**5.4 Chilling sensitivity of teleost embryos**

Medaka (*Oryzias latipes*) embryos at early cleavage stage, i.e. 2-4-cell stage, are very sensitive to cooling at 0°C for 40 minutes. Only 38% of the embryos survived the chilling treatment. However, the same chilling treatment did not affect the survival of embryos in early gastrula stage (Valdez et al., 2005). Similarly, zebrafish (*Brachydanio rerio*) at cleavage stage are more sensitive to chilling than embryos at epiboly and at three-somite stage (Hagedorn et al., 1997c). This indicates that embryos at later developmental stages are more resistant to chilling. A similar phenomenon was found on other teleost species such as red sea bream, olive flounder and multicolorfin rainbowfish. In the same study, it was found that cleavage stage embryos responded to chilling by obstructing mitotic division and early gastrula stage embryos responded by delayed development at epiboly (Sasaki et al., 1998).

#### **5.5 Cryoprotectant toxicity on teleost embryos**

Incubation of the three-somite stage zebrafish (*Brachydanio rerio*) embryos in 1.5M DMSO and methanol for 30 minutes at room temperature did not adversely affect their survival. While propylene glycol is moderately toxic to the embryos, similar treatment with ethylene glycol or glycerol is lethal to all treated embryos. Of the cryoprotecting agents tested, ethylene glycol solution specifically led to the blastoderm being dissociated from the yolk (Hagedorn et al., 1997c). A similar phenomenon was observed on 14 to 20-somite stage embryos of *Danio rerio* (Higaki et al., 2010b). Treating the embryos with glycerol at a

The low permeability of the chorion and the perivitelline is evident from retarded permeation of radio-labeled DMSO from the external medium into the embryos by several folds (Harvey et al., 1983). Even after removal of the outermost barrier, the embryos were poorly permeated by cryoprotectants. By chemical shift selective magnetic resonance microscopy and magnetic resonance spectroscopy, kinetics of permeation of cryoprotectants methanol, DMSO and propylene were measured. While methanol can permeate the entire six-somite zebrafish (*Brachydanio rerio*) embryos in 15 minutes, DMSO and propylene are relatively poor in permeating into the embryos when applied to the medium (Hagedorn et at., 1996). Similar findings were obtained by osmometric measurements of volume changes in the embryos tested (Hagedorn et al., 1997c). Also, magnetic resonance imaging on the distribution of cryoprotectants, delivered to the external medium or injected into the yolk, in the three-somite stage *Brachydanio rerio* embryo revealed that the yolk is far less permeable than the blastoderm and the yolk syncytial layer is the major barrier to the cryoprotectants

To artificially promote permeation of cryoprotectants into fish embryos, ultrasound of 175V was used to increase the permeability by methanol to *Danio rerio* 50% epiboly (Wang et al., 2008). A high-intensity femtosecond laser was also used to introduce transient pores on blastomeres and the blastoderm-yolk boundary. Successful delivery of large molecules such as fluorescein isothiocyanate, streptavidin-conjugated quantum dots and DNA plasmid was detected on pec-fin stage (Kohli et al., 2007). Whether the physically induced permeation method can help cryopreservation of the whole teleost embryos requires experimental

Medaka (*Oryzias latipes*) embryos at early cleavage stage, i.e. 2-4-cell stage, are very sensitive to cooling at 0°C for 40 minutes. Only 38% of the embryos survived the chilling treatment. However, the same chilling treatment did not affect the survival of embryos in early gastrula stage (Valdez et al., 2005). Similarly, zebrafish (*Brachydanio rerio*) at cleavage stage are more sensitive to chilling than embryos at epiboly and at three-somite stage (Hagedorn et al., 1997c). This indicates that embryos at later developmental stages are more resistant to chilling. A similar phenomenon was found on other teleost species such as red sea bream, olive flounder and multicolorfin rainbowfish. In the same study, it was found that cleavage stage embryos responded to chilling by obstructing mitotic division and early gastrula stage

Incubation of the three-somite stage zebrafish (*Brachydanio rerio*) embryos in 1.5M DMSO and methanol for 30 minutes at room temperature did not adversely affect their survival. While propylene glycol is moderately toxic to the embryos, similar treatment with ethylene glycol or glycerol is lethal to all treated embryos. Of the cryoprotecting agents tested, ethylene glycol solution specifically led to the blastoderm being dissociated from the yolk (Hagedorn et al., 1997c). A similar phenomenon was observed on 14 to 20-somite stage embryos of *Danio rerio* (Higaki et al., 2010b). Treating the embryos with glycerol at a

embryos responded by delayed development at epiboly (Sasaki et al., 1998).

**5.3 Permeability of teleost embryos** 

(Hagedorn et at., 1996; Hagedorn et al., 1997a).

**5.4 Chilling sensitivity of teleost embryos** 

**5.5 Cryoprotectant toxicity on teleost embryos** 

verification.

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 (Higaki et al., 2010b).
