Apigenin in freezing medium improves frozen-thawed sperm quality and fertilization competence of Oura-type Tibetan sheep

Apigenin (API), a naturally occurring flavonoid with potent antioxidant activity, has received limited attention regarding its protective effects on frozen-thawed sperm. Here, API was added to the freezing medium at 0-100 μM to determine the optimal concentration by assessing post-thaw survival and kinetic parameters. Dose-response screening identified 10 μM API as the most effective level, and this concentration was used to evaluate sperm quality, antioxidant capacity, fertilization potential and protein expression. Ten micromolar API significantly enhanced acrosomal and head membrane integrity and mitochondrial membrane potential, whereas tail membrane integrity and DNA integrity remained unchanged. Reactive oxygen species and malondialdehyde levels declined, superoxide dismutase activity and total antioxidant capacity increased; IVF cleavage rate improved, while blastocyst rate and blastocyst cell number remained unaffected, and AI pregnancy rate rose. iTRAQ profiling indicated that API primarily modulates proteins linked to energy metabolism, motility and mitochondrial function. API at 10 μM markedly improves the quality of frozen-thawed Oura-type Tibetan sheep sperm.