4.4. Intestinal Histology

To determine intestinal histology, three paraformaldehyde-fixed intestinal segements (from duodenum, jejunum, and ileum) were dehydrated and embedded in paraffin. Five-μm sections were cut and then stained with hematoxylin and eosin. Intestinal histology was determined using a light microscope (Leica, Germany) with Leica Application suite image analysis software (Leica, Germany). Only vertically oriented villi and crypts were measured [12]. Histological indices, such as villus height (from the tip of the villi to the villus crypt junction), villus width at half-height, and crypt depth (defined as the depth of the invagination between adjacent villi) were determined from ten adjacent villi [33]. The villus:crypt ratio and villous surface area were calculated. All intestinal histological analysis was done by the same person, who was blinded to the treatments.