6
Control of Stress and Distress

This chapter provides guidance on the control of stress and distress in laboratory animals pharmacologically—presenting information on the clinical uses, effects, and dosages of four groups of drugs (see Tables 6-1 and 6-2)—and nonpharmacologically.

Distress in laboratory animals is usually unnecessary and unwanted. Despite an inability to define or measure distress in laboratory animals precisely, distress is used to describe a point at which adaptation to a stressor (environmental, psychologic, or physiologic) is not sufficient to maintain equilibrium and maladaptive behaviors appear. Users of animals are responsible for the prevention, alleviation, or elimination of distress. The possibility of distress is best considered before laboratory animals are used experimentally; that is, careful consideration should be given during experimental design to means by which non-pain-induced distress can be avoided (ideally) or minimized. Stressors that can lead to distress should be understood and identified (see Chapter 3). If one is to deal adequately with distress in laboratory animals, one must be able to recognize it (see Chapter 4). That requires that species-typical behaviors associated with well-being be understood and that the normal behavior and appearance of the animals being used be known. Distress can be subtle; so too can its influence on experimental outcomes.

Distress results from stress to which animals cannot adequately adapt. The stressor can be an external or internal event that causes physical or psychologic trauma. For its purpose, the committee identified these stressors as pain-induced and environmentally induced. Nominal stress is usually cause for alarm only if an animal is unable to adapt properly to it. When that occurs and distress results,



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Recognition and Alleviation of Pain and Distress in Laboratory Animals 6 Control of Stress and Distress This chapter provides guidance on the control of stress and distress in laboratory animals pharmacologically—presenting information on the clinical uses, effects, and dosages of four groups of drugs (see Tables 6-1 and 6-2)—and nonpharmacologically. Distress in laboratory animals is usually unnecessary and unwanted. Despite an inability to define or measure distress in laboratory animals precisely, distress is used to describe a point at which adaptation to a stressor (environmental, psychologic, or physiologic) is not sufficient to maintain equilibrium and maladaptive behaviors appear. Users of animals are responsible for the prevention, alleviation, or elimination of distress. The possibility of distress is best considered before laboratory animals are used experimentally; that is, careful consideration should be given during experimental design to means by which non-pain-induced distress can be avoided (ideally) or minimized. Stressors that can lead to distress should be understood and identified (see Chapter 3). If one is to deal adequately with distress in laboratory animals, one must be able to recognize it (see Chapter 4). That requires that species-typical behaviors associated with well-being be understood and that the normal behavior and appearance of the animals being used be known. Distress can be subtle; so too can its influence on experimental outcomes. Distress results from stress to which animals cannot adequately adapt. The stressor can be an external or internal event that causes physical or psychologic trauma. For its purpose, the committee identified these stressors as pain-induced and environmentally induced. Nominal stress is usually cause for alarm only if an animal is unable to adapt properly to it. When that occurs and distress results,

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Recognition and Alleviation of Pain and Distress in Laboratory Animals treatment should begin with an identification of the underlying cause. Pain-induced stress should then be alleviated by removal of the cause of the pain or through administration of analgesics, but non-pain-induced stress is seldom amenable to pharmacologic treatment alone. Rather, environmental stressors or factors should be addressed. The use of tranquilizers can sometimes help an animal adapt to necessary changes in its environment, but is seldom sufficient in itself. This report places considerable emphasis on the importance of recognizing maladaptive behaviors resulting from stress with which an animal is unable to cope effectively as evidence of distress. Some conditions of acute stress in which an animal's behavior is normal and adaptive also suggest that intervention is warranted. Such conditions are brought on typically when an animal is strongly motivated to avoid or escape a stimulus or set of conditions. Such behaviors, like maladaptive ones, should be interpreted as causing harm to the animal and producing unwanted variability in research data. PHARMACOLOGIC CONTROL OF STRESS AND DISTRESS The tranquilizers and sedatives used in animals today include drugs in four groups: phenothiazines, butyrophenones, benzodiazepines, and α2-Adrenergic agonists. Phenothiazines and butyrophenones have many common properties, especially general sympatholytic activity. They used to be considered "major" tranquilizers in human medicine; currently preferred terms are antipsychotics and neuroleptics. Benzodiazepines, once considered "minor" tranquilizers, are now thought of as antianxiety-sedative agents. α2-Adrenergic agonists have emerged as a very important group of drugs for tranquilizing and sedating animals. PHENOTHIAZINES Common Examples Promazine (Sparine®) and acetylpromazine (Acepromazine®). Clinical Use Phenothiazines depress many physiologic functions, decrease motor activity, produce mental calming, and increase the threshold of response to environmental stimulation. Thus, they are useful for animal restraint. They do not produce sleep, analgesia, or anesthesia. The sedation produced by phenothiazines differs from the state produced by barbiturates and opioids, in that sedation occurs without hypnosis and the effects produced in animals can be reversed with an adequate stimulus. In animals, adequate doses produce a quieting effect that includes sedation, ataxia, an increase in the threshold of response to environmental stimuli, relaxation

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Recognition and Alleviation of Pain and Distress in Laboratory Animals of the nictitating membrane in some animals, and abolition of conditioned reflexes. It must be emphasized that animals under the sedation produced by tranquilizers can still react in a coordinated manner. A large animal can still kick with full force in reaction to painful stimuli, a vicious animal can still bite, and nonhuman primates can become aroused unexpectedly. The degree of sedation and inactivity produced by the tranquilizers in many instances depends on the excitability of the animal being treated. That is especially true in wild animals: the tranquilization of free-living and captive undomesticated animals might not be possible, and more potent drugs might be necessary (Graham-Jones, 1960, 1964). It is also true of vicious animals; i.e., tranquilizers might produce insufficient restraint for safe management of extremely high-strung nervous animals. In those circumstances, neuroleptanalgesia might have to be administered with a combination of a tranquilizer and an opioid. In stallions, many phenothiazine tranquilizers cause erections and temporary or permanent prolapse of the penis. In horses, phenothiazines and butyrophenones cause involuntary and hallucinatory activity (Muir et al., 1989). Pharmacologic Effects The major action of the phenothiazines is antagonism of the central dopamine receptors. In addition to their sedative properties, the phenothiazines and the related butyrophenones (e.g., droperidol) produce a dose-dependent decrease in motor activity. At greater doses, they produce a cataleptic state that includes rigidity, tremor, and akinesia. The phenothiazines are also useful in some species as antiemetics. However, they have important anticholinergic, antiadrenergic, and antihistaminic effects, which often lead to undesirable or unanticipated side effects and unpredictable drug interactions. The two most commonly used phenothiazines are promazine and acetylpromazine. They produce numerous cardiovascular effects through central and peripheral actions on the sympathetic nervous system and the CNS and direct actions on vascular and cardiovascular smooth muscle. The CNS manifestation is inhibition of centrally mediated pressor reflexes, which reduces both vascular tone and the ability to respond reflexively to alterations in the cardiovascular system. The peripheral effects are related to α2-adrenergic receptor blockade. Phenothiazines are commonly administered intravenously to animals in the standing position, especially farm animals. The cardiovascular actions have a more rapid onset than the sedative actions, and orthostatic hypotension might explain the occasional collapse. The extent of hypotensive effects of a tranquilizer varies and depends on the state of the cardiovascular system and the sympathetic tone when the drug is administered. Fatigue, hypovolemia, excitement, and trauma can increase sympathetic tone as a part of the adaptive homeostatic process. The administration of a sympatholytic drug under those circumstances can have a profound effect (Bahga and Link, 1966). Phenothiazines lessen the ability of the cardiovascular system to compensate for changes in vascular volume, changes in position, and

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Recognition and Alleviation of Pain and Distress in Laboratory Animals stress. They can produce hemodilution by causing splenic sequestration of red blood cells (Collette and Meriwether, 1965); the effect is especially noted in horses. Dose Recommendations (Table 6-1) Under some circumstances, phenothiazine doses that do not produce obvious overt behavioral manifestations (lower than those recommended in Table 6-1) could be used to alter behavioral patterns slightly and should be considered as adjuncts for the treatment of abnormal behaviors. Phenothiazines are useful in managing postanesthetic emergence delirium, especially after barbiturate anesthesia. A combination of a tranquilizer and an TABLE 6-1 Doses of Tranquilizers in Various Species Species Drug Dose, mg/kg (Routea) Reference Cat Promazine 2.2–4.4 (iv, im) Soma, 1971   Acetylpromazine 0.03–0.05 (iv, im) Gleed, 1987 Cattle Promazine 0.4–1.1 (iv, im) Soma, 1971   Acetylpromazine 0.1 (im) Gleed, 1987 Dog Promazine 2.2–4.4 (iv, im) Soma, 1971   Acetylpromazine 0.03–0.05 (iv, im) Gleed, 1987 Guinea pig Promazine 0.5–1.0 (im) CCAC, 1980 Hamster Promazine 0.5–1.0 (im) CCAC, 1980 Horse Promazine 0.44–1.1 (iv, im) Soma, 1971   Acetylpromazine 0.02–0.05 (iv, im) Gleed, 1987 Mouse Promazine 5 (ip) Vanderlip and Gilroy, 1981   Acetylpromazine 2–5 (ip) Flecknell, 1987 Primate Acetylpromazine 0.2 (im) Flecknell, 1987 Rabbit Promazine 1–2 (im) CCAC, 1980   Acetylpromazine 1 (im) McCormick and Ashworth, 1971 Rat Promazine 0.5–1.0 (im) Kruckenberg, 1979   Acetylpromazine 1.0 (im) Flecknell, 1987 Sheep and goat Promazine 0.44–1.1 (iv, im) Soma, 1971   Acetylpromazine 0.04–0.06 (iv, im) Soma, 1971 Swine Acetylpromazine 1.1–2.2 (im) Benson and Thurman, 1979 a iv = intravenous; im = intramuscular; ip = intraperitoneal.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals opioid might facilitate management during the immediate postanesthetic and postoperative period. BUTYROPHENONES Common Examples Azaperone (Stresnil® and droperidol. Clinical Use Azaperone is approved for swine, in which it is used mainly to prevent fighting and as a preanesthetic agent. It is a more potent sedative and less hypotensive than the phenothiazines, but has no analgesic effect (Flecknell, 1987). Droperidol is incorporated with fentanyl in Innovar-Vet® (see Chapter 5). Pharmacologic Effect Like the phenothiazines, butyrophenones exert general sympatholytic activity that probably accounts for many of their common properties. Butyrophenones seem more likely to produce extrapyramidal signs of rigidity, tremors, and catalepsy. Dose Recommendations In pigs, azaperone at 2.2 mg/kg intramuscularly produces sedation, but has no analgesic effect. Combined at 5 mg/kg with metomidate (10 mg/kg) intramuscularly, it produces sedation and analgesia suitable for minor surgical procedures (Flecknell, 1987). In horses, azaperone administered intravenously at 0.22–0.44 mg/kg might cause excitement and extrapyramidal effects and is not recommended (Muir et al., 1989). BENZODIAZEPINES Common Examples Diazepam (Valium®), zolazepam, and midazolam (Versed®). Clinical Use Benzodiazepines induce a mild calming effect and have therapeutically useful anticonvulsant, muscle-relaxant, and hypnotic effects in animals; they have no analgesic activity. They are commonly used with analgesic drugs (e.g., xylazine, opioids, or ketamine) to enhance muscle relaxation.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Pharmacologic Effects Barbiturates and benzodiazepines share many pharmacologic actions (e.g., sedation, muscle relaxation, anticonvulsant activity, and hypnosis) because of their interaction with the GABA-chloride ionophore receptor complex. GABA (y[γ]aminobutyric acid) is an inhibitory amino acid neurotransmitter. Benzodiazepines appear to increase the frequency of opening of the GABA-activated chloride ion channel in nerve membranes; barbiturates enhance the binding of GABA to its receptor and increase the time that the same GABA-activated ion channel is open. Thus, barbiturates and benzodiazepines both facilitate GABA-mediated inhibitory effects on the CNS. The sedative and anxiolytic effects of the benzodiazepines are produced by doses that also produce muscle relaxation. The most commonly used benzodiazepine is diazepam. As with other drugs, there is great species variability in its effects. The effects in dogs, cats, and horses (Muir et al., 1982) are not the anxiolytic effects noted in people. Excitement, tremors, ataxia, dysphasia, and sometimes sedation occur in animals. There is no known explanation for the major species differences noted in response to the benzodiazepines in people and animals. Dose Recommendations Diazepam, usually administered intravenously, is painful if given intramuscularly. In rats, it is used as an anxiolytic at 1 mg/kg to lessen stress-induced increases in blood pressure, but not changes in heart rate (Conahan and Vogel, 1986). It can be used in ruminants for sedation at 0.2–0.5 mg/kg. In small ruminants, it is used as a premedication before ketamine anesthesia. Diazepam and midazolam are usually used with other drugs in animals. The water solubility of midazolam, as opposed to the water insolubility of diazepam (compounded with propylene glycol), might be advantageous in some drug combinations, and midazolam is less irritating to tissues. However, it is more expensive. Tables 5-4 and 5-5 list combinations of diazepam with ketamine for surgical anesthesia in several species. The preanesthetic administration of the benzodiazepines with ketamine provides good muscle relaxation and eliminates tremors produced by ketamine. α2-ADRENERGIC AGONISTS Common Examples Xylazine (Rompun®) and detomidine (Dormosedan®). Clinical Uses Xylazine is often used alone or with ketamine as a sedative and preanesthetic in ruminants and horses (Clarke and Hall, 1969; Hoffman, 1974; Klein and Baetjer,

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Recognition and Alleviation of Pain and Distress in Laboratory Animals 1974; Klide et al., 1975; Muir et al., 1979). In horses and ponies, xylazine given alone produces a mild degree of CNS depression. There is some ataxia, but animals are able to stand and walk. Horses and ponies can still respond to painful stimuli, so surgical procedures should not be attempted without opioid supplementation or local analgesia. Xylazine suppresses the excitatory effects of opioids in horses when these drugs are administered together for analgesic or preanesthetic purposes. Xylazine is used extensively in other species with other drugs, especially the dissociative anesthetic ketamine. Xylazine is not recommended for use alone to produce analgesia or anesthesia in dogs and cats, but is commonly used with ketamine. Vomiting occurs in dogs and cats after intravenous or intramuscular administration of xylazine (Klide et al., 1975), and sedative effects occur in dogs within 5–10 minutes of administration. Sedative effects include lying down, lack of response to the environment, medial rotation of the eyes, and prolapse of the nictitans. Some degree of analgesia is apparent, but xylazine is not sufficient for surgery. Spontaneous arousal can occur, and the degree of sedation is inconsistent. Pharmacologic Effects Xylazine is a potent adrenergic receptor a[α]2-agonist. The major CNS effect of the a[α]2-agonists is a decrease in sympathetic outflow from the medullary pressor center; this accounts for the sympatholytic actions of this class of drugs. Actions of xylazine at the central a[α]2-agonist adrenergic receptors produce a variety of effects, including sedation, analgesia, hypotension, bradycardia, hypothermia, mydriasis, and relief of anxiety. Cardiovascular changes in dogs and horses have been attributed to them (Klide et al., 1975; Muir et al., 1979). After intravenous administration of xylazine in dogs, heart rate and aortic flow decreased, blood pressure changes were variable, and peripheral resistance increased; there were no significant changes in blood gases and pH; atrioventricular block and nonrespiratory sinus arrhythmia were seen; and atropine did not alter the changes in cardiac rhythm. Cardiovascular changes in horses were similar, except that a transient increase in blood pressure was followed by a decrease. Other a[α]2-agonists have similar actions. Detomidine has recently been introduced in this country as Dormosedan® for use in horses. Detomidine(its intravenous and intramuscular dose is 0.02–0.04 mg/kg) is more potent than xylazine and can produce more profound analgesia, sedation, and bradycardia for a longer period (Kamerling et al., 1988). Dose Recommendations (Tables 5-4, 5-5, and 6-2) Dogs, cats, and ruminants (sheep, goats, and cattle) are more sensitive to xylazine than horses. Doses at the low end of the recommended range produce immobilization; recumbency can occur at higher doses (Table 6-2). Surgery is possible in depressed ruminants at 0.09–0.35 mg/kg. As with any agent that produces recumbency in ruminants, passive regurgitation can occur.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 6-2 Dose of Xylazine and Adjunctive Drugs Other Than Ketamine a in Various Species Species Dose, mg/kg (Routeb) Adjunct Drug, mg/kg (Routeb) Comments References Cat 1.1–4.4 (iv, im, sc) None Sedation, analgesia, emesis Moye et al., 1973; Yates, 1973 Cattle 0.09–0.35 (im) None Sedation, analgesia Hopkins, 1972 Dog 0.5–4.4 (iv, im, sc) None Sedation, analgesia, emesis Moye et al., 1973; Yates, 1973; Klide et al., 1975 Horse 0.5–1.0 (iv, im) None Sedation Hoffman, 1974 Horse 0.5–1.0 (iv, im) Morphine, 0.2–0.5 (iv, im) Sedation,analgesia Klein and Baetjer, 1974 Horse 0.1–0.5 (iv) Butorphanol, 0.01–0.04 (iv) Preanesthesia restraint Orsini, 1988 Rabbit 5.0 (sc) Pentobarbital, 11–28 (iv) Anesthesia Hobbs et al., 1991 a Tables 5-4 and 5-5 list doses for xylazine in combination with ketamine. b iv = intravenous; im = intramuscular; sc = subcutaneous.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals NONPHARMACOLOGIC CONTROL OF STRESS AND DISTRESS This section considers nonpharmacologic ways of preventing, minimizing, and alleviating non-pain-induced distress in laboratory animals through husbandry and management practices, socialization and handling, environmental enrichment, and experimental design. (The pharmacologic management of pain, a major stress that often leads to distress, is discussed in Chapter 5 and will not be discussed here, except to emphasize that the use of drugs to alleviate non-pain-induced distress is generally inappropriate.) As a general rule, nonpharmacologic approaches to the prevention or minimization of distress are more desirable than pharmacologic approaches (Wolfle, 1987). HUSBANDRY AND MANAGEMENT PRACTICES Control of non-pain-induced distress centers around three of the most common causes in laboratory animals: husbandry, environment, and experimental design. Management practices in animal care and housing can contribute to such stressors as fear, anxiety, loneliness, and boredom, which, if not prevented or minimized, have the potential to lead to distress and the appearance of maladaptive behaviors. Hence, understanding and meeting the social and physical needs of animals are essential to the prevention or minimization of distress. Table 4-4 identifies situations and practices that can contribute to distress and adversely affect an animal's well-being. Two points require emphasis: a state of well-being is more than just good health and the absence of pain, and needs are species-specific. It is helpful to keep in mind that no environment is free of stressors. Furthermore, even if a stress-free environment could be achieved, it would not necessarily be desirable. Stress is not always abnormal or harmful to well-being. Stressors are common in the lives of animals in their natural environments, and a captive animal that had never experienced stress would be quite different in its behavior and physiology from the typical members of its species. Whether stress will lead to distress, with the appearance of maladaptive behaviors and physiologic and pathologic changes, and create a serious risk to an animal's well-being depends on the intensity and duration of the stress and the animal's adaptability. Rather than strive to keep a captive environment free of stressors, it is more realistic, and will serve animals' interests better, to try to identify and eliminate extreme forms of chronic or acute stress. That can be achieved partly by designing the physical environment, caretaking regimens, and research procedures from the animals' perspective. It is also helpful to consider the kinds of experiences that animals can be given to help them cope with stressors in situations they are likely to encounter in a captive setting.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Animal-centered approaches to stress and distress are essential, but they can easily be carried to an extreme. There are no animal utopias in nature or in artificial environments. No environments are entirely animal-centered. Even if they were, the biologic makeup of animals includes paradoxes and contradictions that environmental conditions cannot fully resolve. The assumption that animals ''know what is best" for them is a charming fiction. The various activities they engage in, the goals they seek to reach, and the functions they carry out do not necessarily constitute a coherent, harmonious, and entirely beneficial whole; more often, they reflect compromises within the individual between conflicting or incompatible needs and tendencies. Some of the compromises can actually be potent sources of stress and can lead to distress. That is likely to be the case, for example, for both mother and offspring in many nonhuman primate species during the period surrounding weaning. The weaning process can, in fact, be so stressful that it increases the infant's vulnerability. Other examples can be found in Chapter 3. Even if measures of stress and distress were wholly objective, concordant, and unequivocal, that would not always provide sufficient information on which to base practical decisions. One price of human stewardship, even if animal well-being were the only concern, is that human knowledge and human values necessarily influence the decision-making process. When data are lacking, purely anthropomorphic considerations are often helpful, if they are based on a solid understanding of the behavior of the species and the context. In some contexts, humans might make decisions that animals themselves would make, as is often the case between human parents and children. To ensure that decisions are as humane as possible, more information on the sources and manifestations of stress and distress in captive environments is helpful. Agreed-on guidelines for the identification and reduction of stress and the prevention and minimization of distress can also serve a useful purpose. In the pursuit of humane concerns, however, it is essential to recognize the need for professional judgment and to preserve as much flexibility as possible in the process by which practical decisions are reached and implemented. The solutions to most of the problems concerning environmental sources of stress and distress in captive animals will eventually be provided by research. In the meantime, it is necessary to be sensitive to signs of stress and to take whatever steps are possible to control them—not only on humane grounds, but also because of the impact of stress on reproduction and research results. Animals that are chronically stressed are altered behaviorally and physiologically to the extent that they can experience reproductive failure. Knowing the species being used and being familiar with the normal appearance and behavior of individual animals are the best preparation for detecting signs of stress and distress (Chapter 4). The next step is to determine their source. Except for pain and illness, stress, and distress in captive environments usually result from some degree of encroachment of the six ecologic dimensions (described in Chapter 3) on species-typical needs and behavioral tendencies. Those dimensions should be considered in designing captive environments and in planning management proce-

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Recognition and Alleviation of Pain and Distress in Laboratory Animals dures, and they should be evaluated when conditions appear to be causing unacceptable stress. Husbandry practices that contribute to distress should be corrected. The environment should be well defined and controlled (e.g., established temperature, humidity, ventilation, and illumination standards should be met, noise reduced, etc.). The housing, feeding, and care of laboratory animals should be appropriate for the species to promote their health and well-being. Personnel that care for and use animals should be adequately trained. Generally, these issues are not a major source of disagreement. The attainment of well-being, however, might require consideration of other factors, such as environmental enrichment and socialization. Given the present state of knowledge, specific recommendations and guidelines are necessarily tentative. It is possible, however, to indicate the kinds of questions that are reasonable to consider when evaluating environmental sources of stress and distress that can be addressed through changes in husbandry practices. With the discussion of the six ecologic dimensions in Chapter 3 as a guide, we offer the following questions as examples for use in assessing the adequacy of husbandry practices. Relationships with Conspecifics Should the animal be housed alone or with others? Does the animal belong to a species that is mainly solitary (such as cats) or that normally lives in social groups (such as dogs, nonhuman primates, and most rodents)? Is the animal is housed with others, is continuous group living characteristic of the species (such as sheep), or are seasonal or other cyclic variations in sociability the rule (such as hamsters)? If animals are housed in groups, are the number of animals and available space such as to prevent crowding? Are the members of the group compatible? Are some animals being picked on or always causing trouble? Are the numbers or proportions of adult males, adult females, and immature animals in the group appropriate? Have all members of the group been adequately socialized with conspecifics during their early development? Are the animals familiar with each other? Is fighting or aggressive dominance a normal feature of social relationships in the species? If so, are physical arrangements—such as the volume of space, the location of barriers, and the placement of food sources—appropriate to minimize aggression? If offspring are to be separated from parents, when should this occur, which sex normally leaves the family group, and at what age? What provisions have been made to keep stress from becoming extreme?

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Predator-Prey Relationships Is the individual in a species that normally relies on other animals as a source of food? If the species is predatory, what is its normal mode of capturing prey and what stimuli are likely to elicit this behavior? Are there any indications that the elicitation and frustration of predatory behaviors is an important significant source of stress or distress? Should steps be taken to reduce or control the stimulation of predatory behaviors? Are there frequent or chronic signs of defensive reactions, such as snarling, hissing, biting, cowering, and trembling? What events or environmental conditions usually produce defensive reactions? Have changes in physical arrangements, caretaking, or experimental procedures that might reduce defensive reactions been considered? Shelter Is the animal in a species that normally uses shelters, dens, or cover? What functions do shelters, dens, and cover normally serve for the species (e.g., protection from elements or from predators or a depository for young)? If shelters, dens, and cover are provided in the captive environment, what purposes are they expected to serve? Are they adequately designed to fulfill these purposes? Is sanitation a problem? Does it appear that the animal's behavior is altered by the presence of shelters, dens, or cover so as to make it more fearful or more difficult to handle or to cause other effects that are undesirable from the standpoint of management and well-being? Does the animal scent-mark? If so, is this considered in the provisions for sanitation of the cage? Spatial Architecture (Volume, Structure, and Topography) Does the volume of space meet the standards for the species recommended by the Guide for the Care and Use of Laboratory Animals (NRC, 1985) and the Animal Welfare Regulations (CFR Title 9)? How much of the available space is actually used by the animal, and how is it used? Does the animal display repetitive and stereotyped motor patterns or other behaviors that point to some inadequacy? Can caging arrangements be improved by adding perches, climbing devices, or other structures?

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Will such additions be consistent with the requirements of animal safety, restraint, and sanitation? If cages of different sizes or containing different kinds of equipment are in use, do they appear to have different effects on signs of stress and distress? Feeding and Foraging Patterns Is the animal's food consistent in amount and quality with recommended standards for the species? What is the normal feeding pattern of the species? Do animals normally meet their nutritional requirements in a single meal with long intervals between feedings, eat intermittently throughout the day, or show some other predictable pattern? Are animals characteristically picky or wasteful feeders? Do they accept standard foods readily and consume them completely? How do they respond to unfamiliar foods? Is intake of the provided food adequate to maintain animals at an appropriate weight and in good health? Is searching for or preparing foods an important part of the normal activity of the species? Are there indications that animals attempt to engage in food-searching activities in the captive environment, even though they have no need to do so? Should feeding and foraging be facilitated by providing special opportunities, or can they be ignored without producing undesirable consequences? Within practical limitations, is the established feeding regimen consistent with the animal's preferred feeding patterns, with respect to scheduling and the kinds and amounts of food that are provided? Environmental Events Novelty, predictability, and control: What events in the captive environment are unpredictable (for example, light cycle, noise, and restraint)? Do any of those events seem to cause stress or distress? If practical steps can be taken to increase the animal's ability to predict those events, what effect is this likely to have on stress or distress? What procedures place the animal in a situation in which it is helpless, is coerced, or loses control over its own behavior (for example, restraint)? What practical steps can be taken to reduce stress and distress caused by such procedures (for example, adaptation with positive reinforcement and personnel training)? Is the animal in a species that characteristically shows high levels of spontaneous activity, interacts vigorously and flexibly with its surroundings, and

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Recognition and Alleviation of Pain and Distress in Laboratory Animals appears curious about moderately novel objects and events (for example, dogs, rodents, and most nonhuman primates)? Does the animal appear to be listless, apathetic, or behaviorally depressed, compared with other members of its species and in the absence of any signs of ill health? Should enrichment devices or socialization be considered? If an enrichment device is used, what needs or behavioral tendencies is it designed to meet? Is the enrichment device consistent with the requirements of animal safety and sanitation? Is the enrichment device economical to construct, install, and maintain? Is the enrichment device actually used by the animal? In what way? How frequently? Under what conditions? What are the indications that the enrichment device contributes to well-being? Is the animal in a species that benefits from socialization with people? SOCIALIZATION AND HANDLING Socialization is achieved through conspecific housing or through interaction with other species, including humans. The benefit to any individual animal, however, should be carefully evaluated before pair-housing is implemented or an animal is introduced to a group, and a suitable period and method of adaptation should be provided. Many laboratory animals benefit from interaction with people, but this should be undertaken with due consideration for the animal's experience and zoonotic potential. There is general agreement about the value of conspecific socialization for the well-being of most laboratory animals, although it is not always easily achieved, because of the requirements of the protocol, space, finances, and other constraints. It is well known that dogs respond favorably to direct interaction with humans and that their well-being can be enhanced by social, conspecific housing of compatible dogs. Socialization of puppies to humans and continued interaction with them might be the most stress-relieving practice for dogs. Human socialization should be included in every dog breeding program and stipulated in contracts for the purchase of dogs for research. Nonhuman primates might receive the greatest benefit from socialization with conspecifics, but direct human interaction can be beneficial under some circumstances, especially for an animal that is immature and singly housed. A predictable cause of maladaptive behaviors in nonhuman primates is social isolation when they are young. If early maternal separation is necessary, young primates will benefit from frequent exposure to cagemates. Continuous housing of very young animals together is not advisable, however, because it produces excessive mutual clinging and emotional dependence that impairs well-being and impedes normal social development. Where possible, infant nonhuman

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Recognition and Alleviation of Pain and Distress in Laboratory Animals primates should be raised, at least through weaning, with their biologic mothers in a stable, species-typical social group in a predictable environment. Conspecific housing of incompatible animals and frequent changes in group composition lead to socially induced stress. Technicians responsible for the day-to-day care of nonhuman primates should not only understand the social behaviors of the species for which they are responsible, but also understand their role in maintaining social stability and controlling stress within and between cages or pens. There is a substantial literature on the effect of human handling on the physiology, behavior, and development of various animals (Newton and Levine, 1968). The effects of handling are influenced by an animal's age and genotype and by the duration and frequency of handling. Most information has been obtained on laboratory rats. Handling pups between birth and weaning has been reported to have effects on many characteristics, including rate of growth and weight gain, learning, exploratory behavior, emotionality, physiology, responses to food and water deprivation, and the occurrence of some diseases or pathogens (Ader, 1967; Denenberg, 1969; Daly, 1973). Handling rats after weaning tends to be less effective. Comparable data on other species are lacking, but there are good reasons to assume that early handling, aimed at gentling animals and accustoming them to contact with humans, is likely to improve the docility and adaptability (and thus decrease stress) of most laboratory animals. ENVIRONMENTAL ENRICHMENT As defined in Chapter 1, the well-being of an animal encompasses more than freedom from pain and distress and is evaluated not just on the basis of growth and reproductive records, but from a global perspective of behavioral and physiologic stability. Because many stressors are of environmental origin, it is often assumed that the well-being of laboratory animals can be improved by environmental enrichment that permits animals an opportunity to express species-typical behaviors. Laboratory animals given the opportunity to perform species-typical behaviors may interact voluntarily with the enriched environment and participate in activities whose cessation could be interpreted as a change in well-being. Enrichment devices and environmental changes to promote well-being of nonhuman primates are being studied extensively. However, what is appropriate for nonhuman primates (and other species) is a matter of some debate, and research is needed to determine which methods actually improve animal well-being (Beaver, 1989). For example, it is assumed that the creation of a more naturalistic environment for nonhuman primates will permit the expression of the normal range of behaviors. But there is some uncertainty about the validity of the assumption, because it has been reported that the "naturalness" of the environment is not as important to an animal's well-being as are events that are arranged to be contingent on the animal's behavior. Feeding puzzles, manipulanda, and artificial appliances

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Recognition and Alleviation of Pain and Distress in Laboratory Animals with which the animals can interact encourage investigation and activity and are generally acknowledged to enrich the environment (see Beaver, 1989). Although attention is being focused on nonhuman primates, methods for environmental enrichment of other species should be evaluated. Concentration on enrichment of the environment by incorporating objects or devices within the cage should cause the extreme importance of social interaction, possibly the most important form of enrichment for most laboratory animals, to be ignored. The proper balance between conspecific and human social interaction, a cage and room environment developed with an understanding of the normal behaviors of the species, and caring personnel trained to handle and care for the species should be the goal. EXPERIMENTAL DESIGN The objectives of some experiments require the production of stress or even distress (e.g., through food and water deprivation, maternal deprivation, social isolation, etc.), and investigators should be sensitive to the ethical concerns raised by such objectives. Experiments should be justified, use the minimal number of animals consistent with an effective design and statistical analyses, and minimize the duration and magnitude of stress. Restriction of food intake to develop appropriate reward-motivated behaviors in behavioral studies, usually in rats, is common. In those experiments, rats are usually maintained at about 80% of their ad libitum feeding weight, which is considered neither unethical nor excessive deprivation. Although novel foods might be used as environmental enrichment (Chapter 3), the response to novel foods, in either an experimental or a husbandry context, can be stressful. Foods usually should not be changed in the course of an experiment. Caging conditions (e.g., single housing of rodents) and restraint (e.g., of rodents and nonhuman primates) produce stress, which can be so extreme or prolonged that an animal is unable to adapt and becomes distressed and maladaptive. Those procedures often can be minimized by handling and appropriate adaptation procedures, respectively. Because the novelty of an experience increases an animal's emotional response to it, habituating laboratory animals to experimental procedures by regular handling and adaptation to potentially stress-producing procedures should be incorporated into experimental protocols. For example, stress is associated with the first experience of dogs introduced to the leash, monkeys restrained in a chair, or cage-reared rats removed from their cage. Whether the stress of those experiences manifests itself in maladaptive behaviors (e.g., twirling on the leash, self-mutilation in the chair, or freezing and immobility, respectively) and thus distress will likely depend on factors external to the procedures themselves, such as previous experiences of the animal that led to expectations of pleasure or stress, familiarity with people, development of coping strategies for other events, and even time of day. Likewise, the biologically adaptive

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Recognition and Alleviation of Pain and Distress in Laboratory Animals response to stressful stimuli has been shown to be subject to the early experience of the animal (Melzack and Scott, 1957; Green, 1978). Adaptation and handling to minimize stress and prevent distress can be applied to many experimental settings and procedures: chair restraining of nonhuman primates (when necessary for short periods) should be preceded by a series of brief introductions to the chair by a familiar person and rewarded by favorite foods either in the chair or immediately on returning to the home cage. Movement of animals to test chambers or laboratories should be preceded by several days or weeks of conditioning trips in which no aversive interaction takes place and food reward is provided. Through such means, a "transfer cage" or leash can signal a pleasurable event for the animal and facilitate a difficult task for the responsible person. In each case, the goal should be the positive association of the desired task favorably with a conditioned stimulus, such as the transfer cage, leash, or familiar technician. The stimulus need not always be a physical entity; the time of day or the ring of a bell can come to convey the same information, if presented in a predictable and routine manner and associated with the event to which the animal is being adapted. Adaptation to strange or unusual objects or environments, before the experimental introduction of the animal to the object or environment, reduces the novelty and stress of the experience and the likelihood that it will affect the experimental results. Other experimental procedures and poor or inappropriate techniques, such as those common in blood withdrawal or antibody production, also can lead to stress. Amyx (1987) summarized procedures for antibody production, emphasizing that a reduction in volumes injected and a change in the site of injection minimizes the pain and distress of immunization procedures. Distress can be further minimized by sedative pretreatment, rather than use of restrainers. Blood withdrawal can lead to stress if the amount removed exceeds 1% of the animal's body weight. Adaptation and socialization are strategies for reducing the distress of laboratory animals, preventing or alleviating distress, and thereby enhancing their well-being.