Abstract
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.