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The aim of the work was to search for genome-wide associations with the number of damaged rooster sperm membranes after freezing. Roosters of 16 breeds bred in the bioresource collection of the "Genetic Collection of Rare and Endangered Breeds of Chickens" (VNIIGRZH, Pushkin, St. Petersburg) were taken for analysis. For genome-wide genotyping, 96 DNA samples were prepared for genotyping on the Affymetrix Axiom®600k Array. The ejaculates were frozen in pellets and stored in liquid nitrogen at -1960С. Sperm motility was assessed using CASA. Membranes were assessed using the Sperm VitalStain dye (Nidacon International AB, Sweden). Staining was carried out in Eppendorf type tubes (50 µl of sperm was mixed with 50 µl of dye) and smears were made on glass slides. The preparations were viewed at x1000 with oil immersion. Cells were evaluated by CASA, module "Live – dead." The Genome-Wide Association studies were conducted using EMMAX. Manhattan-type plot was obtained using the qqman package in R software. Localization of SNPs was determined and candidate genes were identified using the NCBI and Ensembl genomic browsers. Motility varied from 5 to 80% and membrane damage was from 3 to 96%. Two suggestive SNPs were found on chromosomes 2 and 3. Nucleotide polymorphism AX-76495998 (P-value=1.93E-06) is located on chromosome 3 in the intergenic space. A putative candidate gene was ARID1B , a chromatin regulator. SNP AX-76063628 (P- value=2.61E-07) on chromosome 2 is located in the intron of the PHF14 gene. The PHF14 gene affects cell proliferation, and knockout of the PHF14 gene in mice resulted in neonatal mortality due to respiratory failure. In our case, the polymorphism observed in this gene may be the reason for the instability of sperm membranes to freeze-thaw in roosters. PHF14 genotype analysis revealed a significant negative effect of the T allele on the safety of rooster spermatozoa membranes during cryopreservation. The identified candidate genes are supposed to be validated on a large number of roosters of various breeds. Authors acknowledge financial support from Russian Science Foundation, Grant No: 18-16-00071. [ABSTRACT FROM AUTHOR]

Sperm morphology is an important indicator of sperm quality. We calculated the rank correlation coefficient between fertility and the number of cells with damaged acrosomes, which was r=-0.6, which is relatively high. The aim of the study was to find genetic associations with semen morphology after cryopreservation in bulls. The quality of spermatozoa was assessed by CASA and GWAS was used for genotype analysis. Calculations were carried out using PLINK and EMMAX programs and a Manhattan-type plot was obtained using the qqman package in R software. The localization of SNPs was determined and candidate genes were identified using the NCBI and Ensembl genomic browsers. The semen of 130 bulls was evaluated. Total sperm motility varied from 25 to 65% and progressive motility ranged from 15 to 60%. There were 13.6 to 39.2% of cells with damaged acrosomes and 1.2 to 14.1% of cells with damage in the tail and neck in frozen bovine semen. There were several significant SNPs associated with the number of missing acrosomes after cryopreservation. For example, a significant SNP on the fourth chromosome, BTB-00566744 (P-value=2.89E-15), is an intron variant of the POU6F2 gene, somatic mutations of which have been identified in human glaucoma and prolactinoma. Prolactin, in turn, significantly reduces the spontaneous acrosomal reaction. There were genomic associations with the percentage of swollen acrosomes after cryopreservation. Thus, the LPCAT4 gene is involved in the biosynthesis and metabolism of lipids and phospholipids. The identified genomic associations with disorders in the tail and neck region mostly repeated genes associated with disorders in the head region. These are the genes: FOXF1, ZBTB40, WNT4, FRMD4A, FAM107B, PTPRU, GLRA3, RABGAP1, EPC2, MBD5, TTK, BCKDHB, PPP3CA, MAP2K5, MAP2K6, CD59, DIP2B, AIG1, ADAT2, TTC29, POU4F2, ENDRA, GRIK1, NEDD4, C10H15orf61, WNT5A, RIMS1, RAD51B, KLHL1, PAPSS1, DKK2, RGS17, U6, ORC4. Thus, the RIM1α isoform can compensate for the lack RIM2α in calcium-induced acrosomal reaction. As a result, a large number of genome-wide associations that affect the preservation of spermatozoa after freezing-thawing have been identified. Authors acknowledge financial support from Russian Science Foundation, Grant No: 18-16-00071. [ABSTRACT FROM AUTHOR]