Theriogenology 39: 111 l-l 120, 1993 IN VITRO SURVIVAL OF MURINE MORULAE AFTER QUICK FREEZING IN THE PRESENCE OF CHEMICALLY DEFINED MACROMOLECULES AND DIFFERENT CRYOPROTECTANTS A. Gutikrrez, J. Garde, C.G. Artiga, I. Mtioz and B. Pintado’ Departamento de Producci6n Animal, CIT-INIA Ctra de La Coruiia Km 5,9; 28040 Madrid, Spain Received for publication: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON July 14, 1992 Accepted: January 15, 1993 ABSTRACT We studied the ability of frozen-thawed mouse morulae to develop in vitro when the cryoprotectant proteins were substituted with one of the following nonorganic macromolecules: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and ficoll. We also determined how these agents interacted with 3 different cryoprotectants: glycerol (GLY), propylene glycol (PG), and ethylene glycol (EG). The influence of both of the above factors was measured on the basis of post-thaw morphological appearance, the percentage of development to the expanded blastocyst stage and the total cell count. Morulae (n=950) were collected from superovulated mice. Those classified as good or excellent were distributed among the 12 different freezing solutions, obtained by combining the 3 cryoprotectants with the 4 macromolecules (the 3 mentioned above, plus a control of 5% fetal calf serum) in phosphate buffered saline (PBS). Embryos frozen in PVA, PVP and ficoll tended to be a little difficult to recover from the straws. Development to the expanded blastocyst stage was significantly lower (PcO.05) in propylene glycol (43.6%) than in ethylene glycol (79.5%) or in glycerol (76.1%). Polyvinyl alcohol provided a higher survival rate when combined with glycerol (90.3) or ethylene glycol (95.0), but when it was combined with propylene glycol, only 56.5% of embryos survived after thawing. A positive interaction was observed between glycerol and PVA and between ethylene glycol and PVA or ficoll. The results indicate that fetal serum could be successfully substituted for any of the 3 chemically defined macro- molecules. However, our findings also suggest that the use of PG as a cryoprotectant should be avoided when mouse morulae are frozen using the quick freezing method. Key words: mouse, embryo, quick freezing, cryoprotectant, macromolecules INTRODUCTION The cryopreservation of embryos has allowed for the mobility of genetic material around the world. However, the protein source used in clyoprotectant solutions can be Acknowledgements This work was supported by project CICYT GAN 90-106, Madrid, Spain ‘Correspondence and reprint requests. Copyright 0 1993 Butterworth-Heinemann