Cryopreservation of rat blastocysts: a comparative study of different cryoprotectants and freezing/thawing methods

Cryopreservation has proved to be a highly successful method for long-term storage of viable embryos. The objective of this study on rat blastocysts was to define conditions for their cryopreservation. Three cryoprotectants, dimethyl sulfoxide, glycerol, and propanediol/sucrose, were compared in two cooling programs (to -30 or -80 degrees C) and two thawing protocols. The cooling was followed by immersion in liquid nitrogen. Programmed thawing was at the rate of 8 degrees C per minute; fast thawing consisted of direct exposure of the frozen embryos to the ambient laboratory temperature. The survival after the freeze/thaw was assessed from the post-thaw embryo morphology and ability to develop into apparently normal offspring in uteri of foster mothers (embryonic survival). The best method for preservation of rat blastocysts proved to be programmed cooling to -80 degrees C followed by fast thawing with glycerol as cryoprotectant (embryonic survival of 28.1%). In all the experimental groups, the proportion of embryos with good to excellent preservation of morphology was high. With dimethyl sulfoxide, after programmed cooling to -80 degrees C, embryonic survival was 9.9% (programmed thawing) and 17.5% (fast thawing). No embryos survived after programmed cooling to -30 degrees C. However, when the cryoprotectant was propanediol/sucrose, no difference was observed between programmed cooling to -80 degrees C with either method of thawing and programmed cooling to -30 degrees C and fast thawing (12.3, 6.2, and 8.0%, respectively).