nature publishing group articles

Methods and Techniques

Accurate Measurement of Body Weight

and Food Intake in Environmentally Enriched
Male Wistar Rats
Kylie E.L. Beale1, Kevin G. Murphy1, Eleanor K. Harrison1, Angela J. Kerton2, Mohammad A. Ghatei1,
Stephen R. Bloom1 and Kirsty L. Smith1

Laboratory animals are crucial in the study of energy homeostasis. In particular, rats are used to study alterations in
food intake and body weight. To accurately record food intake or energy expenditure it is necessary to house rats
individually, which can be stressful for social animals. Environmental enrichment may reduce stress and improve
welfare in laboratory rodents. However, the effect of environmental enrichment on food intake and thus experimental
outcome is unknown. We aimed to determine the effect of environmental enrichment on food intake, body weight,
behavior and fecal and plasma stress hormones in male Wistar rats. Singly housed 5–7-week-old male rats were
given either no environmental enrichment, chew sticks, a plastic tube of 67 mm internal diameter, or both chew sticks
and a tube. No differences in body weight or food intake were seen over a 7-day period. Importantly, the refeeding
response following a 24-h fast was unaffected by environmental enrichment. Rearing, a behavior often associated
with stress, was significantly reduced in all enriched groups compared to controls. There was a significant increase in
fecal immunoglobulin A (IgA) in animals housed with both forms of enrichment compared to controls at the termination
of the study, suggesting enrichment reduces hypothalamo-pituitary-adrenal (HPA) axis activity in singly housed rats.
In summary, environmental enrichment does not influence body weight and food intake in singly housed male Wistar
rats and may therefore be used to refine the living conditions of animals used in the study of energy homeostasis
without compromising experimental outcome.

Obesity (2011) 19, 1715–1721. doi:10.1038/oby.2010.331

Introduction to house each animal separately. Feeding is a complex ­behavior

Laboratory animals are vital in the study of energy homeosta-
sis because the physiological mechanisms involved in the con-
trol of food intake and energy expenditure are highly complex.
Due to the huge economic burden induced by obesity and its
comorbidities it is essential to gain a greater understanding of
the pathways controlling food intake to aid the development of
new treatments for this condition.
Wistar rats are well characterized animal models used to
investigate food intake, and rats and humans have comparable
hypothalamic structures and neuroendocrine circuits which
control food intake and energy expenditure (1).
Rats are social creatures, and isolation has been shown to
increase anxiety and induce behavioral abnormalities (2,3).
European guidelines state that “animals should be socially
housed whenever possible”, unless deemed inappropriate by the
scientific protocol (4). In order to accurately record the food
intake or energy expenditure of individual rats, it is necessary
affected by many factors, including stress. Anxiety induced
by housing conditions may therefore be a confounding factor
when conducting feeding studies (5,6).
As social housing is not possible in experiments involving the
measurement of food intake and/or energy expenditure, envi-
ronmental enrichment may be added to cages to overcome the
stressful effects of single housing. Environmental enrichment
is defined as the use of objects to improve the quality of life
of a caged animal and promote species-specific behavior (7).
Laboratory rats consistently show a preference for enriched
cages over basic cages, for example choosing an environment
containing paper towels, wooden platforms or wood chips
over a barren cage (8). When presented with different objects,
rats show a preference for objects they can chew, allowing
them to exercise an important species-­typical ­behavior (9).
Among numerous benefits, ­environmental enrichment has
been shown to improve cognition and memory, increase

The first two authors contributed equally to this work.

1
Section of Investigative Medicine, Division of Diabetes, Endocrinology and Metabolism, Imperial College London, London, UK; 2CBS Department, Imperial College
London, London, UK. Correspondence: Kevin G. Murphy (k.g.murphy@imperial.ac.uk)
Received 11 August 2010; accepted 6 December 2010; published online 13 January 2011. doi:10.1038/oby.2010.331

obesity | VOLUME 19 NUMBER 8 | August 2011 1715

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Methods and Techniques
cortical thickness and induce faster ­recovery from brain
injury (10–12).
The response to stress is predominantly mediated by the
hypothalamo-pituitary-adrenal (HPA) axis. Under stressful
conditions, corticotrophin-releasing hormone is produced by
the hypothalamus and acts at the anterior pituitary gland to
stimulate the synthesis and release of adrenocorticotropic hor-
mone (ACTH). ACTH acts at the adrenal cortex to drive the
release of corticosterone, the major glucocorticoid in rodents.
Plasma corticosterone is therefore the primary measure of
HPA axis activity in rodents. Fecal immunoglobulin A (IgA)
may also be used as a noninvasive marker of long-term well-
being, as chronic stress induces immunosuppression (13). Rats
housed singly in enriched cages have lower baseline plasma
corticosterone and ACTH than rats housed in standard cages
and display less abnormal, maladaptive behaviors, indicating
that enrichment may reduce stress in these conditions (7,14).
Housing conditions have also been shown to affect fecal IgA,
although the effect of environmental enrichment on this
parameter has not been well characterized (15).
Despite the benefits of environmental enrichment, its effect
on experimental variability is unclear and concerns have been
raised that by disrupting standardization, enrichment may
reduce the precision of results (16). In particular, the effect of
environmental enrichment on the feeding behavior of rats has
not been well studied.
The provision of environmental enrichment has been shown
to affect numerous processes within the central nervous sys-
tem, for example cerebral development, and neuronal ­plasticity
(17). Animals housed with enrichment have been shown to
adapt more quickly to new environments than those housed
in barren cages, potentially due to improvements in learning,
memory, and information processing (18,19). As feeding is
sensitive to environmental conditions, enrichment may there-
fore influence food intake and body weight (5).
We hypothesized that environmental enrichment would
improve welfare without compromising experimental out-
come in singly housed male Wistar rats. We aimed to deter-
mine the effect of environmental enrichment on food intake,
body weight, behavior, and the refeeding response to a 24-h
fast. We also measured fecal IgA before and after enrichment
and plasma corticosterone and ACTH at the termination of the
study.
Methods and Procedures
Animals and housing
Animal procedures undertaken were approved by the British Home
Office Animals (Scientific Procedures) Act 1986 (Project License
70/6402).
Male Wistar rats (specific pathogen free; Charles River Laboratories,
Margate, UK) of 5–7 weeks of age weighing 150–200g were maintained
in individual open cages with solid floors and sawdust bedding under
controlled temperature (21–23 °C) and light (12:12 light-dark cycle; lights
on at 0700) conditions with ad libitum access to food (RM1 diet, Special
Diet Services, Witham, UK) and tap water. Relative humidity was ~55%.
Cage dimensions were: width 245 mm, length 415 mm, height 185 mm.
Following a 7-day acclimatization period during which food intake
and body weight were monitored, animals were randomly assigned by
1716
body weight into one of four groups (n = 6–8 per group). The control
group received no environmental enrichment in their home cage. The
second group was given two wooden chew sticks measuring 150 mm ×
20 mm × 1 mm, which were replaced daily. Rats in the third group were
given a black plastic tube (200 mm length × 67 mm internal diameter)
and the fourth group received both wooden chew sticks and a tube. The
tubing was sufficiently large to allow rats weighing up to 450 g to comfort-
ably enter. Cages were changed when enrichment was assigned and once
weekly thereafter. The entire study period was 40 days.
The effect of environmental enrichment on food
intake and body weight
Animals were left to acclimatize to their environmental enrichment
for 5 days before measurements began. Food intake and body weight
were then measured daily for 7 days. Food pellets were stored in mesh
hoppers within each cage to prevent spillage and cages were visually
inspected daily to check for loose pellets.
The effect of environmental enrichment on behavior
Behavior was continuously monitored for 1 h by two observers in the
early dark phase, as this is when rats are most active, on day 21 of the
study using methods adapted from Fray et al. (20,21). Each rat was
observed for 15 s every 5 min. Each 15-s period was subdivided into
three 5 s periods and the behavior of the rat was noted in each 5 s period.
Behavior was classified into the following categories: feeding, drinking,
rearing, locomotion, grooming, burrowing, head down, and sleeping.
For animals supplied with tubing, it was noted if an animal was within
the tubing and the behavior it was performing was recorded to compare
against the control group.
The effect of environmental enrichment on
fasting-induced refeeding
Animals were fasted from 0900 for 24 h on day 25 of the study. The
following morning at 0900 a predetermined quantity of food was
returned to the cages. Food intake was measured at 1, 2, 4, 8 and 24 h
after refeeding. Body weight was measured prior to the fast and 24 h
after refeeding.
Fecal IgA analysis
Fecal IgA extraction was performed as previously described (22). Fresh
fecal samples were collected at 0900 on two occasions: one day prior
to enrichment assignment (day 6) and on the final day of the study
(day 40). Samples were stored on ice then frozen at −20 °C until extrac-
tion. Samples were dried at 50 °C and homogenized. One milliliter
0.01 mol/l phosphate buffered saline was added to 0.1 g of sample and
vortexed for 1 hour. Samples were then centrifuged at 2,500 rpm for
20 min and the supernatant transferred to a clean test tube. Samples
were assayed at a 1 in 2000 dilution using a Rat IgA Enzyme-Linked
Immunosorbent Assay Quantitation Kit purchased from Bethyl
Laboratories. (Montgomery, Texas).
Plasma corticosterone and ACTH
Rats were killed by decapitation in the early light phase on day 40
of the study. Trunk blood was collected in plastic EDTA tubes for
ACTH analysis and in plastic lithium heparin tubes containing 1,000
Kallikrein Inactivator Units of aprotinin (Nordic Pharma, Reading,
UK) for corticosterone analysis. Plasma was separated by centrifuga-
tion, snap frozen in liquid nitrogen and stored at –20 °C until analysis
by radioimmunoassay.
Plasma corticosterone was measured using a radioimmunoassay
kit from MP Biomedicals (Orangeburg, NY) for which the intra- and
inter-assay coefficients of variation were <10% and 7%, respectively.
Plasma ACTH was measured using an immunoradiometric assay pur-
chased from BioSource Europe S.A. (Nivelles, Belgium) for which the
intra- and inter-assay coefficients of variation were 6.4% and 6.2%,
respectively.
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Methods and Techniques

Statistics a 160 Control

The study was powered to detect a 10% change in food intake and body Chew sticks
weight with power of 80% at a level of significance of 0.05. All data are 140
Tubing
presented as mean ± s.e.m. other than data from behavioral ­studies, 120
Chew sticks and tubing
which are presented as median (inter-quartile range). Cumulative

Body weight change (g)

food intake and body weight data were analyzed using the general- 100
ized estimating equation (Stata 9; StataCorp LP, College Station, TX).
80
Behavioral studies were analyzed using the Kruskall–Wallis test (Systat,
Evanston, IL) as data were nonparametric. Acute food intake studies 60
and radio­immunoassay data were analyzed using a one-way ANOVA
with Tukey’s post hoc test (GraphPad Prism version 5 for Windows; 40
GraphPad Software, San Diego, CA). A one-way ANOVA with Tukey’s
20
post hoc test was used to compare fecal IgA data between groups at
individual time points, and changes within groups over time were 0
compared using a paired t-test (GraphPad Prism; GraphPad Software). 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
P values <0.05 were taken as significant. Day

b Control
600 Chew sticks
Results
Tubing
The effect of environmental enrichment on food 500 Chew sticks and tubing

Cumulative food intake (g)

intake and body weight
There was no difference in body weight between groups at the 400

beginning of the study. Environmental enrichment had no sta-

tistically significant effect on body weight or cumulative body
weight gain during the 5-day acclimatization period or over
the 7-day study period (Figure 1a). At the end of the entire
40-day experimental period there was no significant difference
in absolute body weight between groups (body weight day 40
(g): 394.0 ± 10.3 (control); 414.8 ± 9.9 (chew sticks); 407.3 ±
12.1 (tubing); 408.0 ± 8.1 (chew sticks and tubing), n = 6–8 per
group).
There was no difference between 24-h food intake the day
prior to (day 6) and 24-h food intake the day following (day 7)
assignment of environmental enrichment (average 24 h food
intake (g): 27.8 ± 1.0 (control day 6), 27.6 ± 0.5 (control day 7);
28.3 ± 0.7 (chew sticks day 6), 27.7 ± 0.8 (chew sticks day 7);
28.4 ± 0.5 (tubing day 6), 28.0 ± 0.8 (tubing day 7); 27.8 ± 0.5
(chew sticks and tubing day 6), 28.5 ± 0.7 (chew sticks and
tubing day 7), n = 6–8 per group). There was no significant dif-
ference in cumulative food intake between groups at any time
point following enrichment (Figure 1b).
The effect of environmental enrichment on behavior
Control animals displayed significantly more rearing behavior
than all animals with environmental enrichment (P < 0.05, n =
6–8 per group) (Table 1).
To examine the general behavioral pattern, behaviors were
subsequently grouped into four categories: feeding and drink-
ing; activity (rearing, burrowing, and locomotion); grooming;
and resting (head down, and sleeping) as previously described
(21,23). The control group displayed significantly more “active”
behavior than the two groups with tubing (P < 0.05, n = 6–8
per group) (Table 1).
If an animal was noted to be sitting within the tubing dur-
ing behavioral analysis, the behavior the rat was performing at
that time was recorded to allow comparison with rats housed
without tubing. In all cases the behavior recorded was head
down. The percentage of time animals spent in their tubing
during the 1-h study period was 4.2% for the group supplied
300
200
100
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Day
Figure 1  Effect of environmental enrichment on cumulative body weight
gain and cumulative food intake in male Wistar rats. (a) Cumulative
body weight gain (g) and (b) cumulative food intake (g) in rats housed
with no environmental enrichment, two chew sticks, plastic tubing or
chew sticks and tubing from days 1 to 19. Vertical arrow represents
enrichment assignment on day 7. Animals were subsequently given
5 days to acclimatize. Body weight and food intake were measured daily
from day 13 to day 19. Data is expressed as mean ± s.e.m. for all groups
(n = 6–8 per group).
with tubing alone and 5.6% for those supplied with both chew
sticks and tubing (n = 6–8 per group).
The effect of environmental enrichment on
fasting-induced refeeding
Following refeeding after a 24 h fast no significant differences
in food intake were observed between groups at any time point
studied. The 0–1 h food intake was comparable between all
groups and decreased at subsequent time points, as expected
(24) (Table 2).
There was no significant difference between groups in the
amount of body weight lost during the 24 h fast (body weight
lost during 24 h fast (g): 18.9 ± 3.3 (control); 22.8 ± 1.8 (chew
sticks); 21.2 ± 1.6 (tubing); 22.1 ± 1.6 (chew sticks and tubing),
n = 6–8 per group).
Fecal IgA
There was no significant difference in fecal IgA between groups
prior to the assignment of enrichment (Table  3). However,

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Table 1 Effect of environmental enrichment on behavior and grouped behavior

Behavior Control Chew sticks Tubing Chew sticks and tubing
Effect of environmental enrichment on behavior a

  Feeding 11.0 (8.5–12.5) 11.5 (9.5–13.5) 15.0 (14.3–17.3) 9.5 (5.8–12.3)

  Drinking 0.0 (0.0–1.0) 1.0 (0.3–1.0) 0.0 (0.0–0.0) 1.0 (0.0–2.0)
  Rearing 13.0 (11.0–14.5) 8.5 (8.0–9.0)* 5.0 (3.3–6.0)** 8.0 (4.0–10.3)*
  Locomotion 3.0 (1.5–3.0) 2.5 (2.0–4.5) 2.5 (1.3–3.8) 1.5 (0.8–3.0)
  Grooming 6.0 (5.0–8.0) 7.0 (6.3–8.5) 9.0 (4.8–12.5) 10.5 (8.0–16.0)
  Burrowing 0.0 (0.0–0.5) 0.0 (0.0–1.5) 0.0 (0.0–0.8) 0.0 (0.0–0.0)
  Head down 0.0 (0.0–0.5) 1.0 (0.0–2.0) 0.5 (0.0–1.0) 1.0 (0.0–5.5)
  Sleeping 0.0 (0.0–0.0) 0.0 (0.0–0.0) 0.0 (0.0–0.0) 0.0 (0.0–0.0)
Effect of environmental enrichment on grouped behaviora,b
  Feeding and drinking 13.0 (0.5–14.0) 13.0 (10.5–14.8) 17.0 (14.3–18.0) 17.0 (14.3–18.0)
  Activity 16.0 (14.0–17.0) 15.0 (11.0–15.8) 8.0 (5.5–9.0)** 9.0 (6.5–11.5)*
  Grooming 6.0 (5.0–8.0) 7.0 (6.3–8.5) 9.0 (4.8–12.5) 11.0 (8.0–16.0)
  Nonactivity 0.0 (0.0–0.5) 1.0(0.0–2.0) 5.0 (1.3–7.8) 4.0 (0.0–7.0)
a
Animals were observed for 15 s every 5 min for 1 h in the early dark phase. Each 15-s period was subdivided into three, and the behavior of the rat during each time
period was scored (n = 6–8 per group). Data is presented as median (inter-quartile range). bBehavior was grouped into the following categories: feeding and drinking;
activity (rearing, burrowing, and locomotion); grooming; and resting (head down and sleeping).
*P < 0.05; **P < 0.01 vs. controls.

Table 2 Effect of environmental enrichment on fasting-induced refeeding

Control Chew sticks Tubing Chew sticks and tubing
Time period Mean s.e.m. Mean s.e.m. Mean s.e.m. Mean s.e.m.
0–1 h 7.54 0.72 7.35 0.71 8.52 1.04 7.49 0.29
1–2 h 1.50 0.68 3.25 0.86 1.18 0.60 3.35 0.55
2–4 h 1.35 0.88 0.87 0.69 1.27 0.93 0.95 0.48
4–8 h 2.29 0.62 2.77 0.86 1.37 0.60 0.44 0.24
8–24 h 26.70 2.01 28.53 1.14 29.50 1.07 28.99 1.36
0–2 h 9.04 1.21 10.60 1.21 9.70 1.52 10.84 0.39
0–4 h 10.40 0.87 11.47 0.82 11.00 1.04 11.79 0.60
0–8 h 12.69 1.00 14.23 0.86 12.33 1.06 12.19 0.27
0–24 h 39.39 2.42 42.77 0.65 41.83 1.46 41.54 1.08
Animals were fasted from 0900 for 24 h on day 25 of the study (n = 6–8 per group). The following morning at 0900 a predetermined quantity of food was returned to the
cages. Food intake was measured at 1, 2, 4, 8, and 24 h after refeeding. Data is presented as mean and s.e.m. food weight in grams.

Table 3 Effect of environmental enrichment on fecal immunoglobulin A (IgA)

Control Chew sticks Tubing Chew sticks and tubing
Acclimatization period (day 6) 45.47 ± 5.68 39.21 ± 6.27 42.64 ± 6.93 32.43 ± 5.29
Termination of study (day 40) 5.33 ± 1.43** 19.52 ± 6.78 21.48 ± 5.74 48.87 ± 18.37*
Fecal samples were collected prior to the assignment of enrichment (acclimatization period) and at the termination of the study. IgA was extracted and quantified by
enzyme-linked immunosorbent assay. Data is expressed as mean ± s.e.m. (µg/ml) for all groups (n = 6–8 per group).
*P < 0.05 vs. controls,**P < 0.01 vs. controls during acclimatization period.

there was a significant increase in fecal IgA in animals housed
with both chew sticks and tubing compared to controls at the
end of the study (P < 0.05, n = 6–8 per group) (Table 3).
Within the control group there was a significant reduc-
tion in IgA in fecal samples collected on day 40 compared to
those collected prior to the assignment of enrichment on day 6
(P < 0.01, n = 7) (Table 3). This change was not observed in
any other groups.
Plasma corticosterone and ACTH
There was no significant difference in plasma corticosterone
(Table 4) or ACTH (data not shown) between groups.

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Table 4 Effect of environmental enrichment on plasma corticosterone
Control
Plasma corticosterone (ng/ml) 34.21 ± 9.47
Trunk blood was collected at the termination of the experiment and plasma corticosterone measured by radioimmunoassay. Data is expressed as mean ± s.e.m. for all
groups (n = 6–8 per group).
Discussion
The aim of this work was to determine the effect of environ-
mental enrichment on body weight, food intake, behavior,
and fecal and plasma stress hormones in male Wistar rats.
Although the welfare benefits of environmental enrichment
have been well characterized, the effects on experimental vari-
ability are not clear. Enrichment provides a significant refine-
ment to animal husbandry because single housing, a necessity
when studying energy homeostasis, is considered stressful for
social animals such as rats.
Chew sticks were chosen as a form of environmental enrich-
ment because rats show a preference for chewable objects
which allow them to express an important species-specific
behavior (9). Tubing was chosen as the second form of enrich-
ment as rats are prey species and show a preference for cages
containing dark, enclosed spaces to hide in (25). Although
these forms of enrichment are not as complex as some used in
other studies, they are cheap, simple to reproduce and easy to
implement. It should be noted that although bedding can be
considered a form of enrichment, all animals in our study were
supplied with sawdust bedding in accordance with European
guidelines (4). Care should be taken when choosing bedding
material for studies measuring food intake or other metabolic
parameters. If an edible bedding material such as corn cob
bedding is provided, rats may eat the bedding material, par-
ticularly during a fast. This has been reported to confound end
point measurements including plasma glucose and triglycer-
ides (26). However, the sawdust bedding provided in this study
was inedible.
Environmental enrichment had no effect on body weight
gain or food intake over the 5-day acclimatization period or
the 7-day study period. Although the growth curves began to
diverge by day 7, at the end of the entire 40-day study there was
no significant difference in body weight between groups. These
findings are in agreement with previous studies in mice which
showed that the addition of plastic refuges or pipes to the cages
of adult animals had no effect on food intake and weight gain
over a 12-day period (27,28).
The effect of environmental enrichment on the refeeding
response to a 24 h fast was investigated because fasting animals
is a standard procedure often used prior to administration of
an anorectic agent (6). Importantly, there was no significant
difference in the refeeding response to a 24 h fast between
groups. This suggests that environmental enrichment does not
affect variability in this experimental paradigm and that estab-
lished acute food intake protocols can be used as normal in
enriched animals.
We found no chronic effects of environmental enrichment
on food intake and body weight. However, acute changes in
obesity | VOLUME 19 NUMBER 8 | August 2011
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Methods and Techniques
Chew sticks Tubing Chew sticks and tubing
34.11 ± 6.23 27.83 ± 5.42 19.38 ± 4.44
the cage environment have previously been shown to reduce
baseline food intake and thus mask the anorectic effect of
intra-peritoneal administration of the gut hormones peptide
YY3-36 and oxyntomodulin in male Wistar rats (5). We found
no difference in 24-h food intake the day prior to and the day
following the addition of enrichment, suggesting that these
forms of enrichment do not acutely alter food intake in rats.
However, further work is required to confirm that enrichment
does not have more subtle effects, for example, influencing
the magnitude of the anorectic or orexigenic effects of other
compounds.
Behavior was studied in the early dark phase as this is
when rats are most active. A significant reduction in rear-
ing was observed in the three groups with enrichment com-
pared to controls. Additionally, when behaviors were grouped
into categories, control rats demonstrated significantly more
“active” behavior (rearing, locomotion, and burrowing) than
the two groups with tubing. This is in agreement with work in
which rats singly housed in barren cages showed significantly
increased locomotion, rearing, and exploratory behavior com-
pared to animals housed with environmental enrichment (18).
These behaviors are hypothesized to be stress-related, as intrac-
erebroventricular administration of corticotrophin-releasing
hormone significantly increases locomotion and rearing (29).
However, rearing and exploratory behavior can also be inter-
preted as reflecting a reduced state of fearfulness (30), while
grooming can be considered a stress-related behavior in rats
(31). Although we did not observe any differences in grooming
behavior between groups, it is possible that the differences in
rearing observed reflect altered fearfulness. There were no dif-
ferences in body weight gain between groups at the end of the
study, suggesting that the differences in behavior observed were
insufficient to significantly alter long-term energy expenditure
and hence body weight.
The percentage of time animals housed with tubing spent
within the tubing was also recorded and found to be relatively
low. This may be because the behavioral analysis was carried
out at night when the animals are relatively active; through-
out the course of the study, animals were observed to use their
­tubing to sleep in during the day.
The reduction in stress-related behavior often observed
when animals are housed with environmental enrichment may
be due to effects on neuronal plasticity and cerebral develop-
ment. By improving learning and memory, enrichment may
allow animals to adapt to new environments more quickly and
hence reduce stress (17).
IgA is an antibody found in saliva, tears, and respiratory and
intestinal secretions. It is well known that chronic HPA axis
activation induces immunosuppression (13). In rats, fecal IgA
1719
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Methods and Techniques
decreases in response to social stress, surgery, and housing in
metabolic cages and it is therefore a useful marker for assess-
ing long-term well-being (15,32). Fecal sampling also confers
advantages over other methods, for example plasma and saliva
analysis, because samples can be collected easily without dis-
turbing the animal.
The effect of environmental enrichment on fecal IgA has
not previously been well characterized. There was no differ-
ence in fecal IgA between groups during the acclimatization
period. However, at the end of the study there was a significant
increase in IgA in animals housed with both types of enrich-
ment, suggesting these animals had lower HPA axis activity
compared to controls. Interestingly, within the control group,
fecal IgA was significantly lower on day 40 compared to dur-
ing the acclimatization period. This effect was not seen in any
other group. This suggests that single housing causes a chronic
stress reaction in male Wistar rats, which is attenuated by envi-
ronmental enrichment.
Corticosterone is released from the adrenal cortex of rodents
in response to stress. It is secreted in a diurnal rhythm; in rats,
plasma corticosterone levels peak in the early dark phase and
reach a nadir in the early light phase. We collected plasma cor-
ticosterone in the early light phase to allow small differences
between groups to be detected (33). Rats housed with both
tubing and chew sticks had the lowest plasma corticosterone,
which suggests that environmental enrichment (particularly
tubing) may reduce stress. However, there was no difference in
plasma ACTH between groups. This mismatch between corti-
costerone and ACTH may be due to the ultradian rhythm of
the HPA axis. In addition to the daily circadian rhythm, bursts
of corticosterone secretion occur approximately once an hour
in the rat and hence the matching of plasma corticosterone and
ACTH pulses is often poor (34).
It has previously been shown that male and female rats
housed singly with environmental enrichment have sig-
nificantly lower baseline plasma ACTH and corticosterone
compared to those housed without enrichment (7). We were
not able to replicate this finding, perhaps because we only
measured plasma corticosterone at one time point, rather
than continuously over several days. Additionally, our
group sizes may not have been sufficiently large to detect
this change, as our study was powered to detect differences
in body weight and food intake rather than changes in the
HPA axis. However, taken together, the changes detected in
behavior and fecal IgA suggest a reduction in HPA axis activ-
ity in animals housed with environmental enrichment and
confirm previously reported benefits of enrichment to animal
welfare.
Young adult male Wistar rats are commonly used to study
energy homeostasis. However, further work is required to
investigate the effect of environmental enrichment on food
intake and body weight in different strains of rats, using a
range of diets. In particular, the effect of environmental enrich-
ment in Sprague Dawley rats supplied on a high fat diet, which
are known to show a wide variation in body weight gain (35),
would be of interest.
1720
In conclusion, this work has shown that the provision of
environmental enrichment to singly housed male Wistar rats
does not influence food intake or body weight over a short
period of time. These data suggest that environmental enrich-
ment should be routinely included in food intake and body
weight assessment protocols in rats.
Acknowledgments
This work was funded by two NC3Rs/LASA Small Awards. The animals
were kindly donated by Charles River Laboratories. K.E.L.B. is supported
by a Jean Shanks Foundation Studentship. Both K.G.M. and K.L.S. hold
Biotechnology and Biological Sciences Research Council New Investigator
Awards. The Department is funded by the National Institute for Health
Research Biomedical Research Centre Funding Scheme. Funded under
FP7-HEALTH-2009-241592 (European Union) EurOCHIP.
Disclosure
The authors declared no conflict of interest.
© 2011 The Obesity Society
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