Permeability of Dechorionated One-cell and Six-somite Stage Zebrafish (Brachydanio Rerio) Embryos to Water and Methanol

The permeability of dechorionated one-cell and six-somite stage zebrafish (Brachydanio rerio) embryos to water and the cryoprotectant methanol at 22 degreesC was studied, using real-time video microscopy to determine the volumetric changes of the embryos during cryoprotectant exposure. The equilibrium volumetric behavior of the embryos and the Boyle-van't Hoff relationships were determined using sucrose as a nonpermeating compound. These showed the embryos to behave as nearly ideal osmometers over the range of 253-1724 mOsm, with osmotically inactive volumes of 72.9 and 82.6% for one-cell and six-somite stage embryos, respectively. The Boyle-van't Hoff relationship of the ovary eggs was also determined for comparison and their osmotically inactive volume was 63.9%. The Kedem-Katchalsky parameters of water permeability (Lp), cryoprotectant permeability (Ps), and reflection coefficient (sigma) were determined using DIFFCHAM software. The parameters reported in this study are phenomenological parameters referring to the overall embryo response. The mean values of these parameters were Lp = 0.34 and 0.35 (micrometer/min*atm), Ps = 0.45 and 0.04 (micrometer/s), and sigma = 0.88 and 0.93 for one-cell and six-somite stage embryos, respectively. While the water permeability of the dechorionated zebrafish embryos at different developmental stages remained relatively stable, the permeability to the cryoprotectant methanol (Ps) appeared to decrease during embryo development. The Ps and sigma values for methanol are the first reported for dechorionated fish embryos at these stages.