Vitrification of mouse embryos in two cryoprotectant solutions

The objective of this study was to compare the efficiency of 2 media on the vitrification of mouse compacted morulae, early blastocysts and expanded b lastocysts after equilibration at room temperature or 4 degrees C. Embryos were equilibrated for 10 min in either 25% VS3 (Rall Equilibration Medium,R EM) or 10% glycerol + 20% propylene glycol (Massip Equilibration Medium, ME M) in DPBS at 20 degrees C or 4 degrees C. For vitrification either 100% VS 3 (Rall Vitrification Medium RVM) or 25% glycerol + 25% propylene glycol (M assip Vitrification Medium, MVM) in DPBS was used. Embryos equilibrated at room temperature were loaded in 20 mu L of vitrification media into 250 mu L straws and then immediately (30 sec) plunged into liquid nitrogen (LN2). Alter equilibration at 4 degrees C the embryos were put into straws with 20 mu L of precooled vitrification medium and after 20 min at 4 degrees C the y were plunged-into LN2. Embryos from both groups were thawed in a 20 degre es C water-bath for 20 sec, transferred to 1.0 M sucrose in DPBS for 5 min and then cultured for 24 to 48 h in Whitten's medium at 37 degrees C in 5% CO2 in air. In the groups of embryos prepared for vitrification at room tem perature the survival rate of compact morulae vitrified in RVM was higher t han those vitrified in MVM (65/70, 93% vs 49/74, 66%; P<0.01). No differenc e was found in the survival rate of early blastocysts and expanded blastocy sts vitrified in RVM or MVM (30/83, 36% vs 25/75, 33% and 4/66, 6% vs 4/76, 5%). No difference was found between the survival rate of compact morulae after equilibration with RVM or MVM at 4 degrees C (62/75, 83% vs 52/74, 70 %). Both the early blastocysts and expanded blastocysts equilibrated at 4 d egrees C MVM yielded a higher survival rate than RVM (28/74, 38% and 40/70, 57% vs 4/75, 5% and 4/77, 5%; P<0.01). We conclude that, of the 3 developm ental stages, compact morulae withstand the vitrification process best, and reduction of the temperature prior to plunging into. LN2 is not required. A 10-fold increase in the survival rate of expanded blastocysts can be achi eved using low temperature equilibration (4 degrees C) and MVM. (C) 1999 by Elsevier Science Inc.