Gut health
At the end of phase 1, one pig from each pen (from 4 barrow pens and 4 gilt pens per treatment, n = 8) were selected and euthanized at 10 am to collect intestinal tissues and digesta samples. After killing, gastrointestinal tract with digesta were removed. The representative digesta from cecum and colon were gently squeezed into a tube and then stored at -80°C until analysis for volatile fatty acids (VFA) concentrations [21, 22]. Intestinal tissue from middle section of jejunum was isolated, flushed with 0.9% salt solution, fixed in 10% formaldehyde-phosphate buffer, and stored at 4°C for microscopic assessment of mucosal morphology. Briefly, the fixed intestinal segments were embedded in paraffin wax. Cross sections of 6 μm thickness of each intestinal segments were obtained, and stained with hematoxylin and eosin. Villus height was determined at 40× magnification as described by Shen et al. [22]. Villous height from the tip to the crypt-villus junction on 10 well-oriented villi were measured in triplicate for each intestinal segment of each pig [22, 23].