43 Resveratrol during the warming process improves the quality of in vitro-produced vitrified embryos of Hartón del Valle cattle

The Hartón del Valle (HV) breed belongs to the set of Colombian Creole bovine breeds that inhabit the tropics, where it expresses benefits through factors such as resistance to typical diseases of the tropics, tolerance to heat stress, and productive and reproductive parameters superior to introduced breeds. Currently, this breed is at high risk of extinction. Vitrification is an ideal conservation alternative for embryos of this species; however, this process alters the oxidative balance and development of the embryo. The objective of the study was to evaluate the effect of supplementing in vitro culture (IVC) media and/or warming solutions (WS) with resveratrol (R) on embryonic development, survival, and oxidative status of HV embryos produced in vitro. Ultrasonography-guided transvaginal follicular aspiration was used on 13 HV females in one session. An average of 10 oocytes per animal were recovered. The oocytes (n = 131) were collected and subsequently in vitro matured for 24 h at 38°C in 5% CO_2. In vitro fertilisation (IVF) was performed using frozen straws from an HV bull previously tested for IVF and a final concentration of 2 × 10⁶ spermatozoa/mL was used. Presumptive zygotes were cultured in SOF medium supplemented with 0.5 μM of R (CR) and without R (C-) until Day 7, where blastocyst rates were assessed and expanding blastocysts were vitrified using the minimum volume method. Subsequently, they were thawed using R in a WS, so a group without R (C-V-: control) and a group with R (CRVR) were included. Embryos were cultured for 48 h to evaluate the survival and hatching rates. Finally, the levels of reactive oxygen species (ROS) and intracellular glutathione (GSH) were evaluated by 2′,7′-dichlorodihydrofluorescein diacetate staining and CellTracker Blue (ThermoFisher) fluorescent stain, respectively. The fluorescence intensity of the embryos was analysed using ImageJ software (version 1.53a, National Institutes of Health) and normalised to control. For the analysis of embryo culture variables, ROS and GSH levels; a completely randomised design was implemented using the ANOVA procedure. For survival and embryonic hatching variables, the nonparametric were analysed by chi-squared test using InfoStat^® 2020 statistical software package (infostat.com.ar). Data are presented as percentage mean ± standard error of the mean (P < 0.05). The results showed that R did not significantly affect cleavage and blastocyst rates compared with the control group (cleavage: CR = 65.32 ± 9.2% and C- = 71.85 ± 4.71%; blastocysts: CR = 51.09 ± 11.18% and C- = 41.55 ± 6.57%). In contrast, the R showed a significant effect (P < 0.05) on survival and hatch rates compared with the control group (survival: CRVR = 96.43 ± 3.57% and C-V- = 60.91 ± 4.18%; hatching: CRVR = 37.50 ± 12.84% and C-V- = 17.05 ± 8.51%). In addition, R reduced levels of ROS (P < 0.05) compared with the control (CRVR = 0.73 ± 0.06 and C-V- = 1 ± 0.1). In conclusion, results revealed that the use of 0.5 μM resveratrol during IVC and warming process, improves survival, cryotolerance, and quality of HV embryos.