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 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.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.

Horses were domesticated later than other farm animals. Horse breeds have been selectively developed by humans to satisfy different needs and purposes. The factory and indigenous breeds are of particular interest, having been bred in purity for many centuries without the addition of foreign blood. Data from 31 stud farms, as well as ranches, located in fifteen regions of the Russian Federation were used in this work. DNA was sampled from 102 stallions of 11 breeds: Arabian, Akhal-Teke, Don, Orlov Trotter, Vladimir Heavy Draft, Russian Heavy Draft, Soviet Heavy Draft, Kabardin, Yakut, Tuva, and Vyatka. Data on the origin of each animal from which the material was collected were taken into account. DNA genotyping was carried out using GGP Equine 70 k ^® array chips (Thermo Fisher Scientific, USA). Genetic diversity of horse breeds was estimated using Admixture 1.3. and PLINK 1.9 software. FROH inbreeding was computed via the R detectRUNS package. The minimum length for ROH was set at 1 Mb to reduce the occurrence of false positives. We conducted PCA analysis using PLINK 1.9, and used the ggplot2 library in R for visualizing the results. Indigenous equine breeds, such as Vyatka, Tuva, and Yakut, are very hardy, and well adapted to local environmental and climatic conditions. They are employed as draft power, as well as for milk and meat. Both the Akhal-Teke breed and the Arabian breed have retained a minimum effective population size over many generations. We note significant accumulations of homozygosity in these breeds. In equestrian sports, performance is a top priority. ADMIXTURE and PCA analyses showed similarities between Don equine breeds and Kabardin, as well as some Arabian breed animals. Earlier research indicated the presence of thoroughbred traits in Don stallions. The Orlov Trotter breed stands out as a separate cluster in the structural and PCA analyses. Considering the small population size of this breed, our study found high FROH in all tested animals. The general reduction in the diversity of the horse breed gene pool, due to numerous crosses for breed improvement with thoroughbreds, has lead to a decline in the differences between the top sporting breeds. Our study presents new opportunities for exploring the genetic factors that influence the formation of adaptive traits in indigenous breeds, and for finding ways to preserve genetic diversity for effective population reproduction.

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.

The results of an electron microscopy study of the ultrastructural characteristics of stallion spermatozoa in native sperm and after freezing-thawing are given. Changes occurring in spermatozoa under the effect of cryopreservation are shown. Maximum damages are found in acrosomes.