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Biology of Reproduction 63, 1294-1302 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Cryobiology of Rat Embryos I: Determination of Zygote Membrane Permeability Coefficients for Water and Cryoprotectants, Their Activation Energies, and the Development of Improved Cryopreservation Methods1

Reinhold T. Pfaffa,b, Yuksel Agcaa,b, Jun Liua, Erik J. Woodsa, Augustine T. Peterb, and John K. Critser2,,a,b

a Cryobiology Research Institute, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202 Department of Veterinary Clinical Sciences, b School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907

ABSTRACT

New rat models are being developed at an exponential rate, making improved methods to cryopreserve rat embryos extremely important. However, cryopreservation of rat embryos has proven to be difficult and expensive. In this study, a series of experiments was performed to characterize the fundamental cryobiology of rat fertilized 1-cell embryos (zygotes) and to investigate the effects of different cryoprotective agents (CPAs) and two different plunging temperatures (Tp) on post-thaw survival of embryos from three genetic backgrounds. In the initial experiments, information on the fundamental cryobiology of rat zygotes was determined, including 1) the hydraulic conductivity in the presence of CPAs (Lp), 2) the cryoprotectant permeability (PCPA), 3) the reflection coefficient ({sigma}), and 4) the activation energies for these parameters. PCPA values were determined for the CPAs, ethylene glycol (EG), dimethyl sulfoxide (DMSO), and propylene glycol (PG). Using this information, a cryopreservation method was developed and the cryosurvival and fetal development of Sprague-Dawley zygotes cryopreserved in either EG, DMSO, or PG and plunged at either -30 or -80°C, were assessed. The highest fetal developmental rates were obtained using a Tp of -30°C and EG (61.2% ± 2.4%), which was not different (P > 0.05) from nonfrozen control zygotes (54.6% ± 3.0%).

FOOTNOTES

First decision: 23 March 2000.

1 Supported by The Cryobiology Research Institute, grant R01-AA120722 from the National Institutes of Health, and Harlan Sprague Dawley, Inc.

2 Correspondence: John K. Critser, Indiana University School of Medicine, Cancer Research Building, Wells Center for Pediatric Research, 1044 West Walnut St., Room 454, Indianapolis, IN 46202. FAX: 317 274 8679; jcritser{at}iupui.edu






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