Image analysis software

Ileum sections of the small intestinal samples were collected from hens after cervical dislocation (n = 5; one sample per replicate) as described by Mehaisen et al. [33 (link)]. Samples were thoroughly washed and soaked for 72 h in 10% neutral buffered formalin. Cross sections (thickness was 3–5 μm) were obtained, by a rotatory microtome, and stained using general staining method with Harris hematoxylin and eosin stain [42 (link)]. The villus height and crypts depth of sample sections were examined and determined under light microscope at 40× magnification using image analysis software (Leica Microsystems, Wetzlar, Germany).

Intracellular lipid accumulation was examined by using both Oil Red O and Nile Red/DAPI staining following the protocol as mentioned earlier^{1 (link),13 (link)}. For Oil Red O staining, cells were fixed with 4% paraformaldehyde in PBS for 15 min and then stained with 0.2% Oil Red O dissolved in 60% isopropanol for 10 min at room temperature followed by washing with distilled water^{1 (link)}. Cells were imaged using a microscope (Carl Zeiss Inc, Germany). Next, cells were incubated with 100% isopropanol solution for 10 min at room temperature to elute Oil Red O reagent. The eluted solution was removed, centrifuged for 1 min at 12000 rpm to remove any cell materials, and measured the absorbance at 540 nm with a microplate reader (BioTek, USA). The result was expressed as percentage over control. For Nile Red/DAPI staining, cells were stained with Nile Red (0.2 mg/mL) for 15 min at room temperature after fixation with paraformaldehyde, washed with PBS, and mounted with cover slip using the mounting media containing DAPI^{13 (link)}. Images were acquired by confocal microscopy with an inverted laser scanning confocal microscope and the cellular fluorescence was quantified using image analysis software (Leica Microsystems, Germany).

Ten birds from each treatment group were sacrificed and the small intestine was separated from the rest of the gastrointestinal tract as a standard procedure. The intestinal length (jejunum and ileum parts) was measured for each treatment group. For histomorphological studies, intestinal samples were cut from the middle part of ileum tissues. Tissue samples were flushed and soaked in 10% neutral buffered formalin for 72 h. Samples were trimmed and processed by dehydration in alcohol, clearing in Xylene, synthetic wax infiltration and blocking out into Paraplast tissue embedding media. At least, 5 cross sections of 3–5 μm per sample with 50 μm in-between were cut by a rotatory microtome. The sections were stained with Harris Hematoxylin and Eosin as a general staining method as outlined by Suvarna et al. [34 ]. The villus length, villus width, villus area and crypts depth of tissue sections were examined for each treatment group at 40X magnification under light microscope equipped with HD microscopic camera and Image analysis software (Leica Microsystems, Germany).