Animals

In total 59 German shepherd dogs were used in this project (Table 1, Supplementary 1). We sampled 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, competition 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 effects but only for correlations with questionnaire results. The 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).

Table 1 Sex and age (in years) distribution of the analysed German shepherds (N = 59). Full size table

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).

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. Three values above 60 pg/mg in (a,c) have been omitted from the figures 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). Full size image

Hair preparation and cortisol extraction

Hair samples were stored in room temperature until preparation and cortisol extraction which was performed according to methods described in detail previously21,26. Briefly, 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 off and lyophilized using a Savant Speed Vac Plus SC210A and the samples were dissolved in radioimmunoassay buffer and analysed as described by Morelius et al.26. Hair samples of 5 mg or more were needed for maintaining a total inter-assay coefficient of variation below 8% for hair extraction and measurement of cortisol by the radioimmunoassay. The intra-assay coefficient 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 little 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 occasions, 3 = 9 or more competition occasions) during the last three months and these variables were later analysed for possible correlation with the hair cortisol levels. The owners also included information about what kind of training they performed. More than half of the companion dogs (10 out of 17 dogs) performed different 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. The 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). The 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 software 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. The 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 effects of season, using repeated measures. Time (three levels: January, May, September) and lifestyle (three levels: companion, competition or professional working dog) were treated as fixed effects together with their interaction. Individual dogs were included as random effects 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 fixed effects 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 differences between companion, competition and working dogs for the C-BARQ scores due to the ordinal nature of data.