Determination of Salivary Cortisol in Donkey Stallions

Salivary cortisol provides information about free plasma cortisol concentration, and salivary sampling is a noninvasive well-tolerated procedure. The aim of this study was to validate a commercial enzyme immunoassay for the determination of salivary cortisol in donkeys. Saliva samples were collected in four donkey stallions on 13 nonconsecutive days at 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva was collected using a swab inserted at the angle of the lips, placed onto the tongue for 1 minute and returned into a polypropylene tube. Tubes were centrifuged, and at least 1 mL of saliva was aspirated from each sample and frozen at −20°C until analysis. A commercial enzyme immunoassay kit without extraction was used for determination of cortisol in saliva. Median cortisol concentrations with minimum and maximum value were calculated. Recovery of cortisol standard in donkey saliva was between 97.3% and 99.7%, and serial dilution of donkey saliva samples with assay buffer resulted in changes in optical density parallel to the standard curve. Cross-reactivity of the antiserum was 10.4% with 11-deoxycortisol, 5.2% with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone, and <0.1% with testosterone, progesterone, and estradiol. The intra-assay coefficient of variation was 10.7%, the interassay variation was 8.0%, and the minimal detectable concentration was 0.01 ng/mL. The results of the present study demonstrate the validity of a commercial kit to determine the concentration of cortisol in donkey saliva as already reported in other species