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CCAC, Guide Vol. 1 (2nd Ed.) 1993

XII. EUTHANASIA

A. INTRODUCTION

The term "euthanasia," is derived from the Greek terms "eu" for "good" and "thanatos" for "death" or an easy death (Bennett, Brown, Schofield et al. 1990). However, the term "euthanize" will be used in this Chapter, rather than the more accurate "euthanatize." Whichever term is used, the method must be "humane": that is, it must be painless, must minimize fear and anxiety, be reliable, reproducible, irreversible, simple, safe and rapid. If possible, it should also be esthetically acceptable for the person carrying out the procedure, as well as for any observer.

In the 1950s, the term "euthanasia" was rarely heard; euphemisms included "sacrifice," "destroy," "put down," or "put to sleep" (Zweighaft, 1990). However, rarely were these terms prefixed with the word "humane"; nor was it considered necessary. Humane Slaughter Acts were not promulgated and applied until the late 1950s and early 1960s in Canada or the USA. Even with the advent of legislation in this regard, many of the meat-producing species, e.g., fowl, were not included under the Regulations.

In the use of animals in research, teaching, and testing it is essential that the scientific community take on the mantle of responsibility for applying scientific judgement and new knowledge to ensure that, when the life of an animal is taken, it is assured of a "good death." Even with non-consumptive or non-invasive studies using animals, there are occasions when it is necessary to euthanize the animal (e.g., return of a wild animal to a hostile habitat) (see also Categories of Invasiveness found elsewhere in this Guide).

In the first edition of this volume of the Guide, the Chapter on euthanasia states: "The most important criterion of acceptance of a euthanasia method as humane is that it have an initial depressive action on the Central Nervous System (CNS) to ensure immediate insensitivity to pain." Although the principle of this criterion remains sound, to the original wording should be added "to produce rapid unconsciousness and thus assurance of insensitivity to pain; this must be followed by cardiac and respiratory arrest."

It is important that Russell and Burch's (1959) "Three R" principle noted elsewhere in this Guide, be equally applied to methods of euthanasia. Refinement of procedures is an often-neglected area and one which should be addressed in order to ensure that the criteria for a humane death are in place.

The application of these euthanasia guidelines requires the use of professional judgement, technical competence, coupled with an understanding of the animal, its behaviour and its physiology, as well as an understanding of the environmental and ecological impact, the sensitivities of other personnel and the concerns of the general public.

B. CRITERIA FOR A HUMANE DEATH

The person applying the method of euthanasia is a most important factor in ensuring that an animal's death is humane. Regardless of whether the procedure is applied to an individual animal or to a group, it must always attempt to meet the following criteria:

a) death without signs of panic, pain or distress;

b) minimum time to loss of consciousness, i.e., shortest lag time;

c) reliability and reproducibility;

d) safety for personnel involved;

e) minimal undesirable physiological and psychological effects on the animal;

f) compatibility with the requirement and the purpose of the scientific study;

g) minimal or no emotional effects on the observer and the operator;

h) minimal environmental or ecological impact;

i) simple, inexpensive mechanical equipment which is relatively maintenance free; and;

j) a location remote and separate from the animal rooms.

It is often difficult to recognize evidence of stress when animals are euthanized in the presence of other animals. Recent information on pheromones provides evidence that animals can communicate with one another through various types of signals. In certain experiments with rats, stress induced by experimental treatment may give rise to the production of signals that affect non-treated animals housed nearby (Duncan and Petherick, 1991; Beynen, 1992; Short and Van Poznak, 1992).

C. PAIN AND STRESS

The control of animal pain is discussed elsewhere in this Guide (see Control of Animal Pain in Research, Teaching and Testing) and should be reviewed by those conducting euthanasia procedures. The literature is growing regarding animal pain (Dawkins, 1980, 1990; Bateson, 1991; Flecknell, 1984; Wall, 1992; Fosse, 1991; Rowsell, 1992). It is sufficient to note that, over the past 25 years, there has been a revolution in our understanding of pain mechanisms which was generated by experimental work on animals. We cannot get inside the head of the animal; thus, an important part of assessing animal pain is empathy coupled with the ethical concerns.

Briefly, it is believed that, for pain to be experienced, the cerebral cortex and sub-cortical structures must be functional. If the cerebral cortex or sub-cortical structures are rendered non-functional by any method, such as hypoxia, pharmacological depression, electric shock or concussion, then the feeling of pain is inhibited. Unfortunately, we have no critical means of determining the adequacy of anesthesia and the assessment of its depth in animals, whereas a range of different measures have been used for determining the adequacy of anesthesia in humans (Whelan and Flecknell, 1992). The crude criteria available to judge unconsciousness in an animal include, for example, the absence of a blinking reflex, toe pinch reflex and tail reflex. Rarely is an electroencephalogram (EEG) available showing complete flattening of the EEG as an indication of brain death. This is the criterion adopted by the American Academy of Neurology as acceptable for establishing brain death in young children after other clinical criteria, such as deep coma, failure to breathe spontaneously, and the absence of reflexes occur (Anon., 1987).

In assessing death, it is important to observe that heart action has stopped, thus ensuring a cessation of blood delivered to the brain, as well as the cessation of respiration. No animal should be considered dead until reflex movement as well as cardiac and respiratory movements, have ceased.

If an animal has been given a curare-like preparation, the absence of reflexes should not be used to indicate unconsciousness and thus insensitivity to pain.

D. MECHANISMS FOR CAUSING DEATH

Death can ensue when the brain is affected by hypoxia, direct or indirect, when there is direct depression of neurons essential for living physiological functions, or there is physical disruption of brain activity to produce unconsciousness.

a) In hypoxia, death must be considered painless and free of stress only when unconsciousness precedes the loss of muscle activity (paralysis). Paralyzing agents (e.g., curariform agents such as curare, succinylcholine, gallamine, nicotine sulphate, magnesium or potassium salts and other neuromuscular blocking agents) must never be used alone for killing animals (Rowsell, 1990). Following unconsciousness due to brain hypoxia, some animals exhibit a degree of reflex motor activity.

b) Direct depression of neurons: the depression of neurons of the brain which produces unconsciousness and then death, is sometimes associated with vocalization and muscle activity. Death is produced by hypoxemia, direct depression of the respiratory centres in the CNS, or cardiac arrest.

c) Physical disruption of brain activity produces immediate unconsciousness; however, there may be marked physical muscular activity due to the depolarization of the nerve cells. While the movement is esthetically unpleasant, it is not a manifestation of pain or distress: the animal feels nothing.

E. METHODS USED FOR EUTHANASIA

1. Physical

Physical methods of euthanasia include stunning, cervical dislocation, electrocution, pithing, decapitation, shooting, maceration, microwave radiation, and exsanguination.

In the laboratory, physical methods are normally restricted to those animals which are easily handled, such as small rodents, poultry, large domestic animals, and some amphibians and reptiles. If the research protocol requires a physical method of euthanasia because other methods could invalidate the scientific study, the use of such methods must be justified by the scientist and approved by the Animal Care Committee (ACC). Prior sedation or tranquillization should take place whenever possible.

Decisions to use physical methods for euthanasia must be based on professional judgement and be undertaken only by experienced individuals. Acquiring (or re-acquiring) the skills to use physical methods of euthanasia may be accomplished by practising the techniques on dead animals, preferably those recently killed, and be subject to close scrutiny by those experienced in the methodology.

a) Stunning is sometimes used with small laboratory rodents. The blow must be delivered to the central skull bones with sufficient force to produce massive cerebral hemorrhage and thus immediate depression of the CNS, producing rapid unconsciousness. This technique should not be undertaken in the presence of casual observers or the uninformed, for it is esthetically unpleasant. However, when properly applied, the animal is immediately rendered unconscious and thus insensitive to pain. Subsequent to stunning, the animal's major blood vessels should be cut, and the chest and the heart opened.

b) Cervical dislocation is suitable for poultry, mice, immature rats or rabbits, or similar small species. The technique consists of separation of the skull and the brain from the spinal cord by pressure applied posterior to the base of the skull (Clifford, 1984). When the separation of the cord occurs, CNS stimulation of respiration and heart beat is interrupted, leading to death. The supply of blood to the brain continues to nourish it because the carotid arteries and jugular veins are intact; however, the blood will rapidly be depleted of oxygen and there will be an increase in carbon dioxide after respiration ceases, leading to brain disfunction.

Studies have demonstrated that the EEG flattens and the blinking reflex disappears immediately after the spinal cord separates, thus indicating that the animal is not sensitive to pain (Allred and Berntson, 1986; Rowsell, 1990; Derr, 1991). In addition, the severed spinal cord does not deliver a painful stimuli from areas posterior to the separation; thus, with the separation of the spinal cord from the brain, painful stimuli cannot be perceived. However, significant muscular movements may take place.

The Report of the AVMA Panel on Euthanasia (AVMA, 1993) differs from its 1986 predecessor in that it notes that data suggest that electrical activity in the brain persists for 13 seconds following cervical dislocation (Vanderwolf, Buzsaki, Cain et al. 1988). In addition to mice, it lists as suitable subjects immature rats weighing less than 200 g, rabbits weighing less than one kg, poultry and other small birds. It also notes that: "In heavier rats and rabbits, the greater muscle mass in the cervical region makes manual cervical dislocation physically more difficult; accordingly, it should be performed only with mechanical dislocators or by individuals who have demonstrated proficiency in euthanatizing heavier animals." The need for proper training is stressed.

c) Decapitation with guillotine is used primarily to euthanize rodents and small rabbits. Used alone, it provides a means of ensuring that tissues and body fluids are chemically uncontaminated as well as providing a means of obtaining an anatomically intact brain and brain tissues for further study.

After consulting the literature (Vanderwolf, Buzsaki, Cain et al. 1988; Derr, 1991; Mikeska and Klemm, 1975), the Canadian Council on Animal Care (CCAC) concurs with the Report of the AVMA Panel on Euthanasia (AVMA, 1993) that pre-sedation before decapitation or cervical dislocation is not necessary. However, the use of cervical dislocation and decapitation with guillotine as euthanasia methods must be scientifically defended by the investigator and approved by the ACC. Well-designed and easily operated guillotines are available from commercial sources. Guillotines should not be used by personnel who have not been properly trained in the methodology and how to properly handle the animal. Decapitation as a means of euthanizing amphibia and reptiles is not recommended (AVMA, 1986; Cooper, Ewbank, Platt et al. 1989).

d) Pithing, which is used to euthanize frogs and turtles by destroying the brain after the frog has been anesthetized, requires considerable skill. A sharp, pointed probe is inserted through the skin between the skull and the atlas. It is then pushed forward through the foramen magna into the cranial cavity, using a twisting motion. The technique should be attempted only after acquiring knowledge of anatomy using skeletons, and after a period of training including practice on dead animals. This method can cause pain and suffering if the proper regions of the brain are not completely destroyed.

e) Shooting by penetrating captive bolt pistol has been used primarily for pre-slaughter stunning of food animals. To a lesser extent, it is used in emergency situations such as roadside injuries or other similar events where no other method, or the expertise necessary to apply it, is available.

Captive bolt pistols are also used in emergency situations in slaughter of horses. However, because of their behavioural/physical response of rearing on their hind legs and falling backwards, unless proper restraints are applied, there is a danger to the operator. It is essential that the captive bolt pistol be withdrawn from the animal's head instantaneously. An extension handle is attached to the pistol to allow the operator to put one or both hands in front of the horse's eyes, thus reducing head tossing. This specially designed captive bolt pistol for killing horses requires knowledge of the placement needed in order to penetrate the deep structures of the brain (Watts, 1976).

Britains' Royal SPCA, following the use of the captive bolt pistol in the abattoir, developed a smaller, hand-held model for emergency euthanasia of injured dogs and cats which was used by some SPCAs and humane societies (UFAW, 1968). In Canada, however, it was considered esthetically repugnant by the general public and therefore its use was limited to those occasions when the injured animal could be removed from public view (UFAW, 1988).

More recently, a captive bolt pistol was developed for killing rabbits and goats (Accles and Shelvoke Ltd., Aston, Birmingham, Eng. B64QD). With this device, the clinical evidence, including the loss of corneal reflex and organized EEG activity, suggested that loss of consciousness and cerebral death occurred almost immediately (Dennis, Dong, Weisbrod et al. 1988).

Only commercially produced penetrating or captive bolt pistols should be used. Proper utilization is essential. Considerable technical competence and precise knowledge of the animal's anatomy are necessary; therefore, this method should be used only by an experienced operator. Exsanguination must follow unconsciousness.

f) Percussion stunning is carried out by means of a non-penetrating form of captive bolt stunning device with mushroom-shaped tip. The effects on brain function will depend on the speed of the bolt on impact, its proper positioning, and the thickness of the cranial bone. Unfortunately, the minimum speeds necessary for effective percussions have not yet been determined. The value of this method is that it can be used to replace some ritual slaughtering methods. Percussion stunning has been used as an alternative method to the penetrating captive bolt by some Canadian abattoirs. Unfortunately, the instruments are more subject to breakdown and malfunctioning than the standard penetrating captive bolt. More recently, a percussion-type instrument has been developed for use in animal control, particularly for dogs. Its feasibility and its humaneness as a technique need to be established in the field operations.

Captive bolt pistols, including percussion stunning devices, require permits for their possession in Canada.

g) Shooting is an effective means of humanely destroying animals in the field. Only experts should carry out this procedure. The subject must be shot at close range and the bullet must strike the brain so as to render the animal immediately insensitive to pain; 12-bore or 20-bore shotguns, or 22-calibre rifles or revolvers may be employed, depending upon the species and size of the animal to be killed. Whether or not prior sedation should be used is debatable (Ebedes, 1988). The use of shooting by firearms in a laboratory setting is prohibited. In such settings, there are always other available methods of euthanasia and individuals with appropriate expertise.

There are advantages of shooting as a euthanasia method in the field, for unconsciousness is instantaneous if the bullet destroys a significant portion of the brain involving particularly the vital centres. In the field, this may be the only possible method to render the animal immediately unconscious and to produce death. The need for expertise of the marksman cannot be overemphasized. For use in emergency situations, Guidelines for Euthanasia of Domestic Animals by Firearms have been prepared by the Animal Welfare Committee of the Canadian Veterinary Medical Association (CVMA) (Longair, Finley, Laniel et al. 1991).

h) Electrocution, used mainly to kill domestic animals (Eikelenboom, 1983) is rarely used in the laboratory setting. If electric shock is used, it must be delivered in two phases: the first electric shock passes through the brain, stunning the animal; the second, delivered a fraction of a second later, produces a fibrillation of the heart, killing the animal. Violent extension of the extremities, part of the seizure induced by electrocution, is esthetically unpleasant.

i) Microwave radiation is a relatively new technique employed mainly by neuroscientists who wish to maintain the anatomic, enzymatic and physiological chemical composition of the animal's brain in an unaltered state (Stavinoha, 1983; Ikarashi, Maruyama and Stavinoha, 1984). Microwave radiation must be delivered specifically to the brain; therefore, standard household microwave ovens must not be used (Stavinoha, Frazer and Modak, 1977); only instruments which have been designed specifically for this purpose and have the appropriate power and microwave distribution may be utilized.

j) High altitude decompression is considered unacceptable by the CCAC. At one time, it was used by some animal control agencies and humane societies for killing unwanted dogs and cats, but is now not recommended by them (White, 1984). When animals have upper respiratory problems or gastrointestinal upsets, the gases in these areas, as well as in the sinuses, expand and cannot be vented, thus producing significant pain and distress (White, 1984).

k) Exsanguination (depriving an animal of blood) is only acceptable as a euthanasia procedure if the animal is first rendered unconscious by physical means such as stunning, or pharmacological means, e.g., injection of an anesthetic (Gregory and Wotton, 1984). The use of this method must be scientifically justified and approved by an ACC, who must have established the technical competence of the person who will conduct the technique.

l) Maceration requires the application of severe restrictions on the size and age of the animals that may be subjected to this process (Ewbank, 1987). For example, it is used to kill newborn mice, and in some poultry operations for killing surplus one-day-old male chicks, where it has been deemed acceptable by Agriculture Canada. Equipment has been specifically designed for this purpose and, although esthetically unpleasant to consider as well as to observe, the method produces instantaneous unconsciousness and death when used properly.

m) High pressure water jet has recently been proposed for stunning slaughter pigs (Schatzmann, Leuenberger, Fuchs et al. 1991). However, its use has not been reviewed by other experts qualified to evaluate humane slaughter techniques.

2. Non-inhalant Pharmacologic Agents

The majority of injectable drugs used as anesthetic agents are acceptable for euthanasia if an adequate overdose is given. The preferred route is intravenous (IV), and should be accompanied by adequate restraint, making the animal as comfortable as possible with minimum distress or anxiety. Pre-sedation or tranquillization may be necessary for wild, feral or fearful animals not accustomed to restraint.

If the animal is too small to receive intravenous injections, or if anatomically suitable veins are not visible or apparent, e.g., in small rodents and guinea pigs, the intraperitoneal (IP) injection of a non-irritating overdose of a pharmacological agent is acceptable.

With most of the available injectable anesthetic agents, the amount to be injected is too large to use the intramuscular, subcutaneous, intrathoracic, intrapulmonary, and intrathecal sites. Administration by a route other than intravenous in most cases results in a delayed onset of the anesthetic effect of the drug. Under these circumstances, it is essential that the animal be placed in a cage or enclosure to ensure prevention of injuries through stumbling and or falling, and in order to make the animal more comfortable and facilitate onset of the overdose of anesthetic.

a) Barbituric acid derivatives (barbiturates) used as anesthetics are effective in producing euthanasia when given as an overdose. The pharmacological action of these drugs is to depress the CNS, starting with the cerebral cortex, and progressing through the stages of anesthesia to produce unconsciousness. With an overdose, deep anesthesia develops, followed by apnea as the respiratory centre is depressed, followed by cardiac arrest and death. Some of the combination barbiturate derivatives have a cardiotoxic effect; however, this is of no consequence because the animal dies before such an effect histologically manifests itself.

Barbituric acids are controlled substances under regulation by the Bureau of Dangerous Drugs, Health and Welfare Canada. As controlled substances, they must be stored in locked cabinets and a written log maintained of date and amount used and the purpose of use. Euthanasia agents containing barbituric acid derivatives are often coloured to make them clearly identifiable. The amount used for euthanasia should be in accordance with the manufacturer's direction. The abuse or misuse, either accidental or deliberate, of such substances creates a significant risk and associated legal liability. Those possessing such combinations for euthanasia must provide adequate security.

b) T-61 is manufactured by Hoechst-Roussel Canada Ltd. (4045 Cote Vertu, Montreal, Quebec, H4R 2E8). It contains a local anesthetic (tetracaine Hcl), a strong hypnotic agent which depresses the CNS causing unconsciousness (brain death), as well as a curariform drug which has a paralytic effect on the respiratory centre and a relaxing effect on skeletal muscles (Rowsell, 1979). A recent study demonstrated that induction of muscle paralysis and unconsciousness occur simultaneously (Hellebrekers, Baumans, Bertens et al. 1990). These authors concluded that the muscular activity and vocal response seen in some dogs was not a conscious response.

T-61 should be administered intravenously at the dose and administration rate directed by the manufacturer. If instructions are not followed, it is possible for T-61 to produce an excitatory phase and vocalization. It is not registered or restricted by the Bureau of Dangerous Drugs and may be used by non-medical, technical personnel. However, it must be ordered by a veterinarian and be shipped directly to the veterinary clinic involved (Clarke, 1990). Its availability in other countries has been affected because of criticism levelled primarily as a result of failure to follow the manufacturer's direction for the dosage and the rate of injection. Although this is not a restricted drug, the same guidelines concerning its safekeeping apply as to any of the barbituric acid derivatives and anesthetics, for its use has been abused (Smith and Lewis, 1989).

c) Chloral hydrate is a dissociative anesthetic and produces no loss of the corneal or blinking reflex. Difficulties include slow onset of action, restraint difficulties, and the amount that must be administered. Death is due to hypoxemia caused by progressive depression of the respiratory centre. It may be preceded by gasping, muscle spasms, and vocalization, causing difficulties in comfortably restraining the animal being euthanized. Therefore, while it should not be used for dogs and cats, it is acceptable for intravenous use in large animals and is an effective euthanasia agent for poultry.

A combination of chloral hydrate, magnesium sulphate, and sodium pentobarbital, administered intravenously to large domestic animals as an overdose, is an acceptable method of euthanasia.

d) Ketamine hydrochloride, also a dissociative anesthetic, cannot be recommended for euthanasia because it is difficult to assess what constitutes an overdose.

e) Magnesium sulphate alone is a neuromuscular blocking agent (Hevner and de Johng, 1973); however, it does not depress the CNS. Administration of magnesium sulphate should only be used in combination with barbituric acid derivatives and administered only by the IV route. The IP route is not acceptable because of the irritating nature of a saturated solution.

3. Inhalant Anesthetics

An overdose of inhalant anesthetics such as ether, halothane, methoxyflurane, isoflurane, and enflurane fulfils the principle of a humane death. Their use, however, poses a risk to human subjects who may be exposed to their vapours; thus, they are considered an occupational hazard (see Anesthesia, as well as Occupational Health and Safety). Chambers are available commercially to expose animals to such anesthetic gases in order to either produce anesthesia or, by overexposure, to produce euthanasia. Scavenging systems to remove excess gases are readily adapted to such enclosures. Additionally, anesthetic masks can be prepared to fit even small rodents. The vapours are inhaled until respiration ceases. The animal is then checked to ensure that it is dead.

The soaking of gauze with inhalant anesthetics and then placing it in a container with the animal(s) to be euthanized may be used only if there are no other methods of delivery of the anesthetic gases. The fact that inhalant anesthetics are liquid makes it essential that animals be exposed to vapours only, as the liquid form is a topical irritant. The delivery system should provide sufficient oxygen with the anesthetic vapour to ensure unconsciousness precedes hypoxia.

a) In the past, chloroform and ether have been commonly used as anesthetics or, when exposure to their vapours is of sufficient concentration and duration, to produce euthanasia. However, chloroform is no longer recommended because of its carcinogenic, hepatotoxic and nephrotoxic potential. Ether is a flammable and explosive agent, and should never be used in the presence of flame or where electrical equipment is not protected and shock resistant.

b) Nitrous oxide is of value as an agent for euthanasia only in combination with other volatile inhalant anesthetics. It is a combustible agent, but is non-flammable and non-explosive. With the exception of ether, most inhalant anesthetic agents are expensive and require special anesthetic delivery mechanisms; thus, their use as euthanasia agents is limited to species where venipuncture is found to be too difficult or impossible. Additionally, use of inhalant anesthetics for euthanasia of large animals is expensive because of the amounts that must be used.

4. Non-anesthetic Gases

Non-anesthetic gases include carbon monoxide, carbon dioxide, nitrogen, argon and cyanide.

a) Carbon monoxide, even in low concentrations, can harm other animals and humans exposed to its fumes. A colourless, odourless gas, it is difficult to detect. Carbon monoxide obtained from the exhaust of a gasoline combustion engine contains impurities and thus can produce irritation and discomfort. Therefore, delivery of an irritant-free carbon monoxide is mandatory if this is the chosen method. Carbon monoxide is rarely used now to destroy unwanted dogs and cats by animal control agencies and humane societies; however, it continues to be used for some of the fur-bearing species. In the laboratory, because of the safety problems associated with its delivery, carbon monoxide is not recommended as a euthanasia agent (Chalifoux and Dallaire, 1983).

b) Nitrogen and argon are inert gases which are both colourless and odourless, non-flammable and non-explosive. They are considered as having a minimal impact on the environment or the atmosphere. Both are used in a closed chamber through a process called "flushing" in which the passage of these gases reduces oxygen levels to a maximum of 1.5%. At such low levels of oxygen, the animal will collapse, and death will be produced by hypoxemia. However, dogs, cats, and rabbits may vocalize at the 1.5% level of oxygen, as well as show increased muscular activity and struggling. Nitrogen and argon do not produce a narcosis prior to the onset of hypoxia which in itself will lead to unconsciousness followed by death resulting from paralysis of the respiratory centre in the anoxic brain. Carbon dioxide, on the other hand, can induce a narcosis because of its physiological effect on the CNS (Herin, Hall and Fitch, 1978).

Quine, Buckingham and Strunin (1988) found that use of acepromazine as a tranquillizer before placing dogs in a nitrogen euthanasia flushing chamber produced longer survival times. However, it was not recorded whether or not these treated dogs showed the same amount of hyperventilation prior to or following the loss of consciousness; yelping, gasping, convulsions and muscular tremors usually accompany this process. Chalifoux and Dallaire (1983), however, concluded that premedication with tranquillizers improved the humaneness of carbon monoxide euthanasia.

c) Argon gas is more dense than air and thus tends to remain in the lower layers; however, both nitrogen and argon have no analgesic or anesthetic properties.

d) Carbon dioxide (CO₂) is frequently used to kill rodents and birds in the laboratory. Although it is a component of room air, pure CO₂ is heavier than air and practically odourless. It concentrates in the lower portion of the euthanasia chamber, and rats and other burrowing animals will tend to keep their noses in the lower zone containing adequate concentrations of the gas.

Its use as a euthanasia agent is dependent (for its humaneness) on whether or not it is in sufficient concentration to produce a narcosis. Maintenance at the correct level requires some manipulation to physiologically achieve, but will produce a narcosis and if oxygen levels are not increased, will lead to death. Carbon dioxide also stimulates the respiratory centre in the brain and in low concentrations of up to 10% of inspired gas is considered a potent respiratory stimulant causing a tenfold increase in the ventilation rate and a feeling of profound respiratory distress. The stimulation of the respiratory centre produces hyperventilation, thus critically affecting the onset of CO₂ narcosis. At approximately 40%, the CO₂ induces anesthesia which is slow in onset and accompanied by involuntary excitement. Eventually, there is apnea, a fall in blood pressure, and death (Ontario Ministry of Agriculture and Food Memo to Pound Operators and Veterinarians in Ontario, August 12, 1987). Britt (1986) found that the slow induction of narcosis is preferable, as using a precharged chamber, or passing CO₂ into the chamber too rapidly, can produce obvious signs of distress in the animals. However, he concluded that "neither (slow nor swift) method was found to be stress-free, so no recommendation can be a counsel of excellence."

McArthur (1976) reported on a method for delivering a carbon dioxide euthanasia of small animals, including cats, puppies, mice, rats, gerbils, guinea pigs and hamsters. In this study, unconsciousness was produced without distress to the animals, and occurred within a minute when levels of oxygen were maintained at 31 to 33%, with CO₂ ranging from 56 to 63%. Unconsciousness was used to describe animals that had collapsed and showed complete relaxation. Deep surgical anesthesia was produced by leaving the animals in this environment for a three-minute exposure period. Once deep surgical anesthesia was reached, the oxygen supply was turned off and CO₂ filled the chamber.

Carbon dioxide may be purchased in cylinders or in the solid state, as dry ice. It is relatively inexpensive, non-inflammable, non-explosive and essentially non-hazardous. It presents no danger to operators or attendants when used with properly designed equipment in an adequately ventilated space.

The essential criteria for producing a CO₂ narcosis is to maintain the oxygen level close to, or slightly below normal air levels, and increase the percentage of carbon dioxide in the air. It is important to understand that newborn animals, having existed in an environment with low oxygen levels prior to parturition, require higher levels of carbon dioxide in order to produce a humane death. Therefore, newborn animals should not be removed from chambers charged with carbon dioxide for approximately one-half hour after all movement has ceased.

Carbon dioxide does not accumulate in tissues; thus, there is no residue in food-producing animals. Neither does it appear to cause any distortion of the cells, which appear normal under microscopic examination.

Dogs, cats and other larger animals with an investigative behaviour will often extend their heads above the zone of effective CO₂ concentrations and thus be exposed to levels that excite rather than depress the CNS; thus, some will hyperventilate, struggle, stagger, and fall. The important element is to maintain an even distribution of CO₂ in the euthanasia chamber; this requires additional equipment.

Carbon dioxide has proven to be non-effective in killing diving mammals that have adapted to a relatively anerobic (oxygen-free) environment; 100% CO₂ is required to kill mink. Carbon dioxide is also used for pre-slaughter stunning in swine (Gregory, Moss and Leeson, 1987).

e) Potassium cyanide is a very potent paralyzing agent of the respiratory centre. It is one of the most rapidly acting poisons. Death appears to be almost instantaneous and irreversible through the production of rapid anoxia with CNS depression. However, death by exposure to cyanide gas is not considered humane because convulsions or seizures occur prior to death. As well, because of the extreme danger associated with its use, cyanide is not recommended as a method in the laboratory.

F. SPECIFIC SPECIES

In addition to the euthanasia methods for common laboratory species noted above, the following apply to:

1. Amphibians, Fishes and Reptiles

The most commonly used method for euthanizing amphibians, fish, and reptiles is by stunning, using the methodology previously outlined for vertebrates and terrestrial mammals. This can be followed by decapitation or crushing of the skull.

Sodium pentobarbital and barbituric acid derivatives may be used intravenously or introduced directly into the abdominal or pleuroperitoneal cavity of most cold blooded animals, if their anatomy permits. Tricaine methanesulfonate (MS-222) may be administered by a variety of routes to induce euthanasia. For aquatic mammals, as well as amphibians and fish, this material may be placed in the water. Alternatively, in the case of large fish, a gill cover is placed over the gills and concentrated MS-222 is flushed over the gills. Benzocaine hydrochloride is a compound similar to MS-222 and may be used as a bath or in a recirculation system for the euthanasia of fish.

It is worth noting that many experts in the study of amphibians and reptiles approve of the use of cold for the anesthesia of these animals. Subsequent freezing or decapitation is not considered to be painful; unfortunately, there is no evidence that cooling to 4^oC will lessen the pain threshold (Cooper, Ewbank, Platt et al. 1989). Cooper (1986) states that hypothermia would have to be instantaneous if it were to be painless.

Inhalant anesthetic agents, such as halothane, methoxyflorane, etc., may be used to kill reptiles and amphibia either in a chamber or by a suitably adapted face mask. Special attention must be paid to ensure they are dead following application of the method.

Carbon dioxide is not suitable for all species and concentrations must be maintained at a high level. Many semi-aquatic and terrestrial mammals, as well as reptiles and amphibia, are accustomed to living without oxygen and have a tolerance for hypoxia; thus, they have exaggerated anaerobic capabilities (Hochachka, 1980) (see also Chapter 22, Wild Vertebrates in the Field and in Laboratory in Vol. 2 of this Guide [CCAC, 1984]).

Decapitation as a means of killing amphibians and reptiles is unacceptable because of the differences in their physiology (Cooper, Ewbank, Platt et al. 1989; AVMA, 1986).

2. Domestic Animals Killed for Food

Although this chapter is concerned primarily with experimental animals, there are times when farm animals in production studies or, in some cases, in biomedical studies, will ultimately be killed for their food value. Although we do not apply the term "euthanasia" to the killing of such animals, the principles enunciated in the foregoing and in other documents apply: it is essential that humane treatment of animals be provided. In North America, the term "humane slaughter" is commonly used; however, in the United Kingdom, the term used is "pre-slaughter stunning" (Cockram and Corley, 1991; Ewbank, Parker and Mason, 1992; Knowles and Warriss, 1992). Pre-slaughter handling, the amount of time the animal is brought to the area to be killed and when it is stunned, and the proper design of restraint equipment affect the level of stress during handling, stunning and slaughter (Grandin, 1992a; 1992b). A comparison has been made between dislocation and percussion in chickens (Gregory and Wotton, 1990).

All killing of food animals must comply with federal legislation (Agriculture Canada's Meat Inspection Act S.C., 1985, C17 [May 1985 Part 2, Item 39, for 851078S14]; the federal Health of Animals Act [C-66 June, 1990, rev. March 1992]; 38-39 Elizabeth II Chapter 21]). As well, slaughter is discussed in Agriculture Canada Codes of Practice for the various livestock species (Agriculture Canada, 1771/E, 1984; 1821/E, 1988b; 1757/E, 1989b; 1833/E, 1990; 1870/E, 1992) as well as mink and foxes (Agriculture Canada, 1819/E, 1988a and 1831/E, 1989a) and provincial legislation (see Legislation). Provincial legislation on humane slaughter, where it exists, as well as municipal and local bylaws must also be followed.

3. Fur-bearing Animals

Mink, foxes, chinchillas, nutria and opossums are often maintained in research facilities. The methods listed above can be applied to the killing of these species. Ranched mink are commonly killed by the use of carbon monoxide, carbon dioxide and nitrogen (Hansen, Creutzberg and Simmonson, 1991) or electrical stunning followed by cervical dislocation.

G. TISSUE EFFECTS OF EUTHANASIA METHODS

Tissue effects of euthanasia methods may be either direct or indirect. They may affect only components in the intravascular compartment or they may affect fixed tissues, thus influencing histological or electronmicroscopic findings. In the majority of cases, death is so rapid that even electronmicroscopic changes are non-existent or minuscule. In most instances, the concern of the investigator that demonstrable histocytotoxic changes may occur is unfounded.

1. Direct Effects

In general, the direct effects of the euthanizing method are subtle or lacking, particularly with the non-inhalant pharmacological agents. The changes produced by methods which cause anoxia depend upon the rapidity of the induction of the anoxic state, and occur as the result of changes in blood gases. For example, pulmonary congestion and edema may be observed on gross observation in the anoxic state; however, the degree is dependent upon rapidity of death.

Lamellar bodies in Purkinje cells of the cerebellum have been observed in some hypoxic anesthetized dogs subjected to rapid decompression (Bowman, Cooke and Carry, 1969). The knowledge of the sequential series of morphological and biochemical events leading to hypoxic injury to neurons and glial cells has not been adequately studied (Kim, 1975). There is nothing to suggest that hypoxic changes produced by carbon dioxide make tissues unsuitable for routine examination of the respiratory tract (Fawell, Thompson and Cooke, 1972).

Barbiturates ionize on injection into the intravascular compartment. The degree of ionization will depend upon the dissociation constant of the drug and the Ph of the blood. Cell penetration can occur only with the undissociated drug. After cell penetration takes place, dissociation again occurs and the binding of the drug to intracellular organelles will take place. Tissue changes due to cellular penetration and intracellular binding of barbiturates have not been described. At the same time as the barbiturates bind with plasma proteins forming an equilibrium of bound and unbound drug in circulating blood, splenic dilation with sequestration of red blood cells produce an enlarged and grossly blue-black spleen (Lumb, 1974).

2. Indirect Effects

The major indirect effects are due to tissue hypoxia brought on by the death of the animal. Thus, it is important that tissues for histological and electronmicroscopy examination be prepared as rapidly as possible following unconsciousness and death of the animal.

The tissue requirements for oxygen vary widely. The damages to the neurons of the CNS occur most rapidly, but are dependent upon the degree of tissue hypoxia and time elapsed after death before tissue preparation. Hypoxia changes to neurons require electronmicroscopic examination.

In those tissues whose requirements for oxygen are not as great as the neurons (e.g., osteocytes, chondrocytes of bone and cartilage, or other less oxygen-sensitive tissues) changes may be difficult to detect even with electronmicroscopy.

Proper handling of the animal prior to death, with immediate processing of tissues after death, are important to obtain optimal electronmicrographs with minimal changes.

H. EFFECT ON OBSERVERS

We must recognize that a significant number of people experience emotional uneasiness, physical and psychological discomfort or distress when animals are killed, even if a humane method is applied. For example, the 1987 Malouf Commission Report on Seals and Sealing stated that, although the method used for killing seals (striking the animal's head with a hakapik) was proven to be humane by numerous veterinary pathologists, the general public considered the act repugnant. For over a decade, the whitecoat seal on the ice on the Gulf of St. Lawrence or at the Front of St. Anthony, Newfoundland, was the only animal that the public had seen being killed in media presentations.

Inexperienced observers might misinterpret any movement, vocalization, or reflex reactions as indicators of pain and distress. Therefore, it is preferable that euthanasia methods, in addition to providing a humane death, minimize or eliminate such involuntary movements.

Over the past several years, recognition has been given to the effect of euthanasia on those required to carry it out (e.g., humane society workers, laboratory animal veterinarians, animal caretakers and others whose jobs involve euthanasia) (Rollin, 1986; Rollin and Kesel, 1990; Grier and Colvin, 1990). Arluke (1992) stated that, understandably, uneasiness was particularly noticeable among newcomers; with seasoned workers, it was most common among animal caretakers; it occurred among technicians, but was relatively rare among veterinarians and scientists. Owens, Davis and Smith (1981) have noted that those who must euthanize animals have developed ways and means of dealing with their emotions by avoiding unnecessary contact with the animals or by believing that, by being killed, the animal is being spared additional suffering.

Workers can (and will) accept that, when an animal is showing pain and distress that cannot be relieved, it must be killed. In this, their attitudes differ little from those of an owner of a companion animal who must face similar decisions. Like the pet owner, the employee can feel grief; therefore, it is important that strong communications be established with all staff members, with a willingness to listen and to provide support for those who may be feeling distressed or disturbed. It is important that such "feelings" not be suppressed in the research setting. Employees should be aware that, regardless of the purpose of the experimental study, at an established time and date, the animal must be humanely destroyed. Seminars or workshops to help staff cope with animal death may be useful.

I. EUTHANASIA STATEMENTS--OTHER AGENCIES

As noted, the Report of the AVMA Panel on Euthanasia has recently been published (AVMA, 1993). Euthanasia has been discussed by the U.S. National Research Council (NRC, 1991). Reference to euthanasia may also be found in the U.K.'s Universities Federation for Animal Welfare (UFAW) Report on Euthanasia of Unwanted, Injured or Diseased Animals for Education or Scientific Purposes (UFAW, 1986), its Humane Slaughter of Animals for Food (UFAW, 1987) and Britain's Animals (Scientific Procedures) Act, 1986 (Balls, 1986; McKie, 1986; Fisher, 1990). The Australian Council for the Care of Animals in Research and Teaching has prepared a bibliography which includes a number of references to euthanasia (ACCART, 1991). Attention is also drawn to Field Research Guidelines (Orlans, 1988) which lists a number of guidelines (ASM, 1987; AOU, 1988; ASIH, HL, SSAR, 1987; ASIH, AFS, AIFR, 1987; Zwart, deVries and Cooper, 1989).

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