Bovine β-defensin 129 (BBD129) polymorphisms associated with differential posttranslational O-glycosylation and phosphorylation modifications perturb the sperm functions and their fertilizing potential

In our earlier finding BBD129 polymorphisms were differentially distributed in the distinct fertility cattle bulls and associated with altered post-translational modifications (PTMs), and sperm function. This study investigated the relationship between in-silico analysis of BBD129 polymorphism and previously used high-fertility (HF) and low-fertility (LF) cattle bulls’ sperm. Using CFDA-PI staining, it depicted similar frozen viable, moribund, and non-viable sperm population profiles between the HF and LF cattle bulls. Phosphorylation quantification via microscopy and flow cytometry revealed significantly higher serine and threonine phosphorylation in LF sperm, particularly in the tail and head regions, aligning with in-silico predictions and suggesting a link to LF bulls. Glycan localization was assessed using a panel of seven lectins, revealing distinct surface glycan patterns between HF and LF sperm. Lectins targeting O-linked glycans (MAL-II, Jacalin, SNA) showed significantly higher binding on HF sperm surface, whereas LCA and PNA were more abundant on LF sperm. These findings were consistently confirmed by both microscopy and flow cytometry. Notably, HF sperm exhibited a greater abundance of surface glycans, correlating with enhanced cervical mucus penetration ability in the cervical mucus penetration test (CMPT); HF sperm traveled an average of 52.77 mm versus 42.30 mm for LF sperm. This study demonstrates that BBD129 polymorphism influences critical PTMs, particularly glycosylation and phosphorylation, which in turn affect sperm membrane integrity, surface glycan composition, and functional competence in traversing the female reproductive tract. These molecular markers may serve as valuable indicators of bull fertility, providing insights for improving reproductive performance in cattle breeding programs.