TRAINING MODULE
EUTHANASIA OF ANIMALS IN SCIENCE
DATE OF PUBLICATION: July 2020
Objectives
The objectives of this module are:
• to highlight the guiding principles of euthanasia of animals in science;
• to ensure proper handling of animals prior to euthanasia;
• to acknowledge the emotional impact of euthanasia on the people involved; and
• to outline considerations in choosing a suitable method of euthanasia.
Contents
Introduction ....................................................................................................................................1
Guiding Principles for Euthanasia .................................................................................................1
Handling of Animals Prior to Euthanasia .......................................................................................3
Emotional and Psychological Impact on Personnel.......................................................................4
Choosing a Suitable Method of Euthanasia ..................................................................................4
Summary .......................................................................................................................................9
References ....................................................................................................................................9
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Introduction
Euthanasia means a gentle death. In the context of animals in science, the term is generally used
to refer to humane killing, or doing what is humanly possible to minimize pain and distress to the
animals, given the circumstances (including the research goals) under which animals’ lives are
terminated.
Animals in science are euthanized for various reasons, such as:
• 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
• to terminate the lives of animals when no further use consistent with the Three Rs tenet (i.e.,
reduction, replacement, and renement) can be found for them (e.g., at the end of a study,
surplus animals generated by a breeding program).
“Whenever an animal is killed in the course of research, teaching, and testing
or production for scientic purposes, it must be done with respect and in a way
that ensures the death is as painless and distress-free as possible. In the use of
animals in science, it is essential that the scientic community take on the mantle of
responsibility for applying scientic judgment and up-to-date knowledge to ensure
that this is achieved.”
CCAC guidelines on: euthanasia of animals in science (CCAC, 2010)
Guiding Principles for Euthanasia
The most important criteria for accepting a method of euthanasia are that:
1) it has a rapid initial depressive action on the central nervous system to ensure immediate
insensitivity to pain; and
2) steps are taken to minimize distress in the animal prior to the procedure.
This is supported by the Canadian Veterinary Medical Association Euthanasia – Position Statement
(CVMA, 2014), which states: “The Canadian Veterinary Medical Association (CVMA) holds that
when animals are euthanized, death must be quick using a method that causes the least possible
pain and distress.”
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The 10 guiding principles for euthanasia, as stated in the CCAC guidelines on: euthanasia of
animals in science (CCAC, 2010) are as follows:
• Whenever an animal’s life is to be taken, the animal must be treated with the highest degree
of respect.
• When performing euthanasia, the intention should be to make the animal’s death as distress-
free and painless as possible. Therefore, the method likely to cause the least distress and pain
to the animal should be selected, consistent with the nature of the experimental protocol.
• Euthanasia should result in rapid loss of consciousness, followed by respiratory and cardiac
arrest and ultimate loss of all brain function.
• Euthanasia should aim to minimize any pain and distress experienced by the animal prior to
loss of consciousness. When appropriate, restraint should be used in such a manner that pain
and distress associated with the entire process are minimized.
• Methods used for euthanasia must be appropriate for the species, age and health status of the
animal.
• Death must be veried following euthanasia and prior to disposal of the animal.
• Personnel responsible for carrying out the euthanasia must be trained to carry it out in the most
effective and humane manner; recognize signs of pain and distress in relevant species; and
recognize and conrm unconsciousness, and subsequently death, in relevant species.
• Human psychological responses to euthanasia should be taken into consideration when selecting
the method of euthanasia, but should not take precedence over animal welfare considerations.
• Animal care committees are responsible for approval of the method of euthanasia for any study
involving the use of animals. This includes euthanasia as part of the experimental protocol,
as well as euthanasia for animals found to be experiencing unrelievable pain and distress or
approaching previously agreed endpoints.
• A veterinarian experienced with the species in question should be consulted when selecting
the method of euthanasia, particularly when little research has been done on euthanasia of that
species.
The application of these principles requires professional judgment and technical competence to
make an assessment based on both the scientic requirements of the study and the welfare of the
animals. It also involves an understanding of the animal, its behaviour, and its physiology, as well
as an understanding of the death process, the environmental and ecological impact of both the
method of euthanasia used and disposal of the carcass, the sensitivities of other personnel, and the
concerns of the general public.
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Handling of Animals Prior to Euthanasia
Any handling and restraint of an animal that is necessary for euthanasia should be done in a gentle,
careful manner to minimize the animal’s fear, distress, and pain. Where restraint may cause fear,
distress, or pain to the animal, the use of tranquillizers or sedatives should be considered.
Competency of Personnel
Personnel must be competent in performing the method of euthanasia on the particular animals
involved, to ensure it is carried out in the most humane manner and that it is done with professionalism
and respect. Competency includes the ability to:
• recognize pain and distress in the animal using behavioural indicators;
• apply appropriate methods of handling and restraining the animal as necessary;
• perform the method of euthanasia using appropriate equipment;
• recognize and assess unconsciousness in the animal;
• perform a secondary method to ensure the death of the animal when necessary; and
• recognize and conrm death.
Practical training is beyond the scope of this module and should be provided separately by the
institution (see the CCAC guidelines on: training of personnel working with animals in science
[CCAC, 2015]).
Equipment Used to Perform Euthanasia
Any instruments or devices used for euthanasia of animals should allow for easy observation of
the animals and should be professionally designed and kept in good repair to effectively produce
rapid unconsciousness and death. Equipment should be cleaned of all animal tissue, blood, and
excreta after each use.
Ensuring the Death of the Animal
While any acceptable method of euthanasia rapidly renders the animal unconscious and insensitive
to pain, the death of the animal must also be assured. The animal should be considered dead 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 reex activity, have ceased. For some
methods of euthanasia, this involves two steps: the application of the method producing initial
unconsciousness, followed by a procedure to ensure the animal cannot regain consciousness or
recover (e.g., exsanguination, opening the chest, decapitation or cervical dislocation after carbon
dioxide euthanasia).
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Carcass Disposal
All experimental animal tissues and carcasses must be disposed of according to institutional policy
and federal, provincial or territorial, and municipal regulations.
Emotional and Psychological Impact on Personnel
There may be emotional and psychological effects on people performing or observing euthanasia
that must be respected and taken into consideration. In research laboratories, personnel may
become attached to the animals and experience uneasiness at having to euthanize them at the end
of a study. It is important to recognize that regular exposure to the task can affect different people
in different ways. Some people may raise defence mechanisms that could result in reduced ability
to empathize, or less respectful handling of the animals. Others may nd that regular conduct
of the procedure increases their condence and competence and reduces their own stress, thus
improving performance of the procedure.
A number of steps can be taken to minimize any negative impact on personnel performing
euthanasia. Positive measures include 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, etc.), and that they are using the most aesthetic
techniques compatible with the welfare of the animals and the scientic objectives. Moreover,
those involved should willingly consent to carry out euthanasia and should not be pressured in
any way. A forum for open discussion of an individual’s concerns about euthanasia and support
for these individuals should be available. Any person who feels uncomfortable with a particular
method of euthanasia, or with performing the procedure, should discuss it with a supervisor or
veterinarian.
Choosing a Suitable Method of Euthanasia
Scientic Concerns Relating to the Choice of Euthanasia Method
It is important to consider whether research results will be impacted by a proposed method of
euthanasia, as some methods may affect animal tissues. For information on the potential effects
of various methods of euthanasia on particular species, see Additional information on effects of
euthanasia methods on research results: Addendum to the CCAC guidelines on euthanasia of
animals in science (CCAC, 2010).
Proper handling of animals prior to death to avoid stress or fear is also important.
Description of a Number of Acceptable and Conditionally Acceptable Methods
The selection of a method of euthanasia should include consultation with a veterinarian. Before a
method of euthanasia is used, it must be approved by the animal care committee during protocol
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review. Appropriate records should be kept of euthanasia, including the method and drug used, and
the personnel involved.
The CCAC guidelines on: euthanasia of animals in science (CCAC, 2010) classies potential
methods of euthanasia as:
• acceptable – methods that are simple to perform and consistently produce death, with minimal
pain and distress when used on conscious or sedated animals; or
• conditionally acceptable – methods that have greater potential for operator error or safety
hazards, might not consistently produce humane death, or are not well documented in the
scientic literature.
Conditionally acceptable methods may be suitable for use in certain circumstances where there
is scientic justication and animal care committee approval, and where there is assurance that
competent personnel are available.
Some common methods of euthanasia and brief descriptions of their use are presented here. Details
of which methods are acceptable or conditionally acceptable for particular species are provided in
the CCAC guidelines on: euthanasia of animals in science (CCAC, 2010) and the CCAC’s animal-
type guidelines.
Chemical Methods – Inhalants
Inhaled euthanasia agents are delivered to the animal, either as vapours (from a liquid) or gases,
usually in a closed chamber to avoid human exposure. Vapours and gases must be properly
scavenged.
Inhalant Anesthetics
An overdose of a commonly used inhalant anesthetic (e.g., halothane or isourane) can be an
effective method of euthanasia for many species. However, time to death with inhalant anesthetics
is quite lengthy, and therefore, the use of a second procedure to ensure death of the animal is
recommended, once the animal is unconscious as a result of the anesthetic. Inhalant anesthetics
are not appropriate for aquatic species or species that breath hold. Inhalant anesthetic agents must
be administered under controlled conditions with calibrated equipment (i.e., using a vapourizer).
Exposure to inhalant anesthetics has been found to be aversive to rodents and may also be stressful
for other species. The level of aversion appears to be species and strain specic, and depends on
previous exposure to the anesthetic. Combining the anesthetic with a sedative may be indicated
where administration of the sedative helps minimize stress.
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Inhalant Gases
Carbon dioxide (CO
2
) is commonly used for the euthanasia of rodents, particularly when large
numbers of rodents are being euthanized. However, CO
2
is only considered conditionally acceptable
for the euthanasia of small laboratory animal species, and should not be used where other methods
are practical for the experiment and the species.
The ethics of any use of CO
2
for killing animals must be reviewed thoroughly by the animal care
committee, taking into account current scientic information in this rapidly evolving eld. When
CO
2
euthanasia is used, it must be done according to a standard operating procedure approved by
the animal care committee, and the competency of the persons performing the procedure must be
assured.
CO
2
is not an acceptable method of euthanasia for:
• aquatic species (sh and amphibians), due to the formation of carbonic acid when CO
2
dissolves
in water; there is also evidence of sustained brain electrical activity in sh exposed to CO
2
;
• breath-holding species (e.g., lagomorphs, reptiles, and diving species); and
• species that have been shown to exhibit signicant aversion to CO
2
levels capable of stunning
and killing (e.g., pigs and mink).
Most studies of the effects of CO
2
euthanasia on rodents have been conducted on rats; however,
there is evidence that mice show similar aversion thresholds.
Both pre-ll and gradual ll methods of exposure to CO
2
have been shown to be aversive to
rodents; however, the current best practice is to rst place the animals in the euthanasia chamber,
and then introduce 100% CO
2
at a ow rate between 30 and 40% of the chamber volume per
minute. Flow rates greater than 40% of chamber volume per minute likely result in pain prior to
loss of consciousness, whereas ow rates less than 30% of chamber volume per minute are too
slow in causing loss of consciousness. The rate should be monitored using a gas ow meter. Flow
rates, and hence concentration of CO
2
in the chamber, can be increased once the animals have lost
consciousness.
Where practical, animals should be anesthetized prior to the use of CO
2
, preferably using inhalant
anesthetics. The use of CO
2
following anesthesia (with isourane) is an approved method of
euthanasia. While inhalant anesthetics, such as isourane, have also been shown to cause aversion
in rodents, there is evidence that rats appear closer to loss of consciousness at the onset of aversive
behaviour with inhalant anesthetics than with CO
2
. However, prior exposure to inhalant anesthetics
may increase the animal’s aversiveness to them.
If CO
2
euthanasia is to be carried out on different groups of animals using the same chamber, the
chamber must be ushed with air between groups. CO
2
is denser than air and will settle at the
bottom of the chamber; therefore, the CO
2
concentration will be greater in the chamber than in the
surrounding environment.
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Chemical Agents – Injectable
Barbiturates
Barbituric acid derivatives are acceptable euthanasia agents for most species if given intravenously
at high doses. In smaller species, intraperitoneal administration of barbiturates is acceptable when
the intravenous route is not practical and would cause distress; however, barbiturates should be
buffered if administered intraperitoneally, to avoid irritation.
Barbiturates act by depressing the central nervous system (anesthetic properties). Concentrated
solutions of sodium pentobarbital are the most widely used barbiturates. The Canadian Veterinary
Medical Association considers intravenous injection of concentrated barbiturates to be the most
humane method of euthanizing companion animals. From the Canadian Veterinary Medical
Association’s Euthanasia – Position Statement (CVMA, 2014): “The intravenous injection of a
concentrated barbiturate with prior sedation is widely considered the most humane method of
euthanizing companion and many non-domestic animals. It causes a comparatively aesthetic death,
is rapid-acting, reliable, and effective.” Records must be kept of all barbiturate use.
T-61
T-61 is an injectable euthanasia agent comprised of three drugs: a local anesthetic, a strong hypnotic,
and a paralytic. It is not a recommended method of euthanasia for any species. When approved
by the animal care committee after careful review, T-61 must be administered intravenously at the
dose and rate recommended by the manufacturer because of the differential rates of absorption
and onset of action of the active ingredients when administered by other routes. Where possible,
a sedative should be administered prior to the use of T-61 to protect the animal from any adverse
effects that may be associated with the accidental failure of the procedure.
Chemical Agents – Immersion
Tricaine Methanesulfonate (TMS or MS 222)
Tricaine methanesulfonate is a benzoic acid derivative that is used for anesthesia of sh and
amphibians, and can also be used for euthanasia in these species. Stock solutions are dissolved in
water (concentration greater than 250 mg/l) and must be buffered. Immersion methods must be
followed by a physical or chemical method to cause brain death.
Other immersion methods (e.g., benzocaine, etomidate, metomidate, or clove oil) must also be
followed by a second method to ensure death.
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Physical Methods of Euthanasia
Whether any physical method is humane strongly depends on the competency of the operator and
on the use of properly functioning equipment. The competency of the operator must be assessed
in advance. The use of any physical method requires justication to the animal care committee.
Cervical Dislocation
Cervical dislocation is a conditionally acceptable euthanasia method for small rodents, rabbits, and
some species of birds. Competency of the operator is essential.
Unless it will interfere with the scientic outcome of the study, animals should be anesthetized
prior to cervical dislocation.
Commercial cervical dislocators and luxators must be used for heavier rats (>200 g) and rabbits
(>2 kg). Manual cervical dislocation should only be performed when the number of animals to be
euthanized is relatively low, to prevent human error due to fatigue, and should only be performed
on small birds (<3 kg), rodents (<200 g), and rabbits (<1 kg).
Decapitation
Decapitation is a conditionally acceptable euthanasia method for birds and rodents. Personnel
must be competent in proper handling of the animal and in performing the procedure. Guillotines
specically designed for the procedure should be used and kept in good repair to ensure that
decapitation is humanely performed. The use of anesthesia prior to decapitation would make this
an acceptable method of euthanasia.
Concussion
A concussive blow to the head is a conditionally acceptable euthanasia method and may be
appropriate for the emergency killing of small or young animals with a soft skull. Death of the
animal must be ensured using a secondary method. The competency of the operator must be
assured in advance.
Concussion is a conditionally acceptable method for euthanizing sh, and as a method of emergency
killing in other species. When used, it should be carried out in such a manner that the animal
is rendered unconscious almost instantaneously. The procedure should be conducted in an area
beyond the sensory range of other animals.
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Penetrating Captive Bolt
A penetrating captive bolt that is applied by competent personnel, using equipment appropriate for
the species, is suitable for the euthanasia of large animals. With accurate placement of the device
against the skull of the animal, there is sudden unconsciousness, progressing to death. Immediately
after the procedure, a second method should be applied to ensure the death of the animal.
Summary
The euthanasia of animals requires respect for the animal, competency, and an understanding
of the many factors to be considered when selecting the most appropriate method. The primary
requirements for a humane method are that it causes very rapid (immediate) unconsciousness and
subsequent death, no pain is experienced by the animal, and distress to the animal is minimized.
References
Refer to the references in Section 8 of the CCAC guidelines on: euthanasia of animals used in
science (CCAC, 2010).
