Zebrafish (Danio rerio) is a popular model species for human disease, but also aquaculture. A series of experiments was conducted to assess the success of applying mitotic physical shocks, heat and pressure, to zebrafish and pearl danio (D. albolineatus) with the aim of producing viable tetraploids.
Six trials addressed the effect of post-fertilization and incubation temperature at 28 °C (high) and two trials were conducted at 22.3 °C (low). Heat shocks of 41.4 °C and pressure shocks of 7000 PSI were applied for a duration of 2 min at various times (minutes) post activation (mpa). Shocks were applied between 2 and 60 mpa for trials with high fertilization temperatures and between 23 and 83 mpa for trials with low fertilization temperatures. Samples of eggs were collected and preserved in a solution of 5% acetic acid and Ringer’s solution corresponding to the initiation of each shock attempted to assess the overall developmental stage of embryos. Fertilization, 24-hour, and hatch percentages were recorded for each treatment. At 4 days post fertilization (dpf) larvae were sampled for flow cytometry to assess ploidy. Survivors of groups which were identified as possessing ploidy deviating from diploid in 4 dpf samples were reared until fish were large enough for fin clips to be collected for reassessment of ploidy via flow cytometry.
Fluorocytometric analysis identified that tetraploids, mosaics, and triploids were produced by application of these physical shocks for both species. Heat shocks initiated between 48 and 56 mpa at high incubation temperature or between 72.5 and 83 mpa at low temperature, resulted in the highest proportion of tetraploid zebrafish identified from 4 dpf larvae. However, these shocked groups also suffered from decreasing rates of survival to hatching as the time to shock initiation increased. Samples of eggs taken during at the initiation time of each physical shock revealed that embryos were all at the 2-cell stage with a small portion beginning to transition to 4-cell stage. Only diploids were produced for pearl danio which were incubated at low temperature. No tetraploids were identified from fish which survived to 21dpf.