EFFECT OF TROLOX ON REACTIVE OXYGEN SPECIES AND REDOX STATE DURING IN VITRO MATURATION OF BOVINE OOCYTES

TOMÁS GADZE; SERGIO MORADO; MARIANA CÓRDOBA; PABLO CETICA

Jornada; XXIV Jornadas de la Sociedad Argentina de Biología; 2022

The production of reactive oxygen species (ROS) is a normal process that occurs in cells, but certain stressful conditions typical of in vitro environments which alter the balance between ROS production and their removal by intracellular antioxidant systems, can produce oxidative damage and an impaired cell function. The role of ROS and/or antioxidant substances in the manipulation and culture of gametes and embryos in vitro is contradictory, being reported both harmful and beneficial effects. New antioxidants such as trolox have emerged and their properties are being analyzed. There is not enough evidence regarding the concentrations at which trolox could exert an effect on in vitro maturation (IVM) of bovine oocytes, nor its subsequent effect on fertilization and embryonic development. The aim was to study the effect of trolox on the levels of ROS production and the redox state in the IVM of bovine oocytes. Ovaries from slaughterhouses were transported to the laboratory, where the aspiration of the antral follicles and the recovery of the oocyte-cumulus complexes (COCs) were performed. COCs were matured in 199 medium supplemented with gonadotrophins and fetal bovine serum for 22 h at a temperature of 39°C with a humidified atmosphere of 5% CO2 in air (control) or supplemented with 25 µM (T1), 50 µM (T2 ) or 100 µM (T3) trolox. Then the COCs were denuded with hyaluronidase at 37°C. To determine ROS production, denuded oocytes were incubated for 30 minutes with 5 µM 2',7'-dichlorodihydro fluorescein diacetate (DCHFDA) and mounted on slides to quantify their fluorescence. Oocyte redox state was determined by the FAD/NAD(P)H ratio, measuring the intensity of the autofluorescent endogenous compounds FAD and NAD(P)H. The digital microphotographs obtained by epifluorescence microscopy were analyzed using IMAGE J, quantifying the individual luminosity of each oocyte. Oocyte nuclear maturation was evaluated by Hoechst 33342 fluorescent staining. Quantitative data were analyzed by ANOVA and qualitative data by Chi-square (p