Optimization of Semen Extender for Mongolian Sheep by Integrating Metabolomics and Sperm Quality Analysis at 17 °C with Antioxidant Supplementation

This study focuses on optimizing the liquid preservation protocol for Mongolian sheep semen at 17 °C. We systematically compared the protective effects of eight extender formulations on sperm quality and analyzed dynamic metabolic changes under optimal storage conditions via LC-MS/MS metabolomics, aiming to provide experimental evidence for improving semen preservation techniques and to establish a foundation for the selection of preservation media in future germplasm conservation efforts. By comparing eight diluents, a semen preservation formulation for Mongolian sheep was optimized. The eighth diluent, including fructose, lactose, and Tris, effectively sustained sperm motility during liquid storage at 17 °C. To clarify the underlying metabolic mechanism, LC-MS/MS metabolomics identified 2669 metabolites in spermatozoa at storage times of 0, 48, 96, and 144 h. Based on the metabolomics analysis of differential metabolites, three antioxidants including vitamin E (acting as the basic antioxidant protection), lycopene (for targeted inhibition of lipid peroxidation) and resveratrol (suggested to have endogenous protective effects) were used for exogenous addition experiments. Subsequently, the optimal concentrations for addition were screened: resveratrol (25 μM), lycopene (5 μM), and vitamin E (1.5 μM). Verification experiments revealed that each of the three antioxidants could independently sustain sperm viability above 50% for up to 7 days. Moreover, the combined supplementation exhibited a synergistic effect, increasing sperm viability to 55.05%. This study not only elucidates the underlying mechanism of sperm preservation at the metabolic level, but also provides a validated formulation basis and theoretical support for the development of efficient sheep semen preservatives.