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Biology of Reproduction, Vol 14, 256-263, Copyright © 1976 by Society for the Study of Reproduction

Freezing Morula Stage Rabbit Embryos

R. R. MAURER 1, and J. K. HASEMAN 1

1 Environmental Toxicology Branch and Environmental Biometry Branch, National Institute of Environmental Health Sciences, Research Triangle Park, N. C. 27709


Optimization procedures were utilized to determine the levels of five factors that maximized survival and development of frozen and thawed morulae collected from superovulated Dutch-Belted rabbits. The factors were DMSO concentration, equilibration time to the cryoprotective agent, cooling rate, temperature at which slow cooling was terminated and warming rate. DMSO concentration and equilibration time were found to be the most critical factors, and cooling rate the least important. After 10 experiments, which utilized 192 samples and 2164 embryos, the optimal values were estimated to be: 2.0 M DMSO; 10.7 min equilibration time; 1.1° C/min cooling rate; -100° C to stop slow cooling and 17° C/min warming rate. Through use of these values, 83 percent of frozen and thawed morulae developed into blastocysts in vitro.

To test survival in vivo of frozen and thawed New Zealand White and Dutch-Belted morulae, embryos subjected to these optimal conditions were transferred to one horn of Dutch-Belted and New Zealand White recipients, while the contralateral horn received nonfrozen morulae of the opposite strain. After freezing the percentage of embryos that implanted and the overall pregnancy rate were significantly (P<0.05) reduced. New Zealand White recipients had higher pregnancy and implantation rates than the Dutch-Belted recipients, but embryo genetics had little apparent effect on these parameters. Twenty-six percent of the frozen and thawed Dutch-Belted morulae developed into viable fetuses, with no apparent developmental aberrations, when New Zealand White recipients were used.

Note:
ACKNOWLEDGMENTS The authors thank Mr. Franklin Schwartz for his excellent technical assistance in this study.

Submitted on August 14, 1975
Accepted on October 28, 1975






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Copyright © 1976 by the Society for the Study of Reproduction.