Decoding bull fertility in vitro: a proteomics exploration from sperm to blastocyst

Male fertility plays a pivotal role in the success rates of in vitro embryo production. While livestock breeding programs rigorously select bulls according to their predicted field fertility, specific traits such as polyspermy rates are not routinely evaluated. Despite the known negative impact of polyspermy on embryo survival, the paternal factors involved remain unclear. In this study, we aimed to address this gap by evaluating the in vitro outcomes of four bulls, focusing on sperm motility, fertilization rates, polyspermy incidence, embryo development and quality. In addition, we analyzed the proteome profiles of sperm, 2-4 cell stage embryos and blastocysts derived from those bulls to identify potential molecular factors associated with male fertility. Bulls with comparable sperm motility parameters displayed varying in vitro fertilization outcomes. Notably, the bull with the highest polyspermy rate achieved blastocyst rates similar to those of bulls with lower polyspermy rates. The number of apoptotic cells in the blastocysts was bull-dependent. Proteomic analysis revealed bull-specific signatures in sperm and blastocysts, with no differences at the 2-4 cell stage. Differences in the sperm proteome suggested that bull-dependent penetration and polyspermy rates might be associated with the ability of the sperm to undergo capacitation and acrosomal reaction. At the blastocyst level, the bull with the highest polyspermy rates produced lower quality blastocysts due to imbalances in key proteins and pathways for embryo development. In conclusion, bulls with similar blastocyst rates may differ in polyspermy rates and resulting embryo quality underscoring the importance of careful bull selection for in vitro embryo production.