The Humane Killing of Zebrafish
Almut Köhler, Uwe Strähle, Jürgen Weiss, Larissa Kaufmann
The Humane Killing of Zebrafish
In recent years, the zebrafish (Danio rerio) has become increasingly important in biomedical
research. Its specific properties make it particularly well suited for research on major human
diseases such as polycystic kidney disease, cancer, myopathies, heart diseases, addictions,
neural and retinal degenerative disorders, infectious diseases, and many others. It is therefore
essential to develop the maximum standardization of experimental procedures for zebrafish,
just as was achieved earlier on for laboratory animal species such as mice and rats. The
maximum standardization of experimental methods is an indispensable prerequisite for the
reproducibility of experimental test results.
At this time, one of the most pressing questions is undoubtedly the humane killing of zebrafish.
This question has become especially relevant because according to appendix IV of the
“2010/63/EU Directive for the protection of animals used for scientific purposes”, it is only the
“overdose of a narcotic”, the “cerebral concussion/blunt trauma to the head”, or the “electrical
anesthetization” that are considered legal procedures to kill zebrafish. The latter two must be
eliminated. This is because, on the one hand, adult zebrafish only attain a total size of 2-3 cm,
which does not allow for correct placement of a blow that would produce unconsciousness. On
the other hand, there is no adequate approved mechanism for the electric anesthetization of
zebrafish. Narcotic overdose raises the serious question of possible effects of the narcotic on
experimental results (type, concentration, and administration modalities of the narcotic, etc.).
Unfortunately, killing through hypothermia is not mentioned in Appendix IV, so that many
German authorities will not authorize this method at all or only in conjunction with various
restrictions.
Since this situation requires urgent amendment from a scientific point of view, the “Institute for
Toxicology and Genetics (ITG)” at the “Karlsruhe Institute of Technology (KIT)” has conducted
a workshop on ”Euthanasia for Zebrafish” on March 9, 2017. The participation of 78 fish
specialists, 24 of which came from other European States, is a testament to the importance of
the topic studied.
During the first part of the workshop, international speakers shed light on various aspects of
zebrafish killing and evaluated their scientific importance. Almut Köhler, the central KIT
representative for animal welfare and a veterinarian, addressed the multiplicity and the genetic
and physiologic diversity of fish. Karin Finger-Baier, a scientist at the Max Planck Institute for
Neurobiology who works with experimental fish, presented the current literature on the topic of
hypothermia in relation to rapid chilling (see appendix for the standard protocol), that is to say
the shock-like cooling in cold water of 1-3° C with a particular focus on zebrafish. Kieran
Pounder, a researcher in Lynn Sneddon’s team at Liverpool University, gave a lecture on the
present state of knowledge about nociception in different species of fish. 1, 2, 3 Zoltan Varga,
Director of the Zebrafish International Resource Center in Oregon (USA), presented study
results regarding the efficiency and reliability of rapid chilling in zebrafish of various ages.
Chereen Collymore, a veterinarian from Toronto trained in laboratory animal science with
Sneddon LU. , Evolution of nociception in vertebrates: comparative analysis of lower vertebrates.
Brain Res Brain Res Rev. 2004 Oct; 46(2): 123-30.
2 Sneddon LU.; Elwood RW; Adamo SA; Leach MC., Defining and assessing animal pain. Animal Behaviour,
Vol. 97, 11.2014, p. 201-212.
3 Sneddon LU, Pain perception in fish: indicators and endpoints, ILAR Journal, 50 (2009), pp. 338–342.
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The Humane Killing of Zebrafish
Almut Köhler, Uwe Strähle, Jürgen Weiss, Larissa Kaufmann
extensive experience in anesthetic procedures, described the substances available for
zebrafish anesthesia and euthanasia and their respective pros and cons. 4 Bettina Bert, BfR
veterinarian specialist in Pharmacology and Toxicology, explained the legal framework as well
as the forms of decision making in the context of German and European legislation. The
specialist lectures were followed by a panel discussion where the six speakers discussed
different aspects of euthanasia and were available for questions and contributions from the
audience. In the course of the discussion and based on current scientific data, the participants
observed that the rapid chilling method works more reliably and faster than an overdose of
anesthetic. The specifics of the different methods were explained.
Pros
Blunt trauma/blow to the
head
Electrical anesthesia
Overdose
agent
of
Rapid chilling
anesthetic
Cons
• fast
• no residues
• simple device
• uncertain performance
• not
appropriate
for
zebrafish due to their small
body size
• rapid and reliable
• no adequate devices
available for zebrafish
• extensive
experience • partially aversive for the
available
animals (stress reactions)
• chemical
residues
(environmental protection,
impact on experiments,
safety of the experimenter)
• increased
need
for
cleaning due to chemical
properties (oily residues)
with endangerment of the
animals
• effect/results slower than
with the other methods
• fastest procedure (few • not suitable for operative
seconds)
interventions
• short stress period
• reliable (no recovery of • cannot be used with all
vital function)
fish species
• no
chemical
effect
(environment, experiment)
• no endangerment of the
experimenter
• simple disposal
This workshop demonstrated that while various methods of killing are possible, it is only the
expert use of hypothermia that allows the exclusion of potential pharmacological effects on
experimental results. Compared to all other killing procedures, hypothermia allows the fastest
possible death. This is valid for the killing of adult animals (from 14 dpf). In larvae (120 hpf –
14 days) and in earlier stages, based on current data, additional use of anesthetics is
recommended. For embryos, the fastest and most efficient procedure is insertion into liquid
Collymore C, Banks EK, Turner PV. 2016. Lidocaine Hydrochloride Compared with MS222 for the
Euthanasia of Zebrafish (Danio rerio). JAALAS 55(6): 816-820.
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The Humane Killing of Zebrafish
Almut Köhler, Uwe Strähle, Jürgen Weiss, Larissa Kaufmann
nitrogen. In both youth stages, tissue dissociation with bleach solution/proteinase K or similar
substances can complete the killing procedure.
An ad hoc survey of workshop participants demonstrated that a majority were in support of
hypothermia as a scientifically appropriate and humane method of killing for adult zebrafish to
be accepted by competent authorities and adopted by regulatory EU agencies as soon as
possible in the sense of a revision to appendix IV of the EU-Directive mentioned above.
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Appendix:
Standard protocol: Rapid chilling 5, 6
A tank filled with water is placed into another tank of the appropriate size, which is filled with
ice water and ice. This cools the water in the upper tank to 1-2°C (monitored by a
thermometer!), and one ascertains that the fish is completely immersed in water.
The fish is then transferred into the upper tank. After 10 seconds, one can already observe
that the fish no longer shows any signs of life due to the abrupt temperature change. It is
nevertheless recommended to leave the fish in the cold water for at least 10 more minutes.
Experimental setup: The fish is transferred into the upper tank after temperature reaches 12°C.
Currie et al. (1998), Critical thermal minima and maxima of three freshwater game-fish species
acclimated to constant temperatures, Environmental Biology of Fishes 51: 187–200.
6 Monte M and Zoltán MV (2012) Anesthesia and Euthanasia in Zebrafish ILAR Journal 53:2, 192-204.
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