Punicalagin enhances cryopreserved sheep sperm quality via activation of the Nrf2/PGC-1α pathway and metabolic reprogramming.

Cryopreservation-induced damage remains a major limitation in ram semen preservation, particularly impairing sperm motility and structural integrity. Punicalagin, a natural polyphenol with potent antioxidant properties, has demonstrated potential in mitigating oxidative stress. However, its role in ruminant sperm cryoprotection remains incompletely understood. This study investigated the effects of punicalagin on cryopreserved sheep sperm and explored its underlying molecular mechanisms. A comprehensive evaluation of cryodamage indicators was conducted before and after cryopreservation. The effects of varying punicalagin concentrations (0-20 μM) on sperm motility, antioxidant pathways, and mitochondrial function were assessed. Untargeted metabolomics was employed to characterize changes in the sperm metabolite profile, and the selective Nrf2 inhibitor (ML385) was employed to validate the core signaling pathway. Cryopreservation significantly reduced progressive motility, mitochondrial membrane potential (ΔΨm, MMP), and adenosine triphosphate (ATP) levels while increasing tail bending rate and acrosomal defects (P < 0.05). Supplementation with punicalagin (15 μM) significantly decreased reactive oxygen species (ROS) levels, enhanced superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, restored ΔΨm, and downregulated apoptotic markers (Bax/Fas) (P < 0.05). Punicalagin activated the Nrf2/PGC-1α pathway, an effect abolished by ML385. Metabolomic analysis revealed punicalagin-regulated lipid metabolism and identified five differential metabolites, including 20-COOH-10,11-dihydro-LTB4, Acylcarnitine 12:2, Acylcarnitine 20:6, Pyridine, and Undecanoic acid, as metabolic biomarkers for cryodamage evaluation (AUC >0.9). These findings demonstrate that punicalagin enhances sperm cryotolerance through dual mechanisms involving antioxidant pathway activation and metabolic reprogramming, providing a novel strategy for improving ruminant sperm cryopreservation.