Maximal metabolic rates during voluntary exercise, forced exercise, and cold exposure in house mice selectively bred for high wheel-running
Department of Biology, University of California, Riverside, California 92521, USA
* Author for correspondence (e-mail: enrico.rezende{at}email.ucr.edu)
Accepted 31 March 2005
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Summary |
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Key words: artificial selection, exercise, experimental evolution, locomotor activity, maximum oxygen consumption, running performance, thermogenesis, mouse
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Introduction |
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We tested whether selection for increased locomotor activity affected
aerobic capacity in four replicate lines of house mice bred for high voluntary
wheel-running (Selected or S lines) as compared with their four random-bred
Control (C) lines (Swallow et al.,
1998a; Garland,
2003
). The S lines have run about 170% more than C
(revolutions/day) since generation 16 (see
Garland, 2003
), and most of
the increase in distance has been achieved by increasing average running speed
rather than time spent running (the relative importance of each component is
sex-dependent, however; Koteja et al.,
1999a
,b
;
Rhodes et al., 2000
;
Swallow et al., 1998b
;
Girard et al., 2001
). Based on
videotape analyses of instantaneous running speeds, Girard et al.
(2001
) concluded that S
females from generation 23 ran twice as fast as C females (about 0.76 m
s1 and 0.38 m s1, respectively), as well
as more intermittently, during the time of peak wheel-running. Intuitively,
O2max might be
expected to evolve in concert with higher running activity because it
determines the ceiling of sustainable exercise. Hence, individuals with higher
O2max
and higher maximum aerobic speeds (MAS) might have been favored by the
selection regime, at least once they had evolved sufficiently high activity
levels to tax their aerobic limits (see
Koteja et al., 1999a
;
Girard et al., 2001
). Indeed,
a study at generation 10 reported a small (6%) but statistically significant
increase in treadmill
O2max of the S
lines as compared with C (Swallow et al.,
1998b
).
Since the analyses of Swallow et al.
(1998b), these lines have been
through more than 20 additional generations of selection. Males from S lines
that ran on average 75% more than C at generation 10
(Swallow et al., 1998b
) now
run about 190% more than C (generation 29;
Rhodes et al., 2003
). It is
possible, therefore, that
O2max has
evolved even further. The first goal of the present study was to test whether
O2max has
continued to coadapt with additional selection for high wheel-running. To
avoid potential problems arising from differences between S and C in
motivation to run, we employed two separate protocols to estimate
O2max: forced
exercise on a treadmill and cold-exposure in a He-O2 (heliox)
atmosphere. The latter protocol does not involve `motivation' in any
conventional sense and, in small rodents, may elicit different values of
oxygen consumption than are attained in forced exercise protocols (e.g. see
Chappell et al., 1995
;
Rezende et al., 2004a
).
Our second goal was to measure directly maximum metabolic rates during
voluntary wheel-running. Since generation 16, running distance by the S lines
has been at an apparent selection limit or plateau (e.g. fig. 2A in
Rhodes et al., 2003;
Garland, 2003
). Because
running speed, rather than running time, was the main factor explaining
differences between S and C, and maximal running speeds in S lines are close
to their predicted MAS (Girard et al.,
2001
), this `ceiling' in running activity might be related to
constrained aerobic capacity. Pharmacological studies support the hypothesis
that physiological and/or biomechanical factors might limit further evolution
of wheel-running, because none of the drugs tested has increased wheel-running
in S lines whereas several increased voluntary running activity in C (Rhodes
et al., 2001
,
2003
,
2005
;
Rhodes and Garland, 2003
;
Li et al., 2004
). We also
tested whether mice from S lines occasionally run voluntarily at speeds that
exceed MAS, i.e. run `wind sprints'. Studies of various vertebrates, including
human beings, indicate that they generally do not choose to exercise at speeds
above their MAS (or anaerobic threshold in humans;
Taigen and Beuchat, 1984
;
Powers and Howley, 2001
;
Chappell et al., 2004
), but
what about animals that have been purpose-bred for high voluntary
activity?
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Materials and methods |
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Wheel-running was quantified as the total number of revolutions run on days 5 and 6 of the 6-day test. In the four S lines, the highest-running male and female from each family were selected as breeders to propagate the lines to the next generation. In the four C lines, a male and a female were randomly chosen from each family. Within all lines, the chosen breeders were randomly paired, except that sibling matings were not allowed. Selection was suspended for generations 3235 as the colony was transferred from the University of Wisconsin-Madison to California.
Protocol
Forty eight females (6 per line, each from a different family) were
measured in the following protocol. After weaning at 21 days of age, two
individuals each from C and S lines were randomly mixed, four per cage, and
maintained with food and water ad libitum. Measurements began at
about 8 weeks of age; the schedule for each female is summarized in
Table 1. In short, animals had
access to wheels for a total of 6 days, mimicking the conditions of the
selection experiment. Mice had access to wheels from days 14
(Table 1), as used routinely
during selective breeding. On day 5, they were placed inside the wheel
metabolic chamber (see below), and
O2 during
wheel-running was recorded during days 5 and 6 (i.e. over a 48 h period).
Measurements were then performed twice for each individual on the treadmill
and in a heliox atmosphere during consecutive days
(Table 1, details below). To
avoid potential circadian effects, treadmill and heliox trials were performed
between 20:00 and 22:00 h, which corresponds to the period of highest
voluntary wheel activity on a 12 h:12 h light cycle with lights on at 7:00 h
(Girard and Garland, 2002
;
Rhodes et al., 2003
).
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Metabolic rate during voluntary activity
Performance during voluntary exercise was measured using the protocol and
equipment reported in Chappell et al.
(2004), which also provides
figures and URLs for photographs of the setup. Briefly, we enclosed one of the
wheels (circumference 1.12 m) and its attached standard plastic mouse cage, as
used in the routine selection protocol, within an airtight Lucite housing.
Mice could enter and exit the wheel at will through an access port cut into
the side of the mouse cage. The mouse cage contained bedding (wood shavings),
a food hopper and a drinking tube. Food and water were available ad
libitum during measurements.
Two such metabolic wheel enclosures were housed in a temperature control
cabinet (range between 18 and 27°C in a daily cycle) and photoperiod (12
h:12 h L:D, dark period 19:0007:00 h, as in the room) housing the
breeding colony. Paired incurrent and excurrent ports provided for airflow,
and an internal fan rapidly recirculated air within the enclosure to
facilitate mixing. The respirometry system and measurements were identical to
those explained in Chappell et al.
(2004). Mice were weighed
(±0.05 g) before entering the wheel (day 0), as well as before and
after trials in the metabolic chamber. At the completion of days 4 or 5 of
wheel-running, mice were transported with their respective cages from their
acclimation wheels to a wheel metabolic enclosure. Given that only two animals
could be measured at once, measurements were randomly scheduled across lines,
except that we roughly attempted to control for age effects (i.e. mice that
were born first were also measured first), and we always measured one S and
one C female concomitantly.
Although each mouse was measured in its own cage (with its own bedding,
etc.), it was logistically difficult to clean the metabolic chambers between
measurements. We tested rotational resistance before and after each
measurement by spinning wheels to high speed (80 revs
min1) with an electric drill fitted with a rubber friction
disk, and then monitoring the time needed for speed to decay to zero. Order of
measurement, wheel number, and resistance did not significantly affect any
variable, however, and were not included in the final statistical
analyses.
We dried subsampled air with magnesium perchlorate (CO2 was not
removed in order to avoid the large volumes of soda lime or frequent scrubber
changes that otherwise would be necessary for long-duration tests), and
calculated O2
(ml min1) as:
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Maximum O2 during exercise
We used open-flow respirometry to determine maximum aerobic performance as
maximum rate of oxygen consumption
(O2max). After
voluntary activity trials on the wheels, we estimated
O2max during
both forced exercise and acute cold-exposure. Each individual was tested once
a day and
O2max
was estimated twice with each method on consecutive days
(Table 1). Hence, all values
were obtained within a 4 daywindow, and less than 6 days apart from
measurements of
O2 in the
wheel.
Mice were run in an enclosed motorized treadmill, as described previously
(Hayes and Chappell, 1990;
Chappell et al., 2003
). The
treadmill had an inclination of 25°, which has been reported to yield
maximal values of
O2 in laboratory
house mice (Kemi et al.,
2002
). Mice were placed in the working section (6 cm wide, 7 cm
high, 13.5 cm long), allowed a 12 min acclimation period, and then run
at increasing speeds, starting at 0.150.2 m s1 and
raised in step increments of about 0.1 m s1 every 45 s,
until the mouse could no longer maintain position and
O2 no longer
increased. The maximum speed that each individual attained on the treadmill
was recorded. Tests lasted from 6 to 17 min, and reference readings of
incurrent gas were obtained at the start and end of measurements. Trial
quality was also assessed using a subjective scale (five categories, from poor
to excellent: Swallow et al.,
1998b
), and poor trials (a single trial for one C female where
trial quality=1) were not included in the final analyses.
Changes in O2 concentration were measured using an
Ametek/Applied Electrochemistry S-3A analyzer (Pittsburg, Pennsylvania, USA),
and recorded on a Macintosh computer equipped with National Instruments A-D
converters and Warthog software. Gas flow (2100 ml min1) was
regulated with Tylan mass flow controllers (Billerica, MA, USA) upstream from
the treadmill. About 100 ml min1 of excurrent gas was
sampled, dried with magnesium perchloride, and scrubbed of CO2
before going to the O2 analyzer. Because of the short duration of
treadmill tests, we applied the `instantaneous' transformation
(Bartholomew et al., 1981) to
resolve rapid changes in metabolism. Effective volume of the treadmill was 903
ml. We calculated
O2 using
Eq. 2 and computed
O2max as the
highest instantaneous
O2 (ml
min1) averaged over continuous 1 min intervals, using
LabAnalyst:
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Maximum O2 during cold-exposure
We measured
O2max in an
atmosphere of heliox (79% He, 21% O2), which is several-fold more
conductive than air (Chappell and Bachman,
1995
), using the same gas flow system described for treadmill
trials. A Plexiglas metabolism chamber (volume 600 ml) was supplied with
heliox at 1700 ml min1. An environmental cabinet controlled
the temperature of the metabolic chamber. Animals were weighed (±0.05
g) and placed in the metabolism chamber at an ambient temperature of about
1°C, and recording began as soon as the system was completely
flushed with heliox (about 1 min).Temperature declined around 0.5°C
min1. We terminated measurements and removed animals when
O2 declined
below initial values for more than 1 min, or did not increase as temperature
decreased more than 2°C. Trials lasted no longer than 15 min.
During these trials we also measured breathing frequency (f;
breaths s1) and tidal volume (VT; ml)
using whole-body plethysmography (Withers,
1977; Chappell,
1985
). Chamber pressure changes caused by warming and
humidification of tidal air were recorded with a pressure transducer (PX
164-010, Omega Instruments, Stamford, Connecticut, USA) connected to the
computer and sampled at 125 Hz. The system was calibrated after each trial by
injecting a known volume of heliox (1.0 ml) into the chamber at rates matching
the kinetics of inhalation cycles. Tidal volume was calculated from
calibration data and pressure changes during inspiration according to Malan
(1973
); we assumed lung
temperature was 37°C and that air in the respiratory tract was 100%
saturated with water vapor. [Although body temperature Tb
probably decreases during heliox trials, 2°C would affect
VT estimates by only about 2% when ambient temperature
Ta
0°C. Hence, 37°C was assumed for
convenience because real Tb at
O2max was
unknown (Mortola and Frappell,
1998
; Rezende et al.,
2004b
).] Oxygen extraction efficiency (O2EE,%) was
calculated as
100
O2max/(0.2095xminute
volume), where minute volume is (fVT). Immediately after
removing an animal from the chamber, Tb was determined to
±0.1°C using a rectal thermocouple connected to a BAT-12
thermometer (Sensortek, Lake Forest, California, USA). Mice were mildly
hypothermic after trials (Tb=35.11±0.16°C, mean
± S.E.M.), indicating that
O2max was
probably achieved.
Eq. 2 was used to calculate
O2, although in
this case we did not employ the `instantaneous' transformation because the
chamber volume was small relative to the flow rate, mice usually remained
still during heliox trials, and steady-state values of
O2 were
obtained.
Statistical analyses
Because we were interested in values of maximum performance for each
individual, we selected the highest 1 min value of voluntary
O2 obtained
during either of the 2 days of wheel-running
(
O2max,W), the
two treadmill
(
O2max,T) or the
two heliox
(
O2max,H)
trials. We also selected the lowest 5 min average
O2 throughout
the 2 days of measurements in the wheels as an estimate of resting metabolic
rates (RMRW). For repeatability analyses, we selected highest or
lowest values within days 1 and 2 separately (see below). Wheel data for one S
female were discarded because of measurement problems (however, records on
treadmill and heliox for this female were included).
Analyses were performed with SPSS for Windows version 11.5 (SPSS Inc.,
Chicago, IL, USA, 2002) and SAS version 8.02 (SAS Institute, Cary, NC, USA,
1996). First, to determine effects of selection, we estimated line-type
effects (S vs C) using a one-way nested analysis of covariance
(ANCOVA) with type III sums of squares, using SAS PROC MIXED (a program in the
SAS statistical package that allows testing simultaneously for fixed- and
random-effects; i.e. `mixed models'). Line type was the grouping variable
(fixed factor) and replicate lines (N=8) were nested within line type
as random factors. Body mass and age were included as covariates, but many
variables were also analyzed without mass in the model (e.g. maximum speeds,
distances) as these were not correlated with mass. Because of differences in
body mass among lines (see Discussion), head-to-rump length (HRL) was
used as an additional indicator of size. Line random effects were determined
by comparing the log likelihoods of the models with and without line (twice
the difference in log likelihoods follows a 2 distribution
with 1 d.f.). Adjusted least-squares means (and S.E.M.) were used
to estimate the difference between S and C lines. Second, regular ANCOVAs were
performed for S and C separately, including line as a random factor (4 lines)
and using the same covariates of the nested model. Although we report
P-values for two-tailed hypotheses for simplicity, we adopted
directional hypothesis whenever it was possible (i.e. S mice are expected to
run faster than C) to increase statistical power.
We also assessed how
O2 changed
between wheel, treadmill and heliox trials, compared running performance
during voluntary vs forced exercise, and whether these changes
differed between line types and lines, employing general linear model for
repeated measures (GLM procedure in SPSS). Individuals were experimental
units, type of measurement (treadmill, heliox or wheels) was the
within-subjects factor, and selection history (S vs C) and lines were
between-subjects factors. To determine how variables differed between trials,
contrasts (i.e. the difference between successive values for each individual)
were compared employing multivariate ANOVAs (test of within-subjects
contrasts).
Repeatability was estimated in two different ways
(Hayes and Jenkins, 1997).
First, residuals from both nested and regular ANCOVAs obtained in the first
and second trial were tested using Pearson productmoment correlation.
Second, we obtained the intraclass correlation with a one-way ANOVA, employing
again the residuals from nested and regular ANCOVA. The intraclass correlation
coefficient
was calculated as (groups MSerror MS)/[groups
MS+(n1) error MS], where MS is mean square, n (=2) is
the number of repeated measures per individual, and `groups' are the
individual mice (Zar, 1999
;
p. 404). Differences between
days 1 and 2 on wheels, or trials 1 and 2 for treadmill and heliox, were
addressed using paired t-tests.
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Results |
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Most variables were significantly repeatable, independent of whether
residuals were obtained from simple linear regressions or from the complete
nested ANCOVA model (Table
2).O2max
obtained in heliox, however, was not significantly repeatable by either
method. Absolute values of
O2max in heliox
(i.e. not residuals) were significantly correlated with each other, although
the relationship was relatively weak (r=0.311, one-tailed
P=0.017). Body temperature after heliox trials also was not
repeatable (after removing two influential points from the nested model;
Table 2), and was not
correlated with body mass (P=0.374).
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Considering all mice,
O2max during
first and second trials did not differ during wheel trials
(t46=1.858, two-tailed P=0.070), treadmill
trials (t47=1.201, P=0.236) or in heliox
(t46=1.679, P=0.100). No significant
differences between days were detected in maximum voluntary running speeds
(t46=0.801, P=0.426). Results remained
unchanged when S and C were analyzed separately. Maximum speed attained during
forced exercise tended to be higher during the first trial
(t47=1.943, P=0.059). In heliox, S mice had a
significantly higher
O2max during the
second trial (6.27±0.83 vs 6.77±1.00, one-tailed
P<0.03), which was not the case in C (P=0.498).
Metabolism, performance, and selection history
Selected and C lines did not differ significantly in either forced-exercise
or cold-exposure
O2max
(Table 3). Because all
metabolic variables increased significantly with body mass, we tested whether
reduction in size in S lines had changed the interaction (i.e. slope) between
mass and
O2max
(massxselection history factor was tested over massxline in SAS
PROC MIXED). There were no significant differences between S and C in the
slopes of mass vs
O2max on the
treadmill (F1,6=0.69, P=0.44), in cold-exposure
(F1,6=4.12, P=0.09) or during wheel-running
(F1,6=1.24, P=0.31). Line effects within each
line type, obtained with conventional ANCOVAs, were significant only for
O2max on the
treadmill in C mice (P<0.04), but not when HRL was
included as an indicator of size instead of mass (P=0.36). As
expected, size increased significantly with age (body mass:
F1,39=21.79, P<0.001; HRL:
F1,39=37.93, P<0.001). Maximum metabolism
during voluntary exercise was significantly higher in S lines
(Table 3). That conclusion was
unchanged when
O2max was
expressed in absolute terms or on a per gram basis (always with body mass as a
covariate).
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Aerobic scope during cold exposure
(O2max in
heliox/RMR during wheel trials) was significantly higher in S lines only after
removing body mass from the model (with mass in the model,
Pselection=0.16, Pbody mass=0.79).
After accounting for body size, Tb following cold-exposure
was marginally higher in S lines after trial 1 (34.3±0.3 vs
35.4±0.3°C for C and S, respectively, N=39,
P<0.033), but not after trial 2 (35.0 vs 35.4°C,
N=44, P=0.37).
Voluntary wheel-running speeds were significantly higher in S, whereas
maximum speeds attained during forced exercise did not differ between line
types (Table 3,
Fig. 1). There were no
significant differences in trial quality (see Materials and methods) on the
treadmill between S and C (P=0.175), and this variable was never a
significant predictor of
O2max. Effects
of selection on maximum treadmill speeds were statistically significant,
however, when size either mass or HRL was included as
a covariate (S>C: one-tailed P<0.028 and P<0.040,
respectively). In addition, repeated measures performed separately for each
line type showed a highly significant effect of measurement (treadmill
vs wheel) on running speeds in C (F1,20=57.19,
P<0.001), but no effect in S (F1,19=0.61,
P<0.416; Fig. 1B).
All three estimates of
O2max differed
significantly from each other: effects of experimental protocol were
significant in the `pooled' analysis or when S and C were analyzed separately.
O2max obtained
in heliox was higher than on the treadmill, which was higher than maximum
voluntary
O2
(Fig. 2), with a significant
trialxline type interaction (tested over trialxline
typexline, F2,12=4.323, P=0.038) probably
due to higher voluntary
O2max in S
lines. The ratio between
O2max during
cold exposure and forced exercise
O2max,H/
O2max,T)
did not differ between S and C (F1,6=0.02,
P<0.90).
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Correlations between metabolism and performance
Many of the predicted correlations between metabolism and running
performance were statistically significant (e.g.
O2max
vs maximum running speed on treadmill and on wheels for `pooled'
data), although results differed slightly depending on selection history
(Table 5).There was only a weak
positive correlation between
O2max obtained
in heliox and on the treadmill, observed when line types were pooled together
(one-tailed P=0.045).
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Discussion |
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Repeatability was high for most of the metabolic traits, which is
consistent with previous studies of food consumption in these same lines under
cold-exposure (Koteja et al.,
2000),
O2max during
forced exercise in deer mice and ground squirrels, Spermophilus
beldingi (Hayes and Chappell,
1990
; Chappell et al.,
1995
), and deer mice measured in the same enclosed wheels used
here (Chappell et al., 2004
).
The low repeatability of
O2max in heliox
between days was unexpected, however. Chappell et al.
(1995
) reported that
repeatability measured over different test periods in S. beldingi was
generally higher in exercise than thermogenic
O2max, although
the later was repeatable over relatively longer periods (e.g. several days).
In addition,
O2max obtained
during cold-exposure was repeatable in deer mice over long periods (over 8
weeks), and across different acclimation temperatures
(Hayes and Chappell, 1990
;
Rezende et al., 2004b
).
Domestication might be a confounding factor, however. Richardson et al.
(1994) showed that maximum
nonshivering thermogenesis (NST) in response to norepinephrine injection
tended to be higher in wild Mus than in mice from the same strain as
used to found the selection experiment, and wild mice had significantly more
interscapular brown adipose tissue than their laboratory counterparts. Thus,
it is possible that laboratory mice cannot sustain high NST for an entire
heliox trial. Another possibility is that overall repeatability of
O2max in heliox
was lower because S and C lines differed in their `training response' to
heliox trials (S mice had higher
O2max in the
second trial whereas C did not; see Results).
Lack of repeatability of
O2max in heliox
is consistent with low or non-significant repeatabilities for final
Tb, depending on the inclusion of two influential points.
Everything else being equal, one would expect animals with higher
O2 to have
higher Tb in these trials. There was a significant but
weak positive relationship between
O2max and final
Tb in trial 1 (F1,36=6.47,
P=0.015), but not in trial 2 (F1,41=1.74,
P=0.19).
Performance on wheel, treadmill and heliox
Many correlations predicted a priori were significant according to
pairwise tests of Pearson product-moment correlations
(Table 5). As expected, maximum
running speeds on the wheels were positively correlated with maximum voluntary
O2 in both S and
C (Pearson, P<0.05). There was a strong positive correlation
between running speed and
O2max in C lines
during forced exercise on the treadmill, but not in S mice
(Table 5). Differences in the
incremental cost of transport (defined as the slope of the linear regression
of
O2 on running
speed) could explain why the strong correlation between
O2max and speed
observed in C is not present in S (see below). Accordingly, S mice tended to
attain lower
O2max at high
speeds on the treadmill (Fig.
3), although the interaction of line typexspeed was never
significant in the nested model, either when tested over line or including an
additional linexspeed term in the model (P>0.20).
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Whether O2max
during forced exercise and cold-exposure are comparable and to what extent
they can be considered physiologically and genetically `the same trait' has
been debated in the literature (e.g.
Rezende et al., 2004a
). If
O2max was
ultimately restricted by pulmonary and cardiovascular systems (central
limitation hypothesis), then
O2max obtained
under different experimental conditions should be similar, and maximum values
on the treadmill and in heliox should be highly correlated
(Hammond and Diamond, 1997
;
Bacigalupe and Bozinovic,
2002
). In contrast, if the physiology underlying each index is not
the same, then
O2max in heliox
and during forced exercise could be quite different, and they should not
necessarily be correlated.
Our results provide partial support to both alternatives. There was a
significant but weak correlation between
O2max on the
treadmill and in heliox in the complete nested model
(Table 5), consistent with
(some) common physiology underlying these traits. Significant correlations
between these traits were reported for Peromyscus
(Hayes and Chappell, 1990
;
Chappell et al., 2003
) and
Spermophilus beldingi (data from
Chappell and Bachman, 1995
;
reanalyzed in Rezende et al.,
2004a
). However,
O2max during
cold-exposure was about 32% higher than during forced exercise, emphasizing
that different tissues and processes are involved in attaining maximum values
during forced exercise and acute cold-exposure. Recruitment of additional
muscles during shivering, and brown adipose tissue for NST
(Heldmaier, 1993
;
Nespolo et al., 2001
), may
explain why
O2max was higher
during cold-exposure, as has been described for some species of small mammals
(Belding's ground squirrels, Chappell et
al., 1995
; deer mice, Chappell
and Hammond, 2003
). In addition, our results show that, at least
on the treadmill,
O2max is not
centrally limited: pulmonary and cardiovascular systems could provide more
oxygen than required by muscles while running at maximum aerobic levels
(however, limitations at the vascular level might occur in localized regions
of the body).
On these wheels, both C and S mice choose to run at speeds below their
maximum aerobically sustainable speed, as is also true for Peromyscus
(Chappell et al., 2004). This
result seems to contradict the theoretical prediction that mice (S lines in
particular) would run close to maximum aerobic levels on the wheels to
maximize running economy (i.e. minimize costs of transport; see Chappell et
al. (2004
). All else being
equal and given enough individual variation, one would expect that mice that
ran voluntarily closer to their MAS eventually would have been favored in the
selection experiment. Several non-exclusive explanations are possible. First,
the protocol of forced exercise may overestimate maximum
O2 during
voluntary running. Recruitment of additional tissues because of stress
responses, for instance, could account for a higher
O2max during
forced exercise (M.A.C., unpublished data). If that is the case, then perhaps
S mice are indeed running at speeds close to their voluntary MAS, and aerobic
performance could be constraining the evolution of higher running speeds on
the wheels. Another possibility is that mice reach their lactate threshold
before attaining
O2max, as is the
case for human beings (Powers and Howley,
2001
). Finally, if animals are energy-limited i.e. if
energy cannot be provided to cells at similar rates as O2
it may simply be impossible to sustain voluntary running at maximum aerobic
levels (i.e. there may have been a trade-off between speed and endurance
during voluntary running on wheels).
Nevertheless, S lines run voluntarily at speeds that come closer to
eliciting their treadmill
O2max (see
above) i.e. selection for longer running distances has produced
individuals tending to run closer to their maximum aerobic capacities.
Accordingly, because there were no differences in energy expenditure between S
and C lines in spite of the 70% difference in running distances at generation
10, Koteja et al. (1999a
)
concluded that `running distance over a given period of time (e.g. 24 h) could
be increased substantially by increasing speed, with only a small increase in
the total cost of activity', supporting the idea that running faster can have
an important effect on running economy in these lines.
Effects of selection on O2max
Our results show that
O2max during
forced exercise and cold exposure have not increased significantly in the S
lines, despite of a 20.3% increase in maximum voluntary
O2 attained
during voluntary wheel-running. Although this seems to contradict the results
obtained by Swallow et al.
(1998b
), who reported a small
(6%) but significant increase in
O2max during
forced exercise in S mice, mean values in the present study were on average
6.6% higher in treadmill trials and 6.0% higher in heliox in S lines, although
neither difference was significant in our study
(Table 3). One possible
explanation for the lack of statistical significance in the present study is
that differences among the replicate lines are now greater (P=0.083
for treadmill values; Table 3)
than at generation 10 (significance of line effects was not reported in
Swallow et al., 1998b
).
Another possibility is that the results are discrepant because we employed
females whereas Swallow et al.
(1998b
) studied only males,
and some responses to selection have been observed to be sex-specific (e.g. S
females have evolved relatively higher running speeds than males, compared to
C). Possible line differences and sex effects on aerobic capacity will be the
subject of future studies with larger sample sizes.
Although mice from the S lines run faster than C on wheels (Koteja et al.,
1999a,b
;
Rhodes et al., 2000
;
Girard et al., 2001
; this
study), they do not run faster during forced treadmill trials (see also
Swallow et al., 1998b
). These
results emphasize the fact that S and C mice have similar aerobic capacities
and that the major differences in running performance on wheels are apparently
a consequence of neurological changes associated with `willingness to run'
(Rhodes et al., 2001
,
2003
,
2005
;
Rhodes and Garland, 2003
),
rather than the physiology whole aerobic performance, at least
involved with `being able to run'. Nevertheless, with long-term access to
running-wheels, differential training effects related to contrasting running
activity in S and C may eventually lead to important physiological differences
between them (i.e. physiological plasticity in a genotype by environment
interaction; Zhan et al.,
1999
; Houle-Leroy et al.,
2000
; Swallow et al.,
2005
).
Conclusions
Aerobic capacity has not coadapted with increased voluntary wheel-running
in our selected lines of mice, and many explanations may account for this lack
of correlated response. First, the base population may have lacked additive
genetic variation for
O2max.
Consistent with this possibility, Dohm et al.
(2001
) obtained very low
estimates of the additive genetic contribution to individual differences in
O2max in the
base population, with heritabilities ranging between 0 and 0.64, depending on
which quantitative genetic model was used. Second, even if substantial
additive genetic variance existed in the base population, the genetic
correlation between
O2max and
wheel-running behavior might be close to zero, suggesting that only few genes
influencing wheel-running also affect
O2max
(Swallow et al., 1998b
;
Roff, 1997
). We emphasize that
the lack of response in
O2max after 35
generations of selection may be strictly dependent on the genetic background
of the base population; other studies have shown than
O2max can be
heritable and evolutionary labile (Rezende
et al., 2004a
; Nespolo et al.,
2005
; Sadowska et al.,
2005
), including in laboratory rats selected for treadmill running
performance (Henderson et al.,
2002
).
Third, aerobic capacity in these lines could have been `excessive' in the
first place, i.e. animals can run at higher aerobic levels than they are
willing to. Indeed, several lines of evidence indicate higher motivation to
run in S lines (Rhodes et al.,
2001,
2003
,
2005
;
Rhodes and Garland, 2003
).
Fourth, the correlated increase in frequency of the `mighty mini-muscle'
allele (individuals that are homozygous have gastrocnemius about 50% lighter
than normal) in two of the four S lines
(Garland et al., 2002
)
suggests that running efficiency may have been a correlated target of
selection. Individuals with the mini-muscle possess gastrocnemius with twice
the aerobic capacity per gram of tissue than normal (Houle-Leroy et al.,
2000
,
2003
), but evidence regarding
the effect (if any) of the mini-muscle allele on
O2max is not yet
available. Thus, the mini-muscle allele might have increased in frequency
because it reduces the overall cost of locomotion (e.g.
Myers and Steudel, 1985
;
Steudel, 1990
;
Garland et al., 2002
). We are
currently testing whether costs of locomotion have evolved in S lines. Fifth,
sex-specific responses to selection might be involved, and there also is the
possibility that significant effects reported by Swallow et al.
(1998b
) could result from a
type I error.
Although our results suggest that activity levels can evolve without a
concomitant change in aerobic capacity in our lines of laboratory mice,
extrapolation of these results to wild species should be performed with
caution. Domestication of Mus strains has apparently led to major
effects on behavior without compromising overall physiology to any great
extent, at least based on limited comparisons of wild and laboratory house
mice and their reciprocal crosses (Dohm et
al., 1994; Richardson et al.,
1994
; Garland et al.,
1995
). Behavioral and/or whole-organism performance traits (e.g.
maximum sprint speeds,
O2 on the
treadmill) differed considerably between laboratory and wild house mice,
despite relatively minor differences in lower-level physiological traits.
Hence, aerobic capacity may have been `excessive' in these lines to the extent
that it did not and does not presently constrain activity
levels or running performance. Therefore, although we have shown that
voluntary running performance and activity levels can evolve independently of
aerobic capacity, results may depend on the animal model employed. It would be
of considerable interest to perform interspecific comparative studies of home
range area, voluntary wheel-running and maximal aerobic capacity in other
rodents.
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