EVALUATION OF DEVELOPMENT AFTER CRYOPRESERVATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

The inefficiency of embryo cryopreservation protocols limits the broad use of in vitro production (IVP) of bovine embryos. The aim of this work was to identify the damage caused by cryopreservation and embryo culture of IVP bovine embryos after thawing by in vitro development before and after cryopreservation. Cumulus–oocyte complexes were in vitro-matured, fertilized, and cocultured on granulosa cells in SOF with amino acids (SOFaa) supplemented with FCS. Expanded blastocysts (n = 600) harvested on Days 7 to 9 were submitted to controlled freezing [controlled group: 10% ethylene glycol (EG) for 10 min and 1.2°C min–1 cryopreservation], quick-freezing [quick group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s], or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. The embryos of the control group were not exposed to cryoprotectant or cryopreservation method, and the hatching rate was evaluated on Day 12 post-insemination. The straws (quick and vitrification groups) were first placed on nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. Embryos were thawed in air for 10 s followed by a 25°C water bath for 20 s. Embryos were rehydrated in PBS + 0.2% BSA + 0.3 m sucrose and PBS + 0.2% BSA for 3 min each. To evaluate development of frozen–thawed embryos, they were cocultured on granulosa cells in TCM-199 or SOFaa both supplemented with FCS for 4 days. Hatching rate of the control group was 46.1%. Data were analyzed by PROC MIXED model of SAS System for Windows®. For TCM-199, the controlled group hatching rate was 44.65 ± 5.94%, quick group did not hatch, and vitrification group showed hatching rates of 9.4 ± 6.8%. For SOFaa, the controlled group hatching rate was 11.6 ± 3.4%, embryos submitted to the quick group did not hatch, and the vitrification group showed hatching rates of 8.7 ± 4.5%. Values were significant at P < 0.05. The controlled group showed a difference compared with the other groups of cryopreservation in both media (TCM-199 and SOFaa). However, TCM 199 showed high rates of re-expansion and hatching. In conclusion, the culture medium influences embryo development after cryopreservation, and TCM-199 is more appropriate than SOFaa. Financial support by FAPESP (04/05335-1).