In-Ovo Glutamine Administration Enhances Intestinal Development and Functions in Broiler Chickens: Insights from Enteroid Models

Early life events play significant roles in tissue development and animal 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. A recently developed enteroids culture technique allows investigations on intestinal epithelial functions that are close to physiological conditions. In this study, we evaluated the short- and long-term effects of in-ovo administration of glutamine (Gln) on intestinal epithelial development and functions by using intestinal enteroids culture and tissue electrophysiological analysis. A hundred eggs of commercial Cobb500 broilers were in-ovo injected with 0.2 mL of either phosphate buffered saline (PBS) or 3% Gln at embryonic day 18 (E18). Chicks were euthanized on the day of hatch, and at 3- and 14-days post hatch. Enteroids were generated from the small intestine. After 4 days of culture, enteroids were harvested for 5-ethynyl-2'-deoxyuridine (EdU) proliferation, fluorescein isothiocyanate-4 kDa dextran (FD4) permeability, and glucose absorption assays. At day 3 (d3) and day 14 (d14), intestinal barrier and nutrient transport functions were measured by Ussing chamber. The gene expression of epithelial cell markers, nutrient transporters, and tight junction proteins were analyzed in both intestinal tissues and enteroids. In comparison with PBS control group, in-ovo Gln increased intestinal villus morphology, epithelial cell proliferation and differentiation, and altered epithelial cell population towards increased number of enteroendocrine and goblet cells while decreasing Paneth cells. Enteroids gene expression of nutrient transporters (B0AT1, SGLT1, EAAT3), tight junction (ZO2), glucose absorption, and barrier functions were enhanced at day of hatch. Long-term increases of intestinal di-peptide and alanine transport were observed at day 14 post hatch. Together our results suggested that the in-ovo injection of Gln stimulated intestinal epithelium proliferation and programmed the epithelial cell differentiation towards absorptive cells.