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

## TL;DR

This study finds that 100 µM hydrogen peroxide effectively induces oxidative stress in cow oocytes before in vitro maturation, offering a reliable model for reproductive research.

## Contribution

The study identifies a specific H2O2 concentration that reliably induces oxidative stress in bovine oocytes without causing irreversible damage.

## Key findings

- Exposure to 100 µM H2O2 significantly reduced oocyte nuclear maturation rates to 32.83%.
- The model provides a reproducible way to simulate oxidative stress in vitro for studying antioxidant treatments.
- Oocytes exposed to 100 µM H2O2 showed a clear dose-dependent decrease in maturation success.

## Abstract

Oxidative stress impairs the quality of oocytes and limits the success of in vitro embryo production in cattle. A reliable model that mimics oxidative damage under controlled laboratory conditions is developed. In this study, bovine oocytes were exposed to different concentrations of hydrogen peroxide (H2O2) to evaluate oxidative stress without irreversible cellular damage. The findings revealed that short-term exposure to a specific concentration of H2O2 could consistently disrupt oocyte maturation, providing a simple and reproducible way to simulate oxidative stress in vitro. Importantly, this pre-in vitro maturation exposure model also reflects the physiological stress that oocytes may commonly experience. This model can serve as a valuable tool for future research, aiming at improving oocyte resilience and optimizing antioxidant treatments in animal reproductive biotechnology.

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.