Sub x

Mid-jejunal segments were collected from 1 bird per replicate with a median BW, flushed with ice-cold 10% phosphate-buffered saline (VWR International, Radnor, PA) and fixed in 10% neutral buffered formalin (VWR International, Radnor, PA) for approximately 30 d. Subsequently, the samples were dehydrated with ethanol (VWR International, Radnor, PA), cleared with Sub-X (Polysciences, Inc., Warrington, PA) and placed in paraffin (Polyfin paraffin, Sigma Polysciences, St. Louis, MO). The segments (5 μm) were stained with hematoxylin and eosin at the Purdue Histology and Phenotyping Laboratory (Purdue University, West Lafayette, IN). The villus height and crypt depth were measured from 5 complete, vertically oriented villi per slide, and subsequently, the villus height-to-crypt depth ratio was calculated. All measurements were performed under a binocular light microscope (National Optical and Scientific Instruments, Inc., Schertz, TX).

On day 11, 21, 42, and 56 after hatching, mid-jejunal segments were collected from 1 bird per replicate with median BW, flushed with ice-cold 10% phosphate-buffered saline (VWR International, Radnor, PA) and fixed in 10% neutral buffered formalin (VWR International, Radnor, PA) for approximately 30 d. Fixed samples were subsequently dehydrated with ethanol (VWR International, Radnor, PA), cleared with Sub-X (Polysciences, Inc., Warrington, PA) and placed in paraffin (Polyfin paraffin, Sigma Polysciences, St. Louis, MO). Segments (5 μm) were stained with hematoxylin and eosin at the Purdue Histology and Phenotyping Laboratory (Purdue University, West Lafayette, IN). Villus height and crypt depth were measured from 4 complete, vertically oriented villi per slide and subsequently, the villus height to crypt depth ratio was calculated. Villus length is defined as the length from the villus tip to the valley between each villus, whereas crypt depth is defined as the length between the crypt opening and base. The histological sections were evaluated using a binocular light microscope (National Optical and Scientific Instruments, Inc., Schertz, TX). Quantitative measurements were performed with a computerized image analyzer software (AmScope version 3.7, Irvine, CA).

On day 21 posthatching, mid-jejunal segments were collected from 1 bird per replicate with median BW, flushed with ice-cold 10% phosphate-buffered saline (VWR International, Radnor, PA) and fixed in 10% neutral buffered formalin (VWR International) for approximately 30 D. Samples were subsequently dehydrated with ethanol (VWR International), cleared with Sub-X (Polysciences, Inc., Warrington, PA) and placed in paraffin (Polyfin paraffin, Sigma Polysciences, St. Louis, MO). The segments (5 μm) were stained with hematoxylin and eosin at the Purdue Histology and Phenotyping Laboratory (Purdue University, West Lafayette, IN). Villus height and crypt depth were measured from 4 complete, vertically oriented villi per slide and villus height to crypt depth ratio was calculated. Villus length is defined as the length from the villus tip to the valley between each villus, whereas crypt depth is defined as the length between the crypt opening and base. All measurements were performed under a binocular light microscope (National Optical and Scientific Instruments, Inc., Schertz, TX).