Generally, UW-Platteville, USDA, and NIH utilize the guidelines of the AVMA Panel on Euthanasia to set policy. The most recent edition was published in 1993. Modes, methods, agents, and precautions are discussed in the report. All vertebrate animals on campus that are to be killed during the course of or at the conclusion of an experiment must be euthanized using an approved method unless justified and approved by the IACUC. Question #20 on the UW-P protocol form asks for the method of euthanasia.

Euthanasia by definition is a quick, painless death. Pain can be defined as the perception that results from nerve impulses reaching the cerebral cortex. Therefore, the unconscious animal cannot experience pain. Rapidly occurring unconsciousness followed by respiratory and cardiac arrest is required for the method to be acceptable. At the conclusion of the euthanasia procedure, a follow-up exam i s required to confirm the absence of a heartbeat.

The investigator has the responsibility of choosing a proper euthanasia method both on ethical grounds and on the compatibility with the experimental data needed from post-mortem examinations. The humanitarian requirements of the method cannot be ignored in the urgency and/or need to obtain significant experimental results.

Euthanasia may be required during the course of an ex periment. Investigators have a moral obligation to alleviate unnecessary pain and suffering. "An animal that is observed to be in a state of severe pain which cannot be alleviated should be immediately euthanized. . . ¹ It is sometimes difficult to recognize pain and suffering in certain species. All personnel working with animals should be familiar with the signs of pain and suffering in their particular species. (Numerous references provide this information.) Clinically obvious depressi on, loss of appetite, and vocalization are possible signs of stress and pain.

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

MODES AND METHODS

Judging the humaneness of a particular euthanasia method can be extremely difficult for an uninformed observer. During the process of euthanasia, movements of an animal's body (muscle contractions) do not necessarily indicate consciousness, nor does lack of movement in response to painful stimuli indicate the absence of pain (such as under the influence of curariform drugs). There are three modes of action for euthanizing agents.
   1. Hypoxia/hypoxemia
   2. Direct depression of neurons
   3. Physical damage to brain (CNS) tissue
The best choice depends on the use of the animal postmortem, safety to personnel, and the ethical considerations already discussed.

METHODS

As in any procedure involving animals, restraint and handling play a vital role. The proper physical control and environment will minimize discomfort and maximize safety and smoothness of operation. The procedure should be performed out of sight and hearing range of other animals and in a calm and preferably familiar environment.

Unacceptable methods:
   1. Strychnine
   2. Curariform drugs alone are condemned
   3. Exsanguination without prior anesthesia
   4. Drowning without prior anesthesia
   5. Electrocution is strictly forbidden unless the current passes directly through the brain and personnel safety is assured

RECOMMENDED METHODS BY SPECIES

RODENTS:

   1. CO2 chamber. CO2 rapidly acts by direct depression of cerebral cortex. Length of time to effect may be prolonged in immature animals. There are no changes in cellular structure; however some involuntary muscle activity after unconsciousness may be notices. There are specific chambers made f or this purpose. It is important that the flow be adequate to eliminate layering of the gas which will cause the animals to seek any air left at the top of the chamber thus causing death from stress and not from hypoxia. This is the preferred method under the proper conditions.
   2. Barbiturate overdose. Given intra peritoneally in rodents, this works quickly, as it does in all species, by depressing CNS functioning beginning in the cerebral cortex and moving to the respiratory center. Cardiac arrest quickly follows. Pentobarbital is the most frequently used barbiturate. It will cause drug residues and an enlarged spleen. Aesthetically it is considered more acceptable due to putting the an imal under general anesthesia first. A disadvantage is that the DEA requires a license and strict accounting of all drug uses.
   3. Inhalation agent overdose (ether, halothane, methoxyflurane). These must be used under an appropriate fume hood. They act by direct depression of vital neurons.
   4. Cervical Dislocation. (Used in mice, hamsters, gerbils, and immature rats.) This technique causes direct depression of the brain. It may be distasteful to some, due to violent muscle contractions which usually follow. The technique requires training and skill. The tissues remain unaltered by chemicals. The best choice for individual animals when the technician has proper training and skills.
   5. Guillotine (decapitation). This is to be used with small rodents only. Some recommend that the head be immediately placed in liquid nitrogen. However, a new study questions the effectiveness of using liquid nitrogen. We now recommend that the animal be sedated or lightly anesthetized. There are few tissue changes with this technique. Since existing data suggests that animals remai n conscious for 13-14 seconds following decapitation, we discourage this technique and will accept it only with scientific justification and scrutiny.
   6. Nitrogen gas. Although effective in a closed chamber system, it is less preferable than other methods. Newborn animals will survive much longer than adults. Rats may exhibit signs of distress and panic before collapsing and dying. This is a hypoxic agent; tissue changes associated with hypoxemia can occur.
   7. Microwave. Only microwave chambers that are specifically designed for use on rats and mice can be used. They direct most of their energy to the head, causing immediate unconsciousness and death. The method is useful for neurobiologists in fixing brain metabolites without loss of anatomical integrity. Do not try in a conventional microwave apparatus.

FISH:
After anesthetization with MS-222, or 4-Stryrlpyridine added to the water, the fish may be exsanguinated, decapitated if small, or placed in a CO² chamber.

AMPHIBIA:
They must be tranquilized by refrigeration and decapitated or anesthetized with MS-222; then either exsanguinated or killed by a physical method.

REPTILES:
Reptiles will become more tractable if they are refrigerated at 40-45F for 15-20 min utes. Ketamine can be given to induce general anesthesia. At this point, decapitation and/or exsanguination is a suitable method for euthanasia. An overdose of pentobarbital or other barbiturate given IP of IV is preferable. Confirmation of death requires more care in reptiles, particularly if they have been refrigerated first.

BIRDS:
Because of the bird's high respiratory rate, an overdose of anesthetic inhalant agents will work well (i.e., ether, halothane, met hoxyflurane). Personnel, however, must take precautions when working with these agents. CO² also works well, but birds may show some distress before losing consciousness. Barbiturates given IV, most often injected in the wing vein, work very well. Cervical dislocation is acceptable only in chickens and small turkeys.

RABBITS, CATS, DOGS, AND HORSES:
Dogs, cats, and horses have close cultural bonding to humans. Euthanizing companion type animals is b est accomplished with an overdose of barbiturates. These drugs must be given IV, and veins in all these species are readily accessible. Horses must often be sedated prior to administering the euthanizing agent.

Non-Human PRIMATES:
Because they tend to be on long-term experiments and are well known to the animal care staff, special care must be taken when euthanizing nonhuman primates. Chemical restraint with ketamine should precede an overdose of pentobarbital giv en IV.

LARGE ANIMALS:
Large food-producing animals used in research and teaching require different considerations. Personal attachments are uncommon due to historic cultural use and the genetic breeding toward a herd animal. These animals however must be euthanized using the same criteria of rapid loss of consciousness. Their physical size and strength require special attention to the safety of the handlers. Drugs m ay leave residues which will make the meat unfit for human consumption.
   1. Captive bolt. This method causes physical damage to the cerebrum and brainstem. A retractable bolt propelled by gunpowder is directe d toward the center of the brain. The animals must be adequately restrained to ensure proper placement of the bolt. This procedure is followed with exsanguination.
   2. Intravenous injection of barbiturates. If the animal is not to be used for food consumption, this is a preferred method.
   3. Electrocution. The only way to produce immediate unconsciousness is to apply electrodes to either side of the head, directing the current directly through the brain. It must be followed with exsanguination. This technique is primarily used on swine. Specific approval from the IACUC veterinarian must be obtained; this is usually done via protocol review. This method can be hazardous to the persons administering the electricity, so appropriate precautions must be taken.