Protection of rabbit embryos against fracture damage from freezing and thawing by encapsulation in calcium alginate gel

Abstract

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.

Hydrocolloid Coating of Xenopus laevis Embryos

“…Alginate was chosen for this study because its coatings are easy to produce and have been used successfully for many products (Nussinovitch and Kampf, 1993;Nussinovitch and Hershko, 1996;Kampf et al, 2000). Moreover, as can be assumed from the vast experience accumulated from cell-entrapment experiments, alginate gels maintain cell viability (Lim and Sun, 1980;Kojima et al, 1990). LMP is similar to alginate in its cross-linking mechanism, making a comparison between the two of interest.…”

“…The difference between coating and entrapping is the thickness of the coating layer, being very thin in the former and thick in the latter. Taking this definition into account, it seems that most, if not all, reports on coating deal, in fact, with entrapment within a gel matrix (Kojima et al, 1990;Redenbaugh et al, 1986;Hatanaka et al, 1994).…”

The use of polymers for coating of cells

“…Cryopreservation combined with the method used by Willadsen results in higher survival percentages of these embryos [41]. Embedded rabbit embryos were also examined for their ability to survive after one cycle of freezing and thawing [42]. All the above embedding systems are actually describing an embedding of a multi-cell organism, since the process was performed at a late stage of the embryo.…”

Alginate Coating of Xenopus laevis Embryos

“…The difference between coating and entrapping is the thickness of the coating layer, being very thin in the former and thick in the latter. Taking this definition into account, it seems that most if not all reports on coating are in fact describing cell entrapment within a gel matrix (Kojima et al, 1990; Redenbaugh et al, 1986; Hatanaka et al, 1994). To obtain a true coating, special microcoating procedures need to be invented, or at least as a start, attempts need to be made with very large cells.…”