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 <i>in vitro</i> 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 <i>in vitro</i> model for future studies, investigating antioxidant supplementation during pre-IVM and IVM phases.