Feeding dihydroquercetin and vitamin E to broiler chickens reared at standard and high ambient temperatures.

The use of natural antioxidants, in particular polyphenols such as dihydroquercetin (DHQ), in animal nutrition has recently increased in popularity. This may partly be due to the risk of increased incidences of heat stress associated with raising livestock in warmer ambient temperatures, facilitated by global warming, reducing antioxidant capacity. The current research demonstrates the effect of dietary DHQ, vitaminEand standard or high ambient temperatures on growth performance, energy and nutrient metabolism, gastrointestinal tract (GIT) development, jejunal villus morphometry and antioxidant status in broiler chickens. Each of the four experimental diets was fed to 16 pens of five birds, which were allocated to four rooms (four pens in each room). The temperature in two rooms was maintained at aconstant 35°C (high temperature; HT), and the temperature in the other two rooms was gradually reduced from 27°C at 7 dof age to 22°C at 20 dof age (standard temperature; ST). Rearing birds at HT reduced feed intake, weight gain, weight of small intestine, total GIT, liver, spleen, heart, villus height, villus surface area and lowered blood glutationperoxidase (GSH-Px). Dietary DHQ increased blood GSH-Px and total antioxidant status, increased heart weight and reduced caecal size. When fed separately, DHQ and vitamin E improved hepatic vitamin E concentration. Feeding vitamin Eincreased spleen and liver weights. When fed together, DHQ and vitamin Ereduced villus height, villus height to crypt depth ratio and villus surface area. Temperature and antioxidants did not affect energy and nutrient metabolism. There were no effects of dietary antioxidants on growth performance of broiler chickens and there were no mortalities. At present, it is unclear if feeding antioxidants (in particular DHQ) at different levels, using different dietary formulations, and rearing birds under arange of environmental conditions may be effective at enhancing production performance and bird health in hot ambient climates.

Liver Antioxidants in Relation to Beak Morphology, Gizzard Size and Diet in the Common Eider Somateria mollissima.

Antioxidants in the liver are particularly abundant in capital breeders that rely on stored resources for egg production. Capital breeders like eider (hereafter common eider) Somateria mollissima have disproportionately large livers with low levels of coenzyme Q10 when compared to other bird species. Concentrations of total carotenoids and vitamin E in the livers of eiders were smaller than predicted for similarly sized bird species. Eiders with high body condition estimated as body mass relative to skeletal body size had high levels of total carotenoids and low levels of coenzyme Q10. The concentration of total carotenoids per gram of liver increased with age, and vitamin E and total carotenoids accumulated during the winter onwards from February to peak at the start of incubation in April. Total vitamin E, total carotenoids, and coenzyme Q10 per gram of liver decreased with increasing beak volume. The size of the empty gizzard increased with increasing liver mass but decreased with total carotenoids and coenzyme Q10. The main components of the diet were blue mussels Mytilus edulis (40%), draft whelk Nassarius reticulatus (27%), and periwinkle Littorina littorea (10%). The concentration of vitamin E increased with the number of razor clams Ensis sp. and draft whelks in the gizzard and the concentration of total carotenoids increased with the number of beach crabs Carcinus maenas. These observations are consistent with the hypothesis that eiders are limited in their levels of antioxidants through food limitation. Furthermore, they imply that diet and morphological characters involved in food acquisition and processing are important determinants of the level of antioxidants in the liver.

Yolk vitamin E positively affects prenatal growth but not oxidative status in yellow-legged gull embryos.

Parental effects occur whenever the phenotype of parents or the environment that they experience influences the phenotype and fitness of their offspring. In birds, parental effects are often mediated by the size and biochemical quality of the eggs in terms of maternally transferred components. Exogenous antioxidants are key egg components that accomplish crucial physiological functions during early life. Among these, vitamin E plays a vital role during prenatal development when the intense metabolism accompanying rapid embryo growth results in overproduction of pro-oxidant molecules. Studies of captive birds have demonstrated the positive effect of vitamin E supplementation on diverse phenotypic traits of hatchling and adult individuals, but its effects on embryo phenotype has never been investigated neither in captivity nor under a natural selection regime. In the present study, we experimentally tested the effect of the in ovo supplementation of vitamin E on morphological traits and oxidative status of yellow-legged gull (Larus michahellis) embryos. The supplementation of vitamin E promoted somatic growth in embryos soon before hatching, but did not affect their oxidative status. Our results suggest that maternally transferred vitamin E concentrations are optimized to prevent imbalances of oxidative status and the consequent raise of oxidative damage in yellow-legged gull embryos during prenatal development.

A comparison of fat-soluble antioxidants in wild and farm-reared chukar partridges (Alectoris chukar).

This study assessed differences in antioxidant (carotenoid, retinol, retinol-ester, vitamin E and coenzyme Q10) composition of egg yolk and tissue in chukar partridges (Alectoris chukar) newly hatched from eggs of birds maintained in captivity on commercial maize-soybean based diets and birds from the wild whose diet was obtained from the natural environment. All eggs were incubated in a commercial hatchery. Day-old chicks from both groups were sacrificed and dissected for antioxidant analysis. Fat soluble antioxidant concentrations of egg yolk and tissues were determined by HPLC. Total carotenoids, retinol, alpha-tocopherol, and total vitamin E concentration of wild egg yolks were significantly higher compared to yolks from farm-reared birds (p<0.05). However, gamma tocopherol, and coenzyme Q10 were not significantly different in the yolks of either wild or farmed birds (p>0.05). The concentration of total carotenoids in all tissues of wild chukar one-day old partridges was significantly higher than in farmed one-day old chukar partridge tissues (p<0.05). Alpha tocopherol, free-retinol, retinol-esters and total vitamin A were significantly higher in most tissues of wild chukar when compared to farmed chicks (p<0.05). Coenzyme Q10 concentrations of heart, kidney and brain tissues of farm-reared chukar day old chicks were significantly higher than tissues from wild birds, although leg and breast tissues of wild chicks were significantly higher than in farmed birds (p<0.05). These findings suggest that maternal access to antioxidants in the diet of farmed chukar partridges could positively influence fat soluble antioxidant concentrations in the egg yolk and tissues of day old chicks.

Antioxidants and embryo phenotype: is there experimental evidence for strong integration of the antioxidant system?

Organisms have evolved complex defense systems against oxidative stress. Bird eggs contain maternally derived antioxidants that protect embryos from oxidative damage. The antioxidant system components are thought to be integrated, but few studies have analyzed the covariation between antioxidant concentrations, embryo 'oxidative status' and morphology. In addition, no study has tested the effects of experimental change in yolk antioxidant concentration on other antioxidants, on their reciprocal relationships and on their relationships with embryo oxidative status or growth, which are expected if antioxidants defenses are integrated. In yellow-legged gull (Larus michahellis) embryos, we analyzed the covariation between several antioxidants, markers of 'oxidative status' [total antioxidant capacity (TAC), concentration of pro-oxidants (TOS), lipid peroxidation (LPO) and protein carbonylation (PC)] in the yolk, liver and brain, and morphology. Yolk and liver antioxidant concentrations were positively correlated reciprocally and with embryo size, and positively predicted TAC but not oxidative status. TOS and LPO were positively correlated in the liver, while TAC and LPO were negatively correlated in the brain. Weak relationships existed between antioxidants and TOS, PC and LPO. The effects of antioxidants on oxidative status and morphology were non-synergistic. An experimental physiological increase in yolk vitamin E had very weak effects on the relationships between other antioxidants or oxidative status and vitamin E concentration, the concentration of other antioxidants or oxidative status; the covariation between other antioxidants and oxidative status, and relationships between morphology or oxidative status and other antioxidants, challenging the common wisdom of strong functional relationships among antioxidants, at least for embryos in the wild.

Antioxidant systems in chick embryo development. Part 1. Vitamin E, carotenoids and selenium.

Chick viability is known to be an important factor determining profitability of the poultry industry. Chick embryo tissues contain a high proportion of highly polyunsaturated fatty acids in the lipid fraction and therefore need antioxidant defence. The antioxidant system of the developing embryo and newly hatched chick includes the antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase), water-soluble antioxidants (ascorbic acid, taurine, carnitine, glutathione, etc.), fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q) as well as selenium (Se). In fact, the high levels of endogenous antioxidants within the egg and embryonic tissues can clearly serve as a major adaptive mechanism for the protection of the tissue during the oxidative stress experienced at hatching. It has been shown that among different nutrients in the maternal diet which could significantly affect chick embryo development and their viability in the early posthatch life, natural antioxidants have been suggested to play a central role. Our data indicate that increased supplementation of the maternal diet can substantially increase concentrations of vitamin E, carotenoids (especially canthaxanthin) and Se in developing chick tissues and significantly decrease susceptibility to lipid peroxidation being effective nutritional tools to deal with various commercial stresses in poultry production.

Is oxidative status influenced by dietary carotenoid and physical activity after moult in the great tit (Parus major)?

In the context of sexual and natural selection, an allocation trade-off for carotenoid pigments may exist because of their obligate dietary origin and their role both in the antioxidant and immune systems and in the production of coloured signals in various taxa, particularly birds. When birds have expended large amounts of carotenoids to feather growth such as after autumn moult, bird health and oxidative status might be more constrained. We tested this hypothesis in a bird species with carotenoid-based plumage colour, by manipulating dietary carotenoids and physical activity, which can decrease antioxidant capacity and increase reactive oxygen metabolite (ROM) concentration. Great tits were captured after moult and kept in aviaries, under three treatments: physical handicap and dietary supplementation with carotenoids, physical handicap and control diet, and no handicap and control diet. We measured plasma composition (antioxidant capacity, ROM concentration, and vitamin A, vitamin E and total carotenoid concentrations), immune system activation (blood sedimentation) and stress response (heterophil/lymphocyte ratio) and predicted that handicap treatment should influence these negatively and carotenoid supplementation positively. Coloration of yellow feathers was also measured. Carotenoid supplementation increased total plasma carotenoid concentration, decreased feather carotenoid chroma and marginally increased ROM concentration. Handicap increased blood sedimentation only in males but had no clear influence on oxidative stress, which contradicted previous studies. Further studies are needed to investigate how physical activity and carotenoid availability might interact and influence oxidative stress outside the moult period, and their combined potential influence on attractiveness and reproductive investment later during the breeding season.

Foraging for carotenoids: do colorful male hihi target carotenoid-rich foods in the wild?

Dietary access to carotenoids is expected to determine the strength of carotenoid-based signal expression and potentially to maintain signal honesty. Species that display carotenoid-based yellow, orange, or red plumage are therefore expected to forage selectively for carotenoid-rich foods when they are depositing these pigments during molt, but whether they actually do so is unknown. We set out to address this in the hihi (Notiomystis cincta), a New Zealand passerine where males, but not females, display yellow carotenoid-based plumage. We measured circulating carotenoid concentrations in male and female hihi during breeding and molt, determined the nutritional content of common foods in the hihi diet, and conducted feeding observations of male and female hihi during molt. We found that although male and female hihi do not differ significantly in plasma carotenoid concentration, male hihi have a greater proportion of carotenoid-rich foods in their diet than do females. This is a consequence of a greater fruit and lower invertebrate intake than females and an avoidance of low-carotenoid content fruit. By combining behavioral observations with quantification of circulating carotenoids, we present evidence that colorful birds forage to maximize carotenoid intake, a conclusion we would not have drawn had we examined plasma carotenoids alone.

A window on the past: male ornamental plumage reveals the quality of their early-life environment.

It is well established that the expression of many ornamental traits is dependent on the current condition of the bearer. However, conditions experienced in early life are also known to be important for an individual's subsequent fitness and therefore, directly or indirectly, for the fitness of their mate. Specifically, a recent hypothesis suggests that sexually selected traits might be sensitive to conditions experienced during early-life development and thereby function as honest indicators of developmental history. Whether this applies to colourful male plumage, however, is largely unknown. We tested this idea with a field experiment by manipulating neonatal nutrition in a sexually dichromatic passerine, the hihi (Notymystis cincta). We found that carotenoid supplementation increased nestling plasma carotenoid concentration, which was in turn correlated with increased yellow saturation in male breeding plumage after moulting. We also found that the post-moult luminance (lightness) of the white ear-tufts tended to be reduced in males that had received an all-round nutritional supplement as nestlings. Black breeding plumage was not affected by neonatal nutritional treatment. Although the mechanisms that generate colourful plumage are evidently diverse, our results show that at least some parts of this display are accurate indicators of environmental conditions during development.

Maternal effects in the highly communal sociable weaver may exacerbate brood reduction and prepare offspring for a competitive social environment.

Maternal effects can influence offspring phenotype with short- and long-term consequences. Yet, how the social environment may influence egg composition is not well understood. Here, we investigate how laying order and social environment predict maternal effects in the sociable weaver, Philetairus socius, a species that lives in massive communal nests which may be occupied by only a few to 100+ individuals in a single nest. This range of social environments is associated with variation in a number of phenotypic and life-history traits. We investigate whether maternal effects are adjusted accordingly. We found no evidence for the prediction that females might benefit from modifying brood hierarchies through an increased deposition of androgens with laying order. Instead, females appear to exacerbate brood reduction by decreasing the costly production of yolk mass and antioxidants with laying order. Additionally, we found that this effect did not depend on colony size. Finally, in accordance with an expected increased intensity of environmental stress with increasing colony size, we found that yolk androgen concentration increased with colony size. This result suggests that females may enhance the competitive ability of offspring raised in larger colonies, possibly preparing the offspring for a competitive social environment.

High levels of liver antioxidants are associated with life-history strategies characteristic of slow growth and high survival rates in birds.

Antioxidants have a large potential to coevolve with life-histories because of their capacity to counteract the negative effects of free radicals on fitness. However, only a few studies have explored the association between antioxidant levels and life-history strategies comparing a large number of species. Here we used an extensive dataset of 125 species of birds to investigate the association between concentrations of antioxidants (carotenoids and vitamin E) in the liver, which is the main storage organ for fat-soluble antioxidants, and life-history and morphology. We found that high liver antioxidant concentrations were associated with life-history strategies characterized by "live slow, die old", in clear contrast to previous studies reporting a relationship between high plasma antioxidants and life-histories characterized by "live fast, die young". Thus, high carotenoid concentrations were present in species with large body, brain and egg sizes, high absolute metabolic rate and a resident lifestyle, while high vitamin E concentrations were present in species with large brain size and long life span and incubation period. Our results indicate that antioxidants and life-histories coevolve, and that this may be mediated by positive fitness consequences of the accumulation of liver antioxidants, as species with higher antioxidant levels live longer.

Maternal effects mediated by egg quality in the Yellow-legged Gull Larus michahellis in relation to laying order and embryo sex.

BACKGROUND: Maternal effects mediated by egg size and quality may profoundly affect offspring development and performance, and mothers may adjust egg traits according to environmental or social influences. In avian species, context-dependency of maternal effects may result in variation in egg composition, as well as in differential patterns of covariation among selected egg components, according to, for example, position in the laying sequence or offspring sex. We investigated variation in major classes of egg yolk components (carotenoids, vitamins and steroid hormones) in relation to egg size, position in the laying sequence and embryo sex in clutches of the Yellow-legged Gull (Larus michahellis). We also investigated their covariation, to highlight mutual adjustments, maternal constraints or trade-offs in egg allocation. RESULTS: Laying sequence-specific patterns of allocation emerged: concentration of carotenoids and vitamin E decreased, while concentrations of androgens increased. Vitamin A, estradiol and corticosterone did not show any change. There was no evidence of sex-specific allocation or covariation of yolk components. Concentrations of carotenoids and vitamins were positively correlated. Egg mass decreased along the laying sequence, and this decrease was negatively correlated with the mean concentrations of carotenoids in clutches, suggesting that nutritionally constrained females lay low quality clutches in terms of carotenoid content. Finally, clutches with smaller decline in antioxidants between first- and last-laid eggs had a larger increase in yolk corticosterone, suggesting that a smaller antioxidant depletion along the laying sequence may entail a cost for laying females in terms of increased stress levels. CONCLUSIONS: Since some of the analyzed yolk components (e.g. testosterone and lutein) are known to exert sex-specific phenotypic effects on the progeny in this species, the lack of sex-specific egg allocation by mothers may either result from trade-offs between contrasting effects of different egg components on male and female offspring, or indicate that sex-specific traits are controlled primarily by mechanisms of sexual differentiation, including endogenous hormone production or metabolism of exogenous antioxidants, during embryonic development.

Changes in broiler chick tissue concentrations of lipid-soluble antioxidants immediately post-hatch.

The antioxidant protection of the chicken (Gallus gallus) embryo during incubation and early postnatal development plays an important role in chick viability. To assess the antioxidant capacity of the newly hatched chick, we determined the concentrations of vitamin A, vitamin E, carotenoids and coenzyme Q10 in the major tissues of chicks which had been held in an incubator for up to 36 h post-hatch. Concentrations of total carotenoids and free retinol and retinol esters in the tissues did not differ significantly over the 36 h period post-hatch (p>0.05). In contrast concentrations of vitamin E (α-tocopherol, γ-tocopherol, and α-tocotrienol and γ-tocotrienol) in various tissues (liver, heart, brain and leg muscle) decreased significantly in chicks that had been held in the incubator for 36 h when compared to younger chicks that were held for up to 18 h. Comparatively high concentrations of coenzyme Q10 were detected in the yolk sac membrane, liver and heart, the concentrations being dependent on age of chicks, the highest value being recorded 18 h post-hatch. In most of the tissues studied, coenzyme Q10 concentrations decreased substantially between 18 and 36 h post-hatch. This study demonstrated that there are tissue-specific changes in the concentrations of the major antioxidants (vitamin E and coenzyme Q10) during the 36 h post-hatch.

Relationship between HbA1c and coronary flow rate in patients with type 2 diabetes mellitus and angiographically normal coronary arteries.

OBJECTIVES: We examined the relationship between glycosylated hemoglobin (HbA1c) level and coronary flow rate in patients with type 2 diabetes mellitus (DM) and angiographically normal coronary arteries. STUDY DESIGN: The study included 54 consecutive patients (36 males, 18 females; age range 37 to 72 years) with type 2 DM, whose coronary arteries were found normal on coronary angiography. All patients underwent echocardiography and plasma HbA1c levels were measured before coronary angiography. To determine slow coronary flow (SCF), coronary flow rates of the left anterior descending (LAD), circumflex (Cx), and right coronary (RCA) arteries were assessed using the TIMI frame count (TFC) method. RESULTS: None of the patients had echocardiographic abnormalities. The mean HbA1c level was 7.4±2.0%, and the mean TFCs were 34.3±6.5, 22.4±3.5, and 20.4±2.2 for the LAD, Cx, and RCA, respectively. HbA1c levels were <7% in 26 patients, and ≥7% in 28 patients. Thirty-eight patients (70.4%) were found to have SCF in at least one coronary artery. TIMI frame counts of all three coronary arteries were significantly greater in patients in whom HbA1c was ≥7% (p<0.001). TIMI frame counts showed significant correlations with the HbA1c level (LAD: r=0.782; Cx: r=0.707; RCA: r=0.515; p<0.001 for all). The mean HbA1c level was significantly higher in patients with SCF compared to patients without SCF (7.8±1.9% vs. 5.6±0.9%; p<0.001). The incidence of SCF was significantly greater in patients with HbA1c ≥7.0% than those with HbA1c <7.0% (96.4% vs. 61.5%; p=0.004). Increased HbA1c (≥7%) significantly increased the risk for SCF in at least one coronary artery (OR=16.875; 95% CI 1.972-144.38). CONCLUSION: Our findings suggest that there is a strong correlation between the HbA1c level and coronary flow rate.

Levels of antioxidants in rural and urban birds and their consequences.

Numerous animals have successfully invaded urban habitats, although the factors associated with invasion success remain poorly understood. Urban areas are characterized by warmer microclimates, higher levels of primary productivity, longer breeding seasons and higher levels of pollutants. All these factors should cause oxidative stress, favoring invasion by species that have access to high levels of antioxidants. We analyzed concentrations of two categories of dietary, fat-soluble antioxidants (total carotenoids, total vitamin E) in the liver, the main storage organ in birds. Individuals killed by cats had lower levels of vitamin E than individuals that died for other reasons, showing natural selection on stored antioxidants. Bird species that had successfully colonized urban areas had significantly higher levels of vitamin E and total carotenoids than species that did not succeed, and rural populations had higher concentrations of vitamin E and total carotenoids than urban populations of the same species. Interspecific differences in concentrations of fat-soluble antioxidants, and differences between rural and urban populations of the same species, were accounted for by diet, but also by time since urbanization and number of generations since urbanization. These findings suggest that antioxidants, and by implication the ability to cope with oxidative stress, have contributed to successful invasion of urban areas by birds, and that the concentration of these antioxidants has changed in response to the urban environment.

Maternal effects mediated by antioxidants and the evolution of carotenoid-based signals in birds.

Bright yellow to red signals used in mate choice or intrasexual competition are based on carotenoid pigments that are hypothesized to be traded between physiological functions and coloration. These signals have recently been shown to be influenced by maternal effects. Indeed, yolk-derived carotenoids are essential for embryos to develop efficient carotenoid metabolism in posthatching life. Maternal effects facilitate adaptation to environmental variability and influence the evolution of phenotypic traits such as secondary sexual signals. Here we propose that maternal investment in yolk carotenoids promotes the evolution of carotenoid-based ornaments. We conducted a comparative analysis of lipid-soluble antioxidants (carotenoids and vitamins A and E) in the eggs of 112 species of bird. Species with large clutch sizes deposited higher yolk concentrations of the three antioxidants. There was a significant positive relationship between yolk carotenoids and the expression of male carotenoid-based signals, but not between yolk carotenoids and sexual dichromatism in these signals. These relationships were specific to carotenoids, as they were not found for vitamins A and E. This provides evidence consistent with the hypothesis that maternal effects mediated by yolk carotenoids play a role in the evolution of carotenoid-based signals as a response to sexual selection, likely based on organizational effects of carotenoids during embryo development.

Maternally invested carotenoids compensate costly ectoparasitism in the hihi.

Dietary ingested carotenoid biomolecules have been linked to both improved health and immunity in nestling birds. Here, we test whether maternally invested egg carotenoids can offset the cost of parasitism in developing nestling hihi (Notiomystis cincta) from the bloodsucking mite (Ornithonyssus bursa). Our results reveal clear negative effects of parasitism on nestlings, and that maternally derived carotenoids compensate this cost, resulting in growth parameters and ultimate mass achieved being similar to nonparasitized young. Our results offer an unique example of a direct positive relationship between enhanced maternal investment of carotenoids and an ability to cope with a specific and costly parasite in young birds. As O. bursa infestations reduce population viability in hihi, our findings also highlight the importance of key nutritional resources for endangered bird populations to better cope with common parasite infestations.

Comparative effects of levosimendan and dobutamine on right ventricular function in patients with biventricular heart failure.

Severe heart failure represents a major source of morbidity and mortality. Poor right ventricular function is an independent prognostic marker for mortality in patients with chronic heart failure. In this study, levosimendan (L) and dobutamine (D) in patients with severe chronic biventricular failure were compared. Forty consecutive patients, who were judged for inotropic therapy by their primary physicians, with acutely decompensated systolic heart failure and having moderate-to-severe right ventricular dysfunction with right ventricular fractional area change of

Initial syncope associated with alternating attacks of supraventricular tachycardia and atrioventricular block long after surgical correction of tricuspid atresia.

The Fontan procedure has become a generic term to define a surgical procedure that orients the systemic venous return directly to the pulmonary arteries, and has been used as a palliative operation for pulmonary atresia, tricuspid atresia, double inlet ventricle and complex single ventricle. The earliest type of Fontan procedure was a simple atriopulmonary anastomosis between the right atrium and the pulmonary artery. Atrial arrhythmias, particularly atrial flutter and sinus node dysfunction may occur in the early and late postoperative period after simple atriopulmonary anastomosis. The case presented here represents a much delayed occurrence of an initial syncope due to alternating attacks of SVT (supraventricular tachycardia) and second degree heart block on admission 21 years after simple atriopulmonary anastomosis performed for the correction of tricuspid atresia.

Condition dependence of nestling mouth colour and the effect of supplementing carotenoids on parental behaviour in the hihi (Notiomystis cincta).

Carotenoids are integument pigments that often reflect foraging efficiency, disease resistance and body condition. In contrast to the widespread attention this relationship has received in adult birds, the condition dependence of nestling colouration remains an understudied component of animal communication. Here we assess the condition dependence of carotenoid pigmentation in nestling hihi (Notiomystis cincta, an endangered New Zealand bird) and examine the influence of carotenoid supplementation on nestling quality and parental visitation rates. Our results show that carotenoids provided to breeding adult hihi were transferred to their offspring and resulted in an intensified orange-yellow flange colour. After accounting for carotenoid supplementation the parameter that most consistently explained variation in nestling flange colour was nestling tarsus length at 23 days, indicating condition dependence of this trait. We did not, however, detect direct effects of carotenoid supplementation on nestling mass or immune response (or any other fitness parameter measured). Carotenoid supplementation did, however, result in an increased paternal provisioning rate.