Hair cortisol varies with season and lifestyle and relates to human interactions in German shepherd dogs

Abstract

It is challenging to measure long-term endocrine stress responses in animals. We investigated whether cortisol extracted from dog hair reflected the levels of activity and stress long-term, during weeks and months. Hair samples from in total 59 German shepherds were analysed. Samples for measuring cortisol concentrations were collected at three occasions and we complemented the data with individual scores from the Canine Behavioural Assessment and Research Questionnaire (C-BARQ). Generalised linear mixed model (GLMM) results showed that hair cortisol varied with season and lifestyle: competition dogs had higher levels than companion, and professional working dogs, and levels were higher in January than in May and September. In addition, a positive correlation was found between the cortisol levels and the C-BARQ score for stranger-directed aggression (r = 0.31, P = 0.036). Interestingly, the factor “playing often with the dog” (r = −0.34, P = 0.019) and “reward with a treat/toy when the dog behaves correctly” (r = −0.37, P = 0.010) correlated negatively with cortisol levels, suggesting that positive human interactions reduce stress. In conclusion, hair cortisol is a promising method for revealing the activity of the HPA-axis over a longer period of time, and human interactions influence the cortisol level in dogs.

Full text

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Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
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Hair cortisol varies with season
and lifestyle and relates to human
interactions in German shepherd
dogs
Lina S. V. Roth1, Åshild Faresjö2, Elvar Theodorsson3 & Per Jensen1
It is challenging to measure long-term endocrine stress responses in animals. We investigated whether
cortisol extracted from dog hair reected the levels of activity and stress long-term, during weeks and
months. Hair samples from in total 59 German shepherds were analysed. Samples for measuring cortisol
concentrations were collected at three occasions and we complemented the data with individual scores
from the Canine Behavioural Assessment and Research Questionnaire (C-BARQ). Generalised linear
mixed model (GLMM) results showed that hair cortisol varied with season and lifestyle: competition
dogs had higher levels than companion, and professional working dogs, and levels were higher in
January than in May and September. In addition, a positive correlation was found between the cortisol
levels and the C-BARQ score for stranger-directed aggression (r = 0.31, P = 0.036). Interestingly, the
factor “playing often with the dog” (r = −0.34, P = 0.019) and “reward with a treat/toy when the
dog behaves correctly” (r = −0.37, P = 0.010) correlated negatively with cortisol levels, suggesting
that positive human interactions reduce stress. In conclusion, hair cortisol is a promising method for
revealing the activity of the HPA-axis over a longer period of time, and human interactions inuence the
cortisol level in dogs.
Cortisol secretion is the result of the activation of the hypothalamic pituitary adrenocortical (HPA) axis, and plays
a crucial part in the body’s response to dierent kinds of biological stress1,2. In dogs, increased cortisol level can
indicate acute stress from sudden fearful stimuli and is possible to determine in real time with blood and saliva
measurements3,4. It has also been shown that a dog park visit is associated with increased salivary cortisol levels
while a normal walk does not necessarily change the dog’s cortisol level5. In addition, results suggest habituation
to novel and potential stressful situations, since the cortisol levels were negatively correlated with dog park visit
frequency5. Similar negative correlation was found with number of days a dog is staying in an animal shelter,
suggesting habituation to the situation6.
Since cortisol levels in blood and saliva correlate well7, non-invasive saliva sampling has become a useful
method to avoid additional stress due to sampling. Nevertheless, both cortisol in blood and saliva reect a
momentary measurement in real time and to measure long-term cortisol secretion, indicating possible long-term
stress, multiple samples are needed. Cortisol has also been shown to have a circadian rhythm both in dogs and
humans8,9 which makes repeated measurements from dierent times of the day imprecise.
erefore, a promising non-invasive method to study prolonged changes in the HPA-axis activity is to meas-
ure cortisol incorporated in hair10–15. Hair cortisol has been extensively studied during the last years and corre-
lates positively with cortisol levels in both saliva13 and faeces16 of dogs. In addition, rhesus macaques exposed to
prolonged stress14 and lynxes that were weekly injected with ACTH17 (corticotrophin that results in secretion of
cortisol from the adrenals) all showed clear increase in hair cortisol. Correspondingly, dogs with hypercortisolism
have higher hair cortisol values than healthy dogs12 and studies on humans show increased hair cortisol levels
in people with chronic pain18, in unemployed or depressed people19,20 and aer major life events21. Hence, hair
cortisol is a promising indicator of long term HPA-axis activity.
1Linköping University, IFM Biology, 581 85 Linköping, Sweden. 2Linköping University, Department of Medical
and Health Sciences, 581 85 Linköping, Sweden. 3Linköping University, Department of Clinical and Experimental
Medicine, 581 85 Linköping, Sweden. Correspondence and requests for materials should be addressed to L.S.V.R.
(email: lina.roth@liu.se)
Received: 15 October 2015
accepted: 16 December 2015
Published: 21 January 2016
OPEN

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However, even though hair cortisol reveals long-term cortisol secretion and can be an indicator of chronic
stress, we also need to consider possible seasonal variations. From salivary cortisol studies it is shown that levels
vary between seasons with high levels during the winter and low during the summer9,22. It is likely that this is the
case also in hair cortisol even though, to our knowledge, there is no study conrming this.
e aim of this study is to investigate potential relationships between hair cortisol and general lifestyle patterns
in dogs and also to reveal possible dierences in cortisol secretion throughout the year. To exclude possible breed
or size dierences, which may inuence cortisol levels23, we focused on one breed, the German shepherd. Hair
was sampled at three occasions from the same dogs and we also analysed dierent types of hair (wool and guard
hair) from two body sites (chest and neck) in both companion, competition and professional working dogs. We
complement the study with results from the validated Canine Behavioral Assessment and Research Questionnaire
(C-BARQ)24 and additional questionnaires to investigate possible causes of dierences in hair cortisol levels.
Material and Methods
Animals. In total 59 German shepherd dogs were used in this project (Table1, Supplementary 1). We sam-
pled 47 in January, 45 in May and 38 in September. Hence, 38 dogs were sampled at all three occasions and these
repeatedly sampled dogs were grouped according to owner opinion into three lifestyle groups: companion, com-
petition or professional working dogs. Note that additional 12 German shepherds were sampled in September
and were not used in the analysis of seasonal and lifestyle eects but only for correlations with questionnaire
results. e additional dogs were therefor not grouped according to lifestyle. Most of the dogs were privately
owned with the exception of those from the Police (N = 6) and Armed forces (N = 8) and all dogs were recruited
through social media or personal contacts. One male dog was later excluded from all cortisol analyses because of
extreme, possibly pathological, cortisol levels (> 500 pg/mg). All experiments in this paper were conducted in line
with ethical approval from the regional ethical committee for animal experiments in Linköping, Sweden (Permit
number: 51–13).
Hair sampling. Hair samples were obtained by cutting approximately 0.5 g hair with a pair of scissors as close
to the skin as possible without injuring the dog. In January hair was sampled from both chest and neck of the
dogs, but since strong correlation was found in hair cortisol between the two sites (See Results; Fig.1a) and to
minimise possible stress and risk of injuring during sampling only neck hair was sampled in May and September.
We also decided to focus on only the guard hair in consistency with other studies25. Even so the January hair
samples were analysed as both total hair (wool and guard hair as obtained from the dog), and separately in guard
hair only (See Results; Fig.1c). In addition, the separated wool was analysed from 10 dogs (See Results; Fig.1b).
Hair preparation and cortisol extraction. Hair samples were stored in room temperature until prepara-
tion and cortisol extraction which was performed according to methods described in detail previously21,26. Briey,
5–10 mg from each sample were cut into small pieces (< 3 mm), frozen 2 min in liquid nitrogen and minced
together with a steel ball using a Retch Tissue Lyser II in 2 min. Methanol (1 ml) was added to each tube and the
samples extracted for at least 10 hours on a moving board. 0.8 ml of the methanol supernatant was pipetted o
and lyophilized using a Savant Speed Vac Plus SC210A and the samples were dissolved in radioimmunoassay
buer and analysed as described by Morelius et al.26. Hair samples of 5 mg or more were needed for maintaining
a total inter-assay coecient of variation below 8% for hair extraction and measurement of cortisol by the radi-
oimmunoassay. e intra-assay coecient of variation for the radioimmunoassay itself was 7% at 10 nmol/L.
Taking the binding of cortisol as 100%, the antiserum cross-reacts 137% with 5α -dihydroxycortisol, 35,9% with
21-deoxycortisol, 35,9% with prednisolone but less than 1% with endogenous steroids. For a detailed description
of the method, see Karlén et al.21.
Questionnaires and C-BARQ. At each sampling occasion for the 47 dogs sampled in January, May and
September, the owners were asked to answer a simple questionnaire. Here, the owners described the main lifestyle
of their dog (companion, competition or professional working dog), and the dogs were later grouped according
to those answers. Other questions were about background information (name, age, sex, castration), medication,
home environment (other animals/dogs or kids in the household). Most questions generated answers with lit-
tle variation and were not included in further statistical analyses. Furthermore, questions were asked about the
frequency of organised training sessions (scale 0–3 where 0 = 0 sessions, 1 = 1–6 sessions, 2 = 7–15 sessions,
3 = 16 or more sessions) and competition frequency (scale 0–3 where 0 = 0 occasions, 1 = 1–3 occasions, 2 = 4–8
Lifestyle group Total N Females Males Age (mean ± SEM)
Companion 17 10 (0) 7 (2) 3.1 ± 0.7
Competition 16 9 (0) 7 (2) 3.6 ± 0.5
Working (including the excluded male) 14 4 (1) 10 (3) 4.8 ± 0.4
Additional unspecied dogs 12 8 4 2.2 ± 0.4
Table 1. Sex and age (in years) distribution of the analysed German shepherds (N = 59). Castrated/
sterilised dogs are shown in brackets. For 12 additional dogs (only sampled in September) no lifestyle group was
determined and neutered status was not available.

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occasions, 3 = 9 or more competition occasions) during the last three months and these variables were later ana-
lysed for possible correlation with the hair cortisol levels. e owners also included information about what kind
of training they performed. More than half of the companion dogs (10 out of 17 dogs) performed dierent forms
of obedience and tracking training while the majority of the competition dogs (12 out of 16 dogs) were trained
in IPO (Internationale Prüfungs-Ordnung) which includes both tracking, obedience and high level of protection
work.
At the last sampling occasion (September), the owners were asked to complete a C-BARQ questionnaire,
which is a validated and frequently used instrument to collect data about dog behaviour and personality in
everyday life24. All but three completed the questionnaire. e C-BARQ included 105 questions or statements,
where the owners rated their dogs on a scale from 0–4 (0 = never, 1 = seldom, 2 = sometimes, 3 = usually, and
4 = always). e scores were later added to form 13 behavioural categories, according to the standards of the test,
and these categories were analysed for possible correlations with the hair cortisol levels. An additional 17 scaled
questions (0–4, where 0 translates into “do not agree” and 4 into “totally agree”) about play, reward, corrections,
cooperation, focus ability, and training were answered in connection with the C-BARQ.
Statistical analyses. All statistics were performed in the soware SPSS (version 23, IBM).
Due to non-parametric distribution of the hair cortisol levels in the neck and chest hair samples and in the
wool and guard hair samples analysed in January, the Spearman’s nonparametric rank-order correlation was used.
e Spearman’s nonparametric rank-order correlation was also used for all correlations between cortisol levels
and C-BARQ scores and the additional 17 questions.
Generalised linear mixed models (GLMM) with cortisol level (pg/mg) as Fixed target and Gamma log as
probability distribution were used to analyse eects of season, using repeated measures. Time (three levels:
January, May, September) and lifestyle (three levels: companion, competition or professional working dog) were
treated as xed eects together with their interaction. Individual dogs were included as random eects in order
to achieve as good model as possible according to Akaike’s Information Criterion (AIC). Training frequency, age
and sex were also tested as xed eects but were excluded based on AIC. Twelve dogs sampled only in September
for which data on lifestyle was not available, were not included in the GLMM but used in correlation analyses
between cortisol and questionnaire results.
Kruskal Wallis tests were used to investigate dierences between companion, competition and working dogs
for the C-BARQ scores due to the ordinal nature of data.
Results
Hair cortisol from dierent body sites and hair types. Cortisol levels from the January chest hair cor-
related positively with the neck hair (Fig.1a, total hair was analysed, i.e. wool and guard hair together, r = 0.70,
P = 0.001, N = 46). In addition, separating ten of the hair samples from the neck into wool and guard hair
revealed a strong positive correlation of cortisol levels in the two hair types (Fig.1b, r = 0.99, P = 0.001). Similarly,
we found a signicant positive correlation between the hair cortisol level in total hair and in the separated guard
hairs, both sampled from the neck of the dog in January (Fig.1c, r = 0.85, P = 0.001, N = 46).
Cortisol variation with season and lifestyle. Both time of the year, lifestyle and the interaction between
the two showed signicant eects on the hair cortisol level (Table2). us, there was a seasonal eect and the
cortisol level also depended on the lifestyle, where the competition dogs (mainly IPO trained dogs) had higher
cortisol levels than both companion and working dogs (Fig.2). However, no correlation between cortisol and
training frequency, as assessed by the owners themselves, was found (January: r = − 0.27, P = 0.40, May: r = 0.03,
P = 0.87, September: r = 0.05, P = 0.77) and no correlation was found with age of the dog (January: r = − 0.18,
P = 0.24, May: r = 0.13, P = 0.41, September: r = 0.04, P = 0.84).
10 20
20
30
30 40
40
50
50
60
60
10
Cortisol in neck hair (pg/mg)
Cortisol in chest hair (pg/mg)
10 20
20
30
30 40
40
50
50
60
60
10
Cor
tisol in guard hair (pg/mg)
Cortisol in wool (pg/mg)
10 20
20
30
30 40
40
50
50
60
60
10
Cor
tisol in guard hair (pg/mg)
Cortisol in total hair (pg/mg)
a b c
Figure 1. Correlations between hair cortisol (pg/mg) in neck and chest hair (a), in wool and guard hair (b), and
in total hair and the separated guard hair (c), all from the January sampling occasion. ree values above 60 pg/
mg in (a,c) have been omitted from the gures for clarity; (a) (neck vs chest): 173 vs 60 pg/mg, 124.4 vs 101.2 pg/
mg and 166.1 vs 372.8 pg/mg; (c) (guard hair vs total hair): 156.0 vs 173.4 pg/mg, 75.3 vs 124.4 pg/mg and 168.5
vs 166.1 pg/mg).

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Cortisol correlations with owner questionnaires. A positive correlation was found between cor-
tisol level and the C-BARQ score “stranger-directed aggression” (r = 0.31, P = 0.036) for the dogs sampled in
September (N = 46) and there was also a negative correlation between cortisol and the C-BARQ score “Chasing”
(r = − 0.38, P = 0.009). Interestingly, correlations between cortisol levels and the responses to additional ques-
tions included in September questionnaires revealed a negative correlation with the scores on “the purpose of the
dog is to have a nice companion dog” (r = − 0.32, P = 0.029), “play oen with the dog” (r = − 0.34, P = 0.019),
and “reward with a treat/toy when the dog behaves correctly” (r = − 0.37, P = 0.010). No other scores on CBARQ
categories or additional questions were signicantly correlated with cortisol levels.
C-BARQ differences between companion, competition and working dogs. Analyses of the
C-BARQ scores in September for the companion, competition and working dogs showed signicant dierences
for “dog-directed aggression” (higher in working dogs; P = 0.04) and “non-social fear” (higher in companion
dogs; P = 0.004, Table3). ere were no other signicant dierences between the groups of dogs with respect to
questionnaire data.
Discussion
To our knowledge this is the rst study that has investigated seasonal variation and variations related to lifestyle
in long-term cortisol secretion in dogs. e results from the study show that both season and lifestyle signicantly
inuence the hair cortisol levels.
50.0
40.0
30.0
20.0
10.0
JanuaryMay September
Mean (SEM) hair cortisol (pg/mg)
Companion
Competing
Working
Figure 2. Mean hair cortisol level (pg/mg) in January, May and September for companion, competition and
working German shepherds. Standard Error of Mean (SEM) is shown with error bars.
C-BARQ score Dog group Median Mean R ank Mean SEM
Dog-directed aggression Companion 1.00 15.21 1.21 0.35
Competition 1.00 15.32 1.11 0.17
Wor k ing 2.25 24.85 2.10 0.26
Non-social fear
Companion 0.33 23.00 0.35 0.07
Competition 0.00 10.41 0.03 0.03
Wor k ing 0.17 19.35 0.22 0.06
Table 3. C-BARQ scores “Dog-directed aggression” and “Non-social fear” for companion (N = 14),
competition (N = 11) and working dogs (N = 10).
F df1 df2 Sign.
Corrected model 9.16 8 118 0.001
Time (Jan, May, Sep) 18.59 2 118 0.001
Lifestyle (companion,
competition, working) 11.04 2 118 0.001
Time * Lifestyle 2.93 4 118 0.024
Table 2. Generalised linear mixed model with cortisol level (pg/mg) as xed target and time, lifestyle and
the interaction Time*Lifestyle treated as xed factors and individual dog treated as random eect (N = 46).

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Earlier studies on other species have suggested that there are dierences in hair cortisol depending on body
location17,25,27. However, in our study, the levels of cortisol correlated strongly between dierent body parts, so the
method appears highly reliable for long-term assessments over time in the same dogs.
Investigating the cortisol levels in wool and guard hair separately revealed high correlation and no signicant
dierence between the hair types. Hence, the method appears to be quite insensitive with respect to which parts
of the fur that is used for hormone extraction. However, dierences have been reported in other species25, so we
continued with analysing only guard hair to make the results as comparable as possible between individuals,
between breeds in future studies, and possibly also with other species with dierent hair compositions. Further
studies and standardisations of the sampling method are likely to guide future comparative studies.
Our results suggest that seasonal variations need to be considered in cortisol studies. We found highest hair
cortisol values in January. Since the hair grows gradually28, these levels reect the dogs’ secretion in the preceding
late fall and early winter in Sweden. e results therefore indicate an increased stress or activity level during this
period, particularly in competition dogs. In a recent study on hair cortisol in foals during three consecutive foal-
ing seasons (January to July) no eects were found of either temperature, or day length29. However, in polar bears
hair cortisol was shown to heavily depend on uctuations in climate and ice cover30, and as mentioned before
some saliva cortisol studies suggest high cortisol levels during the winter9,22. Hence, possible seasonal variations
need to be considered when sampling and comparing cortisol levels irrespectively of method used.
Dierent lifestyles and human demands on dogs might aect their activity pattern and HPA-axis activity. We
found that competition dogs had higher hair cortisol levels than both companion and working dogs and this
was especially obvious in January. Since a few millimetres proximal to the hair follicle were not included in hair
sample (some part is even below the skin) the novel situation of obtaining hair (which lasted less than a minute)
could not have induced this increase. e cortisol increase could possibly be related to variations in the amount of
training since competition dogs usually train less during the winter and more during spring and in connection to
competition season. A sudden decrease in training and competing during the late fall and winter, which is a com-
mon situation for many competition dogs, could possibly be experienced as an unpredictable stress experience2.
Or , the dogs might simply be less exercised aer competition season which might induce restlessness. However,
no correlation or interaction between cortisol and training frequency was found, but it should be noted that the
information and estimation of the training intensity was relatively crude in our experiments and we have no data
on general exercise. Other studies have found relationship between cortisol and training, for example, increased
cortisol levels were found in a study on faecal cortisol during training activities in avalanche dogs31. Still, an
interesting question for future studies is whether large unpredictable changes in activity level i.e. from a day of
rest to an intensive training session could be perceived as unpredictable and stressful, unlike the situation for
professional working dogs that are more or less active throughout the day or companion dogs where the contrasts
between rest and walks or light training sessions is small.
An additional possibility is that the dogs used for competition are chosen because of certain personality traits
that could be accompanied by high cortisol levels. For example, hair cortisol correlates positively with the person-
ality trait “reactivity” in dogs32. e majority (75%) of the competition dogs in the present study were trained and
competed in IPO (Internationale Prüfungs-Ordnung), which includes both tracking, obedience and high level of
protection work, and thereby requires dogs with high levels of reactivity and assertiveness. It could also be that
the IPO dogs are exposed to dierent training methods than other German shepherd dogs, but more studies are
needed to evaluate this.
From the C-BARQ results we found a positive correlation between ”stranger-directed aggression” and cor-
tisol levels, corroborating previous studies33. Since competition dogs showed higher cortisol levels, one possi-
bility could have been that they would therefor also show more stranger-directed aggression, but in fact the
C-BARQ results for companion, competition and working dogs only diered for “Dog-directed aggression”
and “non-social fear” where competition dogs showed low scores. No signicant dierence between the groups
was found for “stranger-directed aggression”. Hence, more studies are needed to elucidate the exact relationship
between cortisol levels and dierent types of aggression and fear.
In addition, a negative correlation between the C-BARQ score for “chasing” and cortisol was found. e ques-
tions behind this score ask how the dog behaves towards e.g. cats, squirrels and birds and whether the dog is prone
to chase them. It could possibly be that dogs that do not chase due to training generate a suppressed motivation
of hunting behaviour, which might, if exposed to the situation frequently, increase the cortisol level. But this is
highly speculative and needs more investigation.
Maybe not surprising but still welcome result is that a negative correlation was found between cortisol level
and how oen the owner played with their dog and also whether the owners used toy/treat when rewarding their
dog. Both these results could reect that friendly and encouraging relationships are related to less stress in the
dogs. Play interactions including aectionate behaviour have earlier been shown to have a direct decreasing eect
on cortisol levels in dogs34, and dogs treated with corticosteroids are also less playful35, which both are in line with
our ndings on long-term cortisol secretion. ese results may be important for understanding the physiological
consequences of dierent types of human-dog relationships.
Conclusion
Hair cortisol is a reliable long-term assessment method in dogs and our results show high consistency between
dierent body sites and hair types. ere were seasonal uctuations in cortisol levels and they also varied in
relation to lifestyle. In addition, cortisol levels were negatively correlated with friendly human interactions and
positively correlated with human-directed aggression.

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References
1. Selye, H. e nature of stress. Basal Facts. 7, 3–11 (1985).
2. oolhaas, J. M. et al. Stress revisited: a critical evaluation of the stress concept. Neurosci. Biobehav. . 35, 1291–1301 (2011).
3. Hydbring-Sandberg, E. et al. Physiological reactions to fear provocation in dogs. J. Endocrinol. 180, 439–448 (2004).
4. Beerda, B., Schilder, M., van Hoo, J., de Vries, H. & Mol, J. Behavioural, saliva cortisol and heart rate responses to dierent types of
stimuli in dogs. Appl. Anim. Behav. Sci. 58, 365–381 (1998).
5. Ottenheimer Carrier, L., Cyr, A., Anderson, . E. & Walsh, C. J. Exploring the dog par: elationships between social behaviours,
personality and cortisol in companion dogs. Appl. Anim. Behav. Sci. 146, 96–106 (2013).
6. Hennessy, M. Plasma cortisol levels of dogs at a county animal shelter. Physiol. Behav. 62, 485–490 (1997).
7. Vincent, I. C. & Michell, A. . Comparison of cortisol concentrations in saliva and plasma of dogs. es. Vet. Sci. 53, 342–345 (1992).
8. Giannetto, C. et al. Parallelism of circadian rhythmicity of salivary and serum cortisol concentration in normal dogs. J. Appl. Biomed.
12, 229–233 (2014).
9. Persson, . et al. Seasonal variation in human salivary cortisol Concentration. Chronobiol. Int. 25, 923–937 (2008).
10. ussell, E., oren, G., ieder, M. & Van Uum, S. Hair cortisol as a biological marer of chronic stress: current status, future
directions and unanswered questions. Psychoneuroendocrinology 37, 589–601 (2012).
11. Bryan, H. M., Adams, A. G., Invi, . M., Wynne-Edwards, . E. & Smits, J. E. G. Hair as a meaningful measure of baseline cortisol
levels over time in dogs. JAALAS. 52, 189–196 (2013).
12. Ouschan, C., uchar, A. & Möstl, E. Measurement of cortisol in dog hair: a noninvasive tool for the diagnosis of hypercortisolism.
Vet. Dermatol. 24, 428–e94 (2013).
13. Bennett, A. & Hayssen, V. Measuring cortisol in hair and saliva from dogs: coat color and pigment dierences. Domest. Anim.
Endocrinol. 39, 171–180 (2010).
14. Davenport, M., Tiefenbacher, S., Lutz, C. ., Nova, M. A. & Meyer, J. S. Analysis of endogenous cortisol concentrations in the hair
of rhesus macaques. Gen. Comp. Endocrinol. 147, 255–261 (2006).
15. Staufenbiel, S. M., Penninx, B. W. J. H., Spijer, A. T., E lzinga, B. M. & van ossum, E. F. C. Hair cortisol, stress exposure, and mental
health in humans: A systematic review. Psychoneuroendocrinology. 38, 1220–1235 (2013).
16. Accorsi, P. et al. Cortisol determination in hair and faeces from domestic cats and dogs. Gen. Comp. Endocrinol. 155, 398–402
(2008).
17. Terwissen, C. V., Mastromonaco, G. F. & Murray, D. L. Inuence of adrenocorticotrophin hormone challenge and external factors
(age, sex, and body region) on hair cortisol concentration in Canada lynx (Lynx canadensis). Gen. Comp. Endocrinol. 194, 162–167
(2013).
18. Van Uum, S. H. et al. Elevated content of cortisol in hair of patients with severe chronic pain: A novel biomarer for stress. Stress. 11,
483–488 (2008).
19. Dettenborn, L., Tietze, A., Brucner, F. & irschbaum, C. Higher cortisol content in hair among long-term unemployed individuals
compared to controls. Psychoneuroendocrinology. 35, 1404–1409 (2010).
20. Dettenborn, L. et al. Introducing a novel method to assess cumulative steroid concentrations: Increased hair cortisol concentrations
over 6 months in medicated patients with depression. Stress. 15, 348–353 (2012).
21. arlén, J., Ludvigsson, J., Frostell, A., eodorsson, E. & Faresjö, T. Cortisol in hair measured in young adults - a biomarer of major
life stressors? BMC Clin. Pathol. 11, 12 (2011).
22. ing, J. A. et al. Sequence and seasonal eects of salivary cortisol. Behav. Med. 26, 67–73 (2000).
23. Sandri, M., Colussi, A., Perrotta, M. G. & Stefanon, B. Salivary cortisol concentration in healthy dogs is aected by size, sex, and
housing context. J. Vet. Behav. 10, 302–306 (2015).
24. Hsu, Y. & Serpell, J. A. Development and validation of a questionnaire for measuring behavior and temperament traits in pet dogs.
JAVMA. 223, 1293–1300 (2005).
25. Macbeth, B. J., Cattet, M., Stenhouse, G. B., Gibeau, M. L. & Janz, D. M. Hair cortisol concentraton as a noninvasive measure of long-term
stress in free-ranging grizzly bears (Ursus arctos): considerations with implications for other wildlife. Can. J. Zool. 88, 935–949 (2010).
26. Morelius, E., Nelson, N. & eodorsson, E. Salivary cortisol and administration of concentrated oral glucose in newborn infants:
improved detection limit and smaller sample volumes without glucose interference. Scand. J. Clin. Lab. Invest. 64, 113–118 (2004).
27. Moya, D., Schwartzopf-Genswein, . S. & Veira, D. M. Standardization of a non-invasive methodology to measure cortisol in hair
of beef cattle. Livest. Sci. 158, 138–144 (2013).
28. Gunaratnam, P. & Wilinson, G. T. A study of normal hair growth in the dog. J Small Anim. Pract. 24 (1983).
29. Montillo, M. et al. e eect of temperature, rainfall, and light conditions on hair cortisol concentrations in newborn foals. J. Equine
Vet. Sci. 34, 774–778 (2014).
30. Bechshø, T. Ø. et al. Polar bear stress hormone cortisol uctuates with the North Atlantic Oscillation climate index. Polar Biol. 36,
1525–1529 (2013).
31. Slotta-Bachamayr, L. & Schwarzenberger, F. Faecal cortisol metabolites as indicators of stress during training and search missions in
avalanche dogs. Vet. Med. Austria. 94, 110–117 (2007).
32. Siniscalchi, M., McFarlane, J. ., auter, . G., Quaranta, A. & ogers, L. J. Cortisol levels in hair reect behavioural reactivity of
dogs to acoustic stimuli. es. Vet. Sci. 94, 49–54 (2013).
33. osado, B. et al. Blood concentrations of serotonin, cortisol and dehydroepiandrosterone in aggressive dogs. Appl. Anim. Behav. Sci.
123, 124–130 (2010).
34. Horváth, Z., Dóa, A. & Milósi, Á. Aliative and disciplinary behavior of human handlers during play with their dog aects
cortisol concentrations in opposite directions. Horm. Behav. 54, 107–114 (2008).
35. Notari, L., Burman, O. & Mills, D. Behavioural changes in dogs treated with corticosteroids. Physiol. Behav. 151, 609–616 (2015).
Acknowledgements
We would like to thank all dedicated GSD owners, the Linköping Police Force and the Swedish Armed Forces F17
in Kallinge that voluntarily contributed to this study. We also thank Nathalie Bjällerhag, Anna Grozelier and Ann-
Charlotte Svensson Holm for help with sample preparations, Lars Westerberg for statistical advice, and Tassarnas
Trim in Linköping for supplying dog hair for methodological development. e project was performed within the
framework of the Swedish Center of Excellence in Animal Welfare Science, nanced by Formas. e project was
funded by the European Research Council (ERC) within the advanced grant “GENEWELL” (322206) and by the
County Council of Östergötland, Sweden.
Author Contributions
e experiment was conceived by L.S.V.R. and P.J. e sampling and data collection was performed by L.S.V.R.
while Å.F. and E.T. performed all laboratory analyses. L.S.V.R. analysed all data, and wrote the paper in
collaboration with P.J. All authors reviewed the manuscript.

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7
Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Roth, L. S.V. et al. Hair cortisol varies with season and lifestyle and relates to human
interactions in German shepherd dogs. Sci. Rep. 6, 19631; doi: 10.1038/srep19631 (2016).
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