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A. Széll and J. N. Shelton

Summary. The time requirements for permeation by glycerol and dehydration by sucrose before rapid freezing of Day-3 mouse embryos by direct transfer to − 180°C were studied. When the embryos were equilibrated in 2·0, 3·0, or 4·0 m-glycerol + 0·25 m-sucrose for 2·5 to 40 min, the post-thaw viability increased (P < 0·001) with the length of equilibration period at 4°C. At 20°C the volume of embryos increased with the duration of equilibration up to 20 min (P < 0·001), but the post-thaw viability was not affected.

The effect of equilibration in glycerol–sucrose was determined at 20°C for embryos which were previously permeated by glycerol, dehydrated by sucrose or left in PBS + 5% FCS. The survival of previously permeated embryos was not affected by equilibration for 1–16 min in glycerol–sucrose. The maximum survival rate was attained after shorter equilibration in glycerol–sucrose for embryos without pretreatment (4 min) than for those previously dehydrated (8 min).

It is concluded that increases in the intracellular glycerol level are beneficial for the viability of rapidly frozen mouse embryos and previous or concommitant exposure to sucrose unfavourably affects glycerol permeation.

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A. Széll and J. N. Shelton

Summary. The toxic effects of sucrose and the conditions of in-straw glycerol removal after freezing and thawing were studied using Day-3 mouse embryos. At 20°C, exposure to ≤1·0 m-sucrose for periods up to 30 min had no adverse effects on freshly collected embryos. At 25 and 36°C, however, ≥ 1·0 m-sucrose significantly reduced the developmental potential (P < 0·001).

In the freezing experiments the embryos were placed in 0·5 ml straws containing 40 μl freezing medium separated by an air bubble from 440 μl sucrose solution. The straws were frozen rapidly in the vapour about 1 cm above the surface of liquid nitrogen. The post-thaw viability was substantially better after sucrose dilution at 20°C than at 36°C. Mixing the freezing medium with the sucrose diluent immediately after thawing further improved the rate of survival relative to mixing just before freezing (P < 0·001). The best survival was obtained when the freezing medium contained 3·0 M-glycerol + 0·25 m-sucrose; it was mixed with the diluent after thawing and the glycerol was removed at 20°C. Under such conditions the sucrose concentration in the diluent had no significant effect on the rate of development (0·5 m, 69%; 1·0 m, 73%; 1·5 m, 64%).

The results show that during sucrose dilution the temperature should be strictly controlled and suggest that intracellular and extracellular concentrations of glycerol are important in the cryoprotection of embryos.