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ACUP 306.02 Fish and Amphibian Euthanasia The intent of this Animal Care and Use Procedure (ACUP) is to describe the acceptablemethods for euthanasia of fish and amphibians. This ACUP is intended for trainedpersonnel that will perform euthanasia on fish or amphibians. This ACUP is approved bythe Cornell Institutional Animal Care and Use Committee (IACUC). Any deviation mustbe approved by the IACUC prior to its implementation.

TABLE OF CONTENTS1. Introduction2. Materials3. Procedures4. Personnel Safety5. Animal Related Contingencies6. References

1. IntroductionThis ACUP defines the materials and methods approved for euthanizing fish andamphibians under protocol use at Cornell University.

2. Materials -dependent upon method used. Topical euthanasia agent [e.g., tricaine methane sulfonate (MS-222)] Mechanical euthanasia tool (e.g., ice & bucket, guillotine, pithing tool)

3. Procedures

Note: Expired surgical materials or pharmaceuticals cannot be used in, or applied to,animals undergoing survival procedures. See ACUP 210 (General Non-SurvivalSurgery) for applicable restrictions with non-survival procedures.

Note: Prior to disposing of euthanized animals: for amphibians, verify the animal isdead by performing a physical method of euthanasia or by assuring the heart hasstopped beating; for fish, perform a physical means of euthanasia or observe thatopercular movement has ceased for at least 10 minutes.

a. External/topical chemical agentsi. Tricaine methane sulfonate (MS-222):

1. MS-222 is acidic in solution and must be buffered by adding anequal weight of sodium bicarbonate or titrating to pH=7.0-7.5

2. Tank immersion method (fish or amphibians):a) Place the animal in a buffered solution of MS-222, at a

concentration of: Fish: minimum concentration of 250 mg/l;

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Amphibian: 2-3 g/lb) Immerse until death is achieved. Time to effect is proportional to

MS-222 solution concentration.3. Alternative method (fish only):

Remove fish from water and flush gills with a buffered,

concentrated solution of MS-222 (>250 mg/L).ii. Benzocaine hydrochloride (fish or amphibians):1. Benzocaine-HCl is acidic in solution and must be buffered by

adding an equal weight of sodium bicarbonate or titrating topH=7.0-7.5

2. Place animal into a bath of benzocaine hydrochloride solution of250 mg/L ; immerse until death is achieved.

iii. 2-phenoxyethanol (fish only): Place fish into a bath of 2-phenoxyethanol solution at a

concentration of 0.50.6 mL/L or 0.30.4 mg/L; immerse untildeath is achieved.

NOTE: Chemical methods of euthanasia may require up to 3 hours beforedeath results. A physical method, as per section 3.b, can be applied onceunconsciousness is achieved.

b. Physical Methods

NOTE: Anesthesia or heavy sedation must be applied prior to the use ofphysical techniques unless otherwise approved by the IACUC.

i. Decapitation (fish or amphibian):

Use sharp equipment of the appropriate size for the species tobe euthanized to ensure that the head is separated from thebody rapidly and completely. Follow decapitation with pithingvia the open spinal canal.

ii. Pithing without decapitation (amphibians only): Flex the neck, identify the foramen magnum, insert a rigid metal

rod cranially, and pivot/rotate the rod within the cranium todestroy the proximal spinal cord and brain.

iii. Thermal shock (only for specimens that are

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d. Use the minimal amount of water for transfer of animalsinto ice water to prevent local warming of ice water byadding room temperature water to the ice.

e. Use a net for adults and a minimal volume of embryomedium for embryos/larvae.

4. Personnel Safetya. Medical emergencies: call 911b. When working with animals wear appropriate PPE, observe proper hygiene,

and be aware of allergy, zoonosis, and injury risks. Refer to the CAREOccupational Health and Safety webpage for more information.

c. MS-222 (tricaine methane sulfonate) safe practices:i. Wear protective clothing, gloves, and goggles when handling MS-222

powder; wear gloves to handle animals exposed to MS-222.ii. If possible, work inside a fume hood to prepare a concentrated stock

solution by mixing an appropriate amount of MS-222 powder in a small

volume of water. Wear gloves and use a utensil to stir until all powderis dissolved. Dilute the stock solution further as required.iii. Dispose of MS-222 wastes by flushing down the drain to a sanitary

sewer with an excess of water. Do not discard MS-222 directly intosurface water, storm water conveyances, or catch basins.

iv. If in a remote location where a sewer may not be readily available,further dilute the solution with water and dump wastes on land, in alocation away from water.

5. Animal Related Contingenciesa. Post contact information for emergency assistance in a conspicuous location

within the animal facility.b. Emergency veterinary care is available at all times including after working

hours and on weekends and holidays through CARE (pager 1-800-349-2456)c. Non-emergency veterinary questions & requests for care, call or email CARE

veterinary staff at 607-253-4378 or care@cornell.edu, respectively.

6. References NIH Guidelines for use of

zebrafish: http://oacu.od.nih.gov/ARAC/documents/Zebrafish.pdf Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods

of euthanasia in zebrafish (Danio rerio). JM Wilson, et al. JAALAS. 2009.

Nov;48(6):785-789. Neiffer, D.L., Stamper, M.A. 2009. Fish sedation, anesthesia, analgesia, andeuthanasia: Considerations, methods, and types of drugs. ILAR Journal, 50 (4),343-360.

AVMA Guidelines on Euthanasia, June 2007.http://www.avma.org/issues/animal_welfare/euthanasia.pdf

Guidelines for Euthanasia of Nondomestic Animals, American Association of ZooVeterinarians; 2006.

http://oacu.od.nih.gov/ARAC/documents/Zebrafish.pdfhttp://oacu.od.nih.gov/ARAC/documents/Zebrafish.pdfhttp://oacu.od.nih.gov/ARAC/documents/Zebrafish.pdfhttp://www.avma.org/issues/animal_welfare/euthanasia.pdfhttp://www.avma.org/issues/animal_welfare/euthanasia.pdfhttp://www.avma.org/issues/animal_welfare/euthanasia.pdfhttp://oacu.od.nih.gov/ARAC/documents/Zebrafish.pdf

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Laboratory Animals: Recommendations for euthanasia of experimental animals .Royal Society of Medicine Press, London, U.K. 1996.

Guidelines for the Use of Fishes in Research (2004), Copyright 2004 by the American Fisheries Society: http://fisheries.org/docs/policy_useoffishes.pdf

**Personal communication: Zoltn Varga, PhD, Director, Zebrafish International

Resource Center (ZIRC). May 11, 2010. B. Blank. Amphibian Medicine and Captive Husbandry , KM Wright & BR Whitaker, 2001;

Krieger Publishing Companty, Malabar, FL. CARE Occupational Health and Safety

webpage. http://www.research.cornell.edu/care/OHS.html Guidance on the housing and care of the African clawed frog Xenopus laevis, BT

Reed. Formulary for laboratory animals . CT Hawk, et al. 3rd ed., 2005

Rev#: Autho r Rev#: Effecti ve date(s) Review date Referee ACUP.01: C. Brown.02: T. Pavek

.01: 2005

.02: 2011Dec 2011Dec 2012

P. Bowser ACUP 306.02

http://fisheries.org/docs/policy_useoffishes.pdfhttp://fisheries.org/docs/policy_useoffishes.pdfhttp://fisheries.org/docs/policy_useoffishes.pdfhttp://www.research.cornell.edu/care/OHS.htmlhttp://www.research.cornell.edu/care/OHS.htmlhttp://www.research.cornell.edu/care/OHS.htmlhttp://www.research.cornell.edu/care/OHS.htmlhttp://fisheries.org/docs/policy_useoffishes.pdf