ASSESSMENT OF ULTRARAPID AND SLOW FREEZING PROCEDURES FOR 1-CELL AND 4-CELL MOUSE EMBRYOS

Three cryopreservation procedures were assessed for the freezing of mouse 1-cell and 4-cell embryos: the slow freezing protocols with dimethylsulphoxide (DMSO, A) and propanediol (PROH, B) and the ultrarapid procedure with DMSO (C), which was described by the Monash University group [A.Trounson et al. (1987) Fertil. Steril., 48, 843 - 850]. The evaluation of the different procedures included survival after freezing and thawing, further development after in-vitro culture to blastocysts and the ability to implant and to form living fetuses after transfer of early blastocysts to pseudopregnant mice. In-vitro development was lower in all frozen embryos than in the unfrozen controls. Procedures A and B induced comparable results and were significantly better than ultrarapid freezing. When unfrozen blastocysts were transferred to pseudopregnant mice, 64% of them implanted in the uterine wall and 59% developed to living fetuses. For zygotes the percentages of implantation sites and living fetuses were 47 and 33% for A, 52 and 44% for B, and 29 and 17% for C, respectively. When 4-cell embryos were cryopreserved, these results were 54 and 46% for A, 60 and 53% for B and 37 and 23% for C, respectively. In the ultrarapid procedure we also looked at the influence of the freezing solution. To set up a control, mouse zygotes and 4-cell embryos were exposed to DMSO as in the ultrarapid procedure except that they were not frozen; the survival and blastocyst formation was not different from controls, but fewer living fetuses were born (31% for zygotes and 41% for 4-cell embryos versus 67% in the controls). This study demonstrated that slow freezing with 1.5 M DMSO or 1.5 M PROH yielded better results than the ultrarapid method with 3.5 M DMSO.