In this study, the toxicity of sucrose to Oplegnathus punctatus embryos was evaluated. Embryos at the 4-6 somite, tail-bud, heart formation, and heart-beating stages were exposed to 0, 0.5, 1,1.5, 2, 2.5, or 3 M sucrose for 1 h. Survival rates of embryos at the tail-bud, heart formation, and heart-beating stages after rehydration for 1 h were not affected by treatment with 2 M sucrose (the maximum concentration). Embryos at the tail-bud, heart formation, and heart-beating stages were exposed to 2 M sucrose for 0, 30, 60, 90, 120, 150, or 180 min. Long-term developmental indicators, including rates of survival, hatching, swimming, and malformation, were evaluated for 4 days after rehydration. Based on the survival rates 10 min after rehydration, the longest tolerance time for embryos at the three stages was 120 min. Based on long-term developmental indicators, the longest tolerance times were 60 min at the tail-bud, 60 min at the heart formation stage and 30 min at the heart beating stage. The malformation rates increased as the treatment time increased. The malformation rates were 100% when embryos were exposed to sucrose for ≥120 min. Malformation was divided into larval and embryonic abnormality. As the exposure time increased for tail-bud stage embryos, the rate of larval malformation increased. Treatment at heart formation and heart-beating stages resulted in higher rates of failure to hatch at exposure time. Based on these results, toxicity tests of non-permeable cryoprotectant in embryos requires the observation of development for at least 2 days after rehydration. Based on long-term observation, it was concluded that dehydration before freezing was not the direct cause of larvae deformity that hatched from frozen-thawing embryo. These results provide a reference for the singly use of representative non-permeable cryoprotectant sucrose.
Keywords: Embryo; Malformation; Non-permeable cryoprotectant; Oplegnathus punctatus; Rehydration; Sucrose; Toxicity.
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