227 SUPPLEMENTATION WITH CYSTEINE, CYSTEAMINE, AND CATALASE DURING IN VITRO MATURATION PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE

Abstract

Menadione (MD), a naphthoquinone used as a vitamin K source in animal feed, can generate reactive oxygen species (ROS) and cause apoptosis. Hence, we examined whether MD reduces development of pre-implantation bovine embryos by inducing ROS production and apoptosis and tested the hypothesis that the anti-developmental actions of MD would be reduced by supplementation of the in vitro maturation (IVM) medium with antioxidants (a mixture of 0.6 mM cysteine + 100 μM cysteamine + 100 UI mL^–1 catalase). Cumulus-oocyte complexes (COC; n = 3334; 50 per well in 11 replications) were matured in IVM medium (TCM-199 with bicarbonate, hormones, and 10% FCS) for 22 h. After fertilization, presumptive zygotes were cultured in synthetic oviduct fluid medium (SOF) with 2.5% FCS and 0.5% BSA up to 7 days (Day 0 = IVF). On Day 6, MD was included in the SOF medium (0 μM: Control; or 5.0 μM: MD5) during 24 h. All cultures were conducted at 38.5°C in 5% CO₂ in air. In a second experiment, COC were matured in TCM-199 supplemented with or without the mixture of antioxidants (Antiox; only during IVM) and with or without MD at Day 6 in a 2 × 2 factorial design (Control, Control/Antiox, MD5, MD5/Antiox). The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively. A sample of the blastocysts (n = 1112) was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA) to detect the apoptotic cells or with 5 μM H₂DCFDA (Molecular Probes, Canada) to evaluate the ROS levels. Stained embryos were evaluated under an epifluorescence microscope and the images of embryos stained with H₂DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. The means (±s.e.m.) were compared by ANOVA followed by Tukey's test (P < 0.05). In the first experiment, and the cleavage rates were similar (P > 0.05) for Control (80.5% ± 0.9) and MD5 groups (78.9% ± 1.2), but the blastocyst rates were lower (P < 0.05) in the MD5 group (25.9% ± 2.1) when compared with Control (36.4% ± 1.5). The rate of apoptotic cells in blastocysts was higher (P < 0.05) in the MD5 group (19.2% ± 0.7) than in Control (10.0% ± 0.5), as well as the fluorescence intensity for ROS quantification (0.7 ± 0.1 v. 1.1 ± 0.1, respectively, for Control and MD5 groups). In an effort to rescue the impaired embryonic development, a mixture of antioxidants was added to the IVM medium and the results were transitional in response in which MD5/Antiox (28.3% ± 3.5) was intermediate (P > 0.05) between the Controls [Control (38.2% ± 2.5) and Control/Antiox (34.7% ± 1.9) groups] and the MD5 (21.4% ± 2.9) group. Principal effects evaluated were the presence of MD (without: 35.9% ± 2.3 v. with: 24.8% ± 2.3; P < 0.05) or Antiox (without: 28.4% ± 2.3 v. with: 32.3% ± 2.3; P > 0.05). In conclusion, addition of MD to embryonic culture reduces the blastocyst development and increases intracellular levels of ROS and the occurrence of apoptosis. Anti-developmental actions of MD on embryonic development can be partially blocked by treating oocytes with a mixture of antioxidants during IVM.

Financial support was provided by FAPESP (2012/10083-8 and 2013/07382-6).