REVIEW
2 Lab Animal
Using Obese Mouse Models in
Research: Special Considerations for
IACUC Members, Animal Care
Technicians, and Researchers
Deborah J. Good, PhD
The mouse is the animal most commonly
used to study the underlying causes of and
treatments for obesity. The author reviews
many of the issues that should be consid-
ered by all involved in research with mice
expressing this phenotype, and describes
some procedures exclusive to obesity
research.
The author is an Associate Professor in the
Department of Veterinary and Animal Sciences, and
IACUC Chair at the University of Massachusetts,
Amherst, MA 01003. Please address
correspondence to the author at
goodd@vasci.umass.edu.
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Volume 34, No.
February 2005
With >50 different rodent models of obesity available, Institutional Animal Care
and Use Committees (IACUCs) are seeing numerous protocols using these
models for basic research projects and drug testing. Although researchers still
use rat and hamster models of obesity, the mouse has become the most preva-
lent animal used to study this phenotype because of the availability of many
transgenics, knockouts (KOs), and spontaneous mutants that have been created
or isolated in the laboratory. We can categorize these various mutants according
to tissue physiology: white fat or adipocyte mutants, central nervous system
mutants, gut and enteric nervous system mutants, and a broad class of ‘others’,
including angiogenesis control mutants, immune function control mutants,
thermogenesis or brown fat mutants, and skeletal muscle control mutants (for
reviews see refs. 1–7). What these mutant animals have in common is the
propensity to gain weight on regular diets. Some show early-onset obesity, usu-
ally characterized by increased body weight gain before puberty; others show
maturity, or adult-onset obesity, characterized by increased body weight after
sexual maturity. This review examines issues that IACUC members should con-
sider during the review of protocols that use obese mice, and describes special-
ized experiments that reviewers will see researchers use in studying obese
rodents. IACUC members, as well as animal care technicians, veterinarians, and
researchers, may be interested in the animal welfare and care issues associated
with the obese phenotype.
Special Welfare Considerations for Using Obese Mice
In addition to standard concerns for reviewing animal protocols, research proto-
cols using obese mice must address the special circumstances involved with using
these models. These circumstances fit into several categories: husbandry, geno-
types, and total animal numbers; housing conditions and enrichment; food
intake; surgery and anesthesia considerations; and humane endpoints. The
researcher planning the project and the IACUC members reviewing the protocol
must take all of these into consideration.
Husbandry, Genotypes, and Total Animal Numbers
The vast majority of the obese mice used in research today are KO mice, which are
animals with a homozygous deletion of a particular gene. Because of this condi-
tion, most obese animal colonies require careful husbandry, along with meticu-
lous genotyping and record keeping. Compared with wild-type (WT) colonies,
the breeding requirements and limitations of many obese KO mice lead to an
increased number of unused animals.
In general, researchers use heterozygous (HET) animals as breeders, resulting
in a 1:2:1 ratio of WT, HET, and homozygous KO mice. Because most researchers
February 2005 Lab Animal Volume 34, No. 2
use the homozygous KO mice in the experiments, with WT mice as
controls, breeding results in a surfeit of HET mice. It is important
to consider this, both for reporting animal numbers in the proto-
col as well as in justification for not meeting the ‘reduction’ crite-
rion for the ‘3 Rs’ (reduction, refinement, and replacement)8.
The use of KO breeders to produce homozygous KO mice and
WT breeders to produce the control mice would be one means by
which to eliminate the overproduction of HET mice. However, in
many obese models, the KO animals are infertile or show reduced
fertility9–11; therefore, they cannot become reliable breeders. These
circumstances offer researchers no choice but to use the HET mice
as breeders, resulting in the production of twice as many animals
as are needed for the proposed experiments. Charts and informa-
tion in Guidelines for the Care and Use of Mammals in Neuroscience
and Behavioral Research can be useful in determining the exact
numbers of animal needed when the male and female homozygous
KOs have reduced fertility12.
There is evidence of a maternal environment effect on the
development of obesity. For example, in mice heterozygous for a
mutation in the leptin receptor (Lepr (db/+)13–16, gestational dia-
betes might contribute to increased adult body weight in WT off-
spring of db/+ mothers, compared with WT offspring from WT
dams17. Other reports show that in mice heterozygous for a dele-
tion in a phospholipid transporter, Atp10c, inheritance of the
mutation from the dam resulted in increased body weight and
serum leptin levels and an increased risk of type 2 diabetes, com-
pared with WT or Atp10c heterozygotes that had inherited the
mutation paternally18. In mice heterozygous for a mutation in the
stimulatory G protein alpha subunit α (Gsα), those inheriting the
mutation maternally are obese, whereas paternal inheritance
results in thinness19. Each of these examples represents cases in
which breeding schemes and therefore animal numbers would
have to be modified further to correctly study the genetic contri-
bution and phenotypic outcomes of the mutant alleles or loci;
moreover, the researcher during the preparation of the IACUC
protocol and the IACUC members during review would have to
take these characteristics into account.
Housing, Temperature, and Enrichment
Although most obese mice do not require specialized individual-
ly ventilated microisolator cages, the type of housing (solid floor-
ing or grid/hanging cages) and the housing conditions (group or
singly housed animals, cage shelf level, room temperature, and
environmental enrichment) can affect behavior, food intake, and
body weight gain in both normal-weight and obese-prone
rodents. Researchers should consider these conditions when
studying obese mice; animal caretakers and IACUC members
should also be cognizant of the need for controlling housing con-
ditions for these animals.
Most mice live in standard solid-floor caging with recycled
paper or wood chip bedding. Some experiments, such as food
intake analysis (discussed later), require the use of wire-grid
REVIEW
hanging cages (Fig. 1). One study using rats found no difference
in food consumption or body weight gain with either cage system,
although rats clearly preferred the solid-floor caging as measured
by the percentage time spent in the solid floor cage (88%) when
given access to both via a perspex tube20. With all caging systems,
there should be monitoring of the housing of obese mice with
respect to density. The Guide for the Care and Use of Laboratory
Animals (Guide)21 provides guidelines, based on body weight and
required floor space per animal, that the researcher can use to
determine the number of animals allowable in a cage. In general,
up to four mice with a combined weight of <125 g can live togeth-
er in a standard 11 × 7 × 5 in. mouse cage. Because some obese
mice can weigh as much as 60–70 g (or more than twice that of
the average mouse), there should be frequent monitoring of obese
animals in group housing to assure that minimum space require-
ments are met.
To address possible animal welfare concerns, researchers study-
ing diabetic animals should consider the bedding type, the amount
of bedding in the cage, and the cage type. Diabetes, which can
accompany the obese phenotype, results in increased urine pro-
duction in both humans and rodent models of the disease22. The
diabetic phenotype may require more frequent cage changes or a
more absorbent bedding to compensate for the increased urine
production. Some IACUCs recommend for diabetic animals the
use of wire-grid hanging cages, which prevent the animals from
lying in the urine-soaked bedding that could be present in solid-
floor caging. However, one study has found that diabetic rats
exhibit distal tibial mononeuropathy earlier than nondiabetic rats
when reared in wire-grid hanging caging23. There should therefore
be careful consideration of choice of caging with regard to animal
needs and experimental design.
There are limited published data on the relationship in mice
between housing conditions and body weight. One study of
FIGURE 1. Wire-grid hanging cages. We include Nestlet pads and
plastic plumbing pipes to provide areas of solid flooring and
enrichment to the mice.
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REVIEW Volume 34, No. 2
nonobese diabetes-prone mice found no difference in the onset of
diabetes if mice were housed singly, or with up to seven other age-
and sex-matched mice24, whereas another study suggests that
singly housed mice show less variation in body weight and body fat
content than mice housed five per cage25. One study by Leiter
shows that group-housed male mice subjected to stress induced by
repetitive handling developed glucose intolerance that could be
ameliorated by adrenalectomy at 3 weeks of age26. Notably, studies
by Van Loo and colleagues suggest that male mice prefer group
housing over single housing, especially in regard to sleeping sites,
but that neither housing condition altered body weight or food
intake27. Conversely, group-housed hamsters show a >30-g
increase in body weight and a 6% increase in body fat compared
with individually housed hamsters28.
Shelf level on the cage rack may also affect body weight, with a
higher shelf level on the cage rack corresponding to a lower inci-
dence of diabetes and the least amount of weight gain28,29. Thus,
the literature suggests that researchers should design their housing
for obese mice with consideration for conditions that will affect
their own testing results, and based on consistency in housing
parameters between obese and normal control mice, keeping in
mind the density limitation specified by the Guide21.
It is clear that conditions that affect the rate of body heat loss,
such as room temperature and group housing, can affect weight
gain in rodents. The Guide suggests housing mice at a room tem-
perature of 18°–26°C (64°–79°F), with minimal temperature fluc-
tuations21. Many obese animals show reduced core body tempera-
tures, and some show an inability to respond effectively to cold
exposure by increasing their own heat production5. One study
examined the ability of the ob/ob mouse, which has a spontaneous
mutation in the adipocyte hormone leptin, to respond to different
environmental temperatures. At room temperatures between 30°C
and 10°C, the body temperature of ob/ob mice is consistently
reduced by up to 2.5°C compared with normal mice, most proba-
bly due to reduced energy expended on thermogenesis30. When
room temperature is reduced to 4°C, ob/ob mice cannot adapt and
die of hypothermia, whereas normal mice maintain their body
temperature through cold-induced thermogenesis30.
Carrying a spontaneous mutation in the leptin receptor, db/db
mice show a similar response to a room temperature of 4°C, and
their body temperature is 1°–2°C lower than normal mice when
maintained at 23°C (ref. 31). At 23°C, db/db mice deposit 75%
more fat than normal mice, because of reduced thermoregulatory
thermogenesis32. Furthermore, at temperatures considered to be
thermoneutral (33°C), db/db mice still deposited 25% more body
fat than normal mice, most probably because the mutant mouse
was unable to show a compensatory reduction in food intake in
response to the reduced energy requirements32. Thus, fluctuations
in room temperature during experimental testing of obese rodents
could have slight, but meaningful effects on weight gain, fat depo-
sition, and core body temperature.
Moreover, the prevailing opinion is that some of the differ-
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Lab Animal February 2005
ences in body weight gain described earlier for group-housed
compared with singly housed animals are due at least in part to
‘huddling’, which reduces thermoregulatory energy needs. A study
that examined the effect of cage enrichment, including parame-
ters such as group housing, nesting materials, nest boxes, gnawing
sticks, and PVC tubes found that the enriched animals (i.e., in
cages containing nesting material or nesting material plus one or
more of the extras) gained more weight than mice that were sim-
ply group housed, even though they showed reduced food intake,
suggesting that the enrichment materials may also reduce energy
needs of the mice33. Thus, researchers working with obese rodents
should monitor housing environment, as well as room tempera-
ture, in their colony maintenance and experimental conditions.
Food Intake
With few exceptions, mice with targeted or spontaneous mutations
in genes that result in obesity show hyperphagia (i.e., increased
appetite)7. In addition, a diet high in fat can result in obesity in nor-
mal mice5. Animal care facilities purchase rodent diets that can have
a crude fat content anywhere between 4.5% and 11% (e.g., Purina
5001, Purina Prolab RMH 2000, Purina 5015, Purina LabDiet, St.
Louis, MO). In animals with which this author works, moving from
the National Institutes of Health (NIH) to the University of
Massachusetts resulted in a slight reduction in body weight, espe-
cially in our older Nhlh2 KO mice. We traced this weight loss to a
change from Purina 5015, containing a minimum of 9% fat, which
was fed to the mice at NIH, to Purina 5001, containing a minimum
of 4.5% fat, which is currently their diet at the University of
Massachusetts. Additional discussions with representatives from
several rodent diet suppliers have led our laboratory to investigate
the possibility that differences between these rodent diets and,
more importantly, between individual lots of the same rodent diet,
which may vary in the maximum amount of fat, could lead to
experimental differences in the body weight gain in our obese KO
mice. Researchers should be aware of the type of rodent food used
by their animal facility and ensure that the animals’ diet most close-
ly fits the particular study needs.
Surgery and Anesthesia of Obese Mice
To this author’s knowledge, there has been no comprehensive or
comparative study of the effect of different anesthesia regimens on
obese mice, although many veterinary medicine textbooks address
dosage calculations that compensate for obesity and the comitial
decrease in water compartmentalization. In general, increased body
weight, in particular the excess fat accumulation that occurs in
obese mammals, alters drug kinetics and may cause cardiopul-
monary complications during surgery under general anesthesia. In
addition, as a result of the function of some genes, KO mice could
show both obesity and changes in sensitivity to anesthesia.
Examples of this sensitivity are the KO mice for the brain NPY
receptors, Y1 and Y2. The Y1-receptor KO mouse displays both
obesity and reduced sensitivity to pentobarbital and avertin34–36,
February 2005 Lab Animal Volume 34, No. 2
whereas deletion of the Y2 receptor results in increased body weight
in females and increased sensitivity to pentobarbital35,37.
When selecting the anesthesia agent to use with obese rodents,
the surgeon should take into account the agent’s fat solubility. For
highly lipophilic drugs, including barbiturates (e.g., pentobarbital)
and cyclohexamines (e.g., ketamine), animals with large amounts
of body fat show delayed recoveries, and sometimes death, due not
only to the uptake and storage of these drugs in the fat, but also to
severely depressed respiration from slower metabolism38. The
inhalant drug isoflurane, which is insoluble in both blood and
fat38, achieves rapid and easily reversible anesthesia with no death
or breathing difficulties in our Nhlh2 obese mice, which contain a
targeted deletion of the neuronal transcription factor. Inhaled
anesthesia requires the use of a mask or nose cone for the rodent,
a vaporizer for the anesthesia, and a regulator to deliver a supply of
oxygen during anesthesia. Workers should have protection from
the inhalant anesthesia, which enters the atmosphere during the
procedure. They most commonly do surgery in a fume hood or
with a scavenger unit (e.g., NP 60-0979 F/AIR Filer canister,
Harvard Apparatus, Inc., Holliston, MA) to remove the vapors
from the surgery suite. IACUC members should make certain that
the choice of anesthesia is appropriate for the animal, especially
ensuring that the researcher understands the potential complica-
tions of anesthesia in obese rodents. If the scientist is choosing
inhaled anesthesia, then the IACUC should ensure that the sur-
geons follow the requirements to protect themselves.
Humane Endpoints
Researchers should establish humane endpoints for all studies
involving animal use, but for obese and diabetic animals, some
guidelines may require implementation to prevent distress or dete-
rioration of the animals due to the nature of their disease39. For a
diabetic animal, if diabetes is ongoing, end-organ effects will occur,
including diabetic retinopathy, diabetic nephropathy, and uncon-
trollable hyperglycemia40. For an obese animal without diabetes,
severe weight gain can result in hypertension, renal problems, and
even respiratory depression41,42. The IACUC should be certain that
the researcher has established a humane endpoint with regard to
the phenotype of the disease model. In particular, there should be
no possibility of the animals developing morbidities associated
with obesity or diabetes, if it is not necessary to the research ques-
tion being asked. Because it is not possible for the animal care staff
or researcher to have a generalized list of clinical signs that will
cover all types of experimental paradigms, one paper has suggested
the use of an individualized score sheet system that would allow
caretakers or researchers to assess the level of pain or distress in
their animals39. The sheet would be available in the animal room
and would list all possible clinical signs, scoring details, and the
steps to take if one or more of the clinical signs are present, includ-
ing scientific steps such as isolation of particular tissues for analy-
sis. In this way, there can be assessment of the welfare of the animals
without compromising the scientific goals of the study.
REVIEW
Specialized Procedures for Studying Body
Weight Regulation in Rodents
IACUC reviewers of protocols from researchers studying body
weight regulation will certainly encounter proposed experiments
that are somewhat unique to the obesity research field. In particu-
lar, food intake and food deprivation studies are common, along
with specialized diets designed to promote either weight gain or
loss in the animals. Body temperature and the effect of different
temperatures to simulate thermoneutrality or cold-induced ther-
mogenesis are probable considerations. Finally, researchers often
propose measurements of energy expenditure through calorimetry
and physical activity. IACUC reviewers should be aware of special
considerations for these types of experiments and ensure that
researchers follow guidelines in their design and implementation
that comply with applicable guidelines and regulations.
Food Intake, Food Deprivation, and Specialized Diets
When a new KO or mutant mouse is determined to have a pheno-
type of increased body weight, studies of food intake are some of
the first to be undertaken. Next on the list are food deprivation and
refeeding studies to analyze changes in gene expression, body
weight, and other physiological and behavioral changes in the
mutant animal, followed by analysis of the response of the mutant
mice to specialized diets with high-fat, low-fat, or pharmacological
additives.
The analysis of food intake usually involves placement of mice
in specialized cages so that daily food intake can be measured, tak-
ing into account both ingested material and spillage. The most
economical and technologically simple method for doing this
analysis involves the use of a hanging metal cage with a wire-grid
bottom. Animals live individually in these cages, with food spillage
collected daily on paper mats beneath the cage. Workers weigh
both the remaining food in the bin and the spillage to determine
the daily intake for each animal. Vendors also sell cages with elec-
tronic and computerized weighing systems, although most still use
a grid flooring in their design to allow the measurement of
spillage. The primary problem with each of these experimental
designs concerns the guidelines for animal cage flooring published
in the 1996 revised version of the Guide12. Although these guide-
lines do not prohibit wire-grid flooring for rodents, they recom-
mend solid flooring on the basis of research showing that rodents
prefer solid flooring and may develop foot lesions on wire-grid
flooring20. In our laboratory, we use wire-grid flooring for food
intake studies, but we include plastic plumbing pipes 4 in. in diam-
eter and ∼5 in. long, as well as Nestlet pads (Ancare Corp.,
Bellmore, NY) to provide some areas of solid flooring and enrich-
ment to the mice (Fig. 1)43.
Following the results of food intake studies, many researchers
continue with analysis of food deprivation and refeeding. They
normally use these experiments to analyze the role of the gene of
interest (i.e., the knocked out or mutant gene) on physiological
and behavioral parameters. The Guide21 specifies that food can be
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REVIEW Volume 34, No. 2
withheld for research purposes but that the animal should be
monitored for distress. Mice can go without food for up to 48 h
(refs. 44–46), but effects on gene expression can occur at time
points ≤24 h (refs. 47,48), so design of the study should allow for
the minimum required time needed for an effect. In addition,
mice should have, and generally do have, unrestricted access to
water during these deprivation periods. Fasting experiments often
take place in cages with wire-grid bottoms, because fasting-
refeeding paradigms are common endpoints. Thus, the same con-
cerns mentioned for grid flooring in food intake studies should
receive consideration in the fasting-refeeding experiments that
use these cage types. In addition, several reports have suggested
that the ambient temperature of the housing conditions influence
the effects of food restriction themselves49. In particular, reports
indicate that standard housing temperatures of 23°C represent
mild cold stress for mice and that this temperature actually results
in elevated food intake. Thus, colder room temperatures in addi-
tion to food deprivation could represent an added stress on the
animal. In addition, as discussed earlier, group housing can affect
the energy requirements of animals because of their tendency to
huddle to conserve energy. Whether the researcher uses single or
group housing will most probably affect the severity of the fasting
regimen. These issues should receive consideration for all food
deprivation studies.
Diet-induced obesity is one of the most common methods for
studying the role of diet in the development of obesity 5.
Specialized diets, formulated in the amount of fat, calories, or
other components, are available from a number of suppliers (e.g.,
Research Diets, Inc., New Brunswick, NJ; Harlan Teklad, Madison,
WI; and Purina LabDiets). Research specifications can also dictate
the formulation of diets, with the addition of specific types of car-
bohydrate or fat sources or drug to modulate food intake. For
IACUC protocols that request the use of a nonstandard diet for
body weight studies, reviewers should request that the expected
effects of this diet be adequately explained and justified. Some of
the more palatable diets, such as the rodent cafeteria diet, com-
posed of cookies, breads, peanuts, cheese, and other ‘delectables’
for mice, can cause animals to overeat or become hyperphagic, in
addition to having a higher overall fat content than a standard
rodent diet5. These diets can lead mice to become extremely
obese, sometimes doubling their total body weight over the nor-
mal 10- to 30-week period of feeding. Because this situation can
lead to cage densities that are higher than those allowed, the
IACUC reviewer should carefully consider the time frame for spe-
cialized diets, as well as the total weight gain expected over that
time period.
Body Temperature Measurements and Experimentally
Induced Energy Expenditure
Most obese mice show reduced energy expenditure, measured by
body temperature, respiration rate, or physical activity (discussed
later)6,50. Although obligatory energy expenditure for body and
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Lab Animal February 2005
organ function represents the majority of energy usage by the
body, adaptive thermogenesis, which is induced by cold exposure
or diet, accounts for ∼25% of the required daily energy expendi-
ture and is usually defective in obese mutant mouse models50.
There are two methods for measuring core body temperature
in mice. First, surgically implanted transmitters (e.g., Mini Mitter
Co., Inc., Bend, OR) can transmit core body temperatures contin-
uously and throughout the lifetime of the mouse. The obvious
concern is that the animal must undergo a survival surgery for the
implant, but because these devices are placed under the skin and
no body cavity opening is required, surgical complications are
usually minimal. The second method for body temperature mon-
itoring is manual, using rectal probes. In our laboratory, we have
used a tissue-implantable thermocouple microprobe (Physitemp
IT-18, Physitemp Instruments, Inc., Clifton, NJ) as a mouse rectal
probe attached to a Physitemp Thermalert TH-5 digital ther-
mometer (Fig. 2)43. To avoid irritation to the mice, we clean the
probe in 70% ethanol between animals, and we coat it with min-
eral oil before insertion. We measure temperatures at a depth of
2 cm, for ∼15 s, or until a stable reading is obtained. We have
marked our probe with a dot of white correction fluid at the 2-cm
depth to ensure the consistency of our reading and animal safety.
Whereas the advantage of this method over the implanted devices
is that animals do not need to undergo surgery for temperature
monitoring, the disadvantage is that rectal measurements can be
stressful to the animal and can result in injury if a probe is insert-
ed into the rectum beyond 3 cm. Although there are only anecdo-
tal reports on the injuries that can occur with rectal probes, there
are many studies on the stress effects of rectal temperature mea-
surements on mice and rats. In mice, repeated measurements
using a rectal probe can result in hyperthermia of >1°C (ref. 51).
In addition, stress-induced hyperthermia can occur when group-
housed mice are measured sequentially, with the core temperature
of the last mouse increased by nearly 2°C (ref. 52). Use of this
method for body temperature reading does therefore have the
potential to confound results. Thus, IACUC members reviewing
protocols in which body temperature measurements will be made
should make sure the researcher is aware of the disadvantages to
each method and has implemented safeguards to protect the wel-
fare of the animal, either from stress or anxiety induced by the
manual method for body temperature measurement, or from pos-
sible surgical complications with the implanted devices.
Indirect calorimetry is the measurement of the O2 consumed
and the CO2 produced by an animal. Researchers then use the
measurements of these variables to calculate the metabolic rate or
energy usage of the animal. Several suppliers provide individual
units for measuring respiration in mice (e.g., Oxymax cages,
Columbus Instruments, Columbus, OH). In these units, mice usu-
ally have ad lib access to food and water, and have plenty of room
for exercise. Thus, there are no animal welfare issues associated
with use of these cages for metabolic measurements. If the animals
are held in calorimetry chambers for >24 h in an area outside of
February 2005 Lab Animal Volume 34, No. 2
FIGURE 2. Core body temperature measurement in an obese
mouse. We use a mouse rectal probe inserted to a depth of 2 cm.
A small amount of correction fluid indicates the depth mark to the
researcher; this is indicated in the figure as the white mark on the
probe stem nearest to the mouse’s anus.
the normal animal room, IACUC reviewers should inspect the
holding area or room, as required by PHS Policy for Satellite Areas.
Once basal measurements of either body temperature or meta-
bolic rate are made, researchers studying body weight regulation
often test the ability of their obesity models to respond to diet- or
cold-induced thermogenesis. Normal animals sense augmented
food or caloric intake, and the body and brain respond with a com-
pensatory increase in thermogenesis. For studies of diet-induced
thermogenesis, mice usually receive a high-fat diet and have their
body weight changes monitored weekly. Many obese mouse models
do not respond with a compensatory increase in thermogenesis and
show massive weight gain on these diets. As discussed earlier,
researchers and IACUCs should monitor the use of high-fat diets to
ensure that maximum cage densities are not exceeded.
For cold-induced thermogenesis, researchers measure core
body temperatures of animals in response to differing lengths of
time at thermonegative temperatures, usually 4°C. Normal mice
can adapt to this temperature by increasing thermogenesis and
maintaining relatively normal core body temperatures, but many
mutant obese mice fail to respond and show severe declines in
body temperature in relatively short time frames. For example,
mice lacking all three β-adrenergic receptors fail to increase ther-
mogenesis after 10–20 h of exposure to 6°C, showing core body
temperature reductions of 4°–10°C (ref. 53). Some of the animals
in the study appeared to have been taken out at earlier time points,
with body temperatures of <27°C after only several hours of expo-
sure54. Fasting animals in conjunction with cold exposure to max-
imize changes in energy balance can also result in a pronounced
REVIEW
FIGURE 3. Running-wheel cages. The mouse running on the
wheel trips a circuit each time it makes one full revolution.
Computer software calculates the number of revolutions in 20-
min time bins.
decrease in body temperature in some mouse models that show
normal response to cold with ad lib food access (e.g., DGAT1
mutant mice54). Accordingly, IACUCs and researchers using this
method to analyze body weight and gene function in mutant mice
should provide adequate monitoring of animals during cold expo-
sure to ensure that death from severe hypothermia is prevented
and is not an unexpected endpoint in their studies. In addition,
IACUC review of the protocol should include inspection of the
cold room housing any rodents for >24 h.
Physical Activity Measurements
The amount of physical activity done by an animal can influence
the amount of energy used by that animal. Mild food restriction or
cold exposure induces in some obese mouse mutants (e.g., ob/ob
mice55) a torpor or a period of physical inactivity accompanied by
reduced core body temperature. Many obese mice, including the
ob/ob, melonocortin-4 receptor KOs, and those that we use in our
laboratory, the Nhlh2 KO mice, show reduced daily physical activ-
ity when given access to a running wheel or when monitored using
beam breaks with open-field testing equipment38,56,57.
Measurement of daily activity with either computerized running
wheels (Fig. 3) or open-field testing equipment does not pose any
animal welfare issue, and, in fact, animals with running-wheel
cages receive an added enrichment benefit.
An extension of physical activity measurements with which
there could be some animal welfare concerns is the use of forced
exercise either to measure gene expression changes in response to
physical activity or to measure the ability of increased physical
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REVIEW Volume 34, No. 2
activity to reduce body weight in obese rodents. In these experi-
ments, which are becoming more commonplace for obesity
researchers, mice exercise on miniature treadmills either at a set
speed or with increasing speed or increasing inclines for a set peri-
od of time. Scientists in the field of skeletal and cardiac muscle
research have used such procedures for quite some time, with
treadmill speeds of up to 20 m/min and times ranging from 15 min
to two 3-h bouts of exercise per day58,59. Laboratories studying body
weight often measured O2 consumption along with exercise, with
moderate exercise protocols consisting of 12 m/min speed and up
to 1 h of running (e.g., Bouthegourd et al.60), or intense bouts of up
to 18 m/min until exhaustion, usually between 17 and 30 min (e.g.,
Faldt et al.61). The Animal Welfare Act (AWA) mandates exercise for
laboratory dogs and stipulates that this exercise must be voluntary
and cannot be in the form of forced exercise on a treadmill, except
when exemptions are given for scientific research62,63. Because lab-
oratory mice are not covered by AWA, no such mandates exist.
Under Public Health Service guidelines, especially in the area of
alternatives and refinement of procedures, there should be assur-
ance to IACUC members reviewing protocols requiring exercise to
the point of exhaustion that this is the minimal amount of exercise
that can be done for the endpoint or measurement, and that the
researcher has justified the amount of distress for the animal with
an adequate literature search and statement.
Summary
Obese mice are powerful models that we can use to explore the
complexity of body weight regulation. These animals vary with
respect to the tissues affected by the genetic mutation or physical
manipulation, but all have the common phenotype of augmented
body weight, increased body fat, or obesity. The phenotype of
obesity presents several additional welfare concerns, including
issues of housing density, enrichment types, room temperature
regulation, and food composition, as well as specialized experi-
ments to test regulation of body weight, food intake, and exercise
in these models. IACUC members, animal care technicians, and
researchers working with obese mice should educate themselves
on the complexities of this phenotype and the many welfare con-
cerns that result from the disease and the experimental proce-
dures used to study it.
Acknowledgments
The author would like to thank Christopher A. Coyle, Franc-Eric
A. Wiedmer, and Hilary Woodcock for careful reading of the
manuscript and helpful suggestions. This work was supported by
grants from the US National Institutes of Health (R01 DK59903,
R03 HD42499, and P30 DK46200).
Received 8/4/04; accepted 11/9/04.
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