Treatment with early life antibiotics in vivo decreases expression of intracellular digestion markers and increases expression of innate defense and enteroendocrine cell markers. Whole-tissue real-time qPCR analysis of (A) intracellular digestion markers CtsL, CtsZ, CtsA, Dab2, and Mcoln3 and of (B) innate defense markers Lyz1, Reg3β, and Reg3ɣ in distal small intestine. (C) Immunohistochemistry of LYZ1 and quantification of amount of lysozyme-1–positive cells per crypt in distal small intestine. Black arrowheads indicate positively stained cells. Scale bars: 100 μm. (D) Alcian blue and periodic acid-Schiff (PAS) staining of proximal and distal small intestine. Scale bars: 100 μm. (E) Whole-tissue real-time qPCR analysis of (trans)epithelial sensing markers Gip, Nts, Gcg, Pyy, Sst, Sct, and Cck in proximal small intestine. Relative expression to reference genes Cyp and Ppib. Statistical analysis was performed by 1-tailed unpaired t test (CtsZ, Dab2, Lyz, Reg3ɣ, Gip, Nts, Gcg, Pyy, Sst, Sct, and Cck) or the Mann–Whitney test because data were not distributed normally when assessed by the D’Agostino and Pearson normality test (CtsL, CtsA, Mcoln3, and Reg3β). Error bars indicate means ± SD (CtsZ, Dab2, Lyz, Reg3ɣ, Gip, Nts, Gcg, Pyy, Sst, Sct, and Cck) or medians with interquartile range (CtsL, CtsA, Mcoln3, and Reg3β). Levels of significance are indicated: ∗P < .05, ∗∗P < .01. n = 8–12 pups per group.