Antioxidant supplementation during in vitro maturation increased oocyte mitochondrial membrane potential and bovine embryo development

Abstract Text: This study evaluated the effects of bovine oocytes IVM medium supplementation with intracellular (cysteine and cysteamine) and extracellular (catalase) antioxidant on the oocyte competence, based on evaluation of nuclear maturation rates, occurrence of apoptosis, mitochondrial membrane potential and the subsequent embryonic development. Oocytes were matured during 22h in TCM-199 medium with bicarbonate, hormones and 10% FCS, without supplementation (Control group) or supplemented with: 0.6 mM cysteine associated with 100 µM cysteamine (C+C group); 100 UI catalase (CAT group); or 0.6 mM cysteine associated with 100 µM cysteamine and 100 UI catalase (C+C+CAT group), at 38.5°C and 5% CO₂ in air. A sample of matured and immatured oocytes were stained with 500 nM of the fluorescent probe MitoTracker Red® (CMXROs, Molecular Probes, Invitrogen, Oregon, USA) or TUNEL (In Situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, IN, USA), in order to evaluate mitochondrial membrane potential (n=344) and apoptotic index (n=565), respectively. Stained oocytes were evaluated under an epifluorescence inverted microscope (excitation 579/510-550nm and emission 599/590nm, respectively for MitoTracker and TUNEL) and the mitochondrial membrane potential (quantified in arbitrary fluorescence units - AFU) were measured by Q-Capture Pro image software. The intensity values of the fluorescence signal obtained from oocytes were subtracted from mean values of "background" in the images. The rest of oocytes were submited to IVF and presumptive zygotes were IVC in SOFaa, at 38.5°C and 5% CO₂ in air, for 7 days. Cleavage and blastocyst rates were evaluated at 72 and 168 hpi, respectively. Were made 10 replicates with 50 oocytes per dish and it was considered the experimental unit. The mitochondrial membrane potential was analyzed by ANOVA followed by Tukey’s test and percentage of apoptosis, cleavage and blastocyst rates by Chi-square test (P<0.05). Data are presented as mean±SEM. The AFU for membrane mitochondrial potential were 1.00±0.05^a (immature oocytes), 1.60±0.05^b (Control), 0.94±0.03^a (C+C), 1.41±0.05^c (CAT) and 1.81±0.07^d (C+C+CAT). The oocyte maturation rates were 0.0% (immature) 76.7%±1.7 (Control), 80.3%±4.1 (C+C), 80.5%±5.2 (CAT) and 78.2%±1.1 (C+C+CAT), and the percentage of apoptotic oocytes were 1.55%^a (immature), 5.83%^ab (Control), 5.45%^ab (C+C), 1.92%^a (CAT) and 10.78%^b (C+C+CAT). Cleavage and embryo development were 72.5%^a and 28.2%^a (Control), 75.7%^a and 31.1%^a (C+C), 75.4%^a and 33.3%^a (CAT) and 73.1%^a and 46.2%^b(C+C+CAT). In conclusion, supplementation with association of cysteine, cysteamine and catalase improved blastocyst development that can be associated with the increase mitochondrial membrane potential and oocyte competence. 

Keywords: Mitochondrial membrane potential, antioxidant, in vitro maturation, blastocyst development