Physiological validation of enzyme immunoassay of fecal glucocorticoid metabolite levels and diurnal variation measured in captive Black-tufted Marmoset Callithrix penicillata (Mammalia: Primates: Callitrichidae)

Measuring stress responses is an important aspect for the conservation of endangered wild species.  Non-invasive measuring of glucocorticoid metabolite levels has become an important tool to measure stress intensity.  The aims of the present study were as follows: to validate the enzyme immunoassay to measure the concentration of fecal metabolites of glucocorticoids (FGM) after stressful stimuli and to determine whether FGM concentrations fluctuate diurnally in Black-tufted Marmosets Callithrix penicillata in captivity.  Eight captive healthy adult Black-tufted Marmosets (four males and four females) were included in the study.  The animals were subjected to three treatments: (1) hormone challenge with adrenocorticotropic hormone (ACTH), (2) saline administration and (3) control treatment to monitor diurnal changes of FGM.  Fecal samples were collected on days -1, 0, +1 and +2, with intramuscular administration of ACTH and saline performed on day 0.  To control diurnal variations, all feces from all animals were collected over six consecutive days and identified using the time of defecation and animal identification number. There were four designated two-hour periods per day (8–10 h, 10–12 h, 12–14 h and 14–16 h), and the samples were grouped for each two-hour period to obtain a representative pool.  The samples were frozen, and the metabolite concentrations were measured by enzyme immunoassay following extraction.  The results show that immunoassay measurements of FGM concentrations in C. penicillata can be validated physiologically.  Diurnal variation of the FGM concentration was observed, with significantly increased FGM levels in the early afternoon in both sexes.  The mean FGM concentration was higher in captive females than in males.  Physical restraint followed by saline administration led to adrenocortical stimulation similar to that observed following ACTH hormone challenge, a finding that has not previously been reported in C. penicillata .  Our results show immunoassay measurements of FGM concentrations provide a valuable tool for the non-invasive study of the endocrine correlates of behavior and well-being of this species.