Antioxidant protection and plasma carotenoids of incubating great tits (Parus major L.) in relation to health state and breeding conditions

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Abstract

Carotenoids are biologically active pigments, which are important for animals due to their dual role in health maintenance and ornamental signalling. In adult birds, immunostimulatory properties of carotenoids have been repeatedly demonstrated while much less is known about the importance of carotenoids as antioxidants. We studied the relationships between plasma carotenoid levels, as well as total antioxidant protection, and various hemato-serological health state indices in female great tits (Parus major L.), incubating their second clutches in two contrasting (coniferous and deciduous) habitats in southwest Estonia. To manipulate reproductive effort, four eggs were removed from half of the clutches during laying to stimulate females to lay additional eggs. However, egg removal had no effect on the final number of eggs laid. Plasma carotenoid levels increased seasonally in parallel with caterpillar food availability. However, no between-habitat differences in carotenoid levels, total antioxidant capacity, or indices of health state could be found despite the apparently better feeding conditions in the coniferous habitat. No correlation was detected between plasma carotenoid levels and measures of total antioxidant capacity, which suggests that at least for the adult birds feeding on naturally carotenoid-rich diet, antioxidant function of carotenoids is not of primary importance. A strong non-linear association between the measures of antioxidant protection and leukocytic markers of inflammation was found, which suggests that measures of total antioxidant capacity deserve further attention in ecophysiological studies as potential indicators of immunopathology.

Introduction

Reproduction in birds demands considerable metabolic investment (e.g., Drent and Daan, 1980), which can be associated with increased production of reactive oxygen species, ROS (Alonso-Alvarez et al., 2004a, Wiersma et al., 2004, Bertrand et al., 2006). ROS are generally unstable and very reactive with a potential to damage nucleic acids, proteins, and lipids (Halliwell and Gutteridge, 1999). To control and neutralise ROS, organisms evolved defence systems based on antioxidants, which deactivate ROS by donating missing electrons. Endogenous antioxidants (uric acid, bilirubin, albumin, and enzymes like catalase, superoxide dismutase, glutathione peroxidase) are synthesized by an organism and exogenous antioxidants such as fat-soluble vitamins E and A, and carotenoids (in many passerines also ascorbic acid) must be obtained from food. Of all these antioxidants, avian ecologists have focussed their primary attention on carotenoids because these are frequently used in integumentary sexual signals and also possess immunostimulatory properties (Lozano, 1994, von Schantz et al., 1999, Møller et al., 2000, McGraw, 2006). Under this view, carotenoid-based ornaments enable individuals to signal their past and/or current health state: carotenoids can be allocated to signalling only if and when they are not needed for immunomodulation at the same time.

Compared to their role in signalling and immunity, the antioxidant function of carotenoids has remained much more poorly understood, even in traditional mammal models (El-Agamey et al., 2004). The situation is even more complicated with birds—given that most avian species live longer than similar-sized mammals despite their higher metabolic rates, the birds are supposed to have evolved unique protective mechanisms against the oxidative damage (Perez-Campo et al., 1998). In birds the protective effect of maternally transferred carotenoids to embryos and hatchlings is well established (Surai, 2002, McGraw et al., 2005), but only two studies (Alonso-Alvarez et al., 2004b, Costantini et al., 2006) have measured the relationships between carotenoids and general antioxidant defences in nestlings or adult birds.

Here we address the question about the role of carotenoids in modulation of antioxidant protection in female great tits, incubating their second clutches in two contrasting habitats in southwest Estonia. Great tit is a small (ca. 20 g) passerine, breeding in the forests of most of the Palearctic region (Gosler, 1993). The yellow ventral plumage coloration of great tits is based on lutein and zeaxanthin (Partali et al., 1987, Stradi, 1998), two carotenoids that are particularly abundant in lepidopteran larvae (Partali et al., 1987) that are the main source of great tits food during the breeding season (Partali et al., 1987, Naef-Daenzer et al., 2000). Birds in our study area breed in two contrasting habitats (deciduous and coniferous), where physiological condition of adults and reproductive parameters differ considerably (Mägi and Mänd, 2004, Mänd et al., 2005, Kilgas et al., 2006a). The results of an unpublished pilot study suggested that during the second broods, caterpillar food is especially abundant in coniferous forests where the clutches are also larger and nestlings heavier than in the deciduous forests at that period (Mägi and Mänd, 2004). We therefore expected the physiological condition of the females during the second breeding attempt to be better in coniferous habitat than in the deciduous habitat. To manipulate reproductive effort, four eggs were removed from the half of the clutches during laying to stimulate females to lay additional eggs (e.g., Visser and Lessells, 2001).

Specifically, we addressed the following questions. Under the hypothesis that increased egg-laying effort leads to deterioration of antioxidant protection and general health state (Blount et al., 2004, Williams, 2005), we predicted that females induced to lay extra eggs reveal lower levels of antioxidant protection, carotenoids, and indices of nutritional state as compared to the un-manipulated females. We also predicted that if dietary carotenoids have any important antioxidant function in incubating birds, then their plasma concentrations should positively correlate with plasma total antioxidant capacity. Additionally, we tested whether plasma carotenoid levels differ between habitat types, expecting higher concentrations in coniferous habitat with presumably higher caterpillar food supply at that time. On the same grounds, we predicted parallel seasonal trends in caterpillar abundance and plasma carotenoid levels. Finally, we asked whether markers of antioxidant protection and plasma carotenoid levels correlate with leukocytic health state indices, predicting negative correlations with markers of inflammation and stress, such as heterophile count and heterophile/lymphocyte (H/L) ratio. We expected negative correlations between markers of antioxidant protection and heterophile hemoconcentration because these inflammatory cells release free radicals which might deplete the antioxidant capacity (e.g., Ames et al. 1993; Kogut et al., 2002, Costantini and Dell'Omo, 2006).

Section snippets

Materials and methods

The study was conducted near Kilingi Nõmme (58°7′N, 25°5′E), southwest Estonia, in June–July 2005, when great tits were laying and incubating their second clutches. Various forest habitats in the 50 km2 study area were classified as deciduous and coniferous (see Mänd et al., 2005, for details). Deciduous woods occur mainly as isolated patches in agricultural landscape or as 250–500-m-wide galleries along roads and stream valleys. They grow on fertile soils and have rich understory. The most

Results

Egg removal had no effect on the final number of eggs laid F1,44 = 1.5; P = 0.2) in ANOVA with habitat as a factor (F1,44 = 4.1; P = 0.048).). Interaction between manipulation and habitat was not significant (F1,43 = 0.5; P = 0.5). Manipulated females did not differ from controls in any of the hemato-serological or antioxidant parameters either (all P-values > 0.1).

The amount of frass falls did not differ significantly between the habitats, although there was a marginally not significant tendency to higher

Discussion

Frass fall samples revealed increase in caterpillar abundance during the study period in both habitats. Total frass fall index tended to be higher in coniferous woods, similarly to the results of a pilot study in the previous year (unpublished); lack of statistical significance for this difference (P = 0.067) can be evidently ascribed to the low sample sizes (n = 5 for both habitats). This conclusion is reinforced by the finding of significantly higher body masses in coniferous habitat, which

Acknowledgements

We thank Lauri Saks, Ulvi Karu, and Rauno Külavee for the field assistance and Indrek Ots for performing and teaching us the plasma analyses. We are grateful to Siiri-Lii Sandre for consultations on carotenoid content in leaves and Mihkel Zilmer for suggesting the assays for total antioxidant capacity. George Lozano and three anonymous reviewers provided thorough and constructive criticism on the manuscript. The study was supported by Estonian Science Foundation Grants 6222 to PH and 5489 to RM.

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