Measurements of salivary alpha-amylase in horse: Comparison of 2 different assays

The aim of this study was to develop and validate 2 different methods (enzymatic vs. time-resolved immunofluorometric assay [TR-IFMA]) for the detection and measurement of salivary α-amylase (s-α) in horses. The analytical validation of the enzymatic assay showed intra-assay and interassay coefficients of variation of 2.5% and 7.2%, respectively, and a limit of detection of 0.045 IU/L. On the other hand, the analytical validation of the TR-IFMA showed intra-assay and interassay coefficients of variation of 5.4% and 9.9%, respectively, and a limit of detection of 0.097 ng/mL. Both assays also demonstrated a high level of accuracy as determined by linearity under dilution. To check the presence of α-amylase in saliva of horses, 3 different models of physical exercises (exercise in a horse walker and 2 different types of dressage) were made in a total of 27 animals. Saliva samples were obtained before, immediately after, and 30 minutes after the physical exercise. Samples were measured by enzymatic assay and TR-IFMA, and both methods were able to detect the enzyme. Only the results of TR-IFMA showed a significant increase (P < 0.05) in s-α concentration in saliva after the performance of one type of physical exercise and a significant decrease (P < 0.05) in s-α concentration in saliva after the performance of other type of physical exercise, but these significances did not exist in the case of enzymatic measures in both types of exercises. Moreover, TR-IFMA had a lower interindividual variability than the enzymatic assay. Both assays validated in this study could measure s-α in saliva of horses in a reliable and robust way, showing the TR-IFMA advantages that could make this test a suitable alternative to enzymatic measures.