Validation of an EIA [enzyme immunoassay] technique for the determination of salivary cortisol in cattle

Abstract

This work involves the development and validation of an enzyme immunoassay technique (EIA) for the measurement of the cortisol concentration in cattle saliva. Saliva samples present several advantages over plasma samples in animal welfare studies. Saliva collection avoids venipuncture as a stress factor. Also, saliva components do not affect EIA as plasma components do. At present, there is no validated commercial method for saliva cortisol determination in cattle. Commercially available radioimmunoassay kits for human plasma (detection range: 10-100 ng ml-1) are not sensitive enough for animals with low concentrations of salivary cortisol (< 4 ng ml-1). Thus, EIA is the method of choice in cattle. Sensitivity, specificity, precision and accuracy EIA tests showed this method to be suitable and reliable. The detection limit was found to be 0.024 ng ml-1, representing an improvement on previously described techniques. Intra-assay and inter-assay variation coefficients were 1.47-7.30% and 2.40-9.78%, respectively. The recovery rates for cortisol added to saliva samples were 91.36-126.5%. Parallelism tests showed that saliva cortisol levels can be determined in cattle samples without extraction. The correlation between saliva and plasma cortisol was positive (r = 0.75) and the saliva/plasma cortisol ratio was around 10%. Therefore, saliva samples are a suitable alternative to plasma samples in bovine HPA (hypothalamic-pituitary-adrenal) axis evaluation.