A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability.
Mouse morulae were exposed to solutions containing 30-50% of permeable agents (ethylene glycol, glycerol, propylene glycol) in modified phosphate-buffered saline (PB1 medium) at 20 degrees C for 20 min. A high percentage of them developed to expanded blastocysts in culture, after exposure to 30% and 40% ethylene glycol (98 and 84%, respectively), or 30% glycerol (88%). Ethylene glycol and glycerol were diluted to 30 and 40% with PB1 medium or with PB1 containing 30% Ficoll or 30% Ficoll + 0.5 M-sucrose, immersed in liquid nitrogen in straws and warmed in 20 degrees C water. Solutions containing 40% of a permeable agent with Ficoll did not crystallize during cooling or warming. Mouse morulae were exposed to 40% ethylene glycol in PB1 medium containing 30% Ficoll (EF) or PB1 medium + 30% Ficoll + 0.5 M-sucrose (EFS) for 5-20 min at 20 degrees C. EFS solution was non-toxic to the embryos during 5 min of exposure. When embryos, equilibrated in EFS solution for 2 or 5 min at 20 degrees C, were vitrified at -196 degrees C and were warmed rapidly, nearly all embryos developed in culture (97-98%), and 51% developed to live young at term after transfer. This method, which results in virtually no decrease in embryonic viability, may be of practical use for embryo preservation.
Kasai
M
M
Laboratory of Animal Science, College of Agriculture, Kochi University, Japan.
Komi
J H
JH
Takakamo
A
A
Tsudera
H
H
Sakurai
T
T
Machida
T
T
D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D003470:Culture Media; D004624:Embryo Transfer; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D005260:Female; D005362:Ficoll; D005990:Glycerol; D051379:Mice; D008813:Mice, Inbred ICR; D019946:Propylene Glycol; D011409:Propylene Glycols; D013395:Sucrose; D014021:Tissue Preservation / Q000379:methods*
D003451:Cryoprotective Agents; D003470:Culture Media; D005026:Ethylene Glycols; D011409:Propylene Glycols; D005362:Ficoll; D013395:Sucrose; D019946:Propylene Glycol; D019855:Ethylene Glycol; D005990:Glycerol