181 RESVERATROL DURING IN VITRO MATURATION IMPROVES THE QUALITY OF BOVINE OOCYTE AND ENHANCES EMBRYONIC DEVELOPMENT IN VITRO

Abstract

It is known that reactive oxygen species (ROS) are accumulated within the oocyte during in vitro maturation (IVM) and have been related to poor quality and decreased embryo development in vitro. The use of antioxidants in culture media is an alternative to overcome oxidative stress damage in the oocyte. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a phenol produced naturally by several plants and has shown protection against oxidative damage in numerous cell types. Two different experiments were performed to evaluate the effect of resveratrol on the quality of bovine oocytes matured in vitro assessed by levels of ROS and intracellular glutathione (GSH) as well as in vitro embryo development rates. Experiment 1 used different concentrations of resveratrol [0 (Control), 1 (R1), 10 (R10), 20 (R20), and 40 (R40) μM] were used to supplement IVM media. Ovaries were collected from Bos indicus cows at a local abattoir and cumulus-oocyte complexes were matured in vitro for 24 h in TCM199 with 6 mg mL⁻¹ of fatty acid-free BSA, 5% fetal bovine serum, 0.2 mM Na-pyruvate, 50 μg mL⁻¹ of gentamicin, 0.5 μg mL⁻¹ of FSH, and 0.5 µg mL⁻¹ of LH at 38°C in 5% CO₂ and 90% humidity. The ROS were evaluated by 2′,7′-dichlorodihydrofluorescein diacetate staining (n = 301) and intracellular GSH levels were determined by Cell Tracker Blue fluorescent stain (n = 310). Denuded oocytes were observed under an epifluorescence microscope. Fluorescence intensities of oocytes were analysed by ImageJ software (Version 1.49v, National Institutes of Health, Bethesda, MD, USA) and normalized to control oocytes. Experiment 2 used cumulus-oocyte complexes (n = 674) collected and matured in vitro under the same conditions described for Exp. 1. In vitro fertilization was performed for 18 h at 38°C in 5% CO₂ in Tyrode’s medium with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, and 6 mg mL⁻¹ of fatty acid-free BSA. Additionally 10 µg mL⁻¹ of heparin and 20 μM d-penicillamine, 10 μM hypotaurine, and 1 μM epinephrine were added. The presumptive zygotes were cultured in vitro in SOFaa medium with 5% fetal bovine serum, at 38°C, in 5% CO₂, 5% O₂, and 90% humidity until Day 7, when embryonic development was assessed. Data were analysed by ANOVA followed by Fisher´s multiple range test using Statgraphics Centurion XVI (Version 16.2.04, Statpoint Technologies Inc., Warrentown, VA). Data are presented as percentage mean ± standard error of the mean (P < 0.05). All concentrations of resveratrol in treated oocytes showed reduced intracellular levels of ROS compared to control (R1: 0.66 ± 0.04, R10: 0.55 ± 0.04, R20: 0.62 ± 0.04, R40: 0.64 ± 0.04, and Control: 1 ± 0.04 pixel/oocyte; P < 0.01). Intracellular levels of GSH were significantly higher for R1 (1.4 ± 0.06; P < 0.01) and R10 (1.3 ± 0.06; P < 0.01) compared with the control. On the other hand, R10 showed a significantly higher blastocyst rate (51% ± 3) compared with R1 (39% ± 4), R20 (39% ± 3), R40 (33% ± 3), and control (38% ± 4). Treatments R1, R20, and R40 showed no significant differences compared to control. These results indicate that resveratrol at 10 μM during IVM improves maturation conditions by decreasing ROS level, increasing intracellular GSH, and improving embryonic developmental competence.