PROTECTION OF RABBIT EMBRYOS AGAINST FRACTURE DAMAGE FROM FREEZING AND THAWING BY ENCAPSULATION IN CALCIUM ALGINATE GEL

Avoidance of fracture damage to the zona pellucida during freezing and thawing is essential for the successful freezing of rabbit embryos. The aim of this study was to examine the effects of encapsulation of rabbit embryos in calcium alginate gel, prior to freezing, on damage to the non‐cellular components (zona and mucin coat) caused by freezing and thawing, and on the survival of embryos after thawing in vivo as well as in vitro. Morulae immersed in 2% sodium alginate in Ringer solution were aspirated into a Pasteur pipette. The contents of the pipette were then ejected into a 110 mM solution of calcium chloride. After 30 min, 1‐ to 2‐mm long semi‐solid cylinders, each including one embryo, were cut off with a surgical blade. In the presence of 11% dimethyl sulfoxide, the encapsulated embryos were cooled by the standard method and stored in liquid nitrogen. After rapid thawing, the alginate gel was mechanically removed from around the embryos, and the embryos were morphologically evaluated and allocated to different groups for examination of their development in vitro or in vivo. The percentage of embryos with an intact zona was higher in the encapsulated group than in the non‐encapsulated group (95.3% vs. 76.5%, P < 0.001). The encapsulation markedly reduced the occurrence of damage to the mucin coat (from 43.1% to 8.5%, P < 0.001). The frozen and thawed embryos that were released from alginate gel developed normally in vitro (86.3%) and in vivo (54.2%). The results suggest that encapsulation of rabbit embryos in alginate gel prior to freezing improves the percentage of transferable embryos after thawing and increases the number of offspring that originate from frozen and thawed embryos. Copyright © 1990 Wiley‐Liss, Inc., A Wiley Company