Cryoprotectant penetration and supercooling in the eggs of salmonid fishes

Abstract

1. 1. The freezing point of isolated, unfertilized eggs of rainbow trout ( Salmo gairdneri) is −1.7 °C. 2. 2. Experiments using radiolabeled cryoprotectants show that the degree of permeation of methanol, dimethyl sulfoxide, and glycerol is inversely proportional to the molecular weights of the compounds; glycerol does not penetrate the egg, while methanol, which penetrates with the greatest rapidity, achieves no more than 23% of the expected equilibrium concentration after 2 hr exposure at 0 °C. 3. 3. Eggs treated with 10% DMSO and 10% sucrose in Fish Ringer resist intracellular freezing longer when cooled at 0.01 °C/min than do those in Fish Ringer alone; intracellular freezing is, however, inevitable with both cryoprotectants, although eggs that have remained unfrozen for several hours appear viable upon slow rewarming. These results make it likely that earlier studies reporting survival of “cryopreserved eggs” have in fact been dealing with supercooled eggs. 4. 4. Scanning electron micrographs of cooled and frozen DMSO-treated eggs show a progressive deterioration of the outermost layer of the zona radiata following cooling and freezing, and suggest that intracellular freezing is occurring following nucleation by way of pore canals in the zona that are exposed as cooling progresses.