Dm3000 microsystem

The TUNEL assay was used to identify jejunal epithelial cell apoptosis. The Leica DM3000 microsystem was used to analyze labeled cells. At least five views of each image from the different groups were taken with the background light kept constant between images. Five random duplications from each group were analyzed, the ratio of apoptotic to nonapoptotic cells was calculated according to the positive brown coloration, and the average values were calculated. The apoptosis index (AI) was calculated according to the following formula:
$\begin{array}{c}\text{AI}=\frac{\text{\hspace{0.17em}the\hspace{0.17em}number\hspace{0.17em}of\hspace{0.17em}apoptosis\hspace{0.17em}cells\hspace{0.17em}}\left(\text{AC}\right)}{\text{AC}+\text{the\hspace{0.17em}number\hspace{0.17em}of\hspace{0.17em}intact\hspace{0.17em}cells\hspace{0.17em}}\left(\text{IC}\right)}\ast 100\%.\end{array}$

For distal ileum histological analyses, formalin-fixed and paraffin-embedded tissues were cut into thick (4 μm) sections, followed by slicing as well as staining with haematoxylin and eosin (H&E). Leica DM3000 Microsystem was used for obtaining the images of the slices. Next, the height of the villi and the depth of the crypt were evaluated by Image-Pro Plus software (IPP; produced by Media Cybernetics Corporation, USA).

To fix the jejunum tissues, 4% paraformaldehyde was used. Afterward, the jejunum tissues were excised, embedded in paraffin, sliced, and stained with hematoxylin and eosin (H&E) (24 (link)). Images of the paraffin sections were obtained and observed with a Leica DM3000 Microsystem (Leica, Wetzlar, Germany). To fix the jejunum samples for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis, 2.5% glutaraldehyde was used, following previous studies (25 (link), 26 (link)). Then the jejunum samples were fixed in osmic acid and embedded in epon. Digital electron micrographs were obtained with a 1,024 × 1,024 pixel CCD camera system (AMT Corp., Denver, MA).

The jejunum samples of mice were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin blocks. Sections of 5 μm thickness were cut and stained with hematoxylin and eosin (H&E), and images were acquired through a Leica DM3000 Microsystem (Leica, Germany). The villus height and crypt depth were measured using a Leica microscope (DM3000; Leica, Wetzlar, Germany) equipped with a CCD camera (DFC420; Leica). All programs were executed three times, and the data presented is the average of the three replicates. The degree of small intestinal injury was evaluated by Chiu's score classification [38 (link)] as follows: grade zero, normal mucosal villi; grade one, well-structured villi but subepithelial spaces; grade two, elevated villous epithelium with increasing subepithelial spaces; grade three, some cast-off villous epithelium; grade four, structural destruction of villi resulting in shedding and telangiectasia; and grade five, destruction of all the mucosa, hemorrhage, and ulceration.

Nutrient digestibility was calculated as described by Wang et al. [22 (link)]. For the digestive enzyme activity of duodenum and jejunum determination, the mucosa of the duodenum and jejunum were collected and homogenized by adding sterile 4% saline solution to prepare 10% (W:V) homogenates. The homogenate was centrifuged at 1000× g for 10 min at 4 °C, then each digestive enzyme activity (protease, amylase, and lipase) in the supernatant was determined by spectrophotometry using a commercial kit according to the manufacturer’s instruction (Sigma-Aldrich Co, Saint Louis, MO, USA). The middle duodenum was collected and fixed in 10% PBS-buffered formalin and embedded in paraffin. The 0.2 μm thick paraffin sections were cut and stained with hematoxylin and eosin (H&E). Images of the H&E-stained sections were acquired through a Leica DM3000 Microsystem (Leica, Germany). Then, the morphology of the duodenum was evaluated by scanning electron microscopy (SEM, TM-1000, Hitachi, Japan) and transmission electron microscopy (TEM, H-7650, Hitachi, Japan) as previously described [23 (link)]. SEM and TEM images were acquired and analyzed using a Hamamatsu ORCA-HR digital camera system operated with AMT software (Advanced Microscopy Techniques Corp., Danvers, MA, USA) and Semicaps 2000 software, respectively.