Effects of Kaempferol Supplementation on the Cryopreservation Quality of Semen from Yuansheng Aite Dairy Rams

Sperm cryopreservation is important for livestock breeding and germplasm conservation, but freeze–thaw injury can impair ram sperm quality through oxidative stress, membrane damage, and metabolic disturbance. This study evaluated the concentration-dependent effects of kaempferol supplementation on the cryopreservation quality of semen from Yuansheng Aite dairy rams. Qualified ejaculates were pooled and randomly allocated to five equally spaced kaempferol treatment groups: 0, 25, 50, 75, and 100 μg/mL. Post-thaw sperm motility, oxidative stress status, ATP-related energy metabolism, acrosome integrity, and multi-omics profiles were evaluated. Data were analyzed using appropriate parametric or non-parametric tests after assessment of normality and homogeneity of variance. Orthogonal polynomial analysis was performed to evaluate linear and nonlinear dose–response patterns across the tested kaempferol concentrations. Kaempferol supplementation significantly affected PM, VCL, and VAP, while RPM, LIN, WOB, and VSL were not significantly affected. No significant linear effect was observed for the motility parameters, whereas VCL exhibited a significant quadratic response to kaempferol concentration. Based on the observed overall responses of sperm motility, antioxidant capacity, oxidative stress markers, ATP content, and acrosome integrity, 25 μg/mL kaempferol showed the most favorable overall profile among the tested concentrations and was selected for subsequent mechanistic analyses. Proteomic and metabolomic analyses suggested that the protective effects of kaempferol may be associated with pathways related to focal adhesion, cytoskeletal organization, oxidative phosphorylation-related energy metabolism, and central carbon metabolism. These findings indicate that moderate kaempferol supplementation may improve the post-thaw quality of Yuansheng Aite dairy ram semen, although further fertility-oriented studies are needed to confirm its practical reproductive benefits.