Stallion Sperm Research Articles

Genome-wide association study for frozen-thawed sperm motility in stallions across various horse breeds.

The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds. Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), progressive motility after freezing (FPM), and the difference between PM and FPM. The respective DNA samples from these stallions were genotyped using Axiom Equine Genotyping Array. We performed a GWAS search for single nucleotide polymorphism (SNP) markers and potential genes related to motility properties of frozen-thawed semen in the stallions of various breeds. As a result of the GWAS analysis, two SNP markers, rs1141327473 and rs1149048772, were identified that were associated with preservation of the frozen-thawed stallion sperm motility, the relevant putative candidate genes being NME/NM23 family member 8 (NME8), olfactory receptor family 2 subfamily AP member 1 (OR2AP1), and olfactory receptor family 6 subfamily C member 4 (OR6C4). Potential implications of effects of these genes on sperm motility are herein discussed. The GWAS results enabled us to localize novel SNPs and candidate genes for sperm motility in stallions. Implications of the study for horse breeding and genetics are a better understanding of genomic regions and candidate genes underlying stallion sperm quality, and improvement in horse reproduction and breeding techniques. The identified markers and genes for sperm cryotolerance and the respective genomic regions are promising candidates for further studying the biological processes in the formation and function of the stallion reproductive system.

Alginate encapsulation of stallion sperm for increasing storage stability

The aim of this study was to establish an alginate encapsulation procedure for stallion sperm, and investigate if sperm encapsulation enhances longevity during cold storage and survival after cryopreservation. First, biocompatibility of the compounds needed for encapsulation was tested and factors determining capsule structure were identified. Sperm encapsulation was realized either by depositing droplets (20 µL) of sperm solution supplemented with barium or calcium chloride (10 mM) in alginate solution (0.25%, w/v), or by adding sperm-alginate droplets in solution containing barium or calcium chloride, and hardening (10 min). The first procedure resulted in structures with sperm residing in a liquid core surrounded by a spherical alginate shell, whereas the second procedure resulted in sperm embedded in solid beads of alginate matrix. It was found that use of calcium for alginate gelation resulted in decreased sperm motility as compared to using barium, and that encapsulation in solid beads had a negative impact on sperm plasma membrane intactness. Percentages of membrane intact sperm in barium-alginate core-shell structures were similar as found for ordinary diluted sperm, and did not change during 4 d storage at 5 °C. Sperm motility was reduced after direct recovery from core-shell structures, however, remained stable during 4 d storage leading to similar values as found for un-encapsulated sperm at this time point. Cryosurvival of sperm encapsulated in solid beads or core-shell structures was found to be lower compared to that of ordinary diluted sperm.

Influence of the concentrations of essential and toxic elements in the blood serum on the indicators of sperm quality in Arabian purebred stallions

Analysis of trace elements in blood serum can be an important tool for monitoring micronutrient supply in stallions in order to maintain reproductive health. In this regard, the purpose of this study was to determine the relationship between the concentrations of macro- and microelements in the blood serum of stud stallions with the qualitative characteristics of fresh sperm and sperm after cryopreservation and thawing. The studies were carried out on purebred Arabian stallions (n=50). As a biomaterial for studies, we used blood serum, fresh and thawed sperm after cryopreservation. The elemental composition of seminal plasma was determined by 25 chemical elements Co, Cr, Cu, Fe, I, Mn, Se, Zn Ca, K, Mg, P, B, Li, Si, V Na, Ni, Sn, Hg, Sr, Al, As, Cd, Pb) by ICP-DRC-MS methods. It was found that the concentrations of Mg and Co in the blood serum positively correlate with the qualitative characteristics of the sperm of Arabian purebred stallions. Wherein, the optimal range of concentrations of Mg in the blood serum, typical for stallions with relatively high indicators of sperm quality, is 22.64 to 27.05 μg/g; Co – 0.001 to 0.0015 μg/g.

КРИОРЕЗИСТЕНТНОСТЬ СПЕРМЫ ЖЕРЕБЦОВ В РАЗНЫЕ МЕСЯЦЫ ГОДА

It is known that the seasonality of reproductive function is much more pronounced in mares than in stallions. The mare goes through an anestrus period in autumn and winter, while spermatogenesis in stallions continues throughout the year. Therefore, studies of cryoresistance of stallion sperm in different months of the year are relevant. The aim of the study was to study the cryoresistance of stallion sperm in different months of the year when creating cryopreservation of genetic material with intensive breeding use of sires. The cryoresistance of the sperm of stallions with their intensive breeding use was the best from September to December during sexual rest. The highest incidence of sperm was in December, which is 0.44% more than in October, 5.12% more than in November, 7.62% more (P<0.05) than in September, 20.53% more (P<0.01) than in January, 27.3% more (P<0.01) compared to February, 52.7% more (P<0.001) than in March, 137.9% more (P<0.05) than in April, 34.12% more (P<0.001) than in May, 23.5% more (P<0.001) than in June and 59.7% more (P<0.001) than in July. The survivability of sperms after thawing in a thermostat at 37°C for less than 3 hours was observed in March, April and July. The lowest sperm motility after defrosting was observed in April, which is 36.4% less (P<0.001) than in July, 38.6% less (P<0.001) than in May, 45.12% less (P<0.001) than in June, 50.1% less (P<0.001) than in in January, 52.6% less (P<0.001) than in February, 43.75% less (P<0.001) than in March, 53.8% less (P<0.001) than in September, 55% less (P<0.001) than in October, 54.4% less (P<0.001) than in November and 54.1% less (P<0.001) than in December. Based on the data obtained on the mobility of deconserved sperm doses, we mat expect the highest fertility from the genetic material that is harvested from September to December.

Efficiency of Ringer B. Braun solution on stallion epididymal sperm motility and viability compared to the commercial extender within 72 hours of storage

Collection of epididymal stallion sperm offers the opportunity to retain and use genetic material from the males after elective castration or evenpost mortem. The aim of the present study was to evaluate the effect of the Ringer B. Braun solution on stallion epididymal sperm viability and motility during liquid storage for 72 h at 4 °C and at 20 °C. Eight stallions (3–10 years old) were used in the study. Sperm from the cauda epididymis was harvested immediately after routine castration. The sperm from epididymis was washed out and diluted with Ringer B. Braun solution and with a commercial semen extender which was used as a control. Samples from each horse were divided into two parts: one part of samples was stored at 4 ± 1 °C as recommended for the commercial semen extender, the other one was stored at 20 ± 1 °C. Sperm viability, motility, and pH were checked one hour after collection and dilution, and after 24, 48 and 72 h of incubation. The results of sperm viability, subjective and progressive sperm motility showed slightly higher results in semen samples diluted with Ringer B. Braun solution in one hour compared to the commercial extender, by 2.40 ± 0.49% for viability (P> 0.05), by 0.30 ± 9.92% for subjective motility (P> 0.05) and by 5.70 ± 6.07% for progressive motility (P> 0.05). We suggest that Ringer solution could be used for a short term storage (1–24 h) of equine epididymal semen at a 4 °C temperature.

Impact of glyphosate and its formulation Roundup® on stallion spermatozoa

The growing and widespread use of glyphosate-based herbicides (GBHs) has raised an intense public debate about the impact of environmental contamination on animal and human health, including male fertility. The aim of this study was to deepen the impact of glyphosate (Gly) and GBHs on mammalian sperm investigating the effect of in vitro exposure of stallion spermatozoa to Gly and to its commercial formulation Roundup® (R).Spermatozoa were incubated at 37 °C with different Gly or R concentrations (from 0.5 to 720 μg/mL Gly or R at the same Gly-equivalent concentrations). After 1 h of incubation motility, viability, acrosome integrity, mitochondrial activity and ROS production were assessed.Gly, at all the concentrations tested, did not induce any detrimental impact on the sperm quality parameters evaluated. Conversely, R starting from 360 μg/mL (Gly-equivalent dose) significantly (P < 0.05) decreased total and progressive motility, viability, acrosome integrity, mitochondrial activity and the percentage of live spermatozoa with intact mitochondria not producing ROS.Our results indicate that the commercial formulation R is more toxic than its active molecule Gly and that the negative impact on stallion sperm motility might be likely due to a detrimental effect mainly at membrane and mitochondrial level and, at least in part, to redox unbalance. Moreover, based on the data obtained, it can be hypothesized a species-specificity in sperm sensitivity to Gly and GBHs as horse spermatozoa were negatively influenced at higher concentrations of R compared to those reported in literature to be toxic for human and swine male germ cells.

Evaluation of stallion sperm concentration by two different methods and its influence on sperm motility assessment

Received: 2020-12-18 | Accepted: 2021-05-03 | Available online: 2021-12-31 https://doi.org/10.15414/afz.2021.24.04.293-296 The aim of this study was to compare two methods for determining the concentration of sperm in stallion ejaculate and to evaluate the influence of different levels of concentration on the determination of sperm motility using an objective method. The total number of evaluated samples was 123. For the experiment, 14 stallions of different breeds housed in the Tlumačov Provincial Stud Farm were used. Sperm concentration was assessed by the hemocytometric method using a Bürker chamber and the automatic method using the Sperm Class Analyzer®. The SCA system was used to evaluate sperm motility. The samples were divided into 6 groups with sperm concentrations of 50, 100, 150, 200, 250 and 300·10 6 sperm ml -1 . The results were statistically evaluated by Tukey’s HSD test. There were statistically significant differences in the determination of sperm concentrations between the hemocytometric method and the SCA system in samples with concentration exceeding 100·10 6 sperm ml -1 . Sperm motility increased in samples with higher concentration, however the effect of sperm concentration on motility parameters has not been statistically significant. The results of this study indicate that samples with sperm concentration higher than 100·10 6 sperm ml -1 reduce the accuracy of the SCA system evaluation and these samples need to be diluted to concentration ≤100·10 6 sperm ml -1 for more accurate evaluation. Keywords:  sperm concentration, sperm motility, hemocytometer, SCA References Amann, R.P. (1989) Can the Fertility Potential of a Seminal Sample Be Predicted Accurately. Journal of Andrology, 10(2), 89–98. Amann, R.P. and Waberski, D. (2014) Computer-assisted sperm analysis (CASA): Capabilities and potential developments. Theriogenology, 81(1), 5–17. Aurich, J.E. (2012) Artificial Insemination in Horses – More than a Century of Practise and Research. Journal of Equine Veterinary Science, 32(8), 458–463. Broekhuijse, M.L.W.J. (2011) Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination. Theriogenology, 76(8), 1473–1486. Didion, B.A. (2008) Computer-assisted semen analysis and its utility for profiling boar semen samples. Theriogenology, 70(8), 1374–1376. Heckenbichler, S. et al. (2011) Quality and fertility of cooled-shipped stallion semen at the time of insemination. Theriogenology, 75(5), 849–856. Jequier, A.M. and Ukombe, E.B. (1983) Errors Inherent in the Performance of a Routine Semen Analysis. British Journal of Urology, 55(4), 434–436. Kenneth, A. et al. (1990) The influence of chamber characteristics on the reliability of sperm concentration and movement measurements obtained by manual and videomicrographic analysis. Fertility and sterility, 53(5), 882–887. Knuth, U.A. and Nieschlag, E. (1988) Comparison of computerized semen analysis with the conventional procedure in 322 patients. Fertility and sterility, 49(5), 881–885. Kuster, Ch. (2005) Sperm concentration determination between hemacytometric and CASA systems: Why they can be different. Theriogenology, 64(3), 614–617. Lannou, D.L. et al. (1992) Effects of chamber depth on the motion pattern of human spermatozoa in semen or in capacitating medium. Human Reproduction, 7(10), 1417–142. Lu, J.C. et al. (2013) Computer-aided sperm analysis: past, present and future. Andrologia, 45(1), 1–10. Malmgren, L. (1997) Assessing the quality of raw semen: a review. Theriogenology, 46(3), 523–530. Mortimer, D. et al. (1995) Workshop Report: Clinical CASA – the Qest for Consensus. Reproduction, fertility and developement, 7(5), 951–959. Mortimer, S.T. et al. (2015) The future of computer-aided sperm analysis. Asian Journal of Andrology, 17(2), 545–553. Prathalingam et al. (2006) The Precision and Accuracy of Six Different Methods to Determine Sperm Concentration. Journal of Andrology, 27(2), 257–262. Rečková, Z. and Filipčík, R. (2020) An Analysis of Selected Aspects of Sperm Quality in Fresh and Cooled-Storage Stallion Semen. Iranian Journal of Applied Animal Science, 10(3), 405–408. Sokol, R.Z. et al. (2000). Comparison of two methods for the measurement of sperm concentration. Fertility and sterility, 73(3), 591–594. Spiropoulos, J. (2001). Computerized semen analysis (CASA): Effect of semen concentration and chambre depth on measurements. Archives of Andrology, 46(1), 37–42. Varner, D.D. (2008) Developments in stallion semen evaluation. Theriogenology, 70(3), 448–462. Verstegen, J. et al. (2002) Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology, 57(1), 149–l79. Věžník, Z. (2004) Repetitorium of spermatology and andrology and methods of spermatoanalysis. Brno: Research Institute of Veterinary Medicine. WHO. (1999) WHO laboratory manual for the Examination and processing of human semen. 4th ed. Cambridge: Cambridge University Press.

Diluent Containing Dimethylformamide Added With Sucrose Improves In Vitro Quality After Freezing/Thawing Stallion Sperm

The aim of this study was to investigate the effects of sucrose on post-thawed equine semen quality. Semen samples (n = 24) were collected from six stallions. They were diluted (200 × 106 sperm/mL) in a freezing medium based on skimmed milk, egg yolk, dimethylformamide, and supplemented with sucrose at concentrations of 0 (Control), 25, 50, and 100 mM and in a commercial extender (BotuCrio). Subsequently, they were filled in straws (0.5 mL) and subjected to freezing and storage (−196°C). Immediately after thawing (37°C, 30 seconds), semen samples were evaluated for kinetics (CASA), plasma and acrosomal membrane integrity, and mitochondrial membrane potential (flow cytometry). The addition of 50 and 100mM sucrose to the freezing extender increased (P < .05) the parameters of TM, PM, VCL, VSL, and VAP, compared to the control group. The WOB parameter of the group supplemented with 100 mM sucrose was higher (P < .05) than the control group. Higher values ​​(P < .05) of ALH and BCF were observed in groups treated with sucrose (25, 50, and 100 mM), compared to BotuCrio. The semen frozen in the presence of 100 mM sucrose presented higher percentages (P < .05) of sperm with intact plasma and acrosomal membranes, and high mitochondrial membrane potential in relation to the other groups. It is concluded that the addition of sucrose to equine semen freezing extender increase motility (50 and 100 mM), plasma and acrosomal membrane integrity preserve, and high sperm mitochondrial membrane potential (100 mM) after thawing.

Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa.

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

Edible bird’s nest supplementation in chilled and cryopreserved Arabian stallion semen

Diluents and various biological products have been used in different animal species, with promising outcomes in post-thaw sperm quality. Nevertheless, only a few reports are available for the semen of Arabian horses. Edible bird’s nest (EBN) – a product of the salivary secretions of swiftlet species is widely known to have both antioxidant and anti-inflammatory properties. Presently, there is no data available on the role of EBN supplemented in different extenders and its effect on semen quality in stallion semen. Two in vitro experiments were conducted to examine the effects of edible bird’s nest (EBN) on the quality of chilled and post-thawed cryopreserved Arabian stallion spermatozoa. In experiment one, 10 ejaculates were collected, divided into two equal parts, diluted using EquiPlus® and INRA 96® and supplemented with 0 % (control), 0.12 %, 0.24 % EBN concentrations. The semen samples were stored at 5 ℃ and observed at 0, 24, and 48 h. Sperm kinetics variables (% total motility [TM] and progressive motility [PM], curvilinear velocity; VCL, straightness; VSL, average path velocity; VAP) were analyzed using computerized assisted sperm analysis. For chilled semen, there was no significant difference in any of the sperm quality parameters between control (0 %), 0.12 %, and 0.24 % EBN supplementation either in INRA96® or EquiPlus®. In experiment two, nine ejaculates were diluted and cryopreserved using EquiPlus Freeze® and INRA Freeze® containing 0 %, 2.4 %, and 4.8 % EBN, and evaluated after thawing. Sperm kinetics, DNA integrity and antioxidant capacity - Biological Anti-oxidant Potential (BAP) and Reactive Oxygen Metabolites (d-ROMs) test were evaluated. In chilled semen, there was no significant difference in any of the sperm quality parameters between control (0 %), 0.12 %, and 0.24 % EBN supplementation either in INRA96® or EquiPlus®. For frozen semen supplemented with 2.4 % and 4.8 % EBN had higher sperm motility parameters compared to control in INRA Freeze® and EquiPlus Freeze®, but the values were not statistically significant (P > 0.05). Also, EBN supplementation had no significant effects on the DNA integrity, biological antioxidant potential, and reactive oxygen metabolites. EBN supplementation had no significant effects on sperm quality and antioxidant status in chilled and frozen Arabian Stallion semen. Future studies might consider different methods of EBN preparation and concentrations to elucidate the potential biological impact of EBN in Arabian stallion semen.

Towards increasing stallion sperm longevity by storage at subzero temperatures in the absence of ice

The aim of cell preservation technologies is to slow down damaging reactions by lowering the storage temperature. Upon dilution in a stabilizing extender, stallion sperm can be stored at refrigerator temperatures for several days. Cryopreservation allows storage for decades, but freezing and thawing cause damage and viability losses. It is assumed that by storing cells at subzero temperatures in a non-frozen supercooled state, the damaging effects of ice formation can be avoided. In this study, we have investigated if stallion sperm can be stored at −10°C in the absence of ice, and compared viability during supercooled storage with that during storage at 5°C. We found that addition of 2% Ficoll-400 to buffered saline and covering with mineral oil depressed the sample freezing point and inhibited surface-catalyzed nucleation. This allowed storage in a supercooled state at −10°C for up to 7 days. Supplementing specimens with sperm, however, increased the incidence of sample freezing. Nonetheless, with 50×106 sperm mL−1, about 40% of the samples turned out to be non-frozen. Adding 100 mM sucrose was found to preserve sperm membrane intactness during supercooled storage, although this resulted in lower percentages as found with refrigerated storage. Sperm motility appeared to be lost during supercooled storage but could be partly restored by substituting buffered saline with a milk-based extender as base medium. Percentages of membrane intact sperm, however, were found to be lower. Supercooled storage holds promise for semen preservation, but further optimization of the storage solution is required to preserve sperm motility.

INRA96 Supplemented With Phospholipids Liposomes, A Promising Approach for Stallion Sperm Chilling

Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions (“bad coolers”) are excluded from such technology.Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes produced from EY phospholipids on stallion sperm storage at 4°C.The sperm of stallions from two studs was diluted in INRA96 extender (as control) or an experimental extender (EE) composed of INRA96 supplemented with liposomes of EY phospholipids. After 24H (D1), 72H (D3), and 6 days (D6) or 7 days (D7), motility parameters were evaluated using Computer Assisted Semen Analyzer.Our results demonstrated that total and progressive motility decreased significantly after dilution and storage in INRA96 between D1 and D3 (P < .05) while no significant decrease was observed between D1 and D3 with EE. Regarding VAP parameter, no significant difference was observed between extenders except at D7 in stud 2.Moreover, total and progressive motility were maintained at a significantly higher level (D3, D6, D7) when sperm was stored in EE compared to INRA96.These promising results demonstrate that the supplementation of INRA96 extender with egg-yolk phospholipids liposomes allows a higher protection to stallion sperm cells.

PSXV-13 The activity of thiol cathepsins in seminal plasma of stallions with different percentages of viable sperm after freezing-thawing

Abstract The aim of this work was to study the activity of cathepsins B, L and H in the seminal plasma of stallions with normal (&amp;gt;25%, n = 11) and low (&amp;lt; 25%, n = 13) percentage of viable sperm after freezing-thawing. Sperm of 24 Arabian stallions aged from 5 to 20 years (12.1 ± 4.8 years on average) was collected during the breeding season (February-May). The activity of cathepsins in spermoplasm was studied by the spectrofluorometric method (System 3 Scanning Spectrofluorometry, Optical technology devices, inc. Elmstord, New York, 10523) by Barrett&amp;Kirschke. The viability of the sperm was determined after its freezing-thawing. Sperm smears were eosin stained and live:dead-ratio was examined using an Olympus BX41 phase contrast microscope (Olympus Corporation, Japan). The significance of differences in the studied groups was determined using the Mann-Whitney U-test. There were no significant differences in the activity of catepsins B and L in the spermoplasm of stallions with normal and low percentages of viable spermatozoa after freezing-thawing. It was found that in the group of stallions with a normal percentage of viable spermatozoa after freezing-thawing, the activity of cathepsin H in the spermoplasm was significantly higher than in the group with a low percentage of viable spermatozoa (P = 0.0219). Free radicals formed during freezing-thawing of sperm can damage cell membranes, leading to loss of sperm viability. Thiol cathepsins are involved in the degradation of oxidatively modified proteins and, apparently, it is cathepsin H that is most actively involved in this process in the sperm of stallions. We assume that the low activity of cathepsin H in the seminal plasma of stallions with a low percentage of viable spermatozoa indicates a high involvement of this enzyme in the protection of sperm membranes from oxidative damage. The research was supported by the Russian Science Foundation grant No. 20-16-00101.

A stallion spermatozoon’s journey through the mare’s genital tract: In vivo and in vitro aspects of sperm capacitation

Conventional in vitro fertilization is not efficacious when working with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, these gametes fail to initiate the acrosome reaction in the vicinity of the oocyte and cannot, therefore, penetrate into the perivitelline space. Failure of sperm penetration most likely relates to the absence of optimized in vitro fertilization media containing molecules essential to support stallion sperm capacitation. In vivo, the female reproductive tract, especially the oviductal lumen, provides an environmental milieu that appropriately regulates interactions between the gametes and promotes fertilization. Identifying these ‘fertilization supporting factors’ would be a great contribution for development of equine in vitro fertilization media. In this review, a description of the current understanding of the interactions stallion spermatozoa undergo during passage through the female genital tract, and related specific molecular changes that occur at the sperm plasma membrane is provided. Understanding these molecular changes may hold essential clues to achieving successful in vitro fertilization with equine gametes.

Cooled storage of semen from livestock animals (part I): boar, bull, and stallion

This review is part of the Festschrift in honor of Dr. Duane Garner and provides an overview of current techniques for cooled storage of semen from livestock animals. The first part describes the current state of the art of liquid semen preservation in boars, bulls, and stallions, including the diluents, use of additives, processing, temperature, and cooling of semen. The species-specific physiology and varying extents of cold shock sensitivity are taken into consideration. In addition, factors influencing the quality of cooled-stored semen are discussed. Methods, trends, and the most recent advances for improving sperm quality during cold-temperature storage are highlighted and their respective advantages and disadvantages are contrasted. There has been much progress in recent years regarding cold-temperature storage of boar sperm and there is great potential for a large-scale use to replace the current 17 °C temperature storage regime and the associated use of antibiotics in the future. For stallion sperm, there is an opposite trend away from previous low-temperature storage towards storage at higher temperatures to increase sperm viability and longevity. In bulls, liquid storage of sperm is mostly used in the seasonal dairy production systems of New Zealand and Ireland, but with further research focusing on shelf-live elongation of liquid preserved sperm, there is potential for an application in breeding programs worldwide.

Glycerophospholipids protect stallion spermatozoa from oxidative damage in vitro.

Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE; a key end product of lipid peroxidation) positive cells (32.9 ± 2.7 vs 20.9 ± 2.3%; P ≤ 0.05) and increased the concentration of 4-HNE within the spent media (0.026 ± 0.003 vs 0.039 ± 0.004 µg/mL; P ≤ 0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility: 2.0 ± 1.0 vs 68.8 ± 2.9%; progressive motility: 0 ± 0 vs 19.3 ± 2.6%; straight line velocity: 9.5 ± 2.1 vs 50.9 ± 4.1 µm/s; curvilinear velocity: 40.8 ± 10 vs 160.7 ± 7.8 µm/s; average path velocity: 13.4 ± 2.9 vs 81.9 ± 5.9 µm/s; P ≤ 0.001), sperm viability (13.5 ± 2.9 vs 80.2 ± 1.6%; P ≤ 0.001) and reduced mitochondrial ROS generation (98.2 ± 0.6 vs 74.8 ± 6.1%; P ≤ 0.001). Supplementation with GPL during 17°C in vitro sperm storage over 72 h improved sperm viability (66.4 ± 2.6 vs 78.1 ± 2.9%; P ≤ 0.01) and total motility (53 ± 5.6 vs 66.3 ± 3.5%; P ≤ 0.05). It is concluded that incubation of stallion spermatozoa with sub-µm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro.Lay summarySperm collection and storage is an important step in many artificial insemination and in vitro fertilization regimes for several species, including humans and horses. The sperm membrane, which acts as a protective outer barrier, is made up of fatty acid-containing molecules – called phospholipids. These phospholipids may become damaged by waste products generated by the cell, such as hydrogen peroxide, during non-chilled sperm storage. We aimed to determine if sperm cells were able to repair this membrane damage by supplementing them with phospholipids during non-chilled storage. Sperm was collected from five miniature stallions by artificial vagina, and then supplemented with phospholipids during 72 h sperm storage at 17°C. Our studies show that when stallion sperm are supplemented with phospholipids in vitro, they are able to remove their damaged membrane phospholipids and swap them for undamaged ones, aiding in resistance to cellular waste and improving cell health and potential fertility.

Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility

Even in stallions with sperm quality within normal reference ranges at ejaculation, subtle differences in sperm quality exist that in many cases lead to reduced time frames for conservation of the ejaculate and/or reduced fertility. The spermatozoon is a cell highly suitable for proteomics studies, and the use of this technique is allowing rapid advances in the understanding of sperm biology. The aim of the present study was to investigate differences among stallions of variable sperm quality (based on motility and sperm velocities), although all horses had sperm characteristics within normal ranges. The proteome was studied using UHPLC/MS/MS and posterior bioinformatic and enrichment analysis; data are available via ProteomeXchange with identifier PXD025807. Sperm motility, linear motility and circular, straight line and average velocities (VCL, VSL, VAP) were measured using computer assisted sperm analysis (CASA). In stallions showing better percentages of motility, circular and average velocity predominated mitochondrial proteins with roles in the Citric acid cycle, pyruvate metabolism and oxidative phosphorylation. Interestingly, in stallions with better percentages of total motility, sperm proteins were also enriched in proteins within the gene ontology (G0) terms, single fertilization (G0: 0007338), fertilization (G0: 0009566), and zona pellucida receptor complex (GO:0002199). The enrichment of this proteins in samples with better percentages of total motility may offer a molecular explanation for the link between this parameter and fertility. SignificanceProteomic analysis identified a high degree of specificity of stallion sperm proteins with discriminant power for motility, linear motility, and sperm velocities (VCL, VAP and VSL). These findings may represent an interesting outcome in relation to the molecular biology regulating the movement of the spermatozoa, and the biological meaning of the measurements that computer assisted sperm analysis (CASA) provide. Of a total of 903 proteins identified in stallion spermatozoa, 24 were related to the percentage of total motility in the sample; interestingly, gene ontology (G0) analysis revealed that these proteins were enriched in terms like single fertilization and fertilization, providing a molecular link between motility and fertility. Field studies indicate that the percentage of total motility is the CASA derived parameter with the best correlation with fertility in stallions.

Sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion spermatozoa

Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen using three commercial semen extenders. Sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated before and after freezing for each aliquot. In addition, the percentage of ASA-bound sperm was evaluated post-thaw. In Experiment 2, semen from 22 stallions was frozen immediately after collection a single formulation of semen extender. Sperm motility and ASA binding were evaluated post-thaw. The results of both experiments showed similar findings. The frequency of ASA-positive samples was higher among stallions with poor sperm cryosurvival (Exp. 1 and 2 = 6/11, 54.5%) than for good sperm cryosurvival (Exp. 1 = 0/16, 0%; Exp. 2 = 1/11, 9.1%). The percentage of IgG- and IgA-bound sperm was also higher in stallions with poor sperm cryosurvival in both experiments (P < 0.05). Post-thaw sperm motility, velocity and distance parameters were lower in ASA-positive than ASA-negative stallions (P < 0.005). No effect of the semen extender used was observed. In addition, stallions with ASAs had a higher percentage of apoptotic sperm than stallions without ASAs. The presence of sperm-bound ASAs was associated with poor cryosurvival for stallion spermatozoa. Thus, it may be beneficial to evaluate stallions for binding of ASAs prior to freezing to offer and indicator of the prognosis for cryosurvival.

Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations

Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spectroscopy was used to identify dehydration and storage induced conformational changes in isolated DNA and sperm chromatin. Furthermore, hydrogen bonding in the preservation solutions associated with storage stability were investigated. Reactive oxygen species and DNA damage in dried sperm samples were found to accumulate with increasing storage temperature and storage duration. Non-reducing disaccharides (i.e., trehalose, sucrose) and albumin counteracted oxidative stress and preserved sperm chromatin during dried storage, whereas glucose increased DNA damage during storage. When sperm was dried in the presence of trehalose and albumin, no spectral changes were detected during storage at refrigeration temperatures, whereas under accelerated aging conditions, i.e., storage at 37 °C, spectral changes were detected indicating alterations in sperm chromatin structure.

A Modified Flotation Density Gradient Centrifugation Technique Improves the Semen Quality of Stallions with a High DNA Fragmentation Index.

Simple SummarySperm DNA fragmentation has a negative impact on reproductive success, because it compromises fertilization and embryo development. Since spermatozoa lack the machinery to repair DNA damage, the likelihood of establishing a healthy pregnancy can be improved by processing ejaculates with a high sperm DNA fragmentation index. The current study examined a modified flotation density gradient centrifugation technique in which semen was diluted with a colloid solution (Opti-prepTM) to increase its density, later layered between colloid layers of lower and higher density. This modified flotation technique was consistent in selecting viable, progressively motile, morphologically normal spermatozoa with intact acrosome and DNA. In reproductively normal stallions, sperm recovery was 57% and spermatozoa with damaged DNA decreased significantly (from 12% to 4%), with increase in the percentage of viable, progressively motile, morphologically normal spermatozoa with intact acrosome. In stallions with a high sperm DNA fragmentation index, this modified flotation technique markedly decreased the DNA fragmentation index (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, the stallion sperm DNA fragmentation index and other sperm quality parameters can be markedly improved using a modified flotation density gradient centrifugation technique employing a 40% Opti-prepTM cushion and a 20% top layer.Sperm DNA fragmentation compromises fertilization and early embryo development. Since spermatozoa lack the machinery to repair DNA damage, to improve the likelihood of establishing a healthy pregnancy, it is preferable to process ejaculates of stallions with a high sperm DNA fragmentation index (DFI) before artificial insemination or intracytoplasmic sperm injection. The aim of this study was to examine a modified flotation density gradient centrifugation (DGC) technique in which semen was diluted with a colloid solution (Opti-prepTM) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prepTM solution (20–60%) for use as the bottom/cushion layer was first determined, followed by a comparison between a modified sedimentation DGC and the modified flotation DGC technique, using different Opti-prepTM solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions (n = 3) with high sperm DFI (>20%). The optimal Opti-prepTM solution for the cushion layer was 40%. The modified sedimentation technique resulted in two different sperm populations, whereas the modified flotation technique yielded three populations. Among the variants tested, the modified flotation DGC using 20% Opti-prepTM as the top layer yielded the best results; the average sperm recovery was 57%; the DFI decreased significantly (from 12% to 4%) and the other sperm quality parameters, including progressive and total motility, percentages of spermatozoa with normal morphology and viable spermatozoa with an intact acrosome, all increased (p < 0.05). In Friesian stallions with high sperm DFI, the modified flotation DGC markedly decreased the DFI (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, stallion sperm DFI and other sperm quality parameters can be markedly improved using a modified flotation DGC technique employing a 40% Opti-prepTM cushion and a 20% top layer.