Skip to main content Accessibility help

Hair cortisol detection in dairy cattle by using EIA: protocol validation and correlation with faecal cortisol metabolites

  • O. Tallo-Parra (a1) (a2), X. Manteca (a1), M. Sabes-Alsina (a2), A. Carbajal (a2) and M. Lopez-Bejar (a2)...


Hair may be a useful matrix to detect cumulative cortisol concentrations in studies of animal welfare and chronic stress. The aim of this study was to validate a protocol for cortisol detection in hair from dairy cattle by enzyme immunoassay (EIA). Seventeen adult Holstein–Friesian dairy cows were used during the milking period. Hair cortisol concentration was assessed in 25-day-old hair samples taken from the frontal region of the head, analysing black and white coloured hair separately. Concentrations of cortisol metabolites were determined in faeces collected twice a week during the same period of time. There was a high correlation between cortisol values in faeces and cortisol in white colour hair samples but such correlation was not significant with the black colour hair samples. The intra- and inter-assay coefficients of variation were 4.9% and 10.6%, respectively. The linearity showed R 2=0.98 and mean percentage error of −10.8±1.55%. The extraction efficiency was 89.0±23.52% and the parallelism test showed similar slopes. Cortisol detection in hair by using EIA seems to be a valid method to represent long-term circulating cortisol levels in dairy cattle.


Corresponding author


Hide All
Aardal, E and Holm, AC 1995. Cortisol in saliva – reference ranges and relation to cortisol in serum. European Journal of Clinical Chemistry and Clinical Biochemistry 33, 927932.
Boumba, VA, Ziavrou, KS and Vougiouklakis, T 2006. Hair as a biological indicator of drug use, drug abuse or chronic exposure to environmental toxicants. International Journal of Toxicology 25, 143163.
Bryan, HM, Adams, AG, Invik, RM, Wynne-Edwards, KE and Smits, JEG 2013. Hair as a meaningful measure of baseline cortisol levels over time in dogs. Journal of the American Association for Laboratory Animal Science 52, 189196.
Buchanan, KL and Goldsmith, AR 2004. Noninvasive endocrine data for behavioural studies: the importance of validation. Animal Behaviour 67, 183185.
Burnett, TA, Madureira, AML, Silper, BF, Nadalin, A, Tahmasbi, A, Veira, DM and Cerri, RLA 2014. Short communication: factors affecting hair cortisol concentrations in lactating dairy cows. Journal of Dairy Science 97, 16.
Carlitz, EHD, Kirschbaum, C, Stalder, T and van Schaik, CP 2014. Hair as a long-term retrospective cortisol calendar in orang-utans (Pongo spp.): new perspectives for stress monitoring in captive management and conservation. General and Comparative Endocrinology 195, 151156.
Cerri, RLA, Tabmasbi, AM and Veira, DM 2012. Hair cortisol concentrations – influence of color and location in Holstein cows. Journal of Dairy Science 95, S574.
Comin, A, Tidu, L, Cornacchia, G, Cappa, A, Renaville, B and Prandi, A 2008. Neonatal period and hair cortisol in cattle as a marker of stress. XVI Congress of the Mediterranean Federation for Health and Production of Ruminants (FeMeSPrum), 23 to 26 April, Zadar, Croatia, pp. 221–225.
Comin, A, Prandi, A, Peric, T, Corazzin, M, Dovier, S and Bovolenta, S 2011. Hair cortisol levels in dairy cows from winter housing to summer highland grazing. Livestock Science 138, 6973.
Comin, A, Veronesi, MC, Montillo, M, Faustini, M, Valentini, S, Cairoli, F and Prandi, A 2012. Hair cortisol level as a retrospective marker of hypothalamic–pituitary–adrenal axis activity in horse foals. The Veterinary Journal 194, 131132.
Comin, A, Peric, T, Corazzin, M, Veronesi, MCC, Meloni, T, Zufferli, V, Cornacchia, G and Prandi, A 2013. Hair cortisol as a marker of hypothalamic-pituitary-adrenal axis activation in Friesian dairy cows clinically or physiologically compromised. Livestock Science 152, 3641.
Cone, EJ 1996. Mechanisms of drug incorporation into hair. Therapeutic Drug Monitoring 18, 438443.
Davenport, MD, Tiefenbacher, S, Lutz, CK, Novak, MA and Meyer, JS 2006. Analysis of endogenous cortisol concentrations in the hair of rhesus macaques. General and Comparative Endocrinology 147, 255261.
De Lima, V, Piles, M, Rafel, O, López-Béjar, M, Ramón, J, Velarde, A and Dalmau, A 2013. Use of infrared thermography to assess the influence of high environmental temperature on rabbits. Research in Veterinary Science 95, 802810.
Dettmer, AM, Novak, MA, Suomi, SJ and Meyer, JS 2012. Physiological and behavioral adaptation to relocation stress in differentially reared rhesus monkeys: hair cortisol as a biomarker for anxiety-related responses. Psychoneuroendocrinology 37, 191199.
Gatti, R, Antonelli, G, Prearo, M, Spinella, P, Cappellin, E and De Palo, EF 2009. Cortisol assays and diagnostic laboratory procedures in human biological fluids. Clinical Biochemistry 42, 12051217.
González-de-la-Vara, MRDR, Valdez, RA, Lemus-Ramirez, V, Vázquez-Chagoyán, JC, Villa-Godoy, A and Romano, MC 2011. Effects of adrenocorticotropic hormone challenge and age on hair cortisol concentrations in dairy cattle. Canadian Journal of Veterinary Research 75, 216221.
Gow, R, Thomson, S, Rieder, M, Van Uum, S and Koren, G 2010. An assessment of cortisol analysis in hair and its clinical applications. Forensic Science International 196, 3237.
Henderson, GLL 1993. Mechanisms of drug incorporation into hair. Forensic Science International 63, 1929.
Hernandez, CE, Thierfelder, T, Svennersten-Sjaunja, K, Berg, C, Orihuela, A and Lidfors, L 2014. Time lag between peak concentrations of plasma and salivary cortisol following a stressful procedure in dairy cattle. Acta Veterinaria Scandinavica 56, 6168.
Ito, N, Ito, T, Kromminga, A, Bettermann, A, Takigawa, M, Kees, F, Straub, RH and Paus, R 2005. Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal (HPA) axis and synthesize cortisol. FASEB Journal 19, 13321342.
Keckeis, K, Lepschy, M, Schöpper, H, Moser, L, Troxler, J and Palme, R 2012. Hair cortisol: a parameter of chronic stress? Insights from a radiometabolism study in guinea pigs. Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology 182, 985996.
Koren, L, Mokady, O, Karaskov, T, Klein, J, Koren, G and Geffen, E 2002. A novel method using hair for determining hormonal levels in wildlife. Animal Behaviour 63, 403406.
Macbeth, BJ, Cattet, MRL, Obbard, ME, Middel, K and Janz, DM 2012. Evaluation of hair cortisol concentration as a biomarker of long-term stress in free-ranging polar bears. Wildlife Society Bulletin 36, 747758.
Meyer, JS and Novak, MA 2012. Minireview: hair cortisol: a novel biomarker of hypothalamic-pituitary-adrenocortical activity. Endocrinology 153, 41204127.
Morrow, CJ, Kolver, ES, Verkerk, GA and Matthews, LR 2002. Fecal glucocorticoid metabolites as a measure of adrenal activity in dairy cattle. General and Comparative Endocrinology 126, 229241.
Möstl, E and Palme, R 2002. Hormones as indicators of stress. Domestic Animal Endocrinology 23, 6774.
Möstl, E, Messmann, S, Bagu, E, Robia, C and Palme, R 1999. Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock. Journal of Veterinary Medicine Series A 46, 621631.
Möstl, E, Maggs, JL, Schro, G, Schrötter, G, Besenfelder, U and Palme, R 2002. Measurement of cortisol metabolites in faeces of ruminants. Veterinary Research Communications 26, 127139.
Moya, D, Schwartzkopf-Genswein, KS and Veira, DM 2013. Standardization of a non-invasive methodology to measure cortisol in hair of beef cattle. Livestock Science 158, 138144.
National Research Council (NRC) 2001. Nutrient requirements of dairy cattle. National Academy Press, Washington, DC.
Negrão, JA, Porcionato, MA, de Passillé, AM and Rushen, J 2004. Cortisol in saliva and plasma of cattle after ACTH administration and milking. Journal of Dairy Science 87, 17131718.
Palme, R, Robia, C, Messmann, S, Hofer, J and Möstl, E 1999. Measurement of faecal cortisol metabolites in ruminants: a non-invasive parameter of adrenocortical function. Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria 86, 237241.
Palme, R, Touma, C, Arias, N, Dominchin, MF and Lepschy, M 2013. Steroid extraction: get the best out of faecal samples. Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria 100, 238246.
Peric, T, Comin, A, Corazzin, M, Montillo, M, Cappa, A, Campanile, G and Prandi, A 2013. Short communication: hair cortisol concentrations in Holstein-Friesian and crossbreed F1 heifers. Journal of Dairy Science 96, 30233027.
Pötsch, L, Skopp, G and Moeller, MR 1997. Influence of pigmentation on the codeine content of hair fibers in guinea pigs. Journal of Forensic Sciences 42, 10951098.
Pragst, F and Balikova, MA 2006. State of the art in hair analysis for detection of drug and alcohol abuse. Clinica Chimica Acta; International Journal of Clinical Chemistry 370, 1749.
Probst, JK, Spengler Neff, A, Hillmann, E, Kreuzer, M, Koch-Mathis, M and Leiber, F 2014. Relationship between stress-related exsanguination blood variables, vocalisation, and stressors imposed on cattle between lairage and stunning box under conventional abattoir conditions. Livestock Science 164, 154158.
Rigalma, K, Duvaux-Ponter, C, Barrier, A, Charles, C, Ponter, AA, Deschamps, F and Roussel, S 2010. Medium-term effects of repeated exposure to stray voltage on activity, stress physiology, and milk production and composition in dairy cows. Journal of Dairy Science 93, 35423552.
Russell, E, Koren, G, Rieder, M and Van Uum, S 2012. Hair cortisol as a biological marker of chronic stress: current status, future directions and unanswered questions. Psychoneuroendocrinology 37, 589601.
Sabés-Alsina, M, Planell, N, Torres-Mejia, E, Taberner, E, Maya-Soriano, MJ, Tusell, L, Ramon, J, Dalmau, A, Piles, M and Lopez-Bejar, M 2015. Daily exposure to summer circadian cycles affects spermatogenesis, but not fertility in an in vivo rabbit model. Theriogenology 83, 246252.
Sharpley, CF, McFarlane, JR and Slominski, A 2012. Stress-linked cortisol concentrations in hair: what we know and what we need to know. Reviews in the Neurosciences 23, 111121.
Slominski, AT, Manna, PR and Tuckey, RC 2014. Cutaneous glucocorticosteroidogenesis: securing local homeostasis and the skin integrity. Experimental Dermatology 23, 369374.
Slominski, A, Zbytek, B, Nikolakis, G, Manna, PR, Skobowiat, C, Zmijewski, M, Li, W, Janjetovic, Z, Postlethwaite, A, Zouboulis, CC and Tuckey, RC 2013. Steroidogenesis in the skin: implications for local immune functions. The Journal of Steroid Biochemistry and Molecular Biology 137, 107123.
Stalder, T and Kirschbaum, C 2012. Analysis of cortisol in hair – state of the art and future directions. Brain, Behavior, and Immunity 26, 10191029.
Tallo-Parra, O, Carbajal, A, Sabes-Alsina, M, Almagro, V, Fernandez-Bellon, H, Enseñat, C, Quevedo, MA, Manteca, X, Abaigar, T and Lopez-Bejar, M 2013. Preliminary results on hair cortisol detection as a tool to evaluate chronic stress in Sahrawi dorcas gazelle (Gazella dorcas neglecta). 9th International Conference on Behaviour, Physiology and Genetics of Wildlife, 18 to 21 September 2013, Berlin, Germany, 193pp.
Taves, MD, Gomez-Sanchez, CE and Soma, KK 2011. Extra-adrenal glucocorticoids and mineralocorticoids: evidence for local synthesis, regulation, and function. American Journal of Physiology. Endocrinology and Metabolism 301, 1124.
Thun, R, Eggenberger, E, Zerobin, K, Luscher, T and Vetter, W 1981. 24-hour secretory pattern of cortisol in the bull – evidence of episodic secretion and circadian-rhythm. Endocrinology 109, 22082212.
Touma, C and Palme, R 2005. Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation. Annals of the New York Academy of Sciences 1046, 5474.
Van Uum, SHM, Sauvé, B, Fraser, LA, Morley-Forster, P, Paul, TL and Koren, G 2008. Elevated content of cortisol in hair of patients with severe chronic pain: a novel biomarker for stress. Stress 11, 483488.
Von Keyserlingk, MAG, Rushen, J, de Passillé, AM and Weary, DM 2009. The welfare of dairy cattle – key concepts and the role of science. Journal of Dairy Science 92, 41014111.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 1751-7311
  • EISSN: 1751-732X
  • URL: /core/journals/animal
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed