Effect of glutathione addition on vitrification of ovine oocytes

The exogenous antioxidants are commonly employed to mitigate vitrification-induced oxidative damage. Glutathione (GSH), a vital antioxidant, plays a significant role in scavenging free radicals, providing antioxidant protection, and maintaining cellular integrity. This study aimed to investigate the effects of GSH supplementation on the vitrification of ovine oocytes. To identify the optimal concentration of exogenous GSH supplementation, the impacts of 2 mM, 4 mM, and 8 mM GSH on the survival and fragmentation of oocytes were evaluated after vitrification. In addition, immunofluorescence (IF) staining was employed to evaluate spindle morphology, chromosome distribution, cortical granule distribution, mitochondrial function, and reactive oxygen species (ROS) levels. The levels of ATP and NADPH, along with the ratio of GSH/GSSH, were also examined. Additionally, evaluations of in vitro fertilization were carried out. The results of oocyte survival rates and fragmentation rates identified that the addition of 4 mM GSH to the vitrification solution was the optimal concentration. The assessment of spindle morphology, chromosome distribution, cortical granule distribution, mitochondrial function, ROS production, ATP activity, and NADPH levels, as well as the GSH/GSSH ratio, confirm the beneficial effect of GSH supplementation against the vitrification-induced oxidative damage. Lastly, incorporating GSH into the vitrification process enhanced the developmental potential of oocytes, resulting in higher cleavage rates, increased blastocyst rates, and improved quality of blastocysts. Overall, this research uncovered the mechanisms through which GSH mitigates the oxidative damage induced by vitrification, thereby leading to the optimization of the vitrification technique for ovine oocytes.