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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.

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