THE EFFECT OF TEMPERATURE AT WHICH SLOW COOLING IS TERMINATED AND OF THAWING RATE ON THE SURVIVAL OF ONE-CELL MOUSE EMBRYOS FROZEN IN DIMETHYL-SULFOXIDE OR 1,2-PROPANEDIOL SOLUTIONS

We investigated the slow freezing of one-cell mouse embryos with eithe r dimethyl sulfoxide (Me(2)SO) or 1,2-propanediol (PROH) as the cryopr otectant. One-cell embryos, collected from superovulated C57BL/6J x CB A/Ca females were exposed to 1.5 M solutions of either Me(2)SO or PROH . The embryos were cooled at 0.3 degrees C/min to temperatures between -10 degrees and -80 degrees C before being plunged into LN(2) and the n warmed at either 20 degrees C/min or 450 degrees C/min. Survival was expressed as the percentage of hatching or hatched blastocysts per fr ozen-thawed embryo. When the slow cooling was in 1.5 M PROH, the tempe rature at which survival rates after slow thawing began to increase wa s -35 degrees C (52.6 +/- 5.2% survival). For slow cooling in 1.5 M Me (2)SO this temperature was -50 degrees C (45.0 +/- 2.9% survival). The addition of sucrose to the 1.5 M PROH solution raised the temperature at which survival rates after slow thawing began to increase to -30 d egrees C (54.8 +/- 3.7% survival). If slow cooling was stopped at high subzero temperatures, embryos survived better after rapid thawing tha n slow thawing. If slow cooling was stopped at low subzero temperature s, the survival rate was not dependent on the thawing rate if freezing was done in 1.5 M PROH. When freezing was in Me(2)SO solutions and to subzero temperatures of -60 degrees and -80 degrees C, slow thawing g ave better survival than rapid thawing. The addition of sucrose to the Me(2)SO freezing solution restored the survival rates at -60 degrees and -80 degrees C. These results indicate that high rates of survival may be obtained from one-cell mouse embryos by a rapid or a slow thawi ng procedure, as has been found for other developmental stages. The re sults also indicate that PROH provides superior protection compared to Me(2)SO against freezing-thawing damage and that the addition of sucr ose to the freezing solutions prior to freezing improves the overall s urvival rates. Embryos that survived freezing and developed in culture implanted and formed normal fetuses at rates similar to those of nonf rozen control embryos (60% vs 68% and 53% vs 58%, respectively). (C) 1 994 Academic Press, Inc.