88 SURVIVAL AFTER FREEZING OF IN VITRO AND IN VIVO BOVINE EMBRYOS

Abstract

The present study was conducted to investigate the survivability of post-thawed bovine embryos for direct transfer. Bovine ovaries were collected at a local slaughterhouse. The cumulus-oocyte complexes (COC) were aspirated from 2 to 8 mm antral follicles using a syringe with an 18-gauge needle. Selected COC were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 15 COC were matured for 22 h in 50-μL droplets of TCM-199 supplemented with 5% FBS, 10 μg mL^-1 of LH, 10 μg mL^-1 of FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 39°C. Mature COC were fertilized with frozen-thawed semen treated with BO medium (Brackett and Oliphants Biol. Reprod. 12, 260-274). All oocytes and embryos were placed in CR1aa medium culture system for in vivo embryo production. The Korean native cows that were between days 8 and 12 of their estrous cycles were superovulated with 28 mg of porcine follicle stimulating hormone (FSH, Antorine-R10; Kawasaki Mitaka Pharmaceutical, Tokyo, Japan) in twice daily i.m. injections, with a gradual decrease over 4 days. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.5 M, and 1.8 M ethylene glycol(EG) was used as a cryoprotectant. Embryo was loaded into 0.25 mL straw and directly into a cooling chamber (CL-863, USA) and kept at -7°C for 10 min, including time for seeding, and further cooled to -35°C at -0.3°C. After 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in water bath at 35°C and 37°C. Embryos were evaluated at 24, 48, and 72 h post thawing. Embryos that survived were recorded as either blastocysts that had expanded or hatched at 24 h or had hatched at 72 h. All of the results were analyzed by ANOVA using the STATVIEW program. After frozen the blastocysts cultured without serum, better survivability for frozen embryos was seen in the 1.8 M EG with 0.5% BSA (bovine serum albumin) group than 1.5 M EG with 0.5% BSA (75.7 v. 72.7). The survivability of frozen-thawed embryos was significantly higher in the 37°C water bath than 35°C (85.7% v. 70.8%). However, there was no difference in the total cell number of thawed embryos (142 ± 13 v. 137 ± 12), and chromosome abnormality higher than in vivo frozen-thawed embryos. In conclusion, the results suggest that the thawing temperature at 37°C may be optimal for better in vitro survival of frozen-thawed embryos produced in vitro and in vivo. Furthermore, the data suggest that embryo freezing system may provide reasonable conditions for embryo transfer.