Euthanasia of Experimental Animals

Table of Contents

Module Objectives

The objectives of this module are:

  • to discuss the principles of euthanasia - the humane killing of animals;
  • to discuss the emotional impact on people of killing animals;
  • to outline the criteria for a humane killing technique;
  • to describe the advantages and disadvantages of a number of methods of euthanasia; and
  • to discuss the importance of choosing the correct method of euthanasia based upon the tissues to be collected for analysis.

Experimental animals are killed for various reasons. The reasons include: to provide cells or tissues for in vitro research; to collect blood, tissues or other samples at the end of a study; to do veterinary pathology or diagnostics; to prevent unnecessary pain and suffering when the approved endpoint is reached and when they are no longer needed or are culled from a breeding program.

Whenever an animal is killed in the course of research, teaching and testing, it must be done with respect and in a way that ensures the death is as painless and distress-free as possible. The CCAC Guide to the Care and Use of Experimental Animals states: "In the use of animals in research, teaching, and testing it is essential that the scientific community take on the mantle of responsibility for applying scientific judgement and new knowledge to ensure that, when the life of an animal is taken, it is assured of a "good death".


The word euthanasia means a gentle death. The derivation of the word is from the Greek: eu - well or good; thanatos - death. Euthanasia is defined as a quiet or easy death, or means of producing one.

We tend to use many different words in an apparent effort to avoid stating plainly that an animal is being humanely killed: "put down"; "put away"; "put to sleep"; "put out (of its misery)"; "sacrifice". The term "sacrifice" is still used in scientific publications and presentations.

Principles for Humane Killing

The main welfare principles for a humane method of killing an animal are:

  • there should be very rapid (immediate) unconsciousness and subsequent death
  • there should be no pain or distress accompanying the procedure

These points are emphasised in the CCAC guidelines and in the Canadian Veterinary Medical Association position statement on euthanasia. The CCAC guidelines state: "The most important criterion of acceptance of a euthanasia method as humane is that it have an initial depressive action on the Central Nervous System (CNS) to ensure immediate insensitivity to pain." (CCAC, Guide Vol. 1 (2nd Ed.) XII. EUTHANASIA)

The CVMA position statement on euthanasia reads: "The Canadian Veterinary Medical Association believes that when an animal has to be killed, its death must be quick, and cause the least possible pain."

In addition, the circumstances leading to the humane killing of the animal should not produce fear or psychological stress on the animal. Other criteria used to evaluate methods of euthanasia are discussed below.

The application of these principles requires professional judgement, well-maintained equipment, and technical competence, coupled with an understanding of the animal, its behaviour and its physiology, and an understanding of the environmental and ecological impact, the sensitivities of other personnel, and the concerns of the general public.

Training and Expertise of Personnel

Personnel must be adequately trained to ensure that euthanasia is carried out in the most humane manner, and that it is done with professionalism and respect. Training should include: recognizing pain and distress in the behaviour of an animal, proper methods of handling and restraining the animal, proper application of the method and use of equipment, recognizing and assessing unconsciousness, methods of ensuring the death of the animal, and recognizing and confirming death. This training is beyond the scope of this module and should be provided by the institution separately.

Handling of the Animal Prior to Euthanasia

Any restraint of the animal necessary for humanely killing it should be done in a gentle, careful manner to minimise fear, distress, and/or pain. Where the restraint may cause fear, distress, or pain, the use of tranquillizers or sedatives should be considered.

Equipment Used to Perform Euthanasia

Any instruments or devices used for euthanasia of animals should allow for easy observation of the animals and be professionally designed and kept in good repair so that their use effectively produces rapid unconsciousness and death. They should be cleaned of all animal tissue, blood, or excreta after each use.

Evaluating the Humane-ness of a Method for Killing an Animal

When expert panels evaluate methods of killing animals, they use a number of criteria to determine whether a given method is humane and therefore acceptable. The following criteria were used by the AVMA Panel on Euthanasia, in their 2007 report:

  • ability to induce loss of consciousness and death without causing pain, distress, anxiety or apprehension
  • time required to induce loss of consciousness
  • reliability
  • safety of personnel
  • irreversibility
  • compatibility with requirement or purpose
  • emotional effect on observers or operators
  • compatibility with subsequent evaluation, examination or use of tissue
  • drug availability and human abuse potential
  • compatibility with species, age, and health status
  • ability to maintain equipment in proper working order
  • safety for predators/scavengers should the carcass be consumed

Most of these criteria are self-explanatory. However, some will be discussed briefly: a) irreversibility; b) scientific concerns relating to compatibility with requirement or purpose, and with subsequent evaluation, examination or use of tissue; c) emotional effects on observers or operators.

Irreversibility - Ensuring the Death of the Animal

While any acceptable method of euthanasia rapidly renders the animal unconscious and insensitive to pain, there must also be assurance of the death of the animal. Only when there is assurance that blood is no longer being delivered to the brain because the heart has stopped and all other movements such as respiration or reflex activity have ceased, should the animal be considered dead. For some methods this involves two steps, the application of the method producing initial unconsciousness, and secondly ensuring that the animal cannot regain consciousness or recover (for example, exsanguination, opening the chest, severing major blood vessels, cervical dislocation after CO2 euthanasia).

Scientific Concerns Relating to Choice of Euthanasia Method

Euthanasia methods may affect tissues and thus impact on subsequent biochemical, histological or electron microscopic analyses. The direct effects are generally subtle or absent. Methods which cause anoxia may produce pulmonary congestion and edema, depending on the rapidity of death. Tissue hypoxia may produce alterations (e.g., metabolic acidosis). Alterations in the central nervous system tissues probably occur more rapidly than to other tissues or organs, so it is important that tissues be prepared as rapidly as possible following unconsciousness and death of the animal. Proper handling of the animal prior to death to avoid stress or fear is also important.

Where there are scientific concerns about the impact on research results of a chemical method of euthanasia and a physical method of euthanasia is proposed, it must be justified and approved by the animal care committee based on scientific evidence. There should be an evaluation of the skills of the personnel, and of the location and equipment to be used, before any physical method is approved.

Emotional and Psychological Impact on Humans

There may be emotional and psychological effects on the people performing the euthanasia, and on observers, that must be acknowledged. In research laboratories staff may become attached to the animals and experience uneasiness at having to euthanize them at the end of a study. Regular exposure to the task may raise defence mechanisms that may result in callousness or rough handling of the animals. A number of steps can be taken to minimize the negative impact of having to perform euthanasia. Euthanizing animals can be made less distressful by ensuring that people are skilled in the techniques, that they have a good understanding of the physiological events associated with dying (assurance of unconsciousness, reasons for body movements), and are using the most esthetically acceptable techniques. A forum for open discussion of an individual's concerns about euthanasia, and support, should be available. Any person who feels uncomfortable with euthanizing an animal should discuss it with his/her supervisor or the veterinarian.

Modes of Action of Euthanasia Agents

Euthanasia agents cause death by: a) brain hypoxia, b) direct depression of neurons necessary for life to function, and c) physical disruption of brain activity and destruction of neurons necessary for life to function.

Choosing an Acceptable Method of Euthanasia

There are many methods available for humanely killing an experimental animal. Before any method is used, it must be considered by the animal care committee during protocol review. The approval of the proposed euthanasia method should always include consultation with a veterinarian. Appropriate records should be kept of euthanasias, method/drug and personnel involved.

A number of the commonly accepted methods of euthanasia, with a few key points related to those methods (advantages, disadvantages, comments), are presented here. Full discussions of a given agent or method are available in the listed references. In particular, Appendix XIV of the CCAC Guide Vol. 1 (2nd Ed.) - Methods For Euthanasia By Species (Methods In Order Of Acceptability) - should be consulted. The reader is encouraged to bookmark that section of the CCAC Guide for future reference.

Unacceptable methods or agents are merely listed in this module without comment. The reader is also referred to the institutional veterinarian for additional information on any euthanasia method.

Classification of Euthanasia Agents

Methods of humanely killing animals are usually grouped as chemical (inhalation, injectable, or other) or physical methods.

Chemical Agents - Inhalation

Inhaled euthanasia agents are delivered either as vapours (from a liquid) or gases to the animal, usually in a closed chamber to avoid human exposure.

Volatile Inhalant Anæsthetic Vapours

Any of the commonly used inhalation anæthetics (e.g., halothane, isoflurane, and others) can be used to overdose and kill an animal. Rodent preference tests have indicated halothane is the least aversive to inhale. Thus halothane is considered the most acceptable, and is the only one discussed here. All these agents must be used in closed chambers (for small animals) and the vapours must be scavenged to avoid human exposure.

Halothane Anæsthetic


  • easily achieve high vapour levels in closed containers
  • quick acting
  • relatively non-irritating to inhale


  • human health risk on exposure to vapours
  • must be used in circumstances that ensure no human exposure to vapours

Inhalant Gases

There are several gases that can be used to kill an animal, carbon dioxide, carbon monoxide, and inert gases such as argon and nitrogen. Of these, carbon dioxide is considered acceptable for euthanasia in small laboratory animal species, and is the only one discussed here.

Carbon Dioxide

Carbon dioxide (CO2) is heavier than air and is nearly odourless. Compressed gas cylinders of CO2 are readily available. At concentrations above 70%, unconsciousness usually occurs in less than a minute.


  • inexpensive and readily available
  • rapid onset of unconsciousness
  • minimal risk for human exposure


  • difficult to ensure optimal concentration in chamber
  • irritating to inhale and to mucous membranes
  • aversion in rodents
  • longer to unconsciousness than inhalant anæsthetics
  • neonatal mammals, amphibians and reptiles have higher CO2 tolerance and should not be euthanized by CO2 exposure
  • should not be used in any animal with the ability of prolonged breath-holding

Welfare concerns regarding the use of CO2 for euthanasia have been raised. Among the issues are questions of what concentration of CO2in the chamber is optimal for rapid unconsciousness with a minimum of distress, avoiding the reflex respiratory reactions to anoxia, optimal procedure for placing the animal(s) in the euthanasia chamber, and the irritant properties of CO2 when inhaled. When CO2 euthanasia is used it should be done according to an approved SOP (Standard Operating Procedure).

Chemical Agents - Injectable

Acceptable Injectable Euthanasia Agents

A number of commercially produced injectable euthanasia agents are available. These are discussed here.


All barbituric acid derivatives are generally excellent euthanasia agents if given intravenously at high doses. Intraperitoneal administration can also be acceptable when the intravenous route would cause distress. They act by depressing the central nervous system (anæsthetic properties). Concentrated solutions of sodium pentobarbital are the most widely used. The Canadian Veterinary Medical Association (CVMA) considers intravenous injection of concentrated barbiturates to be the most humane method of euthanizing companion animals. From the CVMA Position on Euthanasia: "Intravenous injection of a concentrated barbiturate is widely considered the most humane method of euthanising companion animals. It is rapid acting, reliable, and effective and is therefore superior to all other forms of euthanasia." Records must be kept of all barbiturate use.


  • speed of action to unconsciousness
  • smooth induction
  • relatively inexpensive


  • controlled drugs available only to licensed veterinarians
  • intravenous injection requires skill in smaller animals
  • drugs persist in the animals and carcasses must be disposed of in a manner that prevents scavenging

Intraperitoneal injection may be acceptable in some circumstances where intravenous injection would be distressful, or where intravenous access is not available.


T-61 is an injectable euthanasia agent comprised of three drugs: a local anæsthetic, a strong hypnotic, and a paralytic. It must be administered intravenously at the dose and rate recommended by the manufacturer.


  • it is not a controlled drug and so availability is less restricted, however it needs to be ordered, stored and used like a controlled drug because of the potential for abuse


  • must be given as recommended; intravenously, slowly
  • in dogs there may be vocalisation and muscle contractions upon injection

In most circumstances the disadvantages outweigh the advantages and barbiturates are the preferred injectable agents.

Chloral Hydrate

Chloral hydrate is a sedative that has a slower action in depressing the central nervous system. Death occurs due to hypoxemia as respiratory centres become depressed. It may be conditionally acceptable in some species in high doses when given intravenously. (CCAC, Guide Vol. 1 (2nd Ed.) XII. EUTHANASIA)

Chemical Agents - Other Routes

Tricaine Methane Sulfonate (TMS or MS 222)

Tricaine methane sulfonate is a benzoic acid derivative that is used for anæsthesia of fish and amphibians, and can also be used for euthanasia in these species. Stock solutions are dissolved in water (concentration greater than 250 mg/litre) and buffered, and the animal is immersed in the solution until dead.

Immersion in benzocaine hydrochloride solution may be an alternative to TMS for euthanasia of fish or amphibians.

Unacceptable Chemical Euthanasia Agents

  • Strychnine (rodent pesticide)
  • Nicotine
  • Magnesium sulfate
  • Potassium chloride
  • Any neuromuscular blocker (paralytic)
  • Other toxic chemicals

Some of these chemicals may be acceptable for killing the animal if it is already deeply anæsthetized, for example at the end of a study when blood sampling is taking place and these chemicals are injected to ensure death. Potassium chloride may be useful in the field to reduce the dose of euthanasia drug needed, where the carcass may be accessible to scavengers.

Physical Methods of Euthanasia

Whether any physical method of killing an animal is humane depends very much on the skill and training of the operator, and on properly functioning equipment. The skill of the operator must be monitored. Properly applied, most physical methods can produce a humane death of the animal. The use of any physical method requires justification to the animal care committee.

Cervical Dislocation

Cervical dislocation may be acceptable in small rodents and some species of poultry. Training and skill of operator is essential.


  • done properly it produces rapid unconsciousness
  • accomplished without equipment
  • avoids tissue contamination with chemicals


  • skill and experience required
  • the procedure may be aesthetically unpleasant
  • limited to small animals


Decapitation involves severing the neck and head. Guillotines specifically designed for the procedure should be used. The devices should be kept in good repair to ensure that decapitation is humanely done.


  • rapid loss of consciousness
  • avoids contamination of brain with chemicals


  • animal must be carefully restrained
  • the procedure may be aesthetically unpleasant
  • personal injury may occur


Stunning the animal by a blow to the head may be acceptable in small or young animals with a soft skull. Death of the animal must be ensured secondarily. Training and skill of operator is essential.

Penetrating Captive Bolt

A penetrating captive bolt "pistol" causes concussion by trauma to the brain. The bolt, spring loaded in the front of the device, is driven out by a gunpowder charge, penetrating the skull and brain. With accurate placement of the device against the skull of the animal, there is sudden unconsciousness progressing to death of the animal due to brain trauma.


  • effective use causes immediate unconsciousness


  • the procedure may be esthetically disturbing
  • animal must be adequately restrained
  • training and experience of personnel essential
  • device must be properly maintained


Shooting an animal appropriately can produce immediate unconsciousness proceeding to death, and is a humane way to kill an animal. Using a firearm to kill an animal should only be done by highly skilled operator, and in accordance with legal use regulations. Guidelines for aiming the firearm to the head to penetrate the brain are available.

Shooting as a means of collecting wild animal specimens is acceptable under certain conditions, depending on the skill of the personnel and use of appropriate firearms. The term "collection" is appropriate for this method of killing wild animals.


  • immediate loss of consciousness if bullet destroys much of the brain
  • may be best option for killing wild or free-ranging animals


  • dangerous to personnel and to other animals in the area
  • may not directly hit the brain in free ranging animals

Kill Traps

Kill traps are occasionally used to collect field specimens of small rodents, but they do not always result in a quick humane death of the animal.

Other Physical Methods of Killing Experimental Animals

The following method may be acceptable under certain conditions, if approved by an animal care committee.

Thoracic (cardio-pulmonary) compression involves forceful pressure on the chest of small birds in the field when alternative methods are not available. This is not an appropriate technique in laboratory settings.

Adjunctive Methods

Some additional methods of physically killing animals are acceptable when the animals are already deeply anæsthetized for other reasons. These adjunctive methods include exsanguination (bleeding out the body) quick-freezing the body in liquid nitrogen, pithing (rendering the animal brain dead by destroying the cerebral hemispheres using a sharp probe introduced into the brain cavity through the foramen magnum).

Carcass Disposal

A Reminder: All experimental animal tissues/carcasses must be disposed of according to institutional policy.

Summary Statement

The humane killing of animals requires knowledge, skill, respect for the animal, and an understanding of the many factors that are part of the choosing a humane method. The primary welfare principles for a humane method of killing an animal require that there should be very rapid (immediate) unconsciousness and subsequent death, and there should be no pain or distress accompanying the procedure.

References on Euthanasia

Refer to the references in section 8 of the CCAC guidelines on: euthanasia of animals used in science.