In ovo injection of vitamin D₃ improves intestinal development of chicks via activating vitamin D receptor

Vitamin D₃ (VD₃) is an essential fat-soluble micronutrient that regulates diverse biological processes. However, its long-term impact on avian intestinal development remains largely unknown. In this study, we detected that the VD₃ content was significantly lower in late-cycle production eggs (68 weeks old, termed "LP eggs") compared to the peak-cycle production eggs (44 weeks old, named "PP eggs") (P < 0.05). Notably, the intestine index was reduced (P < 0.05), and the intestinal expression of tight junction (TJ) proteins (Occludin and ZO-1) was decreased for chicks hatched from LP eggs versus those from PP eggs (P < 0.05). Thus serial dosages of VD₃ (2.5, 5.0, or 7.5 μg VD₃/egg) were injected into the amniotic cavity of embryos hatched from LP eggs on embryonic day 17 (E17). This intervention effectively improved hatchability, promoted intestinal development that persisted through 14-days post-hatch, and showed immuno-modulatory effects manifesting as Interleukin-6 (IL-6) downregulation and Interleukin-10 (IL-10) upregulation. Further in vivo and in vitro analysis revealed that VD₃ improved intestinal development by activating TJ-related genes (P < 0.05), with vitamin D receptor (VDR) mediating these effects. Chromatin immunoprecipitation (ChIP) assays confirmed that 1,25-(OH)₂D₃ strengthened VDR binding to promoter regions of Occludin and ZO-1 but decreased the interaction degree of VDR with the IL-6 promoter (P < 0.05). These findings establish that in ovo injection of VD₃ is an efficacious perinatal intervention strategy, which significantly mitigates developmental constraints in late-phase poultry production through VDR-dependent intestinal barrier potentiation and immuno-homeostasis modulation.