Corticosterone in feathers of laying hens: an assay validation for evidence-based assessment of animal welfare

Abstract

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.

Corticosterone in feathers: Inter- and intraindividual variation in pullets and the importance of the feather type

“…Animals were kept on a commercial farm in Germany, in accordance with local legislation ( Council of Europe, 1995 ; Minimum Requirements for Pullets, 2000 ; Recommendations for preventing feather pecking & cannibalism, 2018 ; TierSchNutztV, 2017 ). Feathers were pulled postmortem ( Häffelin et al., 2020 ; Monclús et al., 2017 ) from pullets ( n = 10) with untrimmed beaks, genetics Lohmann Brown, reared in the same flock ( N = 728) in floor husbandry with a modified aviary system and without free range. Pullets were slaughtered at an abattoir as part of another project at the end of the rearing period with 19 weeks of age.…”

“…All selected feathers had to be full-grown which was verified through the absence of blood and feather pulp in the calamus ( Jenni, Ganz, Milanesi & Winkler, 2020 ), and the completeness of the vane. The contour feathers ( n = 3 – 5 / pullet) were taken from the region on the back between the scapulae ( Carbajal, Tallo-Parra, Sabes-Alsina, Mular & Lopez-Bejar, 2014 ; Häffelin et al., 2020 ; Monclús et al., 2017 ) and are subsequently named as interscapular feathers ( Häffelin et al., 2020 ; Monclús et al., 2017 ). Flight feathers taken from the tail ( Rectrices, n = 2 – 3 / pullet) are subsequently named as tail feathers ( Aharon-Rotman, Buchanan, Klaasen, & Buttemer, 2017 ; Häffelin et al., 2020 ; Harms et al., 2015 ; Jenni-Eiermann et al., 2015 ; Lendvai, Giraudeau, Németh, Bakó & McGraw, 2013 ; López-Jiménez et al., 2016 ; Robertson, Muir, Hurd, Hing & Quinn, 2017 ).…”

“…Additionally, the manual scoring requires labor, is time-consuming and birds need to be handled in order to carry out an exact assessment. The measurement and evaluation of corticosterone concentrations in feathers ( CORTf 1 ) was shown to be a suitable, non-invasive tool to detect and monitor birds that had to cope with adverse environmental situations over a long-term period ( Bortolotti, Marchant, Blas, & German, 2008 ; Häffelin et al., 2020 ). Therefore, detecting CORTf 1 may be a promising approach to assess animal welfare in single birds and flocks, respectively.…”

“…Contrary, CORT 2 levels in the blood or GCM 3 in feces are subjected to circadian rhythm ( Möstl, Rettenbacher & Palme, 2005 ; Touma & Palme, 2005 ) and short-term liabilities, such as capturing ( Bortolotti, Marchant, Blas, & German, 2008 ; Dehnhard et al., 2003 ; Romero & Reed, 2005 ), immobilization by hand and heat stress ( Beuving & Vonder, 1978 ). In older pullets or young laying hens, CORTf 1 may be a useful indicator to assess birds' ability to cope with their rearing conditions, and thus, to evaluate their susceptibility to develop behavioral disorders, such as feather pecking and cannibalism before their appearance ( Häffelin et al., 2020 ), because they often emerge with a time lag to the period that is responsible for them. Thus, CORTf 1 would be beneficial regarding the evaluation of pullets before rehousing for production and the implementation of an adapted and preventive flock management ( Weimer et al., 2018 ).…”

“…Thus, CORTf 1 would be beneficial regarding the evaluation of pullets before rehousing for production and the implementation of an adapted and preventive flock management ( Weimer et al., 2018 ). Furthermore, CORTf 1 measurements may also be suitable to be implemented in breeding schemes in order to provide stress-resilient animals ( Häffelin et al., 2020 ). However, prior of any interpretation of CORTf 1 values, there is a need to establish species-specific reference values ( Fairhurst, Marchant, Soos, Machin & Clark, 2013 , 2012 ; Kennedy, Lattin, Romero & Dearborn, 2013 ; Kouwenberg, Hipfner, McKay & Storey, 2016 ).…”