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 a local abattoir, and cumulus–oocyte complexes (COCs) were aspirated, selected, and allocated into four groups, respectively exposed to 0, 50, 75, or 100 µM H₂O₂ for 1 hour in collecting medium. This was followed by IVM in TCM-199 at 38.5 °C in humidified atmosphere of 5% CO₂ for 23 hours. 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 (56.5±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 finding indicated that expose to 100 µM H₂O₂ for 1 hour 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.