Rm2045

Histomorphometry of gut was performed according to the procedure as designated by Alshelmani et al. [49 (link)]. Approximately 5 cm intestinal portions of duodenum, ileum, and jejunum were obtained, followed by flushing with buffered saline before to be fixed in formalin solution (10%). The intestinal segments were cut approximately into 3.5 mm sections. These tissue sections of the intestine were then subjected to a series of dehydration sequences using tissue processor with automation (ASP300, Leica biosystem, Wetzlar, Germany). The embedding of gut sections was performed using a paraffin embedding station (EG1150 II, Leica, Wetzlar, Germany). The intestinal sections were trimmed up to 4–5 μm with a sectioning rotary microtome (RM2045, Leica Wetzlar, Germany). The staining of tissue sections was performed using haematoxylin and eosin staining procedures. The gut morphometric parameters were measured using the light microscope mounted with a digital camera (Leica DM LB2, Wetzlar, Germany). The 12 villi and crypts per each tissue were examined for analysis and observations. A method designated by Touchette et al. [53 (link)] was used to measure the villus height (VH) and crypt depth (CD) using the Image-Pro Plus software.

For each morphological study, at least five adults (n ≥ 3 from each experimental group) were processed and examined [24 (link)]. Animals were briefly cryoanesthetized and dissected in insect saline solution (0.1 M NaCl, 0.1 M Na₂HPO₄ and 0.1 M KH₂PO₄) under a stereomicroscope. The midguts were dissected and fixed in a solution of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 24 h. After fixation, midguts were dehydrated through graded ethanol (70–95%), the midguts were embedded in glycol methacrylate historesin (Leica Historesin Embedding Kit, Leica Biosystems, Wetzlar, Germany), and sections of 3 µm were cut on Leica RM 2045 [24 (link)]. The midgut section slides were stained with 0.5% toluidine blue and 1% borax and analyzed and photographed with a Leica DM500 microscope (installed with LAS EZ-V2.0.0 software) using a ×40 objective.