It takes several hours for mammalian sperm to migrate from the ejaculation or insemination site to the fertilization site in the female reproductive tract in which glucose, amino acids, and fatty acids are regarded as the primary substrates for ATP generation. The present study was designed to investigate whether oleic acid and palmitic acid were beneficial to boar sperm in vitro; and if yes, to elucidate the mechanism that regulates sperm motility. Therefore, the levels of oleic acid and palmitic acid, motility, membrane integrity, acrosome integrity, and apoptosis of sperm were evaluated. Moreover, the enzymes involved in mitochondrial β-oxidation (CPT1: carnitine palmitoyltransferase 1; ACADVL: long-chain acyl-coenzyme A dehydrogenase) were detected with immunofluorescence and Western blotting. Consequently, the ATP content and the activities of CPT1, ACADVL, malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were also measured. We observed that CPT1 and ACADVL were expressed in boar sperm and localized in the midpiece. The levels of oleic acid and palmitic acid were decreased during storage at 17 °C. The addition of oleic acid and palmitic acid significantly increased sperm motility, progressive motility, straight-line velocity (VSL), membrane integrity, and acrosome integrity with a simultaneous decrease in sperm apoptosis after seven days during storage. When sperm were incubated with oleic acid and palmitic acid at 37 °C for 3 h, the activities of CPT1 and ACADVL, the ATP level, the mitochondrial membrane potential, the activities of MDH and SDH, as well as sperm motility patterns were significantly increased compared to the control (p < 0.05). Moreover, the addition of etomoxir to the diluted medium in the presence of either oleic acid or palmitic acid and the positive effects of oleic acid and palmitic acid were counteracted. Together, these data suggest that boar sperm might utilize oleic acid and palmitic acid as energy substrates for ATP production via β-oxidation. The addition of these acids could improve sperm quality.

We conducted pot experiments to investigate the effects of brassinolide on 1-year-old Xanthoceras sorbifolia B. seedlings. In the experiment, roots were soaked in 0-0.4 mg/l brassinolide. After the seedlings were established, the soil water content in the pots was regulated to simulate drought conditions and various physiological parameters were measured. The results showed that the treatment with 0.2 mg/l brassinolide decreased the malondialdehyde content and electrolyte leakage of seedlings growing under moderate or severe water stress when compared with untreated seedlings. Leaf water content, relative water content, soluble sugar content, soluble protein content, free proline content, ascorbic acid content, glutathione content and superoxide dismutase, peroxidase, catalase and ascorbate peroxidase activities were all greater in water-stressed seedlings in the 0.2 mg/l brassinolide treatment as compared to the control. The results indicate that the application of brassinolide can ameliorate the effects of water stress and enhance drought resistance of Xanthoceras sorbifolia seedlings.

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 °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 °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.