Efeito da adição do fulerol ao meio de maturação in vitro de oócitos bovinos

Abstract

Oxidative stress is the main cause of low efficiency in oocyte maturation and embryo development in in vitro embryo production, due to an imbalance between the amount of reactive oxygen species (ROS) and antioxidants. The in vitro atmosphere has a high oxygen tension, which associated with other factors such as light interference, presence of sperm and absence of maternal antioxidants lead to a higher production of ROS, when compared to the in vivo atmosphere. The present study was aimed to evaluate the effect of the addition of fullerenol, in distinct concentrations to the in vitro maturation media of bovine oocytes (IVM), on the production rates and quality of the embryos produced. Fullerenol is recognized as a powerful antioxidant, a very electronegative and stable molecule that is able to reach to specific targets. Four distinct IVM media were tested: group 1 – control (CO) (n=461 oocytes): TCM 199 bicarbonate media; group 2 (F1) (n=461): TCM 199 bicarbonate media with 1nM of fullerenol; group 3 (F10) (n=439): TCM 199 bicarbonate media with 10nM of fullerenol; group 4 (F50) (n=451): TCM 199 bicarbonate media with 50nM of fullerenol. Oocytes were matured in incubator during 24 hours at 38oC, 5% of CO2 and humidity of 95%. Subsequently, the oocytes were fertilized and cultivated in the same media of fertilization and cultivation, respectively. On the second day of culture (D2) the cleavage rate was evaluated, and on the seventh day (D7) the blastocyst rate by the total of cleavage embryos and the rate of blastocyst production by the total number of matured oocytes were evaluated. In addition, after observation and definition of those rates, embryos produced were fixed for posterior TUNEL assay. Nuclear maturation was evaluated by meiotic status at the end of oocyte maturation using HOECHST staining. There were no statistical difference (P>0.05) between cleavage rates (73.96%; 73.53%; 73.12%; 76.94%) for CO, F1, F10 and F50, respectively. Blastocyst rate differed between treatments. CO, F1 and F50 showed statistically similar (P>0.05) results (CO=30.15%, F1=27.11%, F50=31.04%). CO and F50 had higher (P<0.05%) blastocyst production than F10 (23.91%), which was equal to F1. The total number of cells per embryo (CO=130.84, F1=129.78, F10=110.72, F50=134.05) and the total number of apoptotic cells (CO=8.52, F1=7.68, F10=7.54, F50=5.85) did not differ (P>0.05) between treatments. The apoptosis rate was higher (P<0.05) in CO (7.43%) than F50 (4.23%). CO, F1 (5.94%) and F10 (6.98%) did not differ (P>0.05), as F1, F10 and F50 were statistically similar (P>0.05). Nuclear maturation (metaphase II) did not differ (P>0.05) between treatments (CO=53.3%; F1=48.2%; F10=48.0%; F50=33.3%). In conclusion, the presence of fullerenol in the IVM media had no effect on nuclear maturation rate and the cleavage rate, while reduced the blastocyst production at 10nM concentration. In high concentration of fullerenol (50nM), cleavage and blastocyst production were similar (P>0.05) to the control group, however reduced apoptosis rate when compared to the same treatment.