Optimization of Hydrogen Peroxide Concentrations for Inducing Oxidative Stress in Bovine Oocytes Prior to In Vitro Maturation

This study determined the optimal concentration of hydrogen peroxide (H2O2) for inducing oxidative stress in bovine oocytes prior to in vitro maturation (IVM). Ovaries were collected from local abattoirs, and cumulus–oocyte complexes (COCs) were aspirated, selected, and allocated into four groups, exposed to 0, 50, 75, or 100 µM H2O2, respectively, for 1 h in collecting medium. This was followed by IVM in TCM-199 at 38.5 °C in humidified atmosphere of 5% CO2 for 23 h. Nuclear maturation was assessed by aceto-orcein staining. Exposure to increasing concentrations of H2O2 resulted in a clear, dose-dependent trend of decreased nuclear maturation rates. The control group (0 µM) exhibited the highest proportion of oocytes reaching the metaphase II (MII) stage (69.23 ± 8.45%), which remained comparable at 50 µM (67.50 ± 12.29%). A mild, though not statistically significant, decrease was observed at 75 µM (56.50 ± 2.33%). In contrast, treatment with 100 µM H2O2 led to a significant reduction in MII rate to 32.83 ± 7.64%, compared to all other groups. These findings indicated that exposure to 100 µM H2O2 for 1 h effectively induces oxidative stress in bovine oocytes and could serve as a standard in vitro model for future studies, investigating antioxidant supplementation during pre-IVM and IVM phases.