Studies on the Assessment of Hair Cortisol Concentration in Dogs by Radioimmunoassay (RIA) 

Serum cortisol measurement is an invasive procedure that can disrupt behavior and stress levels. Urinary cortisol is a widely used indicator of stress in dogs, as it is collected without significant stress. Naturally voided urine samples can study stress hormones in animals, but they only represent the average concentration in blood during urine formation. Cortisol measurements in serum, urine and saliva reflect systemic concentrations but cannot assess enduring levels. Non-invasive techniques are increasingly used to assess hypothalamic-pituitary-adrenal axis activity in humans. Measurement of hair cortisol offers the merit of a minimally invasive sampling procedure and simple storage (Corradini et al., 2013). During the last two decades, hair cortisol concentration (HCC) has proven to be a promising marker for the evaluation of increased hypothalamic-pituitary-adrenal axis activity caused by repeated or long-term stressful conditions (Heimburge et al., 2020).
 
Radioimmunoassay (RIA) is a sensitive in vitro method for the assessment of antigens i.e., hormones, minerals, vitamins, etc. from biological fluids. 125 I is a commonly used radioisotope among others for RIA due to its long half-life (t1/2 = 60 days). This method offers a convenient assay of large numbers of samples with excellent precision. The practice of RIA in the measurement of hormones for veterinary clinical use has been extensively studied in India (Dadke et al., 2018; Roopali et al., 2020; Galdhar et al., 2021; Jayabhaye et al., 2021; Galdhar et al., 2022 and Salutgi et al., 2023).
 
Hair cortisol is often used as a measure of chronic stress responses in humans, nonhuman primates and companion animals across the globe (Corradini et al., 2013; Meyer et al., 2014; Vives et al., 2015). HCC is not yet widely used in India. The purpose of this paper is to report the development of RIA enabled novel protocol to measure HCC in hair samples from dogs for stress assessment.

Statutory approval
 
The present study was initiated after permission from the Institutional Ethics Committee for Veterinary Clinical Research (IEC-VCR) and Institutional Bio-safety Committee (IBSC) of Mumbai Veterinary College, Maharashtra Animal and Fishery Sciences University (MAFSU), Mumbai-India.
 
Selection of healthy and hospitalized dogs
 
A total of 20 non-descript clinically healthy dogs from the clinical setup of Mumbai Veterinary College, Parel-Mumbai, were ethically enrolled to measure HCC. They consisted of 14 males and 06 females, with a mean age of 5.63±0.39 years and mean body weight of 19.33±0.59 respectively. Health checks, anamnesis and laboratory tests (blood count, liver function test, kidney function test) were conducted to assess health, ensuring normal limits were met.
 
A total of 20 non-descript hospitalized dogs, admitted for various illnesses at the clinical setup of Mumbai Veterinary College, Parel-Mumbai, were ethically enrolled to measure HCC. They consisted of 13 males and 07 females, with a mean age of 4.65±0.57 years and mean body weight of 17.60±1.63 respectively.
 
Hair sample collection and hormone determinations by RIA
 
One gram hair sample was collected from the nape region of the dog by blunted surgical scissors and weighed 250 mg. The samples were then washed with isopropanol and dried at room temperature for 3-5 days. After powdering, 30 mg of powdered hair was transferred to 2 ml eppendorf tube and 1.5 ml High liquid pressure chromatography (HPLC) grade methanol was added. The samples were incubated for 16 hours and sedimented for 2 hours, then 1ml of supernatant was transferred into another 2 ml epindoff tube and complete drying was done by evaporation at room temperature for 24 hours. The dried samples were mixed with 200 µL of phosphate buffer saline (PBS of pH 7.5) and used as hair sample aliquots. Nist et al., (2020) used enzyme-linked immunoassay to assess HCC, as the concentration of cortisol is very small in hair samples and not detected by the assay. In the present study, marginal modifications were undertaken. In our study, we used RIA-enabled dissevered spiking and detectable concentration-based modifications to measure HCC. Five known cortisol concentration standards were used for the spiking of hair samples. The standard concentrations used were 19.0 nmol/l, 47.9 nmol/l, 190 nmol/l, 238.66 nmol/l and 713.33 nmol/l respectively. The first three standards viz. 19.0 nmol/L, 47.9 nmol/L and 190 nmol/L were readily available with a cortisol RIA kit, while the remaining two standards were created by dilution/serial dilution of known standards with zero calibrators. In our case, after pipetting 50 µL of standard and 50 µL of hair aliquot samples into the appropriate antibody-coated test tube, the samples were spiked with 50 µL of the known standard (random any one among-19.0 nmol/l, 47.9 nmol/l, 190 nmol/l, 238.66 nmol/l and 713.33 nmol/l) during the assay procedure. We maintained the manufacturer’s protocol for the rest of the assay procedure. The samples were analysed in a gamma counter to take counts per minute (CPM) followed by extraction of HCC on the semi-log graph paper. Further, the known standards concentration was subtracted and the final concentration of hair samples was measured in nmol/l. Quality control parameters viz. magnitude of control samples and recovery percentage were studied to validate every assay. The HCC in pg/mg was derived as per the method and formula outlined by (Nist et al., 2020).
 
Statistical analysis
 
Mean and standard error for each parameter of collected data was calculated and analysed statistically for comparison as per the methods suggested by Snedecor and Cochran (2004). A nonparametric statistical analysis was also used for the analysis of the results.

Estimation of HCC concentrations of healthy (n=20) and hospitalized (n=20) dogs was done using RIA by employing dissevered spiking and detectable concentration-based modifications. Assay passed all recommended quality control parameters viz. magnitude of control samples provided with kits and percent recovery. The standard curve of the assay was plotted and the HCC was interpolated from the standard curve. The Mean, interquartile range (i.e., 25^th to 75^th percentile) and median of HCC in healthy dogs and hospitalized dogs are presented in Table 1 and Fig 1.



@figure1
 
The mean value of HCC of healthy dogs was recorded as 6.24±0.84 pg/mg and the hospitalized dog was 31.65±5.87 pg/mg, respectively (Table 1). A statistically significant (p<0.05) deviation was recorded in HCC in healthy and hospitalized dogs.
 
A box and whisker graph (Fig 1) was plotted for comparison of HCC between healthy and hospitalized dogs. The ‘T’ bar represents the data which is equal to the range. In the present study, the observed range for healthy dogs was 0.36 to 13.78 pg/mg and for hospitalized dogs was 8.70 to 65.25 pg/mg respectively. The box represents the middle half of the data. The present study reports the interquartile range (25^th to 75^th percentile) as 3.55 to 9.24 pg/mg for healthy dogs and 14.43 to 46.22 for hospitalized dogs, respectively. The Horizontal bar in the box is the median of the data. In the present study, the observed median for healthy dogs was 6.16 pg/mg and for hospitalized dogs was 22.62 pg/mg respectively.
 
The present study reports, a statistically significant (p<0.05) difference in the mean HCC of healthy and hospitalized dogs. These findings are in close agreement with Corradini et al., (2013) and Van der Laan et al., (2022).  Corradini et al., (2013) evaluated HCC in the diagnosis of hypercortisolism in dogs. They mentioned that the evaluation of cortisol from hair samples offered the advantage of easy sample collection. Van der Laan et al., (2022) studied hair cortisol levels in dogs in shelters and after adoption. They found that HCC analysis is a reliable, feasible non-invasive method for evaluating cortisol levels in shelter dogs, particularly when comparing levels over longer periods.
 
The promising study by Packer et al., (2019), highlighted that hair cortisol is a promising indicator of chronic stress in dogs. They emphasized its responsiveness to various stressors, including behavior, disease, medication, lifestyle and the social environment. Ouschan et al., (2013) reported elevated hair cortisol levels in dogs with hypercortisolism, proposing hair cortisol analysis as a non-invasive diagnostic tool. Expanding on this, Park et al. (2016) identified increased hair cortisol in dogs with Canine Atopic Dermatitis, suggesting its potential for linking stress to specific health conditions. Furthermore, Bryan et al., (2013) advocated tracking hair cortisol over time to monitor gradual changes associated with disease progression, especially in conditions related to adrenal hypo- or hyperfunction in dogs.
 
Radioimmunoassay (RIA) is a gold standard method for detecting hormone concentrations and assessing antigens in biological fluids. It uses I125 radioisotope for its long half-life, offering convenient and accurate assays for large samples. The investigation utilized RIA-enabled dissevered spiking and detectable concentration-based modifications to measure HCC in dogs. This promising approach can be used to study stress in hospitalized and confined dogs, potentially defining stress. In the present investigation, we used RIA-enabled dissevered spiking and detectable concentration-based modifications to measure HCC. This offered a promising approach for the detection of HCC. This modified method may be widely used to study stress not only in hospitalized dogs but also to appraise stress-reducing measures in canine welfare.

The study demonstrates RIA-enabled measurement of HCC in healthy and hospitalized dogs, marking the first report from India and Central East Asia. We recommend that hair sample collection is an easy method for assessing stress and RIA offers a promising method for detecting HCC.

The authors express gratitude to Maharashtra Animal and Fishery Sciences University-Nagpur (India)and Mumbai Veterinary College, Parel, Mumbai-400 012 (India) for providing radiation facilities and support for RIA kits for hormonal estimation. The authors also acknowledge the support of the Board of Radiation and Isotope Technology and the Department of Atomic Energy, Govt. of India.

The authors declare that there is no conflict of interest.