122 COMPARISON OF TWO CRYOPROTECTANT DILUTION TREATMENTS FOR QUICK FROZEN IN VITRO-PRODUCED BOVINE EMBRYOS
J.A. Visintin A , A.C. Nicácio A , C. Yamada A , H.V.C. Amaral A , R. Simões A , M. Milazzotto A , M.G. Marques A and C.M. Mendes AReproduction, Fertility and Development 16(2) 183-184 https://doi.org/10.1071/RDv16n1Ab122
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
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).
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