Effects of Chlorogenic Acid on In Vitro Maturation and Vitrification Cryopreservation of Sheep Oocytes

Chlorogenic acid (CGA) has strong antioxidant properties. In order to improve the low maturation rate and poor vitrification freezing effect of sheep oocytes caused by oxidative stress. In this study, oocytes from 200 2-3-year-old Kazakh sheep were collected, and different concentrations of CGA were added to the maturation medium and vitrification freezing solution to study the effects of CGA on the maturation rate, cleavage rate, blastocyst rate, reactive oxygen species (ROS) and glutathione (GSH) levels, mitochondrial membrane potential, and the expression levels of oxidation and apoptosis-related genes in sheep oocytes. The results showed that adding 40 μmol/L CGA to the oocyte in vitro maturation solution significantly increased the maturation rate of oocytes, adding 50 μmol/L CGA to the vitrification cryopreservative solution significantly increased the cleavage and blastocyst rates of mature oocytes activated by parthenogenetic activation after freezing. During in vitro maturation and vitrification freezing in sheep oocytes, CGA significantly reduced the level of ROS and the expression of apoptosis-related genes (Caspase-3 and Bax/Bcl-2), and significantly increased the level of glutathione (GSH), mitochondrial membrane potential, and the expression of antioxidant and anti-apoptosis-related genes (SOD-2 and GPX-3). In addition, CGA significantly increased the expression of the anti-apoptotic gene (AKT) and anti-stress gene (FOXO) during vitrification freezing of sheep oocytes. In conclusion, 40 μmol/L CGA improves the maturation rate of sheep oocytes, and 50 μmol/L CGA improves the quality of parthenogenetic activation embryos after vitrification freezing of mature oocytes in sheep. These results provide a basis for the production of sheep in vitro embryos and the establishment of a germplasm resource bank.