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Some parameters should be evaluated before the analysis of cortisol hormone using  enzyme-linked immunosorbent assay  ( ELISA ). The most important one is to test the validity of the ELISA kit itself since most of the commercially available ELISA kit is not designed for animal use. The time of sample collection is also another important parameter needed to be evaluated due to a known diurnal variation of cortisol concentrations. This study aims to validate a commercial ELISA kit and evaluate the sampling time on the diurnal variations of cortisol concentration in blood and feces of Aceh cattle. Blood and fecal samples were collected in the morning and afternoon from 8 Aceh cows aged 2-3 years. A commercial ELISA Kit (Cat. No. EIA-K003-H5, Arbor Assays ® ) was validated using analytical (parallelism, accuracy, and precision/% CV of intraand inter-assay) and biological validations (by analyzing sample preand post-transportation). The results of a parallelism test showed the diluted sample curve from the fecal extract was not significantly different (parallel) to the standard curve of EIA-K003-H5 kits (P&gt;0.05), the accuracy of the assay: 99,76 ± 3.77%, and the % CV of intra-and inter-assay less than 10%. The results from the biological validation test showed that the concentrations of fecal cortisol post-transportation were significantly higher compared to the pre-transportation (P 0.05). In conclusion, ELISA Kit (EIA-K003H5, Arbor Assays ® ) is a reliable assay for measuring cortisol in the feces of Aceh cows. Plasma cortisol concentrations in Aceh cows show a diurnal variation which is higher in the morning than afternoon, but the concentration of cortisol in the fecal sample did not.

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Validation of Commercial ELISA kit for Non-Invasive Measurement of Cortisol Concentrations and the Evaluation of the Sampling Time of Blood and Fecal Sample in Aceh Cattle