The purpose of the present study is to examine the effects of melatonin on apoptosis and oxidative stress in mouse Leydig cells and to elucidate the mechanisms responsible for these effects. Our results indicated that 10 ng/mL of melatonin significantly promoted cell viability, the ratio of EdU-positive (5-Ethynyl-2′-deoxyuridine) cells, and increased the mRNA expression of proliferating cell nuclear antigen (PCNA), cyclin D1(CCND1), and cell division control protein 42 (CDC42) (p < 0.05). We also observed that melatonin inhibited apoptosis of mouse Leydig cells, accompanied with increased B-cell lymphoma-2 (BCL-2) and decreased BCL2 associated X (BAX) mRNA and protein expression. Moreover, addition of melatonin significantly decreased the reactive oxygen species (ROS) production and malondialdehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, while it increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels (p < 0.05). In addition, we also found that melatonin increased the expression of SIRT1 (Silent information regulator 1) (p < 0.05). To explore the role of SIRT1 signaling in melatonin-induced cells, mouse Leydig cells were pretreated with EX527, an inhibitor of SIRT1. The protective effects of melatonin on mouse Leydig cells were reversed by EX527, as shown by decreased cell proliferation and increased cell apoptosis and oxidative stress. In summary, our results demonstrated that melatonin inhibited apoptosis and oxidative stress of mouse Leydig cells through a SIRT1-dependent mechanism.

Cadmium (Cd) is a common environmental heavy metal contaminant of reproduction toxicity. Cd accumulation in animals leads to the damage of granulosa cells. However, its mechanism needs to be elucidated. This research found that treating granulosa cells with Cd resulted in reduced cell viability. The flow cytometry results showed that Cd increased the degree of apoptosis and level of superoxide anion (O₂⁻) in granulosa cells. Further analysis showed that Cd treatment resulted in reduced expression levels of nuclear factor erythroid 2-related factor-2 (Nrf2), superoxide dismutase (SOD), catalase (CAT) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and an increased expression level of malondialdehyde (MDA); the expression levels of Bcl-2 associated X (Bax) and caspase-3 increased, whereas that of B-cell lymphoma 2 (Bcl-2) decreased. Changes in m⁶A methylation-related enzymes were noted with Cd-induced damage to granulosa cells. The results of transcriptome and MeRIP sequencing revealed that the AKT pathway participated in Cd-induced damage in granulosa cells, and the MAX network transcriptional repressor (MNT) may be a potential target gene of fat mass and obesity-associated protein (FTO). FTO and YTH domain family member 2 (YTHDF2) regulated MNT expression through m⁶A modification. FTO overexpression alleviated Cd-induced apoptosis and oxidative stress through the activation of the AKT/Nrf2 pathway; this process could be reversed using siMNT. Overall, these findings associated m⁶A with Cd-induced damage to granulosa cells and provided insights into Cd-induced granulosa cell cytotoxicity from a new perspective centered on m⁶A modification.

Melatonin has profound antioxidant activity and numerous functions in humans as well as in livestock and poultry. Additionally, melatonin plays an important role in regulating the biological rhythms of animals. Combining melatonin with scientific breeding management has considerable potential for optimizing animal physiological functions, but this idea still faces significant challenges. In this review, we summarized the beneficial effects of melatonin supplementation on physiology and reproductive processes in cattle, including granulosa cells, oocytes, circadian rhythm, stress, inflammation, testicular function, spermatogenesis, and semen cryopreservation. There is much emerging evidence that melatonin can profoundly affect cattle. In the future, we hope that melatonin can not only be applied to cattle, but can also be used to safely and effectively improve the efficiency of animal husbandry.

Endometritis is persistent inflammation caused by bacteria, which can lead to infertility. Although traditional Chinese medicine (TCM) has been used to treat endometritis, the underlying mechanism is still unclear. Here, Baogong Decoction (BGD), a TCM compound, was used to treat mouse endometritis induced by Escherichia coli ( E. coli ), and then 16S rRNA sequencing and non‐targeted metabolomics were used to investigate the change of uterine microbiota and metabolomes in serum and uterine after BGD treatment. Finally, the therapeutic effect of potential metabolites for treating mouse endometritis screened by combined omics analyses was verified using pathological model. The results showed that BGD treatment could effectively treat endometritis associated with the increasing relative abundance of Firmicutes, Bacteroides , Lactobacillus and Lactococcus , and the decreasing relative abundance of Cupriavidus and Proteobacteria. 133 and 130 metabolites were found to be potential biomarkers in serum and uterine tissue respectively. In serum and tissues, dehydroepiandrosterone (DHEA) and catechol were significantly increased in the BGD treatment versus the inflammation group. Results of combined omics analyses demonstrated that DHEA was positively correlated with changes in microbiota. Results of pathological model demonstrated that DHEA could cure endometritis effectively associated with the decreasing infiltration of inflammatory cells and expression of inflammatory factors in the uterus. In summary, our results demonstrated that BGD could cure endometritis in mice by modulating the structure of the uterine microbiota and its metabolites, in which DHEA may be one of the main components of the therapeutic effect of BGD.

The purpose of this study was to determine the effects of apigenin and astragalus polysaccharides on the cryopreservation of bovine semen. Apigenin, astragalus polysaccharides, or their combination were added to a frozen diluent of bovine semen. Afterwards, Computer Assisted Semen Analysis (CASA), membrane functionality, acrosome integrity, mitochondrial integrity, CAT, SOD, GSH-Px, MDA, and ROS detection were conducted. The results showed that adding 0.2 mmol/L AP or 0.5 mg/mL APS could improve the quality of frozen sperm. Compared to 0.2 mmol/L AP alone, the combination of 0.2 mmol/L AP and 0.3 mg/mL APS significantly increased the total motility (TM), average path distance (DAP), straight line distance (DSL), average path velocity (VAP), curvilinear velocity (VCL), wobble (WOB), and sperm CAT and SOD levels (p < 0.05), while reducing the ROS and MDA levels (p < 0.05). These results indicated that the addition of 0.2 mmol/L AP or 0.5 mg/mL APS alone has a protective effect on the freezing of bovine semen. Compared to the addition of 0.2 mmol/L AP, a combination of 0.2 mmol/L AP and 0.3 mg/mL APS could further improve the quality of frozen semen.

It is important to inhibit oxidative stress to maintain sperm motility during cryopreservation. The present study was performed to investigate the effects of supplementing oligomeric proanthocyanidins (OPC) and bamboo leaf flavonoids (BLF) or their combination as an extender for Simmental bull semen freezing. OPC, BLF, or their combination were added to the frozen diluent of bovine semen. Afterwards, computer-assisted semen analysis (CASA), detection of membrane functionality, acrosome integrity, mitochondrial integrity, CAT, SOD, GSH-PX, MDA, and ROS were conducted. The results showed that adding 50 mg/L OPC or 4 mg/L BLF could improve the quality of frozen sperm. Compared with 50 mg/L OPC alone, the combination of 50mg/L OPC and 2 mg/L BLF significantly increased the kinematic parameters of sperm, and sperm CAT, GSH-PX and SOD levels (p < 0.05), whereas the MDA of sperm was decreased (p < 0.05). These results indicated that compared to the addition of 50 mg/L OPC alone, a combination of 50 mg/L OPC and 2 mg/L BLF could further improve the quality of frozen semen. The results could provide theoretical data support for the development of a new protective agent and are significant for the cryopreservation of bovine semen in the future.