Energetics of a long-distance migrant shorebird (Philomachus pugnax) during cold exposure and running
1 Biology Department, University of Ottawa, Ottawa, Ontario,
Canada
2 Biological Sciences, University of Montana, Missoula, Montana,
USA
* Author for correspondence (e-mail: jmweber{at}science.uottawa.ca)
Accepted 15 November 2004
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Summary |
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Key words: oxidative fuel utilization, fuel selection, lipid, carbohydrate, animal energetics, indirect calorimetry, energy expenditure, shivering, exercise, ruff sandpiper, Philomachus pugnax
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Introduction |
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Migration flights are energetically very demanding, but other activities
than flying also offer significant physiological challenges. For instance,
metabolic rate must be increased when low temperatures are encountered (during
the night or at high altitude), or when the birds are rapidly building fat
reserves and spend a lot of time running while feeding. In some species, leg
muscles are even known to hypertrophy during stopovers
(Piersma et al., 1999).
Therefore, shivering thermogenesis and terrestrial locomotion are two
ecologically relevant activities regularly performed by ruff sandpipers.
Acclimation to cold environments has been the subject of many bird studies
(Ballantyne and George, 1978;
Block, 1994
). For acute cold
exposure, however, most of the work has been carried out on juvenile birds,
and it has been shown that they cannot thermoregulate
(Østnes et al., 2001
).
As they age, shivering thermogenesis develops in leg and pectoral muscle, and
the latter becomes a major site of heat production
(Marjoniemi and Hohtola,
1999
), although few studies have determined which oxidative fuels
are being used. One study provides indirect information on metabolic fuels
during cold exposure, and reports respiratory exchange ratios (RER) of 0.70
and 0.77
(RER=
CO2/
O2)
in fasted and fed Arctic terns Sterna paradisaea, suggesting that
lipid oxidation is dominant (Klaassen et
al., 1989
). However, the RER values given were not corrected for
protein oxidation, and results from a single species exposed to two
temperatures cannot be generalized. It is possible that the relative use of
carbohydrates (CHO) for thermogenesis is higher in other species and at lower
temperatures. For example, the use of glycogen as a thermogenic fuel during
extreme cold exposure was investigated in the pectoral muscle of pigeons
(Parker and George, 1975
). The
authors concluded that carbohydrates could become a significant fuel for heat
production during high-intensity shivering.
Most of the information available on avian fuel metabolism during exercise
is based on measurements of metabolite concentrations and total body
composition in captive birds, or in wild animals caught at various stages of
migration (Guglielmo et al.,
2002; Jenni and
Jenni-Eiermann, 1998
;
Jenni-Eiermann et al., 2002
).
All these studies have shown that birds can use lipids at very high rates, but
they have not provided much information on carbohydrate metabolism, on
individual birds (i.e. only groups of animals are compared), or on the time
course of changes in fuel utilization (only start and end points have been
measured). In a few cases, it has been possible to exercise birds in flying
wheels (red junglefowl, Chappell et al.,
1996
; Hammond et al.,
2000
; house sparrow, Chappell
et al., 1999
) or in wind-tunnels (pigeon,
Rothe et al., 1987
;
Rothe and Nachtigall, 1987
;
thrush nightingale, Klaassen et al.,
2000
; Lindström et al.,
1999
; European starling, Ward
et al., 2001
; barnacle and bar-headed geese,
Ward et al., 2002
; red knot,
Jenni-Eiermann et al., 2002
;
Kvist et al., 2001
). Changes
in metabolite concentration reported in many of these studies are very useful,
but they only provide an estimate of fuel utilization, and conclusions based
on such measurements can be misleading (e.g. see
Haman et al., 1997
). In
contrast, dynamic changes in substrate oxidation over time can be followed
using indirect calorimetry, and this is the main reason it was selected for
our experiments. Unfortunately, this method has rarely been applied to bird
exercise studies in the past and the RER values reported have never been used
to calculate fuel oxidation (Brackenbury
and Vincent, 1988
; Rothe et
al., 1987
; Ward et al.,
2002
,
2001
). In this study, our goal
was to quantify the rates of lipid, carbohydrate and protein oxidation in ruff
sandpipers during cold exposure and terrestrial locomotion, using indirect
calorimetry and nitrogen excretion measurements. Even though it is well
established that long-distance flight is predominantly supported by lipid
oxidation, the fuel selection patterns of running and shivering birds are not
known. Information presently available shows that the neural and hormonal
pathways regulating body temperature and thermogenesis are very different
between birds and mammals (Hissa,
1988
). Therefore, we anticipated that fuel selection of birds and
mammals would also be different. The experimental approach selected in our
study has enabled characterization of how the duration and the intensity of
running and cold exposure affect fuel metabolism.
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Materials and methods |
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Indirect calorimetry
For cold exposure, rates of oxygen consumption
(O2) and carbon
dioxide production
(
CO2) were
measured using an Oxymax system (Columbus Instruments, Columbus, OH, USA)
supplied with room air at 23 l min1 as detailed
previously (Weber and O'Connor,
2000
). For exercise, gas exchange was measured with Applied
Electrochemistry analyzers (models S-3A/II and CD-3A; Pittsburgh, PA, USA),
using an air flow rate of 2 l min1. Small fans ensured that
the air was continuously mixed in the measuring chambers. Air flow rate was
controlled by a mass flow regulator accurate to within 1% of full scale and
calibrated using a reference volume meter (Porter Instruments, Edgemont, PA,
USA). Oxygen and CO2 concentrations were measured in the inflow and
outflow air after removing water vapor through calcium sulphate columns
(Drierite; W. A. Hammond, Xenia, OH, USA). New calcium sulfate was always
exposed to air for 5 min before use to avoid CO2 absorption during
measurements. All analyzers were calibrated with known gas mixtures before and
after each experiment.
O2 and
CO2 were
corrected for dry gas under standard temperature and pressure conditions
(STPD). Both experimental systems were accurate to within
±3% after bleeding known amounts of CO2 or N2, or
within ±2% by burning 99% ethanol in the respirometers. The method of
indirect calorimetry was selected for these experiments because it is
non-lethal, non-invasive, and allows us to measure changes in the rates of
metabolic fuel utilization in individual animals over time (i.e. rates of
carbohydrate and lipid oxidation after correcting for protein oxidation,
itself estimated by measuring the rate of nitrogen excretion). These
advantages are not provided by alternative methods commonly used in this field
(i.e. changes in blood or tissue metabolite concentrations, or whole body
composition analyses).
Cold exposure experiments
Shivering experiments were carried out in a closed respirometer (38 cm
x 26 cm x 21 cm) connected to a cooling bath (PolyScience, Niles,
IL, USA) containing Canadian windshield washer fluid. The refrigerated fluid
was recirculated within the respirometer walls. All the cold exposure
measurements were started between 9:00 and 11:00 h, and no food or water was
available in the respirometer. While shivering, the birds stood quietly in
place. No walking or jumping was observed. A piece of perforated Plexiglas
covered the respirometer floor to protect the animal's feet. Before collecting
data, each bird was placed in the respirometer for 5 h at 22°C on two
separate occasions to familiarize it with the experimental set-up. After
familiarization, each animal was measured at four temperatures in random order
(22, 15, 10 and 5°C) with a minimum of 3 days between measurements. Each
experiment included a 60 min baseline period at 22°C, a transition period
of 3060 min to reach the test temperature, and a 3 h period at the test
temperature. Ambient respirometer temperature was recorded every 5 min.
Exercise experiments
The metabolism of running birds was quantified on a motorized treadmill
enclosed in an acrylic respirometer (50 cm x 28 cm x 14 cm) at an
incline of 8% (modified Simplex II, rat treadmill respirometer from Columbus
Instruments). This incline was selected to reach as high a metabolic rate as
possible without wing flapping. The animals were familiarized with the
experimental set-up by running at different speeds for at least three practice
sessions of 1530 min. Baseline gas exchange values (speed 0) were
obtained by leaving each animal quietly in the respirometer for 60 min in the
dark. The last 10 min of this resting period were used to quantify
pre-exercise O2
and
CO2. In each
exercise session, two different running speeds were monitored until steady gas
exchange values were reached (i.e. when the coefficient of variation remained
<10% for at least 10 min). Each bird was measured at speeds of 15, 20, 25,
30, 35 and 40 m min1. The order of speeds tested was
randomized and successive sessions for the same animal were separated by at
least 24 h.
Nitrogen excretion
The rate of nitrogen excretion was measured by collecting excreta for 6 h
in eight fasting individuals kept at 22°C, and by quantifying the
concentrations of uric acid (Marquardt,
1983) and urea (Kit 640-A; Sigma, St Louis, MO, USA). A mean value
of 0.534±0.056 mg N min1 kg1 body
mass (N=8) was measured and used for all birds in our calculations.
Therefore, we have assumed that the rate of protein oxidation was not affected
significantly by cold exposure or running.
Calculations and statistical analyses
Rates of carbohydrate and lipid oxidation (CHOox and
FATox, respectively) were calculated from
O2,
CO2 and the rate
of nitrogen excretion using the equations of Frayn
(1983
) modified for uricotelic
animals (Walsberg and Wolf,
1995
), and for the units used in our study:
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Percentages were transformed to the arcsine of their square root before
analysis. For cold exposure, mean
O2,
CO2, RER and
percentages were compared using one- or two-way, repeated-measures analyses of
variance (ANOVA). For running data, one-way ANOVA was used because sample size
differed between speeds. For each bird, the cost of transport was measured and
compared to values calculated from allometric equations for shorebirds only
(Bruinzeel et al., 1999
) or for
birds in general (Taylor et al.,
1982
). Mean observed and predicted values were compared using a
one-way ANOVA. Comparisons between test and control means were performed using
Bonferroni's adjustment. Decisional threshold was set at P<0.05
and all the values presented are means ± S.E.M.
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Results |
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Changes in the rates of carbohydrate and lipid oxidation (CHOox
and FATox) during cold exposure are presented in
Fig. 2. At all times,
FATox was more than 3.2-fold higher than CHOox (the
average ratio of FATox/CHOox was 5.6). Overall,
CHOox was higher at 5°C than at all other temperatures
(P<0.05). Cold exposure had no effect on CHOox over
time (P>0.05), except between 45 and 75 min at 5°C, when it
was higher than baseline (P<0.05). FATox was increased
in proportion with the intensity of cold exposure. All treatment temperatures
were significantly different from each other (P<0.001), and each
treatment showed a significant increase in FATox over time, except
for the control group kept at 22°C (P<0.001 for 5, 10 and
15°C; P>0.05 for control). Regression analysis shows that the
slope of the relationship between FATox and
O2 is very
different from 0 (slope=0.841; r2=0.920;
P<0.001). The relationship between CHOox and
O2 is also
significant, but the slope is much shallower than for FATox
(slope=0.174; r2=0.338; P<0.01).
|
All the parameters measured in the cold exposure experiments reached
steady-state values (see Fig.
1B), and are summarized in
Table 1. These values were
calculated by averaging measurements over the last 25 min at each temperature,
and they are presented for gas exchange
(O2,
CO2 and RER) and
fuel utilization (CHOox and FATox). Steady-state values
were progressively higher for
O2,
CO2 and
FATox as cold exposure intensified. However, RER and
CHOox were not affected by temperature
(Table 1).
|
Exercise experiments
Oxygen consumption and RER of birds running at various speeds are shown in
Fig. 3. Baseline values for
animals resting on the treadmill at 22°C were 51.7± 4.0 ml
min1 kg1 body mass for
O2 and
0.761±0.008 for RER (N=11). These resting rates were not
different from baseline values measured in the cold exposure experiments
(P>0.05; Fig. 1).
O2 increased
progressively with running speed (P<0.05;
Fig. 3A), and the highest
exercise
O2 of
78.9±3.5 ml O2 min1 kg1
body mass was measured at a speed of 35 m min1. RER was
elevated from baseline at low and intermediate speeds (P<0.05 at
15, 30 m min1; P<0.001 at 25 m
min1), but it was not different from baseline at the two
highest speeds (P>0.05; Fig.
3B). The slope of the regression line for RER vs speed
was not different from zero (P>0.05).
|
Fig. 4 shows CHOox and FATox as a function of speed. At all times, FATox was more than 1.6-fold higher than CHOox (the average ratio of FATox/CHOox was 2.5). Baseline CHOox in animals resting on the treadmill was 9.07± 1.28 ml O2 min1 kg1 body mass. The slope of the regression line for the CHOox vs running speed was not different from zero (P>0.05). In contrast, FATox increased progressively as speed increased (slope of regression line higher than zero; P<0.01).
|
Fig. 5 shows the
relationship between the energy cost of locomotion per unit time
(Emetab) and running speed. Mean resting rate of energy
expenditure was 2.21±0.23 J s1 (speed=0), and the
slope of the linear regression between Emetab and speed
was different from 0 (P<0.01). The slope of this relationship, or
cost of transport (energy cost per unit distance) for running ruff sandpipers
was 1.29 J m1. This value is significantly lower than
predicted from the allometric equation for birds in general (2.49 J
m1; P<0.01;
Taylor et al., 1982). However,
it is not different from the value predicted from the allometric equation
specifically derived for shorebirds (1.68 J m1;
P>0.05; Bruinzeel et al.,
1999
).
|
Fuel selection during cold exposure and exercise
The relative contributions of carbohydrate, lipid and protein oxidation to
O2 during cold
exposure and running are summarized in Fig.
6. In the cold exposure experiments
(Fig. 6A), lipid oxidation
accounted for more than 80% of
O2, the same
value observed in the control animals held at 22°C. Protein oxidation
(47%) and carbohydrate oxidation (1014%) had much lower relative
contributions. The importance of carbohydrates and lipids did not vary between
temperatures (P>0.05), but the percentage contribution of proteins
decreased with temperature (P<0.001).
|
In the exercise experiments (Fig.
6B), lipid oxidation contributed 75% of
O2 at speed 0.
This contribution was lower at 15, 25 and 30 m min1
(5862%; P<0.05) but did not differ from the resting value
at the other speeds measured (6572%; P>0.05). Exercise
caused an increase in relative carbohydrate oxidation above resting levels
(P<0.001), but only at the lower running speeds (1530 m
min1). The relative contribution of protein oxidation to
O2 was decreased
during exercise compared to rest (P<0.001).
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Discussion |
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Cold exposure
The metabolic rate of ruff sandpipers is stimulated in proportion with the
intensity of cold exposure (Fig.
1B) and lipids are responsible for fueling shivering thermogenesis
(except for a minor, transient contribution from carbohydrates; see Figs
2,
6A, and
Table 1). This conclusion is
further supported by the observation that the slope of the regression line
between FATox and
O2 is much
higher than for CHOox vs
O2 (0.841 and 0.174,
respectively). This study is the first to quantify the use of oxidative fuels
for heat production in an avian species exposed to cold. It shows that the
relative use of lipids and carbohydrates is not affected by shivering, and is
therefore independent of environmental temperature
(Fig. 6A). More than 80% of
O2 is accounted
for by lipid oxidation at all temperatures, even during intense shivering at
5°C. At the highest FAT measured here (
62 ml O2
min1 kg1 body mass), and assuming that the
same pattern of fuel utilization is maintained, we can calculate that a 110 g
ruff sandpiper with lipid reserves of 20% body mass (or half the maximal value
observed just before migration; Van Rhijn,
1991
) could shiver continuously for 4.5 days.
How does this fuel selection pattern compare with what has been observed in
other cold-exposed endotherms? In the absence of information on birds, we
looked for shivering studies on mammals of similar size (and surface to volume
ratio) as our sandpipers, but without much success. Using arteriovenous
differences in substrate concentrations, the only study we were able to find
reports that lipid oxidation accounts for more than 90% of
O2 in
cold-exposed rats (Adán et al.,
1995
). However, comparing these results with ours is not
appropriate because the rats were acclimated to 22°C and kept in
individual cages. Under such housing conditions, rats are known to produce
significant amounts of brown adipose tissue, because they are well below their
thermoneutral zone (2931°C for Wistar rats;
Romanovski et al., 2002
) and
cannot use social thermoregulation. Therefore, heat production from brown
adipose tissue may explain the high FATox reported for rats by
Adán et al. (1995
),
whereas the presence of this specialized thermogenic tissue has never been
demonstrated in birds (Saarela et al.,
1991
). In addition, we have recently carried out experiments on
group-housed rats acclimated to 28°C, to eliminate the thermogenic
contribution of brown adipose tissue. Under these conditions, lipids were
responsible for 52%
O2 and
carbohydrates for 37%
O2 during
prolonged exposure to 5°C (E. Vaillancourt, F. Haman and J.-M. Weber,
unpublished).
Surprisingly, the only other mammal whose fuel metabolism has been well
characterized during shivering is the adult human. In this experimental model,
it was established that lipids play a much less important role than observed
in ruff sandpipers. For cold exposure conditions eliciting a twofold increase
in metabolic rate, lipid oxidation is responsible for 50% of
O2 in humans
(Haman et al., 2002
). The
exact reasons for these discrepancies are unknown, but may be due to
differences in the fiber composition of shivering muscles. In humans, it has
been shown recently that CHOox for thermogenesis depends on the
specific recruitment of type II, fast glycolytic fibers, responsible for
`burst shivering' (Haman et al.,
2004
). In long-distance migrant birds, large pectoral muscles
produce most of the heat, and the metabolic machinery of their fibers is
specifically geared for lipid oxidation. Therefore, the high metabolic
capacity of flight muscles for fat catabolism could explain why, on their own,
lipids account for over 80% of
O2 in
cold-exposed ruffs. This high capacity for lipid oxidation and the large lipid
reserves available in migrant birds mean that carbohydrates and proteins only
play minimal roles during shivering, each accounting for less than 14% of
O2. Taken
together, these results show that lipids probably play a much more prominent
role for shivering thermogenesis in migrant birds than in mammals.
At the lowest temperature tested in our study, metabolic rate appeared to
fluctuate with a period of 90 min
(Fig. 1B) and this pattern may
be related to a well-known heat-saving strategy previously observed in birds.
Similar cyclic variation in
O2 and leg
temperature has been observed in cold-exposed pigeons; these changes are
caused by vasoconstriction/vasodilation cycles of the leg geared to decrease
heat loss from this uninsulated region of the body. Interestingly, pigeons
exposed to a lower temperature than tested here (10°C) showed a
shorter period of only 20 min
(Østnes and Bech,
1998
). Further research is needed to establish whether this
energy-saving strategy is found in all birds or only in species regularly
exposed to cold conditions.
Exercise
As experimental models, long-distance migrant birds provide a unique
vantage point to study the extreme performance of metabolic systems during
exercise. Unfortunately, making physiological measurements during migration or
simulating prolonged flight in the laboratory are extremely difficult and
rarely attempted. As a compromise, and because little in vivo
information is presently available on avian exercise, we reasoned that
investigating land locomotion would be an important first step towards a
better understanding of fuel metabolism in exercising migrants. However, it is
clear that running does not simulate migration because leg and flight muscles
are different, and maximal metabolic rates achievable during running are low
compared to flight.
Indirect calorimetry measurements in running or flying birds have only been
performed in a few studies that report
O2 and
CO2 (or RER),
but these parameters have never been used to determine oxidation rates of
metabolic fuels (Brackenbury and Vincent,
1988
; Rothe et al.,
1987
; Suarez et al.,
1990
; Tucker,
1968
; Vincent and Brackenbury,
1988
; Ward et al.,
2002
,
2001
). In the present study,
we have quantified
O2,
CO2 and nitrogen
excretion to calculate absolute rates of fat, carbohydrate and protein
oxidation as well as the relative contribution of each fuel to
O2 during
running (Figs 3,
4 and
6B). Results show that, as for
cold exposure, lipids play a dominant role in energy metabolism during running
(Fig. 6B). When exercise
intensity is increased, FATox is augmented whereas CHOox
remains independent of running speed (Fig.
4). The overall fuel selection pattern of exercising ruff
sandpipers stays relatively constant across speeds, with lipids providing more
energy than all other fuels combined (5872%
O2) and
carbohydrates being responsible for about half the contribution of lipids
(2438%
O2;
Fig. 6B). The increased
reliance on carbohydrates associated with higher running speeds commonly
observed in mammals does not occur in birds, showing that the fuel selection
patterns of exercising birds and mammals are different. Even though lipids are
well known to provide most of the energy for sustained flying in long-distance
migrant birds (Guglielmo et al.,
2002
; Jenni and Jenni-Eiermann,
1998
, 2002), the present study
is the first to investigate running, and to provide detailed information about
the use of all oxidative fuels under controlled exercise conditions of
different intensities.
For comparison, we have calculated FATox and CHOox
from published bird exercise studies reporting
O2 and
CO2 values, but
without correcting for protein oxidation (because rates of nitrogen excretion
are not available). The only two treadmill studies we could find show that CHO
oxidation accounts for >80% of
O2 in running
chickens (Brackenbury and Vincent,
1988
; Vincent and Brackenbury,
1988
). The divergent fuel selection patterns observed in highly
aerobic ruff sandpipers and sedentary, domesticated chickens can probably be
explained by differences in the fiber composition of leg muscles (and
associated enzymatic machinery), a parameter known to vary with species, age
and gender (Guglielmo et al.,
2002
; Olson,
2001
). From studies on flying birds, we calculated that lipid
oxidation accounts for >65% of
O2 (budgerigar,
Tucker, 1968
; pigeon,
Rothe et al., 1987
; European
starling, Ward et al., 2001
;
barnacle and bar-headed goose, Ward et
al., 2002
), except for hovering hummingbirds that can temporarily
rely entirely on carbohydrates while feeding on nectar
(Suarez et al., 1990
). Migrant
birds must clearly rely predominantly on lipids during non-stop, long-distance
flights because alternative fuels are only stored in very small quantities,
and would therefore be rapidly depleted.
Land locomotion is particularly important for long-distance migrant
shorebirds because it allows them to replenish energy reserves rapidly during
short stopovers. Strong selection pressure for decreasing the cost of walking
and running may therefore be responsible for the very low cost of transport
(energy cost per unit distance) observed here in ruff sandpipers. This cost is
48% lower than predicted from the allometric equation established for birds in
general (Taylor et al., 1982),
but it is not different from the value predicted from the allometric equation
for shorebirds only (Bruinzeel et al.,
1999
). This observation suggests that economical running is not an
exclusive attribute of migrants, but that it is a common feature of all
shorebirds, possibly related to their particular leg morphology.
Conclusions
The energy necessary to support shivering and running in ruff sandpipers is
provided almost exclusively by the oxidation of lipid reserves. Their pattern
of oxidative fuel selection does not depend on shivering or running intensity.
During shivering, total ATP production is unequally shared between lipids
(82%), CHO (12%) and proteins (6%). During land locomotion, lipids remain the
dominant substrate (66%), with CHO (29%) and proteins (5%) playing more minor
roles. The prevailing use of lipids during intense shivering and high-speed
running is not consistent with the fuel selection pattern observed in
exercising and cold-exposed mammals. Long-distance flying is well known to be
supported primarily through lipid oxidation and this study shows that the same
source of fuel is also dominant during other activities like intense running
and shivering. The exact mechanisms allowing birds to use lipids at extremely
high rates are still largely unexplored, and quantifying the exact importance
of proteins and carbohydrates during long-distance flight remains a major
challenge for future research.
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Acknowledgments |
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