Guidelines for Euthanasia

(Material adapted from the University of Iowa Animal Care Unit and Stanford University)

• General
• Approved Euthanasia Methods for Rodents
• Table of Euthanasia Methods
• Comments on Recommended Agents and Methods of Euthanasia

The euthanasia method chosen must be appropriate for the species and research use described by the protocol, and must be consistent with the current recommendations of the American Veterinary Medical Association Guidelines on Euthanasia.

General

The NIH Guide defines euthanasia as "the procedure of killing animals rapidly and painlessly". University of California, Riverside euthanasia guidelines, summarized in the Office of the Campus Veterinarian follow those established by the American Veterinary Medical Association Panel on Euthanasia. Euthanasia techniques must be reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) during review and approval of the Animal Use Protocol Form.

Euthanasia must be carried out by personnel properly trained in the procedure being used. While decapitation and cervical dislocation may be humane when administered by properly trained personnel, animal use protocols proposing these techniques without sedation or anesthesia must include the scientific rationale justifying this exclusion. Measures should be taken to ensure that euthanasia is performed in a way that minimizes reactions among other animals that may be present.

Gentle, careful handling of subject animals is of the utmost importance during the procedure in order to minimize distress to the animal, as well as to the operator. An unconscious animal does not perceive pain. Appropriately conducted procedures which render the cerebral cortex nonfunctional by means such as hypoxia or drug induced anesthesia eliminate perception of pain. Reflex motor activity may be present in an animal with a nonfunctional cerebral cortex, but pain is not perceived.

Proper euthanasia technique includes a follow-up exam to confirm the absence of a heartbeat, which is a reliable indicator of death. Monitoring respiration is not considered sufficient since with some euthanasia techniques heartbeat may be maintained after visible respiration has ceased. Decapitation, cervical dislocation or thoracotomy should be used after administration of euthanatizing drugs to insure that animals do not revive.

The need to minimize fear and apprehension must be considered in determining the method of euthanasia. Distress vocalizations, fearful behavior, and release of certain odors or pheromones by a frightened animal may cause anxiety and apprehension in other animals. Therefore, whenever possible, animals should not be exposed to euthanasia of others, especially of their own species. The resultant distress may lead to physiologic changes in other animals, such as a release of hormones, which may effect research results. The acceptable methods of euthanasia vary aesthetically. Personnel's perception must often be considered in addition to experimental requirements when a method of euthanasia is chosen.

The following are methods and procedures which are accepted by the IACUC for humane killing of animals. In general these are the same as recommendations of the American Veterinary Medical Association (AVMA) Panel on Euthanasia, 1986. Methods other than those generally approved may be approved with adequate justification based on scientific need and demonstration that the method chosen produces the minimum amount of animal pain and distress consistent with experimental requirements. Also included is a list of unacceptable methods deemed inhumane or dangerous to personnel and to other animals.

Many of the approved methods of euthanasia require technical proficiency for proper conduct and should not be attempted without prior training (e.g., intravascular injections and physical methods). Other methods, such as carbon dioxide inhalation and electrocution, may only be used with properly designed equipment. Trained personnel in the ACU are available to perform or assist in the performance of animal euthanasia.

Approved Euthanasia Methods for Rodents

• Carbon Dioxide
• Anesthesia
• Physical Methods

Carbon Dioxide

Carbon dioxide inhalation can be suitable for all species, provided acceptable equipment is used. Practically, its use is limited to rodents and other mammals weighing less than about 500 grams. Compressed CO2 from cylinders is the only acceptable source. Dry ice is no longer permitted as a CO2 source.

Chamber method - This method employs a transparent top-opening chamber which is charged with CO2 prior to introducing the animal(s). Please note: An opaque chamber is not acceptable as this prevents the visual assessment of the animal and may prolong the suffering of the animal. After the animal(s) are placed in the chamber, a slow flow of CO2 should be continued for a few minutes to maintain a high concentration at the bottom of the chamber.

After breathing has stopped and the animal(s) are unconscious, euthanasia may be completed by any of the following procedures:

• continued exposure to CO2 for 10-30 minutes after breathing has stopped (newborn of most species are more resistant than adults to CO2 and will require exposure times 2-3 times as long to assure death.);
• exsanguination;
• cervical dislocation;
• administration of injectable anesthetic or euthanasia preparation.

Anesthesia

Induction of general anesthesia followed by death without regaining consciousness is acceptable. Inhalation of anesthetic gas or injection of parenteral general anesthetic agents are suitable for most species including pig, dog, cat, rodents, rabbits, birds and primates. Ether is extremely flammable and not acceptable unless used with adequate protection against the risk of explosion. After anesthesia is induced by usual means, euthanasia may be completed by any of the following procedures:

• continued exposure to CO2 for 10-30 minutes after breathing has stopped (newborn of most species are more resistant than adults to CO2 and will require exposure times 2-3 times as long to assure death.);
• exsanguination;
• cervical dislocation (in small animals less than 200 gm);
• administration of injectable anesthetic or euthanasia preparation.

Barbiturates such as pentobarbital or barbiturate combinations formulated for animal euthanasia are suitable for most species. Such agents include Beuthanasia-D (Burns-Biotec) and Sleepaway (Fort Dodge Laboratories). Detailed records of use of these combinations must be maintained because these are controlled substances. Typically the dosage for euthanasia is three times the dose required for anesthesia.

Physical Methods

Cervical dislocation (dislocation of the neck) is a simple and humane method of killing mice and small rats (<125 gms). Although not required, the use of sedation or anesthesia prior to euthanasia is encouraged. If sedation or anesthesia prior to cervical dislocation is not used the investigator must provide scientific justification for its exclusion. In the case of mice and small rats, the animal is held by its tail and placed on a surface that it can grip, then it will stretch itself out so that a pencil or similar object can be placed firmly across the back of the neck. A sharp pull on the base of the tail will then dislocate the neck.

Decapitation is acceptable for rodents, rabbits and birds, and other animals of similar size. This requires special equipment and is aesthetically unacceptable to many. Because of electroencephalographic evidence suggesting continued cortical activity after decapitation the Report of the American Veterinary Medical Association Panel on Euthanasia recommends that animals be anesthetized when decapitated or that the investigator provide scientific justification for the use of this technique without anesthesia or sedation. While the use of anesthetics prior to decapitation is preferred, the scientific justification for decapitation may precludes their use. In lieu of the use of anesthetics, prior recommendations have required the immediate placement of the decapitated head into liquid nitrogen. This is still an acceptable alternative however it is not required in part because of the human safety risk.

Table of Euthanasia Methods

• Rabbits & Rodents <1kg

• Rabbits & Rodents >1kg

• Avian

• Amphibians

• Fish

• Reptiles

• Barbiturate euthanasia dosages

Amphibians

• Inhalant anesthetics
• CO₂
• Barbiturates
• Tricaine methane sulfonate (MS222)
• Double pithing
• Benzocaine
• Conditionally acceptable - Single pithing; stunning and decapitation; decapitation and pithing

Fish

• Tricaine methane sulfonate (MS222)
• Benzocaine
• Barbiturates
• Inhalant anesthetics
• CO₂
• 2-phenoxyethanol
• Conditionally acceptable - stunning followed by decapitation/pithing; decapitation and pithing

Reptiles

• Barbiturates
• Inhalant anesthetics (in appropriate species)
• CO₂ (in appropriate species)
• Conditionally acceptable - stunning and decapitation; decapitation and pithing

Barbiturate euthanasia doses (mg/kg):

NOTE: Euthanasia with barbiturates usually requires approximately 3X the anesthetic dosage.

Adapted from the report of the American Veterinary Medical Association Panel on Euthanasia (J. Am. Vet. Med. Assn. 202:229-249, 1993). The JAVMA article provides the rationale for these recommendations. The University Animal Care and Use Committee reviewed approved this table.

These methods are in accordance with humane euthanasia as defined by the Federal Animal Welfare Act (54 FR 36112-36163).

Methods always unacceptable in an awake animal include: potassium chloride, magnesium sulfate, strychnine, neuromuscular blocking agents, exsanguination, air embolism, and chloroform.

Comments on Recommended Agents and Methods of Euthanasia

• Anesthetics
• Pharmacological Agents
• Physical Methods

INHALANT ANESTHETICS - Because in the liquid state most inhalant anesthetics act as topical irritants, animals should be exposed to the vapors of the anesthetic only. Air or oxygen must be provided during the induction period. All agents are given "to effect" until respiratory and cardiac arrest occurs.

Halothane and isoflurane have the most rapid action, and since halothane is better tolerated, it is preferred. Methoxyflurane is less suitable, due to its slow effect and poor market availability. Care should be taken to minimize personnel exposure to vapors.

Ether is acceptable but not recommended because it poses an explosive hazard and is a respiratory irritant that is considered stressful to animals. It cannot be used in the UCR centralized facilities, and special precautions must be taken when used elsewhere. Administration should be performed in a fume hood, and signs indicating that ether is present or in use should be posted conspicuously. To avoid explosions, the carcasses of ether-killed animals should be stored in explosion-safe refrigerators or freezers, and should not be incinerated until the ether is removed by aeration in a hood. Methoxyflurane is a similar but non-explosive and less irritating agent which is recommended as a substitute for ether.

NON-ANESTHETIC GASES - Most agents in this category require the use of special equipment.

CO₂ - Carbon dioxide is the preferred technique for euthanizing rodents and other small laboratory animals. Use of a sealed chamber filled by a compressed gas cylinder is required. CO₂ generated by other methods, such as from dry ice, is unacceptable because gas flow can't be regulated precisely. Chambers should not be overcrowded. CO₂ concentration of 70% or more should be utilized for euthanasia. Because CO₂ can act as a reversible anesthetic, it is imperative that the animals be kept in the chamber for several minutes after respiratory arrest. Where possible, death should be verified by absence of a palpable heart beat. Due to physiologic characteristics, neonates require prolonged exposure to the gas. For more information, see the IACUC guidelines on using CO₂ for rodent euthanasia.

Nitrogen or carbon monoxide may be acceptable but are not recommended and require special equipment.

PHARMACOLOGICAL AGENTS - Use of these agents requires adequate restraint and mastery of appropriate injection techniques.

Barbiturates such as pentobarbital are acceptable for mammalian species and birds. These drugs should be administered intravenously (IV) except in rodents where intraperitoneal (IP) administration is an acceptable alternative. Sodium pentobarbital (Nembutal) is the most common barbiturate agent for euthanasia. The dosage is usually at least twice that required for anesthesia, and ranges from 85 mg/kg for larger species to 200 mg/kg for some rodents. A dosage of 120 mg/kg is sufficient for most species, but more should be given if death does not ensue. Commercial barbiturate euthanasia formulations as are also appropriate, and should be used following label directions (e.g., 1 ml/lb for Beuthanasia-D*). Sodium pentobarbital is a Class II drug which is regulated by the Drug Enforcement Agency. Personnel using this agent are required to store it in a locked location and maintain records which include the date and amount of use.

Chloral hydrate is not recommended, but may be used in ruminants and swine when administered I.V. at 900 mg/kg, but only after sedation with another drug.

Neither magnesium sulfate nor potassium chloride (KCl) can be used as a sole agent of euthanasia. Overdose with KCl is permissible in an anesthetized animal. Concentrated KCl should be given rapidly IV until rising serum potassium levels result in cardiac arrest.

Tricaine methane sulfonate (MS222) can be used either as an injectable agent (200-300 mg/kg of a 1% buffered solution) or as an immersion bath (2 mg/ml in H2O) for amphibians and fish. The immersion time needed to assure death can range from 20 minutes to three hours, so it may be advantageous to use MS222 as an anesthetic followed by a physical method of euthanasia. Benzocaine immersion (100-200 mg/liter H2O) is also acceptable.

Neuromuscular blocking drugs are absolutely condemned for use as euthanasia agents.

PHYSICAL METHODS - These methods require that the user have experience and skill in the techniques to be used.

Exsanguination is acceptable for all species if animal is first rendered unconscious by another agent.

Cervical dislocation is acceptable for mice, birds, rats (< 200 gm) and rabbits (< 1 Kg), but proper technique is essential. It is therefore recommended that animals be first sedated with another agent (carbon dioxide, pentobarbital or halothane are suggested). Its use as a sole means of euthanasia requires scientific justification and IACUC approval for more information, see the IACUC guidelines on the use of cervical dislocation for euthanasia of rodents.

Decapitation with proper equipment may be performed on small mammals or birds after the animal has been sedated or lightly anesthetized (carbon dioxide, pentobarbital or halothane are suggested). Decapitation of fish, amphibians and reptiles should be followed by pithing. Use as a sole means of euthanasia in any species requires scientific justification and IACUC approval. Decapitation should generally be used only when study design requires it due to the potential hazard to personnel. Many species react adversely to the smell of blood, so animals should not be decapitated in the presence of other animals and the person performing decapitation should change gloves and/or wash hands between animals.

Pithing of both the brain and the spinal cord (double pithing) may be used as the sole means of euthanasia in frogs of the genus Rana or other amphibians with anatomic features that facilitate easy access to the central nervous system. In all other amphibian and reptile species pithing should be followed by decapitation.

Under very specialized circumstances, stunning, rapid freezing or air embolism (under anesthesia) may be allowed in small species if research needs make it necessary and there are no available alternatives.