Survival of mouse embryos frozen and thawed rapidly

Abstract

Mouse morulae were frozen rapidly to -196 degrees C in the presence of 1.0-2.5 M-DMSO by a 3-step procedure; the samples were seeded at -4 to -8 degrees C, held at -20 degrees C in an ethanol bath for 10 min, suspended over liquid nitrogen at approximately -100 degrees C for 10 min and then plunged directly into liquid nitrogen at -196 degrees C. The cooling rate between -20 and -75 degrees C was approximately 17 degrees 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 degrees C/min) than after slow thawing (25 degrees 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 + 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 degrees 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.