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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
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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.
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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;
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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.
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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;
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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.
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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
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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
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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
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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.
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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
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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
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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.
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