Corticosterone sampling and measurement
We sampled 75 µl blood from each subject’s brachial vein within 3 min of
capture (mean = 157.9 sec, min = 85, max = 216, SD = 30.877; for two
birds their blood collection ended after the 3 min mark). The time to
end blood collection was not correlated with natural log-transformed
baseline corticosterone levels (Pearson’s r = 0.03, df = 41, p = 0.87)
and we did not account for it in the statistical models.
Blood was kept on ice until centrifugation 2-8 hrs later at +4 °C for 10
min at 8000 rpm. Following centrifugation, blood plasma was removed and
stored at -80 °C until analysis. Plasma corticosterone was measured
using enzyme immunoassay (Cayman Chemical, catalogue number 501320; Ann
Arbor, MI, USA) which has been validated and optimized for American
robins (Abolins-Abols & Hauber 2020b). We extracted the non-polar
components of plasma using diethyl ether extraction, described in
(Abolins-Abols & Hauber 2020b). Briefly, 10 µl plasma was suspended in
200 µl ultrapure water and mixed with 1 ml diethyl ether. After passive
phase separation, the mixture was flash-frozen, and the ether phase was
decanted. Ether was then evaporated using nitrogen gas at 40 °C. Ether
was added to the aqueous portion and decanted two more times. The
extract was suspended in 600 µl assay buffer overnight at 4 °C. The
concentration of corticosterone in the extract was measured according to
the manufacturer’s instructions. Each extracted sample was analyzed in
triplicate. We measured absorbance at 405 nm using Biotek 800TS plate
reader (Winooski, VT, USA) and analyzed the data using an 8-point
logistic curve using the Cayman Chemical analysis spreadsheet. The
average intraplate coefficient of variation (CV) was 5.78%, while
inter-plate CV was 6.19%; we therefore averaged values from the three
replicates for each sample and standardized concentrations across places
using the mean concentration of a standard control sample, for use in
the statistical models.