Fig. 3.

In-ovo feeding (IOF) increases post–hatch small intestinalepithelial cell quantities. (A, B) Immunofluorescence of total villus epithelial cells by DAPI staining (blue), merged with proliferating cell nuclear antigen (PCNA)+ staining (red) at day of hatch (DOH) (A) and d 7 post-hatch (D 7) (B) in control (non-injected), IOF-NaCl, IOF-Leu and IOF-Gln treated chicks. Images were captured at X200 magnifications and automatically stitched. Arrowhead outlines indicate PCNA+ proliferating cells at the bottom regions of the villi. (C, D) Immunofluorescence of total crypt epithelial cells by DAPI staining (blue), merged with PCNA staining (Red) and SRY-box transcription factor 9 (Sox9) staining at DOH (C) and D 7 (D) in control, IOF-NaCl, IOF-Leu and IOF-Gln treated chicks. Images were captured at X400 magnifications. Multipotent Sox9+ cells were located at the base of each crypt and co-localized with PCNA+ cells (C, D, arrowheads). PCNA+/Sox9- progenitor cells were scattered throughout the crypt epithelium (C, D, arrowhead outlines). Scale bars, 50 μm. (E) Quantification of total villus cells and villus proliferating cells from DOH to D 7 in control (non-injected) and IOF-treated chicks. (F) Quantification of total crypt cells, Sox9+ multipotent cells and PCNA+/Sox9- progenitor cells from DOH to D 7 in control (non-injected) and IOF-treated chicks. Values are means ± SEM. Different uppercase letters mark significant differences between age/treatment group for each cell type by Tukey–Kramer HSD, P < 0.05.