Conditioned place avoidance of zebrafish (Danio rerio) to three chemicals used for euthanasia

Zebrafish are increasingly used as a vertebrate model organism for developmental and biomedical research. These fish are commonly euthanized at the end of an experiment with an overdose of tricaine methanesulfonate (TMS), but to date little research has assessed if exposure to this or other agents meets the criteria of a “good death”. Clove oil and metomidate hydrochloride are alternatives to TMS and have been approved for use in several countries. The aim of my thesis was to use a conditioned place avoidance paradigm to compare aversion to TMS, metomidate, and clove oil. Zebrafish showed a natural preference for the light environment (in a 900-s trial, they spend 95% of their time in the light compartment); by exposing them to anaesthetics in the light side of a light-dark box we were able to show a difference in preference after exposure. Conditioned place avoidance was less pronounced for fish exposed to metomidate and clove oil than for TMS; fish exposed to the former reduced the time spent in the preferred light side by 131 ± 68 s and 165 ± 97 s, respectively, versus a reduction of 591 ± 88 s for those exposed to the TMS. Complete rejection, where no attempted entries were made into the light side, were recorded after exposure to anaesthetics. Nine of 17 fish exposed to TMS did not re-enter the previously preferred side, versus 2 of 18 fish and 3 of 16 fish for metomidate and clove oil, respectively. These results suggest that the use of metomidate and clove oil are humane alternatives to TMS and should be considered when euthanizing zebrafish.Land and Food Systems, Faculty ofGraduat