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. 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 knocked down in boar sperm via siRNA electro-transfection, and differential metabolites were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results showed that SIRT5/IDH2 knockdown 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 hr; cryopreserved: ∼+10%), progressive motility, ATP content, and mitochondrial function, while maintaining acrosomal and plasma membrane integrity. Notably, L-methionine reduced reactive oxygen species levels by 9% (p < 0.05) in fresh sperm at 48 hr 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 success rates.