Vitrification of Immature Oocytes for the Production of Equine Embryos by ICSI: Protocol Effect on Maturation, Embryo Development, Mitochondrial Distribution and Functionality

The present study aimed to examine the effects of adding different concentrations of resveratrol during in vitro culture (IVC) alone and during both in vitro maturation (IVM) and IVC on ovine blastocyst yield and quality. Therefore, this study was conducted in two separate experiments. The first experiment was carried out to test the effect of different concentrations of resveratrol (0, 0.1, 0.25, 0.5, 2.0, and 5.0 μM) in the IVC medium on cleavage, morula, developmental potential of blastocyst, and total cell number (TCN) of the embryos. Addition of 0.25 and 0.5 μM of resveratrol during IVC significantly enhanced morula and blastocyst rates as compared with other groups (P < 0.05). Also, supplementation of the IVC medium with 0.5 μM of resveratrol had beneficial effects on trophectoderm cells (TE), inner cell mass (ICM), and TCN of blastocysts. In the second experiment, the same concentrations of resveratrol (0, 0.1, 0.25, 0.5, 2.0, and 5.0 μM) were applied during IVM and IVC. Therefore, oocytes were matured in vitro in the presence of different concentrations of resveratrol for 22–24 h. After in vitro fertilization, presumptive zygotes were cultured in media containing 0, 0.1, 0.25, 0.5, 2.0, and 5.0 μM of resveratrol for 8 d. No significant difference was found in the percentage of oocytes developed to MII (0, 0.1, 0.25, 0.5, and 2.0 μM of resveratrol), but the percentage of oocytes developed to MII were significantly lower in 5.0 μM of resveratrol in comparison with other groups. Addition of 0.5 μM of resveratrol to the maturation and culture media significantly increased morula and blastocyst rates compared with other groups (P < 0.05). However, a too high concentration of resveratrol (5.0 μM) during IVM and IVC decreased cleavage, morula, and blastocyst rates compared with low concentrations (P < 0.05). Treatment with 0.5/0.5 μM of resveratrol during IVM/IVC significantly improved the TE, ICM, and TCN of blastocysts. In conclusion, sequential treatment with 0.5 μM of resveratrol during IVM and IVC and during IVC alone improved the developmental competence of oocytes, which was reflected in higher blastocyst rates and TCN of blastocysts.