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On the morning that each egg was laid, it was removed and replaced with a dummy egg. Clutches were considered complete once laying had stopped for 3 days or six eggs had been laid (20). Each pair of birds was housed in individual cages, without visual contact with other birds. The room was lit with full-spectrum fluorescent tubes. The failure of some studies to replicate an effect of band color on male attractiveness is most likely due to inadequate housing conditions without ultraviolet (UV) light (21). Our experiment was done under the required conditions of UV light.
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The whole yolk was removed, homogenized in 0.5 ml of distilled water, and frozen on the morning of laying. Free steroids were extracted twice from yolk- water homogenate with 3 ml of ether. Ether fractions were decanted from the snap-frozen egg yolk-water phase, pooled, and then dried under a stream of nitrogen. The dried extract was redissolved in 90% ethanol and kept overnight at -20°C. Precipitated proteins and lipids were separated by decanting after 5 min of centrifugation at 1300g, and the ethanol was dried under a stream of nitrogen. The dried extract was redissolved in 1.5 ml of assay buffer. Extraction efficiencies were assessed in each assay and were on average 70%. Testosterone (T) + 5α-dihydrotestosterone (DHT) concentrations were measured by radioimmunnoassay. The established intraassay coefficient of variation is 5.0%, and the interassay coefficient of variation is 11.3% [BIOTRAK testosterone/dihydrotestosterone (3H) assay system Code TRK 600; Amersham Pharmacia Biotech]. The measured intraassay coefficient of variation was 3.9% for T + DHT and 2.0% for DHT alone. Disintegrations per minute was used throughout.
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We thank S. Buckland, M. Ritchie, and P. Gardner for statistical advice and comments on the manuscript. D.G. was supported by a grant to J.G. from the School of Environmental and Evolutionary Biology, University of St. Andrews, UK.
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