Sucrose dilution of frozen mouse embryos: interaction of glycerol and sucrose concentrations.

Several concentrations of glycerol for cryoprotection and several concentrations of sucrose for cryoprotectant dilution were examined with frozen, thawed and cultured mouse embryos. Four hundred and eighty late morulae to early blastocyst stage embryos were collected from 35 superovulated mice (B6D2 x Swiss Webster crosses back-crossed to Swiss Webster males) 3-1/2 days after breeding. The embryos were transferred through increasing concentrations of glycerol in modified Dulbecco's phosphate buffered saline (MDPBS) to reach three final concentrations of 1.0 M, 1.4 M and 1.8 M. The embryos were loaded in 0.5-ml French straws appropriately filled with the cryoprotectant and sucrose solutions for each treatment. The straws were cooled with a standard fast-freezing program to -35 degrees C, then plunged into liquid nitrogen. After 58 days of storage at -196 degrees C the straws were thawed in a 37 degrees C water bath. Cryoprotectant dilution was accomplished with a standard step-wise procedure or in the straw with one of three concentrations of sucrose solution (0.25 M, 0.5 M, 1.0 M) in MDPBS. The embryos were then washed twice in MDPBS, twice in Whitten's media for embryo culture and then placed in microdrops of Whitten's media under paraffin oil in a water saturated 5% CO2 in air atmosphere at 37 degrees C. Embryos were observed 24 hours later for development to the expanded blastocyst stage. The proportion of embryos developing in vitro from the three glycerol concentrations were not significantly different with standard step-wise dilution procedures for glycerol removal. After step-wise cryoprotectant removal, blastocyst expansion occurred in 49%, 44% and 52% of embryos frozen in 1.0 M, 1.4 M and 1.8 M glycerol, respectively. The 1.0 M sucrose dilution of 1.0 M glycerol showed the highest development (60.5%) in vitro but was not significantly different from any of these three step-wise diluted glycerol concentrations. The step-wise dilution of the three glycerol concentrations and dilution of the 1.0 M glycerol and 1.0 M sucrose were all superior (P<0.01) to any other dilution procedure examined.