High in vitro and in vivo survival of day 3 mouse embryos vitrified or frozen in a non-toxic solution of glycerol and albumin.
A vitrification solution consisting of 6.5 mol glycerol l-1 and 6% (w/v) BSA in a modified Dulbecco's PBS (designated solution VS3a) was examined for the cryopreservation of 8-12-cell mouse embryos. Solution VS3a vitrified when cooled to -196 degrees C at rates of 10-2500 degrees C min-1 and vitrified suspensions did not crystallize when warmed at 200 or 2000 degrees C min-1. However, slow cooling at 5 degrees C min-1 or slow warming at 20 degrees C min-1 resulted in visible crystallization of solution VS3a. Embryos were equilibrated in solution VS3a in three steps at room temperature and placed into a 0.25 ml plastic straw in a way that permitted in-straw dilution with 1 mol sucrose l-1. Embryos equilibrated in solution VS3a and diluted immediately exhibited high rates of development in vitro to blastocysts (> 90%) if the total time of exposure to 100% solution VS3a did not exceed 5 min. Embryos exhibited high rates of development in vitro (75-97%) when equilibrated in 100% solution VS3a for 1 min and then cryopreserved using all combinations of three rates of cooling (5200 or 2500 degrees C min-1) and three rates of warming (20,000 or 2000 degrees C min-1). Although embryo suspensions visibly crystallized during slow cooling at 5 degrees C min-1, the rate of cooling was not a significant source of variance (P > 0.26). However, the rate of warming was found to have a small but significant effect on embryo survival (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Veterinary Resources Program, National Center for Research Resources, National Institutes of Health, Bethesda, MD 20892.
W F
WF
Rall
M J
MJ
Wood
D000818:Animals; D001755:Blastocyst; D015925:Cryopreservation / Q000379:methods*; D003470:Culture Media; D004624:Embryo Transfer; D005328:Fetal Viability; D017110:Freeze Substitution / Q000379:methods*; D005990:Glycerol; D051379:Mice; D008813:Mice, Inbred ICR; D012710:Serum Albumin, Bovine
D003470:Culture Media; D012710:Serum Albumin, Bovine; D005990:Glycerol