Verbascoside treatment during in vitro maturation improves the embryo development of prepubertal oocytes.

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a prooxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at ?-molar concentrations in a continuative 24 hours 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 (2h versus 24h) 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 one-month old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1 IU/ml of FSH/LH and 100 µM cysteamine, in 5% CO2 in air at 38.5°C for 24h. Based on our previous results (1), VB was added in the IVM medium at 1.03 nM, and two incubation times (24h and 2h) were tested. In the 2h exposure group, after 2h exposure to VB, oocytes were washed and cultured up to 24h 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 22h and zygotes were cultured in vitro for 8 days. Metaphase II (MII), cleavage and blastocyst rates were analyzed by Chi-square 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 24h exposure had no effect on MII rates (196/268, 73% versus 226/323, 70% MII/cultured oocytes; P>0.05), but it allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% vs 165/226, 73%; P<0.05), blastocyst yield/cleaved embryos (59/156, 38% vs 45/165, 27%; P<0.05) and total blastocyst cell number (108.62±19.87 vs 89.61±26.32; P<0.05). VB treatment at the same concentration but for 2h induced only significantly higher cleavage rate (196/210, 93% vs 165/226, 73%; P<0.05). In conclusion, our results showed that VB treatment at 1.03 nM during 24h 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.