Sperm-TLR2 activation enhances bovine embryo developmental competence via modulation of intracellular calcium and autophagic flux

The optimization of in vitro embryo production (IVP) is critical for bovine biotechnology, particularly for maximizing the yield of high-quality embryos suitable for transfer. This study investigated how Toll-like receptor 2 (TLR2) activation in sperm, using the agonist Pam3CSK4 (Pam), influences fertilization outcomes, metabolic activity, and cryotolerance. Japanese Black sperm were treated with 50 ng/ml Pam for 30 min before fertilizing Holstein oocytes. Embryos were cultured for 7 days, with quality assessed via total cell counts, apoptosis, calcium signaling, and autophagy markers. For cryopreservation trials, only high-quality (grade A) blastocysts were selected for slow-freezing to evaluate re-expansion and hatching rates. Our results demonstrate that TLR2 activation in sperm significantly enhanced cleavage and blastocyst rates, increasing the overall efficiency of embryo production by approximately 10% compared to the control. Embryos derived from Pam-treated sperm exhibited superior biological quality, characterized by increased total cell numbers in blastocysts and reduced apoptosis in expanded blastocysts. Mechanistically, TLR2 activation elevated basal calcium levels and modulated autophagic flux, indicating a beneficial shift in metabolic programming. Notably, while the absolute number of embryos increased, the proportion of grade A blastocysts and their post-thaw survival metrics remained comparable to the control. This suggests that while TLR2 signaling effectively scales up IVP efficiency, the resulting developmental gains represent a hidden physiological strength that conventional morphological grading may not fully capture. These findings indicate that pre-IVF sperm treatment with Pam effectively primes the embryo for superior cellular development while maintaining the essential cryo-survival standards required for commercial applications.