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Veterinary schools rely on live animals to assist in teaching veterinary students clinical procedures. The aims of the study were to (1) quantify a potential physiological stress response of teaching mares during veterinary student transrectal palpation of their reproductive tracts using fecal glucocorticoid metabolites (fGCM) and salivary glucocorticoid concentrations (sGC) and (2) to determine the relationship between the glucocorticoid concentrations and both the ages and palpation experience of the study animals. Twenty-nine, adult, nonpregnant Nooitgedacht mares, which were accustomed to the routine procedure of palpation, were subjected to a palpation of 20 minutes per mare by a single student. Median sGC concentrations before palpation (collected in paddocks) were not significantly different from those determined at 10, 40, and 70 minutes after palpation (χ2 = 1.69, df = 3, P = .64). Median fGCM concentrations before palpation (start of palpation) were not significantly different from those determined at 26 hours after palpation (W = −89.0; P = .34). Neither prepalpation sGC nor postpalpation fGCM concentrations correlated with either age of the mares (Pearson's r = −0.02, n = 28, P = .91; Pearson's r = 0.30, n = 29, P = .11) or practical experience (number of transrectal palpations conducted before the study; Pearson's r = −0.008, n = 28, P = .97; Pearson's r = 0.26, n = 29, P = .18) of the mares, respectively. This study suggested that student transrectal palpations of the reproductive tract in habituated mares did not activate the hypothalamic-pituitary-adrenal axis.Twenty-minutes student transrectal palpation of the reproductive tract in habituated mares did not activate the hypothalamic-pituitary-adrenal axis.Basal fecal glucocorticoid metabolite concentrations were not influenced by age or palpation experience of the habituated mares.Basal salivary glucocorticoid concentration was not influenced by age or palpation experience of the habituated mares.