Potential for Unintended Consequences of Environmental Enrichment
for Laboratory Animals and Research Results
Kathryn Bayne
Abstract
Many aspects of the research animal’s housing environment
are controlled for quality and/or standardization. Of recent
interest is the potential for environmental enrichment to
have unexpected consequences such as unintended harm to
the animal, or the introduction of variability into a study that
may confound the experimental data. The effects of enrichment provided to nonhuman primates, rodents, and rabbits
are described to illustrate that the effects can be numerous
and may vary by strain and/or species. Examples of parameters measured where no change is detected are also included because this information provides an important
counterpoint to studies that demonstrate an effect. In addition, this review of effects and noneffects serves as a reminder that the provision of enrichment should be evaluated
in the context of the health of the animal and research goals
on a case-by-case basis. It should also be kept in mind that
the effects produced by enrichment are similar to those of
other components of the animal’s environment. Although it
is unlikely that every possible environmental variable can
be controlled both within and among research institutions,
more detailed disclosure of the living environment of the
subject animals in publications will allow for a better comparison of the findings and contribute to the broader knowledge base of the effects of enrichment.
Key Words: enrichment; experimental variable; nonhuman
primates; rabbits; rodents
S
ubmission of an animal study proposal (protocol) to
the institutional animal care and use committee
(IACUC1), in general, requires a rationale for the species selected for the study. Often, the investigator is also
asked to indicate whether the research animals need to be
excluded from part or all of the environmental enrichment
Kathryn Bayne, M.S., Ph.D., D.V.M., DACLAM, CAAB, is Senior Director and Director of Pacific Rim Activities for AAALAC International,
Hilo, Hawaii.
1
Abbreviations used in this article: AAALAC International, Association
for Assessment and Accreditation of Laboratory Animal Care; BDNF,
brain-derived neurotrophic factor; Guide, Guide for the Care and Use of
Laboratory Animals; IACUC, institutional animal care and use committee;
NK, natural killer; PHS, Public Health Service; SIB, self-injurious behavior; SIV, simian immunodeficiency virus.
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2005
program and if so, the reason. The importance of the first
question—the appropriateness of the animal model—is obvious. The investigator’s response to the second question—
regarding enrichment—may not receive the same degree of
consideration. The researcher may know that the provision
of certain environmental enrichments to the animal’s primary enclosure is a standard housing practice at his/her
institution; or the researcher may be aware of the numerous
publications regarding positive effects of providing enrichment to animals, and wants to provide optimal care for the
animals. Adding to the overall inclination to provide research
animals with enrichment is the obligation of the IACUC to
attend to the regulatory requirement: to promote the psychological well-being of nonhuman primates through an environmental enhancement program; and to implement a
behavioral management program that addresses the animals’ structural and social environment, as well as cognitive
and physical activity, as recommended in the Guide for the
Care and Use of Laboratory Animals (Guide1) (NRC 1996).
Following (or not following) the recommendations of the
Guide has implications for compliance with the Public
Health Service (PHS1) Policy on Humane Care and Use of
Laboratory Animals (OLAW 2002) for institutions that receive PHS funding for research and for accreditation with
the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC1) International.
The investigator’s consideration of the best animal
model for his/her research should also include factors that
have the potential to affect the quality of the animal and the
research. For example, it is important to evaluate the quality
and amount of the food and water provided. To assist in
achieving the research goals, a special diet may be provided
to the animals, or the water quality may be specially treated
to eliminate certain minerals, adjust the pH, or prevent bacterial contamination. Alternatively, the amount of food or
fluid provided to the animals may be restricted for either
health or research reasons. Other environmental factors that
colony managers and investigators reliably evaluate for impacts on research animals include the following: the temperature, relative humidity, and ventilation conditions of the
animal holding room; the quality and schedule of the room’s
illumination (e.g., whether a reverse light cycle is important
for the animals’ well-being or for the research goals); the
presence of extraneous noise in the facility that may negatively effect the animals’ reproductive success or other aspects of their well-being; the use of any chemicals in the
animal facility (e.g., in conjunction with the pest control
129
program, or through the use of cleaning agents containing
volatile oils as deodorizers); and the quality of the bedding
used with some species of animals.
Recently, more attention has been directed at understanding the various effects that enrichment, as one more
component of the housing environment, can have on research animals. Depending on the species in question, the
concern for the effect of enrichment on the research animal
has ranged from inadvertent physical harm to the animal to
subtle physiological changes in the animal that may have an
impact on the study goals. Environmental enrichment is
generally considered to imply an increase in the complexity
of the environment in which the animal lives, with the goal
of enhancing the animal’s welfare. These changes in the
animal’s environment can encompass the variety of food
items offered to the animal; whether or not the animal is
housed in a bedded cage (i.e., rodents); and additional
“structural” enhancements such as nest-building materials,
shelves/perches, hiding areas, manipulanda (toys), exercise
wheels, climbing/swinging apparatuses, water features, access to the outdoors, and much more. Social enrichment is
usually meant to imply conspecific interactions, but in a
broader sense can include positive interactions with facility
personnel and occasionally contraspecific interactions (e.g.,
young chimpanzees and dogs). The potential impact of each
of these interactions depends on the animal model and the
research goals. Ultimately, the decision to include a particular type of enrichment should be based on a consideration
of the safety of the animal and staff, whether the enrichment has a demonstrable beneficial effect on the animal,
and whether the potential effects of the enrichment are experimentally relevant. However, if one considers the provision of enrichment to be similar to other components of the
basic animal husbandry program, the same component
can and should be considered for all aspects of the animal’s
environment.
The scientific literature is becoming replete with examples of changes in animals (and thus potentially changes
in the data they generate) resulting from the increasing use
of enrichment with laboratory animals. Indeed, a metaanalysis of the published data has been recommended regarding the potential impact of enrichment on the research
animal (Clausing 2004). In part, such an analysis is necessary because conflicting reports have been published regarding the impact of enrichments on animals. Thus, the
investigator who is considering whether or not to permit the
provision certain of enrichments to his/her animals is faced
with sifting through literature that concludes beneficial effects, negative consequences, or no change to the animal.
From the perspective of the IACUC, these sometimes
conflicting reports are important because they illustrate the
potential error of relying exclusively on one published report in making sweeping judgments about animal housing
conditions.
The organization of this article is based on a separation
of the literature by the principal species for which there is a
substantial body of published data regarding the effects of
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enrichment—namely, nonhuman primates and rodents/rabbits.
For each of these species, examples of research are provided
that describe physical, physiological, and/or immunological
changes; altered susceptibility to disease; changes in brain
function or anatomy; or, in some cases, specific behavioral
changes in the animals that could influence either the health
and well-being of the animal or the research. A broaderbased discussion of the behavioral effects of enrichment is
outside the scope of this article.
Nonhuman Primates
Physical Harm or Change
One of the most serious potential outcomes to the provision
of enrichment is associated with physical harm to the animal. The irony of inadvertent and accidental physical harm
coming to a research subject when the intent was to do
something beneficial for the animal’s well-being makes this
circumstance all the more poignant and regrettable. Unfortunately, not all incidents of harm resulting from enrichments are published, thereby allowing some mistakes to be
perpetuated. For this reason, publication of descriptions of
the circumstances surrounding negative health consequences directly related to an enrichment device or strategy
should be encouraged.
A striking example of animal harm resulting from an
enrichment effort is the report by Line and colleagues
(1990a) describing the death of the highest ranking female
rhesus monkey following the formation of a group of 13
animals, of which 12 were wild caught and thus had prior
social experience. Line et al. (1990a) also described other
injuries associated with this group formation, including lacerations on the tail, head, and/or hand as well as a fracture
of the metacarpals on the hand of one animal. Some animals
required hospitalization due to their injuries, others required
temporary removal from the home cage for treatment with
immediate return to the cage, and a third category of animals required no treatment. One male animal was reportedly
severely depressed and was permanently removed from the
socialization effort. The group formation was conducted to
enhance the social lives of these animals. The authors concluded that the group formation was a success, despite the
death of one animal and injuries to most of the other animals
in the group. They based this conclusion, in part, on the
ethical choice of providing research animals with as much
protection from physical harm as possible versus providing
them the opportunity to engage in more diverse behaviors.
Changes in the social environment can also lead to selfinflicted injuries, sometimes referred to as self-injurious
behavior (SIB1). Common manifestations of SIB include
hair-pulling and self-biting. The extent of SIB can range
from quite mild, when the animal inflicts no damage on its
body because the behavior is inhibited (e.g., the animal may
touch its mouth to its arm, but not bite or otherwise break
the skin), or can produce significant damage, sometimes
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necessitating euthanasia. For example, Chamove and colleagues (1984) demonstrated that macaques who were
moved from a social group to individual housing exhibited
a higher incidence of self-aggression. The authors concluded that the individual housing precluded social redirection of aggression, and the animals therefore vented their
aggression on themselves. Alternatively, Reinhardt (1999)
notes that self-biting was completely eliminated in individually housed rhesus monkeys after pair formation. More variable success with the reduction or elimination of self-biting
behavior through pair formation has been reported for cynomolgus monkeys. In one instance, SIB was eliminated in
five monkeys after pair formation (Line et al. 1990b); however, in another study, similar results were not observed,
and no animals evidenced a reduction in SIB (Line et al.
1991). Despite these different findings, it is clear that
changes in the social environment can have a profound effect on individual nonhuman primates, whether moving the
animal from a social environment to individual housing or
the reverse.
Hahn and coworkers (2000) report the occurrence of an
intestinal linear foreign body in a cynomolgus monkey after
it ingested sisal rope that had been suspended on the outside
of the cage for enrichment purposes. Reportedly, the macaques would “groom” the rope, chew on it, and pull on it.
In this case, the animal had multiple ulcerations, perforations, and septic peritonitis. The rope was later removed
from all of the animals’ cages. Similarly, the provision of
long rope or chains has been reported to lead occasionally to
the death of a nonhuman primate. Indeed, Mahoney (1992)
notes that “facilities must exercise caution when installing
such climbing devices as vertically hanging or horizontally
suspended ropes and chains . . . because an animal can
strangle its neck, limbs, or other body parts . . .” (p. 27).
This caution to ensure animal health and safety as items,
intended as enrichments, are introduced into the animal’s
environment cannot be overstated.
Other physical changes have been documented as resulting from modifications to the housing environment of macaques. Line and colleagues (1990a) reported an increase in
the rate of nail growth of aged rhesus monkeys after formation of a group of animals. In general, the rate of nail
growth is reduced in older monkeys (Short et al. 1987).
Slow nail growth combined with a low level of natural killer
(NK1) cell activity in older macaques is correlated with
earlier mortality (Coe and Ershler 2001). Line and coworkers (1990a) posed two possible mechanisms for the heightened nail growth rate they observed in the socially housed
older macaques: (1) the immune system was activated due
to the incidence of wounds sustained by the animals, or
(2) the immune status was improved due to the positive
changes in the animals’ social and physical environment.
Coe and Ershler (2001) have shed some light on the answer
to this question, reporting that the highest NK response
occurred in old monkeys housed with one other older animal compared with an older monkey housed with a young
animal or living alone.
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In addition, providing nonhuman primates with extensive space in the primary enclosure can influence the
progression of joint disease and mobility. A comparison of
free-ranging rhesus monkeys with caged animals of the
same age shows that the free-ranging animals have a significantly higher prevalence and severity of degenerative
joint disease in the knee and significantly more restricted
passive knee joint extension (Kessler et al. 1986). The potential for larger housing conditions to influence studies of osteoarthritis in macaques therefore becomes a consideration.
Physiological Changes
Several parameters have been monitored to assess nonhuman primates for physiological changes following an alteration in their environment that would be considered
enriching. One early report documents increases in the level
of zinc in rhesus monkeys housed in group cages that were
made of galvanized (zinc-coated) wire resulting from the
animals chewing on the wire bars (Stevens et al. 1977).
However, in some cases no change in the animal’s physiology has been documented. For example, there was no
change in the heart rate of rhesus monkeys that were moved
into a cage that was 40% larger than their home cage (Line
et al. 1989), nor was a change in body weight observed after
pair housing compatible female rhesus monkeys (Reinhardt
et al. 1988).
Alternatively, statistically significant reductions in heart
rate have been documented in individual pigtail macaques
that are the recipients of grooming from other animals, although the reduction was not found for animals that initiated
grooming or engaged in self-grooming (Boccia et al. 1989).
Similarly, allogrooming causes a faster deceleration of the
heart rate of individual rhesus monkeys that had recently
experienced an increase in heart rate due to a stressful situation, such as the approach of a dominant animal (Aureli
et al. 1999). The presence of a familiar, compatible animal
has been shown repeatedly to buffer perceived stressors of
the subject animal (e.g., Cohen et al. 1992; Epley 1974;
Gerber et al. 2002; Gonzalez et al. 1982, 1996; Gust et al.
1994; Stanton et al. 1985). The converse is also true; separation from a familiar animal can result in increases in heart
rate and body temperature (Reite et al. 1978), and in some
cases this stress is not ameliorated even if the animals maintain visual, olfactory, and auditory contact (Smith and
French 1997). Thus, forming pairs or groups of animals,
which are later separated for research purposes, may have
consequences for the animals that are reflected in physiological changes. The duration of these changes may be
relatively brief, but would require assessment for the specific circumstances.
Immunological Changes
Perhaps the most widely researched immunological response measured in nonhuman primates is in association
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with concomitant changes in the social environment of the
animal. In some cases an animal undergoes social separation, and in other cases the animal is placed into a novel
social environment. Specific paradigms evaluated include
the separation of infant monkeys from their mothers, peer
separation, response to removal from and return to the social
group, and response to living with a compatible cage mate.
The changes in an animal as a result of social separation are
important to a discussion of the effects of enrichment because as animals are socially housed for enrichment purposes, they also may be separated from their cage mate(s)
for varying periods of time during the experiment, depending on the research. Often there is a physiological response
to these changes, mediated through the hypothalamicpituitary-adrenal system, that are commonly measured by
changes in levels of cortisol (e.g., Laudenslager et al. 1982;
Levine 1993; Reinhardt et al. 1989). However, other immunologically based indices of the stress response to social
changes include decreases in lymphocyte proliferation in
response to mitogen stimulation (Coe 1993; Laudenslager
et al. 1985), variability in counts of T cell subsets (Gust
et al.1993), rapid eye movement sleep (Boccia et al. 1989),
and reduced levels of the animal’s neutralizing antibody to
a bacteriophage (Coe et al. 1987).
Coe (1993) concisely reviewed the influence of psychosocial factors on the immune system of nonhuman primates,
and expanded the data by comparing the impact of social
enrichment and inanimate enrichment on various immune
parameters. Shapiro and colleagues (2000) found that individually housed rhesus monkeys had lower CD4(+):CD8(+)
ratios than did either pair housed or group housed rhesus,
and group housed monkeys had the highest levels of the
cytokines interferon-gamma and interleukin-10. Measures
of lymphocyte proliferation in response to various gastrointestinal pathogens and nonspecific mitogens showed a
trend (p ⳱ 0.08) toward a difference among individually,
pair-, or group-housed animals. The authors concluded that
the socially housed animals had an enhanced immune response. Shapiro and coworkers (1998) found no effect when
they evaluated several immunological parameters resulting
from the provision of inanimate enrichment.
Elevations of cortisol in nonhuman primates exposed to
new social stimuli have been reported to be more closely
related to fear experienced by the animal than as a reflection
of aggression exhibited by the animal (Chamove and Bowman 1978). Based on this information, the measurement of
cortisol values in monkeys exposed to other common procedures is of interest for placing in context a discussion of
the impact of enrichment techniques on the well-being of
the animal. To that end, the impact of cage size, cage level,
room change, tethering, and sedation on the urinary cortisol
response in macaques and baboons has been evaluated
(Bentson et al. 2003; Crockett et al. 1993, 2000). To summarize their findings, neither cage size nor cage level (upper
or lower tier of cages) produced a significant change in
cortisol level (Crockett et al. 1993, 2000). However, ketamine sedation did produce elevations in urinary cortisol in
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both cynomolgus (male and female) and pigtailed (female)
macaques.
Of interest is Bentson and colleagues’ (2003) finding
that in male rhesus monkeys, ketamine had no effect on
(blood) cortisol level, although Telazol® resulted in reduced cortisol in the morning, but not in the afternoon. The
authors also found that baboons exhibited increased cortisol
levels after ketamine administration, but no significant increase in cortisol was observed following sedation with
Telazol®. Clarke and coworkers (1988) showed similar species variability in corticosteroid measurements of bonnet,
cynomolgus, and rhesus macaques after confinement in a
transport cage. The rhesus monkeys had the least adrenocortical response to confinement in a transport cage, and the
cynomolgus macaque had the highest response. These results underscore the need to assess each species for changes
in response to its environment, whether those changes involve routine husbandry procedures or the addition of environmental enrichment. Indeed, the data suggest that not
many aspects of the primate’s environment are controlled,
and that the effects of enrichment provision do not differ
significantly from other animal care and use procedures.
Response to Disease and Other Conditions
In general, the response of nonhuman primates, principally
macaques, to the progression of a disease or other condition,
or the response to disease challenge, is studied under altered
social environment conditions. In many cases, a monkey is
removed from its social environment either intermittently
during the experiment or for the duration of the experiment.
Vulnerability to addiction has also been assessed in
these types of studies. Juvenile rhesus monkeys have been
shown to consume more alcohol when intermittently separated than when they were socially housed, and the intermittently separated monkeys drank more alcohol than
continuously separated monkeys (Kraemer and McKinney
1985). Individual differences were also noted; some monkeys were more likely to drink larger quantities of alcohol
than other animals. The authors concluded from these findings that social stressors can elicit alcohol abuse and addiction in primates.
Social housing has also been found to have an impact on
cocaine addiction. A relationship has been postulated between drug addiction and monoamine concentrations in the
brain. Measurements of dopaminergic function in individually and socially housed cynomolgus monkeys via positron
emission tomography revealed no differences in individually housed monkeys, but higher levels or more available
dopamine D2 receptors were detected in dominant monkeys
housed socially compared with subordinate animals (Morgan et al. 2002). The following observations were then
linked to these findings: (1) cocaine served as a reinforcer
for subordinate, but not dominant, animals (Morgan et al.
2002); and (2) animals living in chronically unstable social
groups, wherein group membership changed on multiple
ILAR Journal
occasions over a protracted period of time, had lower concentrations of serotonin and monoamine metabolites in the
prefrontal cortex (Fontenot et al. 1995).
The implications of the studies cited above are numerous. The definition for a “social group” is not entirely clear.
It is possible that the social relationships among individually housed animals that have been maintained in the same
room for a long period of time could be well established, to
the point where they function to some degree as a social
group. In such a case, transfer of animals in or out of the
room on a routine basis could have an impact similar to that
described for socially housed animals that are in physical
contact. Similarly, moving an animal from a social group to
individual housing for the purpose of an experiment, or
putting a previously singly housed animal in a social group,
could have an influence on addiction studies.
Several studies evaluating the social environment of a
nonhuman primate and that animal’s susceptibility to disease have demonstrated differences between animals maintained in individual housing and group housing, as well as
differences between dominant and subordinate individuals
within a group. An evaluation of cardiovascular disease in
animals experiencing social changes has shown that chronic
individual housing of female cynomolgus monkeys augments the incidence of atherosclerosis (Watson et al. 1998).
In group-housed cynomolgus monkeys, atherosclerosis is
more prominent in dominant males housed in unstable social groups (although not when the group membership is
held stable) and subordinate females, regardless of the stability of the group membership (Kaplan and Manuck 1999).
More consistently, rhesus monkeys living in an unstable
social group that exhibited more agonism and less affiliative
behavior have been reported to have a reduced survival rate
after infection with simian immunodeficiency virus (SIV1)
compared with animals living in a stable social group (Capitanio et al. 1998). Interestingly, rhesus monkeys that engaged in affiliative social interactions had lower plasma
concentrations of the virus whereas those animals receiving
more social threats had higher concentrations. A detailed
review of animal records that include data regarding animal
housing conditions and SIV inoculation date has shown that
social disruptions (e.g., housing relocation, social separation, or placing into a social group) occurring in close temporal juxtaposition to the inoculation result in reduced
survivability of the animals (Capitanio and Lerche 1998). In
addition, although there was no effect on the antibody response of male rhesus monkeys related to the timing of a
tetanus vaccination and social disruption (placement into
individual housing), the behavioral dimension of “high sociability” and “low sociability” expressed by different
animals was associated with the strength of the antibody
response, with the high-sociable animals having a significantly higher antibody response (Maninger et al. 2003).
The role of inanimate environmental enrichments in
causing disease in nonhuman primates has been evaluated
only preliminarily. Colleagues and I (1993) have documented that microbial growth persisted on a type of enrichVolume 46, Number 2
2005
ment toy even after sanitation in a mechanical cagewasher.
Because the bacterial contamination was limited principally
to bacteria commonly found in the environment (Bacillus,
Staphylococcus, Micrococcus, nonenterics, and mixed coliforms), we concluded that the potential for toys to cause
disease is low. However, the possibility of this contamination
confounding infectious disease work should be considered.
Rodents and Rabbits
Physical Harm
The most significant unexpected impact occurs when an
item intended to enhance the animal’s welfare results in
harming the animal in some way. Although not many examples of such an occurrence have been reported in the
literature for rodents and rabbits, there are two instances of
note. Shomer and coworkers (2001) described an injury to a
New Zealand White rabbit from a whiffle ball (a hollow
plastic ball with numerous holes in its surface) provided to
the rabbit for play activity. Although the whiffle ball was
considered by facility staff to be too hard to be broken by
the rabbits, and too large to be swallowed, a ball unexpectedly became entrapped in the incisors of one rabbit. The
outcome of this event was that the animal could not eat or
drink for about 12 hr , and there was trauma to the gums.
Athymic nude mice have reportedly been adversely affected
by cotton nesting material (Nestlets®) placed in their cages
(Bazille et al. 2001). In this case, all animals provided with
this nesting material manifested conjunctivitis within 2 wk
of exposure. In some animals, portions of the nesting material were trapped in the conjunctival sac. The severity of
the condition continued to increase until the cotton material
was removed. The authors suggested that this finding was
limited to athymic nude mice due to the absence of eyelashes in these animals that would otherwise help to exclude
particulate material derived from the Nestlets®. Clearly,
these cases represent a minority of incidences, but they
nevertheless illustrate the point that implementation of environmental enrichment should include an assessment of the
potential risk to the animal associated with the item.
Physical Changes
Since the early 1950s, investigators have documented
physical changes in rodents resulting from changes to their
environment. These studies initially focused on how the
handling of rats (also referred to as gentling) resulted in a
significantly higher average weight gain for the handled
rodents compared with the nonhandled, control animals
(Bernstein 1952; Weininger 1956; Weininger et al. 1954).
This greater weight gain among handled rats was consistent
whether the animals were individually or group housed
(Ruegamer et al. 1954). This increase in weight was later
shown to be related to greater amounts of fecal pellets ex133
creted by the control animals, with a presumed increased
“food utilization” by the handled rats because the amount of
food consumed did not differ significantly between handled
and unhandled rats (Ruegamer et al. 1954). Handled animals also tended to have a greater skeletal length than control animals (Ruegamer and Silverman 1956).
The finding of weight gain in rodents exposed to a more
complex environment has been extended to include influences from nonsocial enrichments. For example, Augustsson and colleagues (2003) found that BALB/c and C57BL/6
mice housed in enriched or “superenriched” cages gained
more weight than control mice (nonenriched cages), even
though food consumption did not differ significantly among
the groups. However, such findings appear to be inconsistent among laboratories evaluating the same strains of mice.
Van de Weerd et al. (2002) found that mice reared in superenriched and enriched conditions gained weight faster
than mice (RIVM:N:NIH, BALB/c) housed in nonenriched
cages, but the superenriched mice also consumed more food
than mice housed in the other conditions. Tsai and coworkers (2002) reported that BALB/c, C57BL/6, and A/J mice
living in enriched (nest box, nesting material, climbing bar)
or nonenriched cages had mean body weights that either
were not different or differed only slightly from each other
(heavier or lighter), but not to a statistically significant degree. Similarly, Smith and colleagues (2000) evaluated the
body weights of CD-1 mice provided with a synthetic gauze
pad as enrichment and found no difference in either body
weight or weight gain from that of control animals (no
gauze pad).
Researchers have also compared the body weights of
New Zealand White rabbits living in enriched and nonenriched environments. Harris and coworkers (2001) evaluated enrichments that included food items (celery, Bunny
Blocks®, Bunny Stix®) as well as nonfood items (Jingle
Ball®, Kong® toy, Nylabone®). Not surprisingly, the foodenriched rabbits showed weight gains, although not to a
statistically significant degree after 15 days, compared with
rabbits receiving only nonfood enrichments.
Organ weights have also been measured for an influence
from environmental enrichment. Tsai et al. (2002) weighed
the heart, liver, kidney, adrenal, spleen, and uterus of three
inbred strains of mice (BALB/c, C57BL/6, A/J) living in
enriched and nonenriched cages. In comparisons with control animals, they found that the weights of the spleen from
enriched animals were slightly, but not significantly, increased; and the weights of the adrenal from enriched animals were slightly, but not significantly, decreased. This
latter finding is further supported by Bonnet and coworkers
(2004), who have documented statistically significant lower
adrenal weights in female B6C3F1 mice housed in stainlesssteel cages with a mat of hemp fibers compared with animals housed in cages without a mat. They also found that
the thymus weight of mice housed in social groups (in plastic cages) was higher than individually housed mice in stainless-steel cages. Smith et al. (2000) found no significant
differences in the organ weights (kidneys, liver, heart,
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spleen, testes, prostrate, adrenals, thyroid and parathyroids,
pituitary, and brain) of CD-1 mice with a gauze pad in the
cage compared with mice without a pad.
Numerous studies have also evaluated the effect of environmental enrichment on recovery from an experimentally induced physical change (e.g., injury) in the animal.
A very early study (Hammett 1921) showed that the relative
mortality subsequent to thyroparathyroidectomy and parathyroidectomy of rats was much lower (13%) in animals
that had been handled compared with animals that had not
been handled (79%). In more recent examples, rats with
experimentally induced traumatic brain injury living in an
enriched environment exhibit a shorter latency to find the
platform in a Morris Water Maze test compared with injured
individually housed rats (Hamm et al. 1996, Passineau et al.
2001). This finding indicates facilitated recovery of some
cognitive functions from exposure to enrichment items. Of
note is the report that the brain injury in the enriched rats
was approximately two times smaller than the individually
housed rats after 2 wk (Passineau et al. 2001). Rats having
undergone ligation of the middle cerebral artery and living
in an enriched environment had significantly better motor
function and improved more quickly than control animals
(Grabowski et al. 1995; Johansson 1996; Ohlsson and Johansson 1995), even when the enrichment was limited to
social housing (no inanimate enrichment) compared with
individual housing (Risedal et al. 2002). Brain injury
induced by a septal lesion in mice living in enriched or
nonenriched environments was also influenced by environmental complexity.
In rodents, a septal lesion produces a phenomenon
known as septal rage, which is characterized by hyperemotionality and aggressiveness by the rodents. Enriched mice
were less “reactive” than nonenriched mice, and mice living
first in an enriched environment and later transferred to the
nonenriched environment showed an immediate increase in
reactivity (Goodlett et al. 1982). In addition, the amount of
exposure to enrichment may not need to be very long to be
effective. For example, Will and coworkers (1977) noted
that rats that received 2 hr/day of enrichment recovered
from bilateral lesions to the occipital cortex to the same
degree as rats exposed to enrichment 24 hr/day.
Neurological Changes
Morphological changes to the brain are perhaps among the
most well-known effect of enrichment on rodents. Walsh
(1981) has reviewed the effects on the brain in response to
environmental complexity. Typically, greater cerebral
weight and length, as well as increased cortical depth, are
measured in rodents living in an enriched environment (e.g.,
Diamond et al. 1976, 1985). Recently, the specific regions
of the rodent brain affected by living in an enriched environment have been determined. In the hippocampus, CA1
and dentate gyrus cells were affected; however, changes
were not observed in layer V pyramidal neurons of the
ILAR Journal
cerebral cortex or in the spiny neurons in the striatum
(Faherty et al. 2003). When the histological changes in the
brains of rats provided with social and inanimate enrichments have been compared with rats that are socially housed
without inanimate enrichments, the number of oligodendrocytes and astrocytes in the occipital cortex have reflected an
increase in the former group. With rats that were handled
daily, only the number of astrocytes increased (Szeligo and
Leblond 1977).
Increases in dendritic spines and branching, synaptic
connections, and neural cell size have long been recognized
to result from enriching the environment of rodents (e.g.,
Globus et al. 1973; Volkmar and Greenough 1972). These
effects on brain morphology cannot be attributed to the
enriching effects of social housing, but rather appear to be
related to the presence of the inanimate enrichments (Renner and Rosenzweig 1986, Rosenzweig et al. 1978) and,
more specifically, to direct contact with the inanimate enrichments (Ferchmin and Bennett 1975). Although enriched
mice move longer distances and spend more time in the
center of an open field testing apparatus (Kohl et al. 2002),
it appears that the increased activity associated with exploring and manipulating inanimate enrichment items is not
responsible for the histological changes in the brain
(Faherty et al. 2003; Kohl et al. 2002).
However, an examination of gene expression in the
brain, relative to the availability of enrichment items in the
home cage, reveals that enrichment affects the expression of
several genes that regulate neuronal structure, synaptic signaling, and brain plasticity (Rampon et al. 2000). In fact, the
transgenic R6/1 and R6/2 mice, which are used to model
Huntington’s disease—a genetic disorder that results in motor dysfunction, dementia, and death—exhibit less decline
in select motor function tasks and delayed loss of cerebral
volume in those transgenic mice living in an environment
that includes both social and inanimate enrichment (Glass
et al. 2004; Hockly et al. 2002). Recent evidence suggests
that the mode of action for enrichment on slowing the
course of the disease is mediated through reducing protein
deficits in the brain (Spires et al. 2004), because nonenriched mice have significant decreases in brain-derived neurotrophic factor (BDNF1) as well as reductions in dopamine
levels. Not only is BDNF higher in the brains of enriched
animals, but so too are nerve growth factor and neurotrophin-3 proteins (Ickes et al. 2000). Hockly and colleagues (2002) note that standardization of the housing
condition should be considered in therapeutic trials. However, simply using R6/2 mice in behavioral testing (rather
than housing them in enriched cages) also improves their
survivability (Carter et al. 2000). So, it appears that there
are a number of potential confounding variables, some of
which may not have even been identified to date, that can
alter the course of experimentally induced or modeled disease research.
A discussion of the possible neurological changes would
not be complete without mentioning the effects of enrichment on memory and learning because these effects reflect
Volume 46, Number 2
2005
the functional change that can occur in rodent brains concomitant with the anatomical changes already described.
Learning rate has been described to be faster in enriched rats
than nonenriched rats (Kobayashi et al. 2002; Wainwright
et al. 1993), Additionally, spatial memory, in general, is
positively affected by an enriched environment (e.g., Martinez-Cue et al. 2002; Nilsson et al. 1999), even when the
enrichment is provided to the animals when they are adults
(Frick et al. 2003). This effect has also been documented in
rats with induced status epilepticus (Faverjon et al. 2002).
Although the precise mechanism of the memory enhancement has not been identified fully, recent evidence suggests
that enrichment affects cAMP-dependent protein kinase
long-term potentiation in the hippocampus (Duffy et al.
2001).
Physiological Changes
The cardiovascular system and hematology of rodents from
enriched and nonenriched environments have also been assessed. Tsai and coworkers (2002) found a nonsignificant
decrease in red blood cell count and hematocrit and a nonsignificant increase in hemoglobin in enriched mice
(BALB/c, C57BL/6, A/J) compared with nonenriched controls. They also observed a nonsignificant increase in the
level of white blood cells in enriched C57BL/6 and A/J
mice, but not in enriched BALB/c mice. Similarly, no effect
on the hematological profile was observed in New Zealand
White rabbits provided with a stainless steel rattle clipped to
the cage (Johnson et al. 2003). Social enrichment alone can
result in an increase in systolic blood pressure (but not
diastolic pressure or heart rate), and nonsocial enrichment
in combination with brief periods of social enrichment (2
hr/day) led to increased systolic and diastolic blood pressures but no change in heart rate. When rats that were previously socially housed were changed to individual housing
in enriched cages, all three dimensions measured (systolic
blood pressure, diastolic blood pressure, and heart rate) increased (Lawson et al. 2000). However, even routine procedures such as cage changing can lead to significant, albeit
brief (45- to 60-min), increases in systolic and diastolic
blood pressure as well as heart rate (Duke et al. 2001).
The effect of enrichment in the environment has been
measured for several other physiological parameters.
Higher levels of testosterone and immunoglobulin G levels
have been detected in enriched mice compared with control
animals, although there is some strain variability in these
findings (Nevison et al. 1999). Plasma triglyceride levels
have been reported to be lower in individually housed rats,
compared with socially housed rats, although individual
housing did not result in a concomitant increase in total
cholesterol levels (Pérez et al. 1997). Lower cholesterol
values were noted, however, in rats housed in large, enriched cages compared with rats housed in standard plastic
cages (Augustsson et al. 2002). The urine corticosterone
135
level of these “pen-housed” rats was higher than that of the
rats housed in standard cages, which the authors attributed
to the greater activity exhibited by the rats in the large pens.
Conversely, the plasma corticosterone of rats in response to
stress (maternal separation) was lower for enriched animals
than for nonenriched animals (Francis et al. 2002), but no
difference in corticosterone (or thyroxine) levels was observed in enriched versus nonenriched DBA/2 mice (Tsai
et al. 2003).
Enrichment items are not the only environmental factors
that have the potential to influence the physiology of a
rodent. Cage size alone is a potentially confounding factor
for some types of research. Specifically, Kuhnen (1999)
found that the basal rectal temperature of hamsters housed
in smaller cages was significantly higher than that of hamsters housed in larger cages, and the animals’ response to a
fever-inducing lipopolysaccharide also varied depending on
the cage size. The depth and type of bedding placed in the
cage also influence body temperature (Gordon 2004). Mice
housed in deep wood bedding were noted to have a significantly higher temperature than comparable mice housed on
a layer of beta chips or thin wood bedding, although this
difference was time dependent because it was observed only
during the daylight hours. As Gordon (2004) notes, such a
difference in body temperature based on bedding depth and
type would be of concern in toxicological studies in which
determination of the endpoint is based in part on the animal’s body temperature.
Discussion
Several key points emerge from this review of the potential
effects of environmental enrichment that bear further discussion. First, there is no consensus regarding what constitutes enrichment for some animal species. For example,
many facility personnel do not consider solid-bottom, bedded cages a form of enrichment. However, some researchers
do believe that this type of cage constitutes enrichment because rodents, for example, can burrow into it, thereby expressing a species-typical behavior. However, bedding also
can affect the research animals. Indeed, contact bedding
(Shred-N-Nest®, a corn-husk product) has been shown to
reduce the aggression exhibited by male BALB/cAnNHsd
mice (Armstrong et al. 1998), although the aggression
among male BALB/cAnNCRLBr mice was not attenuated
by the provision of Lignocel® 3⁄4 sawdust bedding alone
(Van Loo et al. 2002).
Bedding can also pose a problem for rats dosed with
buprenorphine, for example after surgery, due to the quantity ingested as a result of pica behavior (Clark et al. 1997).
Some types of bedding (e.g., pine, cedar) emit volatile hydrocarbons, which can interfere with the induction of cytochrome P450 enzymes in hepatic microsomes (Vesell et al.
1973), while others can modify the mucosal immune response, as evidenced by increased numbers of Peyer’s
136
patches in the intestinal mucosa (Sanford et al. 2002).
Weichbrod and colleagues (1986) have reviewed many
of the effects of different bedding materials on laboratory
animals.
Similarly, some studies refer to the provision of increased cage space as a form of enrichment with positive
results for the animals (Augustsson et al. 2002), although
the provision of increased cage size alone has been found to
promote the expression of undesirable behaviors by some
species of animals (Bayne and McCully 1989). Thus, although the type of bedding used and the cage size provided
the animals may be standardized in an animal facility, this
very standardization may result in unexpected or undetected
confounding variables in the research.
A second key point is that numerous factors in the animal’s proximate environment can affect the animal and the
research data. Some environmental variables of importance
include the quality and quantity of illumination in the animal room (e.g., Lanum 1979; Novak and Drewsen 1989),
sound (Kaplan and Miezejeski 1972; Novak and Drewsen
1989), food and water quality, air quality, temperature, how
much the animals (especially rodents) are handled, and even
the demeanor of the people interacting with the animal. So,
of the innumerable environmental variables that can have an
impact on the animal research model, enrichment techniques are simply a part of a larger picture of the overall
housing environment that should be considered.
Characterizing a particular feature in the research animal’s environment as an enrichment is an artificial delineation that perhaps should be abandoned. Rather, it is more
appropriate to assess the gestalt of the animal’s environment. Questions that should be addressed include whether
the animal’s well-being is enhanced by including a particular feature in the environment, whether a better animal
model results, or whether increased variability is introduced
into the study. If the latter, then a second tier of decisionmaking should occur because different aspects of the principles of refinement, reduction, and replacement (“the 3
Rs”) are involved. In providing certain elements such as
enrichments, to the animal’s environment, a refinement is
introduced. However, if the enrichment also causes more
variability in the study, if more animal subjects are needed
to achieve appropriate statistical power, or if studies must be
duplicated, then the goal of using fewer animals is not
achieved. Investigators and IACUC members need to balance the issues of enhanced animal welfare with the potential for reduced animal numbers used in the research.
In some cases, the very finding that an enrichment technique has modified the response of an animal to a disease,
addiction, or condition has proven to be of interest (Gordon
2004; Hockly et al. 2002; Kuhnen 1999). This result suggests that preventing or delaying the onset of clinical signs,
or possibly even reversing some signs, as a result of living
in an enriched environment (Francis et al. 2002; Ohlsson
and Johansson 1995; Passineau et al. 2001) may have implications for the human condition (Glass et al. 2004;
Hockly et al. 2002; Kobayashi et al. 2002).
ILAR Journal
Concluding Remarks
As illustrated by the preceding review, the effects of environmental enrichment are similar to those of other environmental components. In some cases, there is no impact on the
animal or research parameter being studied, and in other
cases there may be an effect on either the animal or the
research data that might be construed as either positive or
negative, depending upon the specific circumstances. Although it is unlikely that every possible variable can be
controlled both within and among research institutions,
more detailed disclosure in journals and other publications
of the living environment of the subject animals in publications will allow for a better comparison of the findings,
and contribute to the broader knowledge base of the effects
of enrichment.
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