Swine Blood Sample Collection and Analysis

Two 3-mL blood samples were collected from the jugular vein from one pig per pen on the last day of phase 2, as well as on the last day of phase 3. An equal number of barrows and gilts were sampled within treatment, and the same pigs were sampled at each time point. Of the two samples of blood obtained per pig, one was collected in a vacutainer containing ethylenediamine-tetraacetic acid as an anticoagulant, immediately placed on ice, and analyzed for complete blood cell count on a multiparameter automated hematology analyzer (CELL-DYN 3700, Abbott Laboratories, Abbott Park, IL, USA). The second sample of blood was collected in a serum vacutainer containing spray-dried silica as a clot activator. Samples were allowed to clot and then centrifuged at 4,000 × g for 13 min at room temperature. Serum was removed from centrifuged tubes and stored at −20°C until analysis. Total protein, blood urea N, and albumin were analyzed using a Beckman Coulter Clinical Chemistry AU analyzer (Beckman Coulter Inc., Brea, CA, USA). The concentration of immunoglobulin G (IgG) in serum samples was determined by enzyme-linked immunosorbent assay following the manufacturer’s instructions (Bethyl Laboratories, Inc., Montgomery, TX, USA). Concentrations of interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-gamma (IFN-γ), and tumor necrosis factor-α in serum samples were measured via a porcine-specific multiplex immunoassay kit (MilliporeSigma, Burlington, MA, USA) and read with a Luminex MagPix instrument (Luminex Corporation, Austin, TX, USA).