Ginseng Peptide Improves the Cryopreservation Efficiency and Fertilization Potential of Yak Semen via FOXO1/PI3K/AKT Axis

Semen cryopreservation is a critical biotechnological approach for preserving superior genetic resources in livestock. Spermatozoa are particularly vulnerable to cryogenic stress during the freeze-thaw process, resulting in impaired structure and function. Therefore, the development of effective cryoprotective additives is essential for improving yak semen cryopreservation. In this study, ginseng peptide (GFREH) was incorporated into the freezing extender at different concentrations (0, 0.25, 0.5, 0.75, and 1.0 mg/mL) to evaluate its effects on post-thaw sperm quality, in vitro fertilization (IVF) capacity, and the underlying regulatory mechanisms. Semen samples treated with 0 and 0.75 mg/mL GFREH were further subjected to proteomic analysis to elucidate the molecular basis of its cryoprotective action. The results demonstrated that GFREH significantly increased total motility (TM), progressive motility (PM), straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), as well as plasma membrane and acrosome integrity of frozen-thawed yak spermatozoa (<i>p</i> < 0.05). GFREH also significantly reduced malondialdehyde (MDA) levels while enhancing antioxidant enzyme activities, mitochondrial membrane potential (MMP), and ATP content (<i>p</i> < 0.05). Moreover, GFREH at concentrations of 0.5, 0.75, and 1.0 mg/mL significantly improved IVF and blastocyst formation rates compared with the control (<i>p</i> < 0.05), with the 0.75 mg/mL group exhibiting the highest fertilization and blastocyst rates. Proteomic analysis further revealed that GFREH modulated the PI3K/AKT signaling pathway and downregulated FOXO1 expression. Collectively, these findings indicate that ginseng peptides enhance yak sperm cryotolerance by coordinating oxidative balance, mitochondrial energy metabolism, and survival-related signaling, with 0.75 mg/mL representing an optimal effective concentration within the functional dose range tested.