Developmental Toxicity Assessment in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 16

treatment as early as possible. Due to the presence of unfertilized eggs and the time
required to select healthy, synchronized eggs prior to compound treatment, the stage
for compound treatment is ordinarily 6hpf and not later than 24hpf.
Since spontaneous mutation and death can take place between 6 and 24hpf,
selecting healthy, synchronized embryos is crucial for establishing a valid devel-
opmental toxicity screen. The earliest stage for distinguishing healthy, synchronized
embryos is 4hpf, leaving a 2 h window for selecting embryos prior to compound
treatment. Figure 2.1 illustrates typical phenotypes of 4hpf embryos observed in a
pool of several clutches. Normal 4hpf embryos that exhibit intact chorion membranes
(Fig. 2.1a and b) are selected and reexamined 2 h later; normal chorionated 6hpf
phenotypes (Fig. 2.1c) are then used for compound treatment. Normal fertilized
embryos are pooled for random selection for subsequent compound treatment.
Six-well microplates are typically used for treating 30 embryos simultaneously
with 4mL compound solution in which embryos are incubated at 28 C in the dark
for 114 h.
Figure 2.1 Phenotypes of normal and abnormal 4 and 6hpf zebrafish embryos. (a and b)
Normal 4hpf embryos. (c) Normal 6hpf embryo. (d-i) Abnormal 4hpf embryos.