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.

Pyrroloquinoline Quinone Improves Ram Sperm Quality through Its Antioxidative Ability during Storage at 4 °C.

Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the PQQ treatment group was much higher than that in the control. In addition, supplementation of PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm-zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm's high ability of fertilization.

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.

Determination of geniposide in rat urine after oral administration of the traditional Chinese medicinal preparation yin-zhi-ku decoction by high-performance liquid chromatography.

A high-performance liquid chromatography method with solid-phase extraction is introduced for the determination of geniposide in rat urine after oral administration of yin-zhi-ku decoction. Geniposide and an internal standard (paeoniflorin) are extracted from urine using Strata cartridges. Analysis of the extract is then performed on a reversed-phase C18 column using acetonitrile-water (14:86, v/v) as eluting solvent system. UV detection is set at 238 nm. The calibration curve for geniposide is linear (r = 0.9996) in the concentration range of 2.0-240 microg/mL. Both intra- and interday precision of the geniposide are determined, and their relative standard deviation does not exceed 10%. The validated method is successfully applied to determine geniposide from rat urine after oral administration of yin-zhi-ku decoction.

HPLC method for the determination and pharmacokinetic studies on geniposide in rat serum after oral administration of traditional Chinese medicinal preparation Yin-Zhi-Ku decoction.

A new HPLC method for the determination of geniposide in rat serum with solid-phase extraction (SPE) for preconcentration is described. Geniposide and an internal standard (paeoniflorin) were extracted from serum by SPE using C18 cartridges. Analysis of the extract was then performed on a reversed-phase C18 column using acetonitrile-water (16:84, v/v) as the eluting solvent system, and UV detection at 238 nm was used to measure the analyte with a limit of quantitation about 0.1 microg/mL. The calibration curve for geniposide was linear (r = 0.9993) in the concentration range 0.1-16.0 microg/mL. The intra- and inter-day precision of the geniposide were determined and their RSD did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of geniposide from rat serum after oral administration of Yin-Zhi-Ku decoction.

Feitai, a Chinese herbal medicine, reduces transforming growth factor-beta1 and monocyte chemoattractant protein-1 expression in bleomycin-induced lung fibrosis in mice.

Feitai, a Chinese medicine formulation, has been shown to protect against lung fibrosis induced by bleomycin (BLM). In the present study, we investigated the effect of Feitai on transforming growth factor (TGF)-beta1 and monocyte chemoattractant protein-1 (MCP-1), which play important roles in the pathogenesis of BLM-induced lung fibrosis. The results demonstrated that Feitai could significantly attenuate BLM-induced acute lung inflammation and subsequent lung fibrosis. Meanwhile, the expression of MCP-1 and TGF-beta1 mRNA in the lungs increased in the BLM-treated group compared with the saline-instilled control group and Feitai treatment significantly decreased cytokine expression in BLM-treated mice. In addition, Feitai diminished the accumulation of MCP-1- and TGF-beta1-positive cells in lung tissues at the time of peak mRNA levels. In summary, the results of the present study indicate that treatment with Feitai ameliorates BLM-induced lung fibrosis, at least in part via the inhibition of MCP-1 and TGF-beta1 expression.