Studies on Cryopreservation of Mammalian Embryos -Induction of Ice Formation with Droplets of Silver Iodide Immobilized in Alginate Gel-

Abstract

The aim of the present study was to develop an automatic and reliable ice nucleation method by using silver iodide (AgI) as the ice nucleating agent. The effect of the introduction of ice nucleation by AgI was examined by observing the temperature rise due to the release of latent heat of fusion at temperatures where spontaneous freezing did not yet occur. AgI in distilled water was loaded into a plastic straw, and was separated from the freezing solution by air bubbles. By using a copper-constantan thermocouple to measure temperature of latent heat released, it was confirmed that AgI could be used for effective ice nucleation in freezing solutions. Rabbit morulae and bovine Day 7 to 8 embryos were frozen by the AgI seeding method. It became clear that AgI was an effective ice nucleating agent for freezing rabbit and bovine embryos. The original method was improved for practical application by immobilizing AgI in alginate gel. This method makes it possible to immerse AgI with embryos in any kind of freezing vessels. The reliability of seeding increased when this method was used, judging from the high post thaw survival of the bovine embryos. The process of ice crystal growth germinating from AgI alginate gel droplets was observed under a cryomicroscope. The droplets suspended in a solution containing 10% glycerol or 11% dimethyl sulfoxide germinated ice crystals at approximately -8°C. The start of freezing in the straw used to be detected indirectly with a thermocouple as a temperature increase due to the emission of the latent heat. Thus, it was uncertain whether the freezing was induced through the aid of AgI alginate gel droplets or not. This problem appeared to be clarified by the cryomicroscopic observation.