Adult zebrafish euthanasia: efficacy of anaesthesia overdose versus rapid cooling

The rapid increase in zebrafish use needs to be accompanied by research into refinement of procedures, such as euthanasia. The EU directive lists three possible euthanasia methods for fish: anaesthetic overdose, electrical stunning and concussion. However, for a small fish like zebrafish, concussion and electrical stunning are difficult to perform, leaving anaesthetic overdose as the only realistic option. Thus, more tailored solutions need to be found for this small tropical water species. Our aim was to test the efficacy of different anaesthetics overdose and of the rapid cooling method to kill adult zebrafish. Adult mixed-sex AB zebrafish (n= 12) were randomly assigned to: 250mg/L MS222; 20 mg/L propofol + 100 mg/L lidocaine; 6 mg/L etomidate; 50 mg/L clove oil; rapid cooling (water at 2-4º C). Two minutes after opercular movement ceased, the animals were transferred to clean water to assess if they could recover. Zebrafish euthanized with rapid cooling ceased the opercular movements significantly quicker compared with the other groups, and etomidate-treated animals took longer to die. No zebrafish recovered after being placed in clean water. All protocols used were efficacious to achieve euthanasia, but rapid cooling was consistently the fastest and so more efficient. Further studies evaluating animal welfare and the quality of the biological samples are needed to refine the rapid cooling protocol before recommendations can be made to change legislation to include this as a method for euthanasia for small tropical fish.