160 Exposure to Cadmium Affects Oocyte and Embryo Competence in Cattle

There is a growing worldwide concern regarding the increased release of the heavy metal cadmium (Cd) in the environment, due to several industrial processes, as it is known to affect health. Among other heavy metals, Cd is widely recognised to influence the reproductive system at different levels, interfering with both gametes and embryo functions in several species (Thompson and Bannigan, 2008 Reprod. Toxicol. 25, 304-315). The in vitro model can be used to mimic environmental conditions allowing us to evaluate their effect on oocyte maturation and early embryo development. Therefore, the aim of this study was to evaluate the influence of different Cd concentrations on nuclear maturation, apoptosis in cumulus cells, and cleavage and blastocyst yields in cattle. For this purpose, abattoir-derived bovine oocytes were in vitro matured, fertilized, and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). In particular, oocytes were matured with 0 (control; n=126), 0.1 μM (n=139), 1 μM (n=134), and 10 μM of Cd (n=135), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. For each replicate, after 22h of maturation, a representative sample of oocytes (n=10 per each group) was used to evaluate nuclear maturation by 4′,6-diamidino-2-phenylindole (DAPI) staining and another sample (n=10 per each group) to assess cumulus-cells complex apoptosis by TUNEL/Hoechst staining (Pocar et al. 2005 Reproduction 130, 857-868). The remaining oocytes were in vitro fertilized and cultured with 0 (n=106), 0.1 μM (n=119), 1 μM (n=114), and 10 μM (n=115) Cd. The experiment was repeated 3 times. On Day 8 post-IVF, the blastocyst yields were recorded. Differences among groups were analysed by ANOVA, with the least significant difference method used as a post hoc test. Data are presented as means±SE. Unexpectedly, the exposure of oocytes to Cd during IVM did not affect the percentage of oocytes undergoing nuclear maturation (on average 96.3±2.3). In contrast, concentrations of 1 and 10 μM Cd increased the percentage of apoptotic cumulus-cells in cumulus–oocyte complexes (COC) compared with the control (3.4±0.4, 10.6±1.8, 15.0±0.9, 16.7±4.0, respectively, with 0, 0.1, 1, and 10 μM; P<0.05). It is worth pointing out that with the highest concentration, cumulus expansion did not occur and cumulus cells appeared detached from the oocyte. Likewise, 1 and 10 μM Cd decreased cleavage rates compared with the control (68.7±1.8, 54.3±5.0, 58.5±4.2 and 2.8±2.6, respectively, with 0, 0.1, 1, and 10 μM Cd; P<0.01). Finally, blastocyst yields decreased when oocytes were treated with 0.1 μM Cd and no development to blastocyst was observed at the 2 higher concentrations (35.1±1.7, 26.2±3.1, 0, 0, respectively, with 0, 0.1, 1, and 10 μM; P<0.01). In conclusion, exposure to Cd during maturation negatively affects bovine COC, as indicated by the increased apoptotic index in cumulus cells, without influencing the nuclear maturation process. Furthermore, the presence of Cd during in vitro fertilization and culture severely impairs both the fertilization and post-fertilization embryo development.