Sigmaplot version 13

Sample sizes were based in previous studies or followed ISO recommendations, and were confirmed to provide power of 90% or better. The data were tested for normal distribution (Anderson-Darling) and homoscedasticity (Levene’s test), followed by parametric statistical tests. A two-way analysis of variance (ANOVA) was performed and multiple comparisons were made using Tukey’s test (95%). All analyses were carried out using statistical package Sigma Plot version 13.1 (Systat Software Inc.).

Blood samples were taken the day before habituation began and the day after the CPP post-test. Approximately 0.5 ml of blood was taken from the brachial (wing) vein of the quail and placed into heparinized tubes. To minimize corticosteroid release, the time between removal of quail from carrier box and collection did not exceed 5 min (Dallman & Bhatnager, 2001 ; Mizrahi, Holberton, & Gauthreaux, 2001 ). Blood was immediately centrifuged at 1500 RPM for 5 min and plasma was stored at −20°C until assayed.
To determine plasma estradiol (E2) levels, samples were measured in duplicate via an enzyme-linked immunoassay kit (DRG Diagnostics; estradiol EIA-2693) according to manufacturer’s instructions and modified from the procedure described by Wilhelms, Cutler, Proudman, Anderson, & Scanes, (2005) . The range of sensitivity for the assay was 9.7 pg/ml – 2000 pg/ml. Six samples from photoregressed quail that were below the limit of detectability were assigned a value equal to the sensitivity of that assay (Henare et al., 2011 (link)). The intra-assay coefficient of variance (CV) for the estradiol ELISA was 2.1%. Results were determined using a four parameter logistic standard curve analysis within Sigma Plot version 13.1 (Systat Software, Inc., San Jose, CA).