Vitamin D3 status of the cows was evaluated by determining liver-derived 25-hydroxyvitamin D3 (25(OH)D3) content in
plasma by using High Pressure Liquid Chromatography (HPLC,
Perkin-Elmer Inc., MA-02451 Waltham, USA) according to
Hymoller et al. (2009). The samples were saponified and
extracted before separation was carried out by reverse phase
gradient HPLC on a C30 column from YMC (Dinslagen,
Germany) and UV detection at 265 nm using 1 α-hydroxyvitamin D3 as an internal standard for quantification. Concentration of the bone formation marker OC was determined in
serum using a competitive 2-step MicroVueTM osteocalcin
enzyme immunoassay (Quidel Corporation, San Diego, Ca,
USA). The intra- and inter-assay coefficients of variation were
10.0% (n ¼ 22) and 9.8% (n ¼ 3), respectively. Concentration of