Abstract
Preimplantation stage (16-celled and morula) rabbit embryos were successfully frozen to-196°C. The cooling rate (from a room temperature to 0°C), the presence of the mucin layer surrounding embryos, the ice-seeding treatment and the thawing procedure were examined to determine their effects on the survival of the frozen embryos of Japanese white, New Zealand white and Dutch-Belted rabbits. A high proportion (51%; 16-celled, 69%; morula) of Dutch-Belted rabbit embryos developedin vitro, when they were frozen to-196°C, applying the ice-seeding at-4°C in the presence of 12.5% DMSO, after being cooled to 0°C at the rate of 7-9°C/min, and were diluted by a stepwise addition of 4 different strength PBS on thawing. The highest rate ofin vitrodevelopment (81%; Japanese white, 75%; New Zealand white, 82%; Dutch Belted embryos) was obtained when the morula stage embryos were frozen to-196°C applying seeding at-4°C after being cooled to 0°C at the rate of 1°C/2.5 min and were diluted, on thawing, by stepwise addition of 6, 3 and l% DMSO solution and a culture medium. No great difference was found in the survival rate between the embryos covered with the mucin layer and those which had not the coat. All the embryos frozen without applying seeding treatment failed to develop in vitro after being thawed and diluted. Nine out of 27 does each of which received 6 reimplantations of the embryos frozen-thawed became pregnant and were found to be carrying 37 normal fetuses on the 12 th day of pregnancy.
