**4.4 The success of insect embryo vitrification**

Adopting and modifying the protocol for vitrifying mouse embryos with ethylene glycol (Rall & Fathy, 1985), Steponkus et al. (1990) first demonstrated successful vitrification of 13- 14 hour *Drosophila* embryos. Instead of permeabilizing the vitelline layer with the currently developed method using an alkane, the eggs were "permeabilized" with a medium containing 2.125M ethylene glycol for 20 minutes. The intracellular concentration of ethylene glycol was further increased by dehydrating the embryos in 8.5M ethylene at 0°C for 8 minutes before plunging the embryos into nitrogen slush (-204°C), using a copper electronic microscopic grid as an open carrier (to achieve a cooling rate of about 400°C per second). After thawing, 18% of the eggs hatched and 3% developed into fertile adults. On the contrary, there were no embryos surviving at a lower cooling rate of 15°C per second when using a polypropylene straw as a carrier. The surrounding cryoprotecting solution vitrified *per se* since no crystals were detected by differential scanning calorimetry. The high lethality is probably due to the suboptimal permeabilization of the vitelline layer by ethylene glycol, leading to a lower concentration of cryoprotectants in the inner part of the multicellular/highly differentiated insect embryos. This may result in crystallization of water in the area with a lower concentration of cryoprotectant which would require a higher cooling rate to induce vitrification. A higher cooling rate was achieved by using a metal grid allowing vitrification to occur at such low concentrations of cryoprotectants, thus partially circumventing this potential permeating defect. Using a similar protocol, other *Dipteral* species such as blowfly (*Lucilia cuprina*, a parasite in sheep) (Leopold & Atkinson, 1999 20), midge (*Culicoides sonorensis*) (Nunamaker & Lockwood, 2001) and screw-worm (Leopold et al., 2001) were reported to be cryopreserved.

On the other hand, Mazur et al. (1992a) made use of the accumulated experience of wax removal by butanol-heptane based procedures to permeabilize the vitelline membrane prior to vitrification. In the optimized procedure, dechorionated *Drosophila* embryos were first permeabilized in 0.3% 1-butanol in n-heptane for 90 seconds. The embryos were then preincubated in 2M ethylene glycol for 30 minutes and then 8.5M ethylene glycol solution containing 10% polyvinylpyrrolidone for 5 minutes at 5°C before rapid cooling, using a filter membrane as an open carrier. The developmental stage of the embryos was also found to be critical for cryo-survival determination. Vitrifying precisely staged 14.5 hour embryos using the above mentioned method resulted in 60% of the cryopreserved embryos hatching and more than 40% of the hatched larvae developed into fertile adults (Mazur et al., 1992a).

The development of cryopreservation on *Drosophila* embryos suggests that permeabilization of the sample to cryoprotectant is the key to success even though the embryos are structurally complex. In nematode larvae, which are also susceptible to being cryopreserved, the organogenesis is even more advanced. This indicates that the body complexity brought about by organogenesis is not associated with the susceptibility of an embryo/larva to be cryopreserved, at least by vitrification. On the other hand, chilling injury can be circumvented by vitrification practically.
