Rapid freezing of the mouse blastocyst: effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen.
This study investigates the effects of time and temperature of exposure to a high concentration (4.5 M) of dimethyl sulfoxide (DMSO), glycerol, 1,2-propanediol (PROH), or a mixture of DMSO and glycerol (DG) in a solution containing 0.25 M sucrose, on the survival and development of rapidly frozen mouse blastocysts. Embryos had significantly (P less than 0.01) higher rates of survival and development when exposed to cryoprotectant at 0 degree C compared with room temperature. The time of exposure to cryoprotectant at either 0 degree C or room temperature before being plunged into liquid nitrogen significantly (P less than 0.01) affected the survival and development of frozen-thawed embryos. Survival and development of blastocysts in vitro and in vivo was significantly (P less than 0.05) higher when exposed at 0 degree C for 10 min to DG, DMSO and glycerol than to PROH. It is concluded that, unlike early-cleavage stage embryos, blastocysts need to be equilibrated at a low temperature (0 degree C) with high concentrations of cryoprotectant before rapid freezing. Exposure of blastocysts to 4.5 M cryoprotectant and 0.25 M sucrose at room temperature either was toxic or else markedly reduced their viability after freezing and thawing, depending on the duration of the initial exposure.
Li
R
R
Centre for Early Human Development, Monash Medical Centre, Clayton, Vic., Australia.
Trounson
A
A
D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000502:physiology*; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005260:Female; D005990:Glycerol / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009584:Nitrogen; D019946:Propylene Glycol; D011409:Propylene Glycols / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D003451:Cryoprotective Agents; D011409:Propylene Glycols; D019946:Propylene Glycol; D009584:Nitrogen; D005990:Glycerol; D004121:Dimethyl Sulfoxide