**3. Results**

#### *3.1. Controlled Experiment with ACTH Challenge on Eight Reindeer (Tverrvatnet)*

#### 3.1.1. Identification of Individual Animals

Of the 303 faecal samples analyzed, 48 (16%) were negative for all eight microsatelite markers and 255 (84%) were positive for at least one STR-marker. Among the 255 samples, eight unique genetic profiles were found, representing the eight individual reindeer. A total of 28 of the positive samples (11%) could not be assigned to one of the eight unique profiles, due to the lesser quality of the samples and/or the lack of private alleles or unique allele combinations. The 227 samples (89%) were given an identity based on the eight unique genetic profiles that make up the eight reindeer individuals (Figure 1). As a result, 54 of the samples were assigned to individual 1; 32 samples to individual 2; 15 samples to individual 3; 39 samples to individual 4; 17 samples to individual 5; 16 samples to individual 6; 34 samples to individual 7; and 20 samples to individual 8.

**Figure 1.** Box-plot graphs of levels of faecal cortisol metabolites of all samples as a function of hours after ACTH administration. Hours with dissimilar letters had significantly different (*p* < 0.05) concentrations. Above the x-axis, the respective numbers of samples are given.

#### 3.1.2. Results from the ACTH Challenge Test

Median (range) FCM levels from two to six hours after injection of ACTH were 505 (34–6408 ng/g). Overall, FCM concentrations peaked at 7 h and slowly decreased afterwards (including high values in some intervals; Figure 1).

When expressed at individual animal levels, the FCM concentrations align with the herd level for five animals where peak levels ranged from 5000 ng to 8000 ng/g faeces. As can be seen in Figure 2, animal numbers 2, 4 and 5 reached their peak values later than the other individuals. There were three unidentified high concentration (6408, 5409 and 3721 ng/g faeces) samples (6–9 h), which could have been those animals' (e.g., animal 5) early peak samples. Individual peak samples were about 6.3 (2.0–16.8) times higher (median 3359; range 2352–5361 ng/g) than respective baselines (0–6 h; 685; 315–1079 ng/g faeces).

**Figure 2.** Concentration of faecal cortisol metabolites (FCMs) from identified samples of the eight individual animals at Tverrvatnet, shown as a function of hours after ACTH challenge.

#### *3.2. Results from the Biological Validation (Normal Handling and Calf Marking at Stjernevatn)*

The highest values of cortisol metabolites were found around eight hours after the human activity started within the fences. FCM concentrations (median; range) in reindeer (Figure 3) were significantly different between morning (480; 212–1159 ng/g faeces) and evening (1469; 605–4673 ng/g faeces) sampling (F = 116.9; *p* < 0.001). Calves and adults did not show any significant differences in FCM levels (F = 1.35; *p* = 0.25). A total of 65 samples were from calves and 49 samples were from adult reindeer. In the first phase (0–2 h) adult samples were from males and females, non-lactating and lactating. In the second phase (8–10 h) samples from adult reindeer came only from lactating females with calves at foot.

**Figure 3.** Concentrations of faecal cortisol metabolites (FCMs) in reindeer collected immediately after fencing (0–2 h; morning, n = 51 where 31 were calves and 20 adults) and later after handling and calf marking (8–10 h; evening, n = 63 where 34 were calves and 29 adults). Dissimilar letters indicate significant (*p* < 0.001) differences.
