Online Course on Laboratory Animal Science

EUTHANASIA OF LABORATORY ANIMALS: RODENTS AND LAGOMORPHS

Summary
Euthanizing laboratory animals may be necessary at the end of an experimental protocol, at the beginning (for organ or tissue
collection) or within breeding context. Any euthanasia technique must be compatible with the animal’s welfare, and therefore it
must be conducted without causing stress, suffering or pain. There are several methods available; these must be adequate to
the animal species in question, as well as with their physiological condition and age; the experimental protocol must also be
taken into account – when euthanasia precedes it – as well as technical conditions, namely in what concerns the equipment
used and operator safety. For each animal species there are recommended, adequate and inacceptable methods of
euthanasia; adequate methods may be used when recommended methods cannot be applied and inacceptable methods should
not be used under any circumstances. Finally, any methods of euthanasia must be followed with the confirmation of the death of
the animal.

Objectives
The death of animals used for scientific or other ends is a very sensitive matter and requires the utmost care to assure that
animal suffering is kept to a minimum i.e., it should be taken all measures to avoid stress, anxiety and fear. The objective of this
section is to provide useful information to all technicians who need to euthanize laboratory animals, namely rats, mice, guinea
pigs, gerbils and rabbits, in accordance with international norms aimed at protecting of animal welfare. The choice of method
must follow rigorous criteria regarding animal species and size, since a method may be adequate in some circumstances but not
in others. For instance, the choice of the method will depend on the experimental protocol used. The advantages and
disadvantages of each method, application method, and also precaution according with experimental protocol, the operator’s
skill, equipment, among other issues will be discussed in this document.

References
AVMA. The AVMA Guidelines on Euthanasia (formerly the 2000 Report of the AVMA Panel on Euthanasia). 2007.
www.avma.org/resources/euthanasia.pdf AVMA. 2000 Report of the AVMA Panel on Euthanasia, 2001, JAVMA, 218(5): 669-696.
Bryony Close (Chair), Dr Keith Banister, DrVera Baumans, Dr Eva-Maria Bernoth, Dr Niall Bromage, Dr John Bunyan, Professor
DrWolff Erhardt, Professor Paul Flecknell, Dr Neville Gregory, Professor Dr Hansjoachim Hackbarth,Professor DavidMorton &Mr
CliffordWarwick. Recommendations for euthanasia of experimental animals: Part 1. Laboratory Animals (1996): 30: 293-316.
www.lal.org.UK/pdffiles/LA1.pdf Bryony Close (Chair), Dr Keith Banister, DrVera Baumans, Dr Eva-Maria Bernoth, Dr Niall Bromage,
Dr John Bunyan, Professor DrWolff Erhardt, Professor Paul Flecknell, Dr Neville Gregory, Professor Dr Hansjoachim
Hackbarth,Professor DavidMorton &Mr CliffordWarwick. Recommendations for euthanasia of experimental animals: Part 2.
Laboratory Animals (1997):31: 1-32. www.lal.org.UK/pdffiles/LA2.pdf Conlee K.M., Stephens M.L., Rowan A. N., King L.A. (2005)
Carbon dioxide for euthanasia: concerns regarding pain and distress, with special reference to mice and rats, Lab. Anim.
39(2):137-61. European Commission, DGXI, Working party (Close B.Chairman ) 2005. Euthanasia of Experimental animals.
Johnson D., Blaszak K. (2005) Carbon Dioxide use for euthanasia of laboratory animals. (review), Bureau of Animal Welfare, DPI
Victoria. Reillly J.S. (Ed.) Euthanasia of Animals Used for Scientific Purposes, 2001, ANZCCART (em revisão).
http://www.adelaide.edu.au/ANZCCART/publications/Euthanasia.pdf Oliveira, H.P., Alves, GES, e Rezende CMF (2002) Eutanásia
em medicina Veterinária. CETEA, Escola Veterinária da UFMG-Dep. Clin. Circ. Vet. Brasil

Euthanasia: definition and reasons for its practice

Description
The word euthanasia means “good death”; therefore, in the context of euthanasia of laboratory animals, a “good death” is a
death with a minimum of stress and suffering, this means a humanely acceptable death. Thus, any situation of suffering or
stress is not compatible with the animal’s welfare and must be avoided before the animal’s death. To be considered humane,
any euthanasia technique must result in the loss of consciousness of the animal followed by respiratory or cardiac arrest and
finally by the loss of brain function.

In experimental animals, the need for euthanasia occurs at the end of an experimental procedure, to avoid suffering or stress
or after the collection of blood or tissues, or when during experimentation the level of pain or suffering goes beyond acceptable
levels; euthanasia could also be needed when the health and well-being of the animal are not good enough or to eliminate
excess animals.

Selecting the appropriate method of euthanasia will depend on several factors, such as animal species, availability of
means of restraining the animal, operator’s skill, availability of techniques and equipment, number of animals, their age, etc.
Thus, the evaluation of the method of euthanasia must comply with the following criteria:

To induce loss of consciousness and death with a minimum of pain and distress
To keep restraint to a minimum
To be appropriate and compatible with age, species and health status of the animal
To be reliable, reproducible and irreversible
To be compatible with the objectives of the study
To be compatible with the subsequent use of the tissues, or animal examination
To take into account the availability of drugs and the possible human use, and/or the capacity to keep the equipment in
proper working order
To be easy to perform
To be safe for the personal
To be acceptable for the operator and observers, whenever possible

Choosing a method of euthanasia comprises two important aspects and should be a compromise between the welfare of the
animal and, the adequacy for the operator and the availability of resources: a technique that is very adequate for the animal
could be difficult to perform by the operator resulting in suffering and pain for the animal. Thus, when evaluating the technical
possibilities, we should choose the method that is the best for the animal and then, if necessary, provide training to the
operator, before performing the euthanasia. In the case of animal experimentation, the current trend is to assign responsibility
of the researcher for choosing the most appropriate method of euthanasia to the researcher.

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Euthanasia of laboratory animals: rodents and lagomorphs
Main methods of euthanasia

Description
According to the principle of “humane death” methods for euthanasia may be classified under three generic categories:
recommended (or acceptable), acceptable with reservations and unacceptable methods[1]. Recommended methods are those
best suited to the animal; methods acceptable with reservations are those, which, despite not being the most adequate, may
be applied whenever the recommended method cannot be used for any reason; unacceptable methods are those that should
not be used, under any circumstances.

[1] See ANNEX I at the end of this document – Table of acceptable or conditionally acceptable methods for rodents and
lagomorphs.

Advantages and disadvantages of each method

Description
Euthanasia agents have different mechanisms of action and can be classified under two large groups: physical and chemical
methods, which include inhalable or injectable methods, regardless the adequacy to the animal species[1].

[1] See ANNEX II – Table of chemical and physical methods recommended or acceptable with reservations for rodents and
lagomorphs.

Physical Methods

Description
Physical methods, whatever they are, they must induce immediate loss of consciousness, by applying physical trauma to the
brain. These are specially used when chemical or pharmacological methods interfere with the experimental protocol. Usually,
even if unpleasant to the observer, physical methods are quicker and cause less stress when properly performed. The great
disadvantage of physical methods is that the operator must be properly trained to minimise animal manipulation and restraint,
since manipulating and immobilising the animal may induce great stress. Furthermore, animals should not be euthanized by
physical methods in the presence of other animals, as this would disturb them. In general, these are the recommended or
acceptable physical methods for use with rodents and adult rabbits: penetrating captive bolt, concussion, cervical dislocation,
decapitation and microwave irradiation. The three first methods must always be followed by confirmation of the animal’s death,
using methods described ahead.

Penetrating captive bolt is a method that can be used on laboratory rabbits, in particular those weighing over than 4 kg. This
method requires adequate equipment to ensure that the dart penetrates at least 3cm into the brain. The way of action is the
concussion and trauma on the brain hemispheres and brainstem causing quick loss of consciousness and subsequent death of
the animal. The animal needs to be restrained to ensure the dart penetrates it correctly, which may be a disadvantage if the
equipment does not work correctly or the operator is not properly trained; it is also aesthetically unpleasant.

Concussion can be performed by several methods depending on the animal’s size, but for rats, gerbils, mice, young guinea pigs
and small rabbits, a blow to the head is sufficient to render the animal insensitive. For larger animals, specialised equipment
must be used. This type of method requires a trained operator, and must be conducted on a single animal at a time; when not
properly performed, this method may cause different degrees of consciousness on the animal, and consequently pain; for this
reason, it should only be used on rodents weighing less than 1kg.

Cervical dislocation can only be used on mice, gerbils, rats, young guinea pigs and neo-natal rabbits, as long as they weigh less
than 150 g; it may also be used on rabbits weighing up to 1 kg if they have been previously sedated. This method, when
correctly performed, will cause immediate loss of consciousness; it is a swift, humane, and therefore an acceptable method of
euthanasia. Whenever possible, animals should be sedated or anaesthetised prior to cervical dislocation. The great advantage
of this method is that it does not chemically contaminate tissues. In a laboratory this method must be used only when its use is
scientifically justified and approved by the Animal Welfare Committee of the institution. The main disadvantages of this method is
that cervical dislocation may be anaesthetically displeasing to personnel performing or observing the method and it requires a
trained operator and master technical skills to induce quick loss of consciousness. For this, cervical dislocation should be
limited to small animals, being difficult to apply in guinea pigs because of the large muscle mass in the cervical region.

Decapitation is generally used on rodents but can also be applied on small rabbits weighing up to 1kg, since it provides a quick
loss of consciousness; the neck of larger and heavier rabbits is too strong and thick for a quick loss of consciousness by
decapitation. Decapitation consists on the separation of the neck of the animal, close to the head, by using a cutting
instrument, such as commercially available guillotine ensuring an uniformly and instantaneous severance. Using scissors is
unadvised, unless the scissor is adequate to the animal’s size and strong enough to cut easily through the neck in a single
snip. There has been some discussion regarding the loss of consciousness after decapitation, and prior sedation or
anaesthesia has been proposed; however, the required manipulation of the animal to inject sedatives or anaesthetics before
euthanasia will induce additional distress, and is therefore not considered a good practice for the animal´s welfare. At this
moment decapitation should be avoided whenever possible. The great advantage of this method is the possibility of not using
anaesthetics that would interfere with the experimental protocol, and being fast and humane for the animal. Thus, decapitation
can be considered as an acceptable method, especially for neurosciences studies. The main disadvantage of this method is
that it might be dangerous and unpleasant for the operator, and that handling and restraining the animal may cause additional
distress.

Microwave irradiation consists in applying a microwave beam targeted at a specific region of the brain. It can only be used on
small laboratory animals such as rats, mice, gerbils and rabbits weighing less than 300 g. This method is used primarily by
neurobiologists to fix brain metabolites in vivo while maintaining the anatomic integrity of the brain. It requires the use of specific
devices, since the use of a domestic microwave units is fully unacceptable. It also requires the operator to be specially trained,
but, when conducted properly, it is a humane method, since death occurs in milliseconds. Manipulation of the animal for correct
placement under the microwave beam should be kept to a minimum in order to avoid distress. Despite of its advantages,
microwave irradiation should not be used as a routine method of euthanasia, since there are associated risks to the operator.

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Electrocution or Electric stunning has been used on rabbits. It requires specialised equipment, correct application of electrodes,
correct selection of the intensity and duration of the voltage to apply, to ensure passage of enough current through the brain
to induce immediate loss of consciousness; this happens usually 15 to 20 seconds after applying the electrical discharge; a
second method of euthanasia should be used to ensure death.

Chemical Methods

Description
Chemical methods comprise, among others, applying anaesthetics in sufficiently high doses to induce death. The anaesthetics
induce, in a controlled manner, absence of perception, loss of consciousness, analgesia, and muscular relaxation. Anaesthetic
overdose induces death without suffering or pain, and is therefore an adequate method of euthanasia. Chemical methods of
anaesthesia require the use of another method to confirm death. There are two types of chemical methods: inhalable and
injectable ones.

Inhaled Agents

Description
Inhaled methods consist in applying a gas or anaesthetic by vaporisation or conduction – in anaesthesia chambers or circuits –,
such as CO2, CO, halothane and isoflurane and, therefore, take time to produce effect. Inhalable anaesthetics are usually liquids
that are vaporised naturally or mechanically; vapours are inhaled until breathing ceases and death occurs. During the induction
period air or oxygen should be provided to the animals to avoid hypoxia. Thus, the adequate use of these methods depends on
whether the animal is distressed between the application of the inhalational agent and the occurrence of loss of
consciousness. All inhalational agents that cause convulsions prior to loss of consciousness are considered unacceptable.
Inhalational methods may be used on most small rodents as well as rabbits, but should not be unpleasant to inhale, since some
may be irritating and therefore be stressful for the animals. The use of inhaled methods requires the use of adequate
equipment to ensure a homogenous distribution of the gas, in order to get the animals quickly exposed to a high concentration
of the agent. However, a high gas displacement rate should be avoided because it produces noises that will frighten the animal.
On the other hand, low fluxes allows for a gradual – and therefore painless – induction of death. Only animals of the same
species and, whenever possible, of the same social group, should be placed together in the inhalation chamber; they should be
kept isolated, not to hurt each other. When inhalational methods are used, death must be confirmed by using another method
of euthanasia, such as decapitation.

When using CO2 or CO as a euthanasia agent in new-born animals, it is important to be aware that they will require a longer
exposure to the agent, because they are more resistant to hypoxia.

Carbon dioxide (CO2) is an effective and humane method for most small animals when used in concentrations above 70%, since
high concentrations of this gas induce loss of consciousness quickly due to the narcotic effect of the CO2 in the brain, without
going through the hypoxia phase. With concentrations of 60%, CO2 will function as an anaesthetic, leading to quick loss of
consciousness. Lower concentrations will stimulate the respiratory centres and causes stress and anxiety, becoming unpleasant
to the observers. Nevertheless, a steady flow of CO2 at 15-35% chamber volume inhalation per minute is often used, a minimal
volume per minute of 20% is recommended. This is also the optimal recommended flow rate for gerbils. Concentrations of 100%
CO2 are not recommended for rats and mice, because this may cause severe dyspnoea and distress on conscious animals,,
although this is the recommended concentration for guinea pigs and large rabbits. When used on rabbits, causes excitement
and stress when the animals are still conscious, and for this reason, CO2 should be avoided. Moreover, CO2 can form carbonic
acid when in contact with the mucosae, becoming irritating for some animal species.

Euthanasia by CO2 must be initiated by progressive exposure to this gas, and must be conducted in a homogenous atmosphere
with a constant gas concentration. This means that, while CO2 is recommended as a method of euthanasia for small groups of
laboratory animals, including mice, gerbils, rats, guinea pigs and rabbits, care should be taken, when simultaneously euthanizing
several animals, to ensure a constant and effective gas atmosphere. After apparent death by CO2, the gas flow should be kept
constant for one more minute and death must be confirmed before removing the animals from the chamber; if the animal is not
dead, a second method of euthanasia should be applied.

This method is both humane for the animal and safe for the operator, since CO2 is neither flammable nor explosive. The great
advantages of this method lie with its fast-acting depressing, analgesic and anaesthetic effects, its commercial availability and
the low cost. Also, as it is not flammable or explosive, the potential danger to the operators is minimal, when using proper
equipment. The greatest disadvantage of this method is that it requires more time than other methods. Thus, despite being
recommended, the use of CO2 as a method of euthanasia must follow strict criteria depending on species, in order to minimise
discomfort, distress and suffering on animals.

CO2/O2 mixtures, which cause death by narcosis rather than hypoxia, do not seem to provide any benefit for the animal’s
welfare, and therefore should not be used; furthermore, obtaining CO2/O2 mixtures is usually difficult.

Carbon monoxide (CO) induces quick death by hypoxia, since this gas binds with haemoglobin instead of with oxygen. This
method is considered a humane method, even for rabbits, because this gas is scentless and does not induce distress in the
animal and it is relatively quick. CO should be used only from commercialised gas bottles and should never be obtained from
combustion of oil derivatives, since this is highly irritating to the respiratory tract of the animals. This method requires
appropriate, high-quality equipment that allows for the separation of the animals. Since it is highly noxious and harmful for the
operator, the equipment should be placed in a well-aired area, and operators must be properly trained; these are the main
disadvantages of this method. It´s great advantage lies with the fact that this gas induces loss of consciousness without pain or
discomfort, and death occurs quickly. Rats, mice, gerbils, guinea pigs and rabbits should only be placed in CO euthanasia
chambers after these chambers have been saturated with a homogenous mixture of 6-10% (from commercially available bottles
of 100% CO), since CO concentrations above 10% can be explosive. The use of this gas in guinea pigs is well documented on
literature: 8% CO causes collapse within 40 seconds to 2 minutes, and death occurs in 6 minutes.

Euthanasia in a nitrogen (N2) or argon (Ar) rich atmosphere consists in inducing death by hypoxia, since these gases replace
O2. These methods require the use of appropriate equipment, but because they are inert gases, colourless and scentless, non-
flammable and non-explosive ones, they can be used as a method of euthanasia of small animals. However, N2 or argon causes

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distress in some species of animals, such as in rats. Regardless, a flow of 39% per minute in the chamber can cause collapse in
3 minutes and respiratory arrest within 5 to 6 minutes; signs of panic and distress will be evident before death occurs. For this
reason, the use of this method in non-sedated or anaesthetized animals is questionable, which is a disadvantage of this
method. Advantages include the commercial availability of these gases and the fact that they are not dangerous to the
operator. Besides being effective, it is better to use other methods of euthanasia than N2 or argon.

Halothane, isoflurane and enflurane are volatile inhalational anaesthetics, with a decreasing power of effect respectively. All
three are commonly used an anaesthetics for small laboratory animals, and when administered in overdose – relative to
anaesthesia – causes a quick and stress-free death by depressing the cardiovascular and respiratory system. Halothane,
despite its strong odour and toxicity to the operator, has a quick-acting anaesthetic effect, and is therefore the inhalational
anaesthetic most suitable for euthanasia, particularly for rats. Isoflurane has an intense odour and should therefore not be
used in animals that can hold their breath; it is particularly useful when it is necessary to collect organs or tissues, such as the
liver, for toxicological or microsome experimentation, since this gas does not interfere with hepatic metabolism. Enflurane, like
isoflurane, is better than halothane when metabolical or toxicological studies are being conducted, since very little of this gas is
metabolised in the liver; enflurane is particularly adequate for use in mice. Other possible inhalational agents include N2O,
sevoflurane, desflurane and methoxyflurane, with the latter being a conditionally accepted or unacceptable agent for use in
rodents. Sevoflurane is less potent than isoflurane and halothane and has little odour. Ether has been used as a stress
model, as it is an irritating to the eyes and nose, induces anaesthesia slowly and is highly flammable and explosive. Ether is an
unacceptable method of euthanasia. N2O can be used in combination with other inhalational agents, but never in isolation,
since it does not induce anaesthesia on animals, even at concentrations of 100%, whenever used isolated, causes stress
before the loss of consciousness occurs.

The great advantage of the inhaled anaesthetics is that they are particularly useful for small rodents (weighing up to 7 kg) and
that, except for ether; they are non-flammable and non-explosive in normal environmental conditions. The only disadvantage is
that vapours can induce irritation and excitation during anaesthesia, and exposure to these agents may cause harm to animals
as well as to the operators.

Injectable Agents

Description
The Injectable agents are the quicker, the safer and the reliable methods of euthanasia of laboratory animals, namely for rats,
mice, gerbils and guinea pigs, because they can be applied without causing fear or stress in the animal. Whenever animal
restraining is necessary to apply the injection, it causes stress in the animal or it is unsafe to the operator, sedation or
anaesthesia should be previously applied, or another acceptable method of euthanasia should be used. The chosen method
should not compromise the experimental protocol, and should not cause suffering or distress to the animal.

Injectable methods consist in using mixtures of specific chemical substances that cause the death of the animal, namely the
use of anaesthetics in far superior doses than those used to induce anaesthesia. For choosing the methodology to be adopted
for general anaesthesia of a given animal species and for euthanasia of an individual or a group of animals a good knowledge of
the action of different drugs in different animal species is required. Drug metabolism is identical in the different animal species,
but their effects on the organism vary considerably depending on dosage, physiology, size, and the temperament of the animal;
it is therefore almost impossible to establish an anaesthesia or euthanasia protocol that can be applied for all laboratory
animals; morphine, for example, causes deep depression in rats and rabbits, but will cause convulsions in other animal species.

Many different chemical substances can be used as euthanasia agents, as long as they do not contain neuromuscular blockers;
this type of agents induce distress on the animal, and animals should therefore be under deep anaesthesia before
neuromuscular blockers begin to produce effect. The use of injectable methods requires previous and considerable knowledge
of the dosage to be used, as well as technical proficiency. Intravenous injection is the best method, since it provides the
quickest and safest effect, but, when not possible to use, intraperitoneal injection is acceptable. Intraperitoneal injections are
easier to apply, particularly in animals with veins that are smaller or difficult to penetrate, but will take longer to produce effect
and may therefore cause irritation, pain and stress. Intracardiac injections should be avoided, as they cause discomfort on
animals and may sometimes not penetrate the heart successfully at the first attempt; this method of euthanasia is only
acceptable when applied to animals that are deeply sedated, anaesthetised or in a deep coma. Intrapulmonary, intrahepatic,
intrarenal, intrathoracic and other non-vascular injection modes cannot be used to inject euthanizing agents. Oral and rectal
administration of agents will take considerable time to produce effect, with variable effect and are potentially irritating to the
tissues. Finally, intramuscular or subcutaneous injections should not be used, since euthanasia effect will take too much time
for the animal. For rabbits, intravenous administration at the ear’s vein, if not damaged, is the recommended method, since it
will induce quick anaesthesia and death; intraperitoneal injection is also acceptable, although it takes longer to produce effect.
In rats, mice and gerbils, intraperitoneal injection is the most commonly used method.

The great disadvantage of these methods for injecting non-inhalational chemical agents is the need for skilled operators, and
care should be taken for the disposal of the death animals, because these will contain chemical substances.

Barbituric acid and its derivates are the most commonly used and more widely accepted agents of euthanasia for most animals.
All of these agents are acceptable when administered intravenously, since they cause quick death with minimal discomfort
(depending on dosage and method of injection). These agents act by depressing the central nervous system, and subsequently
lead to cardiac and respiratory arrest. They include sodium pentobarbital, thiopentone, and other mixtures. Sodium
pentobarbital is the most widely recommended and adequate agent, because of its quick effect on rats, mice, gerbils, guinea
pigs and rabbits; a triple dose of the anaesthetising dose should be used. Intravenous injection is the best method because of
its quicker effect; however, intraperitoneal injection may be simpler to apply to many species, reducing distress by animal’s
handling. On the other hand, pentobarbital may cause irritation of the peritoneum, which can be avoided by using the drug in
low concentrations. The dose of pentobarbital to apply by intravenous or intraperitoneal injection, to have euthanizing effects,
is generally in a concentration of 200mg/ml in a dose of 100 mg/kg and 200 mg/kg of weight of the animal, for rats and rabbits,
respectively. The great advantage of using these agents is that they induce euthanasia smoothly with minimal discomfort for the
animal; these chemicals are also cheaper than most other euthanasia agents. The great disadvantage of these methods is that
since they require an intravenous injection, trained operators will be needed, as each animal will have to be restrained and then
euthanized individually. Using combinations of pentobarbital with other anaesthetising agents such as lidocaine is allowed, but
neuromuscular blockers should never be used. Sodium pentobarbital is the most adequate method for euthanizing rabbits.

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T-61 is a solution composed of three drugs that acts very quickly, causing a quick loss of consciousness by depressing the
central nervous system. The intravenous administrating route should be used in a very slowly way, not to be painful, which is not
always easy to perform on rodents and rabbits: prior sedation may be necessary to restrain the animal during intravenous
injection. However, T-61 should never be used in any other way when using these two species, and T-61 is no longer used in the
USA, and is considered inhumane by most of the people. T-61 should be kept under close surveillance due to its powerful
narcotic effects.

Methods of euthanasia considered acceptable for use on unconscious animals

Description
Apart from the methods already described, other methods of euthanasia considered acceptable for use on
unconscious animals include rapid freezing, exsanguination, ethanol, potassium chloride and intravenous injection of air. All
of these methods, when used on conscious animals, will cause suffering and stress, convulsions, vocalisation and are always
unpleasant to the operator. Whenever the animal is not anaesthetised these are considered unacceptable methods[1] of
euthanasia and should not be used.

[1] For more details on these and other methods considered unacceptable when applied on conscious animals, see the section
on unacceptable methods of euthanasia.

Selecting criteria according to model, age and physiological status of the animal

Description
Any method of euthanasia has advantages and disadvantages. Therefore, selecting the technique to be used for euthanasia
requires professional evaluation or judgement; when in doubt, the animal’s best interest should prevail in the choice of the
method.

Professional evaluation requires a good knowledge of the animal species in order to recognise animal behaviours showing
stress and suffering. Stress can be defined as an effect of physical, physiological or emotional factors that induce changes in
animal’s homeostasis or adaptive state. These are often related to behavioural changes, but may also include changes in the
neuroendocrine system or the autonomous nervous system. Animal’s responses to stress will depend on the species, age, and
physiological and psychological state of each animal.

Selecting the most appropriate method will depend primarily on the specie of the animal to be euthanized, but also on available
methods of restraining the animal, the training of the operator in charge of performing the euthanasia and the number of
animals to be killed among other factors.

Pre-natal and neo-natal euthanasia requires special care according to the embryonic stage of development because these
animals are more resistant to hypoxia than more developed or mature animals. For example, a 6-days old rabbit will survive
about 13 minutes in a nitrogen atmosphere, while at 19 days of age the same animal would only survive 90 seconds. For this
reason, inhalational agents cannot be used alone on animals with ages under 16 weeks, except in order to induce loss of
consciousness prior to the application of another method of euthanasia. On the other hand, foetal animals or new-born animals
will metabolise drugs more slowly than adult animals.

In embryos and foetuses, euthanasia must be humane, as in the case of the already born animals, if performed in a stage of
embryonic development in which the animal can already feel pain. It is generally considered that at 60% of gestation, pain can
be felt, but this will depend on the animal species. Thus, whenever an embryo is removed from an anaesthetised mother and is
rendered insensitive, euthanasia may be executed by cervical displacement, decapitation or removal of the heart. On the other
hand, non-anaesthetised foetuses of rodents and rabbits can be euthanized by rapid freezing in liquid nitrogen, as long as they
weigh less than 4 g.

New-born rodents have different stages of development: rats, mice and gerbils are totally dependent at birth, while guinea pigs
born fully developed. For rodents and rabbits up to 10 days of age, decapitation and concussion are adequate methods of
euthanasia, despite there are no guillotines commercially available for those ages. For rats, mice, gerbils and rabbits, the
injection of anaesthetics such as pentobarbital in lethal doses is an acceptable method of euthanasia. Due to the relative
resistance of new-born animals to hypoxia, the use of CO2 chambers is not recommended, since loss of consciousness takes
time in these animals and may induce distress and suffering, thus being considered not a humane method of euthanasia

In conclusion, selecting the most appropriate method of euthanasia will depend on the physiological state of the animal and his
age.

Selecting criteria according to user /researcher

Description
When choosing a method of euthanasia, besides the aspects concerning the animal itself, one should also consider the users,
i.e., the researchers and technicians who will perform euthanasia. As we saw in the previous section, concerning the user or the
researcher, the experimental protocol will determine the selection of the most adequate method. With respect to the person
who will perform the euthanasia, besides minimising animal suffering, the method should be chosen with regards to his skill in
the frame of the less dangerous appropriated methods for the animal. Thus, inhalational agents, while highly recommended for
the animals, are in most cases extremely dangerous, because of the risk of narcosis (halothane), hypoxia (nitrogen and CO),
addiction (NO) or other effects on the operator’s health resulting from chronic exposure to, as for example, NO or CO. For
instance human exposure to halothane, enflurane and isoflurane, should be inferior to 2 ppm, and in the case of N2O, it should
be inferior to 25 ppm.

In certain circumstances it is necessary to perform mass euthanasia of animals, such as in animal facilities. In these cases, the
best interest of the animals and the operators should be considered when choosing a method of euthanasia, not forgetting as
well as its applicability. Acceptable methods include CO2 chambers and physical methods such as cervical displacement or dart
projection, among others.

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With respect to people who are engage of performing euthanasia continuously, there are references in literature for more or
less serious, psychological disturbances in the operator.

Selecting the most appropriate method according to the experimental design

Description
The method of euthanasia may interfere with experimental results directly or indirectly. Therefore, the general principle of
selecting the most humane method of euthanasia according with the animal might be overridden if there will be some conflicts
with the scientific validity of the research. In such a case, instead of using the recommended method of euthanasia, it is
possible to use an acceptable method with reservations, as long as the negative impact to the animal’s welfare is kept to a
minimum.

Typically, chemical methods of euthanasia may interfere not only with the experimental protocol, but also with
experimental results, since many anaesthetics have pharmacological side effects. Whenever there is a chance that this might
happen, a rigorous selection of the types and routes of administering of these anaesthetics should be done, and those best
suited to each animal species, should be done in order to minimise or suppress any interference with the protocol or the results
of the experiment. When this is not possible, another method of euthanasia should be used. Usually physical methods, even if
not recommended, could be used as long as they are in accordance with the established rules for particular species and
animals.

Under no circumstances should be used methods considered unacceptable, even when the experimental design makes
it difficult to choose an appropriate method.

Unacceptable methods of euthanasia

Description
Unacceptable methods of euthanasia are those that, in any way, are not in accordance with the principles of animal´s welfare
causing discomfort, suffering, pain, or severe stress.

Besides those previously referred, the following are also considered unacceptable methods of euthanasia for application in
rodents or rabbits, when used isolated or in animals that are not unconscious: vacuum or decompression, asphyxiation,
nitrogen, NO, hypo and hyperthermia, chemical or thermal burn, neuromuscular blockers, narcotics, strychnine and cyanides,
ether and chloroform, any other substances and domestic solvents such as acetone, disinfectants and cleaning products,
ammonia-derived products, other toxins and salts. We will now discuss some of them.

Rapid freezing, performed always after rendering the animal insensitive, is of particular interest when collecting tissues for
biochemical studies, since this method reduces enzymatic activity. This technique is generally performed by completely
immersing the animal in liquid nitrogen, or by decapitating and then immersing the animal in liquid nitrogen. As a euthanasia
technique, it can only be performed in very particular circumstances, namely when experimental protocol demands it. It can only
be used in small animals, such as embryos or new-born rats, gerbils, mice, guinea pigs and rabbits.

Exsanguination is the incision and perforation of deep blood vessels of the animal. As this procedure causes extreme
hypovolaemia, pain and distress, it should only be conducted after the animal being insensible, and should be performed far
away from other animals. For an aesthetical point of view, this method is always very unpleasant.

Ethanol is used by an intraperitoneal injection of a 70% ethanol solution in small animals. Ethanol depresses the central
nervous system and respiratory arrest, and may be irritating to the peritoneum; for this reason, ethanol can only be used on
unconscious animals, since concentrations of ethanol above 10% are irritating to the animal. Potassium chloride is cardiotoxic,
causing convulsions, muscular spasms, vocalisations and other reactions, being both unpleasant to the user and a cause of
distress to the animal; it can only be applied intravenously or intracardiacly, by a properly trained operator. This method may be
used in almost any animal species, but only when the animal is under general anaesthesia. Intravenous injection of air is
extremely painful for the animal and causes the pupils to dilate, vocalisations and other undesirable effects; it is also an
unreliable method of euthanasia.

Resorting to vacuum and decompression not only caused suffering to the animal before losing consciousness, but it is also an
extremely unpleasant method for the operator, as it causes bleeding, convulsions and uncontrolled defecation and urination.
Further, immature animals may take a considerable amount of time to stop breathing, and accidental recompression may occur,
with the consequent recovery of consciousness by injured animals. Asphyxia takes a long time until the animal loses
consciousness, and therefore causes suffering and distress. Nitrogen kills rodents by hypoxia, but this is a slow process. With a
concentration of 39%, rats will be unconscious only after 3 minutes, and will display signs of panic and distress during the
process; also, it does not cause death on small animals. NO requires concentrations nearing 100% and even with this
concentration it is a slow and stress-inducing process; the animal will convulse and the whole process is unpleasant to the
operator. Hypothermia and hyperthermia will cause extreme pain and a slow death, although hypothermia can be used, in a
certain degree, as anaesthesia. Any agent that acts as a neuromuscular blocker (nicotine, magnesium sulphite, potassium
chloride) or that does not induce loss of consciousness before death, such as sedatives, cannot be used as euthanasia
agents. Furthermore, death by sedation requires very large volumes of sedatives to be used, and these do not have any
analgesic or anaesthetic effects. Narcotics cause death by depressing the respiratory centres with variable effects between
species with effects on animal’s welfare not fully understood; narcotics should not be used as a method of euthanasia.
Strychnine will cause strong convulsions and painful muscular contractions, and for this it is not a humane method of
euthanasia. Complete immersion of animals in formalin, should not be used as a method of euthanasia. Another method
considered not humane and therefore considered unacceptable for euthanasia is the use of cyanides (namely, hydrogen
cyanide gas); death by cyanides is unpleasant, as well as dangerous, for the operator. Propane cycle, trichloroethylene, ether
and chloroform are high flammable and explosiveness when in contact with air. Because of this, these agents, besides being
humane for the animals, are unacceptable methods for euthanasia as they are very dangerous for the operator.
Trichloroethylene only produces mild anaesthesia in the animal and is very dangerous to the operator, as this substance is a
carcinogen and causes respiratory insufficiency. Ether and chloroform are very dangerous to the operator and should be used
under no circumstances, not even on small rodents, as ether acts as an irritant to the mucosa and, in high concentrations, may
cause distress to the animals; chloroform induces excitement prior to loss of consciousness. In addition, ether is explosive and
chloroform is carcinogen and is also toxic for the liver and the kidneys.

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The addition of substances to drinking water is also an unacceptable method of euthanasia because of their slow effect and the
risk that not all the animals receive sufficient and adequate concentrations to cause death; these substances are also usually
dangerous to the operators. Other unacceptable methods of euthanasia include the use of methoxyflurane, magnesium
sulphite and chloral hydrate, especially in small animals, since side effects on animals can be severe, including vocalisation,
muscular spasms and other effects also unpleasant to the operator.

Performing and confirming euthanasia

Description
Besides choosing the most appropriate method of euthanasia, the death of the animal should occur in a quiet environment,
always far from animals that will not be euthanized. Records of euthanasia procedures should be carefully elaborated and kept.

Once euthanasia has been performed, it is necessary to perform the recognition and the confirmation of the death of the
animal before the disposal of the remains. An animal after the application of an inhalational or injectable agent may seem dead
but eventually recover. In rats, gerbils, mice and guinea pigs, respiratory arrest and absence of reflexes (cornea and eyelids)
are good indicators of irreversible death; in small animals, reduced body temperature to values below 25 o C is also a good
indicator. For rabbits, cessation of breathing and heartbeat and absence of reflexes are good indicators of irreversible death.
The death of rodents and rabbits can be confirmed by several methods, such as cervical displacement, decapitation or
destruction of the brain, exsanguination or extraction of the heart, removal of entrails, and partial substitution of O2 for
nitrogen, intraperitoneal injection of ethanol or potassium chloride, or deep freezing. The choice of the method to confirm death
will depend on the species, and when in doubt, a second method of euthanasia should be applied; cervical displacement is the
most frequently used in rats, gerbils and mice. It is never too much to emphasise the need for properly trained operators who
are able to recognise death in these animals.

After euthanasia has been performed and the death of the animal has been confirmed, the carcasses should be immediately
disposed of; when this is not possible, they should be frozen. Whenever the animals that had been euthanized are carriers of
infectious organisms, special measures should be taken for their discarding. Whenever possible, the tissues of killed animals
should be shared with other researchers.

ANNEX I - Summary table of acceptable or conditionally acceptable methods for rodents and lagomorphs

Description
ANNEX I - Summary table of acceptable or conditionally acceptable methods for rodents and lagomorphs[1]

Acceptable Methods Conditionally Acceptable Methods

· Barbiturates;

· Inhalational anaesthetics; · N2;

RODENTS · CO2 Ø · Ar;

(rats, gerbils, mice and guinea · · Cervical displacement (<150g);

CO;
pigs)
· decapitation;
· Potassium chloride ( only when used
together with general anaesthesia;

· Microwave irradiation¤

· Barbiturates;

· Inhalational anaesthetics; · N2;

· CO2◙ ; · Ar;
LAGOMORPHS
· Cervical displacement (<1Kg);
(rabbits) · CO;
· decapitation;
· Potassium chloride ( only when used
together with general anaesthesia; · captative bolt (>4Kg).

Ø CO2 has been the most widely recommended and used agent on rodents, despite increasing evidence showing that exposure
to this gas affects rodents, and other animals, beyond transitory suffering and stress, and cannot therefore be considered a
humanely acceptable method.[For revision: Johnson & Blaszak, 2005 and Conlee et al., 2005]. Requires long exposure in new-
born and immature animals.

¤ Cannot be used as a routine method; domestic microwaves should never be used.

◙ For large rabbits, a method of euthanasia other than CO2 should be used whenever possible, since these animals display
signs of distress at an initial stage, prior to loss of consciousness.

[1] Adapted from the AVMA Guidelines on Euthanasia,2007. While this paper considers ether as a conditionally acceptable
method for small rodents, we have decided not to include it on the table, since most literature consider that ether should not
be used under any circumstances as a method of euthanasia [see “Recommendations for euthanasia of experimental animals”–
Report of a Working Party, Bryony Close (Chair), 1996 (p. 307), 1997 (p.17,20) and “Euthanasia of experimental animals”-
European Commission, 1995, p.19-20,44]

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ANNEX II – Detailed table of recommended or acceptable with reservations chemical and physical methods for
rodents and lagomorphs

Description
ANNEX II – Detailed table of recommended or acceptable with reservations chemical and physical methods for
rodents and lagomorphs[1]

Acceptable with
Methods Recommended/ Acceptable
reservations

· CO
· Inhalant · N2
· CO2 preceded by
Isoflurane, Halothane or · Air
Enflurane
Chemical
· Sodium pentobarbital by
intraperitoneal injection
RATS, (60mg/ml to 200mg/Kg)
GERBILS AND · Injectable ----
MICE · T-61 by intravenous
injection

· Cervical displacement
(<150g);
Physical · Microwave irradiation
· Decapitation (<200g)

· Dart projection (>4Kg)

· Halotane (4%)
· Inhalant · CO2
Chemical · NO (only with another
inhalant agent).

· Sodium pentobarbital by
intraperitoneal injection
· Injectable (60mg/ml to 90mg/Kg)
GUINEA PIGS
· T-61 by intravenous
----
injection

· Cervical displacement

· Electric shock with

Physical ----
exsanguination.

· · Halothane
Inhalant
---- · NO (only with another
inhalant agent).
Chemical
· Sodium pentobarbital
administered intravenously or
intraperitoneally.
· Injectable ----
· T-61 by intravenous
RABBITS
injection

· Dart projection

· Electric shock with

· Microwave irradiation (only cervical displacement
Physical
for small rabbits)
· Cervical displacement
or decapitation (<1Kg) with
prior sedation

[1] Based on the AVMA Guidelines on Euthanasia, 2007; Euthanasia of animals used for scientific purposes - ANZCCART 2001;
Recommendations for euthanasia of experimental animals 1996, 1997.

Whenever discrepancies were observed in the literature regarding a certain method, we have decided not to include it on this
table, considering that it needs further scientific evidence regarding to its use.

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