Techniques for euthanasia usually should follow current guidelines established by the American Veterinary Medical Association Panel on Euthanasia.

A process of killing that renders the animal unconscious (and thus insensitive to pain) as rapidly as possible, without fear and anxiety (CCAC, 1980).

It is imperative that death be verified after euthanasia and before disposal of the animal. Death must be confirmed by checking for the absence of vital signs, such as a heartbeat. An animal in deep narcosis from a chemical agent may appear to be dead but it may eventually recover if its respiration is allowed resume. The absence of respiration does not always indicate death.

Criteria should be developed for deciding when the level of pain and distress is such that euthanasia is warranted, and the person responsible for making that decision must be identified in the experimental protocol. (Ad hoc Committee on Animal Research, 1988; Everitt and Griffin, 1988)

Moribund Animals: Animals should be killed as soon as the objectives of the study have been achieved to avoid unnecessary suffering.

Solid Tumor Studies: If consistent with the purposes of the experiment, animals should be killed when one of the following conditions occur rather than waiting for the animal to die spontaneously.
 

Euthanasia also is often a stressor to the person performing the procedure. The degree of distress experienced by observing or performing animal euthanasia is influenced by the person's background, personal philosophies and ethics. (Arluke, 1988)

The stress associated with performing animal euthanasia may cause a strong sense of work dissatisfaction or alienation among people regularly performing euthanasia. That stress might be expressed by absenteeism, belligerence, careless or callous handling of animals, and high personnel turnover rates.

Dealing Effectively with Euthanasia-Associated Stress

A supervisory awareness of and sensitivity to people's concerns over animal euthanasia must exist and the facts related to euthanasia must be discussed.

The choice of a method of euthanasia, however, must be based primarily on humane concerns for the animal rather than on the sensitivities of the technician who performs the euthanasia or the people who observe the procedure. Some physical methods may be aesthetically unpleasant but quite humane.

The efficacy of various agents and methods are evaluated on the basis of scientific data, professional judgement, and experience. Some of the reported disadvantages and controversy about certain practices are based on sentiment and anthropomorphic assessments rather than on sound scientific data.

Involuntary movements and vocalization can occur after an animal is unconscious and neither activity necessarily indicates that the animal is feeling pain.
 

Be appropriate for the age, species, and health of the animal being euthanized

Cause no fear, anxiety, or panic for an animal before it is killed or disturb other animals in the room

Be compatible with the requirements and purpose of a study and not interfere with postmortem evaluations

Be safe for operators and observers to use and cause minimal emotional stress for them

Be available, economically feasible to use and produce irreversible effects.

Pharmacologic Methods:
 

Inhalant Agents

The agent is breathed into the lungs where it either displaces air and causes a loss of consciousness by hypoxia or is diffused into the bloodstream and enters the central nervous system to cause loss of consciousness and death.

Inhalant agents can be quick and simple to use thereby decreasing handling-associated stress in animals. They are particularly valuable in animals (eg. rodents, birds, cats, and small dogs) in which venipuncture is difficult.

The vapors associated with these agents can be irritating and can induce excitement. Vapor exposure can be harmful to personnel and to other animals so a gas scavenging system or fume hood is necessary for conducting the process.

Rapid rates of flow of the gas into the euthanasia chamber can produce a noise that frightens animals.

NOTE: Newborn animals are often resistant to hypoxia and take longer to die from inhalant agents.
 
Halothane, Methoxyflurane, and Isoflurane

The agents are nonflammable and nonexplosive under ordinary environmental conditions.

These agents are relatively expensive and occupational exposure to inhalant anesthetics constitute a human health hazard.

Carbon Dioxide (CO2)

Oxygen is displaced from the respiratory system and the hypoxia produces CNS depression and death.

Carbon dioxide produces rapid central nervous system depression and anesthesia. It is inexpensive, nonflammable, nonexplosive and it does not persist as tissue residues and which could distort cellular architecture.

Any discomfort associated with sudden hypoxia can be reduced by adding CO₂ to the chamber at a rate of 20% per minute. CO₂ use presents a minimal hazard to personnel when used with properly designed equipment and it is effective for small laboratory animals such as rodents.

Carbon dioxide is heavier than air so incomplete filling of an euthanasia chamber may permit tall or climbing animals to avoid exposure to the gas. This can be avoided by precharging the chamber to full capacity.

Chloroform: Not recommended for use

Chloroform causes CNS depression and it is a potent hepatotoxin and a suspected carcinogen.

It can produce phosgene gas in the presence of a flame.

Nitrogen:

It displaces oxygen and produces hypoxia which occurs before unconsciousness in most species.

Nitrogen constitutes a minimal hazard to humans because it mixes easily with room air.

An overdose with nitrogen does not kill very young animals rapidly and it can produce a manner of death which is aesthetically objectionable in any age animal. If used, animals should be sedated or anesthetized before nitrogen use.

Carbon Monoxide (CO):

Displaces oxygen on hemoglobin and produces death by CNS hypoxia.

CO overdose induces a rapid death without pain or discernible discomfort. It is acceptable for small animals, including dogs and cats, provided that precautions are taken as prescribed by the AVMA Panel on Euthanasia (1993).

Safeguards must be taken to prevent exposure of personnel. If CO is generated by gasoline combustion engines, it must be filtered and cooled to prevent discomfort to the animals.

Diethyl Ether:

Quickly and efficiently causes CNS hypoxia of brain and loss of consciousness.

It is highly flammable and explosive. Special precautions are required not only while the agent is being used, but also during dead animal disposal since fur and tissue retain gas and remain combustible.

Noninhalant Pharmacologic Agents:

Generally, intravenous injections are the preferred route for these agents because their effects are achieved most rapidly and reliably. There is a wide range in lethal doses however, and the agents are potentially irritating to tissues surrounding blood vessels.

Oral, rectal, and intraperitoneal routes of administration are inadvisable because of the prolonged onset of drug action.

The intracardiac and intrathecal route are not recommended except in anesthetized or comatose animals. Intrapulmonic injections should be avoided.

Barbiturates: (barbituric acid derivatives)

These agents depress the central nervous system in descending order, beginning with the cerebral cortex, with unconsciousness progressing to anesthesia and death.

Euthanasia can be achieved rapidly with minimal discomfort, depending on the dose of agent and route of injection. The preferred route is intravenous administration.

These agents must be used under the supervision of personnel registered with the U.S. Drug Enforcement Agency (DEA).

An aesthetically objectionable terminal gasp may occur in some unconscious animals.

Drugs That Should Never Be Used Alone:

Chloral Hydrate: The drug depresses the cerebrum very slowly and death may be preceded by gasping, muscle spasms, and vocalization.

Magnesium Sulfate: Lacks analgesic or anesthetic effects.

Potassium Chloride: Lacks analgesic or anesthetic effects.

Curariform Drugs: Animals remain fully conscious while the muscles used for respiration become paralyzed and the animal suffocates.

Drugs Never Used for Euthanasia:

Strychnine: Excites the central nervous system causing violent convulsions and the animal remains conscious until it dies from suffocation.

Nicotine: Produces respiratory arrest before unconsciousness. The animal may perceive pain after it is immobilized.

Hydrocyanic Acid (Cyanide): Extremely hazardous to humans, and death is aesthetically objectionable.
 

Persons performing physical methods of euthanasia must be thoroughly trained in the procedure to be used. Improper performance of these procedures can cause severe pain or distress to the animal.

NOTE: These procedures have a high potential for being aesthetically displeasing to observers.

Cervical Dislocation:

When properly executed, manual cervical dislocation is a humane technique for euthanasia of poultry, other small birds, mice, rats weighing <200g, and rabbits weighing <1 kg.

In heavier rats and rabbits, the greater muscle mass in the cervical region makes manual cervical dislocation physically more difficult to achieve and thus potentially painful for the animal.

Personnel who perform cervical dislocation techniques must be properly trained and demonstrate their proficiency before using the technique.

Requests to use this method should be reviewed by the institutional animal care and use committee on a case-by-case basis.

Decapitation by Guillotine:

This method is usually limited to euthanasia of rodents and small rabbits because of the difficulty in handling and restraining larger animals. Decapitation should only be used when scientifically justified and specifically approved by the institutional animal care and use committee.

The procedure facilitates collection of brain tissue that is not contaminated with extraneous chemicals but it is accompanied by the inherent possibility for severe injury to personnel.

Focussed-Beam Microwave Irradiation:

This method is humane for laboratory rats and mice and is used primarily by neurobiologists to fix brain metabolites in vivo while maintaining the anatomic integrity of the brain.

The instruments differ in design from kitchen units and may vary in maximal power output from 1.3 to 10 kilowatts.

This is the most effective method to fix brain tissue in vivo for subsequent assay of enzymatically labile chemicals.

Adjunctive Physical Methods:

Exsanguination:

Can be used to ensure death subsequent to stunning or in otherwise unconscious animals

Because anxiety is associated with extreme hypovolemia, exsanguination must not be used as a sole means of euthanasia.

Stunning and Pithing do not necessarily cause immediate death, and must be followed with a second procedure (e.g., exsanguination, decapitation, thoracotomy) to ensure that death occurs without pain or distress.

Stunning:

Small laboratory animals with thin craniums may be stunned by a blow to the head.

A nonpenetrating captive bolt may be used to induce unconsciousness in ruminants, horses and swine.

Alternating electrical current has been used for stunning in species such as dogs, cattle, sheep, goats, hogs and chickens. Experiments in dogs have shown the necessity of directing the electrical current through the brain in order to induce rapid loss of consciousness.

Pithing:

In general, it is used as an adjunctive procedure to ensure death in an animal that has been rendered unconscious by other means.

For some species, such as frogs, with anatomic features that facilitate easy access to the central nervous system, pithing may be used as a sole means of euthanasia, but anesthetic overdose is a more suitable method.
 

Personnel handling carcasses should be advised of any potential human hazard associated with the disposal of experimental animals, such as infectious agents, carcinogens, radioisotopes or toxic chemicals.