**1. Introduction**

The source of stress experienced by animals can be grouped into three categories: (i) physical due to disease or injury; (ii) physiological, for example, due to hunger or temperature control; and (iii) behavioural or psychological, for example, due to a change in living environment. Regardless of the cause, mammals exposed to stress are equipped with defense mechanisms, whereby catecholamines and glucocorticoids (GCs) are released from the adrenals [1]. In spite of their main task to eliminate

the e ffects of the stressor, GCs, when secreted at high amounts over a prolonged time, may pose a risk to animal welfare. Therefore, measurement of cortisol as an indicator of stress is a useful approach in determining changes in health and biophysical parameters, even before the symptoms occur [2,3].

However, cortisol, like many other hormones, is secreted in an episodic and diurnal pattern in many animal species, making it di fficult to draw conclusions from single samples. Further, handling animals during the collection of blood samples exposes them to additional stress and may produce results that may reflect the consequences of handling procedures. Therefore, non-invasive and "feedback-free" sampling methods, such as collection of faeces, are preferred [2,4]. Concentrations of faecal cortisol metabolites (FCMs) reflect secreted GCs better than plasma cortisol concentrations estimated at the time of blood collection, providing a reliable approach for measuring adrenocortical function [5,6].

Reindeer (*Rangifer Tarandus Tarandus L.*) are free-ranging, semi-domesticated animals that are not accustomed to being handled by humans in the same way as cattle, sheep or pigs [7]. Gathering, sorting and handling may, therefore, result in increased levels of blood cortisol and urea concentrations [8]. Stress results in increased pH levels and reduced glycogen reserves in the meat, compromising the meat quality [9] and, hence, the shelf life of reindeer products [10,11].

A physiological validation of a non-invasive method aims at specifically stimulating the adrenal glands to release GCs into circulation, which should be well captured in the excreted faecal metabolites [12]. Such validations also yield information about the species-specific time delay between ACTH stimuli and detection of increased FCM levels—a time delay that also corresponds roughly to gu<sup>t</sup> passage time [5]. Species di fferences in metabolism and excretion of glucocorticoids require that each method is validated for each species and sex [12]. The determination of faecal stress hormone metabolites in mammals and birds has been performed in many species [5,13]. However, it appears that only Ashley et al. [14] have previously performed an ACTH test in reindeer. Since their study did not show an expressed response in FCMs even after a high dose of ACTH, a full validation of such a method in reindeer still appears to be lacking. Thus, this paper aims to evaluate the physiological relevance of an enzyme immunoassay (EIA) for determining FCMs in reindeer through an ACTH challenge, in addition to assessing FCM concentrations of reindeer during calf marking as a biological validation.

#### **2. Materials and Methods**

In order to obtain enough reference data to validate the FCMs, two separate studies were performed. The ACTH challenge test was done on eight animals in a controlled fence facility at Tverrvatnet and collection of faecal samples for biological validation was performed for a whole flock of reindeer, representing di fferent ages and both sexes, at Stjernevatn.

#### *2.1. Reindeer (Both Regions) and ACTH Challenge Test (Only Tverrvatnet)*

At Tverrvatnet, eight reindeer males (≥1.5 years of age) were injected intramuscularly with 1 mL adrenocorticotrophic hormone (ACTH; 0.25 mg/mL: Synacthen ®; CD Pharma Srl & CD Pharmaceuticals AB, Sweden) in the neck area in front of the shoulder. After the injection of ACTH, animals were moved to a designated fenced area where they were not disturbed except for the collection of samples or refilling of food. At Stjernevatn, on the other hand, the animals were gathered for calf marking. The herd consisted of females (lactating and non-lactating), males, yearlings and calves.

#### *2.2. Study Area and Field Conditions*

The physiological validation study was carried out at Tverrvatnet (66◦ N) in Rana municipality in Nordland County in Norway in January 2018. The area was covered with snow and the ambient temperature ranged from −10 to −15 ◦C. In addition, a biological validation was performed at Stjernevatn (70◦ N) in Tana municipality in Finnmark County in Norway in August 2017. The ambient temperature at Stjernevatn ranged from 5 to 13 ◦C.

#### *2.3. Faecal Pellet Collection*

In both locations, faecal samples were collected opportunistically, resulting in a challenge to quickly discriminate the samples belonging to adults or calves. Flasko et al. [15] sugges<sup>t</sup> that pellet size could be used as a significant criterion to distinguish calves from adults in wild Canadian caribou (*Rangifer tarandus caribou*). Therefore, to overcome the problem, we allocated the samples with an apparent small pellet size to calves.
