Studies on Chilling Sensitivity of Zebrafish ( Brachydanio rerio) Embryos

Abstract

Two related studies on chilling and nonfreezing storage of zebrafish ( Brachydanio rerio) embryos were undertaken. In the first part of the investigation, 10 developmental stages of zebrafish embryos were studied for chilling sensitivity. Early developmental stages were the most sensitive to chilling, with approximately 50% of 3-, 7-, 15-, 20-, and 27-h (heartbeat-stage) embryos being killed by exposure to 0°C for periods of 0.2, 4, 14, 16, and 20 h, respectively. Heartbeat-stage embryos tolerated a temperature of 0°C for up to 10 h without adverse effect, but embryo survival was significantly reduced at subzero temperatures. While 27- to 40-h stage embryos showed similar sensitivity to chilling with 56.9 ± 12.1 to 54.5 ± 5.5% survival, 45- to 49-h (prehatch) stage embryos showed increased sensitivity with 28.4 ± 6.3 to 11.7 ± 4.6% survival after 18 h at 0°C. In the second part of the investigation, the cryoprotective effects of various media on chilling sensitivity were studied. The presence of sucrose or trehalose slightly enhanced cooling tolerance of the embryos. Methanol proved to be an optimal cryoprotectant for nonfreezing storage of embryos at zero and subzero temperatures. The optimal methanol concentration, when supplemented with sucrose (0.1 M), was temperature dependent, being 1 M at 0°C, 2 M at - 5°C, 3 M at -10°C, and 5 M at - 15°C, with embryo survival of 30.2 ± 3.5, 28.6 ± 5.8, 27.3 ± 12.1, and 14.3 ± 4.2% after storage for 48, 24, 6, and 1 h, respectively. Survival of supercooled embryos decreased with storage time and temperature. No embryo survived exposure for 72 h at 0°C or 1 h at -20°C. The loss of embryo viability may be related to chilling injury, cryoprotectant toxicity, osmotic stress, or other factors.