CRYOPRESERVATION OF MURINE ZYGOTES FOR USE IN TESTING CULTURE ENVIRONMENTS

Developing one-cell mouse zygotes are more sensitive to in vitro envir onmental conditions than are cleavage-stage embryos. However, for conv enience and reproducibility, cryopreserved two-cell zygotes are routin ely used for such assays, Concern over the possibility of inducing dam age by exposing one-cell zygotes to cryoprotective agents and freeze-t haw procedures during syngamy led us to examine one cell zygotes, with and without visible pronuclei, in an effort to minimize or avoid thes e effects and obtain the highest possible developmental rate. In vivo fertilized mouse zygotes were collected 21 to 43 h after administratio n of human chorionic gonadotropin (hCG). Suspensions of zygotes in 2M ethylene glycol were aspirated into 0.25-ml plastic insemination straw s and slowly cooled at -0.5 degrees C/min to -40 degrees C before bein g plunged int liquid nitrogen for storage. Zygotes were thawed, rinsed , and placed in culture, Zygotes were examined initially for damage fr om the freeze-thaw procedure. Daily in vitro development was recoded. In this group of zygotes, no damage was apparent immediately after tha wing, and a high degree of development in vitro was observed. Thus, us efulness of a cryopreservation method for one-cell murine zygotes has been confirmed.