Preparation of animals for use in the laboratory: issues and challenges for the Institutional Animal Care and Use Committee (IACUC)

Preparation of animals is important for optimization of animal welfare as well as to minimize interanimal variation, thereby strengthening the quality of data for in vivo studies. These issues are important in the work of institutional animal care and use committees (IACUCs), but they pose many challenges as well. This article provides IACUC members with a resource for use in determining whether and how preparation of animals for research affects the IACUC's responsibilities. The topics addressed are intended to serve as a starting point for consideration and discussion. Questions related to subject selection and acclimation of subjects to experimental housing and procedures are emphasized and should provide IACUC members with a framework for discussion of relevant questions. Guidelines are provided for promoting the acclimation of a number of species to experimental settings. Additional, potentially controversial points are also raised, including the effects on longitudinal data sets of changing subject preparation procedures. The roles of the IACUC in the research endeavor are multifaceted and continuously evolving. As empirical data are produced that affect additional aspects of animal care and use, it is important for these committees to be able to evaluate and, when appropriate, stimulate the implementation of improved procedures and strategies.     

Selection, acclimation, training, and preparation of dogs for the research setting

Dogs have made and will continue to make valuable contributions as animal models in biomedical research. A comprehensive approach from time of breeding through completion of in-life usage is necessary to ensure that high-quality dog models are used in studies. This approach ensures good care and minimizes the impact of interanimal variability on experimental results. Guidance related to choosing and developing high-quality laboratory dogs and managing canine research colonies is provided in this article. Ensuring that dogs are healthy, well adapted, and cooperative involves good communication between vendors, veterinarians, care staff, and researchers to develop appropriate dog husbandry programs. These programs are designed to minimize animal stress and distress from the postweaning period through the transfer and acclimation period within the research facility. Canine socialization and training programs provided by skilled personnel, together with comprehensive veterinary health programs, can further enhance animal welfare and minimize interanimal and group variability in studies.     

Preparing chimpanzees for laboratory research

The chimpanzee is the only representative of the Great Apes that is extensively involved in biomedical research in primate laboratories. These apes are used as animal models in a variety of studies, including research on infectious disease, parasitic disease, pharmacokinetic studies, neuroscience, cognition, and behavior. Chimpanzees used in biomedical research in the United States reside largely in six specialized research and holding facilities, and most of the research with them is conducted at these sites. Given the relatively small population of chimpanzees and its importance to biomedical research, it is imperative that we carefully manage the care, production, and use of these animals in biomedical research studies. Selection criteria and preparation techniques are reviewed in this article in an effort to begin a discussion on best practices for choosing and handling chimpanzees participating in biomedical research. The use of routine health assessment information is described for subject selection, as are behavioral issues to be considered. Due to the relatively small number of chimpanzees available, issues related to experimental design and multiple uses of chimpanzees are discussed. Practices related to the transportation and acclimation of chimpanzees are described. Finally, behavioral conditioning procedures are discussed, including habituation, desensitization, and positive reinforcement training that have been applied to reduce animal distress and improve the quality of the science being conducted with chimpanzee subjects.     

An hourly variation in zoo visitor interest: measurement and significance for animal welfare research

A methodological difficulty facing welfare research on nonhuman animals in the zoo is the large number of uncontrolled variables due to variation within and between study sites. Zoo visitors act as uncontrolled variables, with number, density, size, and behavior constantly changing. This is worrisome because previous research linked visitor variables to animal behavioral changes indicative of stress. There are implications for research design: Studies not accounting for visitors' effect on animal welfare risk confounding (visitor) variables distorting their findings. Zoos need methods to measure and minimize effects of visitor behavior and to ensure that there are no hidden variables in research models. This article identifies a previously unreported variable—hourly variation (decrease) in visitor interest—that may impinge on animal welfare and validates a methodology for measuring it. That visitor interest wanes across the course of the day has important implications for animal welfare management; visitor effects on animal welfare are likely to occur, or intensify, during the morning or in earlier visits when visitor interest is greatest. This article discusses this issue and possible solutions to reduce visitor effects on animal well-being.     

An Hourly Variation in Zoo Visitor Interest: Measurement and Significance for Animal Welfare Research

A methodological difficulty facing welfare research on nonhuman animals in the zoo is the large number of uncontrolled variables due to variation within and between study sites. Zoo visitors act as uncontrolled variables, with number, density, size, and behavior constantly changing. This is worrisome because previous research linked visitor variables to animal behavioral changes indicative of stress. There are implications for research design: Studies not accounting for visitors' effect on animal welfare risk confounding (visitor) variables distorting their findings. Zoos need methods to measure and minimize effects of visitor behavior and to ensure that there are no hidden variables in research models. This article identifies a previously unreported variable—hourly variation (decrease) in visitor interest—that may impinge on animal welfare and validates a methodology for measuring it. That visitor interest wanes across the course of the day has important implications for animal welfare management; visitor effects on animal welfare are likely to occur, or intensify, during the morning or in earlier visits when visitor interest is greatest. This article discusses this issue and possible solutions to reduce visitor effects on animal well-being.     

The Use of Positive Reinforcement Training Techniques to Enhance the Care,Management, and Welfare of Primates in the Laboratory

Handled frequently and subjected to a wide range of medical procedures that may be particularly invasive, nonhuman animals in a laboratory setting have unique needs. To produce the most reliable research results and to protect and enhance the well-being of the animals, it is desirable to perform these procedures with as little stress for the animals as possible. Positive reinforcement training can use targeted activities and procedures to achieve the voluntary cooperation of nonhuman primates. The benefits of such work include diminished stress on the animals, enhanced flexibility and reliability in data collection, and a reduction in the use of anesthesia. Training also provides the means to mitigate social problems, aid in introductions, reduce abnormal behavior, enhance enrichment programs, and increase the safety of attending personnel. This article describes the application of operant conditioning techniques to animal management.     

The use of positive reinforcement training techniques to enhance the care, management, and welfare of primates in the laboratory

Handled frequently and subjected to a wide range of medical procedures that may be particularly invasive, nonhuman animals in a laboratory setting have unique needs. To produce the most reliable research results and to protect and enhance the well-being of the animals, it is desirable to perform these procedures with as little stress for the animals as possible. Positive reinforcement training can use targeted activities and procedures to achieve the voluntary cooperation of nonhuman primates. The benefits of such work include diminished stress on the animals, enhanced flexibility and reliability in data collection, and a reduction in the use of anesthesia. Training also provides the means to mitigate social problems, aid in introductions, reduce abnormal behavior, enhance enrichment programs, and increase the safety of attending personnel. This article describes the application of operant conditioning techniques to animal management.     

Environmental enrichment for laboratory rodents

Modernization of housing and husbandry techniques for rodents has minimized confounding variables. The result has been vastly improved health maintenance and reproducibility of research findings, advances that have decreased the numbers of animals needed to attain statistically significant results. Even though not all aspects of rodent manipulation have been strictly defined, as housing and handling procedures have become increasingly standardized, many animal care personnel have recognized the lack of complexity of the rodents' environment. Concern for this aspect of animal well-being has led many research facilities to provide "environmental enrichment" for rodents. Additionally, regulatory agencies in the United States and Europe have also been increasingly concerned about this issue relative to laboratory animal husbandry. However, little is known about the influence such husbandry modifications may have on biological parameters. In this article, laws and guidelines relating to rodent enrichment are reviewed, the natural behaviors of select rodent species are discussed, and an overview of widely used types of enrichment in laboratory rodent management is provided. The literature evaluating effects of rodent enrichment is reviewed both in terms of neurological development and as an experimental variable, and results of a study evaluating the effect of enrichment on immune and physiological parameters are reported. Survey data on current enrichment practices in a large multi-institutional organization are presented, and practical aspects requiring consideration when devising a rodent enrichment program are discussed.     

High temperature acclimation alters the emersion behavior in the crab Neohelice granulata

An increase in environmental temperature can deleteriously affect organisms. This study investigated whether the semiterrestrial estuarine crab Neohelice granulata uses emersion behavior as a resource to avoid thermal stress and survive higher aquatic temperatures. We also examined whether this behavior is modulated by exposure to high temperature; whether, during the period of emersion, the animal loses heat from the carapace to the medium; and whether this behavior is altered by the temperature at which the animal has been acclimated. The lethal temperature for 50% of the population (LT₅₀) was determined through 96-h mortality curves in animals acclimated at 20 °C and 30 °C. The behavioral profile of N. granulata during thermal stress was based on monitoring crab movement in aerial, intermediary, and aquatic zones. Acclimation at a higher temperature and the possibility of emersion increased the thermotolerance of the crabs and the synergistic effect of acclimation temperature. The possibility of leaving the hot water further increased the resistance of these animals to thermal stress. We observed that when the crab was subjected to thermal stress conditions, it spent more time in the aerial environment, unlike under control conditions. Under the experimental conditions, it made small incursions into the aquatic environment and stayed in the aerial environment for a longer time in order to cool its body temperature. The animals acclimated at 20 °C and placed into water at 35 °C remained in the aerial zone. The animals acclimated and maintained at 30 °C (control) that were placed in water at 35 °C with the possibility of emerging into hot air transited more frequently between the aquatic and aerial zones than did the animals that were put in water at 35 °C with the possibility of emerging into a cooler air environment. We conclude that emergence behavior allows N. granulata to survive high temperatures and that this behavior is influenced by acclimation temperature.     

IACUC issues associated with amphibian research

Numerous species of amphibians are frequently utilized as animal models in biomedical research. Despite their relatively common occurrence as laboratory animals, the regulatory guidelines that institutional animal care and use committees (IACUCs) must employ provide little in the way of written standards for ectothermic animals. Yet, as vertebrates, laboratory amphibians are covered by the National Research Council Guide for the Care and Use of Laboratory Animals and the Public Health Service (PHS) Policy for federally funded research. This article focuses on three issues that are relevant to IACUC oversight of the use of amphibians in research: (1) recommended educational requirements of investigators and animal care staff engaged in research with amphibians, (2) zoonoses and other issues of occupational health importance, and (3) indicators of stress and disease. Addressing these issues should enable investigators, IACUCs, and animal care staff to meet the regulatory expectations of the PHS and accrediting bodies such as the Association for Assessment and Accreditation of Laboratory Animal Care International.     

Use of biophotonic imaging as a training aid for administration of substances in laboratory rodents

Dosing of experimental animals and the removal of blood are two of the most frequent procedures performed in biomedical research using live animals. Despite the apparently simple nature of these procedures, they can, if not correctly carried out, have significant effects on the welfare of the animals and the scientific value of the results. There are several methods by which research staff may obtain training in the administration of substances. These include practical demonstrations during teaching courses; observation of techniques; videos and educational computer programs and practising on recently killed animal cadavers or plastic animal models. Each method has its own advantages and disadvantages. A common factor encountered during training is the difficulty in assessing competency. This paper reports a pilot study on the use of bioluminescent imaging technology to assess competency in the administration of substances to rodents. Bioluminescence was rapidly detected after dosing of animals with a bioluminescent substance. However, living animals were required for a signal to be generated. The data presented suggest that this technology is ideal for use as a teaching aid and may also prove valuable in assessing the effectiveness of 'complex' and novel administration routes in 'realtime'.     

Anesthesia and other considerations for in vivo imaging of small animals

The use of small animal imaging is increasing in biomedical research thanks to its ability to localize altered biochemical and physiological processes in the living animal and to follow these processes longitudinally and noninvasively. In contrast to human studies, however, imaging of small animals generally requires anesthesia, and anesthetic agents can have unintended effects on animal physiology that may confound the results of the imaging studies. In addition, repeated anesthesia, animal preparation for imaging, exposure to ionizing radiation, and the administration of contrast agents may affect the processes under study. We discuss this interplay of factors for small animal imaging in the context of four common imaging modalities for small animals: positron emission tomography (PET) and single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging. We discuss animal preparation for imaging, including choice of animal strain and gender, the role of fasting and diet, and the circadian cycle. We review common anesthesias used in small animal imaging, such as pentobarbital, ketamine/xylazine, and isoflurane, and describe techniques for monitoring the respiration and circulation of anesthetized animals that are being imaged as well as developments for imaging conscious animals. We present current imaging literature exemplifying how anesthesia and animal handling can influence the biodistribution of PET tracers. Finally, we discuss how longitudinal imaging studies may affect animals due to repeated injections of radioactivity or other substrates and the general effect of stress on the animals. In conclusion, there are many animal handling issues to consider when designing an imaging experiment. Reproducible experimental conditions require clear, consistent reporting, in the study design and throughout the experiment, of the animal strain and gender, fasting, anesthesia, and how often individual animals were imaged.     

Use of Transgenic Systems to Investigate Oligodendrocyte Differentiation and Function

Transgenic techniques are generating new strains of animals that are of great importance for many neurological research projects. This includes new animal models of human diseases that should allow analysis of disease etiology and treatment. The insertion of new genetic material into the mouse genome enables the investigator to study the effects of overexpression of normal or mutated genes under a variety of experimental conditions. The use of cell-specific and/or developmentally regulated promoters permits studies on the expression of the specific DNA in selected cells within the nervous system at important developmental stages. This article focuses on the techniques for generating transgenic mice, noting specific advantages or problems that should be considered when designing a transgenic project. The use of reporter genes such as the LacZ gene is discussed, using the particular example of the myelin proteolipid protein promoter directing expression of the LacZ gene in differentiating oligodendrocytes.     

水产动物行为及其在渔业中的应用研究进展

水产动物的行为研究是渔业领域的重要研究方向, 对理解水产动物的行为表型与功能, 对提升渔业生产效果有着重要的理论和应用价值。现阶段水产动物行为学的研究成果已被广泛应用于水产动物人工养殖、渔业资源保护和捕捞渔业等多个领域。文章综述了水产动物行为的研究现状, 水产动物主要的行为类型, 每种行为的发生及其机理, 行为间的相互影响及行为在渔业中的应用状况。同时, 针对当前水产动物行为学研究存在的问题, 提出了今后水产动物行为学的研究方向和研究重点。文章旨在为水产动物行为学领域的研究提供借鉴与启发, 并为水产动物行为学研究成果在渔业中的应用提供理论参考。     

Breeding for robustness: the role of cortisol

Robustness in farm animals was defined by Knap as 'the ability to combine a high production potential with resilience to stressors, allowing for unproblematic expression of a high production potential in a wide variety of environmental conditions'. The importance of robustness-related traits in breeding objectives is progressively increasing towards the production of animals with a high production level in a wide range of climatic conditions and production systems, together with a high level of animal welfare. Current strategies to increase robustness include selection for 'functional traits', such as skeletal and cardiovascular integrity, disease resistance and mortality in various stages. It is also possible to use global evaluation of sensitivity to the environment (e.g. reaction norm analysis or canalization), but these techniques are difficult to implement in practice. The hypothalamic-pituitary-adrenocortical (HPA) axis is the most important stress-responsive neuroendocrine system. Cortisol (or corticosterone) released by the adrenal cortices exerts a large range of effects on metabolism, the immune system, inflammatory processes and brain function, for example. Large individual variations have been described in the HPA axis activity with important physiopathological consequences. In terms of animal production, higher cortisol levels have negative effects on growth rate and feed efficiency and increase the fat/lean ratio of carcasses. On the contrary, cortisol has positive effects on traits related to robustness and adaptation. For instance, newborn survival was shown to be directly related to plasma cortisol levels at birth, resistance to bacteria and parasites are increased in animals selected for a higher HPA axis response to stress, and tolerance to heat stress is better in those animals that are able to mount a strong stress response. Intense selection for lean tissue growth during the last decades has concomitantly reduced cortisol production, which may be responsible for the negative effects of selection on piglet survival. One strategy to improve robustness is to select animals with higher HPA axis activity. Several sources of genetic polymorphism have been described in the HPA axis. Hormone production by the adrenal cortices under stimulation by adrenocorticotropin hormone is a major source of individual differences. Several candidate genes have been identified by genomic studies and are currently under investigation. Bioavailability of hormones as well as receptor and post-receptor mechanisms are also subject to individual variation. Integration of these different sources of genetic variability will allow the development of a model for marker-assisted selection to improve animal robustness without negative side effects on production traits.     

The effects of the stress response on immune function in invertebrates: An evolutionary perspective on an ancient connection

This article is part of a Special Issue "Neuroendocrine-Immune Axis in Health and Disease." Stress-induced changes in immune function occur in animals across phyla, and these effects are usually immunosuppressive. The function of this immunomodulation remains elusive; however, the existence of specialized receptors on immune cells suggests that it is adaptive. A comparative approach may provide a useful perspective. Although invertebrates have simpler endocrine/neuroendocrine systems and immune systems than vertebrates, they have robust stress responses that include the release of stress hormones/neurohormones. Stress hormones modify immune function in mollusks, insects, and crustaceans. As in vertebrates, the effects of stress hormones/neurohormones on invertebrate immune function are complex, and are not always immunosuppressive. They are context-, stressor-, time- and concentration-dependent. Stress hormone effects on invertebrate immune function may help to re-align resources during fight-or-flight behavior. The data are consistent with the hypothesis that stress hormones induce a reconfiguration of networks at molecular, cellular and physiological levels that allow the animal to maintain optimal immunity as the internal environment changes. This reconfiguration enhances some immune functions while suppressing others. Knowing the molecular details of these shifts will be critical for understanding the adaptive function of stress hormones on immune function.     

Habituation and desensitization as methods for reducing fearful behavior in singly housed rhesus macaques

Operant conditioning using positive reinforcement techniques has been used extensively in the management of nonhuman primates in both zoological and laboratory settings. This research project was intended to test the usefulness of counter-conditioning techniques in reducing the fear-responses of singly housed male rhesus macaques living in the laboratory environment. A total of 18 male rhesus macaques (Macaca mulatta) were selected for this project and randomly assigned to one of three groups: a desensitization training group, a husbandry training group, or a control group. Behavioral data were collected before and after a 6 weeks training and/or habituation period during which the first two groups received a total of 125 min of positive reinforcement training (and also were assumed to undergo habituation to the environment) and the control group experienced only simple habituation to the environment. Based on a Wilcoxon Matched-Pairs Sign Test, we found that a significant proportion of animals exposed to desensitization training showed a reduction in the rate at which they engaged in cringing toward humans (exact significance=0.016, one-tailed, N-ties=6), cringing in general (exact significance=0.016, one-tailed, N-ties=6), and in stress-related behaviors (exact significance=0.016, one-tailed, N-ties=6). This was not the case for animals exposed to basic husbandry training or animals in the control group. A significant proportion of desensitization-exposed animals also showed a reduction in the duration of time spent cringing toward humans (exact significance=0.016, one-tailed, N-ties=6), but not in cringing behaviors in general or in stress-related behaviors. There were not a significant proportion of animals in either the husbandry training group or the control group that showed a decrease in duration of these behaviors. Results of this study could enhance both laboratory animal welfare and laboratory animal research, and could be a first step in developing techniques for reducing fearful behavior in rhesus monkeys in the laboratory environment.     

土壤跳虫适应低温环境的策略与机制研究进展

低温和干燥是极地及寒温带地区动物的主要环境胁迫因子.为适应恶劣的环境,此区域的动物必须运用有效的方式来适应其栖息环境.土壤跳虫是典型的以避免体液结冰为主要策略来适应低温环境的动物类群,其抗低温的生态策略和模式与昆虫存在相似之处,表现在其利用冷驯化及与干旱协同作用,降低冰晶形成的伤害.在抗低温过程中,其体内甘油等小分子物质含量迅速增加,海藻糖、葡萄糖等分子迅速降低,低温保护蛋白--抗冻蛋白(AFP)产生,并存在脂质的相互转化,提高包膜的流动性,以保护细胞免受低温的伤害;同时也存在与其他昆虫不同的特异性策略与模式,主要包括在雪被保护下的垂直迁移行为,以及将冰核剂随蜕皮而排除体外等.这些土壤跳虫特异的抗冻模式与机制表明其低温生物学研究具有一定的科学意义.本文综述了土壤跳虫抵抗低温环境的模式、类型及机制,总结了近几年其生理生态机制研究进展,讨论了土壤跳虫抗低温研究存在的问题,并就全球变化背景下土壤跳虫低温生态研究前景进行了展望.     

Hierarchical modelling of acclimatory processes

The term acclimation has been used with several connotations in the field of acclimatory physiology. An attempt has been made, in this paper, to define precisely the term “acclimation” for effective modelling of acclimatory processes. Acclimation is defined with respect to a specific variable, as cumulative experience gained by the organism when subjected to a step change in the environment. Experimental observations on a large number of variables in animals exposed to sustained stress, show that after initial deviation from the basal value (defined as “growth”), the variables tend to return to basal levels (defined as “decay”). This forms the basis for modelling biological responses in terms of their growth and decay.Hierarchical systems theory as presented by Mesarovic, Macko & Takahara (1970) facilitates modelling of complex and partially characterized systems. This theory, in conjunction with “growth-decay” analysis of biological variables, is used to model temperature regulating system in animals exposed to cold. This approach appears to be applicable at all levels of biological organization. Regulation of hormonal activity which forms a part of the temperature regulating system, and the relationship of the latter with the “energy” system of the animal of which it forms a part, are also effectively modelled by this approach. It is believed that this systematic approach would eliminate much of the current circular thinking in the area of acclimatory physiology.     

The impact of huts on physiological stress: a refinement in post-transport housing of male guineapigs (Cavia porcellus)

The ideal animal model would contribute no confounding variables in experimental science. Variables affect experimental design resulting in increased animal use or repeated studies. We demonstrated a simple refinement which may reduce the number of animals used experimentally while simultaneously improving animal welfare. The objective of this study was to determine if the presence of a hut was an impact on physiological stress levels, as determined by faecal cortisol concentration, during a routine four-day acclimatization period of newly received male Hartley-Outbred guineapigs. We hypothesized that those animals provided with huts would have decreased physiological stress compared with animals not provided with huts. We examined this effect within both paired and single-housed animals. A between-subjects one-way analysis of variance revealed that pair-housed animals with a hut had significantly lower faecal cortisol concentration than pair-housed animals without a hut and the presence and absence of a hut had no significant impact on faecal cortisol concentration in single-housed animals. These findings show that presence of a hut is beneficial in reducing physiological stress when pair housing male guineapigs and does not appear to have an impact when single housing male guineapigs. In addition, we have shown that faecal cortisol, and therefore physiological stress, is still increasing on study day 4 suggesting a longer acclimatization period is necessary. A simple refinement in housing environment and acclimatization time can both reduce the number of animals used experimentally and improve animal welfare.