Mitochonic Acid 5 Increases Ram Sperm Quality by Improving Mitochondrial Function during Storage at 4 °C.

Semen preservation involves lengthening sperm's fertile lifespan without any detrimental effects on its biochemical, functional, and ultrastructural properties. Liquid storage at 4 °C is a ram sperm preservation method. However, this method of storage causes irreversible damage due to cold shocks, osmotic stresses, oxidative stresses, and reductions in sperm metabolism. The present study aims to investigate whether the supplementation of mitochonic acid 5 (MA-5) in a sperm extender could improve chilled ram sperm quality and elucidate its mechanism of action. Ram sperm were diluted with a tris-citrate-glucose extender containing different concentrations of MA-5 (0, 0.1, 1, 10, and 100 nM) and stored at 4 °C for up to 48 h. Sperm motility, membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) level, ATP content, and the expression of NADPH dehydrogenase subunits 1 (MT-ND1) and NADPH dehydrogenase subunits 6 (MT-ND6) were evaluated. It was observed that compared to the control, the 10 nM MA-5 treatment significantly (p < 0.05) increased total motility (82 ± 3.5% vs. 76 ± 5.9%), progressive motility (67.6 ± 8.2% vs. 51 ± 8.3%), and other parameters (straight-line velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL)). In addition, 10 nM MA-5 supplementation also improved ram sperm membrane integrity and acrosomal integrity as well increased mitochondrial membrane potential (51.1 ± 0.7% vs. 37.7 ± 1.3%), reduced ROS levels, and elevated adenosine triphosphate (ATP) contents. Furthermore, a Western blot analysis demonstrated that the addition of MA-5 significantly (p < 0.05) increased the expression of MT-ND1 and MT-ND6 proteins in ram sperm, with the 10 nM MA-5 treatment resulting in the highest expression level. These results suggest that MA-5 improves ram sperm quality by maintaining high sperm mitochondrial function during liquid storage at 4 °C.

Resveratrol Improves the Frozen-Thawed Ram Sperm Quality.

Cryopreservation generates a substantial quantity of ROS in semen, leading to a decline in sperm quality and fertilization capacity. The objective of this study was to investigate the effects of resveratrol and its optimal concentration on ram sperm quality after cryopreservation. Ram semen was diluted with a freezing medium containing different concentrations of resveratrol (0, 25, 50, 75, and 100 µM). After thawing, various sperm parameters such as total motility, progressive motility, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, glutathione (GSH) content, glutathione synthase (GPx) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, lipid peroxidation (LPO) content, malondialdehyde (MDA) content, ROS level, SIRT1 level, DNA oxidative damage, and AMPK phosphorylation level were assessed. In addition, post-thaw sperm apoptosis was evaluated. Comparatively, the addition of resveratrol up to 75 µM significantly improved the sperm motility and sperm parameters of cryopreserved ram sperm. Specifically, 50 µM resveratrol demonstrated a notable enhancement in acrosome and plasma membrane integrity, antioxidant capacity, mitochondrial membrane potential, adenosine triphosphate (ATP) content, SIRT1 level, and AMPK phosphorylation levels compared to the control group (p < 0.05). It also significantly (p < 0.05) reduced the oxidative damage to sperm DNA. However, detrimental effects of resveratrol were observed at a concentration of 100 µM resveratrol. In conclusion, the addition of 50 µM resveratrol to the cryopreservation solution is optimal for enhancing the quality of cryopreserved ram sperm.

Impact of lipopolysaccharide administration on luteinizing hormone/choriogonadotropin receptor (Lhcgr) expression in mouse ovaries.

Lipopolysaccharide (LPS) is isolated from the genital tract of animals suffering from uterine damage and ovarian dysfunction. This study provides direct molecular evidence about the mechanism through which endotoxins cause reproductive disorders. Granulosa cells and ovaries were collected from immature mice treated with eCG or with eCG and LPS injection intraperitoneally. Normal large antral follicles were observed in ovaries obtained from eCG and LPS coinjected mice, and the morphology of the ovaries was similar to that observed in the control group. These antral follicles were not deemed atretic because few TUNEL-positive cells were observed. However, the granulosa cells of large antral follicles did not acquire the ability to respond to hCG stimulation. The number of ovulated oocytes was significantly lower in LPS-injected mice after superovulation compared to mice that were not exposed to LPS. The low reactivity was caused by the limited expression of the Lhcgr gene, which encodes the LH receptor in granulosa cells as well as an LPS-induced increase in the level of Dnmt1 expression. The methylation rate of the Lhcgr promoter region was significantly higher in granulosa cells obtained from the LPS treatment group compared with the control group. Together, these findings demonstrated that the decrease in the expression of Lhcgr due to LPS was a result of the epigenetic regulatory action of LPS. Our studies suggest that ovarian follicular cysts that is characterized by bacterial infection in humans and animals, is closely connected to the level of methylation of the Lhcgr promoter region.