Preparation of Animals for Research—Issues to Consider for Rodents and Rabbits Laura A. Conour, Kathleen A. Murray, and Marilyn J. Brown Abstract Key Words: acclimation; environment; nonexperimental variable; rabbit; refinement; rodent; stress; welfare Introduction A central theme in designing research projects is to minimize nonexperimental variables. Many experiences in a research animal’s lifetime can be expected to cause some degree of stress, such as birth, weaning, transport, husbandry practices, environmental changes, and experimental procedures. Stress may be defined as “the biological responses an animal exhibits in an attempt to cope with threats to its homeostasis” (Carstens and Moberg 2000, p. 65). Responses may be physiological and/or behavioral. These responses to stress are a natural coping mechanism. When this natural coping mechanism fails, distress noted as clinically relevant physiological and behavioral changes may be observed. It has been proposed that an environment Laura A. Conour, D.V.M. DACLAM, is Director, Veterinary Services, Transgenic Services, at Charles River Laboratories (Charles River), Wilmington, MA. Kathleen A. Murray, D.V.M., M.S., DACLAM, is Senior Director, Animal Program Management, Charles River, Wilmington, MA. Marilyn J. Brown, D.V.M., M.S., DACLAM, Dipl. ECLAM, is Executive Director, Animal Welfare and Training, Charles River Laboratories, East Thetford, VT. Address inquiries and reprint requests to Dr. Conour, 251 Ballardvale Street, Wilmington, MA 01887, or email Laura.Conour@crl.com. Volume 47, Number 4 2006 Acquisition of Animals: Planning Considerations When preparing an animal shipment, order, or request, a number of considerations require planning, policies, and 283 Downloaded from http://ilarjournal.oxfordjournals.org/ at CINVESTAV on February 28, 2013 This article provides details to consider when preparing to use animals in biomedical research. The stress of transport and receipt of animals into a new environment mandate the need for a period of stabilization and acclimation. This allotment of time often occurs in conjunction with the quarantine period and permits a stress “recovery” period. Discussions in the article include specific effects of the environment on the animal, such as housing and environmental enrichment. Suggestions are offered regarding how to minimize the effects of procedures and equipment through the use of preconditioning techniques. Guidelines for these techniques and for acclimation should be instituted by the institutional animal care and use committee. Stress and distress are placed in perspective as they relate to the preparation of laboratory animals for research. without any stress can actually be detrimental to an animal (Claassen 1994). Establishing a stress-free environment is both unrealistic and may not be in the animal’s best interest. Minimizing stress and enhancing the animal’s ability to cope with stresses decrease the potential for distress and safeguard both animal welfare and research results. Preparing animals for research is one way scientists can minimize animal stress. Preparation most commonly includes quarantine, acclimation, and stabilization. Other preparations specific to the animal model or research goals might include surgery, disease induction, and training to perform on experimental equipment. Quarantine for the protection of colony health is a well-described and recognized concept. From an animal welfare perspective, acclimation and stabilization are important to prevent overwhelming the animal’s normal coping mechanisms, which maintain homeostasis and minimize animal distress. The need to allow animals to stabilize and acclimatize to new surroundings and procedures before collecting research data is intuitive. A number of studies have shown that activities and environments (e.g., transportation, social or single housing, cage housing density, environmental enrichment, and handling) can affect both physiological and behavioral parameters (Tables 1 and 2). Many of the mechanisms by which such activities and environments affect specific study parameters and animal welfare as well as the duration of such effects have not yet been identified. Although many studies cited in this article highlight changes in behavioral tests, hematology and serum chemistries, and even morphology, most of these changes do not produce clinically relevant outcomes. Nevertheless, such changes may be important to consider for their potential effect on research results. Awareness of the possible effects of common activities and environments will allow scientists to monitor affected parameters and determine appropriate baselines to account for such influences. Similarly, awareness of such effects will permit inclusion of appropriate control groups exposed to the same activities and environments without the imposition of the study variables, thus allowing more accurate interpretation of study data. Table 1 Factors to consider for evaluation of shipping or transportation stress Table 2 Factors to consider for evaluation of stress related to novelty of housing environment Shipping/transportation stressors References (see text) Housing environment stressors Landi, Kerider et al. 1982 Caging type Total time out of home cage environment Time in transit Mode of transport Exposure to temperature extremes Available food and water Short duration transport (e.g., animal facility to laboratory) procedures to protect current animal populations and provide animals with an environment suitable for acclimation after transport. When investigators or staff place or receive an animal order, the extent of inquiry and approval depends on the source of the animals (i.e., approved vendor vs. collaborative institution). With the increased number of shipments of genetically modified animals from collaborative institutions, it is advisable (and common) to use an import form. We recommend compiling all documents that describe health monitoring and animal husbandry and care programs to expedite processing animal shipments. It is essential to review current and past health reports to determine which pathogens or opportunists are present or have not been tested for, and to be aware of any recent outbreaks that have occurred in the room, barrier, or facility. A description of the health monitoring program should include details of the following critical components: (1) duration and type of sentinel exposure (contact vs. soiled bedding); (2) number of sentinels screened per room or rack; (3) strain of sentinel used; (4) extent to which principal animals are screened; (5) the agents screened; (6) frequency of screening; (7) type of sample screened; and (8) test/assay performed. A complete treatment history for endoparasites, ectoparasites, or other agents (e.g., opportunistic bacteria) of the source colony or specific animals to be shipped is advised. In total, this information permits a quick assessment of an institution’s health monitoring program and provides the requisite information for designing a quarantine plan that will minimize biosecurity risks to the receiving institution’s existing colonies. 284 Bedding type Animal density in cage Change in personnel handling animals Temperature Light/dark cycle (intensity, duration) Noise Water treatment Diet Gordon 2004; Kuhnen 1999; Tuli et al. 1995; Whary et al. 1993 Gordon 2004; Kuhnen 1999; Tuli et al. 1995 Baer 1971; Everett 1984; Plaut and Friedman 1982; Strange et al. 2000; Tuli et al. 1995; Whary et al. 1993 Tuli et al. 1995 Everett 1984 Bellhorn 1980; Furudate et al. 2005; Mrosovsky and Salmon 1987; van Ruiven et al. 1998; Winfree 1987 Nayfield and Besch 1981 Fidler 1977; Hall et al. 1980 Everett 1984; Tou et al. 2004 In addition to health status information, it is important to review details of any experimental manipulations (i.e., one or more surgical procedures) that have been performed on the animals before shipment. This information should include details of the manipulation, date of the technique, any reported complications, and whether further postoperative care (e.g., staple or suture removal) is needed. Other information regarding potential effects on the health of the animals in the shipment (e.g., malocclusion requiring teeth trimming or approximate parturition date of pregnant animals) should be provided and carefully reviewed. It is possible to facilitate the animal’s acclimation to its new environment with additional knowledge of the source colony environmental conditions, including illumination parameters, bedding, feed, water, social housing, enrichment, and husbandry practices. Animals with special dietary requirements should be shipped with the diet packed as the food source. It is advisable to ship extra diet with the animals to ensure the continuation of special feeding. Similarly, any required parenteral treatment should also be shipped at the same time or before the animal shipment. The receiving institution should receive material specifications including vendor/ supplier source sufficiently ahead of time so that critical diets and treatments may be ordered and received before animal receipt. Examples of animals that may have special requirements include inducible transgenic models that are maintained on a medicated diet (e.g., doxycycline or tamoxifen) to maintain a gene in the “on”/“off” position; ILAR Journal Downloaded from http://ilarjournal.oxfordjournals.org/ at CINVESTAV on February 28, 2013 Light/dark cycle (changes, duration, phase shift) Aguila et al. 1988; Landi et al. 1982; van Ruiven et al. 1998 Aguila et al. 1988; Landi et al. 1982; van Ruiven et al. 1998 Baer 1971; Hirvonen et al. 1978; Oratz et al. 1967; Trapani 1966 Mrosovsky and Salmon 1987; van Ruiven et al. 1998; Winfree 1987 Landi et al. 1982 Drozdowicz et al. 1990; Tuli et al. 1995 References (see text) animals with a toothless phenotype that require powdered or liquid diet; and diabetic animals that require insulin. Acquisition of Animals: Shipping Considerations 1 Abbreviations used in this article: IACUC, institutional animal care and use committee; Guide, Guide for the Care and Use of Laboratory Animals; NK, natural killer; PHS, Public Health Service; SD, Sprague-Dawley; SOP, standard operating procedure. Volume 47, Number 4 2006 285 Downloaded from http://ilarjournal.oxfordjournals.org/ at CINVESTAV on February 28, 2013 When considering the shipping arrangements, it is important to balance the potential stress of transport, the risks to biosecurity, the current clinical condition of the animals, and the sensitivities of the particular research model. The shipping process begins with the removal of the animal from its home cage at the originating institution and ends when the animal is unpacked and placed into its new home cage at the receiving institution. Table 1 provides a list of potential stressors to consider as a result of shipment. Landi and colleagues (1982) examined the effects of air transport (24- to 36-hr in duration) and truck transport (36to 48-hr duration) on acute corticosterone plasma levels in CD-1® mice. Mice exposed to both modes of transport showed increased levels of plasma corticosterone for 48 hr after arrival. Additionally, immunosuppression was evident as detected by foot pad test, hemagglutination assay, and plaque forming assay. A return to baseline levels occurred by 48 hr. Aguila and colleagues (1988) also examined the effects of transportation on the immune system of C57BL/ 6J mice transported via air and truck. Splenic natural killer (NK1) cell activity was measured on days 0, 1, 3, and 5 after arrival. Increased plasma corticosterone and decreased NK cell activity were noted on arrival and returned to baseline levels by 24 hr after arrival. Van Ruiven and colleagues (1998) confirmed the Landi et al. findings through evaluation of corticosterone levels, food and water consumption, body weight, and performance in rats tested in an open field activity test over a 3-wk period following transportation by air or car (15-hr duration). Reductions in plasma corticosterone levels, blood glucose, free fatty acids, and blood urea nitrogen were observed 24 hr after transport in conjunction with an increase in total cholesterol. All parameters returned to normal by 3 days following transport and remained normal when screened 3 wk later. While it is important to be aware that the stress of transit may result in physiological changes, it is equally as important to understand that a period of acclimation of 48 to 72 hr is necessary for most values to return to normal ranges. For this reason, we recommend that facilities establish acclimation guidelines that include 2 to 3 days of stabilization before initiation of research studies. Shipment by air may pose an increased risk of exposure to disease. Animals maintained under uncontrolled or conventional conditions could be shipped in nonfiltered crates that are placed in direct proximity to other animals on commercial carriers. Dry intact crate filters protect animals from cross-contamination even if the outside surfaces of the crate become exposed to infectious agents. To minimize biosecurity risks, it is important to carefully inspect for filter integrity and to perform surface disinfection on arrival. One advantage of air travel is that it generally takes less time than ground transport, thereby reducing the duration of the stress event. Both truck and air transport are of equivalent significance with respect to the magnitude of stress induced when transit times are equal (Aguila et al. 1988; Landi et al. 1982). Alternatively, shipment by dedicated truck may decrease risk of infection but often increases the duration of travel. In the absence of dedicated delivery of animals in environmentally controlled vehicles, arrangements such as air shipment do pose a greater risk in exposing animals to a less rigorously controlled environment. The receiving facility must weigh the benefits of reduced transit time against the potential for increased biosecurity risks associated with air transit and develop a plan that considers all factors, including any model specific requirements. It may be possible to prevent exposure of animals to temperature extremes by noting forecasted temperatures along the transport route and by delaying shipping during times of extreme high and low temperatures. Such prevention is important for many species. For example, rats exposed to cold temperature extremes are reported to have experienced increased production of thyroid hormone and mineralocorticoids and increased sensitivities to certain pharmacological agents such as isoprenaline (Baer 1971). Rabbits, guinea pigs, and mice exposed to cold temperature extremes demonstrate decreased production of antibody and increased antibody decay (Trapani 1966). Acute extreme cold exposure in guinea pigs (4ºC) resulted in erosions of the gastric mucosa and depletion of cellular stores of histamine and 5-hydroxytryptamine as well as decreased survival time in response to propranolol or reserpine administration (Hirvonen et al. 1978). Exposure to extreme heat in rabbits resulted in decreased food consumption, body weight, and serum protein metabolism (i.e., albumin and fibrinogen) (Oratz et al. 1967). International shipment across several time zones can result in phase shifts in the light/dark cycle. Hamsters that had experienced an 8-hr shift in the light/dark cycle required 1 wk to adjust to a new light/dark cycle (Mrosovsky and Salmon 1987; Winfree 1987), and mice required a 2-wk acclimation period (van Ruiven et al. 1998). It is important to consider the long-distance transport of animals from a vendor or collaborative institution. Equal consideration should also be given to the transport of animals between animal facilities on campus, between rooms within an animal facility, or from the animal facility to the laboratory. Such “in-house” transport induces stress similar to that of long distance travel. Tuli and colleagues (1995) measured the effects of room-to-room movement on BALB/c mice by evaluating corticosterone levels and behavioral parameters. Plasma corticosterone levels increased Acquisition of Animals: Receipt of Shipment Upon receipt of a shipment of animals, it is essential to reconcile the number of shipping crates with the enclosed invoice and to carefully inspect each crate for any defects or damage before signing transport carrier documents. Additionally, it is advisable to have an established plan to handle damaged crates in advance (Figure 1). If animals were exposed to the external environment during transport, the approach to quarantine or facility access may require alternative arrangements. The damage should be documented via notation or by digital photograph, especially if financial reparations and/or animal replacements are warranted. The method of unpacking animal shipments should be consistent with a method that is documented in a standard operating procedure (SOP1). An important component of unpacking is the provision of adequate disinfection to avoid cross-contamination between shipments of animals whose health status varies. For example, it is possible to schedule shipments from different sources to arrive on different days (Loew 1980). After the quantity, sex, age, weight, and strain/stock received have been confirmed with the order, it is important to note the following: • The source of food and water present within the shipping containers; 286 Figure 1 Damage to shipping container during transport. The barrier between the animal’s microenvironment and the external environment is compromised, exposing the animals to unknown biosecurity risks. • • Whether the animals had free access to food and water and whether they consumed it; Any clinical abnormalities at the time of receipt, which will require implementing treatment plans after diagnosis and scheduling re-examinations. Any animal received in severe distress should be euthanized and a postmortem diagnostic evaluation performed that includes gross and histological evaluation. Any abnormal findings should be reported to the responsible animal health care provider at the originating institution or supplier, preferably before animals are manipulated or euthanized. This immediate action will allow for a more comprehensive investigation to be conducted. To ensure that animals adapt to new living conditions and to monitor their coping response, we recommend that investigators, veterinarians, and/or care staff conduct frequent animal observations within the first 24 to 48 hr of receipt. For example, water consumption should be confirmed by monitoring the animal’s water intake or hydration status. Monitoring is especially important if there has been a change in the treatment of the drinking water or the type of watering device used. Additionally, the animals should be observed for any clinical abnormalities as a result of transit or fighting due to altered cage populations. Preparing Animals for Research Use: Quarantine, Stabilization, and Acclimation Quarantine for health monitoring purposes, stabilization of an animal to a new environment, and acclimation to new procedures are all necessary to prepare an animal for optimal use as a research model. Some of these events may occur concurrently following receipt; however, care must be taken not to overwhelm the animal’s coping mechanisms. ILAR Journal Downloaded from http://ilarjournal.oxfordjournals.org/ at CINVESTAV on February 28, 2013 immediately after movement and returned to baseline within 24 hr. Increased activity was also noted in the form of an increased number of rearing and climbing behaviors and decreased grooming time. These activities decreased dramatically by the second day after movement but were not completely normalized at 4 days. Additional stressors in short-distance transport (i.e., between the animal room and the laboratory) may occur as a result of changes in environmental conditions such as the light/dark cycle, temperature, and relative humidity. Unrestricted local travel of animals may also result in potential biosecurity issues if animals of varying health statuses are housed within the facility. As evidenced above, stress occurs with all modes of transportation. The number of unknown stressors may also vary depending on the mode of transportation. In the case of animal transfers, whether between vehicles or aircraft, holding times and conditions become factors that contribute to transport stress. Likewise, each conveyance introduces a new set of variable environmental conditions (e.g., light, temperature, pressure, vibration, noise, presence of other animals). The inability to quantify the stress that occurs during every step of animal transport does not negate the need to consider these factors when preparing an animal for use in research but instead, provides additional support for the establishment of a standard acclimation/stabilization period to allow mitigation of these effects. As stated above, we recommend a minimum of a 2- to 3-day acclimation period. Quarantine Program Volume 47, Number 4 2006 Stabilization and Acclimation to Environmental Variables Differences in environment and housing practices between the source and the receiving institution may also serve as stressors (Table 2). Whary and colleagues (1993) report that in “rodent stress studies, the results suggest that the most important stress-inducing factor is a sudden change in housing method rather than the method itself” (p. 331). For example, it has been reported that food and water consumption is affected during transfer to a different cage type and requires 3 to 5 days to normalize (Tuli et al. 1995). Differences in environment and housing practices should also be considered as variables that may influence experimental 287 Downloaded from http://ilarjournal.oxfordjournals.org/ at CINVESTAV on February 28, 2013 Many institutions draft pathogen exclusion lists and establish health monitoring protocols for quarantine of newly received animals. This standard commonly applies to the institution as a whole but may vary between animal facilities on site or by areas within a facility. Investigator awareness of these standards and laboratory animal staff involvement in the preliminary stages of animal acquisition may prevent the delay of a study due to incompatibility of the incoming animals’ health status with institutional requirements. The length of quarantine and the extent of the health monitoring protocol often vary according to the source of the animals and the reliability of that source (Loew 1980). For example, many institutions do not perform separate health monitoring on vendors but instead, accept the health monitoring program of the vendor as adequate to determine whether unwanted infectious agents are present in the colony. Alternatively, some institutions periodically perform diagnostic evaluations of samples of animals from commonly used vendor production barriers rather than imposing quarantine on each shipment from that barrier. When defining quarantine parameters, it is important to consider mode of transportation, incubation period of viruses and bacteria, duration of sentinel exposure versus direct screening of received animals, and health history (Loew 1980). When reviewing health documents, investigators and staff should remember that a health report is always retrospective and thus not necessarily indicative of current health status. Each animal’s clinical status and potential disease exposure during transport are related to the exact steps of the animal’s journey, which are often unknown. Receipt of rodents that have been implanted with transplantable tumors warrants additional concern due to the existing potential for viral contamination of the animal by the tumor cells (Loew 1980). Before animal shipment, it is essential to request documentation of viral screening of the tumor cell line or any other biological product administered to the animal. If viral screening of the tumor cell line or biological product was not performed, then consideration should be given to maintaining these animals in an extended quarantine and/or containment housing system, until testing can be done to ensure that these animals do not pose a biosecurity risk to the facility. Depending on the operation of the quarantine area and the established quarantine procedures, the particular type of animal housing may affect quarantine duration as well as risk cross-contaminating other animals within the same quarantine area. When quarantine of multiple shipments occurs within one room, we advise considering caging such as ventilated racks or static microisolators along with implementation of husbandry techniques that will avoid crosscontamination. When a pathogen that was not previously noted on the incoming health reports during quarantine health monitoring is identified, it is imperative to notify the veterinary staff at the originating institution. Conversely, if an outbreak occurs at the originating institution, that institution should alert all facilities that recently received animals. Many institutions also include prophylactic treatments for endo- and ectoparasites as a component of the quarantine process, often without any diagnostic screening results. Such treatments include fenbendazole medicated diet for the treatment of pinworms in rats and mice (Coghlan et al. 1993; Huerkamp et al. 2000) or application of Dichlorvos (Atgard) into rodent bedding for the treatment of fur mites (Csiza and McMartin 1976; Toth et al. 2000; Weisbroth et al. 1976). If it has been decided that diagnostic screening will be performed before prophylactic treatments, it will be necessary to schedule the treatment according to timing that will ensure proper sentinel exposure and/or sample collection before treatment. Failure to schedule appropriately may result in false-negative results. Both international and domestic investigator collaborations continue to increase, resulting in larger numbers of transgenic rodents undergoing quarantine. Some institutions require rederivation via embryo transfer before entry into the animal facility, regardless of incoming health status. In this scenario, shipment of frozen embryos, sperm, or ovaries is a preferable alternative, although these alternatives are not always available. Accessibility to research animals by investigators during quarantine and acclimation varies according to each institution’s standards. However, some manipulated animals (e.g., timed pregnant animals or animals with particular surgical modifications) cannot be subjected to a lengthy quarantine process. Common sense and animal welfare considerations clearly suggest the need for viable options that may include designating a procedural space outside or at the periphery of the animal facility. This course of action permits access to the animals and provides accommodations for performing experimental techniques while preventing exposure of animals of unconfirmed health status to the larger animal population. In these situations, particular attention must be given to the designation of and adherence to SOPs, traffic flow patterns, and controlled access to this area. Acclimation: Preparation for Special Procedures and/or Equipment Acclimation of the animal following transport and exposure to a new housing environment provides a more stabilized animal for research use. However, additional stressors may occur after acclimation in animals that are naive to equipment and experimental techniques such as handling, dosing, and restraint. Numerous studies demonstrate the beneficial effects of frequently handling research animals before initiation of study protocols as well as in early life (Avishai-Eliner et al. 2002; Bale 2005; Chapillon et al. 2002; Lehmann and Feldon 2000; Levine 2005; Meaney et al. 1991; Tuli et al. 1995). Tuli and colleagues (1995) suggest that “Animals habituated to a handler or which are gentled in early life show less handling stress in later life and react only to the particular experimental stimuli used in the study, whereas nonhandled animals are much more likely to react to a new handler as well as to the test situation” (p. 132). Su and coworkers (2004) discuss the concept of “stress preconditioning” in a model of decompression in rabbits. The authors postulate that exposure to stress before an unavoidable stressful event diminishes the negative effect of the secondary stress event and generates a potentially protective effect. This effect is preferable to a significant primary stress event in a naive animal never exposed to any type of stress. This concept has also been referred to as “bioprotection” (Su et al. 2004), or the “bidirectional” effects of stress (Dhabhar 2000). Plaut and Friedman (1982) confirm this theory by demonstrating the effects of stress-inducing experimental techniques, which are protective against subsequent, more significant stress events. The type of special diet, the ingredients, and the diet 288 form (e.g., pelleted vs. powdered, crumbly vs. hard) should be evaluated and may result in additional requirements for the care of the animal. For example, in our experience, regularly scheduled teeth trimming may be necessary with diets that are powdered, liquid, or soft. Additionally, diets high in fat (atherogenic diets) often cause an animal’s coat to become greasy and may delay wound healing whereas diets high in salt (hypertensive diets) increase an animal’s water consumption and require more frequent refilling of the water bottle. Conclusion: Quarantine, Stabilization, and Acclimation Among the many experiences to which an animal must adapt during its research life, some are related to the experiment and others, to the environment. Additionally, the animal’s response and ability to cope are influenced by its species, stage of development, strain/stock, and gender. All of these factors require consideration while identifying which parameters are likely to have an impact on particular research goals and how such an impact may be minimized through acclimation and stabilization. The optimal duration of acclimation for rodents and rabbits is difficult to establish based on reported studies (Table 1). The literature varies regarding the animals studied, environmental conditions, experimental protocols, and parameters measured. These variations compound the difficulty in determining the time the animals need to recover from the various stressors of shipping. The adverse effect of shipping on the animal as a research model mandates the need for an acclimation period (Aguila et al. 1988; Landi et al. 1982; Tuli et al. 1995; van Ruiven et al. 1998). Multiple factors should be considered when evaluating the severity of stress on the animal and the time required for recovery. Many sources (Aguila et al. 1988; Landi et al. 1982; van Ruiven et al. 1998) indicate that a 3-day acclimation period is the minimum length of time required for adaptation based on the most common parameters studied. However, certain research models might necessitate a longer acclimation period depending on research procedures and organ systems or physiological indices studied. The use of an unprepared animal in an experimental protocol has a potentially significant effect on the data collected. Failure to plan for acclimation and stabilization of the animals may result in a need for increased animal numbers to determine statistically significant differences in experimental results and may place collected data at risk for inaccuracy and irreproducibility (Furudate et al. 2005). The negative aspect of this risk also underlines the importance of controls in each study compared with the use of historic controls. The representation of a control group in each experimental protocol makes it possible to identify potential effects due to the presence of unexpected or unplanned stressors. ILAR Journal Downloaded from http://ilarjournal.oxfordjournals.org/ at CINVESTAV on February 28, 2013 data. For example, the presence of phytoestrogens in the diet of rodents may cause a dramatic effect on the reproductive cycle of rodents as well as constitute an epigenetic factor to stress (Tou et al. 2004). The maintenance of body temperature in mice (Gordon 2004) and hamsters (Kuhnen 1999) is affected by cage construction (solid bottom vs. wire bottom) and type of bedding, factors that could influence various toxicology and infectious disease studies due to the animals’ continual need to adapt to changes in the ambient environment. Environmental monitoring is equally important to avoid stressors, possibly as a result of undetected, malfunctioning climate controls. For example, persistent estrus (Furudate et al. 2005), cataracts (Bellhorn 1980), and phototoxic retinal degeneration (Bellhorn 1980) may result from prolonged light exposure (as few as 4 days) in rats. Monitoring of climate controls is important to detect and document malfunctions. Tracking deviations will make it possible to retrospectively investigate possible causes for unexpected research results. Environmental Enrichment and Potential Effect on Experimental Data Behavioral Characteristics of Different Species To minimize the stresses of environment, handling, and experimental procedures in research animals, we believe that it is advisable to begin by looking at a species’ typical behavioral characteristics. Because wide behavioral variations exist among species and strains, such considerations may pose a particular challenge. An understanding of an animal’s normal behavior is the basis for decisions regarding the most appropriate environment. Environmental enVolume 47, Number 4 2006 289 Downloaded from http://ilarjournal.oxfordjournals.org/ at CINVESTAV on February 28, 2013 In the Guide for the Care and Use of Laboratory Animals (Guide1), it is recommended that “Animals should be housed with the goal of maximizing species specific behaviors and minimizing stress-induced behaviors” (NRC 1996, p. 22). Environmental enrichment is a combination of complex inanimate and social stimulation (Rosenzweig et al. 1978). Programs for social housing and environmental enrichment may be met with some resistance from scientists who wish to minimize the effects of ever-elusive nonexperimental variables. However, the stresses associated with limiting an animal’s ability to express basic species-specific behaviors and the subsequent potential effects of that stress on both animal welfare and research results must also be considered. Many articles have addressed the environmental effects that influence the animal’s ability to express speciesspecific behaviors using measurements in the following areas of research: neuroanatomy and functional learning and memory (Wurbel 2001), stereotopies (Wurbel 2001), behavioral profiles (Wolfer et al. 2004), and stress (Sharp et al. 2002). Interpretation of these articles is difficult because there is a great deal of interaction and overlap related to how a given enrichment specifically affects the measurement in question. For example, provision of nesting material gives an animal the ability to hide from potential threats (humans or cage mates), changes thermoregulation, affects exposure to light, and provides for increased activity, thus leading to complex mechanistic and quantitative determinations of the effect of bedding material on the animal. The literature on the effect of environmental enrichment on data variability and animal numbers has been conflicting. Mering and colleagues (2001) found that in rats, some physiological parameters (e.g., weights of adrenal glands, interscapular brown adipose tissue, and epididymal adipose tissue) were significantly different depending on exposure to gnawing blocks, solid bottom cages versus wire grid floors, and population density. The authors concluded that the “variation of different parameters may vary from one experiment to another and between different environments thus hindering the estimations of appropriate numbers of animals” (p. 80). Similarly, Knight (2001) found that well-implemented enrichment may reduce variability. richment considerations should include social housing and other elements that may stimulate normal behavior including nesting materials, gnawing materials, bedding, shelters, and other forms of biologically relevant enrichment. What is biologically relevant to one species or strain may have no effect, or even a negative effect, on another. Plaut and Freidman (1982) discuss “strain effect” at length and conclude that in many instances, “An environment that is maladaptive for one species or strain of animal may not be for another” (p. 278). It is therefore necessary to search any available literature for the specific stock and strain of interest when designing an experiment and considering appropriate enrichment. Although it is beyond the scope of this article to discuss in detail all of the behavioral characteristics of rabbits and different species and strains of rodents, we mention a few of these key traits below. Rodents. Rodents are generally social species; however, some strains are particularly aggressive, depending on gender and age (Smith and Hargaden 2001). Normal mouse behavior usually includes digging, gnawing, investigating, territorial scent marking, climbing, nesting, and foraging (Jennings et al. 1998). Rats exhibit similar behaviors but are less inclined to build nests (Hurst 1999; Kohn and Clifford 2002; Koolhaas 1999). Guinea pigs. Unlike rats and mice, guinea pigs generally do not raise up on their hind legs, are not nest builders, and are poor climbers. Guinea pigs are quite social, docile, and easily frightened. In addition, like most rodents, guinea pigs are thigmotaxic (Harkness et al. 2002; Harper 1976; North 1999). Hamsters. Hamsters are generally regarded as less social than other commonly used rodents. Hamsters have a tendency to fight, to dig when given the opportunity, and, like many other rodents, to build nests (Hankenson and Van Hoosier 2002; Mohr and Heinrich 1987). Rabbits. Rabbits are often considered social animals as well; however, aggression can be a problem, particularly among males (Jenkins 2001). Foraging, exercise, and the use of elevated resting surfaces should be considered for rabbits. When considering environmental enrichment, it is important for the scientist to consider the potential effects on research results in addition to the impact of animal welfare on the enhancement of species-specific behaviors (Bayne 2005). Much has been written about the effects of environmental enrichment on areas such as tumor growth, neurophysiology, neuroanatomy, behavior, learning, and other various physiological parameters. For example, social housing conditions and novelty stress affects differential growth rates of tumors in mice (Kerr et al. 1999). Van Praag and colleagues (2000) extensively reviewed the neural consequences of environmental enrichment, which may serve as a starting reference for scientists interested in this area. The effects of enrichment may be dictated by the model under investigation. In the Morris Water Maze, enrichment (multilevel cages, activity wheel, and plastic and wooden toys) improved spatial memory performance in partially tri- Potential Effects of Housing Social housing produces different effects on various animal research models. In addition, there are degrees of sociality from simple aggregations to animals with highly intertwined and dependent social structures. Sociality also varies within a species due to many factors such as strain, age, and gender. It has been suggested that in some circumstances, individually housed animals do not represent as robust a research model when compared with group-housed animals (two or more per cage) (Baer 1971; Strange et al. 2000). The effects of stress on mice and rats as a result of long-term isolation include decreased food consumption, reduced weight gain, decreased adrenal weights, leucopenia, eosinopenia, lower splenic weights, and increased adrenal and liver weights. Abnormal behavior such as aggression and excitement may occur when singly housed rodents are exposed to a cage cohort in a group housing situation. Additional abnormal behaviors include decreased male mating behavior and decreased maternal care in long-term singly housed mice and rats. Study results are influenced by the strain/stock of rodent used as well as the duration of isolation stress (Baer 1971). The effects of stress as a result of short-distance or short-duration transport have been shown to be diminished by group housing as a standard procedure (Tuli et al. 1995). The effect of social housing on physiological parameters in Sprague-Dawley (SD1) rats housed four per cage showed a weaker and shorter stress response 290 (elevated heart rate and blood pressure) to common husbandry and experimental procedures than SD rats housed singly or in pairs. Measurements returned to baseline within 120 min (Sharp et al. 2002). Although group housing appears to result in a more stable research animal, the effects of social hierarchies may result in decreased testicular weights, increased adrenal cortical function, and greater splenic weights in subordinate male rats. Any change in animal cage densities may result in stress and require stabilization of the animals during an additional acclimation period. Handling rodents housed individually or after changes in animal cage densities may accelerate the acclimation process (Baer 1971). Reports of social housing in rabbits have documented variable results. Whary and colleagues (1993) reported that housing rabbits in small social groups had no effect on commonly measured stress indices and the immune response and resulted in increased exercise, social contact, and exploratory behavior compared with individually housed rabbits. However, there have been other reports of problems with aggression when rabbit social housing was attempted (Love 1991; Swanson and McNitt 2006). Conclusion: Environmental Enrichment and Potential Effect on Experimental Data Despite attempts documented in the literature to isolate the effects of individual environmental variables, an interaction of multiple factors likely occurs and results in biological and behavioral effects on animals. For this reason, we believe that searching the literature for specific environmental treatments for specific species, stock, and strain while considering specific variables of research interest will likely lead only to an approximation of total environmental effects for a given research project. Even when every attempt is made to standardize environments between laboratories, differences in research results have been noted (Crabbe et al. 1999). This outcome again emphasizes the importance of appropriate concurrent controls maintained under the same environmental conditions, subjected to the same manipulative procedures, and handled by the same laboratory and husbandry staff. Institutional Animal Care and Use Committee (IACUC1) Considerations The IACUC is responsible for overseeing and evaluating all aspects of animal care and use within an institution. Two major IACUC functions that enable the IACUC to fulfill their oversight responsibilities are the semiannual evaluation of the institution’s programs and facilities for activities involving animals and the review and approval of all animal use protocols. During the program and facility review, IACUC members review the institution’s policies and SOPs, which should include those involved with animal ILAR Journal Downloaded from http://ilarjournal.oxfordjournals.org/ at CINVESTAV on February 28, 2013 somic (Ts65Dn) female mice and their euploid female littermate controls but had no effect on control male mice and had a deteriorating effect on Ts65Dn male mice (MartinezCue et al. 2002). In an example of strain variation, enrichment (nestbox, plastic tube, tissue and wool nesting material) as measured by the hole board resulted in increased reactivity and alertness, cage emergence, and open field tests in male C57BL/6JIcoU mice compared with controls housed in standard bedded cages, whereas male BALB/cAnCrRyCpbRivU mice exhibited increased levels of anxiety (van de Weerd et al. 1994). In contrast, however, some studies report that particular variables are unchanged when a specific enrichment is added. Compared with rabbits given no devices, rabbits given manipulanda showed no significant differences in body weight, food consumption, or various hematological parameters. Although animals interacted with the device, variation was large and interaction decreased (average = 83%) over the 8-wk observation period indicating the importance of novelty (Johnson et al. 2003). Other physiological parameters and their potential interactions with environmental enrichment include variables such as testosterone and immunoglobulin G (Nevison et al. 1999), plasma triglycerides (Perez et al. 1997), cholesterol (Augustsson et al. 2002), and blood pressure and heart rate (Lawson et al. 2000), to name only a few. It is therefore important that scientists review the literature specific to their field of study. Summary Laboratory animals are exposed to a variety of experiences to which they must adapt starting in the breeding colony and Volume 47, Number 4 2006 lasting until the end of a given experiment. These experiences are both experimental and nonexperimental. The need for awareness and control of nonexperimental variables when designing a research project is a well-recognized and accepted concept. It may be difficult, however, to isolate individual variables to identify the impact on animal welfare and study results. This review has outlined some variables that may have an impact on the laboratory animal. We have identified parameters that have been documented to have an effect on either the animal or the research data. We have suggested an approach that attempts to consider an animal’s coping mechanisms and emphasizes the importance of carefully monitoring the shipping, receipt, quarantine, acclimation, and stabilization of the animal in ways that minimize overwhelming the coping mechanism. During this discussion we have tried to place “stress” in perspective. All stress cannot be eliminated, but every effort should be made to minimize the number of stressors and the severity of the induced stress. We acknowledge that to control every possible nonexperimental variable is an impossible task. Nevertheless, we emphasize the importance of appropriate control groups, which are exposed to the same activities and environments. It is the responsibility of the entire research team (the investigator, veterinarian, IACUC, and animal care staff) to provide an environment that promotes animal well-being balanced with the practice of sound scientific principles. To enable better comparison of experimental data both within and between research institutions, it is critically important to include fully detailed information in the Materials and Methods descriptions pertaining to quarantine, stabilization, acclimation, environmental parameters, and procedures when reporting scientific results in the literature. Acknowledgments The authors thank Ms. Judy Murray for her invaluable administrative assistance in the preparation of this manuscript, Ms. Suzann Ordile for her expertise in grammar and punctuation in an editorial capacity, and Mr. Matthew Bouchard for provision of the digital image. References Aguila HN, Pakes SP, Lai WC, Lu YS. 1988. The effect of transportation stress on splenic natural killer cell activity in C57BL/6J mice. Lab Anim Sci 38:148-151. ARENA [Applied Research Ethics National Association]. 2002. Institutional Animal Care and Use Committee Guidebook. Bethesda: Office for Laboratory Animal Welfare, National Institutes of Health. Augustsson H, Lindberg L, Hoglund AU, Dahlborn K. 2002. Humananimal interactions and animal welfare in conventionally and penhoused rats. Lab Anim 36:271-281. Avishai-Eliner S, Brunson KL, Sandman CA, Baram TZ. 2002. Stressedout, or in (utero)? Trends Neurosci 25:518-524. Baer H. 1971. 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