Resveratrol supplementation promotes recovery of lower oxidative metabolism after vitrification and warming of in vitro-produced bovine embryos

Abstract

Although vitrification is the current method of choice for oocyte and embryo cryopreservation, it may have detrimental effects on reduction–oxidation status and mitochondrial activity. The aim of this study was to evaluate the effect of supplementing invitro culture (IVC) media and/or vitrification solutions with the antioxidant resveratrol on active mitochondria, mitochondrial superoxide production and lipid peroxidation. Abattoir-derived oocytes were matured and fertilised invitro using standard procedures. Following IVF (21h later), zygotes were cultured in IVC medium supplemented with 0 or 0.5µM resveratrol. On Day 7, blastocysts were vitrified using the Cryotech Vitrification Kit (Cryo Tech Laboratory) with or without 0.5µM resveratrol. After warming, active mitochondria, mitochondrial superoxide production and lipid peroxidation were evaluated using Mito Tracker Green FM, MitoSOX Red and BODIPY581/591 C11 staining respectively. The vitrification–warming process significantly increased active mitochondria and mitochondrial superoxide production in bovine embryos (P<0.05, ANOVA). The addition of 0.5µM resveratrol to the IVC medium or vitrification solutions significantly attenuated the increase in active mitochondria (P<0.05), but not in mitochondrial superoxide production, whereas embryos cultured and vitrified with resveratrol showed the highest values for both parameters (P<0.05). Regarding lipid peroxidation, no significant differences were detected between treatments. In conclusion, resveratrol supplementation of IVC medium or vitrification solutions contributes to recovery of an embryo’s ‘quieter’ state (i.e. lower oxidative metabolism) after vitrification. However, supplementation of both solutions with resveratrol seemed to have a pro-oxidant effect.