Cryoprotectant microinjection toxicity and chilling sensitivity in gilthead seabream (Sparus aurata) embryos

Cryopreservation of fish embryos requires an optimal distribution of cryoprotectants inside all embryo compartments. Traditional techniques for the incorporation of cryoprotectants (CPAs) have failed to protect all fish compartments, especially the yolk sac which has been considered the principal point of embryo chilling sensitivity. In the present study, microinjection was used to incorporate cryoprotectants into the yolk sac of gilthead seabream (Sparus aurata) embryos at tail bud stage. The effect of microinjection viability, cryoprotectant toxicity and chilling resistance was evaluated through the hatching rate. Larval survival at first feeding was also determined in microinjection viability and cryoprotectant toxicity studies. Permeabilized seabream embryos were microinjected with 2.35 nl dimethyl sulfoxide (Me₂SO), methanol (MeOH), ethylene glycol (EG) (5 M, 10 M and pure) or sucrose (10% and 15%). In a second experiment, 29.5 nl and 154.0 nl of the highest concentration of each cryoprotectant were used in the same embryo stage. To test the effect of microinjected cryoprotectants on embryo chilling resistance, 29.5 nl of pure Me₂SO or 15% sucrose was microinjected into the yolk sac of tail bud stage embryos and then at a later stage, (tail-bud-free), were exposed to 3 M Me₂SO solution at − 10 °C for 30 min. Our results showed that microinjection technique did not affect the viability of tail bud stage embryos as is shown by the high hatching and survival rates. Hatching and larval survival rate at first feeding were not affected with any of the CPAs tested, showing percentages higher than 75% and 90%, respectively, when embryos were microinjected with a smaller quantity of cryoprotectant. Sucrose was the cryoprotectant better tolerated at higher concentration and volume. Cryoprotectant concentration inside the yolk higher than 1.18 M for Me₂SO, 1.5 M for EG and 2 M for methanol decreased the hatching rate. Microinjection allowed the delivery of high concentrations of CPAs into the yolk sac without deleterious effects on the embryo, but did not provide a significant level of protection for the whole embryo against chilling injury.