J. Anim. Sci. 2006. 84:1481-1488
© 2006 American Society of Animal Science
Effects of 25-hydroxyvitamin D3 and manipulated dietary cation-anion difference on the tenderness of beef from cull native Korean cows1
Y. M. Cho*,2,
H. Choi
,2,
I. H. Hwang
,
Y. K. Kim¶ and
K. H. Myung,3
* Hanwoo Experiment Station, National Livestock Research Institute, Pyungchang, Gangwon 232-950;
and
Department of Animal Science, Chonnam National University, Gwangju 500-757;
and
Department of Animal Resources and Biotechnology, Chonbuk Naional University, Jeonju, Jeonbuk 561-756; and
and
¶ Animal Genetic Resources Station, National Livestock Research Institute, Namwon, Jeonbuk 590-832, Korea
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Abstract
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In this study, we characterized the effects of 25-hydroxyvitamin D3 (25-OH D3) and manipulated dietary cation-anion difference (DCAD) on the performance, urine pH, serum constituents, carcass traits, tissue residual vitamin D and its metabolites, beef tenderness, and mRNA and protein concentrations of Ca-dependent proteinases in LM using 24 cull native Korean cows. The cows were divided into 3 groups of 8: control, 25-OH D3 supplemented (25-OH D3), and manipulated DCAD plus 25-OH D3 supplemented (DCAD+25-OH D3). Cows receiving 25-OH D3 or DCAD+25-OH D3 were dosed with 125 mg of 25-OH D3 6 d before slaughter. The manipulated DCAD (–10 mEq/ 100 g of DM) diet was fed from 20 to 6 d (14 d) before slaughter. The DCAD+25-OH D3 treatment decreased urine pH and increased serum Ca concentrations. Although the vitamin D concentrations in LM, liver, and kidney were not affected by 25-OH D3 or DCAD+25-OH D3, muscle tissue 25-OH D3 concentrations were increased by both regimens. Serum 25-OH D3 concentrations were increased by 25-OH D3 supplementation, and the increase was even greater for DCAD+25-OH D3. The same pattern was observed for serum 1,25- (OH)2 D3. However, the LM concentration of 1,25-(OH)2 D3 was less for DCAD+25-OH D3 than for control. Although Ca concentrations of LM increased numerically in response to 25-OH D3 supplementation, no statistical differences in Warner-Bratzler shear force or sensory traits of LM were detected. The LM of cows receiving 25-OH D3 with or without manipulated DCAD had greater concentrations of µ-calpain and m-calpain mRNA, whereas the reverse was observed for calpastatin mRNA. Expression of µ-calpain protein was increased relative to control by DCAD+25-OH D3. The amount of 25-OH D3 and manipulated DCAD administered to cull native Korean cows was insufficient to improve tenderness of beef by increasing muscle Ca concentration. However, DCAD+25-OH D3 induced greater expressions of µ-calpain protein as well as mRNA.
Key Words: anion • beef • calcium • 25-hydroxyvitamin D3 • tenderness
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INTRODUCTION
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The average parity of cull native Korean cows varies widely, depending upon the market price of the calves. Korean farmers normally cull cows at parity 2 or 3, primarily as the result of low calf prices and the tendency for mature cull cows to have inferior meat palatability. The inverse relationship between the physiological maturity of the carcass and the tenderness of the beef has been well documented (Boleman et al., 1996
). Preslaughter feeding of 25-hydroxyvitamin D3 (25-OH D3) seems to exert beneficial effects on postmortem proteolysis and the tenderness of the beef, without accumulation of large concentrations of residual vitamin D3 and its primary metabolite, 25-OH D3, in beef (Foote et al., 2004). The manipulation of acid-base balance has been extensively investigated in studies of the health and productivity of dairy cows; a low prepartum dietary cation-anion difference (DCAD) seems to exert a mitigating effect on hypocalcemia peripartum via increases in urinary Ca, blood-ionized Ca, and responsiveness to Ca-homeostatic hormones (Block, 1994). Very little attention, however, has been focused on the possibility of increasing Ca availability to the proteases responsible for the muscle tenderness of beef cattle, as has been demonstrated with vitamin D3, its metabolites, or both (Montgomery et al., 2000; Foote et al., 2004; Rider Sell et al., 2004), by use of a low DCAD regimen. In the current study, we hypothesized that manipulation of DCAD and administration of 25-OH D3 (DCAD+25-OH D3) to multiparous cull native Korean cows before slaughter might effect an increase in the concentrations of Ca2+ in both the blood and muscle of the cows and would, therefore, result in the production of more tender beef, via an increase in myofibril proteolysis, than was observed in cows supplemented with only 25-OH D3. This hypothesis was assessed via the evaluation of tenderness, postmortem proteolysis, vitamin D metabolite residue concentrations, and the gene and protein expression of Ca-dependent proteases.
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MATERIALS AND METHODS
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Animals and Feeding Management
All of the experimental procedures conducted in this study were approved by the Chonnam National University Animal Ethics Committee. Twenty-four cull native Korean cows (approximately 94 mo of age, and weighing between 361 and 540 kg) were stratified according to parity and weight, and 8 cull cows were randomly assigned to each of 3 treatment groups: 1) control, 2) 25-OH D3 (as ROVIMIXHy·D, DSM Nutritional Products Inc., Belvidere, NJ), and 3) manipulated DCAD+25-OH D3 groups. Beginning 2 wk before the study, cows were adapted to the concentrate diet (Woosung Feed Co. Ltd., Cheonan, Korea; Table 1), which was fed at 1.8% of BW (on a DM basis) twice daily, with rice straw being provided separately on an ad libitum basis during the entire concentrate feeding period of 4 mo. The concentrate diet met the requirements of finishing steers (NRC, 2000; KMAF, 2002). The nutritive composition (DM basis) of the rice straw was as follows: 5.7% CP, 0.3% Ca, 0.1% P, 75.1% NDF, and 50.8% ADF.
The control animals were maintained on the concentrate diet until the end of the experiment, whereas both the 25-OH D3 and DCAD+25-OH D3 groups were dosed with 25-OH D3 (125 mg) once, on d 6 before slaughter. We conducted a force-feeding to provide the cows with the 25-OH D3, which was prepared via the dissolution of 10 g of ROVIMIXHy·D (12.5 g of 25-OH D3/kg of DM) in 150 mL of drinking water, using a bottle to yield the appropriate amount. The DCAD+25-OH D3 cow group received a manipulated DCAD (–10 mEq/100 g of DM) diet between 20 and 6 d (14 d) before slaughter; the diet contained Animate (Mosaic Co., South Riverview, FL), which is composed of corn distillers dried grains with solubles, NH4Cl, MgSO4, CaSO4, and cane molasses, and contained 0.17% Na, 0.53% K, 11.90% Cl, and 4.65% S. The concentrate portion of the diet and Animate contained 5.68 and –711.5 mEq/100 g of DM, respectively, based on DCAD = (Na + K) – (Cl + S). We therefore fed the cows at the ratio of 97.8:2.2, on a DM basis, in a mixture to generate the manipulated DCAD (–10 mEq/100 g of DM).
Blood and Urine Sample Preparation and Analyses
Blood was collected from all of the experimental cows 48 h after 25-OH D3 treatment. The blood was collected via jugular venipuncture into serum Vacutainer tubes that contained a clot activator of micronized silica (Becton Dickinson, Franklin Lakes, NJ). The sera were separated by centrifugation at 179 x g for 15 min and stored at –20°C for later analysis.
A Roche Cobas Integra Blood Chemistry Analyzer (F. Hoffmann-La Roche Ltd., Basel, Switzerland) was used to measure the amounts of Ca, Mg, Cl, and P in serum. Serum Na and K concentrations were evaluated with a Bayer ion-selective electrode module (Bayer Ltd., Ibaraki, Japan). A Bayer Advia Blood Chemistry analyzer (Bayer Ltd.) was used to determine serum triglyceride and glucose concentrations. The cows were manually stimulated to urinate on the penultimate day of DCAD treatment, and a sample of midstream urine was collected into a 30-mL container, and pH was measured within 30 min of collection.
Tissue Sample Preparation and Analysis
The cows were fasted for 24 h before slaughter, and the live weight of each of the cows was determined. Liver, LM, and kidney samples were collected at slaughter and were stored immediately in liquid nitrogen for later analyses. After a 24-h period of chilling (4°C), the carcasses were ribbed, and the Korean Ministry of Agriculture and Forestry quality and yield grade information was recorded for each of the samples (KMAF, 2003).
After the grade information was collected, 1 strip loin was removed from each carcass. Five steaks (2.54 cm thick) were fabricated from each strip loin subprimal. Steaks were vacuum-packed, and the first steak was aged for 1 d and used for sensory evaluation by trained evaluators. The second, third, and fourth steaks were aged for 1, 7, and 14 d, respectively, and were used in the determinations of Warner-Bratzler Shear Force (WBSF). The fifth steak was used to determine the concentrations of muscle-extractable free Ca, which was determined using a Ca-selective electrode (Cole-Parmer Instrument Co., Vernon Hills, IL) coupled with an ion meter (Ion 510, Okaton, Singapore) as described by Hopkins and Thompson (2001) and modified by Hwang et al. (2004).
Vitamin D3, 25-OH D3, and 1,25-(OH)2 D3 in Tissues and Sera
The tissue vitamin D3, 25-OH D3, and 1,25-(OH)2 D3, and serum vitamin D3 were extracted and vacuum-dried according to Horst et al. (1981), whereas the serum 25-OH D3 and 1,25-(OH)2 D3 were extracted by the method described by Hollis et al. (1993). Vitamin D3 was quantified by HPLC (Kontron Instruments, Milan, Italy) via the comparison of the peak areas of the unknowns with those of the standards, as described in the study of Horst et al. (1981). Vitamin D3 was separated on an Agilent Zorbax Sil HPLC column (0.46 x 25 cm; Agilent Technologies, Palo Alto, CA), with a mobile phase of hexane:isopropanol (99:1). The 25-OH D3 and 1,25-(OH)2 D3 concentrations were quantified using RIA kits (BioSource Europe, Nivellis, Belgium) and a gamma counter (Perkin-Elmer Corp., Beltsville, MA). The 25-OH D3 and 1,25-(OH)2 D3 intraassay variations were 1.5 and 4.0%, and interassay variations were 0.2 and 0.6%, respectively. Recovery estimates averaged approximately 87 and 79% for 25-OH D3 and 1,25-(OH)2 D3, respectively.
Warner-Bratzler Shear Force Determination for Tenderness and Sensory Evaluation
The WBSF was measured on LM steaks (2.54 cm thick) after cooking in a preheated water bath for 60 min to a core temperature of 70°C, then cooled in running water (ca. 18°C) for 30 min. Eight cores, each with a diameter of 1.27 cm, were made for each of the samples, and the peak force was determined using a V-shaped shear blade with a cross-head speed of 400 mm/min (Wheeler et al., 2000). The palatability characteristics of LM steaks aged for 1 d were evaluated by a trained 8-member panel, in accordance with NLRI (2004) guidelines.
RNA Isolation and Real-Time Reverse Transcription-PCR
The RNA from the LM was isolated according to the method described by Chomezynski and Sacchi (1987). Thereafter, RNA concentrations were determined by absorbance at a wavelength of 260 nm, and the RNA integrity was determined via electrophoresis in 1% agarose-formaldehyde gel, followed by ethidium bromide staining for the visualization of 28 S and 18 S ribosomal RNA. Real-time reverse transcription PCR was conducted to compare the quantities of µ-calpain, m-calpain, and calpastatin mRNA relative to the quantity of cyclophilin mRNA in the total RNA isolated from the LM. Measurements of the relative quantities of the complementary DNA (cDNA) of interest were conducted using SYBR Green RT-PCR Master Mix (Qiagen, Valencia, CA), appropriate forward and reverse primers (0.5 µM each; Table 2), and 0.2 µg of RNA, according to Kim et al. (2005). The assays were conducted in a Rotor-Gene 2000 Real-Time Cycler, using the appropriate analysis software (Corbett Research, Sydney, Australia), at the thermal cycling settings recommended by the manufacturer (40 cycles, each consisting of 15 s at 94°C and 30 s at 55°C). The titration of cyclophilin, µ-calpain, m-calpain, and calpastatin (0.5 µM) forward and reverse primers against increasing cDNA quantities yielded linear responses, with slopes of –0.24.
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Table 2. Forward and reverse primers for real-time PCR for bovine µ-calpain, m-calpain, calpastatin, and cyclophilin mRNA
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Western Blot Analysis
The proteins from LM were extracted using tris-EDTA buffer (pH 8.3), and were assayed for protein content with a BCA protein assay kit (Pierce Biotechnology Inc., Rockford, IL) or subjected to SDS-PAGE on a 7.5% gel gradient, and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked for 2 h in blocking buffer (20 mM Tris-HCl, pH 7.6; 137 mM NaCl; 0.05% Tween20; 1% polyvinylpyroiodide; and 0.1% BSA), then incubated overnight with monoclonal anti-µ-calpain (1:2,000; Clone 9A4H8D3, Affinity BioReagents, Golden, CO), monoclonal anti-m-calpain (1:500; Clone 107-82, Affinity BioReagents), monoclonal anticalpastatin (1:5,000; Clone 1F7E3D10, Sigma, Saint Louis, MO), and monoclonal anti-ß-actin (1:5,000; Clone AC5, Sigma) at 4°C in incubating buffer (20 mM Tris-HCl, pH 7.6; 137 mM NaCl; 0.05% Tween20; 0.1% polyvinylpyroiodide; and 0.1% BSA). After a series of washes (20 mM Tris-Base, pH 7.6; 137 mM NaCl; 0.5% Tween50), the membranes were incubated in incubating buffer containing goat anti-mouse antibodies (1:8,000 in incubating buffer) conjugated with horseradish peroxidase (Product No A9917, Sigma), washed 3 times (20 mM Tris-Base, pH 7.6; 137 mM NaCl; 0.5% Tween50; 30 min each), sprayed with WEST-1 chemiluminescence solution (iN-tRON Biotechnology, Sungnam, Gyunggi, Korea), and exposed for 1 to 2 min in a dark box (Intelligent Dark box II, LAS-1000 plus, Fujifilm Microdevices Co. Ltd., Miyagi, Japan). The quantities of µ-calpain, m-calpain, and calpastatin proteins were measured relative to the quantity of ß-actin protein from same tissues.
Statistical Analyses
Data were analyzed according to a completely randomized design, using PROC ANOVA of SAS (SAS Inst. Inc., Cary, NC). When the ANOVA revealed a significant effect of treatment, the treatment differences were separated with Duncan’s at a P-value of 0.05. The cows served as the experimental units in all data analyses except for DMI and G:F.
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RESULTS AND DISCUSSION
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Performance
The BW of the cows fed on the control and treated diets are shown in Table 3. No significant treatment responses were detected in response to the 125 mg of 25-OH D3 administered 6 d before slaughter or manipulated DCAD plus 125 mg of 25-OH D3. The average initial weights of the cows were similar among treatments. The manipulated DCAD diet, administered between d 20 and 6 d (14 d) before slaughter showed the highest feed intake of 11.19 kg/d. The ADG during the 4-mo period before slaughter did not differ between groups. Wertz et al. (2004) showed that the oral administration of a single dose of 62.5 or 125 mg of 25-OH D3 had no adverse effect on feedlot performance of steers.
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Table 3. Performance of cull cows fed control, supplemental 25-hydroxyvitamin D3 (25-OH D3), or manipulated dietary cation-anion difference diet plus supplemental 25-OHD3 (DCAD+25-OH D3)
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Urine pH and Blood Serum Contents
Urine pH and concentration of blood serum constituents are shown in Table 4. The urine pH was lower for cows treated with the manipulated DCAD diet (pH 8.15, 7.94, and 6.72 for control, 25-OH D3, and DCAD+25-OH D3, respectively). This suggests that the manipulated DCAD diet (–10 mEq/100 g of DM) induced a more acidic condition (Lomba et al., 1978). This result was also consistent with the findings of Roche et al. (2003), in which the urine pH declined in a curvilinear fashion with decreases in dietary DCAD. The blood serum profile revealed that 125 mg of 25-OH D3 supplementation alone was insufficient to elevate the blood Ca concentration, but manipulated DCAD diet increased the concentration of serum Ca compared with the other treatments. This was consistent with the findings of Foote et al. (2004), who reported that, although Ca2+ concentrations in plasma were increased in steers treated with vitamin D3 and 1,25-(OH)2 D3, 25-OH D3 did not elevate plasma Ca2+ concentrations. However, because low prepartum concentrations of DCAD are known to exert a mitigating effect on hypocalcemia peripartum via an increase in urinary Ca, ionized Ca in blood, and responsiveness to Ca homeostatic hormones in dairy cattle (Block, 1994), the effects of 25-OH D3 might be altered by manipulated acid-base status in the cull cows. The 25-OH D3 and DCAD+25-OH D3 treatments reduced Mg concentrations compared with the control, as was also reported in the study of Swanek et al. (1999), in which 5 x 106 IU of vitamin D3 was administered to feedlot steers for 7 d before slaughter. The manipulated DCAD+25-OH D3 resulted in a reduction of serum Cl concentrations, as compared with the control cows, as in Roche et al. (2003). Concentrations of P were substantially elevated in the 25-OH D3 and DCAD+25-OH D3 cows. We observed no significant differences among the treatments in blood Na, K, triglyceride, and glucose concentrations.
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Table 4. Urine pH and blood serum constituents of cull cows fed control, supplemental 25-hydroxyvitamin D3 (25-OH D3), or manipulated dietary cation-anion difference diet plus supplemental 25-OH D3 (DCAD+25-OH D3)
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Carcass Characteristics, and Muscle and Sera Vitamin D Metabolites
Korean standard carcass measures, including cold carcass weight, LM area, 13th-rib fat thickness, marbling scores, quality grades, and yield grades are shown in Table 5. No carcass traits were affected by treatments.
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Table 5. Carcass characteristics for cull cows fed control, supplemental 25-hydroxyvitamin D3 (25-OH D3), or manipulated dietary cation-anion difference diet plus supplemental 25-OH D3 (DCAD+25-OH D3) treatment
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The administration of 25-OH D3 and DCAD+25-OH D3 had no effect on vitamin D3 concentrations in LM, liver, kidney, or serum (Table 6), a finding consistent with the reports of Foote et al. (2004) who detected no differences in the tissue and serum vitamin D concentrations of steers fed 25-OH D3. The fact that the feeding of supplemental 25-OH D3 to cull cows did not increase vitamin D3 concentrations in the LM, liver, and kidney, compared with the controls, was noteworthy because hypervitaminosis D can be a concern, as Foote et al. (2004) previously reported. Cows fed 25-OH D3 with or without manipulated DCAD exhibited increases in 25-OH D3 concentrations in LM, liver, and kidney. Furthermore, the 25-OH D3 supplement increased serum 25-OH D3 concentration, and the increase in response to 25-OH D3 was greater when the manipulated DCAD diet was fed. The muscle, liver, kidney, and serum 25-OH D3 concentrations of 25-OH D3-supplemented cows were similar to those in studies conducted by Foote et al. (2004) and Wertz et al. (2004).
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Table 6. Concentrations (as-is basis) of vitamin D3, 25-hydroxyvitamin D3 (25-OH D3), and 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) in LM steak, liver, kidney, and serum of cull cows fed control, supplemental 25-hydroxyvitamin D3 (25-OH D3), or manipulated dietary cation-anion difference diet plus supplemental 25-OH D3 (DCAD+25-OH D3)
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Although serum 1,25-(OH)2 D3 concentrations demonstrated responses to treatment that were similar to those for serum 25-OH D3 concentrations, the manipulated DCAD diet reduced the 1,25-(OH)2 D3 concentrations of LM, as compared with either the control or the 25-OH D3 supplemented cows. The manipulated DCAD diet, however, effected an increase in the hepatic and sera 1,25-(OH)2 D3 concentrations, as compared with 25-OH D3 supplemented cows. On the other hand, supplemental 25-OH D3 with or without DCAD manipulation increased renal concentration of 1,25-(OH)2 D3. The possible mechanism responsible for this may involve the fact that the production of renal 1,25-(OH)2 D3 is an enzyme-dependent process, which is pH-sensitive such that changes in intracellular pH might be more favorable for generation of hepatic 1,25-(OH)2 D3, as was hypothesized by Gaynor et al. (1989) and Block (1994). However, this was not consistent with the findings of Wertz et al. (2004), who observed that the exogenous administration of 25-OH D3 did not elevate the concentrations of plasma 1,25-(OH)2 D3, most probably due to the conversion of excess 25-OH D3 to inactive 24,25-dihydroxyvitamin D3. The exact reason for this discrepancy, however, is currently unexplainable.
Muscle Ca, Tenderness, and Sensory Evaluation
The effects of 25-OH D3 and manipulated DCAD+25-OH D3 on the muscle Ca concentrations, WBSF, and sensory traits of the LM steaks are shown in Table 7. Although we noted numerical increases of approximately 10% in Ca concentrations for treatments providing supplemental 25-OH D3, we detected no statistically significant differences among treatments. Our baseline concentrations (8.9 µg/g) of extractable free Ca in the LM were less than those reported in the study of Swanek et al. (1999). The WBSF values of steaks were similar among the 3 treatments. Although postmortem aging improved the tenderness of the steaks, tenderization during the postmortem aging process was not enhanced by either the administration of 25-OH D3 or the manipulated DCAD. Platter et al. (2003) concluded that the threshold at which tenderness influences the acceptability of beef is 4.5 kg of shear force. The average WBSF of the LM steaks was far above this threshold for steaks aged for 1 or 7 d. The lack of improvement in tenderness in response to the treatments could be attributable to the similarities in muscle Ca concentrations among the treatments. The lack of statistical differences in WBSF, in spite of modest numerical improvements with treatment, probably was due to the small sample size or the greater proportion of connective tissue in old cows. Hwang et al. (2004) reported that more than 6 kg of shear force was mainly derived from connective tissue rather than muscle fiber itself, especially in old beef cattle. It therefore would not be easy to achieve a substantial improvement of WBSF with manipulated Ca metabolism in such old cows. No discernible effects of treatments were evident for sensory-panel-evaluated juiciness, tenderness, and beef-flavor intensity.
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Table 7. Calcium concentrations, Warner-Bratzler shear force (WBSF), and sensory traits of LM steak for cull cows fed control, supplemental 25-hydroxyvitamin D3 (25-OH D3), or manipulated dietary cation-anion difference diet plus supplemental 25-OH D3 (DCAD+25-OH D3)
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Calpain and Calpastatin Gene, and Protein Expressions in Tissues
The concentrations of µ-calpain mRNA in the LM of cows supplemented with 25-OH D3, with or without manipulated DCAD, were about 400% of concentrations observed for control cows (Table 8). Moreover, the LM m-calpain mRNA concentrations for the 25-OH D3 and DCAD+25-OH D3 treatments were 497 and 300%, respectively, of those of the control (Table 8). Expression of LM calpastatin mRNA was notably attenuated to 14 and 29% by the 25-OH D3 and DCAD+25-OH D3 treatments, respectively, as shown in Table 8. This is the first report in which the effect of vitamin D metabolites on the bovine muscle gene expressions of Ca-dependent proteinases has been addressed. On the whole, the 25-OH D3 regimen clearly increased the expression of the calpain genes but effected a reduction in the expression of the calpastatin gene in the LM. However, no synergistic effects of manipulated DCAD and 25-OH D3 were detected with regard to the gene expressions of the Ca-dependent proteinases.
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Table 8. Concentrations of mRNA and protein for µ-calpain, m-calpain, and calpastatin in LM of cull cows fed control, supplemental 25-hydroxyvitamin D3 (25-OH D3), or manipulated dietary cation-anion difference diet plus supplemental 25-OH D3 (DCAD+25-OH D3)
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The amounts of muscle µ-calpain, m-calpain, and calpastatin, which were measured immediately after slaughter, are also shown in Table 8
. Although no differences were detected among treatments in the quantities of m-calpain and calpastatin proteins, the amount of µ-calpain protein was increased above control concentrations in the muscle tissues of the DCAD+25-OH D3 group. Because cytosolic concentrations of free Ca2+ in the skeletal muscle (0.05 to 0.10 uM at rest and 10 to 20 uM during action potentials; Konishi, 1998) are less than the concentration of free Ca2+ required for half-maximal activity (3 to 50 uM for µ-calpain and 200 to 1,000 uM for m-calpain; Barrett et al., 1991; Goll et al., 1992), it is reasonable that the 4-fold increase in the µ-calpain mRNA concentrations detected for DCAD+25-OH D3 would translate to a 40% increase in the concentration of calpain protein (Table 8). Changes in mRNA levels generally are followed by changes in the same protein (Pfaff et al., 1990). The acidogenic environment of body fluid induced by the manipulated DCAD, as was demonstrated by the low urine pH (Table 4), also may have contributed to the increase in µ-calpain concentrations because µ-calpain may have been autolyzed less extensively under the more acidogenic situation, thereby resulting in a greater concentrations of active µ-calpain in the assay. In previous work, the activities of the muscle Ca-dependent proteinases were decreased (Swanek et al., 1999) or not affected (Montgomery et al., 2002) by the feeding of vitamin D to beef steers. The contrast between our data and those of Swanek et al. (1999) and Montgomery et al. (2002) could be due to the early sampling conducted in our study, as we collected samples immediately after the animals were killed, whereas previous researchers have measured them at 24 h postmortem, by which time the muscle Ca concentrations had been elevated, and the autolysis of the enzymes had occurred to a greater degree. Increased expressions of µ-calpain proteins have been also reported in conjunction with the application of 1,25-(OH)2 vitamin D3 to a leukemia cell line (Berry and Meckling-Gill, 1999).
Under the conditions of our study, supplementing 25- OH D3 or manipulating DCAD between 20 and 6 d before slaughter to cull cows is not a viable method to increase beef tenderness. The more acidic urine pH and the increased sera Ca and LM µ-calpain protein concentrations in manipulated DCAD cows indicate, however, that there may be an important relationship between DCAD and µ-calpain enzyme. Further investigation of this relationship is warranted.
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Footnotes
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1 This research was funded by the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea (grant No. 104036-2). 
2 These two authors contributed equally to this work.
3 Corresponding author: khmyung{at}jnu.ac.kr
Received for publication September 23, 2005.
Accepted for publication January 12, 2006.
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K. M. Carnagey, E. J. Huff-Lonergan, S. M. Lonergan, A. Trenkle, R. L. Horst, and D. C. Beitz
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Abstract
INTRODUCTION
