Applied research of frozen preservation for mammalian embryo.

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A micro-computer controlled freeze-thaw apparatus for embryo was developed. The apparatus can be freely programmed to choose the freezing temperature and the ice seeding can be smoothly performed with temperature increase held to within 1.0゜C.The effects of various cryoprotective agents namely dimethyl sulfoxide, methyl alcohol, ethylene glycol, glycerol, erythritol, adonitol and xylitol on the survival of frozen-thawed mouse embryos were examined. The effects of the stage of development and the dilution method of cryoprotective agent were also examined. Cryoprotective agent was added to the embryos in 5 equal increments 5 minutes apart, giving a final concentration of 1.5 M. Embryos were then loaded in 0.25 ml plastic straws and placed vertically in the cooling chamber of a programmable freezer. The embryos were cooled to -6゜C at 1゜C/min and seeding was induced at -6゜C. After being held for a further 5 minutes at the seeding temperature, the samples were cooled to -35゜C at 0.3゜C/min and then transferred to liquid nitrogen. Embryos in the early stages of development survived better than those in the late stages of development when glycerol was used. However, embryos in the late stages of development survived better when DMSO was used. Morulae and early blastocysts survived best when erythritol and xylitol was used, respectively. Obvious effect of the dilution method was observed when erythritol or xylitol was used. In this study it was observed that the sucrose method was superior to the PBS method especially when erythritol was used.