Incorporating Laboratory Animal Science into Responsible Biomedical Research

Abstract

Biomedical research has made great strides in the past century leading to rapid advances in human life expectancy, all derived from improved understanding, prevention, and treatment of many diseases and conditions. Research involving laboratory animals has played a significant role in this medical progress. However, there continues to be controversy surrounding the use of animals in research, and animal models have been questioned regarding their relevance to human conditions. While research fraud and questionable research practices could potentially contribute to this problem, we argue that a relative ignorance of laboratory animal science has contributed to the “uncontrolled vivarium experiment” that runs parallel to the more controlled scientific experiment. Several variables are discussed, including husbandry, animal environment, social housing, and more, that can contribute to this uncontrolled experiment, and that can simultaneously decrease quality of life for rodent test subjects when ignored. An argument is put forward that laboratory animal veterinarians and scientists can and should play an important role in better controlling such variables. Similarly, the laboratory animal veterinarian and scientist should play an important role in responsible science by addressing complex interdisciplinary challenges.

Animal models, Environment, Husbandry, Laboratory animal veterinarian, Gastrointestinal microbiota, Reproducibility, Mouse, Rat

Introduction

Scientists have made great progress since the beginning of the twentieth century understanding and treating many human diseases and conditions, and laboratory animals have played a prominent role in this success. However, there is ongoing controversy surrounding the use of animals for research, in particular, focusing on apparent poor reproducibility of preclinical research involving laboratory animals.^1,2 Most scientists agree that true research misconduct (fabrication, falsification or plagiarism) is relatively rare or at least uncommon.³ However, there is likely a higher percentage of questionable research practices that do not fit the definition of research misconduct, which contribute to variable and undependable research outcomes.¹ Some practices may relate to experimental design and approach—this paper will address practices pertaining to how animals are cared for, treated, and worked with in research settings.

Animal models of human disease are just that—conditions occurring spontaneously or induced in animals that mimic aspects of specific conditions in humans—sometimes superficially based only on external appearances and time course of progression, sometimes quite similar based on pathogenesis, epidemiology, or molecular marker expression. Animal models have limitations that must be considered and addressed to better interpret research outcomes.⁴ By improving our understanding of research animals and their needs we can better design studies and understand the strengths and limitations of the models worked with. Vivarium- and research animal–related issues that impact model use have been studied for decades by laboratory animal scientists and veterinarians. Some of these areas are only now receiving increased attention from biomedical scientists as they may impact their research results.^5–8 There are numerous papers and book chapters dedicated to specifics surrounding intrinsic and extrinsic factors influencing research animals and the reader is referred to other more detailed reviews for general information.^9,10 Examples of how some of these basic and sometimes poorly considered parameters can impact animal well-being, behavior, physiology, and performance will be discussed in this paper. The discussion will be limited to mice and rats, since use of these rodents is common within in vivo research.

An argument can be made that many preclinical research laboratories are in fact conducting two parallel experiments within every in vivo study. The first involves conducting procedures and collecting data related to their scientific discipline (eg, ECG, blood pressure, enzyme assay, tumor growth, etc.), which are, hopefully well-controlled. The second experiment involves vivarium- or animal-related issues that are either poorly controlled, uncontrolled or not documented. Biomedical scientists and preclinical researchers need to first recognize this “parallel laboratory,” and second, actively recognize and incorporate laboratory animal veterinarians and scientists as an integral part of the research team to adequately consider the impact of animal biology on research outcomes.

By necessity, approved guidelines and legislation for laboratory animal care and husbandry can supply only general information. While many items contained within current laboratory animal guidelines remain relevant, aspects of their content have become dated as new information has become available. Cost, conservatism and concern about historical control data, sociozoologic preference for and societal value of large animal species, and convenience drives much of what occurs in biomedical research, sometimes resulting in a lack of attention to current information in laboratory animal science as it impacts rodent care and well-being (which ultimately impacts validity of research findings). In the following paragraphs, examples of how laboratory animal science can be better incorporated into rodent care and research use will be explored to improve research quality by considering animal environment, behavioral management, pain management, and consideration of the gut microbiota.

Animal Environment and Husbandry

Ambient Temperature

Over the last 40 years, there has been a significant shift from large to small animal species in research, in large part because of the ability to genetically modify research mice (see, for examples^11,12).¹³ The eighth edition of the ILAR Guide for the Care and Use of Laboratory Animals (hereafter referred to as The Guide) introduces some basic thermoregulatory concepts relevant to housing (thermoneutral zone, upper and lower critical temperatures) that impact physiologic stress. However, significant research has been published since the last edition of The Guide evaluating the effect of ambient temperature on research rodent health and physiology. The marked increase in surface area:body weight ratio of mice (eg, 0.15 for a mouse vs. 0.03 for a Beagle dog) means that these animals have the capacity to lose more heat to the environment than some other species, particularly if not provided with conspecifics, and sufficient bedding and nesting material to maintain their body temperatures.¹⁴ This need for thermal adjustments in their husbandry has either not been fully appreciated or has been ignored in many experiments.^14,15 As a result, mice and rats are often housed up to 10°C colder (ie, 20–22°C) than their thermoneutral zone (approximately 30°C).^16,17 This chronic cold stress may lead to elevated metabolism, food intake, oxygen consumption, heart rate, and blood pressure, with abnormal responses to lipopolysaccharide and infectious organisms.^18,19 Chronic mild cold-stress can also depress immune responses and enhance or reduce tumor growth rates, depending on the tumor cell lines and induction protocol used.^20–23 Mice housed at optimal temperatures (ie, within their thermoneutral zone) have significantly smaller internal organs—specifically, hearts, livers, and kidneys, which may dramatically influence research outcomes evaluating cardiovascular and metabolic effects.²³ Ambient housing temperature can have complex interactions with sex-dependent disease models; Giles et al (2017) have demonstrated that female mice are no longer resistant to nonalcoholic fatty liver disease when housed at thermoneutrality.²⁴ Although group housing of rodents and provision of shelters and adequate amounts of bedding and nesting materials may help to reduce cold stress in mice,^25–27 these effects may not completely restore physiology and behavior to that seen when rodents are housed at preferred optimal temperatures.²⁸ Few biomedical research papers describe the type and volume of substrate and nesting material provided to rodents, despite journals and editors agreeing that animal husbandry information is essential when considering experimental reproducibility.^29,30 Ambient temperature in the vivarium microenvironment is a variable that requires further attention.

Relative Humidity

Relative humidity control in the vivarium is considered less important by some than temperature control,³¹ and yet this parameter has a significant effect on animal comfort but varies widely depending on the caging systems used for rodents.³² Modern heating ventilation and cooling systems are capable of providing more tightly controlled relative humidity and yet when it is reported, relative humidity is highly variable.⁸ It has been known for decades that relative humidity can alter the infectivity of viruses,³³ but it also alters drinking behaviors in mice, thereby altering overall water consumption and urine osmolality.³² This in turn can impact drug-in-water consumption and interpretation of metabolic and pharmacokinetic parameters when urine is collected for analysis. This is an area of laboratory animal science that requires closer evaluation.

Lighting and Photoperiod

Illumination in animal facilities has potentially profound effects on the physiology and behavior of a variety of species. As an environmental variable, lighting includes daily duration of exposure to light or photoperiod, wavelength or spectral quality, and intensity. Further, these parameters should be assessed at the animal level to determine actual versus estimated exposures. Of these specific parameters, only duration of daily light exposure (ie, photoperiod) is commonly reported in publications. Albino rats were first used to establish vivaria housing room illumination levels,³⁴ since these animals are more sensitive to retinal photoxicity (for a full review, see De Vera Mudry et al³⁵).³⁶ Traditionally, retinal components are thought of in terms of having a primary role in image generation; however, intrinsically photosensitive retinal ganglion cells (ipRGCs) also play a major role in regulation of circadian rhythms and overall metabolism and functioning of animals.³⁷ The chromophores of the classic photoreceptors and ipRGC’s together determine biological responses to different wavelengths; these spectral peaks vary between these different classes of photosensitive cells and by species.^38,39 Damage to the retina from inappropriate light levels (eg, albino rodents housed on the top shelf of cage racks close to ceiling lights) can alter these biological responses.

Light exposure also influences rodent metabolism, reproduction, and behavior (see Behavior Management section below for a discussion of impact on sleep). Even a small amount of light pollution in the dark phase of rodent light cycles is thought to be sufficient to disrupt their circadian rhythm and metabolism.^40,41 Use of longer wavelengths (eg, red lighting in the dark phase) does not necessarily prevent this disruption, but use of blue shifted LED during the day does help to better entrain circadian rhythms.⁴² Since rodents are a nocturnal species, an obvious suggestion has been to conduct studies during the dark phase when they are more active. However, there is limited data on how such testing improves reproducibility or experimental translation.⁴³ The practical considerations regarding light and mouse husbandry have been recently reviewed.⁴³ A key conclusion from this area of research is that improved descriptions of light variables (eg, photoperiod, wavelength, and intensity) is needed in publications to improve scientific reproducibility.

Noise and Vibration

Noise originating from many possible sources is an ever present variable in animal facilities.⁴⁴ While the intensity of the sound may be reduced through careful facility planning,^45,46 it is unlikely to be eliminated, and noise in animal facilities is a variable that is uncommonly measured or reported. This is particularly troublesome considering the large number of non-auditory changes induced with excessive noise in humans and other animals (eg, increased stress hormones, blood pressure, cardiac hypertrophy, along with decreased body weight and fertility; reviewed elsewhere^47,48). There are significant differences as to which sounds are audible to humans versus other animals,⁴⁹ and even within rodent strains.⁵⁰ Some sounds clearly audible to human caregivers might not be perceived by mice.⁵¹ Conversely, rodents emit and hear vocalizations and other sounds beyond the 20 kHz limit of human hearing.^52,53 Personnel can generate significant levels of noise when working in animal rooms, something that many researcher teams are completely unaware about.⁵⁴ Radios and speakers are commonly employed in vivaria—sometimes for human caregivers, but often in the name of animal welfare, for example, to provide background white noise. This noise can interfere with conspecific animal communication and preference studies in rats demonstrate that the majority prefer silence to artificial sounds.⁵⁵ Finally, there are some sources of noise that are ignored due to pressure level or frequency, such as fluorescent light ballasts and alarms.⁵⁶

Many sources of noise will also generate vibration, such as top of rack mounted blowers on IVC caging. This vibration is easily detected by rodents,⁵¹ and can have a profound effect on reproduction, metabolism, and general stress (decreased litter size, muscle angiogenesis, increased blood pressure, heart rate and stress hormones; reviewed previously⁵⁷). The potential effects of these variables are likely to become exacerbated when institutions attempt to renovate old vivaria and bring in heavy equipment, generators, etc.

Transportation and Post-Transport Acclimation

While animal transportation standards have improved over the past several decades, even short shipping periods (ie, <24 h) can cause significant effects in rodents, impacting sympathetic tone, body weight, food consumption, and other aspects of behavior and metabolism.^58–60 It is important to note that transportation is used as both an acute and chronic inducer of stress in rodent models⁶¹ and due respect and consideration should be given to this process. These effects can occur regardless of the type of caging that animals are placed into upon arrival.⁶² Certain types of research in mice and rats, such as study of hypertension, may require acclimation periods of at least 3–6 weeks following transport before animals regain pre-transport blood pressure levels.⁵⁹ Even basic studies of stress and metabolism in rodents may require 2–4 weeks of acclimation^60,62 for resetting of homeostasis. While longer acclimation periods, such as 30 days, are routinely considered necessary and acceptable for large animal species, such as nonhuman primates,⁶³ similar consideration is not afforded to rats and mice, with potential detrimental effects on research outcomes.

Space, Caging, and Other Resources

Rodents are typically housed within vivaria to maximize density and efficiency while minimizing cost and ergonomic issues. Within the limits of a standard rodent housing room and husbandry provisions, there is minimal evidence that incremental increases in cage sizes beyond current EU levels⁶⁴ provides recognizable advantages to research mice and rats.^65,66 Increases in cage size (while maintaining stocking density ratios) can destabilize social hierarchies and promote aggression in mice.⁶⁶ Similarly, a recent study in rats has suggested that increased cage sizes promote increased territoriality in dominant rats, resulting in increased risk of attacks on subordinates.⁶⁵

Certain types of caging systems can have a profound effect on animal behavior, phenotype, and comfort.^67,68 For example, neither rats nor mice acclimate well to standard metabolism cages^69,70 and this type of housing should be used sparingly and for the shortest periods of time possible. Individually ventilated cages (IVCs) are widely used within research facilities because they permit increased stocking densities within a fixed footprint. Air change rates can approach 60 air changes per minute with some units, resulting in chronic cold stress in mice.^71,72 As discussed, cold stress is an important consideration for rodent comfort and study outcome.

Optimizing the internal cage environment by adding preferred resources can be beneficial to rodent health and well-being, but also may have a profound impact in maintaining animals in an optimal state for research. Rats maintained in pairs or singly housed in a barren cage environment without resources to promote locomotion and other activities lose circadian variation in fecal corticosterone output in as short as 12 weeks, likely because of reduced physical activity.⁷³ Adding cage resources, such as a tube, a small amount of wood wool, and a piece of toasted oat cereal given three times weekly restores the normal circadian pattern of corticosterone secretion⁷⁴ and this is maintained for at least 6 months as long as the resources are present. It is likely that the novelty of these resources is gone within 30 minutes of putting them in the cage but they do contribute to cage complexity, add novel texture, provide something to chew on, act as visual barriers, and represent structures or materials that need to be navigated when moving from one part of the cage to another. This emphasizes that provision of in-cage resources is not really an optional consideration, but a necessity to maintain normal physiologic states in rodent models. As further examples of resources that can be used for rodents, Wheeler et al, demonstrated that access to multi-level caging is beneficial for rats⁷⁵ while partial cage division using cardboard dividers was found to reduce fighting between sexually mature male mice, enhancing tolerance for group living.⁷⁶

Mice and rats also demonstrate strong preferences for types and amounts of bedding substrate,⁷⁷ as this impacts animal comfort and sleep. Although corncob bedding absorbs more ammonia than cellulose bedding,⁷⁸ housing rats and mice on corncob bedding is less preferred and less comfortable than housing on wood sawdust or shavings.⁷⁹ Further, rats housed on corncob bedding show less slow wave sleep patterns, suggesting disrupted sleep. This effect is reversible if rats are moved onto hardwood chip bedding.⁸⁰ As in humans, disrupted sleep patterns in rats can affect their performance on tasks involving learning and memory, and animal comfort should be a key consideration for behavior and learning studies (reviewed by McCoy and Strecker⁸¹). In addition, changes in sleep patterns impact glucose metabolism and hormone physiology (reviewed by Carley and Farabi⁸²), thus those working in endocrine and metabolism research should closely consider animal comfort as it impacts animal physiology.

Many long-term and large-scale studies and reviews have demonstrated that enhancing the environments of laboratory mice and rats with adequate bedding substrate, nesting materials, shelters, and other common and inexpensive resources significantly increases animal comfort and decreases agonistic behaviors without impacting most physiologic variables.^74,83–85 Providing comfortable spaces for rodents to live in is relatively cheap compared to other research costs but is often not sufficiently considered as it relates to animal model validity.

Sanitation Practices.

Bedding and cage changes, as well as room sanitation represent significant stressors for laboratory rodents.⁵ Husbandry practices have evolved over time, but show inter- (and in some cases intra-) institutional variability regarding the length of time that animals are disturbed in addition to the type and magnitude of the disturbance.

The introduction of individually ventilated cage systems with high air changes per cage has reduced the frequency of bedding and cage changes. Reduced cage change frequency might reduce experimental variability, as cage changing results in significant behavioral⁸⁶ and cardiovascular⁸⁷ alterations lasting for hours on the day of cage change. This needs to be weighed against the impact of caging on animal comfort. Similar changes (with sex-based differences in recovery) have been noted in mice,⁸⁸ together with increased aggression noted following cage changes, which can be attenuated by simple husbandry practices, such as transfer of nesting material.⁸⁹ It seems logical to argue that all experimental animal procedures should be avoided on bedding/cage change days (or at least for 2–3 hours following a cage change). However, we suspect that many investigators are unaware of the cage changing day, frequency, or impact on rodent behavior or physiology.

Enhancing Behavioral Management of Rodents

An intimate understanding of the behavioral biology of an animal is essential for respecting its capabilities and ensuring that it is fit for the research being planned. Data collected from animals that are behaving normally is likely more reflective of population responses on which relevant conclusions can be based than data collected from animals with significant stereotypies,⁹⁰ which may be indicative of underlying physiologic or psychologic abnormalities. Significant research has been devoted to understanding the behavioral and psychological well-being of many large animal species used in research, such as nonhuman primates⁹¹ and dogs,⁹² including consideration of personality (reviewed by Freeman and Gosling, and Fratkin, et al)^93,94 and compatibility when socially housing in research facilities. In contrast, minimal attention is given to assessing the behavioral well-being or readiness of rats and mice for biomedical research. For many years, rodents have been known to have stable differences in responsiveness,⁹⁵ and yet this is rarely factored into biomedical research design. Presence of stereotypic behavior has been suggested to be a cause of significant variability in mice⁹⁶ and, again, this is never recorded in the methods section of biomedical publications. While it may not be convenient or timely to evaluate personalities of all rodents individually, some rapid and general surrogate measures of behavioral well-being may be applied, such as evaluating nest building quality or burrowing in mice.^97,98 Similarly, greater consideration of overall animal well-being and preparation for study is likely needed for rats and mice. Rodent habituation and training in preparation for procedures is an area that needs to be better explored, as a number of studies suggest that both rats and mice can be readily trained for various procedures in a short period of time.⁹⁹

Rodents are social species and should be socially housed in research facilities except when justified by research requirements or issues surrounding compatibility. Sufficient cage resources should be provided at the outset of cohousing animals to minimize the later development of agonistic behaviors; however, it may be that some mice, and in particular, male mice of certain strains, are harmed by social housing, which may induce variability in research outcomes.¹⁰⁰ Researchers and institutions need to be flexible when considering how best to house rodents, and ultimately, some strains of male and female mice may need to be housed differently to best meet their needs.

Handling and Restraint

Rodent handling stress is a documented cause of experimental variability.¹⁰¹ Similarly, restraint for various procedures in unhabituated rodents often induces as much stress as the actual procedure.^102,103 The use of low-stress handling techniques for mice and rats has been widely promoted in publications and at meetings and yet is still not broadly taught or practiced within research facilities. For example, tunnel or whole body cupping techniques have been shown to reduce anxiety in mice^104,105 and reduce experimental variation,¹⁰⁶ and this approach should be more widely implemented. A recent study also suggests that rats are less stressed¹⁰⁷ when less restrictive handling methods are used for injections. Rats are also known to respond positively to gentle handling and tickling (reviewed elsewhere¹⁰⁸) and these methods can be used to reduce fearfulness and stress in some study animals, helping to ensure that animals are behaviorally normal during studies. Gentle handling combined with refinement of technical procedures and practices, such as selecting optimal dose volumes, dosing vehicles, and blood collection sites^109,110 are likely to improve animal model translatability and reduce procedure-related stress.

Pain Management

Public acceptability of research involving animals is contingent upon minimizing pain and distress that animals may experience (for an example of survey responses on this topic, see¹¹¹). Similarly, consistent recognition, reporting, and treatment of pain in laboratory rodents remain areas of ethical concern for IACUCs, veterinarians, and researchers (reviewed in^112–114). Unmanaged pain during experimental study influences model relevance and translatability due to variation in animal response.¹¹⁴ It is not always a straightforward matter to recognize pain in a prey species; however, algorithms have been developed to assist researchers in determining when to employ pain medication.¹¹⁴ Pain recognition in rats and mice and clinical management of pain in rodents are areas of active research with recent promising results. For example, a facial grimace scale has recently been validated in rats to permit live time assessment of acute pain based on scoring of several facial action units.¹¹⁵ Nesting material consolidation (in which mice are scored for bringing together pieces of nesting material and building a nest) and grooming transfer (in which grooming and removal of a drop of fluorescent oil from the head is scored) have both been validated as useful cage-side pain assessment techniques in mice.¹¹⁶ It is hoped that the development of more relevant tools for monitoring rodent pain will lead to improved clinical management of laboratory mice and rats after painful procedures. Anesthesia and analgesia use during experimentation should always be reported in publications. This is important for reproducibility but also to emphasize the ethical consideration and care being afforded to research rodents undergoing painful procedures.

Consideration of the Gut Microbiota

The gut microbiota has received increasing interest in the past decade because of its potential impact on health and disease states. Perhaps not surprisingly, alterations in the gut microbiota are common between institutions housing mice from the same sources^117,118 and these differences may have a significant impact on rodent model reproducibility (reviewed previously¹¹⁹). Simple transportation of mice from a commercial vendor to a research facility or even across a campus may induce transient changes in the mouse gut microbiota.^120,121 Additionally, changes in bedding, caging, and diet may impact mouse and rat gut microbiota in subtle ways.^122,123 Evaluating the gut microbiota is not a technique readily available to most biomedical researchers at this point, but some of these variations can be dealt with by using germ-free mice inoculated with known bacterial flora¹¹⁹ or even by biobanking feces from experimental animals, which can be analyzed at a later date if gut microbiota changes are suspected. All of this research only serves to further emphasize the need for carefully documenting all aspects of husbandry and procedural details in scientific publications.

Getting Help—Collaborating with Experts in Laboratory Animal Science

Throughout this article, we have provided a number of compelling examples of factors affecting laboratory rodent research outcomes. It is not our intention to suggest that every biomedical researcher become an expert in laboratory animal science, but rather to emphasize that the potential pitfalls for researchers are many and responsible scientists should look to collaborate with those who specialize in laboratory animal science when developing their experiments and preparing grants. Veterinarians with research training and specialization in laboratory animal science and medicine are an excellent possible resource, often bringing applied clinical knowledge about the animal models (ie, comparative medicine) together with a mechanistic understanding of the impact of external and internal variables on animal physiology, behavior, and welfare.^124,125 Developing an interdisciplinary team with the correct expertise can have a synergistic effect on research outcome and translatability of findings.^125–127

Conclusions

There are many factors that can impact research that involves laboratory mice and rats. To avoid running parallel experiments during in vivo studies, biomedical researchers have a responsibility to develop a more thorough understanding of laboratory animal science-related parameters and their potential effects on their research models. It may not be impossible for researchers to know about all of these issues in depth, thus there is a need to recognize the value of laboratory animal veterinarians and scientists as key partners and collaborators during experimental planning. Because there are many potential impacts of housing, husbandry, analgesia, etc., on research outcome, more detailed reporting is needed concerning housing, husbandry and other experimental parameters in research publications. This permits closer replication of experiments, but also helps readers better scrutinize and understand how the research was conducted, including evaluating study quality. At minimum, the ARRIVE Guidelines²⁶ provide a basis for describing experimental details to promote reproducibility, transparency, and improved assessment of experimental bias and quality.

R. Wayne Barbee, Ph.D. is Professor of Physiology & Biophysics and IACUC Chair at the Virginia Commonwealth University in Richmond, Virginia.

Patricia V. Turner M.S., D.V.M, D.V.Sc. is a laboratory animal veterinarian and pathologist, and animal welfare scientist who works as Corporate Vice President, Global Animal Welfare for Charles River in Wilmington, Massachusetts.