Yolk Carotenoid Concentrations Research Articles

Applied Research Note: “The impact of orange corn in laying hen diets on yolk pigmentation and xanthophyll carotenoid concentrations on a percent inclusion rate basis”

Consumers associate nutritional quality and potential health benefits with yolks showing deeper colors produced by healthy layers fed a well-balanced, nutrient-rich diet. However, cereals and oilseed meals generally used in poultry feed do not provide sufficient carotenoids to achieve deeper yolk colors, thus carotenoid additives may be supplemented. High carotenoid corn genotypes have been developed, and when included in poultry diets increase carotenoid concentrations in yolks. Seven different diets were fed to 210 Novogen White laying hens over 21 d. Each diet had 5 replicates of 6 hens housed in conventional cages. The only difference in diet treatments was percent inclusion rate of orange corn or inclusion of a carotenoid additive (Oro Glo). Yolk color was assessed by colorimeter and DSM YolkFan. Yolk carotenoid concentrations were determined by high performance liquid chromatography. Yolks from hens fed diets with a 100% orange corn inclusion rate had the highest YolkFan scores and total xanthophyll levels at the end of the study. Orange corn inclusion rate showed a linear relationship with colorimeter a* values (r = 0.96) and YolkFan scores (r = 0.90) for egg yolks. Orange corn inclusion of 50 and 75% produced yolks with a* axis color value equivalent to that found in yolks from hens fed 100% yellow corn plus 35 g/kg of Oro Glo. Data indicates that orange corn can be a suitable replacement for carotenoid additives. A 50 to 75% orange corn inclusion could be sufficient to achieve yolk colors similar to diets that involve supplementation with a carotenoid additive.

Effects of a probiotic containing Bacillus subtilis on the gut microflora, yolk quality and blood lipid concentrations of laying Pharaon quails

The current literature is not comprehensive concerning the influence of Bacillus subtilis on lipid metabolism, egg chemical characteristics, and intestinal microbiota of Japanese quail. The aim of this study was to evaluate the effects of a Bacillus subtilis strain on yolk quality, gut bacterial populations, and total cholesterol and triglyceride concentrations in the plasma of organically farmed Pharaon quail layers. Forty-five-day-old female quails were randomly distributed into four groups of 10 birds each: a no-treatment control group and three test groups receiving powder of Bacillus subtilis DSM 32424 at a minimum rate of 1 × 106 colony forming units per gram, dissolved in drinking water at doses of 50, 75 and 100 mg per kg body weight, daily during 30 days. The use of the probiotic at doses of 50 and 100 mg resulted in significantly reduced faecal staphylococci count on day 30 (P<0.05), while no significant changes were detected in lactobacilli or coliform bacteria. This study gives the first evidence of the influence of Bacillus subtilis on acid value and carotenoid levels in egg yolks laid by quails. Carotenoids were significantly elevated in the 75 mg group on day 30 (P<0.05), but significantly decreased in the 50 and 100 mg groups (P<0.05). Acidity, triglyceride and total cholesterol concentrations in quail serum were not affected significantly by the treatment throughout the study, though their values were reduced. The inclusion of Bacillus subtilis DSM 32424 in drinking water for laying Pharaon quails inhibited faecal staphylococci proliferation and enhanced yolk carotenoid content. Therefore, it can be proposed that including this probiotic in laying quail diets may have beneficial outcomes for both layers and the second generation.

Non‐analytical methods for the estimation of total yolk carotenoids in passerine eggs

Carotenoids are essential antioxidant micronutrients. Oviparous species acquire carotenoids from their food and deposit them in the egg yolk, where they support embryonic development. The total carotenoid concentration in the egg yolk is typically measured analytically, which requires time, equipment and expertise, and can limit the sample available for other measurements, at least in species laying small eggs. Here we evaluate whether yolk colour can be used as a reliable alternative measure for total yolk carotenoid concentration. We compare two non‐analytical methods, digital photography and visual colour scoring, using eggs from a wild population of Blue Tits Cyanistes caeruleus. Yolk hue estimated from digital photographs correlated more strongly with total yolk carotenoid concentration measured by high‐performance liquid chromatography (HPLC) than did the visually assessed colour score based on a Yolk Colour Fan. Previous results based on HPLC measurements of total carotenoid concentration could be reproduced using yolk hue measurements. The results suggest that measuring yolk hue is a suitable proxy for assessing natural variation in total yolk carotenoid concentration in eggs of free‐living birds.

Poultry diets containing (keto)carotenoid-enriched maize improve egg yolk color and maintain quality

The raw materials used in poultry feed do not provide sufficient carotenoids to achieve the yolk pigmentation demanded by consumers, so color additives are included usually in the form of carotenoid-rich dried plant extracts. We fed thirty-two laying hens (ISA Brown) on diets supplemented with two genetically engineered maize lines (enriched in carotenoids or ketocarotenoids) to assess egg productivity and quality traits. Four different maize-supplemented diets were compared based on two genetically engineered maize lines (Ph3 and BKT), the white near isogenic wild-type line (M37W) and a commercial feed based on standard commercial (SC) yellow maize. Line M37W-Ph3 (Ph3) accumulates high levels of β-carotene, zeaxanthin and lutein, whereas line NSL76*BKT (BKT) is rich in astaxanthin and other ketocarotenoids. There were no significant differences among the diet groups in terms of egg weight, breaking strength, albumen height, Haugh units, or shell thickness, but there were significant differences in DSM scale values for yolk pigmentation (P < 0.001) that were higher in the Ph3 (10.08) and BKT (11.38) groups than the SC (4.22) and M37W (≤1) groups. At the end of the trial, the total yolk carotenoid content was highest in the Ph3 diet group, followed by BKT, SC and M37W diet groups (56.5, 26.18, 11.54 and 1.81 μg/g freeze-dried yolk, respectively). The Ph3 yolks were enriched in β-carotene and β-cryptoxanthin (pro-vitamin A carotenoids) whereas the BKT yolks accumulated astaxanthin and other ketocarotenoids. The diets had no detrimental effects on the health of the hens. We conclude that (keto)carotenoid-enriched maize enhances the (keto)carotenoid content and color of yolks and can replace the color additives used routinely in poultry feed formulations.

Alfalfa meal as a source of carotenoids in combination with ascorbic acid in the diet of laying hens

The purpose of the experiment was to determine how the addition of dehydrated alfalfa (DA) and ascorbic acid (AA) into the diet of hens influences the performance characteristics and egg quality characterised by physical parameters, yolk carotenoid content, and the oxidative stability of yolks. Two hundred and forty Hisex Brown hens were divided into 6 treatment groups according to the level of DA (0 and 40 g/kg) and concentration of AA (0, 100, and 200 mg/kg) in the diet. Hen-day egg production and egg mass production were reduced by the supplement of DA (P &amp;lt; 0.001) and AA (P = 0.017). The addition of DA itself increased the feed intake per egg (P &amp;lt; 0.001) and the feed conversion ratio (P &amp;lt; 0.001). The heaviest eggs (P &amp;lt; 0.001) were laid by hens fed a diet with 100 mg/kg of AA (67.6 g). The AA supplement improved egg content quality, i.e., Haugh units (P &amp;lt; 0.001), albumen index (P &amp;lt; 0.001), and yolk index (P &amp;lt; 0.001), whereas the addition of DA increased values of yolk colour (P &amp;lt; 0.001) and had a negative effect on shell quality. The interaction of DA and AA was detected in the zeaxanthin concentration in the yolks (P = 0.002). The highest concentration was found in eggs from hens fed a diet with a DA supplement and 100 or 200 mg/kg of AA (17.33 or 17.25 mg/kg dry matter of yolk). In addition, 40 g/kg of DA significantly increased the concentration of other carotenoids, lutein (P &amp;lt; 0.001) and β-carotene (P &amp;lt; 0.001) in the yolks and increased the oxidative stability of fresh eggs (P &amp;lt; 0.001). The results suggest that adding DA in the amount of 40 g/kg to the hen diet provides a good source of carotenoids and, in combination with AA, a better deposition of carotenoids in egg yolks. On the other hand, DA deteriorates performance and shell quality.

Eggshell biliverdin concentration does not sufficiently predict eggshell coloration

Avian eggshell coloration may have arisen due to selection on the biological, chemical, or physical properties of the pigments embedded within the eggshell, or due to selection on the coloration that emerges due to pigment deposition. Within both hypothetical frameworks, pigment‐based eggshell coloration would be related to metrics of egg quality; however, no one has evaluated the relative strength of coloration and pigment concentration in predicting egg quality. Here, we examined 66 European starling Sturnus vulgaris eggs and quantified eggshell biliverdin concentration (an antioxidant that produces the eggshell's blue coloration) and used 28 different coloration metrics derived from both photographic and spectrophotometric data. We also measured egg size, eggshell thickness, concentration of carotenoids in the yolk, and concentration of lysozyme in the albumen to capture variation in egg quality. We found that throughout the laying sequence, biliverdin concentration increased while eggshell thickness, yolk carotenoid concentration, and lysozyme concentration in the albumen all decreased, but this variation was not captured by any eggshell coloration metric. Both eggshell coloration and biliverdin concentration were negatively associated with yolk carotenoid concentration, but only eggshell biliverdin concentration was negatively associated with yolk mass. Lastly, biliverdin concentration explained, at most, only 46% of the variation for all eggshell coloration metrics. Our results suggest biliverdin concentration is a better predictor of egg quality than egg coloration in European starlings, supporting the hypothesis that eggshell pigment concentration per se may be the target of selection.

Lutein, zeaxanthin and meso-zeaxanthin content of eggs laid by hens supplemented with free and esterified xanthophylls.

The xanthophyll carotenoids lutein (L), zeaxanthin (Z) and meso-zeaxanthin (MZ) are found at the macula, the central part of the retina, where they are referred to as macular pigment (MP). MP is studied in human subjects because of its proven role in enhancing visual function and its putative role in protecting against age-related macular degeneration. These benefits are probably due to the antioxidant and short-wavelength filtering properties of MP. It is known that eggs are a dietary source of L and Z. This experiment was designed to measure the egg yolk carotenoid response to hen supplementation with L, Z and MZ. A total of forty hens were used in the trial and were divided into eight groups of five hens. Each group was supplemented (with about 140mg active xanthophylls/kg feed) with one of the following oil-based carotenoid formulations for 6 weeks: unesterified L (group 1); L diacetate (group 2); unesterified Z (group 3); Z diacetate (group 4); unesterified MZ (group 5); MZ diacetate (group 6); L-MZ (1:1) diacetate mixture (group 7); L-MZ diacetate (1:3) mixture (group 8). Yolk carotenoid content was analysed weekly (in four randomly selected eggs) by HPLC. We found that hens supplemented with Z diacetate and MZ diacetate produced eggs with significantly greater carotenoid concentrations than their free form counterparts. This finding potentially represents the development of a novel food, suitable to increase MP and its constituent carotenoids in serum.

A Comparison of Natural and Restored Wetlands as Breeding Bird Habitat Using a Novel Yolk Carotenoid Approach

Restored wetlands are commonly assessed as avian breeding habitat using surveys, but data on reproductive parameters are needed to better understand how restoration affects wetland-dependent bird populations. A novel way to assess differences in breeding habitat quality is to compare the maternal allocation of carotenoids (i.e., biologically active yellow, orange and red pigments) to egg yolks, which is both diet and habitat dependent. We compared yolk carotenoid concentrations of red-winged (Agelaius phoeniceus) and yellow-headed blackbirds (Xanthocephalus xanthocephalus) that bred in natural, recently restored (i.e., restored ≤ 6 yrs ago) and restored (i.e., restored 9–16 yrs ago) wetlands to determine if restored wetlands provided critical reproductive resources at levels similar to natural sites. We also measured emerging insect abundances, water chemistry, and landscape-level parameters for study wetlands and related these variables to yolk carotenoid concentrations. Moreover, to understand the importance of damselflies to the diet of blackbirds, we measured the carotenoid content of damselflies and compared this to the yolk carotenoid content found in blackbird eggs. Bird abundances were also measured at each wetland to compare the traditional method of assessing breeding habitat to our novel yolk-carotenoid approach. In 2008, red-winged blackbirds in natural wetlands had higher carotenoid concentrations than birds in recently restored wetlands, suggesting that natural wetlands provided better breeding habitats. In 2009 there was a severe drought and we found contrasting results, with red-winged blackbirds in restored wetlands having higher carotenoid concentrations. Adult damselflies contained five carotenoids commonly found in avian tissues, suggesting they are likely an important source of carotenoids for wetland-breeding blackbirds. In contrast to yolk carotenoids, neither avian species richness nor abundance was significantly related to wetland type. Based on model selection results for water chemistry parameters, variation in concentrations of yolk carotenoids in red-winged blackbirds was best explained by conductivity, whereas variation in avian abundance was explained by total nitrogen, and avian species richness by chlorophyll-a concentration. Model selection results for landscape-level parameters revealed that variation in concentrations of red-winged blackbird yolk carotenoids was best explained by percent water in a 500 m buffer and variation in both avian abundance and avian species richness by wetland surface area. Our novel yolk carotenoid approach allowed us to identify differences in breeding bird habitat quality across wetland types, which we failed to detect with traditional bird survey data; our results highlight the need for additional studies comparing methods for assessing wetlands as breeding habitat for wetland-dependent birds.

Fat provisioning in winter impairs egg production during the following spring: a landscape‐scale study of blue tits

1. Provisioning of garden birds is a growing phenomenon, particularly during winter, but there is little empirical evidence of its true ecological impacts. One possibility is that winter provisioning could enhance subsequent breeding performance, but this seems likely to depend on the types of nutrients provided. For example, whereas effects of macronutrients such as fat are unlikely to be carried over to influence breeding in small passerines, micronutrients such as dietary vitamin E (an antioxidant) may be stored or have lasting health benefits. 2. Here, we examine the carry-over effects of winter food supplements on egg production in wild populations of blue tits (Cyanistes caeruleus). Over three consecutive years, birds were provisioned with fat, fat plus vitamin E or remained unfed (controls). 3. The provision of fat in winter resulted in smaller relative yolk mass in larger eggs and reduced yolk carotenoid concentrations in early breeders. However, these effects were not seen in birds provisioned with fat plus vitamin E. Lay date, clutch size, egg mass and yolk vitamin E concentrations were not significantly affected by winter provisioning treatment. 4. Our results indicate that winter provisioning can have important downstream consequences, in particular affecting investment in egg production several weeks or months later. 5. Provisioning is widely applied to support garden bird populations and for the conservation management of endangered species. However, our results challenge the assumption that such practices are always beneficial at the population level and emphasize how the ecological impacts can depend on the specific nutritional profile of provisioned foods.

Avian eggshell pigments are not consistently correlated with colour measurements or egg constituents in two Turdus thrushes

The colourful appearance of avian eggshells is a prominent aspect of maternal reproductive effort in birds. Some differences in eggshell coloration have been reported to co‐vary with various measures of maternal condition and these patterns support the hypothesis that, in some bird species, several aspects of eggshell colour (i.e. primary chroma and brightness) function as a signal of maternal and offspring quality to induce greater paternal investment. We directly quantified eggshell pigment concentrations of blackbird Turdus merula and song thrush T. philomelos eggshells and tested how the two key pigments (protoporphyrin IX and biliverdin) co‐varied with other eggshell traits and egg constituents as measures of maternal reproductive investment, including total yolk carotenoid concentration, total lipid concentration, yolk mass, and shell thickness. Contrary to predictions, we detected few statistical patterns overall. We found that protoporphyrin IX concentration was negatively associated with blue‐green chroma in blackbirds but not in song thrush. The concentration of protoporphyrin IX was significantly greater in blackbirds and also showed different patterns of association with total yolk lipids and yolk carotenoid concentrations between these two species (significant species interaction terms). Our results reveal that it is not appropriate to simply assume in these two avian species that reflectance‐based eggshell colour measures are a suitable proxy for eggshell pigment concentrations or can be used as consistent predictors of maternal reproductive investment. These results highlight the need to assess and validate the strength and direction of the statistical relationships between eggshell colour measures, pigment concentrations, and maternal resource deposition in the egg for other species of birds.

Effect of Restricted Preen-Gland Access on Maternal Self Maintenance and Reproductive Investment in Mallards

BackgroundAs egg production and offspring care are costly, females should invest resources adaptively into their eggs to optimize current offspring quality and their own lifetime reproductive success. Parasite infections can influence maternal investment decisions due to their multiple negative physiological effects. The act of preening – applying oils with anti-microbial properties to feathers – is thought to be a means by which birds combat pathogens and parasites, but little is known of how preening during the reproductive period (and its expected disease-protecting effects) influences maternal investment decisions at the level of the egg.Methodology/Principal FindingsHere, we experimentally prevented female mallards (Anas platyrhynchos) from accessing their preen gland during breeding and monitored female immunoresponsiveness (e.g., plasma lysozyme concentration) as well as some egg traits linked to offspring quality (e.g., egg mass, yolk carotenoid content, and albumen lysozyme levels). Females with no access to their preen gland showed an increase in plasma lysozyme level compared to control, normally preening females. In addition, preen-gland-restricted females laid significantly lighter eggs and deposited higher carotenoid concentrations in the yolk compared to control females. Albumen lysozyme activity did not differ significantly between eggs laid by females with or without preen gland access.Conclusion/SignificanceOur results establish a new link between an important avian self-maintenance behaviour and aspects of maternal health and reproduction. We suggest that higher yolk carotenoid levels in eggs laid by preen-gland-restricted females may serve to boost health of offspring that would hatch in a comparatively microbe-rich environment.

Geographical trends in the yolk carotenoid composition of the pied flycatcher (Ficedula hypoleuca).

Carotenoids in the egg yolks of birds are considered to be important antioxidants and immune stimulants during the rapid growth of embryos. Yolk carotenoid composition is strongly affected by the carotenoid composition of the female’s diet at the time of egg formation. Spatial and temporal differences in carotenoid availability may thus be reflected in yolk concentrations. To assess whether yolk carotenoid concentrations or carotenoid profiles show any large-scale geographical trends or differences among habitats, we collected yolk samples from 16 European populations of the pied flycatcher, Ficedula hypoleuca. We found that the concentrations and proportions of lutein and some other xanthophylls in the egg yolks decreased from Central Europe northwards. The most southern population (which is also the one found at the highest altitude) also showed relatively low carotenoid levels. Concentrations of β-carotene and zeaxanthin did not show any obvious geographical gradients. Egg yolks also contained proportionally more lutein and other xanthophylls in deciduous than in mixed or coniferous habitats. We suggest that latitudinal gradients in lutein and xanthophylls reflect the lower availability of lutein-rich food items in the northern F. hypoleuca populations and in montane southern populations, which start egg-laying earlier relative to tree phenology than the Central European populations. Similarly, among-habitat variation is likely to reflect the better availability of lutein-rich food in deciduous forests. Our study is the first to indicate that the concentration and profile of yolk carotenoids may show large-scale spatial variation among populations in different parts of the species’ geographical range. Further studies are needed to test the fitness effects of this geographical variation.

Maternal investment of female mallards is influenced by male carotenoid-based coloration.

The differential allocation hypothesis predicts that females modify their investment in a breeding attempt according to its reproductive value. One prediction of this hypothesis is that females will increase reproductive investment when mated to high-quality males. In birds, it was shown that females can modulate pre-hatch reproductive investment by manipulating egg and clutch sizes and/or the concentrations of egg internal compounds according to paternal attractiveness. However, the differential allocation of immune factors has seldom been considered, particularly with an experimental approach. The carotenoid-based ornaments can function as reliable signals of quality, indicating better immunity or ability to resist parasites. Thus, numerous studies show that females use the expression of carotenoid-based colour when choosing mates; but the influence of this paternal coloration on maternal investment decisions has seldom been considered and has only been experimentally studied with artificial manipulation of male coloration. Here, we used dietary carotenoid provisioning to manipulate male mallard (Anas platyrhynchos) bill coloration, a sexually selected trait, and followed female investment. We show that an increase of male bill coloration positively influenced egg mass and albumen lysozyme concentration. By contrast, yolk carotenoid concentration was not affected by paternal ornamentation. Maternal decisions highlighted in this study may influence chick survival and compel males to maintain carotenoid-based coloration from the mate-choice period until egg-laying has been finished.

Stable Isotopes Link Diet to Avian Yolk Carotenoid Allocation: A Comparative Study of Five Auk Species (Charadriiformes: Alcidae)

The allocation of important but environmentally limited nutrients, such as carotenoids, often represents a trade-off between homeostasis and reproduction. However, key questions remain about how diet and species traits influence carotenoid allocation. We studied yolk carotenoid profiles and yolk color in relation to trophic level (based on delta(15)N values) in five species of seabirds belonging to the family Alcidae: common murre (Uria aalge), pigeon guillemot (Cepphus columba), Cassin's auklet (Ptychoramphus aleuticus), rhinoceros auklet (Cerorhinca monocerata), and tufted puffin (Fratercula cirrhata). In three species, which ranged from low (tufted puffin) to high (pigeon guillemot) trophic level, yolks had pink to red hues and contained exclusively astaxanthin, while yolks of species from a high trophic level (common murre) and from a generalist forager (rhinoceros auklet) had yellow to orange hues and contained astaxanthin, lutein, and zeaxanthin. The absence of a systematic relationship between trophic level and carotenoid types suggests that these species possess adaptations for the uptake and use of specific carotenoids. In contrast, total yolk carotenoid concentrations were best explained by the combination of species and trophic level: lower-trophic-level feeding was linked to production of carotenoid-rich yolks, both across species and within the one generalist species. We conclude that both behavioral and physiological traits can play strong roles in the acquisition and allocation of critical nutrients from mothers to their offspring.

Growth of yellow‐headed blackbird Xanthocephalus xanthocephalus nestlings in relation to maternal body condition, egg mass, and yolk carotenoids concentrations

Condition‐dependent resource allocation to eggs can affect offspring growth and survival, with potentially different effects on male and female offspring, particularly in sexually dimorphic species. We investigated the influence of maternal body condition (i.e., mass‐tarsus residuals) and two measures of female resource allocation (i.e., egg mass, yolk carotenoid concentrations) on nestling mass and growth rates in the polygynous and highly size dimorphic yellow‐headed blackbird Xanthocephalus xanthocephalus. Egg characteristics and carotenoid concentrations were obtained from the third‐laid egg of each clutch and were correlated with the mass and growth rates of the first two asynchronously hatched nestlings. Maternal body condition was associated with the growth of first‐hatched, but not second‐hatched nestlings. Specifically, females in better body condition produced larger and faster growing first‐hatched nestlings than females in poorer body condition. As predicted for a polygynous, size‐dimorphic species, females that fledged first‐hatched sons were in better body condition than females that fledged first‐hatched daughters. Associations between egg mass, yolk carotenoid content, and nestling growth were also specific to hatching‐order. Egg mass was positively correlated with the mass and growth rates of second‐hatched nestlings, and yolk concentrations of β‐carotene were positively correlated with second‐hatched nestling mass. Surprisingly, the relationship between yolk lutein and hatchling growth differed between the sexes. Females with high concentrations of yolk lutein produced larger and faster growing first‐hatched sons, but smaller first‐hatched daughters than females with lower lutein concentrations. Mass and growth rates did not differ between first‐ and second‐hatched nestlings of the same sex, despite asynchronous hatching in the species. Results from this study suggest that maternal body condition and the allocation of resources to eggs have carotenoid‐, sex‐, and/or hatch‐order‐specific effects on yellow‐headed blackbird nestlings.

Compensatory investment in zebra finches: females lay larger eggs when paired to sexually unattractive males

The classical version of the differential allocation hypothesis states that, when females reproduce over their lifetime with partners that differ in their genetic quality, they should invest more in reproduction with high-quality males. However, in species with lifetime monogamy, such as the zebra finch, partner quality will typically remain the same. In this case, the compensatory investment (CI) hypothesis predicts higher investment for low-quality males, because low genetic quality offspring are more dependent on maternal resources. Here, we show that female zebra finches invested more resources, both in terms of egg volume and yolk carotenoid content, when paired to a low genetic quality male, as judged from his previous ability to obtain extra-pair paternity in aviary colonies. We also found that females deposited slightly larger amounts of testosterone into eggs when paired to a low parental quality male, as judging from his previous success in rearing offspring. This is, to our knowledge, the first experimental support for the CI hypothesis in a species with lifetime monogamy. We stress that in more promiscuous species, the benefits of classical differential allocation may partly be neutralized by the supposed benefits of CI.

Phenotypic Correlates of Yolk and Plasma Carotenoid Concentration in Yellow‐Legged Gull Chicks

Abstract Carotenoids perform important biological actions in animal tissues, including contributing antioxidant protection. However, the function of transmission of maternal carotenoids to bird eggs is still largely unknown. We made a yolk biopsy of yellow-legged gull (Larus michahellis) eggs and found that the concentration of lutein declined with laying date and across the laying order and increased with egg mass. The concentration of all the main carotenoids (lutein, zeaxanthin, and dehydrolutein) pooled also declined with date and increased with egg mass. We also performed a partial reciprocal cross-fostering of eggs between clutches and investigated the covariation between morphology, T cell-mediated immunity, and plasma carotenoid concentrations of the chicks and carotenoid concentrations in their original eggs. Absolute plasma carotenoid concentrations did not covary with those in the yolk, whereas a positive covariation was found for relative concentrations. Yolk and absolute plasma carotenoid concentrations positively predicted chick body mass and size but not the intensity of the cell-mediated immune response. Thus, yolk carotenoid concentrations may affect chick carotenoid profile and growth, possibly mediating early maternal effects. However, rearing conditions also contributed to determining relative concentrations of circulating carotenoids. Since yolk or plasma antioxidant capacity did not correlate with carotenoid concentrations, future studies of maternal effects mediated by antioxidants should integrate information on carotenoids with information on other components of the antioxidant systems.

Egg Yolk Carotenoids in Relation to Habitat and Reproductive Investment in the Great TitParus major

Maternal allocation of antioxidants to egg yolk has been shown to affect early embryonic development and nestling survival. In environments with high levels of anthropogenic pollution, antioxidants (such as carotenoids) are important to protect the body from elevated oxidative stress. Thus, female allocation of antioxidants to yolk may be traded off against self-maintenance. Here we investigate maternal reproductive investment with respect to yolk carotenoid content and composition in relation to subsequent female condition and carotenoid status in urban and rural great tits Parus major. We found no differences between the urban and rural populations in total yolk carotenoids, egg mass, clutch size, hatching success, or female carotenoid status. Interestingly, however, rural eggs contained more zeaxanthin, a more potent antioxidant than lutein, which suggests that rural embryos have better antioxidant protection than urban embryos. Whether rural females actively transfer more zeaxanthin to the yolk or whether it passively reflects differences in dietary access or uptake needs to be further investigated. This highlights the importance of carotenoid identity and composition in future studies of carotenoid physiology, ecology, and signaling.

A nondestructive method for extracting maternally derived egg yolk carotenoids

Maternally deposited carotenoids are a prominent component of egg yolk and are vital for the development and growth of the embryo. In most studies of avian yolk carotenoids, eggs are destructively sampled and this may limit both the number of clutches studied and the research questions addressed. We describe an empirical field trial for a nondestructive biopsy method to extract small samples (0.05 ml) of egg yolk for high- performance liquid chromatography (HPLC) analysis of yolk carotenoid concentrations. We sampled 180 clutches (N = 44 biopsies) of two species of introduced thrushes (genus Turdus) from agricultural habitats in central North Island, New Zealand. Once the protocol was established, all biopsied eggs from clutches that were not depredated or deserted before candling were found to be developing normally after 3-5 d of incubation (N = 28) and all hatched. Biopsy samples (>0.02 g) produced concentrations of yolk carotenoids (and variances) that were statistically indistinguishable from whole yolk destructive samples. In addition, our samples (>0.02 g) confirmed previously reported differences in yolk carotenoid concentrations between the two thrush species and revealed a significant decline in yolk carotenoid concentration with laying order. Further examination of how variability in yolk carotenoid concentration and identity influences offspring sex, success, and survival or, later in life, reproductive success and ability to efficiently incorporate dietary carotenoids into both integument and immune tissues will require larger sample sizes. Studies to date have been restricted by the number of destructive samples that investigators are willing (or permitted) to obtain from wild species. Thus, we hope that our nondestructive method of sampling yolk will promote further examination of the links between carotenoid uptake from the environment and maternal investment in the avian yolk.

Parental effects on carotenoid-based plumage coloration in nestling great tits, Parus major

Carotenoid pigments have attracted much interest in behavioural and evolutionary ecology because of their dual function in immune physiology and as color signals. In vertebrates, carotenoids must ultimately be obtained from the diet, and the mechanisms and magnitude of this environmental dependence are central for understanding carotenoid signal functions and evolution. In the present cross-fostering experiment with great tits Parus major, we investigate pre- and postnatal parental effects (egg yolk carotenoids, parental coloration) on nestling size and carotenoid coloration, using HPLC analysis of egg yolk carotenoids, and a reflectance-based measure of ‘chroma’ that reflects the plumage pigment concentration. Both rearing environment and origin influenced offspring size and plumage chroma. Maternal allocation of carotenoids to eggs had a weak positive effect on nestling plumage chroma, whereas we found no prenatal maternal effects (egg size or yolk carotenoid concentration) on size. Nestling plumage chroma was also significantly predicted by the chroma of the rearing father, but not by the color of the rearing mother or either of the original (genetical) parents. Thus, both prenatal maternal effects and postnatal paternal effects influence the carotenoid-based plumage coloration of nestling great tits. Future studies will reveal if parental effects have long-term consequences for plumage development and associated fitness components.