[Zbrafish] Re: Back once more for IACUC help

Burdine, Rebecca D via zbrafish%40net.bio.net (by rburdine from Princeton.EDU)
Wed Feb 23 14:15:01 EST 2011

Hi everyone,

Please keep the answers coming both on and offline!  I can summarize what I hear on this for the entire list.

I did want to comment on the euthanasia issue. I recognize that MS-222 is commonly advocated, but I have resisted for a few reasons that are admittedly not scientific, but from personal observations.

1 - It is impossible for me to imagine under any circumstance how instant death (ice water submersion) isn't preferable to suffocating to death without being able to move (MS-222).  I have a hard time euthanizing anything because I am just that much of a wimp. But if I have to, I want the fish to be dead instantly.

2 - I have personally seen fish sitting in MS-222 for really long periods of time recover.  So I shudder to think of fish having sat in MS-222 but not died, then being put into a freezer or garbage disposal.

3 - I have never seen a fish recover from being put into ice water.  (We make an ice slurry with ice and water, move the ice back to make a water pocket, with no ice, and put the fish into that so they don't touch the ice.)

That's just me, and it's hard to argue with the AVMA.

But this paper does a pretty good job of it.  I have had it forwarded to me several time in this discussion and wanted to be sure everyone had the reference.

Thanks again for all the help!!


1. J Am Assoc Lab Anim Sci. 2009 Nov;48(6):785-9.

Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of
euthanasia in zebrafish (Danio rerio).

Wilson JM, Bunte RM, Carty AJ.

University Laboratory Animal Resources, University of Pennsylvania, Philadelphia,
Pennsylvania, USA. jolainer from upenn.edu

Despite the progressively increasing use of zebrafish (Danio rerio) in research,
the most humane method of euthanasia for these fish has not been determined.
Contemporary guidance documents state that hypothermia is an unacceptable method
of euthanasia. The goal of this study was to compare rapid cooling and tricaine
methanesulfonate (MS222) for zebrafish euthanasia. Zebrafish (n = 46) were
euthanized by immersion in either an ice-water (4 degrees C or less) bath or
unbuffered MS222 solution (250 mg/L; 25 to 30 degrees C). Another cohort (n = 10)
was exposed to buffered MS222 to determine whether the acidity of the water alone
caused distress. The times from exposure until the animals became unable to swim,
right themselves, and death were measured, and signs of distress were recorded.
Fish then were placed in a 'recovery tank' of system water to verify that
recovery did not occur. Tissues were examined histologically. The mean time for
euthanasia was longer and the number of fish exhibiting signs of distress was
greater for fish exposed to MS222 than those exposed to chilled water. In
addition, 4 of the 23 fish exposed to MS222 regained consciousness in the
recovery tank, whereas none of 23 fish exposed to chilled water recovered. No
histopathologic changes or evidence of ice crystal formation were seen in either
group. In light of the faster time to death and fewer signs of distress in
zebrafish euthanized by rapid cooling, we advocate this method as a humane
veterinary practice.

PMCID: PMC2786934
PMID: 19930828 [PubMed - indexed for MEDLINE]

Rebecca D. Burdine, Ph.D.
Assistant Professor
Dept. of Molecular Biology
Princeton University
Washington Road Mof 433
Princeton, NJ 08544

Phone: (609) 258-7515
Fax: (609) 258-6730
Email: rburdine from princeton.edu<mailto:rburdine from princeton.edu>
Admin Assistant: Anna Schmedel (609) 258-5028

From: zbrafish-bounces from oat.bio.indiana.edu [mailto:zbrafish-bounces from oat.bio.indiana.edu] On Behalf Of Rachel Tell
Sent: Tuesday, February 22, 2011 12:57 PM
To: zbrafish from oat.bio.indiana.edu
Subject: [Zbrafish] Re: Back once more for IACUC help

Becky -

I cannot speak directly to questions one or two in your message, but I hope that they generate some good on-list discussions, as they are questions that I have had, as well.
3)     How do people euthanize their zebrafish?  We use ice water followed by deep freezing, and I had this set with IACUC previously and had some references to back it up, but there is a new sheriff in town and they aren't happy again.  Any thoughts?
Many of us in the aquatic veterinary community utilize overdose of MS-222 (tricaine methanosulfate, trade name Finquel) as our primary euthanasia method for small fish. It is one of very few AVMA-approved humane euthanasia methods for fin fish and is supported by a body of literature. MS-222 overdose is the method of euthanasia used in all of the other established fish labs that I have heard of previously to this as well. This method is simple, quick, and easy.

As far as cooling then freezing, the AVMA's position is that this is not a humane method of euthanasia for fish or other animals, including other ectotherms, in part because the cooling leads to formation of ice crystals on the skin and in the cells that may be painful and cause distress to the animal. While quick-freezing of animals already in a deep plane of anesthesia is acceptable, with zebrafish that seems inefficient and unnecessary to me because we could simply up the exposure time to or dosage of MS-222 to euthanize without the need to place a live fish in the freezer.

The AVMA is currently working on updating and revising their human euthanasia guidelines for fish with lots of input from the aquatic veterinary community, but the current standards document, from 2007, can be found here: www.avma.org/issues/animal_welfare/euthanasia.pdf<http://www.avma.org/issues/animal_welfare/euthanasia.pdf>

If you want more information on protocols, etc, I would be happy to help.
Best of luck!


Rachel Tell, DVM
Research Assistant, PhD Student
Biomedical Sciences
Immunobiology Interdepartmental Graduate Studies Program
Iowa State University
rtell from iastate.edu<mailto:rtell from iastate.edu>

On Tue, Feb 22, 2011 at 11:03 AM, <zbrafish-request from oat.bio.indiana.edu<mailto:zbrafish-request from oat.bio.indiana.edu>> wrote:
Back once more for IACUC help
Re: Zbrafish Digest, Vol 69, Issue 7
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