Microinjection of the antifreeze protein type III (AFPIII) in turbot (Scophthalmus maximus) embryos: Toxicity and protein distribution

Fish embryo cryopreservation has not been achieved. Different methods and alternative cryoprotective agents (CPAs) should be explored in order to succeed in this purpose. Antifreeze proteins (AFPs) are naturally expressed in sub-arctic fish species, and they inhibit the growth of ice crystals as well as recrystallization during thawing. Therefore, their introduction into embryos can be highly beneficial for vitrification purposes. In this study, AFP type III was introduced into turbot embryos, by microinjection into the yolk sac and the perivitelline space at F stage (tail bud).Toxicity and distribution of protein in microinjected embryos were established before testing the protein effect on embryo cryopreservation. AFP-FITC distribution within the embryo was analyzed by confocal microscopy at 5 min and 24 h after microinjection in F stage embryos. To test the sensitivity of microinjected embryos to CPAs, embryos were subjected to a protocol for the incorporation of a vitrifying solution that was specially designed for turbot embryos. Hatching rates after CPA incorporation were determined. Results indicate that embryos at late developmental stages are more resilient to microinjection, with embryo survival rates between 60 and 82%. Confocal microscopic images demonstrated that the protein was homogeneously distributed within the microinjected embryo compartment, but did not enter any other compartment. On the other hand, microinjected embryos successfully surmounted their incubation in the CPAs. This study explores new alternatives for cryopreservation suggesting the use of natural cryoprotectants (AFPs) in the protection of intra-embryo compartments, which are usually unprotected with the conventional cryopreservation protocols for fish embryos.