In-ovo injections of Glutamine and Sodium Butyrate Regulate Intestinal Epithelium Development in a Broiler Intestinal Organoid Model

Early life events play significant roles in the tissue development and animal’s health in their later life. Early nutrition, through in ovo delivery, has shown beneficial effects on improving intestinal health in broiler chickens. However, the underlying mechanism is not fully investigated. The proliferation and differentiation of intestinal epithelial stem cells (IESCs) and progenitor cells govern the rapid development and functions of intestinal epithelium. A recently developed 3D intestinal organoids culture technique allows investigations on intestinal epithelial functions that are close to the physiological conditions. In this study, we evaluated the effects of in ovo feeding of two well-known gut beneficial nutrients, glutamine (Gln) or sodium butyrate (SB), on intestinal epithelial development and functions by using intestinal enteroids culture. A hundred eggs of commercial Cobb500 broilers were injected with 0.2 ml of either PBS, 3% Gln, or 1%SB on day E18. Chicks were euthanized at the day of hatch. The small intestines were sampled for epithelial isolation and enteroids culture. The enteroids formation and growth were monitored by microscope. After 4 days of culture, the enteroids were harvested for FITC-dextran (FD4) permeability analysis, and gene expression analysis of epithelial cell markers and functional proteins. One-way ANOVA and Student’s T test were used for statistical analysis. Our results indicated that in ovo injection of both Gln and SB significantly (P < 0.05) increased the enteroids formation rate compared with PBS control. In ovo SB injection significantly (P < 0.05) increased the gene expressions of nutrient transporters (B0AT1, SGLT1, EAAT3) and tight junction proteins (TJP1, ZO2) compared with the PBS control group. Moreover, both Gln and SB treatments significantly (P < 0.01) reduced the FD4 permeability of the cultured enteroids. The gene expressions of epithelial cell markers were analyzed. In comparison with PBS control, ChgA (enteroendocrine cell) was upregulated (P < 0.1) in Gln group, MUC2 (goblet cells) was upregulated (P < 0.05) in SB group, while LYZ (Paneth cells) was down regulated in SB group, Lgr5 (IESCs) were numerically downregulated in both of Gln and SB groups. Together, our results suggested that the in ovo injection of Gln or SB simulated intestinal epithelium proliferation, and programmed the epithelial cell differentiation towards absorptive cells, which may improve the early post-hatch feed adaptation. Understanding the programming effects of early nutrition would shed lights on developing novel feeding strategies for improving gut health.