132 RESVERATROL DURING IN VITRO CULTURE IMPROVES CRYOTOLERANCE OF IN VITRO PRODUCED BOVINE EMBRYOS

Abstract

Despite the great improvement of in vitro embryo production (IVEP) efficiency recorded over the years in cattle, the in vitro produced (IVP) embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. One of the major factor impairing in vitro embryo development is oxidative stress. Resveratrol is an important antioxidant polyphenolic compound found in several vegetal sources, that contributes to red wine’s beneficial effects on the prevention of human cardiovascular disease. Recently, the interest in resveratrol has increased exponentially following the major findings that this molecule has positive effects on cancer chemoprevention, cardioprotection, inflammatory processes, several aspects of metabolism, leading to increased lifespan of various organisms from yeasts to vertebrates (Pirola et al. 2008 IUBMB Life 60, 323–332). A positive effect of resveratrol on in vitro embryonic development was demonstrated in swine (Lee et al. 2010 J. Reprod. Dev. 56, 330–335). The aim of this study was to evaluate whether supplementation of culture medium with resveratrol improves in vitro blastocyst development and the embryo resistance to cryopreservation in cattle. A preliminary dose response trial indicated that the optimal concentration in the range tested (from 0.5 to 10 µM) was 0.5 µM, with evident toxic effects at concentration higher than 5 µM. Abattoir-derived oocytes (n = 581, over 5 replicates) were matured and fertilized in vitro according to our standard procedure (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium, supplemented with 5% bovine serum, in the absence (control, n = 271) or presence of 0.5 µM resveratrol (n = 310) at 39°C under humidified air with 5% CO₂, 7% O₂, and 88% N₂. On Day 7 (IVF = Day 0), embryo yields were assessed and the blastocysts (except the hatched blastocysts) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5M sucrose (Rubessa et al. 2011). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and hatching rate after 48 h culture. Data were analyzed by chi-square test. Resveratrol supplementation during culture did not affect either cleavage (69.1 v. 72.0%, in the control and resveratrol groups, respectively) or blastocyst yields (38.3 v. 36.3%, in the control and resveratrol groups, respectively). However, treatment with resveratrol increased the cryotolerance of IVP embryos, as indicated by higher survival rates (74.7 v. 88.4%, in the control and resveratrol groups, respectively; P < 0.05) and hatching rates (35.1 v. 53.8%, in the control and resveratrol groups, respectively; P = 0.06) at 48 h. In conclusion, these results demonstrated that resveratrol supplementation during culture improves the quality, and hence the resistance to cryopreservation, of IVP bovine embryos.