Survival of mouse embryos frozen and thawed rapidly

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Abstract
Mouse morulae were frozen rapidly to -196.degree. C in the presence of 1.0-2.5 M-DMSO [dimethyl sulfoxide, a cytoprotective agent] by a 3-step procedure; the samples were seeded at -4 to -8.degree. C, held at -20.degree. C in an ethanol bath for 10 min, suspended over liquid N at .apprx. 100.degree. C for 10 min and then plunged directly into liquid N at -196.degree. C. The cooling rate between -20 and -75.degree. C was .apprx. 17.degree. C/min. In all concentrations of DMSO significantly higher proportions of embryos developed to fully expanded blastocysts after 48 h in culture after rapid thawing (360.degree. C/min) than after slow thawing (25.degree. C/min). The highest survival rates were obtained for the embryos frozen rapidly in the presence of 1.5 and 2.0 M-DMSO (36 and 53%, respectively). Various methods for removal of DMSO (2.0 M) were tested with the 3-step freezing and rapid thawing procedures. The best results for development to fully expanded blastocysts were obtained with PBS [phosphate-buffered saline] + 2.0 M DMSO + 0.5 M-sucrose (2 min), followed by PBS + 0.5 M-sucrose (2 min) at room temperature (82%) and with stepwise dilution in PBS at 30.degree. C (70%). When 26 embryos developed to blastocysts in culture after rapid freezing and thawing were transferred into 2 recipients; 11 newborn young (42%) were obtained.