**4. Discussion**

#### *4.1. Non-Invasive Versus Non-Disturbing*

Even though faeces collection is a non-invasive approach compared to blood collection, it may not be non-disturbing, because sample collection can trigger a stress response in animals that are not used to close human presence [4]. This is especially true with herd animals such as reindeer. Since stress is conducive to changes in welfare, production, physiology, health and mortality [23], even slow but repetitive entries into the fence, prompting sudden movements or alertness of the animals, may increase cortisol levels. This will not affect FCM levels in the sample collected immediately but may very well result in higher levels in serial samples collected later on [5]. Thus, the experimental animals receiving ACTH in Tverrvatnet may have already had higher cortisol levels, due to the earlier handling by the reindeer herder moving the animals in the fence. The delayed decrease in FCM levels may be attributed to such a disturbance, because, during the first phase, samples were taken from the herd every hour.

Even though the sample collection at Stjernevatn started once the animals were moved to the working fence, the gathering earlier in the morning may also have influenced or even exacerbated the stress levels of the animals. However, since the time lag between the time the work started in the working fence and the gathering was less than seven hours, the 0–2 h samples most likely reflect true FCM baseline levels.

Sampling protocols, based on earlier experience in similar studies, were prepared in detail before the data collection started. A total number of 317 samples were collected over 44 h in the ACTH challenge test, which should capture any peak levels in FCMs. Earlier studies have demonstrated a time lag of FCM in reindeer of 8 to 24 h [14]. In the biological validation, a total number of 114 samples were collected over 10 h, which could be too short to detect late peak levels. After handling was completed, it was usual practice to release the animals back to pasture. Holding them much longer in fence would, therefore, not be justified in the private herd visited.

#### *4.2. Peak FCM Levels*

Following the ACTH challenge, all animals showed well expressed peak FCM levels which were about 630% above their respective baseline levels. This is the first time such a non-invasive method to measure FCM levels in reindeer has been successfully validated. Comparing absolute values in the present study with earlier studies not using the same immunoassays is not relevant, as the di fferent antibodies pick up di fferent cortisol metabolites and to a di fferent degree [5]. The FCM levels peaked seven to eight hours after stimulating the adrenals. This was quicker than reported delay times for deer or goats [24,25], but closer to those found in other ruminants (10–12 h), such as sheep or cattle [6]. However, our results correspond well with findings in caribou, where a significant (but not comparably expressed) increase in FCM was found eight hours after an ACTH challenge [14].

In the biological validation experiment performed at Stjernevatn, the FCM levels eight to ten hours after handling showed more than a three-fold rise compared to baseline levels (samples collected after 0–2 h). This indicates that our method is sensitive enough to detect the e ffect of handling stress. However, the peak FCM levels were lower when compared to the ACTH challenge test. A study by Carlsson et al. [26] found no increase in FCM levels eight hours after handling stress, but their method was not validated for this species. This underlines the importance of performing validation studies in every animal species and validating each method of FCM analysis against baseline values found under practical and representative conditions.

#### *4.3. E*ff*ects of Sex and Individual*

At Tverrvatnet, only males were used, because the females were pregnant. Ashley et al. [14] found that female reindeer showed a prolonged response to a very high ACTH dose (8 IU/kg; possibly about 15–16 times higher than the one used here (about 25 IU/animal), assuming the animals weighed about 45–50 kg, whereas males did not have this response. Faecal samples were collected from both males and females in the first collection period at Stjernevatn. In the second period, only lactating females and calves were present. Sex and age may have an influence on FCM levels [5,12]. However, the variation in our dataset from the biological validation was small, as shown in Figure 3. Furthermore, the statistical analysis showed no di fference in FCMs between calves and adults, thus making such an influence less likely.

There were quite large individual di fferences in the FCM response for the ACTH challenge test at Tverrvatnet. Some animals (animals 1, 3, 6, 7, 8) had an early peak (about seven hours after ACTH injection), in others (animals 2, 4 and 5) it occurred later (after 12, 14 and up 24 h, respectively). The reasons for this might be manifold. Di fferences in the resorption of the ACTH could have played a role. If the needle hits the muscle close to a blood vessel, the response should be quicker (immediate) than if the ACTH injection was further away from a vessel. In case of the latter, the increase could be delayed. Another explanation might be that earlier peaks were missed in those animals. This is likely true in animals (e.g., animal 5) where samples were probably missed, especially as high concentration samples of unknown identity were present in that time window (7–8 h). In addition, the enterohepatic recirculation, which has been proven in ruminants [27], could have caused secondary peaks, especially when they were smaller (e.g., animals 1 and 3). However, median delay times in FCM peaks found in the biological validation at Stjernevatn were also seven hours, which suggests that shorter delay times in faecal peak excretion are more likely found in reindeer.

#### *4.4. Identification of Unknown Samples*

Being able to document the individual responses by using DNA analysis added extra value to the results because these individual patterns would have been hidden in anonymous sampling. Non-invasive genetic sampling methods have become widely used in genetic studies and have been shown to be a reliable approach for genetic studies in reindeer [28–30]. It has also been shown that combining genetic analysis with FCM measurements is a useful approach to document individuals

and their sex, and that ignoring this information could lead to erroneous conclusions when addressing stress levels in animal populations [31,32].

In this study, we have used non-invasive genetic sampling without any previous knowledge of the genotypes of the eight individuals involved in the experiment and achieved a high success rate in assigning samples to genotypes/individuals across the duration of the study. This shows that the technique can be a useful addition to other scientific fields, to ensure reliable tracking of individuals across time and reduced degree of disturbance (i.e., less human-induced stress responses in the animals). Since we already knew the sex of the eight reindeer individuals, a molecular sexing was not performed, but is advised in future studies without this knowledge, whenever sex can be expected to be an important explanatory factor.

Samples collected by non-invasive methods, such as faecal samples, are less likely to give complete profiles due to low quality and/or quantity (i.e., allelic dropout or fragmented DNA) compared to blood and tissue samples [33–35]. When possible, we therefore recommend including control samples (tissue or blood) from all individuals, collected either before or after the experiment, to confirm their genetic profiles. This would facilitate unambiguous assignment of samples to genetic profiles/individuals and thus contribute to a higher percentage of the samples being available for the primary analysis, e.g., FCM or other applications.
