Histomorphometric Analysis of Ileum

A 1-cm ileal specimen per bird was prepared as the procedure described by Jiang et al. (2020) (link). Briefly, the formalin-fixed samples were dehydrated in graded ethanol solutions from 70 to 100%, cleared with xylene, then embedded in paraffin. Thereafter, 5.0-µm thick sections were sliced using a Leica RM 2145 microtome (Leica, Nussloch, Germany). The sections were stained with hematoxylin and eosin (Thermo, Waltham, MA), then examined using an Olympus BX40F-3 microscope (Olympus Cooperation, Tokyo, Japan). Three tissue sections containing intact lamina propria were selected from each bird, and an average of two readings (villus height, VH and crypt depth, CD, both measured in μm) were made from each section (total 6 counts per bird, 42 counts per group per time point). Image J software (NIH, Bethesda, MD) was used to measure VH and CD. The VH and CD per tissue sample were averaged, and the VH/CD ratio was calculated.