Evaluating Total Plasma Carotenoids in Wild Birds

carotenoids (Hill et al. 1994; Hill 1995a, 1995b; Figuero and Gutié rrez 1998). Because of its simplicity, color-chart matching The study of the role of carotenoids on the physiology and was proposed as a quick assay for field biologists (Hill 1995a). evolutionary ecology of birds demands methods for their quanHowever, no study has compared the scores derived from color tification in the bloodstream. We compared color-chart scores charts with the actual carotenoid concentrations objectively of plasma hue with the actual concentration of plasma carotmeasured by spectrophotometry. Our aim is to assess the validenoids obtained by spectrophotometry in 356 wild birds from ity and accuracy of color charts for quantifying total plasma 26 species. Repeatability of chart scores between three indepencarotenoids in both intraand interspecific studies of wild dent observers was high. However, color scores did not correbirds. late with the spectrophotometric results in interspecific analyses. Within species (n Å 3), one showed no relationship and two showed weak but significant positive correlations. HemoMaterial and Methods globin, and probably other substances, may mask the color of The study was conducted in the surroundings of La Paz (Baja carotenoids, making the accurate use of color charts difficult. California Sur, Mé xico) between November 15 and December Spectrophotometry should be the method of choice as it per9, 1996. Wild birds were trapped and blood samples (0.1 – 0.5 mits precise quantifications of total plasma carotenoids and mL) were taken from the brachial or jugular veins. Heparinized objective comparisons among studies. syringes or microcapillary tubes were used for collecting the blood. All individuals were freed after blood collection. The samples were transported to the laboratory (Centro de InvestiIntroduction gaciones Bioló gicas del Noroeste, La Paz) within 8 h after blood The role of carotenoids in the physiology and sexual selection withdrawal, and they were centrifuged at 1,500 g for 10 min. of birds is the focus of an increasing forum in evolutionary The plasma was frozen at 070°C until analysis 1 – 6 d later. The concentration of plasma carotenoids was first estimated *To whom correspondence should be addressed; E-mail: [email protected]. visually. Just after thawing the plasma samples, plasma hue csic.es. was independently scored by three of the authors, using a sixoption color chart derived from paint samples. The extremes (1 Å pale yellow, 6 Å red-orange) were determined on the basis of our experience after previously sampling the plasma and both variables log-transformed) did not show a significant of thousands of birds (J. L. Tella et al., unpublished data) and relationship (r Å 0.04, P Å 0.50). correspond to the following Munsell’s color chips: 1 Å 5Y 9/ Intraspecific correlations were also obtained for the three 6, 2 Å 5Y 8.5/10, 3 Å 2.5Y 8/12, 4 Å 8.75YR 7/14, 5 Å species with larger sample sizes, excluding the clearly hemo3.75YR 6/12, 6 Å 10R 5/12 (see Munsell Color Company 1976). lyzed samples. The results differed between species: while in Second, the concentration of total plasma carotenoids was American kestrels and white-crowned sparrows (Zonotrichia measured spectrophotometrically (see, e.g., Allen 1987; Bortoleucophrys) plasma-hue scores correlated significantly with aclotti et al. 1996; Negro et al. 1998). We diluted a variable tual plasma concentrations (rs Å 0.49, P Å 0.0001, n Å 62 and amount of plasma (20 – 100 mL) in acetone obtaining dilutions rs Å 0.31, P Å 0.03, n Å 50, respectively), there was no correlaranging from 1 : 10 to 1 : 40. After the plasma was well mixed tion in common ground doves (rs Å 0.042, P Å 0.73, n Å 67; with 100% acetone, the flocculent protein was precipitated by Fig. 2). centrifuging the sample at 1,500 g for 10 min. We examined the supernatant in a Beckman Du-70 spectrophotometer and Discussion determined the optical density of the carotenoid peak at 476 nm. Xantophylls, the most common carotenoids in birds Our color chart covered all natural variability found in the (Brush 1990) show two absorption maxima in acetone, at 445 – plasma of a wide phylogenetic range of wild birds, and the 450 nm and 474 – 478 nm, respectively. We choose the upper scores of independent observers were highly repeatable. Howpeak following previous researchers (e.g., Allen 1987). Carotever, color scores did not accurately reflect the actual concenenoid concentration (mg/mL plasma) was derived from a stantration of total carotenoids in plasma. dard curve of lutein (alpha-carotene-3,3Ì-diol, SIGMA).