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Tierärztliche Hochschule Hannover
Messung von Corticosteron in Federn
von Jung- und Legehennen
INAUGURAL-DISSERTATION
zur Erlangung des Grades einer
Doktorin der Veterinärmedizin
- Doctor medicinae veterinariae -
(Dr. med. vet.)
vorgelegt von
Katharina Elisabeth Häffelin, geb. Fleig
Villingen
Hannover 2021
Wissenschaftliche Betreuung: Prof. Dr. med. vet. Nicole Kemper
Institut für Tierhygiene, Tierschutz und Nutztierethologie
Stiftung Tierärztliche Hochschule Hannover
Prof. Dr. agr. Robby Andersson
Fachgebiet Tierhaltung und Produkte
Fakultät Agrarwissenschaften und Landschaftsarchitektur
Hochschule Osnabrück
1. Gutachterin: Prof. Dr. med. vet. Nicole Kemper
Institut für Tierhygiene, Tierschutz und Nutztierethologie
Stiftung Tierärztliche Hochschule Hannover
2. Gutachter: Prof. Dr. med. vet. Silke Rautenschlein
Klinik für Geflügel
Stiftung Tierärztliche Hochschule Hannover
Tag der mündlichen Prüfung: 20.05.2021
Diese Arbeit wurde vom Niedersächsischen Ministerium für Wissenschaft und Kultur durch
ein Stipendium im Rahmen des Promotionsprogrammes „Animal Welfare in Intensive
Lifestock Production Systems“ sowie der Hochschule Osnabrück finanziell gefördert.
Dem Tierwohl
Abkürzungsverzeichnis
Inhaltsverzeichnis
1. Einleitung ................................................................................................... 1
2. Eingebundene Manuskripte ..................................................................... 5
2.1. Corticosterone in feathers of laying hens: an assay validation
for evidence-based assessment of animal welfare ........................ 6
2.2. Corticosterone in feathers: Inter- and intraindividual variation
in pullets and the importance of the feather type ....................... 16
3. Übergreifende Diskussion ....................................................................... 22
3.1. Diskussion des eigenen Vorgehens und Studiendesigns ....................... 22
3.2. Mögliche Einflussfaktoren auf den Corticosteron-Wert einer Feder . 26
3.2.1. Einflussfaktoren vor der Probenahme ....................................................... 26
3.2.2. Einflussfaktoren nach der Probenahme ..................................................... 31
3.3. Stärken und Schwächen anderer Matrizes ........................................... 34
3.3.1. Blutplasma oder -serum............................................................................. 35
3.3.2. Kot ............................................................................................................. 36
3.3.3. Eier ............................................................................................................ 37
3.4. Federwachstum ........................................................................................ 38
3.4.1. Junghennenmauser .................................................................................... 39
3.4.2. Mauser während einer induzierten Legepause .......................................... 39
3.5. Was kann CORTf leisten? ...................................................................... 40
4. Schlussfolgerung ...................................................................................... 43
5. Zusammenfassung ................................................................................... 45
6. Summary .................................................................................................. 47
7. Literaturverzeichnis ................................................................................ 49
8. Weitere Publikationen ............................................................................ 67
9. Danksagung.............................................................................................. 69Abkürzungsverzeichnis
Abkürzungsverzeichnis
Abkürzungsverzeichnis
ACTH Adrenocorticotropes Hormon
bzw. beziehungsweise
cm² Quadratzentimeter
CORT Corticosteron
CORTf Corticosteron in Federn
CV Variationskoeffizient (engl. Coefficient of variation)
ELISA Enzyme-linked Immunosorbant Assay
engl. englisch
FCM engl. faecal cortisol/corticosterone metabolites
g Gramm
HFP engl. High Feather Pecking
HPA-Achse Hypothalamus-Hypophysen-Nebennieren-Achse
(engl. Hypothalamic-pituitary-adrenal axis)
i. p. intraperitoneal
i. v. intravenös
LB Lohmann Brown
LFP engl. Low Feather Pecking
LSL Lohmann Selected Leghorn
LW Lebenswoche
ml Milliliter
mm Millimeter
ng Nanogramm; 10-9 Gramm
pg Pikogramm; 10-12 GrammAbkürzungsverzeichnis
s. c. subcutan
SD Standardabweichung (engl. Standard Deviation)
TBS Puffersubstanz bestehend aus Tris(hydroxymethyl)aminomethan
und Natriumchlorid (engl. Tris-buffered saline)
u. a. unter anderem
z. B. zum BeispielEinleitung 1 1. Einleitung Der Grundsatz des deutschen Tierschutzgesetzes nimmt den Menschen in die Verantwortung, nicht nur das Leben, sondern auch das Wohlbefinden des Tieres als Mitgeschöpf zu schützen (§ 1 TierSchG 2020). Zudem hat mit Einführung des § 2, der sogenannten Tierhalternorm, in das Tierschutzgesetz, jeder, der Nutztiere zu Erwerbszwecken hält, im Rahmen betrieblicher Eigenkontrollen Tierschutzindikatoren zu erheben und zu bewerten (§ 11 (8) TierSchG 2020). Damit soll sichergestellt werden, dass der Tierhalter oder -betreuer das Management seiner Tiere so gestaltet, dass er Probleme frühzeitig erkennt und darauf reagieren kann. Im Jahr 2019 wurden in der Bundesrepublik Deutschland auf 1.965 landwirtschaftlichen Betrieben (> 3.000 Hennenhaltungsplätze) über 42 Millionen Jung- und Legehennen gehalten, die insgesamt etwa 12,5 Milliarden Eier produzierten (DESTATIS 2019). Bei der Haltung und dem Management von Jung- und Legehennenherden stellt das Auftreten von Federpicken und Kannibalismus eine große Herausforderung dar (SPINDLER et al. 2016). Es wird als Ausdruck von Verhaltensstörungen mit belastenden Einwirkungen auf die Tiere in Verbindung gebracht (SPINDLER et al. 2016; GIERSBERG et al. 2017) und kann gleichermaßen bei allen Haltungsformen auftreten (GUNNARSSON 1999; SHERWIN et al. 2010). Um die Auswirkungen der Schäden, die sich die Tiere hierdurch gegenseitig zufügen können, gering zu halten, wurde bislang bei den Küken für die konventionelle Legehennenhaltung die Kürzung der sensiblen Schnabelspitze vollzogen (SEPEUR et al. 2015; SPINDLER et al. 2016). Hierbei handelt es sich nach § 6 (1) TierSchG (2020) um eine schmerzhafte und daher grundsätzlich verbotene teilweise Amputation von Körperteilen. Sofern nicht im Einzelfall eine zeitlich befristete Genehmigung durch die zuständige Behörde zum Schutz der Tiere erteilt wurde (§ 6 (3.1) TierSchG 2020), steht dies in Konflikt mit dem deutschen Tierschutzgesetz, einem Tier nicht ohne vernünftigen Grund Schmerzen, Leiden oder Schäden zufügen zu dürfen (§ 1 TierSchG 2020). Dies hat aktuell eine äußerst hohe Relevanz, sowohl beim zunehmend an Tierwohl interessierten Konsumenten (HEISE 2016), als auch für die Landwirte. Da seit dem 01. August 2016 auf das routinemäßige Schnabelkürzen bei Legehennenküken in Deutschland verzichtet wird, können die Folgen in Form von Gefiederschäden und Hautverletzungen aufgrund des spitzen Schnabels gravierender ausfallen (SEPEUR et al. 2015; SPINDLER et al. 2016). Es ist somit auch hier von einer tierschutzrelevanten Beeinträchtigung des Wohlbefindens der betroffenen Tiere auszugehen (RODENBURG et al. 2004; SPINDLER et al. 2016). Auch die ökonomischen Auswirkungen für den Landwirt durch das Auftreten dieser Verhaltensstörungen sind von Bedeutung (WECHSLER et al. 1998). Zur Unterstützung der Landwirte in Bezug auf die angesprochenen Themen hat das Niedersächsische Ministerium für Ernährung, Landwirtschaft und Verbraucherschutz „Empfehlungen zur Verhinderung von Federpicken und Kannibalismus bei Jung- und Legehennen“ (ML 2018) veröffentlicht. Zur Früherkennung von Federpicken und Kannibalismus wird derzeit vornehmlich eine systematische Integumentbonitur mittels manueller Boniturschemata zur Bewertung des Gefieder- und Hautzustandes durchgeführt (TAUSON et al. 2005; Welfare Quality® 2009;
Einleitung 2 SEPEUR et al. 2015; GIERSBERG et al. 2017; CAMPE et al. 2018; KAESBERG et al. 2018; SCHMIDT et al. 2019; SPINDLER et al. 2020). Bezüglich Objektivität und Reproduzierbarkeit ist dies aus wissenschaftlicher Sicht nicht zufriedenstellend. Auch ist der mit intensiverer Tierbetreuung verbundene ökonomische Mehraufwand (SPINDLER et al. 2016) aus Sicht der Praxis nicht zu vernachlässigen. Daher besteht weiterhin der Bedarf, messbare Indikatoren für eine aussagekräftige Beurteilung des Zustandes einer Jung- bzw. Legehennenherde sowie einer objektiven Einschätzung von Tierwohl zu finden. Zur Erfassung von Reaktionen auf Belastungen hat sich bei Tieren die Quantifizierung von Glucocorticoiden im Blut (u. a. BEUVING u. VONDER 1977; ALAM u. DOBSON 1986; KORTE et al. 1997; CARROLL et al. 2006; MCILWRICK et al. 2017; HOFMANN et al. 2019) sowohl in der experimentellen Tierhaltung als auch bei der Durchführung von Feldstudien mit landwirtschaftlichen Nutztieren etabliert. Bei Belastungen wird über die Hypothalamus- Hypophysen-Nebennieren-Achse (HPA-Achse) die Bildung des Hormons ACTH angeregt, infolgedessen speziesspezifisch die Cholesterinabkömmlinge Cortisol und Corticosteron im Blut ansteigen (ROMERO u. FAIRHURST 2016; PALME 2019). Dieser Zustand der gesteigerten Glucocorticoid-Sekretion aufgrund einer Stimulation der HPA-Achse als Antwort auf einen Stressor stellt nach COCKREM (2007) die Definition für „Stress“ dar. In der Vergangenheit wurde zwischen Eustress und Dystress unterschieden (SELYE 1975), dabei wird erstgenannte Form „positiven“ Belastungen, an die sich ein Organismus anpassen kann, zugeordnet. Letztgenannte wird mit „negativen“ Belastungen, an die eine Anpassung nicht möglich ist und zu Frustration bis hin zu körperlichen Auswirkungen führen kann, verbunden. Neuere Publikationen berufen sich darauf, dass Tiere in verschiedenen Situationen positive oder negative „feelings“ (BROOM 2011) oder „emotions“ (REIMERT et al. 2013) erfahren. Da Anstieg und Abfall des Hormonspiegels innerhalb weniger Minuten bzw. über Stunden erfolgen, erlauben Blutuntersuchungen lediglich eine kurzfristige Momentaufnahme über den Cortisol- bzw. Corticosteron-Spiegel eines Tieres (BEUVING u. VONDER 1978; ROMERO u. REED 2005; MORMÈDE et al. 2007; BORTOLOTTI et al. 2008). Jedoch können beide Hormone in keratinisierten Geweben nachgewiesen werden, wo sie während deren Wachstum über längere Zeit eingelagert werden (BORTOLOTTI et al. 2008; DAVENPORT et al. 2006; BERKVENS 2012; BAXTER-GILBERT et al. 2014; HUNT et al. 2014; ROMERO u. FAIRHURST 2016). Das beim Vogel dominierende Glucocorticoid ist Corticosteron (ROMERO u. WINGFIELD 2001; PALME 2019). Abgeleitet von dem Vorgehen bei Haaranalysen (THIEME et al. 2003; KINTZ 2004) wurde das Vorgehen zur Isolierung und Analyse von Corticosteron (CORT) aus Federn erstmalig von BORTOLOTTI et al. (2008) unter Verwendung der Federn von Rothühnern (Alectoris rufa) publiziert. Weitere Studien bei Wildvögeln (u. a. BORTOLOTTI et al. 2009; LATTIN et al. 2011; FAIRHURST et al. 2012; LENDVAI et al. 2013; HARMS et al. 2015; KOUWENBERG et al. 2016; AHARON-ROTMAN et al. 2017; FREEMAN u. NEWMAN 2018; MONCLÚS et al. 2020) geben einen Hinweis darauf, dass sich die Analyse von Corticosteron in Federn (CORTf) retrospektiv für eine Aussage über länger andauernde,
Einleitung 3
belastende Einwirkungen auf den Vogel während des Federwachstums nutzen lässt. Erste
Untersuchungen bei Wirtschaftsgeflügel (z. B. CARBAJAL et al. 2014; JENNI-EIERMANN
et al. 2015; JOHNS et al. 2018; WEIMER et al. 2018; VON EUGEN et al. 2019; NORDQUIST
et al. 2020) unterstützen diese Hypothese. Ein großer Vorteil wird darin gesehen, dass dabei ein
Wert ausgegeben wird, der keinen kurzfristigen Schwankungen unterliegt, sondern CORT über
einen längeren Zeitraum in der Feder akkumuliert (BORTOLOTTI et al. 2008). Federanalysen
bei Jung- und Legehennen der Genetik Lohmann Brown sind zum derzeitigen Stand des
Wissens nicht bekannt.
Bei der Haltung von Jung- und Legehennen könnten in den Federn gemessene CORT-Werte
einen wertvollen Hinweis darüber geben, ob eine Herde während des Federwachstums
dauerhaften Belastungen ausgesetzt war und damit ein erhöhtes Risiko zur Entwicklung von
Verhaltensstörungen aufweist (HÄFFELIN et al. 2020a). Es ist davon auszugehen, dass die
Bedingungen für eine Herde während der Aufzucht – in der die Federn physiologischerweise
wachsen – einen Einfluss auf ihr späteres Verhalten in der Legeperiode haben (SCHREITER
2020). Die Messung von CORTf könnte im Sinne eines non-invasiven Frühwarnsystems dazu
genutzt werden, das Risiko für das Auftreten von Verhaltensstörungen in der Legeperiode,
bedingt durch Belastungen in der Aufzucht, vorherzusagen. Infolgedessen könnten
prophylaktische Maßnahmen eingeleitet und die Herde intensiver betreut werden, mögliche
Belastungsfaktoren aufgedeckt und die Haltung dementsprechend verbessert werden. Ferner
ist, bei entsprechender Varianz, CORTf als Selektionskriterium in der Zucht stressresilienter
Hybridlinien denkbar (HÄFFELIN et al. 2020a).
Modifikationen sowie Unterschiede in Versuchsaufbau und -auswertung erschweren einen
Vergleich zwischen verschiedenen Studien (ROMERO u. FAIRHURST 2016). BORTOLOTTI
(2010) rät von voreiligen Interpretationen von Ergebnissen ab, solange die physiologischen
Vorgänge zur Einlagerung von CORT in die Feder sowie technische Einflüsse bei der
Extraktion ungeklärt sind. Fehlende Vergleichbarkeit könnte durch eine Standardisierung des
Protokolls gelöst werden. Bei der Anwendung neuer Methoden für neue Spezies oder eines
neuen Substrates ist zudem vor der Durchführung von Feldstudien zunächst eine Validierung
der Methodik unabdingbar (BUCHANAN u. GOLDSMITH 2004; TOUMA u. PALME 2005;
PALME 2019).
Ziel der Arbeit war daher, zu untersuchen, ob sich Corticosteron in Federn von Jung- und
Legehennen valide und wiederholbar messen lässt, um eine Grundlage für einen möglichen
objektiven und non-invasiven Tierschutzindikator (TierSchG 2020) zu schaffen. Daraus leiten
sich folgende Fragestellungen ab, die im Rahmen der vorliegenden Arbeit untersucht wurden:
Lässt sich Corticosteron in Federn von Jung- und Legehennen valide und wiederholbar
messen?
Welche Methodik und welcher Federtyp bzw. welche Federflur (Pteryla) eignen sich
zur repräsentativen Bestimmung von Corticosteron in Federn von Jung- bzw.Einleitung 4
Legehennen?
Mit welcher inter- und intra-individuellen Varianz ist bei der Messung von
Corticosteron in Federn von Jung- bzw. Legehennen zu rechnen?
Den Fragestellungen wurden im Rahmen des Promotionsvorhabens insgesamt zwei
Publikationen gewidmet.Eingebundene Manuskripte 5 2. Eingebundene Manuskripte Darlegung des selbständigen Anteils an den vorliegenden Untersuchungen gemäß § 8 (3) der Promotionsordnung der Tierärztlichen Hochschule Hannover für die Erteilung des Grades eines Doctor medicinae veterinariae: HÄFFELIN, K. E., LINDENWALD, R., KAUFMANN, F., DÖHRING, S., SPINDLER, B., PREISINGER, R., RAUTENSCHLEIN, S., KEMPER, N. u. ANDERSSON, R. (2020a): Corticosterone in Feathers of Laying Hens: An Assay Validation for Evidence-based Assessment of Animal Welfare. Poultry Science 99 (10), 4685-4694 Der Eigenanteil umfasst die Erstellung des Studiendesigns unter der wissenschaftlichen Betreuung von R. Andersson, R. Lindenwald und S. Döhring, die Durchführung sämtlicher Labortätigkeiten und Datenauswertung nach Einarbeitung durch R. Lindenwald, sowie die Verfassung des Manuskriptentwurfes. HÄFFELIN, K. E., KAUFMANN, F., LINDENWALD, R., DÖHRING, S., SPINDLER, B., PREISINGER, R., RAUTENSCHLEIN, S., KEMPER, N. u. ANDERSSON, R. (2020b): Corticosterone in feathers: Inter- and intraindividual variation in pullets and the importance of the feather type. Veterinary & Animal Science, in press. doi: 10.1016/j.vas.2020.100155 Der Eigenanteil umfasst die Erstellung des Studiendesigns unter der wissenschaftlichen Betreuung von R. Andersson und F. Kaufmann, die Durchführung sämtlicher Labortätigkeiten, Datenauswertung sowie die Verfassung des Manuskriptentwurfes.
Corticosterone in feathers of laying hens: an assay validation for
evidence-based assessment of animal welfare
K. E. H€affelin,*,1 R. Lindenwald,y F. Kaufmann,* S. D€ohring,* B. Spindler,z R. Preisinger,x
S. Rautenschlein,y N. Kemper,z and R. Andersson*
*Faculty of Agriculture Sciences and Landscape Architecture, Osnabr€uck University of Applied Sciences, 49090
Osnabr€uck, Germany; yClinic for Poultry, University of Veterinary Medicine Hannover (Foundation), 30559
Hannover, Germany; zInstitute for Animal Hygiene, Animal Welfare and Farm Animal Behavior, University of
Veterinary Medicine Hannover (Foundation), 30173 Hannover, Germany; and xEW GROUP GmbH, 49429
Visbek, Germany
ABSTRACT Studies indicate that the evaluation of respectively. The serial dilution showed linearity and
animal welfare in birds may be carried out with the parallelism. Examining the hormone extraction effi-
measurement of the stress-related hormone corticoste- ciency by using different methanol volumes resulted in no
rone in feathers. However a standardized procedure for statistical differences (P . 0.05). Pulverized feathers
corticosterone measurements in feathers is lacking, a showed higher corticosterone values than minced
validation needs to be carried out for each new species feathers (P . 0.05). Differences were shown between 2
before implementation. The aim of the present study was feather types (tail vs. interscapular feathers; P , 0.05),
to establish a valid method to measure corticosterone as well as between vane and rachis (P , 0.05). Perfor-
concentrations in feathers of laying hens in a precise and mance of a freeze–thaw cycle led to a decrease of corti-
repeatable manner using an established and commer- costerone concentrations in the samples. A possible effect
cially available ELISA. Validation was performed with of UV-A radiation on the stability of corticosterone in the
feather pools of tail and interscapular feathers of feathers was not found (P . 0.05). With the present
commercial Lohmann Brown laying hens. Assessment study, a valid protocol, feasible for analyzing feather
groups, consisting of 5 replicates, were created. All rep- pools of laying hens, was developed. It may provide
licates of an assessment group were processed at the same fundamentals for further investigations on corticosterone
time. Each replicate was run in 4 repetitions by ELISA. in feathers as a noninvasive indicator to evaluate aspects
Intra-assay and interassay CV was 7.5 and 6.4%, of animal welfare.
Key words: glucocorticoid, HPA axis, indicator, stress, domestic chicken
2020 Poultry Science 99:4685–4694
https://doi.org/10.1016/j.psj.2020.06.065
INTRODUCTION measurement and evaluation of environmental and
animal-related signals (Mormede et al., 2007). In Ger-
Animal welfare in livestock production has become many, farmers bearing the responsibility for commercial
increasingly important in recent years (Broom, 2010; livestock are legally obligated to evaluate the state of their
Butterworth, 2013; Sandøe et al., 2020). As a consequence, animals using welfare-associated indicators (TierSchG,
animal welfare and especially the assessment of animal 2020). However, monitoring and evaluating animal wel-
welfare is a focal point in various research fields fare in farm animals needs to be feasible under commercial
(Mormede et al., 2007). There is consensus among conditions and requires a competent and, at best, an objec-
different stakeholders and the academic landscape that tive and evidence-based view (Giersberg et al., 2017). In
animal welfare assessment heavily relies on the laying hens, the condition of the plumage and the integu-
ment acts as an indicator for feather pecking and canni-
balism, both being behavioral disorders caused by
Ó 2020 The Authors. Published by Elsevier Inc. on behalf of Poultry various challenges the birds had or have to cope with
Science Association Inc. This is an open access article under the CC BY (Sepeur et al., 2015; Giersberg et al., 2017). Recent studies
license (http://creativecommons.org/licenses/by/4.0/).
Received April 15, 2020.
showed that the evaluation of animal welfare in birds may
Accepted June 25, 2020. be carried out objectively and noninvasively with the mea-
1
Corresponding author: k.haeffelin@hs-osnabrueck.de surement of the stress-related hormone corticosterone in
46854686 H€aFFELIN ET AL.
feathers (Bortolotti et al., 2008; Bortolotti et al., 2009; procedure, authors applied different methods to detect
Fairhurst et al., 2011; Carbajal et al., 2014; Ganz et al., and quantify corticosterone in feathers. These varia-
2018; Johns et al., 2018; Weimer et al., 2018; Alba et al., tions, such as variations in the amount of feather mate-
2019; von Eugen et al., 2019; Nordquist et al., 2020). rial or methanol volume for the extraction, crushed vs.
When exposed to certain stressors, the hypothalamic– grind up feathers, and different methods for filtration
pituitary–adrenal axis responds with the secretion of or different assays, make it rather impossible to compare
corticosterone in birds (Touma and Palme, 2005; the results, properly (Romero and Fairhurst, 2016).
Palme, 2019) and cortisol in most of the mammals As investigations on corticosterone in feathers are
(Palme, 2019). Consequently, the concentrations of rather new in laying hens, a method validation is
corticosterone in the blood increase within min in required, which includes the determination of precision,
captured wild birds of different species (Romero and specificity, sensitivity, and accuracy (Buchanan and
Reed, 2005) as well as in laying hens (Beuving and Goldsmith, 2004; Touma and Palme, 2005; Palme,
Vonder, 1978) and decrease within h, depending on the 2019). This is essential, given that, to the best of our
initial stressor they have been exposed to (Beuving and knowledge, no proper validation for commercial laying
Vonder, 1978). In humans, a half-life of circulating corti- hens has been performed before and, especially, because
costerone of about 1 h is reported (Doggui, 2012); how- noninvasive measurements of corticosterone and related
ever, no values are found for birds. The quantification hormones are finding their way into field studies, applied
of hormone levels influenced by the hypothalamic–pitu- by researchers being new in the field of noninvasive endo-
itary–adrenal axis has been applied over y as an indica- crine assessments (Buchanan and Goldsmith, 2004).
tor for stress and animal welfare in farm animals Alba et al. (2019) validated another method for domestic
(Beuving and Vonder, 1978; Dehnhard et al., 2003; chickens, using a keratinase to digest the protein matrix
Rettenbacher et al., 2004; Odihambo Mumma et al., in the first step. Berkvens (2012) validated a modified
2006; Mormede et al., 2007; Palme, 2019) and others method for Barred Rock hens. Carbajal et al. (2014)
(Bortolotti et al., 2008; Sheriff et al., 2011; Fairhurst evaluated a method for broilers. Thus, the objective of
et al., 2013; Schmaltz et al., 2016; Robertson et al., the present study was to establish a reliable and valid
2017; Peric et al., 2018; Palme, 2019). In addition to method to measure corticosterone concentrations in
blood, several matrices, such as feces (Rettenbacher feathers of laying hens. Therefore, we focused on the
et al., 2004; M€ostl et al., 2005; Touma and Palme, assay validation and extraction efficiency first, using rep-
2005; Weimer et al., 2018; Palme, 2019) and eggs licates, and thereafter, further technical influences
(Rettenbacher et al., 2005; Schmaltz et al., 2016), are (based on Bortolotti, 2010; Romero and Fairhurst,
used to detect and quantify corticosterone or its metab- 2016) were examined, such as the manner of crushing
olites in birds, whereas in other species, the use of saliva the feathers (Newman and Freeman, 2018), different
(Mormede et al., 2007), urine (Hay and Mormede, 1997), parts (Newman and Freeman, 2018) and types of
milk (Tucker and Schwalm, 1977), and hair (Arnon feathers (Moncl us et al., 2017; Weimer et al., 2018), as
et al., 2016; Peric et al., 2018) is reported. Bortolotti well as an effect of defrosting samples several times.
et al. (2008) succeeded in detecting circulating cortico- Furthermore, as it was recommended by Romero and
sterone deposited in feathers of adult red-legged par- Fairhurst (2016), the effect of UV radiation on feather
tridges (Alectoris rufa), which were exposed to corticosterone stability was investigated briefly as hens
stressors over wk during growth, when feathers are sup- are exposed to UV radiation in outdoor runs and also
plied with blood. Thereby, a promising tool was found, in floor husbandry systems where artificial light with a
in contrast of measuring blood parameters, which react natural daylight spectrum is getting increasingly imple-
within a short period of time and therefore are less suit- mented because of animal welfare issues (K€ammerling
able to evaluate long-term liabilities (Mormede et al., et al., 2017; TierSchNutztV, 2017).
2007; Bortolotti et al., 2008), as we assume for poor an-
imal welfare.
Subsequent studies on corticosterone in feathers were
MATERIALS AND METHODS
performed mostly in wild birds (e.g., Bortolotti et al., Subjects
2009; Koren et al., 2011; Lattin et al., 2011; Fairhurst
et al., 2012; Lendvai et al., 2013; Harms et al., 2015; Generally, body feathers from the interscapular area
Kouwenberg et al., 2016; Aharon-Rotman et al., 2017; (Carbajal et al., 2014; Moncl us et al., 2017), hereinafter
Freeman and Newman, 2018; Moncl us et al., 2020), us- referred to as interscapular feathers (Moncl us et al.,
ing feather corticosterone as a retrospective view on 2017), and rectrices (Aharon-Rotman et al., 2017;
challenges the birds had to cope with during feather Robertson et al., 2017; Freeman and Newman, 2018),
growth. Despite the wild birds, results of first investiga- hereinafter referred to as tail feathers (Aharon-Rotman
tions in poultry (Berkvens, 2012; Carbajal et al., 2014; et al., 2017; Robertson et al., 2017), were pulled from
Jenni-Eiermann et al., 2015; Zeinstra et al., 2015; 11 adult laying hens. Feathers were collected from com-
Johns et al., 2017; Weimer et al., 2018; Alba et al., mercial Lohmann Brown laying hens (Lohmann Tier-
2019; von Eugen et al., 2019; Nordquist et al., 2020; Lin- zucht GmbH, Cuxhaven, Germany), a commonly used
denwald and Rautenschlein, unpublished data) are also genotype in Germany, as soon as discovering the bird’s
encouraging; however, in the absence of a standardized death. The animals originated from 7 flocks ofANIMAL WELL-BEING AND BEHAVIOR 4687
commercial farms in Germany, where they were kept in Assay Validation
accordance with local legislation (TierSchNutztV,
2017). Collected feathers were stored dark and dry in pa- The validation of the assay was performed in consider-
per envelopes at room temperature as recommended by ation of the recommendations by Buchanan and
Bortolotti et al. (2009) and Monclus et al. (2017). Every Goldsmith (2004), Sheriff et al. (2011), and Palme
feather was thoroughly cleaned (based on Jenni- (2019). Feather corticosterone concentrations were
Eiermann et al., 2014; von Eugen et al., 2019) with analyzed using the commercial Enzo Life Sciences Cortico-
distilled water and degreased by bathing it in HPLC- sterone ELISA Kit ADI-901-097 (Enzo Life Sciences Inc.),
grade methanol (Carl Roth GmbH 1 Co. KG, Karls- a competitive immunoassay, also used by Bourgeon et al.
ruhe, Germany) for 2 to 4 s (based on Robertson et al., (2014), Harris et al. (2016), and Harris et al. (2017),
2017). Based on the studies by Lattin et al. (2011) and whereby samples were incubated with a sheep polyclonal
Freeman and Newman (2018), different feather pools antibody to corticosterone (Corticosterone ELISA Anti-
were prepared, consisting of the same feather type of body by Enzo Life Sciences Inc.) over 2 h, first. After a
one animal or different animals, depending on the washing procedure (Wash Buffer Concentrate by Enzo
research question, as described in the chapters that Life Sciences Inc.), a p-nitrophenyl phosphate (p-Npp
follow, and subsequently processed. Table 1 gives an Substrate by Enzo Life Sciences Inc.) was added, followed
overview over the created pools. by a 1-h incubation. Finally, the Stop Solution (Enzo Life
Sciences Inc.) completed the reaction.
Every sample was analyzed in 4 repetitions each. To
Corticosterone Extraction validate the assay, all replicates related to the same
Feather corticosterone extraction was undertaken us- research question were run in the same assay, with the
ing a modified procedure of that described by Bortolotti exception of samples intended to calculate the interassay
et al. (2008). In general, after removing the calamus of variation and the validation of the freeze–thaw cycle.
every feather, feathers of one pool (see Table 1) were Precision of the ELISA was expressed via intra-assay
crushed simultaneously, vortexed to homogenize the par- and interassay CV. Intra-assay CV was calculated over
ticles, and then aliquoted to samples of 10.0 mg (range of all samples (n 5 70 samples, each 4 repetitions). Interas-
9.5 mg to 10.5 mg; precision balance Mettler; Spoehrhase say CV was examined by analyzing 2 replicates (each 4
A.G., Giessen) each (based on Freeman and Newman, repetitions) of an interscapular feather pool consisting
2018). Up to 5 replicates were related to 1 assessment of 25 feathers of 1 animal (Table 1). The 2 replicates
group, which went through the same treatment, depend- were stored at 280 C and defrosted separately when
ing on the research questions described as follows (see also analysis was performed.
Table 1). Replicates used for serial dilution and interas- Specificity of the ELISA was tested by examining the
say variation, as well as the freeze–thaw cycle, amounted linearity of a serial dilution (Carbajal et al., 2014) and
50.0 mg (range of 49.5 mg to 50.5 mg) and 100.0 mg (range the parallelism of the serial dilution and the standard
of 99.5 mg to 100.5 mg), respectively, for being expected curve of each assay (Bourgeon et al., 2014; Carbajal
to decrease in their levels. HPLC-grade methanol (Carl et al., 2014; Glucs et al., 2018). Therefore, a replicate of
Roth GmbH 1 Co. KG) was added to each sample, and a pool of interscapular feathers (17 feathers of 1 laying
extraction was then initiated with an ultrasonic bath hen, Table 1) was used and diluted 1:2, 1:4, 1:5, and 1:10
(VWR International, LLC, Radnor) for 30 min, followed with the assay buffer (Tris-buffered saline) before
by an incubation of 12 h (Freeman and Newman, 2018). analyzing.
Samples therefore were placed on a moving vortex plat-
form at 50 C (Aharon-Rotman et al., 2017). Subse- Technical Issues
quently, feather particles of each sample were separated
from methanol by pressure filtration using polyether sul- Extraction Efficiency To examine the required quan-
fone syringe filters with a mesh diameter of 22 mm (Carl tity of methanol for a complete feather corticosterone
Roth GmbH 1 Co. KG). To avoid loss of extracted corti- extraction (Romero and Fairhurst, 2016), a pool of 38
costerone, sample vials were washed twice using 1.0 mL of interscapular feathers plucked from 1 laying hen was
HPLC-grade methanol (Carl Roth GmbH 1 Co. KG) pulverized using a ball mill (MM-400; Retsch, Germany;
that was subsequently filtered and added to formerly also used by Ganz et al., 2018; see Table 1). Twenty-five
filtered methanol from the sample. To evaporate the replicates were created; of which, 5 were treated with
methanol, samples were placed into a water bath at 0.5 mL, 1.0 mL, 5.0 mL, 10.0 mL, or 15.0 mL HPLC-
40 C until complete evaporation. Based on the studies grade methanol (Carl Roth GmbH 1 Co. KG) each
by Harris et al. (2016), Harris et al. (2017), and (based on the study by Newman and Freeman, 2018).
Moncl us et al. (2017) samples were resuspended in Processing and analyzing of the replicates followed the
500 mL of Tris-buffered saline, which was provided by procedure as described previously.
the ELISA kit (Assay Buffer 15 by Enzo Life Sciences Mincing vs. Pulverizing Investigations regarding the
Inc., New York). Samples were frozen at 240 C for up influence of the crushing method (Newman and
to 12 h until examination; samples for long-term investi- Freeman, 2018) were performed using a pool of 10 tail
gations, such as the interassay variation and the freeze– feathers taken from 1 animal (see Table 1). All feathers
thaw cycle, were stored at 280 C. were minced using scissors (following Bortolotti et al.,4688 H€aFFELIN ET AL.
Table 1. Feather pools created to corresponding research question.
N
Pools Replicates Feather type Feathers Laying hens
Interassay CV 2 Interscapular 25 1
Serial dilution 1 Interscapular 17 1
Methanol volume 25 Interscapular 38 1
Mincing 5 Tail 10 1
Pulverizing 5 Tail
Tail 5 Tail
Interscapular 5 Interscapular 17
Vane 5 Tail 14 8
Rachis 5 Tail
Freeze–thaw 3 Interscapular 25 1
UV-A radiation 10 Interscapular 80 3
2008) and then vortexed. Half of the amount of the (Table 1), was used to create 2 different groups, one for
minced and vortexed feathers were further processed and an UV-A treatment, and one as a control group. The ma-
pulverized using a ball mill (MM-400; Retsch, Germany; terial of each group was spread into a petri dish. Consid-
also used by Ganz et al., 2018). Therefore, the replicate ering the total amount of radiation laying hens are exposed
was placed into a metal container, frozen in liquid ni- to with a lighting system for poultry during feather growth
trogen for 3 min to embrittle, and then pulverized for in the rearing period, the treatment group was placed 1 m
1 min at 30 Hz. The minced and pulverized samples were beneath UV lights (LEDfactory B.V., Leeuwarden, the
divided into 5 replicates each and enriched with 5.0 mL Netherlands) emitting a wavelength of 315 nm to 380 nm
HPLC-grade methanol (Carl Roth GmbH 1 Co. KG) to and a radiation power of 0.0676 Watt/m2 at room tem-
extract feather corticosterone as mentioned previously. perature for 18 D. The control group was placed in an
Tail vs. Interscapular Feathers To investigate the ef- opaque box and stored for 18 D beneath the treatment
fect of feather type (Monclus et al., 2017; Weimer et al., group. After 18 D, 5 replicates were created out of the
2018), a pool of 10 tail feathers and a pool of 17 inter- groups and examined for feather corticosterone concen-
scapular feathers of the same laying hen (Table 1) were trations following the aforementioned procedure.
pulverized. Five replicates each were taken, as described
before. Extraction was performed using 5.0 mL of Statistical Analyses
HPLC-grade methanol (Carl Roth GmbH 1 Co. KG) for
each replicate. Calculation of feather corticosterone concentrations
Vane vs. Rachis To examine potential differences of was performed as per the product manual of Enzo Life Sci-
feather corticosterone concentrations within one feather, ences Corticosterone ELISA Kit ADI-901-097 (Enzo Life
the rachis and vane (Newman and Freeman, 2018) of 14 Sciences Inc.), whereby the standard curve fitting was per-
tail feathers, distinguished and pooled from 8 different formed using a 4-parameter logistic curve to interpolate
laying hens (Table 1), were analyzed. After separating feather corticosterone concentrations (also used by
the vane and rachis of feathers with a scalpel, the vane Gurung et al., 2018) by means of the Magellan data anal-
and rachis pools were pulverized separately and then ysis software 7.2 (Tecan Group Ltd., M€annedorf,
aliquoted before feather corticosterone extraction was Switzerland), after measurements of the optical density
performed using 5.0 mL HPLC-grade methanol (Carl at 405 nm with an absorbance microplate reader (Tecan
Roth GmbH 1 Co. KG) for each replicate. Group Ltd., M€annedorf, Switzerland). Data management
Freeze–Thaw Cycle A pool of 25 interscapular and calculations regarding descriptive statistics were
feathers of 1 animal was pulverized, and 3 replicates performed using Microsoft Excel 2019 (Microsoft Corpo-
were created (Table 1) and extracted applying the ration, Redmond). Statistical analyses were carried out
aforementioned method. They were stored at 280 C. To using the software package Minitab 16.2.3 (Minitab
examine the effect of the freeze–thaw cycle on feather LLC., State College). Feather corticosterone value of
corticosterone extraction and concentration, all repli- 1 sample was calculated as the arithmetic mean over the
cates were defrosted 24 h after freezing, as part of the 4 repetitions. Generally, repetitions having a CV less
original protocol. While replicate 1 was analyzed after than 20% were included in statistical analyses (based on
these 24 h, the remaining 2 replicates were frozen again Kinn Rød et al., 2017). Feather corticosterone values
and both defrosted after another 2 wk. While replicate 2 were converted from the unit pg/mL, given by the ELISA,
was then examined, the third replicate was frozen again to pg/mg, except for testing linearity and parallelism
until examination after another 16 wk. Therefore, the 3 (based on Carbajal et al., 2014). Values of the diluted sam-
replicates underwent a freeze–thaw cycle once, twice, or ples were plotted against the calculated corticosterone
thrice and were frozen for 1, 15, and 113 D, respectively. concentrations (Carbajal et al., 2014). For the parallelism
All analyses were carried out as mentioned previously. test, results were logarithmized to the base 10, and a linear
Effect of UV-A Radiation A pool of 80 pulverized inter- regression was calculated (based on Carbajal et al., 2014).
scapular feathers, taken from 3 different laying hens To assess distribution, Anderson–Darling normality testANIMAL WELL-BEING AND BEHAVIOR 4689
Figure 1. Linearity of the serial dilution. Figure 3. Effect of different methanol volumes (n 5 5 replicates each
group; *: 2 values, 108.9 pg/mg and 108.8 pg/mg, are overlapping).
was calculated. To show possible differences, a Kruskal–
Wallis test was performed for the methanol groups. Technical Issues
With the Mann–Whitney U-test, differences between the Extraction Efficiency Varying the methanol volume
other groups (mincing vs. pulverizing, tail vs. interscapu- for feather corticosterone extraction did not show any
lar feathers, vane vs. rachis, effect of UV-A radiation) were significant differences (P 5 0.204; Figure 3).
tested. Significance was assumed at the level of P , 0.05. Mincing vs. Pulverizing Although not being signifi-
cant, feather corticosterone concentrations of the sam-
ples being crushed by scissors resulted in lower values
RESULTS (19.3 pg/mg, SD 5.00 pg/mg, n 5 5) than those of
samples treated with the ball mill (23.3 pg/mg, SD
Assay Validation 3.13 pg/mg, n 5 5; P 5 0.296; see also Table 2).
Tail vs. Interscapular Feathers Feather corticosterone
Intra-assay CV over all samples was in average 7.5%
concentrations in tail feathers were significantly
(median, n 5 70 samples), whereas interassay CV was
(P 5 0.012) lower (23.3 pg/mg, SD 3.13 pg/mg, n 5 5)
6.4% (n 5 2 samples). Linearity of the serial dilution
than those of interscapular feathers (80.0 pg/mg, SD
(n 5 4 diluted samples) was R2linearity 5 0.997, described
18.14 pg/mg, n 5 5; Table 2).
by the formula ylinearity 5 0.1352x 1 95.58 (Figure 1).
Vane vs. Rachis Feather corticosterone concentra-
Parallelism of the serial dilution and the standard curve
tions of the vane and rachis were assessed separately
is shown in Figure 2, with R2dilution 5 0.873
and showed significant differences (P 5 0.012) with
(ydilution 5 0.5358x 1 0.8315) and R2standard 5 0.989
61.7 pg/mg (SD 15.06 pg/mg, n 5 5) in the vanes and
(ystandard 5 1.103x – 119.3), respectively.
23.3 pg/mg (SD 3.43 pg/mg, n 5 5) in the rachises
(Table 2).
Freeze–Thaw Cycle The initial feather corticosterone
concentration of the measurement was 25.3 pg/mg
(SD 1.05 pg/mg, 4 repetitions). The concentration
decreased within the cycle, amounting 17.2 pg/mg (SD
1.25 pg/mg, 4 repetitions) after defrosting twice, up to
a final value of 8.0 pg/mg (SD 0.56 pg/mg, 4 repetitions)
after defrosting thrice.
Effect of UV-A Radiation Exposure to UV-A radiation
did not affect concentrations or traceability of cortico-
sterone in feathers (P 5 0.403). Mean feather cortico-
sterone concentration of the UV-A–treated samples was
49.4 pg/mg (SD 10.51 pg/mg, n 5 5), whereas the
control group samples had a mean of 42.6 pg/mg (SD
10.58 pg/mg, n 5 5; Table 2).
DISCUSSION
Measuring corticosterone in feathers of laying hens
may be a suitable tool to evaluate birds’ welfare. More-
Figure 2. Parallelism test of the serial dilution and the standard over, feather corticosterone measurements may be valu-
curve. able for an indicator-based flock management as flocks4690 H€aFFELIN ET AL.
Table 2. Feather corticosterone concentrations of different pools in pg/mg,
each pool consisting of n 5 5 replicates.
Pools Mean SD Median Minimum Maximum
Tail feathers (minced) 19.3 5.00 22.5 11.1 24.3
Tail feathers (pulverized) 23.3 3.13 24.2 18.2 26.8
Interscapular feathers 80.0 18.14 79.5 48.8 92.2
Vane 61.7 15.06 54.9 43.5 87.2
Rachis 23.3 3.43 25.4 19.1 27.8
UV-A group 49.4 10.51 44.0 37.9 67.0
Control group 42.6 10.58 40.2 32.1 61.2
and individuals, who had to cope with adverse husband- Palme, 2005; Odihambo Mumma et al., 2006), showing
ry conditions during feather growth in the rearing period a responsive hypothalamic–pituitary–adrenal axis. How-
and are therefore susceptible to develop behavioral disor- ever, the ACTH challenge test is based on reactions
ders (e.g., feather pecking and cannibalism), can be iden- within h; thus, a method to perform an adequate physi-
tified and treated accordingly. In addition, a correlation ological validation of corticosterone in feathers growing
between altered feather corticosterone levels and behav- over wk is desired (Berk et al., 2016).
ioral disorders of individual birds would enable breeders Results of intra-assay and interassay CV being lower
to implement corticosterone in feathers in breeding than 10%, as calculated in the present study, indicate a
schemes to provide stress resilient genetics, to address good precision of the assay (Carbajal et al., 2014). Using
the occurrence of feather pecking, which is a heritable the same ELISA kit as in the present study, Bourgeon
trait but difficult to quantify directly (Grams et al., et al. (2014), Harris et al. (2016), and Harris et al.
2015). The present study suggests a reliable protocol (2017) achieved comparable results. In addition, a more
for measuring corticosterone in feathers; however, there precise assessment could be done using low and high
are still unclear aspects when quantifying it, such as the concentrated feather corticosterone samples (Palme,
deposition of corticosterone into feathers (Bortolotti 2019). Yet, the present values were in the range of what
et al., 2010) per se. Jenni-Eiermann et al. (2015) can be achieved for intra-assay and interassay CV as
addressed this issue in their research with feathers of pi- per the product manual for the assay (Enzo Life
geons and were able to recover injected and labeled corti- Sciences Inc., 2019). The linearity of the serial dilution
costerone. Additional “unresolved technical issues” indicated a good specificity, also shown in broilers by
(Romero and Fairhurst, 2016), such as influences on Carbajal et al. (2014). Furthermore, it showed that
the deposition (Romero and Fairhurst, 2016) and the measured feather corticosterone concentrations are in
so-called small sample artifact (Lattin et al., 2011; the quantitative range of the assay. The parallelism test
Berk et al., 2016), which describes the appearance of led to acceptable results: R2 for the standard curve was
higher feather corticosterone concentrations in small comparable with that of the study by Carbajal et al.
sample masses, compared with larger ones, are discussed. (2014), who achieved 0.988. However, their modified
Therefore, besides studies on effects of stressors influ- standard curve showed a higher R2 than that of the pre-
encing feather corticosterone levels, different validation sent study (0.934 vs. 0.873). Serial dilutions not being
studies have already been performed in different species parallel with the standard curve may be affected from
(Lattin et al., 2011; Carbajal et al., 2014; Berk et al., interfering substances (Bourgeon et al., 2014; Freeman
2016; Harris et al., 2016; Robertson et al., 2017; and Newman, 2018). Reference values regarding cross
Freeman and Newman, 2018). Furthermore, an official reactivity and sensitivity of the assay were taken from
abbreviation for corticosterone should be established the product manual of the ELISA kit (Arnon et al.,
(Raff, 2016), such as ACTH for adrenocorticotropic 2016), mentioning 28.6% for deoxycorticosterone, 1.7%
hormone. for progesterone, and several derivatives of cholesterol
having a cross reactivity lower than 0.28%, and the lowest
Assay Validation detection limit for corticosterone is represented at
26.99 pg/mL (Enzo Life Sciences Inc., 2019). To the
The present study performed an analytical validation, best of our knowledge, no recombinant deoxycorticoster-
through the determination of precision, specificity, sensi- one of chickens is available to check the cross reactivity
tivity, and accuracy (Palme, 2019) and the investigation for them. Cross reactivity is only described for unsatu-
on few technical issues. Yet, a physiological or biological rated steroids but not for 5a- or 5b-reduced corticosterone
validation to fulfill a complete validation as recommen- metabolites (Enzo Life Sciences Inc., 2019). This has to be
ded by Palme (2019) is missing, owing to the lack of a considered when interpreting results. On the other hand,
suitable method (Berk et al., 2016): Studies analyzing the slope of the serial dilutions being smaller than the
corticosterone concentrations in serum, plasma, or slope of the standard curve (Figure 2) may indicate
excreta commonly use an ACTH challenge test for phys- that, instead of unspecific binding, there may be less bind-
iological validation (Palme, 2019). In domestic chickens, ing of actually available corticosterone. This can be
several studies using ACTH are reported (Dehnhard explainable by the results of Kinn Rød et al. (2017)
et al., 2003; Rettenbacher et al., 2004; Touma and finding less corticosterone in the Enzo Life SciencesANIMAL WELL-BEING AND BEHAVIOR 4691 ELISA Kit compared with others. The affinity for evaluating and comparing results are birds’ genotype chickens’ corticosterone coming from feathers may be and age: Jenni-Eiermann et al. (2015) showed that the lower in some binding sites of the polyclonal antibody. deposition of corticosterone into feathers may also be Considering parallelism, an inappropriate sample mass affected by melanism, which has to be taken into account should be taken into account, which emphasizes the when comparing results from white and brown layers, importance of a consistent sample mass. Freeman and respectively. Monclus et al. (2017) did not find different Newman (2018) determined the optimal sample mass values in birds of different ages. for feathers of the wild turkey (Meleagris gallopavo), Can- ada jay (Perisoreus canadensis), and black-capped chick- adee (Poecile atricapillus) by serial dilutions of different Technical Issues on Corticosterone sample masses. Applying this in further investigations Extraction on corticosterone in feathers of laying hens may improve the present procedure. Regarding accuracy, spike recov- Extraction Efficiency An appropriate validation re- ery by the application of the Enzo Life Sciences Cortico- quires an efficient hormone extraction (Buchanan and sterone ELISA kit was assessed by Aharon-Rotman et al. Goldsmith, 2004). In the present study, 5 different vol- (2017) using plasma samples of house sparrows (Passer umes of methanol were used for extraction purposes; domesticus), spiked with tritiated corticosterone. They however, no differences in feather corticosterone values achieved an average accuracy of 92.2 6 2.1% (Aharon- were found, which is in accordance with the study by Rotman et al., 2017). Spike recovery for this kit using Freeman and Newman (2018), using 5.0 mL and 10.0 mL feathers of chickens is lacking. of methanol (P . 0.05), respectively. Thus, we conclude, Unlike Bortolotti et al. (2008) who recommend the a saturation of methanol was not achieved, and corti- unit pg/mm for feather corticosterone concentrations, costerone was extracted completely from the feathers. samples used in the present study were standardized The decision of using 5.0 mL for the further group on mass, using pg/mg (Moncl us et al., 2017; Robertson treatments was based on the fact that this volume et al., 2017; Freeman and Newman, 2018). This seems showed the smallest variation of feather corticosterone to be reasonable for the authors as the aim was to values (see Figure 3) combined with practical issues, compare the same sample under different treatments such as handling the samples and the duration of and to determine their repeatability, rather than investi- evaporation. gate feather corticosterone concentrations within a sin- Mincing vs. Pulverizing Freeman and Newman (2018) gle feather under consideration of its growth rate. found higher feather corticosterone concentrations when Consequently, replicates were created of feather pools feathers were pulverized than those when feathers being (Lattin et al., 2011; Freeman and Newman, 2018). minced by scissors (P , 0.05). Although no significant Applying this methodology in the present study avoided difference between the groups was found in the present the small sample artifact as every sample had the same study, results show the same tendency (P . 0.05). Pul- weight (Lattin et al., 2011; Berk et al., 2016). On the verization increases the surface of the sample and thus other hand, weighing variations may also lead to high may explain the increased feather corticosterone values variance in results (Bortolotti, 2010), which has to be (Sheriff et al., 2011; Romero and Fairhurst, 2016; taken into account when applying this method. In addi- Freeman and Newman, 2018). In contrast to Freeman tion, it should be considered that comparing absolute and Newman (2018), results of the minced samples values between studies based on modified procedures showed a higher variability in the present study, which could lead to incorrect conclusions (Palme, 2019). Inves- may simply be explained with the lack of homogeny tigations on different commercial ELISA kits showed when compared with pulverized samples. Consequently, that comparisons can be carried out based on relative the ball mill treatment was applied for the other groups values at most (Abelson et al., 2016; Kinn Rød et al., in this study. 2017) but not on “true values” (Kinn Rød et al., 2017). Tail vs. Interscapular Feathers As expected, different An external standard for analyzing corticosterone in feather types of the same bird showed significant differ- feathers via an ELISA is not known. These results ences in feather corticosterone concentrations emphasize the need for researchers to evaluate the pro- (P , 0.05). Different feather types grow during different cedure they use in their own standardized way (Berk periods of time with different growth rates (Rohwer and et al., 2016) and to describe and point out modifications Rohwer, 2013; H€affelin and Andersson, unpublished as long as an official standardization is lacking. Never- data) and thus are exposed to corticosterone over theless, the range of corticosterone concentrations in different durations (Moncl us et al., 2017). In addition, broiler feathers measured by Carbajal et al. (2014) via the difference in structure between feather types may an ELISA in ng/mL is comparable with the values given have an impact on the deposition of corticosterone into in pg/mL from the ELISA used in the present study. feathers (Romero and Fairhurst, 2016). Moncl us et al. This underlines the necessity of investigations on (2017) did not find a correlation between primary and species-specific values (Fairhurst et al., 2012, 2013; interscapular feathers. Weimer et al. (2018) found “a Kouwenberg et al., 2016) or a species-specific curve of strong correlation” between corticosterone in primary sample mass vs. corticosterone in feathers (Lattin feathers and body feathers from the interscapular area, et al., 2011). Other aspects to be considered when grown at the same time, administering synthetic
4692 H€aFFELIN ET AL. corticosterone to broilers via the drinking water for 72 h. Nevertheless, the current results indicate that the sta- Surprisingly, elevated feather corticosterone concentra- bility of corticosterone already deposited into feathers is tions could be measured beginning 6 h after application not influenced by UV-A radiation. This finding allows to (Weimer et al., 2018), which is hardly comprehensible compare results of feathers of hens being exposed to UV- when considering growth rates of feathers to be around A light through the lighting system with hens that were wk (Serra and Underhill, 2006; Butler et al., 2008; not exposed to UV-A radiation. However, the aforemen- Oschadleus and Underhill, 2008; Rohwer and Rohwer, tioned effects of light parameters remain to be 2013; H€affelin and Andersson, unpublished data). investigated. Consequently and based on the studies by Romero and Fairhurst (2016) and Moncl us et al. (2017), it is recom- mended to use the same type of feather when analyzing CONCLUSION and comparing feather corticosterone concentrations as the feathers have the same structure and time of growth. The aim of the present study was to establish a reliable When drawing comparisons, feathers should origin from and valid method to measure corticosterone concentra- the same replacement generation as Moncl us et al. tions in feathers of laying hens. The presented results indi- (2020) found different levels of feather corticosterone cated that the applied technique and methodology, and in the same bird but from different seasons. Concerning thus the extraction procedure and assay kit, qualifies as the most suitable feather type for feather corticosterone valid. In this study, the groundwork for future investiga- analysis, very small feathers, such as body feathers from tions of reference values for laying hens was laid. To draw the belly and the flanks, were ruled out, as they were comparisons and gain information about response of birds broken frequently. Previous investigations were also and flocks to and in different environmental conditions, performed using the same feather types as in the present the use of the same extraction procedure and assay kit study (Carbajal et al., 2014; Aharon-Rotman et al., proposed in the present study is essential. A possible 2017; Moncl us et al., 2017; Robertson et al., 2017; correlation between corticosterone concentrations in Freeman and Newman, 2018). feathers and in the blood should be clarified. Further Vane vs. Rachis Showing higher feather corticosterone investigations should be performed on whether feather levels in the vane than in the rachis of tail feathers is in corticosterone is suitable as an indicator for animal accordance with the findings of Freeman and Newman welfare in laying hens. With additionally obtained infor- (2018; P , 0.05). Owing to the fact that the total weight mation on those issues, the present method may have po- of a feather is mainly made up of the rachis, one must be tential for an evidence-based assessment of animal welfare aware of choosing feathers not only of the same feather in laying hens, which can be applied noninvasively. As type but with the same weight. Another possibility is corticosterone in feathers is related to animal welfare it to refer the feather corticosterone concentration on may also be suitable to assess and enhance husbandry feather length in pg/mm (Bortolotti et al., 2008; conditions and production systems regarding animal Romero and Fairhurst, 2016). welfare. Moreover, corticosterone in feathers may also Freeze–Thaw Cycle All samples of the present study be implemented in breeding schemes to provide stress were frozen until examination (Bortolotti et al., 2008). resilient strains (genotype ! environment interaction). This procedure was mainly performed because of prac- Samples from individual marked and pedigreed birds tical reasons and is however a fixed part of the protocol. need to be analyzed for estimating genetic parameters Studies on freeze–thaw cycles using corticosterone orig- for feather corticosterone values at different ages and inated for example from mouse serum (Kang et al., 2013) management conditions. In the last decade, examinations or mouse plasma (Li et al., 2015) showed that levels do of corticosterone in feathers have been performed basi- not alter. However, comparisons of the values measured cally in wild birds, yet the potential has not been in the present study after freeze–thaw cycles showed exhausted for commercial poultry. decrease of feather corticosterone concentrations up to a third from the initial value. The buffer coming from the ACKNOWLEDGMENTS ELISA kit used in the present study, and in which the hormone was stored frozen, may not be an adequate The authors gratefully acknowledge funding from the matrix to freeze and thaw feather corticosterone sam- Ministry of Science and Culture in Lower Saxony, Ger- ples. Thus, researchers should avoid freezing samples many. They also thank Alina Uhlenkamp, University that have been defrosted, when planning their experi- of Applied Sciences Osnabr€ uck, Germany, for her tech- mental design. nical support, as well as Thomas Bartels, Federal Effect of UV Radiation Samples being exposed to UV-A Research Institute for Animal Health in Celle, Germany, radiation did not differ from the control group samples and Chadi Touma, University of Osnabr€ uck, Germany, regarding feather corticosterone concentrations. Howev- for their helpful suggestions. er, the effect of UV radiation remains to be studied in Ethics Statement: The current study was undertaken detail for free range hens as natural UV radiation per- in accordance with the German legislation centage varies during seasons, time of day, and location (TierSchNutztV, 2017; TierSchG, 2020) and the “Guide (K€ammerling et al., 2017) and may have an effect on for the Care and Use of Agricultural Animals in corticosterone deposition during growth of feathers. Research and Teaching” (Ag Guide, 2010).
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