Intestinal permeability was measured using fluorescein isothiocyanate dextran (FITC-d; MW 4000; Sigma-Aldrich, St. Louis, MO, USA), modified by the method described by a previous study [61 (link)]. Briefly, on Day 19, one bird was randomly selected from each cage and orally inoculated with 1 mL of the FITC-d solution (2.2 mg/mL). Blood was collected from the birds two hours post-inoculation and allowed to clot for two hours in the dark at room temperature. After centrifugation at 1500× g for 15 min, serum was collected. A standard curve was generated using serial dilutions of the FITC-d stock, while a dilution buffer was created using pooled serum from birds on a basal diet. Standards and samples were loaded onto black 96-well plates (Greiner BIO-ONE, Monroe, NC, USA), and the FITC-d concentrations were quantified using a spectrophotometer (VICTOR Nivo Multimode Microplate Reader, PerkinElmer, Shelton, CT, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.
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