7
Euthanasia

GENERAL CONSIDERATIONS

Euthanasia is the act of inducing death without pain. Humane death of an animal may be defined as one in which the animal is rendered unconscious, and thus insensitive to pain, as rapidly as possible with a minimum of fear and anxiety.

The 1986 Report of the AVMA Panel on Euthanasia (AVMA, 1986) is a comprehensive review of euthanasia, and persons involved in euthanasia of animals should refer to it. (The AVMA panel report is being revised.) This chapter will not duplicate the AVMA report, but will summarize and supplement it with emphasis on animals used in research, teaching, and testing.

EUTHANASIA AS AN OPTION FOR ALLEVIATION OF PAIN

Euthanasia is an acceptable method for relieving pain or distress that cannot be controlled by other means. For studies in which death of the animal can be anticipated or is an inevitable part of the protocol, the investigator should specify the end point of the experiment and alternative situations in which termination of the experiment would be mandatory to avoid distress. Such specifications may be based on pathologic, physiologic, or behavioral considerations. Unless euthanasia would interfere with the experimental protocol, animals should be humanely killed*

*  

Following the convention of the 1986 Report of the AVMA Panel on Euthanasia, the verb euthanatize is used in this report. Other acceptable terms are euthanasia and humanely kill, but not put to sleep, put down, or other common euphemisms.



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Recognition and Alleviation of Pain and Distress in Laboratory Animals 7 Euthanasia GENERAL CONSIDERATIONS Euthanasia is the act of inducing death without pain. Humane death of an animal may be defined as one in which the animal is rendered unconscious, and thus insensitive to pain, as rapidly as possible with a minimum of fear and anxiety. The 1986 Report of the AVMA Panel on Euthanasia (AVMA, 1986) is a comprehensive review of euthanasia, and persons involved in euthanasia of animals should refer to it. (The AVMA panel report is being revised.) This chapter will not duplicate the AVMA report, but will summarize and supplement it with emphasis on animals used in research, teaching, and testing. EUTHANASIA AS AN OPTION FOR ALLEVIATION OF PAIN Euthanasia is an acceptable method for relieving pain or distress that cannot be controlled by other means. For studies in which death of the animal can be anticipated or is an inevitable part of the protocol, the investigator should specify the end point of the experiment and alternative situations in which termination of the experiment would be mandatory to avoid distress. Such specifications may be based on pathologic, physiologic, or behavioral considerations. Unless euthanasia would interfere with the experimental protocol, animals should be humanely killed* *   Following the convention of the 1986 Report of the AVMA Panel on Euthanasia, the verb euthanatize is used in this report. Other acceptable terms are euthanasia and humanely kill, but not put to sleep, put down, or other common euphemisms.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals when death is certain or predictable, before they become moribund, rather than allowed to die and possibly be cannibalized by cagemates or allowed to undergo autolysis. Tissue autolysis, especially in rodents and other small mammals, can reduce the opportunity to evaluate some aspects of a study. Euthanasia can avoid or terminate unnecessary and severe pain and distress and allow for a complete necropsy. Developing uniform methods to assess morbidity as part of the protocol can contribute to the validity and uniformity of experimental data. Regardless of the cause of morbidity (i.e., spontaneous or experimentally induced illness, pain, or distress), euthanasia should be considered. No guidelines are available that provide specific information on when to euthanatize animals in pain or distress, and such guidelines would be unrealistic, because animal species, types of studies, experimental needs, and end points vary so widely. Nevertheless, for studies in which death of animals is a necessary part of the protocol, efforts should be made to determine an acceptable end point when euthanasia may be performed to prevent or minimize unnecessary prolongation of pain and distress. WHEN TO PERFORM EUTHANASIA Some factors to consider in deciding whether to perform euthanasia are weight loss; emaciation; failure to gain weight (in a growing animal); severe pain that cannot be controlled; inordinate tumor growth or ascites; prolonged self-trauma; generalized alopecia caused by disease; prolonged diarrhea for which treatment is precluded in the protocol; coughing, wheezing, or severe nasal discharge; shallow and labored breathing; prolonged lethargy associated with rough hair coat, hunched posture, abdominal distention, or impaired movement; severe anemia or leukemia; icterus; CNS signs, such as convulsions, paralysis, paresis, tremors, and progressive head tilt; uncontrolled hemorrhage; urinary dysfunction (polyuria or anuria); lesions that interfere with eating or drinking; infectious disease (in terminal phases); hypothermia; and impairment of function, disablement, and other behavioral and physiologic signs of distress. Information on signs of pain, distress, morbidity, and moribund condition in previous chapters of this volume should be used to determine when euthanasia is appropriate and justified. Another factor in the decision is the differentiation between a condition from which animals might recover and a moribund condition that is likely to progress to death. Principal investigators, study directors, attending veterinarians, and institutional animal care and use committees (IACUCs) should collaborate in determining general policies regarding end points of studies and when to perform euthanasia. In specific instances when euthanasia should be considered for humane reasons, attending veterinarians should consult with principal investigators and study directors in making a final decision. The IACUC at the University of Texas M. D. Anderson Cancer Center has considered this issue for rats and mice used in the center's programs (Tomasovic et al., 1988). Such guidelines, applied case by case, provide a rationale for determining whether potentially lethal

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Recognition and Alleviation of Pain and Distress in Laboratory Animals experiments are justified in accordance with institutional policy and the merits of the research. AESTHETICS OF EUTHANASIA: TRAINING, SKILL, AND EMOTIONAL IMPACT ON PEOPLE Objective information on euthanasia of animals is sparse. Much of the information on the effects of the various agents and methods is subjective and based on professional judgment, experience, and intuition. Some of the reported disadvantages and arguments against particular practices are based in part on sentiment and human aesthetic considerations, rather than sound scientific data (Lumb, 1974). Some physical methods are aesthetically unpleasant but quite humane. Because unconsciousness and death do not necessarily occur simultaneously, it might be difficult for an untrained attendant to determine whether an animal is in distress or is unconscious and vocalizing and moving involuntarily without pain. Although the choice of a means of euthanasia should be based on humane concern for the animal being killed, the sensitivity of the attendant and observers should not be dismissed. Those performing or observing euthanasia can experience stress. People's emotions regarding this matter vary, and co-workers should be empathetic and sensitive to their feelings and attitudes. The possible necessity for euthanasia and untoward situations that might require euthanasia should be included in protocols and should be planned for and discussed among all personnel involved. Supportive discussion groups led by persons knowledgeable about grief and death can be useful in dealing with this difficult procedure (CCAC, 1980; Owens et al., 1981; Bustad, 1982; Wolfle, 1985; Arluke, 1990). Euthanasia should be conducted professionally and compassionately by skilled persons using means that are optimal for the circumstances. Persons performing euthanasia should verify that the animals presented are those scheduled for euthanasia. When properly performed by competent persons using appropriate techniques, euthanasia is humane. However, if inappropriate procedures are used or personnel are incompetent, attempts at euthanasia can result in inhumane treatment of animals. Therefore, it is imperative that IACUCs and attending veterinarians provide for the training of personnel who will perform euthanasia and carefully monitor the procedures. RESEARCH CONSIDERATIONS Well-designed objective studies of euthanasia are needed and recommended. The assessment tools and measures to be considered for such studies include electroencephalograms, electrocardiograms, electromyograms, arterial blood pressure, respiration and heart rates, serum biochemical characteristics, pupil diameter, and behavior. Although decapitation is considered by many knowledgeable

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Recognition and Alleviation of Pain and Distress in Laboratory Animals persons to produce instantaneous unconsciousness and death (Allred and Berntson, 1986; Vanderwolf et al., 1988), just how quickly death by decapitation occurs has been questioned (Mikeska and Klemm, 1975). Recommendations for sedation of all animals before decapitation, unless the head will be immediately frozen in liquid nitrogen (AVMA, 1986), are controversial. Decapitation below the level of the atlanto-occipital joint should be avoided, because it fails to interrupt all afferent fibers. If restraint is a problem or one is not confident that severance will be above this level, prior sedation is recommended. AVOIDING FEAR IN OTHER ANIMALS Distress vocalizations, fearful behavior, and release of odors or pheromones by a frightened animal can cause anxiety and apprehension in other animals (AVMA, 1986). That can affect the well-being of nearby animals and the validity of experimental data on animals to be euthanatized later by compromising their physiologic stability. Therefore, it is recommended that, whenever possible, animals be euthanatized in an area separated from other live animals, especially of their own species. ADJUNCTS TO EUTHANASIA Relief of pain and distress is of primary concern during the euthanasia procedure, so it might be necessary to administer drugs other than those used expressly for euthanasia, especially to nervous or intractable animals. Thus, tranquilizers, analgesics, or narcotics may be given before euthanasia to minimize apprehension and assist in animal control. VERIFICATION OF DEATH It is imperative that death be verified. Proper techniques of euthanasia should include a followup examination to confirm the absence of a heartbeat, which is a reliable indicator of death. Monitoring respiration is not sufficient. In some animals, particularly under deep CO2 anesthesia, heartbeat can be maintained after visible respiration has ceased, and the animal might eventually recover. SELECTION OF EUTHANATIZING AGENTS AND METHODS A means of euthanasia is chosen on the basis of animal species, size, tractability, excitability, presence of painful injury, distress, disease, restraint of the animal, protocol requirements for tissue collection or analysis, and other considerations. Suitable physical control of the animal being killed is critical for satisfactory euthanasia to minimize fear, anxiety, and pain and to ensure the safety of attending

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Recognition and Alleviation of Pain and Distress in Laboratory Animals persons and other nearby persons and animals. The selection of a procedure also depends on the skill of the personnel performing euthanasia and the number of animals to be killed. The criteria that should be used in selecting an agent or method for euthanasia include: Lack of pain perception by the animal. Relative rapidity and irreversibility. Minimal stress, apprehension, and fear in the animal or in nearby animals. Minimal interference with experimental protocol. Simultaneous interruption of consciousness and reflex mechanisms. Safety of personnel. Reliability. Efficiency and ease of procedure. Cost and availability. Minimal psychologic stress for attending personnel and observers. Minimal adverse environmental impact. Minimal potential for human drug abuse. Each means of euthanasia has advantages and disadvantages. It is unlikely that any agent or method will meet all the above criteria in a given situation. An acceptable means of euthanasia must have an initial depressant action on the CNS that causes relatively rapid unconsciousness and insensitivity to pain. Therefore, an overdose of a chemical anesthetic is a desirable means of euthanasia. Once the animal is anesthetized, the method used to kill it is less important. In some research protocols, the use of chemical agents is contraindicated, and physical methods must be used. When the animal is unconscious, it is desirable to stop its heart action as soon as possible, to reduce the flow of blood to its brain. A technique need not be considered inhumane merely because the heartbeat persists for a longer period, as long as the animal is unconscious (CCAC, 1980). There are three basic causes of death: hypoxia, either direct or indirect; direct depression of neurons vital for life functions; and damage to brain tissue (Sawyer, 1988). Attitudes, sensitivities, and scientific knowledge regarding euthanasia are changing. A number of agents or methods formerly used are now considered undesirable, because they are inhumane, dangerous to personnel, or unaesthetic. Use of strychnine, nicotine, magnesium sulfate, potassium hydrochloride, hydrocyanic acid, curare, or succinylcholine is not considered acceptable (Lumb and Moreland, 1982). Others have sufficient disadvantages to exclude them from a list of recommended agents and methods. Agents and methods recommended for euthanasia (Table 7-1) can be grouped into three categories: inhalational agents, physical methods, and noninhalational pharmacologic agents. The selection of the appropriate technique depends on an evaluation of the factors and criteria previously discussed.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 7-1 General Recommendations for Euthanasia (Categorized by Agent) Agent or Method Suitable Species Remarks Carbon dioxide Small laboratory animals, small dogs and puppies, cats and kittens, birds, small swine, and terrestrial reptiles and amphibians Not recommended for newborn animals Barbiturates Most species Agent of choice Methoxyflurane and halothane Most laboratory animals Expensive Stunning Small laboratory animals, snakes, lizards, turtles, terrapins, tortoises, frogs, toads, newts, salamanders, and fish Follow by immediate killing Cervical dislocation Small laboratory animals   Decapitation Rodents and birds Aesthetically unpleasant and dangerous for operator Captive bolt Large farm animals Follow by immediate killing Gunshot Large animals and crocodilians Useful in field studies, on farms, and in emergencies Microwave irradiation Mice and rats Requires focused-beam instrument; expensive Tricaine methanesulfate Frogs, toads, newts, salamanders, and fish   INHALATIONAL AGENTS A number of inhalational agents have been used for euthanasia, including ether, chloroform, halothane, methoxyflurane, enflurane, isoflurane, cyclopropane, nitrous oxide, and the nonanesthetic gases carbon monoxide (CO), carbon dioxide (CO2), nitrogen, argon, and hydrogen cyanide. Inhalational agents are usually administered in closed containers or chambers and have been used for small animals, such as rodents, rabbits, birds, puppies, kittens, and cats. Adequate ventilation and a means of exhausting waste gases should be provided for the safety of attendants using these agents.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals The replacement of air with nonanesthetic gases produces a deficiency of available oxygen, and animals develop hypoxia. Without sufficient oxygen, the brain becomes depressed, and the animals lapse into unconsciousness. These gases are not recommended for euthanasia of newborn animals, because newborn animals have been accustomed to low oxygen concentration in the uterus and are more resistant to hypoxic conditions (Rowsell, 1981). Although some of the agents have been widely used, most have sufficient disadvantages that preclude their common use for euthanasia of laboratory animals. Two disadvantages in some instances are that excitement and struggling are associated with the initial action on the CNS and initiating vapors can cause anxiety during induction. Most of the agents are hazardous to attending personnel and other animals (AVMA, 1986). Halothane, methoxyflurane, enflurane, and isoflurane are expensive and thus impractical for routine use. Ether is explosive and therefore not recommended. Carcasses of animals killed by ether require special storage, handling, and disposal, because ether fumes are retained in the bodies. Chloroform should not be used, because it is extremely hepatotoxic and potentially carcinogenic for humans. Nitrous oxide alone is not potent enough to be useful. Hydrogen cyanide and CO are dangerous to personnel and are not recommended. Carbon Dioxide CO2 is a well-accepted, commonly used gas for euthanasia of laboratory animals other than newborns. Inhalation of at least 40% CO2 has a rapid anesthetic effect that proceeds to respiratory arrest and death if exposure is prolonged. It is effective for use in small laboratory animals, including rodents, rabbits, cats, poultry, small dogs, and swine. It is rapid, painless, humane, easy to use, relatively inexpensive, nonflammable, nonexplosive, nearly odorless, and heavier than air. If used in well-ventilated areas, it is much safer than most of the other inhalational agents. Waste gas poses no substantial environmental hazard. CO2 is available in cylinders as a compressed gas or in a solid state as ''dry ice." The compressed-gas form is preferable. Animals should be kept out of direct contact with dry ice, to prevent chilling or freezing. CO2 is heavier than air, so chambers used for euthanasia should be filled from the bottom. Anxiety and struggling are minimized if the chamber is precharged. Relatively slow introduction of the gas into the chamber tends to minimize animal anxiety; high-speed flow causes turbulence and noise. Animals typically become unconscious within 45–60 seconds of the beginning of CO2 exposure. With continued depression of vital centers, hypoxia and death occur soon. Animals should remain in the chamber for at least 5 or 6 minutes and then examined closely to determine that all vital signs have ceased before they are removed from the chamber and disposed of.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Consideration of Behavior Burrowing animals, such as most rodents, tend to keep their noses in the lower portion of the chamber. Thus, narcosis, anesthesia, and death are relatively rapid when CO2 is used. Investigative animals, such as dogs and rabbits, might extend their heads upward when put into the chamber. Some animals might attempt to climb up the sides. Unless the chamber is precharged, nonburrowing animals will initially be exposed to CO2 at a lower concentration, and some might hyperventilate, struggle, and stagger (CCAC, 1980). The number of animals placed in a chamber at a given time depends on the size and temperament of the species, but the animals should not be crowded. PHYSICAL METHODS Physical methods of euthanasia include stunning, cervical dislocation, decapitation, gunshot, electrocution, decompression, use of a captive bolt, microwave irradiation, exsanguination, rapid freezing, and pithing. Physical methods are appropriate in three general situations: with easily handled small animals whose anatomic features are compatible with the method used; with large farm animals; and in situations in which other methods might invalidate the experimental results. Some people consider physical methods of euthanasia aesthetically displeasing or repulsive. However, some physical methods cause less fear and anxiety and can be more rapid, painless, humane, and practical than other forms of euthanasia. The skill and experience of the attendant are paramount when physical methods are used, because trauma is often involved. If they are not performed correctly, animals might be injured, have various degrees of consciousness, and suffer pain and distress. Some of the methods, particularly stunning and pithing, do not ensure death and therefore necessitate followup measures, such as exsanguination or decapitation. Before using physical methods, inexperienced persons should practice on carcasses or anesthetized animals that are scheduled for euthanasia until they are proficient in performing the methods properly and humanely. Stunning Stunning is used primarily on small laboratory animals with relatively thin craniums. A sharp blow must be delivered to the central skull bones with sufficient force to produce massive hemorrhage and thus immediate depression of the CNS. When it is done properly, unconsciousness is immediate. After stunning, the animal should be killed immediately by another procedure, such as exsanguination or decapitation.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Properly performed stunning is humane. However, the probability of improper stunning is rather high for inexperienced personnel. Stunning should be used only by properly trained persons and when other means are inappropriate or unavailable. Stunning can affect the release and the concentration of catecholamines and other neurotransmitters and perhaps other brain metabolites. Captive-bolt guns are commonly used for stunning large farm animals. After stunning with a captive bolt, euthanasia must be performed by another means. A penetrating captive bolt has been described for euthanasia of dogs and rabbits (Dennis et al., 1988). Cervical Dislocation Cervical dislocation is commonly used to euthanatize small laboratory animals (e.g., poultry, mice, and immature rats and rabbits). It causes almost immediate loss of consciousness because of cerebral shock, and all voluntary motor and sensory functions cease because of damage to the spinal cord. This method can cause considerable involuntary muscle activity. However, there is no evidence that animals feel pain, if it is performed correctly. The technique consists of a separation of the skull and brain from the spinal cord by anteriorly directed pressure applied to the base of the skull. Decapitation Decapitation with a guillotine (or heavy shears) is used primarily when pharmacologic agents and CO2 are contraindicated, e.g., in pharmacologic and biochemical studies. The procedure causes rapid death if properly performed. An animal should be properly restrained, and its head must be completely severed from its body at the atlanto-occipital joint. Decapitation is often aesthetically unpleasant and dangerous for the operator. The guillotine must be kept in good operating condition, and the blade must be sharp. The equipment should be thoroughly cleaned after each use. Gunshot Gunshot is an effective and practical means of humanely killing some animals in particular circumstances, such as field studies, rural settings (farm animals), and emergencies. Microwave Irradiation Euthanasia by microwave irradiation with focused beam instruments is used by neurobiologists to fix brain metabolites without losing the anatomic integrity of the brain. Unconsciousness and death occur in less than a second. Instruments are expensive, and only animals the size of mice or rats can be euthanatized with

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Recognition and Alleviation of Pain and Distress in Laboratory Animals currently available equipment (AVMA, 1986). Standard kitchen microwave appliances are not appropriate, because their beams are diffuse and animals might experience severe pain or distress. Double Pithing Double pithing is an effective method of killing some poikilotherms (cold-blooded animals). A sharp needle with a diameter appropriate to the size of the animal is quickly inserted in a cranial direction through the foramen magnum and is rotated so as to crush the brain bilaterally. The needle is then inserted in a caudal direction at the same point of entry and rotated to destroy the spinal cord. Pithing requires dexterity and skill and should be conducted only by trained personnel. Rapid Freezing Rapid freezing is used by neurobiologists, because it instantaneously inactivates enzymes in the brain. Immersion of the entire animal in liquid nitrogen achieves rapid death of an animal that weighs less than 40 g. Heavier animals should be anesthetized before this technique is used. Suitable equipment and properly trained personnel are required. Exsanguination Because of the anxiety associated with extreme hypovolemia, exsanguination should be done only in sedated, stunned, or anesthetized animals (AVMA, 1986). NONINHALATIONAL PHARMACOLOGIC AGENTS Several noninhalational agents are used for euthanasia. Although death can be induced by administering these drugs by several routes, intravenous administration is preferred, because of its smooth induction, rapid action, and reliability. Intrapulmonic injection should be avoided. Intracardiac injection should be used discriminatingly, because it requires considerable skill; the injection might be painful, and, if the drug is accidentally discharged outside the heart, death will be slow and painful. Intracardiac injection of drugs is recommended only for moribund, anesthetized, or comatose animals. Intraperitoneal administration requires higher doses and leads to prolonged dying, and induction can be marked by ataxia and struggling. Barbituric Acid Derivatives The barbiturates are used extensively and are considered the agents of choice for most euthanasia on both aesthetic and scientific bases. Barbiturates are humane, safe, and efficient when properly administered. Intravenous administration of a

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Recognition and Alleviation of Pain and Distress in Laboratory Animals barbiturate produces a smooth and rapid onset of unconsciousness without disturbing behavior before death. Sodium pentobarbital has been used as a standard against which other agents and methods for euthanasia are often compared. Other barbiturates are available, and overdoses of them will produce similar results. But none has any advantage over sodium pentobarbital, and most are considerably more expensive. There are several disadvantages in the use of barbiturates. Animals must be handled individually. Because there is a potential for human drug abuse, barbiturates are controlled substances that require Drug Enforcement Agency registration and record-keeping. Trained personnel are required to administer the drugs intravenously. There can be aesthetically unpleasant behavior and some animal discomfort when the drugs are not administered intravenously. For nervous and intractable animals, preinduction with tranquilizers or sedatives might be appropriate. An overdose of barbiturates causes death by depression of vital medullary respiratory and vasomotor centers. Within seconds of intravenous administration, the animal simply relaxes, closes its eyes, and stops breathing. Cardiac arrest follows, but the heart can continue to beat for a few minutes. The effect depends on dose, concentration, and rate of injection. Commercial preparations of sodium pentobarbital for euthanasia contain 260–390 mg/ml and are usually given intravenously at a dose of approximately 100 mg/kg of body weight. T-61 T-61 is a nonbarbiturate, non-narcotic euthanatizing agent. It was formerly used for euthanasia, but its commercial production was discontinued in Great Britain in 1962 and in the United States in 1989. T-61 can be used humanely with close adherence to the recommended rate of administration. It is included here because supplies of the agent exist and some still use it. Other Drugs Other injectable pharmacologic agents are available for restraint, immobilization, analgesia, and anesthesia of animals, such as ketamine hydrochloride, xylazine, and opioids. Although it might be possible to cause death with high doses of these drugs, they are not recommended for euthanasia, because such use would be impractical and might produce convulsions before death. RECOMMENDATIONS FOR SPECIFIC ANIMALS Table 7-2 lists recommendations for euthanasia of particular species.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 7-2 General Recommendations for Euthanasia (Categorized by Species) Species Agent or Method Dogs and cats Barbiturates Carbon dioxidea (small dogs) Ferrets Barbiturates Rabbits Barbiturates Stunningb Cervical dislocation (small rabbits) Carbon dioxidea Rodents Carbon dioxidea Barbiturates Cervical dislocation (< 200 g) Stunningb Decapitation Nonhuman primates Barbiturates Birds Carbon dioxidea Barbiturates Cervical dislocation Decapitation Stunningb Snakes and lizards Barbiturates or inhalant anesthetics Stunningb Turtles, terrapins, and tortoises Barbiturates or inhalant anesthetics Stunningb Crocodilians Barbiturates Gunshot Frogs, toads, newts, and salamanders Stunningb Anesthetic overdose Tricaine methanesultate (MS 222) Fish Stunningb Tricaine methanesultate (MS 222) a Not suitable for newborn or newly hatched animals. b Followed by immediate killing by another method.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals DOGS AND CATS Intravenous administration of sodium pentobarbital at 90–100 mg/kg, 3 times the anesthetic dose, should ensure respiratory and then cardiac arrest in dogs and cats. This is the procedure of choice for these species. Intracardiac injection can be used in anesthetized or moribund dogs and cats. High concentrations (40–70%) of CO2 can also be used. FERRETS Intraperitoneal administration of sodium pentobarbital (100–120 mg/kg) is a practical means of euthanatizing ferrets. RABBITS The preferable means of euthanatizing rabbits is intravenous injection of sodium pentobarbital (100 mg/kg). The intracardiac route should be used only in anesthetized or moribund animals. Rabbits can also be killed by cranial concussion (stunning) or by cervical dislocation (recommended only for small rabbits). Extreme care should be used to ensure that these techniques are properly performed by trained personnel. CO2 at concentrations over 40% is safe and effective for euthanatizing rabbits. LABORATORY RODENTS CO2 is considered acceptable for euthanatizing rodents. Newborn animals are more resistant to CO2 than older animals. Sodium pentobarbital (150–200 mg/kg) is also commonly used. Intraperitoneal administration is acceptable, because intravenous injection is difficult. Intracardiac and intrapulmonic administration are recommended only for anesthetized animals. Cervical dislocation can be used to kill animals that weigh less than 250 g. Hamsters and guinea pigs are more difficult to kill with this method, because of their short necks, stronger neck muscles, and loose skin over the neck and shoulders. Stunning by cervical concussion can cause unconsciousness, but extreme care should be used to ensure that this technique is performed properly. After stunning, the animal should be killed immediately by another procedure, such as exsanguination or decapitation. Decapitation with a guillotine is recommended only when use of drugs is contraindicated. Successful decapitation, like cervical dislocation, is more difficult in hamsters and guinea pigs than in rats, because of the anatomy of their necks and shoulders.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals NONHUMAN PRIMATES Nonhuman primates are usually euthanatized by an intravenous overdose of sodium pentobarbital (100 mg/kg). BIRDS Euthanasia in a CO2 chamber is effective for birds and is safe. Sodium pentobarbital (100 mg/kg) can be administered intravenously or intraperitoneally. Cervical dislocation of birds is rapid and inexpensive. It can be done manually with small birds. In large fowl, like turkeys and geese, an instrument like Burdizzo forceps is needed. Decapitation with a guillotine or shears is effective with small birds. Small birds can also be stunned (see guidelines on stunning). AMPHIBIANS, FISH, AND REPTILES Euthanasia of amphibians, fish, and reptiles has been studied less than euthanasia of other animals, and guidelines are less available. When euthanasia of poikilothermic animals and aquatic animals is performed, the differences in their metabolism, respiration, and tolerance of cerebral hypoxia might preclude some procedures that would be acceptable in terrestrial mammals (AVMA, 1986). Anatomic differences should also be considered. For example, veins can be hard to find. Some animals have a carapace. For physical methods, access to the CNS can be difficult, because the brain is relatively small and hard for inexperienced persons to find. Euthanasia of Amphibians and Reptiles (UFAW/WSPA, 1989) suggests that, when physical methods of euthanasia of poikilothermic species are used, cooling to 4°C decreases metabolism and might facilitate handling; but there is no evidence that it raises the pain threshold. That report provides line drawings of the heads of various amphibians and reptiles with recommended locations for captive-bolt or firearm penetration. Most amphibians, fishes, and reptiles can be euthanatized by cranial concussion followed by decapitation or some other physical method. Decapitation with a guillotine or heavy shears is effective in some species that have appropriate anatomic features. It has previously been assumed that stopping blood supply to the brain by decapitation causes immediate unconsciousness followed by rapid loss of sensation. That view has recently been questioned, because the CNS of reptiles and amphibians tolerates hypoxic and hypotensive conditions (UFAW/WSPA, 1989). Decapitation should therefore be followed by pithing.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Pithing is an effective method of killing some poikilotherms, but death might not be immediate, unless both the brain and spinal cord are pithed, which is recommended. Pithing of only the spinal cord should be followed by decapitation or another appropriate procedure. The anatomic features of some species preclude this method. Pithing requires some dexterity and skill and should be conducted only by trained personnel. Snakes and turtles have been killed by freezing after immobilization by cooling. However, the 1989 report of the Universities Federation for Animal Welfare and World Society for the Protection of Animals (UFAW/WSPA, 1989) does not recommend that method; formation of ice crystals on the skin and in tissues of an animal can cause pain or distress. Sodium pentobarbital (60 mg/kg) or other barbiturates can be administered, depending on anatomic features, intravenously, intracardially, or into the abdominal or pleuroperitoneal cavity of most cold-blooded animals. Tricaine methanesulfate (MS 222) can be administered by various routes to induce euthanasia. For aquatic animals, it can be placed in the water—an effective but expensive means of euthanasia that is not hazardous to personnel. Snakes, lizards, turtles, frogs, and toads can be killed by overexposure to gaseous anesthetics, such as halothane or methoxyflurane, in a chamber or by mask. CO2 can be used for terrestrial animals. For additional reading on this subject, refer to Amphibians: Guidelines for the Breeding, Care, and Management of Laboratory Animals (NRC, 1974), The Relief of Pain in Cold-Blooded Vertebrates (Arena and Richardson, 1990), and Anesthesia in Fish (Brown, 1988).