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Comparative Medicine
Copyright 2016
by the American Association for Laboratory Animal Science
Vol 66, No 5
October 2016
Pages 384–391
384
The visual system of mammals encompasses the anatomic
structures of the eye and the primary optic tract. Because rats
are nocturnal animals, their retinas are rod-dominated but also
contain cones. This visual system contains 2 main classes of cones,
which contain photopigment with spectral sensitivity in either
the 359-nm (UV) or 509-nm (M cone) wavelengths. Rudimen-
tary color discrimination is believed to be supported by these 2
cone classes, but this discrimination may be only dichromatic in
nature.27 This dichromatic nature is a characteristic of the visual
system and is reected in rats’ preference for a dark environment:
darkness likely is preferred due in part to the effects of light on
retinal degeneration over time, which perhaps is painful, causing
rats to shy away from light.33
Changes in lighting intensity, duration, and wavelength (visu-
ally perceived as color or tint) at various times of the day can
disrupt circadian rhythms of metabolism and endocrine hormone
concentrations.7,12,13,15,41 These responses are primarily elicited by
means of a nonvisual system, also located in the retina, through
intrinsically photosensitive retinal ganglion cells5,8,11,17,21,24,25,32 and
not through the visual primary optic tract. Circadian rhythms
are entrained by the nonvisual light–dark cycle through signals
from the retinohypothalamic tract to the suprachiasmatic nucle-
us (SCN). The SCN regulates circadian rhythms and is termed
the ‘master biologic clock.’5,7 Wavelengths between 450 and 484
nm (that is, blue light) are the strongest inciters of neuroendo-
crine and circadian responses in mammals, however high-in-
tensity, longer-wavelength (that is, red) light can have this effect
also.7,14,15,23,35,41 There is now overwhelming evidence of the inu-
ence of the retinohypothalamic tract system on the regulation of
behavior20 and metabolism.7,22,30,41
Prior research12-16,41 from our laboratory revealed significant
disruptions of various circadian rhythms and increased tumor
growth from light leaks around doorways at night. In addition,
we investigated the effect of spectral transmittance (cage tint)
through standard laboratory caging in female pigmented athymic
nude rats and male Sprague–Dawley rats12,41 and showed that
these animals developed chronobiologic disruptions in various
plasma measures of physiology and metabolism. Although the
circadian rhythm of total fatty acids (TFA) in plasma remained
unchanged, all other metabolic and physiologic rhythms were
signicantly altered when exposed to amber-, red-, or blue-tinted
caging as compared with clear caging. We also have shown that
Original Research
Effects of Colored Enrichment Devices on
Circadian Metabolism and Physiology in Male
Sprague–Dawley Rats
Melissa A Wren-Dail,1,2,* Robert T Dauchy,2 Tara G Ooms,3 Kate C Baker,4 DavidEBlask,2 Steven M Hill,2 Lynell M Dupepe,1
and Rudolf P Bohm, Jr4
Environmental enrichment (EE) gives laboratory animals opportunities to engage in species-specic behaviors. However, the ef-
fects of EE devices on normal physiology and scientic outcomes must be evaluated. We hypothesized that the spectral transmittance
(color) of light to which rats are exposed when inside colored enrichment devices (CED) affects the circadian rhythms of various
plasma markers. Pair-housed male Crl:SD rats were maintained in ventilated racks under a 12:12-h light:dark environment (265.0
lx; lights on, 0600); room lighting intensity and schedule remained constant throughout the study. Treatment groups of 6 subjects
were exposed for 25 d to a colored enrichment tunnel: amber, red, clear, or opaque. We measured the proportion of time rats spent
inside their CED. Blood was collected at 0400, 0800, 1200, 1600, 2000, and 2400 and analyzed for plasma melatonin, total fatty acids,
and corticosterone. Rats spent more time in amber, red, and opaque CED than in clear tunnels. All tubes were used signicantly
less after blood draws had started, except for the clear tunnel, which showed no change in use from before blood sampling began.
Normal peak nighttime melatonin concentrations showed signicant disruption in the opaque CED group. Food and water intakes
and body weight change in rats with red-tinted CED and total fatty acid concentrations in the opaque CED group differed from
those in other groups. These results demonstrate that the color of CED altered normal circadian rhythms of plasma measures of
metabolism and physiology in rats and therefore might inuence the outcomes of scientic investigations.
Abbreviations: CED, colored enrichment devices; EE, environmental enrichment; SCN, suprachiasmatic nucleus; TFA, total fatty acids
Received: 11 Nov 2015. Revision requested: 05 Jan 2016. Accepted: 12 May 2016.
1Departments of Comparative Medicine and 2Structural & Cellular Biology, Tulane
University School of Medicine, New Orleans, Louisiana, 3Section of Laboratory Animal
Medicine, IIT Research Institute, Chicago, Illinois and 4Division of Veterinary Medicine,
Tulane National Primate Research Center, Covington, Louisiana
*Corresponding author. Email: mwren@tulane.edu
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Effects of colored enrichment devices on circadian rhythms
385
Mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse
adenovirus types 1 and 2, Hantaan virus, Encephalitozoon cuniculi,
cilia-associated respiratory bacillus, parvovirus NS1, rat parvo-
viruses, rat murine virus, and rat theilovirus as well as external
and internal parasites; all test results were negative. Rats had
free access to a commercial diet (no. 5053 Irradiated Laboratory
Rodent Diet, Purina, Richmond, IN) and acidied water. Qua-
druplicate determinations of this diet’s TFA composition were
reported previously.41 Food and water intakes were measured 2 or
3 times weekly (every 2 to 3 d), and body weight was measured
weekly throughout the 36-d experimental period. Food and water
were measured (500 g and 500 mL, respectively) and placed in
the stainless steel holder or water bottle. After 2 or 3 d, each was
removed from each cage and measured. Any food on the cage
oor was added to the remaining amount (counted as uneaten).
Each of the 2 rats were assumed and recorded to have consumed
half, but for statistical analysis, each counted as one measure-
ment, such that each treatment group had 3 measurements per
time point (n = 3).
Caging, enrichment devices, and lighting regimen. Upon arrival,
rats were housed in standard translucent ventilated laboratory
rat cages (2 rats per cage; 10.5 in. × 19.0 in. × 8.0 in.; wall thick-
ness, 0.1 in.) with identical stainless steel lids (for cradling food
and the water bottle; catalog no. 10SS, Ancare, Bellmore, NY) that
were covered with matching polysulfone translucent microlter
tops (catalog no. N10MBT, Ancare) for a 1-wk acclimation pe-
riod. After this time, subjects were assigned at random to 1 of 4
treatment groups that contained either an amber, red (no. K3326,
polycarbonate translucent amber, and no. K3325, polycarbonate
translucent red; BioServe Flemington, NJ), clear (polycarbonate
translucent clear, model no. R20, Pharmacia, Uppsala, Sweden),
or opaque (PVC ASTM F891-10, Charlotte Pipe, Charlotte, NC)
tunnel (length, 6 in.; inner diameter, 3 in.; wall thickness, 1/8
in.). Rats were housed in pairs so that each animal had ample
opportunity to rest inside the tunnel; thus, each treatment group
(n = 6 animals) incorporated 3 separate cages. The CED was con-
tinuously in the cage throughout the 36-d experiment and was
replaced weekly during cage changes with a clean CED of the
same tint. The rats were maintained in climate-controlled rooms
(21 to 24 °C; 50% to 55% humidity) with diurnal lighting (12:12-h
light:dark photoperiod; lights on, at 0600). As described previous-
ly,12 animal rooms were lighted with overhead white uorescent
lamps and were completely devoid of light contamination during
the dark phase. All cages were rotated daily from left to right on
Sprague–Dawley rats have signicant circadian disruptions in
melatonin, corticosterone, leptin, insulin, and glucose concentra-
tions when exposed to various color caging during the day.12,41
These disruptions in plasma levels included duration, phasing
(timing), amplitude, or some combination of these elements.
On inspection of items currently placed into the animal cage for
environmental enrichment (EE), we noticed that many of these
items are color-tinted. Most are made from transparent durable
plastic for ease of cleaning and sanitation. The premise for us-
ing these color-tinted items is that they allow for monitoring and
health checking yet afford rodents the opportunity to retreat from
stressful stimuli or cage mates and to regulate body tempera-
ture.27 However, some objects added to the animals’ environment
are known to induce stress.27 Although some studies1,4,18,26,37,38,40
have explored the effect of EE on behavior, few studies4,40 have
examined the inuence of EE, particularly colored enrichment
devices (CED), on plasma measures of circadian metabolism and
physiology in laboratory animals. Therefore, we sought to deter-
mine whether various CED affected animal measures of metabo-
lism or neuroendocrine hormones.
For the current study, we explored the effect of spectral trans-
mittance exposure from commonly used CED (amber, clear, red,
and opaque; Figure 1) during the light period in male albino
Sprague–Dawley rats. We chose this rat stock because it is among
the most widely used in laboratory research, comprising approxi-
mately 53% of EE studies.37 Melatonin, TFA, and corticosterone
were chosen to examine common circadian rhythms through
plasma measures of metabolism and physiology. We hypothe-
sized that changing the tinting (spectral transmittance or quality)
of EE by using various CED affects circadian rhythms of these
measures in male Sprague–Dawley rats.
Materials and Methods
Reagents. HPLC-grade chloroform, ethyl ether, methanol, gla-
cial acetic acid, heptane, and hexane were purchased from Fisher
Scientific (Pittsburg, PA). Free fatty acids, rapeseed oil methyl
ester standards, boron–triuoride–methanol, potassium chloride,
sodium chloride, and perchloric and trichloroacetic acids were
purchased from Sigma Scientic (St Louis, MO). Ultrapure water
(catalog no. 400000) was purchased from Cayman Chemical (Ann
Arbor, MI).
Animals, housing condition, and diet. Male Sprague–Daw-
ley rats (age, 4 to 5 wk) were purchased from Charles River
(Crl:CD[SD]; Kingston, NY) and certied by the vendor to be free
of all known rodent bacterial, viral, and parasitic pathogens. Ac-
cording to the vendor, rats at this age were provided with only
group social enrichment during rearing. Animals were main-
tained in Tulane University School of Medicine Vivarium, which
has been an AAALAC-accredited facility since 1962. All animal
use and accompanying procedures were in accordance with The
Guide for the Care and Use of Laboratory Animals27 and were IACUC-
approved.
Rats were maintained in ventilated cages using hardwood
maple bedding (no. 7090, Sanichips, Harlan Teklad, Madison,
WI; 1 bedding change weekly). Serum samples from sentinel
animals on combined soiled bedding from project animals were
tested quarterly (Multiplex Fluorescent Immunoassay 2, IDEXX
Research Animal Diagnostic Laboratory, Colombia, MO) for rat
coronavirus, Sendai virus, pneumonia virus of mice, sialodacryo-
adenitis virus, Kilham rat virus, Toolan H1 virus, reovirus type 3,
Figure 1. Colored enrichment devices used in this experiment were
(from left to right) translucent amber, clear, red (polycarbonate) and
opaque (polyvinyl chloride) and had identical internal diameters and
lengths.
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October 2016
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Kodak, Rochester, NY) to preserve the normal nocturnal mela-
tonin surge. Exposure at rodent eye level was 45.0 to 179.5 µW/
cm2, depending on the number and position of safelights used,
but all measurements remained in the low, safe range that does
not affect melatonin.39
Melatonin analysis. Plasma melatonin levels were measured
by using the melatonin I125 radioimmunoassay kit (Bühlmann,
Schönenbuch, Switzerland) and analyzed by using an automated
gamma counter (Cobra 5005, Packard, Palo Alto, CA) as previ-
ously described.12 Briey, C18 reverse-phase extraction columns
included in the kit were used to extract the melatonin from the
samples, by using 0.125 mL of plasma for the 2400 and 0400 time
points from 3 animals chosen at random. For the 0800, 1200, 1600,
and 2000 time points, 0.125 mL of plasma from each of 2 animals
within the same treatment group were pooled to make a 0.25-mL
sample, due to the low levels of melatonin at these time points,
giving a total n number of 3. The functional sensitivity for the as-
say was 0.9 pg/mL. The intraassay precision of column extraction
and radioimmunoassay combined is 8.2%.
Fatty acid extraction and analysis. Plasma free fatty acids were
extracted as previously described12 from 0.05-mL samples in du-
plicate by using an internal standard consisting of heptadecanoic
acid (100 µg) dissolved in chloroform. A gas chromatograph t-
ted with a ame ionization detector, auto injector (both adjusted
to 220 °C), and integrator (model 58990A, Hewlett Packard, Palo
Alto, CA) was used to analyze the methyl esters of fatty acids ac-
cording to their retention time compared with known standards.
A 0.25 mm × 30 cm capillary column with helium as the carrier
gas was used for separations. The minimum detection level for
the assay was 0.05 µg/mL.
ELISA of corticosterone. Plasma samples were prepared in du-
plicate for measurement of corticosterone levels by using chemi-
luminescent ELISA diagnostic kit (catalog no. 55-CORMS-E01,
ALPCO, Salem, NH) and measured with a microplate reader (450
nM; VersaMax, Molecular Devices, Sunnyvale, CA) as previously
described.12 Detection sensitivity for corticosterone was 4.5 ng/
mL, the lower limit of detection was 15 ng/mL and the coefcient
of variation was less than 4.0%.
Statistical analysis. All data are compared by using one-way
ANOVA followed by the Bonferroni multiple comparison test
and 2-tailed paired or unpaired t test by using Prism software
(GraphPad, La Jolla, CA) and presented as mean ± 1 SD unless
otherwise noted. Figures are double-plotted to facilitate visualiza-
tion of circadian time. The sample size for TFA analysis is n = 6,
except for the following time points due to low sample volume:
0800: amber, n = 5; red n = 4; 1200: amber, clear, and red, n = 5
each; 1600: amber, red, and clear, n = 5 each; 2000: amber n = 5;
red and clear, n = 4 each. Statistical signicance among the group
means was set at a P value of less than 0.05.
Results
Animal room illumination and CED measurements. Mean day-
time animal room illumination at the left, right, and center of the
rack 1 m above the oor with the detector facing the ceiling had
relatively small variance and measured 265.0 ± 43.2 lx (n = 21 [7
wk, 3 positions]). Measurements in the inside front of the cages
were averaged for all cage positions and had little variance
(57.6 ± 10.5 lx, n = 84 [7 wk, 12 cages]). Laboratory CED used in this
experiment are shown in Figure 1. Measurements of radiometric
irradiance (lx) from inside the CED positioned in the middle of
the 2 ventilated rack rows to ensure that there were no signicant
differences in lighting intensity inside the cages (at rodent eye
level). Weekly at 0800 during the course of this experiment, the
animal room lighting intensity (spectral power distribution) was
measured at 1 m above the oor in the right, left, and center of
the individually ventilated cage rack, and at the front within the
animal cages, as well as facing up inside the enrichment device,
by using a radiometer–photometer and radiance detector with
lter and diffuser which were calibrated regularly, as previously
reported.12 Cage cleaning procedures12 did not cause any varia-
tions in light-intensity measurements throughout the course of
the study.
Data collection timeline. During days 1 through 7, rats were ac-
climated to the facility. On day 8, animals were housed with their
respective CED; behavioral observations were recorded on days
10 and 11 (phase 1) and 24 and 25 (phase II). On days 14, 18, 22,
26, 30, and 34, blood samples were collected at a single circadian
time point each day. The experimental period was 36 d in total.
Behavioral observations. Instantaneous scan sampling2 with a
15-min intersample interval was used to quantify time spent in-
side each respective CED during 2 separate phases of the experi-
ment. On experimental days 10 and 11, before any blood draws
were started (phase 1), 1 of 3 coinvestigators observed the rats for
120 min; animals were coded as being inside the tunnel when the
entire head and shoulders were within it. Every 15 min for 2-h
segments, we recorded the animal’s position (inside or outside of
CED) for all rats during the 12-h light phase. On day 10, recording
occurred from 0800 to 1000, 1200 to 1400, and 1600 to 1800. On
day 11, alternate times were recorded (0600 to 800, 1000 to 1200,
1400 to 1600). For phase 1 sampling, rats had their respective CED
inside the cage for 3 and 4 d (experimental days 10 and 11, respec-
tively) and had not undergone any other experimental manipula-
tions at this point. Dark-phase measurements were not taken in
complete darkness because there is no spectral transmittance of
any light through the CED that might affect the animals. Phase
2 occurred on experimental days 24 and 25, when instantaneous
scan sampling was repeated. A total of 720 min (12-h light phase)
was recorded for each observation phase. Behavioral data were
collected no sooner than 2 d after any blood draw.
Blood collection. After a 2-wk exposure under the respec-
tive CED and lighting conditions, blood was collected from rats
through cardiocentesis under gas anesthesia for a total of 6 low-
volume collections (0400, 0800, 1200, 1600, 2000, and 2400; 0.5 to
1.0 mL, less than 5% total blood volume per blood draw); draws
were 3 to 5 d apart, as previously reported.12,16 Briefly, a 70%
CO2–30% air mixture was passed into an acrylic gas anesthesia
chamber.6,19,31 Upon loss of righting reex, each rat was removed
and allowed to breathe room air while blood was collected by
using a 3-mL luer-lock syringe (Tyco Healthcare, Manseld, MA)
and 25-gauge, 3/8-in. needle (Tyco) moistened with sodium
heparin (5000 USP U/mL, Sagent Pharmaceuticals, Schaumburg,
IL). At our facility over the last decade of cardiocentesis in rats,
mortality and morbidity have been less than 5%.16 Whole-blood
samples were centrifuged at 12,000 × g for 10 min at 4 °C (model
Micro17R, accuSpin centrifuge, Fisher Scientic, Fair Lawn, NJ)
for plasma collection. Plasma samples were stored at −20 °C until
assayed for melatonin, TFA, and corticosterone.
Dark-phase sampling was performed in less than 1 min (from
removal until recovery) in the home cage under 1 or 2 low-intensity
safelight red lamps (120 V, 15 W; 1A model B, catalog no. 1521517;
cm15000139.indd 386 10/7/2016 10:00:58 AM
Effects of colored enrichment devices on circadian rhythms
387
male Sprague–Dawley rats with free access to food displayed a
signicant rhythm in all CED treatment groups (Figure 6), which
reached the nadir at 1600 (1041 ± 43 µg/mL) and peaked at 0400
(5598 ± 161 µg/mL).
Plasma corticosterone. Plasma corticosterone circadian rhythms
are depicted in Figure 7. Values for plasma corticosterone in rats
of all CED treatment groups followed diurnal rhythms, which
increased continuously from the nadir at 0800 until their peak at
2000. At the 2400 and 0400 time points, the red CED group’s val-
ues were signicantly lower (P = 0.0121) than those for the clear
CED control group (2400: red, 12.1 ± 2.2 ng/mL; clear, 20.6 ± 1.4
ng/mL; 0400: red, 2.2 ± 1.2 ng/mL; clear, 12.3 ± 0.5 ng/mL; P =
0.025), n = 3 per treatment group).
each cage were made daily (amber, 2.4 ± 0.7 lx [n = 15, P = 0.0046];
red, 1.3 ± 0.5 lx [n = 12, P < 0.0001]; opaque, 1.8 ± 0.5 lx [n = 7,
P < 0.0001); and clear, 3.9 ± 1.8 lx [n = 14]), and the measurements
for amber, red, and opaque CED differed from that for the control
clear CED. In addition, irradiance differed (P < 0.0001) between
amber and red CED. Sample sizes (n) for statistical analysis dif-
fered due to the exclusion of measurements taken when only the
running lighting (1/2 task lighting) was illuminated in the room,
which was corrected for subsequent measurements.
Behavioral observations. Figure 2 depicts the average amount
of time that male rats (n = 6 per treatment group; 3 cages) chose
to spend inside each CED, recorded after a 10-d exposure (before
blood draws) and again after a 24-d exposure, which was 2 or 3 d
after a scheduled blood draw. Before beginning scheduled blood
sampling (phase 1, days 10 and 11), rats spent signicantly more
time in the red (mean ± SEM, 290 ± 74 min, P = 0.0059), amber
(110 ± 28 min, P = 0.008), and opaque (68 ± 20 min, P = 0.0248)
CED compared with the clear CED tunnel (8 ± 5 min). Rats used
all tunnels significantly less during the blood-sampling phase
(phase 2, days 24 and 25; red: 38 ± 22 min, P = 0.0312; amber: 10 ±
9 min, P = 0.0305; opaque: 8 ± 3 min, P = 0.0367) than before blood
collection, except for the clear tunnel (8 ± 5 min), which showed
similar use during both phases.
Dietary and water intakes and body weight. Daily dietary intake
(P < 0.0001) and water intake (P = 0.006) differed significantly
between the red and clear CED groups (Figure 3), and dietary
intake differed between the opaque and clear CED groups (P =
0.0225; food intake [n = 168; 14 measurements, 3 cages, 4 treat-
ment groups]: clear, 27.0 ± 2.6 g; amber, 27.0 ± 2.5 g; red, 30.6 ± 2.9
g; and opaque, 28.6 ± 3.4 g; water intake [n = 168]: clear, 43.1 ± 3.7
mL; amber, 42.0 ± 4.1 mL; red, 47.3 ± 7.4 mL; and opaque, 43.4 ±
4.7 mL). In addition, body weight change differed signicantly
between amber (P = 0.0023) and red (P = 0.0022) CED groups
compared with the clear control CED group (clear, 269.3 ± 40.8 g;
amber, 260.5 ± 40.0 g; red, 280.2 ± 42.6 g; opaque, 274.5 ± 44.0 g;
n = 36 [6 measurements, 6 rats per group]) over the 36-d experi-
mental period (Figure 4).
Plasma melatonin. Figure 5 depicts diurnal rhythms of plasma
melatonin concentrations (mean ± SEM) for each CED treatment
group (n = 3 for all groups). Melatonin concentrations were low
for all treatment groups at 0800, 1200, and 1600 (23.2 ± 6.8 pg/
mL), as expected for rats during the light phase, although the
hormone levels were signicantly higher at the 0800 time point
for the red CED group (32.5 ± 0.50 pg/mL; P = 0.035) as compared
with the clear group (24.9 ± 4.13 pg/mL). All rats in all 4 CED
groups demonstrated significant circadian rhythms in plasma
melatonin concentrations. At 2000, melatonin levels in amber
(26.9 ± 1.8 pg/mL; P = 0.012) and opaque (23.5 ± 1.6 pg/mL;
P = 0.035) CED groups were signicantly higher than in the clear
CED group (15.3 ± 4.2 pg/mL). Peak levels of melatonin occurred
at 2400 for all CED groups, but the concentration in the opaque
CED group (83.5 ± 33.3 pg/mL; P = 0.20) was signicantly lower
than that for the clear CED group (247.0 ± 68.0 pg/mL). At 0400,
the amber CED group melatonin level (252.0 ± 42.2 pg/mL)
was signicantly higher than the value for the clear CED group
(78.2 ± 57.1 pg/mL).
Plasma TFA. Significant differences for plasma TFA concen-
tration between clear and opaque CED groups (P = 0.0231)
occurred at 0400 (clear, 5387 ± 77 µg/mL; opaque: 5598 ± 160
µg/mL, n = 6 per treatment group). All plasma TFA values in
Figure 2. Instantaneous scan sampling for the time (mean ± SEM) that
rats spent inside either the amber, clear, red, or opaque colored enrich-
ment device (CED) before (phase I) and during (phase II) the experi-
ment. During phase 1, rats spent signicantly (*) more time in the red
(290 ± 74 min, P = 0.0059), amber (110 ± 28 min, P = 0.008), and opaque
(68 ± 20 min, P = 0.0248) than in the clear (8 ± 5 min) CED. During phase
2, rats spent less time in all tunnels (red: 38 ± 22 min, P = 0.0312; amber:
10 ± 9 min, P = 0.0305; opaque: 8 ± 3 min, P = 0.0367) except for the
clear CED (8 ± 5 min), for which there was no difference compared with
phase 1 data.
Figure 3. Dietary (bottom, g) and water (top, mL) intakes (mean ± 1
SD) for rats housed with either amber, clear, red, or opaque colored en-
richment devices (CED). The integrated mean dietary intake differed
between the red and clear CED groups (P < 0.0001) and between the
opaque and clear CED groups (P = 0.0178), and water intake differed
(P = 0.006) between the red and clear CED groups. Food intake (n = 168,
14 measurements, 3 cages, 4 treatment groups): red, 30.5 ± 2.7 g; opaque,
28.6 ± 3.4 g; amber, 27.2 ± 2.8 g; clear, 27.2 ± 2.05 g. Water intake (n =
168): red, 47.3 ± 7.4 mL; amber, 42 ± 4.1 mL; opaque, 43.4 ± 4.7 mL; clear,
43.1 ± 3.7 mL.
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scientic validity.27 EE is an independent variable27 and refers to
the provision of a more naturalistic environment and to the ad-
dition of objects or resources to the environment for increasing
sensory and motor stimulation, the expression of species-typical
behaviors, and the repression of stereotypies.27,37 The success of
EE can be measured by using parameters of normal health, in-
cluding growth, development, and reproduction.27,40 For rodents,
EE can include bedding depth and type, which can promote spe-
cies-typical foraging, burrowing, and nest-building behaviors in
addition to opportunities for thermoregulation, as well as nest-
building materials, novel food stuffs, chew toys, tunnels, igloos
or houses, social housing, and cage modications.4,27 However,
Discussion
Animal health and wellbeing are affected by the environment,
housing, and management of the species, and these components
should account for their physical, physiologic, and behavioral
needs.27 Recent methods have been suggested to standardize ro-
dent care, so that research models are better dened and more
stable, thus requiring fewer animal subjects40 and increasing
Figure 4. Integrated body weight change (mean ± SEM, n = 6) over the
36-d experimental period differed (2-tailed t test) between the clear con-
trol group and both the amber (P = 0.0023) and red (P = 0.0022) CED
groups (red, 280.2 ± 42.6 g; amber, 260.5 ± 40.0 g; opaque, 274.5 ± 44.0 g;
clear, 269.3 ± 40.8 g).
Figure 5. Circadian rhythm of plasma melatonin concentration (mean ±
SEM) in rats housed with either amber, clear, red, or opaque colored en-
richment devices (CED). Black bars represent the dark phase; concentra-
tions with asterisks differ (P < 0.05) compared with the clear CED. Data
are plotted twice to show rhythmicity. Melatonin concentrations were
low for all treatment groups at 0800, 1200, and 1600 (23.2 ± 6.8 pg/mL)
during the light phase as expected, although the hormone levels were
signicantly higher (P = 0.0348) at the 0800 time point in the red CED
group (32.5 ± 0.50 pg/mL) as compared with the clear group (24.9 ± 4.13
pg/mL). At 2000, values for the amber (26.9 ± 1.76 pg/mL, P = 0.0116)
and opaque (23.5 ± 1.62 pg/mL, P = 0.0351) CED groups were signi-
cantly higher than for the clear (15.3 ± 4.21 pg/mL) CED group. Peak
levels of melatonin occurred at 2400 for all CED groups, but the opaque
CED group (83.5 ± 33.3 pg/mL) was signicantly lower than the clear
CED group (247 ± 68 pg/mL, P = 0.0202). At 0400, the amber CED group
melatonin level (252.0 ± 42.2 pg/mL) was signicantly higher than the
clear CED group (78.2 ± 57.1 pg/mL, P = 0.0163).
Figure 6. Circadian rhythm of plasma total fatty acids (mean ± 1 SD)
concentrations for rats housed with either amber, clear, red, or opaque
colored enrichment devices (CED). Black bars represent the dark phase;
concentrations with asterisks differ (P < 0.05) when using the clear CED
as the control. Data are plotted twice to show rhythmicity. Signicant
differences for plasma total fatty acids between clear and opaque CED
groups (P = 0.0231) occurred at 0400 (clear, 5387 ± 77 µg/mL; opaque,
5598 ± 161 µg/mL). All CED treatment groups displayed a nadir at 1600
(1041 ± 43 µg/mL) and a peak at 0400 (5598 ± 161 µg/mL).
Figure 7. Circadian rhythm of plasma corticosterone (mean ± SEM)
concentrations for rats housed with either amber, clear, red, or opaque
colored enrichment devices (CED). Black bars represent the dark phase;
concentrations with asterisks differ (P < 0.05) when using the clear CED
as the control. Data are plotted twice to show rhythmicity. All CED
groups nadir at 0800, and rise continuously until their peak at 2000 and
were not signicantly different. However, at 2400 and 0400 time points,
the red CED group’s values were signicantly lower (P = 0.0121) when
compared with those for the clear CED (2400: red, 12.1 ± 2.19 ng/mL;
clear, 20.6 ± 1.38 ng/mL; 0400: red, 2.17 ± 1.15 ng/mL; clear, 12.3 ± 0.493
ng/mL; P = 0.025).
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Effects of colored enrichment devices on circadian rhythms
389
Several difculties have been encountered in implementing EE
for laboratory rodents. First, EE can have negative effects, includ-
ing the induction of aggressive behaviors in male mice and rats,1
changes in fecundity or breeding pattern, and litter size;40 aller-
gies and skin rashes caused by bedding type; and the ingestion of
bedding or EE material.27,40 Second, difculties have been encoun-
tered with the interpretation of behavioral and chemical effects as
being positive, negative, or neutral in terms of their inuence on
health and wellbeing.40 For this reason, we believe that includ-
ing physiologic and metabolic measures in behavioral studies
is imperative so that all parameters of health and wellbeing can
be taken into account. In addition, more studies are needed to
determine whether any of these changes are biologically or clini-
cally signicant.40 Last, the literature demonstrates a lack of detail
concerning the reporting all aspects of the environment and EE.40
Reviews show the great diversity in control housing conditions,
the amount of space provided in cages, social group number,
rat strain and sex, age at onset of EE,26 duration of exposure to
EE, types of physical objects used for EE, and behavioral tests
used.37,40
Social companionship has been found to act as a form of EE.4
Social enrichment has a greater effect when compared with EE or
isolation or impoverished conditions.18 More importantly, these
affects are lasting,18 unlike the acclimation seen with physical en-
richment. Reports have demonstrated that toys used for EE have
a very short (1 d) period of interest, whereas toys related to food
retain their attraction longer, likely due to the primary reinforce-
ment of food.4,42 Given that rats in all groups spent less time in the
CED, regardless of color, during phase II compared with phase
I, acclimation to EE could have occurred. In addition, animals’
social needs should be considered,27 given that studies report ad-
verse behaviors in isolation or impoverished conditions.1,37 Single
housing for social animals requires justication and review by the
IACUC and veterinarian, and time alone should be minimized to
the shortest duration possible.27 Providing extra EE for animals
housed in isolation, in small spaces or providing visual, audi-
tory, olfactory, or tactile contact with conspecics have been rec-
ommended.27 EE can help animals escape aggression and avoid
social conicts.27 However, when reports yield evidence of object-
induced changes in normal physiology, alternatives should be
sought.
Melatonin is a neurohormone released primarily at night in re-
sponse to rhythms generated in the SCN and is the body’s biolog-
ic clock.36 As expected, melatonin concentrations were low for all
CED groups during the light phase, although the hormone levels
were statistically (but perhaps not clinically) signicantly higher
at the 0800 time point in the red CED group. None of the CED
affected the normal circadian rhythm of melatonin, as evidenced
by the coinciding peaks and troughs. At 2000, melatonin concen-
trations of the amber and opaque CED groups rose more rapidly
than did that of the clear CED group. Interestingly, the opaque
CED group’s melatonin concentration did not peak as high as
those of the other groups. These results suggest that the low level
of exposure was sufficiently significant to affect the circadian
rhythm. Alternatively, the light–dark cycle might be altered in
these rats, due to the presence of the CED, perhaps providing a
darker area in the cage, which would affect overall sensory per-
ception of light. Finally, the instantaneous scan-sampling method
used for behavioral observations may not accurately reect the
true behavior of the rats and might otherwise be measured by
little is known about the effects of these EE items on scientific
outcomes.4 We chose to evaluate the commonly used tinted rat
enrichment tunnels in this study.
We used 4 types of CED: the 2 colors (amber and red) common-
ly available from manufacturers of rat enrichment tubes; opaque
tubes, which were included because CED produced inhouse are
often constructed of PVC; and clear tunnels, which eliminated
the mere presence of an EE device as an independent variable;
several studies have found that the presence of EE, when com-
pared with isolation or impoverished conditions, has effects on
cognition, development, and performance.18,37,38,40 In circadian
light studies, 3 major variables include duration and timing of
exposure, intensity, and tint (or wavelength). Lighting intensi-
ties inside each cage were measured at all cage locations and had
little variance. However, radiometric irradiance measured inside
each CED was signicantly different between clear and colored
(red, amber, and opaque) tunnels as well as between amber and
red CED. Adherence to a strict 12:12-h light:dark environmental
lighting cycle throughout the study, with complete darkness at
night, ensured that any observed effects result only from varia-
tions in color tint.
We found signicant differences in daily food and water in-
take between animals with red compared with clear CED and in
dietary intake between groups in opaque and clear CED. Food
intake for rats with red CED was higher, and correspondingly,
average body weight changes were signicantly greater in the
red CED group when compared with the clear CED group over
the 36-d experimental period. The amber CED group had signi-
cantly lower average body weight changes. Other investigators
reported significant increases in food and water consumption
for rats under isolation or impoverished conditions compared
with cages with EE and standard social conditions (4 rats per
cage) but found signicantly heavier body weights only after the
rst week of differential housing and no signicant differences
in body weight between EE and standard-condition treatment
groups.38 The current study used social housing of rat pairs with
EE, which resulted in the red CED group consuming more food
and water with a corresponding increase in body weight. More
studies should be conducted to reveal the mechanisms underly-
ing this nding—perhaps by adding activity monitoring, record-
ing food and water intake at shorter intervals, and measuring
other neurohormones, such as plasma leptin and ghrelin, blood
glucose, and insulin concentrations—to elucidate any possible
central circadian disruption as a cause.
Monitoring locomotor activity would be helpful in determining
whether the signicant decreases in use of the amber, red, and
opaque CED are a result of these rats acclimating to the novel
environment. In a behavioral context, locomotor activity is a reli-
able and consistent index of learning or information processing,
and persistently high levels suggest a lack of information pro-
cessing or the absence of acclimation to the novel environment.18
Results from the current study suggest that rats spend more time
inside red CED as compared with all other CED tested. Because
intensities were held constant, our results indicate that the tint of
the CED causes this effect. However, the total time spent in each
CED was markedly lower approximately 2 wk after presentation
(phase 2) compared with 2 d after introduction (phase 1). There
was no feasible way of controlling for this animal-dependent vari-
able. However, this reduction in use and associated brief expo-
sure times might have mitigated signicant effects in some cases.
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Vol 66, No 5
Comparative Medicine
October 2016
390390
An animal’s coping to environmental changes is a complex behav-
ioral reaction revealed by their responses to changes in novelty
(well-known territory) and can have 2 differently motivated reac-
tions. That is, animals respond to changes in novelty by displaying
fear-associated defensive reactions, such as freezing and escape,
or by mounting exploratory associated reactions, such as curiosity
(neotic preference) and active exploration. Typically, fear-evoking
objects are avoided at rst, and this behavior may reect a need to
seek immediate safety. If the fear level is low or when fear subsides
with time or distance, subsequent active exploration of the object
and information-gathering allows animals to adapt to the envi-
ronmental change (novelty). When exploration brings no signs of
danger and after information is consolidated into memory, animals
habituate to the changed environment, resulting in reduction or re-
moval of fear and curiosity.34 This typical behavioral process is hard
to predict and varies between species, sexes, housing conditions,
and even EE objects.4,37,38 Other limitations in terms of our modi-
ed experimental design likely would be recovery time and blood
sample volumes and thus would require larger groups to collect
all of the circadian time points for comparison. Other variables
include strain, sex, and species, as discussed earlier.
Our next steps will be to add individual behavioral measure-
ments by using activity monitors, which would require indi-
vidual housing, and to include time-recorded food and water
measurements to elucidate individual dosages for each CED type.
In studies now underway, we are evaluating the effects of other
commonly used CED, such as the popular plastic hut for mice,26
which is manufactured in amber, red, and blue plastics and as
disposable cardboard (opaque) items, to evaluate whether differ-
ences due to the color of these enrichment devices emerge.
The effects of colored EE tunnels we used—and likely other
CED such as huts—cannot be completely controlled, because we
can only provide the animal the opportunity to use the device
and cannot hold constant the dose of each CED received, given
that this variable is behavior-dependent. Regardless of this limi-
tation, circadian rhythms of metabolism and physiology were
signicantly disrupted depending on the CED in the cage. There-
fore, CED used for enrichment in laboratory rat cages might sig-
nicantly confound scientic outcomes, and their use should be
thoroughly reported or pilot-studied. Further testing of these pre-
viously assumed harmless EE objects is warranted.
Acknowledgments
We thank Michael Webb, Patricia Beavers, and Katie Castillo for their
outstanding care of the animals and Erin Dauchy for her technical
training and statistical expertise. This work was supported by NIH
grants 1R25RR032028 (MAWD and RPB) and 1R56CA193518-01 (DEB
and SMH).
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Peak plasma TFA levels (0400) differed between the opaque
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