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


Abstract and Figures

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.
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Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
Hair cortisol varies with season
and lifestyle and relates to human
interactions in German shepherd
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.
Received: 15 October 2015
accepted: 16 December 2015
Published: 21 January 2016
Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
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.
Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
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
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 Spearmans nonparametric rank-order correlation was used.
e Spearmans 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 Akaikes 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.
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
30 40
Cortisol in neck hair (pg/mg)
Cortisol in chest hair (pg/mg)
10 20
30 40
tisol in guard hair (pg/mg)
Cortisol in wool (pg/mg)
10 20
30 40
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).
Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
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.
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.
JanuaryMay September
Mean (SEM) hair cortisol (pg/mg)
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).
Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
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.
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.
Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
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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.
Scientific RepoRts | 6:19631 | DOI: 10.1038/srep19631
Additional Information
Supplementary information accompanies this paper at
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).
is work is licensed under a Creative Commons Attribution 4.0 International License. e images
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... Welfare and stress are closely related, and therefore assessing longterm stress levels can give an indication of the animal's welfare. A reliable method to assess stress levels is by measuring cortisol concentrations in for example saliva or hair (Russell et al., 2012;Roth et al., 2016;Heimbürge et al., 2019;Sauveroche et al., 2020). While saliva cortisol is a biomarker for acute stress responses (Russell et al., 2012;Vieira de Castro et al., 2020), hair cortisol concentration is a non-invasive method to assess cortisol release over an extended period forming a retrospective calendar of long-term stress levels (Roth et al., 2016;Heimbürge et al., 2019;Sundman et al., 2019;Sauveroche et al., 2020). ...
... A reliable method to assess stress levels is by measuring cortisol concentrations in for example saliva or hair (Russell et al., 2012;Roth et al., 2016;Heimbürge et al., 2019;Sauveroche et al., 2020). While saliva cortisol is a biomarker for acute stress responses (Russell et al., 2012;Vieira de Castro et al., 2020), hair cortisol concentration is a non-invasive method to assess cortisol release over an extended period forming a retrospective calendar of long-term stress levels (Roth et al., 2016;Heimbürge et al., 2019;Sundman et al., 2019;Sauveroche et al., 2020). Hence, by analysing hair cortisol concentrations in the mane we can assess long-term stress in horses (Roth et al., 2016;Heimbürge et al., 2019;Sauveroche et al., 2020). ...
... While saliva cortisol is a biomarker for acute stress responses (Russell et al., 2012;Vieira de Castro et al., 2020), hair cortisol concentration is a non-invasive method to assess cortisol release over an extended period forming a retrospective calendar of long-term stress levels (Roth et al., 2016;Heimbürge et al., 2019;Sundman et al., 2019;Sauveroche et al., 2020). Hence, by analysing hair cortisol concentrations in the mane we can assess long-term stress in horses (Roth et al., 2016;Heimbürge et al., 2019;Sauveroche et al., 2020). ...
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Horses are commonly trained using negative reinforcement. However, a growing body of scientific evidence supports positive reinforcement as an efficient training method for horses. In this study we investigated the effects of adding a small but regular amount of positive reinforcement training to horses trained with negative reinforcement. A total of 36 privately owned horses not previously trained with positive reinforcement were divided into a training (N=17) and a control (N=19) group. The owners in the training group were asked to follow a training plan based on positive reinforcement for eight to nine weeks, in addition to their normal negative reinforcement training. The control horses continued with their usual negative reinforcement training. All horses were subjected to behavioural tests before and after the training period: a motionless human test to assess contact-seeking behaviour and a cognitive bias test to assess emotional state. Mane hair samples were obtained from all horses at the start and at the end of the training period to analyse hair cortisol concentrations as an expression of long-term stress. In addition, all owners filled out a questionnaire about their perceived relationship with their horses before and after the training period. We found that horses in the training group engaged in more physical contact (P=0.050) with an unfamiliar person after the training period compared to before. The training group also tended to improve their owner-assessed relationship score (P=0.072). They did not, however, show changes in their emotional state as assessed by the cognitive bias test (P>0.1). Furthermore, we found no difference between the training and control groups in terms of hair cortisol concentrations. We conclude that a small but regular addition of positive reinforcement training can increase horses’ contact-seeking behaviour towards humans but is not enough to improve their emotional state or long-term stress levels.
... A 3 × 3 cm area was shaved or cut bald on T1 and pre-adoption to allow a re-shave of newly grown hair at 6 weeks in-shelter and 6 weeks post-adoption. Hairs were collected following a standard protocol in the dorsal neck region as this region is often used in other studies and is easy to access [23][24][25]28 . The researcher or owner wore nitrile gloves and shaved (Wahl® professional Cordless Trimmer-Super Trim, Type 1592) or cut (blunted scissors) the dog's hair as close to the skin as possible. ...
... Hair selection, processing, and analysis. Both wool and guard hair were used for analysis, as these are strongly correlated 23 . From samples T2 and T3, longer and therefore older hairs were removed to prevent contamination of hair outside of the shave/re-shave area. ...
... Other main effects. Overall, low weight dogs (i.e., smaller dogs, < 10 kg, 20.2 ± 7.9 pg/mg, n = 18 or 35% of all dogs) had higher HCCs than larger weight dogs (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) 2 Estimated ratio of mean of specified sample collection moment and mean at T1. 3 Estimated ratio of mean of specified weight class and mean in reference weight class. 4 Estimated ratio of mean of specified sex and mean in reference sex. 5 Estimated ratio of mean of specified relinquishment reason and mean in reference relinquishment reason. ...
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Shelter dogs are exposed to a variety of stressors. Among non-invasive techniques, hair cortisol concentration (HCC) is suggested an easy to collect biomarker for giving insight into long-term stress responses. We evaluated HCC as an indicator of long-term cortisol responses in dogs in an animal shelter over different chronological time points during sheltering and after adoption. Hair samples were collected from the neck region following a shave/re-shave protocol of shelter dogs (total n = 52) at four different time periods: T1 intake at shelter (pre-shelter period, n = 51); T2 after 6 weeks in the shelter (n = 23); T3 6 weeks after adoption (n = 24); T4 6 months after adoption (n = 22). HCC at T2 was significantly higher than HCC at T1, T3 and T4 (effect of sample collection moment: F3,41 = 12.78, p < 0.0001). The dog’s weight class, age class, sex, reason for admission, kennel history and melanin type also explained HCC variability. No significant difference in HCC was found between shelter dogs T1 and control pet dogs in their own homes (n = 20, one sample, t = − 1.24, p = 0.219). A significant but moderate positive correlation between HCC and urinary cortisol:creatinine ratios was found (т = 0.3, p < 0.001). As HCC increased in the shelter, the use of this non-invasive parameter appears a useful additional tool in dog welfare research.
... Combined these results suggest that dogs have a capacity to grow accustomed to their working environment when they encounter it more frequently, and as a result mount a lower cortisol response to working situations compared to less experienced dogs. This conclusion is further supported by results of Roth, Faresjö, Theodorsson, and Jensen (2016) who observed little difference between the hair cortisol levels of companion and working (police/military) dogs over the course of three different seasons. Whether these results would also hold true for therapy or even service dogs however, remains to be seen as the longitudinal effect of assistance work in both these types of dogs has yet to be studied. ...
... One of the ways in which the longitudinal effect of assistance work in service dogs could be explored is by following the example of Roth et al. (2016) and compare the level of hair cortisol in service dogs with that of companion dogs. Hair cortisol is a fairly new analysis method to determine physiological stress in an individual human or animal over extended periods of time. ...
... It therefore takes a varied period of time before the new segment of hair arrives at the skin surface and its cortisol contents can be measured (Harkey, 1993;Udo, 1978). This period depends mainly on the growth rate of the hair, which in turn may be affected by lifestyle, social interaction, month or season, sex, age, hair color, species, and the body region it is taken from (Bennett & Hayssen, 2010;Dettenborn, Tietze, Kirschbaum, & Stalder, 2012;Mesarcova, Kottferova, Skurkova, Leskova, & Kmecova, 2017;Roth et al., 2016;Terwissen, Mastromonaco, & Murray, 2013). Hair cortisol has nonetheless been successfully linked to changes in diurnal salivary (D'Anna-Hernandez, Ross, Natvig, & Laudenslager, 2011;Papafotiou et al., 2017;Vanaelst et al., 2012), and 24-hour urinary cortisol (Russell, Koren, Rieder, & Van Uum, 2012) within an individual, which makes it a valuable tool for assessing longitudinal physiological stress experience on an individual level. ...
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Service dogs are trained to assist humans. This assistance potentially exposes them to stressors To investigate if service dogs are exposed to more stressors than companion dogs we questioned whether hair cortisol levels differed between both groups. We studied this by cutting a tuft of hair from the neck of 19 companion and 11 service dogs. Cortisol levels were subsequently analyzed via immunoassay and compared via a simple linear regression model. The influence of coat color, season, sex, other dogs, pets, or mental health diagnoses in the household was also checked . Results showed that cortisol values did not differ between service and companion dogs. Furthermore, none of the additional variables had an influence on cortisol levels. This lead to the conclusion that the service dogs in this study did not have higher hair cortisol levels than companion dogs Further study should be conducted as to why no difference did occur between groups and if this difference is persistent over time given that we only studied a period of up to two months' worth of hair cortisol.
... Measuring cortisol metabolites in feces or urine enable retrospective measures, unaffected by the sampling procedure, but are influenced by passage rate and require the collection of all feces or urine excreted from the animal during the time period of interest, which causes practical difficulties (Möstl and Palme, 2002). Hair cortisol, on the other hand, is a non-invasive technique used for studying retrospective cortisol levels (Lee et al., 2015;Roth et al., 2016;Burnard et al., 2017). As cortisol is integrated with the hair during its growth, this method gives a picture of the total amount of circulating cortisol over time and the sampling is not subjected to the problems of circadian variations or the interference of momentary stress during sampling (Lee et al., 2015). ...
... From 318 sampled animals, 196 samples including both individuals colonized by VTEC O157:H7 and randomly selected negative controls (1-3 controls per colonized individual) were selected for analysis of hair cortisol. The protocol for preparing and analyzing hair was based on previous studies on cattle and other species (Meyer et al., 2014;Tallo-Parra et al., 2015;Roth et al., 2016) ...
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Using levels of the stress hormone cortisol as an indicator for welfare is a common, but debated practice. In this observational study, hair cortisol concentration (HCC) of samples from 196 dairy calves from 7 to 302 days of age collected from 12 Swedish farms was determined using a commercially available ELISA. An assessment of animal welfare, assessed using animal-based indicators, was performed on the day of sampling. First, methodological factors with the potential to impact HCC and the effect of age were analyzed using generalized additive models. This revealed a significant peak in hair cortisol in young calves (around 50 days of age) and an association between fecal contamination of hair samples and the level of cortisol extracted. Second, associations between welfare indicators and HCC were explored using cluster analysis and regularized regression. The results show a complex pattern, possibly related to different coping styles of the calves, and indicators of poor welfare were associated with both increased and decreased hair cortisol levels. High cortisol levels were associated with potential indicators of competition, while low cortisol levels were associated with the signs of poor health or a poor environment. When running the regularized regression analysis without the contaminated hair samples and with the contaminated samples (including a contamination score), the results did not change, indicating that it may be possible to use a contamination score to correct for contamination.
... Measuring steroids in hair is non-invasive and the samples can be stored at room temperature. Although hair cortisol concentrations (HCCs) have been assessed in several species [8,[10][11][12][13], to the best of our knowledge, no study has analysed HCCs in dairy donkeys. ...
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The increased interest in donkeys because of their milk has led to changes in their farm management. Little is known about the effect of the farming systems on donkey health and welfare. Measuring hair cortisol concentrations is an emerging method to assess stress in animals. To the best of our knowledge, no cortisol assessment has been done on dairy donkeys; similarly, only a few studies have investigated donkey haematological values. The aim of this study was to evaluate the effects of the lactation phase, parity and season on blood parameters, milk yield and quality and hair cortisol in dairy donkeys. Individual samples of milk, blood and mane hair were taken from twenty jennies at 1, 6 and 10 months after parturition. Higher values of hair cortisol were found in the first sampling, suggesting temporary stress during the peri-parturition. The parity influenced the number of blood cells, which was lower in the pluriparous jennies. The season affected milk quality and mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. The latters might represent the adaptation to the environmental conditions. This study contributes to a better understanding of the biochemical processes occurring in lactating jennies, and to their physiological and wellbeing status.
... In the present study, no difference in hair cortisol concentrations was found between securely and insecurely attached dogs. Although there is some evidence that aspects of the dog-owner relationship, such as positive interactions [64,65] or the owner's reduced perception of the costs of caring for the dog [38] may be associated with lower hair cortisol concentrations, this is the first study to investigate a possible association of the latter with dog attachment insecurity. In human literature, attachment insecurity seem to be a predictor of chronic stress-related health problems, such as cardiovascular diseases [66] and inflammatory-related illnesses [67]. ...
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The quality of the attachment bond towards the caregiver may affect the dog’s physiological responses to stressful stimuli. This study aimed to measure chronic and acute physiological parameters of stress in ten securely and ten insecurely attached dogs. The twenty experimental subjects were selected from a sample of dogs that participated with their owners in the Strange Situation Procedure. Saliva samples were collected before (T0) and after (T1) the test. Blood pressure, heart rate, respiratory rate, and rectal temperature were measured after the test, only. At this time, a hair sample was also collected. RM ANOVA was used to analyse cortisol concentrations between secure and insecure dogs at T0 and T1. Mann–Whitney U test or T test were used for other physiological parameters. Insecure dogs had significant higher salivary cortisol concentrations than secure dogs at T1 (p = 0.024), but only a non-significant trend towards higher cortisol concentrations at T0 (p = 0.099). Post-test heart rate also tended to be higher in insecure compared to secure dogs (p = 0.077). No significant differences in hair cortisol concentration were found. The quality of attachment may affect the dog’s physiological response to acute stress, at least when related to separation from the caregiver. The effect of attachment on chronic stress requires further investigation.
... HCC is influenced by multiple factors of different origin-internal, external, physiological, and environmental. Hair cortisol levels were found to vary with sex (Lafferty et al. 2015), age (Laudenslager et al. 2012;del Rosario et al. 2011), body condition (Macbeth et al. 2012), personality (Sauveroche et al. 2020), pregnancy (Fairbanks et al. 2011), season of the year (Roth et al. 2016), or even features of the hair itself such as hair color (Yamanashi et al. 2013). However, their effects on HCC are inconsistent across species and may be caused by different mechanisms (Heimbürge et al. 2019). ...
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Hair cortisol concentration (HCC) has recently gained popularity as an easy-to-measure biomarker of long-term stress in wild and domestic animals. Hair integrates cortisol over long time periods within a single sample and it can be collected non- invasively, which makes its use particularly interesting for wildlife studies. Interpreting HCC values, however, is challenging, because they are determined by the interplay of multiple factors. Here, were explore potential determinants of HCC in the Alpine marmot Marmota marmota. We tested the relationship of sex, age class, physical condition and body temperature with the hair cortisol concentration of free-ranging marmots. We found marked sex difference in HCC, with higher levels in females. This might be related to sex-specific variation in social stress or resulting from physiological difference, e.g., in baseline and stress-induced levels of cortisol secretion. Interestingly, body temperature was also positively related to HCC, possibly hinting at individual short- and long-term stress reactivity as part of coping styles. Although further work is needed to entangle possible mechanisms underlying the neuro-endocrinological modulation on HCC, our results emphasize that determinants such as sex and body temperature in Alpine marmots should be accounted for, when using HCC as marker of chronic stress.
... Proximity seeking is also related to affiliative behavior during, for example, reunion after separation [52,55]. Secure attachment, strong emotional bond and positive interactions between the dog and the owner are associated with reduced level of stress in dogs [57][58][59]. The quality of the dog-owner relationship seems to modulate the dog's long-term stress coping [60,61]. ...
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We evaluated the effect of the dog–owner relationship on dogs’ emotional reactivity, quantified with heart rate variability (HRV), behavioral changes, physical activity and dog owner interpretations. Twenty nine adult dogs encountered five different emotional situations (i.e., stroking, a feeding toy, separation from the owner, reunion with the owner, a sudden appearance of a novel object). The results showed that both negative and positive situations provoked signs of heightened arousal in dogs. During negative situations, owners’ ratings about the heightened emotional arousal correlated with lower HRV, higher physical activity and more behaviors that typically index arousal and fear. The three factors of The Monash Dog–Owner Relationship Scale (MDORS) were reflected in the dogs’ heart rate variability and behaviors: the Emotional Closeness factor was related to increased HRV (p = 0.009), suggesting this aspect is associated with the secure base effect, and the Shared Activities factor showed a trend toward lower HRV (p = 0.067) along with more owner-directed behaviors reflecting attachment related arousal. In contrast, the Perceived Costs factor was related to higher HRV (p = 0.009) along with less fear and less owner-directed behaviors, which may reflect the dog’s more independent personality. In conclusion, dogs’ emotional reactivity and the dog–owner relationship modulate each other, depending on the aspect of the relationship and dogs’ individual responsivity.
Background: There are no studies which evaluate hair cortisol as a biological marker of stress and anxiety in pruritic dogs during atopic dermatitis therapy. Objectives: A longitudinal evaluation of hair cortisol concentrations, the severity of disease and the QoL in dogs with cAD during therapy with lokivetmab. Animals: Ten client-owned dogs with cAD. Materials and methods: Dogs were assessed at three time points: at the initial visit at day (D) 0 and at D28, when lokivetmab (2.2-3.2 mg/kg) was administered, and at D56 for one further evaluation. At all time points, pruritus and lesion severity was assessed using the pruritus Visual Analog Scale (PVAS) and Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04). Dog owners filled out a validated QoL questionnaire and hair cortisol concentrations were measured from samples collected from the same area on each dog. Results: There was a significant reduction in PVAS (p < 0.001) and improvement in QoL of dogs (QoL1) and owners (QoL2) after lokivetmab administration, with a positive correlation of the PVAS with QoL1 and QoL2 (r = 0.71 and 0.52, respectively). There was no difference in CADESI-04 scores at the different time points (p = 0.515). A significant reduction in hair cortisol levels at D56 was measured compared with D28 (p < 0.001). Conclusions and clinical relevance: Hair cortisol may be a useful marker of stress in dogs with cAD. These results highlight the negative impact of cAD on the QoL of dogs and their owners, and the positive benefit of lokivetmab therapy.
Animal welfare is the quality of life as perceived by the animal itself. It is also the state of an animal in its attempt to cope with its environment. Animal welfare has high ethics and economic importance. Thus the need to develop parameters for assessing animal welfare. An acute increase in glucocorticoid (GC) concentration is necessary for adaptation to a stressful situation. Glucocorticoids also play a significant role in metabolic, cardiovascular, and immune systems. Glucocorticoid enhances effective learning through the hippocampus and other normal body functions. That is why we remember events (either positive or negative) associated with strong emotions. Long-term secretion of GCs has catabolic effects. Thus, affecting animal health. Measuring GC is one of the ways of assessing animal welfare. But, high GC concentration does not only indicate pain or suffering. We report that stress and emotion trigger similar physiological responses. So, measuring GC levels cannot differentiate between positive and negative states. We conclude that GC shows circadian rhythms and episodic spikes in some species. Values from a single sample point are not reliable to make conclusions about a condition. Training animals for blood collection may reduce stress. Thus not causing bias in the GC concentration measured.
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Avalanche dogs are specially trained to use their sensitive sense of smell to find buried people in avalanches. Emotional and physical stress factors before and during a search, as well as nervousness felt by the dog handler, negatively influence the efficiency of search dogs. To improve the performance of avalanche dogs and to enable the dogs to operate over longer time periods, a better understanding of the physiological consequences of possible arousal, disturbance and stress occurring during a search mission are needed. In this study faecal glucocorticoid metabolites were analysed. Faecal samples from privately owned avalanche dogs (n = 11) of different breed and age before, during and after 2 separate training camps, each 1 week in length, were collected and the results combined. An enzymeimmunoassay to analyse faecal cortisol metabolites was used to evaluate the stress response during training and search. Compared to baseline concentrations (measured during the weeks before and after the training camp, when dogs were at home), cortisol metabolites increased when dogs were in the training camp. The type of activity in the training camp influenced stress hormone levels significantly (Friedman test, p < 0.001). In descending order, helicopter flights, actual training and the days of arrival/departure caused levels to rise, but there was no significant difference between basal cortisol metabolite concentrations and resting days in the camp. In a real emergency, an experienced dog did show a 2.5 times increase in cortisol metabolite concentration, but 12 h later levels had returned to normal. Age, temperament in terms of being prone to stress, as well as previous experience with training camps were considered to be important factors influencing cortisol metabolite concentrations. Basal hormone levels were significantly and positively correlated with a temperament more prone to stress (r = 0.817, p = 0.002). The number of previously attended training courses affected mean cortisol metabolite concentrations during training, but the observed negative correlation was statistically not significant. A questionnaire was used to investigate whether the dog handlers could realistically estimate the arousal and amount of stress the dogs were exposed to. However, no correlation was found between faecal cortisol metabolites and the estimated stress, indicating that handlers overestimated the amount of stress for the dogs most of the time. In summary results of this study indicate that experience and training are the primary factors in reducing stress during search missions in avalanche dogs.
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A study was conducted to (a) determine if beef cattle hair contains cortisol at measurable concentrations, and (b) identify the effect of hair location and collection method on hair cortisol concentrations. Hair samples (0.5 g) from the head, neck, shoulder, hip, and switch were collected from twelve Angus cross bulls (313.1±14.7 kg BW) using two sampling methods: plucking, to ensure collection of the hair follicles; and clipping, using an electric razor to ensure collection of the hair as close as possible to the skin. After two washings with isopropanol, hair samples were ground with a ball mill for 5 min at 22 Hz, sonicated with methanol for 30 min, and incubated on a shaker for 18 h, at 50 °C and 100 rpm. The supernatant was pipetted off and evaporated in a block heater, at 45 °C under a stream of nitrogen. Samples were reconstituted with phosphate buffered saline before quantification of cortisol with a competitive immunoassay. The described method was successful in detecting cortisol in all the hair samples, with concentrations ranging from 0.30 to 5.31 pg/mg. The intra-assay coefficient of variation (CV) ranged from 3.6% to 6.0%, while the inter-assay CV ranged from 5.4% to 11.2%. The cortisol concentration was greater (P<0.05) in the hair from the tail (1.99±0.189 pg/mg) compared with the head and the shoulder (1.14 and 0.82±0.189 pg/mg, respectively), and in the hair from the neck and the hip (1.50 and 1.59±0.189 pg/mg, respectively) compared with the shoulder (0.82±0.189 pg/mg). Cortisol concentration was greater (P<0.01) in hair samples collected by clipping (2.35±0.176 pg/mg) than by plucking (1.75±0.176 pg/mg). There was a day×location interaction (P=0.01), where the hair from the head, neck and shoulder had a lower cortisol concentration at d 28 than at d 1 of the experiment. Data show a significant positive association between cortisol concentration in saliva samples and its level in hair from the hip (r=0.52) and the tail (r=0.63). There was also a trend for a positive association between fecal glucocorticoid metabolites and cortisol concentration in the hair from the neck and the tail (r=0.46 and 0.47, respectively). Results indicate that hair can be used as matrix to measure cortisol levels in beef cattle. Clipping hair from the tail seems to be the most suitable way for measuring cortisol concentration in hair.
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Polar bears are heavily dependent on sea ice for hunting sufficient prey to meet their energetic needs. When the bears are left fasting, it may cause a rise in the levels of the stress hormone cortisol. Cortisol is the major corticosteroid hormone in most mammals, including polar bears. Production and regulation of this stress hormone are vital for the body as it is part of a myriad of processes, including in relation to metabolism, growth, development, reproduction, and immune function. In the present study, we examined the correlation between East Greenland polar bear hair cortisol concentration (HCC), a matrix that reflects longer-term hormone levels, and the fluctuations of the North Atlantic Oscillation (NAO) index, a large-scale climate phenomenon applied as a proxy for sea ice extent in the Greenland Sea along the coast of East Greenland. In doing so, a significant positive correlation (r = 0.88; p = 0.0004) was found between polar bear hair cortisol and the NAO, explaining 77 % of the variation in HCC observed between years over the period 1989–2009. This result indicates that interannual fluctuations in climate and ice cover have a substantial influence on longer-term cortisol levels in East Greenland polar bears. Further research into the implications and consequences inherent in this correlation are recommended, preferably across multiple polar bear populations.
Background: Unemployment and financial strain are chronic stressors that have been shown to be associated with an increase in mean salivary and serum cortisol levels. The impact of chronic stress on cortisol excretion is best measured using a summary index of cortisol excretion over longer periods of time rather than momentary assessments. Hair analysis for cortisol content is a new promising tool by which hair segmental analysis may provide a retrospective calendar of cumulative cortisol exposure. Methods: Participants of this study were 31 unemployed and 28 employed individuals (n=46 women). Hair segmental analysis was conducted using 3cm -long segments starting with the scalp-near segment. Due to differing hair length, n=52 individuals had values for the second segment and n=33 individuals had values for the third segment. Results: Univariate analysis of variance indicated that unemployed individuals had higher cortisol content in the first (p<0.05, eta2=0.071) and second (p<0.05, eta2=0.085) hair segment (a total of 6cm long hair representing the preceding 6 months of collection). Duration of unemployment was related to hair cortisol content (r=0.415 and r=0.430 for segment 1 and 2, respectively). Interestingly, BMI was related to hair cortisol content (r=0.430) but was not different between groups. Consistent with other data from our laboratory, there was a wash-out effect for the third segment (p<0.05 for segment 3 vs. segment 1 and 2). Conclusions: We conclude that hair analysis for cortisol content may be a valid method to detect differences in cumulative cortisol exposure between chronically stressed individuals and healthy controls. Due to a wash-out effect, retrospective ascertainment of cortisol exposure may be limited to the preceding 6 months of specimen collection.
In human medicine, psychiatric side effects among patients on corticosteroid therapy are widely reported, but this appears to have been largely overlooked in the animal literature despite glucocorticoids being widely used in veterinary medicine. Therefore the aim of the current study was to identify possible psycho-behavioural changes in dogs treated with corticosteroids. Two different methodologies were used. Firstly, dog owners were asked to fill a 12 item questionnaire aimed at further validating the initial results of a previous survey relating to changes seen when their dog was receiving corticosteroid treatment. In a second study, a population of dogs undertook behavioural tests aimed at objectively identifying changes when receiving corticosteroid therapy.
Fear is a common behavioral problem in dogs. In this paper, we studied the association between behavioral and physiological responses in two potentially fear-eliciting situations. The aim was to establish whether it is possible to separate dogs of the collie breed that are fearful of floors and gunshots from those that are not by studying changes in heart rate and hematocrit, plasma cortisol, progesterone, testosterone, vasopressin, and -endorphin concentrations. Thirteen privately owned male dogs of the collie breed were studied during a floor test, using different types of floors, and a subsequent gunshot test. Seven of the dogs were identified as being fearful of floors and six were declared as fearless. Out of the 13 dogs, seven were fearful of gunshots and six were fearless of gunshots. Since fear of floors did not always occur concomitantly with fear of gunshots, there were consequently four different groups of dogs. The heart rate increased during the floor test in all groups, but dogs that were fearful of floors had higher heart rates than dogs that were fearless of floors. Dogs that were fearful of gunshots had higher heart rates, higher hematocrit levels and higher plasma concentrations of cortisol, progesterone, vasopressin, and -endorphins during the gunshot test than did dogs that were found to be fearless of gunshots. Plasma cortisol and progesterone increased drastically during the gunshot test in dogs identified as being fearful of gunshots. In fearful dogs, the testosterone concentration increased after completion of the floor test and before the gunshot test started, but there were no significant differences in testosterone between the groups. Since dogs fearful of gunshots had increased levels of several physiological parameters, the results demonstrated that this fear is a serious stress for the individual, a fear which it is possible to register with physiological variables.
The aim of the paper was to investigate the effect of site of sampling, size and gender on the variations of salivary cortisol of healthy dogs. Samples of saliva were collected from dogs of private owners (n. 13), kennels (n. 4) and shelters (n. 2). For each dog, samples were collected at the first interaction of the day with man (T0) before the morning meal (6:00 to 8:00 am), 30 minutes after the meal (T1) and 30 minutes after the last interaction of the day with man (T2), when dogs were resting and apparently relaxed. A total of 92 dogs belonging to 17 different pure breeds or crossbred were eligible for the study, being 19 dogs privately owned, 47 recruited in kennels and 26 hosted in shelters. Salivary cortisol concentrations of the dog population were not normally distributed and data were transformed to natural logarithm. The mean values ranged from -0.70 to 3.40 ln ng/ml, with an average of 0.90±0.76 ln ng/ml, corresponding to 0.50, 30.00 and 3.48±4.05 ng/ml. Mean salivary cortisol was significantly higher for dogs hosted in shelters than those privately owned or in the kennels (P < 0.05). Cortisol values from intact dogs did not differ between males and females, while for castrated males and spayed females significantly lower values were found (P < 0.01 intact Vs castrated males; P < 0.05 intact Vs spayed females). Mean salivary cortisol concentration was significantly lower for giant and large size dogs than for small size dogs (P < 0.01), whilst mean cortisol for medium size dogs was not significantly different from the other sizes. The interaction of site with time of sampling was significant (P < 0.05), with the highest cortisol concentration at T2 for dogs privately owned and housed in kennels and at T0 for dogs hosted in the shelters.
To evaluate parallel circadian rhythms in salivary and serum cortisol concentrations during 48-h period, sampling was performed in six clinically healthy dogs of various breeds housed under natural photoperiod in spring (sunrise 05:20, sunset 20:20). Saliva and blood samples were taken every 3 h for a 48-h period to determine the daily changes in salivary and serum cortisol concentrations. The relationship between salivary and serum cortisol level was determined as well. In the two-day period of monitoring, salivary and serum cortisol concentrations showed the same trend. Their levels started to increase at sunrise and reached their peak in the middle of the photophase. Both parameters showed a high robustness of rhythm. A positive correlation between salivary and serum cortisol concentration was observed during the day 1 and 2. Acrophase and robustness of rhythm showed no statistically significant difference between salivary and serum cortisol concentrations. We can claim that salivary cortisol, a measure of free cortisol, follows the circadian rhythm of serum cortisol. Therefore, saliva sampling is a valid and non-invasive technique useful in chronomedicine to estimate free cortisol.
The aim of this study was to investigate the possible effects of environmental factors such as temperature, rainfall and light conditions on hair cortisol concentrations in foals during the perinatal period. The study, performed during 3 consecutive foaling seasons from January to July, enrolled 219 foals from one farm. Hair samples were collected from each foal immediately after birth and at 30 days of age, and the samples were analyzed by RIA (radioimmunoassay) to measure the cortisol concentrations. The mean cortisol concentration of hair collected at 30 days of age was significantly (p< 0.01) lower than that found at birth, but none of the evaluated environmental factors (temperature, rainfall or day length) influenced the hair cortisol concentrations.