4
Avoiding, Minimizing, and Alleviating Distress

The simplest approach to avoiding, minimizing, and alleviating distress in laboratory animal care and use is to follow the principles of the Three Rs—refinement, reduction, and replacement. It is important, however, to strike a balance between the integrity of research outcomes and the welfare of animals used. Investigators, veterinarians, and animal care personnel should function as a team and base their decisions on professional judgment, best practices, and thorough clinical evaluation of distressed animals. Refining aspects of housing, husbandry, enrichment, and socialization helps alleviate or prevent distress. Refining the experimental design, utilizing humane or surrogate endpoints, and using the appropriate statistical analyses (including an accurate calculation of sample size) helps reduce the numbers of animals used and alleviate some of their distress. A team approach is necessary to address treatment options for distressed animals. When using procedures that intentionally result in distress, the investigator should, in consultation with the veterinarian and the IACUC, develop a plan that will establish limits to the levels of distress allowed.

INTRODUCTION

Established ethical, regulatory, and scientific practices, standards, and policies mandate avoiding animal distress whenever possible. However, if research or regulatory testing objectives cause a research animal to experience distress, it is incumbent upon the animal user to identify the cause(s) of distress, attempt to minimize its duration and intensity, and/or provide the



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4 Avoiding, Minimizing, and Alleviating Distress The simplest approach to avoiding, minimizing, and alleviating dis- tress in laboratory animal care and use is to follow the principles of the Three Rs—refinement, reduction, and replacement. It is important, how- ever, to strike a balance between the integrity of research outcomes and the welfare of animals used. Investigators, veterinarians, and animal care personnel should function as a team and base their decisions on profes- sional judgment, best practices, and thorough clinical evaluation of dis- tressed animals. Refining aspects of housing, husbandry, enrichment, and socialization helps alleviate or prevent distress. Refining the experimental design, utilizing humane or surrogate endpoints, and using the appropriate statistical analyses (including an accurate calculation of sample size) helps reduce the numbers of animals used and alleviate some of their distress. A team approach is necessary to address treatment options for distressed animals. When using procedures that intentionally result in distress, the investigator should, in consultation with the veterinarian and the IACuC, develop a plan that will establish limits to the levels of distress allowed. INTRODuCTION Established ethical, regulatory, and scientific practices, standards, and policies mandate avoiding animal distress whenever possible. However, if research or regulatory testing objectives cause a research animal to experi- ence distress, it is incumbent upon the animal user to identify the cause(s) of distress, attempt to minimize its duration and intensity, and/or provide the 63

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64 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS means for the animal to cope. The simplest approach is to follow the prin- ciples developed by Russell and Burch known as the Three Rs (Russell and Burch 1959). The Three Rs are (1) refinement of the protocol to minimize or eradicate distress for the species used (e.g., by employing nonclinical [e.g., molecular measurements] or defined [e.g., tumor growth instead of survival] endpoints; giving positive rewards; changing or refining the data/sample collection methods; or instituting species-specific husbandry refinements such as enrichment); 2) reduction of the number of animals used to the absolute minimum necessary (based on appropriate statistical sample size determination or other field-specific methods), particularly if they are likely to experience unavoidable distress; and (3) replacement of an animal with a nonanimal model or a less sentient species, usually of a lower phylogenetic order, such as a primitive invertebrate. As discussed in Chapters 2 and 3, distress may result from a single intense or prolonged stressful experience or from several simultaneous stressors that might individually cause stress but not likely distress. There- fore, mitigating some potentially stressful circumstances, such as husbandry schedules, may allow an animal to better adapt to other stressors such as experimental procedures. It is important to weigh any possibly adverse impact of replacement, refinement, and reduction on scientific outcome against both the negative impact of failure to avoid, minimize, or allevi- ate stress and distress on the research data and the numbers of potentially wasted animal lives. This chapter identifies approaches to avoid or minimize distress through the alleviation and minimization of stress, in both the care and use of laboratory animals. The chapter also suggests ways to alleviate distress that cannot be avoided or minimized because of scientifically justified research protocols, and addresses the challenges and compromises that arise when the object of research itself is the study of stress and distress. This chapter is not intended to be comprehensive but to provide investigators and IACUC members with an awareness of common problems and useful strategies. The Committee encourages all who are involved in laboratory animal care and use to think creatively when considering solutions to specific circumstances. AvOIDINg OR MINIMIZINg DISTRESS IN LAbORATORy ANIMAL CARE Most animals are able to cope with a relatively wide range of naturally occurring environments, but such environments are not usually character- istic of laboratory animal facilities. Research environments are generally designed as a compromise between the needs of the animal, the user, and

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6 AVOIDING, MINIMIZING, AND ALLEVIATING DISTRESS the husbandry staff. While most animals continue to function normally1 in animal care facilities, some do not adapt successfully to a laboratory environment (see Chapter 3) and it is important to consider the effects of their maladaptation on both the research and the animal’s welfare. The Committee notes the lack of consensus among the scientific community as to the impact and quantification of the effects of the major stressors on an animal’s welfare and the most appropriate modifications in response to these perturbations. The Committee also notes that relevant research data to answer many of the issues undertaken in this chapter are still inadequate and often absent. Therefore the text below is a combination of available scientific literature and professional judgment and expertise. Housing Potential environmental stressors that may lead to stress and distress include levels of ambient light, noise, vibrations, fluctuations or extremes in temperature, husbandry practices, and facility maintenance (e.g., construc- tion, vibration). The degree to which these stressors can lead to distress is highly variable; in many cases, signs of distress appear only after prolonged stimulation, as noted in Chapters 2 and 3. Most laboratory rodents, for example, are nocturnal but their housing environment may make it difficult for them to withdraw from brightly lit areas, they may be handled during their somnolent period, or they may be exposed to sudden or loud noises. It has been demonstrated that exposing rodents (normal, albino, or trans- genic) to excessively bright or continuously high levels of light can cause permanent damage such as retinal injury (Kaldi et al. 2003; Wasowicz et al. 2002). In these cases it is appropriate to reduce the levels of light (for more information see pages 34-35 of The Guide for the Care and Use of Laboratory Animals [NRC 1996]) and to provide refuges that enable the animals to hide from it. Similarly, just as noise can be a stressor and affect the health of humans (Passchier-Vermeer and Passchier 2000; Tomei et al. 2000), sound and ultrasound can be stressful and cause external variation 1 In the interest of uniformity and adherence to commonly accepted normative values, the Committee chose the word “normal” to describe the life of a laboratory animal that, if not subjected to experimental procedures, would live mostly undisturbed. Implicit in this position is the notion that many laboratory animals are “artificial constructs” for which the question of whether they could successfully adapt or live in the natural world is a philosophical exercise. The Committee, however, recognizes the argument put forth over the last 15 years that any artificial environment is abnormal, because it does not allow the laboratory animal to function according to its natural predisposition, and as such, behavioral variations among laboratory animals are only degrees of abnormality (Garner 2005, 2006; Würbel 2000). However, the definition of “natural predisposition” is not entirely clear when the subject matter is laboratory animals, especially those that have been domesticated over many generations.

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66 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS in animal data (Clough 1982; Milligan et al. 1993; Sales et al. 1999; Shoji et al. 1975). Noise in dog kennels can reach levels that can damage human hearing, and that may well have an impact on dog hearing and physiology as well (Hubrecht et al. 1997; Sales et al. 1997). Vibration has been dem- onstrated to be a stressor in both poultry (Abeyesinghe et al. 2001) and pigs (Perremans et al. 1998); investigators should, therefore, strive to minimize common sources of vibration such as ventilation machinery in adjacent rooms or on cage racks (Clark 1997). In addition to such continuous back- ground disturbances, the effects of isolated environmental insults may also cause distress in some species and models. For experimental and comfort reasons it is best to maintain animals in their thermoneutral zone (NRC 2006, pages 39-45). The Committee notes the discrepancy between the temperature recommendations for housing rodents in the Guide for the Care and Use of Laboratory Animals (NRC 1996, page 32) and those put forth in the Guidelines for the Humane Transportation of Research Animals (NRC 2006, pages 39-45) due to new evidence used in the later publication. Rabbits are very susceptible to heat stress (Marai et al. 2002). Singly housed mice prefer ambient temperatures of 28-30°C while group-housed mice prefer a slightly colder environment with ambient temperatures of 24-27°C (NRC 2003b, page 97). The provi- sion of nesting materials, refuges, or nest boxes for rodents, or areas within an enclosure with different levels of heating or cooling (i.e., heated areas for dogs) allows the animals to control their microclimate. Moreover, long- term housing in cages with mesh floors where adequate bedding or nesting materials cannot be provided can also result in stress, distress, or more obvious deleterious effects, such as foot ulcerations and arthritis. Enrichment Barren environments may not meet the species-specific needs of an animal. In addition to their impact on welfare these conditions can adversely affect the validity of experimental data (Sherwin 2004). Such environments can cause distress as shown by the development of abnormal behaviors (see Chapter 3) or by experiments in which animals, when given the choice to self-medicate with anxiolytics, consume larger proportions of midazolam-water solution than their littermates housed in enriched envi- ronments (Sherwin and Olsson 2004). In contrast, supporting evidence has shown that biologically relevant enrichment can help avoid the develop- ment of abnormal behaviors (see Chapter 3), although it may not alleviate previously established patterns. Moreover, as Olsson and Dahlborn have shown, some animals exhibit clear preferences and will work to access these enrichments (Olsson and Dahlborn 2002). In mice, environmental

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6 AVOIDING, MINIMIZING, AND ALLEVIATING DISTRESS enrichment may attenuate anxiety and stress and restore immune response (Benaroya-Milshtein et al. 2004). It can also slow disease progression, a consequence that in some circumstances might interfere with the research aims but that may also provide insights into new or better treatments or new research avenues (Hockly et al. 2002). Enrichment can thus improve welfare, reduce stress, and improve the quality of data obtained from the animals in situations where such enrichment does not compromise the anticipated research outcomes. However, the effect of environmental enrichment on stress responses can vary depending on species or strain, the type of enrichment used, the stressor employed, and the type(s) of stress response(s) evaluated (Bardo et al. 2001; Belz et al. 2003; Green et al. 2002; Lawson et al. 2000; Marashi et al. 2003; Moncek et al. 2004; Roy et al. 2001; Schrijver et al. 2002; Sharp et al. 2005; Tsai et al. 2002). Ideally, enrichment devices or strategies should draw on previous literature or research that shows that they are beneficial to the animals and have no unexpected adverse effects on their health, and that their use does not jeopardize experimental outcomes and research goals through the introduction of uncontrolled variables, increased variability, and/or inter- experimental variance leading to a need for more animal studies (Baumans 2005; Bayne 2005; FELASA Working Group Standardization of Enrichment 2006). Benefiel and colleagues suggest the need for evidence-based evalu- ation of “mandatory” enrichment practices for all laboratory animal species (Benefiel et al. 2005). Meier and colleagues have shown that enrichment (in the form of various housing supplements) can increase the acute stress response (as evidenced by elevated heart rate and body temperature) of individually housed mice (Meier et al. 2007). Recent evaluation of the effect of enriched environment on genetically engineered fibulin-4 knockout mice (fibulin-4+/–) has shown that knockouts in enriched cages had fewer dis- organized regions on their arterial walls than knockout littermates housed in standard cages. These results suggest that the type of housing environment may interfere with the expected phenotype of genetic manipulations and with the experimental outcomes (Cudilo et al. 2007). However, despite a lack of adequate pilot studies, background data, or published information, even such highly controlled conditions as toxicology studies have effec- tively adopted appropriate enrichment enhancements (Dean 1999; Turner et al. 2003). Faced with the absence of unequivocal scientific evidence for data-driven enrichment standards and aware of the potential for unexpected consequences by the indiscriminate use of enrichment strategies, the Com- mittee makes its recommendations guided by best practices and expert pro- fessional judgment in an attempt to balance the need to safeguard animal welfare while maintaining scientific excellence.

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68 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS Socialization It is generally appropriate to house naturally gregarious animals in compatible social groups unless there are scientific or welfare reasons not to do so (National Health and Medical Research Council 2004; Canadian Council on Animal Care 1993; Council of Europe 2006; NRC 1996). Social housing can activate stress responses involving the HPA axis in rats, but when a wider range of measures is taken into account, overall, social hous- ing is neither stressful nor harmful (Hurst et al. 1997, 1998). For example, even macaques fitted with a cranial implant could be paired with another compatible macaque without it inflicting damage to the device or interfering with the research goals (Roberts and Platt 2005). Furthermore, a consider- able body of evidence indicates that housing naturally sociable animals (e.g., rats, mice, dogs, primates) in solitary conditions can result in stress and harm (Baker 1996; Eaton et al. 1994; Hetts 1991; Hubrecht 1995; Novak 2003; Patterson-Kane 2002; Sharp and Lawson 2003; Van Loo et al. ; 2004). Even cats, which are not particularly gregarious, can benefit from ). social housing (Council of Europe 2006). It is therefore important to provide thorough scientific rationale for solitary housing. Disruption of established social groups, pairing (for additional informa- tion see Appendix), or the introduction of animals to larger preformed units are all potential causes of aggression or stress. As a husbandry refinement, therefore, social groups should be established early, and disruption of established groups should be minimal, as demonstrated in studies of mice, rabbits, and cats (Bradshaw and Hall 1999; Jennings et al. 1998; Morton et al. 1993; Sharp et al. 2002b). Close cooperation with the supplier or breeder may be necessary to promote group formation and ensure minimal disruption of group dynamics. Adequate socialization to both humans and conspecifics at an early age may also help prevent subsequent stress and distress (Council of Europe 2006). Animals housed in social groups generally need adequate space as well as objects in their enclosure to allow them to modulate their social interactions. However, some structures can actually trigger aggression, as shown in certain strains of male mice (Haemisch and Gartner 1994). Because competition for resources often triggers aggression, the provision of sufficient or separate feeding devices for some species (e.g., dogs, cats, pigs) can help minimize the risk of fights during feeding. For other species, such as mice and marmosets, that regulate social interactions through olfactory markings, appropriate cage changing and cleaning routines can minimize social disruption. For example, decreasing the frequency of cage clean- ing or leaving some older bedding can help maintain tolerance between familiar male mice (Hurst et al. 1993) and transferring nesting material between cages can positively influence several stress-related physiological

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6 AVOIDING, MINIMIZING, AND ALLEVIATING DISTRESS parameters (Van Loo et al. 2004), while retaining scents in certain areas of the cage (e.g., the top grill) may increase aggression (Gray and Hurst 1995). Housing animals in groups that are not compatible (e.g., certain strains of postpubertal male mice) can result in aggression, stress, distress, injuries, and even death. While all social groups should be monitored for compat- ibility, this is particularly important immediately after the formation of the group. Animals that require individual housing may benefit from visual, auditory, olfactory, and even tactile contact with other animals, as such interactions are thought to improve the welfare of all animals involved. Husbandry While predictable variations in housing conditions can be a useful com- ponent of enrichment, unpredictability in animal care can be stressful and potentially distressing if prolonged or extreme. Even routine cage cleaning and changing can be stressful or become distressful if not consistently and routinely performed in a gentle manner (for more details see Chapter 3). Cardiovascular and behavioral changes, such as elevated blood pressure, heart rate, and movement, lasted up to 60 min after changing the cages of adult male Sprague Dawley rats (Duke et al. 2001). Cage and room clean- ing also disrupt olfactory environments that are important to animals that depend on their sense of smell to socialize (Gray and Hurst 1995). Because husbandry procedures can be stressful to the laboratory animal, performing more than one simultaneously (e.g., weighing animals at the time of transfer to clean cages) may decrease the handling stressor in some species if such arrangements are possible. Alternatively, more frequent, gentle, predictable handling may habituate an animal and thus minimize handling stress. In species such as dogs and primates, strategies such as positive rewards and operant conditioning techniques can minimize stress and thus the potential for distress for both animals and handlers (Prescott and Buchanan-Smith 2003; Weed and Raber 2005). Many techniques that minimize stress in husbandry—such as combining husbandry handling with habituation and handling for research purposes, acclimation to new envi- ronments, positive reinforcements, operant conditioning, and well-trained staff—can be helpful tools for the overall reduction of stress and distress; for further information see ILAR Journal 47(4). It is extremely important to involve both research personnel who are knowledgeable and skilled in current methods and well-trained and attentive animal care employees. Individuals who understand the normal behavior and appearance of animals and have mastered the appropriate handling and restraint techniques are quick to identify abnormal clinical signs that may be indicators of distress. Rapid identification and prompt attention to

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0 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS the stressors facilitates avoidance, minimization, and alleviation of distress, if such interference is not incompatible with the objectives of the research protocol. AvOIDINg OR MINIMIZINg DISTRESS IN LAbORATORy ANIMAL uSE Refining the Experimental Design A variety of strategies to refine the research protocol can help minimize animal stress and distress. A thorough literature review is vital for a criti- cal analysis of the suitability, applicability, and validation of the proposed methodology. This section addresses the importance of correct statistical methods on the number of animals used. Choosing an earlier stage for an intervention (mild severity) or employing a different approach to arrive at the same research objective might work as effectively as waiting for later impacts of high severity and substantial distress. Examples of less stressful approaches include not allowing a tumor to grow to the point that it affects mobility before starting an experimental treatment, replacing long fasts as a motivating factor with the work-by-reward method, selecting a smaller stimulus to elicit a response before high-intensity stimuli are employed for the evaluation of a novel analgesic, and keeping the withdrawal of food and water in learning experiments to the minimum time necessary (Morton 1998; Morton and Hau 2002; NRC 2003a). If a potential source of distress is the data-gathering or sample collection process itself, a less invasive method may be appropriate. For example, if the experimental design justifies it, the one-time surgical implantation of vas- cular lines and sensors can replace manual restraint for frequent blood col- lection or other physiological measurements, to avoid repeatedly subjecting the animal to stressful experiences (proper aseptic techniques and frequent peridermal maintenance is required when handling such surgical implants; for more information see chronically instrumented nonhuman primates in Broadbear et al. 2004). This is a common strategy for animals in chronic studies. However, it may be necessary to strike a balance if repeated surgery is necessary in order to replace batteries or sensors (Hawkins et al. 2004; Morton et al. 2003). Obviously, the constraints of the study will determine the appropriateness of alternative techniques, which may not be suitable for some types of studies or housing systems (Vahl et al. 2005). Further examples of less stressful options (more information on the severity of stress caused by these methods is included in Chapter 3) include the use of oral or rectal swabs, plucked hair, or tissue from ear punches in place of tail tip amputation for the purpose of genotyping (Hawkins et al. 2006; Pinkert 2003; Robinson et al. 2003), and the measurement of cortisol

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1 AVOIDING, MINIMIZING, AND ALLEVIATING DISTRESS and other steroids in samples from saliva (Aardal and Holm 1995; Kiess et al. 1995) and plucked hair (Davenport et al. 2006). Even less handling is involved when samples are taken from voided urine, feces, expired air, and shed hair (Poon and Chu 1999), if these methods are validated for the species under study. Other noninvasive techniques for data collection include sound recordings (Holy and Guo 2005), cameras (Hobbs et al. 1997), or noninvasive, sensor-laden apparel simply worn by the animal (Jarrell et al. 2005). Humane Endpoints Validated endpoints that occur earlier in the course of the protocol and involve no detectable indication of disease, injury, or abnormal behavior can prevent or minimize distress in experimentation and testing. The use of humane endpoints (i.e., “end a study earlier to avoid or terminate unrelieved pain and/or distress”; Stokes 2000) or surrogate endpoints (i.e., those that can reliably substitute for more distressing or painful phenomena) is espe- cially applicable in scientific disciplines that focus primarily on molecular and cellular phenomena associated with disease (Morton 2000; Hendriksen and Morton 1999). In these cases, biochemical changes may be detectable at early stages in the disease process, prior to the manifestation of clinical signs consistent with distress. For example, elevated white blood cell counts are detectable in leukemia models before illness becomes obvious and serum biochemical values often change in early stages of toxicity before animals appear ill (Poon and Chu 1999). Thus, taking measurements or collecting samples from animals before the appearance of any clinical signs (including all clinical manifestations, not only those related to distress) is desirable, especially when the signs themselves are not the study’s focus. In such cases, the predictive value of validated endpoints may permit early euthanasia of these animals and postmortem collection of data or samples (for additional information see Appendix). Alternatively, a clinically normal animal could be anesthetized before a distressful procedure and euthanized before regaining consciousness. Familiarity with certain procedures or experimental protocols often allows for predicting the course of adverse clinical signs and distress. In many instances death results from indirect effects such as dehydration and is not related to the response variable under study. In mice, for example, progressive hypothermia due to low food intake will cause an animal’s death over several days. However, distress can be minimized through the use of validated humane endpoints, such as euthanizing the animals at the first recording of low body temperature (Morton 1998; Soothill et al. 1992). The choice and use of endpoints should be part of the experimental proto- col whenever possible.

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2 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS The value of Statistics Pilot Studies For certain experimental procedures (e.g., acute toxicity protocols), the scientific literature or the complexity of the biological system under study suggests that distress is possible but not predictable. Distress may also result from investigator inexperience, the use of technically demanding procedures, or the establishment of a new animal model. In those cases, a pilot study with fewer animals may be appropriate in order to establish proof-of-concept or to achieve a learning curve before seeking approval for the use of more animals. Other benefits of pilot studies include the col- lection of useful preliminary data to better estimate the appropriate sample size, the identification of unanticipated adverse effects, and opportunities for refinements (e.g., endpoint determination and monitoring schedules). Pilot studies, however, are not appropriate for all protocols, as they can also lead to an increase in the number of animals needed or the unnecessary consumption of valuable reagents and other limited resources. Sample Size Determination Appropriate statistical analyses are useful for the reduction of the numbers of animals used and determination of the desired statistical power and minimum sample size values (n) needed to discriminate between sig- nificantly different groups or endpoints (NRC 2003a). Several publications reviewing the use of animals in experimental protocols found that the majority of studies evaluated did not have adequate statistical power to detect even a large difference between experimental groups (Chung et al. 2002; Dirnagl 2006; Gold et al. 2005; Riley et al. 1998). In the preferred method of sample size determination, the “power analysis”, the experiment should be designed so that there is at least an 80 percent probability (i.e., a minimal statistical power of 0.8) of detecting a difference (“the effect size”) of a specific magnitude between experimental groups. According to Shaw and colleagues, the “effect size is the magnitude of the difference between treatment and control means, which the experiment is to be designed to detect” (Shaw et al. 2002). An adequate sample size determination is nec- essary to ensure that the effect size achieves both scientific validity and statistical significance. It is crucial for researchers to perform the sample size calculation before initiating a study in order to reduce the number of animals utilized and ensure that the number of animals (sample size, n) will provide scientifi- cally valid data. This calculation derives the sample size necessary to detect a statistically significant effect at the desired power level. There are four

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3 AVOIDING, MINIMIZING, AND ALLEVIATING DISTRESS factors that must be known or estimated to calculate a sample size (Dell et al. 2002): 1. Effect size: the difference between experimental groups (see above); 2. Population standard deviation: the variability within a population; 3. Power level (1-β): the probability that a difference of specific mag- nitude between groups will be detected (at least 80 percent); and 4. Significance level (α): the probability that a difference between groups is due to chance alone (classically defined as 0.05 or 5 percent). Power analysis cannot be applied in all situations. For example, in cases in which experiments measure many variables, it is quite difficult to specify and almost impossible to calculate the effect size for each one. In micro- array analyses, where thousands of observations per animal are collected, it is not possible to postulate an effect size for each one. Furthermore, power analysis requires an estimate of the standard deviation of the population, which may not always be available. In these situations, other methods of sample size determination may be more appropriate (Festing et al. 2002; Mead 1988). After calculating the sample size, researchers should consider additional ways to further reduce it. For example, because the power and significance levels have been set a priori (i.e., prior to the sample size determination), increasing the effect size or decreasing the population standard deviation could result in a smaller sample size without sacrificing power. A sample of the various methodologies that have been described includes: 1. Decreasing measurement error (will decrease sample variance and increase sensitivity); 2. Choosing appropriate animal strains (helps control variation; for example, the use of isogenic or inbred murine strains may be more appropriate than outbred ones in some experimental designs (Festing and Altman 2002; Festing et al. 2001); 3. Utilizing endpoints that are continuous rather than dichotomous (continuous data require smaller sample sizes to detect a desired difference between experimental groups); 4. Utilizing the repeated measures experimental design approach (i.e., each animal acts as its own control, decreasing the overall popula- tion variability); 5. Decreasing the number of experimental groups (i.e., utilizing the minimum data needed to disprove the null hypothesis; for example, by reducing the number of points of a dose-response curve). This method should be considered in relation to the type of statistical

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6 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS TABLE 4-1 Example of a decision and response algorithm for unanticipated distress in laboratory animals al. 1998), while Coleman and colleagues demonstrated that the ability of individual monkeys to respond to conventional training methods is closely correlated with their unique temperament (exploratory or inhibited person- alities; Coleman et al. 2005). The actual causes of distress may also lead to sequelae that require attention even if the underlying cause is not treatable. For example, the clinical signs of a distressed animal often include dehydration and weight loss resulting from anorexia. Provision of supplemental fluids and nutrition may relieve the compounding impact of dehydration or poor body condi- tion on the compromised animal. Supplemental heat, cooling, bedding, social housing, and human companionship are other strategies that make a

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 AVOIDING, MINIMIZING, AND ALLEVIATING DISTRESS distressed animal more comfortable. Regardless of the approach selected, it is essential to maintain the dialogue between the investigator, veterinarian, and animal care personnel throughout the treatment phase, because the prognosis and the status of the animal’s condition may change. Distress resulting from behavioral problems resistant to the relatively simple and straightforward approaches listed above can be especially dif- ficult to treat. It may be appropriate to consider psychotropic medications such as anxiolytics, tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), and neuroleptics if they are compatible with the research protocol. SSRIs and TCAs have been effective in the treatment of animals with repetitive, self-injurious, and anxiety-based behaviors. A firm diagnosis will aid in the choice of medication, as these drugs have been used to treat assorted behavioral problems in multiple species with varying levels of success (for studies on monkeys see Fontenot et al. 2005; Tiefenbacher et al. 2003, 2005; Weld et al. 1998). Taylor and colleagues used a combination of chlorpromazine, buprenorphine, and environmental enrichment to successfully treat a self-injurious behavior in a rhesus monkey (Taylor et al. 2005). Hugo and colleagues showed that fluoxetine had some efficacy in the reduction of stereotypies in captive vervet monkeys (Hugo et al. 2003). Recent studies have shown that stereotypic behavior in mice responded to self-administered anxiolytics (Olsson and Sherwin 2006). Furthermore, opioid antagonists have been used to treat behaviors with a self-rewarding effect in sows (Cronin et al. 1985). An accurate diagnosis and the preparation of a behavior modification plan should precede the initiation of therapy with any psychotropic medications. The Committee notes that, while interest in the use of psychopharmacological treatment for behavioral modifications is growing, limited research data exist relevant to the effects of these drugs on animal behavior. The Committee cautions that there should be appropriate justification for their use (which should not be the first line of defense), that other behavioral modification measures should be implemented, and that these should be accompanied by careful monitoring of the animal. Decisions to treat, not treat, or euthanize animals with a severe condi- tion or a poor prognosis should involve the entire research and veterinary support team, whose members should make every possible effort to achieve consensus on the decision regarding the fate of the animal. Regulations, however, mandate that the institution’s Attending Veterinarian retain the ultimate responsibility and authority over the final disposition of the animal (see Figure 4-1). Decisions that call for euthanasia should follow approved methods, which are regularly updated and published (AVMA 2007). Only skilled, compassionate persons, with properly maintained equipment, should perform euthanasia. Proper handling of animals prior to euthanasia is important to avoid inducing further and unnecessary distress. Sources of

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78 RECOGNITION AND ALLEVIATION OF DISTRESS IN LABORATORY ANIMALS FIGURE 4-1 Distressed animal: Team dialogue on decision making. The decision to not treat an animal would depend on the cause of the distress and the severity of the animal’s condition. If the distress is appropriately caused by the research protocol, then the animal will either remain on the study without treatment or—if severely compromised—euthanized. If the distress is caused by an external perturbation, such as husbandry issues, that can be corrected without a direct therapeutic inter- vention on the animal (which might interfere with protocol), then again the animal would remain in the study without treatment, but the environmental causes would have to be addressed. distress include, but are not limited to, improper grouping with incompat- ible conspecifics or other species; lack or withdrawal of food, water, or clean bedding; and inappropriate noise levels and light cycles, particularly if the interval before euthanasia is long. Last, not least, it is essential to ensure that the animals are truly dead before their disposal.

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 AVOIDING, MINIMIZING, AND ALLEVIATING DISTRESS STuDyINg DISTRESS Distress in humans may be more widespread (or at least more readily recognized) than that observed in nonhuman animals because of unique human cognitive capacities, such as the ability to clearly communicate threatening, dangerous, or painful conditions; to remember these circum- stances and their consequences over extended periods of time; and to apply the emotions engendered to other stimuli on the basis of verbal or categorical concepts (Sapolsky 1994). A substantial proportion of the Ameri- can population will at some point suffer from an illness that is distressing or even incapacitating (e.g., depression or a severe anxiety disorder). Many of those afflicted present with no specific experiential basis for their disorder, which suggests that our society’s efforts to prevent and/or control intense and chronic stressors, even if relatively successful, may not prevent these maladies. A significant portion of research with laboratory animals deals with pathology resulting in distress, incapacitation, or death for the animals. While it is often possible to study incapacitating or lethal conditions while using palliative agents or euthanasia in order to alleviate or preclude animal distress, it is not possible to adequately investigate distress itself without allowing it to occur. While it is therefore desirable to reduce distress in laboratory animals, this should not extend to eliminating all of it. Animal models have provided insight into the anatomical and molecular bases of various human distresses (Blanchard and Blanchard 2005; Herman et al. 2005; Maier and Watkins 2005; Phelps and LeDoux 2005). An attempt to totally eliminate the study of distress would imply abandoning the major goal of biomedical research: to understand and find therapeutic solutions for conditions that continue to plague a significant portion of humanity as well as nonhuman animals. With care and attention, it should be possible to attain the optimum goal of reducing distress even while continuing to investigate it. When using procedures that intentionally result in distress, the investigator, in consul- tation with the veterinarian and the IACUC, should develop a plan that will establish limits to the levels of distress allowed. Appropriate methods include measures to alleviate distress following completion of the proce- dures or attainment of the research aims (e.g., maximum allowable weight loss as a percentage of normal body weight). In line with the important goal of extrapolating such research to specific human conditions or disease states, the limits chosen should be sensitive to the goals of the research project and the wider scope of distress-related phenomena to which the project is potentially relevant.

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