Determination of Salivary Cortisol in Donkey Stallions.

This research investigates the use of a commercial enzyme immunoassay to accurately measure salivary cortisol levels in donkey stallions, a noninvasive method used for assessing stress levels.

Methodology

• Four donkey stallions served as subjects for the study. To account for potential circadian variation in cortisol levels, the researchers collected samples at a fixed time (8:30 AM) over 13 non-consecutive days.
• The researchers obtained saliva samples using a swab, which was inserted at the angle of the lips and left on the tongue for one minute.
• The saliva-laden swabs were then placed into polypropylene tubes. The tubes were subsequently centrifuged, and a minimum of 1ml of saliva was drawn from each sample and frozen at -20°C in preparation for analysis.

Analysis

• A commercial enzyme immunoassay kit designed for saliva cortisol testing was used. This type of kit doesn’t require an extraction process, making it a more straightforward method for cortisol detection.
• The researchers calculated the median cortisol concentrations and identified the minimum and maximum values.

Results

• Recovery of cortisol standard in the donkey saliva ranged from 97.3% to 99.7%, indicating a high accuracy in detected amounts of cortisol.
• Serial dilution of the saliva samples with buffer showed changes in optical density that were consistent with the standard curve. This suggests a reliable reproducibility of results.
• The testing kit’s antiserum had varying degrees of cross-reactivity with other hormones like 11-deoxycortisol, corticosterone, and 11-deoxycorticosterone, among others. The cross-reactivity rates were relatively low, indicating specificity of the assay for cortisol.
• The intra-assay and interassay coefficients of variation were 10.7% and 8.0% respectively, demonstrating reasonable consistency and reliability of the enzyme immunoassay kit for cortisol detection.
• The minimum detectable concentration was 0.01 ng/mL, which indicates a high sensitivity of the assay.

Conclusion

• The study found that the commercial enzyme immunoassay kit used for cortisol detection in saliva was valid for use in donkeys, as it has been in other species. Pavíng the way for non-invasive stress monitoring in these animals.