100 Resveratrol reduces oxidative stress after a six-hour incubation of in vivo-derived sheep embryos

The use of molecules, such as resveratrol, a natural substance with antioxidant and anti-inflammatory action, has been applied as a strategy to protect cells from the adverse effects of oxidative stress, capable of reducing embryonic viability. The supplementation of antioxidants in culture media and cryopreservation solutions have been studied to increase embryo viability and competence of development. The exposure to resveratrol before the slow freezing of bovine embryos was able to increase their hatching and pregnancy rate. However, the beneficial effect of resveratrol on sheep embryos has not yet been completely elucidated. This study aimed to evaluate, within the same embryo, the possible protective effect of 6 h of exposure to resveratrol in culture before embryo slow freezing as a subsequent beneficial effect in the cryopreservation process. To achieve that, embryos (n = 29) were nonsurgically recovered and classified according to their stage of development and quality. The GI and GII embryos were kept in a holding medium (phosphate-buffered saline +20% of fetal bovine serum), supplemented with 1 uM of resveratrol, and incubated at 38.5°C and 5% CO₂ for 6 h. The structures were stained before (PRE) and after (POST) incubation, evaluated under a fluorescence microscope, and photographed for analyses of glutathione (GSH; CMF₂HC, Cell Tracker Blue, Invitrogen™, C12881), reactive oxygen species (ROS; H₂DCHFDA, Invitrogen, D399), and mitochondrial activity (MitoTracker Red, Invitrogen, M7512) following manufacturer’s instructions. To perform the analysis, the same embryos were measured at both times (PRE and POST), compared by average fluorescence intensity per embryo with ZEN 3.8 Blue Edition software (ZEISS Microscopy), and normalized to the background average intensity. Data were checked for normality (Shapiro–Wilk test), and paired t-test (GSH) and Wilcoxon test (ROS, mitochondrial activity, and ROS/GSH) were performed. Results are presented as relative fluorescence intensity levels (mean ± standard error of the mean of arbitrary units). When compared to the PRE time point, both GSH (1.00 ± 0.05 vs 0.55 ± 0.04) and ROS (1.00 ± 0.09 vs 0.33 ± 0.07) exhibited a reduction (P < 0.05) after incubation (POST), decreasing (P < 0.05) the ROS/GSH ratio (1.00 ± 0.07 vs 0.48 ± 0.09) as well. However, interestingly, the mitochondrial activity was maintained through time, and there was no difference (P > 0.05) between PRE and POST (1.00 ± 0.03 vs 0.91 ± 0.04, respectively). In conclusion, exposure of sheep embryos produced in vivo to resveratrol for 6 h reduced their levels of oxidative stress and did not impair their mitochondrial activity. These findings may help maintain embryo redox homeostasis, making this strategy a valuable tool for embryo development by mitigating cryopreservation-induced ROS and supporting cryopreservation outcomes.

This research was supported by Capes (001), FAPERJ, CNPq.