206 Graphene-oxide toxicity evaluation and its effects on porcine oocyte in vitro maturation

The use of 2-dimensional nanocarbons to improve reproductive biotechnologies is still in its earliest stages, and much is yet to be done to elucidate if these materials can be employed safely when manipulating gametes and embryos. For this, we first synthesised graphene oxide (GO) according to a modified Hummer’s method and changed the oxidation degree of the developed material by heating while stirring it at 80°C until a maximum period of 2 weeks to obtain GO with reduced oxygen contents (rGO). After that, we characterised the GO and rGO structures by the following methods: atomic force microscopy, thermal gravimetric analysis, X-ray photoelectron spectroscopy as well as their reactive oxygen species activity by electron spin resonance (ESR). The ESR results indicated that reactive oxygen species activity of pristine GO was very low, which is a desirable characteristic for nanomaterials so the cells are not damaged by creating additional oxidative-stress conditions when added to culture media. Further analysis of the material showed that the maximum level of GO oxidation was 34% until reaching a maximum reduction of 26%. Decreasing the oxidation level of GO strongly decreased the material solubility in water, making the rGOs not adequate to apply for in vitro culture. Finally, we evaluated the toxicity of pristine GO on oocytes when added as a supplement for oocyte IVM. For this, groups of ~45 cumulus–oocyte complexes derived from small (<3 mm in diameter, SF) or medium follicles (3 to 6 mm in diameter, MF) were cultured in a 400-µL drop of modified porcine oocyte medium with the following concentrations of GO (0, 100 μg mL⁻¹, 1 μg mL⁻¹ or 10 ng mL⁻¹) at 39°C and 5% CO₂ in air. During the first 20 h after the start of IVM, the medium was supplemented with 1 mM dibutyryl cAMP, 10 IU mL⁻¹ eCG and 10 IU mL⁻¹ hCG. After the first period of IVM, the culture continued in the same conditions but in the absence of dibutyryl cAMP and gonadotropins for another 24 h. At the end of IVM, viability was assessed by trypan blue and meiotic progression of the oocytes was examined by Hoechst staining. Our results indicated that there was no significant difference on the viability of SF and MF oocytes, which was always kept to a minimum value of at least 80%. The maturation rates of MF oocytes were significantly higher than that of SF ones, independent of the concentration of GO applied. Surprisingly, the addition of GO did not negatively affect oocyte viability, even to the highest concentration evaluated (100 μg mL⁻¹). In summary, the addition of GO to the IVM medium up to a concentration of 100 μg mL⁻¹ is not detrimental to oocyte viability, but it is unable to further promote meiotic progression in MF- and SF-derived gilt oocytes. Further analyses on the capacity of GO to protect oocytes from oxidative stress need to be evaluated.