Baicalein and Neohesperidin Dihydrochalcone Improve Post-Thaw Sperm Quality and Modulate the Semen Supernatant Metabolome in Qinchuan Bulls

Oxidative stress during cryopreservation compromises post-thaw sperm quality in bulls. This study evaluated the effects of baicalein (BAI), neohesperidin dihydrochalcone (NHDC) and their combined supplementation on Qinchuan bull sperm cryopreservation and the associated metabolic responses. Concentration screening identified 40 μg/mL BAI and 30 μg/mL NHDC for subsequent experiments. Compared with the control, BAI, NHDC and BAI + NHDC increased post-thaw sperm motility, plasma membrane integrity, acrosome integrity, adenosine triphosphate (ATP) content and mitochondrial membrane potential (MMP). The supplemented groups also showed lower reactive oxygen species (ROS) and malondialdehyde (MDA) levels; higher activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); and higher total antioxidant capacity (T-AOC). The combined treatment showed higher total motility, plasma membrane integrity, and acrosome integrity than either single treatment. Untargeted metabolomics identified 96, 123 and 197 differential metabolites in the BAI, NHDC and BAI + NHDC groups, respectively. Differential metabolites from all three treatments were enriched in lipid-related pathways, including glycerophospholipid metabolism and linoleic acid metabolism. Metabolites identified in the BAI + NHDC group were additionally enriched in nucleotide metabolism, purine metabolism, and ubiquinone-related pathways. BAI × NHDC interaction analysis identified 200 metabolites showing non-additive responses, with enrichment in lipid-related pathways, including glycerophospholipid metabolism and linoleic acid metabolism. Together, BAI and NHDC improved post-thaw sperm quality in Qinchuan bulls, accompanied by changes in oxidative status, mitochondrial function, and lipid- and energy-related metabolites.