Sperm Quality Variables Research Articles

Effect of exogenous sperm capacitation inducers on stallion sperm

Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse.

Sperm melatonin receptors, seminal plasma melatonin and semen freezability in goats

Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season.

Canine sperm motility is associated with telomere shortening and changes in expression of shelterin genes

BackgroundMotion quality is a critical property for essential functions. Several endogenous and exogenous factors are involved in sperm motility. Here, we measured the relative telomere length and evaluated the gene expression of its binding-proteins, shelterin complex (TRF1, TRF2, RAP1, POT1, TIN2, and TPP1) in sperm of dogs using relative quantitative real-time PCR. We compared them between two sperm subpopulations with poor and good motion qualities (separated by swim-up method). Telomere shortening and alterations of shelterin gene expression result from ROS, genotoxic insults, and genetic predisposition.ResultsSperm kinematic parameters were measured in two subpopulations and then telomeric index of each parameter was calculated. Telomeric index for linearity, VSL, VCL, STR, BCF, and ALH were significantly higher in sperms with good motion quality than in sperms with poor quality. We demonstrated that poor motion quality is associated with shorter telomere, higher expression of TRF2, POT1, and TIN2 genes, and lower expression of the RAP1 gene in dog sperm. The levels of TRF1 and TPP1 gene expression remained consistent despite variations in sperm quality and telomere length.ConclusionData provided evidence that there are considerable changes in gene expression of many shelterin components (TRF2, TIN2, POT1and RAP1) associated with shortening telomere in the spermatozoa with poor motion quality. Possibly, the poor motion quality is the result of defects in the shelterin complex and telomere length. Our data suggests a new approach in the semen assessment and etiologic investigations of subfertility or infertility in male animals.

Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Deleterious impact of COVID-19 pandemic: Male fertility was not out of the bag.

The emergence and the spread of coronavirus disease (COVID-19) induced by the SARS-CoV-2 virus has multiple consequences in all countries around the world. Male germ cells of infertile patients which are shown to be vulnerable to many environmental conditions, could be particularly vulnerable to such an exceptional pandemic situation. We aimed through the current study to investigate the potential variations in sperm quality of infertile patients during the COVID-19 pandemic in Tunisia. This was a cohort study including 90 infertile patients addressed to Laboratory of Cytogenetics and Reproductive Biology of Monastir Department of Maternity and Neonatology in Monastir, during the two first COVID-19 waves in Tunisia and who already have a spermogram before the pandemic period. We have pointed out a significant decrease in both total and progressive sperm motility during COVID-19 pandemic (p<0.0001 and p = 0.001 respectively). The percentage of morphologically abnormal spermatozoa increased from 90.99±7.38 to 93.67±4.55% during the pandemic (p< 0.001). The remaining sperm parameters were similar between the two compared timepoints. Interestingly, the univariate analysis didn't show any other associated factor to the observed impairment in sperm mobility and morphology. These data highlight the severe impact of the pandemic of the male reproductive health of hypofertile patients. Delaying infertility investigations and management after pandemic waves is recommended to hope a better gamete quality and hence to improve conception potential.

Ureaplasma urealyticum and Mycoplasma hominis urogenital infections associate with semen inflammation and decreased sperm quality.

Ureaplasma urealyticum and Mycoplasma hominis are among the most prevalent sexually transmitted infections proposed to induce urogenital inflammation and impair sperm quality. However, the topic remains controversial since contradictory findings have been reported. Herein, we performed a comprehensive analysis of U. urealyticum and M. hominis urogenital infections and their association with urogenital inflammation (i.e., leukocyte subsets and inflammatory cytokines in semen,) and sperm quality parameters in a cohort of men with couple's primary infertility undergoing initial infertility evaluation or with lower urinary tract symptoms and no infertility-related complaints. Overall, U. urealyticum and M. hominis infection was detected in 17.0% and 23.6% of patients, respectively, whereas the coinfection was detected in 3.8% of patients only. Remarkably, similar infection frequencies were found in the different patient subpopulations analyzed. Moreover, infections were associated with elevated semen levels of TNF, IL-1β, and IL-6 and/or increased counts of total leukocytes and their subsets, including CD4 and CD8 T lymphocytes and neutrophils. In addition, M. hominis infection and the coinfection with U. urealyticum were associated with impairments in sperm quality variables. Our results indicate that U. urealyticum and M. hominis male urogenital infections induce urogenital inflammation and decrease sperm quality, thus impairing male fertility potential. Screening for U. urealyticum and M. hominis infections and performing a comprehensive analysis of different leukocyte subsets and inflammatory cytokines in semen may be clinically helpful in the diagnosis and follow-up of male urogenital infection.

Characterization of boar semen microbiome and association with sperm quality parameters.

Elevated levels of bacteria within fresh extended boar semen are associated with decreased sperm longevity, therefore reducing the fertility of a semen dose. The objective of this study was to characterize the bacterial communities using 16S rRNA sequencing in freshly extended boar semen samples and relate the prevalence and diversity of the microbial population to sperm quality parameters 1) between studs, 2) between pooled and single-sire doses, and 3) over a 5-day period. Eight single-sire (n = 4 per stud) and eight pooled (n = 4 per stud) non-frozen extended semen doses were obtained from two boar studs (A and B). Pooled doses were the composite of the boar's ejaculates used in single-sire doses. Doses were subsampled for 5 d post-collection. Ten negative controls of each pooled dose (n = 2) and single-sire dose (n = 8) remained sealed until the last day. Microbiome analysis was achieved by examining the V4 hypervariable region of the 16S rRNA gene of flash-frozen samples. Two evaluators determined the average sperm motility and agglutination (0: no adhesion to 3: >50% adhesion) by averaging their estimates together at 10 random locations per slide. Stud A had greater sperm agglutination (1.6 vs. 1.0 ± 0.1; P < 0.01) than stud B. Sperm motility decreased over the 5-day period (P < 0.01) and tended (P = 0.09) to be greater in stud B than A (67.4% vs. 61.5% ± 0.02%). Compared with stud A, stud B had a greater relative abundance of Proteobacteria (60.0% vs. 47.2% ± 1.5%; P < 0.01) and a lower relative abundance of Firmicutes (22.5% vs. 31.9% ± 1.4%; P < 0.01). Moreover, stud A had a greater relative abundance of Bacteroidetes (6.3% vs. 5.3% ± 0.4%; P < 0.01) and Actinobacteria (11.5% vs. 10.1% ± 0.5%; P = 0.05) than stud B. Differences were found in alpha diversity for both Chao1 (P < 0.01) and Shannon (P < 0.01) diversity indexes among days 2, 3, 4, and 5 post-collection to day 1. For beta diversity, unweighted UniFrac metric on days 2, 3, 4, and 5 post-collection differed from those on day 1 (P < 0.01). There were significant correlations between sperm motility and relative abundance of Prevotella (r = -0.29), Ruminococcus (r = -0.24), and Bacteroides (r = -0.32). Additionally, there were significant correlations between sperm motility and Chao1 (r = -0.50) and Shannon's index (r = -0.36). These results demonstrate that differences in bacterial communities over time and between boar studs can be associated with variation in sperm quality.

Sperm parameters in the Great Dane: Influence of age on semen quality

Not all sires have sperm suitable for chilled or frozen storage, and success in artificial insemination (AI) varies highly among individual dogs and breeds. Fertilizing potential is further complicated as sperm quality declines with the aging process. Due to the rapidity of aging and senescence in large breed dogs, associated health and fertility changes may be observed over a shorter period, though this period remains undefined for any breed. Working with a population of purebred Great Danes (GD), our aims were (1) to characterize the distribution of a series of sperm parameters, (2) to distinguish sources of variation in sperm quality within this rapidly aging breed, and (3) to identify changes in sperm quality that may accompany aging. Ejaculates collected from young, middle-aged, and senior Great Dane dogs (n = 50) were evaluated for semen volume, total sperm number and viability, and reactive oxygen species (ROS), in addition to sperm morphology and kinematic parameters. Total testicular volume was also determined using ultrasonography. Testicular volume was not a predictor of sperm production in the GD, however, significant differences between coat colors were identified. Age was negatively associated with total motility, progressive motility, and amplitude of lateral head displacement (ALH) (p < .05). We identified significant relationships between GD male age and TM, PM, and immotility with −9.9%, −9.0%, and +8.3% change per year of age, respectively, which support the anecdotal reports of decline of the fertility with the advance of age in this breed. Sperm of younger GD dogs aged 12 ≤ x < 24 months had significantly higher TM, PM, ALH, and nonlinear motility (p < .05) than older dogs (x ≥ 48 months). High ROS levels were positively associated with TM and PM, average pathway distance (DAP) and straight line distance (DSL), average pathway velocity (VAP), straight line velocity (VSL), and the presence of hairpin tails (p < .05). While age and ROS have significant influences on sperm parameters in the GD, the influence of selection for breed specific phenotypes could help explain the functional significance of the diversity among GD males.

289 Sperm Quality Assessments Using Flow Cytometry do not Differ between Angus and Charolais Breeds of Yearling Bulls Meeting Breeding Soundness Exam Threshold Requirements

Abstract Flow cytometry has been used as a reliable and objective tool for bull sperm quality assessments and identification of sub-fertility. Our objective was to determine if breed differences exist in sperm quality assessments using flow cytometry when evaluating semen from yearling Angus and Charolais bulls that passed breeding soundness exam (BSE) semen evaluation thresholds. Ejaculates were collected via electroejaculation on one of three consecutive days from yearling (403.15 ± 11.19 d age) Charolais (n=23) and Angus (n=23) bulls as part of a BSE. BSEs were conducted by one veterinarian and all bulls met all requirements as set by the Society for Theriogenology. An additional sperm motility analysis was conducted with the iSperm analyzer. Ejaculates were diluted in BoviFree to 70 million cells/mL and sent overnight for flow cytometry analyses. Sperm flow cytometry analyses included: acrosome and cell membrane integrity, mitochondrial energy potential, and oxidation status. The GLIMMIX procedure of SAS with bull as experimental unit, bull breed as the main effect and collection date as a random variable was used to assess potential differences in sperm quality variables. Neither iSperm (P = 0.26) nor visual assessment (P = 0.66) of sperm motility differed among breeds. Bull breed did not influence (P = 0.83) total percentage of viable cells, nor percentage of viable cells with intact acrosomes (P = 0.83). When evaluating oxidation status by measuring reactive oxygen species, bull breed did not influence (P = 0.92) percentage of live sperm cells with positive reactive oxygen species status. There was a tendency (P = 0.10) for a greater percentage of sperm from Charolais bulls (31.1% ± 3.35) to have positive mitochondrial energy potential as compared with Angus bulls (17.6% ± 3.35). Bull breed appears to have little influence on sperm quality assessments among yearling bulls meeting threshold requirements for passing BSEs.

Transcriptome Analysis Reveals Key Gene Expression Changes in Blue Catfish Sperm in Response to Cryopreservation.

The hybrids of female channel catfish (Ictalurus punctatus) and male blue catfish (I. furcatus) account for >50% of US catfish production due to superior growth, feed conversion, and disease resistance compared to both parental species. However, these hybrids can rarely be naturally spawned. Sperm collection is a lethal procedure, and sperm samples are now cryopreserved for fertilization needs. Previous studies showed that variation in sperm quality causes variable embryo hatch rates, which is the limiting factor in hybrid catfish breeding. Biomarkers as indicators for sperm quality and reproductive success are currently lacking. To address this, we investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm. Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. The present RNA-seq study identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage: MnSOD was induced to control ROS production; chaperones and ubiquitin ligases were upregulated to correct misfolded proteins or direct them to degradation; negative regulators of apoptosis, amide biosynthesis, and cilium-related functions were also enriched. Our study provides insight into underlying molecular mechanisms of sperm cryoinjury and lays a foundation to further explore molecular biomarkers on cryo-survival and gamete quality.

Sperm freezability is neither associated with the expression of aquaporin 3 nor sperm head dimensions in dromedary camel (Camelus dromedarius)

The expression of aquaglyceroporin 3 (AQP-3) has been demonstrated in the spermatozoa of several mammalian species and its role has been associated with cryotolerance. Post-thaw sperm quality from individual dromedary males with different response to freezing-thawing process was evaluated through sperm head morphometry. In order to understand the cellular mechanisms affected by cryoinjury we have explored the presence and distribution of sperm AQP-3 using western blotting and immunocytochemistry. WB showed different intensity of the specific signal bands at 28 kDa. Immunofluorescence assessments allowed us to identify five different and clear AQP-3 distribution patterns of labelling in the sperm plasma membrane; acrosome, post-acrosome, mid-piece, and principal and final tail. Although expression of AQP-3 varied among male ejaculates, the individual sperm response to freeze-thawing was not associated with AQP-3 expression. Thus, AQP3 expressions do not seem like a reliable predictor of sperm response to freeze-thawing process in this species. This work is the first to describe the morphometric characteristics of the heads of dromedary spermatozoa. No correlation was found between sperm head dimensions and sperm quality variables after freeze-thawing suggesting that dromedary camel sperm head morphometry is also not a reliable predictor of cryosurvival.

Telomere Length in Pig Sperm Is Related to In Vitro Embryo Development Outcomes.

Simple SummaryUnderstanding how gamete chromatin influences fertilization is highly important not only to improve animal production, but also to develop new biomarkers helping in the selection of those animals with higher fertility potential. In this regard, sperm telomere length has been pointed out as a putative biomarker in human infertility, but no studies have been conducted into its influence in pig fertility. Here, we determined that sperm telomere length is independent from the conventional sperm quality parameters and, through the production of in vitro embryos, we showed that it is indicative of the percentage of morulae and blastocysts, thus becoming useful to be used as biomarker in this species.Telomere length has attracted much interest as a topic of study in human reproduction; furthermore, the link between sperm telomere length and fertility outcomes has been investigated in other species. This biomarker, however, has not been much explored in other animals, such as pigs, and whether it is related to sperm quality and fertility outcomes remains unknown. The present work aimed to determine the absolute value of telomere length in pig sperm, as well as its relationship to sperm quality parameters and embryo development. Telomere length was determined through quantitative fluorescence in situ hybridization (qFISH) in 23 pig sperm samples and data were correlated to quality parameters (motility, morphology, and viability) and in vitro fertilization outcomes. We found that the mean telomere length in pig sperm was 22.1 ± 3.6 kb, which is longer than that previously described in humans. Whilst telomere length was not observed to be correlated to sperm quality variables (p > 0.05), a significant correlation between telomere length and the percentage of morulae 6 days after in vitro fertilization was observed (rs = 0.559; 95% C.I. = (−0.007 to 0.854); p = 0.047). Interestingly, this correlation was not found when percentages of early blastocysts/blastocysts (rs = 0.410; 95% C.I. = (−0.200 to 0.791); p = 0.164) and of hatching/hatched blastocysts (rs = 0.356; 95% C.I. = (− 0.260 to 0.766); p = 0.233) were considered. Through the separation of the samples into two groups by the median value, statistically significant differences between samples with shorter telomeres than the median and samples with longer telomeres than the median were found regarding development to morula (11.5 ± 3.6 vs. 21.8 ± 6.9, respectively) and to early blastocyst/blastocysts (7.6 ± 1.4 vs. 17.9 ± 12.2, respectively) (p < 0.05). In the light of these results, sperm telomere length may be a useful biomarker for embryo development in pigs, as sperm with longer telomeres lead to higher rates of morulae and blastocysts.

Oxytocin in pig seminal plasma is positively related with in vivo fertility of inseminated sows

BackgroundIdentification of relevant in vivo biomarkers for fertility remains a challenge for the livestock industry. Concentrations of the small peptide hormone oxytocin (OXT), involved in male reproductive function and present in the seminal plasma (SP) of several species could be a robust one. This study characterized concentrations of SP-OXT in ejaculates from boars used in artificial insemination (AI) programs aiming to evaluate its relationship with sperm quality variables and in vivo fertility of their liquid-stored AI-semen. Seminal OXT concentrations (ng/mL) were measured in 169 ejaculates from 61 boars of the Duroc, Pietrain, Landrace and Large White breeds using a direct competitive immunoassay test based on AlphaLISA® technology. Ejaculate (ejaculate volume, sperm concentration, total sperm count) and sperm parameters (motility, viability, intracellular generation of reactive oxygen species, plasma membrane fluidity) were assessed at 0 h and 72 h in AI-semen samples stored at 17 °C. In vivo fertility included only 18 Large White and Landrace boars whose AI-semen was used to inseminated > 100 sows and evaluated both farrowing rate and litter size of 3,167 sows.ResultsThe results showed that SP-OXT differed between boars and between ejaculates within boar (P < 0.05) but not between breeds (Duroc, Pietrain, Landrace and Large White). Ejaculates with higher SP-OXT concentration/mL (hierarchically grouped; P < 0.001) had larger volume and came from younger boars (P < 0.05). Ejaculates of boars showing positive farrowing rate deviation exhibited higher (P < 0.05) SP-OXT concentration/mL than those with negative farrowing rate deviation.ConclusionThe SP concentrations of OXT are boar, ejaculate and age dependent, and positively related with ejaculate volume and farrowing rates of liquid-stored semen AI-doses.

Evaluation of dextran for rabbit sperm cryopreservation: Effect on frozen–thawed rabbit sperm quality variables and reproductive performance

Effects were analysed of dextran supplementation to Me2SO and acetamide rabbit semen freezing extenders on quality characteristics of rabbit spermatozoa and reproductive performance. The final concentration of cryoprotectants in pooled semen samples was 12.4 % Me2SO for the A extenders, 10.7 % Me2SO and 2.9 % acetamide for the D extenders and 8.9 % Me2SO and 2.9 % acetamide in F extenders, with a supplementation of 1.7 % sucrose in all cases. There was not inclusion of dextran in the A0, D0, F0; while 5 % dextran was included in A5, D5, F5 and 10 % dextran in A10, D10 and F10 extenders. Sperm motility and viability rates were similar with use of the different extenders. Acrosome integrity after the freeze-thawing processes, however, was markedly greater when there was dextran supplementation of D and F extenders. Prolificacy was affected by extender composition. When there was artificial insemination (AI) using semen cryopreserved in the A extenders, number of kits born was similar to when there was AI with fresh semen when there was inclusion of 5% dextran for cryopreservation, while there was no effect on prolificacy when there was cryopreservation of semen using the D and F extenders. In conclusion, dextran supplementation of extenders containing Me2SO and acetamide resulted in greater acrosome integrity. Furthermore, when there was AI using sperm preserved in cryo-diluents containing an intermediate concentration of Me2SO, combined with inclusion of 5 % dextran, there was a marked beneficial effect on rabbit doe reproductive performance.

Effect of orally administrated letrozole on reproduction performance and gene expression of FOXJ1, LPR2 and PVRL3 in reproductive tract in aged roosters

letrozole is an aromatase inhibitor that stops the production of estrogen through interrupting the entrance of hormone androgen into a small amount of estrogen. Therefore, the current study was developed to estimate orally administrated Letrozole on the reproductive performance and relative abundance of Foxj1, PVRL3, and LPR2 mRNA in aged roosters. Fifty-five-week old ROSS 308 breeder roosters (n = 18) were orally treated using letrozole. Primarily, the body weight of the animals was recorded, and they were randomly classified into three groups (n = 6 birds/group) receiving different doses of Letrozole, including 0, 0.015, and 0.03 mg/kg body weight/day for three weeks. At the end of the trial, seminal traits, plasma, testicular hormone levels (testosterone, estradiol, and FSH), histopathological studies, in vitro fertility, and relative abundance of testis PVRL3, epidydimal Foxj1, and LPR2 mRNA were evaluated. Based on the results, the sperm quality variables were statistically higher in the 0.03 group compared to the controls. Greater histologic parameters, such as diameter of seminiferous tubules, thickness of seminiferous epithelium, categorized epididymal region, and in vitro fertility rates were estimated for the treated groups(p < 0.001). Plasma and testicular testosterone, estradiol concentrations, and plasma FSH levels were significantly influenced by letrozll treatment (p < 0.001). Relative mRNA transcript abundance increased for PVRL3 and decreased for Foxj1 and LPR2 in treated groups. Overall, aromatase inhibitors can enhance the reproductive performance of aged commercial broiler breeder roosters. However, it can impact endocytosis and ciliogenesis events via reducing estradiol.

Estimate of in vitro embryo production based on sperm subpopulations in Senepol bulls

In cattle, in vitro embryo production (IVEP) is an important reproductive biotechnology responsible for the rapid expansion of the Senepol breed in our country. This breed has shown important results when used in crossbreeding and estimate IVEP in Senepol based on seminal analysis would be valuable for the semen cryopreservation industry, research institutes and breeders. Combining the evaluation of sperm subpopulations with analysis of other sperm attributes may help to improve fertility predictions in cattle. Therefore, the objectives of the present study were to: 1) identify and characterize motile sperm subpopulations in cryopreserved Senepol semen following the washing process carried out before in vitro fertilization, and 2) to determine an model for estimate IVEP based on sperm subpopulations in conjunction with other sperm quality analyzes. Samples of 38 cryopreserved semen from 28 Senepol bulls, chosen based on retrospective data from 386 IVEP routines, underwent the semen washing and were evaluated by the computer-assisted sperm analysis system. Sperm morphology was evaluated by wet preparation technique, and plasma and acrosomal membranes integrity, mitochondrial potential, oxidative status and chromatin resistance were analyzed by flow cytometry. After multivariate analysis of principal components and grouping, three sperm subpopulations were identified: SBP1 (fast and progressive motility), SBP2 (hyperactivated motility) and SBP3 (slow non-progressive motility). After categorization of IVEP in high, medium and low embryo yield, logistic regression analysis was applied to associate the results of subpopulations and other sperm quality variables with IVEP. The SBP1 and SBP2 variables affected embryo production, and an IVEP estimation model was generated for Senepol bulls based on these two subpopulations: embryo yield = 0.1563 + 0.0328 (SBP1) + 0.0173 (SBP2). SBP1 and SBP2 represents the absolute value of the percentage of subpopulations in semen. If the calculated value (by this equation) is close to 1, the embryo yield will be low; if is close to 2, will be medium; if is close to 3, will be high. In conclusion, three subpopulations were found for Senepol semen and, despite all analyzed variables, only SBP1 and SBP2 were included in the model to estimate IVEP in this breed.

Proteomic comparison of non-sexed and sexed (X-bearing) cryopreserved bull semen

It is widely recognized that quality of spermatozoa in sexed semen (SS) samples is not as great as for conventional, non-sexed semen (NS). There are differences in qualitative and biochemical variables between spermatozoa in NS and SS. Information, however, is lacking on molecular differences, especially concerning spermatozoa proteomic differences is SS and NS. The objective of this study was to compare commercially available NS and SS bull semen by evaluating sperm quality variables in conjunction with a proteomics approach. Results from flow cytometry and computer-assisted sperm analyses indicated there was less sperm motility, viability, mitochondrial potential and acrosome integrity in sperm from SS. Results from proteomic analysis indicated sperm from NS and SS samples were characterized by different protein profiles. There was identification of 70 sperm proteins that differed in abundance and six protein spots that were different in extent of carbonylation. Sperm from SS had altered structures of enzymes involved in glycolysis, oxidative phosphorylation and maintenance of a constant adenylate energy charge. Furthermore, sperm from SS had alterations of several flagella substructures, especially outer dense fiber proteins, which were less abundant and more carbonylated than in sperm from NS. In sperm of SS, compared with NS, there were differences in abundance of proteins involved in capacitation, acrosome reaction and sperm-egg fusion as well as lesser abundances of sperm surface proteins. Results enable a greater understanding of differences between sperm from NS and SS samples, thereby contributing to development of improved protocols for more effective protection of sexed spermatozoa during processing.

Effect of astaxanthin in extenders on sperm quality and functional variables of frozen-thawed boar semen

The aim of the study was to determine whether the presence of astaxanthin (ASX) protects boar spermatozoa against damage related to cryopreservation. Pooled ejaculates extended in Beltsville Thawing Solution (BTS) were used. Three experiments were conducted: 1) sperm samples were pre-incubated overnight (17 °C) with ASX (0, 0.5, 5, 15 μM) prior to freezing and then frozen using cooling and thawing extenders supplemented with ASX (0, 0.5, 5, 15 μM); 2) sperm samples were treated with ASX (0, 0.5, 5, 15 μM) only during overnight pre-incubation (17 °C) prior to cryopreservation; and 3) a thawing extender was supplemented with ASX (0, 0.5, 5, 15 μM). The groups were as follows: control (C; no treatment), ASX 1 (0.5 μM), ASX 2 (5 μM) and ASX 3 (15 μM). Total (TM) and progressive (PM) motility was analyzed using CASA, while sperm viability, reactive oxygen species generation, lipid peroxidation and apoptoticlike changes were analyzed using flow cytometry. Sperm variables were evaluated prior to freezing as well as 30 and 150 min after thawing. In Experiment 1, the values of TM and sperm viability post-thaw were less in the ASX 3 than C group. In Experiment 2, there was no effect of ASX on any of the sperm variables evaluated, while in Experiment 3, apoptotic-like changes were less in the ASX 1 than C group. In conclusion, there was a subtle beneficial effect on cryopreserved boar spermatozoa after addition of ASX to thawing media.

Effects of selection by the Percoll density gradient method on motility, mitochondrial membrane potential and fertility in a subpopulation of Atlantic salmon (Salmo salar) testicular spermatozoa

The aim of this study was to evaluate effects of selection using the Percoll density gradient method on motility, mitochondrial membrane potential (ΔΨMit) and fertility in a subpopulation of testicular spermatozoa obtained from Atlantic salmon (Salmo salar). Samples were divided into three groups: Control (C), T1 (45/90 % Percoll®) and T2 (45/60 % Percoll®). Sperm motility was evaluated using CASA (Computer-Assisted Sperm Analysis), ΔΨMit using flow cytometry, and fertility evaluating whether cleavage of fertilised eggs had occurred after 16 h of incubation at 10 °C. Results indicate that motility was greater in T1 (92 ± 2.91 %) and T2 (89 ± 2.88 %) than in the Control (83.2 ± 2.04 %). The percentage of ΔΨMit was 88.3 ± 0.58 % and 85 ± 2% for T1 and T2, respectively, compared to 35 ± 6.24 % for the control. The fertility rates were 76 ± 9.1 % and 70 ± 8.1 % for T1 and T2, respectively, compared with 66 ± 12 % for the control. The kinetic characteristics for T1 were curvilinear velocity (VCL): 92.44 ± 21.12 μm/s, average path velocity (VAP): 85.87 ± 21.83 μm/s; and for T2 VCL was 78.69 ± 17.63 μm/s and VAP was 73.62 ± 17.08 μm/s. The results indicate sperm motility and ΔΨMit were greater in T1 and T2 compared with the control (P < 0.05). Similarly, there was an increase in the fertilisation rate compared to the control. The results from this study are the first where sperm quality variables were evaluated for Salmo salar testicular sperm using the Percoll® density gradient method.

Effect of relaxin on semen quality variables of cryopreserved stallion semen

The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 106 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferred into 0.5 mL straws, equilibrated at 5 °C for 30 min, and placed in liquid nitrogen (LN2) vapour for 15 min before being plunged into LN2. After thawing, sperm samples were evaluated for motility and velocity variables, mitochondrial membrane potential, apoptosis, and plasma membrane and DNA integrities. For sperm motility variables, there were dose- and time-dependent effects, with the largest values recorded when 12.5 and 25 ng/mL relaxin were used for 0–120 min of incubation. Furthermore, at all of the concentrations at which there were evaluations, relaxin additions to semen diluent led to a marked improvement in sperm mitochondrial membrane potential and a lesser percentage of apoptotic cells compared to the control group. Plasma membranes and DNA integrities were not affected by relaxin supplementations to the diluent. In conclusion, supplementation of relaxin in extender before semen cryopreservation, especially at 12.5 and 25 ng/mL, had a positive effect on the sperm quality variables.