**5.2 Complexity of teleost embryos**

Fish embryos are composed of several components with distinct physical properties. They include the highly dynamic cellular part on the animal pole which contributes entirely to the future animal body, the yolk surrounded by the yolk syncytial layer occupying the majority of the early stage embryo and the chorion as the outermost mechanical protective shield. From 40-100% epiboly, the blastoderm and the yolk occupy 18% and 82% of the dechorionated embryo, respectively, in zebrafish (*Brachydanio rerio*). At the six-somite stage, the volume of the blastoderm increases to about 40%, leaving the yolk occupying 60% of the embryo (Hagedorn et al., 1997c). Between the chorion and the embryo is the perivitelline space filled with liquid with a chemical composition virtually identical to the surrounding medium (Rawson et al., 2000). The complexity of the teleost embryos is further increased by the unbalanced partial density of water in different compartments. At the six-somite stage, the blastoderm occupies about 40% of the dechorionated embryos but water constitutes 82% of its volume. Conversely, the yolk occupies about 60% of the volume of the dechorionated embryos but only 42% of this is constituted by water (Hagedorn et al., 1997b). It was estimated that the osmotically inactive volume in the onesomite stage and six-somite stage embryos are 72.9% and 82.6%, respectively (Zhang & Rawson, 1998).

Cryopreservation of Embryos from Model Animals and Human 277

concentration of 2M for 30 minutes did not yield any viable cells in the embryos. The rest of the cryoprotectants tested (including methanol, ethylene glycol, DMSO, propylene glycol and 1,3-butylene glycol) gave a survival rate of between 90 to 100%. Treating the embryos with cryoprotectants at a higher concentration revealed that ethylene glycol is the next most toxic cryoprotectant, after glycerol; it kills all cells in the embryos at a concentration of 3M. In comparison, methanol and DMSO are moderately toxic. Propylene glycol and 1,3 butylene glycol are mildly toxic, killing only 58-78% of cells even at a concentration of 5M

So far, there have been no successful examples of live fish recovery after cryopreservation. The difficulty in controlling the dynamics of cryoprotecting agents and water in the highly structurally complex embryos may be the cause. However, studies have been conducted to assess the degree of protection provided by the cryoprotectant in vitrification. In a study, five-somite stages of turbot and zebrafish embryos were treated for 5 minutes with incremental concentrations of DMSO and then for a total time of 4 minutes in mixtures containing 5M DMSO, 2M methanol and 1M ethylene glycol, before being loaded into plastic straws and plunged into liquid nitrogen for vitrification. Although 50% of the overall glucose-6-phosphate dehydrogenase activity was retained, no embryo hatched after thawing

The yolk and the surrounding syncytial layer were suggested to be a major reservoir of osmotically inactive water and a barrier to permeation of cryoprotectant to the blastoderm. After vitrifying yolk-removed zebrafish (*Danio rerio*) embryos at 14 to 20-somite stage in 20% ethylene glycol, 20% DMSO and 0.5M sucrose, no living embryos were obtained, but 87% of the cells survived after vitrification and up to 90% of the primordial germ cells were viable (Higaki et al., 2010a). Removal of yolk is deleterious to the development of the embryos. Eliminating the solute and water barrier by yolk removal is not the ultimate solution for

cryopreserving fish embryos unless an artificial replacement of yolk is made feasible.

Due to the lack of progress in development of cryopreservation of fish embryos, isolated somatic cells are being explored as a means to preserve diploid genetic materials. The blastomere becomes one of the attractive candidates because of its abundance in embryos and its pluripotent property in the chimeric animals generated by blastomere implantation. The blastomere from genetically pigmented zebrafish embryos at mid-blastula stage were transplanted into an albino recipient embryo of the same developmental stage. In five out of the twenty-eight chimeric fish produced, blastomeres from the donor contributed to the germline, transmitting the pigmented phenotype to the next generation at a frequency of 1% to 40% (Lin et al., 1992). Slowly cooled zebrafish (*Danio rerio*) blastomeres, isolated from 50% epiboly, were cryopreserved with 1.5M DMSO and 0.1M sucrose in 0.25 ml straws by a programmable freezer. A survival rate of 70% was obtained after thawing (Lin et al., 2009). Combining these technologies, the germline transmission of the cryopreserved genetic materials through blastomeres-embryo chimera seems to be possible. More optimization, e.g., the stages from which the blastomeres are to be isolated, is needed to maximize germline transmission and to minimize operations to be conducted in a recovery procedure.

**5.7 Alternatives for whole embryo cryopreservation** 

(Higaki et al., 2010b).

(Robles et al., 2004).

**5.6 Attempts in vitrifying teleost embryos** 
