127 IN VITRO DEVELOPMENT OF IVF CRYOPRESERVED BOVINE EMBRYOS SUPPORTED BY TCM-199 OR SOFaa CULTURE MEDIUM

Abstract

In vitro bovine embryo production is commercially applied around the world. However, the cryopreservation of these embryos is not yet possible, which raises difficulties for the expansion of this biotechnology. The aim of this work was to evaluate the influence of the culture media on embryo development after cryopreservation. Cumulus–oocyte complexes obtained from slaughterhouse bovine ovaries were in vitro-matured, fertilized, and cultured. A total of 600 expanded blastocysts (between 7 and 9 days of culture) were cryopreserved by slow freezing, quick freezing, or vitrification methods. For slow freezing (slow group), the embryos were exposed to 10% ethylene glycol (EG) for 10 min and cryopreserved at 1.2°C per minute. For quick freezing (quick group), the embryos were exposed to 10% EG for 10 min and to 20% EG + 20% glycerol (Gly) for 30 s. For vitrification (vitrification group), the embryos were exposed to 10% EG for 10 min and to 25% EG + 25% Gly for 30 s. The straws (quick and vitrification groups) were placed in nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. The embryos were thawed in air for 10 s and in a water bath at 25°C for 20 s. For warming, embryos were washed in PBS + 0.2% BSA + 0.3 M sucrose for 3 min and in PBS + 0.2% BSA for 3 min. To evaluate development after thawing, the embryos were cultured on a granulosa cell monolayer with TCM-199 or SOFaa for 4 days. The embryos of the slow group showed re-expansion rates of 55.55% (55/99) and 29.00% (29/100), respectively, for TCM-199 and SOFaa. The quick group showed re-expansion rates of 4.85% (5/103) and 7.22% (7/97), and the vitrification group 10.89% (11/101) and 14.00% (14/100), respectively, for TCM-199 and SOFaa. The slow group showed hatching rates of 47.47% (47/99) and 11.00% (11/100), respectively; the quick group did not show hatching rates in either medium. The vitrification group showed hatching rates of 7.92% (8/101) and 6.00% (6/100), respectively, for TCM-199 and SOFaa. The results were analyzed by chi-square test, and all values were significant at P < 0.05. The slow group showed difference in re-expansion and hatching rates when the different media were compared. The quick and vitrification groups did not show differences in re-expansion and hatching rates when the different media were compared. The slow group showed higher re-expansion and hatching rates than the quick and vitrification groups. In conclusion, the culture medium influences embryo development after slow freezing, and the TCM-199 is more appropriate than SOFaa.

This work was supported by FAPESP 04/05335-1