M.Sc. / Various researchers have found that supplementing extremely high levels of dietary vitamin D₃ for a limited time prior to slaughter improved meat tenderness due to the increase in blood calcium levels caused by vitamin D, which play an important role in activating the calpain protease system. Vitamin D₃ is metabolised in the liver into 25-hydroxyvitamin D₃ , which controls calcium and phosphate homeostasis, a process regulated by parathyroid hormone. It is hypothesised that ultra-high vitamin D₃ supplementation should alleviate negative effects of beta-agonist supplementation (which reduces meat tenderness), but these levels might be toxic for human consumption. The aim of this study was to determine the levels of vitamin D₃ and its metabolites after feeding of high levels of vitamin D₃ to beef animals to alleviate the effects of beta-agonist supplementation in feedlot cattle and to determine the safety of tissues after ultra-high levels of supplementation of vitamin D₃ . In this study, 20 young steers received neither beta agonist nor vitamin D₃ (control, C), 20 animals each received zilpaterol hydrochloride (Z) and vitamin D₃ . Various levels of vitamin D₃ was administered (xM=x million units), for a given number (y) of days (yD) in some cases withdrawn (N) for a period of z days prior to slaughter (zN). Thus the following treatment groups were examined in this study: C, Z, Z3D7M, Z6D7M, Z6D7M7N and Z9D1M. Samples of liver, meat and fat were analysed for vitamin D₃ and 25-hydroxyvitamin D₃ . An HPLC method for quantification of vitamin D₃ and its metabolites was developed and used to quantify and compare vitamin D₃ and its metabolites from liver, meat and fat. Blood serum was analysed for calcium, phosphorus and parathyroid hormone. Serum calcium concentrations were analyzed using a colorimetric assay kit whereas plasma parathyroid hormone levels (PTH) were determined by an electrochemiluminescence immunoassay employing a sandwich test principle. Levels of vitamin D₃ and 25- hydroxyvitamin D₃ differed with the amount fed - levels in the meat were generally lower than the RDA. Vitamin D₃ was most abundant in liver, especially in the group supplemented with 7 million IU of vitamin D₃ for six days (Z6D7M), accumulating at an average level of 74 μg/100 g, followed by fat (8.39 μg/100 g accumulated vitamin D₃ ), which is higher than the average RDA. Vitamin D₃ supplementation resulted in significant lowering of the M. longissimus lumborum Warner-Bratzler shear force (WBS) and myofibril fragmentation (MFL). There were several positive and negative ix correlations between levels of 25-hydroxyvitamin D₃ and calcium, phosphate, parathyroid hormone and vitamin D₃ , WBS and MFL of M. longissimus lumborum and the muscle proteolytic degradation system (calpastatin, μ-calpain and mcalpain). Controversy remains regarding the upper limit (toxic level) of vitamin D₃ for human consumption. New studies indicate that the current RDA of vitamin D is too low for sufficient health benefits. Vitamin D₃ levels measured in this study were higher than the current RDA, but within the limit proposed by recent clinical studies. Vitamin D₃ as a tool for a meat tenderizer seems not providing reliable results because in this study the control group exhibited lower shear force than the treated groups who received vitamin D₃ zilpaterol supplementation. This is an indication that the tenderazation mechanism by vitamin D₃ still needs further elucidation before vitamin D₃ supplementation can confidently be used as a tool for meat tenderization.