Effect of supplementing high levels of vitamin D 3. on calcium homeostasis of steers fed barley-based finishing diets

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1 Effect of supplementing high levels of vitamin on calcium homeostasis of steers fed barley-based finishing diets G. Aranda-Osorio 1, A. A. Olkowski 1, T. A. McAllister 2, A. Van Kessel 1, and J. J. McKinnon 1,3 1 Animal and Poultry Science Department, University of Saskatchewan. 51 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A8; 2 Agriculture and Agri-Food Canada, Research Centre, Box 3000, Lethbridge, Alberta, Canada T1J 4B1. Received 7 July 2003, accepted 29 September Aranda-Osorio, G., Olkowski, A. A., McAllister, T. A., Van Kessel, A. and McKinnon, J. J Effect of supplementing high levels of vitamin on calcium homeostasis of steers fed barley-based finishing diets. Can. J. Anim. Sci. 84: The objective of this study was to evaluate the effect of supplementing high levels of vitamin to steers fed barley-grain-based finishing diets on Ca metabolism. Fifteen Hereford steers (607 ± 12 kg) were individually penned and fed at 0800 and 1600 with a ration consisting of 90% barleygrain-based concentrate and 10% barley silage [dry matter (DM) basis]. Steers were randomly assigned to one of three treatments: 0, 2.5 or 5 million IU (MIU) of vitamin steer 1 d 1 for 7 d. Daily blood samples were obtained for measurement of serum for total and ionized Ca, parathyroid hormone (PTH) and calcitonin and plasma for vitamin and 25(OH). Data were analyzed by repeated measures analysis and single degree of freedom contrasts. Feed intake was depressed relative to control animals by 18 and 37% for the 2.5 and 5 MIU treatments, respectively. Relative to controls, total and ionized Ca increased (P < 0.05) by 8 and 19% and 6 and 18% for the 2.5 and 5 MIU treatments, respectively. Serum PTH concentrations were reduced (P < 0.05) by vitamin feeding with the greatest reduction seen with animals fed the 5 MIU treatment. Calcitonin values were not (P > 0.05) affected. Relative to controls, plasma vitamin concentrations increased (P < 0.05) in a quadratic fashion over time with vitamin supplementation, while that of 25(OH) increased (P < 0.05) in a linear fashion. It was concluded that the increase in serum calcium was stimulated by a rise in plasma vitamin and the resulting increased 25(OH) concentrations, which have been shown to influence calcium absorption either directly or via induced synthesis of 1,25(OH) 2. Key words: Calcium homeostasis, vitamin D3, cattle, beef tenderness Aranda-Osorio, G., Olkowski, A. A., McAllister, T. A., Van Kessel, A. et McKinnon, J. J Incidence des suppléments de vitamine sur l homéostasie du calcium chez les bouvillons recevant une ration de finition à base d orge. Can. J. Anim. Sci. 84: L étude devait servir à préciser l incidence d un fort supplément de vitamine sur le métabolisme du calcium (Ca) chez les bouvillons de finition recevant une ration à base d orge. Quinze bouvillons Hereford (607 ± 12 kg) ont été placés dans des enclos distincts et ont reçu une ration composée à 90 % d orge en grain et à 10 % d ensilage d orge (selon la matière sèche) à 8 h et à 16 h. Les animaux ont été affectés au hasard à l un de trois traitements : 0, 2,5 ou 5 millions d UI (MUI) de vitamine D3 par bête et par jour pendant 7 jours. Les auteurs ont prélevé des échantillons de sang afin d établir la concentration sérique de Ca total et de Ca ionisé, la PTH et la calcitonine ainsi que la concentration plasmatique de vitamine D3 et de 25(OH)D3. Les résultats ont fait l objet d une analyse par mesures répétées et par contraste à un degré de liberté. Comparativement aux sujets témoins, les animaux testés ingèrent moins d aliments, soit 18 % et 37 % de moins pour l administration de 2,5 et de 5 MUI de vitamine D3, respectivement. L administration de 2,5 et de 5 MUI de vitamine D3 augmente (P < 0,05) la concentration de Ca total et de Ca ionisé respectivement de 8 et de 19 % et de 6 et 18 % comparativement à celle relevée chez les témoins. La vitamine D3 réduit la concentration sérique de PTH (P < 0,05), l administration de 5 MUI entraînant la plus forte baisse. La concentration de la calcitonine n est pas affectée (P > 0,05). Comparativement aux sujets témoins, le supplément vitaminé augmente la concentration plasmatique de la vitamine D3 (P < 0,05) de façon géométrique dans le temps, alors que la concentration de 25(OH)D3 s accroît (P < 0,05) de manière linéaire. On en conclut que la hausse de la concentration de vitamine D3 dans le plasma et la hausse résultante de celle de 25(OH)D3 entraînent un relèvement du calcium sérique, le 25(OH)D3 exerçant une influence sur l absorption du calcium directement ou indirectement par la synthèse de 1,25(OH)2D3. Mots clés : homéostasie du calcium, vitamine D3, bovins, tendreté du bœuf In an effort to maintain and improve market share, the beef industry has focused on methods of improving beef tenderness. One such effort has been the feeding of high levels of vitamin (i.e., 5 to 7 MIU head 1 d 1 ) for 7 to 10 d prior to slaughter (Swanek et al. 1997, 1999; Montgomery et al. 1998, 2000). The proposed mechanism of action is thought to involve increased intestinal Ca absorption as a result of vitamin feeding, which in turn leads to higher intracellular Ca concentrations post-slaughter. High intra-cellular Ca 3 To whom correspondance should be addressed. 81 levels are known to enhance the activity of Ca-dependant proteolytic enzymes responsible for post-rigor myofibril degradation, which in turn leads to improved beef tenderness (Owens et al. 1998). Calcium plays a key role in many physiological processes. Fluctuations in the normal blood Ca concentration can cause serious metabolic alterations. High serum Ca concentrations can cause progressive depression of the nervous system, conversely, low Ca levels can result in tetany (Kaneko et al. 1997). Abbreviations: DM, dry matter; DMI, dry matter intake; MIU, million IU; PTH, parathyroid hormone

2 82 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 1. Ingredient composition and formulated nutrient levels of the basal diet Item DM (%) Total mixed diet Concentrate 87.2 Barley silage 12.8 Concentrate Barley grain 89.4 Canola meal 3.9 Tallow 3.9 Limestone 1.2 Rumensin z 0.6 Vitamin y 0.4 Salt 0.6 Formulated composition Digestible energy (Mcal kg 1 ) x 3.60 Crude protein Calcium 0.52 Phosphorus 0.36 Ca:P ratio 1.49 z Premix: Monensin (20% active) mixed with barley grain. y Premix: Vitamin A ( IU kg 1 ) and vitamin D ( IU kg 1 ). x Predicted TDN 4.4 Mcal kg 1 (Weiss et al. 1992). As a result, maintenance of a constant Ca concentration in blood is one of the primary homeostatic functions of the body. The metabolism of Ca is regulated by the interaction of three hormones: parathyroid hormone (PTH) secreted by the parathyroid glands; calcitonin, secreted by the C-cells of the thyroid gland; and vitamin in its hormonal form, 1,25-dihydroxyvitamin [1,25(OH) 2 ], produced in the kidneys (Cunningham 2000). The role of PTH and 1,25(OH) 2 is to stimulate Ca retention by kidneys, mobilization from bone and absorption from the intestinal tract when plasma Ca levels fall below normal. In contrast, when plasma Ca levels are elevated, the action of calcitonin results in Ca excretion in urine and decreased bone mobilization (Bhagavan 1992; Rhoades and Tanner 1995). The rise in serum Ca as a result of supplemental vitamin feeding is hard to explain in light of the tight regulatory control mechanisms that govern systemic Ca levels. Little effort has been made to this point to explain the observed changes in serum Ca from an underlying metabolic point of view. Understanding the hormonal control of serum Ca levels is of interest in animals fed high levels of vitamin as it will possibly provide insight into management practices that may lead to improved meat tenderness. The objectives of this study were to evaluate the effect of supplementing high levels of vitamin in cattle fed barley-based finishing diets on serum Ca levels and those of the principle hormones involved in its regulation. MATERIAL AND METHODS Experimental Design Fifteen Hereford steers (607 ± 12 kg) were housed in individual pens ( m) in the Livestock Research Barn of the Department of Animal and Poultry Science at the University of Saskatchewan. Animals were cared for according to the guidelines of the Canadian Council on Animal Care. The cattle were adapted to experimental conditions for 18 d (A1 to A18). The cattle were fed at 0800 and 1600 with a ration consisting of 90% barley grain-based concentrate and 10% barley silage (DM basis) formulated to meet nutrient requirements (National Research Council, 1996; Table 1). Individual voluntary feed intake was measured from day A5 to A11. Subsequently, the cattle were fed at this level from day A12 to A18 in an effort to equalize intakes. The steers were then randomly assigned to one of the following treatments: 0, 2.5, or 5 MIU of vitamin steer 1 d 1 for 7 d (day S1 to S7). The vitamin supplement (12.90 ± 0.46 MIU kg 1 ; New Jersey Feed Laboratory, Inc. Trenton, NJ) was prepared from vitamin concentrate ( IU g 1. Rhone-Poulenc Canada Inc.), using ground barley as a carrier. The vitamin supplement for each steer was weighed out daily and fed in equal amounts at each feeding by hand mixing into the ration. During the supplemental period, daily blood samples were obtained by jugular venipunture, from all steers at Three sets of samples were taken. For the first and second sets, blood was collected into 7-mL evacuated (Vacutainer ) tubes without anticoagulant. The blood was allowed to clot at room temperature for approximately 2 h. One set was centrifuged at 2060 g for 15 min, and the serum was immediately used to determine total and ionized Ca concentrations. The second set was centrifuged at 2060 g for 10 min at 4 C, transferred to 1.8-mL clear centrifuge vials and stored at 70 C until analysis for PTH and calcitonin. For the third set, blood was collected into 7-mL evacuated tubes with anticoagulant (100 USP units of lithium heparin), placed in crushed ice immediately after collection and then centrifuged at 2060 g for 10 min at 4 C. The plasma was collected and transferred to 1.8 ml centrifuge clear vials and stored at 70 o C until analysis for vitamin and 25-hydroxyvitamin [25(OH) ]. No attempt was made to analyze for 1,25(OH) 2 as plasma levels of this hormone are very tightly regulated (DeLuca 1980; Guyton and Hall 2000). This regulation is illustrated by the results of Montgomery et al. (2000) who did not find a significant change in 1,25(OH) 2 plasma concentration in steers supplemented with 5 MIU for 9 d. Following day S7, the vitamin supplement was withdrawn and the steers were fed the basal diet for a further 5 d (day W1 to W5) to determine post-treatment effects. Blood samples were taken as described. Following this period, the cattle were sent to the Beef Cattle Research Unit of the University of Saskatchewan. A final blood sample was taken 9 d after supplementation ended (W9). Laboratory Analyses Serum samples were analyzed for total and ionized Ca by indirect potentiometry utilizing a Ca ion selective electrode (Radiometer ABL 700 Series Analyzer. Copenhagen, Denmark). Serum concentrations of PTH were analyzed by a two-site immunoradiometric assay (Intact PTH- Parathyroid Hormone. Nichols Institute Diagnostics. San Juan Capistrano, CA). Serum calcitonin concentration was determined by a double-antibody radioimmunoassay (Double Antibody Calcitonin, Diagnostic Products Corporation, Los Angeles, CA.). Vitamin and 25(OH) levels in plasma were determined using reverse-phase high-performance liquid chromatography (HPLC) according to the procedure of Olkowski et al. (2003).

3 ARANDA-OSORIO ET AL. CALCIUM HOMEOSTASIS IN BEEF STEERS 83 Table 2. Effect of high levels of vitamin supplementation on dry matter intake (DMI) of finishing steers Vitamin level (MIU steer 1 d 1 ) Dry matter intake (kg) Contrasts (P value) z Days SEM C vs. D 2.5 vs. 5 Adaptation period A Supplemental period S S S S S S S Withdrawal period W W W W Repeated measures analysis P value Treatment Time < Time treatment < z Contrasts: C vs. D = control vs. the average of the two vitamin treatments; 2.5 vs. 5 = 2.5 vs. 5 MIU vitamin treatment. Statistical Analysis The feed intake and blood sample data were analyzed using the Analysis of Variance Technique including Repeated Measures Analysis using the General Linear Model procedure of SAS (SAS Institute, Inc. 1989) to compare treatments within days and over time. The following model for the repeated measures analysis was used: Model: Y ijk = µ + α i + τ ij + δ k + (αδ) ij + ε ijk, where: Y ijk = is the response at time k on animal j in treatment i, µ is the overall mean, α i is a fixed effect of treatment I, τ ij is a random effect of animal j in treatment i, δ k is a fixed effect of time k, (αδ) ij is a fixed interaction effect of treatment i with time k, ε ijk is the random error at time k on animal j in treatment i. Contrast of interest included: (1) Control versus the average response of the two vitamin treatments. (2) Comparison between the 2.5 and 5 MIU treatments. RESULTS AND DISCUSSION Feed Intake (DMI) During the adaptation period, feed intake averaged 8.99 ± 1.21 kg of DM (Table 2). During the initial days of supplementation, feed intake was not different (P > 0.05) between control and supplemented steers, with the exception of a short-lived depression noted on d S2 (P < 0.05) for the 5 MIU treatment. Feed intake was reduced (P < 0.05) by vitamin as the supplementation period progressed with the greatest effect seen with the 5 MIU treatment, although there was no significant difference in intake between animals fed the two supplemental levels of vitamin. Feed intake tended to recover after supplementation ceased, following an inverse relationship with the level of vitamin supplemented, but had not recovered to pretreatment levels 4 d post-treatment. Minimum intakes were attained for the 2.5 and 5 MIU treatments on days S5 and S6, and represented 18 and 37% decreases relative to control animals, respectively (Table 2). This depression in feed intake in animals fed high levels of vitamin was seen in a preliminary study undertaken in our laboratory (Aranda- Osorio et al., unpublished data) and has been observed in other studies (Karges et al. 1999; Berry et al. 2000). As a result of this reduced intake, the average daily vitamin consumption for the 2.5 and 5 MIU treatments was 85.4 (2.14 MIU d 1 ) and 68.4% (3.41 MIU d 1 ) of the targeted intakes, respectively. Total and Ionized Serum Ca Concentrations On day S1, total and ionized serum Ca concentrations for the 0, 2.5 and 5 MIU treatments were 10.11, and mg dl 1 and 4.92, 5.08 and 5.06 mg dl 1, respectively, with no difference (P > 0.05) between treatments (Tables 3 and 4). These initial serum Ca concentrations, as well as the range over the whole experiment for the control animals (9.90 to mg dl 1 for total and 4.78 to 5.11 mg dl 1 for ionized Ca) were within the normal range for cattle (Rosol and Capen 1997; Cunningham 2000). Total and ionized serum Ca concentrations increased (P < 0.05) with vitamin supplementation. However, the response was greater from day S3 to W5 for total and from day S4 to W9 for ionized Ca. Differences due to treat-

4 84 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 3. Effect of high levels of vitamin supplementation on total serum Ca concentrations of finishing steers Total Ca (mg dl 1 ) Vitamin level (MIU steer 1 d 1 ) Contrasts (P values) z Days SEM C vs. D 2.5 vs. 5 Supplemental period S S S S S S < S < Withdrawal period W < W W W W < W Repeated measures analysis P value Treatment Time < Time treatment < z Contrast: C vs. D = control vs. the average of the two vitamin treatments 2.5 vs. 5 = 2.5 vs. 5 MIU treatment. Table 4. Effect of high levels of vitamin supplementation on ionized serum Ca concentrations of finishing steers Ionized Ca (mg dl 1 ) Vitamin level (MIU steer 1 d 1 ) Contrasts (P values) z Days SEM C vs. D 2.5 vs. 5 Supplemental period S S S S S S S Withdrawal period W W W W W W Repeated measures analysis P value Treatment Time < Time treatment z Contrasts: C vs. D = control vs. the average of the two vitamin treatments. 2.5 vs. 5 = 2.5 vs. 5 MIU treatment. ment (2.5 vs. 5 MIU ) were evident only (P < 0.05) during the withdrawal period (day W2 to W9). Maximum values were obtained on day W4 for total serum Ca (11.02 and mg dl 1 ). These values represent an increase relative to control animals of 8 and 19% for the 2.5 and 5 MIU treatments, respectively (Table 3). Maximum values (5.36 and 5.98 mg dl 1 ) were also obtained on day W4 for ionized Ca and represented an increase relative to control animals of 6 and 18% for the 2.5 and 5 MIU treatments, respectively (Table 4). Supplementation of 5 MIU steer 1 d 1 for 7 d increased total and ionized Ca by approximately 2 and 1 mg dl 1, respectively, results that agree with preliminary results

5 ARANDA-OSORIO ET AL. CALCIUM HOMEOSTASIS IN BEEF STEERS 85 Table 5. Effect of high levels of vitamin supplementation on serum parathyroid hormone (PTH) concentrations of finishing steers PTH (pg dl 1 ) Vitamin Level (MIU steer 1 d 1 ) Contrasts (P values) z Days SEM C vs. D 2.5 vs. 5 Supplemental period S S S S S S S Withdrawal period W < W W W W < W Repeated measures analysis P value Treatment Time < Time treatment z Contrast: C vs. D = control vs. the average of the two vitamin treatments. 2.5 vs. 5 = 2.5 vs. 5 MIU treatments. Table 6. Effect of high levels of vitamin supplementation on serum calcitonin hormone concentrations of finishing steers Calcitonin (pg ml 1 ) Vitamin level (MIU steer 1 d 1 ) Contrasts (P values) z Days SEM C vs. D 2.5 vs. 5 Supplemental period S S S Withdrawal period W W W Repeated measures analysis P value Treatment Time Time treatment z Contrasts: C vs. D = control vs. the average of the two vitamin treatments. 2.5 vs. 5 = 2.5 vs. 5 MIU treatment. from our laboratory (Aranda-Osorio and McKinnon, unpublished data) as well as others who have found that such increases in serum Ca as a result of vitamin supplementation were associated with improved beef tenderness (Swanek et al. 1997, 1999; Karges et al. 2001; Montgomery et al. 1998, 2000). Serum Parathyroid Hormone Concentrations Serum PTH concentrations for the 0, 2.5 and 5 MIU treatments were 147.1, and pg ml 1, respectively, on day S1. Over the course of the supplemental period, PTH values dropped in all steers (Table 5). Minimum values for the 0, 2.5 and 5 MIU treatments were 80.8 (W5), 19.4 (W5) and 8.2 (W4) pg ml 1, respectively. These values represented decreases of 45, 87 and 96% with respect to values obtained on day S1 (Table 5). PTH is involved in the shortterm regulation of serum Ca concentration in blood (Rosol et al. 1995). When serum Ca reaches levels above normal, PTH secretion is reduced. There is no clear explanation as to why PTH values in control steers declined over the course of the sampling period. However, the reduction (P < 0.05) in serum PTH of supplemented steers relative to the control animals is consistent with current thinking as to how PTH responds to a rise in serum calcium (Rosol and Capen 1997).

6 86 CANADIAN JOURNAL OF ANIMAL SCIENCE Fig. 1. Effect of vitamin supplementation (0, 2.5 and 5 MIU for 7 d) on plasma vitamin concentrations of finishing steers during supplemental (S1 to S7) and withdrawal (W1 to W9) periods. Repeated measures analysis indicates effect (P < ) of time, treatment and time treatment interaction. Serum Calcitonin Concentrations Serum calcitonin concentrations of the experimental steers were determined on days S1, S3, S6, W1, W3 and W5 (Table 6). On day S1, there was no difference (P > 0.05) in calcitonin concentrations between control and supplemented steers, although steers fed the 5 MIU treatment had lower (P < 0.05) values ( vs pg ml 1 ) than those fed the 2.5 MIU treatment (Table 6). On day S3, the situation was inversed, that is, there was a difference (P < 0.05) between control and supplemented steers, but not between vitamin treatments. From the end of the supplementation period (S6) and throughout the withdrawal period, there were no differences (P > 0.05) between control and vitamin treatments. Calcitonin functions to reduce the blood Ca concentration (Bhagavan 1992). This is achieved by increasing urinary Ca excretion, by decreasing absorption from bone osteoclasts and by preventing the formation of new osteoclasts (Guyton and Hall 2000). However, even though serum Ca concentrations were increased by vitamin supplementation, and PTH values were reduced in these animals, calcitonin levels were not significantly altered throughout the experiment. Rosol et al. (1995) indicated that calcitonin inhibition of osteoclastic bone resorption is transitory, as the osteclasts rapidly become refractory to its effect. For this reason, the calcitonin regulatory mechanism is considered weaker than the PTH system (Rhoades and Tanner 1995). This may explain why calcitonin levels did not react to the prolonged increased in serum Ca concentration in supplemented steers, even though PTH levels were markedly reduced in an attempt to control plasma Ca levels. Plasma Vitamin and 25-hydroxyvitamin Concentrations Concentrations of vitamin and 25(OH) in plasma on day S1 were 4.92, 6.70 and ng ml 1 and 50.13, 50.11, and ng ml 1 for the 0, 2.5 and 5 MIU treatments, respectively. Plasma vitamin values for control animals were similar to reported values of 1 to 3 ng ml 1 for cattle (DeLuca 1979; McDermott et al. 1985; Horst 1986). Slightly elevated levels for the two treatment groups likely reflect the initial feeding of the vitamin supplement that morning. Similarly, the 25(OH) values were in good agreement with the normal range of 20 to 60 ng ml 1 for dairy cattle (Littledike and Horst 1982; Horst and Reinhardt

7 ARANDA-OSORIO ET AL. CALCIUM HOMEOSTASIS IN BEEF STEERS 87 Fig. 2. Effect of vitamin supplementation (0, 2.5 and 5 MIU for 7 d) on plasma 25-hydroxyvitamin [25(OH) ] concentrations of finishing steers, during supplemental (S1 to S7) and withdrawal (W1 to W4) periods. Repeated measures analysis indicates effect (P < ) of time, treatment and a time treatment interaction. 1983; Horst et al. 1994) and identical to that reported for finishing steers by Montgomery et al. (2000). Vitamin and 25(OH) were increased (P < 0.05) in a dose-dependent manner as a result of vitamin supplementation (Figs. 1 and 2) although the nature of the response varied for the two forms of the hormone. The changes in plasma vitamin levels resembled a bell-shaped curve, with values increasing until maximum levels were reached on days S5 through S7. Values then declined and ultimately plateaued over days W3 to W9 (Fig. 1). Plasma vitamin levels of the control steers did not change over this period. In contrast, the increase in plasma 25(OH) levels of supplemented steers was linear, with maximum values obtained toward the end of the withdrawal period (Fig. 2). As with vitamin levels, 25(OH) did not change in control animals. Maximum vitamin levels were and ng ml 1, which represented a and 36.9-fold increase relative to the control animals for the 2.5 and 5 MIU treatments, respectively (Fig. 1). Maximum 25(OH) levels were and ng ml 1. These values represent a 4.9- and 6.9-fold increase relative to the control animals for the 2.5 and 5 MIU treatments, respectively (Fig. 2). Horst and Reinhardt (1983) injected 15 MIU of vitamin in a single dose to dairy cows. Despite differences in dose and route of administration, these authors reported a similar pattern to changes in plasma vitamin and 25(OH) levels as observed in this study. Montgomery et al. (2000) supplemented steers with 5 MIU for 9 d and reported plasma vitamin and 25(OH) concentrations of 3.1 and ng ml 1 and 48.1 and ng ml 1 for control and supplemented steers, respectively. It is evident that these workers obtained considerably higher peaks to plasma vitamin and 25(OH) levels than reported in the present study. The difference is likely due to the route of vitamin administration. Montgomery et al. (2000) administered the vitamin by intraruminal bolus, while in the present trial vitamin was top-dressed to the ration. The depression in feed intake noted in the current study influenced actual vitamin intake with steers fed the 5 MIU treatment consuming only 68.4% of targeted intake. However, despite differences in vitamin intake, the relationship between vitamin supplementation and plasma vitamin and 25(OH) concentrations were very similar in both studies. It is clear that vitamin supplementation induced a series of metabolic responses in supplemental animals.

8 88 CANADIAN JOURNAL OF ANIMAL SCIENCE These included sharp increases in plasma vitamin and 25(OH) levels, and increases in total and ionized serum Ca. PTH levels were reduced, while that of calcitonin were unchanged. The rise in total and ionized serum Ca levels are likely a result of the changes in the principle hormones involved with Ca homeostasis. The drop in PTH in the face of elevated serum Ca is fully consistent with its metabolic function (Rosol and Capen 1997). The increase in plasma 25(OH) levels is associated with the rise in plasma vitamin levels and indicates that the first hydroxylation in the liver was carried out relatively unregulated by the 25- hydroxylase enzyme. This is consistent with known functions of this enzyme (Guyton and Hall 2000). The rise in 25(OH) levels could lead to elevated serum Ca levels in one of two fashions. The first is increased synthesis in the kidney of 1,25(OH) 2, which in turn stimulates the synthesis of Ca binding protein in the intestinal epithelial cells. This protein leads to increased active transport of Ca across the epithelium. However, it is unlikely that this route is the explanation for the observed increase in serum Ca in steers fed supplemental vitamin. The final hydroxylation to 1,25(OH)2D3 in the kidney is a very tightly regulated step, with 1,25(OH) 2 levels maintained in a narrow range (DeLuca 1980; Guyton and Hall 2000). Supporting this reasoning, Montgomery et al. (2000), did not find a significant change in 1,25(OH) 2 plasma concentration in steers supplemented with 5 MIU for 9 d. The second mechanism that may have led to increased serum Ca levels in vitamin supplemented steers is that the sheer abundance of 25(OH) in the plasma may allow it to override the normal metabolic control of plasma calcium. It has been shown that synthesis of Ca-binding protein is a result of a series of events that begin with binding of 1,25(OH) 2 to intestinal epithelial receptors. These receptors have a much greater affinity for 1,25(OH) 2 ; however, they will bind 25(OH) when present in excess, allowing 25(OH) to mimic the role of 1,25(OH) 2 and as a result stimulate Ca absorption (Smith et al. 1983; Bhagavan 1992; Rhoades and Tanner 1995). In view, of the results of Montgomery et al. (2000), where 1,25(OH) 2 levels were not influenced by supplementation of vitamin, it is likely that this mechanism led to the increased serum Ca levels seen in this study. The results of this study clearly illustrate that plasma calcium levels of growing steers fed barley-based finishing diets can be increased by vitamin supplementation, results similar to that observed with corn-based diets (Swanek et al. 1997, 1999; Montgomery et al. 2000, 2002; ) It has been shown, however, that in addition to improved beef tenderness via manipulation of plasma and tissue Ca levels, feeding high levels of vitamin to finishing steers can result in marked increases in vitamin levels in muscle and in other organs (liver) and tissues (adipose) (Montgomery et al. 2000, 2002). High levels of vitamin in edible tissues represent a significant human health concern. As a result, it is unlikely that regulatory agencies such as the Canadian Food Inspection Agency would allow such a practice to be implemented on a commercial basis. As such, research should be directed at exploring alternative mechanisms to increase serum Ca levels that may work either alone or in combination with lower levels of vitamin supplementation to improve beef tenderness. The results of this study provide a starting point for such work. CONCLUSION Vitamin supplementation at levels used in this experiment (2.5 and 5 MIU steer 1 d 1 for 7 d) caused a significant depression in feed intake. The degree of depression and the rate of recovery were inversely related to the level of vitamin fed. Supplemental vitamin increased total and ionized serum Ca concentrations in a dose-dependent manner. Maximum serum Ca concentrations were attained several days after supplementation ended. This suggests that the cascade of events leading to synthesis of Ca binding protein and increased Ca absorption where activated; however time is required for full expression. Vitamin supplementation at both levels induced a significant increase in plasma vitamin and 25(OH) concentrations. Vitamin was increased only during supplementation, whereas 25(OH) levels increased in a linear fashion throughout the supplemental and withdrawal periods. The increase in plasma vitamin and 25(OH) and subsequently in the total and ionized serum Ca caused a depression in serum PTH levels, although calcitonin levels were not affected. These results suggest that increased serum Ca levels as a result of vitamin feeding are due to elevated levels of 25(OH). If the beef industry is going to implement such strategies to manipulate serum Ca levels in an effort to improve beef tenderness, research is required to develop approaches that will not result in potentially harmful residues. ACKNOWLEDGEMENTS The authors express appreciation to the Saskatchewan Agriculture Development Fund for support of this work. 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