3
General Animal-Care Concerns

TRAINING AND SUPERVISION

Oversight and training of all individuals associated with animal care and use (PI, research personnel, students, animal-care staff, veterinary staff, and IACUC members) is critical for the success of research. Gaining consensus on the importance of training is easy; implementation and participation present challenges. Neuroscience research often involves situations in which the research team and the animal-care staff must work in close cooperation to optimize both animal welfare and research outcomes. The diversity in education and experience of these multi-disciplinary teams adds to the training challenge.

Proper training is fundamental in ensuring animal welfare, and is recognized by regulatory agencies. For example, both the AWRs and PHS Policy require institutions to ensure that every person who works with animals is appropriately qualified (AWR 2.32(a) and PHS Policy IV.C.1.f.). There are several good references that provide guidance on training, including Essentials for Animal Research: A Primer for Research Personnel (Bennett, Brown, & Schofield, 1994) and Education and Training in the Care and Use of Laboratory Animals: A Guide for Developing Institutional Programs (NRC, 1991).

Although the PI is ultimately responsible for ensuring that appropriate training has been provided to the research staff, it is an institutional responsibility to make available training in animal anesthesia, surgery, experimental manipulations, and occupational health and safety. The ultimate responsibility for overseeing training rests with the IACUC, which must consider the qualifications of personnel involved in conducting research as part of its protocol review and approval process (AWR 2.31 (d)(1)(viii); NRC, 1996).



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3 General Animal-Care Concerns TRAINING AND SUPERVISION Oversight and training of all individuals associated with animal care and use (PI, research personnel, students, animal-care staff, veterinary staff, and IACUC members) is critical for the success of research. Gaining consensus on the importance of training is easy; implementation and participation present challenges. Neuroscience research often involves situations in which the research team and the animal-care staff must work in close cooperation to optimize both animal welfare and research outcomes. The diversity in education and experience of these multi-disciplinary teams adds to the training challenge. Proper training is fundamental in ensuring animal welfare, and is recognized by regulatory agencies. For example, both the AWRs and PHS Policy require institutions to ensure that every person who works with animals is appropriately qualified (AWR 2.32(a) and PHS Policy IV.C.1.f.). There are several good references that provide guidance on training, including Essentials for Animal Research: A Primer for Research Personnel (Bennett, Brown, & Schofield, 1994) and Education and Training in the Care and Use of Laboratory Animals: A Guide for Developing Institutional Programs (NRC, 1991). Although the PI is ultimately responsible for ensuring that appropriate training has been provided to the research staff, it is an institutional responsibility to make available training in animal anesthesia, surgery, experimental manipulations, and occupational health and safety. The ultimate responsibility for overseeing training rests with the IACUC, which must consider the qualifications of personnel involved in conducting research as part of its protocol review and approval process (AWR 2.31 (d)(1)(viii); NRC, 1996).

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Training of research personnel should include procedure-specific training in neuroscience-research techniques, which the PI or senior research staff are usually best suited to teach, and more general training in such subjects as regulation, aseptic technique, anesthesiology, euthanasia techniques, and animal handling, which members of the veterinary or animal-care staff are generally most qualified to teach. The extent of training can depend on the duties and responsibilities of the staff involved. If the procedures to be used have the potential to cause pain and/or distress, mechanisms must be in place to ensure that the research staff can perform them competently. The selection of a trainer should be flexible and adaptive because it will depend on who is best qualified and prepared to provide training. A consortium of individuals from various disciplines may be necessary for complex projects (Kreger, 1995). Training should be a continuing process. Open communication and cooperation between the veterinary staff and the investigative staff regarding innovations in technique are essential to ensure the most up-to-date and refined use of animals. Evaluation of outcomes and results is critical in assessing technical experience and the need for training. The IACUC must be prepared to re-review training and experience whenever problems occur in projects. MONITORING EXPECTED AND UNEXPECTED CONSEQUENCES Assessing the nature and context of the clinical problems that an animal may experience during neuroscience experiments can be challenging for both researchers and veterinarians. For example, some strains of genetically modified mice spontaneously develop severe and debilitating disease unrelated to experimental manipulation. In some models, animals may develop substantial or exacerbated neurologic abnormalities because of drug treatment or experimental lesions. The assessment of postprocedure pain, distress, and general health is a matter of subjective clinical judgment that depends on evaluating a variety of measures, including behavioral factors, and recognizing that the interpretation of these measures differs greatly among species; for example, some species mask pain or distress from the observer. However, how a trained animal performs a behavioral task can be a sensitive index of its general condition. Changes in baseline experimental measures can also be informative. In many cases, a change in a specific behavioral measure, rather than changes in a general repertoire of behaviors, is particularly informative. Accordingly, thorough record keeping is essential in any behavioral monitoring program, and the frequency and method of record keeping should be described in detail in the animal-use protocol. Review of proposed experiments that involve the care and use of animals with induced neurologic deficits poses special concerns for IACUCs. Depending on the nature and extent of the deficits, animals with induced neurologic disease may be limited in their ability to ambulate, obtain food and water, groom, urinate, or defecate, or they may experience pain, behavioral depression, or fear. The

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experimental induction of debilitating neurologic deficits must be well grounded in scientific need, the animals must receive appropriate specialized care as needed, the number of animals exposed to a debilitating deficit must be minimized, the experimental end point must be well defined (for example, as to the length of time that an animal may be debilitated or the degree of debility), and the experimental protocol should be refined to reduce or eliminate pain, distress, discomfort, and mortality to the greatest extent consistent with valid experimental and statistical design. Studies of neural injury and disease necessitate stringent requirements for the assessment and alleviation of animal pain and distress. Prolongation or repetition of many treatments, chronic alteration of neural activity, or the destruction of a population of neurons can cause substantial or permanent neurologic deficits. Neuroactive agents and even treatments themselves can cause adverse side effects or toxicity. Evaluating the likelihood of such adverse outcomes and designing strategies for avoiding or alleviating them without compromising the scientific goal of an experiment can be challenging for investigators, veterinarians, and IACUCs. For example, in some studies, the repeated application of an agent or a treatment might require multiple major survival surgeries. In such cases, the stress of undergoing general anesthesia repeatedly, the level of necessary asepsis, and the need to perform the procedures in a surgical setting may be special considerations. The personnel in a research laboratory usually have some knowledge or expectation about the likely effects of a specific neuroscience procedure on animal health and well-being. Such information is typically solicited as part of the protocol evaluation. A structured approach to developing a profile of anticipated pain, distress, or disease should consider whether any major body systems are likely to be substantially affected during a study. Such an assessment can also guide the development of a systematic approach to animal monitoring and record keeping. The plan should incorporate a list of variables to be assessed and a timetable of observations. Three general considerations apply to research projects that require animal monitoring and maintenance to promote animal well-being (NIH, 1991): Consultation. Consultation between neuroscientists and veterinarians is essential for the design and implementation of monitoring and maintenance procedures. Achieving appropriate solutions to problems that arise in neuroscience experiments requires continuing discussion and collaboration between the PI and veterinarians. Interaction should begin before experiments are initiated. The interaction between the research team and the veterinary staff should provide an opportunity for mutual education and support. Responsibility. Periodic or regular veterinary assessment of both the animal and the experimental records is important in ensuring adequate veterinary care. Animal monitoring and maintenance are conducted and should be documented by neuroscientists as a routine part of their experiments. Documentation should in-

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clude objective data to identify clinical trends. The records should be readily available to the attending veterinarian acting on behalf of the IACUC. Veterinary oversight is essential to the process for two reasons. First, laboratory-animal veterinarians are trained specialists in the recognition and management of animal health problems, whether spontaneous or iatrogenic. Second, regulatory responsibility for providing appropriate veterinary care rests with the veterinarian. Record keeping. Good record keeping is essential. Records should be written as soon as practical after the animal observation is conducted and should not be phrased with excessive jargon or abbreviations. They should be dated and signed by the record keeper. Observations should be clearly understood by all persons who may have reason to read the records. This documentation serves at least four purposes: It facilitates detection of gradual changes in health that might not be obvious during a single observation period. A change in condition (such as weight loss) can sometimes be more informative than the condition at any given time. It requires an advance decision regarding the characteristics that will be assessed and the frequency of monitoring. Completing a form or checklist designed for a particular study promotes diligence and consistency. It becomes an archive that can be used to improve future study design and animal management. It documents that appropriate monitoring and maintenance activities were conducted. All personnel who use animals should be trained to recognize health problems in their animals. That requires knowledge of the appearance and behavior of normal and abnormal animals and a solid understanding of what conditions are acceptable and unacceptable. Animals should be observed initially in an undisturbed state in their home cage. Making such observations can be difficult in some modern high-density caging systems, but attempts should nonetheless be made to evaluate the animals for general activity levels, posture, the condition of the hair coat, signs of self-induced trauma, pattern of respiration, and the general condition of the cage. Next, the animals should be examined, especially if it is suspected that they have problems. The frequency of individual examination depends on the nature of the debility or disease and the expected rate of progression. For example, for general rodent-colony health surveillance, evaluations should probably be done only on a scheduled basis, such as during a cage change. This limits the number of times that the cages are opened as opening rodent cages properly can be time-consuming and is not risk-free, particularly in light of the possibility of subclinical infectious disease. Characteristics that can be assessed through manipulation are the response to handling; tremors, seizures, vocalization; ulceration; masses; injury; abnormalities of the eyes, ears, nose, or mouth; hyperthermia or hypothermia; and general body condition. Body-condition assessment in rodents requires

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that personnel learn to palpate the vertebral column to look for emaciation (Ullman-Cullere and Foltz, 1999). Body-condition scoring can be superior to simply weighing animals (especially rodents) because it minimizes the potential for the spread of disease through a shared scale, a reference weight is not needed to calculate a percentage of weight loss for assessment of health status, and body condition can be evaluated more rapidly than body weight. Neuroscience preparations can cause various degrees of debility that may be predictable in both severity and duration. Sedating the animals at critical post-procedural intervals may prevent discomfort and even inadvertent injury. If debility is unexpectedly severe or prolonged, the PI and the attending veterinarian must intervene to ensure the animal’s welfare. For example, a necessary intervention for animals that are not drinking is fluid replacement to prevent dehydration. Similarly, an anorectic animal may be encouraged to eat by being provided easy access to soft, rather than hard, food or a highly palatable food rather than the standard diet. Appropriate scheduling of procedures that are potentially debilitating, painful, or stressful is important. It may be challenging to provide adequate veterinary care at night, on the weekend, or over holidays because of a shortage of trained staff, closure of diagnostic laboratories, or an inability to obtain specific drugs or equipment. For this reason, it is recommended that researchers schedule experimental procedures that may necessitate supportive or interventional care so that the time during which the animals would be expected to experience distress falls during normal operating hours. Detection, assessment, and alleviation of pain and distress are additional critical aspects of animal monitoring. Both pharmacologic and nonpharmacologic interventions can be used to alleviate pain and distress. The research and animal-care staff must ensure that instances of animal pain and distress are reported promptly to a veterinarian. Research personnel and animal-care staff must be trained to recognize signs of pain and distress in the species they care for or use. In summary, appropriate monitoring of animals and maintenance of clinical and experimental records are essential for maximizing the well-being of experimental animals in neuroscience research. Training of personnel and good communication among the research personnel, animal-care staff, and the veterinarian are key components of success. ANIMAL HUSBANDRY AND NURSING CARE Many animal models used in neuroscience research demand exceptional attention to daily care. Induction of neural injury and disease may compromise animals’ basic coping and survival mechanisms, as well as their ability to eat, drink, and defecate. Communication, coordination, and creativity in implementation of basic nursing support by the research team and veterinarian are necessary for successful outcomes in these challenging animal models.

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The research team should describe the model to the IACUC in the animal-use protocol and should review the approved protocol with the animal-care and veterinary team before beginning the study. The process should include an overview of the scientific benefits that could be achieved from the study and a frank discussion of the challenges involved in maintaining the comfort of the animals after development of a deficit. This front-end investment will go a long way toward creating a team approach to maintaining what are, in effect, intensive-care patients. The clear delineation of responsibility for monitoring animals is fundamental in ensuring adequate postprocedure care. The Guide’s general recommendation for daily observation may be inadequate in many cases. Ideally, frequent observation and the opportunity for intervention constitute a team effort involving both the research group and the animal-care and veterinary staffs. Clearly defined and well-understood scientific goals allow informed intervention (as opposed to inaction) by the caregivers to manage the animals optimally without compromising research goals. A planned strategy for undertaking defined nursing interventions benefits both the animals and the research. The basics of animal husbandry that are so readily provided in modern housing systems—bedding, food and water, waste-handling—may require extensive modifications or personnel intervention for animals with impaired nervous system function. Enlisting the animal-care group early to consider strategies that will meet basic needs and maximize well-being presents an opportunity to build a team approach. Generally, recovery from neurosurgery involves the same considerations as recovery from other surgical procedures. Cranial surgery is typically well tolerated by laboratory species. Postoperative analgesia should be used whenever it would not compromise scientific goals. Moistening of chow or providing a diet of softer or more palatable foods for several days postoperatively may make eating more comfortable for the animal and promote food intake, but nutritional modifications are often unnecessary. It may be necessary to consider the use of specialized or modified caging for animals with implanted devices, for example, it may be necessary to remove hanging food bins from rodent cages and place the food on the floor of the cage as hanging food bins could potentially damage a cranial implant. Special considerations with respect to social housing may be warranted for animals that have had devices implanted for neuroscience research. Animals recovering from such surgery should generally be housed individually during recovery. If damage to implanted devices by a cagemate is unlikely, most animals can then be gradually reintroduced to social housing after their behavior returns to normal. Some neuroscientists study animal models of human disease. Thus, some surgery is intended to alter the normal physiologic functions of the animal systematically and can affect the psychologic or behavioral state of the animal during postsurgical recovery.

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Such procedures include those that reduce a subject’s ability to interact socially or with the environment. Examples are procedures that result in impairment of sensory perception, limit an animal’s movement capacities, and impair cognitive abilities. After those procedures, appropriate accommodations should be made in an animal’s housing environment or access to enrichment devices to maximize the extent to which it can interact socially and with the environment. Such accommodations can include housing the animal in a social group where it will not be subject to aggressive attacks, giving it manipulanda that can be used with a particular sensory or motor deficit, and giving increased personal attention to an animal that can no longer be put in social housing (NRC, 1998). On occasion, changes in standard husbandry practices are warranted by the scientific goals of an experiment. For example, cats may be reared in total darkness to determine the influence of visual experience on the development of the visual system (Lein and Shatz, 2000; Mower and Christen, 1985) or animals with lesions of the labyrinth may be housed in the dark to prevent visual compensation for altered vestibular cues (Fetter et al., 1988; Zennou-Azogui et al., 1996). In each of those types of neuroscience research, the animal protocol must ensure appropriate care and monitoring of the animal while maintaining the environmental requirements of the experiment; for example, food and water might be provided in the same locations before and after the lesion is produced. Care of animals used in neuroscience or behavioral research often requires creativity and exceptions to an institution’s normal husbandry procedures. For example, the research team often provides all or much of the daily care of animals used in behavioral studies because of protocol-specific issues or special housing situations. If husbandry responsibilities (including cleaning and sanitization) are to be shared by the animal-care staff and the research staff, the role of each group must be clearly delineated and the care must be documented and freely available to both parties. Integrated husbandry responsibility can work well but only when all members of the team know and accept their roles. The IACUC is authorized to approve exceptions to standard husbandry practices that deviate from the Guide’s recommendations when the exceptions have a sound justification and appropriate performance standards are met. SPECIAL ENVIRONMENTS AND ENCLOSURES AND HOUSING OF MULTIPLE SPECIES Experimental designs for neuroscience or behavioral studies may involve the use of special environments, including periodic or chronic housing of animals in unusual, nontraditional settings; for example, animals may be reared in total darkness or exposed to omnidirectional sound, microgravity or hypergravity, hyperbaric, or magnetism-free environments. The need to use a special environment may require housing multiple species in close proximity. The Guide recommends “physical separation of animals by species to prevent interspecies disease transmission

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and to eliminate anxiety and possible physiologic and behavioral changes due to interspecies conflict” (p. 58). However, the well-defined health status of most research animals allows the risk of interspecies disease transmission to be reasonably assessed. The possibility of interspecies physiologic and behavioral stressors must also be evaluated. Occasionally, those stressors are an integral part of an experimental design. The veterinarian and IACUC should carefully evaluate such factors and work with the investigator to develop reasonable compromises that allow a balance between animal welfare and research objectives. Neuroscience or behavioral research may also require the use of nontraditional primary enclosures or caging. Special configurations may allow less space than the standard minimal recommendations in the Guide. The Guide (p. 25) encourages the use of professional judgment and performance outcomes in assessing space needs for animals with special research needs. It is important that deviations from the Guide’s space recommendations be evaluated continuously, not just approved prospectively. SURGERY AND PROCEDURES Frequently, surgical procedures are required to meet the scientific needs of neuroscience research, and it is the responsibility of PIs, veterinarians, and IACUCs to ensure that the procedures are designed and conducted in a manner that complies with applicable animal-welfare guidelines and regulations. Interpreting the guidelines and regulations and applying them to a specific neuroscience procedure can be complicated, and it is important for all concerned to be cognizant of the relevant guidelines and regulations. The Guide states that: In general, surgical procedures are categorized as major or minor and in the laboratory setting can be further divided into survival and nonsurvival. Major survival surgery penetrates and exposes a body cavity or produces substantial impairment of physical or physiologic functions (such as laparotomy, thoracotomy, craniotomy, joint replacement, and limb amputation). Minor survival surgery does not expose a body cavity and causes little or no physical impairment (such as wound suturing; peripheral-vessel cannulation; such routine farm-animal procedures as castration, dehorning, and repair of prolapses; and most procedures routinely done on an “outpatient” basis in veterinary clinical practice) [pp. 61–62]. Minor procedures are often performed under less-stringent conditions than major procedures but still require aseptic technique and instruments and appropriate anesthesia. Although laparoscopic procedures are often performed on an “outpatient” basis, appropriate aseptic technique is necessary if a body cavity is penetrated [p. 62]. The definition of a major operative procedure in the AWRs is almost identical with that in the Guide except that it refers to permanent, rather than substan-

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tial, impairment of functions (AWR 1.1). Both the Guide and the AWRs offer additional language related specifically to the conduct of survival surgical procedures—those in which the animal is allowed to awaken from surgical anesthesia. The Guide provides detailed recommendations regarding facility requirements for survival surgery (pp. 62–63, 78–79) and also states: In general, unless an exception is specifically justified as an essential component of the research protocol and approved by the IACUC, nonrodent aseptic surgery should be conducted only in facilities intended for that purpose [p. 62]. The relative susceptibility of rodents to surgical infection has been debated; available data suggest that subclinical infections can cause adverse physiologic and behavioral responses (Beamer, 1972; Bradfield et al., 1992; Cunliffe-Beamer, 1990; Waynforth, 1980, 1987) that can affect both surgical success and research results. Some characteristics of common laboratory-rodent surgery—such as smaller incision sites, fewer personnel in the surgical team, manipulation of multiple animals at one sitting, and briefer procedures—as opposed to surgery in larger species, can make modifications in standard aseptic techniques necessary or desirable (Brown, 1994; Cunliffe-Beamer, 1993). Useful suggestions for dealing with some of the unique challenges of rodent surgery have been published (Cunliffe-Beamer, 1983, 1993) [p. 63]. The AWRs stipulate: Activities that involve surgery include appropriate provision for pre-operative and post-operative care of the animals in accordance with established veterinary medical and nursing practices. All survival surgery will be performed using aseptic procedures, including surgical gloves, masks, sterile instruments, and aseptic techniques. Major operative procedures on non-rodents will be conducted only in facilities intended for that purpose which shall be operated and maintained under aseptic conditions. Non-major operative procedures and all surgery on rodents do not require a dedicated facility, but must be performed using aseptic procedures. Operative procedures conducted at field sites need not be performed in dedicated facilities, but must be performed using aseptic procedures [AWR 2.31 (d)(1)(ix)]. When preparing animal-use protocols for neuroscience experiments that require surgical procedures, PIs must take care to describe all aspects of their proposed perioperative procedures accurately and completely. In reviewing the protocols, veterinarians and IACUCs must ensure that proposed surgical procedures are properly classified as major or minor, survival or nonsurvival, and rodent or nonrodent. Furthermore, veterinarians and IACUCs must ensure that the surgical procedures are performed in a manner that complies with the Guide and the AWRs; for example, major nonrodent survival surgery should be conducted in a dedicated surgical suite. Although that sounds relatively straightforward, the complexities of contemporary neuroscience research demand that professional judgment, guided by outcome or performance-based considerations (NRC, 1996, p. 3) be used at each

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step of the process. Both the PHS Policy and the AWRs permit a great deal of flexibility in their application to research by allowing the IACUC to grant exceptions to their recommendations when acceptable justification is provided. Thus, the PHS Policy states: The IACUC shall confirm that the research project will be conducted in accordance with the Animal Welfare Act insofar as it applies to the research project, and that the research project is consistent with the Guide unless acceptable justification for a departure is presented [Policy IV.C.1]. The AWRs state: In order to approve proposed activities or proposed significant changes in ongoing activities, the IACUC shall conduct a review of those components of the activities related to the care and use of animals and determine that the proposed activities are in accordance with this subchapter unless acceptable justification for a departure is presented in writing; [AWR 2.31 (d)(1)]. A common exception to the AWRs and to the PHS Policy surgical requirements that IACUCs allow is to permit major surgery to be performed in a modified laboratory setting when necessary equipment (such as electrophysiologic recording equipment) cannot be moved to a dedicated surgical suite (see section on “Asepsis and Physical Environment,” below). One area of confusion for IACUCs, veterinarians, and researchers alike is the definition of what actually constitutes a major surgery. Neuroscience research often involves procedures that do not meet the strict definitions of major survival surgery given in the Guide and AWRs. Some procedures do not involve both penetration and exposure of a body cavity (for example, endoscopic surgery), or they do not penetrate or expose a body cavity at all (for example intravenous infusion or injection of neuroactive or neurotoxic substances, closed-head trauma, or peripheral neurectomy). Determining whether such procedures meet the definitions of major survival surgery hinges on whether they seem likely to produce “substantial impairment of physical or physiological functions” (NRC, 1996, pp. 11-12, 61) or “permanent impairment of physical or physiological functions” (AWR 1.1). The IACUC must assess whether a proposed minimally invasive procedure seems likely to result in an impairment of physical or physiologic functions that is substantial or permanent. If so, the procedure must be categorized as a major surgical procedure and reviewed as such by the IACUC to ensure compliance with the provisions of the Guide and the AWRs. However, both the Guide and the AWRs expect the IACUC to exercise professional judgment in applying their criteria to the review of surgical protocols. For example, the Guide does not define what constitutes a “substantial impairment of physical or physiologic functions,” and does not require that an induced impairment be permanent to be considered major surgery. The AWRs stipulate that a noninvasive procedure should result in a “permanent” impairment to be classified as major surgery, but

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they do not require the impairment to be substantial. Because minimally invasive procedures like those mentioned above can result in impairments whose severity ranges from no apparent loss of function to obvious major functional deficits or impairments whose severity changes markedly (either lessening or worsening) over time, IACUC review of these kinds of protocols can be challenging. Rather than debate the extent to which a particular neuroscience procedure meets the various regulatory criteria for a major surgical procedure, the authors of this report strongly recommend that PIs, veterinarians, and IACUCs collaborate on the development of animal-use protocols that are designed to safeguard animal welfare and address the scientific needs of the research. The Guide and the AWRs provide for sufficient flexibility in the application of their standards for major surgical procedures to allow those involved to craft such protocols. Careful attention should be given to the outcomes of earlier neuroscience studies that used the same or similar procedures. In the absence of precedents for a particular minimally invasive procedure, consideration should be given to obtaining preliminary data from a pilot study performed under direct veterinary supervision and with appropriate reporting to the IACUC. Multiple Major Survival Surgeries In general, multiple major survival surgeries are discouraged, but they may be conducted if they are scientifically justified, related components of a research project approved by the IACUC (NRC, 1996). For example, animals that receive a unilateral visual cortex lesion neglect visual stimuli presented to them on the side opposite the lesion (contralateral visual neglect). Subsequent lesioning of the tectum can ameliorate this neglect (Sprague, 1966). A physical or chemical lesion of the basal ganglia causes a Parkinson’s-like tremor in animals that can be reduced or eliminated by a second lesion (Bergman et al., 1990; Wichmann and DeLong, 1996) or by stereotaxic implantation of a stimulating device (Benazzouz et al., 1993, 1996; Boraud et al., 1996). The use of cranial implants for experimental restraint, recording chambers, or implanted monitoring devices is another situation where multiple survival surgeries may be justified. Some neuroscience-research designs involving multiple surgeries and procedures may have special requirements for asepsis and facilities that will be discussed later. Careful monitoring of the animal, in consultation with the veterinary staff, is necessary. Techniques should be continually refined to minimize pain and/or distress and the monitoring program should be appropriately matched to the anticipated level of pain and/or distress. Multiple major survival surgeries may also be used to conserve scarce animal resources or if the multiple major surgeries are needed for clinical reasons (NRC, 1996). When a research project involves species covered by the AWA, a waiver must be obtained in writing from USDA for multiple major surgeries that are not related components of a research project (APHIS/AC Policy 14). The

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related anorexia. Second, the consumption of food associated with water allows animals to consume solutes that will help retain water in the circulation, correct volume deficits, and avoid excessive hemodilution that will cause urinary excretion of the ingested water (Toth and Gardiner, 2000). Other Influences on Fluid Homeostasis In some situations, fluid reinforcers (such as fruit juice) are used because they may maintain behavioral performance when access to fluid is restricted; for example, some monkeys prefer fruit juice when performing long behavioral sessions in which many reinforcements are delivered (NIH, 2002). Investigators, veterinary personnel, and IACUCs should consider and monitor for any potential physiologic ramifications of total substitution of solute-containing fluids for water in a fluid-restricted protocol. Sweetened milk or juices may be unfavorable choices for use in a long-term study in which an animal will participate for many months or years, because of the potential for dental caries (NIH, 2002). Provision of treats, such as fruits or vegetables, is recommended when appropriate to provide variety and nutritional balance to an experimental animal’s diet (NRC, 1996). The water content of these dietary supplements can be difficult to estimate, so their potential contribution to hydration should not be considered in determining the minimal ration of fluids to be given to the animal (see Pennington et al., 1998, for data on water content of fruit and vegetable supplements). However, investigators, veterinary personnel, and caretakers should be aware of the potential need for restriction or substitution of supplemental food items in fluid-regulated animals. Variability Between Individuals When presented with the homeostatic challenge of dehydration, animals can respond by conserving water and excreting concentrated urine (physiologic regulators) and/or by drinking more fluid and excreting dilute urine (behavioral regulators) (Kanter, 1953; Toth and Gardiner, 2000). Animals on fluid restriction or scheduling protocols may implement different compensatory mechanisms to different extents. Animals that are physiologic regulators may be problematic when used in behavioral studies, because they often tend to accommodate to the consumption of a minimal volume of fluid by excreting more concentrated urine instead of consuming more fluid (Toth and Gardiner, 2000). In contrast, the behavioral regulator tends to modify its behavior during the experimental task to obtain more fluid as a reward. In both instances, nonhuman primates often supplement fluid consumption by licking water from cages after washing. Therefore, the assessment of each animal on a fluid-regulation protocol is prudent.

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Training Considerations The difficulty of the behavioral task that an animal must learn and the goals of the experiment often influence the degree to which the animal must be motivated to perform the task and thus the degree of fluid regulation that is necessary. When possible, palatable rewards rather than regulation should be used to motivate behavior. However, if fluid regulation is determined to be the preferred method of motivating a particular behavior, consultation with veterinary personnel and a review of recent literature regarding animal training may be appropriate (NIH, 2002). In training of a naïve subject to perform a new task with a fluid regulation, gradual introduction to the concept that fluid availability is restricted or context-dependent (for example, earned while in the experimental apparatus) is important (Toth and Gardiner, 2000). After the animal has experienced the absence of ad libitum fluid, its motivation to learn or perform tasks to earn fluid usually increases. The degree of restriction may require periodic adjustment to generate adequate motivation to learn or perform difficult phases of a task (Toth and Gardiner, 2000). However, the restriction often may be reduced after the animal learns the task and becomes proficient at it. As noted in Methods and Welfare Considerations in Behavioral Research with Animals (NIH, 2002): When the study begins, be prepared to consider and address a range of behavioral, environmental, or equipment-related variables that might hinder training or disrupt performance. Inexperienced personnel may presume that a source of problems in training or maintaining a food- or fluid-motivated behavior is that the restriction is not strict enough (or, in some cases, that it is too strict). The other types of variables that should be considered first, however, are equipment malfunctions, programming errors, task criteria that are raised rapidly or set too high for the animal’s level of training, illness, or nonprogrammed water restriction (in the case of food-motivated behavior). Furthermore, experimental animals, like humans, may have deficits, such as myopia, that impair performance on tasks because of perceptual limitations. “In all circumstances, careful monitoring of animals under food or fluid control is necessary every day to avoid additional nonprogrammed restriction” (NIH, 2002). Assessment of Animal Subjects as Individuals The previous paragraphs emphasize that animals on food or fluid regulation schedules are individuals whose performance is likely to vary from day to day. Variations between individual animals in performance on a given task are also expected. The differences between individuals and even within an individual during different phases of an experiment may occasionally necessitate some adjustment of food or fluid scheduling to maintain homeostatic balance and achieve the desired experimental goals. The frequency of observations should therefore

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be adjusted according to how fast an animal can be compromised in the experimental situation. Diligent record keeping on daily food or fluid volume consumed, hydration status, appearance, general affect, experimental performance, and routine weighing are reliable for identifying changes in behavior patterns. Those records should be reviewed regularly and kept readily accessible to the veterinary staff and others who may have a need to evaluate them, such as the IACUC during its semiannual inspections. The need for intervention or reassessment of the hydration needs of an experimental animal can thus be recognized before adverse physiologic consequences develop. Methods of Assessing Nutrition and Hydration A system of daily monitoring procedures is essential for animals that are food or fluid regulated. Records should be kept of the amount of food or fluid earned in the behavior task as well as any supplements given. Careful observation of the animal’s behavior and regular clinical monitoring of the animal’s health are critical to ensuring successful application of food or fluid regulation (NIH, 2002). Clinical monitoring should include assessments of the nutritional and hydration state of experimental animals whose access to food or fluid is regulated. There are various methods for assessing a food- or water-regulated animal and reliance on a single variable is discouraged. Instead, investigators, caretakers, and veterinary staff should use several methods concurrently to ensure the health and well-being of the animals. Variables that can be monitored to assess the nutritional or hydration status of experimental subjects include the following. Weight and food intake Experimental animals on food or fluid regulation should be weighed several times a week, ideally before experimental sessions (NIH, 2002). Some accommodations in the frequency of weighing may be necessary if experimental animals require sedation or anesthesia to be weighed (NIH, 2002). Often, animals can be trained to cooperate with the procedures. Despite conditioning, however, the process of weighing may be very stressful to some experimental animals. In such cases, an animal can be weighed less frequently, and other reliable methods of hydration monitoring can be used. Aside from daily fluctuations in weight due to fluid gain and loss, animals on fluid regulation may lose weight as a result of decreased food ingestion. Using a percentage weight-loss criterion during fluid deprivation as an endpoint for determining when an animal should be removed from a fluid restriction paradigm and their fluid requirements reassessed can indicate not only a proper level of motivation, but also health (Bolles, 1975). The amount of food consumed by experimental animals is a good measure of general health and of hydration status and should be monitored by caretakers or investigators, or

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both. Persistent decreases in food consumption should be brought to the attention of appropriate veterinary personnel. Skin turgor The texture and elasticity of skin are important indicators of an animal’s hydration status. Ordinarily, dehydration will cause a slow return of the skin to its normal position after it has been lifted. However, that characteristic is less reliable in obese animals because their skin tends to maintain its elasticity even in the presence of dehydration (Kirk and Bistner, 1985). Solid and fluid waste output and moistness of feces As part of the long-term adaptation to fluid restriction, healthy animals produce concentrated urine and feces that are less moist than normal (Toth and Gardiner, 2000). Regular observation of the quantity and qualities of the excrement produced by an animal on a fluid or food regulation provides information about both hydration status and physiologic compensation for fluid regulation. General appearance and demeanor and quality of fur and skin Investigators and veterinary personnel share the responsibility for observing behavior, general appearance, and demeanor of experimental animals, which can be valuable indicators of their health status. For example, dry mucus membranes and sunken eyes are indications of dehydration (Aiello, 1998a). If signs indicate that an animal is developing problems related to dehydration, hemoglobin content or hematocrit and blood urea nitrogen can be measured to determine its physiologic status. A plan of action, complete with endpoints for therapeutic intervention, should be considered when the experimental animal protocol is being developed. The plan should include standard operating procedures to be used if an animal develops diarrhea or vomiting that would prompt the return to an ad libitum fluid or food schedule and application of a schedule for veterinary evaluation to prevent serious health consequences due to dehydration or malnutrition. GENETICALLY MODIFIED ANIMALS Genetically modified animals have induced mutations that are human-made alterations in their genetic code. The generic phrase genetically modified includes both transgenic and targeted mutations that are created to study the expression, overexpression, or underexpression of a specific gene (ARENA-OLAW, 2002). A transgenic animal has genes from another organism or species incorporated into its genome, whereas an animal with a targeted mutation has had the coding sequence of a gene in its own genome altered. For a genetic modification to be useful in research animals, the introduced or altered gene must be transmitted to

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the offspring. Most induced mutations have been made in laboratory strains of mice (Mus) or rats (Rattus). Although mice are used as examples in the following discussion, the general considerations are applicable to induced mutants of any species (ARENA-OLAW, 2002). Genetically modified animals are used to test hypotheses in several ways: the phenotype of the modified animal is evaluated to determine the pathogenesis of disease or gene influences on development, the modified animal is used to test interventions to treat its condition, or the animal is used as a tool to study the pathogenesis of other conditions. Transgenic Animals A transgenic animal has exogenous (foreign) deoxyribonucleic acid (DNA) inserted into its cells. Typically, transgenic animals are created by the ”pronucleus method,” in which complimentary deoxyribonucleic acid (cDNA) made from specific messenger ribonucleic acid (mRNA) is inserted into cells by using microinjection, electroporation, or nonpathogenic viruses. Each of those methods has been used to insert new DNA into the pronucleus of a fertilized mouse egg to create viable transgenic mice. The manipulated fertilized eggs may be cultured in vitro for several days before they are surgically implanted into the oviducts or uterus of pseudopregnant female mice. The successful production of a transgenic animal will be affected by several events: the inserted DNA will incorporate into the chromosomes of only a percentage of the embryos developing from the microinjected eggs; the DNA will incorporate at different genetic locations; and different numbers of copies of the DNA will incorporate in different embryos. Therefore each embryo has the potential to become a unique transgenic mouse even though the same quantity and type of DNA was injected into genetically identical fertilized eggs. Not all manipulated, fertilized eggs become live-born transgenic mice. Losses occur at every step from injection through gestation and delivery (ARENA-OLAW, 2002). Although an individual mouse may carry transgenes, it cannot transmit the transgene to its offspring, unless the cDNA incorporates into germ cells. A “founder” is a mouse that passes the transgene to its descendants. Thus, many fertilized eggs must be microinjected to obtain a few transgenic mice, and only a few of the transgenic mice will be founders of a particular transgenic line (ARENA-OLAW, 2002). Knockout and Knockin Mutants: Animals with Targeted Mutations Targeted mutation refers to a process whereby a specific gene is made non-functional (“knockout”) or, less frequently, made functional (“knockin”). A mouse with a targeted disruption, or knockout, of a specific gene is typically created through the embryonic-stem-cell method. This arduous method requires

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the occurrence of several low-probability events. First, the gene in question must be identified, targeted, and marked precisely. This has been accomplished for an astounding number of murine genes during the last several years (Harris and Ford, 2000; Takahashi et al., 1994). Second, mouse embryonic stem cells must be harvested and cultured. Third, a mutated form of the gene of interest is created (the mutation, or altered order of nucleotides, renders the gene inactive). Fourth, the mutated gene is introduced into the cultured stem cells by using microinjection or electroporation transfection (Tonegawa, 1994); a very small number of the altered genes will be incorporated into the DNA of the stem cells through recombination (Sedivy and Sharp, 1989). Fifth, the mutated embryonic stem cells are inserted into otherwise normal mouse embryos (blastocysts), which are then implanted into a surrogate mother (Boggs, 1990; Le Mouellic et al., 1990; Steeghs et al., 1995). All the descendant cells from the mutated stem cells will have the altered gene; the descendants of the original blastocyst cells will have normal genes. Thus, the newborn animals will have some cells that possess only a copy of the mutant gene and some cells that only possess the normal (wild-type) gene. This type of animal is called a chimera. If the mutated stem cells are incorporated into the germ line (the cells destined to become sperm or ova), some of the gametes will contain the mutant gene. If the chimera is bred with wild-type mice, some of the offspring will be heterozygous for the mutation (possess one copy of the mutant gene). If the heterozygous mice are interbred, about one-fourth of their offspring will be homozygous for the mutation. The homozygous mice become the founders and can be interbred to produce pure lines of mice with the gene of interest “knocked out” (Galli-Taliadoros et al., 1995). As a result, the product that the gene typically encodes will be missing from the progeny (Sedivy and Sharp, 1989). For technical reasons, most of the stem cells used in targeted-gene deletion studies were derived from mice of the 129/SV strain. The 129/SV stem cells were typically implanted into C57BL/6 blastocysts (Soriano, 1995). The resulting “mixed” offspring are often backcrossed to the C57BL/6 (background) strain. After 10 backcrosses, the mutated strain is considered a congenic strain, identical with the C57BL/6 background strain except at the site of the altered gene. There are several important advantages of using knockout mice: (1) disabling a gene often results in a precise and “clean” ablation, (2) the effects of the gene product can be abolished without the side effects of drugs, and (3) genetic manipulation may be the only way to determine the precise role of the gene product particularly in behavior. The use of new inducible knockouts, in which the timing and placement of the targeted gene disruption can be controlled, will refine and extend the usefulness of genetically modified animals in neuroscience and behavioral research. One drawback in the use of knockout animals is lethal mutations. The products of many genes are essential for normal function, and inactivating a gene may prove lethal because of gross morphologic or physiologic abnormalities. For

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example, knockout mice with targeted disruption of either the parathyroid hormone-related peptide, ß-1 integrin subunit, or ß-glucocerebrosidase genes die in utero or immediately after birth (Karaplis et al., 1994; Stephens et al., 1995). Development of Animal Protocols Involving Genetically Modified Animals Disease Profiling The first step in developing a protocol to produce or use genetically modified animals is to determine the disease profile that any particular animal or group of animals is likely to experience during the course of normal life or as a result of experimental use. Some genetically modified animals are created to develop a disease spontaneously, but others may develop a severe or debilitating disease even if the disease is not the intended outcome. Genetically modified animals are used in a wide array of experimental studies. They can be used in studies of the pathogenesis and therapy of a primary disease, of a concurrent disease or associated clinical problems, or of aging and longevity. However, many of the animals will never be used in experimental studies but rather are maintained as breeders. The PI, IACUC, and veterinarian all need to develop a general health profile of a given strain that is relevant even to nonexperimental animals (breeders and animals intended for but not yet included in a study). The potential adverse effects of the genetic modification itself have to be considered. For an established strain of genetically modified animal, the literature may provide a good description of the expected phenotype and the course of its development. However, the full repertoire of a gene’s effects may not be envisioned, or a gene’s functions may be unknown at the time a knockout or transgenic animal is created. That uncertainty can make the health-related consequences of developing a knockout or transgenic strain difficult to predict. Many modified mice are generated and maintained for purposes of discovery rather than hypothesis-testing. An example of the “discovery” approach is the use of random mutagenesis, which may create animals whose individual phenotypes are theoretically unpredictable. Because some of the new strains of mice may spontaneously develop problems that are painful or debilitating, assessment strategies and endpoints for these animals must be considered before their generation or their experimental use. Such information is typically solicited by the IACUC as part of the animal-use protocol evaluation. Animal-Number Estimates When submitting an animal-use protocol to develop a genetically modified animal, neuroscientists must include an estimate of the number of animals to be

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used (not including experimental manipulations). Determining the number can be challenging because the process to develop a genetically modified animal is subject to unpredictable outcomes. A detailed presentation of a method for estimating the number of animals needed to develop a genetically modified founder mouse can be found in Appendix B. After founder mice have been identified, 80–100 mice may be needed to maintain and characterize a line. That assumes that up to five breeder pairs per line are needed, that there is no unusual infertility, and that adequate numbers of weanlings are produced for genotypic and phenotypic characterization (ARENA-OLAW, 2002) Appendix B also contains extensive information on calculating animal estimates for colony breeding and experimental use. Breeding of a congenic strain by using “speed congenics” requires a significant number of animals. Speed congenics is the process by which the DNA of each mutated animal is screened to select animals with the most genetic similarity to the background strain; this reduces the number of back-crosses necessary to develop the congenic strain. Usually, at least 750 mice are required, assuming a breeding colony of 10–12 breeding pairs and adequate progeny for phenotypic and genotypic characterization. If the homozygous mutant is infertile, the congenic strain must be developed by using intercross matings, and the number of mice needed is about 1,200 (ARENA-OLAW, 2002). Because development and maintenance of genetically modified animal colonies require large numbers of animals, animals may be produced that are determined not to be useful for a particular project. Those animals may be useful for another project and should either be transferred to that project or culled from the colony. Animal-Welfare Considerations The debility that genetically modified animals may experience is a cause of concern. It is important to provide as much support and comfort for mutant animals as possible. Some strains may require specific husbandry interventions to enable or promote well-being. For example, mice with targeted deletion of the gene for neuronal nitric oxide synthase (NOS-1 -/-) develop defects that model the clinical idiopathic voiding disorders that can affect to 10–15% of men and women. These mice have hypertrophic dilated bladders, dysfunctional urinary outlets, and increased urinary frequency (Burnett et al., 1997). They require extra bedding and more frequent cage changes than wild-type mice. Other examples of special husbandry interventions are those prone to audiogenic seizures, which must be housed in quiet environments, and those with ataxia or paralysis, which may require special provisions to enable access to food and water. Close scrutiny of genetically modified animals during routine daily observation by the animal-care personnel may be warranted. Animal-care personnel often discover disabilities and abnormalities in genetically modified animals (such

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as motor deficits or anorexia) and should be trained to recognize them. Additional training of the animal-care staff to include practical information on the special needs and common problems associated with specific strains is recommended. The dramatic growth in the use of genetically modified rodents, primarily mice, creates substantial challenges for timely and effective assessment of animal health and well-being. Many institutions house large populations of genetically modified mice with a wide array of deficits that affect physiologic homeostasis and behavior. The popularity of high-density, individually ventilated caging systems for housing these valuable mice adds barriers and challenges for effective observation and increases the importance of a careful and systematic examination of individual animals during scheduled cage-maintenance activities. General Health Assessment The general health of novel genetically modified animals should be assessed soon after their availability and before the onset of complex behavioral analyses (Crawley, 1999). Identifying potential health problems early is critical to providing appropriate care. Undetected health problems can also skew the assessment of more complex behaviors—such as learning and memory, aggression, mating, and parenting, so it is essential to identify problems before behavioral phenotyping (see “Behavioral Screening of Genetically Modified Animals” in Chapter 9). For mice, a general health assessment starts with a brief evaluation of body mass, core body temperature, and appearance of the pelage (fur). Neurologic reflexes should be assessed, including the righting reflex, the eye blink, and the ear and whisker twitch in response to tactile stimuli (Crawley, 1999). Any of the following symptoms should be recorded, treated if necessary, and considered when behavioral phenotyping is later conducted: self-mutilation, guarding, vocalization (with or without stimuli associated with pain), hunched posture, inactivity, lethargy, rough hair coat, no response to mild stimuli, increased heart or respiratory rate, anorexia for longer than 24 hours, weight loss greater than 20%, decrease in weight gain compared with aged-matched controls, and lesions (such as swelling, redness, and abnormal discharges). Any obvious deviations from the typical naturally occurring behaviors (ethogram) of mice should be noted. The mouse ethogram includes such behaviors as sleeping, resting, locomotion, grooming, ingestion of food and water, nest-building, exploration, foraging, and fear, anxiety, and defensive behavior (Brown et al., 2000). After an initial health assessment, daily observation of the genetically modified animal should include an assessment of general activity levels, posture, haircoat condition, the presence of scratching or self-mutilation, and the general condition of the cage. When the cage is manipulated, as during cleaning, animals can be more closely examined for additional characteristics, such as response to handling; unexpected vocalization; ulceration; masses; abnormalities of the eyes,

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ears, nose, and mouth; palpable hyperthermia or hypothermia; and general body condition. Behavioral Assessment Subsequent to the general health assessment, sensory and motor testing should be carried out followed by behavioral testing (e.g., anxiety behaviors). Behavioral assessment should proceed as soon as sufficient numbers of transgenic animals are available to identify sensory, motor, or motivational deficits that may compromise the well-being of the animals. Behavioral screening is discussed at length in Chapter 9, “Behavioral Screening of Genetically Modified Animals.” Pain, Distress, and Endpoints The elimination of all pain and distress from all affected animals is unlikely, inasmuch as the diseases being modeled in genetically modified animals are often associated with pain or distress that cannot be relieved in human patients. Achieving a balance between animal well-being and research objectives is essential to obtaining valid answers to questions about the causes, treatment, and preventions of such diseases in humans. When a neuroscientist initiates assessment of a new genetically modified animal, information about clinical abnormalities associated with the phenotype and special husbandry requirements usually are not available. The investigator must, however, include general humane endpoints in case a severe debilitating phenotype develops and should provide the IACUC with this information in writing when the new mutant has been developed or at the next annual review of the animal-use protocol. When a genetically modified animal strain has been characterized, the standard of “normal” for a mutant animal may or may not be the same as that for a nonmutant animal (ARENA-OLAW, 2002); therefore, clinical signs that would be used as humane endpoints in normal animals may be inappropriate as endpoints in genetically modified animals. If the mutant phenotype does not affect the general welfare or clinical well-being of an animal, the same standard of “normal” may be used for mutant and nonmutant animals. In the case of mutants whose phenotype involves clinical abnormalities, the standard for “normal” may have to be modified to include the expected phenotype. For example, 8-month-old mice lacking the gene for a key enzyme that encodes ganglioside biosynthesis (GM2/GD2 synthase) develop substantial neuropathologies, motor incoordination and an abnormal gait (Chiavegatto et al., 2000). As these mice age, muscular weakness progresses, and the standard of “normal” for GM2/GD2 synthase knockout mice includes difficulties in locomotion, which in a nongenetically modified animal might be one criterion of a humane endpoint.

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Humane endpoints for mutant animals should be established on the basis of the ability of the mutant to access and consume food and water, the response of the mutant to stimuli, and the general condition of the mutant (for example, it is excessively underweight, it shows progressive weight loss, it doesn’t groom, it has a hunched posture, or it has sensorimotor deficits). The specific use of a genetically modified animal will influence the type of endpoint that is described in the animal-use protocol and the circumstances in which an endpoint decision will be implemented. For example, an animal that develops a clinical problem while in a study of the prevention of disease development could potentially be euthanized earlier than one involved in a study of disease therapy. A nonexperimental animal (a breeder or an animal intended for but not yet part of a study) that develops a substantial clinical problem should be euthanized. A maximum holding period should be set to avoid the development of predictable problems in strains of mice that have debilitating phenotypes. Endpoint issues generally apply to the entire life of genetically modified animals. Therefore, endpoints become relevant both in the context of experimental procedures and with regard to the potential pain or distress that is caused by the genetic modification itself. Care must be taken to provide general endpoints in the animal-use protocol for the period in which the initial colony is being developed and the phenotype of the animals is first characterized, as well as for experimental and nonexperimental animals.