122 Comparison of two cryoprotectant dilution treatments for quick frozen in vitro-produced bovine embryos

Abstract

The aim of this study was to compare the viability of in vitro-produced bovine embryos following quick freezing in ethylene glycol (EG) and subsequent dilution of EG by either a two- or a three-step procedure. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39�C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n�=�544) were selected 7 and 9 days after insemination and randomly distributed to one of three EG equilibration treatment groups. Embryos were exposed to 10% EG for 10�min, and then to 17%, 22% or 28% EG for 30�s (respectively referred to as EG 17, EG 22 and EG 28). In all treatment groups, EG solutions were prepared in PBS�+�0.2% BSA, and embryos were exposed to EG solutions at 22�C. Embryos were loaded into 0.25-mL straws which were then heat-sealed. Straws were cooled in liquid nitrogen vapor for 2�min, and then plunged and stored in liquid nitrogen. Straws were thawed in room temperature air for 10�s, and then in 25�C water for 20�s. The thawed embryos of the EG 17, EG 22 and EG 28 groups were randomly assigned to one of two EG dilution procedures. Two-step dilution consisted of transfer of embryos into PBS�+�0.2% BSA�+�0.3�M sucrose solution for 3�min, and then PBS�+�0.2% BSA for 3�min. Three-step dilution consisted of transfer of embryos into PBS�+�10% EG�+�0.2% BSA�+�0.3�M sucrose for 3�min, PBS�+�0.2% BSA�+�0.3�M sucrose for 3�min, and then PBS�+�0.2% BSA for 3�min. Embryos were co-cultured on a granulosa cell monolayer in TCM199 and evaluated after 24�h for blastocyst re-expansion (EXP), and again at 48, 72 and 96�h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the Table. No significant differences were found between the two- and three-step dilution procedures (P�>�0.05) for in vitro-produced bovine embryos cryopreserved by quick freezing. This project was supported by FAPESP (01/11266-4).