Abstract
Simple SummaryAlthough zebrafish are used in vast numbers in laboratories all over the world, no consensus has been reached in the scientific community on a humane, consistent, and effective method for euthanasia of this species. Here, we screened commonly used anaesthetic drugs to see if an overdose could induce loss of reflexes of adult zebrafish in a rapid and reliable manner, and without causing distress. The tested anaesthetics were isoeugenol, clove oil, 2-phenoxyethanol, tricaine, benzocaine, lidocaine hydrochloride, and etomidate. We found that lidocaine hydrochloride, buffered with sodium bicarbonate and ethanol to increase its efficacy, induces loss of reflexes in a fast, predictable, and relatively peaceful manner. We recommend its use for adult zebrafish euthanasia.Zebrafish are often euthanized by overdose of anaesthesia. However, fish may have aversion towards some anaesthetics, and protocol efficacy varies between species. Using wild type adult Danio rerio, we assessed time to loss of opercular beat, righting, and startle reflexes during induction of anaesthetic overdose by either tricaine (0.5 g/L or 1 g/L), benzocaine (1 g/L), 2-phenoxyethanol (3 mL/L), clove oil (0.1%), isoeugenol (540 mg/L), lidocaine hydrochloride (1 g/L), or etomidate (50 mg/L). Initial screening demonstrated that benzocaine and buffered lidocaine hydrochloride achieved the fastest loss of reflexes. The rapid induction times were confirmed when retesting using larger batches of fish. The fastest induction was obtained with 1 g/L lidocaine hydrochloride buffered with 2 g/L NaHCO3, in which all adult zebrafish lost reflexes in less than 2 min. Next, we monitored signs of distress during benzocaine or buffered lidocaine hydrochloride overdose induction. The results indicated that buffered lidocaine hydrochloride caused significantly less aversive behaviors than benzocaine. Finally, we tested several buffers to refine the lidocaine hydrochloride immersion. The most efficient buffer for euthanasia induction using 1g/L lidocaine hydrochloride was 2 g/L NaHCO3 with 50 mL/L 96% ethanol, inducing immobility in less than 10 s and with only 2% of adult zebrafish displaying aversive behaviors during treatment.
Highlights
It is likely that the extensive use of zebrafish as a research model will only continue to grow in the future; a safe, humane, and effective method of euthanasia for this species is necessary
In the present study, screening of commonly used anaesthetic agents for overdose euthanasia revealed 1 g/L lidocaine hydrochloride buffered with 2 g/L NaHCO3 could induce loss of all reflexes within 2 min, be reliable and provoke little aversive behavior in adult zebrafish
It is possible that some anaesthetic agents tested here, slower at inducing euthanasia, would cause a more peaceful death, with even less signs of aversion than buffered lidocaine HCl
Summary
On welfare grounds or due to animals reaching experimental endpoints, fish are euthanized every day in the laboratories. If the body tissue is to be used for further experimental analysis, some physical methods and some anaesthetic compounds may not be suitable, as they may damage cells or affect physiological and experimental data [2,3,4]. Additional factors such as safety for personnel, operator’s experience, cost, and environmental impact should be taken into consideration when deciding upon the optimal euthanasia protocol.
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