Effect of Testosterone, Dihydrotestosterone, Estradiol and Cortisol on the Quality of Fresh and Cryopreserved Stallion Sperm

The effect of steroid hormones on the quality of fresh and cryopreserve sperm has not been fully understood. This study aimed to evaluate the effect of testosterone, dihydrotestosterone, estradiol, and cortisol on the quality of fresh and cryopreserved stallion sperm. The study was conducted on 40 Equus caballus stallions, including Arab (n=20), Oryol trotting (n=4), Standardbred (n=4), and Soviet Heavy Draft (n=12) breeds. The average age of the experimental animals was 9.9 ± 0.7 years. We determined standard quality indicators in fresh and cryopreserved sperm and the concentration of steroid hormones in the blood plasma of stallions. Results of the study suggested a negative correlation between the level of testosterone with total (r=-0.41; p<0.01) and progressive (r=-0.44; p<0.01) sperm motility in cryopreserved sperm as well as in fresh sperm (r=-0.38; p<0.05 and r=-0.39; p<0.05 correspondingly). While the level of estradiol showed a positive correlation with survival rate in cryopreserved (r=0.35; p<0.05) and in fresh (r=0.33; p<0.05) sperm. Further, the level of cortisol in the blood plasma of stallions did not show any statistically significant correlations with the qualitative characteristics of sperm.  A positive relationship was found between the concentration of dihydrotestosterone with the volume of ejaculate (r=0.37; p<0.05) and the total number of sperm in the ejaculate (r=0.43; p<0.01). Results of the study can be concluded that steroid hormones have different effects on the quality indicators of fresh and cryopreserved sperm of stallions and their concentration in the blood should be considered when selecting stallions for cryopreservation of sperm

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

Objective: 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. Methods: 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. Results: 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 NME8, OR2AP1 and OR6C4. Potential implications of effects of these genes on sperm motility are herein discussed. Conclusion: 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

Reproductive Characteristics of Thawed Stallion Sperm

The main goal of our study was to determine a set of thawed stallion sperm characteristics that have predictive value for the pregnancy rate (PR) of mares after artificial insemination (AI). DNA fragmentation and survival of sperm during hypothermic storage were studied in addition to routinely determined semen characteristics such as concentration, percentage of motile spermatozoa, and morphology. To estimate DNA fragmentation, a modified hallo assay was applied. Sperm survival was determined within hours as the ability of spermatozoa to maintain progressive motility (PM) during the storage of ejaculate diluted with lactose-chelate-citrate-yolk (LCCY) medium at +4 °C. Strong positive correlation between PR and thawed sperm motility (r = 0.90, p < 0.05) as well as between PR and sperm survival (r = 084, p < 0.05) was revealed. There was also a strong negative correlation between PR and DNA damages in spermatozoa (r = −0.94, p < 0.05). We found no dependence of PR on normal morphology spermatozoa percentage in thawed semen. We concluded that the sperm activity, survival, and DNA fragmentation should be considered as the sufficient reproductive characteristics of semen to evaluate the quality of frozen/thawed sperm and prediction of PR