Apoptosis rate after vitrification / rewarming of pure breed Gyr embryos produced in vitro

Gyr breed is the main representative Bos indicus in dairy systems in South America, especially in Brazil. Due to significant improvement of this breed regarding milk production, combined to its resistance to tropical conditions and ectoparasites, it has become one of the main IVP donors breed. Embryo cryopreservation is an important tool for IVP, and singularities among Bos Taurus and Bos indicus resistance to cryopreservation has been described. Despite the importance of Gyr breed in Brazil, little is known about their parameters for tolerance in cryopreservation. Thus, the objective of the study was to assess the apoptosis rate associated with cryotolerance of pure breed Gyr embryos produced in vitro. COCs recovery was performed by OPU in Gyr donors. Viable oocytes were matured, fertilized with sexed semen of Gyr bull, and presumptive zygotes were cultured for 7 days. An incubation system at 38.5o C, and 5% CO2 in atmospheric air and high humidity was used. MIV, FIV and CIV media were purchased from Bioklone® (Jaboticabal, Brazil). On day 7 of culture, embryos were vitrified using a protocol developed by Vajta (Vajta, Mol Reprod Dev, 51, 53-58, 2008) with minor modifications. The vitrified embryos were rewarmed and cultured for another 3 hours, when they were evaluated regarding the re-expansion of the blastocoele. Vitrified/warmed embryos presenting re-expanded blastocoele (V+) or not (V-) were stained for evaluation of the total number of cells (TNC) with Hoechst and for apoptosis evaluation by immunofluorescence detection of caspase-3. The comparison between the NTC and apoptosis rate between groups were performed using the Mann Whitney Test. Statistical analyzes were performed at INSTAT GraphPad software, with 5% significance level. A total of 38 vitrified / rewarmed embryos were obtained in 3 replicates (V + n = 10, V n = 28). There was no difference (P > 0.05) between groups, both for NTC analysis (V +: 82,4a ± 27.1; V-: 79,5a ± 26), and for the apoptosis rate analysis (V + 5, 9%: V: 15.4% a). It is noteworthy that, although this effect have not been significant, we observed in the group of embryos that did not show re-expansion of blastocoele, a greater number of embryos displaying more than 20% of apoptotic cells. However, the rate of apoptosis does not seem to be the cause of failure in re-expanding the blastocoele of the vitrified Gyr embryos that are not selected for transfer.