**5.6 Attempts in vitrifying teleost embryos**

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 (Robles et al., 2004).

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.
