Research in Veterinary Science 93 (2013) 49–54

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Research in Veterinary Science

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Cortisol levels in hair reflect behavioural reactivity of dogs to acoustic stimuli

M. Siniscalchi a, J.R. McFarlane b, K.G. Kauter b, A. Quaranta a,⇑, L.J. Rogers c

a Department of Animal Production, University of Bari ‘‘A. Moro’’, Bari, Italyb Centre for Bioactive Discovery in Health and Aging, University of New England, Armidale, NSW 2351, Australiac Centre for Neuroscience and Animal Behaviour, University of New England, Armidale, NSW 2351, Australia

a r t i c l e i n f o

Article history:Received 12 September 2011Accepted 29 February 2012

Keywords:DogsBehaviourCortisolHairAcoustic stimuli

0034-5288/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.rvsc.2012.02.017

⇑ Corresponding author. Address: Department of AVeterinary Medicine, University of Bari ‘‘A. Moro’’, StrKm 3 – 70010 Valenzano, Italy. Tel.: +39 0805443948

E-mail address: a.quaranta@veterinaria.uniba.it (A

a b s t r a c t

Cortisol levels in hair samples were examined in fourteen domestic dogs and related to the dogs’responses to different acoustic stimuli. Stimuli were playbacks of species-typical vocalizations recordedduring three different situations (‘‘disturbance’’, ‘‘isolation’’ and ‘‘play’’ barks) and the sounds of a thun-derstorm. Hair samples were collected at 9:00 h and 17:00 h two weeks after the behavioural tests.Results showed that behavioural reactivity to playback of the various stimuli correlates with cortisol lev-els in hair samples collected at 9:00 h, and the same was the case for the separate measures of behaviour(i.e. hiding, running away, seeking attention from the tester, panting and lowering of the body posture).Hence, levels of cortisol in hair appear to reflect the dog’s chronic state of emotional reactivity, ortemperament.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction response to hearing playbacks of the sounds of a thunderstorm

Behavioural and physiological measures have both been used tomeasure the effects of stressful situations on domestic dogs(Beerda et al., 1998; Dreschel and Granger, 2005; Blackwell et al.,2010). Different sounds such as recordings of thunderstorms orsound blasts have been used as psychological stressors that influ-ence behaviour and activation of the hypothalamic–pituitary–adrenal (HPA) axis. However, despite the fact that changes incortisol levels are well known to be a major physiological responseto stress (Coppola et al., 2006), the relationship between cortisollevels and stress-induced behaviour remains unclear. The behav-ioural response may differ between dogs depending on the differ-ent types of stimuli presented and differences in temperamentbetween dogs (Rooney et al., 2007). Studies in different environ-ments have delineated a range of behaviour indicative of the dog’swelfare status (Beerda et al., 1999; Hiby et al., 2006). Licking,lowered body posture, panting, body shaking and paw liftingare generally displayed by dogs in response to short-termstress, whereas the repetitive performance of a particularbehaviour, a stereotypy, is usually expressed during prolongedstress (Hetts et al., 1992; Beerda et al., 1999. 2000).

Regarding reactivity to acoustic stimuli, Dreschel and Granger(2005) reported that dogs with phobia to thunderstorms exhibitbehavioural signs of anxiety (i.e., pacing, whining, hiding) in

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nimal Production, Faculty ofada Prov.le per Casamassima,; fax: +39 0805443883.. Quaranta).

and these responses correlated with an increase in salivary cor-tisol levels. Hydbring-Sandberg et al. (2004), in addition, foundthat dogs with high scores of the startle reaction to the soundof a gunshot had higher blood cortisol levels five minutes afterthe second gunshot test than dogs with low startle reactionsto the same gunshot. Moreover, Beerda et al. (1998) reportedthat, in dogs, a very ‘‘low posture’’ may indicate intense, acutestress since dogs display this posture concomitant with elevatedcortisol levels in saliva after hearing stimuli that could not beanticipated, such as sound blasts.

In dogs, cortisol levels have been found to increase not onlyafter exposure to acoustic stimuli that are disturbing but alsoduring transportation, after electric shock and during social iso-lation and spatial restriction (Beerda et al., 1998, 1999). Previousstudies of the HPA activity in dogs have used blood, saliva, faecalor urine samples (Gordon and Lavie, 1985; Beerda et al., 1998;Accorsi et al., 2008; Castillo et al., 2009). A more recent andpromising technique for monitoring adrenal activity is the mea-sure of cortisol levels in the hair (Accorsi et al., 2008; Bennettand Hayssen, 2010). Bennet and Hayssen reported that, in dogsin their home environment, cortisol concentrations in samplesof hair and saliva are correlated positively, suggesting that hairsampling may provide a means of measuring basal levels of cor-tisol secretion. In addition, these authors confirmed that therewere no significant effects of age, breed, weight, or neuter statuson cortisol in hair, supporting the claim that hair samplingallows comparisons of baseline cortisol with individual traitssuch as temperament or social status (Bennett and Hayssen,2010; Koren et al., 2002). Furthermore, in humans, recent studies

50 M. Siniscalchi et al. / Research in Veterinary Science 93 (2013) 49–54

reported a considerable degree of chemical stability in hair cor-tisol levels which highlights the utility of this method for obtain-ing trait estimates of long-term cortisol secretion (Webb et al.,2010; Stalder et al., 2011).

Measurement of cortisol in hair could be useful in studies ofcanine welfare, especially since collection of hair samples is anon-invasive technique.

A recently study found that, in common marmosets, hair cor-tisol levels are correlated inversely with a measure of socialmobbing behaviour (Clara et al., 2008). Hence, we decided tosee whether there is an association between cortisol levels inthe hair of dogs and behaviour performed in response to hearingplaybacks of species-typical vocalizations (social calls) with dif-ferent emotional valences and thunderstorm sounds (usuallyperceived as fearful by dogs). We have reported previously thatthe same dogs showed biases to use the left hemisphere to pro-cess the vocalizations of co specifics and the right hemisphere toprocess the sounds of thunderstorms, unless the dog was verynervous, in which case it used the right hemisphere to processall of the sounds (Siniscalchi et al., 2008). Here we report on cor-tisol levels in hair samples collected from these dogs after theperiod of behavioural testing.

2. Material and methods

This project was conducted in accordance with the AustralianCode of Practice for the Care and Use of Animals for Scientific Pur-poses (National Health and Medical Research Council, 1997) and itwas approved by the University of New England Animal EthicsCommittee (Approval ID: AEC07/120).

2.1. Animals

The animals tested were 14 domestic dogs of various breeds (5Rhodesian Ridgebacks, 2 Boxers, 2 Labrador Retrievers, 2 BorderCollies, 1 Dachshund and 2 mixed-breed dogs), 8 females and 6males, aged 2–13 years (5.6 ± 1.03 years; mean + S.E.M.). All dogswere pets living in households. All were neutered, apart fromone male and one female. These dogs were also included in thebehavioural tests discussed in Siniscalchi et al. (2008).

2.2. Behavioural testing

The dogs were tested in their home environment by playingthem a range of their typical vocalizations and the sounds of athunderstorm. Vocalizations were of three different categoriesaccording to the work of Yin and McCowan (2004): (1) ‘‘distur-bance’’ barks recorded during a situation in which an unknownperson knocked on the door of the owner’s house; (2) ‘‘isolation’’

Table 1Cortisol values in hair of the 14 dogs at 9:00 h and 17:00 h (values are means of the dupl

Name Breed Age Gen

1 Rhodesian Ridgeback 2.4 F2 Border Collie 2.4 M3 Boxer 2.4 F4 Labrador retriever 4 F5 Boxer 6 F6 Labrador retriever 13 M7 Rhodesian Ridgeback 9.5 F8 Rhodesian Ridgeback 10 M9 Mixed breed 3 M

10 Rhodesian Ridgeback 0.8 M11 Dachshund cross breed 10 F12 Dachshund 2 F13 Border Collie 8 F14 Rhodesian Ridgeback 7 M

barks in which the dog was in a room isolated from its ownerand (3) ‘‘play’’ barks, produced when two dogs or a human and adog played together. The sound of a thunderstorm was taken froma commercial CD (‘‘Loud noises, to calm your dog’’, Sound DesignStudios, 2000). An audio recording of the sounds was played tothe dogs while they were eating their favourite dry pellets givenin a bowl. Each sound was played in a random order for 3 s at a vol-ume of 60–80 db (measured with a Precision Sound Level Meter,Type 2206, Brüel & Kjær, Nærum, Denmark at 2.5 m from thespeakers in a sound proof room). Identical sounds were playedfrom two speakers one on the left side of the dog and the otheron the right side, placed at 2.5 m from the dog head and balancedin amplitude (see Siniscalchi et al. (2008) for details). A 1-mininterval was allowed between each playback provided that thedog remained at the food dish. The playback was stopped immedi-ately when the dog ceased feeding and resumed only after the doghad resumed feeding. Acoustic stimuli were presented to the dogsduring one-hour sessions per week until 10 playbacks for eachstimulus had been presented to all of the dogs. The average timeto complete the test for each dog was 6 ± 2 weeks (mean ± S.D.).

The dog’s behavioural response to playbacks was video re-corded continuously during sessions with a Handycam Video8 mm camera mounted on a tripod at a distance of 6 meters fromthe dog. The video recordings were analysed subsequently and thefollowing behaviour was scored: vocalization (barking or whining),panting, salivating, ears back, shaking of the body, urinating, defe-cating, tail between the legs, running away, hiding, seeking atten-tion from the tester, adopting a lowered body posture and freezing.Each behavioural response performed was allocated a score of 1and the total score represented the reactivity index. The highestpossible score was 15 and the lowest score was 0.

2.3. Hair sampling

Since in dogs, Bennett and Hayssen (2010) reported that theaverage amount of cortisol did not differ between proximal anddistal hair sections suggesting that hormone levels in clipped, orcut hairs are the same as those found in full-length hair, we clippedapproximately 300 mg of hair from the ischiatic region to withinabout 5 mm of the skin. Furthermore Wheeler et al. (1998) foundthat steroid hormones in human hair did not vary significantly be-tween different regions of the body. The experimenter wore glovesand used scissors.

As the hair samples were collected to provide a general measureof dog’s hormonal state and to link it with its state of emotionalreactivity, or temperament, collection of hair samples was deliber-ately separated in time from the behavioural tests. The hair sampleswere collected at 9:00 h and 17.00 h on the same day two weeksafter the behavioural testing had been completed (two weeks rep-

icate hair samples).

der Cortisol 9 a.m. (pM/g) Cortisol 5 p.m. (pM/g)

141.6 234.489.5 295.8

253.0 421.9128.1 260.5201.2 284.8222.7 286.6290.0 321.1153.6 182.0229.4 255.7100.1 106.1364.7 283.0567.6 400.1397.9 200.7340.8 112.6

M. Siniscalchi et al. / Research in Veterinary Science 93 (2013) 49–54 51

resented for us a good sampling time to avoid acute stress due tothe last playbacks experiments). At the time of collection, each hairsample was placed into a plastic bag and stored at room tempera-ture for three weeks until analysis. Each dog contributed one sam-ple of hair at each time of sampling (9:00 h and 17:00 h).

2.4. Extraction from hair samples

The hair was not washed prior to the extraction process basedon the finding of Yang et al. (1998) and Davenport et al. (2006)showing no significant difference between the sex steroid levelsextracted from washed and unwashed hair. The extraction tech-nique by (Sharpley et al., 2009) was modified after a number of pi-lot tests had been performed. Duplicate samples of between 27 and187 mg of full-length hairs of each sample were placed in a 20 mlglass vial. The exact weight of each hair sample was recorded. Scis-sors were used to mince the hair into 1–3 mm length fragments in-side the glass vial and then 3 ml methanol was added. A controlsample of 3 ml aliquot of methanol was put into a 20 ml glass vialwithout hair and also analysed. The vials were placed on a tray andgently agitated for 48 h. They were then removed from the agitatorand the methanol was poured into a 3 ml polypropylene tube(Röhre tube), leaving the residue of hair in the glass tube. Themethanol was then evaporated in a vacuum oven at 37 �C for threehours. Phosphate buffered saline (PBS, 100 ll) containing 0.1% gel-atine was added to each Röhre tube, which was vortexed to recon-stitute the extract.

2.5. Cortisol assay

Cortisol was measured using a radioimmunoassay describedpreviously (McFarlane et al., 1990). Briefly, the antiserum

Fig. 1. Presents a comparison of 9:00 h a.m. cortisol values in hair versus latency to replayback. A positive and statistically significant correlation was found and a line of best

(#C-3368) used was raised in sheep against cortisol-3CMO-BSA(Minitube Australia, Smythesdale, Victoria Australia) at a final con-centration of 1:20,000. The tracer was tritiated cortisol(NET396001, Perkin Elmer, Springvale VIC Australia) used at afinal activity of 10,000 cpm. Cross reactivity with other likelysteroids were less than 1% except for cortisone (21%), 17-hydroxyprogesterone (14%) and corticosterone (1%). The minimum concen-tration of cortisol detectable in the assay was 40 pg and cortisolcould be detected in as little at 10 mg of hair. The recovery effi-ciency of cortisol from the hair could not be determined becauseinternal standards could not be run, but repeated extractions ofhair did not yield detectable amounts of cortisol. The intra- andinter-assay variation was 7.5% and 11% respectively.

2.6. Analysis of data

The cortisol levels determined in the hair samples collected at9:00 h and 17:00 h were tested against latency to resume feed-ing and the reactivity index after presentation of acoustic play-backs. Pearson’s Correlation Tests were used to assess thestrength of the association. For all statistical tests, SPSS softwarewas used, and the results were considered to be significant ifP < 0.05.

3. Results

3.1. Hair cortisol

The hair cortisol levels at 9:00 h and 17:00 h are presented inTable 1. The mean levels of cortisol varied between 89.5 pM/gand 567.6 pM/g. Basal hair cortisol values in dogs sampled in their

sume feeding (A) and reactivity index (B) after presentation of the thunderstormfit is plotted.

52 M. Siniscalchi et al. / Research in Veterinary Science 93 (2013) 49–54

own homes ranged between 12.6 pM/g and 74.8 pM/g (Bennettand Hayssen, 2010). Although, on visual inspection of Table 1 itcan be seen that in 11 of the 14 dogs the mean cortisol level washigher at 17.00 h than at 9:00 h (the mean values + sems were180.8 + 21.49 pM/g at 9:00 h and 264.89 + 26.40 pM/g at 17.00 h),statistical analyses of the 9:00 h versus 17:00 h levels of cortisolfor all 14 dogs revealed no significant difference (t(13) = �0.329,P = 0.748, two-tailed paired t-tests).

3.2. Hair cortisol level and behavioural response to acoustic stimuli

A significant positive correlation was identified between corti-sol levels in the hair samples collected at 9:00 h and the latencyto resume feeding after presentation of the playback of the soundsof a thunderstorm (r = 0.729, p = 0.003) and the reactivity to thethunderstorm (r = 0.656, p = 0.011) (Fig. 1A and B). No significantassociation was found between the 9:00 h levels of cortisol in hairand latency to resume feeding after playbacks of the various vocal-izations (contact barks, r = 0.176, p = 0.547; play barks, r = 0.365;p = 0.200 and barks given to an intruder, r = 0.280, p = 0.332).

Analysing the behavioural response to vocalizations, a positiveand statistically significant Pearson’s correlation was found be-tween levels of cortisol in hair at 9:00 h and reactivity to playbacksof play barks (r = 0.575; p = 0.032) and the barks given to an intru-der (r = 0.537; p = 0.048); Fig. 2A and B). No correlation between

Fig. 2. Presents a comparison of 9:00 h cortisol values in hair versus reactivity to playcorrelation was found and a line of best fit is plotted.

9:00 h hair cortisol levels and reactivity to the isolation bark wasfound (r = 0.316; p = 0.271).

We looked at each component of the reactivity score to seewhether it correlated with cortisol levels at 9:00 h since this mightallow us to determine which measure might be used as a behav-ioural indicator of a long term activation of the hypothalamic–pitu-itary–adrenal (HPA) axis. Cortisol levels correlated positively withhiding (r = 0.705, P = 0.005), running away (r = 0.679, P = 0.008),seeking attention from the tester (r = 0.586, P = 0.028), panting(r = 0.594, P = 0.025) and lowering of the body posture (r = 0.597,P = 0.024); Fig. 3A–E.

No significant associations were found between the cortisol lev-els in the samples collected at 17:00 h and any of the behaviouralresponses to the acoustic stimuli (P > 0.05 in all measures).

4. Discussion

We found in 11 of the 14 dogs higher cortisol levels at 17:00 hcompared to 9:00 h. This result is similar to that observed in stud-ies of diurnal plasma cortisol variations in both rats and dogs (Orthand Kovacs, 1998). In particular a recent study showed that, in apopulation of clinically healthy dogs, plasma cortisol levels in-creased slightly throughout the day, reaching a maximum between16:00 h and 18:00 h (Castillo et al., 2009). Similarly, Gordon andLavie (1985) reported that, in four adult, female mongrel dogs,

backs of ‘‘Play’’ (A) and ‘‘Intruder’’ (B) barks. A positive and statistically significant

Fig. 3. Presented are comparisons of behaviour that dogs performed in response to acoustic stimuli versus levels of cortisol in hair at 9:00 h. A positive statistically significantPearson’s correlation was found in all of the presented samples and a line of best fit is plotted.

M. Siniscalchi et al. / Research in Veterinary Science 93 (2013) 49–54 53

plasma cortisol levels exhibited temporal variations with a peak at16:00 h. At present there are no published data on the presence ofdiurnal cycle in hair cortisol levels. However, according to our re-sults, individual variations occur since three dogs displayed higherhair cortisol levels at 9:00 h than at 17:00 h and no significantdiurnal cycle was present in our complete sample of dogs. It shouldbe noted that the lack of significant variations in cortisol hair levelsduring the day in our sample is supported also by the fact thatadrenocorticotropic hormone (ACTH) secretion in the dog, whichregulates cortisol production, is sporadic and not rhythmic or pul-satile. Therefore, it does not follow a typical circadian rhythm(Kempanien and Sartin, 1984). Furthermore individual variationscould be explained by the fact that, in normal dogs, the hypotha-lamic–pituitary–adrenal axis (HPA) shows two different types ofactivation: one that depends on the stress generated by stimulipresented (e.g. acoustic sounds heard by the dog prior to collectionof the hair samples) and the other that depends directly on activityof the suprachiasmatic nucleus (SCH), essential for maintaining en-ergy balance and dependent directly on how active the dog is dur-ing the day and on feeding behaviour (Castillo et al., 2009).

The experiment was designed to study in domestic dogs associ-ations between behavioural reactivity and hair cortisol levels.Reactivity was scored in response to playbacks of typical vocal-

izations of dogs and to the sounds of a thunderstorm (stressfulsound). We found that cortisol levels in hair samples collectedat 9:00 h correlated positively and significantly with latency toresume feeding from the bowl and with reactivity (behaviouralscore) after exposure to playback of the sounds of a thunder-storm. This finding supports previous work of Dreschel andGranger (2005), who reported that, when exposed to playbackof the sounds of a thunderstorm, dogs exhibited classic signsof fear (pacing, whining, trembling, and either hiding) followedby an increased saliva cortisol level. In this work cortisol in-creased in saliva 20 min after playback of the thunderstorm,reflecting short-term activation of the HPA axis. Our results indi-cate that heightened reactivity to the sounds of a thunderstormis associated with higher levels of cortisol in hair samples col-lected in the morning two weeks after the behavioural testing,which suggests that 9:00 h cortisol levels may reflect a chronicbehavioural state of reactivity to certain sounds. Since by after-noon the cortisol reflect the day’s activities, with greater vari-ability than occurs after a night’s sleep, afternoon cortisol maybe less reliable as an indicator of the basal cortisol concentrationthan morning levels of cortisol (‘‘in vivo’’ rapid change in haircortisol levels were possible due to the fact that cortisol in haircomes not only from the general circulation but also from the

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skin and hair follicles, (Sharpley et al., 2009). These results seemto be aligned with previous work on rhesus macaques (Daven-port et al., 2006) obtained under controlled laboratory conditionsin which hair cortisol content was found to correlate positivelywith salivary cortisol and to increase as a result of prolongedstressful conditions. Furthermore, in humans, a recent study re-ported that cortisol levels in hair were significantly related toserious life events occurring during the last 3 months beforethe hair was sampled, showing that cortisol measured in haircould serve as a retrospective biomarker of increased cortisolproduction reflecting exposure of major life stressors (Karlénet al., 2011).

Our results also showed that hair cortisol levels at 9:00 h cor-relate with reactivity to barks that dogs produce when playingor when disturbed by an intruder. Heightened reactivity to thesestimuli is also associated with elevation of cortisol levels sam-pled two weeks after testing. No association was found betweencortisol levels in the hair and reactivity to the barks that dogsmake when they are isolated. Isolation barks are more tonaland have more modulation in both pitch and amplitude (Ohl,1996) and (Rogers and Kaplan, 2003) than Disturbance (harsh,low-pitched barks) and Play barks. These different acoustic fea-tures may affect the arousal state of the animal hearing them.Possibly the dogs reacted least to the Isolation bark because itis a signal intended for the dog’s owner and not directed atconspecifics.

By looking separately at each behaviour contributing to themeasure of reactivity in relation to cortisol levels in the hair, wefound that cortisol levels into the hair measured at 9:00 h is asso-ciated with hiding, running away, seeking attention from the tes-ter, panting and lowering of the body posture. Hiding andlowering of the body posture, in particular, seems to indicate se-vere states of acute stress in dogs (Beerda et al., 1998) and (Dre-schel and Granger, 2005), suggesting that these two types ofbehaviour represent a valid indicator of the HPA axis activationoverall. In addition, all of the other measures that we collectedindicate HPA axis activation.

In conclusion, our findings show that hiding, running away,seeking attention from the tester, panting and lowering of thebody posture are behavioural markers of long-term activationof the HPA axis. It appears that chronic elevation of activity ofthe HPA-axis, as shown by hair samples collected in the morningbut not later in the afternoon, is associated with a behaviouraltemperament that is more reactive to sounds of different emo-tional valences.

Conflict of interest statement

No conflict of interest is declared by any of the authors.

Acknowledgments

This research was conducted at the University of New England,NSW, Australia, and was supported by grant from the GiovanniPutignano Educational Award to M.S.

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