213 USE OF ACTIVE CASPASE 3 AND TUNEL ASSAYS TO ESTIMATE EMBRYONIC QUALITY IN IN VIVO SANTA INES EWE EMBRYOS
Authors: Oliveira M. E. F. , Oliveira C. S. , Lima M. R. , Barros F. F. P. C. , Perini A. P. , Feliciano M. A. R. , Oliveira L. G. , Fonseca J. F. , Vicente W. R. R. 
Published: December 2013

This study was designed to quantify the percentage of apoptotic cells using active caspase 3 and TUNEL assays, in order to estimate the quality of ovine embryos produced in vivo. For that, 60 Santa Ines ewes (n = 10 per group) were submitted to superovulation with FSH treatment started near the different follicular wave emergence of the protocols (G-1 or G-2), during breeding season, transition, and nonbreeding season. Follicular wave emergence days were defined in a previous study that evaluated the follicular dynamic in oestrus synchronization treatments (Oliveira et al. 2011 Acta Sci. Vet. 38, 361). On Day 0, all ewes received a P4 device (CIDR®) and 37.5 μg of D-cloprostenol. The P4 device was replaced by a new one on Day 7 just for G-2 in the transition period. The superovulation treatment started on Day 4, 4, and 6 of protocol for G-1 and on Day 10, 10, and 11 for G-2 in nonbreeding, transition, and breeding season, respectively. The FSH treatment consisted of eight injections of pFSH administrated twice a day in descending order (40, 30, 20, and 10 mg of pFSH). The P4 device was removed two days after beginning of FSH treatment. At these times, all ewes received another injection of 37.5 μg of D-cloprostenol and a dose of 200 IU of eCG. Ewes were mated with a fertile ram for 4 days after P4 device removal. Embryo recoveries were carried out by laparotomy, 7 days after CIDR withdrawal. Embryos were morphologically classified. Grade I to III morulas and blastocysts were considered viable. A representative sample of each treatment was fixed and stained by active caspase 3 and TUNEL assays to assess the apoptotic cells percentage. Data were analysed by GLIMMIX using SAS comparing mean values (±s.e.m.) between groups at each season (P = 0.05). Pearson correlation was estimated between active caspase 3 and TUNEL assays. No effect was detected between treatments in each season on the number of viable embryos (3.2 ± 0.8 v. 1.8 ± 0.8, 3.9 ± 1.9 v. 5.7 ± 1.4, and 3.8 ± 1.5 v. 3.4 ± 0.8 for G-1 v. G-2 in nonbreeding, transition and breeding season, respectively). The treatment G-2 increased (P < 0.05) apoptotic cells percentage in nonbreeding season group, assessed by active caspase 3 (G-1: 3.1 ± 1.6% and G-2: 12.8 ± 4.3%) and TUNEL (G-1: 1.6 ± 0.5% and G-2: 11.1 ± 3.5%) assays. The apoptotic cells percentage remained unaltered for Transition and Breeding season groups, assessed by either active caspase 3 (G-1: 6.0 ± 0.9% and 5.6 ± 1.5%; G-2: 5.6 ± 1.1% and 5.1 ± 0.5%) and by TUNEL (G-1: 7.5 ± 1.3% and 5.2 ± 1.0%; G-2: 5.0 ± 0.9% and 6.4 ± 1.1%). The Pearson correlation between active caspase 3 and TUNEL tests was r2 = 0.436 (P < 0.0001). In conclusion, the active caspase 3 and TUNEL assays presented similar results for apoptosis level assessment in Santa Ines ewes in vivo produced embryos, and both assays were considered appropriate for this purpose. The increased apoptosis levels detected in the G-2 nonbreeding season group suggest that this treatment is harmful for Santa Ines ewe embryos.
Financial support was provided by FAPESP and FUNDUNESP.