There is a potential relationship between cortisol levels in hair and those in saliva, urine, and feces, but each of these has its limitations [9,35]. Furthermore, correlations between different samples of materials have not proven useful, especially those reflecting different time windows. Nonetheless, the trend (of increased or decreased) stress hormones in various matrices provide insights into interpreting the results of studies which they can be studied by comparing the level of hormones in different matrices (e.g., saliva vs. blood, hair, etc.) [9]. Given the variety of the research analyzed, meta-analyses of correlation coefficients showed significant variability between studies [36]. There are a number of factors that tend to bias the result of blood cortisol and its metabolites rather than the treatment effect, which can also be used in the same manner to alter saliva values. It should be noted that while the use of urine and feces for hormone measurement is promising and associated with hormone production over an extended period, collecting urine and feces samples from each individual and the difficulty of storing these samples pose some difficulty [9]. It seems that the use of hair, wool, and feathers from pets to better identify hormonal changes over time is a better approach to overcoming the above difficulties [9]. Earwax has been recently presented as a promising matrix that can be reliably used to measure stress by extracting cortisol. Earwax has the same advantages of hormone measurement and sampling in hair/wool/feathers over traditional biological fluids (blood, plasma, serum, and saliva) and eliminates ethical concerns because sampling is non-invasive [37]. The recent indicator of cortisol in earwax as a non-stressful sampling method in both humans [38] and animals [no published data] is also considered to provide cumulative retrospective measurements of stress hormones (up to a few weeks) and sampling is simple and not painful. Earwax is thought to represent an accumulation of cortisol output over weeks or months [9]. In addition, the collection and storage of earwax and hair are simple, which may facilitate their use in chronic stress research. In addition, the scales, fins, and jawbones of aquatic animals appear to be promising matrices to show stress levels by measuring cortisol levels [9,21]. However, further studies are needed to validate these matrices as a reliable indicator of stress. A comparison of the hair matrix with other biomatrices that have been used to study chronic stress in animals is shown in Table 1 [37,39]. In addition, a classical biomarker should have the following characteristics: (1) ease of collection and processing of the appropriate biological sample; (2) stability and durability of the marker throughout the storage and evaluation period; (3) availability of assays with sufficient specificity and sensitivity for the marker in question. Additionally, the identification of non-invasive methods for biomarker assessment has the potential to provide accurate data concerning induced stress stimuli to ensure a standard measurement in response to stress.

Using blood cortisol and corticosterone (serum and plasma) as a biomarker of stress can be confounded and problematic due to its possible affection by various factors [40,41,42,43,44,45,46], including circadian rhythms [43]; sampling [47,48]; restraint [42,43,49]; stage of lactation [50]; milking [42,49,51], degree of habituation [52,53], other hormones (e.g., vasopressin can potentiate ACTH secretion [51]; infections as well as endotoxins [3,51]. The phenomenon could also be extended to salivary cortisol. Thus, in the case of the use of catheters, the restraining issue can be solved, while the use of other biomarkers can contribute to a better interpretation of the results. The use of saliva as a biological sample has advantages including being non-invasive in some animal species [54], particularly in pigs where blood collection is stressful and painful for both the animal and the staff in charge of the sampling [54,55]. Cortisol exists in two fractions in blood: protein-bound cortisol and free cortisol, whereas free cortisol is the active fraction only in saliva [54,55]. In aquatic animals, recent studies focused on the measurement of cortisol in golden fish [56] and sturgeon [21] and indicated the usability of fins and scales as matrices to extract and show cortisol levels. Recently, Ghassemi Nejad et al. [21] took a step forward and successfully extracted cortisol from the jawbones of sturgeons. They also identified the type of washing solvent and its effect on cortisol detection and found that there were no differences in cortisol levels in fins of three sturgeon species or cortisol levels in the fins and jawbones between washing solvents. Overall, they suggested that the sturgeon jawbone matrix is a promising alternative stress indicator to non-liquid matrices in dead fish.