FIGURE 1.

Study design. (A) Forty-eight 68-wk Hy-Line Brown hens were used in the study and were randomly assorted into 1 of 6 treatment groups and fed the diet with the corresponding dietary vitamin D₃ supplementation concentration. (B) Hens were individually housed in cages of a 2-leveled battery cage in a randomly assigned complete block design (n = 8/diet). (C) The experimental timeline in which hens were fed the same basal diet (400 IU D₃/kg) for 4 wk as a washout period (W-4). Hens were started on the experimental diets at week 0 (W0), eggs were collected daily, and weekly body weight was taken until the end of the study. On weeks 0, 3, 6, and 10, blood was collected from the brachial (wing) vein from all hens to measure ionized blood calcium using an i-STAT blood analyzer. Blood was centrifuged down, and plasma was collected to measure vitamin D₃ metabolite concentrations. The study ended on week 10, and all hens were sacrificed, and sample tissues were collected.