Identification of molecular genetic causes of bull infertility

This is a cumulative thesis that includes three different topics around male infertility in cattle and canine diseases. Whole genome association analysis (GWAS) and whole genome sequencing (WGS), as well as additional experimental approaches, were used to identify the candidate genes for these disorders. One of the current challenges facing the dairy industry is to predict and improve the fertility of bulls. We have detected a Holstein bull, who had been approved for artificial insemination based on his semen characteristics, but did not produce offspring after 412 first inseminations, resulting in a non-return rate (NR-Abw) of -29. To identify causative mutations underlying this idiopathic infertility, GWAS and WGS were performed on different non-return rate bulls followed by genotyping of candidate variants in a large population. Finally, a nonsense mutation (AC_000170.1: g.54429815G>A, rs468948776) in α/β-Hydrolase D16B (ABHD16B) on chromosome 13 was identified as a potential candidate variant. Protein analysis revealed expression of ABHD16B in the testes and epididymis of control bulls, and lipidomic analysis showed significant differences in lipid content of the plasma membrane between carriers and controls. We conclude that ABHD16B may play a role in lipid changes during spermatogenesis or sperm maturation and that the altered lipid content may explain the reduced fertilization capacity of mutant sperm. Hemophilia B is a monogenic, X-chromosome recessive bleeding disorder caused by genetic variants in the coagulation factor IX gene (F9). Here we identify a Hovawart family with hemophilia B Leyden caused by a deletion (NC_006621.3:g.109501492delC) in the F9 promoter within the conserved binding region of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). Using EMSA we found that the deletion only eliminated the binding of HNF-4α to the promoter, but did not affect the binding of AR. In vitro approach utilizing luciferase reporter gene assay revealed a significant expression reduction of the mutated promoter compared to wild-type. Our results suggest that dogs provide a natural occurrence model for understanding hemophilia B. Congenital deafness is prevalent in many modern dog breeds, but few causative genes have been identified to date. Here, we attempt to find the genetic cause of congenital deafness around three deaf Australian Stumpy Tail Cattle Dogs (ASCD) using GWAS and WGS. A GWAS was performed on 3 bilateral deaf ASCDs, 43 herding dogs, and one unaffected ASCD, resulting in the identification of 13 significantly associated loci on 6 chromosomes (CFA3, 8, 17, 23, 28, and 37). More than half of the significantly associated signals were localized on CFA37, containing the most significantly associated variant. Further WGS was conducted on same three deaf ASCDs and one control, whereupon a missense variant (NC_006619.3:g.15562684G>A; XP_022270984.1:p.Leu173Phe) of kruppel-like factor 7 (KLF7) on CFA 37 was considered as a candidate variant. Genotyping of the KLF7 variant in an additional cohort of 55 ASCD samples (28 deaf and 27 normal hearing dogs) indicated that the variant was still associated with deafness (p = 0.014), with a calculated penetrance of 0.75. In conclusion, KLF7 is a promising candidate gene for the etiology of deafness in ASCD.2021-11-2