135 LEVELS OF REACTIVE OXYGEN SPECIES (ROS), APOPTOSIS AND CRYORESISTANCE OF BOVINE EMBRYOS PRODUCED IN VITRO IN THE PRESENCE OF ANTIOXIDANTS

Abstract

The production of ROS is a normal process that occurs in cellular mitochondrial respiratory chain. However, the increase in ROS due to the high oxygen tension during in vitro production of bovine embryos induces oxidative stress, leading to embryonic development failure. Thus, the aim of this study was to evaluate the effects of supplementation with antioxidants (cysteine – CIST; β-mercaptoethanol – βME and catalase – CAT) during in vitro maturation (IVM) and in vitro culture (IVC) on the embryo development, cryoresistance, and quality, as well as the amounts of intracellular ROS produced during embryo culture. Cumulus–oocyte complexes (n = 565) were IVM in medium TCM-199 supplemented with 0.6 mM CIST, 100 µM βME, 100 UI CAT, or without antioxidants (Contr). After fertilization, zygotes were IVC in SOF medium during the first 72 h (up to Day 3) with or without addition of the same antioxidant used for IVM, and then all the embryos were transferred to SOF medium without antioxidants. All cultures were conducted at 38.5°C in 5% CO₂ in air. The cleavage and blastocysts rates were evaluated, respectively, at 72 and 168 h post-insemination. Part of obtained blastocysts (n = 133) was vitrified (Ingámed^®, Maringá, PR, Brazil). The remaining was stained (n = 46) with 5 µM of the fluorescent probe 6-carboxy-2′7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Molecular Probes, Invitrogen, Oakville, Canada) or stained (n = 62) for TUNEL (In situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, IN, USA). Stained embryos were immediately evaluated under an epifluorescence inverted microscope and the images of embryos stained with H₂DCFDA were analyzed by Q-Capture Pro image software (QImaging, Surrey, BC, Canada) for determining the fluorescent intensity. The vitrified embryos were thawed and cultured for 24 h to evaluate the re-expansion rates. The difference between groups was compared by ANOVA followed by Tukey test, and re-expansion rates by chi-square test (P < 0.05). The cleavage rates were 82.9% ± 3.2^a (Contr), 80.4% ± 2.5^a (CIST), 87.0% ± 1.8^a (βME) and 81.2% ± 3.0^a (CAT). The blastocyst rates were 48.7% ± 3.4^ab (Contr), 34.4% ± 4.2^b (CIST), 36.0% ± 5.6^ab (βME), and 56.8% ± 7.5^a (CAT). The re-expansion rates were 76.0%^ab (Contr), 66.7%^b (CIST), 50.0%^b (βME), and 66.1%^b (CAT). The fluorescent intensity was 1.0 ± 0.07^a (Contr), 0.8 ± 0.05^bc (CIST), 0.6 ± 0.05^c (βME) and 0.7 ± 0.07^bc (CAT). The total number of cells was 85.7 ± 3.5^a (Contr), 89.6 ± 4.8^a (CIST), 99.5 ± 5.4^a (βME), and 81.5 ± 4.3^a (CAT), and the rate of apoptotic cells was 4.3 ± 1.2^a (Contr), 1.5 ± 0.4^b (CIST), 1.6 ± 0.4^b (βME), and 2.4 ± 0.6^ab (CAT). Supplementation with CAT improved embryo development, compared with CIST (P < 0.05). The fluorescent intensity was lower for all embryos produced with antioxidants (P < 0.05) and the rate of apoptosis was reduced in CIST and βME (P < 0.05). In conclusion, supplementation with antioxidants is an interesting strategy to improve the embryo quality since it reduces the cell death caused by oxidative stress. However, such improvement in embryo quality did not affect embryo cryotolerance.

Financial support was provided by CNPq and FAPESP 01/18257-2.