Biphasic Responses of Porcine Oocytes to Metformin: Concentration-Dependent AMPK Activation and Nrf2-Mediated Antioxidant Regulation

Simple SummaryPoor porcine oocyte and embryo quality limits pig breeding efficiency. This study tested metformin supplementation in culture media to improve oocyte maturation and embryo development. Low-dose metformin enhanced maturation and blastocyst formation by activating AMPK through a non-canonical pathway, reducing oxidative damage and improving mitochondrial function, while high doses impaired oocyte quality. Appropriate metformin use optimizes in vitro culture systems, supporting more efficient pig embryo production for agriculture and reproductive research.Metformin (MET) plays crucial regulatory roles in mammalian oocyte meiosis, yet the concentration-dependent biphasic impacts of MET on porcine oocyte in vitro maturation (IVM) and the related molecular mechanisms remain poorly clarified. This study aimed to explore the distinct effects and underlying pathways of low- and high-dose MET in porcine oocytes. Different concentrations of MET (0, 7.5, 15, 30, 150, and 300 μM) were supplemented during oocyte IVM, with phenotypic detection, untargeted metabolomic analysis, and Nrf2 inhibitor (ML385) intervention performed for mechanism exploration. Results showed that 15 μM low-dose MET facilitated oocyte maturation, mitochondrial function and redox balance, while 300 μM high-dose MET caused obvious developmental damage. Mechanistically, low-dose MET triggered noncanonical AMPK activation independent of the AMP/ATP ratio and enhanced AMPK–Nrf2 antioxidant signaling, whereas high-dose MET induced energy stress and oxidative injury via inhibiting mitochondrial complex I. Blockade of Nrf2 further abolished the protective effects of low-dose MET. Collectively, this finding illustrates the biphasic actions of MET on porcine oocytes and provides a theoretical reference for optimizing porcine in vitro embryo production.