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Cryopreservation is a widely used method of semen conservation in animal breeding programs. This process, however, can have a detrimental effect on sperm quality, especially in terms of its morphology. The resultant sperm disorders raise the risk of reduced sperm fertilizing ability, which poses a serious threat to the long-term efficacy of livestock reproduction and breeding. Understanding the genetic factors underlying these effects is critical for maintaining sperm quality during cryopreservation, and for animal fertility in general. In this regard, we performed a genome-wide association study to identify genomic regions associated with various cryopreservation sperm abnormalities in Holstein cattle, using single nucleotide polymorphism (SNP) markers via a high-density genotyping assay. Our analysis revealed a significant association of specific SNPs and candidate genes with absence of acrosomes, damaged cell necks and tails, as well as wrinkled acrosomes and decreased motility of cryopreserved sperm. As a result, we identified candidate genes such as POU6F2 , LPCAT4 , DPYD , SLC39A12 and CACNB2 , as well as microRNAs ( bta-mir-137 and bta-mir-2420 ) that may play a critical role in sperm morphology and disorders. These findings provide crucial information on the molecular mechanisms underlying acrosome integrity, motility, head abnormalities and damaged cell necks and tails of sperm after cryopreservation. Further studies with larger sample sizes, genome-wide coverage and functional validation are needed to explore causal variants in more detail, thereby elucidating the mechanisms mediating these effects. Overall, our results contribute to the understanding of genetic architecture in cryopreserved semen quality and disorders in bulls, laying the foundation for improved animal reproduction and breeding.

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.