In ovo vitamin D3 injection leads to a long-term improvement of bone development in laying chicks.

Laying hens are prolific egg producers, and the nutrient composition of breeder eggs critically influences the growth and development of offspring chicks. This study investigated the long-term effects of in ovo vitamin D3 injection (IOI-VD3) on bone development in chicks hatched from eggs laid during the late laying stage (68 weeks old, termed eggs-late). Compared to eggs from the peak laying period (44 weeks old, eggs-peak), eggs-late exhibited significantly lower phosphorus (↓7 %, P = 0.077) and vitamin D3 (↓26.9 %, P < 0.001). Consequently, chicks from eggs-late demonstrated inferior bone development at both 1 day (lower leg bone weight by 18 %, P < 0.05) and 4 weeks (reduced bone mineral content by 10 %; P < 0.05) of age relative to eggs-peak chicks. Notably, IOI-VD3 efficiently improved embryonic and long-term bone growth in late-egg-derived chicks, increasing bone mass by 14 % and accelerating growth rate by 10 % (P < 0.05). Further in vivo and in vitro analyses revealed that vitamin D3 promoted bone development by activating osteogenesis-related genes (P < 0.05) and suppressing osteoclastogenic markers (P < 0.05), with the vitamin D receptor (VDR) mediating these effects. Chromatin immunoprecipitation (ChIP) assays confirmed that 1,25-(OH)2D3 strengthened VDR binding to promoter regions of RANKL (P = 0.016) and RUNX2 (P = 0.031). In conclusion, this study highlights the disparity in egg quality between late and peak laying periods and its impact on chick bone development. IOI-VD3 ameliorated bone deficits in eggs-late chicks via VDR-dependent mechanisms.