Rapid freezing of mouse embryos in ethylene glycol at different preimplantation stages

The objectives of this study were to examine the effects of a rapid freezing protocol on the survival and in vitro development of mouse embryos cryopreserved in ethylene glycol (EG) at different preimplantation stages, and secondly, to investigate the effect of exposure to 3.0 M EG with 0.25 M sucrose on the survival and in vitro development of mouse embryos without freezing at different developmental stages. To perform the rapid freezing procedure, embryos were equilibrated in Dulbecco's phosphate buffered saline (DPBS) containing 3.0 M EG and 0.25 M sucrose (freeze medium) for 20 min and loaded into 250 microliters straws in a single column of freeze medium. The straws were held in liquid nitrogen (LN2) vapour for 2 min and immersed into LN2. Embryos were thawed in a 37 degrees C water bath for 20 sec and transferred to DPBS supplemented with 0.5 M sucrose (rehydration medium) for 10 min and cultured for 24 to 96 h in HTF (Human Tubal Fluid) plus 4 mg/ml BSA (Bovine Serum Albumin). Significant differences were found in the survival and development of mouse embryos at different developmental stages rapid frozen in EG and sucrose: two cell 43/84 (51%), 4-8 cell 44/94 (47%), morula and early blastocyst 56/70 (80%), expanding and expanded blastocysts 10/59 (17% (p < 0.05). These data indicate that the developmental stage in which mouse embryos are subjected to this quick freeze protocol affects survival and development in vitro and the majority (80%) of morula and early blastocyst stage embryos survive the procedure. No significant differences were observed in the in vitro developmental capacity of embryos at different developmental stages after treatment with high concentrations (3.0 M) of EG solution without freezing. Further investigations are underway to better understand the reasons for different survival rates of embryos frozen at different developmental stages using the present procedure.