Histo star embedder

The method described by Daneshmand et al.^{59 (link)} was used to prepare samples for morphometry analysis. In summary, jejunal and ileal samples were stored in a 10% formaldehyde phosphate buffer for 48 h. Then, the samples were trimmed and processed on a tissue processor (Excelsior™ AS, Thermo Fisher Scientific, Loughborough, UK), fixed in paraffin using an embedder (Thermo Fisher Histo Star Embedder, Loughborough, UK) and cut with a microtome (Leica HI1210, Leica Microsystems Ltd., Wetzlar, Germany) to a slice of 3 μm, placed on a slide and dehydrated on a hotplate (Leica ASP300S, Leica Microsystems Ltd., Wetzlar, Germany). Then, the prepared samples were dyed with hematoxylin and eosin and examined under a microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan). A total of 8 slides were prepared from the jejunal segment per bird, and 10 individual well-oriented villi were measured per prepared slide (80 villi/bird). The average of slide measurements per sample was stated as a mean for each bird. Villus width (VW) was measured at the base of each villus; villus height (VH) from the top of the villus to the villus-crypt junction, crypt depth (CD) from the base of the adjacent villus to the sub-mucosa, the ratio of VH to CD and villus surface area were calculated.

The method used to prepare samples for morphometry analysis was previously described by Daneshmand et al.^{24 (link)}. Briefly, jejunal samples were stored in a 10% formaldehyde phosphate buffer for 48 h. The samples were then processed on a tissue processor (Excelsior AS, Thermo Fisher Scientific, Loughborough, UK), fixed in paraffin using an embedder (Thermo Fisher Histo Star Embedder, Loughborough, UK), and cut with a microtome (Leica HI1210, Leica Microsystems Ltd., Wetzlar, Germany) to a length of 3 cm per slice. The slices were placed on a slide and dehydrated on a hotplate (Leica ASP300S, Leica Microsystems Ltd., Wetzlar, Germany) and dyed with hematoxylin and eosin. Finally, the dyed slices of jejunal were examined under a microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan). A total of 8 slides were prepared from the jejunal segment per bird, and ten individual well-oriented villi were measured per slide (80 villi/bird). The average of each measurement per sample was reported as the respective a mean for each bird. Villus width (VW) was measured at the base of each villus; villus height (VH) from the top of the villus to the villus-crypt junction, crypt depth (CD) from the base of the adjacent villus to the sub-mucosa, the ratio of VH/CD and villus surface area were calculated.