Factors affecting survival of frozen-thawed mouse embryos

Optimization procedures were utilized to determine the levels of six factors thought to be important for survival and development of frozen and thawed 8-cell and blastocyst stage embryos from superovulated CD-1 and NMRI mice. For 8-cell embryos optimal values were estimated to be: 0.94 M dimethyl sulfoxide (DMSO); 9.7 min equilibration time; 1.6 degrees C/min cooling rate; -80 degrees C to stop slow cooling; 20 degrees C/min warming rate and 20 to 37 degrees C to dilute the DMSO. Blastocyst stage embryos showed optimal values when 1.54 M DMSO; 9.8 min equilibration time; 0.5 to 1.0 degree C/min cooling rate; -80 degrees C to stop slow cooling; 20 degrees C/min warming rate and 20 to 37 degrees C to dilute the DMSO were used. Only DMSO concentration and equilibration time to DMSO were found to have critical values within the ranges tested. To test survival in vivo of frozen-thawed 8-cell embryos from CD-1 mice and of frozen-thawed blastocysts from NMRI mice, embryos were frozen and thawed using optimal conditions, cultured for 24 h and transferred to pseudopregnant recipients. Forty-four percent of the frozen-thawed 8-cell embryos and 58% of the nonfrozen embryos developed into viable fetuses. The percentage of viable fetuses for the blastocyst stage was 37% for frozen-thawed embryos and 44% for nonfrozen embryos.