Boldenone, Boldione, and Milk Replacers in the Diet of Veal Calves: The Effects of Phytosterol Content on the Urinary Excretion of Boldenone Metabolites

Twenty-six veal calves were split into two groups and fed two milk replacers with a different content of phytosterols for 26 days; then, 14 calves (7 animals from each diet) were kept as controls and 12 calves (6 per diet) received daily, per os, a combination of 17beta-boldenone (17beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) for 38 days. The urinary elimination of 17 alpha-/17beta-boldenone conjugates (17 alpha/beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) was followed by liquid chromatography-tandem mass spectrometry from all of the animals until slaughtering. In urine from treated animals, 17 alpha-Bol concentrations, despite a great variability, were greater than 17beta-Bol, both detected always as conjugates. At days 1, 2, and 3, the mean urine concentration of 17 alpha-Bol was higher than 12 ng/mL. A remarkable decrease was observed during the following days, but the 17 alpha-Bol concentration was still higher than the attention level of 2 ng/mL in 58% of the samples; the concentration of 17beta-Bol was around the action level of 1 ng/mL; two days after treatment withdrawal, no 17beta-Bol was detected in the urine. In urine from control animals, the 17 alpha-Bol concentration was strictly related to the phytosterol content of the diet, while, in urine from treated animals, the much higher 17 alpha-Bol levels were not modified by the production from diet precursors. The results confirmed that a 17 alpha-Bol level higher than 2 ng/mL should be considered as evidence of suspected illegal treatment and that the urinary excretion of 17beta-Bol is due to exogenous administration of 17beta-Bol. The discontinuous rate of elimination of both 17 alpha- and 17beta-Bol, despite the daily administration of 17beta-Bol plus ADD, indicates the necessity for further research to detect other urinary boldenone metabolites to strength surveillance strategy.