Study species and captive setting
In total, 18 and 48 Himalayan Black Bulbuls were bought from a pet-shop in Taipei in 2010 and 2011 respectively. Each bird was housed individually in the laboratory. We collected 150 μL blood from each individual for molecular sex typing. A drop of blood, approximately 5 μL, from each bird was used as the blood smear for the oxidative stress test. Each bird’s brightness measurement and oxidative stress test were carried out in March of 2010 or 2011, which was the beginning of the breeding season.
Molecular sex typing
Gross DNA was extracted from blood samples with traditional proteinase K digestion followed by LiCl extraction [modified from the procedure of Gemmell and Akiyama (1996)]. Extracted DNA was re-suspended in ddH2O and stored at −20 °C. Less than 100 ng of genomic DNA was added to 12.5 μL of PCR (polymerase chain reaction) mix containing 0.5 mmol/L of each of the dNTPs, 0.3 μmol/L of each PCR primer (2550F/2718R, Fridolfsson and Ellegren 1999), 10 mmol/L Tric-HCL, 50 mmol/L KCL, 1.5 mmol/L of MgCl2 and 0.4 U of Taq DNA polymerase (Amersham Biosciences). The PCR profile was 94 °C for 3 min, followed by 40 cycles of 95 °C for 20 s, 46 °C for 30 s and 72 °C for 40 s, finishing at 72 °C for 2 min. The PCR reactions were carried out in iCyclers (Bio-Rad, Hercules, CA, USA). After the PCR reactions, we conducted electrophoresis with 1.2 % agarose gel to determine the birds’ sex. We identified eight females and ten males in 2010 and 18 females and 30 males in 2011.
Brightness measurements
For each individual, the reflectance of eight regions of melanin-based plumage- including the forehead, nape, back, breast, belly, tail, remige and scapular feathers—was measured using an USB2000 spectrometer (Ocean Optics) with a HL2000 deuterium-halogen light source (Ocean Optics). A R600-7-UV/125F probe (Ocean Optics) was held perpendicular to the surface of the feathers with a cylindrical cap at the end to standardize the distance (5 mm) measured and to shield ambient light. To calculate relative reflectance, a white standard (Labsphere) was used. To collect the dark reference, the light source was capped by a black plastic plate. Each part was measured three times to calculate repeatability (repeatability >90 %, Lessells and Boag 1987). The brightness of each region was defined as the average of total reflectance within the range of 300–700 nm. McGraw et al. (2005) demonstrated that the concentrations of both eumelain and phaeomelanin in feathers were significantly and positively correlated with brightness: a brighter measurement indicates more melanin in the feathers.
Oxidative stress test
We calculated the ratio of lymphocytes (L) to heterocytes (H) in total of 100 leukocytes as a measure of oxidative stress for each individual bulbul following the methods used by Vleck et al. (2000). The blood smear was first dyed with a Wright-Giemsa stain for 3 min and then with 5 % PBS for 70 s. After drying, the numbers of lymphocytes and heterocytes were counted under a microscope at a magnification of 100× with oil immersion, and the H/L ratio calculated. The H/L ratio has been proved to be a good indicator of oxidative stress: higher levels of corticosterones can cause higher oxidative stress and also increase the number of heterocytes in the blood (Davis et al. 2008; Gross and Siegel 1983). Because leukocyte numbers change more slowly in response to stress than corticosterone does (Maxwell 1993), H/L ratios provide a more useful and accurate measure of long-term stress than a single measure of plasma corticosterone (McFarlane and Curtis 1989; Vleck et al. 2000).
Statistical analysis
Two-way ANOVA was conducted to test whether each individual’s oxidative stress level and the brightness of each its body parts differed significantly between years. In these tests, sex, year and the interaction between sex and year were taken into account. Multiple regression was used to test whether the brightness of each body part correlated with oxidative stress (H/L ratio) while taking into account the birds’ sex and the interaction between sex and oxidative stress. In the tests, the H/L ratio was log transformed to fit the normal distribution.
Ethics statement
Live birds were housed in the Animal Care House of the National Taiwan Normal University and cared for using procedures approved by the Institutional Animal Care and Use Committee of the Department of Life Science (IACUC Approval No 96026).