fa
aT
IM
mi
de
3 Juine
invalid and reliable alternative to existing non-invasive
techniques for monitoring the hypothalamicpituitaryadrenal (HPA)
axis activity.
has stimulated research on the eects of the shelter
environ-ment, thereby promoting studies aimed at the validation
of
2000). Recently, several authors (Koren et al., 2002; Dav-enport
et al., 2006) reported on a novel method for deter-
the hair of the rock hyrax. Davenport et al. (2006) vali-dated a
simple procedure for measuring cortisol concentra-
tain material that may bias extraction. The main character-istic
that makes hair assay particularly appealing is that itprovides a
long-term endocrine prole, a measure of hor-monal activity averaged
over the chosen period. The mea-sure is insensitive to the impact
of acute stress includingthat caused by handling during sampling
procedures. A
* Corresponding author. Fax: +39 051 2097899.E-mail address:
[email protected] (P.A. Accorsi).
Available online at www.sciencedirect.com
General and Comparative Endocrinoalternative, non-invasive
techniques for monitoring adrenalfunction. The decision to use
non-invasive techniques toassess control of the HPA axis activity
in animals may beessential (ethical reasons), imperative (logistic
reasons) andfurther grounded on experimental considerations.
Restraintand handling required for blood sampling may be
stressorsby themselves, thus causing sharp increases in peripheral
glu-cocorticoid concentrations within minutes (Beerda et al.,1996;
Carlstead et al., 1992, 1993; Willemse et al., 1993).
Currently applied methodologies include measurementof faecal,
urinary or salivary corticoids (Cook et al.,
tions in the hair of rhesus macaques. The analysis of
hairsteroidal hormones could be useful in studies of chronicstress
and welfare that require monitoring of adrenal func-tion for
extended periods.
Hair assays are used for a variety of purposes: for trac-ing
pollutants, drugs, anabolic steroids and othercompounds, and for
determining sex steroids and glucocor-ticoids (Koren et al., 2002;
Yang et al., 1998). Hair sam-pling is relatively easy, its
collection does not entailserious health hazards, hair is very
easily preserved, isnot aected by variations in water content, nor
does it con-To this aim, 56 new hair growth samples and 870 faecal
samples from 27 domestic cats and 29 domestic dogs were collected
and cortisolcontent was assessed. A signicant positive association
was observed in both species between the concentrations of cortisol
determined inhair and faeces. This nding is discussed in the light
of the existing knowledge of hair physiology and in the perspective
of its applicationto studies on chronic stress. 2007 Elsevier Inc.
All rights reserved.
Keywords: Non-invasive cortisol assessment; Cats; Dogs; Hair;
Faeces
1. Introduction
In the last decade increasing attention to animal welfare
mining endogenous levels of steroidal hormones in thehair. Koren
et al. (2002) showed that male rank is associ-ated with
testosterone but not cortisol levels measured inCortisol
determination in hair and
Pier A. Accorsi a,*, Elena Carloni b, PMatteo Gamberoni a,
Carlo
a Dipartimento di Morfosiologia Veterinaria e Produzioni Animali
(D
40064 Ozzano Eb Dipartimento di Biologia Evolutiva e Funzionale,
Universita`
Received 20 April 2007; revisedAvailable onl
Abstract
The present study explored the feasibility of a hair cortisol
assay0016-6480/$ - see front matter 2007 Elsevier Inc. All rights
reserved.doi:10.1016/j.ygcen.2007.07.002eces from domestic cats and
dogs
ola Valsecchi b, Roberta Viggiani a,amanini a, Eraldo Seren
a
ORFIPA), Universita` degli Studi di Bologna, Via Tolara di Sopra
50,
lia (BO), Italy
gli Studi di Parma, via G. Usberti 11/A, 43100 Parma, Italy
ly 2007; accepted 13 July 200726 July 2007
domestic cats (Felis silvestris catus) and dogs (Canis
familiaris) as a
www.elsevier.com/locate/ygcen
logy 155 (2008) 398402
trends, but even shed hair may still provide precious indi-
collected cats and dogs faecal samples ad libitum through-
aratout the study period to get an overall estimate of
individualendocrine patterns to be compared with the hormonal
con-tent determined in newly grown hair.
In contrast to the methods employed by Davenportet al. (2006)
who sampled saliva, we chose to comparethe concentration of
cortisol in hair and faeces. The deci-sion was made on the ground
of several considerations: col-lection of faecal samples is less
stressful than salivacollection for non-trained animals. Faecal
cortisol concen-trations accurately reect adrenocortical responses
tostressors in canids (Sands and Creel, 2004) and felids(Brown et
al., 1994; Schatz and Palme, 2001; Wasseret al., 2000) and this
stress assessment technique has predic-tive and explanatory value
(Mostl and Palme, 2002; Schatzand Palme, 2001; Wasser et al.,
2000). However, if long-term average measures are needed, faecal
assay may notbe the best solution.
In the present study we expected that a signicantlypositive
association between the two measures would oper-ationally validate
the hair assay method in both cats anddogs.
2. Materials and methods
2.1. Animals
Cats. Twenty-seven neutered domestic cats, 19 females and 8
males,aged 210 years, were randomly selected from a shelter colony.
All catswere group living and kept in similar conditions. Shelter
facilities com-prised an open-air grassy compound and indoor
accommodations.
Dogs. Twenty-nine domestic dogs, 8 females and 21 males, aged17
years, were selected from two dierent contexts: a rescue shelter
anda utility and defence class. Dogs (females: n = 5; males: n = 9)
fromthe rescue shelter were mixed breeds and only females were
neutered. Dogsfrom the utility/defence class were intact German
shepherds (females:n = 3; males: n = 12).
2.2. Hair and faecal sampling
Cats. Hair from the ischiatic region was manually shaved to the
level ofcation of the individuals hormonal prole. On the con-trary,
due to its slow growth hair does not provide nemonitoring over
short periods of time since it does notreect daily or hourly
uctuations in circulating hormones(Koren et al., 2002). The main
limits of this method residein the incomplete information on hair
physiology, and thelack of laboratory validation to date. In
summary, thismethod could be best suited for studies of chronic
stressand to assess the hormonal substrate of social trends,
butawaits validation and calls for deeper understanding ofthe way
in which steroids are incorporated into and metab-olised in hair.
We decided to verify whether this method isapplicable to domestic
cats and dogs. To this purpose, wetwo-point sampling (shave and
resample) is hence sucientto determine the endocrine background of
behavioural
P.A. Accorsi et al. / General and Compthe skin 12 days before
commencement of the study to eliminate old-growth hair. The same
patch of skin was resampled at the end of the sam-pling period:
once again hair was manually clipped to collect new hairgrowth.
Hair samples were identied, labelled and stored at room
temper-ature until analysis. Faeces were collected
opportunistically upon evacua-tion throughout the study period,
whenever defecation was detected,between 7:00 a.m. and 9:00 p.m.
Faecal samples were collected avoidingdebris and
cross-contamination, immediately identied, labelled andstored in
PPL bags at 20 C until assay.
Hence, each cat contributed one sample (n = 27) of new hair
growth,with hair growth span corresponding to faecal sampling
period. However,number of faecal samples and duration of sampling
period varied acrosscats. On the whole, the 27 cats contributed on
average 5.89 0.72(mean SEM) faecal samples over a mean period of
94.96 9.36 days(22 cats supplied 5.36 0.67 faeces over 72.45 1.73
days, whereas 5 indi-viduals provided 8.20 2.50 faeces over 194
days). Dierences in theduration of sampling periods were caused by
adoption or relocation ofpart of the cats.
Dogs. Hair samples were collected and processed as detailed for
cats.Faecal samples were collected approximately every 3 days, and
then pro-cessed as described for cats. Each sheltered dog provided
one sample ofnew hair growth (n = 14) and on average 14.5 0.91
faecal samples col-lected along 78 days. Dogs from the
utility/defence class supplied one sam-ple of new hair growth (n =
15) and 33.86 4.86 faecal samples over aperiod of 87.93 2.15
days.
The study started in March 2004 and nished in December of the
sameyear.
2.3. Cortisol determination
A total of 56 hair (27 feline and 29 canine) and 870 faecal (159
felineand 711 canine) samples were processed for steroid assay.
Cortisol(17-a-hydroxycorticosterone) concentrations were determined
by RIAbased on binding of 3H-steroid by competitive adsorption
(Fenske andSchonheiter, 1991). All concentrations were expressed in
pg/mg of hairshaft and faecal matter.
2.4. Extraction from hair
Extraction methodology was modied from Koren et al. (2002).
Hairwas rst minced into 13 mm length fragments and 60 mg of trimmed
hairwere put in a glass vial. Five millilitre methanol (Carlo Erba,
Rodano, MI,Italy) were added, and vials were incubated at +50 C
with gentle shakingfor 18 h. The vial content was then ltered to
separate the liquid phasewhich was evaporated to dryness under an
air-stream suction hood at37 C. Dry residue was then dissolved into
0.6 ml of phosphate-bueredsaline (PBS) 0.05 M, pH 7.5. A recovery
test on ve replicates was per-formed by adding 125, 250, 500 or
1000 pg of 3H-cortisol (PerkinElmerLife Sciences Inc., Boston, MA,
USA) to 60 mg of trimmed hair and incu-bating for 18 h at room
temperature. The extraction was performed asdescribed above. The
mean percentage of recovery was 90.61 2.48.
2.5. Extraction from faeces
Extraction methodology was modied from Schatz and Palme
(2001).Five millilitre of a methanol:water (v/v 4:1) solution were
added to 500 mg(wet weight) of faeces in capped glass tube vials.
Vials were then vortexedfor 30 min using a multitube pulsing
vortexer. Following centrifugation(1500g for 15 min), 5 ml ethyl
ether (BDH Italia, MI, Italy) and 0.2 mlNaHCO3 (5%) (Sigma Chemical
Co., St. Louis, MO, USA) were addedto 1 ml supernatant. This
preparation was vortexed for 1 min on multitubepulsing vortexer and
centrifuged for 5 min (1500g). The ether portion wasthen separated
by sucking it with a pipet, and evaporated under an air-stream
suction hood at 37 C. Dry residue was nally redissolved into0.5 ml
PBS 0.05 M, pH 7.5. A recovery test on ve replicates was per-formed
by adding 125, 250, 500 or 1000 pg of 3H-cortisol to 500 mg of
fae-ces and incubating for 30 min at room temperature. The
extraction was
ive Endocrinology 155 (2008) 398402 399performed as described
above yielding a mean percentage recovery of89.74 2.64.
(Sigma Chemical Co.) and endogenous cortisol in cats and dogs,
hair
1.16 0.23 pg/mg. The Spearman Correlation Test
1 10 100 1000Log pg / tube-vial
1 10 100 1000Log pg / tube-vial
0
20
40
60
80
100
B/B
0 %
0
20
40
60
80
100
B/B0
%
b
Fig. 1. Parallelism between cortisol standards (circles;
range7.811000 pg/100 ll tube vial) and serially diluted samples.
Each data-point (squares) isthe average of three hair (a) and
faecal (b) samples, containing highendogenous cortisol diluted to
obtain volumes of 100, 50, 25, 10 and 5 ll.
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0mean faecal cortisol
(pg/mg)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
hair
cor
tisol
(pg/m
g)
Fig. 2. Mean individual faecal cortisol values plotted against
individual haircortisol content for the feline sample (N = 27).
Values are expressed in pg/mg. The condence interval 95% around the
best-t slope is shown.
ratand faecal samples from three dierent animals, containing
high concen-trations of endogenous cortisol (100 ll), were serially
diluted with PBS0.05 M, pH 7.5 to obtain volumes of 50, 25, 10 and
5 ll. Parallelismwas assessed between these serial dilutions and
cortisol standards (rangingfrom 7.81 to 1000 pg/100 ll tube vial,
prepared in buer).
2.7. Determination of concentration
Radioactivity was determined using a liquid scintillation b
counter andusing a linear standard curve (ad hoc designed software
program: Mottaand Degli Esposti, 1981) the concentration of
cortisol in the unknownsamples was determined.
2.8. Statistical analysis
Individual cortisol contents determined in the hair were tested
againstmean individual faecal cortisol levels over the period of
hair growth.Spearman Rank Correlation Test was used to assess the
strength of theassociation. A non-linear regression test was used
to assess parallelismbetween standard and endogenous hormones.
For all statistical tests alpha value was set at 0.05
(statistical packageStatistica 6.0).
3. Results
A high degree of parallelism (P < 0.01) was observedbetween
the standard and the diluted samples cortisolcurves, with a
parallel drop in percent binding as samplevolumes and cortisol
standard concentrations increased(Fig. 1).
Cats. Mean cortisol content in 27 hair samples was3.32 0.27
pg/mg whereas mean cortisol content deter-mined in 159 faecal
samples was 0.83 0.08 pg/mg.
Hair and faecal values were signicantly positively cor-related
(rs = 0.902, P < 0.001; Fig. 2).
Dogs. Correlation between hair and faecal cortisol levelswas
checked for each dogs group. Since signicant associ-ations were
detected for the two subsets of animals, statis-tical analysis is
detailed only for pooled data.
Mean cortisol content in 29 hair samples was2.6. Cortisol
assay
Assay in both hair and faeces was carried out according to
Tamaniniet al. (1983). Analysis was performed in duplicate: 100 ll
of 3H-cortisol (spe-cic activity 100 Ci/mmol, amount 30 pg/tube
vial, 12,771 dpm/100 ll)and 100 ll of an anti-cortisol antibody
(dilution 1:20,000) were added to100 ll of the solution obtained
from glucocorticoid extraction. Afterincubation at +4 C for 18 h,
free steroid was separated from bound bythe addition of 1 ml of a
solution of charcoal 1% (Sigma ChemicalCo.) + 0.025% dextran (Sigma
Chemical Co.), and incubation at +4 Cfor 15 min followed by
centrifugation (4000g) for 4 min at+4 C.The super-natant containing
thehormonebound to its antibodywas thendecanted intoscintillation
vials and measured in a liquid scintillation b counter (PerkinElmer
Life Science Inc.). Validation parameters of the analysis were:
sensi-tivity 0.26 pg/mg, intra-assay variability 6.8%, inter-assay
variability 9.3%,specicity (%): cortisol 100; corticosterone 9.5;
11a-hydroxyprogesterone8.3; cortisone 5.3; 11a-desoxycortisol 5.0;
progesterone 0.6; desoxycorticos-terone0.5;
20a-dihydrocortisone0.4; testosterone0.3; aldosterone0.1;
dehy-droepiandrosterone, 5a-pregnenolone, 17b-estradiol,
cholesterol:
rat2001; De Palma et al., 2005). Similarly, our cats mean
val-ues were lower than those determined in other studies(Schatz
and Palme, 2001; Young et al., 2004). Most prob-ably these
dierences are to be ascribed to the dierent liv-ing conditions of
the subjects and, possibly, to the dierentmethodologies applied
(RIA versus EIA). All these aspectsdeserve further
investigation.
In conclusion, we established a positive correlationbetween
individual hair and faecal cortisol concentrationsin cats and dogs.
On the basis of these ndings and in con-sideration of the
practicability of both the laboratorymethod and the sampling, we
believe that the use of hairis promising and deserves further
investigations both inthe laboratory and in the eld.
Acknowledgments
We are grateful to Valentina Beretta, Jenny Bertozzi,Eva Leroy,
MatteoMestieri, Manuela Stru, Sara Zannoniand Cristian Linguerri
for their assiduous collection andhelp in the analysis of
biological samples. Special thanksto the sta and to the volunteers
of the Canile Municipaledi Cella (RE, Italy) and of Villanova (FC,
Italy) cat shelterAmici dei cani di Bagnolo, and to the owners of
the dogstrained in the defence/utility class. Also, we are
indebtedwith two anonymous reviewers, whose comments, correc-tions
and discussions have greatly improved the manu-script. The study
was supported by Universita` di Bologna(FIL 2004) and MIUR (PRIN
2004) grants to Pier AttilioAccorsi, and by Universita` di Parma
(FIL 2003) andMIUR(PRIN 2004) grants to Paola Valsecchi.
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Cortisol determination in hair and faeces from domestic cats and
dogsIntroductionMaterials and methodsAnimalsHair and faecal
samplingCortisol determinationExtraction from hairExtraction from
faecesCortisol assayDetermination of concentrationStatistical
analysis
ResultsDiscussionAcknowledgmentsReferences
