186 Protective effect of resveratrol on invitro fertilization of ovine oocytes matured under cadmium exposure

Cadmium (Cd) is one of the most toxic environmental and industrial heavy metals, with adverse effect on female reproduction. Earlier in vitro studies have demonstrated that exposure to Cd during IVM impairs oocyte fertilization through the induction of oxidative stress (Martino et al. 2017 Reprod. Toxicol. 69, 132-145). The aim of this study was to evaluate the effect of supplementing IVM medium with resveratrol (Res), a phytoalexin with antioxidant activity, on IVF of ovine oocytes matured under Cd exposure and to evaluate whether this effect could be mediated by alterations of cortical granules (CGs) distribution and/or intracellular levels of reactive oxygen species (ROS). Cumulus-oocyte complexes collected from slaughterhouse-derived ovaries of prepubertal ewes were in vitro matured with 0 μM CdCl2 (control), 2 μM CdCl2 (Cd group), or 2 μM CdCl2 + 1 μM Res (Cd-Res). Groups of MII oocytes were in vitro fertilized with ram semen (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817). After 16 h, oocytes were fixed with absolute ethanol, stained with Hoechst 33342, and classified as follows: normally fertilized (two pronuclei), polyspermic (more than two pronuclei), and abnormally fertilized (asynchronous pronucleus formation). Levels of ROS and distribution of CG in MII oocytes (n = 25 each group) were evaluated by confocal laser scanning microscopy after staining with 2’,7’dichlorodihydrofluorescein diacetate and Alexa Fluor 488-conjugated lectin peanut agglutinin, respectively (Jiao et al. 2013 PLoS One 8, 58018). Data of fertilization and CGs distribution were analysed by Chi-squared test and ROS levels (mean ± standard error) by Kruskal-Wallis test with STATA\IC 11.0 (Stata Corp.). Oocytes matured in presence of Res had higher normal fertilization and lower polyspermy rates than those matured without Res (Table 1). Levels of ROS in oocytes were lower in the Cd-Res group than in the Cd group (74.4 ± 1.3 vs. 122.4 ± 1.5 pixel/oocyte; P < 0.05), and similar to those of the control (71.1 ± 2.3 pixel/oocyte). The ratio of oocytes of Cd-Res group with CGs normally distributed in the peripheral region was higher than that of Cd group (91.3% vs. 65.2%; P < 0.05) and comparable to that of control oocytes (95.8%). Collectively, our results showed that resveratrol protects oocytes from Cd-induced alteration of normal fertilization by preventing oxidative stress and improving the major mechanism responsible for blockage of polyspermic fertilization. Table 1.Effect of resveratrol on IVF rate of oocytes matured under cadmium exposure Group No. of total oocytes No. of MII oocytes No. of fertilized oocytes (%) Normal Polyspermy Abnormal Control 136 105 (77.2) 59 (56.2)a 23 (21.9)a 6 (5.7) Cd 146 106 (72.6) 39 (36.8)b 38 (35.8)b 7 (6.6) Cd-Res 114 88 (77.2) 47 (53.4)a 16 (18.2)a 7 (7.9) a,bValues in the same column with different superscripts differ significantly (P < 0.05). This project was supported by FDS 2016 (CUP J86C18000780005).