ABSTRACT: The study applied a targeted metabolomics approach that uses a direct injection and tandem mass spectrometry (DI-MS/MS) coupled with a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics of plasma to evaluate the effects of supplementing clay with or without Saccharomyces cerevisiae fermentation product (SCFP) on the metabolic status of dairy cows challenged with aflatoxin B1. Eight healthy, lactating, multiparous Holstein cows in early lactation (64 ± 11 DIM) were randomly assigned to one of four treatments in a balanced 4 × 4 duplicated Latin square design with four 33 d periods. Treatments were control, toxin (T; 1725 µg aflatoxin B1 (AFB1)/head/day), T with clay (CL; 200 g/head/day), and CL with SCFP (YEA; 35 g of SCFP/head/day). Cows in T, CL, and YEA were dosed with aflatoxin B1 (AFB1) from days 26 to 30. The sequestering agents were top-dressed from day 1 to 33. On day 30 of each period, 15 mL of blood was taken from the coccygeal vessels and plasma samples were obtained from blood by centrifugation and analyzed for metabolites using a kit that combines DI-MS/MS with LC-MS/MS-based metabolomics. The data were analyzed using the GLIMMIX procedure of SAS. The model included the effects of treatment, period, and random effects of cow and square. Significance was declared at p ? 0.05. Biomarker profiles for aflatoxin ingestion in dairy cows fed no sequestering agents were determined using receiver-operator characteristic (ROC) curves, as calculated by the ROCCET web server. A total of 127 metabolites such as amino acids, biogenic amines, acylcarnitines, glycerophospholipids, and organic acids were quantified. Compared with the control, T decreased (p < 0.05) plasma concentrations of alanine, leucine, and arginine and tended to decrease that of citrulline. Treatment with CL had no effects on any of the metabolites relative to the control but increased (p ? 0.05) concentrations of alanine, leucine, arginine, and that of citrulline (p = 0.07) relative to T. Treatment with YEA resulted in greater (p ? 0.05) concentrations of aspartic acid and lysine relative to the control and the highest (p ? 0.05) plasma concentrations of alanine, valine, proline, threonine, leucine, isoleucine, glutamic acid, phenylalanine, and arginine compared with other treatments. The results of ROC analysis between C and T groups revealed that the combination of arginine, alanine, methylhistidine, and citrulline had sufficient specificity and sensitivity (area under the curve = 0.986) to be excellent potential biomarkers of aflatoxin ingestion in dairy cows fed no sequestering agents. This study confirmed the protective effects of sequestering agents in dairy cows challenged with aflatoxin B1.
