INVITRO SURVIVAL OF MURINE MORULAE AFTER QUICK FREEZING IN THE PRESENCE OF CHEMICALLY DEFINED MACROMOLECULES AND DIFFERENT CRYOPROTECTANTS

We studied the ability of frozen-thawed mouse morulae to develop in vi tro when the cryoprotectant proteins were substituted with one of the following nonorganic macromolecules: polyvinyl alcohol (PVA), polyviny l pyrrolidone (PVP), and ficoll. We also determined how these agents i nteracted with 3 different cryoprotectants: glycerol (GLY), propylene glycol (PG), and ethylene glycol (EG). The influence of both of the ab ove factors was measured on the basis of post-thaw morphological appea rance, the percentage of development to the expanded blastocyst stage and the total cell count. Morulae (n=950) were collected from superovu lated mice. Those classified as good or excellent were distributed amo ng the 12 different freezing solutions, obtained by combining the 3 cr yoprotectants with the 4 macromolecules (the 3 mentioned above, plus a control of 5% fetal calf serum) in phosphate buffered saline (PBS). E mbryos frozen in PVA, PVP and ficoll tended to be a little difficult t o recover from the straws. Development to the expanded blastocyst stag e was significantly lower (P<0.05) in propylene glycol (43.6%) than in ethylene glycol (79.5%) or in glycerol (76.1%). Polyvinyl alcohol pro vided a higher survival rate when combined with glycerol (90.3) or eth ylene glycol (95.0), but when it was combined with propylene glycol, o nly 56.5% of embryos survived after thawing. A positive interaction wa s observed between glycerol and PVA and between ethylene glycol and PV A or ficoll. The results indicate that fetal serum could be successful ly substituted for any of the 3 chemically defined macromolecules. How ever, our findings also suggest that the use of PG as a cryoprotectant should be avoided when mouse morulae are frozen using the quick freez ing method.