Copy Number Variation of β-defensin Genes and Bull Fertility

β-defensin are cationic peptides traditionally viewed as antimicrobial molecules. However, their role is emerging as some β-defensins have documented roles in important traits like fertility in mammals. In humans and in mice, DEFB126 has been shown to protect sperm against the female immune system by mediating the attachment of glycoproteins to the sperm surface and thereby enhance their survival. Earlier work has also documented a similar role for the bovine orthologous protein in sperm attachment to oviductal epithelium as well as in the reduction in sperm agglutination. Genes encoding β-defensin exhibit extensive copy number variation (CNV), including both duplications and deletions in mammals. This CNV presents potential as biomarkers of fertility. Despite the critical importance of bull fertility in cattle breeding, the extent and nature of β-defensin CNV remain unclear. This Ph.D study utilized the latest cattle reference genome (ARS-UCD1.2) to investigate the evolutionary relationships, CNV, and functional implications of β-defensin genes in fertility. Analysis revealed 55 cattle beta-defensin genes, with 35 having human orthologs, and identified significant CNV, with DEFB103 showing substantial multiallelic variation. While no direct correlation with fertility was found, breed-specific CNV patterns were observed. Furthermore, a comparison of β-defensin expression levels in the testes of calves and adult bulls revealed that the expression of 14 β-defensin genes, including DEFB103, increased during sexual maturation. In a study of 94 matched samples from the caput of the adult bull epididymis, β-defensin gene expression levels and copy number were correlated in four β-defensins, including DEFB103. In vitro experiments suggested that bulls with low DEFB103 CN exhibited increased sperm motility across all samples (n=20, p<0.05). Interestingly, genetic diversity in DEFB103 CN was primarily observed in bulls with low fertility (LF). Functional analysis was performed on three bulls from each CN class (low, normal, and high). Sperm from LF bulls with low CN exhibited higher binding to oviduct epithelium in vitro, while high CN was associated with altered sperm membrane fluidity under non-capacitating conditions (p<0.05). To further explore the functional effects of DEFB103 CNV in vivo, 18 heifers were inseminated with sperm from bulls with low, normal, and high CN. Transcriptomic analysis of uterine tissue 12 hours post-insemination revealed the differential expression of 58 genes (FDR<0.1), associated with sperm migration, immune signaling, and chemotaxis. DEFB103 CN may alter the uterine immune response to sperm from bulls with LF, and therefore affecting early pregnancy establishment. These findings underscore the significant role of DEFB103 CN in sperm function and the uterine response to bull sperm, suggesting its potential impact on pregnancy outcomes in cattle.