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First published online March 2, 2006
Journal of Experimental Biology 209, 1004-1015 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02089
Carotenoid availability in diet and phenotype of blue and great tit nestlings
1 Laboratoire de Parasitologie Evolutive, CNRS UMR 7103, Université
Pierre et Marie Curie–Paris 6, Bât. A–Case 237, 7 quai Saint
Bernard, F-75252 Paris Cedex 05, France
2 Lipid and Antioxidant Group, Department of Biochemistry and Nutrition,
Avian Science Research Centre, Scottish Agricultural College, Auchincruive,
Ayr, KA6 5HW, UK
* Author for correspondence at present address: Equipe Ecologie Evolutive, UMR CNRS 5561 Biogéosciences, Université de Bourgogne, 6 bd Gabriel, F-21000 Dijon, France (e-mail: Clotilde.Biard{at}u-bourgogne.fr)
Accepted 11 January 2006
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Summary |
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Key words: antioxidants, early development, feather colour, fledgling body mass, immune function
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Introduction |
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;
Surai, 2002). In animals, they
are acquired from food and used for prevention of oxidative stress, regulation
of immune function and development of bright yellow to red coloration of
feathers and skin (reviewed by
Møller et al., 2000).
Antioxidants and particularly carotenoids have been shown to be of crucial
importance during embryo development, at hatching and during the nestling
phase, as this period of intense growth is associated with increased oxidative
stress (Surai et al., 1999).
Efficient antioxidant protection is essential for normal development of
embryos and chicks, and any impairment in antioxidant defences leads to
profound, and in many cases, irreversible damage to physiological functions
(for a review, see Surai,
2002). For example, chicks reared in carotenoid-poor environments
have a compromised ability to assimilate carotenoids from the diet in later
life (Blount et al., 2003a;
Hõrak et al., 2000;
Koutsos et al., 2003).
Furthermore, in several passerine species carotenoids are involved in the
development of coloured signals like gape colour
(Hunt et al., 2003), which may
mediate parent-offspring communication
(Saino et al., 2000).
Carotenoids may also be transferred into growing feathers, and determine
yellow to red coloration, as well as green and blue coloration when associated
with proteins (Ong and Tee,
1992). Carotenoid availability during early stages of development
may thus modulate several functions of potential importance for nestling
fitness.
Carotenoids may be limiting in the environment as a result of environmental
scarcity or individual variation in foraging ability
(Møller et al., 2000;
Olson and Owens, 1998). Thus
parents able to provide their nestlings with a carotenoid-rich diet should
enhance their offspring's antioxidant protection, body condition and immunity,
and modulate their phenotype. Parental provisioning with carotenoids takes
place very early in nestling life when females invest carotenoids in egg yolk
(Blount et al., 2000), but also
later when parents feed nestlings. However, very little is known in natural
populations about the possible effects of carotenoid availability in the diet
on growing nestlings. To our knowledge, the effect of dietary carotenoids has
only been investigated in nestling great tits (Parus major) in a
population breeding in deciduous woodland
(Fitze et al., 2003;
Tschirren et al., 2003).
Nestlings in the study by Fitze et al. were provided with extra carotenoids on
two feeding occasions, either shortly after hatching or later during the
nestling period. An increase in carotenoid supply affected feather colour only
if occurring before 6 days of age, and no effect of carotenoid supply was
found on nestling growth independent of age at which carotenoids were provided
(Fitze et al., 2003). A
similar increase in yellow feather colour was observed in carotenoid-fed
nestlings in the study by Tschirren et al.
(Tschirren et al., 2003).
The aim of the present study was to investigate the potential effects of
carotenoid availability in the diet for growing nestlings. We experimentally
manipulated dietary intake by nestlings of two closely related species, the
blue tit (Parus caeruleus) and the great tit during the nestling
period, and measured several aspects of nestling condition and phenotype
before fledging. Blue and great tits are small hole-nesting passerines of
similar ecology but differing in life-history traits
(Blondel et al., 1990;
Newton, 1989). In both
species, nestling diet is composed of various insects and arachnids
(Minot, 1981). We hypothesised
that increasing the amount of carotenoids in the diet could modulate one or
several of the following four types of parameters. First, an increase in
circulating carotenoids could be expected in nestling blood, but also an
increase in the concentration of other lipid-soluble antioxidants, as
interactions such as recycling and mutual protection are known to occur
between different antioxidants (e.g. Surai
and Speake, 1998; Surai et
al., 2001b). We thus measured the concentration of carotenoids and
the two other major antioxidants for birds, vitamin E and vitamin A, in
nestling plasma following carotenoid supplementation.
Second, an increase in general body condition of nestlings could be
expected because of the antioxidant properties of carotenoids. Indeed, an
increase in dietary carotenoids has been found to lead to an increase in
antioxidant protection (Blount et al.,
2002; Surai,
2002), which could be beneficial to rapidly growing chicks under
intense oxidative stress produced by metabolism. Thus an increase in dietary
intake of carotenoids could facilitate nestling growth and lead to better
condition of nestlings, and/or to a better utilisation of nutrients. Body mass
at fledging is a good predictor of immediate post fledging and overwinter
survival in tits (Naef-Daenzer et al.,
2001; Tinbergen and Boerlijst,
1990), and body mass with tarsus length as a covariate was
therefore used as a measure of nestling body condition.
Third, carotenoids play important roles in immunoregulation in vertebrates.
For example, they are known to enhance T and B lymphocyte proliferation,
enhance macrophage and cytotoxic T cell capacities, and stimulate the
production of various cytokines and interleukins in humans and other animals
(reviewed by Chew, 1993;
Møller et al., 2000).
An increase in carotenoid availability could be expected to help clear
infections, reduce the intensity of current infections, and enhance the
ability to respond to an antigen challenge. The state of development and
activation of the immune system was measured using red blood cell
sedimentation rate, amount of leukocytes circulating in blood and
cell-mediated immune response. Blood sedimentation is useful for detecting
elevated levels of immunoglobulins and fibrinogen, high sedimentation rate
being indicative of acute infections and inflammatory diseases (e.g.
Sturkie, 1986;
Svensson and Merilä,
1998). The number of leukocytes increases in case of stress and
infection (e.g. Ots et al.,
1998; Sturkie,
1986), and in chicken Gallus gallus and quail
Coturnix sp. offspring the number of leukocytes in blood increases to
reach adult levels by three weeks of age
(Sturkie, 1986). Nestlings
were tested for their ability to raise a cell-mediated immune response to
phytohemagglutinin (PHA). This test reflects the combined responses of
T-cells, cytokines and inflammatory cells
(Davison et al., 1996).
Fourth, feather colour may mirror the amount and type of carotenoids
available for an individual at the time of feather growth, and individual
nestlings must trade allocation of pigments deposited in feathers against
their use for other physiological functions. Both species show a carotenoid
based yellow plumage on the breast
(Partali et al., 1987), and we
therefore investigated the effect of dietary carotenoids on development of
juvenile plumage coloration.
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). Both species
lay large clutches (i.e. generally more than seven eggs), although blue tit
females lay on average more eggs than great tit females
(Blondel et al., 1990;
Newton, 1989). Nestlings of
both species have a similar diet of arthropods, mainly lepidopteran larvae and
spiders (Minot, 1981). The
study area (about 250 ha) contained 400 nestboxes evenly distributed among a
homogenous deciduous old woodland composed mainly of oak (Quercus
spp.), hornbeam (Carpinus betulus) and beech (Fagus
sylvatica). Nests were regularly inspected to determine laying date,
clutch size, start date of incubation, hatching date and number of hatchlings
and fledglings.
Carotenoids were obtained from Kemin Foods (Nantes, France; OroGlo Layer
Dry 20) in the form of a dietary supplement made of crystalline lutein derived
from marigold flowers. Xanthophyll concentration in the product was 1.8%
lutein and 0.2% zeaxanthin, confirmed with HPLC (high-performance liquid
chromatography) analysis, and both carotenoids were in free alcohol forms
readily available for absorption. It is generally accepted that efficiency of
carotenoid absorption in birds is about 20% of the used dose as determined
from supplementation experiments (Surai,
2002; Surai et al.,
2001a). The quantity of carotenoids to be given with each
supplementation was therefore fixed to be five times the total quantity of
carotenoids circulating in nestling plasma, which was estimated as follows,
based on data collected in 2000 in the same population. Mean fledgling body
mass was 10.5 g and 16.5 g and mean carotenoid concentration in plasma was 37
µg ml–1 and 56 µg ml–1 for blue and
great tit nestlings, respectively. In nestling birds, blood volume represents
about 10% of body mass (Sturkie,
1986), thus total quantity of circulating carotenoids can be
estimated as 38 µg and 92 µg, respectively. Therefore, carotenoid
treatment consisted of 200 µg and 500 µg of carotenoids diluted in 0.05
ml sunflower seed oil per feeding, for blue and great tit nestlings,
respectively. Control treatment consisted of 0.05 ml pure sunflower seed oil
per feeding. Sunflower seed oil was chosen because it is rich in mono-(24%)
and polyunsaturated (65%) fatty acids, and contains natural vitamin E. Vitamin
E will ensure protection of carotenoid pigment against oxidation before
ingestion, and unsaturated fatty acids will enhance their absorption from the
food matrix (Surai et al.,
2001a). Supplemental food was freshly prepared each evening and
stored at 4°C and in darkness until use the following day. We started
feeding nestlings when they were sufficiently large to be identified with a
numbered aluminium ring (mean nestling age ± s.e.m., blue tit:
6.64±0.25 days, N=14 nests and 141 nestlings, great tit:
5.89±0.18 days, N=19 nests and 182 nestlings). All broods used
in this experiment were first broods. In all nests, half the brood was
randomly attributed to each treatment, and nestlings were fed every 2 days
until just before fledging (mean number of feedings ± s.e.m., blue tit:
4.82±0.04, great tit: 5.47±0.05). Supplementary food was
delivered into the nestlings' throat with a graduated 1-ml syringe, and
complete swallowing was checked.
Nestlings were measured, and feather and blood sampled on the day after the
last supplemental feeding (mean nestling age ± s.e.m., blue tit:
15.35±0.1 days, N=108 nestlings from 12 nests, great tit:
15.58±0.16 days, N=82 nestlings from 10 nests). Sample size
was reduced as a result of early fledging or predation. We measured tarsus
length to the nearest 0.1 mm with a calliper and weighed nestlings to the
nearest 0.25 g with a Pesola spring balance. A sample of five to eight yellow
feathers was plucked from the centre of the yellow breast from each bird, and
stored in individual plastic bags in the dark until later colour analysis. A
blood sample (50–100 µl) was taken from the brachial vein in
heparinized micro-haematocrit tubes. Blood samples were stored in a cooling
bag in the field, and when back in the lab, stored at 4°C and upright to
measure sedimentation after 8 h [for a randomly chosen sub-sample of nests:
seven nests (N=59) for blue tit, eight nests (N=64) for
great tit]. All blood samples were then centrifuged for 5 min at 2800
g. Length of the red blood cell layer was measured to the
nearest 0.5 mm and length of the `buffy coat' layer was measured to the
nearest 0.01 mm with a graduated magnifying ocular. Plasma was then separated
from blood cells and stored at –20°C. Sedimentation and haematocrit
were measured as the amount of red blood cells divided by the total length of
the tube filled with blood. In the same way, the relative amount of leukocytes
in total blood volume was measured as the ratio of the `buffy coat' layer to
total length. Sedimentation rate and relative proportion of leukocytes are
routinely measured with this method to describe different aspects of health
both in physiological ecology (e.g.
Hõrak et al., 1998;
Ots et al., 1998;
Svensson and Merilä,
1998) and veterinary studies
(Coles, 1997).
After capture and measurements, nestlings were injected subcutaneously with
0.2 mg phytohemagglutinin (PHA; Sigma L-8754, Saint-Quentin Fallavier, France)
in 0.04 ml sterile PBS within the patagium first plucked of feathers and
marked for injection with permanent ink. Following recommendations by
(Smits et al., 1999), no
control injection of PBS was made on the other wing web. PHA first induces an
acute response 4 h after injection, primarily characterised by oedema. Then it
induces a delayed-type hypersensitivity response through stimulating
heterophils, basophils, granulocytic and mononuclear cell infiltration in
dermis and dense perivascular infiltration of T lymphocytes at the site of
injection (Parmentier et al.,
1998; Sharma,
1990). This late response generally peaks 18 h after injection,
and may last up to 36 h. Wing web thickness was measured with a spessimeter
(Alpa S.p.A., Milan, Italy; the spring was removed from the spessimeter and
replaced with a fixed weight of 15 g) with an accuracy of 0.01 mm just before
injection and at least 19 h after injection to assess the intensity of the
immune response (mean time ± s.e.m., blue tit: 26.78±0.41 h,
N=48, great tit: 21.24±0.37 h, N=27). The immune
response index was calculated as the difference between thickness after and
before injection.
Plasma antioxidant analysis
Antioxidants were extracted from plasma as follows. 20 µl of plasma was
mixed with 40 µl ethanol, then extracted twice with 500 µl hexane.
Hexane extracts were pooled and evaporated at 60–65°C under nitrogen
flow and the residue was dissolved in 0.1 ml dichloromethane and 0.1 ml
methanol. Carotenoid composition and concentration, and vitamin A and E
concentration were determined using reverse phase HPLC following previously
published procedures (Surai,
2000; Surai et al.,
2001c) (for details see Biard
et al., 2005). All nestling plasma samples of at least 20 µl
(blue tit N=99, great tit N=82) were analysed for total
carotenoid concentration, and a random sub-sample was used for carotenoid
composition and vitamin analysis (one nestling of each treatment for blue tit
N=10 nests, great tit N=7 nests). Concentrations are given
in µg ml–1.
Feather colour analysis
Nestling breast feather colour was analysed blindly with respect to
treatment, in the laboratory, using a spectrometer (Ocean Optics, Duiven, The
Netherlands) following methods described previously
(Hõrak et al., 2000;
Saino et al., 1999). Feathers
were illuminated at an angle of 90° with a deuterium-halogen lamp, and
reflected light was measured at an angle of 45°. Percentage of reflectance
at each 1 nm interval was calculated between 300 and 700 nm, with respect to
white and dark references, as
R(
)=100x[(sample–white)/(white–dark)].
The reflectance spectra of breast feathers were similar for both species
(Fig. 1A,B) and typically show
two peaks, in the ultraviolet and the yellow parts of the spectrum [for blue
tit nestlings (see Johnsen et al.,
2003)]. Variation in reflectance spectra was summarised performing
a principal component analysis (PCA) on raw spectra
(Cuthill et al., 1999;
Endler, 1990;
Hill et al., 2005) separately
for each species. Based on the scree plot of eigenvalues, the three first
principal components that together explained more than 99% of the variation in
reflectance were retained. PC1 explained 87% and 89.7%, PC2 10.6% and 8.8% and
PC3 1.6% and 1% of the variance in reflectance spectra in blue and great tit,
respectively. Factor loading for principal components was qualitatively
similar for both species (Fig.
1C,D). PC1 described mean reflectance, as it is generally the case
in a PCA on raw spectra: brightness is the main source of variation between
spectra, and subsequent principal components describe variation in spectral
shape (Cuthill et al., 1999).
Factor loading for PC2 was positive below and negative above 475 nm,
suggesting that it represents the relative importance of ultraviolet and
yellow peaks. Factor loading for PC3 coefficient was positive in both short
and long wavelengths and negative in medium wavelengths. Feathers with high
values of PC3 coefficients will show greater differences among parts of the
spectrum and will therefore appear more chromatic
(Endler, 1990). Two feathers
were analysed for each individual, with four measures per feather.
Repeatability of measurements calculated as the intra-class correlation
coefficient (Lessells and Boag,
1987) was always highly significant (all P<0.0001)
with the following values (blue tit; great tit): (a) repeatability within
feathers, PC1: 0.57; 0.69, PC2: 0.66; 0.73, PC3: 0.61; 0.61; and (b)
repeatability within individuals, PC1: 0.42; 0.48, PC2: 0.55; 0.62, PC3: 0.53;
0.40. Average values for the eight measures per individual were used in
subsequent statistical analyses.
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Statistical analyses
All statistical analyses were made using SAS v8.2 (SAS Institute Inc.
1999–2001, Cary, NC, USA). Tests of the residuals for normality and
homoscedasticity were used to check the validity of the model. In order to
control for the effect of the proportion of red blood cells on sedimentation,
size on body mass, and time between measures of initial and post-reaction wing
web thickness on immune response, haematocrit, tarsus length and time were
entered as covariates in all models with sedimentation rate, body mass, and
cell-mediated immune response as dependent variables, respectively
(Freckelton, 2002). As the
effect of supplementation may differ according to body size, tarsus length and
its interaction with treatment were initially included in all models. Hatching
date, brood size and nestling age were also first included in all models to
account for seasonal variation, sibling competition and nestling growth,
respectively. Retinol and vitamin E concentrations in nestling plasma were
investigated including carotenoid concentration and its interaction with
treatment in the models. Indeed, it is known that physiological interactions
occur between these different antioxidants, and we may therefore expect plasma
levels of vitamin A and E to be related to plasma levels of carotenoids, and
these relationships to be modulated by experimental treatment. Interactions
and main effects were dropped from the models when not significant. The age at
first experimental feeding and the total number of feedings received were both
initially included as covariates in all models, but they never explained a
significant amount of variance, and thus were not retained in the models.
Effect of treatment on plasma carotenoid composition was investigated using
a multivariate analysis of variance (MANOVA) model with the procedure GLM, on
log-ratio-transformed proportions
(Reyment, 1989). Effect of
treatment on nestling characteristics was tested using the MIXED procedure of
mixed linear models (Goldstein,
2003), with treatment as fixed effect and nest as random effect to
account for non-independence of nestlings belonging to the same nest. The null
model likelihood ratio 
2 test was used to assess whether the
model with random effect provided a significantly better fit than the same
model without random effect. If that was not the case, the model was
constructed without random effect. Models were compared by Akaike's
Information Criterion (AIC), and the most parsimonious was retained [lowest
AIC (Burnham and Anderson,
1998)]. Effect of treatment on nestling retinol and vitamin E was
tested using the GLM procedure of generalised linear models.
Significant models were followed by comparisons of means or least square means between treatments with adjusted values of P using the Tukey-Kramer method. Values are reported as mean ± 1 s.e.m.
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Results |
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=0.55,
F6,12=1.62, P=0.22)
(Table 1). Total carotenoid
concentration in plasma was not significantly affected by feeding treatment
(F1,86=1.00, P=0.32; carotenoid supplemented:
35.8±4.4 µg ml–1, control: 42.8±4.9 µg
ml–1) (Fig.
2). Retinol was not significantly affected by feeding treatment
(F1,17=0.45, P=0.51; carotenoid supplemented:
1.0±0.1 µg ml–1, control: 1.3±0.1 µg
ml–1) (Fig.
2), but was marginally and positively related to carotenoid
concentration (F1,17=2.18, P=0.16; when removing
feeding treatment from the model: F1,18=4.36,
P=0.05, R2=0.19, slope estimate ± s.e.m. =
0.0064±0.0030). Total vitamin E (summed 
-, 
-,
-tocopherol) concentration was affected by the interaction between
feeding treatment and total carotenoid concentration (carotenoid main effect:
F1,16=0.01, P=0.94, feeding treatment main
effect: F1,16=10.94, P=0.004, interaction:
F1,16=9.99, P=0.006). There was a positive
relationship between vitamin E and carotenoids in carotenoid-fed nestlings,
but that was not the case for control-fed nestlings
(Fig. 3). However, there was no
significant difference in plasma vitamin E concentration between treatments
(carotenoid supplemented: 3.6± 1.3 µg ml–1,
control: 6.9±1.3, difference of least squares means: t=2.04,
P=0.06) (Fig. 2).
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Body condition
Nestling body mass was significantly affected by feeding treatment and its
interaction with tarsus length (Table
2). Overall, nestlings from the carotenoid-supplemented group were
slightly heavier than nestlings from the control group (least square mean
± s.e.m., carotenoid supplemented: 10.8±0.1 g, control:
10.6±0.1 g, t=2.04, P=0.04)
(Fig. 4). Feeding treatment
modified the relationship between body mass and size
(Fig. 5A): small (i.e. tarsus
length below mean tarsus length) carotenoid-fed nestlings were heavier than
small control-fed nestlings, this difference increased with decreasing tarsus
length. The smallest carotenoid-fed nestlings were between 0.50 and 0.75 g
heavier than similarly sized control-fed nestlings
(Fig. 5A).
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Immune function
Nestling blood sedimentation rate increased with haematocrit
(F1,53=6.78, P=0.01), hatching date
(F1,53=11.72, P=0.001) and nestling age
(F1,53=12.79, P=0.0008), but did not depend on
feeding treatment (F1,53=0.08, P=0.77; carotenoid
supplemented: 72.9±0.9%, control: 72.4±0.9%)
(Fig. 4). The relative amount
of leukocytes in nestling blood increased with brood size
(F1,101=3.83, P=0.05), but was not significantly
affected by feeding treatment (F1,101=2.31,
P=0.13; carotenoid supplemented: 1.45±0.05%, control:
1.56±0.06%) (Fig. 4).
Cell-mediated immune response decreased with nestling age
(F1,44=22.87, P<0.0001) and hatching date
(F1,44=24.56, P<0.0001), without any
significant effect of feeding treatment (F1,44= 0.00,
P=0.95; carotenoid supplemented: 0.25±0.04 mm, control:
0.25±0.03 mm) (Fig.
4).
Breast feather colour
Juvenile plumage colour scores for PC1, PC2 and PC3 did not differ
significantly with feeding treatment (Table
3; Fig. 6).
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Great tit nestlings
Carotenoid profile and antioxidant concentration in plasma
Feeding treatment did not significantly modify relative concentrations of
individual carotenoids in nestling plasma (Wilks' =0.30,
F6,7=1.62, P=0.11)
(Table 1). Total carotenoid
concentration in plasma was not significantly affected by feeding treatment
(F1,80=0.00, P=0.97; carotenoid supplemented:
45.5± 4.7 µg ml–1, control: 45.2±3.4 µg
ml–1) (Fig.
2). Retinol concentration in plasma was found to decrease with
increasing hatching date (F1,9=13.14, P=0.005),
and to increase with brood size (F1,9=17.00,
P=0.002), with no significant effect of treatment
(F1,9=0.55, P=0.48; carotenoid supplemented:
0.23±0.11 µg ml–1, control: 0.39± 0.25 µg
ml–1) (Fig.
2). Total vitamin E concentration in plasma decreased with
nestling age (F1,11=6.38, P=0.03), but was not
significantly affected by treatment (F1,11=0.08,
P=0.79; carotenoid supplemented: 11.8±2.9 µg
ml–1, control: 12.5±1.3 µg ml–1)
(Fig. 2).
Body condition
Body mass was significantly affected by feeding treatment in interaction
with tarsus length, in a model controlling for nestling age
(Table 2), although there was
no difference in mean body mass between feeding groups (least square mean
± s.e.m., carotenoid supplemented: 16.96±0.16 g, control:
16.91±0.16 g, t=0.33, P=0.74)
(Fig. 4). Smaller than average
carotenoid-fed nestlings were heavier than similarly sized control-fed
nestlings and the reverse was true for nestlings larger than the average
(Fig. 5B). The smallest
carotenoid-fed nestlings were about 1 g heavier than similarly sized
control-fed nestlings, while the largest carotenoid-fed nestlings were about 1
g lighter than similarly sized control-fed nestlings
(Fig. 5B).
Immune function
Nestling blood sedimentation rate increased with haematocrit
(F1,60=14.17, P=0.0004) and brood size
(F1,60=4.31, P=0.04), but was not significantly
affected by feeding treatment (F1,60=0.63,
P=0.43; carotenoid supplemented: 68.9±0.7%, control:
70.3±1.2%) (Fig. 4).
There was no significant effect of feeding treatment on the relative amount of
leukocytes in nestling blood (F1,80=0.35, P=0.55;
carotenoid supplemented: 1.29±0.09%, control: 1.35±0.07%)
(Fig. 4). Cell-mediated immune
response was not significantly affected by feeding treatment
(F1,25=0.09, P=0.77; carotenoid supplemented:
0.16±0.03 mm, control: 0.17±0.03 mm)
(Fig. 4).
Breast feather colour
Feeding treatment influenced all colour parameters
(Table 3;
Fig. 6). PC1 was significantly
greater, i.e. feathers were brighter in carotenoid-fed than control-fed
nestlings. PC2 was smaller for carotenoid-fed nestlings than for control-fed
nestlings: feathers from carotenoid-fed nestlings showed a proportionally
higher peak in the yellow than in the ultraviolet part of the spectrum, as
compared to control-fed nestlings. Finally, PC3 was marginally greater in
carotenoid-fed than control nestlings: feathers from carotenoid-fed nestlings
appeared slightly more chromatic than feathers from control fed nestlings.
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;
Negro et al., 2000;
Slagsvold and Lifjeld, 1985;
Tschirren et al., 2003). In
both species, increasing carotenoid intake had a positive effect on body
condition (body mass relative to body size), but this was not paralleled by an
effect on immune function. Although carotenoid supplementation did not result
in an increase in plasma carotenoids in either species, in blue tits a
positive relationship between plasma carotenoids and vitamin E was observed in
carotenoid-fed nestlings, while no such relationship was present in control
nestlings. Increasing carotenoid intake enhanced plumage colour in great tit,
but not in blue tit nestlings.
Carotenoids are hypothesised to be limiting in the environment
(Olson and Owens, 1998), but
studies investigating availability of these pigments in the natural food in
different populations and species are very scarce
(Partali et al., 1987). Plasma
carotenoid levels in wild birds are documented mainly for adults (e.g.
Biard et al., 2005;
Blount et al., 2002;
Bortolotti et al., 2000;
Hõrak et al., 2004;
Ninni et al., 2004;
Tella et al., 2004;
Wallace et al., 1996). In our
population, plasma carotenoid concentration in breeding great tit females was
similar to that found in females of a rural population studied by Hõrak
et al. (Hõrak et al.,
2004) (also our unpublished data). However, both physiology and
diet may differ between nestlings and adults, and it would be hazardous to
extrapolate plasma carotenoid levels of nestlings from plasma carotenoid
concentrations of adults. There is very little information available on plasma
carotenoid levels in nestlings (Biard et
al., 2005; Negro et al.,
2000). Therefore, it is difficult to determine whether and how
much carotenoids may be limiting for nestlings in our population.
Carotenoid composition of supplementary food was 90% lutein and 2%
zeaxanthin. However, natural food during nestling growth for blue and great
tit is composed of various insects and arachnids rich in lutein and
zeaxanthin, but also in ß-carotene [example of composition of
lepidopteran larvae: lutein: 80%, zeaxanthin: 3%, ß-carotene: 17%
(Partali et al., 1987)]. There
was no difference in plasma carotenoid profile in carotenoid-fed nestlings
compared to control-fed nestlings, although our carotenoid supply was
deficient in ß-carotene. Therefore, it is unlikely that the experimental
protocol induced any modification in carotenoid absorption or metabolism that
we would expect to be mirrored by changes in plasma carotenoid profile in
carotenoid-fed nestlings. There was no detectable increase in plasma
carotenoids after treatment in carotenoid-fed nestlings of either species
despite regular supply of dietary carotenoids. In a study on white storks,
Ciconia ciconia, plasma carotenoid concentration was found to be five
times higher in nestlings from a population feeding mainly on a
carotenoid-rich diet (crayfish Procambarus clarkii) as compared to
nestlings from a population having access to low carotenoid food only
(Negro et al., 2000).
Possibly, carotenoid concentration in plasma is maintained at a stable level
preventing increase above a certain optimum, which may already be attained
naturally in our population that was breeding in a rich habitat. Excess
dietary carotenoids may be stored, with the most important storage organ being
the liver, and then released in circulation when needed
(Surai, 2002;
Surai and Speake, 1998).
Although plasma vitamin E concentrations were not significantly different in
carotenoid-supplemented as compared to control nestlings, there was a tendency
for control nestlings to show higher plasma vitamin E levels. The mechanism of
carotenoid absorption in the intestine is similar to that of tocopherols
(Surai, 2002;
Woodall et al., 1996).
Therefore, it could be speculated that increased carotenoid supplementation
would have induced competitive interactions between carotenoids and tocopherol
during absorption, and this may explain why some carotenoid-fed nestlings had
low carotenoid and vitamin E levels. This particular question requires further
study. Indeed, studies investigating the possible negative interactions
between vitamin E and carotenoids in dietary supplementation experiments have
yielded contradictory results (Furr and
Clark, 1997). However, in carotenoid-fed blue tit nestlings
vitamin E concentration increased with carotenoid concentration in plasma,
while that was not the case for control nestlings. Dietary supplementation
with carotenoids thus had an effect on plasma vitamin E and carotenoids,
probably through interactions between them. This may have arisen because
carotenoids were provided in oil containing vitamin E. However, we find this
unlikely, because both groups received the same quantity of vitamin E in oil,
and carotenoids are present in the nestlings natural food. In addition, when
carotenoids were provided to female blue tits in low vitamin E vegetable oil,
a similar enhancement of vitamin E availability without increase in plasma
carotenoid level was found (Biard et al.,
2005). Therefore, an increase in dietary availability of
carotenoids may cause synergistic interactions between vitamin E and
carotenoids even in the absence of a detectable increase in plasma carotenoid
concentration. Such synergistic effects among antioxidants are described in
the poultry literature (e.g. Surai and
Speake, 1998; Surai et al.,
2001b), but have generally been neglected in studies of wild
birds. However, there was no such effect on plasma antioxidants in great tit
nestlings. This may be linked to the increase in pigment deposition into
growing feathers in carotenoid-fed nestlings. Our data indicate that mean
(±s.e.m.) carotenoid concentration in blue tit nestling plumage is
640±37 µg g–1 (N=4). For comparison, mean
carotenoid concentration in the liver of the same nestlings was 145±62
µg g–1 (our unpublished data). Therefore, the amount of
pigments deposited into plumage may not be negligible compared to circulating
or stored carotenoids for a nestling. In conclusion, we suggest that
assessment of treatment effects can only be made when carefully considering
other antioxidants, different sites of storage, and the use of antioxidants in
production of signals that will not allow subsequent use for physiological
functions.
In both species a strong effect of carotenoid supplementation was found on
body condition through a modification of the relationship between body mass
and size, with an increase in body mass for small carotenoid-fed nestlings
compared to similarly sized control nestlings. In great tit nestlings only, a
decrease in body mass was also observed for large carotenoid-fed nestlings.
Body mass at fledging is a good predictor of immediate post fledging and
overwinter survival in tits (Naef-Daenzer
et al., 2001; Tinbergen and
Boerlijst, 1990). Thus parents able to provision their brood with
a carotenoid-rich diet would increase the probability of survival of both
smaller and larger nestlings. An increase in the availability of carotenoid
pigments may enhance mass gain in nestlings by regulating oxidative stress
resulting from rapid growth. The mechanism through which an increase in
carotenoid availability could reduce mass gain in large great tit nestlings
could be an increase in intensity of sib competition that may
disproportionately affect large siblings.
In both great and blue tits, there was no effect of carotenoid
supplementation on any of the variables used to describe activation of the
immune system (sedimentation rate, relative amount of circulating leukocytes)
or ability to raise a cell-mediated immune response. These were mostly
influenced by hatching date, nestling age and brood size. A positive effect of
an increase in dietary carotenoids was indeed expected because these pigments
are involved in activation and regulation of immune function (reviewed by
Chew, 1993;
Møller et al., 2000).
Cell-mediated immune response to PHA is localised at the site of injection and
thus any effect of carotenoids on this immune response should be mediated
through carotenoid concentration in plasma. However, carotenoid plasma levels
were not enhanced by dietary supplementation, which could explain why there
was no difference in cell-mediated immune response between treatments. In
adult zebra finches Taeniopygia guttata, birds fed with a
carotenoid-enriched diet showed an increase in plasma concentrations of
carotenoids and an increase in both cell-mediated and humoral immune responses
(Blount et al., 2003b;
McGraw and Ardia, 2003). In
nestlings, carotenoids may influence the development and maturation of the
immune system and/or the immune response, depending on when they are
available. In barn swallows Hirundo rustica, nestlings originating
from experimentally carotenoid-enriched eggs had a stronger cell-mediated
immune response than controls, and this response predicted survival to
fledging (Saino et al., 2003).
This suggests that an increase in carotenoid availability, if occurring during
early embryo and hatchling development (i.e. maternally derived yolk
carotenoids) may improve the development of an efficient immune system,
whereas this may not be the case if carotenoids are provided later during
nestling growth, as in our experiment. Alternatively, our population may not
suffer from carotenoid scarcity in the environment, with the natural diet
providing sufficient amounts of carotenoids to allow nestlings to efficiently
mount an immune response. If that was the case, further increasing dietary
availability of carotenoids for nestlings would not be expected to have an
effect on the magnitude of immune responses.
In the great tit, carotenoid-supplemented nestlings grew brighter yellow
feathers than control nestlings. In addition, in carotenoid-fed nestlings
feathers showed a proportionally higher peak in the yellow than in the
ultraviolet, and a tendency to be more chromatic than feathers from
control-fed nestlings. These results show that dietary pigments were deposited
into growing feathers, and that the development of juvenile plumage colour
depends on nutritional conditions. This confirms the findings of previous
studies showing that nestling great tits from carotenoid-poor habitats grew
paler yellow plumage than nestlings from carotenoid-rich habitats
(Slagsvold and Lifjeld, 1985).
An enhancement of yellow plumage colour was also obtained in previous
supplementary feeding experiments (Fitze
et al., 2003; Tschirren et
al., 2003). These studies did not investigate plasma carotenoid
levels following supplementation. In our study, enhancement of plumage colour
occurred in the absence of a detectable increase in plasma carotenoids. We may
hypothesise that transitory increases in plasma carotenoid occurring after
ingestion and absorption of supplementary carotenoids have stimulated
follicles and increased the rate of pigment uptake and deposition into growing
feathers. All great tit nestlings in this study were fed when older than 5
days, suggesting that in great tit nestlings, feather colour may be determined
after an age of 6 days (contrary to what was stated by
Fitze et al., 2003). In great
tit fledglings, plumage colour has been shown to reflect rearing conditions in
terms of habitat quality, year effects, and experimental reduction in brood
size (Hõrak et al.,
2000; Tschirren et al.,
2003). However, at present nothing is known about the possible
function of juvenile plumage colour in parent-offspring and/or
offspring/offspring communication after fledging. Although there was an effect
of dietary supplementation on feather colour in great tit nestlings, no such
effect was found in blue tit nestlings. A different timing of feather
development could explain this interspecific difference in response to
carotenoid supplementation. However, blue and great tit nestlings show a very
similar pattern of feather growth
(Schoppe, 1977). Blue tits lay
larger clutches, and thus invest relatively more in reproduction than great
tits in terms of total clutch mass relative to female body mass
(Blondel et al., 1990;
Gosler, 1993;
Newton, 1989). Beyond the
energetic costs of producing larger clutches, female blue tits also need far
more carotenoids for investment in their eggs. Carotenoid availability in the
environment may indeed be lower at the time of egg production than at the time
of rearing nestlings, when caterpillars reach peak abundance. This may cause
egg yolk carotenoid concentration to be lower, and consequently embryos and
hatchlings to develop in less favourable conditions in blue tits than in great
tits. Indeed, data collected in 2001 and 2002 in this population indicate that
mean (±s.e.m.) yolk carotenoid concentration for blue tits was
18.03±0.7 µg g–1 (N=169) while it was
26.21±1.6 µg g–1 (N=98) for great tit; a
mean difference of 45% (our unpublished data). In addition, growth rate is
reported to be higher in blue tit nestlings than in great tit nestlings [mean
growth rate for blue tit=0.41 for an adult body mass of 11 g, mean growth rate
for great tit=0.36 for an adult body mass of 19 g; a mean difference of 14%
(Starck and Ricklefs, 1998)].
Blue tit nestlings may thus be subject to more intense oxidative stress than
great tit nestlings. Therefore, blue tit nestlings are probably more in need
of carotenoids for physiological functions than are great tit nestlings, and
if provided with extra carotenoids blue tit nestlings may primarily invest
them in antioxidant function rather than plumage colour.
To our knowledge, this is the first study showing that an increase in dietary availability of carotenoids influences fledgling body mass independently of immune function or plasma carotenoid concentration. As body mass at fledging is a key parameter for offspring survival in tits, parental availability of dietary carotenoids may have important fitness consequences for their offspring. Furthermore, plasma carotenoids correlated positively with vitamin E levels in carotenoid-supplemented but not in control blue tit nestlings, while dietary supplementation with carotenoids enhanced plumage colour in great tit nestlings. We hypothesise that the differences in effect of experimental carotenoid supplementation on the two species may be due to a relatively larger clutch size and a higher growth rate in blue tits compared to great tits. This hypothesis of a differential need for antioxidants depending on early developmental conditions (i.e. maternal investment of carotenoids to eggs) and life history traits would require extensive comparative analyses taking phylogenetic relationships into account.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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