Search for genome-wide associations with the number of damaged membranes in rooster semen after freezing

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 (50µl of sperm was mixed with 50µl 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.