CHAPTER 1 - Ethics and Public Perception of Animal Care and Use	CHAPTER 7 - Animal Housing and Care
CHAPTER 2 - Replacement, Reduction of Animal Care and Use	CHAPTER 8 - Handling Common Laboratory Species
CHAPTER 3 - Policies and Regulations	CHAPTER 9 - Research Animal Diagnostic and Investigative Laboratory
CHAPTER 4 - Animal Welfare Act	CHAPTER 10 - Control of Animal Pain and Distress
CHAPTER 5 - University of Missouri-Columbia Animal Care and Use Committee	CHAPTER 11 - Surgery and Postoperative Care
CHAPTER 6 - Sources of Information	CHAPTER 12 - Animal Euthanasia

Animal Euthanasia

Euthanasia can be defined as the intentional induction of death by a procedure which causes rapid and painless loss of consciousness and death. Current moral, scientific and legal principles dictate that experimental animals be killed only by methods which fulfill the definition of euthanasia. 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, drug induced anesthesia, electric shock or concussion eliminate perception of pain. Reflex motor activity may occur for a short time in an animal with a nonfunctional cerebral cortex but pain is not perceived. After unconsciousness has been induced, it is important to ensure by positive means (decapitation, cervical dislocation, or bilateral pneumothorax) that death has occurred to avoid recovery after disposal.

Euthanasia must be performed only by trained personnel familiar with the procedure. When animals utilized on research protocols are euthanatized, it is the responsibility of the principal investigator to ensure that proper procedures for euthanasia are carried out.

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 others. Therefore, whenever possible, animals should not be exposed to euthanasia of others, especially of their own species. The resultant distress may lead to release of hormones in other animals which may serve to confuse research results.

The following are methods and procedures which are accepted by the ACUC 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 isa list of nonacceptable 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 OAR are available to perform or assist in the performance of animal euthanasia.

Approved Animal Euthanasia Methods

1. Carbon dioxide inhalation is 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. In most cases, compressed CO2 from cylinders is the preferred source. Dry ice may be used with precautions to protect animals from contact and cold.
   1. Plastic bag method - Place the animal cage, containing the animals, inside a clear plastic bag with a volume at least four times that of the cage. The bag must be large enough to ensure that the animal(s) will be kept in the bottom half of the bag after it is inflated.

      After the animal(s) have settled down, press the plastic bag tightly against the sides of the cage to remove as much air as possible and introduce the hose from the C02 cylinder. Slowly inflate the plastic bag to about four times the volume of the cage and tie off the opening of the bag.
   2. Chamber method: This method employs a top-opening chamber which is charged with C02 prior to introducing the animal(s). After the animal(s) are placed in the chamber, a slow flow of C02 should be continued for a few minutes to maintain a high concentration at the bottom of the chamber.

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

1. continued exposure to C02 for 10-30 minutes after breathing has stopped. (Newborn of most species are more resistant than adults to C02 and will require 2-3 times as long to assure no recovery.);
2. exsanguination;
3. cervical dislocation,
4. Administration of injectable anesthetic or euthanasia preparation.

2. Induction of general anesthesia followed by causation of 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:
   1. continued administration of anesthetic agent until cardiac and respiratory activity has ceased. This may require artificial ventilation with some of the anesthetic gases;
   2. exsanguination;
   3. cervical dislocation,
   4. administration of injectable anesthetic or euthanasia preparation.

 

3. T-61 is a mixture of three drugs that, in combination, produce general anesthesia, local anesthetic and curariform effects. T-61 may be administered by intravenous or intracardiac routes. Administration by other routes (such as intraperitoneal, intramuscular, or subcutaneous injection) is not acceptable.
   The dose for T-61 is 0.3 mI/kg. Two-thirds of the calculated dose must be given slowly (over 1 5-20 seconds). The remaining 1/3 may be given rapidly. Failure to follow this technique may cause the animal to go through an excitement phase and may result in the production of pulmonary edema and other tissue lesions.
   Note: A major advantage of T-61 is that it is not controlled by the Drug Enforcement Administration so records are not necessary. Major disadvantages are that it is expensive, and that artifactual histologic lesions are produced.
4. Barbiturate combinations formulated for animal euthanasia are suitable for most species. Such agents include Beuthanasia - D (regular, Burns-Biotec) and Sleepaway, (Fort Dodge Laboratories). Detailed records of use of these combinations must be maintained because these are controlled substances.
   Small amounts of injectable anesthetic agents or euthanasia combinations are available through the OAR. Investigators euthanatizing large animals or many animals should arrange to purchase their own agents.
5. Physical Methods of Euthanasia:

   1. Cervical dislocation, dislocation of the neck is a simple and humane method of killing mice and small rats. 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.
   2. Decapitation is acceptable for rodents, rabbits and birds, and other animals of similar size. This requires special equipment and is aesthetically unacceptable to many. The Report of the AVMA Panel on Euthanasia recommends that animals be anesthetized when decapitated, or that the animal’s head be placed immediately into liquid nitrogen, because of electroencephalographic evidence that suggests continued cortical activity after decapitation. The ACUC has determined that decapitation, without anesthetics or placing the head in liquid nitrogen, is acceptable where there is sufficient technical justification for its use. While the use of anesthetics prior to decapitation is preferred, the technical justification for decapitation often precludes their use. The immediate placement of the decapitated head into liquid nitrogen is not required, in part because of the human safety risk.
6. Methods for killing laboratory animals which are specifically NOT acceptable without specific justification include:

   1. Drowning - inhumane;
   2. Air embolism - inhumane and unacceptable except in the anesthetized animal;
   3. Paralyzing agents (curare, gallamine, decamethonium, etc.) - inhumane and unacceptable except in the anesthetized animal;
   4. Chloroform - extremely toxic to other animals and presents undue toxicity and carcinogen hazard to personnel,
   5. Other toxic agents, such as strychnine, magnesium sulfate, potassium chloride, and nicotine are unacceptable except in the anesthetized animal.

Recommendations for Euthanasia of Reptiles

1. Inhalation of volatile anesthetic agents - may be used, but reptiles may hold their breath for long periods. Ether should not be used.
2. Overdose of injectable anesthetic agents - barbiturates - given by IV injection or cardiac puncture are technically difficult and very stressful if not carried out expertly, therefore they are not usually advocated for the induction of euthanasia.
3. Physical procedures - a skull-crushing blow, decapitation or cervical dislocation will all produce instant death and may be the most humane method when experimental requirements and considerations of human sensitivities will permit.
4. Chilling to the point of torpor (1-4oc) prior to the application of other procedures for euthanasia may be useful. The animal may subsequently be killed by further lowering the temperature to induce death by freezing.

CCAC Recommendations for Euthanasia of Amphibians:

1. MS-222 (place in a 0.1 to 1 %) - trizone methane sulfonate;
2. A blow to the head, crushing the skull, or pitching preferably after immobilizing by chilling the animals to 4oc,
3. Amphibians, even larvae, should never be killed by placing them directly in formaldehyde solution.

REFERENCES:

Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc, Vol. 1 88, No. 3, pages 252-268, February 1, 1986.

Code of Federal Regulations, Title 9, Part 1, 2 and 3.

Guide for the Care and Use of Laboratory Animals. National Research Council, 1985, NIH Publication No. 85-23:38-39.

Guide to the Care and Use of Experimental Animals, Vol. 1, 1980. Canadian Council on Animal Care. Ottawa, Ontario.