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A study was conducted to understand the effects of 25-hydroxyvitamin D 3  (25OHD) and 1,25-dihydroxyvitamin D 3  (1,25OHD) administration on the expression of key genes related to osteogenesis, adipogenesis, myogenesis, and vitamin D 3  metabolism in the chicken embryo. A total of 120 fertilized Cobb 500 eggs were used in the current study and were reared under standard incubation conditions. On embryonic day 3 (ED 3), PBS (C), PBS with 40ng 1,25OHD (1,25D-L), 200ng 1,25OHD (1,25D-H), 40ng 25OHD (25D-L), or 200ng 25OHD (25D-H) were injected into the dorsal vein of developing embryos. Whole embryos were harvested at 1, 3, and 6h post-injection for gene expression analyses ( n =8). Gene expression for key osteogenesis markers ( RUNX2 : runt-related transcription factor 2;  BMP2 : bone morphogenetic protein 2;  COL1A2 : collagen type I alpha 2 chain;  BGLAP : bone gamma-carboxyglutamate protein; SPP1: secreted phosphoprotein 1; and  ALP : alkaline phosphatese), adipogenesis markers ( PPAR-γ : peroxisome proliferator-activated receptor gamma;  FASN : fatty acid synthase; and  FABP4 : fatty acid binding protein 4), myogenesis markers ( MYOG : myogenin;  MYOD1 : myogenic differentiation 1; and  MYF5 : myogenic factor 5), and the enzyme responsible for vitamin D 3  inactivation ( CYP24A1 : cytochrome P450 family 24 subfamily A member 1) were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data were normalized by the ΔΔCT method and analyzed using a one-way ANOVA. Results indicated that at 1h post-injection, no differences were found among treatments. At 3h, the early osteogenesis differentiation marker,  ALP , was increased by 1,25D-H and 25D-H, and 25D-H also stimulated the expression of adipogenesis markers ( FAPB4  and  FASN ). In contrast, the expression of myogenesis markers ( MYOD1  and  MYF5 ) was suppressed by 25OHD or 1,25OHD treatments, respectively. At 6h, a late osteogenic differentiation marker,  SPP1 , was increased by 25D-H.  MYOD1  and  MYF5  were continuously suppressed by 25OHD treatments or 1,25D-H. The evidence of vitamin D 3  metabolite retention was assessed by measuring  CYP24A1  expression. At 1h, there were no differences in  CYP24A1  expression. At 3h, all treatments upregulated  CYP24A1  expression relative to control (PBS) embryos. However, at 6h, only the 25D-H group retained higher  CYP24A1  expression compared to the other treatments. In conclusion, the results suggested both 1,25OHD and 25OHD induced chicken embryo osteogenesis and adipogenesis, but inhibited myogenesis during early chicken embryo development. The higher dosage of 25OHD showed a possibility of a longer retention time in the embryos.

Role of 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 in Chicken Embryo Osteogenesis, Adipogenesis, Myogenesis, and Vitamin D3 Metabolism