Impact of β-defensin 103 (DEFB103) copy number variation on bull sperm parameters and post-insemination uterine gene expression

Pregnancy rates for elite bulls used in artificial insemination (AI) can vary significantly and therefore the identification of molecular markers for fertility and targets to improve bull selection is important. β-defensin peptides have diverse regulatory roles in sperm function across multiple species but the role of copy number variation (CNV) on fertility parameters has not been previously evaluated. In this study, Holstein-Friesian bulls were screened based on reliable field fertility data to identify two groups (High and Low fertility (HF and LF, respectively)) of n = 10 bulls/group which were genotyped for β-defensin 103 (DEFB103) gene CNV by droplet digital PCR. Overall, low DEFB103 copy number (CN) was associated with increased sperm motility across all bulls (n = 20, p < 0.05). As genetic diversity of DEFB103 CN was only apparent in the LF group, three bulls per CNV class (low, intermediate and high CN) were chosen for more detailed comparative functional analysis. Sperm from low CN bulls exhibited higher binding to the oviductal epithelium, while high CN increased sperm membrane fluidity in vitro (p < 0.05). To investigate the functional effect of DEFB103 CNV on the uterine response in vivo, 18 heifers were inseminated with sperm from bulls with low, intermediate and high CN. Transcriptomic analysis on uterine tissue harvested 12 h post-insemination showed differential expression of 58 genes (FDR < 0.1) involved in sperm migration, immune signalling and chemotaxis. Although field fertility results from a complex number of interactive factors, these novel results suggest a contributory role for DEFB103 CN in both sperm function and the uterine response to bull sperm, thereby potentially contributing to pregnancy outcomes in cattle. Further analysis of the role of CNV in additional β-defensin genes in bull fertility is now warranted.