Plating larval zebrafish prior to the day of experimentation has no impact on spontaneous swimming and startle responses.

Behavioural testing in larval zebrafish often involves pipetting the larvae into well plates for individual testing. Transferring larvae into plates the day prior to experimentation can increase efficiency of testing. Furthermore, pharmacological and toxicological studies can require a prolonged dosing period requiring the larvae to be pre-plated into the well plate the day prior to experimentation. Here, we compared the behavioural impact of pre-plating larval zebrafish at 4 days post-fertilization (dpf) to fish transferred at 5 dpf on the day of testing. Motion-tracking software was used to examine locomotion and zone preference, and responses to light, dark, and mechanical startle stimuli. We found no significant differences in distance moved, time spent in the thigmotaxis zone (outside edge of the arena), high mobility, immobility, light startle, dark startle, and mechanical startle responses. This data suggests that pre-plating larval zebrafish one day prior to testing does not have a significant impact on behaviour in a spontaneous swim task, dark startle test, light startle test, or mechanical startle test. Pre-plating larval zebrafish can increase the efficiency of behavioural testing.•Compare plating larvae one day prior to testing to plating day of testing.•Test the behaviour in a spontaneous swimming test, and measure light-, dark-, and mechanical-startle responses.•There were no significant differences in locomotion or startle responses.