Wanying Li, Renjian Lin, Shengming Wang, Peichu Zhou, Weijun Pang, Tongying Feng, Yi Zheng
During cryopreservation, boar sperm are readily subjected to cold shock, ice crystal formation, osmotic changes, and oxidative stress, leading to a significant decline in the post-thaw sperm quality. The addition of cryoprotective agents to the freezing extender can effectively alleviate cryodamage and improve sperm motile performance. In this study, we first employed a metabolomic analysis to identify metabolites potentially associated with sperm cryoinjury, and then explored their roles and underlying mechanisms in amelioration of sperm cryodamage. Briefly, by way of a LC-MS-based comparative analysis among fresh semen, once frozen-thawed samples, and repeatedly frozen-thawed samples, we identified ergothioneine (EGT) as a metabolite strongly correlated with sperm cryoinjury. Supplementation of the freezing extender with different concentrations of EGT (0, 100, 200, 500, and 1000 μM) significantly improved post-thaw sperm motility, motion parameters, and the acrosome and plasma membrane integrity, with 200, 500, and 1000 μM EGT notably enhancing sperm antioxidant capacity and energy metabolism, thereby effectively alleviating sperm cryoinjury. The subsequent mechanistic investigation revealed that EGT exerted its protective effects by activating the AMPK signaling pathway, while the addition of an AMPK inhibitor markedly attenuated the protective effects. Collectively, this study demonstrates that the endogenous metabolite EGT alleviates the cryodamage of boar sperm via the AMPK signaling pathway, providing robust scientific evidence for introduction of endogenous antioxidant metabolites into the freezing extender and shedding light on development of safer and more effective strategies for the sake of semen cryopreservation.