Studies indicate that the evaluation of animal welfare in birds may be carried out with the measurement of the stress-related hormone corticosterone in feathers. However a standardized procedure for corticosterone measurements in feathers is lacking, a validation needs to be carried out for each new species before implementation. The aim of the present study was to establish a valid method to measure corticosterone concentrations in feathers of laying hens in a precise and repeatable manner using an established and commercially available ELISA. Validation was performed with feather pools of tail and interscapular feathers of commercial Lohmann Brown laying hens. Assessment groups, consisting of 5 replicates, were created. All replicates of an assessment group were processed at the same time. Each replicate was run in 4 repetitions by ELISA. Intra-assay and interassay CV was 7.5 and 6.4%, respectively. The serial dilution showed linearity and parallelism. Examining the hormone extraction efficiency by using different methanol volumes resulted in no statistical differences ( P > 0.05). Pulverized feathers showed higher corticosterone values than minced feathers ( P > 0.05). Differences were shown between 2 feather types (tail vs. interscapular feathers; P < 0.05), as well as between vane and rachis ( P < 0.05). Performance of a freeze–thaw cycle led to a decrease of corticosterone concentrations in the samples. A possible effect of UV-A radiation on the stability of corticosterone in the feathers was not found ( P > 0.05). With the present study, a valid protocol, feasible for analyzing feather pools of laying hens, was developed. It may provide fundamentals for further investigations on corticosterone in feathers as a noninvasive indicator to evaluate aspects of animal welfare.

Measuring corticosterone concentrations in feathers of poultry may be suitable to determine birds' exposure to stress. It is thinkable, that in laying hens such information could be helpful as an animal welfare indicator to evaluate adverse husbandry conditions and to predict the risk of developing behavioral disorders, such as feather pecking and cannibalism. Yet, there are some fundamental issues which remain unclear. Therefore, the objective of the current pilot study was to examine the inter- and intraindividual variation of pullets at the end of the rearing period, when most of the feathers are fully grown and animals are reaching sexual maturity. Flight feathers from both wings ( n = 4), the tail ( n = 2 – 3), and body feathers ( n = 1 pool of 3 – 5 feathers) were taken from pullets ( n = 10), genetics Lohmann Brown, at an age of 19 weeks who were reared in the same flock ( N = 728). Corticosterone analysis was performed applying a validated protocol for laying hens. Results indicate not only high intraindividual, but also high interindividual variation. Mean over all samples was 75.2 pg/mg (± 38.58 pg/mg, n = 76), showing higher intraindividual variation (between feather types; SD: 23.75 pg/mg – 49.38 pg/mg; n = 10 pullets) than interindividual variation (within feather types; SD: 11.91 pg/mg – 49.55 pg/mg; n = 6 feather types). The variation between different feather types within one bird was higher than the variation within one feather type between different birds, indicating that birds a) may respond differently when exposed to stressors and b) corticosterone measurements should be done with the same feather type.