Proline Protects Boar Sperm against Oxidative Stress through Proline Dehydrogenase-Mediated Metabolism and the Amine Structure of Pyrrolidine

Simple Summary Reactive oxygen species that accumulate during liquid storage of boar semen lead to oxidative stress to sperm. In this study, we found that proline significantly improved boar sperm quality and protected sperm against oxidative damages during liquid storage at 17 degrees C. Using the model of artificially induced oxidative stress, we found that proline exerted an antioxidative role by modulating redox homeostasis in boar sperm. The secondary amine structure of proline and proline dehydrogenase-mediated metabolism are involved in the antioxidative role. We suggest that addition of proline to the extender would be beneficial to improve boar sperm quality. Proline was reported to improve sperm quality in rams, stallions, cynomolgus monkeys, donkeys, and canines during cryopreservation. However, the underlying mechanism remains unclear. The aim of this study was to investigate the effect of proline on boar semen during liquid storage at 17 degrees C and explore the underlying mechanism. Freshly ejaculated boar semen was supplemented with different concentrations of proline (0, 25, 50, 75, 100, 125 mM) and stored at 17 degrees C for nine days. Sperm motility patterns, membrane integrity, ATP (adenosine triphosphate), reactive oxygen species (ROS), and GSH (glutathione) levels, and the activities of catalase (CAT) and superoxide dismutase (SOD) were evaluated after storage for up to five days. It was observed that boar sperm quality gradually decreased with the extension of storage time, while the ROS levels increased. Addition of 75 mM proline not only significantly improved sperm membrane integrity, motility, and ATP levels but also maintained the redox homeostasis via increasing the GSH levels and activities of CAT and SOD. When hydrogen peroxide (H2O2) was used to induce oxidative stress, addition of proline significantly improved sperm quality and reduced ROS levels. Moreover, addition of proline also improved sperm quality during the rapid cooling process. Notably, addition of DL-PCA (DL-pipecolinic acid) rescued the reduction of progressive motility and total motility caused by H2O2, and THFA (tetrahydro-2-furoic acid) failed to provide protection. Furthermore, addition of proline at 75 mM increased the activity of proline dehydrogenase (PRODH) and attenuated the H2O2-induced reduction in progressive motility. These data demonstrate that proline protects sperm against oxidative stress through the secondary amine structure and proline dehydrogenase-mediated metabolism.