49 Resveratrol exposure improves oxidative metabolism of vitrified immature feline oocytes

The use of domestic cats as an experimental model for the application of reproductive biotechnologies has provided advances for investigation of reproductive physiology and gamete cryopreservation focused on the conservation of endangered felids. However, oocyte vitrification still leads to mitochondrial damage and increases the amount of reactive oxygen species (ROS), generating a reduction in oocyte developmental competence. The aim of this study was to: (i) evaluate the effect of resveratrol (RESV) on glutathione (GSH) and ROS levels, and (ii) identify the best moment for its exposure during vitrification of immature feline oocytes. Cumulus-oocytes complexes (COCs) were recovered from feline ovaries obtained in elective surgeries. COCs presenting homogeneous cytoplasm and surrounded by at least one layer of cumulus cells were selected and vitrified in eight replicates according to the following groups: vitrified without RESV (VIT, n = 20), exposed for 90 min to RESV before vitrification (RESV-VIT, n = 25), or exposed for 90 min to RESV after vitrification (VIT-RESV, n = 22). In addition, two fresh groups were performed, with (CONT-RESV, n = 27) or without (CONT, n = 32) RESV. Exposure was carried out in TCM 199 supplemented with 1 mmol/L of pyruvate, 4 mg/mL of bovine serum albumin, 100 µL/mL of penicillin-streptomycin, and 1 µM of resveratrol, for 90 min at 38.5°C, 5% CO₂ in atmospheric air and maximum humidity. After the exposure to RESV and vitrification, oocytes were warmed, denuded, and incubated with 2′,7′-dichlorodihydro-fluorescein diacetate (H2DCHFDA, Invitrogen™, D399) and 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC, CellTracker Blue [ThermoFisher], Invitrogen™, C12881) to detect intracellular ROS and GSH levels as green and blue fluorescence (arbitrary unit /µm²), respectively. The data were submitted to the homoscedasticity and normality tests, then evaluated using the Kruskal-Wallis test, followed by the Dunn test at a significance level of 5%. RESV-VIT (0.71 ± 0.09), CONT (0.61 ± 0.05), and CONT-RESV (0.57 ± 0.05) groups had similar (P > 0.05) ROS levels. The first two groups had higher ROS levels when compared with VIT (0.30 ± 0.08) and VIT-RESV (0.39 ± 0.14), while CONT-RESV was similar to VIT and VIT-RESV. Regarding GSH, the RESV-VIT group (0.62 ± 0.06) presented similar (P > 0.05) levels to the CONT-RESV group (0.39 ± 0.04), and the former had higher (P < 0.05) levels than all the other treatments (VIT: 0.19 ± 0.03, VIT-RESV: 0.27 ± 0.05, and CONT: 0.36 ± 0.05). The CONT-RESV, CONT, and VIT-RESV groups were similar to each other. The CONT group (2.16 ± 0.29) had a higher (P < 0.05) ratio between ROS and GSH when compared with RESV-VIT (1.15 ± 0.13) and VIT-RESV (1.14 ± 0.27) groups. On the other hand, VIT (1.45 ± 0.31) and CONT-RESV (1.72 ± 0.22) groups showed intermediary results, similar to CONT, RESV-VIT, and VIT-RESV. These results suggest that RESV is not able to prevent the damage caused by cryopreservation; however, regardless of the moment of exposure (either before or after vitrification), it supports the cell to recover when it is challenged by stressors.

This research was supported by FAPERJ and CAPES.