**5.7 Alternatives for whole embryo cryopreservation**

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.

Cryopreservation of Embryos from Model Animals and Human 279

recipient blastulae have to be sterilized, e.g., by injecting a *dead end* antisense morpholino (Ciruna et al., 2002). This demands additional procedures in the entire cryopreservation/recovery cycle, making the primordial germ cell-base approach less attractive than the existing sperm cryopreservation. Delivery of antifreezing proteins to directly minimize water crystal formation or aquaporins to increase permeability to cryoprotectants and movement of water (Chauvigne et al., 2011) through transgenesis

Successful cryopreservation relies on a number of conditions and properties of the embryos or larvae to be fulfilled. The conditions, which may be interdependent on each other, are (1) the chilling sensitivity of the embryos/larvae; (2) the permeability of the embryos/larvae to cryoprotectant and water; and (3) the sensitivity of the embryos/larvae to the cryoprotectant toxicity. The permeability of the embryos/larvae can be a function of size and structural heterogeneity. The toxicity of the cryoprotectant to the embryos/larvae can be a function of permeability at a particular developmental stage. Although a cryopreservation protocol can be as simple as slow freezing *Caenorhabditis elegans* in 15% glycerol, most of the other organisms require extensive optimization before being cryopreserved efficiently. Understanding the behavior of the interacting conditions can help initiate the development

*Chilling injury* We learned from classical model organisms that chilling sensitivity coupled with a slow cooling procedure could be detrimental and vitrification can be a shortcut or even a better starting point to achieve the same goal. Vitrification of highly chilling-sensitive insect embryos is an excellent example. On the other hand, we have to keep in mind that vitrification requires a relatively high concentration of permeating cryoprotectant(s). If a new model organism to be cryopreserved is highly sensitive to the cryoprotectant(s) and has relatively low permeability, vitrification may not be feasible. Slow cooling, which requires a lower concentration of cryoprotectant, thus also allowing longer time for permeation, may

*Permeability* The permeability of a sample towards cryoprotectants is the major barrier to cryopreservation of *Drosophila melanogaster*, *Danio rerio* and probably some other model organisms. Understanding the complexity and structural properties of the embryo/larvae can make cryopreservation possible by developing a corresponding strategy to manage the flow of cryoprotecting agents and water at will. Although the studies on *Danio rerio* embryo complexity and development did not bring about successful cryopreservation of the whole embryo, they helped development of alternatives for cryopreserving diploidic germplasms. Cryopreservation of blastomeres and primordial germ cells using the optimized conditions

*Toxicity of cryoprotectants* Knowing the toxicity of cryoprotectants at different developmental stages of an organism is critical in determining the combination of cryoprotectants with embryonic/larval stages to be chosen for effective cryopreservation during protocol development. For example, glycerol and dimethylformamide are very toxic to fish embryos

leads to generation of germline-transmitting chimera after transplantation of cells.

and mammalian morulae, respectively (Higaki et al., 2010b; Kasai et al., 1981).

similarly complicate the cryopreservation procedures.

of cryopreservation of other model animals.

**6. Concluding remarks** 

be considered.

Another alternative diploid material often sought to be cryopreserved is the primordial germ cells. Compared with the blastomere, primordial germ cells are developmentally closer to the cell type to be differentiated *in vivo*, i.e. the germ cells. The first success in transplantation of primordial germ cells was demonstrated on rainbow trout (*Oncorhynchus mykiss*), a model with a relatively larger body size. Green fluorescent protein expressing primordial germ cells isolated from the genital ridge of hatchlings were injected into the peritoneal cavities of a wild type hatchling. The maker-labeled primordial germ cells were able to colonize the genital ridge of the recipient animal and transmit the donor characteristic to the next generation through sperm and eggs at a rate of up to about 4% (Takeuchi et al., 2003).

A similar operation in the smaller teleost species such as zebrafish is more challenging. A single primordial germ cell isolated from the pearl *Danio* (*Danio albolineatus*) at ten- to fifteen-somite stage was transplanted into the marginal region of each zebrafish (*Danio rerio*) embryo at the blastula stage and *vice versa*. The development of host germ cells was prevented in advance by injection of an antisense *dead end* morpholino oligonucleotide at an earlier embryonic stage (Slanchev et al., 2005). In the host, the transplanted primordial germ cell developed into a single gonad, making the animal regain fertility and transmit the donor genotype to the progenies. This complete germline replacement procedure can be applied to both goldfish (*Carassius auratus*) and loach (*Misgurnus anguillicaudatus*) (Saito et al., 2008). The success of these cases suggests that cryopreservation of primordial germ cells is a feasible approach to preserve the diploid germplasm. As the reservoirs of primordial germ cells, genital ridges from Rainbow trout (*Oncorhynchus mykiss*) embryos were cryopreserved by cooling in dry ice and then liquid nitrogen after treating with 1.8M ethylene glycol. About 51% of primordial germ cells survived. Fifteen to twenty surviving primordial germ cells were transplanted to the peritoneal cavity of each newly hatched animal. Germline transmission of the donor genotype could be found in 7.8% of the hosts and the germline transmission frequency was from 0.1 to 13.5%. (Kobayashi et al., 2007).

Later, Higaki et al. (2010b) vitrified whole zebrafish (*Danio rerio*) embryos at 14- to 20-somite stage with an optimized vitrification solution to cryopreserve primordial germ cells. With the use of 3M ethylene glycol and 0.5M sucrose, about 4 primordial germ cells, about 40% of all, survived in each embryo, after thawing. To increase cryo-survival, yolk-removed zebrafish (*Danio rerio*) embryos were vitrified in 20% ethylene glycol, 20% DMSO and 0.5M sucrose. Up to 90% live primordial germ cells were obtained. Half of the primordial germ cells retained pseudopodial movement. After transplanting the motile primordial germ cells into sterilized golden-type zebrafish blastulae, about 2.8% of the recipients developed normally and produced progenies with the donor's genotype (Higaki et al., 2010a).

Unless there is a breakthrough in cryopreserving and recovering whole fish embryos, cryopreservation of blastomeres or primordial germ cells seem to be the only methods for cryopreserving the fish diploid germplasm. Blastomeres may have advantages over primordial germ cells in generating germline transmitting chimera. Firstly, identification and isolation of primordial germ cells relies on a readily observable transgenic marker (Higaki et al., 2010b; Kobayashi et al., 2007). Breeding of a strain to a marker transgenic strain or freshly injecting DNA constructs is required before cryopreservation procedures, making the procedures more complicated. Removal of the marker from the recovered animals may also be required in some applications. Secondly, for germ-line replacement, the recipient blastulae have to be sterilized, e.g., by injecting a *dead end* antisense morpholino (Ciruna et al., 2002). This demands additional procedures in the entire cryopreservation/recovery cycle, making the primordial germ cell-base approach less attractive than the existing sperm cryopreservation. Delivery of antifreezing proteins to directly minimize water crystal formation or aquaporins to increase permeability to cryoprotectants and movement of water (Chauvigne et al., 2011) through transgenesis similarly complicate the cryopreservation procedures.
