, C(2)EN, displays paternal transmission well below Mendelian expectations (NOVITSKI et al. 1981). Because C(2)EN stocks also show higher-than-expected rates of zygotic lethality, it was proposed that this reduced paternal inheritance might be wholly or partially due to postfertilization events. Efforts to investigate this phenomenon have been hampered because the progeny of crosses between C(2)EN-bearing individuals and those with normal karyotypes die during embryogenesis. We have circumvented this obstacle by employing fluorescence in situ hybridization to directly karyotype early embryos from crosses involving C(2)EN-bearing individuals. This analysis reveals that the distortion in paternal transmission is established before fertilization. Moreover, measurement of the sperm ratios within both the male and female reproductive organs demonstrates that C(2)EN-bearing sperm are selectively lost after sperm transfer to the female and before storage of sperm in the seminal receptacles and spermathecae. Our results are consistent with a model of meiotic drive in which aberrations occuring early in meiosis lead ultimately to sperm dysfunction. PMID:8844151

Selective loss of sperm bearing a compound chromosome in the Drosophila female.

PubMed

Dernburg, A F; Daily, D R; Yook, K J; Corbin, J A; Sedat, J W; Sullivan, W

1996-08-01

The Drosophila compound entire second chromosome, C(2)EN, displays paternal transmission well below Mendelian expectations (NOVITSKI et al. 1981). Because C(2)EN stocks also show higher-than-expected rates of zygotic lethality, it was proposed that this reduced paternal inheritance might be wholly or partially due to postfertilization events. Efforts to investigate this phenomenon have been hampered because the progeny of crosses between C(2)EN-bearing individuals and those with normal karyotypes die during embryogenesis. We have circumvented this obstacle by employing fluorescence in situ hybridization to directly karyotype early embryos from crosses involving C(2)EN-bearing individuals. This analysis reveals that the distortion in paternal transmission is established before fertilization. Moreover, measurement of the sperm ratios within both the male and female reproductive organs demonstrates that C(2)EN-bearing sperm are selectively lost after sperm transfer to the female and before storage of sperm in the seminal receptacles and spermathecae. Our results are consistent with a model of meiotic drive in which aberrations occurring early in meiosis lead ultimately to sperm dysfunction.

Red light improves spermatozoa motility and does not induce oxidative DNA damage

NASA Astrophysics Data System (ADS)

Preece, Daryl; Chow, Kay W.; Gomez-Godinez, Veronica; Gustafson, Kyle; Esener, Selin; Ravida, Nicole; Durrant, Barbara; Berns, Michael W.

2017-04-01

The ability to successfully fertilize ova relies upon the swimming ability of spermatozoa. Both in humans and in animals, sperm motility has been used as a metric for the viability of semen samples. Recently, several studies have examined the efficacy of low dosage red light exposure for cellular repair and increasing sperm motility. Of prime importance to the practical application of this technique is the absence of DNA damage caused by radiation exposure. In this study, we examine the effect of 633 nm coherent, red laser light on sperm motility using a novel wavelet-based algorithm that allows for direct measurement of curvilinear velocity under red light illumination. This new algorithm gives results comparable to the standard computer-assisted sperm analysis (CASA) system. We then assess the safety of red light treatment of sperm by analyzing, (1) the levels of double-strand breaks in the DNA, and (2) oxidative damage in the sperm DNA. The results demonstrate that for the parameters used there are insignificant differences in oxidative DNA damage as a result of irradiation.

HT-COMET: a novel automated approach for high throughput assessment of human sperm chromatin quality.

PubMed

Albert, Océane; Reintsch, Wolfgang E; Chan, Peter; Robaire, Bernard

2016-05-01

Can we make the comet assay (single-cell gel electrophoresis) for human sperm a more accurate and informative high throughput assay? We developed a standardized automated high throughput comet (HT-COMET) assay for human sperm that improves its accuracy and efficiency, and could be of prognostic value to patients in the fertility clinic. The comet assay involves the collection of data on sperm DNA damage at the level of the single cell, allowing the use of samples from severe oligozoospermic patients. However, this makes comet scoring a low throughput procedure that renders large cohort analyses tedious. Furthermore, the comet assay comes with an inherent vulnerability to variability. Our objective is to develop an automated high throughput comet assay for human sperm that will increase both its accuracy and efficiency. The study comprised two distinct components: a HT-COMET technical optimization section based on control versus DNAse treatment analyses ( ITALIC! n = 3-5), and a cross-sectional study on 123 men presenting to a reproductive center with sperm concentrations categorized as severe oligozoospermia, oligozoospermia or normozoospermia. Sperm chromatin quality was measured using the comet assay: on classic 2-well slides for software comparison; on 96-well slides for HT-COMET optimization; after exposure to various concentrations of a damage-inducing agent, DNAse, using HT-COMET; on 123 subjects with different sperm concentrations using HT-COMET. Data from the 123 subjects were correlated to classic semen quality parameters and plotted as single-cell data in individual DNA damage profiles. We have developed a standard automated HT-COMET procedure for human sperm. It includes automated scoring of comets by a fully integrated high content screening setup that compares well with the most commonly used semi-manual analysis software. Using this method, a cross-sectional study on 123 men showed no significant correlation between sperm concentration and sperm DNA damage, confirming the existence of hidden chromatin damage in men with apparently normal semen characteristics, and a significant correlation between percentage DNA in the tail and percentage of progressively motile spermatozoa. Finally, the use of DNA damage profiles helped to distinguish subjects between and within sperm concentration categories, and allowed a determination of the proportion of highly damaged cells. The main limitations of the HT-COMET are the high, yet indispensable, investment in an automated liquid handling system and heating block to ensure accuracy, and the availability of an automated plate reading microscope and analysis software. This standardized HT-COMET assay offers many advantages, including higher accuracy and evenness due to automation of sensitive steps, a 14.4-fold increase in sample analysis capacity, and an imaging and scoring time of 1 min/well. Overall, HT-COMET offers a decrease in total experimental time of more than 90%. Hence, this assay constitutes a more efficient option to assess sperm chromatin quality, paves the way to using this assay to screen large cohorts, and holds prognostic value for infertile patients. Funded by the CIHR Institute of Human Development, Child and Youth Health (IHDCYH; RHF 100625). O.A. is a fellow supported by the Fonds de la Recherche du Québec - Santé (FRQS) and the CIHR Training Program in Reproduction, Early Development, and the Impact on Health (REDIH). B.R. is a James McGill Professor. The authors declare no conflicts 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.

Membrane proteins associated with sperm-oocyte interaction: A proteomic comparison between Kedah Kelantan (Bos indicus) and Mafriwal (Bos taurus × Bos indicus) sperm

NASA Astrophysics Data System (ADS)

Ashrafzadeh, Ali; Nathan, Sheila; Othman, Iekhsan; Yee, Tee Ting; Karsani, Saiful Anuar

2013-11-01

Production performance of European cattle breeds has significantly improved through various breeding programs. However, European breeds are more susceptible to heat stress compared to zebu cattle (Bos indicus) as their conception rate can range between 20 to 30% in hot seasons compared to winter. To identify cattle sperm proteins associated with zebu cattle higher fertility and heat tolerance in tropical environments, we utilised a proteomics-based approach to compare sperm from the highly fertile Malaysian indigenous breed, Kedah Kelantan (Bos indicus), with sperm from the sub-fertile crossbreed, Mafriwal (Bos taurus × Bos indicus). Frozen semen of three high performance bulls from each breed was processed to obtain live and pure sperm. Proteins were separated and gel bands were processed by in-gel tryptic digestion. For each breed, mass spectrometry data was acquired over 11 replicates. The analyzed data identified peptides with different expression levels (99% confidence level) and protein identification was determined by targeted MS/MS. Among the identified proteins associated with sperm-oocyte interaction, two proteins were up-regulated in Kedah Kelantan sperm and 7 proteins were up-regulated in or specific to Mafriwal. Our results suggest that the higher fertility of zebu cattle in tropical areas may not be related to more efficient sperm-oocyte interaction. Further analysis of the other regulated proteins in these two breeds may contribute further knowledge on the physiological reason/s for higher fertility and heat tolerance of Zebu cattle in tropical areas.

Modified expression of several sperm proteins after chronic exposure to the antiandrogenic compound vinclozolin.

PubMed

Auger, Jacques; Eustache, Florence; Maceiras, Paula; Broussard, Cédric; Chafey, Philippe; Lesaffre, Corinne; Vaiman, Daniel; Camoin, Luc; Auer, Jana

2010-10-01

Little is known about the molecular impact of in vivo exposure to endocrine disruptors (EDs) on sperm structures and functions. We recently reported that the lifelong exposure of rats to the antiandrogenic compound vinclozolin results in low epididymal weight, changes in sperm kinematic parameters, and immature sperm chromatin condensation, together with the impairment of several fertility end points. These results led us to focus specifically on possible molecular abnormalities in sperm. Sperm samples were recovered from the frozen epididymides of rats exposed during the previous study. The proteins present in the samples from six exposed and six control rats were analyzed in pairs, by two-dimensional fluorescence difference gel electrophoresis, to investigate possible exposure-induced changes to sperm protein profiles. Twelve proteins, from the 380 matched spots observed in at least five gels, were present in larger or smaller amounts after vinclozolin exposure. These proteins were identified by mass spectrometry, and several are known to play a crucial role in the sperm fertilizing ability, among which, two mitochondrial enzymes, malate dehydrogenase 2 and aldehyde dehydrogenase (both of which were present in smaller amounts after treatment) and A-kinase anchor protein 4 (larger amounts of precursor after treatment). Finally, Ingenuity Pathway Analysis revealed highly significant interactions between proteins over- and underexpressed after treatment. This is the first study to show an association between in vivo exposure to an ED and changes to the sperm protein profile. These modifications may be at least partly responsible for the reproductive abnormalities and impaired fertility recently reported in this rat model of vinclozolin exposure.

Simultaneous determination of nicotine, cotinine, and nicotine N-oxide in human plasma, semen, and sperm by LC-Orbitrap MS.

PubMed

Abu-Awwad, Ahmad; Arafat, Tawfiq; Schmitz, Oliver J

2016-09-01

Nicotine (Nic) distribution in human fluids and tissues has a deleterious effect on human health. In addition to its poisoning profile, Nic may contribute to the particular impact of smoking on human reproduction. Although present in seminal fluid, still nobody knows whether nicotine is available in sperm or not. Herein, we developed and validated a new bioanalytical method, for simultaneous determination of Nic, cotinine (Cot), and nicotine N'-oxide (Nox) in human plasma, semen, and sperm by LC-ESI-orbitrap-MS. Blood and semen samples were collected from 12 healthy smoking volunteers in this study. Sperm bodies were then separated quantitatively from 1 mL of semen samples by centrifugation. The developed method was fully validated for plasma following European and American guidelines for bioanalytical method validation, and partial validation was applied to semen analysis. Plasma, semen, and sperm samples were treated by trichloroacetic acid solution for protein direct precipitation in single extraction step. The established calibration range for Nic and Nox in plasma and semen was linear between 5 and 250 ng/mL, and for Cot between 10 and 500 ng/mL. Nic and Cot were detected in human sperm at concentrations as high as in plasma. In addition, Nox was present in semen and sperm but not in plasma. Graphical abstract Nicotine correlation between plasma and semen a; Nicotine correlation between semen and sperm c; Cotinine correlation between plasma and semen b; Cotinine correlation between semen and sperm d.

Structure and function of the undulating membrane in spermatozoan propulsion in the toad Bufo marinus

PubMed Central

1980-01-01

Accessory fibers in most sperm surround the axoneme so that their function in propulsion is difficult to assess. In the sperm of the toad Bufo marinus, an accessory fiber is displaced from the axoneme, being connected to it by the thin undulating membrane in such a way that the movement of axoneme and accessory fiber can be viewed independently. The axoneme is highly convoluted in whole mounts, and the axial fiber is straight. Cinemicrographic analysis shows that it is the longer, flexuous fiber, the presumed axoneme, that move actively. The accessory fiber follows it passively with a lower amplitude of movement. The accessory fiber does not move independent of the axoneme, even after demembranation and reactivation of the sperm. On the basis of anatomical relations in the neck region, it appears that the accessory fibers of amphibians are analogous to the dense fibers of mammalian sperm. SDS polyacrylamide gel electrophoresis of demembranated toad sperm tails reveals two principal proteins in addition to the tubulins, the former probably arising from the accessory fibers and the matrix of the undulating membrane. The function of displacing an accessory fiber into an undulating membrane may be to provide stiffness for the tail without incurring an energy deficit large enough to require a long middle piece. A long middle piece is not present in toad sperm, in contrast to those sperm that have accessory fibers around the axoneme. However, the toad sperm suffers a reduction in speed of about one- third, compared with the speed expected for a sperm without an undulating membrane. PMID:6771299

Outdoor air pollution and sperm quality.

PubMed

Lafuente, Rafael; García-Blàquez, Núria; Jacquemin, Bénédicte; Checa, Miguel Angel

2016-09-15

Exposure to air pollution has been clearly associated with a range of adverse health effects, including reproductive toxicity, but its effects on male semen quality are still unclear. We performed a systematic review (up to June 2016) to assess the impact of air pollutants on sperm quality. We included 17 semi-ecological, panel, and cohort studies, assessing outdoor air pollutants, such as PM2.5, PM10, NOx, SO2, and O3, and their effects on DNA fragmentation, sperm count, sperm motility, and sperm morphology. Thirteen studies assessed air pollution exposure measured environmentally, and six used biomarkers of air pollution exposure (two did both). We rated the studies using the Newcastle-Ottawa Scale and assessed with the exposure method. Taking into account these factors and the number of studies finding significant results (positive or negative), the evidence supporting an effect of air pollution on DNA fragmentation is weak but suggestive, on sperm motility is limited and probably inexistent, on lower sperm count is inconclusive, and on sperm morphology is very suggestive. Because of the diversity of air pollutants and sperm parameters, and the studies' designs, we were unable to perform a meta-analysis. In summary, most studies concluded that outdoor air pollution affects at least one of the four semen quality parameters included in the review. However, results lack consistency, and furthermore, studies were not comparable. Studies using standardized air pollution and semen measures are required to obtain more reliable conclusions. CRD42015007175. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. 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

Incubation of boar spermatozoa in viscous media by addition of methylcellulose improves sperm quality and penetration rates during in vitro fertilization.

PubMed

González-Abreu, David; García-Martínez, Soledad; Fernández-Espín, Vanesa; Romar, Raquel; Gadea, Joaquín

2017-04-01

This work was designed to study whether viscous media can improve the in vitro sperm functionality in pigs by using methylcellulose as a thickener. Viscosity of porcine oviductal fluid (POF) was compared with culture medium (Tyrode's) supplemented with methylcellulose (MET 0, 0.5 and 1% w/v). Spermatozoa were incubated in the different media (0, 1 and 2 h) and sperm motion parameters, lipid membrane disorder, plasma membrane integrity and reactive oxygen species (ROS) formation were assessed. Fertilization results were assessed i) preincubating spermatozoa in the viscous media followed by gamete coculture in a non-viscous medium; and ii) gamete coculture in the viscous media. Viscosity of POF from early luteal phase was higher than late follicular phase. Medium without methylcellulose presented constant viscosity with increased shear rate, while viscosity of the POF and media with methylcellulose was reduced by increased shear rates. Methylcellulose improved sperm linearity, straightness and the proportion of fast-linear spermatozoa. Moreover, methylcellulose increased the rate of viable spermatozoa with intact acrosome and low lipid disorder, reducing the ROS generation. Preincubation in viscous media increased the penetration rate and the mean number of spermatozoa bound to the zona pellucida (both with 0.5 and 1% MET) and reduced monospermy with 1% MET. On the other hand fertilization in the viscous media reduced penetration rate and increased monospermy. The efficiency of the IVF system was not improved with the use of viscous media. The results show the relevance of increasing viscosity thus making the in vitro media more comparable to physiological conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

The post-thaw quality of ram sperm held for 0 to 48 h at 5 degrees C prior to cryopreservation.

PubMed

Purdy, P H

2006-06-01

The effects of holding diluted ram semen at 5 degrees C for up to 48 h prior to cryopreservation were investigated. Semen from six rams was collected by electro-ejaculation in the autumn and again from six different rams in the spring. The sperm concentration and motility were determined using spectrophotometry and computerized automated semen analysis, respectively. Samples were diluted at 23 degrees C to 400 x 10(6)cells/ml in a one-step Tris-egg yolk-glycerol (5%, v/v) media, cooled to 5 degrees C over 2h and maintained at 5 degrees C for the duration of the experiments. Aliquots were loaded into 0.5 ml French straws at 0, 24 or 48 h after cooling, frozen in liquid nitrogen vapor for 12-13 min, 4.5 cm above the liquid nitrogen, and plunged into liquid nitrogen for storage. After thawing, autumn samples frozen after 0, 24, or 48 h of storage exhibited similar percentages of motility (29, 31, 36%, respectively), progressively motility (16, 15, 17%, respectively), plasma membrane integrity (28, 35, 29%, respectively) and live acrosome-reacted cells (0.4, 0.6, 0.8%, respectively; P>0.05). In addition, the quantity of sperm that bound to hen's egg perivitelline membranes after being held at 5 degrees C for 0, 24, or 48 h was not significantly different when the values were expressed as means of the quantity of sperm (155, 177, 106 sperm, respectively) or as the proportion of sperm inseminated (0.39, 0.49, 0.34, respectively; P>0.05). Likewise, ram sperm collected in the spring and frozen at 0, 24 and 48 h after cooling had similar (P>0.05) total motility (21, 25, 20%, respectively), progressive motility (14, 15, 11%, respectively), plasma membrane integrity (26, 33, 31%, respectively) and live acrosome-reacted cells (3.7, 3.5, 3.2%, respectively; P>0.05). The 0 h holding time had significantly less sperm bound to a hen's egg perivitelline membrane compared to the 48 h holding time (250 and 470 sperm, respectively) although the 24h holding time was not different from the 0 or 48 h holding time (281 sperm; P<0.05) but analysis of the proportion of the total sperm inseminated resulted in no significant differences observed (P>0.05). These results indicate that ram sperm can be held at 5 degrees C for up to 48 h prior to freezing with no injurious effects on motility, membrane integrity, or fertilizing potential as indicated by membrane binding ability.

Analysis of mutational changes at the HLA locus in single human sperm.

PubMed

Huang, M M; Erlich, H A; Goodman, M F; Arnheim, N

1995-01-01

Using a simple and efficient single sperm PCR and direct sequencing method, we screened for HLA-DPB1 gene mutations that may give rise to new alleles at this highly polymorphic locus. More than 800 single sperm were studied from a heterozygous individual whose two alleles carried 16 nucleotide sequence differences clustered in six polymorphic regions. A potential microgene conversion event was detected. Unrepaired heteroduplex DNA similar to that which gives rise to postmeiotic segregation events in yeast was observed in three cases. Control experiments also revealed unusual sperm from DPB1 homozygous individuals. The data may help explain allelic diversity in the MHC and suggest that a possible source of human mosaicism may be incomplete DNA mismatch repair during gametogenesis.

Advances in understanding paternally transmitted Chromosomal Abnormalities

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

Marchetti, F; Sloter, E; Wyrobek, A J

2001-03-01

Multicolor FISH has been adapted for detecting the major types of chromosomal abnormalities in human sperm including aneuploidies for clinically-relevant chromosomes, chromosomal aberrations including breaks and rearrangements, and other numerical abnormalities. The various sperm FISH assays have been used to evaluate healthy men, men of advanced age, and men who have received mutagenic cancer therapy. The mouse has also been used as a model to investigate the mechanism of paternally transmitted genetic damage. Sperm FISH for the mouse has been used to detect chromosomally abnormal mouse sperm, while the PAINT/DAPI analysis of mouse zygotes has been used to evaluate themore » types of chromosomal defects that can be paternally transmitted to the embryo and their effects on embryonic development.« less

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.

Utility of light scatter in the morphological analysis of sperm

EPA Science Inventory

We were able to differentiate the morphologically diverse sperm nuclei of four animal species by using an Ortho flow cytometer to detect the forward light scatter from a red (helium-neon) laser. Cytograms depicting the axial light loss and forward red scatter signals revealed uni...

INDIVIDUAL VARIATION IN THE FREQUENCY OF SPERM ANEUPLOIDY IN HUMANS

EPA Science Inventory

To examine interindividual differences in sperm chromosome aneuploidy, repeated semen specimens were obtained from a group of ten healthy men, aged 20-21 at the start of the study, and analyzed by multi-color fluorescence in situ hybridization (FISH) analysis to determine the fre...

EFFECT OF CRYOPRESERVATION AND THEOPHYLLINE ON MOTILITY CHARACTERISTICS OF LAKE STURGEON (ACIPENSER FULVESCENS) SPERMATOZOA

EPA Science Inventory

Computer-assisted motility analysis (CASA) was used to evaluate the effect of cryopreservation and theophylline treatment on sperm motility of lake sturgeon (Acipenser fulvescens).Motility was recorded at 0 and 5 min postactivation.The effect of cryopreservation on sperm acrosin-...

Initial analysis of sperm DNA methylome in Holstein bulls

USDA-ARS?s Scientific Manuscript database

Aberrant DNA methylation patterns have been associated with abnormal semen parameters, idiopathic male infertility and early embryonic loss in mammals. Using Holstein bulls with high (Bull1) or low (Bull2) fertility rates, we created two representative sperm DNA methylomes at a single-base resolutio...

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

Abundance and Distribution of Sperm Whales in the Canary Islands: Can Sperm Whales in the Archipelago Sustain the Current Level of Ship-Strike Mortalities?

PubMed

Fais, Andrea; Lewis, Tim P; Zitterbart, Daniel P; Álvarez, Omar; Tejedor, Ana; Aguilar Soto, Natacha

2016-01-01

Sperm whales are present in the Canary Islands year-round, suggesting that the archipelago is an important area for this species in the North Atlantic. However, the area experiences one of the highest reported rates of sperm whale ship-strike in the world. Here we investigate if the number of sperm whales found in the archipelago can sustain the current rate of ship-strike mortality. The results of this study may also have implications for offshore areas where concentrations of sperm whales may coincide with high densities of ship traffic, but where ship-strikes may be undocumented. The absolute abundance of sperm whales in an area of 52933 km2, covering the territorial waters of the Canary Islands, was estimated from 2668 km of acoustic line-transect survey using Distance sampling analysis. Data on sperm whale diving and acoustic behaviour, obtained from bio-logging, were used to calculate g(0) = 0.92, this is less than one because of occasional extended periods when whales do not echolocate. This resulted in an absolute abundance estimate of 224 sperm whales (95% log-normal CI 120-418) within the survey area. The recruitment capability of this number of whales, some 2.5 whales per year, is likely to be exceeded by the current ship-strike mortality rate. Furthermore, we found areas of higher whale density within the archipelago, many coincident with those previously described, suggesting that these are important habitats for females and immature animals inhabiting the archipelago. Some of these areas are crossed by active shipping lanes increasing the risk of ship-strikes. Given the philopatry in female sperm whales, replacement of impacted whales might be limited. Therefore, the application of mitigation measures to reduce the ship-strike mortality rate seems essential for the conservation of sperm whales in the Canary Islands.

Abundance and Distribution of Sperm Whales in the Canary Islands: Can Sperm Whales in the Archipelago Sustain the Current Level of Ship-Strike Mortalities?

PubMed Central

Fais, Andrea; Lewis, Tim P.; Zitterbart, Daniel P.; Álvarez, Omar; Tejedor, Ana; Aguilar Soto, Natacha

2016-01-01

Sperm whales are present in the Canary Islands year-round, suggesting that the archipelago is an important area for this species in the North Atlantic. However, the area experiences one of the highest reported rates of sperm whale ship-strike in the world. Here we investigate if the number of sperm whales found in the archipelago can sustain the current rate of ship-strike mortality. The results of this study may also have implications for offshore areas where concentrations of sperm whales may coincide with high densities of ship traffic, but where ship-strikes may be undocumented. The absolute abundance of sperm whales in an area of 52933 km2, covering the territorial waters of the Canary Islands, was estimated from 2668 km of acoustic line-transect survey using Distance sampling analysis. Data on sperm whale diving and acoustic behaviour, obtained from bio-logging, were used to calculate g(0) = 0.92, this is less than one because of occasional extended periods when whales do not echolocate. This resulted in an absolute abundance estimate of 224 sperm whales (95% log-normal CI 120–418) within the survey area. The recruitment capability of this number of whales, some 2.5 whales per year, is likely to be exceeded by the current ship-strike mortality rate. Furthermore, we found areas of higher whale density within the archipelago, many coincident with those previously described, suggesting that these are important habitats for females and immature animals inhabiting the archipelago. Some of these areas are crossed by active shipping lanes increasing the risk of ship-strikes. Given the philopatry in female sperm whales, replacement of impacted whales might be limited. Therefore, the application of mitigation measures to reduce the ship-strike mortality rate seems essential for the conservation of sperm whales in the Canary Islands. PMID:26999791

Novel Insights into DNA Methylation Features in Spermatozoa: Stability and Peculiarities

PubMed Central

Sayols, Sergi; Chianese, Chiara; Giachini, Claudia; Heyn, Holger; Esteller, Manel

2012-01-01

Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic and cancer cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research. PMID:23071498

Rapid evolution of asymmetric reproductive incompatibilities in stalk-eyed flies.

PubMed

Rose, Emily G; Brand, Cara L; Wilkinson, Gerald S

2014-02-01

The steps by which isolated populations acquire reproductive incompatibilities remain poorly understood. One potentially important process is postcopulatory sexual selection because it can generate divergence between populations in traits that influence fertilization success after copulation. Here we present a comprehensive analysis of this form of reproductive isolation by conducting reciprocal crosses between variably diverged populations of stalk-eyed flies (Teleopsis dalmanni). First, we measure seven types of reproductive incompatibility between copulation and fertilization. We then compare fertilization success to hatching success to quantify hybrid inviability. Finally, we determine if sperm competition acts to reinforce or counteract any incompatibilities. We find evidence for multiple incompatibilities in most crosses, including failure to store sperm after mating, failure of sperm to reach the site of fertilization, failure of sperm to fertilize eggs, and failure of embryos to develop. Local sperm have precedence over foreign sperm, but this effect is due mainly to differences in sperm transfer and reduced hatching success. Crosses between recently diverged populations are asymmetrical with regard to the degree and type of incompatibility. Because sexual conflict in these flies is low, postcopulatory sexual selection, rather than antagonistic coevolution, likely causes incompatibilities due to mismatches between male and female reproductive traits. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

Test of Fruit Extract Pare (Momordica charantia L.) to Quality of Ejaculated Spermatozoa Mice (Mus musculus L.)

NASA Astrophysics Data System (ADS)

Fifendy, M.; Indriati, G.

2018-04-01

Pare (Momordica charantia L.) can be used in the treatment of various diseases, such as influenza, cancer, anti-inflammatory, anti-HIV, antimitotic and antifertilitas. This study aimed to determine the effect of the herbal bitter (Momordica charantia L.) to ejaculated sperm quality mice (Mus musculus L.). This research was conducted using Completely Randomized Design (CRD) with 4 treatments and 6 replications, water and fed adlibitum. First treatment is given solvent extract. Second treatments extract were given 0.2 gram, third treatment were given 0.4 gram of extracts and fourth treatment were treated exstrac 0.6 gram were orally for 30 days. After the mice decapitated, dissected and take sperm from vas deferens. Then, the sperm preparation determined using the improved Neubauer. Data were analyzed by ANOVA (Analysis of Varians). The results shoured at doses of 0,2 gram, the average sperm count was 19.89. decrease significant when compared with the control in which the average number of sperm 29.13. So with this research the effective doses to decrease sperm count and can be used as a contraception medication dosage was 0,2 gram. It can be conclude that the extract of bitter (Momordica charantia L.) can decrease the quality of the ejaculated sperm of mice (Mus musculus L.)

Comparison of intracytoplasmic sperm injection outcomes between spermatozoa retrieved from testicular biopsy and from ejaculation in cryptozoospermic men.

PubMed

Amirjannati, N; Heidari-Vala, H; Akhondi, M A; Hosseini Jadda, S H; Kamali, K; Sadeghi, M R

2012-05-01

The infrequent presence of spermatozoa in cryptozoospermic men ejaculate is a limiting factor in the treatment of them. Sometimes, this consideration impels us to apply meticulous microscopic search in ejaculate or testicular sperm extraction (TESE) method. The aim of this study was to assess putative effectiveness of sperm origin, ejaculated or testicular, in cryptozoospermia treatment. In this context, were evaluated intracytoplasmic sperm injection (ICSI) outcomes in two parameters including fertilisation rate (2PN) and embryo quality, independently. We compared the outcome in two groups: patients who underwent ejaculate/ICSI and ones who underwent TESE/ICSI process. Nineteen ICSI cycles performed with testicular spermatozoa and the rest of cycles (n = 208) carried out with ejaculated spermatozoa. Result analysis showed similar fertilisation rate between testicular and ejaculated spermatozoa (respectively, 60% versus 68%, P ≥ 0.05). Also, on the other hand, embryo quality did not show significant differences between two groups, except grade A with low significance. With regard to almost equal performance of both methods in results and being invasive of TESE as surgical sperm retrieval method, the use of ejaculated sperm more than testicular sperm should be recommended in patients with cryptozoospermia whenever possible. © 2011 Blackwell Verlag GmbH.

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.

Monoclonal antibodies, immunofluorometric assay, and detection of human semenogelin in male reproductive tract: no association with in vitro fertilizing capacity of sperm.

PubMed

Koistinen, Hannu; Soini, Tuuli; Leinonen, Jari; Hyden-Granskog, Christel; Salo, Jaakko; Halttunen, Mervi; Stenman, Ulf-Håkan; Seppälä, Markku; Koistinen, Riitta

2002-03-01

Semenogelin plays an important role in sperm clotting and is degraded into smaller fragments by prostate-specific antigen (PSA) during clot liquefaction. Semenogelin and its fragments inhibit sperm motility in vitro. We studied the expression of semenogelin I mRNA and its localization in various tissues of the male genital tract. We also studied semenogelin concentrations with respect to sperm parameters and the outcome of in vitro fertilization. Semenogelin protein was detected by immunohistochemical staining and semenogelin I mRNA was detected by Northern blot analysis in the seminal vesicles and ampullary part of the vas deferens, whereas specimens from the prostate, epididymis, testis, and the female genital tract were negative. Using monoclonal antibodies against semenogelin, an immunofluorometric assay was developed to measure semenogelin levels in seminal plasma and to evaluate possible correlations with sperm parameters and fertilization in vitro. No correlation was found between the semenogelin concentration and the volume of the ejaculate, sperm concentration, sperm motility, or in vitro fertilization rate. Semenogelin levels were positively correlated with the total protein concentration in seminal plasma, and there was an inverse correlation between the concentration of semenogelin and that of PSA. The levels of semenogelin appear to bear no relationship to the in vitro fertilization capacity of the spermatozoa.

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

Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization.

PubMed

Tavares, R S; Silva, A F; Lourenço, B; Almeida-Santos, T; Sousa, A P; Ramalho-Santos, J

2013-11-01

Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post-prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff-Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p < 0.001), but no correlations were found with fertilization or embryo development rates (p > 0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p < 0.05) and clinical pregnancy among IVF couples (p < 0.05). Furthermore, regression analysis confirmed the clinical value of Diff-Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871-0.985, p = 0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9-fold when the percentage of abnormal dark staining was ≥34.25% (p = 0.05). In conclusion, the Diff-Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of 'natural' sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low-cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well-established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling) are available. © 2013 American Society of Andrology and European Academy of Andrology.

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.

Manual vs. computer-assisted sperm analysis: can CASA replace manual assessment of human semen in clinical practice?

PubMed

Talarczyk-Desole, Joanna; Berger, Anna; Taszarek-Hauke, Grażyna; Hauke, Jan; Pawelczyk, Leszek; Jedrzejczak, Piotr

2017-01-01

The aim of the study was to check the quality of computer-assisted sperm analysis (CASA) system in comparison to the reference manual method as well as standardization of the computer-assisted semen assessment. The study was conducted between January and June 2015 at the Andrology Laboratory of the Division of Infertility and Reproductive Endocrinology, Poznań University of Medical Sciences, Poland. The study group consisted of 230 men who gave sperm samples for the first time in our center as part of an infertility investigation. The samples underwent manual and computer-assisted assessment of concentration, motility and morphology. A total of 184 samples were examined twice: manually, according to the 2010 WHO recommendations, and with CASA, using the program set-tings provided by the manufacturer. Additionally, 46 samples underwent two manual analyses and two computer-assisted analyses. The p-value of p < 0.05 was considered as statistically significant. Statistically significant differences were found between all of the investigated sperm parameters, except for non-progressive motility, measured with CASA and manually. In the group of patients where all analyses with each method were performed twice on the same sample we found no significant differences between both assessments of the same probe, neither in the samples analyzed manually nor with CASA, although standard deviation was higher in the CASA group. Our results suggest that computer-assisted sperm analysis requires further improvement for a wider application in clinical practice.

Semen cryopreservation in pubertal boys before gonadotoxic treatment and the role of endocrinologic evaluation in predicting sperm yield.

PubMed

van Casteren, Niels J; Dohle, Gert R; Romijn, Johanens C; de Muinck Keizer-Schrama, Sabine M P F; Weber, Robertus F A; van den Heuvel-Eibrink, Marry M

2008-10-01

To evaluate the feasibility of semen cryopreservation in pubertal boys before they receive gonadotoxic therapy and to identify which pretreatment parameters might predict successful cryopreservation. Retrospective data analysis. Tertiary fertility center, academic children's hospital. Between 1995 and 2005, 80 boys (median age 16.6 years, range 13.7-18.9 years) consulted the outpatient clinic of andrology for semen cryopreservation before a potentially gonadotoxic treatment. We assessed the pretreatment semen parameters, hormone levels, and patients' characteristics. Measurement of the number of adolescents able to cryopreserve semen. Thirteen boys were unable to produce semen by masturbation. In 53 boys semen quality was adequate for cryopreservation. In 14 patients semen analysis did not show motile spermatozoa, and therefore semen cryopreservation could not be performed. Although inhibin B showed a strong correlation with sperm count, no significant difference was found in serum T, inhibin B, LH, and FSH levels in the patients with or without successful sperm yield. Moreover, median age was not different between patients with and without a successful sperm yield. Semen cryopreservation in boys is a feasible method to preserve spermatozoa before gonadotoxic therapy is started and should be offered to all pubertal boys despite their young age. Serum hormone levels do not predict sperm yield.

Unlocking the secrets of multi-flagellated propulsion: drawing insights from Tritrichomonas foetus

PubMed Central

Lenaghan, Scott C.; Nwandu-Vincent, Stefan; Reese, Benjamin E.; Zhang, Mingjun

2014-01-01

In this work, a high-speed imaging platform and a resistive force theory (RFT) based model were applied to investigate multi-flagellated propulsion, using Tritrichomonas foetus as an example. We discovered that T. foetus has distinct flagellar beating motions for linear swimming and turning, similar to the ‘run and tumble’ strategies observed in bacteria and Chlamydomonas. Quantitative analysis of the motion of each flagellum was achieved by determining the average flagella beat motion for both linear swimming and turning, and using the velocity of the flagella as inputs into the RFT model. The experimental approach was used to calculate the curvature along the length of the flagella throughout each stroke. It was found that the curvatures of the anterior flagella do not decrease monotonically along their lengths, confirming the ciliary waveform of these flagella. Further, the stiffness of the flagella was experimentally measured using nanoindentation, allowing for calculation of the flexural rigidity of T. foetus's flagella, 1.55×10−21 N m2. Finally, using the RFT model, it was discovered that the propulsive force of T. foetus was similar to that of sperm and Chlamydomonas, indicating that multi-flagellated propulsion does not necessarily contribute to greater thrust generation, and may have evolved for greater manoeuvrability or sensing. The results from this study have demonstrated the highly coordinated nature of multi-flagellated propulsion and have provided significant insights into the biology of T. foetus. PMID:24478286

Finite cell lines of turkey sperm storage tubule cells: ultrastructure and protein analysis

USDA-ARS?s Scientific Manuscript database

Cell lines of turkey sperm storage tubule (SST) epithelial cells were established. Turkey SSTs were dissected from freshly obtained uterovaginal junction (UVJ) tissue and placed in explants culture on various substrates and media. Primary cultures of SST epithelium only survived and grew from SST ex...

EVALUATION OF SPERM CHROMATIN STRUCTURE ASSAY (SCSA REGISTERED TRADEMARK) IN HUMAN SPERM AFTER SIMULATED OVERNIGHT SHIPMENT

EPA Science Inventory

Home semen collection kits allow men to collect a sample at their convenience and send it via overnight mail to the laboratory. Benefits of this approach include facilitated sample collection from different geographic locations, minimized variability through analysis by a central...

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

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.

The Effects of Cell Phone Waves (900 MHz-GSM Band) on Sperm Parameters and Total Antioxidant Capacity in Rats.

PubMed

Ghanbari, Masoud; Mortazavi, Seyed Bagher; Khavanin, Ali; Khazaei, Mozafar

2013-04-01

There is tremendous concern regarding the possible adverse effects of cell phone microwaves. Contradictory results, however, have been reported for the effects of these waves on the body. In the present study, the effect of cell phone microwaves on sperm parameters and total antioxidant capacity was investigated with regard to the duration of exposure and the frequency of these waves. This experimental study was performed on 28 adult male Wistar rats (200-250 g). The animals were randomly assigned to four groups (n=7): i. control; ii. two-week exposure to cell phone-simulated waves; iii. three-week exposure to cell phonesimulated waves; and iv. two-week exposure to cell phone antenna waves. In all groups, sperm analysis was performed based on standard methods and we determined the mean sperm total antioxidant capacity according to the ferric reducing ability of plasma (FRAP) method. Data were analyzed by one-way ANOVA followed by Tukey's test using SPSS version 16 software. The results indicated that sperm viability, motility, and total antioxidant capacity in all exposure groups decreased significantly compared to the control group (p<0.05). Increasing the duration of exposure from 2 to 3 weeks caused a statistically significant decrease in sperm viability and motility (p<0.05). Exposure to cell phone waves can decrease sperm viability and motility in rats. These waves can also decrease sperm total antioxidant capacity in rats and result in oxidative stress.

Lactoferrin increases sperm membrane functionality of frozen equine semen.

PubMed

Martins, H S; da Silva, G C; Cortes, S F; Paes, F O; Martins Filho, O A; Araujo, Mss; Stahlberg, R; Lagares, M A

2018-06-01

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents. © 2018 Blackwell Verlag GmbH.

Effect of 655 nm laser different powers on dog sperm motility parameters

NASA Astrophysics Data System (ADS)

Corral-Baqués, M. I.; Rigau, T.; Rivera, M. M.; Rodríguez-Gil, J. E.; Rigau, J.

2006-04-01

Introduction: One of the most appreciated features of the sperm is its motility, which depends on a big energy consumption despite differences among species. Laser acts direct or indirectly on mitochondria increasing ATP production. Material and method: By means of a Computer Aided Sperm Analysis (CASA) we have studied the effects of a 655 nm continuous wave diode laser irradiation at different power outputs with a dose of 3.3418 J on sperm motility. After an eosine-nigrosine stain to establish its quality, the second fraction of fresh beagle dog sperm was divided into 5 groups, 1 control and four to be irradiated respectively with an average output power of 6.84 mW, 15.43 mW, 33.05 mW and 49.66 mW. At times 0 and 45 minutes from irradiation pictures were taken and analysed with the Sperm class Analyzer SCA2002 programme. The motility parameters of 4987 spermatozoa studied were: curvilinear velocity (VCL), progressive velocity (VSL), straightness (STR), wobble (WOB), average path velocity (VAP), linearity (LIN), mean amplitude of lateral head displacement (ALHmed), beat cross frequency (BCF) and the total motility (MT). At time 15 minutes after irradiation a hypoosmotic swelling test (HOST) was done. Results: Several motility parameters that affect the overall motile sperm subpopulation structure have been changed by different output powers of a 655 nm diode laser irradiation, and prevents the decrease of the sperm motility properties along time.

Comparison of in vitro fertilization/intracytoplasmic sperm injection outcomes in male factor infertility patients with and without spinal cord injuries.

PubMed

Kathiresan, Anupama S Q; Ibrahim, Emad; Aballa, Teodoro C; Attia, George R; Ory, Steven J; Hoffman, David I; Maxson, Wayne S; Barrionuevo, Marcelo J; Lynne, Charles M; Brackett, Nancy L

2011-09-01

To determine if outcomes after in vitro fertilization with intracytoplasmic sperm injection (IVF/ICSI) using sperm from men with spinal cord injury (SCI group) differ from those of other etiologies of male factor infertility (non-SCI group). In men with SCI, to determine if IVF/ICSI outcomes differ with sperm obtained by penile vibratory stimulation (PVS group) versus electroejaculation (EEJ group). Retrospective analysis. University medical center and major infertility center. Couples with male factor infertility due to SCI versus other etiologies. PVS, EEJ, surgical sperm retrieval, and IVF/ICSI. Rates of fertilization, pregnancy, and live birth. A total of 31 couples in the SCI group underwent 48 cycles of IVF/ICSI, and a total of 297 couples in the non-SCI group underwent 443 cycles of IVF/ICSI. The SCI group had lower fertilization rates but similar pregnancy and live birth rates compared with the non-SCI group. These rates, however, did not differ significantly when the PVS group was compared with the EEJ group. IVF/ICSI of sperm from men with SCI yield lower fertilization rates but similar pregnancy and live birth outcomes as IVF/ICSI of sperm from men with other etiologies of male factor infertility. Sperm collected by PVS versus EEJ in men with SCI appear to result in similar IVF/ICSI success rates. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Reliable collection of Caspian brown trout (Salmo trutta caspius) sperm using a catheter.

PubMed

Aramli, M S; Golshahi, K; Banan, A; Sotoudeh, E

2016-10-01

The traditional stripping procedure for collecting fish semen is associated with the risk of urine contamination, which may significantly affect semen quality and quantity. The use of a catheter as an alternative method for semen collection may overcome this problem. Therefore, this study compared Caspian brown trout (Salmo trutta caspius) semen parameters (i.e. sperm density, seminal plasma osmolality, motility parameters of spermatozoa analysed using computer-assisted sperm analysis and fertility) between the traditional stripping method and the use of a catheter. All parameter values of the semen collected with a catheter were significantly higher (p < .05; density = 7.67 ± 1.02 × 10(9)  ml(-1) and osmolality = 279.28 ± 32.84 mOsm kg(-1) ) than those collected with stripping method (density = 4.85 ± 0.47 × 10(9)  ml(-1) and osmolality = 216.42 ± 20.75 mOsm kg(-1) ). Semen collected with a catheter was characterized by higher spermatozoa motility compared with sperm collected via stripping. Similarly, the fertilization ability of sperm collected with a catheter was significantly greater (p < .05) than sperm collected with the traditional stripping method. In conclusion, collection of sperm with a catheter was shown to effectively reduce urine contamination and is therefore recommended for the collection of Caspian brown trout sperm. © 2016 Blackwell Verlag GmbH.

Mono or bilateral inflammatory postmicrobial prostato-vesciculo-epididymitis: differences in semen parameters and reactive oxygen species production.

PubMed

La Vignera, S; Calogero, A E; Cannizzaro, M A; Vicari, E

2006-12-01

The aim of this study was to analyse possible differences in sperm characteristics (including seminal leukocyte concentration, number of spermiophagies, and sperm reactive oxygen species [ROS] production) between patients with monolateral and bilateral prostate-vesciculo-epididymitis (PVE). Seventy-eight selected consecutively enrolled patients (mean age 34 years, range 24-40) with chronic inflammatory postmicrobial PVE and 30 infertile (aged-matched) patients (control group) with inflammatory postmicrobial prostatitis underwent sperm analysis, including seminal leukocyte concentration, number of spermiophages, and ROS production. Sperm concentration and total sperm number were significantly lower in the patients with bilateral PVE than in those with monolateral PVE. The percentage of cases with oligozoospermia or hyperspermia was significantly higher among patients with bilateral PVE with than those with monolateral PVE. Although the remaining cytologic and physicochemical sperm characteristics and ROS production (both in 45% and 90% Percoll fractions) were not significantly different between the 2 PVE groups, both groups showed median and relative percentages of frequency of abnormal sperm characteristics worse than those in the control group. Although sperm abnormalities (including seminal leukocyte concentration, number of spermiophages, and ROS production) can distinguish patients with PVE from those with prostatitis alone, the abnormalities did not discriminate between unilateral and bilateral PVE. In diagnostic suspicion of PVE, didymo-epididymal and prostato-vescicular ultrasonography can help discriminate monolateral from bilateral forms of PVE, and is a useful aid in the follow-up of these patients.

Heritability of semen traits in German Warmblood stallions.

PubMed

Gottschalk, M; Sieme, H; Martinsson, G; Distl, O

2016-07-01

The objectives of the present study were to evaluate genetic parameters for semen quality traits of 241 fertile German Warmblood stallions regularly employed in artificial insemination (AI). Stallions were owned by the National Studs Celle and Warendorf in Germany. Semen traits analyzed were gel-free volume, sperm concentration, total number of sperm, progressive motility and total number of progressively motile sperm. Semen protocols from a total of 63,972 ejaculates were collected between the years 2001 and 2014 for the present analysis. A multivariate linear animal model was employed for estimation of additive genetic and permanent environmental variances among stallions and breeding values (EBVs) for semen traits. Heritabilities estimated for all German Warmblood stallions were highest for gel-free volume (h(2)=0.28) and lowest for total number of progressively motile sperm (h(2)=0.13). The additive genetic correlation among gel-free volume and sperm concentration was highly negative (rg=-0.76). Average reliabilities of EBVs were at 0.37-0.68 for the 241 stallions with own records. The inter-stallion variance explained between 33 and 61% of the trait variance, underlining the major impact of the individual stallion on semen quality traits analyzed here. Recording of semen traits from stallions employed in AI may be recommended because EBVs achieve sufficient accuracies to improve semen quality in future generations. Due to favorable genetic correlations, sperm concentration, total number of sperm and total number of progressively motile sperm may be increased simultaneously. Copyright © 2016 Elsevier B.V. All rights reserved.

Quality of Bovine Chilled or Frozen-Thawed Semen after Addition of Omega-3 Fatty Acids Supplementation to Extender

PubMed Central

Abavisani, Abbas; Arshami, Javad; Naserian, Abbas Ali; Sheikholeslami Kandelousi, Mohammad Ali; Azizzadeh, Mohammad

2013-01-01

Background: This study was conducted to evaluate the potential protective effects of omega-3 poly unsaturated fatty acids (Ω-3 PUFAs) on bovine sperm quality in response to cooling and cryopreservation. Materials and Methods: In this experimental study included ejaculates from five proven fertile bulls, allocated to the control and the four experimental groups. For group 1, polyethylene glycol (PEG) as a solvent was added alone to the extender, while for groups 2, 3 and 4, different concentration of omega-3 PUFAs (1, 2.5 and 5%, respectively) in combination with PEG were added to the semen extender. Motility [using computer aided sperm analysis (CASA)], viability and morphology of bovine sperm were investigated after 24 and 48 hours in both cold liquid storage and frozen-thawed conditions. Results: Our findings showed that PEG has some detrimental effects on sperm quality. Cooling as well as cryopreservation decreased significantly most of measured variables of sperm as compared to fresh semen, whereas the treatments did not improve sperm quality. Furthermore, levels of some variables were decreased significantly during treatments (p<0.05). Conclusion: Addition of Ω-3 PUFAs to semen extenders cannot be effectively introduced to conservation media as well as sperm membrane in order to protect spermatozoa in response to cooling and freezing. It can be suggested if Ω-3 PUFAs is supplemented to the diet of bulls in order to modify the fatty acid compositions of sperm, they might perform their preventive properties. PMID:24520481