Abstract
Semen cryopreservation is a crucial technology for enhancing reproductive efficiency in livestock production; however, oxidative stress-induced sperm damage during the freeze-thaw process remains a significant challenge. In this study, metabolomics was used to analyze the differences in metabolites in semen from Qinchuan cattle with different freezing tolerance, and to screen out the candidate markers of sperm freezing tolerance. The metabolomics results indicate that a total of 264 differential metabolites were identified, and KEGG pathway annotation revealed that amino acid metabolism (15.07%) were prominently represented, and L-glutamine (L-Gln) showed a particularly high abundance in high freezability group (HFG) compared to the low freezability group (LFG). Further experiments demonstrated that L-glutamine supplementation significantly improved post-thaw sperm motility, plasma membrane integrity, and acrosomal integrity (p < 0.05). It also enhanced sperm antioxidant capacity by increasing the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), while reducing malondialdehyde (MDA) content (p < 0.05). Additionally, L-Gln maintained mitochondrial function and energy homeostasis by elevating mitochondrial membrane potential (MMP) and promoting AMPK phosphorylation (p < 0.05). These results indicate that L-glutamine alleviates oxidative damage during cryopreservation and enhances semen freeze tolerance.