L-Methionine Enhances Cryosurvival and Antioxidant Activity of Boar Sperm

Cryopreserved boar sperm yields lower conception rates and litter sizes than liquid-stored semen due to oxidative damage from elevated reactive oxygen species (ROS). Our prior study demonstrated that mitochondrial regulators, SIRT5 and IDH2, were significantly downregulated during cryopreservation; however, how this dysregulation alters sperm metabolism remains unknown. Here, SIRT5 and IDH2 were knock down in boar sperm via siRNA electro-transfection, and differential metabolites were identified by LC-MS/MS. Results showed that SIRT5/IDH2 knock-down dysregulated antioxidant pathways and significantly downregulated L-methionine (P < 0.05). Supplementation with 80 µM L-methionine significantly increased total sperm motility (fresh: ∼+6% at 48 h; cryopreserved: ∼+10%), progressive motility, ATP content, and mitochondrial function while maintaining acrosomal and plasma membrane integrity. Notably, L-methionine reduced ROS levels by 9% (P < 0.05) in fresh sperm at 48 h and 13% in post-thawed sperm. This study elucidates a mechanism-driven strategy wherein the SIRT5/IDH2 axis maintains sperm antioxidant capacity by regulating L-methionine and associated metabolites. These findings enhance our understanding of energy metabolism in sperm and offer a targeted approach to improve cryopreserved semen quality, potentially increasing artificial insemination (AI) success rates.