Role of equilibration before rapid freezing of mouse embryos

Abstract

The time requirements for permeation by glycerol and dehydration by sucrose before rapid freezing of Day-3 mouse embryos by direct transfer to -180.degree. 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.degree. C. At 20.degree. 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.degree. 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 unfavorably affects glycerol permeation.