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First published online May 21, 2007
Journal of Experimental Biology 210, 2006-2012 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.001420
Metabolic rate of nocturnal incubation in female great tits, Parus major, in relation to clutch size measured in a natural environment
Animal Ecology Group, Centre for Ecological and Evolutionary Studies, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands
* Author for correspondence (e-mail: m.e.de.heij{at}rug.nl)
Accepted 13 March 2007
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: clutch size manipulation, energy expenditure, natural selection, oxygen consumption
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Drent and Daan, 1980;
Walsberg, 1983;
Daan et al., 1990), resulting
in a wide range of studies on limits to parental care during this period
(Lindén and Møller,
1989; Dijkstra et al.,
1990; Vanderwerf,
1992). As a consequence, the energetic demands during other
reproductive phases – such as egg laying and incubation – have
long been ignored (Williams,
1996; Monaghan and Nager,
1997).
During incubation, avian eggs need external heat provisioning, regular
turning and favourable humidity for proper embryonic development
(Deeming, 2002); care that is
often provided by one or both of the parents. The energetic costs of providing
heat to the eggs have long been thought to be negligible. Increasing evidence
suggests, however, that below thermo-neutrality the metabolic rate (energy
spent per time unit) of an incubating female is higher than that of a
nonincubating female at rest (Williams,
1996; Thomson et al.,
1998; Tinbergen and Williams,
2002). Since temperatures are normally below thermo-neutrality at
temperate latitudes, energetic costs of incubation may substantially add to
the overall daily energy expenditure of attending parents.
It is now known that, over 24 h, energy expenditure in the incubation phase
is of the same order of magnitude as expenditure in the nestling phase for a
number of small passerines (Williams,
1996; Tinbergen and Williams,
2002). Whether these costs are related to clutch size remains to
be investigated. The most accurate way to measure this is through the use of
respirometry. For species breeding in nest-boxes, this technique can be
applied in the field to determine the energetic costs of nocturnal
incubation, because at night the attending parent remains in the nest-box. To
put possible effects of clutch size variation within the context of other
factors influencing the energy expenditure of incubating birds, measurements
should preferably be determined in free-living individuals under field
conditions.
Several studies have measured metabolic rate during nocturnal incubation in
relation to clutch size (Biebach,
1981; Biebach,
1984; Haftorn and Reinertsen,
1985; Weathers,
1985). Of these studies, only the study by Haftorn and Reinertsen
(Haftorn and Reinertsen, 1985)
was under field conditions. All of these studies support the idea that clutch
size manipulation affects metabolic rate during nocturnal incubation.
In the present study, we measured metabolic rate during nocturnal incubation (MRinc; J s–1) in free-living female great tits, Parus major, using mobile oxygen analysers. We attempted to establish the causal relationship between clutch size and nocturnal energy expenditure by repeatedly measuring the same individual experiencing different clutch size manipulations. We performed the measurements during first clutches in one year and second clutches in another year. The natural variation in ambient temperature experienced by the incubating birds during the two experiments enabled us to estimate the temperature dependence of energy expenditure during nocturnal incubation. In one of the two years, we additionally measured nest thickness and body mass. These two variables may explain variation in the metabolic rate of nocturnal incubation.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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General procedure
Nest-boxes were checked at least once a week from the beginning of April to
estimate laying date assuming that one egg was laid per day. Onset of
incubation was defined as the first day that the female was found incubating
or that the eggs were uncovered and warm and was determined by daily nest
visits from the seventh egg onwards during first clutches and from the third
egg onwards during late clutches. Further weekly nest checks were made to
determine the success of the nests. Females were caught for identification and
measurement of individual characteristics, such as body mass, when nestlings
were between 7 and 10 days old (day of hatching=0).
Measuring oxygen consumption
Experimental set-up
To study the energetic costs during nocturnal incubation in relation to
clutch size, the same experiment was performed twice in two different years:
2001 and 2004. In 2001, the study area was closed during the first few weeks
of the breeding season due to an outbreak of foot-and-mouth disease.
Consequently, the experiment was performed during late clutches
(June–July) that year. To increase the natural range of ambient
temperatures that incubating females experienced during the measurements, the
experiment was repeated during first clutches (April–May) in 2004. As a
result, measurements were performed during different breeding attempts in
different years. Consequently, variation in oxygen consumption could not be
attributed to variation between years or variation between breeding attempts;
we refer to this as `year effect'. The experiment was performed as early as
possible during the incubation period because the oxygen consumption of
embryos rises exponentially during the second half of incubation; prior to
this it is negligible (Vleck et al.,
1980; Prinzinger et al.,
1995).
Individuals that participated in the experiment were randomly selected. No
individual was used in both years. Each individual was measured on two or
three consecutive nights, with the same oxygen analyser, while incubating
either manipulated or original clutch sizes. Females were assumed to respond
to the experimental treatment as if the resulting clutch size were a result of
their own decision (Lessells,
1993). In total, the oxygen consumption of 30 individuals was
measured: 10 individuals during late clutches (between 14 June and 10 July) in
2001 and 20 individuals during first clutches (between 19 April and 12 May) in
2004.
Clutch size manipulation
Clutch sizes were manipulated during daytime (around noon) prior to the
night that the oxygen measurements were performed. In 2001, clutches (clutch
size 7.0±0.7 eggs; range 6–8) experienced a sequence of three
treatments involving reduction, enlargement or control (i.e. original clutch
size laid by the female). The sequence of manipulations was randomised in such
a way that the sequence was either `reduced – control – enlarged'
or `enlarged – control – reduced'. In 2004, clutches (clutch size
8.8±1.4 eggs; range 5–11) were only enlarged or kept constant:
clutches were not reduced in order to prevent nest desertion
(de Heij et al., 2006). In this
year, the sequence of manipulations was randomised. Clutches were
experimentally manipulated by the addition and removal of three eggs (about
one-third of the original clutch). In this, we followed previous studies on
brood size manipulations in the same population
(Sanz and Tinbergen, 1999;
Wiersma and Tinbergen, 2003).
Clutches of six eggs were reduced by two eggs to prevent nest desertion. Eggs
that were added to the clutch for enlargement came from donor nests with eggs
at about the same incubation stage.
Oxygen measurements
The nest-box (inner size of nest-box 8.5x25x12 cm; total volume
2.6 litres) was converted into a metabolic chamber in the days before the
measurements by making the nest-box airtight
(Fig. 1). Five small holes
(diameter 5 mm) in the bottom of the nest-box ensured that air entered the
nest-box from below, passing the incubating female and leaving the nest-box
via a tube from which air was drawn from the nest-box for
analysis.
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Oxygen consumption was calculated using equation 6 of Hill
(Hill, 1972). The total
metabolic rate of nocturnal incubation (MRtot) included
the metabolic rate of incubation by the incubating females
(MRinc) and that of the embryos
(MRemb). MRtot was calculated assuming
a respiratory quotient of 0.75 and an energy equivalent of 19.9 kJ
l–1 oxygen consumed
(Tinbergen and Dietz, 1994).
Per sample period, data from at least the first three minutes were discarded
to allow stabilising of the measurement. MRtot was
corrected for MRemb. Knowing the incubation stage of the
embryos of a clutch, MRemb could be derived using data
from Vleck et al. (Vleck et al.,
1980). In fig. 1c
in their study, they summarised the relative MRemb in
relation to the relative incubation stage for altricial birds. By using the
maximum MRemb of great tits [J. A. L. Mertens,
unpublished; cited in Vleck et al. (Vleck
et al., 1980)], MRemb could be derived for
each egg at a particular incubation stage. Eggs from abandoned nests were
assumed to be dead or only briefly incubated: their metabolic rate was not
included in our calculations. MRinc was calculated by
subtracting the total MRemb of a clutch from the
MRtot measured in the field;
MRinc=MRtot–MRemb.
MRemb was, on average, 0.010 J s–1 and
0.004 J s–1 per clutch in 2001 and 2004, respectively. Per
night of measurement, mean values of MRinc were used in
the analysis.
Additional measurements
Besides oxygen measurements, ambient temperature and incubation behaviour
of the attending females were recorded. Female body mass and nest thickness
were only measured in 2004.
Ambient temperature was recorded once every minute in the vicinity of the nest-box. The mean temperature over the measurement period was used in the analysis.
To determine the behaviour of an incubating female, a temperature sensor (HOBO logger; Mulder-Hardenberg b.v., Haarlem, The Netherlands) was placed between the eggs to register the temperature of the nest every 15 s. All but one bird incubated normally during the oxygen measurements (see later).
We estimated female body mass, since catching incubating females at night led to high rates of nest desertion (in a pilot study in 2002, nest desertion was 40%; N=10). Estimates were derived from measurements of body mass from females caught during late incubation in 2004 (N=35). Females were captured with a hand-net on leaving the nest-box during the day. Using this technique, only 9% of the birds abandoned their nests. Of this sample, 18 females were also involved in the present study. Body mass during late incubation (Minc; mean ± s.d. 20.2±1.0 g) was highly related to that of the same females during the nestling phase (Mnest): Minc=3.15±5.63(s.e.m.)+0.96·Mnest (r2=0.43, P<0.01, N=14). This calculation was used to estimate body mass during early incubation from body mass during the nestling phase.
Nest thickness was recorded once during the incubation period in 2004, using a knitting needle as the measuring tool. Both the height of the nest rim (drim; distance from nest rim to bottom of the nest-box) and the thickness of the nest cup (dcup; the distance between the bottom of the nest cup and the bottom of the nest-box) were recorded to the nearest mm (Fig. 1).
Statistical analysis
Due to missing values, there is some variation in the sample size between
analyses (in 2001, missing values for oxygen consumption = 2; in 2004, missing
values for body mass = 2 and for nest thickness = 1). One bird did not
continuously incubate the eggs, as judged from measurements of nest
temperature; excluding this individual in the model did not change the
results.
All analyses were performed with a hierarchical linear regression model in
MLwiN version 2.02 (Rasbash et al.,
2000) to account for repeated measurements. All variables and
their two-way interactions were tested by backward elimination from the model.
Three different analyses were performed, because of slightly different
experimental procedures in the two years. Firstly, we tested whether in 2001
clutch size manipulation (reduced, control, enlarged) affected
MRinc. Original clutch size, date, ambient temperature and
oxygen analyser (unit A or B) were included in the model covariates. Secondly,
we tested whether clutch size manipulation (control and enlarged) affected
MRinc in 2004 but controlled for additional covariates:
body mass and nest thickness. Thirdly, data from both years were used to test
consistency between years.
All values are presented as means ± s.d., unless stated otherwise.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Second experiment; first clutches in 2004
The metabolic rate during nocturnal incubation was 0.65±0.09 J
s–1 for females incubating control clutches that contain on
average 8.8±1.4 eggs and at ambient temperatures of
9.3±2.4°C. Height of the nest rim and thickness of the nest cup
were strongly related:
drim=38.26±4.36(s.e.m.)+1.37±
0.30(s.e.m.)·dcup (r2=0.56,
P<0.00, N=19). Analyses of MRinc were
therefore performed with dcup as covariate. Experimental
treatment, mean ambient temperature and thickness of the nest cup, all
explained part of the variation in MRinc
(Table 1B). Females had higher
MRinc when incubating experimentally enlarged clutches as
compared with control clutches (Fig.
2B). The effect of clutch size manipulation on
MRinc was similar to that in the experiment in 2001.
MRinc was negatively related to mean nocturnal temperature
(Fig. 3) and thickness of the
nest. Variation in body mass did not explain variation in
MRinc.
Combined results
The experimental treatment, ambient temperature and year explained a
significant part of the variation in MRinc
(Table 1C). The effect of
clutch enlargement relative to the mean MRinc in each of
the two years (6–10%; three additional eggs) was similar in both years
(treatment x year; 
2=0.29, d.f.=1, P=0.59;
N=29). The effect of ambient temperature did not differ for the two
experiments (temperature x year; 2=0.09, d.f.=1,
P=0.79; N=29). The effect of ambient temperature on
MRinc was strong (43–49% per 10°C; mean ±
s.d. 11.1±3.4°C). Year (or breeding attempt) explained an
additional part of the variation in MRinc.
MRinc of females incubating late clutches in the year 2001
was higher than that of females incubating first clutches in 2004, when
controlled for ambient temperature and clutch size manipulation.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Biebach,
1984). The absence of an effect of clutch reduction is surprising.
Assuming that each egg requires a constant amount of energy, we expected
MRinc to be linearly related to clutch size. Also,
previous studies found an effect of clutch size reduction
(Haftorn and Reinertsen, 1985;
Weathers, 1985) and/or found
MRinc to be linearly related to clutch size
(Biebach, 1981;
Biebach, 1984).
We think these results can be explained when the number of eggs that can be
in direct contact with the brood patch is limited. This number is referred to
as a `threshold clutch size'. Above such a threshold clutch size, eggs that
are not in contact with the female's brood patch are likely to cool
(Mertens, 1977a).
Consequently, incubating birds will repeatedly rearrange the eggs to rewarm
the cooled eggs. Rewarming has been shown to be energetically more costly than
maintaining eggs at incubation temperatures
(Vleck, 1981;
Biebach, 1986). This may
potentially cause an increase in energetic costs when clutch size is above the
threshold clutch size.
To explain the difference in relationship between MRinc
and clutch size found in our study (non-linear) with that in previous studies
(linear), we looked in more detail at previous studies
(Table 2). Haftorn and
Reinertsen (Haftorn and Reinertsen,
1985) measured MRinc of a female blue tit
after a clutch reduction from 13 to eight eggs. Under the hypothesis about the
threshold clutch size, the effect of clutch reduction will depend on the
manipulated clutch size relative to the threshold clutch size. If the clutch
size after manipulation is above the threshold, clutch reduction can be
expected to lower energy expenditure, whereas if it is below, no such effect
is to be expected. This explanation may be valid for the results of Haftorn
and Reinertsen but not for those of Biebach
(Biebach, 1984). He reported a
linear relationship between MRinc and clutch size for
female starlings at three different temperatures that were below
thermo-neutrality. A closer look at the data, however, suggests that a
non-linear effect of clutch size on MRinc may also exist
in his study (Fig. 4). We
reanalysed the data of Biebach (Biebach,
1984), taking values from his
fig. 2. When in addition to
clutch size a quadratic term was included in the analysis to test for a
non-linear effect of clutch size, this term was significant (0°C,
F1,12=4.98, P=0.05) or approached significance
(10°C, F1,19=3.73, P=0.07; –10°C,
F1,12=3.98, P=0.07). This analysis makes it
plausible that the non-linear effect of clutch size on
MRinc may be a general phenomenon.
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Metabolic rate of nocturnal incubation
Several variables, other than clutch enlargement, explained variation in
MRinc. We will briefly discuss each of them.
As in previous studies (Biebach,
1984; Bryan and Bryant,
1999; Weathers et al.,
2002), ambient temperature was strongly related to
MRinc. Fig.
3 shows that a change in ambient temperature of 10°C, which is
equal to a change in MRinc of 43–49%, is likely to
occur during a breeding attempt. Although temperature variation will affect
MRinc considerably, for the energetic consequences of the
timing of reproduction the mean ambient temperature is of prime importance. A
shift in timing of two weeks results in a change in mean ambient temperature
of about 2°C (M.E.d.H., unpublished). This is equal to a change in
MRinc of about 9%. The strong fluctuation in ambient
temperature may nevertheless affect the peak demand in energy expenditure of
parents during reproduction.
The change in MRinc with ambient temperature was
similar in both years (i.e. the interaction term was non-significant).
Controlling for ambient temperature, there was still a year effect on
MRinc. The direction of this effect was counterintuitive.
When corrected for ambient temperature, females spent more energy during late
clutches in 2001 than during first clutches in 2004. Several explanations may
apply. Firstly, parents may incubate more intensively during late clutches as
compared with first clutches to accelerate embryonic development and thereby
shorten the incubation period (Smith,
1989). Secondly, nests may be proportionally thinner during late
clutches than during first clutches, because having a thick nest may be
disadvantageous later in the season due to the risk of hyperthermia of
nestlings (Mertens, 1977b).
Thirdly, parents of late clutches are likely to be a selection of the
population; they may be the ones that invest more during incubation. Fourthly,
any year difference (for instance humidity) may be explanatory. All the above
explanations are worthy of further study.
In the year we measured nest thickness, birds with thicker nests had lower
energy expenditure. A probable explanation for this effect is that thicker
nests were better insulated (Hoi et al.,
1994; Szentirmai et al.,
2005). Nest insulation is known to be of importance for the
incubation behaviour of attending parents
(Reid et al., 1999;
Cresswell et al., 2003). The
fact that not all birds build well-insulated nests suggests that there is a
cost to thick nests (Hansell,
2000).
Implications
Our finding that females expend more energy during nocturnal incubation
when incubating experimentally enlarged clutches is a first step towards the
detection of a potential mechanism underlying negative selection on clutch
size during the incubation phase. The finding is consistent with the survival
cost associated with enlarged, but not reduced, clutches in this population
(de Heij et al., 2006).
Nevertheless, measurements on energy expenditure over a full 24 h are needed
in order to judge how important energy expenditure can be in explaining
fitness consequences of incubating experimentally enlarged clutches.
List of abbreviations
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Acknowledgments |
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