2.3.2. Stjernevatn

The sample (n = 114) collection took place from 10:30 to 20:35, forming a total of around 10 h, during two phases: (i) separation of males and non-lactating females from lactating females and calves; and (ii) earmarking of calves. In the first phase, a subgroup of 12 to 30 reindeer were moved into the handling fence where animals were caught by hand and examined by the reindeer herders. The females, which were lactating, were marked with a colour spray and later released back into the subgroup where they eventually remained together with all calves. The males or non-lactating females were moved to adjacent holding pens for separation. Each batch took around 2–8 min to process, depending on the batch size. This phase took approximately five hours, during which a total of 800–900 animals were processed. Faecal samples collected during the first two hours of the separation process were considered as baseline (n = 51, of which n = 31 from known calves).

The second phase of faeces collection was done when calves and lactating females were brought back to the fence for earmarking. In the same way as before, smaller subgroups were taken into the work fence in batches and processed there. Between batches, researchers collected fresh faecal samples from the ground (n = 63, of which n = 34 from known calves) to determine potential changes in FCM concentrations as an e ffect of handling. This phase lasted 2.5 h. At Stjernevatn, faecal samples could not be attributed to individual reindeer, but stools with smaller pellets were marked as belonging to calves, as described above.

#### *2.4. Analysis of FCMs*

Some of the samples from Tverrvatnet were already covered in snow and, to avoid the extra weight snow adds, all samples were oven-dried at 75 ◦C for 24 h. The dried samples were ground and homogenized using a mortar and pestle, and 0.2 g of each sample was weighed. Steroid extraction was performed according to Palme et al. [16] and Palme [17]. For suspension of samples, 4 mL 99.9% methanol was mixed with 1 mL distilled water and added to the 0.2 g faecal sample. The mixture was hand-vortexed for 1–2 min followed by centrifugation at 2.500 g for 15 min. A 0.5 mL aliquot of each supernatant was transferred to microtubes and stored at −18 ◦C until analysis.

FCMs were measured in aliquots (after further 1:10 dilution with assay bu ffer) of the extracts, utilizing a group-specific enzyme immunoassay (11-oxoaetiocholanolone EIA), previously described

in detail by Möstl et al. [18], which was found to be well suited in various ruminant species [5]. Intraand inter-coe fficients of variation were below 10 and 15%, respectively.

## *2.5. DNA Analysis*

To identify the individual in the ACTH challenge test that the faecal samples belonged to, we performed DNA analysis on 303 of the 317 samples from Tverrvatnet. A total of 14 samples were discarded at this point, due to low quality.

#### 2.5.1. DNA Extraction

One faecal pellet from each of the 303 faecal samples was transferred to a stool collection tube containing 8 mL stool DNA stabilizer. The DNA was then extracted using PSP Spin Stool DNA Plus Kit (Stratec, Birkenfeld, Germany) following the manufacturers protocol.
