Short Communication Hair cortisol level as a retrospective marker of hypothalamic–pituitary–adrenal axis activity in horse foals Antonella Comin a , Maria C. Veronesi b,⇑ , Marta Montillo a , Massimo Faustini c , Silvia Valentini d , Fausto Cairoli b , Alberto Prandi a a Department of Food Sciences, University of Udine, Udine, Italy b Department of Veterinary Clinical Sciences, Università degli Studi di Milano, Milan, Italy c Department of Veterinary Sciences and Technologies for Food Safety, Università degli Studi di Milano, Milan, Italy d Veterinarian, Allevamento Toniatti, S. Michele al Tagliamento (VE), Italy article info Article history: Accepted 5 April 2012 Keywords: Horse Foal Hair Cortisol HPA axis abstract Stimulation of the hypothalamic–pituitary–adrenal (HPA) axis and elevated cortisol concentrations in fetal plasma are associated with foal maturity, viability and adaptation to independent life. However, non-invasive measurement of cortisol in hair samples has not yet been validated in horses. The current study developed a radioimmunoassay to analyse cortisol in horse hair and was used to measure cortisol hair concentration at birth and at 30 and 60 days of age as a retrospective study of HPA axis activity. Cor- tisol was detectable in the hair of foals from birth until 2 months, but decreased with time and varied greatly between individuals. Analysis of hair cortisol could be useful for non-invasive retrospective stud- ies of HPA axis activity in perinatal horse. Ó 2012 Elsevier Ltd. All rights reserved. The stimulation of the hypothalamic–pituitary–adrenal (HPA) axis and increased cortisol concentrations in fetal plasma are asso- ciated with neonatal maturity, viability, and adaptation to inde- pendent life in horses. However, collection of blood from a fetus is potentially hazardous and non-invasive techniques to assess cor- tisol activity are therefore required. Cortisol from the plasma is incorporated into the hair over a period of several days (Gow et al., 2010) and measurement of cortisol in the hair of neonatal foals may reflect the incorporation that occurred from the time prenatal hair first developed until birth (Kirschbaum et al., 2009). To the author’s knowledge, there have been no studies to inves- tigate cortisol in the hair of horses and in this study we aimed to verify the reliability of a cortisol assay of the hair of foals and to use this assay to measure cortisol concentration in hair at birth and at 30 and 60 days old. This would also provide a retrospective understanding of HPA axis stimulation in prenatal, neonatal, and early paediatric periods using non-invasive sampling techniques. The study was carried out in accordance with EC Directive 86/ 609/EEC for animal experiments. The animals included were at-term, multiparous mares that were spontaneously foaling. The foals were only included if they were mature and viable, with a normal time to stand up (TSU) and to first suck (TFS). Information concerning each mare’s age and parity, and the foal’s sex and birth weight were recorded. Foals were checked daily for health and normal development until 60 days after birth. Hair samples were collected from each foal at birth by shaving the withers to the level of the skin at each sampling time. Dry hair samples were stored at room temperature until analysis. The hair strands were washed in 5 mL isopropanol (Davenport et al., 2006) at birth to remove traces of steroids originating in sweat and sebum and to remove cortisol remaining from amniotic fluid. Hair samples (1 cm in length) were analysed for cortisol by a pre- viously described method (Koren et al., 2002) with minor modifica- tions (Accorsi et al., 2008). A radioimmunoassay (RIA) was used to measure cortisol concentration (Comin et al., 2011). Intra-assay and inter-assay coefficients of variation were 5.4% and 9.6%, respectively, while the sensitivity of the method was 0.21 pg/mg. The associations between the mare’s age (<10 or P10 years), parity (2–5 or >5 foalings), foal gender and hair cortisol concentra- tion at birth were analysed with ANOVA, with differences between the three sampling times analysed using the Kruskal–Wallis test, followed by Bonferroni-corrected Mann–Whitney test for multiple comparisons. The a level was set at 0.05. Statistical analysis was performed with SS9.1 for Windows. One hundred and two foals (45 females, 57 males) were in- cluded in the study and had hair samples collected at birth. Descriptive data about the mares’ age and parity, and the foals’ Apgar score, birth weight, TSU, and TFS are reported in Table 1. Daily clinical checks showed normal neonatal development in all 1090-0233/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tvjl.2012.04.006 ⇑ Corresponding author. Tel.: +39 02 50318149. E-mail address: maria.veronesi@unimi.it (M.C. Veronesi). The Veterinary Journal 194 (2012) 131–132 Contents lists available at SciVerse ScienceDirect The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl