134 CONCEPTION RATES OF IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED IN 6% GLYCEROL AND TRANSFERRED BY THE DIRECT METHOD

Abstract

Ethylene glycol has been used as the standard cryoprotectant for direct transfer of bovine embryos due to its high permeability. But Merton et al. reported that cryoprotectivity of glycerol for bovine embryos was superior to that of ethylene glycol (2001 Theriogenology 55, 312 abst). We previously reported that nonsurgical transfer of in vivo-derived bovine embryos cryopreserved in a lower concentration (5%) of glycerol and thawed by stepwise method resulted in a 55.4% conception rate, whereas direct transfer without removal of cryoprotectant showed only a 45.1% conception rate (Takada et al. 2005 Jpn. J. Embryo Transfer 27, 59–64). In this experiment, survival and conception rates of in vitro-produced (IVP) bovine embryos cryopreserved in 6% glycerol solution (GLY) were compared to those of embryos cryopreserved in 10% ethylene glycol plus 0.1 M sucrose solution (EG). Cumulus–oocyte complexes were matured and fertilized according to Numabe et al. (2000 Theriogenology 54, 1409–1420). Presumed zygotes were cultured in mSOF supplemented with 5% calf serum (CS) and 0.25% linoleic acid albumin at 38.5°C under 5% CO₂, 5% O₂, 90% N₂ for 7 days. At the expanded blastocyst stage, embryos were placed in GLY or EG in PBS supplemented with 20% CS for 15 min at room temperature and loaded into 0.25-mL straws. Straws were placed directly into an alcohol freezer. When the cryoprotectant was GLY, straws were seeded at -4.0°C, held for 10 min, cooled at 0.5°C min to -30.5°C, and then plunged into liquid nitrogen. When the cryoprotectant was EG, the seeding point was -7.5°C, and the plunging point was -34.0°C, but the rest of the protocol was the same as for GLY. In Exp. 1, thawing in both groups was done in a 30°C water bath, and the contents were directly rehydrated in PBS with 20% CS. Thawed embryos were cultured in mSOF with 5% CS for 24 h to assess embryo survival rate, based on the re-expansion of the blastcoele and on their hatching ability. In Exp. 2, embryos in both groups were thawed and transferred to synchronous recipients without removing the cryoprotectant. Data were analyzed using chi-square analysis. In Exp. 1, the developmental rates of post-thaw embryos were similar in GLY (46/52, 88.5%) and EG (45/52, 86.5%); however, the hatching rate was significantly higher (P < 0.05) in embryos cryopreserved in EG (26/52, 50.0%) than in GLY (15/52, 28.8%). In Exp. 2, the conception rates of embryos were similar in both groups, GLY (7/15, 46.7%) and EG (6/15, 40.0%). In conclusion, after direct rehydration of embryos, the developmental ability of IVP bovine embryos cryopreserved in EG was superior to that of embryos cryopreserved in GLY in vitro. However, conception rates in vivo were similar in both groups. These results suggest that a lower concentration of glycerol might be still useful as a cryoprotectant for direct transfer of IVP bovine embryos.