Cold storage of alarm substance reduces the behavioural stress response of zebrafish ( <i>Danio rerio</i> )

Alarm substance (AS) is widely used in zebrafish (Danio rerio, Hamilton, 1822) antipredator research to elicit an alarm reaction. This alarm reaction consists of stereotypical behaviours, including freezing, erratic swimming and a tendency to dwell at the bottom of the tank (geotaxis). Because the active compound in AS is known to degrade rapidly, many investigators choose to freeze AS until use, whereas others argue for collecting AS freshly the morning of an experimental session. As part of efforts to reduce the number of donor fish necessary for an experiment, we sought to investigate whether AS maintained overnight under cold storage could elicit an equivalent alarm response to AS of equivalent dilution collected freshly. Our results indicate that exposure to AS stored overnight at -20°C elicited a diminished alarm response compared to fresh AS of equivalent dilution. Although frozen AS may still be sufficient for many studies, our results suggest that AS is most potent when collected fresh the morning of experimentation.