ß-Nicotinamide mononucleotide improves chilled ram sperm quality in vitro by reducing oxidative stress damage.

OBJECTIVE: The present study aimed to investigate the effect of ß-nicotinamide mononucleotide (NMN) supplementation on ram sperm quality during storage at 4°C in vitro. METHODS: Tris-citric acid-glucose solution containing different doses of NMN (0, 30, 60, 90, and 120 µM) was used to dilute semen collected from rams and it was stored at 4°C. Sperm motility, plasma membrane integrity as well as acrosome integrity were evaluated at 0, 24, and 48 h time points after storage at 4°C. In addition, sperm mitochondrial activity, lipid peroxidation (LPO), malondialdehyde (MDA) content, reactive oxygen species (ROS) content, glutathione (GSH) content, superoxide dismutase (SOD) activity, and apoptosis were measured at 48 h time point after storage at 4°C. RESULTS: Results demonstrate that the values obtained for sperm motility, acrosome integrity, and plasma membrane integrity in the NMN treatments were significantly higher than control (p<0.05). The addition of 60 µM NMN significantly improved ram sperm mitochondrial activity and reduced LPO, MDA content, and ROS content compared to control (p<0.05). Interestingly, sperm GSH content and SOD activity for the 60 µM NMN treatment were much higher than those observed for control. NMN treatment also decreased the level of Cleaved-Caspase 3, Cleaved-Caspase 9, and Bax while increasing Bcl-2 level in sperm at 48 h time point after storage at 4°C. CONCLUSION: Ram sperm quality can be maintained during storage at 4°C with the addition of NMN at 60 µM to the semen extender. NMN also reduces oxidative stress and apoptosis. Overall, these findings suggest that NMN is efficient in improving the viability of ram sperm during storage at 4°C in vitro.

Effect of Zearalenone-Induced Ferroptosis on Mice Spermatogenesis.

Male reproductive health is critically worsening around the world. It has been reported that the mycotoxin ZEA causes reproductive toxicity to domestic animals and affects spermatogenesis, thereby inhibiting male reproductive function. Ferroptosis is a newly identified type of programmed cell death that is different from apoptosis and it depends on iron accumulation and lipid peroxidation. Whether ferroptosis is linked to ZEA's detrimental effect on spermatogenesis needs to be further explored. This study clarifies ferroptosis's involvement in ZEA-induced damage on spermatogenesis. The reproductive injury model used in this study was induced by gavaging male mice in the ZEA treatment group with 30 µg/kg of ZEA for five weeks. Results show that ZEA treatment reduced mouse sperm motility and concentration, destroyed the structure of the seminiferous tubules of the testis, damaged the antioxidant defense system, and blocked spermatogenesis. Ferrostatin-1 (Fer-1) inhibition of ferroptosis partially alleviated ZEA-induced oligozoospermia in mice. In addition, ZEA treatment was found to activate a signaling pathway associated with ferroptosis in mouse testis. ZEA also downregulated the expression of Nrf2, SLC7A11, and GPX4, and decreased the protein expression of SLC7A11 and GPX4, resulting in the accumulation of lipid peroxides and an increase in the level of 4-HNE protein in the testis. Importantly, these changes were accompanied by an increase in the relative contents of Fe2+ and Fe3+. Iron accumulation and lipid peroxidation are the causes of ferroptosis in spermatogenic cells, leading to a decrease in sperm motility and concentration. While the administration of Fer-1 at 0.5 and 1 mg/kg also increased the expression of SLC7A11 and GPX4 proteins by upregulating Nrf2 expression, reducing iron accumulation, and reversing ZEA-induced ferroptosis, Fer-1 at 1.5 mg/kg had the best repairing effect for all parameters. In conclusion, ZEA-induced ferroptosis may be mediated by a notable reduction in Nrf2, SLC7A11 and GPX4 expression levels. Overall, ferroptosis is a novel therapeutic target for mitigating ZEA-induced reproductive toxicity.

Beneficial Effect of Proline Supplementation on Goat Spermatozoa Quality during Cryopreservation.

Sperm cryopreservation contributes to the extensive utilization of artificial insemination (AI) in the daily livestock industry. However, due to the presence of few sperm with good biological function in post-thaw goat sperm, its use has been limited for AI purposes. Hence, its improvement has been the focus of many research studies. This study aimed to investigate the effects of proline supplementation of the freezing medium on goat sperm. The goat semen was cryopreserved with freezing medium supplementation of different concentrations of proline (0, 0.5, 1, 2 and 4 mM). The post-thaw sperm motility patterns, membrane integrity, acrosome integrity, lipid peroxidation (LPO) levels, malondialdehyde (MDA) levels, total antioxidant capacity (T-AOC), proline dehydrogenase (PRODH) activity, superoxide dis-mutase (SOD) activity, glutathione (GSH) levels and GSH/GSSG were evaluated. Likewise, the expression and immunofluorescent localization of PRODH in post-thaw goat sperm was also detected. It was observed that addition of 2 mM proline to the freezing medium significantly enhanced post-thaw goat sperm total motility, progressive motility, straight-linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), straightness (STR), linearity (LIN), membrane integrity and acrosome integrity. Interestingly, PRODH was expressed in post-thaw goat sperm, especially in the post-acrosome and sperm tail. Addition of 2 mM proline also significantly increased the post-thaw sperm PRODH activity compared to the control. Moreover, post-thaw goat sperm LPO levels and MDA levels were reduced by supplementation of 2 mM proline. Furthermore, compared to the control, the values of post-thaw goat sperm T-AOC, SOD activity, GSH level and GSH/GSSG were also significantly increased in 2 mM proline treatment. Reduction of post-thaw goat sperm apoptosis in 2 mM proline treatment was also observed as the levels of Caspase3 and Caspase9 were decreased by the supplementation with 2 mM proline. These observations suggest that the addition of 2 mM proline to the freezing medium increased post-thaw goat sperm quality by reducing oxidative stress during cryopreservation. These findings also provide novel insights into the use of proline as an efficient additive to enhance post-thaw goat sperm quality during cryopreservation.