Survival of zebrafish, Brachydanio rerio (Hamilton-Buchanan), embryo after immersion in methanol and exposure to ultrasound with implications to cryopreservation

摘要

This study examined the viability of embryos after immersion in highly concentrated methanol solutions (40-60%) and exposing embryos to ultrasound to enhance efficient transport of the cryoprotectant. The exposure to ultrasound, methanol concentrations. duration of treatment and the stages of embryonic development was found to have measurable effects on embryo viability. The effect of ultrasound was more evident at high voltage ( > 440 V) settings and at early developmental stages (30 and 60% epiboly stage). Older embryos were more resistant to cryoprotectant toxicity and ultrasound-induced mortality. The high concentration of methanol (60%) was more toxic to embryos than the low concentration (40%). When methanol treatment and ultrasound were applied simultaneously the optimum concentration was found to be 45% methanol (45% survival: P < 0.05) in a 3 min treatment. Although there was no significant difference between the 2 and 3 min treatments, embryos treated for 4 min had a significantly lower survival rate (P < 0.05). These findings provide initial results to select the developmental stage of the embryo, the concentration of methanol for the preparation of a vitrification solution and duration of ultrasound treatment for cryopreservation. Furthermore, it indicates the potential use of ultrasound to enhance the transport of methanol intracellularly with minimum mortality of the developing embryos.