Synergistic effects of supplementation of dietary antioxidants during growth on adult phenotype in ring-necked pheasants, Phasianus colchicus

Authors

  • Josephine M. Orledge,

    1. Centre for Ecology and Conservation, Biosciences, College of Life and Environmental Sciences, University of Exeter, Cornwall Campus, Penryn, Cornwall, TR10 9EZ, UK
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  • Jonathan D. Blount,

    1. Centre for Ecology and Conservation, Biosciences, College of Life and Environmental Sciences, University of Exeter, Cornwall Campus, Penryn, Cornwall, TR10 9EZ, UK
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  • Andrew N. Hoodless,

    1. Game and Wildlife Conservation Trust, Fordingbridge, Hampshire, SP6 1EF, UK
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  • Thomas W. Pike,

    1. Centre for Ecology and Conservation, Biosciences, College of Life and Environmental Sciences, University of Exeter, Cornwall Campus, Penryn, Cornwall, TR10 9EZ, UK
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    • Present address: Department of Biological Sciences, University of Lincoln, Riseholme Park, Riseholme, Lincoln, LN2 2LG, UK.

  • Nick J. Royle

    Corresponding author
    1. Centre for Ecology and Conservation, Biosciences, College of Life and Environmental Sciences, University of Exeter, Cornwall Campus, Penryn, Cornwall, TR10 9EZ, UK
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Correspondence author. E-mail: n.j.royle@exeter.ac.uk

Summary

1. Oxidative stress may provide a proximate link between investment in growth and/or reproduction and investment in self-maintenance. Dietary antioxidants, such as carotenoids and vitamin E, provide potentially important roles in regulating these trade-offs. Recent work suggests that carotenoids may have synergistic effects in combination with non-pigmentary antioxidants (e.g. vitamin E) on the expression of sexually selected traits in adulthood. However, these studies involved the supplementation of antioxidants to adults so did not take account of early life-history effects.

2. Here, we test the independent and combined roles of supplementation of carotenoids and vitamin E during early growth in regulating the expression of traits in adulthood, in ring-necked pheasants, Phasianus colchicus. Individuals supplemented with a combination of carotenoids and vitamin E were larger at adulthood than individuals receiving other treatment diets (including vitamin E or carotenoids alone), but there were no differences in ornament expression, immune function, the swelling response to phytohaemagglutinin or levels of oxidative damage.

3. This shows that there are synergistic early life-history effects of these dietary antioxidants on body size at adulthood and suggest that the allocation of limited antioxidant resources are prioritized towards traits that increase competitive ability rather than sexual attractiveness in this strongly sexually selected species.

Introduction

Life-history trade-offs result from competition for a limited and shared resource by multiple traits (Stearns 1989), such that allocation to one trait results in a reduction in the resources available for investment in another (Zera & Harshman 2001). Resource allocation trade-offs may be particularly acute for strongly sexually selected species, where significant investment in sexual characteristics can only be achieved at a cost to other traits (Royle, Lindström & Metcalfe 2005). Little is known about the mechanistic costs underlying such trade-offs, although there is growing evidence for the role of oxidative stress as a potentially unifying mechanism (Costantini 2008; Monaghan, Metcalfe & Torres 2009; Hall et al. 2010). Oxidative stress results from an imbalance between the production of damaging reactive oxygen species (ROS) and antioxidant defences, in favour of the former (Sies 1997). An important level of antioxidant defence involves fat-soluble antioxidants such as vitamin E and carotenoid pigments, which cannot be synthesized de novo by vertebrates and are therefore limited by dietary intake. Vitamin E is a colourless and highly effective antioxidant (Surai 2002), which may also have roles in signal transduction and gene expression (Brigelius-Flohé 2009). Carotenoids, on the other hand, are highly pigmented antioxidants that are expressed in many sexually selected traits and also have immunoenhancing properties (McGraw & Ardia 2003).

A large proportion of the research on the role of oxidative stress in life-history trade-offs has focused exclusively on carotenoid supplementation (Catoni, Peters & Schaefer 2008), often producing conflicting results. In particular, recent analyses have questioned the effectiveness of carotenoids as antioxidants in vivo, concluding that they have only minor antioxidant capabilities (Costantini & Møller 2008; Cohen & McGraw 2009), and at high concentrations, carotenoids can have detrimental effects on health (Huggins et al. 2010). However, animals ingest a cocktail of antioxidants, which may interact either antagonistically (e.g. competition during absorption) or synergistically (e.g. carotenoid recycling by vitamin E; Surai 2002; Catoni, Peters & Schaefer 2008), and several supplementation studies have now demonstrated experimentally such synergistic effects of antioxidants on the expression of sexually selected traits (e.g. Pike et al. 2007; Pérez, Lores & Velando 2008). However, these studies have all been conducted at adulthood so do not take account of early life-history effects on the expression of traits in adulthood.

Somatic growth results in the production of high levels of ROS (Stoks, De Block & McPeek 2006) and the period of rapid growth and development early in life constitutes a period of heightened vulnerability to oxidative stress (Surai 2002). Individuals often experience variation in resource availability during early development and selection favours rapid growth when competition for resources is acute, so trade-offs between allocation of resources to growth and self-maintenance are expected (Hall et al. 2010). Such early life-history effects are therefore likely to play an important role in the expression of sexually selected traits at adulthood (Blount et al. 2003a; Walling et al. 2007). Sexually selected traits are known to have an increased susceptibility to environmental stress during development (Hunt & Simmons 1997; David et al. 2000), and nutritional quality can influence circulating antioxidant levels at adulthood (Blount et al. 2003a). If antioxidant availability during development has long-term affects on the ability of adults to assimilate antioxidants, it is therefore also likely to affect the expression of sexually selected traits at adulthood.

Trade-offs between growth and self-maintenance during development are expected to be particularly strong in sexually dimorphic species of birds such as the ring-necked pheasant, Phasianus colchicus (Fig. 1). In this species, males have bright plumage, conspicuous wattles, long tail feathers, spurs and ear tufts. Females are smaller, with a duller yellowish buff plumage with a mottled chestnut pattern and long banded tail. Pheasants have a harem polygyny mating system, and females choose mates based on multiple sexual ornaments (Hill & Robertson 1988). These ornaments include facial wattles (Hillgarth 1990), the colour of which is likely to be carotenoid mediated (Czeczuga 1979), long spurs on the legs (Göransson et al. 1990) and long tails (Mateos & Carranza 1995). The bright wattle of males varies in shape and is expanded during sexual displays to attract females (Hill & Robertson 1988); females have been shown to prefer larger wattles (Hillgarth 1990). Body mass has also been found to be an important determinant of success in mating and is correlated with spur length (Göransson et al. 1990). Previous work has shown that a low intake of dietary protein during the early growth and development of males resulted in reduced wattle size and brightness at adulthood (Ohlsson et al. 2002), demonstrating that the expression of at least one sexual ornament in pheasants is sensitive to environmental conditions experienced post-hatching in this species.

Figure 1.

 An adult male ring-necked pheasant [Phasianus colchicus] showing sexually selected ornament, the facial wattle. Photo credit N.J. Royle.

In the current study, we manipulated the dietary antioxidant availability of pheasants during early life and quantified effects on growth and self-maintenance (oxidative damage and immunity) in relation to the expression of multiple sexual ornaments of males at adulthood. More specifically, we supplemented pheasants with either carotenoids, vitamin E or a combination of both vitamin E and carotenoids, or provided a control diet, during the first 8 weeks of life. This allowed us to examine the synergistic and independent effects of carotenoids and vitamin E in regulating trade-offs during growth and development that affect the expression of traits important in reproductive success as adults. If oxidative stress is an important mediator of trade-offs during growth and development (Hall et al. 2010), we predict that supplementary antioxidants provided during early development will be preferentially allocated towards traits most important to reproductive success at the expense of investment in self-maintenance. In particular, in the light of recent work that indicates that carotenoids are relatively poor antioxidants in birds in vivo (e.g. Cohen & McGraw 2009), we tested whether carotenoids and vitamin E are only effective antioxidants in combination with one another (vitamin E can recycle oxidized carotenoids, for example; Catoni, Peters & Schaefer 2008), and whether such synergistic effects during development lead to an increase in resources allocated to achieving higher reproductive success (increased sexual ornament expression and/or body size).

Materials and methods

General protocol

Two hundred and forty 1-day old ring-necked pheasants of mixed genetic stock (Holme Park Game Hatcheries, Wokingham) were used in the experiment, at the Game and Wildlife Conservation Trust, Hampshire, UK. The game farm that supplied the pheasants maintains breeding stock in groups of 30 hens with three cock pheasants (i.e. replicating the natural harem polygyny mating system). As a result, males and females encounter multiple potential copulation partners. The pheasants are not intensively farmed or artificially selected for traits such as high egg production or disease resistance either, so there is no evidence that the phenotypes of the pheasants are uncoupled from past natural and sexual selection pressures. For the first 8 weeks (commencing in early May), birds were housed in groups of 30 in indoor pens (1·8 m × 1·5 m) under infra red heat lamps within a semi-intensive brooder hut system. Lighting levels during the first 8 weeks were limited to the light emitted by the heat lamps, and windows were painted to minimize light entering the pens as per standard husbandry practice (GCT 2004). Additional birds were reared, and chicks that died (N = 8) during the experimental period (8 weeks) were replaced with non-experimental, similar-aged birds to maintain consistent rearing densities. On day 1, the birds were allocated randomly to one of four equal-sized diet treatment groups (n = 60 birds per group): (i) Carotenoid supplemented (group C); (ii) α-tocopherol (vitamin E) supplemented (group V); (iii) α-tocopherol (vitamin E) with carotenoid supplemented combined (group CV); and (iv) a control diet (Control). These diets were fed for the first 8 weeks. An 8-week period of dietary manipulation was chosen to include the early developmental window identified by previous studies on pheasants (Ohlsson & Smith 2001; Ohlsson et al. 2002). After 2 weeks, birds were given daily access to outdoor pens with wire floors (3 m × 1·5 m). At 8 weeks of age, the birds were sexed and then transferred to one of two outdoor single-sex pens (30 m × 27 m) with access to grass for the remainder of the experiment. The diet provided after 8 weeks was identical for all birds (maintenance pellet 13% protein; Duke’s and Botley Ltd, Botley, Southampton, Hampshire, UK).

Morphometric measurements were taken at day 1 and then at weekly intervals until 10 weeks of age, then again at 21 and 22, 36 and 37, 46 and 47 weeks of age. Blood samples were taken from all birds at 8 and 47 weeks of age. At 8 weeks of age, samples were used to assay plasma concentrations of vitamin E, carotenoids and oxidative damage [determined by measuring the concentration of a biomarker of lipid peroxidation, malondialdehyde (MDA) in all individuals]. A biomarker of lipid peroxidation was measured because carotenoids and vitamin E are lipid soluble and the most abundant scavengers of hydroxyl-free radicals within the lipid membrane of cells (Mascio, Murphy & Sies 1991). At 47 weeks of age, oxidative damage (MDA) was assayed in all individuals again, and plasma concentrations of vitamin E and carotenoids were measured for males only. Phytohaemagglutinin was used to measure immune response of all individuals at 21 weeks of age (Smits, Bortolotti & Tella 1999; Vinkler, Bainova & Albrecht 2010). In addition, wattle colour, size and shape, and spur length were measured at 21 weeks (a period when body size growth was still occurring) and at 47 weeks of age before the breeding season by which stage wattles were fully developed (Ohlsson & Smith 2001). A previous study on protein manipulation in ring-necked pheasants showed significant differences in wattle size at 20 and 40 weeks of age (Ohlsson et al. 2002).

Dietary supplementation

All birds received a custom-made basal diet, based on standard commercial pheasant pellets, but with no added vitamin E, and low levels of vitamin A (10·0 mg kg−1) and selenium (0·20 mg kg−1) (Target Feeds Ltd., Shropshire, UK). The basal diet was manipulated for each treatment group as follows: Carotenoid-supplemented birds received 100 mg carotenoids in the form of ORO-GLO® brand 11 liquid pigmenter (lutein and zeaxanthin, 20:1 w/w) (Kemin industries Inc., Des Moines, Iowa, USA) per kg of feed; birds supplemented with vitamin E received supplemental all-trans α-tocopherol (Sigma-Aldrich T36634, St. Louis, Missouri, USA) at a concentration of 100 mg kg−1 of feed; birds receiving both carotenoids and vitamin E received 50 mg per kg of feed; control birds received an unsupplemented diet. Little is known about the concentrations of antioxidants in the natural diet of pheasants, but the concentration of vitamin E and carotenoids supplemented was consistent with what are considered to be moderate concentrations (100 mg kg−1; Surai 2002) successfully used in previous studies on poultry (vitamin E: Bartov & Frigg 1992; Guo et al. 2001; carotenoids: Woodall, Britton & Jackson 1996; Surai, Speake & Sparks 2001). Supplements were added to the feed daily, by spraying with a 5-L spray pump. α-Tocopherol was sprayed in soybean oil onto the feed, and ORO-GLO® xanthophylls were mixed in water and stored in refrigerated vacuum pumped containers until given to the birds. Soybean oil was selected as a medium for α-tocopherol supplementation because it contains comparatively low levels of α-tocopherol (0·07 μg mg−1) compared to other naturally occurring oils (Carpenter 1979). Equal volumes of soybean oil and water were sprayed onto the other feeds. Every afternoon, the feed was replenished with fresh refrigerated treatment feed stored in vacuumed pumped containers. Four diets were provided over the 8-week period of supplementation, all with medium levels of protein, in line with standard pheasant rearing practice (starter crumb 1–2 weeks: 29·8% protein, starter pellets 3–4 weeks: 25·5% protein, rearer pellets 5–6 weeks: 21·4% protein, grower pellets 7–8 weeks: 18·1% protein). Feed, grit and water were provided ad libitum.

Morphometric measurements

To calculate growth body mass, tarsus length, head-to-bill length and wing length was measured at each week from 0 to 10 weeks, at 21 and 22, 36 and 37, 46 and 47 weeks of age. Body mass was measured using a Pesola® spring balance (30, 60, 100, 300, 600, 1000, 2500 g depending on age). Tarsus length and head-to-bill length was measured using callipers (to an accuracy of + or −0·01 mm), and wing length was recorded using a wing rule (accuracy + or −0·1 mm). Spur length was measured using dial calliper measurements of the tarsus width just above the spur and by subtracting this from a measurement of the tarsus width and spur length (Ohlsson et al. 2002).

Oxidative stress assays and α-tocopherol concentration of plasma

Blood samples were taken at 8 weeks (at the end of the supplementation period) and at 47 weeks of age. Whole blood (up to 0·3 mL) was collected from the brachial vein under Home Office licence in 5/8” 26 gauge MicrolanceTM needles (Fisher Scientific UK Ltd., Loughborough, Leicestershire, UK) and BD PlastipakTM 1 mL syringes (Fisher Scientific UK Ltd., Hastings, East Sussex, UK) flushed with heparin (Sigma-Aldrich Inc.) and microhaematocrit EDTA-coated capillary tubes (Bilbate Ltd., Hastings, East Sussex, UK). Syringe samples were transferred to 1·5-mL EDTA-coated microtubes (Sarstedt) and stored in a dark cool bag. The samples were centrifuged, and plasma was removed and stored at −20 °C within 1 h of collection. The samples were then transferred to a −80 °C freezer within 5 days before biochemical analysis.

α-Tocopherol was measured within a month using high-performance liquid chromatography (HPLC). Plasma (50 μL) was mixed with 5% sodium chloride (50 μL) and ethanol (100 μL). The mixture was vortexed for 20 s. Hexane (600 μL) was added to the solution and vortexed for 20 s and centrifuged for 4 min (13·8 g). The hexane layer was removed, and the absorbance measured at 450 nm using a spectrophotometer (Nicolet Evolution 500) to determine total carotenoid concentration using 2500 as an average extinction coefficient for all carotenoids. The hexane (400 μL) was dried down, and samples were redissolved in methanol (150 μL), centrifuged for 4 min, then injected (50 μL) into a Dionex HPLC system (Dionex Corporation, Sunnyvale, CA, USA) fitted with a 3 μ C18 reverse-phase column (15 cm × 4·6 mm) (Spherisorb S30DS2; Phase separations, Clwyd, UK) and using a mobile phase of methanol/distilled water (97:3) at a flow rate of 1·1 mL min−1. Fluorescence detection was carried out at 295 nm (excitation) and 330 nm (emission). Known concentrations of α-tocopherol (Sigma-Aldrich T36634) dissolved in methanol were used for calibration.

To measure plasma concentrations of MDA, 20 μL butylated hydroxytoluene (BHT) (0·05% w/v in 95% ethanol), 160 μL of phosphoric acid (0·44 m) solution and 20 μL of 2-thiobarbituric acid (TBA) (42 mm) were added to either 20 μL of plasma or 1,1,3,3-tetraethoxypropane (TEP), which was used for the calibration. The mixture was vortexed for 10 s and heated in a dry bath incubator for 1 h at 100 °C. Samples were then cooled on ice for 5 min. Eighty micro litres of n-butanol (HPLC grade) was added, and the mixture was vortexed for 20 s and centrifuged for 3 min at 4 °C (13·8 g) and 20 μl of the butanol phase containing MDA-TBA adduct was injected into a Dionex HPLC system fitted with a Hewlett-Packard Hypersil 5 μm ODS 100 × 4·6 mm column and a 5 μ ODS guard column maintained at 37 °C. The mobile phase was 50 mm potassium monobasic phosphate (pH 6.8 adjusted using 5 m potassium hydroxide) mixed with methanol (HPLC grade) running isocratically at 60:40 (v/v), at a flow rate of 1 mL min−1. Fluorescence detection was performed at 515 nm (excitation) and 553 nm (emission). For calibration, a standard curve was prepared using a TEP stock solution (5 mm in 40% ethanol) serially diluted using 40% ethanol.

Wattle size, shape and colour

An image of the right wattle at 46 weeks of age was taken with the head held on the same plane as a fixed scale. Image J software (Rasband, W.S., ImageJ; U. S. National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij, 1997–2009) was used to calibrate the scale of the image, and a polygon was drawn around the wattle to calculate area. The calculated shape outlines of the wattles for all individuals were included in a common elliptic fourier analysis (EFA) (Rohlf 1992) using Morpheus et al. software (D. E. Slice, Morpheus et al.: Software for Morphometric Research. Revision 01-31-00 Department of Ecology and Evolution, State University of New York). The EFA decomposed the curved edges of the polygon into a sum of 15 harmonically related ellipses (to produce 60 Fourier coefficients). Normalization allowed for the variation in the size, position and the rotation of images taken of each wattle. The 60 fourier coefficients were then used as variables in principal component analyses. Two principal components that described over 95% of the wattle shape variation as calculated by EFA (PC1 = 78%, PC2 = 20%) were used for analyses (South & Arnqvist 2009).

Wattle reflectance data were collected using a USB2000 UV-Visible spectrophotometer and OOIBase32 Software (Ocean Optics Inc., Dunedin, FL, USA). The spectrophotometer was fitted with a 90° probe pointer to ensure perpendicular contact with the wattle surface and to exclude ambient light (Mougeot, Redpath & Leckie 2005). Reflected radiance was measured across a spectral range of 260–680 nm at 0·3 nm resolution relative to a WS-1 white standard (Ocean Optics Inc., Dunedin, Florida, USA). The probe was held against the wattle and the spectra allowed to stabilize before capture (Keyser & Hill 1999). Three spectra were collected for the left wattle and three for the right wattle. The brightness of the wattle has been identified as important in female mate choice (Hillgarth & Wingfield 1997). We calculated brightness as it would be perceived by a conspecific female, using the method detailed in Endler & Mielke (2005). In galliforms, brightness is likely to be perceived by the double cones (Osorio, Vorobyev & Jones 1999). Because no data on photoreceptor spectral sensitivity have been collected for ring-necked pheasants, we used data for the closely related pheasant species, the blue peafowl (Pavo cristatus) (Hart 2002). The peafowl’s double cone has a peak sensitivity at 567 nm and is associated with a carotenoid-coloured oil droplet (Hart 2002). Effective double-cone sensitivity functions were modelled using the visual pigment template of Govardovskii et al. (2000) and incorporating the transmittance spectra of the combined ocular media for peafowl (Hart 2002), and estimated oil droplet transmission spectra was calculated using the equations of Hart & Vorobyev (2005) and data from Hart (2002). The birds were reared outdoors, so a standard daylight-simulating illumination spectrum (D65) was used in the model (Wyszecki & Stiles 1982).

Immune response

Immune response was measured in all birds at 21 weeks of age. Phytohaemagglutinin (PHA), a lectin from the red kidney bean (Phaseolus vulgaris), is used as a standard measurement of in pro-inflammatory potential in avian studies (Vinkler, Bainova & Albrecht 2010). An area of feathers (c. 1 cm2) from the patagium of both wings for each bird was plucked and sterilized with ethanol. The web diameters were then measured with a digimatic micrometer (0·01 mm, Mitutoyo APB-2D). In the right patagium, 0·2 mg of PHA (Sigma-Aldrich Inc.) in 0·1 mL of sterilized phosphate buffer solution (PBS) (Sigma-Aldrich Inc.) was injected subcutaneously using 5/8” 26 gauge MicrolanceTM needles (Fisher Scientific UK Ltd.) and BD PlastipakTM 1 mL syringes (Fisher Scientific UK Ltd.). 0·1 mL of sterilized PBS was injected into the left wing patagium. The thickness of the wing patagium of each wing was measured using the digimatic micrometer (0·01 mm) directly before injection. 24 h (±10 min) after the injection, the thickness of the patagium of the wings was measured again. The original thickness measurement was subtracted from this measurement to identify the pro-inflammatory potential to PHA 24 h after exposure (Vinkler, Bainova & Albrecht 2010).

Statistical analyses

Normality checks were carried out in SPSS, and variables were log-transformed where necessary (SPSS Inc., Chicago, IL, USA). Dependent variables conforming to a normal distribution were analysed using generalized linear mixed models (GLMMs) with supplementary treatment group, sex and/or age included as fixed effects and hatch date as a random factor. GLMMs were run in R version 2.9.2 (R Development Core Team 2009), using the lme function. Treatment was included as a single factor with 4 levels and sex as a factor with two levels. Males and females were approximately equally represented at all levels of the factor ‘treatment’. The date on which the HPLC assay was run for each sample was also included as a covariate but was dropped from all models during simplification. Where repeated measures were taken (vitamin E plasma concentration and wattle brightness) or male secondary sexual signals were measured, treatment diet and age were included in the maximal model, and two-way interactions were also included. Hatch date and bird identification number were included as random factors in models with repeated measures. Only measurements from birds that survived to a year of age were used to avoid measuring the effects that were the result of illness rather than treatment effects. Model simplification was achieved by the backward stepwise deletion of non-significant terms from the model, until variables could not be removed without causing an increase in deviance of P < 0·05. For model simplification, we removed the highest-order interactions, followed by lower-order terms in turn from the maximal model using maximum likelihood tests. For post hoc tests involving treatment groups GLMMs in which the focal treatment groups were paired were compared to the original GLMM (i.e. with unpaired treatments) using anova model comparison. Principal components produced using the coefficients calculated by elliptic fourier analysis of wattle shape were used in multivariate analyses of covariance (mancova), and the PCs were included as dependent variables.

Results

Concentrations of plasma antioxidants

The minimum adequate model of a GLMM with the concentration of carotenoids in plasma in males as the response variable included all explanatory variables entered into the initial model, with significant main effects of age and treatment and a significant interaction between age and treatment (Table 1, Fig. 2a,b). The concentration of carotenoids in the plasma decreased considerably with age from a mean across groups of 20·77 at 8 weeks to 1·99 μg mL−1 by 47 weeks of age, with the greatest declines shown by males in the treatment groups that received carotenoid supplements in the diet during the first 8 weeks of life (i.e. groups C and CV; Fig. 2b). GLMMs run as above but separated by age showed that males in groups C and CV had higher concentrations of plasma carotenoids at 8 weeks of age than males in the control and V groups (GLMM comparing treatment diets for males at 8 weeks of age: χ2 = 291·7, d.f. = 3108, P < 0·0001; Fig. 2b), but there were no differences between treatment groups in plasma carotenoid concentrations at 47 weeks (GLMM comparing treatments: χ2 = 0·68, d.f. = 3108, P = 0·88; Fig. 2b). Males did not differ from females in the concentrations of carotenoids circulating in plasma at 8 weeks of age (Table 1).

Table 1.   Results of glmm models of total antioxidant plasma concentrations and oxidative stress at 8 and 47 weeks of age
 χ2d.f. P-value
  1. MDA, malondialdehyde.

  2. *** < 0.001

Plasma concentration of carotenoids of males at 8 and 47 weeks of age
 Treatment × age134·93108<0·0001***
 Treatment58·723107<0·0001***
 Age346·981111<0·0001***
Plasma concentration of carotenoids at 8 weeks
 Treatment × sex5·7332130·13
 Treatment442·573216<0·0001***
 Sex0·9012160·34
Plasma concentration of α-tocopherol of males at 8 and 47 weeks of age
 Treatment × age64·183108<0·0001***
 Treatment54·463107<0·0001***
 Age321·331111<0·0001***
Plasma concentration of α-tocopherol at 8 weeks
 Treatment × sex3·0632130·08
 Treatment303·453216<0·0001***
 Sex4·1012160·25
Plasma concentration of MDA at 8 and 47 weeks
 Treatment/age134·93108<0·0001***
 Treatment58·723107<0·0001***
 Age346·981111<0·0001***
Figure 2.

 Mean plasma carotenoid concentrations (μg mL−1) in males in relation to treatment and age at (a) 8 weeks of age (b) 47 weeks of age (n = 112). Mean plasma α-tocopherol concentrations (μg mL−1) of males in relation to treatment and age at 8 (c) and 47 (d) weeks of age (n = 112). Mean plasma malondialdehyde concentrations (μg mL−1) of both males and females in relation to treatment and age at 8 (e) and 47 (f) weeks of age (n = 118). Error bars show 95% confidence intervals. Note that the scale on the y-axis differs at 8 weeks compared to 47 weeks of age. Results are presented on separate graphs because of scale differences. Means with the same letter do not differ significantly to one another using post hoc comparisons.

There were significant main effects of treatment and age and a significant interaction between age and diet treatment on concentrations of α-tocopherol in plasma (Table 1, Fig. 2c,d). Males in groups V and CV had higher concentrations of plasma α-tocopherols at 8 weeks of age than males in C and control groups (GLMM comparing treatment diets for males at 8 weeks of age: χ2 = 59·32, d.f. = 3108, P < 0·0001). Plasma concentrations of α-tocopherols in birds that received a diet supplemented with α-tocopherol up to 8 weeks of age (i.e. groups V and CV) remained significantly higher at 47 weeks than controls and birds supplemented with carotenoids only (Table 1, Fig. 2d). The concentration of α-tocopherols decreased between 8 and 47 weeks of age most in those birds that received α-tocopherols in their diet up to 8 weeks of age. Males did not differ from females in the concentrations of α-tocopherol circulating in plasma (Table 1).

Oxidative damage

The minimum adequate model of a GLMM with plasma MDA concentration as the response variable and age, treatment and sex as main effects included significant main effects of age and treatment and a significant interaction between treatment and age (Table 1). The mean concentration of plasma MDA across groups decreased from 5·07 μg mL−1 at 8 weeks to 2·28 μg mL−1 at 47 weeks of age (see Fig. 2e, f). GLMMs on data separated by age showed a significant main effect of treatment diet on concentration of MDA at 8 weeks of age (treatment: χ2 = 291·7, d.f. = 3216, P < 0·0001), with the birds given a control diet having a higher concentration of plasma MDA than birds given the other diets (Percentage difference between circulating MDA levels in the control group and other treatment groups: C = 23·4%, V = 20·4%, CV = 13·73%, Fig. 2e). However, by 47 weeks, there were no differences in plasma MDA concentrations among treatment groups (GLMM: χ2 = 5·59, d.f. = 3213, P = 0·13; Fig. 2f).

Growth

Repeated measures GLMs with mass at 0, 8, 21, 47 weeks as the response variable and sex, age and treatment group as explanatory variables showed that males grew faster than females and that there were significant differences between treatments in growth (Table 2). Post hoc analyses showed that birds supplemented with carotenoids and vitamin E (CV) grew faster from 21 to 47 weeks of age than those birds given only carotenoids only (C), vitamin E only or controls (Cont) (GLMM comparing CV with C: χ2 = 16·41, d.f. = 3212, P = 0·0005, CV with V: χ2 = 5·19, d.f. = 3212, P = 0·042, CV with Cont: χ2 = 16·32, d.f. = 3212, P = 0·0005) (Fig. 3).

Table 2.   Results of repeated measures glmm models of growth and glmm models of mass
 χ2d.f. P-value
  1. *< 0.05, **< 0.01,***< 0.001

Mass growth
 Treatment × sex × age0·3036550·96
 Treatment2·9132150·035*
 Sex247·701215<0·001**
 Age634·71662<0·001**
Tarsus length growth
 Treatment × sex × age0·836550·85
 Treatment2·2732150·082
 Sex820·671215<0·001**
 Age450·61662<0·001**
Head-to-bill length
 Treatment × sex × age1·5036550·67
 Treatment2·8832150·037*
 Sex30·391215<0·001**
 Age557·51662<0·001**
Wing Length
 Treatment × sex × age0·093655<0·99
 Treatment1·5632150·20
 Sex140·881215<0·001**
 Age566·551662<0·001**
Mass aged 1 day
 Treatment × sex8·4832100·37
 Treatment1·2732130·74
 Sex0·1512160·70
Mass at 8 weeks of age
 Treatment × sex1·0232100·80
 Treatment12·663213<0·01*
 Sex88·031213<0·001**
Mass at 21 weeks of age
 Treatment × sex5·3332100·15
 Treatment2·7632130·43
 Sex91·001216<0·001***
Mass at 47 weeks of age
 Treatment × sex2·0132100·57
 Treatment12·503213<0·01*
 Sex165·681213<0·001**
Figure 3.

 Mean mass growth of females (a) and males (b) in relation to treatment and age (8, 21 and 47 weeks, females n = 118, males n = 112). Error bars show 95% confidence intervals. Individuals receiving a diet supplemented with both carotenoids and vitamin E had a significantly greater mass (males and females) than all other treatment groups at 8 and 47 weeks of age.

As expected, there were no initial differences in the mass of chicks allocated to different dietary treatments (Table 2). However, by 8 weeks of age (i.e. the immediate end of the antioxidant supplementation period), males were heavier than females and there was a significant effect of treatment on mass (Table 2; Fig. 3). Birds receiving the combined carotenoid and α-tocopherol supplement (CV group) were heavier than those supplemented with carotenoids only (GLMM comparing C and CV: χ2 = 8·548, d.f. = 3212, P = 0·0035). At 21 weeks, at the time of immune response measurement, males remained heavier than females but there was no difference between diet treatment groups in mass (Table 2; Fig. 3). However, by 47 weeks of age, mass of birds varied according to the diet received during the first 8 weeks of life. Birds supplemented with carotenoids and α-tocopherol (CV) had a significantly higher mass than birds in other groups (GLMMs comparing carotenoid and vitamin E treatment to the other treatment diets: mass: χ2 = 12·50, d.f. = 3212, P = 0·009; Fig. 3).

Immune function

Immune response did not vary in relation to either sex or treatment. The minimum adequate model of a GLMM with wing patagium inflammation following immune challenge as the response variable and treatment and sex as main effects included just the intercept, with all other variables dropping out of the model (GLMM Treatment/Sex χ2 = 5·87, d.f. = 3213, P = 0·12, Sex: χ2 = 1·48, d.f. = 3216, P = 0·22, Treatment: χ2 = 1·58, d.f. = 3216, P = 0·66).

Secondary sexual signals

The expression of secondary sexual signals by males did not vary among treatment groups (Fig. 4). GLMMs with the sexual signal parameter as the response variable and age, head-to-bill length and treatment as explanatory variables in the initial models included only age in the minimum adequate model in all cases (Table 3). There was a trend towards a significant interaction between treatment and age in spur length between 21 and 46 weeks of age (Fig. 4b), with the mean growth (mm) of the spurs of birds that received vitamin E (i.e. CV and V males) higher than birds that received a control diet (CV = 3·08, V = 3·23, Cont = 1·63, C = 2·02), but the effect was not significant (Table 3). There was no difference in wattle size or brightness in relation to dietary treatment (Table 3). A manova of the 5 principal components that collectively described 75% of the shape variation calculated by EFA analysis indicated that there was also no difference in the shape of the wattles of males in relation to treatment (GLMM: = 0·66, d.f. = 3104, P = 0·82).

Figure 4.

 There were no significant differences in the expression of sexual signals at adulthood. Wattle Area at 47 weeks of age (a) Spur length at 21 and 47 weeks of age (dark grey = 21 weeks of age, light grey = 47 weeks of age (b) and wattle brightness at 47 weeks of age (c) with 95% confidence intervals (n = 112)).

Table 3.   Results of Glmm models of secondary sexual signals
 χ2d.f. P-value
Spur length
 Treatment × head-to-bill length0·1531070·96
 Treatment2·3831060·50
 Head-to-bill length2·5611090·11
Wattle Size
 Treatment × head-to-bill length3·2631070·35
 Treatment6·0231060·11
 Head-to-bill length1·8511090·17
Wattle shape
 Treatment0·6731060·93
 Wattle brightness
 Treatment0·1531060·93

Discussion

The results of this study provide evidence that male ring-necked pheasants preferentially allocate supplementary antioxidants to achieving a large size rather than to the expression of sexually selected traits. Moreover, there were synergistic combined effects of supplementation of carotenoids and α-tocopherol (vitamin E) on growth. Individuals that received a diet of carotenoids combined with vitamin E had a faster growth rate and reached a larger size than individuals given other treatment diets, including either carotenoids or vitamin E alone. We also found that supplementation with vitamin E over the early developmental period resulted in increased vitamin E plasma content at adulthood. In contrast, early supplementation with carotenoids on their own had no affect on carotenoid plasma concentrations at adulthood. These results provide evidence for an effective antioxidant role in vivo. Antioxidant availability affected oxidative damage at the end of the experimental period (8 weeks), with birds that received a control diet having higher levels of oxidative stress than individuals that had received a diet supplemented with antioxidants (either alone or in combination). However, antioxidant supplementation did not reduce oxidative damage at adulthood, and secondary sexual characteristics and immune function were not influenced by the availability of antioxidants during early growth and development.

In contrast with the prediction that supplementation with both non-pigmentary and pigmentary antioxidants would result in increased allocation to sexual ornaments, extra resources were instead allocated to growth. Those birds that were supplemented with a combination of carotenoids and vitamin E showed a greater synergistic growth response than those supplemented with either carotenoids or α-tocopherol (vitamin E) alone, demonstrating that there were synergistic benefits of supplementing non-pigmentary antioxidants along with carotenoids. Oxidized antioxidants can be recycled and regenerated to their active reduced form by reacting with other antioxidants (Catoni, Peters & Schaefer 2008). Studies have documented the regenerative properties of vitamin E and C on carotenoids (Mortensen, Skibsted & Truscott 2001; Amorati et al. 2002), and similarly vitamin E can be recycled by carotenoids (Surai 2002). Our results provide clear evidence for synergistic effects of dietary antioxidants on somatic development, but not the expression of sexual ornaments.

Individuals supplemented with a combination of carotenoids and vitamin E were significantly heavier at 8 weeks of age, and at adulthood (47 weeks) were heavier, had a longer tarsus length, head–bill length and wing length than birds in other groups. Investment of additional antioxidant resources towards increased growth rather than sexually selected traits may appear surprising unless there are longer-term benefits of achieving a large size, because increased rates of growth can result in higher levels of oxidative stress (Brown-Borg & Rakoczy 2003; Alonso-Alvarez et al. 2007). However, when resources are finite, competition may lead to selection favouring traits that increase competitive ability (Wolf, Harris & Royle 2008). This may explain why supplementary antioxidants were preferentially allocated to attaining a greater size rather than towards sexual ornaments. Significant competition for scarce resources would favour allocation to growth rather than self-maintenance to increase the competitive ability of an individual, thereby increasing their subsequent ability to acquire disproportionate amounts of further resources for both growth and self-maintenance (Wolf, Harris & Royle 2008). In contrast, allocating resources primarily to maintenance at the expense of growth may result in these individuals being out-competed for scarce resources by larger, more competitive individuals, reducing competitiveness and therefore the relative amount of resources subsequently acquired, in a negative feedback process (Hall et al. 2010). In ring-necked pheasants, attaining a larger body size could have beneficial downstream effects. Smith et al. (2007) found that pheasants in better body condition, measured as residual mass, showed increased wattle colour when carotenoid supplemented as adults. By maintaining a better body condition, it is possible that birds may be able to capitalize on environmental fluctuations in carotenoid availability as adults (Smith et al. 2007), and it also has a positive effect on dominance (Göransson et al. 1990; Grahn & von Schantz 1994). Allocating supplementary antioxidant resources to increasing body size and therefore dominance could lead to more reproductive benefits than allocating resources preferentially to sexually selected traits, by increasing the ability of a male to maintain control of territories and to acquire mating opportunities (Grahn & von Schantz 1994).

In contrast to treatment effects of antioxidant supplementation on growth, there were no detectable effects of supplementation on immune response. Although numerous previous studies have shown that carotenoids have immunoenhancing properties (Amar et al. 2000; Saks, Ots & Hõrak 2003; Chew & Park 2004; McGraw & Ardia 2005), we found no evidence for a greater pro-inflammatory potential following PHA injection at 21 weeks of age. This contrasts with responses to immune challenge during antioxidant supplementation of adult diets in many other species [e.g. zebra finches, Taenopygia guttata, (Blount et al. 2003b) guppies, Poecilia reticulata, (Grether et al. 2004), grey partridges, Perdix perdix, (Cucco et al. 2006)]. Hõrak et al. (2007) found no evidence for enhanced immune response to PHA injection in greenfinches following supplementation with vitamin E, but the basal diet of sunflower seeds provided to all control and supplemented birds during the experiment had a relatively high antioxidant content which may have masked the effects of antioxidant supplementation (Hõrak et al. 2007). During these studies, immune response was measured during or directly after antioxidant supplementation. In contrast, in this study, immune response was measured 13 weeks after antioxidant supplementation had ceased. Our results suggest that supplemented birds did not store antioxidants for later use following the costly growth period. Smith et al. (2007) found that pheasants in better body condition, measured as residual mass, had a higher pro-inflammatory immune response to PHA injection. In our experiment, there was no significant difference in mass between treatment groups at 21 weeks, so it is possible that had the immune challenge been carried out at 8 or 47 weeks of age, when there were mass differences between treatment groups, there may also have been differences in immune function among treatment groups. Alternatively, it may be that the basal diet supplied sufficient resources to maintain adequate immune function, allowing supplementary antioxidants to be available for allocation to other functions, such as growth.

Circulating antioxidants were increased at the end of the 8-week period of supplementation (both carotenoids and α-tocopherol), supporting the results of previous studies in a range of avian species (e.g. Alonso-Alvarez et al. 2004; Aguilera & Amat 2007). More particularly, supplementation with α-tocopherol during the first 8 weeks resulted in increased circulating α-tocopherol defences at adulthood, supporting previous work on zebra finches, Taeniopygia guttata, by Blount et al. (2003a). This suggests that availability of α-tocopherol during early development can have a long-term affect on the capacity of individuals to assimilate α-tocopherol in adulthood and indicates that the lipoproteins required to assimilate antioxidants are primarily produced early in life (Blount et al. 2003a). In contrast to the long-term effects of supplementation with α-tocopherol, there was no evidence that early exposure to dietary carotenoids had an effect on circulating levels of carotenoids in blood plasma at adulthood.

In addition to affecting blood plasma concentrations of antioxidants, supplementation also resulted in reduced oxidative damage in the form of lipid peroxidation during the supplementation period, as levels of malondialdehyde (a robust measure of oxidative damage; Monaghan, Metcalfe & Torres 2009) were significantly higher for control birds at 8 weeks of age. However, despite the higher levels of circulating α-tocopherol (vitamin E) in birds that received a supplement of vitamin E during early life when measured at 47 weeks of age, there was no difference in the levels of oxidative damage in relation to treatment group. In particular, compared to supplementation with carotenoids supplementation with vitamin E did not provide any benefits with respect to mitigating oxidative damage, despite the antioxidant role of carotenoids not generally being considered to be as effective in birds. For example, a recent fixed effect meta-analysis study found that carotenoids could account for as little as <0·002% of antioxidant activity in birds (Costantini & Møller 2008). However, this meta-analysis included studies carried out at all periods of the life cycle. In contrast, our results indicate that carotenoids can be effective antioxidants, especially in combination with other antioxidants as supplementation with a combination of vitamin E and carotenoids during early development resulted in increased size without elevated costs of oxidative damage.

Our study aimed to address the longer-term effects of early antioxidant supplementation on adult phenotype. However, it is possible that measurements over the first 47 weeks of life were still insufficient to be able to detect differences difference treatment groups. For example, spur length in ring-necked pheasants is known to be the most important predictor of harem size, but continues to grow throughout the second year of life (Göransson et al. 1990), and spur length at 1 year of age has less influence on female mate choice than the spur length of older males (Grahn & von Schantz 1994). We found that the relative spur length of individuals was not significantly enhanced in males given vitamin E (and/or carotenoids) during development. However, it is possible that beyond 1 year of age, significant differences in spur length could develop if there is continued growth. In addition, the effects of higher circulating vitamin E at 47 weeks found in birds supplemented with vitamin E during development on the accumulation of ROS beyond 47 weeks on the rapidity of ageing and longevity are also unknown. The greater antioxidant protection available at 47 weeks of age in individuals supplemented with vitamin E during early development could result in reduced costs of self-maintenance and therefore a higher availability of antioxidant resources for allocation to the subsequent development of sexual signals. Consequently, the lack of a difference between treatments in the expression of sexually selected traits may not be indicative of longer-term effects.

The results show that antioxidant availability during early development can have a substantial effect on adult phenotype, providing support for the role of oxidative stress as a unifying mechanism in life-history trade-offs (e.g. Costantini 2008), and a synergistic effect of non-pigmentary and pigmentary antioxidants supplementation on growth and development. However, there was no support for the ‘carotenoid protection theory’ as there were no synergistic effects of antioxidants on the expression of secondary sexual traits (Hartley & Kennedy 2004). Future studies should further explore the role of synergistic effects of ingesting and absorbing dietary antioxidants during development on the expression of adult phenotypes in other species as it is likely to provide considerable insights into the evolution of life-history traits among species.

Acknowledgements

We would like to thank Chris Davis (MRCVS) and Matt Ford at the GWCT. We would also like to thank field volunteers: James Connell, Matthew Cooke, Nia Denman, Marc Edwards and John Simper. This research was funded by a NERC studentship (NE/F007450/1) to JMO, supervised by NJR and JDB, in CASE partnership with the GWCT. JDB was funded by a Royal Society Research Fellowship.

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