Ultrasound enhanced permeation of methanol into zebrafish, Danio rerio, embryos

Only vitrification is likely to result in successful cryopreservation of teleost embryos, requiring a high level of cryoprotectant penetration into the cell. This study determined the effect of ultrasound on methanol (MeOH) penetration in three types (dechorionated, soft chorion and intact embryos) and three stages (90% epiboly, bud and 4-somite stages) of zebrafish (Danio rerio) embryos. Three types of embryos were first exposed to varying levels of methanol and optimized for survival. Dechorionated embryos were then exposed to 20%, soft chorion embryos were exposed to 30% and the intact embryos were exposed to 40% methanol for 3 min with or without ultrasound treatment. High performance liquid chromatography (HPLC) was used to measure methanol levels in the treated embryos. The total MeOH recovery from zebrafish embryo samples spiked with MeOH was 100.62 ± 3.99%. The overall penetration of methanol into zebrafish embryo ranged from 85.3 ± 8.1 μmol (in soft chorion embryos at 90% epiboly stage treated with ultrasound) to 3.1 ± 1.0 μmol (in dechorionated embryos at 90% epiboly stage treated without ultrasound). Ultrasound clearly helped to improve the permeation level of methanol into zebrafish embryos (p < 0.05). A high level of methanol was found in soft chorion embryos. Dechorionated embryos had the highest relative difference in methanol levels between ultrasound and controls. Although, ultrasound significantly affected survival (p < 0.05), survival was generally high (64 ± 2% to 100%). Even the highest level of methanol (85.3 ± 8.1 μmol) in the best treatment was far lower than that needed for vitrification. Further studies using novel and non-invasive methods are needed to achieve greater methanol penetration for successful vitrification.