Non-thermal plasma enhances boar sperm quality via FOXO3-PINK1/Parkin-mediated mitophagy

Boar sperm quality is a key intrinsic factor of reproductive success, and its optimization is crucial for improving pig production efficiency. Here, this study investigated the effect of non-thermal dielectric barrier discharge (DBD) plasma on boar sperm quality and the underlying regulatory mechanism. Results showed that optimized DBD plasma improved boar sperm viability and motility, adenosine triphosphate level, mitochondrial membrane potential, and mitochondrial respiratory chain enzyme activity, while reducing the opening degree of mitochondrial permeability transition pore. Transcriptome sequencing revealed significant changes in sperm mitophagy-related genes after DBD plasma treatment. Correspondingly, mitophagy-related proteins including Forkhead box O3 (FOXO3), PTEN induced kinase 1 (PINK1), Parkin, BCL2 interacting protein 3 (BNIP3), and LC3 II/LC3 I ratio exhibits increased at protein levels, while the protein levels of P62 and TOM20 were decreased in DBD plasma-treated spermatozoa. Mitophagy inhibitor Mdivi-1 decreased sperm mitophagy level, mitochondrial function and sperm quality, but these effects were rescued by DBD plasma. Moreover, DBD plasma increased the binding interaction of FOXO3-PINK1 and PINK1-Parkin. Silencing FOXO3, PINK1, and Parkin by using siRNAs inhibited mitophagy level, mitochondrial function, and sperm quality, but these effects were alleviated by DBD plasma. These findings illustrated appropriate DBD plasma enhanced mitochondrial function by activating FOXO3-PINK1/Parkin-mediated mitophagy, leading to improved boar sperm quality without disrupting sperm DNA integrity and acrosome integrity. Therefore, non-thermal plasma technology provides a novel strategy to enhance sperm quality through the regulation of targeted mitophagy, offering promising potential for its application in livestock reproduction.