Content uploaded by Carlos Mejías
All content in this area was uploaded by Carlos Mejías on Jun 19, 2022
Content may be subject to copyright.
Body Composition and Energy Savings by Hibernation: Lessons
from the South American Marsupial Dromiciops gliroides*
Juan G. Navedo
Lida M. Franco
Roberto F. Nespolo
Instituto de Ciencias Ambientales y Evolutivas, and Magister
en Ecología Aplicada, Facultad de Ciencias, Universidad
Austral de Chile, Valdivia, Chile;
Instituto de Ciencias
Marinas y Limnológicas, Universidad Austral de Chile,
Departamento de Ciencias Ecológicas,
Facultad de Ciencias, Universidad de Chile, Santiago,
Facultad de Ciencias Naturales y Matemáticas,
Universidad de Ibagué, Carrera 22, Calle 67, Ibagué,
Millennium Nucleus of Patagonian Limit of Life
(LiLi) and Center of Applied Ecology and Sustainability
(CAPES), Departamento de Ecología, Facultad de Ciencias
Accepted 12/13/2021; Electronically Published 4/20/2022
Hibernation (i.e., seasonal or multiday torpor) has been described
in mammals from ﬁve continents and represents an important
adaptation for energy economy. However, direct quantiﬁcations
of energy savings by hibernation are challenging because of the
complexities of estimating energy expenditure in the ﬁeld. Here,
we applied quantitative magnetic resonance to determine body fat
del monte). During an experimental period of 31 d in winter, fat
was signiﬁcantly reduced by 5:72 50:45 g, and lean mass was
signiﬁcantly reduced by 2:05 50:14 g. This fat and lean mass
consumption is equivalent to a daily energy expenditure of hi-
, representing 13.4% of
basal metabolic rate, with a proportional contribution of fat and
lean mass consumption to DEE
of 81% and 18%, respectively.
During the deep heterothermic bouts of monitos, body temperature
remained 0.417C50.27C above ambient temperature, typical of
hibernators. Animals shut down metabolism and passively cool
down to a critical defended temperature of 5.07C50.17C, where
they begin thermoregulation in torpor. Using temperature data
loggers, we obtainedan empirical estimation of minimum thermal
conductance of 3:37 50:19 J g
, which is 107% of the
expectation by allometric equations. With this, we parameterized
body temperature/ambient temperature time series to calculate
torpor parameters and metabolic rates in euthermia and torpor.
Whereas the acute metabolic fall in each torpor episode is about
96%, the energy saved by hibernation is 88% (compared with the
DEE of active animals), which coincides with values from the
literature at similar body mass. Thus, estimating body compo-
sition provides a simple method to measure the energy saved by
hibernation in mammals.
Keywords: quantitative magnetic resonance, body composi-
tion, lean mass, hibernation energy savings, allometric scaling,
The study of metabolic depression in endotherms (i.e., seasonal or
multiday torpor) has attracted the attention of physiologists for
more than a century (Pembrey and White 1896; Lyman 1948).
Today, it is known that the physiological changes that occur
during episodes of torpor and hibernation are diverse and or-
chestrated, beginning with the abandonment of euthermic ther-
moregulation (Heller and Colliver 1974); apneas, which can last
from seconds to minutes (Wang and Hudson 1970; Milsom and
Jackson 2011); and active suppression of metabolic rate (Geiser
et al. 2014). Indeed, there is consensus that the most important
beneﬁt of torpor is energy savings, as torpid animals could reduce
their resting metabolic rate by up to 95% when torpid (the classic
case of the hamster; Lyman 1948; but see also Melvin and An-
drews 2009; Ruf and Geiser 2015; Geiser 2020; Giroud et al. 2021).
Using percentage of basal metabolic rate (BMR; i.e., the metabolic
reduction from normothermia to torpor), the metabolic reduc-
tion of torpor ranges from 5.1% of BMR in daily heterotherms to
29.5% in hibernators (Ruf and Geiser 2015). But the real impact
*This paper was submitted in response to the Focused Collection call for
papers “Time-Out for Survival: Hibernation and Daily Torpor in Field and Lab
†Corresponding author; email: firstname.lastname@example.org.
Physiological and Biochemical Zoology, volume 95, number 3, May/June 2022. q2022 The Universityof Chicago. All rights reserved. Published by The University of
Chicago Press. https://doi.org/10.1086/719932
of hibernation on an animal’s energy balance depends on the
averaged reduction in expenditure during an extended period, which
is particularly difﬁcult to estimate. Indirect estimates of the energy
savings of hibernation, calculated from metabolic measurements in
the laboratory extrapolated to the time budget observed in the ﬁeld,
suggest that these savings range from 87.7% to 96% (Wang 1978;
Kenagy et al. 1989; Thomas et al. 1990; Geiser 2007).
Given that hibernators rely on fat stores, and to some extent
proteins, during torpor, a good measure of the average energy
consumption of hibernation could be obtained from changes in
body composition over time. Here, we applied quantitative mag-
netic resonance (qMR) to estimate such changes in the marsupial
monito de monte (Dromiciops gliroides). We estimated the daily
energy expenditure of hibernation (DEE
) using fat and lean mass
consumption, which is informative of energy consumption during
long-term hibernation. The qMR is a rapid and simple method to
quantify body composition in live animals that permits repeated
sampling with minimal disturbance. It has often been used for
biomedical research (Taicher et al. 2003; Gibert-Ramos et al. 2020;
Han et al. 2020; Snelson et al. 2020), as well as for analyzing lean
and fat mass changes after long-term migration in birds (Gug-
lielmo et al. 2011; Kennedy et al. 2017; Kelsey and Bairlein 2019).
To the best of our knowledge, just one author has used qMR in
hibernating animals, ﬁnding lean tissue preservation throughout
hibernation (Hindle et al. 2015). At least four other methods, with
various advantages and difﬁculties, have been applied to estimate
changes in body composition, such as total body electrical con-
ductance (Toien et al. 2011), isotopic dilution (Hilderbrand et al.
2000), dissections and fat extraction (Cranford 1978), and indirect
estimations based on bioenergetic models (Jonasson and Willis
2012). We also provide a suite of hibernation parameters for mo-
nitos under outdoor conditions, obtained by simultaneous mea-
surements of body and environmental temperatures. With these,
we predicted the metabolic changes during torpor and euthermia,
together with torpor patterns, in a period of 31 d.
Material and Methods
All procedures presented in this study were approved by the
Chilean Agriculture and Livestock Bureau (permits 4371/2019
and 3393/2019) and by the Bioethics Committee of the Uni-
versidad Austral de Chile (resolution 313/2018annexe 2019). The
experiments described here were performed in individuals of
Dromiciops gliroides (np26;body mass [M
]range:30–43 g). All
results were obtained with this sample size except for hiberna-
tion patterns, which were obtained in only three individuals (see
details below). Monitos were captured in San Martin Biological
W) in February 2019 during
nighttime using 100 Tomahawk traps attached to trees 2 m above-
ground and baited with banana. Animals were transported to the
outdoor enclosures after capture (see next paragraph), which were
located 20 m away from the trapping site, and maintained with ad
lib. food (apples and puppy/cat pellet) and water. No animal was
harmed in these procedures. Each captured individual was tagged
using a passive integrated transponder subcutaneous chip (BTS-
ID, Helsingborg, Sweden) to allow subsequent identiﬁcation. At
the end of the experiments, all monitos were released at the site of
To reproduce the physiological and thermoregulatory responses
of monitos under ﬁeld conditions, ﬁve seminatural cylindrical
enclosures were built, which were distributed within the forest
with a separation of 5 m between each one, covering a total area of
80m². Eachenclosurehad an internalvolumeof 2 m³,composedof
1.8-m-long zinc plates curved to generate a cylinder, and was
buried 10 cm in the ground, leaving a heightof 0.8 m aboveground.
Each enclosure was covered by a removable upper lid with mesh
that allowed the entrance of light and ventilation. The mesh was
chosen to let light enter similarly as in old-growth forests (range:
1,090–2,500 lx during the day, measured by a TASI 21 lumino-
meter). Within each enclosure, branches andplants were included,
imitating the microenvironment of monitos. A data logger (HOBO)
was located in each enclosure in the shadowat 50 cm above the soil
for the measurement of air temperature every 5 min.
We released the monitos in the enclosures at the beginning
of winter (June 20, 2019), without food but with water ad lib.,
for 31 d. As indicated before, monitos do not exhibit a ﬁxedpattern
of hibernation. Instead, they normally increase the duration of
torpor episodes gradually, as ambient temperatures and photo-
period decrease, and they can suppress hibernation if food is
abundant (Nespolo et al. 2021). Previously, the animals had been
kept in the enclosures with food ad lib. for 2 mo after capture,
during which they experienced the typical fattening of autumn,
in February: 30:25
2:1 g). Before the release in enclosures and after 24 h of fasting
in the laboratory, the body composition of each individual was
determined using the qMR scanner. Then animals were placed
in the enclosures in three groups of ﬁve and two groups of six
animals per enclosure. Thus, the animals’last food intake
occurred 24 h before the qMR measurement, which is long
enough to empty the intestine of an omnivorous marsupial
(Hume et al. 1997). After 31 d, all individuals were collected and
transported to the laboratory for the second qMR determina-
tion. All animals aroused from torpor during the transportation
to the laboratory. Arousals, which are common in monitos
during hibernation (see “Results”), take about 2 h under deep
torpor. After qMR measurements, animals were acclimated to
the laboratory to 207C, food and water were provided ad lib. for
24 h, and the qMR determination was repeated to quantify
recovery. This procedure was repeated 48 h after to measure a
second period of recovery. After ﬁnishing the experiment, ani-
mals were maintained with food and water ad lib. until spring
and then released in the place of capture.
Quantitative Magnetic Resonance
The qMR scanner we used was an EchoMRI 500 (Houston, TX),
which has been validated several times in wild animals (Riley et al.
2016; Kraft et al. 2019; Eastick et al. 2020). It gives instantaneous
measures of body composition (fat mass, lean mass, free water,
and total water [g]; free water represents mainly the water in the
bladder) in less than 1 min per animal. We calculated the hydration
index as (total water 2free water)/lean mass. At each measurement,
the animal was placed in an acrylic cylinder (5 cm in diameter,
60 cm long) and immobilized by a Velcro-secured plunger. Then
it was introduced into the magnetic resonance module, which
was previously programmed for a certain number of scans. We
performed three scans per animal, which were achieved in a total
of 1.5 min. Each time the coefﬁcient of variation of these repeti-
tions exceeded 6% (usually due to movement of the animal within
the probe), the measurement was discarded and repeated. We
calibrated the qMR scanner daily before every batch of measure-
ments, according to the manufacturer’s recommendation, using a
known sample of canola oil located in the antenna.
Given that during torpor animals rely almost exclusively on
fat metabolism (Storey and Storey 2010; Hindle et al. 2015;
Giroud et al. 2021), we calculated the total amount of energy
available (i.e., heat and metabolic work) as 39.7 kJ g
mer et al. 2005 , p. 126). Thus, this value includes both the usable
energy content of fat and the unusable energy content of fat.
This approach has been used in birds (Walsberg and Wolf 1995;
McNab 2002) and to calculate energy consumption in mam-
malian hibernators (Harlow et al. 2002; Geiser 2007). When fat
stores are depleted, hibernators start to consume lean mass
(Lopez-Alfaro et al. 2013; Hindle et al. 2015), which produces a
total amount of energy equal to 23.6 kJ g
. As with fat energy
content, this number is also very general and standard and
includes the heat lost by utilization (Robbinset al. 2012; Lopez-
Alfaro et al. 2013). Thus, with the obtained changes in lean
mass and fat reduction after the experimental hibernation of 31 d,
we calculated DEE
#fat reduction in
grams 123.6 kJ g
#lean mass reduction in grams. These
coefﬁcients were obtained from previous works assuming respi-
ratory quotient (RQ) p0:71 for fat metabolism (Walsberg and
Wolf 1995) and RQ p0:74 for protein metabolism (Harlow et al.
Intraperitoneal Data Loggers and Metabolic Predictions
We used three miniature data loggers (Star-Oddi DST nano,
to record body temperature (T
) every 5 min. The devices were
surgically implanted into the abdomen (intraperitoneal) of
three individuals, which were among the 27 animals used in
the qMR experiment. This was performed 2 mo before the ex-
periment (April). According to the manufacturer, the devices are
calibrated at factory over a temperature range of 57C–457C.
Additionally, the data loggers were calibrated by us in a beaker
with water at 407C that was allowed to cool to room temperature
(107C), with temperature records made every 2 min using a la-
boratory thermometer (alcohol). The linear regression between
water and data logger temperature (20 points) was highly signiﬁcant
(R2<0:99, Pp0:001). For both implantation and removal, we
used subcutaneous tramadol 5 mg kg
and inhalation anesthesia
for induction(isoﬂurane, 5%) and maintenance (isoﬂurane, 2.5%).
We then administered subcutaneous meloxicam 0.5 g kg
surgical approach consisted of a small incision (3 mm) on the
abdominal region in their median plane, from the xiphoid process
to the marsupium. The device was delicately placed perpendicu-
larly to the body axis between the layers of the peritoneum, and
the wound was closed with a stitch using sutures that are self-
absorbing, both in the muscular plane and in the skin. The whole
procedure lasted less than 5 min per animal. After this, the animal
was maintained in the clinic for 5 d for recovery (under outdoor
conditions), with food and water ad lib.
Calculating Resting Metabolism during Torpor/Euthermia
We associated the T
time series obtained withthe intraperitoneal
data loggers with the ambient temperature (T
) records of the
HOBO data loggers in each enclosure, which permitted us to
estimate the thermal gradient of each animal every 15 min. Under
steady-state conditions, metabolic rate is proportional to this
thermal gradient, scaled by minimum thermal conductance (C
Thus, the Scholander-Irving model permitted us to predict resting
metabolic rate (RMR) bymultiplying the thermal gradient by C
(McNab 2002; Rezende and Bacigalupe 2015). Thus, RMR was
calculated for every point in the time series of T
RMR pCmin(TB2TA). According to several authors (McNab
1980; Nicol and Andersen 2007; Rezende and Bacigalupe 2015),
is approximately constant below thermoneutrality. Then
using the average DEE
obtained from the qMR data, for each
individual we calculated C
This gives an approximate value of C
during the experimental
period, which was close to the allometric predictions (see “Dis-
cussion”). This procedure assumes that the animals are ther-
moregulating at steady state (i.e., metabolic heat production
equals heat loss), which is mostly true for the periods of torpor
and euthermia (Nicol and Andersen 2007; Rezende and Baci-
galupe 2015; see also ﬁg. 1). Later we used this empirical value of
, which was very similar to what has been reported for this
species before (Bozinovic et al. 2004) and to the allometric
predictions (see “Discussion”), to estimate RMR in each T
graph. This permitted us to estimate the changes in RMR in tor-
por and euthermia and to contrast them with what was seen with
the qMR and also with literature.
Rewarming, however, does not meet the steady-state con-
dition, as during these episodes animals produce heat at a
greater rate than heat dissipation (Geiser and Baudinette 1990;
Stone and Purvis 1992). Thus, we calculated the cost of re-
) for each bout in each record as the amount of
energy needed to warm up an animal from the measured T
Flexible Hibernation in a Marsupial 241
) at the beginning of rewarming to the ﬁnal
recorded euthermic T
Rcost pHtissue(TBeu 2TBtorpor ):ð2Þ
is the caloric capacity of living tissue (Htissue p
We performed standard statistical analyses (ANOVA, ANCOVA,
linear regressions, residual analyses). To calculate the time con-
sistency of the fat and lean mass proportions of M
, we estimated
repeatabilities as the intraclass correlation coefﬁcient (Lessells and
Boag 1987), using variance components, of both measurements
(i.e., before and after). Thus, the repeatability is computed as the
ratio of between-individual variance over total variance for each
trait (percentage of fat and percentage of lean mass) after an
ANOVA. It represents the maintenance of the ranking of the
individuals during the measurements. We used Statistica (2006;
ver. 7.0) for the statistical analyses.
Fat Consumption and Daily Energy
Expenditure of Hibernation
Field visits revealed that all of the animals entered torpor within
24 h after release into the enclosures, and they remained in packed
clusters within small cavities on the ground (ﬁg. 2). In three of the
six enclosures, monitos changed their hibernating position within
the enclosure during the ﬁrst week, revealing some movements
during the euthermic intervals.However, from the second week to
the end of the experiment, the clusters remained in the same
position (within the enclosure), thus indicating that during inter-
bout euthermic episodes, animals did not move. In total, monitos
reduced their fat content from 19:851:7g(44%ofM
), which represents a signiﬁcant reduction
(ﬁg. 3a;F3, 78 p88:8, P<0:001, repeated-measures ANOVA
from the parameters of table 1 gives 8:950:60 kJ d
Overall Changes in Body Composition
The general variation in body composition before, after, and
during the 2 d of recovery is presented in ﬁgure 3. The ﬁrst day
of recovery (i.e., day 32 of the experiment) did not induce a
signiﬁcant increase in fat content (ﬁg. 3a;day32;Pp0:46,
Tukey post hoc test), and during the third day of recovery (i.e.,
day 33; ﬁg. 3a), animals reduced their fat content signiﬁcantly
(Pp0:001, Tukey post hoc test).
Lean mass experienced a signiﬁcant net reduction during
the 31 d of the experimental hibernation (ﬁg. 3b;F3, 78 p15:3,
P<0:001, repeated-measures ANOVA and Tukey post hoc
test). During the recovery period, the amount of lean mass did
not change (nonsigniﬁcant differences between days 32 and 33,
Tukey post hoc test; ﬁg. 3b). The hydration index was maintained
constant during the whole experiment (ﬁg. 3c; nonsigniﬁcant
effects after a repeated-measure ANOVA). The relationship be-
and initial fat content was signiﬁcant (R2p0:55,
P<0:001; ﬁg. 4a) and remained signiﬁcant after removing M
effects by using residuals (R2p0:23, Pp0:014; ﬁg. 4b). This
suggests that comparatively fatter individuals consumed their fat
stores at a faster rate than lean ones.
The linear regression between fat and lean mass (as pro-
portion of M
) before and after the hibernation experiment is
presented in ﬁgure 4cand 4d. These relationships were both
negative, and the slopes of these two regressions were not signif-
icantly different after an ANCOVA (slope initial p20:50 5
0:087, slope final p20:47 50:086, F1, 49 p0:89, Pp0:35,
separate-slopes model ANCOVA), thus indicating that the rela-
tionship persisted after the experimental period of hibernation.
Also the repeatabilityof fat and lean mass(as percentageof M
signiﬁcant (intraclass correlation coefﬁcient for fat p65:6%,
lean p89:0%, F26, 26 p4:7, P<0:001, ANOVA), suggesting
time consistency in the body composition of monitos.
Torpor Patterns from Data Loggers
The torpor patterns of hibernating monitos (np3, MBp
30:8–40 g) are shown in ﬁgure 5, from which detailed hiberna-
tion parameters were extracted and presented in table 2. These
parameters revealed a mean torpor duration of 31:253:6h,with
maximum multiday torpor episodes of 127.9 h (5.3 d). Animals
aroused from torpor in 2:61 50:3 h, and the energetic cost of
rewarming per bout was 3:51 50:2 kJ, which gave a net rewar-
ming cost per individual of 54:755:5 kJ (table 2). The empir-
ically estimated C
and equation (1) was 3:375
0:19 J g
, which permitted us to predict a RMR in torpor
(i.e., the mean RMR values during deep torpor) of 3.21 kJ d
and a euthermic RMR of 72.4 kJ d
, which represents an acute
Figure 1. Schematized torpor cycles during hibernation, indicating
the hibernation parameters extracted in this study from simultaneous
ambient temperature (T
) and body temperature (T
metabolic reduction of 96% (table 2). With these empirically
values, we plotted RMR for each individual (ﬁg. 5b,
5d,5f). Each plot reproduces two metabolic curves, one for
euthermia and the other for heterothermia, with intermediate
points given by intermediate states of cooling or rewarming.
The heterothermic metabolic curves (ﬁg. 5b,5d,5f; lower limit in
the data point distribution) suggest that animals increased their
RMR with T
reductions, with a torpor defended temperature of a
little less than 57C(ﬁg. 5b,5d,5f).
In this study, we used qMR to determine changes in body com-
position (fat, lean tissue, and water) in fasted monitos during an
Figure 2. Clustered hibernating Dromiciops gliroides in our outdoor enclosures. The thick tail is typical of autumn-fatted animals.
Flexible Hibernation in a Marsupial 243
experimental hibernation period of 31 d in the ﬁeld. It is well-
known that hibernators reduce their energy needs as winter prog-
resses, lengthening episodes of torpor and shortening euthermic
periods (Cranford 1978; Geiser 2007; Jonasson and Willis 2012);
thus, our preliminary results warrant a complete monitoring of
the hibernation period of this marsupial. Still, we think this work
is novel and interesting because (1) monitos, together with pygmy
possums and feathertail gliders, are considered one of the few
true hibernating marsupials (Geiser 1994); (2) we provide de-
tailed hibernation parameters, including an estimation of energy
savings for the overall period as well as the torpor-arousal cycles
and costs; and (3) the study shows a viable alternative to esti-
mate ﬁeld energetic parameters of hibernators using the qMR
In the experimental hibernation period considered here
(31 d), there was a reduction in fat of 5.6 g, a reduction in lean
tissue of 1.3 g, and maintenance of the hydration percentage of
the animals to a nearly constant level of 95% (ﬁg. 3). The three
individuals with data loggers (i.e., a subsample of the 26 ani-
mals measured with the qMR; see ﬁg. 5) spent approximately
21.33 d in torpor (72% of the time; this includes cooling), 1.9 d in
rewarming (6% of the time), and 6.6 d in euthermia (22% of the
time). The small, observed reduction in fat and lean mass con-
tent of the animals was equivalent to a DEE
of 8.9 kJ d
this rate, a monito starting the hibernation period with 20 g
of fat will survive without food for 111 d (3.7 mo). However,
this calculation is conservative and assumes a constant DEE
through hibernation, which is variable. For instance, the little
brown bat (Myotis lucifugus; lean adult mass: 8–9 g) hibernates
6 mo per year and consumes fat at a rate of 0.0063 g d
reduced from 0.008 to 0.0049 g d
as hibernation proceeds
(Jonasson and Willis 2012). Similarly, in the jumping mouse
(Zapus princeps; lean adult mass: 20–30 g), which hibernates
9 mo per year, fat consumption during (laboratory) hibernation
is on average 0.07 g d
but is reduced from 0.28 to 0.04 g d
at different T
’s and across the different phases of hibernation
(Cranford 1978). On the other hand, for the pygmy possum (Cer-
cartetus nanus; lean adult mass: 21 g), which can pass a complete
year in hibernation, fat consumption during (laboratory) hi-
bernation decreased from 0.40 to 0.04 g d
(Geiser 2007). Thus,
our estimation of energy consumption in monitos is coincident
with the expected value for the ﬁrst month of hibernation. Im-
portantly, variation in this parameter was high (0.03–0.37 g d
and a detailed inspection of the data also revealed a large initial
variation in the proportion of fat content in the initial sample
(range: 24.4%–63.7%). This produced a signiﬁcant relationship
between initial fat content and DEE
that remained signiﬁcant
after removing M
effects (ﬁg. 4).
Our results suggesting that individuals with good fat stores
had proportionally less DEE
(ﬁg. 3) are interesting and suggest
that body condition is an important predictor of torpor use.
Some hibernators are known to reduce torpor frequency and
depth when food is abundant (Landry-Cuerrier et al. 2008; Vuarin
et al. 2015), but others show the opposite response (Stawski and
Geiser 2010). In Dromiciops gliroides, the cycle of adiposity is
associated with circulating levels of leptin, a lipostatic hormone
(Franco et al. 2017). Thus, it is likely that fat stores, via leptin
secretion, induce a net increase in energy consumption. This
could happen by three ways: (1) fat animals (compared with
lean animals) reduced torpor metabolic rate, (2) their rewarm-
ing costs were comparatively lower, or (3) these animals spent
less time torpid. We believe that the ﬁrst option is unlikely, since
Figure 3. Body composition as percentage of body mass (M
[a], le an mass [b], hydration index [c]) of animals during the hibernation
experiment (days 0–31)and during recovery (days 32–33), as given by the
quantitative magnetic resonance equipment. The hydration index was
calculated as (total water –free water)/lean mass. *P!0.05; **P!0.01;
results indicate that above minimum T
moconformers (thus, they do not regulate metabolism most of
the time). The second option is also unlikely, as rewarming costs
do not vary much among individuals and depend mostly on M
and the thermal gradient (which is fairly constant; see table 2;
see also Stone and Purvis 1992). The third option (i.e., well-
fed individuals spend less time in torpor) is the most likely, as it
is known that monitos could even suppress hibernation if re-
ceiving ad lib. food (Nespolo et al. 2021, 2022). This result
supports the view that D. gliroides opportunistically regulates the
frequency of torpor, depending on the trophic supply and energy
The maximum rewarming rate (RR) from torpor has been
considered an important parameter describing the thermo-
genic capacity of hibernators (Geiser and Baudinette 1990;
Stone and Purvis 1992). In eutherians, there is a heating organ
(brown adipose tissue [BAT]) that proliferates during cold ac-
climatization that is absent in marsupials (Gaudry and Camp-
bell 2017; Jastroch et al. 2021); however, marsupials do not
seem to show differences in RRs and costs or in acclimation capac-
ities compared with eutherian mammals (Geiser and Baudinette
1990; Stone and Purvis 1992; Opazo et al. 1999; Nespolo et al.
2002). As we argue in the next paragraph, our results maintain
In our monitos, we estimated maximum RRs (0.217Cmin
TBtorpor p9:17C; from table 2), which are somewhat lower
than what has been reported for other marsupials of similar
, such as the didelphid Thylamys elegans (RR p0:337Cmin
MBp25 g; Opazo et al. 1999) or the dasyurid Sminthopsis la-
niger (RR p0:757C min
,MBp27:4 g; Geiser et al. 1986).
Compared with the expected value using the allometric equa-
tion of RR in np16 marsupial species (log10 RR p0:006 2
0:174log10MB;r2p0:41; Geiser and Baudinette 1990), our value
is only 38% of the expected value by allometry (0.557Cmin
However, this is probably explained by the fact that all of the
other heterothermic species considered in Geiser and Baudi-
nette (1990) exhibit relatively mild torpor T
Rewarming rates from lower T
’s have been estimated in the
arctic ground squirrel (MB∼800 g), which are 0.0977Cmin
(TBtorpor p2:377C), 0.0847Cmin
(TBtorpor p20:177C), and
(TBtorpor p21:447C; Karpovich et al. 2009).
Thus, monitos, without BAT, rewarm from torpor as fast as other
cold-adapted mammals. Thus, identifying the thermogenic tissue
of marsupials remains an open question (see a review of this topic
in Jastroch et al. 2021).
Our data could also clarify rewarming costs, which have been
estimated in up to 85% of the total energy spent during hi-
bernation (Geiser 2007). However, this amount is dependent on
the frequency of arousals, which is reduced as the cold station
progresses and at different temperatures (Geiser 2007). For
instance, in cold-acclimated Chilean mouse opossums (Thylamys
elegans;MBp40 g), the cost of rewarming from TBtorpor p197C
is 5.5 kJ bout
, whereas in warm-acclimated animals this cost
is 6.86 kJ bout
(Opazo et al. 1999). Moreover, the efﬁciency of
rewarming should consider the rewarming rate. Thus, with a re-
warming cost of 3.51 kJ bout
, a 34.6-g monito rewarms from
torpor in just 2.61 h (0.039 kJ g
), for a thermal gradient of
277C (table 2). In comparison, the rewarming cost empirically
measured by Oelkrug et al. (2011) in wild-type mice was 2.3 kJ
(0.048 kJ g
), for a thermal gradient of 13.77C (see
Table 1: Descriptive statistics of fat consumption in Dromiciops gliroides (np26) during hibernation in outdoor
enclosures for 31 d in winter
Variable Mean SE Min to max
Initial body mass (M
; g) 45.4 2.3 29.3–77.0
(g) 37.8 1.9 24.4–63.7
Initial fat content (g) 19.8
Final fat content (g) 14.1 1.4 4.5–35.1
Initial lean mass content (g) 21.7
Final lean mass content (g) 19.6 .60 14.9–27.3
Daily lean mass consumption (g d
) .07 .004 .016–.11
Daily energy consumption from lean mass (kJ d
Hibernation daily energy consumption (DEE
Proportional contribution of fat to DEE
(%) 81.3 1.6 60.4–94.5
Proportional contribution of lean tissue to DEE
(%) 18.0 1.6 5.5–39.6
Note. Fat consumption was measured before and after this period using quantitative magnetic resonance (qMR). No food was available. Mean
air temperature during the period was 8.17C547C.
Determined using qMR after 24 h of fasting and before release.
Calculated as daily fat consumption#39.7 kJ g
(Walsberg and Wolf 1995).
Calculated as lean mass consumption#23.6 kJ g
(Harlow et al. 2002).
Sum of values from fat and lean mass consumption.
Daily energy expenditure of reference, obtained using doubly labeled water in (np8; MBp35 g) winter-acclimated euthermic D. gliroides
(Nespolo et al. 2022).
Flexible Hibernation in a Marsupial 245
ﬁg. 4 in Oelkrug et al. 2011). Thus, this calculation suggests that
monitos rewarm from torpor with less energy and cover a larger
thermal gradient than this similarly sized eutherian mammal.
winter, rewarming costs were 54:68 55:5 kJ, which represents
only 25% of the total energy expenditure in hibernation and not
85% as indicated by Geiser (2007).
The interpretation of our data should consider two important
caveats. First, our experiments were performed during a limited
window of time in a species that is known to hibernate at least
5 mo per year. Thus, we analyzed probably the ﬁrst part of the
cycle, and it is known that several physiological changes occur
in the long-term during hibernation (discussed below). Second,
our estimations of metabolic variations assume passive cooling
and steady-state conditions, which are sensitive to the empirical
estimation of C
. Several authors have stressed that the value
below the thermoneutral zone is approximately constant
(McNab 1980; Rezende and Bacigalupe 2015). For instance, Nicol
and Andersen (2007) computed C
for the cooling phase enter-
ing torpor in echidnas (Tachyglossus aculeatus;MBp3–5 kg;
Cmin p0:024 50:003 J g
) and found it similar to the
value measured in cold-exposed, nonhibernating individuals
(0:013 50:0005 J g
; McNab 1984). Also, when
of dormice (Glis glis) during cooling, no change
is found in this parameter during entry into torpor and during
hibernation or between daily and seasonal torpor (Wilz and Held-
maier 2000). Interestingly, our estimation of Cmin p3:375
0:19 J g
is 107% of the expected value using the
allometric equation Cmin p0:755MB0:559 (np43; R2p0:95;
Rezende and Bacigalupe 2015) and is 97% of what Bozinovic
et al. (2004) estimated from the
VO2=TAcurve in euthermic
monitos (3.48 J g
). Thus, we seem to have obtained
a relatively accurate estimation of C
Given that C
is the calibration parameter we used to esti-
mate euthermic and torpor RMR from the T
can compare these predictions with empirical values obtained
Figure 4. a, Hibernation daily energy expenditure (DEE
) calculated from lean and fat mass consumption (averaged; see table 1) using quantitative
magnetic resonance in Dromiciops gliroides during 31 d in outdoor enclosures. b, Residuals of linear regressions with body mass (M
size effects. c,d, Linear regressions of lean and fat mass compositions before and after the experiment.
previously (Nespolo et al. 2010). This author used ﬂow-through
respirometry to record CO
production (MBp38:9 g) and re-
ported a RMR (TAp207C) of 44.6 kJ d
and a torpor metab-
olism of 4.36 kJ d
, which represents a metabolic fall of 90.2%.
These values are very close to our predicted RMRs in torpor and
euthermia here, thus again validating our C
summary, in this article we calibrated a procedure to estimate the
energy savings of hibernation involving animals in seminatural
Figure 5. Torpor patterns in three Dromiciops gliroides implanted with intraperitoneal data loggers to measure body temperature (T
) was also measured continuously using meteorological data loggers. a, Time series of T
for the ﬁrst individual. b, Respective
predictions of resting metabolic rate (RMR) using equation (1) for the ﬁrst individual. c,d, Results for the second individual. e,f, Results for the third
Flexible Hibernation in a Marsupial 247
conditions, quantitative magnetic resonance, and time series of
This work was funded by Fondo Nacional de Desarrollo Cien-
tíﬁco y Tecnológico (FONDECYT) grant 1180917 to R.F.N.,
Agencia Nacional de Investigación y Desarrollo PIA/BASAL
FB0002 to F.B. and P.S., the LiLi Millenium Nucleus to R.F.N.,
and Fondo de Equipamiento Cientíﬁco y Tecnológico (FON-
DEQUIP) grant EQM180055 to J.G.N. We thank Macarena
Alvarado and Claudia Pereira for their ﬁeld and laboratory as-
sistancewith the monitos. R.F.N.conceived the studyand designed
and wrote the ﬁrst draft of the manuscript. C.M. performed the
statistical analyses and contributed with ﬁgures. J.G.N., P.S., F.B.,
and M.F. contributed with statistical analyses and manuscript
editions. We declare that we have no conﬂicts of interest.
Bozinovic F., G. Ruiz, and M. Rosenmann. 2004. Energetics
and torpor of a South American “living fossil”,themicro-
biotheriid Dromiciops gliroides. J Comp Physiol B 174:293–297.
Cranford J.A. 1978. Hibernation in the western jumping mouse
(Zapus princeps). J Mammal 59:496–509.
Eastick D.L., A.M. Edwards, S.R. Grifﬁths, S.J. Spencer, and
K.A. Robert. 2020. Validation of quantitative magnetic reso-
nance as a non-invasive measure of body composition in an
Australian microbat. Aust Mammal 43:196–202.
Franco M., C. Contreras, N.J. Place, F. Bozinovic, and R.F.
Nespolo. 2017. Leptin levels, seasonality and thermal acclima-
tion in the microbiotherid marsupial Dromiciops gliroides:does
photoperiod play a role? Comp Biochem Physiol A 203:233–
Gaudry M.J. and K.L. Campbell. 2017. Evolution of UCP1
transcriptional regulatory elements across the mammalian
phylogeny. Front Physiol 8:670.
Geiser F. 1994. Hibernation and daily torpor in marsupials: a
review. Aust J Zool 42:1–16.
———. 2007. Yearlong hibernation in a marsupial mammal.
———. 2020. Seasonal expression of avian and mammalian
daily torpor and hibernation: not a simple summer-winter
affair. Front Physiol 11:436.
Geiser F. and R.V. Baudinette. 1990. The relationship between
body-mass and rate of rewarming from hibernation and
daily torpor in mammals. J Exp Biol 151:349–359.
Geiser F., R.V. Baudinette, and E.J. McMurchie. 1986. Seasonal
changes in the critical arousal temperature of the marsupial
Sminthopsis crassicaudata correlate with the thermal transi-
tion in mithochondrial respiration. Experientia 42:543–547.
GeiserF.,S.E.Currie,K.A.O’Shea, and S.M. Hiebert. 2014.
Torpor and hypothermia: reversed hysteresis of metabolic
rate and body temperature. Am J Physiol 307:R1324–R1329.
Gibert-Ramos A., H. Palacios-Jordan, M.J. Salvado, and A. Cre-
scenti. 2020. Consumption of out-of-season orange modulates
Table 2: Summary of hibernation parameters in Dromiciops gliroides (np3; body mass p34.6 52.7 g) recorded
using temperature data loggers during 31 d in outdoor enclosures and without access to food
Mean SE Minimum Maximum
No. torpor bouts 17.3 2 16 21
Torpor bout duration (h) 31.18 3.6 .48 127.9
Duration of interbout euthermic periods (h) 8.57 2.1 .8 90.1
Rewarming rate (7Cmin
.01 .1 3.6
Rewarming time (h) 2.61 .3 2.1 3.1
Rewarming cost per arousal (kJ) 3.51
.2 2.9 4.4
Net rewarming cost per individual (kJ) 54.68
5.5 44.9 64.0
Mean cooling rate (7Cmin
.0 2.1 2.1
Mean torpid body temperature (7C) 9.1 1.0 2.1 10.1
.2 .2 .7
Mean euthermic body temperature (7C) 36.1 .6 36.1 36.7
Minimum thermal conductance (C
.19 3.13 3.75
Resting metabolic rate (RMR) in torpor (kJ d
1.1 1.48 5.68
RMR in euthermia (kJ d
8.5 60.9 93.2
Metabolic reduction during torpor (%) 96
Estimated from the linear phase.
Calculated using the caloric capacity of tissues and eq. (2).
Calculated from the sum of all rewarming episodes in each record.
Calculated as body temperature (T
) minus ambient temperature (T
), where T
is the temperature of air at 40 cm aboveground.
Calculated as Cmin pRMR=(TB2TA), where RMR is the average energy consumption from fat, estimated using the quantitative magnetic resonance
approach (see “Material and Methods”for details).
After comparing each torpor episode with the next euthermic episode in each individual record (see ﬁg. 4).
fat accumulation, morphology and gene expression in the
adipose tissue of Fischer 344 rats. Eur J Nutr 59:621–631.
Giering K., L. Lamprecht, and O. Minet. 1996. Speciﬁcheat
capacities of human and animal tissues. Proc SPIE 2624:
Logan, R.H. Henning, and J.M. Storey. 2021. The torpid
state: recent advances in metabolic adaptations and protec-
tive mechanisms. Front Physiol 11:623665.
Guglielmo C.G., L.P. McGuire, A.R. Gerson, and C.L. Seewagen.
2011. Simple, rapid, and non-invasive measurement of fat,
lean, and total water masses of live birds using quantitative
magnetic resonance. J Ornithol 152:75–85.
Cancer causes metabolic perturbations associated with re-
duced insulin-stimulated glucose uptake in peripheral tissues
and impaired muscle microvascular perfusion. Metab-Clin
Body mass and lipid changes by hibernating reproductive
and nonreproductive black bears (Ursus americanus). J Mam-
Heller H.C. and G.W. Colliver. 1974. CNS regulation of body
temperature during hibernation. Am J Physiol 227:583–589.
Hilderbrand G.V., C.C. Schwartz, C.T. Robbins, and T.A.
Hanley. 2000. Effect of hibernation and reproductive status
on body mass and condition of coastal brown bears. J Wildl
Manag 64: 178–183.
Hindle A.G., J.P. Otis, L.E. Epperson, T.A. Hornberger, C.A.
Goodman, H.V. Carey, and S.L. Martin. 2015. Prioritization
of skeletal muscle growth for emergence from hibernation.
J Exp Biol 218:276–284.
Hume I.D., M.J. Runcie, and J.M. Caton. 1997. Digestive physi-
ology of the ground cuscus (Phalanger gymnotis), a New Guinean
phalangerid marsupial. Aust J Zool 45:561–571.
Jastroch M., E.T. Polymeropoulos, and M.J. Gaudry. 2021.
Pros and cons for the evidence of adaptive non-shivering
thermogenesis in marsupials. J Comp Physiol B 191:1085–
Jonasson K.A. and C.K.R. Willis. 2012. Hibernation energetics
of free-ranging little brown bats. J Exp Biol 215:2141–2149.
Karpovich S.A., O. Toien, C.L. Buck, and B.M. Barnes. 2009.
Energetics of arousal episodes in hibernating arctic ground
squirrels. J Comp Physiol B 179:691–700.
Kelsey N.A. and F. Bairlein. 2019. Migratory body mass increase
in northern wheatears (Oenanthe oenanthe) is the accumu-
lation of fat as proven by quantitative magnetic resonance.
J Ornithol 160:389–397.
cycle of energy and time expenditure in a golden-mantled
ground squirrel population. Oecologia 78:269–282.
Kennedy L.V., Y.E. Morbey, S.A. Mackenzie, P.D. Taylor, and
C.G. Guglielmo. 2017. A ﬁeld test of the effects of body com-
position analysis by quantitative magnetic resonance on song-
bird stopover behaviour. J Ornithol 158:593–601.
Kraft F., S.C. Driscoll, K.L. Buchanan, and O.L. Crino. 2019.
Developmental stress reduces body condition across avian
life-history stages: a comparison of quantitative magnetic res-
onance data and condition indices. Gen Comp Endocrinol
Landry-Cuerrier M., D. Munro, D.W. Thomas, and M.M.
Humphries. 2008. Climate and resource determinants of
fundamental and realized metabolic niches of hibernating
chipmunks. Ecology 89:3306–3316.
Lessells C.M. and P.T. Boag. 1987. Unrepeatable repeatabil-
ities: a common mistake. Auk 104:116–121.
Lopez-Alfaro C., C.T. Robbins, A. Zedrosser, and S.E. Nielsen.
2013. Energetics of hibernation and reproductive trade-offs
in brown bears. Ecol Model 270:1–10.
Lyman C.P. 1948. The oxygen consumption and temperature
regulation of hibernating hamsters. J Exp Zool 109:55–78.
McNab B.K. 1980. On estimating thermal conductance in endo-
therms. Physiol Zool 53:145–156.
———. 1984. Physiological convergence amongst ant-eating
and termite-eating mammals. J Zool (Lond) 203:485–510.
———. 2002. The physiological ecology of vertebrates: a view
from energetics. Cornell University Press, Ithaca, NY.
Melvin R.G. and M.T. Andrews. 2009. Torpor induction in
mammals: recent discoveries fueling new ideas. Trends Endo-
crinol Metab 20:490–498.
Milsom W.K. and D.C. Jackson. 2011. Hibernation and gas
exchange. Compr Physiol 1:397–420.
Nespolo R.F., L.D. Bacigalupe, P.A. Sabat, and F. Bozinovic.
2002. Interplay among energy metabolism, organ masses and
digestive enzyme activity in the mouse opossum, Thylamys
elegans: the role of thermal acclimation. J Exp Biol 205:2697–
Nespolo R.F., F.E. Fontúrbel, C. Mejias, R. Contreras, P.
Gutierrez, E. Oda, P. Sabat, C. Hambly, J.R. Speakman and
F. Bozinovic. 2022. A mesocosm experiment in ecological
physiology: adaptive modulation of energy budget in a hiber-
nating marsupial under chronic caloric restriction. Physiol Bio-
chem Zool 95:66–81.
Nespolo R.F., C. Mejias, A. Espinoza, J.F. Quintero-Galvis,
E.L. Rezende, F.E. Fonturbel and F. Bozinovic. 2021.
Heterothermy as the norm, homeothermy as the exception:
variable torpor patterns in the South American marsupial
monito del monte (Dromiciops gliroides). Front Physiol 12:
Nespolo R.F., C. Verdugo, P.A. Cortes, and L.D. Bacigalupe.
2010. Bioenergetics of torpor in the microbiotherid mar-
supial, monito del monte (Dromiciops gliroides): the role of
temperature and food availability. J Comp Physiol B 180:
Nicol S.C. and N.A. Andersen. 2007. Cooling rates and body
temperature regulation of hibernating echidnas (Tachyglossus
aculeatus). J Exp Biol 210:586–592.
Oelkrug R., G. Heldmaier, and C.W. Meyer. 2011. Torpor
patterns, arousal rates, and temporal organization of torpor
entry in wildtype and UCP1-ablated mice. J Comp Physiol
Flexible Hibernation in a Marsupial 249
Opazo J.C., R.F. Nespolo, and F. Bozinovic. 1999. Arousal from
torpor in the chilean mouse-opposum (Thylamys elegans): does
non-shivering thermogenesis play a role? Comp Biochem Physiol
Pembrey M.S. and W.H. White. 1896. The regulation of tem-
perature in hybernating animals. J Physiol 19:477–495.
Rezende E.L. and L.D. Bacigalupe. 2015. Thermoregulation in
endotherms: physiological principles and ecological con-
sequences. J Comp Physiol B 185:709–727.
Riley J.L., J.H. Baxter-Gilbert, C.G. Guglielmo, and J.D. Litzgus.
2016. Scanning snakes to measure condition: a validation of
quantitative magnetic resonance. J Herpetol 50:627–632.
Robbins C.T., C. Lopez-Alfaro, K.D. Rode, O. Toien, and O.L.
Nelson. 2012. Hibernation and seasonal fasting in bears: the
energetic costs and consequences for polar bears. J Mammal
Ruf T. and F. Geiser. 2015. Daily torpor and hibernation in
birds and mammals. Biol Rev 90:891–926.
Snelson M., S.M. Tan, G.C. Higgins, R.S.J. Lindblom, and M.T.
Coughlan. 2020. Exploring the role of the metabolite-sensing
receptor GPR109a in diabetic nephropathy. Am J Physioogy
Statistica. 2006. Statistica (data analysis software system).
Version 6.1. StatSoft, Tulsa, OK. http://www.statsoft.com.
Stawski C. and F. Geiser. 2010. Fat and fed: frequent use of summer
torpor in a subtropical bat. Naturwissenschaften 97:29–35.
Stone G.N. and A. Purvis. 1992. Warm up rates during arousal
fro m torpor in heterothermic mammals: physiological correlates
and a comparison with heterothermic insects. J Comp Physiol B
Storey K.B. and J.M. Storey. 2010. Metabolic rate depression:
the biochemistry of mammalian hibernation. Pp. 77–108 in
G.S. Makowski, ed. Advances in clinical chemistry. Vol. 52.
Elsevier, San Diego, CA.
Taicher G.Z., F.C. Tinsley, A. Reiderman, and M.L. Heiman.
2003. Quantitative magnetic resonance (QMR) method for
bone and whole-body-composition analysis. Anal Bioanal
energy budgets and cost of arousals for hibernating little
brown bats, Myotis lucifugus. J Mammal 71:475–479.
Toien O., J. Blake, D.M. Edgar, D.A. Grahn, H.C. Heller, and
B.M. Barnes. 2011. Hibernation in black bears: indepen-
dence of metabolic suppression from body temperature.
Vuarin P., M. Dammhahn, P.M. Kappeler, and P.Y. Henry.
2015. When to initiate torpor use? food availability times
the transition to winter phenotype in a tropical heterotherm.
Walsberg G.E. and B.O. Wolf. 1995. Variation in the respiratory
quotient of birds and implications for indirect calorimetry
using measurements of carbon-dioxide production. J Exp Biol
Wang L.C.H. 1978. Energetic and ﬁeld aspects of mammalian
torpor: the Richardson’s ground squirrel. J Therm Biol 3:87.
Wang L.C.H. and J.W. Hudson. 1970. Some physiological
aspects of temperature regulation in the normothermic and
torpid hispid pocket mouse, Perognathys hispidus.Comp
Biochem Physiol A 32:275–293.
physiology of animals. Blackwell, Malden, MA.
Wilz M. and G. Heldmaier. 2000. Comparison of hibernation,
estivation and daily torpor in the edible dormouse, Glis glis.
J Comp Physiol B 170:511–521.