Cryoprotective effects of antifreeze proteins delivered into zebrafish embryos

Fish embryo cryopreservation, which is useful in aquaculture or biodiversity conservation, is still far from being achieved. Structural barriers reduce the entrance of cryoprotectants into embryo compartments. Previous studies demonstrated a better ability for freezing in Arctic species which naturally express antifreeze proteins (AFPs). In this study, AFPs were delivered in early zebrafish embryos by incubation in media containing protein. Their cryoprotective effects were then analyzed. Chilling sensitivity was evaluated at 4 degrees C and -10 degrees C. Survival rates significantly increased in embryos incorporating AFPI and kept at -10 degrees C. To analyze their effects on cryopreservation, 5-somite embryos were vitrified. Incorporation of AFPI reduced the percentage of embryos that collapsed at thawing (14.2% of AFPI-treated embryos and 48.9% of controls). Cellular damage caused by vitrification was assessed after thawing by cell dissociation and further analysis of cell survival in culture (SYBR-14/IP labeling). The percentage of viable cells at thawing ranged from 25 to 50%, considered incompatible with embryo development. Cells recovered from frozen-control embryos did not survive in culture. However, the incorporation of AFPs allowed survival similar to that of cells recovered from non-frozen embryos. Blastomere cryopreservation trials incorporating AFPI in the extender also demonstrated a significant increase in viability after freezing. Our findings demonstrated that delivery of AFPs into zebrafish embryos by incubation in media containing protein at early stages is a simple and harmless method that increases cryoprotection of the cellular compartment. This beneficial effect is also noticed in blastomeres, encouraging their use in further protocols for embryo cryopreservation.