Abstract

Aquatic invertebrates may need to be euthanized for a variety of reasons in clinical settings or to collect samples for research projects. Clinical situations that may require euthanasia include poor animal health, controlling disease outbreaks, population management, or the animal’s status as an invasive or destructive species. Due to concerns over sample quality for research such as advanced molecular testing, invertebrates in research projects are frequently sampled terminally without being rendered insensible prior to invasive sampling. Drinkwater et al. identified collection of individual animals and euthanasia practices as two of the largest ethical dilemmas in invertebrate research.¹ Cephalopod mollusks and decapod crustaceans were recently recognized as sentient in the United Kingdom.² As invertebrate welfare draws increasing attention from the scientific community and the public, improvements to the treatment of invertebrates are much needed. The objective of this report is to offer specific best practice techniques for euthanasia methods in invertebrates based on the latest literature, an online survey, and recent regulatory advances.

The American Veterinary Medical Association Guidelines recommend using a two-step approach for euthanasia of aquatic invertebrates, where the first step results in rendering the animal insensible and the second step destroys the brain or major ganglia chemically or physically. For a method to be considered euthanasia, it must cause rapid death and not result in trauma prior to loss of consciousness. Unacceptable methods for euthanasia of aquatic invertebrates include removal from water to die by desiccation, freezing, or immersion in caustic chemicals (including tissue fixative) as a solo or first step. Appropriate methods for the first step of euthanasia include immersion in magnesium salts, clove oil, eugenol, or ethanol.³ In decapod crustaceans, injection of potassium chloride into the hemolymph sinus can be used as a solo or first step.⁴ Other injectable anesthetics for the first step include ketamine, xylazine, tiletamine-zolazepam, ketamine-xylazine⁵ and alfaxalone.⁶ Appropriate chemical methods for the second step include immersion in 70% alcohol or 10% formalin, or physical methods such as pithing, freezing, or boiling.³ In cephalopod mollusks, immediate mechanical destruction of the brain is recommended for the second step.⁷ The choice for the second step should be driven by the reason of euthanasia and by whether and what type of samples are to be collected. For example, placement in 10% formalin as a second step is ideal if only histological samples are desired but precludes the use of tissues for molecular diagnostics. In research settings, the requirements for optimal sample quality need to be weighed against animal welfare considerations. For example, high quality RNA was extracted from the pyloric ceca of common sea stars immersed in magnesium chloride as a first step then sharply dissected as a physical method for the second step.⁸ Jellyfish euthanized by immersion in magnesium chloride were successfully used for NMR-based metabolomics.⁹ Two-step methods for aquatic invertebrate euthanasia are humane, inexpensive, practical, still allow for high quality molecular diagnostics, and present an advancement in animal welfare.

Literature Cited

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