308 VERBASCOSIDE TREATMENT DURING IN VITRO MATURATION IMPROVES THE EMBRYO DEVELOPMENT OF PREPUBERTAL OVINE OOCYTES

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a pro-oxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at micromolar concentrations in a continuative 24-h in vitro maturation (IVM) exposure protocol (1). The aim of the present study was to determine whether a lower (nanomolar) VB concentration and a shorter exposure time (2 v. 24 h) during IVM may improve the maturation rates of prepubertal ovine oocytes and their subsequent embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered 1-mo-old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1 IU mL–1 of FSH/LH, and 100 µM cysteamine, in 5% CO2 in air at 38.5°C for 24 h. Based on our previous results (Dell'Aquila et al. 2014 Biomed. Res. Int. 2014, 878062), VB was added in the IVM medium at 1.03 nM, and 2 incubation times (24 and 2 h) were tested. In the 2-h exposure group, after 2 h of exposure to VB, oocytes were washed and cultured up to 24 h without VB. A group of oocytes were cultured in absence of VB, as controls. Matured oocytes were fertilized with frozen-thawed ram semen in SOF medium for 22 h and zygotes were cultured in vitro for 8 days. Metaphase II (MII) cleavage and blastocyst rates were analysed by Chi-squared test. Embryo quality was evaluated by staining and total cell count of the blastocyst and analysis of variance (ANOVA) was applied. Differences were considered to be significant when P < 0.05. Compared to controls, VB treatment at the concentration of 1.03 nM and 24 h of exposure had no effect on MII rates (196/268, 73% v. 226/323, 70% MII/cultured oocytes; P > 0.05). However, this treatment allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% v. 165/226, 73%; respectively; P < 0.05), blastocyst yield/cleaved embryos (59/156, 38% v. 45/165, 27%, respectively; P < 0.05), and total blastocyst cell numbers (108.62 ± 19.87 v. 89.61 ± 26.32, respectively; P < 0.05) compared to control oocytes. The VB treatment at the same concentration but for 2 h induced only significantly higher cleavage rate (196/210, 93% v. 165/226, 73%; P < 0.05). In conclusion, our results showed that VB treatment at 1.03 nM during 24 h of IVM exerted a positive effect on in vitro embryo development of prepubertal ovine oocytes by increasing the blastocyst yield and their quality. The hypothesis that VB at nanomolar concentrations may improve cumulus-oocyte energy/redox status is under investigation. The authors acknowledge support by the Regione Autonoma della Sardegna (LR 7, Agosto 2007, no. 7, CRP-17602). The authors thank Dr D. Bebbere and L. Falchi, Dept. Veterinary Medicine, Sassari, for statistical analysis.