Evaluation of hair and nail cortisol concentrations and associations with behavioral, physical, and environmental indicators of chronic stress in cats

Abstract Background Chronic stress is implicated in behavioral and health issues in cats, but methods for recognition, evaluation, and measurement of stress are lacking. Cortisol concentration is typically used as an indicator of stress. Objectives To evaluate use of an enzyme immunoassay to quantitate hair and nail cortisol concentrations (HCC and NCC) in cats and evaluate associations between HCC and NCC and behavioral, physical, and environmental correlates of chronic stress in cats. Animals Forty‐eight adult, owned or community cats. Methods Cross‐sectional study. Nail clippings and hair were collected from cats. Medical history and cat daily lifestyle questionnaires were completed by owners or caretakers. A commercial laboratory performed cortisol extraction and quantification using a validated enzyme immunoassay kit. Correlational and regression analyses were used to evaluate associations between HCC and NCC and behavioral, environmental, and medical factors. Results Hair and nail cortisol concentrations were significantly associated (r s = 0.70; P < .001), but HCCs varied widely within and among cats. Cats with litterbox issues had significantly increased HCC (P = .02) and NCC (P = .001) as compared to cats without litterbox issues. Cats with groomed coats had lower HCCs (P = .02) as compared to cats without groomed coats, whereas cats with dander and mats had higher NCCs (P = .01) as compared to cats without dander and mats. Conclusions and Clinical Importance The quantification of NCCs might improve identification and evaluation of chronic stress in cats. The variability of HCCs in individual cats warrants caution using this measurement in chronic stress studies.


| INTRODUCTION
Stress contributes to illnesses in cats including upper respiratory infection, elimination disorders, dysrexia, grooming alterations, and gastrointestinal disturbances, often leading to additional medical sequelae. [1][2][3][4] Chronic stress also can initiate and potentiate common behavioral problems in cats, such as house soiling or aggression. 3,[5][6][7] Cats frequently are surrendered to shelters because of these issues and may experience chronic stress when housed long-term in a shelter. [8][9][10][11] Owners, however, might not notice signs of stress in their cats, and they might not attribute certain behaviors or illnesses to chronic stress, and thus it can go unrecognized. 12,13 Methods for recognition, evaluation, and physiological measurement of chronic stress in the cat are lacking. Quantitative stress measures in the cat historically have been based on serum cortisol concentration and more recently salivary, urinary, and fecal cortisol concentrations. [14][15][16][17][18] None of these cortisol measurements as single results is indicative of chronic stress, however, because each gauges either instantaneous cortisol concentrations that can vary within minutes (as in serum and saliva), or concentrations that reflect the previous several to 24 hours (as in urine or feces). 16,19,20 Furthermore, some of the procedures used to obtain the samples can induce stress in cats. 14,21 The slow growth rate and accumulation of cortisol within hair and nails allow for a single measurement of cortisol that reflects bodily concentrations of the hormone over an extended period of time. [22][23][24][25] Hair cortisol concentration (HCC) increasingly is being evaluated as an indicator of long-term chronic stress in other species, and nail cortisol concentration (NCC) also is being evaluated. 22,[26][27][28][29] Enzyme immunoassays (EIAs) and radioimmunoassays (RIAs) have been used to measure HCCs in dogs, 16,30,31 and RIA has been used to measure HCCs in cats. [32][33][34] Coat color 31 and body location 35 might influence HCCs; these factors have not been assessed in cats. No published data are available regarding EIA assessment of HCC in cats. Nail cortisol concentrations have been found to be associated with HCCs in dogs 24,36 and had less variability than HCC. 24 Nail cortisol concentrations have not been evaluated in cats.
Noninvasive objective measurements of chronic stress are needed in feline medicine. Hair and nail cortisol assays combined with observations, such as coat condition, 11 might assist with evaluation of chronic stress in cats. Such measurements would more effectively guide prevention, intervention, and management strategies. Our aims were to evaluate use of EIA for the quantitation of HCC and NCC in cats and to determine if either could be used to evaluate chronic stress in cats. The primary hypotheses were that HCCs from different body locations would be similar and that HCCs would be positively associated with NCCs. The secondary hypotheses were that behavioral and physical factors potentially indicative of chronic stress in cats would be associated with HCC or NCC, and NCC would be more consistent than HCC. 24 2 | MATERIALS AND METHODS

| Cats and samples
The subjects were either owned or community cats. Participants were recruited in 3 stages. At the request of the commercial laboratory, the first 3 cats were chosen based on owner report of being likely chronically stressed; this request was to potentially extract high concentrations of cortisol from the 3 samples to perform assay calibrations. The next 16 cats were healthy adult cats from teaching hospital staff and a spay/neuter clinic ( Figure 1). Inclusion criteria were the following: generally healthy, adult, and no known glucocorticoid administration or contact within the past month, and if a cat had a nail trim performed within the past 2 weeks, it was excluded from participation.
To enroll cats with a variety of owner-reported health and behavioral conditions, participation next was sought from adult cats (n = 33) that had any of the following: geriatric age, unkempt appearance, exces-

| Cortisol extraction and assay
Cortisol extraction and laboratory validation of the assay were performed by UC BIEL using previously published methods. 22,[40][41][42] Between 20 and 40 mg from each sample was used for analysis. Each sample was washed with 100% isopropanol, dried, ground, and extracted. [40][41][42] A commercial high sensitivity EIA kit (Salimetrics, LLC) was used; the lower limit of sensitivity was 0.007 μg/dL. Cortisol concentrations were determined according to the manufacturer's protocol and were presented as picograms of cortisol/milligram (pg/mg) of hair or nail. To perform laboratory assay validation calibrations, samples from 3 cats were used. Pooled samples were processed to create low and high internal assay controls for determination of intra-assay and interassay coefficients of variation (CV). The mean CVs were 10.6% and 9.3% for the low and high controls, respectively, and intra-assay CV was 1.5%. Assay cortisol extraction recovery was evaluated by spiking 3 hair samples and 1 nail sample. The mean cortisol extraction recoveries were 97.1% for the hair and 89.2% for the nails. For comparison to the expected values of the assay standard curve, hair samples were serially diluted from 1 : 1 to 1 : 32, and nail samples were serially diluted from 1 : 1 to 1 : 8. Linear regressions showed strong positive correlations between expected and observed cortisol concentrations for hair (mean R 2 = 0.989) and nails (R 2 = 0.997).

| Owner questionnaires
The owners or caretakers of the cats completed a medical history questionnaire and a daily lifestyle questionnaire based on previous studies. 12 posts, rubs affectionately on owner, fights with or hides from other cats in home, and plays. Last nail trim also was categorized into 3 categories (between 2 and 4 weeks; >1 month; never). The following were reclassified into the dichotomous category of yes or no: children in household, pheromone use, litterbox issues (defecating or urinating outside of box or both), water spray punishment, pica, hisses or bites, F I G U R E 1 Flow diagram outlining cats recruited for study (ovals) and hair and nail sample inclusion and exclusion in different parts of the study and analyses (rectangles) greets when entering home, and chronic illness (including kidney disease, emaciated community cat euthanized, chronic rhinitis, chronic gingivostomatitis, severe periodontal disease, untreated asthma, thyroid disease, and obesity).
Significance was defined as P < .05. For the first 19 cats, the concordance correlation coefficient 45

| Cats and general characteristics
Samples were collected from 52 cats (Figure 1). Four cats were excluded from analyses because of topical corticosteroid contamination in the home (n = 3) and PO prednisolone administration (n = 1).
Of the 48 cats that were used for the study, 5 were intact males

| HCCs and body locations (n = 19)
Samples were obtained from at least 1 body location in the first 19 cats (Table 1)  The amount of cortisol released into the hair or nail depends partly on the growth rate of the hair or nail. 16 35,38,54,55 The HCC of the Canada lynx was found to vary among foot, leg, location on foot or leg, and by individual, 55 and significantly different HCCs in body regions also have been found in grizzly bears, with neck having the highest cortisol concentration and rump the lowest. 35 In contrast, 1 study that evaluated HCC in dogs did not report variation between right and left shoulder HCCs. 30 Variation in HCCs also might be partly attributable to different concentrations in guard vs undercoat hairs. 35,56,57 Variation also might be a consequence of >1 source of cortisol from both the skin's localized stress response as well as from systemic circulating cortisol. 32,[58][59][60] Neither hair segment length nor color were responsible for the variation in HCC in our study, because those variables were not significant in analyses, but sample sizes were too small within each color category to draw conclusions. Conflicting findings have been reported regarding these factors in other studies. 31,35,54 It was found that dogs had higher HCC in the distal segment of hair as compared to the proximal segment, 31 whereas chimpanzees had significantly decreasing HCC toward the distal end of the hair shaft. 54 Conflicting results also have been reported regarding the relationship between hair color and HCC. 31,35 The final HCC model in our study was driven, in large part, by a  30 The reliability of any singular measurement of HCC in the cat is questionable considering the matrix of potentially different cortisol concentrations depending on source, variable hair growth rates, types, amounts, densities, and physiology.
The amount of cortisol extracted from nails was lower than that extracted from hair in our study. Lower NCCs as compared to HCCs also have been reported in dogs, and it was suggested that there might be a slower rate of diffusion from circulation into the nail as compared to the hair follicle. 24 Cat nails are unique in morphology, function, and structure with a complex architecture of multiple layers, vasculature, growth rates, and shedding mechanisms. 23,63,64 The diffusion of hormones to the nails might not occur at the same rate and volume as in the hair. 65 As the amount of cortisol released into and accumulated in nails depends, in part, on nail growth rate, the rapid growth rate might be partly responsible for lower cortisol concentrations in nails vs hair in cats. Because the unique structure of the feline nail likely leads to difficulties in extracting large amounts of cortisol, improved methods of extraction should be explored.
Cats' front limbs and paws differ from the rear limbs and paws in function, musculature, and innervation. 66 The variable, groomed and soft hair coat, was associated with decreased HCC, whereas the variable, dander and matted hair coat, was associated with increased NCC. Chronically stressed cats often have unkempt appearances because they discontinue performing regular behaviors such as grooming; lack of grooming can indicate a medical or stress-related issue or both. 11,71,72 One shelter study found that poor hair coat was associated with lack of enrichment, limited access to resources, overcrowding, and longer shelter stays. 11 A feral cat colony study found poor hair coat was associated with larger colony size and number of cats per feeding station, indicating heightened social tensions and less access to resources. 73 High behavioral stress scores also have been found in cats with decreased grooming behaviors. 74 Some cats cannot groom because of medical conditions including chronic pain, and these cats also might be experiencing chronic stress because of those conditions. Therefore, hair coat condition should be monitored as an indicator of overall welfare in cats.
Our study had several limitations. The small sample size could lead to type 2 errors, whereas using multiple variables to predict a best fit model with a small sample size could result in type 1 errors. 75,76 Another limitation was that it was not an experimental study evaluating presumably stressed as compared to nonstressed cats, but an observational study, lacking a dedicated control group, and the subjects had varied histories. If future studies further evaluate hair from different body areas, hair should be collected more systematically so that improved comparisons among body areas can be made.
Although cat HCC has been evaluated in 4 other studies, understanding the timeframe over which cortisol accumulates in the hair in cats is still not validated, and no other study of cat NCC has been published to our knowledge.
Another limitation was that the medical, behavioral, and environmental data were obtained only through owner reporting, which could have resulted in bias and inaccurate reporting. 77,78 Many of the subjective owner-assessments of cats in our study were not associated with cortisol concentrations. Results might suggest that some owners were unaware of their cats' affective states or stress levels. In a study of the perception of stress in cats, owners did not recognize some important indicators, and most considered their cats to have a very low stress level. 12 Another study found that owners did not utilize many recommended environmental modifications and enrichment modalities and were unaware of the adverse consequences. 77 It is also possible that owner reporting is inaccurate when describing environmental enrichment modalities. So that owners know when to seek further care for their cat, it is important that