Daily Rhythms of Serum Vitamin D-Metabolites, Calcium and Phosphorus in Horses

Many physiological processes of domestic animals exhibit daily rhythmicity. The goal of the present study was to investigate the daily rhythms of calcium, inorganic phosphorus and 24,25-(OH)2-D3, 25-(OH)-D 3 and 1,25-(OH)2-D3 in the blood serum of horses. Five Thoroughbred mares from the same farm, clinically healthy and placed in individual stalls, at the same environmental temperature and photoperiod were used. For 30 days prior to the study, the animals underwent the same pattern of daily activity. Blood samples were collected at 4 h-intervals for 48 consecutive h, starting at 08:00 h of the first day and finishing at 04:00 h of the second day, via intravenous cannula inserted into the jugular vein. Each individual sample was assessed for serum concentration of calcium and inorganic phosphorus by means of a UV spectrophotometric test, and serum concentration of 24, 25-(OH)-D3, 25-(OH)-D3, and 1,25-(OH)2-D3 were assessed by means of HPLC method. Data analysis was conducted by one-way repeated measures analysis of variance (ANOVA) and by the single cosinor method. ANOVA showed a significant effect of time on all the variables studied (p < 0.0001) and post-hoc test (SNK) showed significant differences (p < 0.001) comparing all the time intervals of 4 h, on either day. The application of the periodic model and the statistical analysis of the cosinor procedure allowed defining the periodic variables and their acrophases (expressed in hours) during the 2 days of monitoring. Calcium showed diurnal acrophases at 15:00 h for the 1st day and at 15:48 for the 2nd day; inorganic phosphorus showed diurnal coincident acrophases at 14:32 h both for the 1st and 2nd day and 25-(OH)-D3 showed diurnal acrophases at 14:08 h for the 1st day and at 15:04 h for the 2nd day. The results obtained could be useful for standardizing blood sampling according to the time of day and for optimizing the administration of these substances according to their circadian or other rhythms.