title: Validation of an assay for quantification of alpha-amylase in saliva of sheep.
abstract: The objective of this study was to develop a time-resolved immunofluorometric assay (TR-IFMA) for quantification of salivary alpha-amylase in sheep. For that purpose, after the design of the assay, an analytical and a clinical validation were carried out. The analytical validation of the assay showed intra- and inter-assay coefficients of variation (CVs) of 6.1% and 10.57%, respectively and an analytical limit of detection of 0.09 ng/mL. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution. For clinical validation, a model of acute stress testing was conducted to determine whether expected significant changes in alpha-amylase were picked up in the newly developed assay. In that model, 11 sheep were immobilized and confronted with a sheepdog to induce stress. Saliva samples were obtained before stress induction and 15, 30, and 60 min afterwards. Salivary cortisol was measured as a reference of stress level. The results of TR-IFMA showed a significant increase (P < 0.01) in the concentration of alpha-amylase in saliva after stress induction. The assay developed in this study could be used to measure salivary alpha-amylase in the saliva of sheep and this enzyme could be a possible noninvasive biomarker of stress in sheep.
authors.affiliation: Animal Science Department, School of Veterinary Medicine, University of Murcia, Murcia, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Fuentes-Rubio
authors.affiliation: Animal Science Department, School of Veterinary Medicine, University of Murcia, Murcia, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Fuentes
authors.affiliation: Animal Science Department, School of Veterinary Medicine, University of Murcia, Murcia, Spain.
authors.forename: Julio
authors.initials: J
authors.lastname: Otal
authors.affiliation: Animal Science Department, School of Veterinary Medicine, University of Murcia, Murcia, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Quiles
authors.affiliation: Animal Science Department, School of Veterinary Medicine, University of Murcia, Murcia, Spain.
authors.forename: María Luisa
authors.initials: ML
authors.lastname: Hevia
mesh_terms: D000818:Animals; D015415:Biomarkers; D015200:Fluoroimmunoassay / Q000379:methods / Q000662:veterinary*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006854:Hydrocortisone / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry / Q000201:enzymology*; D012756:Sheep / Q000378:metabolism*; D013312:Stress, Physiological / Q000502:physiology; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D000516:alpha-Amylases; D006854:Hydrocortisone