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General and Comparative Endocrinology 155 (2008) 398–402

Cortisol determination in hair and faeces from domestic cats and dogs

Pier A. Accorsi a,*, Elena Carloni b, Paola Valsecchi b, Roberta Viggiani a,Matteo Gamberoni a, Carlo Tamanini a, Eraldo Seren a

a Dipartimento di Morfofisiologia Veterinaria e Produzioni Animali (DIMORFIPA), Universita degli Studi di Bologna, Via Tolara di Sopra 50,

40064 Ozzano Emilia (BO), Italyb Dipartimento di Biologia Evolutiva e Funzionale, Universita degli Studi di Parma, via G. Usberti 11/A, 43100 Parma, Italy

Received 20 April 2007; revised 3 July 2007; accepted 13 July 2007Available online 26 July 2007

Abstract

The present study explored the feasibility of a hair cortisol assay in domestic cats (Felis silvestris catus) and dogs (Canis familiaris) as avalid and reliable alternative to existing non-invasive techniques for monitoring the hypothalamic–pituitary–adrenal (HPA) axis activity.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 significant positive association was observed in both species between the concentrations of cortisol determined inhair and faeces. This finding 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 welfarehas stimulated research on the effects of the shelter environ-ment, thereby promoting studies aimed at the validation ofalternative, 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.,2000). Recently, several authors (Koren et al., 2002; Dav-enport et al., 2006) reported on a novel method for deter-

0016-6480/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2007.07.002

* Corresponding author. Fax: +39 051 2097899.E-mail address: pierattilio.accorsi@unibo.it (P.A. Accorsi).

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 inthe hair of the rock hyrax. Davenport et al. (2006) vali-dated a simple procedure for measuring cortisol concentra-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 affected by variations in water content, nor does it con-tain material that may bias extraction. The main character-istic that makes hair assay particularly appealing is that itprovides a long-term endocrine profile, 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

P.A. Accorsi et al. / General and Comparative Endocrinology 155 (2008) 398–402 399

two-point sampling (shave and resample) is hence sufficientto determine the endocrine background of behaviouraltrends, but even shed hair may still provide precious indi-cation of the individual’s hormonal profile. On the con-trary, due to its slow growth hair does not provide finemonitoring over short periods of time since it does notreflect daily or hourly fluctuations 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, wecollected cats’ and dogs’ faecal samples ad libitum through-out 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 reflect 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 significantlypositive 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 2–10 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, aged1–7 years, were selected from two different 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 ofthe skin 1–2 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 identified, 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 identified, 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). Differences 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 finished 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 modified from Koren et al. (2002). Hairwas first minced into 1–3 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 filtered 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-bufferedsaline (PBS) 0.05 M, pH 7.5. A recovery test on five replicates was per-formed by adding 125, 250, 500 or 1000 pg of 3H-cortisol (Perkin–ElmerLife 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 modified 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 finally redissolved into0.5 ml PBS 0.05 M, pH 7.5. A recovery test on five 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 wasperformed as described above yielding a mean percentage recovery of89.74 ± 2.64.

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/B

0%

a

b

Fig. 1. Parallelism between cortisol standards (circles; range7.81–1000 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.

8.0

400 P.A. Accorsi et al. / General and Comparative Endocrinology 155 (2008) 398–402

2.6. 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-cific 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 the hormone bound to its antibody was then decanted intoscintillation vials and measured in a liquid scintillation b counter (Perkin–Elmer 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%,specificity (%): cortisol 100; corticosterone 9.5; 11a-hydroxyprogesterone8.3; cortisone 5.3; 11a-desoxycortisol 5.0; progesterone 0.6; desoxycorticos-terone 0.5; 20a-dihydrocortisone 0.4; testosterone 0.3; aldosterone 0.1; dehy-droepiandrosterone, 5a-pregnenolone, 17b-estradiol, cholesterol: <0.0001.

In order to determine the parallelism between cortisol standards(Sigma Chemical Co.) and endogenous cortisol in cats and dogs, hairand faecal samples from three different 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 buffer).

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).

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

mean faecal cortisol (pg/mg)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

hair

cor

tiso

l (pg

/mg)

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 confidence interval ±95% around the best-fit slope is shown.

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 significantly 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 significant 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 was2.10 ± 0.22 pg/mg whereas mean cortisol content deter-mined in the 711 faecal samples from 29 dogs was

1.16 ± 0.23 pg/mg. The Spearman Correlation Testrevealed a significant positive correlation between hairand faecal cortisol levels (rs = 0.67, P < 0.001; Fig. 3).

-1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0mean faecal cortisol (pg/mg)

-0.5

0.0

0.5

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1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

hair

cor

tiso

l (pg

/mg)

Fig. 3. Mean individual faecal cortisol values plotted against individualhair cortisol content for the canine sample (N = 29). Values are expressedin pg/mg. The confidence interval ±95% around the best-fit slope isshown.

P.A. Accorsi et al. / General and Comparative Endocrinology 155 (2008) 398–402 401

4. Discussion

The aim of the present study was to evaluate the reliabil-ity and practicality/utility of a method for measuring haircortisol as an index of HPA axis activity in domestic catsand dogs. To achieve this goal we verified whether cortisolcontent in hair reflects its level in faeces by collecting sam-ples in field conditions. Our findings show that, in both spe-cies, a significant positive association exists between theconcentrations of cortisol determined in hair and in faeces.This correlation seems to support the hypothesis that bothfaecal and hair cortisol measurements reflect at least in partthe same adrenal activity.

Our results are in agreement with those by Davenportet al. (2006) on rhesus macaques obtained under controlledlaboratory conditions. In that species hair cortisol contentwas found to positively correlate with salivary cortisol andto increase as a result of prolonged stressful conditions.The fact that hair cortisol content correlates with faecalor salivary cortisol across a variety of subjects and livingconditions encourages the employment of this non-invasivetechnique to monitor stress responses over protracted peri-ods. Nevertheless, a major question remains. To whatextent is the cutaneous HPA axis function related to thesystemic HPA axis stress response, i.e. to which extent isit directly modulated by the systemic levels determined bythe adrenergic response.

It is well-known that glucocorticoids and androgens aswell as local stimuli induce various processes at the hairgerm level (papilla, sebaceous glands, follicular epithelium,etc.) (Stenn and Paus, 2001). The hair follicle is definitely atarget of various hormones, but the actual location (bothintra- and extra-cellular) and scope (function, origin, rateof uptake and deposition) of hormones measured in hairshafts and possibly in the adhering organic debris areunknown. A number of studies have reported the presenceof either steroid hormones or their precursors (Botchkarev,

2003), their receptors (Ahsan et al., 1998; Hoffmann, 2003;Oh and Smart, 1996; Sawaya and Price, 1997) or enzymesinvolved in their metabolism (Hoffmann, 2003; Sawaya andPenneys, 1992; Sawaya and Price, 1997; Stenn and Paus,2001) in several structures or epidermal compartments ofthe hair organ, even in the hair matrix cells (Bratka-Robiaet al., 2002). It is known that there is a direct connectionbetween the nervous system and the skin (Slominski andWortsman, 2000), and particularly between the nervoussystem and the hair follicle, that is a target of the stressresponse via circulating and local mediators. Furthermore,the pilosebaceous unit itself is a source of hormones(including ACTH and cortisol). Therefore, the skin is con-sidered to operate as a ‘‘local equivalent of the hypotha-lamic–pituitary–adrenal axis’’ by performing (in vitro)cortisol synthesis and secretion and negative feedback reg-ulation on CRH expression (Botchkarev, 2003; Slominskiet al., 2007). Furthermore, Ito and colleagues (2005) haveconclusively demonstrated that the human hair follicleitself (in vitro) is effective at producing cortisol followingCRH stimulation and thus is indeed a functional equiva-lent of the HPA axis. All these studies have demonstratedthat the hair follicle has a local stress response system ofendocrine, paracrine, juxtacrine and nervous nature.Hence, for all these reasons hair seems to be an ideal can-didate for stress-assessment, but the pilosebaceous unit is afunctionally complex system. This makes the presence ofcortisol in the hair shaft difficult to interpret. Thus, at pres-ent we cannot ascertain whether cortisol determined in hairshaft is either of systemic or local origin or both.

This uncertainty about the origin of cortisol does notinvalidate the meaning and the scope of our findings, butmerely suggests caution in its application to studies onchronic stress and stimulates further investigations to clar-ify this topic. In fact, all published studies point towards aprofitable application of this methodology to the evalua-tion of the stress response. Yamada and collaborators(2007) found that hospitalized infants had higher levels ofhair cortisol than healthy term infants and that hair corti-sol levels were sensitive to exposure to a potential stressor.Similar results were obtained—as previously reported—byDavenport and colleagues (2006) in rhesus. Accordingly,we are exploring in depth the connections between hair cor-tisol levels and stress-related behaviours in cats and dogsconfined in animal shelters.

Finally, the concentrations of faecal and hair cortisol wedetermined in cats and dogs deserve some considerations.As far as hair is concerned we have no references to otherworks in these species. Therefore, our findings represent astarting point for future investigations that could, amongother aspects, clarify the relationship between environmen-tal conditions and hair cortisol content. On the other side,there are few published works examining the faecal concen-trations of cortisol in cats and dogs, at least to our knowl-edge. Thus, we can hardly compare our results to others’.The faecal levels we determined in our dogs were lowerthan those reported by other authors (Schatz and Palme,

402 P.A. Accorsi et al. / General and Comparative Endocrinology 155 (2008) 398–402

2001; 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 differences are to be ascribed to the different liv-ing conditions of the subjects and, possibly, to the differentmethodologies 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 findings 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 field.

Acknowledgments

We are grateful to Valentina Beretta, Jenny Bertozzi,Eva Leroy, Matteo Mestieri, Manuela Struffi, Sara Zannoniand Cristian Linguerri for their assiduous collection andhelp in the analysis of biological samples. Special thanksto the staff and to the volunteers of the Canile Municipaledi Cella (RE, Italy) and of Villanova (FC, Italy) cat shelter‘‘Amici 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) and MIUR(PRIN 2004) grants to Paola Valsecchi.

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