AWIC

1993 Report of the AVMA Panel on Euthanasia

Provided by the Animal Welfare Information Center
United States Department of Agriculture
National Agricultural Library

This document appears in the AWIC Electronic Library by permission of the American Veterinary Medical Association. It may be reproduced in its entirety, including the citation (J. Am. Vet. Med. Assoc. 1993;202:229-249), so long as it is used for educational purposes and distributed without charge. Neither the American Veterinary Medical Association nor the Animal Welfare Information Center would be responsible for any alterations to the document after downloading from AWIC. Reprints of the 1993 Report of the AVMA Panel on Euthanasia are available for $0.50 each, prepaid, from the AVMA, 1931 N. Meacham Rd., Suite 100, Schaumburg, IL 60173-4360, (708) 925-8070, ext. 250.

1993 REPORT OF THE AVMA

PANEL ON EUTHANASIA

JAVMA, Vol 202, No. 2, pp 229-49, January 15, 1993
Report of the AVMA Panel on Euthanasia

Table of Contents

Members of the Panel

Edwin Andrews, VMD, PhD (Chairman), Dean, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6044

B. Taylor Bennett, DVM, PhD, Director of Biological Resources Laboratory, 1840 W. Taylor St., M/C 533, University of Illinois, Chicago, IL 60612

J. Derrell Clark, DVM, MS, DSc, Director of Animal Resources, 206 Animal Resources, College of Veterinary Medicine, University of Georgia, Athens, GA 30602

Katherine A. Houpt, VMD, PhD, Department of Physiology, D-121 Schurman Hall, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401

Peter J. Pascoe, BVSc, Department of Surgery, School of Veterinary Medicine, University of California, Davis, CA 95616-8745

Gordon W. Robinson, VMD, American Society for the Prevention of Cruelty to Animals, 441 E. 92nd St., 2nd Floor, New York, NY 10128

John R. Boyce, DVM, PhD, Staff Coordinator, American Veterinary Medical Association, 1931 N. Meacham Rd., Suite 100, Schaumburg, IL 60173-4360

iii

Preface

In 1992, at the request of the AVMA Council on Research, the Executive Board of the AVMA convened a Panel on Euthanasia to review and make necessary revisions to the fourth Panel Report, published in 1986.1 Since 1986, the panel has become aware of a need for additional information on some aspects of euthanasia. In this report, the panel has updated information on euthanasia of animals in research and animal care and control facilities, expanded information on poikilothermic, aquatic, and fur-bearing animals, added information on horses and wildlife, and deleted methods or agents considered unacceptable. Some euthanasia methods and agents are not discussed, because this report is limited to those methods and agents supported by data from scientific studies. Predator control and depopulation and slaughter of animals for food are not addressed in this report. The report is intended to be used primarily by veterinarians, but will be understandable by a broad segment of the general population. Although the interpretation and use of this report cannot be limited, the panel's overriding commitment is to give professional guidance for relieving pain and suffering of animals that are to be euthanatized. The recommendations in this report are intended to serve as guidelines requiring the use of professional judgment for application to the various settings where animals must be euthanatized.

Introduction

The term euthanasia is derived from the greek terms eu meaning "good" and thanatos meaning "death."2 A "good death" would be one that occurs without pain and distress. In the context of this report, euthanasia is the act of inducing humane death in an animal. Euthanasia techniques should result in rapid unconsciousness followed by cardiac or respiratory arrest and ultimate loss of brain function. In addition, the technique should minimize any stress and anxiety experienced by the animal prior to unconsciousness. Stress may be minimized by technical proficiency and humane handling of the animals to be euthanatized.

Emotional uneasiness, discomfort, or distress experienced by people involved with euthanasia of animals may be minimized by assuring that the person performing the euthanasia procedure is technically proficient. Uninformed observers may mistakenly relate any movement of animals with consciousness and lack of movement with unconsciousness. Although these are not adequate criteria, euthanasia techniques that preclude movement of animals are those aesthetically acceptable to most people. Pain must be defined before criteria for painless death can be established. Pain is that sensation (perception) that results from nerve impulses reaching the cerebral cortex via specific nociceptive neural pathways. The term nociceptive is derived from noxious stimuli, which threaten to, or actually do, destroy tissue. The stimuli initiate nerve impulses by acting on a specific set of receptors, called nociceptors. Nociceptors respond to mechanical, thermal, or chemical stimuli. Endogenous chemical substances such as hydrogen ions, serotonin, histamine, bradykinin, and prostaglandins as well as electrical currents are capable of generating nerve impulses by nociceptors.

Nerve impulse activity generated by nociceptors is conducted to the spinal cord or the brain-stem via nociceptor primary afferent fibers. In the spinal cord or brainstem, nerve impulses are transmitted to two sets of neural networks. One set is related to nociceptive reflexes that are mediated spinally, and the second set consists of ascending pathways to the reticular formation, thalamus, and cerebral cortex for sensory processing. The transmission of nociceptive neural activity is highly variable. Under certain conditions, both the nociceptive reflexes and the ascending pathways may be suppressed, as, for example, in deep surgical anesthesia. In another set of conditions, nociceptive reflex actions may occur, but the activity in the ascending pathways is suppressed; thus, the noxious stimuli are not perceived as pain, as, for example, in a light plane of surgical anesthesia. It is incorrect to use the term "pain" for stimuli, receptors, reflexes, or pathways because the term implies perception, whereas all of the above may be active without consequential pain perception.3-7

Pain is divided into two broad categories: (1) sensory-discriminative, which indicates the site of origin and the stimulus giving rise to the pain; and (2) motivational-affective in which the severity of the stimulus is perceived and the animal's response is determined. Sensory-discriminative processing of nociceptive impulses is most likely to be accomplished by subcortical and cortical mechanisms similar to those utilized for processing of other sensory-discriminative input that provides the individual with information about the intensity, duration, location, and quality of the stimulus. Motivational-affective processing involves the ascending reticular formation for behavioral and cortical arousal. It also involves thalamic input to the forebrain and the limbic system for perceptions such as discomfort, fear, anxiety, and depression. The motivational-affective neural networks also have strong inputs to the hypothalamus and the autonomic nervous system for reflex activation of the cardiovascular, pulmonary, and pituitary-adrenal systems. Responses activated by these systems feed back to the forebrain and enhance the perceptions derived via motivational-affective inputs. On the basis of neurosurgical experience in human beings, it is possible to separate the sensory-discriminative components from the motivational-affective components of pain.4

For pain to be experienced, the cerebral cortex and subcortical structures must be functional. An unconscious animal cannot experience pain because the cerebral cortex is not functioning. If the cerebral cortex is nonfunctional because of hypoxia, depression by drugs, electric shock, or concussion, pain is not experienced. Therefore, the choice of the euthanasia agent or method is of less importance if it is to be used on an animal that is anesthetized or unconscious, provided that the animal does not regain consciousness prior to death.

An understanding of the continuum that represents stress and distress is essential for evaluating techniques that minimize any distress experienced by an animal being euthanatized. Stress has been defined as the effect of physical, physiologic, or emotional factors (stressors) that induce an alteration in an animal's homeostasis or adaptive state.8 The response of an animal to stress represents the adaptive process that is necessary to restore the baseline mental and physiologic state. These responses may involve changes in an animal's neuro-endocrinologic system, autonomic nervous system, and mental state that may lead to overt behavioral changes. An animal's response varies according to its experience, age, species, breed, and current physiologic and psychologic state.9

Stress and the resulting responses have been divided into three phases.10 Eustress results when harmless stimuli initiate adaptive responses that are beneficial to the animal. Neutral stress results when the animal's response to stimuli causes neither harmful nor beneficial effects to the animal. Distress results when an animal's response to stimuli interferes with its well-being and comfort.

As with many other procedures involving animals, some methods of euthanasia require physical handling of the animal. The amount of control and kind of restraint needed will be determined by the animal species, breed, size, state of domestication, presence of painful injury or disease, degree of excitement, and method of euthanasia. Proper handling is vital to minimize pain and distress in animals, to assure safety of the person performing euthanasia, and, frequently, to protect other animals and people.

Personnel who perform euthanasia must have appropriate certification and/or training and experience with the techniques to be used, to assure that animal pain and distress are minimized during euthanasia. This training and experience should include familiarity with the normal behavior of the species being euthanatized, how handling and restraint affects that behavior, and an understanding of the mechanism by which the selected technique induces unconsciousness and death. Prior to being assigned full responsibility for performing euthanasia, all personnel must have demonstrated proficiency in the use of the technique in a closely supervised environment. References provided at the end of this document may be useful for training personnel.11-13

An in-depth discussion of euthanasia procedures is beyond the scope of this report; however, several excellent euthanasia procedural manuals are particularly applicable to animal care and control agencies.11,13,14

Selection of the most appropriate method of euthanasia in any given situation depends on the species of the animal involved, available means of animal restraint, skill of personnel, numbers of animals, and other considerations. This report deals primarily with domestic animals, but the same general considerations should be applied to all species.

This report includes four tables that summarize information from the text. Table 1 lists acceptable and conditionally acceptable methods of euthanasia, categorized by species. Tables 2 and 3 provide summaries of characteristics for acceptable and conditionally acceptable methods of euthanasia. Table 4 provides a summary of some unacceptable euthanasia agents and methods.

General Considerations

In evaluating methods of euthanasia, the panel used several criteria: (1) ability to induce loss of consciousness and death without causing pain, distress, anxiety, or apprehension; (2) time required to induce unconsciousness; (3) reliability; (4) safety of personnel; (5) irreversibility; (6) compatibility with requirement and purpose; (7) emotional effect on observers or operators; (8) compatibility with subsequent evaluation, examination, or use of tissue; (9) drug availability and human abuse potential; (10) age and species limitations; and (11) ability to maintain equipment in proper working order.

Several issues were discussed by the panel as a result of comments solicited from a broad spectrum of individuals with special expertise in the many and varied settings in which euthanasia must be performed. Inasmuch as an in-depth consideration of these issues was beyond the expertise of the panel members and not directly related to the panel's charge, they were not included in the body of the report, but will be mentioned briefly in this section.

The panel discussed the definition of euthanasia used in this report as it applies to circumstances when the needed control over the animal makes it difficult to assure death without pain and distress. The slaughter of animals for food, fur, or fiber and techniques commonly used to control wild and feral animal populations may represent such situations. While recognizing these interactions with animals, the panel does not believe the term euthanasia is appropriate in some of these cases.

Animals for food should be slaughtered as specified by the US Department of Agriculture.15 Painless death can be achieved by properly stunning animals, followed immediately by exsanguination. Preslaughter handling of animals should be as stress-free as possible. Electric prods or other devices to encourage movement of animals should not be used. Chutes and ramps should be properly designed to enable animals to be moved and restrained without undue stress.16,17 Animals must not be restrained in a painful position before slaughter.

Animals raised for fur are not included in the Humane Slaughter Act, but will be addressed in this report.

The ethical considerations that must be made when euthanatizing healthy and unwanted animals raises both professional and societal issues.18,19 These issues are complex and warrant thorough consideration by both the profession and all those concerned with the welfare of animals. While the panel recognizes the need for those responsible for the euthanasia of animals to be cognizant of these issues, it does not believe that this report is the appropriate forum for an in-depth discussion of this topic.

It is the intent of the panel that these recommendations be carried out in accordance with applicable federal, state, and local laws governing drug acquisition and storage, occupational safety, and methods used for euthanasia and disposal of animals. However, space does not permit a review of current federal, state, and local regulations.

The panel is aware that circumstances may arise that are not clearly covered by this report. Examples are euthanasia of neonatal or prenatal animals or uncommonly encountered species. Whenever such situations arise, a veterinarian or other experienced professional should use professional judgment and knowledge of clinically acceptable techniques in selecting an appropriate euthanasia technique. Essential to the application of professional judgment is the consideration of the animal's size and its species-specific physiologic and behavioral characteristics. In all circumstances, the euthanasia method should be selected and used with the highest ethical standards and social conscience.

It is imperative that death be verified after euthanasia and before disposal of the animal. To a casual observer, an animal in deep narcosis following administration of an injectable or inhalant agent may appear dead, but may eventually recover. Death should be confirmed by examining the animal for cessation of vital signs. Professional judgment should be used, in consideration of the animal species and method of euthanasia, to determine the means of confirming death.

Animal Behavioral Considerations

The facial expressions and body postures that indicate various emotional states of animals have been described.20-22 Behavioral and physiologic responses to noxious stimuli include distress vocalization, struggling, attempts to escape, defensive or redirected aggression, salivation, urination, defecation, evacuation of anal sacs, pupillary dilatation, tachycardia, sweating, and reflex skeletal muscle contractions causing shivering, tremors, or other muscular spasms. Some of these responses can occur in unconscious as well as conscious animals. Fear can cause immobility or "playing dead" in certain species, particularly rabbits and chickens. This immobility response should not be interpreted as unconsciousness when the animal is, in fact, conscious.

The need to minimize animal distress, including fear, anxiety, 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, other animals should not be present when euthanasia is performed, especially euthanasia of the same species. This is particularly important when vocalization or release of pheromones may occur during induction of unconsciousness. Gentle restraint, preferably in a familiar environment, careful handling, and talking during euthanasia often have a calming effect on companion animals. However, some of these methods may not be effective with wild animals or animals that are injured or diseased. When struggling during capture or restraint may cause pain, injury, or anxiety to the animal or danger to the operator, the use of tranquilizers, analgesics, and/or immobilizing drugs should be considered.

Human Behavioral Considerations

The psychologic issues involved with euthanasia of animals are within the purview of this discussion. Moral and ethical imperatives associated with individual animal or mass euthanasia should be consistent with acceptable humane practice. Grief at the loss of an animal's life is the most common reaction.23 There are three circumstances in which we are most aware of the effects of euthanasia on people. The first of these is the clinical setting. The owner will have had to make the decision about whether and when to euthanatize, and, although many owners rely heavily on their veterinarian's judgment, others may have misgivings about their own decision. This is particularly apt to be the case if the owner feels responsible for allowing an animal's medical or behavioral problem to go unattended so that euthanasia became necessary. Counseling services for grieving owners are now available in some communities24 and telephone counseling is available at some veterinary schools.25 Owners are not the only people affected by euthanasia of animals. Veterinarians and their staffs may also be attached to patients they have known and treated for many years.

The second circumstance in which people are affected by euthanasia is at humane societies and animal control facilities where unwanted, homeless, diseased, and injured animals must be euthanatized in large numbers. Distress may develop among personnel directly involved in performing euthanasia repetitively. Constant exposure to, or participation in, euthanasia procedures can cause a psychologic state characterized by a strong sense of work dissatisfaction or alienation, which may be expressed by absenteeism, belligerence, or careless and callous handling of animals.26 This is one of the principal reasons for turnover of employees directly involved with repeated animal euthanasia. This should be recognized as a bona fide personnel problem related to animal euthanasia, and management measures should be instituted to decrease or eliminate the potential for this problem. Specific coping strategies can make the task more tolerable. Some of these strategies are: adequate training programs so that the method of euthanasia is performed competently; peer support in the workplace; focusing on animals that are successfully adopted or returned to owners; devoting some work time to educational activities; and providing time off when workers feel stressed.

The third setting in which people are affected by euthanasia of animals is in the laboratory. Researchers, technicians, and students may become attached to an animal that must be euthanatized.27 The same considerations given to pet owners or shelter employees should be afforded to those working in such facilities.

Human attitudes and responses should be considered whenever animals are euthanatized, including animals in zoos, at sites of roadside or racetrack accidents, and in cases of stranded marine animals. However, these considerations should not outweigh the primary responsibility of using the most rapid and painless euthanasia method possible under the circumstances.

Modes of Action of Euthanatizing Agents

Euthanatizing agents cause death by three basic mechanisms: (1) hypoxia, direct or indirect; (2) direct depression of neurons vital for life function; and (3) physical disruption of brain activity and destruction of neurons vital for life.

Agents that induce death by direct or indirect hypoxia can act at various sites and can cause unconsciousness at different rates. For death to be painless and distress-free, unconsciousness should precede loss of motor activity (muscle movement). This means that agents that induce muscle paralysis without unconsciousness are absolutely condemned as sole agents for euthanasia (eg, curare, succinylcholine, gallamine, strychnine, nicotine, magnesium or potassium salts, pancuronium, decamethonium, vecuronium, atracurium, pipecuronium, and doxacurium). With other techniques that induce hypoxia, some animals may have motor activity following unconsciousness, but this is reflex activity and is not perceived by the animal.

The second group of euthanatizing agents depress nerve cells of the brain, inducing unconsciousness followed by death. Some of these agents "release" muscle control during the first stage of anesthesia, resulting in a so-called "excitement or delirium phase," during which there may be vocalization and some muscle contraction. These responses do not appear to be purposeful. Death follows unconsciousness, and is attributable to hypoxemia following direct depression of respiratory centers and/or cardiac arrest.

Physical disruption of brain activity, caused by concussion, direct destruction of the brain, or electrical depolarization of the neurons, induces rapid unconsciousness. Death occurs because of destruction of midbrain centers controlling cardiac and respiratory activity or by adjunctive methods (e.g. exsanguination) used to kill the animal. Exaggerated muscular activity can follow unconsciousness and, although this may disturb some observers, the animal is not experiencing pain or distress.

Inhalant Agents

Any gas that is inhaled must reach a certain concentration in the alveoli before it can be effective, therefore, euthanasia with any of these agents takes some time. The suitability of a particular agent depends on whether an animal experiences distress between the time it begins to inhale the agent and the time it loses consciousness. Some agents may induce convulsions, but these generally follow unconsciousness. Agents inducing convulsions prior to unconsciousness are unacceptable for euthanasia.

Certain considerations are common to all inhalant agents. (1) In most cases, onset of unconsciousness is more rapid, and euthanasia more humane, if the animal is rapidly exposed to a high concentration of the agent. (2) The equipment used to deliver and maintain this high concentration must be in good working order. Leaky or faulty equipment may lead to slow, distressful death and/or be hazardous to other animals and to personnel. (3) Most of these agents are hazardous to the health of personnel because of the risk of explosions (eg, ether), narcosis (eg, halothane), hypoxemia (eg, nitrogen, carbon monoxide), addiction (eg, nitrous oxide), or health effects resulting from chronic exposure (eg, nitrous oxide, carbon monoxide). (4) Alveolar concentrations rise slowly in an animal with decreased ventilation, making agitation more likely during induction. Other non-inhalant methods of euthanasia should be considered for such animals. (5) Neonatal animals appear to be resistant to hypoxia, and because all inhalant agents ultimately cause hypoxia, neonatal animals take longer to die than adults. Therefore, these agents should not be used in neonates unless the animal can be exposed long enough to ensure death. Glass et al12 reported that newborn dogs, rabbits, and guinea pigs survived a nitrogen atmosphere much longer than adults. Dogs, at 1 week of age, survived for 14 minutes compared with 3 minutes at the age of weeks or as adults. Guinea pigs survived for 4.5 minutes at 1 day of age, compared with 3 minutes at 8 days and as adults. Rabbits survived for 13 minutes at 6 days of age, 4 minutes at 14 days, and 1.5 minutes at 19 days and as adults. Until more reliable data are available, the panel recommends that inhalant agents not be used alone in pups and kittens less than 16 weeks of age. Inhalants may be used to induce unconsciousness, followed by use of some other method to kill the animal.(6) Rapid gas flows can produce a noise that frightens animals. If high flows are required, the equipment should be designed to minimize noise.(7) Animals placed together in chambers should be of the same species, and, if needed, should be restrained so that they will not hurt themselves or others. Chambers should be kept clean to minimize odors that might distress animals subsequently euthanatized.

Inhalant Anesthetics

Inhalant anesthetics (eg, ether, halothane, methoxyflurane, isoflurane, and enflurane) have been used to euthanatize many species.29 Ether has high solubility in blood and induces anesthesia slowly, is irritating to the eyes and nose, and poses serious risks associated with its flammability. Although ether is acceptable for euthanasia, other agents may be preferable. Methoxyflurane also has high solubility and the slow anesthetic induction with its use may be accompanied by agitation. It is more acceptable than ether, but other agents may be preferable. Halothane induces anesthesia rapidly and is the most effective inhalant anesthetic for euthanasia. Enflurane is less soluble in blood than is halothane, but, because of its lower vapor pressure and lower potency, induction rates may be similar to those for halothane. At deep anesthetic planes, animals often have a seizure. It is an effective agent for euthanasia, but the seizure activity may be disturbing to personnel. Isoflurane is the least soluble of the potent inhalant anesthetics and it should induce anesthesia more rapidly. However, it has a slightly pungent odor and animals often hold their breath, delaying the onset of unconsciousness. Isoflurane also may require more drug to kill an animal, compared with halothane. Although isoflurane is acceptable as a euthanasia agent, halothane is preferred.

With inhalant agents, the animal is placed in a closed receptacle containing cotton or gauze soaked with the anesthetic.30 The anesthetic also may be introduced from a vaporizer, but this usually results in longer induction time. Vapors are inhaled until respiration ceases and death ensues. Because the liquid state of most inhalant anesthetics is irritating, animals should be exposed only to vapors. Also, sufficient air or oxygen must be provided during the induction period to prevent hypoxemia.30 In the case of small rodents placed in a large container, there will be sufficient oxygen in the chamber to prevent hypoxemia. Larger species placed in small containers may need supplemental air or oxygen.

Nitrous oxide (N2O) may be used with the other inhalants to speed the onset of anesthesia, but it alone does not induce anesthesia in animals, even at 100% concentration. If N2O is used as a sole euthanasia agent, hypoxemia develops before respiratory or cardiac arrest, and animals may become distressed prior to unconsciousness.

Occupational exposure to inhalant anesthetics constitutes a human health hazard. Spontaneous abortion and congenital abnormalities have been associated with exposure of women to trace amounts of inhalation anesthetic agents in early stages of pregnancy.31 In human exposure to inhalant anesthetics, the concentration of ether, halothane, methoxyflurane, enflurane, and isoflurane should be less than 2 ppm, and less than 25 ppm for nitrous oxide.32 There are no controlled studies proving that such concentrations of anesthetics are "safe", but these concentrations were established because they were shown to be attainable under hospital conditions. Effective procedures must be used to protect personnel from anesthetic vapors.

Advantages - (1) Inhalant anesthetics are particularly valuable for euthanasia of smaller animals (< about 7 kg) or in animals in which venipuncture may be difficult. (2) Halothane, enflurane, isoflurane, methoxyflurane, and N2O are nonflammable and nonexplosive under ordinary environmental conditions.

Disadvantages - (1) Struggling and anxiety may develop during induction of anesthesia because anesthetic vapors may be irritating and can induce excitement. (2) Ether is flammable and explosive and should not be used near an open flame or other ignition sources. Explosions have occurred when animals, euthanatized with ether, were placed in an ordinary (not explosion proof) refrigerator or freezer and when bagged animals were placed in an incinerator. (3)Nitrous oxide will support combustion. (4) Personnel and animals can be injured by exposure to these agents. (5) There is a potential for human abuse of some of these drugs, especially N2O. Recommendations - In order of preference, halothane, enflurane, isoflurane, methoxyflurane, and ether, with or without nitrous oxide, are acceptable for euthanasia of small animals (< about 7 kg). Nitrous oxide should not be used alone, pending further scientific studies on its suitability for animal euthanasia. Although acceptable, these agents are generally not used in larger animals because of their cost and difficulty of administration.

Carbon Dioxide

Room air contains 0.04% carbon dioxide (CO2), which is heavier than air and nearly odorless. Inhalation of CO2 in concentrations of 7.5% increases the pain threshold, and higher concentrations of CO2 have a rapid anesthetic effect.33-37 Leake and Waters35 reported the experimental use of CO2 as an anesthetic agent in dogs. At concentrations of 30% to 40% CO2 in oxygen, anesthesia was induced within 1 to 2 minutes, usually without struggling, retching, or vomiting. The signs of effective CO2 anesthesia are those associated with deep surgical anesthesia, such as loss of withdrawal and palpebral reflexes.38 In cats, inhalation of 60% CO2 results in loss of consciousness within 45 seconds, and respiratory arrest within 5 minutes.39 Carbon dioxide has been used to euthanatize groups of small laboratory animals, including mice, rats, guinea pigs, chickens, and rabbits,3,40-44 and to render swine unconscious before humane slaughter.15,45,46 Several investigators have suggested that inhalation of high concentrations of CO2 may be distressing to animals45-48 because of mucosal irritation and ventilatory stimulation. However, the degree of distress appears to be mild, and it is unlikely that it is anymore than inhalation of volatile anesthetics. The combination of 40% CO2 and approximately 3% CO has been used experimentally for euthanasia of dogs.47 Carbon dioxide has been used in specially designed chambers to euthanatize cats50,51 and other small laboratory animals.30,40,49

Studies in day-old chickens have shown that CO2 is an effective euthanatizing agent. Inhalation of CO2 caused little distress to the birds, suppressed nervous activity, and induced death within 5 minutes.41 Because respiration begins during embryonic development, the unhatched chicken's environment may normally have a CO2 concentration as high as 14%. Thus, CO2 concentration for euthanasia of newly hatched chickens and neonates of other species should be especially high. A CO2 concentration of 60% to 70% with a 5-minute exposure time appears to be optimal.41 A similar technique was used in mink and, although 70% carbon dioxide induced unconsciousness, it did not kill the animals.52 These and other diving animals may have physiologic mechanisms for coping with high concentrations of CO2. It is necessary, therefore, to have a high enough concentration of CO2 to kill the animal by hypoxemia following the induction of anesthesia with CO2.

Carbon dioxide is used for preslaughter anesthesia of swine. The undesirable side effect of CO2, as used in commercial slaughterhouses, is that swine experience a stage of excitement with vocalization for about 40 seconds before they lose consciousness.45,46,53 For that reason, CO2 preslaughter anesthesia may appear less humane than other techniques.

Advantages - (1) The rapid depressant and anesthetic effects of CO2 are well established. (2) Carbon dioxide may be purchased in cylinders or in solid state as "dry ice." (3) Carbon dioxide is inexpensive, nonflammable, and nonexplosive, and poses minimal hazard to personnel when used with properly designed equipment. (4) Carbon dioxide does not result in accumulation of tissue residues in food producing animals. (5) Carbon dioxide euthanasia does not distort cellular architecture.54

Disadvantages - (1) Because CO2 is heavier than air, incomplete filling of a chamber may permit tall or climbing animals to avoid exposure and to survive. This appears to be very distressful to the animals. (2) Some species may have extraordinary tolerance for CO2.

Recommendations - Carbon dioxide is acceptable for euthanasia. Compressed CO2 gas in cylinders is preferable to dry ice because the inflow to the chamber can be regulated precisely. If dry ice is used, animal contact must be avoided to prevent freezing or chilling. Carbon dioxide generated by other methods such as from a fire extinguisher or from chemical means (eg, Alka-Seltzer) are unacceptable. With an animal in the chamber, an optimal flow rate should displace at least 20% of the chamber volume per minute.55 Unconsciousness may be induced more rapidly by exposing animals to a CO2 concentration of 70% or more by prefilling the chamber. It is important to verify that an animal is dead before removing it from the chamber. If an animal is not dead, CO2 narcosis must be followed with another method of euthanasia. Larger animals, such as rabbits, cats, and swine, appear to be more distressed by CO2 euthanasia; therefore, other methods of euthanasia are preferable.

Nitrogen, Argon

Nitrogen (N2) and argon (Ar) are colorless, odorless gases that are inert, nonflammable, and nonexplosive. Nitrogen comprises 78% of atmospheric air, whereas Ar is present at less than 1% of atmospheric air.

Euthanasia is induced by placing the animal in a closed container into which N2 or Ar is rapidly introduced or prefilled at atmospheric pressure. Nitrogen/Ar displaces oxygen in the container, thus inducing death by hypoxemia.

In studies by Herin et al,56 dogs become unconscious within 76 seconds when N2 concentration of 98.5% was achieved in 45 to 60 seconds. The electroencephalogram (EEG) became isoelectric (flat) in a mean of 80 seconds, and arterial blood pressure was undetectable at a mean of 204 seconds. Although all dogs hyperventilated prior to unconsciousness, the investigators concluded that this method induced death without pain. Following loss of consciousness, vocalization, gasping, convulsions, and muscular tremors occurred in some dogs. At the end of a 5-minute exposure period, all dogs were dead.56 These findings were similar to those for rabbits57 and mink.52,58

With N2 flowing at a rate of 39% of chamber volume per minute, rats collapsed in approximately 3 minutes and stopped breathing in 5 to 6 minutes. Regardless of flow rate, signs of panic and distress were evident before the rats collapsed and died.55 Insensitivity to pain under such circumstances is questionable.59 Tranquilization with acepromazine, in conjunction with N2 euthanasia of dogs, was investigated by Quine et al.60 Using ECG and EEG recording, they found that these animals had much longer survival times than animals not given acepromazine before the administration of N2. In one dog the ECG activity continued for 51 minutes. Quine also addressed the issue of distress associated with exposure to N2 by removing cats and dogs from the chamber following unconsciousness and allowing them to recover. When these animals were put back into the chamber, they did not appear afraid or apprehensive.

When Ar was used to euthanatize chickens, exposure to a chamber prefilled with Ar, with an oxygen concentration of <2%, led to EEG changes and collapse in 9-12 seconds. Birds removed from the chamber at 15-17 seconds failed to respond to comb pinching. Continued exposure led to convulsions at 20-24 seconds. Somatosensory-evoked potentials were lost at 24-34 seconds and the EEG became isoelectric at 57-66 seconds. The onset of convulsions appeared after the loss of consciousness (collapse and loss of response to comb pinch), so this would appear to be a humane method of euthanasia in chickens.62

Advantages - (1) Nitrogen and Ar are readily available as compressed gases. (2) Hazards to personnel are minimal.

Disadvantages - (1) Unconsciousness is preceded by hypoxemia and ventilatory stimulation, which may be distressing to the animal. (2) Reestablishing a low concentration of O2 (ie, 6% or greater) in the chamber before death will allow immediate recovery.

Recommendations - Nitrogen and Ar can be distressful in some species (eg, rats), therefore, this technique is acceptable only if oxygen concentrations <2% are achieved rapidly and the animal is heavily sedated or anesthetized. With heavy sedation or anesthesia, it should be recognized that death may be delayed. In dogs, cats, and chickens, this appears to be a humane method of euthanasia. Although N2 and Ar are effective, other methods of euthanasia are preferable.

Carbon Monoxide

Carbon monoxide (CO) is a colorless, odorless gas that is nonflammable and nonexplosive until concentrations exceed 10%. It combines with hemoglobin to form carboxyhemoglobin and blocks the uptake of oxygen by erythrocytes, leading to fatal hypoxemia.

In people, the most common symptoms of early CO toxicosis are headache, dizziness, and weakness. As concentrations of carboxyhemoglobin increase, these signs may be followed by decreased visual acuity, tinnitus, nausea, progressive depression, confusion, and collapse.63 Because CO stimulates motor centers in the brain, unconsciousness may be accompanied by convulsions and muscular spasms.

Carbon monoxide is a cumulative poison.64 Distinct signs of CO toxicosis are not evident until the concentration is 0.05% in air, and acute signs do not develop until the concentration is approximately 0.2%. In human beings, exposure to 0.32% CO and 0.45% CO for one hour will induce unconsciousness and death, respectively.65 Carbon monoxide is extremely hazardous for personnel because it is highly toxic and difficult to detect. Chronic exposure to low concentrations of carbon monoxide may be a health hazard, especially with regard to cardiovascular disease and teratogenic effects.66-68 An efficient exhaust or ventilatory system is essential to prevent accidental exposure of human beings.

In the past, mass euthanasia has been accomplished by using three methods for generating CO: (1) chemical interaction of sodium formate and sulfuric acid; (2) exhaust fumes from idling gasoline internal combustion engines; and (3) commercially compressed CO in cylinders. The first two techniques are associated with a number of problems, such as production of other gases, inadequate concentrations of carbon monoxide achieved, inadequate cooling of the gas, and maintenance of the equipment; therefore, the only recommended source is compressed CO in cylinders.

In a study by Ramsey and Eilmann,69 8% CO caused guinea pigs to collapse in 40 seconds to 2 minutes, and death occurred within 6 minutes. Carbon monoxide has been used to euthanatize mink52,58 and chinchillas. These animals collapsed in one minute, breathing ceased in 2 minutes, and the heart stopped beating in 5 to 7 minutes.

In a study evaluating the physiologic and behavioral characteristics of dogs exposed to 6% CO in air, Chalifoux and Dallaire70 could not determine the precise time of unconsciousness. Electroencephalographic recordings revealed 20 to 25 seconds of abnormal cortical function prior to unconsciousness. It was during this period that agitation and vocalization occurred. It is not known whether animals experience distress; however, human beings in this phase reportedly are not distressed.64 Subsequent studies have shown that tranquilization with acepromazine significantly decreases behavioral and physiologic responses of dogs euthanatized with CO.71

In a comparative study, CO (gasoline engine exhaust) and 70% CO2 + 30% O2 were used to euthanatize cats. Euthanasia was divided into 3 phases. Phase I was the time from initial contact to onset of clinical signs (eg, yawning, staggering, or trembling). Phase II extended from the end of phase I until recumbency, and phase III from the end of phase II until death.33 The study revealed that signs of agitation before unconsciousness were greatest with CO2 + O2. Convulsions occurred during phases II and III with both methods. However, when the euthanatizing chamber was prefilled with CO (ie, "exhaust fumes"), convulsions did not occur in phase III. Time to complete immobilization was greater with CO2 + O2 (approximately 90 seconds) than with the CO alone (approximately 56 seconds).33 In neonatal pigs, excitation was more likely to precede unconsciousness if the animals were exposed to a rapid rise in CO concentration. This agitation was decreased at lower flow rates, or when CO was combined with N.72

Advantages - (1) Carbon monoxide induces unconsciousness without pain and with minimal discernible discomfort. (2) Hypoxemia induced by CO is insidious, so that the animal appears to be unaware. (3) Death occurs rapidly if concentrations of 4-6% are used.

Disadvantages - (1) Safeguards must be taken to prevent exposure of personnel. (2) Any electrical equipment exposed to CO (eg, lights and fans) must be explosion proof.

Recommendations - Carbon monoxide used for individual animal or mass euthanasia is acceptable for small animals, including dogs and cats, provided that commercially compressed CO is used and the following precautions are taken: (1) Personnel using CO must be instructed thoroughly in its use and must understand its hazards and limitations. (2) The CO source and chamber must be located in a well-ventilated environment, preferably out of doors. (3) The chamber must be well lit and have viewports that allow personnel direct observation of the animals. (4) The CO flow rate should be adequate to rapidly achieve a uniform CO concentration of at least 6% after animals are placed in the chamber, although some species (eg, neonatal pigs) are less likely to become agitated with a gradual rise in CO concentration.72 (5) If the chamber is inside a room, CO monitors must be placed in the room to warn personnel of hazardous concentrations.

Noninhalant Pharmaceutical Agents

Intravenous administration is the most rapid and reliable method of performing euthanasia with injectable euthanasia agents. It is the most desirable method when it can be performed without causing fear or distress in the animal. Sedation of aggressive, fearful, wild, or feral animals should be accomplished prior to intravenous administration of the euthanasia agent.

When intravenous administration is considered impractical or impossible (eg, in animals weighing 7 kg, intraperitoneal administration of a nonirritating euthanasia agent is acceptable, provided that it does not contain neuromuscular blocking agents. Intrahepatic administration has also been described for use in cats.73 Intracardiac administration is not considered acceptable in awake animals, owing to the difficulty and unpredictability of performing the injection accurately. Intracardiac injection is acceptable only when performed on heavily sedated, anesthetized, or comatose animals. Intramuscular, subcutaneous, intrathoracic, intrapulmonary, intrarenal, intrasplenic, intrathecal and other nonvascular injections are not acceptable methods of administering injectable euthanasia agents.

When injectable euthanasia agents are administered other than intravenously, animals may be slow to pass through stages I and II of anesthesia. Accordingly, they should be placed in small cages in a quiet area to minimize excitement and trauma.

Barbituric Acid Derivatives

Barbiturates depress the central nervous system in descending order, beginning with the cerebral cortex, with unconsciousness progressing to anesthesia. With an overdose, deep anesthesia progresses to apnea, owing to depression of the respiratory center, which is followed by cardiac arrest.

All barbituric acid derivatives used for anesthesia are acceptable for euthanasia. Induction of unconsciousness by barbiturates results in minimal or transient pain associated with needle puncture, therefore satisfying the basic criterion for classifying an agent as acceptable for euthanasia. Barbiturates have rapid onset of action, which is a desirable characteristic for a euthanasia agent. Desirable barbiturates are those that are potent, long-acting, stable in solution, and inexpensive. Sodium pentobarbital fits these criteria and is most widely used, although others such as secobarbital are acceptable.

Advantages - (1) A primary advantage of barbiturates is speed of action. This effect depends on the dose, concentration, and rate of injection. (2) Barbiturates induce euthanasia smoothly, with minimal discomfort to the animal. (3) Barbiturates are less expensive than many other euthanasia agents.

Disadvantages - (1) Intravenous injection is necessary for best results, necessitating trained personnel. (2) Each animal must be restrained. (3) Current federal drug regulations require strict accounting for the barbiturates and these must be used under the supervision of personnel registered with the US Drug Enforcement Administration (DEA). (4) An aesthetically objectional terminal gasp may occur in unconscious animals.

Recommendations - The advantages of using barbiturates for euthanasia in small animals far outweigh the disadvantages. The intravenous injection of a barbituric acid derivative is the preferred method for euthanasia of dogs, cats, other small animals, and horses. Intraperitoneal injection may be used in situations wherein these approaches would cause less distress than intravenous injection.

Pentobarbital Combinations

Several euthanasia products are formulated to include a barbituric acid derivative (usually sodium pentobarbital), with added local anesthetic agents or agents that metabolize to pentobarbital. Although some of these additives are slowly cardiotoxic, this pharmacologic effect is inconsequential. These combination products are listed by the DEA as Schedule III drugs, making them somewhat simpler to obtain, store, and administer than Schedule II drugs such as sodium pentobarbital.

The pharmacologic properties and recommended usage of combination products presently available (which combine sodium pentobarbital with lidocaine or phenytoin) are interchangeable with those of pure barbituric acid derivatives. A combination of pentobarbital with a neuromuscular blocking agent is not an acceptable euthanasia agent.

Chloral Hydrate

Chloral hydrate depresses the cerebrum slowly; therefore, restraint may be a problem in some animals. Death is caused by hypoxemia resulting from progressive depression of the respiratory center, and may be preceded by gasping, muscle spasms, and vocalization.

Recommendations - Chloral hydrate is acceptable for euthanasia of large animals only when administered intravenously, preferably after sedation to decrease the aforementioned undesirable side effects. Chloral hydrate is not acceptable for dogs, cats, and other small animals because the side effects may be severe and are aesthetically objectionable.

Combination of Chloral Hydrate, Magnesium Sulfate, and Sodium Pentobarbital

A commercially available combination of chloral hydrate, magnesium sulfate, and sodium pentobarbital has been used for anesthesia of large animals, and is an acceptable large animal euthanasia agent when an overdose is administered intravenously.

T-61

T-61 is an injectable nonbarbiturate, non-narcotic mixture of three drugs used for euthanasia. These drugs provide a combination of general anesthetic, curariform, and local anesthetic actions. T-61 has been withdrawn from the market and is no longer manufactured or commercially available in the United States, although it is available in Canada. T-61 should be used only intravenously, because there is some question as to the differential absorption and onset of action of the active ingredients when administered by other routes.1

Unacceptable Injectable Agents

The injectable agents listed in Table 4 (strychnine, nicotine, caffeine, magnesium sulfate, potassium chloride, and all neuromuscular blocking agents), when used alone, are unacceptable and are absolutely condemned for use as euthanasia agents.

Physical Methods

Physical methods of euthanasia include captive bolt, gunshot, cervical dislocation, decapitation, electrocution, microwave irradiation, exsanguination, stunning, or pithing. However, some of these procedures, namely exsanguination, stunning, and pithing, are not recommended as a sole means of euthanasia, but are adjuncts when used in association with other agents or methods. Some consider physical methods of euthanasia aesthetically displeasing. However, some of these methods cause less fear and anxiety, and may be more rapid, painless, humane, and practical than other forms of euthanasia when properly used by skilled personnel with well-maintained equipment.

Physical methods are appropriate in three general situations: (1) easily handled small animals with anatomic features compatible with the method used; (2) large farm, wild, or zoo animals; and (3) in research when other methods might invalidate experimental results or interfere with subsequent use of tissues or body fluids.

Given that most physical methods involve trauma, there is inherent risk for animals and human beings; therefore, extreme care and caution should be used. Skill and experience of the personnel are of paramount importance when using physical methods. If the method is not accomplished correctly, animals may be injured and may have varying degrees of consciousness, resulting in pain and distress. Before using physical methods, inexperienced persons should be trained by experienced persons and should practice on carcasses or anesthetized animals to be euthanatized until they are proficient in performing the method properly and humanely. In general, physical methods are recommended for use only after other acceptable means have been excluded; in sedated or unconscious animals when practical; and when scientifically or clinically justified. Consequently, the panel considers all physical methods, except microwave irradiation, conditionally acceptable.

Penetrating Captive Bolt

A penetrating captive bolt is used for euthanasia in ruminants, horses, and swine and has recently been developed for use in laboratory rabbits and dogs.74 Its mode of action is concussion and trauma to the cerebral hemisphere and brain-stem.75,76 Captive bolts are powered by gunpowder or compressed air. Animals must be adequately restrained to ensure proper placement of the captive bolt. The correct placement of the captive bolt on the animal's head is critical. It is imperative that a cerebral hemisphere and the brainstem are sufficiently disrupted by the projectile to induce sudden unconsciousness and subsequent death. Accurate placement of captive bolts for various species has been described.75-78 A multiple projectile has been suggested as a more effective technique, especially on large cattle.75

A nonpenetrating captive bolt only stuns animals and, therefore, should not be used as a sole means of euthanasia. See the section regarding stunning.

Advantage - The penetrating captive bolt is an effective method for use in slaughterhouses and in research facilities when the use of drugs is inappropriate.

Disadvantages - (1) It is aesthetically displeasing. (2) Death may not occur.

Recommendations - Use of the penetrating captive bolt is a practical method of euthanasia for horses, ruminants, and swine when chemical agents cannot be used. It is strongly recommended that other adjunctive measures (eg, exsanguination) be used to ensure rapid death. Except for unusual circumstances, there are more acceptable methods of euthanasia for dogs and rabbits. The nonpenetrating captive bolt is not recommended as a method of euthanasia.

Gunshot

In some circumstances, gunshot may be the only practical method of euthanasia. It should be performed by highly skilled personnel using a firearm appropriate for the situation. For captive animals, the firearm should be aimed so that the projectile enters the brain, causing instant unconsciousness.30,78-80 For wildlife and other freely roaming animals, the preferred target area should be the head or neck.

Advantages - (1) Unconsciousness is instantaneous if the projectile destroys most of the brain. (2) Under field conditions, gunshot may be the only effective method available.

Disadvantages - (1) It may be dangerous to personnel. (2) It is aesthetically unpleasant. (3) Under field conditions, it may be difficult to hit the vital target area.

Recommendations - When other methods cannot be used, an accurately delivered gunshot is an acceptable method of euthanasia. When the animal can be appropriately restrained, the penetrating captive bolt is preferred to gunshot. Gunshot should not be used for routine euthanasia of animals in animal control situations, such as municipal pounds or shelters.

Cervical Dislocation

Cervical dislocation is used to euthanatize poultry, other small birds, mice, and immature rats and rabbits. For mice and rats, the thumb and index finger are placed on either side of the neck at the base of the skull or, alternatively, a rod is pressed at the base of the skull. With the other hand, the base of the tail or hind limbs are quickly pulled, causing separation of the cervical vertebrae from the skull. For immature rabbits, the head is held in one hand and the hind limbs in the other. The animal is stretched and the neck is hyper-extended and dorsally twisted to separate the first cervical vertebra from the skull.40,77 In poultry, cervical dislocation by stretching is a common method for mass euthanasia, but unconsciousness may not be instantaneous.81

Advantages - (1) Cervical dislocation is a technique that may induce rapid unconsciousness.82 (2) It does not chemically contaminate tissue. (3) It is rapidly accomplished.

Disadvantages - (1) Cervical dislocation may be aesthetically displeasing to personnel. (2) Data suggest that electrical activity in the brain persists for 13 seconds following cervical dislocation.82 (3) Its use is limited to poultry, other small birds, mice, and immature rats and rabbits.

Recommendations - When properly executed, manual cervical dislocation is a humane technique for euthanasia of poultry, other small birds, mice, rats weighing <200 g, 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; accordingly, it should be performed only with mechanical dislocators or by individuals who have demonstrated proficiency euthanatizing heavier animals.

Until additional information is available to better define the nature of the persistent EEG activity,82 this technique should be used in research settings only when scientifically justified by the user and approved by the Institutional Animal Care and Use Committee.

Those responsible for the use of this technique must determine that personnel who perform cervical dislocation techniques have been properly trained to do so.

Decapitation

Decapitation is most often used to euthanatize rodents and small rabbits. It provides a means to recover tissues and body fluids that are chemically uncontaminated. It also provides a means of obtaining anatomically undamaged brain tissue for study.54

Guillotines that are designed to accomplish decapitation in a uniformly instantaneous manner are commercially available.

Advantages - (1) Decapitation is a technique that may induce rapid unconsciousness.82-84 (2) It does not chemically contaminate tissues. (3) It is rapidly accomplished.

Disadvantages - (1) The handling and restraint required to perform this technique may be distressful to animals.85 (2) Data suggest that electrical activity in the brain persists for 13-14 seconds following decapitation. (3) Personnel performing this technique should recognize the inherent danger of the guillotine and take adequate precautions to prevent personal injury. (4) Decapitation may be aesthetically displeasing to personnel performing or observing the technique.

Recommendation - Until additional information is available to better define the nature of the persistent EEG activity,82,86 this technique should be used in research settings only when scientifically justified by the user and approved by the Institutional Animal Care and Use Committee. Decapitation of amphibians, fish, and reptiles is addressed elsewhere in this report.

Those responsible for the use of this technique must determine that personnel who perform decapitation techniques have been properly trained to do so.

Electrocution

Electrocution, using alternating current, as a form of euthanasia has been used in species such as dogs, cattle, sheep, swine, foxes, and mink.79,87-92 Electrocution induces death by cardiac fibrillation, which causes cerebral hypoxia.89,91,93 However, animals do not lose consciousness for 10 to 30 seconds or more after onset of cardiac fibrillation. It is imperative that animals be unconscious before being electrocuted. Therefore, euthanasia by electrocution must be a two-step procedure. First, an animal must be rendered unconscious by any acceptable means, including electrical stunning. If electrical stunning is used, the electrical current must pass through the brain (see the section addressing this procedure in Adjunctive Methods).

Advantages - (1) Electrocution is humane if the animal is first rendered unconscious. (2) It does not chemically contaminate tissues. (3) It is economical.

Disadvantages - (1) Electrocution may be hazardous to personnel. (2) It is not a useful method for mass euthanasia because so much time is required per animal. (3) It is not a useful method for dangerous, intractable animals. (4) It is aesthetically objectionable because of violent extension and stiffening of the limbs, head, and neck. (5) It may not result in death in small animals (< 5 kg) because ventricular fibrillation and circulatory collapse do not always persist after cessation of current flow.

Recommendation - Electrical stunning and euthanasia by electrocution require special skills and equipment that will assure passage of sufficient current through the brain to induce unconsciousness followed by electrically induced cardiac fibrillation. Although the method is conditionally acceptable if the aforementioned requirements are met, its disadvantages far outweigh its advantages in most applications. Techniques that apply electric current from head to tail or head to foot are unacceptable.

Microwave Irradiation

Heating by microwave irradiation is used primarily by neurobiologists to fix brain metabolites in vivo while maintaining the anatomic integrity of the brain.94 Microwave instruments have been specifically designed or modified for use in euthanasia of laboratory mice and rats. The instruments differ in design from kitchen units and may vary in the maximal power output from 1.3 to 10 kw. All units direct their microwave energy to the head of the animal. The power required to rapidly halt brain enzyme activity depends on the efficiency of the unit, the ability to tune the resonant cavity and the size of the rodent head.95 There is considerable variation among instruments in the time required to induce unconsciousness and euthanasia. A 10-kw, 2,450-MHz instrument operated at a power of 9 kw will increase the brain temperature of 18- to 28-g mice to 79C in 330 msec, and the brain temperature of 250- to 420-g rats to 94C in 800 msec.96

Advantages - (1) Unconsciousness is achieved in less than 100 msec, and death in less than one second. (2) This is the most effective method to fix brain tissue in vivo for subsequent assay of enzymatically labile chemicals.

Disadvantages - (1) Instruments are expensive. (2) Only animals the size of mice and rats can be euthanatized with commercial instruments that are currently available.

Recommendations - Microwave irradiation is a humane method to euthanatize small laboratory rodents if instruments that induce rapid unconsciousness are used. Only instruments that are designed for this use and have appropriate power and microwave distribution can be used. Microwave ovens designed for domestic and institutional kitchens are absolutely unacceptable for euthanasia.

Adjunctive Methods

Stunning and pithing, when properly done, induce unconsciousness but do not ensure death. Therefore, these methods should be used in conjunction with other procedures such as pharmacologic agents, exsanguination, or decapitation to kill the animal.

Exsanguination

Exsanguination can be used to ensure death subsequent to stunning, electrical stunning, or in otherwise unconscious animals. Because anxiety is associated with extreme hypovolemia, exsanguination must not be used as a sole means of euthanasia.97 Animals may be exsanguinated to obtain blood products, but only when they are sedated, stunned, or anesthetized.98

Stunning

Animals may be stunned by a blow to the head, use of a nonpenetrating captive bolt, and electric current. With stunning, evaluation of unconsciousness is difficult, but it is usually associated with a loss of the menace or blink response, pupillary dilatation, and a loss of coordinated movements. Specific changes in the electroencephalogram and a loss of visually evoked responses are also thought to indicate unconsciousness.38,99

Blow to head - Stunning,9,100-102 by a blow to the head is used primarily in small laboratory animals with thin craniums. A single sharp blow must be delivered to the central skull bones with sufficient force to produce immediate depression of the central nervous system. When properly done, unconsciousness is rapid.

Nonpenetrating captive bolt - A nonpenetrating captive bolt may be used to induce unconsciousness in ruminants, horses, and swine. The signs of effective stunning by captive bolt are immediate collapse and a several-second period of tetanic spasm, followed by slow hind limb movements of increasing frequency.38 Other aspects regarding use of nonpenetrating captive bolt are similar to use of a penetrating captive bolt. Refer to the captive bolt section for additional information.

Electrical stunning - Alternating electrical current has been used for stunning in species such as dogs, cattle, sheep, goats, hogs, and chickens.87,88,103-105 Experiments in dogs have shown the necessity of directing the electrical current through the brain in order to induce rapid loss of consciousness. In the dog, when electricity passes only between fore- and hindlimbs or neck and feet, it causes the heart to fibrillate but does not induce sudden unconsciousness.93 For electrical stunning of any animal, an apparatus that applies electrodes to opposite sides of the head, or in another way directs electrical current immediately through the brain, is necessary to induce rapid unconsciousness. Attachment of electrodes and animal restraint can pose problems with this form of stunning. The signs of effective electrical stunning are extension of the limbs, opisthotonos, downward rotation of the eyeballs, and tonic spasm changing to clonic spasm, with eventual muscle flaccidity.

Electrical stunning should be followed promptly by electrically induced fibrillation of the heart, exsanguination, or other appropriate methods to ensure death. Refer to the section on electrocution for additional information. Water jet stunning - A stunning and slaughter method for swine using water under high pressure has been described recently.106

Pithing

In general, pithing 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.

Special Considerations

Equine Euthanasia

Pentobarbital or a pentobarbital combination is the best choice for equine euthanasia. Because a large volume of solution must be injected, a catheter should be placed in the jugular vein. To facilitate catheterization of an excitable or fractious animal, a tranquilizer such as acepromazine, or an alpha-2-adrenergic agonist can be administered, but these drugs may prolong the time to unconsciousness because of their effect on circulation. Opioid agonists or agonist/antagonists in conjunction with alpha-2 adrenergic agonists may further facilitate restraint.

In certain emergency circumstances, it may be difficult to restrain a dangerous horse or other large animal for intravenous injection, and the animal might cause injury to itself or to bystanders before a sedative could take effect. In such cases, which might include euthanasia of a horse with a serious injury at a racetrack, the animal can be given an immobilizing agent such as succinylcholine, but an anesthetic must be administered as soon as the animal can be controlled. After the animal is anesthetized, an overdose of the anesthetic can be used to accomplish euthanasia. Succinylcholine alone or without sufficient anesthetic must not be used for euthanasia.

Precautions Concerning Use of Euthanatizing Agents in Animals Intended For Human or Animal Food

In euthanasia of animals intended for human or animal food, agents that result in tissue residues cannot be used, unless they are approved by the US Food and Drug Administration.107 Carbon dioxide is the only chemical currently used in euthanasia of food animals (primarily swine) that does not lead to tissue residues.

Carcasses of animals euthanatized by barbituric acid derivatives or other chemical agents may contain potentially harmful residues. These carcasses should be disposed of in a manner that will prevent them from being consumed by human beings or animals.

Euthanasia of Nonconventional Species: Zoo, Wild, Aquatic, and Poikilothermic Animals

Compared with objective information on companion, farm, and laboratory animals, euthanasia of species such as zoo, wild, aquatic, and poikilothermic animals has been studied less, and guidelines are more limited.

In selecting a means of euthanasia for these species, factors and criteria in addition to those previously discussed must be considered. The means selected will depend on the species, size, safety aspects, location of the animals to be euthanatized, and experience of personnel. Whether the animal to be euthanatized is in the wild, in captivity, or free roaming are major considerations. Anatomic differences must be considered. For example, amphibians, fish, reptiles, and marine mammals differ anatomically from domestic species. Veins may be difficult to locate. Some species have a carapace. For physical methods, access to the central nervous system may be difficult because the brain may be small and difficult to locate by inexperienced persons.

Zoo animals- For captive zoo mammals and birds with related domestic counterparts, many of the means described previously are approprIate. However, to minimize injury to persons or animals, additional precautions such as handling and physical or chemical restraint are important considerations.108

Wildlife- For wild and feral animals, many of the recommended means of euthanasia for captive animals are not feasible. In field circumstances, wildlife biologists generally do not use the term euthanasia, but use terms such as killing, collecting, or harvesting, recognizing that a distress-free death may not be possible.

For many field studies, the only practical means of animal collection are those involving direct killing of the animal.12,109-113 Under these conditions, methods must be as age- species- or taxonomic/class-specific as possible. Commonly used methods include gunshot and kill trapping. Gunshot is the most effective or only way to collect some species. When shooting is used as the means of animal collection, the firearm and ammunition should be appropriate for the species and purpose of the study. Personnel should be sufficiently skilled to be able to accurately hit preferred target organs for the particular species of animal. Personnel should be experienced in the proper and safe use of firearms and must comply with laws and regulations governing their possession and use. For killing larger wildlife with gunshot, preferred target areas are the head or neck.

Kill traps are practical and effective for animal collection when used in a manner that minimizes the potential for attraction and collection of nontarget species. Traps should be checked at least once daily. In those instances when an animal is wounded or captured but not dead, the animal must be killed quickly and humanely.

Amphibians, fish, and reptiles- When euthanasia of poikilothermic animals is performed, the differences in their metabolism, respiration, and tolerance to cerebral hypoxia may preclude some procedures that would be acceptable in homeothermic animals. Additionally, it is often more difficult to ascertain when an animal is dead. Euthanasia of amphibians, fishes, and reptiles has been addressed.12,30,114

Sodium pentobarbital (60 mg/kg of body weight) or other barbiturates can be administered intravenously, intraabdominally, or intrapleuro-peritoneally in most cold-blooded animals, depending on anatomic features.

Tricaine methanesulfonate (TMS, MS-222) may be administered by a variety of routes to induce euthanasia. For aquatic animals, including amphibians, this chemical may be placed in the water. Large fish may be removed from the water, a gill cover lifted, and a concentrated solution from a syringe flushed over the gills. This is an effective but expensive means of euthanasia, and is not hazardous to personnel. Benzocaine hydrochloride, a compound similar to TMS, may be used as a bath or in a recirculation system for euthanasia of fish113,115 or amphibians.12

Species such as snakes, lizards, turtles, frogs, and toads may be killed by over-exposure to gaseous anesthetics such as halothane or methoxyflurane in a chamber or via face mask. Carbon dioxide gas may be used for terrestrial animals. Some reptiles can stop or reduce their breathing for long periods without overt ill effects, and may not die even after prolonged exposure.

It has been suggested that, when using physical methods of euthanasia in poikilothermic species, cooling to 4C will decrease metabolism and facilitate handling, but there is no evidence that it raises the pain threshold. Line drawings of the head of various amphibians and reptiles, with recommended locations for captive bolt or firearm penetration, are available.12

Most amphibians, fishes, and reptiles can be euthanatized by cranial concussion (stunning) followed by decapitation or some other physical method.

Decapitation with heavy shears or guillotine is effective in some species that have appropriate anatomic features. It has been assumed that stopping blood supply to the brain by decapitation causes rapid unconsciousness. Recently, this view has been questioned because the central nervous system of reptiles and amphibians is tolerant to hypoxic and hypotensive conditions.12 Consequently, decapitation should be followed by pithing.

Severing the spinal cord behind the head by pithing is an effective method of killing some poikilotherms. Inasmuch as death may not be immediate unless both the brain and spinal cord are pithed, double pithing is recommended. Pithing of the spinal cord should be followed by decapitation and pithing of the brain or other appropriate procedure. The anatomic features of some species preclude effective use of this method. Pithing requires dexterity and skill and should be done only by trained personnel.

Snakes and turtles, immobilized by cooling, have been killed by subsequent freezing. However, this method is not recommended.12 Formation of ice crystals on the skin and in tissues of an animal may cause pain or distress. Quick freezing of deeply anesthetized animals is acceptable.

Crocodilians and other large reptiles can be shot through the brain.30

Marine mammals - For smaller pinnipeds and cetaceans, barbiturates or potent opioids (eg, etorphine hydrochloride [M 99] and carfentanil) are recommended. An accurately placed gunshot may also be an acceptable method for euthanasia of stranded marine mammals.30 For beached whales or other large cetaceans or pinnipeds, succinylcholine chloride in conjunction with potassium chloride, administered intravenously or intraperitoneally, has been used.116 This method, which is not a method of euthanasia as defined in this report, should be used only as a last resort. Although it leads to complete paralysis of the respiratory musculature, and death due to hypoxemia, it may be more humane than allowing the stranded animal to suffocate over a period of hours or days.

Euthanasia of Animals Raised For Fur Production

Animals raised for fur are usually killed individually at the location where they are raised. Although any handling of these species constitutes a stress, it is possible to minimize this by euthanatizing animals in or near their cages. For the procedures described below, please refer to the previous sections for a more detailed discussion.

Carbon monoxide - In the case of the smaller species (eg, mink), CO appears to be an adequate method for euthanasia. Compressed CO is delivered from a tank into an enclosed cage that can be moved adjacent to holding cages. Using the apparatus outside reduces the risk to human beings; however, people using this method should still be made aware of the dangers of CO. Animals introduced into a chamber containing 4% CO lost consciousness in 64 14 seconds and were dead within 215 45 seconds.52 In a study involving electroencephalography of mink being euthanatized with 3.5% CO, the animals were comatose in 21 7 seconds117. Only one animal should be introduced into the chamber at a time, and death should be confirmed in each case.

Carbon dioxide - Carbon dioxide (CO2) is also a good euthanasia method for the smaller species and is less dangerous than CO for personnel operating the system. Using compressed CO2 from a tank is likely to be more reliable and efficient than using solid CO2. When exposed to 100% CO2, mink lost consciousness in 19 4 seconds and were dead in 153 10 seconds. When 70% CO2 was used with 30% CO2, the animals were unconscious by 28 seconds, but they were not dead after a 15-minute exposure.52 Therefore, if animals are first stunned by 70% CO2, they should be killed by exposure to 100% CO2 or by some other means. As with carbon monoxide, only one animal should be introduced into the chamber at a time.

Barbiturate overdose - Barbiturate overdose is an acceptable procedure for euthanasia of many species of animals raised for fur. The drug is injected intraperitoneally and the animal slowly loses consciousness. It is important that the death of each animal be confirmed following barbiturate injection. Barbiturates will contaminate the carcass; therefore the skinned carcass cannot be used for animal food.

Electrocution - Electrocution has been used for killing foxes and mink.89 The electric current must pass through the brain to induce unconsciousness before electricity is passed through the rest of the body. Use of a nose-to-tail or nose-to-foot89 method may kill the animal by inducing cardiac fibrillation, but the animal may be conscious for a period before death; therefore, these techniques are unacceptable. Electrical stunning may be followed by cervical dislocation in mink and other small animals. It is recommended that cervical dislocation be done within 20 seconds of electrical stunning.118

Postface

This report summarizes contemporary scientific knowledge on euthanasia in animals and calls attention to the lack of scientific reports assessing pain, discomfort, and distress in animals being euthanatized. Many reports on various methods of euthanasia are either anecdotal, testimonial narratives, or unsubstantiated opinions and are, therefore, not cited in this report. The panel strongly endorses the need for well-designed experiments to more fully determine the extent to which each procedure meets the criteria used for judging the methods of euthanasia. Each means of euthanasia has advantages and disadvantages. It is unlikely that, for each given situation, any means will meet all desirable criteria. It is also impractical for this report to address every potential circumstance in which animals are to be euthanatized. Therefore, the use of professional judgment is imperative.

Failure to list or recommend a means of euthanasia in this report does not categorically condemn its use. There may occasionally be special circumstances or situations in which other means may be acceptable. For research animals, these exceptions should be carefully considered by the attending veterinarian and the Institutional Animal Care and Use Committee. In other settings, professional judgment should be used.

The references cited in this report do not represent a comprehensive bibliography on all methods of euthanasia. Persons interested in additional information on a particular aspect of animal euthanasia are encouraged to contact the Animal Welfare Information Center, National Agricultural Library, 10301 Baltimore Blvd, Beltsville, MD 20705.

The Panel on Euthanasia is fully committed to the concept that, whenever it becomes necessary to kill any animal for any reason whatsoever, death should be induced as painlessly and quickly as possible. It has been our charge to develop workable guidelines for addressing this need, and it is our sincere desire that these guidelines be used conscientiously by all animal care providers.

Acknowledgment: The panel acknowledges the assistance of Jennifer Klages in coordinating the preparation and circulation of various drafts of the report. The panel also acknowledges and thanks the 130 individuals and organizations that provided valuable review, criticism, and input to the panel through the many drafts of the report. The research and humane communities were especially helpful in shaping important changes and additions to the report.


Table 1 - Agents and methods of euthanasia by species

(Refer to Table 4 for unacceptable agents and methods.)

Species

I. Acceptable (Refer to Table 2)
II. Conditionally acceptable (Refer to Table 3)

Amphibians

I. Inhalant anesthetics, CO, CO2, barbiturates, tricaine methanesulfonate, double pithing, benzocaine

II. Pithing, gunshot, penetrating captive bolt, stunning and decapitation, decapitation and pithing

Birds

I. Inhalant anesthetics, CO, CO2, barbiturates

II. N2, Ar, cervical dislocation, decapitation

Cats

I. Inhalant anesthetics, CO, CO2, barbiturates
II. N2, Ar

Dogs

I. Inhalant anesthetics, CO, CO2, barbiturates

II. N2, Ar, electrocution, penetrating captive bolt

Fish

I. Tricaine methanesulfonate, benzocaine, barbiturates

II. Stunning and decapitation, decapitation

Horses

I. Barbiturates, chloral hydrate, chloral hydrate/ MgSO4/pentobarbital

II. Penetrating captive bolt, gunshot, electrocution

Marine mammals

I. Barbiturates, etorphine hydrochloride

II. Succinylcholine chloride and potassium chloride, gunshot

Mink, fox and other mammals produced for fur

I. Inhalant anesthetics, CO, CO2, barbiturates

II. N2, Ar, electrocution followed by cervical dislocation

Non-human primates

I. Barbiturates

II. Inhalant anesthetics, CO, CO2, N2, Ar

Rabbits

I. Inhalant anesthetics, CO, CO2, barbiturates

II. N2, Ar, cervical dislocation, decapitation, penetrating captive bolt

Reptiles

I. Barbiturates, inhalant anesthetics, CO2

II. Gunshot, penetrating captive bolt, stunning and decapitation, decapitation and pithing

Rodents and other small mammals

I. Inhalant anesthetics, CO, CO2, microwave irradiation, barbiturates

II. N2, Ar, cervical dislocation, decapitation

Ruminants

I. Barbiturates

II. Penetrating captive bolt, gunshot, electrocution, chloral hydrate

Swine

I. Barbiturates, CO2

II. Inhalant anesthetics, CO, penetrating captive bolt, gunshot, electrocution, chloral hydrate

Zoo animals

I. Inhalant anesthetics, CO2, CO, barbiturates

II. N2, Ar, penetrating captive bolt, gunshot


Table 2 - Summary of acceptable agents and methods of euthanasia - characteristics and modes of action

Agent

Classification
Mode of action
Rapidity
Performance ease
Personnel safety
Species suitability
Efficacy

Barbiturates

Hypoxia due to depression of vital centers
Direct depression of cerebral cortex, subcortical struc- tures and vital centers; direct depression of heart muscle
Rapid onset of anesthesia
Animal must be restrained; personnel must be skilled to perform IV injection
Safe except human abuse potential;DEA-controlled substance
Most species Highly effective when appropriately administered; acceptable IV and IP in small animals

Inhalant anesthetics

Hypoxia due to depression of vital centers
Direct depression of cerebral cortex and subcortical struc- tures and vital centers
Moderately rapid onset of anesthesia, some excitation may occur during induction
Easily performed with closed container; can be administered to large animals by means of a mask
Must be properly scavenged or vented to minimize exposure to personnel
Amphibians, birds, cats, dogs, fur bearing animals, rabbits, reptiles, rodents and other small animals, zoo animals
Highly effective provided that subject is sufficiently exposed

Carbon dioxide

Hypoxia due to depression of vital centers
Direct depression of cerebral cortex, subcortical struc- tures and vital centers; direct depression of heart muscle
Moderately rapid
Used in closed container
Minimal hazard
Small laboratory animals, birds, cats, small dogs, mink, zoo animals, am- phibians
Effective, but time required may be prolonged in immature and neonatal animals

Carbon monoxide (bottled gas only)

Hypoxia
Combines with hemoglobin, preventing its combination with oxygen
Moderate onset time; but insidious so animal is unaware of onset
Requires appropriately operated equipment for gas production
Extremely hazardous, toxic and difficult to detect
Most small species including dogs, cats, rodents, mink, chinchillas, birds, reptiles, amphibians and zoo animals
Effective; acceptable only when equipment is properly designed and operated

Microwave irradiation

Brain enzyme inactivation
Direct inactivation of brain enzymes by rapid heating of brain
Very rapid
Requires training and highly specialized equipment
Safe
Mice and rats
Highly effective for special needs

Tricaine methanesulfonate

Hypoxia due to depression of vital centers
Depression of CNS
Very rapid, depending on dose
Easily used
Safe
Fish and amphibians
Effective but expensive

Benzocaine

Hypoxia due to depression of vital centers
Depression of CNS
Very rapid, depending on dose
Easily used
Safe
Fish and amphibians
Effective but expensive


Table 3 - Summary of conditionally acceptable agents and methods of euthanasia - characteristics and modes of action

Agent

Classification
Mode of action
Rapidity
Ease of performance
Safety
Species suitability
Efficacy/comments

Cervical dislocation

Hypoxia due to disruption of vital centers
Direct depression of brain
Moderately rapid
Requires training and skill
Safe
Poultry, birds, lab mice and rats less than 200g or rabbits less than 1kg Irreversible. Violent muscle contractions can occur after cervical dislocation

Decapitation

Hypoxia due to disruption of vital centers
Direct depression of brain
Moderately rapid
Requires training and skill
Guillotine poses potential employee injury hazard
Lab rodents, small rabbits, birds, fish, amphibians, reptiles
Irreversible. Violent muscle contraction can occur after decapitation

Penetrating captive bolt

Physical damage to brain
Direct concussion of brain tissue
Rapid
Requires skill, adequate restraint and proper placement of captive bolt
Safe
Ruminants, horses,swine, dogs, rabbits,zoo animals, reptiles
Instant unconsciousness but motor activity may continue

Gunshot

Hypoxia due to disruption of vital centers
Direct concussion of brain tissue
Rapid
Requires skill and appropriate firearm
May be dangerous
Large domestic and zoo animals, reptiles, and wildlife
Instant unconsciousness but motor activity may continue

Electrocution

Hypoxia
Direct depression of brain and cardiac fibrillation
Can be rapid
Not easily performed in all instances
Hazardous to personnel
Used primarily in foxes, sheep, swine and mink
Violent muscle contractions occur at same time as unconsciousness

Pithing

Hypoxia due to disruption of vital centers, physical damage to brain
Trauma of brain and spinal cord tissue
Rapid
Easily performed but requires skill
Safe
Some poikilotherms Effective but death not immediate unless double pithed

Nitrogen, Argon

Hypoxic hypoxemia
Reduces partial pressure of oxygen available to blood
Rapid
Use closed chamber with rapid filling
Safe if used with ventilation
Cats, small dogs, birds, rodents, rabbits, other small species, mink, zoo animals
Effective except in young and neonates; an effective agent, but other methods are preferable; not acceptable in most animals less than 4 months old


Table 4 - Summary of some unacceptable agents and methods of euthanasia

Agent

Comments

Exsanguination

Because of the anxiety associated with extreme hypovolemia, exsanguination should be done only in sedated, stunned or anesthetized animals.

Decompression

Decompression is not a recommended method for euthanasia because of the numerous disadvantages. (1) Many chambers are designed to produce decompression at a rate 15 to 60 times faster than that recommended as optimum for animals, resulting in pain and distress due to expanding gases trapped in body cavities. (2) Immature animals are tolerant of hypoxia, and longer periods of decompression are required before respiration ceases. (3) Accidental recompression, with recovery of injured animals can occur. (4) Bloating, bleeding, vomiting, convulsions, urination, and defecation, which are aesthetically unpleasant, may occur in the unconscious animal.

Rapid freezing

Rapid freezing as a sole means of euthanasia is not considered to be humane. If used, animals should be anesthetized prior to freezing.

Air embolism

Air embolism may be accompanied by convulsions, opisthotonos and vocalization. If used, it should be done only in anesthetized animals.

Drowning

Drowning as a means of euthanasia is inhumane.

Strychnine

Strychnine causes violent convulsions and painful muscle contractions.

Nicotine, magnesium sulfate, potassium chloride, all curariform agents (neuromuscular blocking agents)

When used alone, these drugs all cause respiratory arrest before unconsciousness, so the animal may perceive pain after it is immobilized.

Chloroform

Chloroform is a known hepatotoxin and suspected carcinogen, and therefore hazardous to human beings.

Cyanide

Cyanide poses an extreme danger to personnel and the manner of death is aesthetically objectionable.

Stunning

Stunning may render an animal unconscious, but it is not a method of euthanasia. If used, it must be followed by a method to ensure death.


References

1. Smith AW, Houpt HA, Kitchell RL, et al. Report of the AVMA panel on euthanasia. J Am Vet Med Assoc 1986; 188:252-268.

2. Webster's ninth new collegiate dictionary. Springfield, Mass: Merriam-Webster Inc, 1990.

3. Breazile JE, Kitchell RL. Euthanasia for laboratory animals. Fed Proc 1969;28:1577-1579.

4. Kitchell RL, Erickson NH, Carstens E, et al, eds. Animal pain: perception and alleviation. Bethesda, Md: American Physiological Society, 1983.

5. Kitchell RL, Johnson RD. Assessment of pain in animals. In: Moberg GP, ed. Animal stress. Bethesda, Md: American Physiological Society, 1983;113-140.

6. Willis WD. The pain system. The neural basis of nociceptive transmission in the mammalian nervous system. Basel, Switzerland: S Karger, 1985;346.

7. Zimmerman M. Neurobiological concepts of pain, its assessment and therapy. In: Bromm B, ed. Pain measurement in man. Neurophysiological correlates of pain. Amsterdam: Elsevier Publishing Co, 1984; 15-35.

8. Kitchen N, Aronson AL, Bittle JL, et al. Panel report on the colloquium on recognition and alleviation of animal pain and distress. J Am Vet Med Assoc 1987;191:1186-1191.

9. National Research Council. Recognition and alleviation of pain and distress in laboratory animals. Washington, DC: National Academy Press, 1992.

10. Breazile JE. Physiologic basis and consequences of distress in animals. J Am Vet Med Assoc 1987;191:1212-1215.

11. Grier RL, Clovin TL. Euthanasia guide (for animal shelters). Ames, Iowa: Moss Creek Publications, 1990.

12. Cooper JE, Ewbank R, Platt C, et al. Euthanasia of amphibians and reptiles. London: UFAW/WSPA, 1989.

13. Greyhavens T. Handbook of pentobarbital euthanasia. Salem, Ore: Humane Society of Willamette Valley.

14. Operational guide for animal care and control agencies. Denver: American Humane Association, 1988.

15. Humane slaughter regulations. Fed Reg 1979;44: 68809-68817.

16. Grandin T. Observations of cattle behavior applied to design of cattle-handling facilities. Appl Anim Ethol 1980;6:19-31.

17. Grandin T. Pig behavior studies applied to slaughter-plant design. Appl Anim Ethol 1982;9:141-151.

18. Tannenbaum J. Issues in companion animal practice. In: Veterinary ethics. Baltimore: The Williams & Wilkins Co, 1989;208-225.

19. Rollin BE. Ethical question of the month. Can Vet J 1992;33:7-8.

20. Beaver B. Veterinary aspects of feline behavior. St Louis: The CV Mosby Co, 1980;217.

21. Houpt HA. Domestic animal behavior for veterinarians and animal scientists. Ames, Iowa: Iowa State University Press, 1991;408.

22. Hart BL. The behavior of domestic animals. New York: WH Freeman and Co, 1985;390.

23. Hart LA, Hart BL, Mader B. Humane euthanasia and companion animal death: caring for the animal, the client, and the veterinarian. J Am Vet Med Assoc 1990;197:1292-1299.

24. Neiburg HA, Fischer A. Pet loss, a thoughtful guide for adults and children. New York: Harper & Row, 1982.

25. Hart LA, Mader B. Pet loss support hotline: the veterinary students' perspective. Calif Vet 1992;Jan-Feb:19-22.

26. Arluke A. Coping with euthanasia: a case study of shelter culture. J Am Vet Med Assoc 1991;198:1176-1180.

27. Wolfle TL. Laboratory animal technicians: their role in stress reduction and human-companion animal bonding. Vet Clin North Am Small Anim Pract 1985; 15:449-454.

28. Glass HG, Snyder FF, Webster E. The rate of decline in resistance to anoxia of rabbits, dogs, and guinea pigs from the onset of viability to adult life. Am J Physiol 1944;140:609-615.

29. Booth NH. Inhalant anesthetics. In: Booth NH, McDonald LE, eds. Veterinary pharmacology and therapeutics. 6th ed. Ames, Iowa: Iowa State University Press, 1988; 181-211.

30. Humane killing of animals. Preprint of 4th ed. South Mimms, Potters Bar, Herts, England: Universities Federation for Animal Welfare, 1988; 16-22.

31. Occupational exposure to waste anesthetic gases and vapors. No. 77-140. Washington, DC: Department of Health, Education, and Welfare (National Institute for Occupational Safety and Health), 1977.

32. Lecky JH, ed. Waste anesthetic gases in operating room air: a suggested program to reduce personnel exposure. Park Ridge, Ill: The American Society of Anesthesiologists, 1983.

33. Simonsen HB, Thordal-Christensen AA, Ockens N. Carbon monoxide and carbon dioxide euthanasia of cats: duration and animal behavior. Br Vet J 1981;137:274-278.

34. Klemm WR. Carbon dioxide anesthesia in cats. Am J Vet Res 1964;25:1201-1205.

35. Leake CD, Waters RM. The anesthetic properties of carbon dioxide. Curr Res Anesthesiol Analg 1929;8:17-19.

36. Mattsson JL, Stinson JM, Clark CS. Electroencephalographic power-spectral changes coincident with onset of carbon dioxide narcosis in rhesus monkey. Am J Vet Res 1972; 33:2043-2049.

37. Woodbury DM, Rollins LT, Gardner MD, et al. Effects of carbon dioxide on brain excitability and electrolytes. Am J Physiol 1958; 192:79-90.

38. Blackmore DK, Newhook JC. The assessment of insensibility in sheep, calves, and pigs during slaughter. In: EikeIenboom G, ed. Stunning of animals for slaughter. Boston: Martinus Nijhoff Publishers, 1983.

39. Glen JB, Scott WN. Carbon dioxide euthanasia of cats. Br Vet J 1973; 129:471-479.

40. Hughes HC. Euthanasia of laboratory animals. In: Melby, Altman, eds. Handbook of laboratory animal science. VoI 3. Cleveland, Ohio: CRC Press, 1976;553-559.

41. Jaksch W. Euthanasia of day-old male chicks in the poultry industry. Int J Stud Anim Prob 1981;2:203-213.

42. Kline BE, Peckham V, Hesic HE. Some aids in handling large numbers of mice. Lab Anim Care 1963;13:84-90.

43. Kocula AW, Drewniak EE, Davis LL. Experimentation with in-line carbon dioxide immobilization of chickens prior to slaughter. Poult Sci 1961;40:213-216.

44. Stone WS. Amiraian K, DueIl C, et al. Carbon dioxide anesthetization of guinea pigs to increase yields of blood and servm. Proc Care Panel 1961;11:299-303.

45. Hoenderken R. Electrical and carbon dioxide stunning of pigs for slaughter. In: Eikelenboom G, ed. Stunning of animals for slaughter. Boston: Martinus Nijhoff Publishers, 1982:59-63.

46. Gregory NG, Moss BW, Leeson RH. An assessment of carbon dioxide stunning in pigs. Vet Rec 1987;121:517-518.

47. Carding AH. Mass euthanasia of dogs with carbon monoxide and/or carbon dioxide: preliminary trials. J Small Anim Pract 1968;9:245-259.

48. Britt DP. The humaneness of carbon dioxide as an agent of euthanasia for laboratory rodents. In: Euthanasia of unwanted, injured or diseased animals or for educational or scientific purposes. UCAW 1987:19-31.

49. Blackshaw JK, Fenwick DC, Beattie AW, et al. The behaviour of chickens, mice and rats during euthanasia with chloroform, carbon dioxide and ether. Lab Anim 1988;22:67-75.

50. Euthanasia (carbon dioxide). In: Report and accounts 1976-1977. South Mimms, Potters Bar, Herts, England: Universities Federation for Animal Welfare, 1977;13-14.

51. Hall LW. The anaesthesia and euthanasia of neonatal and juvenile dogs and cats. Vet Rec 1972;90:303-306.

52. Hansen NE, Creutzberg A. Simonsen HB. Euthanasia of mink (Mustela vison) by means of carbon dioxide (CO2), carbon monoxide (CO) and nitrogen (N2). Br Vet J 1991:147:140-146.

53. Laursen AM. Choosing between CO2 and electrical stunning of pigs. A preliminary examination of stress and ethics. In: Eikelenboom G, ed. Stunning of animals for slaughter. Boston: Martinus Nijhoff Publishers, 1983:64-72.

54. Feldman DB, Gupta BN. Histopathologic changes in laboratory animals resulting from various methods of euthanasia. Lab Anim Sci 1976:26:218-221.

55. Hornett TD, Haynes AP. Comparison of carbon dioxide/air mixture and nitrogen/air mixture for the euthanasia of rodents. Design of a system for inhalation euthanasia. Anim Technol 1984:35:93-99.

56. Herin RA, Hall P. Fitch JW. Nitrogen inhalation as a method of euthanasia in dogs. Am J Vet Res 1978:39:989-991.

57. Noell WK, Chinn HI. Time course of failure of the visual pathway in rabbits during anoxia. Fed Proc 1949:8:119.

58. Vinte, FJ. The humane killing of mink. London: Universities Federation for Animal Welfare, 1957.

59. Stonehouse RW. Loew FM. Quine JP, et al. The euthanasia of dogs and cats: a statement of the humane practices committee of the Canadian Veterinary Medical Association. Can Vet J 1978:19:164-168.

60. Quine JP, Buckingham W. Strunin L. Euthanasia of small animals with nitrogen; comparison with intravenous pentobarbital. Can Vet J 1988;29:724-726.

61. Quine JP. Euthanasia by hypoxia using nitrogen. A review after four years of operation involving 20,500 animals (letter). Can Vet J 1980;21:320.

62. Raj ARM, Gregory NG, Wotton SR. Changes in the somatosensory evoked potentials and spontaneous electroencephalogram of hens during stunning in Argon-induced anoxia. Br Vet J 1991:147:322-330.

63. Lowe-Ponsford FL. Henry JA. Clinical aspects of carbon monoxide poisoning. Adverse Drug React Acute Poisoning Rev 1989:8:217-240.

64. Haldane J. The action of carbonic oxide in man. J Physiol 1895:18:430-462.

65. Bloom JD. Some considerations in establishing divers' breathing gas purity standards for carbon monoxide. Aerosp Med 1972:43:633-636.

66. Norman CA. Halton DM. Is carbon monoxide a workplace teratogen? A review and evaluation of the literature. Ann Occup Hyg 1990;34:335-347.

67. Eechter LD. Neurotoxicity of prenatal carbon monoxide exposure. Research report. Health Effects Institute, 1987;3-22.

68. Wojtczak-Jaroszowa J, Kubow S. Carbon monoxide, carbon disulfide, lead and cadmium-four examples of occupational toxic agents linked to cardiovascular disease. Med Hypotheses 1989;30:141-150.

69. Ramsey TL, Eilmann HJ. Carbon monoxide acute and chronic poisoning and experimental studies. J Lab Clin Med 1932;17:415-427.

70. Chalifoux A, Dallaire A. Physiologic and behavioral evaluation of CO euthanasia of adult dogs. Am J Vet Res 1983; 44:2412-2417.

71. Dallaire A, Chalifoux A. Premedication of dogs with acepromazine or pentazocine before euthanasia with carbon monoxide. Can J Comp Med 1985;49:171-178.

72. Lambooy E. Spanjaard W. Euthanasia of young pigs with carbon monoxide. Vet Rec 1980; 107:59-61.

73. Grier RL. Schaffer CB. Evaluation of intraperitoneal and intrahepatic administration of a euthanasia agent in animal shelter cats. J Am Vet Med Assoc 1990;197:1611-1615.

74. Dennis MB, Dong WK, Weisbrod KA, et al. Use of captive bolt as a method of euthanasia in larger laboratory animal species. Lab Anim Sci 1988;38:459-462.

75. Blackmore DK. Energy requirements for the penetration of heads of domestic stock and the development of a multiple projectile. Vet Rec 1985;116:36-40.

76. Daly CC, Whittington PE. Investigation into the principal determinants of effective captive bolt stunning of sheep. Res Vet Sci 1989;46:406-408.

77. Clifford DH. Preanesthesia, anesthesia, analgesia, and euthanasia. In: Fox JG, Cohen BJ, Loew FM, eds. Laboratory animal medicine. New York: Academic Press Inc, 1984;528-563.

78. Australian Veterinary Association. Guidelines on humane slaughter and euthanasia. Aust Vet J 1987:64:4-7.

79. Carding T. Euthanasia of dogs and cats. Anim Reg Stud 1977;1:5-21.

80. Longair JA, Finley GG, Laniel M-A, et al. Guidelines for euthanasia of domestic animals by firearms. Can Vet J 1991:32:724-726.

81. Gregory NG, Wotton SB. Comparison of neck dislocation and percussion of the head on visual evoked responses in the chicken's brain. Vet Rec 1990;126:570-572.

82. Vanderwolf CH, Buzak DP, Cain RK, et al. Neocortical and hippocampal electrical activity following decapitation in the rat. Brain Res 1988:451:340-344.

83. Derr RF. Pain perception in decapitated rat brain. Life Sci 1991:49:1399-1402.

84. Holson RR. Euthanasia by decapitation: evidence that this technique produces prompt, painless unconsciousness in laboratory rodents. Neurotoxicol Teratol 1992:14:253-257.

85. Urbanski HF. Kelly SF. Sedation by exposure to gaseous carbon dioxide-oxygen mixture: application to studies involving small laboratory animal species. Lab Anim Sci 1991; 41:80-82.

86. Mikeska JA, Klemm WR. EEG evaluation of humaneness of asphyxia and decapitation euthanasia of the laboratory rat. Lab Anim Sci 1975:25:175-179.

87. Warrington R. Electrical stunning, a review of the literature. Vet Bull 1974:44:617-628.

88. Lambooy E. van Voorst N. Electrocution of pigs with notifiable diseases. Vet Q 1986:8:80-82.

89. Loftsgard G, Rraathen S, Helgebostad A. Electrical stunning of mink. Vet Rec 1972:91:132-134.

90. Hatch RC. Euthanatizing agents. In: Booth NH. McDonald LE, eds. Veterinary pharmacology and therapeutics. 6th ed. Ames, Iowa: Iowa State University Press, 1988:1143-1148.

91. Croft PG, Hume CW. Electric stunning of sheep. Vet Rec 1956:68:318-321.

92. Roberts TDM. Electrocution cabinets. Vet Rec 1974:95:241-242.

93. Roberts TDM. Cortical activity in electrocuted dogs. Vet Rec 1954:66:561-567.

94. Stavinoha WR. Study of brain neurochemistry utilizing rapid inactivation of brain enzyme activity by heating and mirowave irradiation. In: Black CL, Stavinoha WB, Marvyama Y, eds. Microwave irradiation as a tool to study labile metabolites in tissue. Elmsford, NY: Pergamon Press, 1983;1-12.

95. Stavinoha WB, Frazer J, Modak AT. Microwave fixation for the study of acetylcholine metabolism. In: Jenden DJ, ed. Cholinergic mechanisms and psychopharmacology. New York: Plenum Publishing Corp, 1978;169-179.

96. lkarashi Y, Marvyama Y, Stavinoha WB. Study of the use of the microwave magnetic field for the rapid inactivation of brain enzymes. Jpn J Pharmacol 1984:35:371-387.

97. Blackmore DK. Differences in behaviour between sheep and cattle during slaughter. Res Vet Sci 1984:37:223-226.

98. Gregory NG, Wotton SB. Time to loss of brain responsiveness following exsanguination in calves. Res Vet Sci 1984:37:141-143.

99. Blackmore DK. Non-penetrative percussion stunning of sheep and calves. Vet Rec 1979:105:372-375.

100. Canadian Council on Animal Care. Guide to the care and use of experimental animals. Vol 1. Ontario, Canada: Canadian Council on Animal Care, 1980.

101. Green C. Euthanasia. In: Animal anaesthesia. London: Laboratory Animals Ltd, 1979:237-241.

102. Clifford DH. Preanesthesia, anesthesia, analgesia, and euthanasia. In: Fox JG, Cohen BJ, Loew FM, eds. Laboratory animal medicine. Orlando, Fla: Academic Press Inc, 1984:527-562.

103. Gregory NG, Wotton SB. Effect of slaughter on spontaneous and evoked activity of the brain. Br Poult Sci 1986:27:195-205.

104. Anil MH, McKinstry JL. Reflexes and loss of sensibility following head-to-back electrical stunning in sheep. Vet Rec 1991;128:106-107.

105. Eikelenboom G, ed. Stunning of animals for slaughter. Boston: Martinus Nijhoff Publishers, 1983.

106. Schatzmann U, Leuenberger T, Fuchs P, et al. Jet injection: the possibility of using a high pressure waterjet for the stunning of slaughter pigs. Fleischwirtschaft 1991:71:899-901.

107. Booth NH. Drug and chemical residues in the edible tissues of animals. In: Booth NH, McDonald LE, eds. Veterinary pharmacology and therapeutics. 6th ed. Ames, Iowa: Iowa State University Press, 1988;1149-1205.

108. Fowler ME, ed. Zoo and wild animal medicine. Philadelphia: WB Saunders Co, 1986.

109. Acceptable field methods in mammalogy: preliminary guidelines approved by the American Society of Mammalogists. J Mammal 1987;68(Suppl 4):1-18.

110. American Ornithologists' Union. Report of committee on use of wild birds in research. Auk 1988:105(Suppl1):1A-41A.

111. American Society of Ichthyologists and Herpetologists, Herpetologist League, Society for the Study of Amphibians and Reptiles. Guidelines for the use of live amphibians and reptiles in field research. J Herpetol 1987:21(Suppl 4): 1-14.

112. American Society of Ichthyologists and Herpetologists, American Fisheries Society, American Instititute of Fisheries Research Biologists. Guidelines for use of fishes in field research. Copeia Suppl 1987:1-12.

113. Cailliet GM. Fishes: a field guide and laboratory manual on their structure, identification, and natural history. Belmont, Calif: Wadsworth, 1986.

114. Zwart P, deVries HR, Cooper JE. [The humane killing of fishes, amphibia, reptiles and birds.] Tijdsehr Diergeneeskd 1989:114:557-565.

115. Brown LA. Anesthesia in fish. Vet Clin North Am Small Anim Pract 1988:18:317-330.

116. Hyman J. Euthanasia in marine animals. In: Dierauf LA, ed. CRC handbook of marine mammal medicine: health, disease, and rehabilitation. Boca Raton, Fla: CRC Press Inc, 1990; 265-266.

117. Lambooy E, RoelofsJA, Van Voorst N. Euthanasia of mink with carbon monoxide. Vet Rec 1985:116:416.

118. Recommended code of practice for the care and handling of mink. Ottawa, Canada: Agriculture Canada, 1988:17.

AWIC

Animal Welfare Information Center
United States Department of Agriculture
National Agricultural Library

USDA Cooperative Agreement No. 58-0520-5-076 - July, 1995