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ANIMAL NUTRITION |
* Departments of Animal Science and
and
Large Animal Clinical Sciences, Michigan State University, East Lansing 48824
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: bone • supplement withdrawal • swine
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Feeding Ca and P in concentrations that exceed growth requirements increases bone mineralization and bone strength (Crenshaw et al., 1981; Maxson and Mahan, 1983; Combs et al., 1991).
In recent years, there has been increased interest in minimizing P in finishing pig diets to reduce nutrient excretion and feed costs. Removing two-thirds or more of dietary inorganic P during the late finishing phase is sufficient to maintain growth performance and carcass quality (O’Quinn et al., 1997; Mavromichalis et al., 1999; Shaw et al., 2002). During this period, the animal may draw upon mineral body reserves present in the bone and other tissues to support metabolic requirements. Consequently, bone strength is decreased (O’Quinn et al., 1997). Reduced dietary mineral additions may alter bone metabolism sufficiently to increase the incidence of bone fractures during the slaughter.
The primary objective of this research was to determine the effects of supplement withdrawal (omission of vitamin and trace mineral premixes and two-thirds reduction of inorganic P) for 28 d before slaughter on bone metabolism, bone strength, and incidence of bone fractures occurring at slaughter. We also evaluated the influence of dietary wheat middling inclusion on these same responses. Treatments and animals used in this research were part of a larger study that evaluated if supplement withdrawal and the feeding of wheat middlings also affected growth performance, carcass characteristics, fecal mineral concentrations, and the nutrient content and oxidative stability of the longissimus dorsi muscle (Shaw et al., 2002).
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Experimental Design
A more detailed description of the experimental design and dietary treatments is provided in Shaw et al. (2002). In summary, 64 crossbred barrows were blocked by weight (average initial BW of 8.5 ± 0.7 kg) and allotted to a 2 x 2 factorial arrangement of treatments, replicated twice over time. The factors were with or without supplement withdrawal (omitting vitamin and trace mineral premixes and 2/3 of the inorganic P) 28-d pre-slaughter (from corn-soybean meal-based diets), and with or without wheat middlings.
Diets
Each pig was individually penned and fed in 7 different phases (nursery 1, 2, and 3; grower 1 and 2; early finisher and late finisher). The nursery 1 diet was provided at weaning. Animals were weighed twice per wk, and subsequent diet changes were made as each animal attained a desired BW of 10, 15, 25, 45, and 65 kg for nursery 2 and 3, grower 1 and 2, and early finisher, respectively. The change to the late-finisher diet was made 28 d before slaughter (BW of 78.8 ± 3.9 kg). Target slaughter age was 145 d.
In the nursery through early-finishing phases, all diets met or exceeded NRC (1998) recommendations for all nutrients, regardless of treatment. The composition of the nursery diets is presented in Table 1, and the compositions of the grower diets and early finisher diet are presented in Table 2. Three vitamin premixes were utilized in this study: 1 for the 3 nursery phases, another for the 2 grower phases, and a third for the 2 finishing phases. The premixes were stored in airtight containers at –20° C until diets were mixed. Complete feeds were mixed weekly and stored at 5° C to maintain vitamin activity. Any feed that remained in the feeders for more than 72 h was weighed and discarded.
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The impact of supplement withdrawal was studied by providing pigs 4 different diets during the late-finishing phase (the 28 d before slaughter). Vitamin and trace mineral premixes and two-thirds of the inorganic P were removed from the diets of one-half of the pigs in both the CSBM and CSBM+WM treatment groups (Table 3). Limestone additions were adjusted to maintain a Ca:available P ratio of 2.5:1. All 4 diets were formulated to contain identical lysine and ME concentrations.
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Serum osteocalcin concentrations were determined in duplicate using commercially available ELISA tests (Metra Osteocalcin, Quidel Corporation, San Diego, CA) according to the manufacturer’s instructions (Catalog number 8002; Metra Biosystems, Mountain View, CA). The antibody used in the osteocalcin assay recognizes only the intact osteocalcin produced by osteoblasts and not the osteocalcin fragments from resorbed bone. Consequently, the analyzed serum osteocalcin concentrations are a reflection of osteoblast activity specifically, and not bone turnover (Lang et al., 2001).
Serum pyridinoline concentrations were determined in duplicate using commercially available ELISA tests (Metra Serum Pyd, Quidel Corporation) according to the manufacturer’s instruction. This assay demonstrates high affinity for free pyridinoline and negligible binding to deoxypyridinoline and deoxypyridinoline peptides, reducing the variability in estimating collagen degradation (Lang et al., 2001).
Radiographs
Twenty-eight days after the initiation of the late finishing diets, and at a live weight of 106.6 ± 7.0 kg, pigs were transported to the Michigan State University Meat Laboratory for slaughter. No visible indications of bone fractures were observed preslaughter. Pigs were electrically stunned (110 V, 420 A, 3 s), hoisted by chain from the right leg, and exsanguinated. After scalding, pigs were mechanically dehaired (approximately 60 s), and the remaining hair and feet were manually removed.
After evisceration and before splitting the spine, a ventrodorsal projection radiograph of the lumbar vertebrae of each carcass was recorded (80 KVp, 17 mA, 0.18 s, 30-cm focal film distance) using a portable radiograph machine (Minxray, Northbrook, IL). After splitting the spine, radiographs were recorded (75 KVp, 17 mA, 0.12 s, 30-cm focal film distance) of the right and left femurs, including the femoral heads. To identify bone fractures, a radiologist, blinded to treatments, examined each radiograph.
Computed Tomography
Bone mineral density of the third metacarpal (MC III) of the right foot was examined by computed tomography (CT) scan. Excised feet were placed on the CT table (GE 9800; GE Medical Systems, Milwaukee, WI) in palmar recumbency on a pad that contained 3 hydroxyapatite bone density standards (0, 75, and 150 mg/cm3; Image Analysis, Inc., Columbia, KY). These standards served as internal controls for each CT image to account for x-ray energy fluctuations that may occur between images. A scout view was produced in the dorsopalmar projection to locate the middiaphyseal level of MC III. A single 10-mm thick image in the transverse plane was acquired at 120 kV, 80 mA, 2 s, 512 x 512 matrix, and small scan field of view, in the bone algorithm. The bone mineral density of the MC III was determined by comparing the x-ray linear attenuation coefficient of the bone to that of the hydroxyapatite standards. Total and cortical cross-sectional areas were measured by tracing the endosteal and periosteal margins of the MC III.
Bone Characteristics
The MC III bones were cleaned of all muscle and connective tissue with a scalpel. Peak force and ultimate shear stress of the MC III were determined with an Instron Universal Testing Machine (Model 4202; Instron, Canton, MA) fitted with a 20-kN load cell that moved at a test speed of 5.0 mm/min according to the procedure described by Combs et al. (1991). However, the shape of the cross-sectional area of the bone was assumed to be that of an elliptical quadrant, and area was calculated using the following equation:
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in which B is the outside major diameter, D is the outside minor diameter, b is the inside major diameter, and d is the inside minor diameter. Ultimate shear stress was calculated according to the following equation: Stress = ultimate load/(2 x area) (ASAE, 1999).
The same bones that were mechanically tested were wrapped in cheesecloth and extracted with ethyl ether for 72 h using a Soxhlet apparatus. Bones were dried at 100° C for 12 h to determine the dry fat-free weight, placed in crucibles, and ashed in a muffle furnace (Thermolyne 30400; Barnstead/Thermolyne, Dubuque, IA) for 16 h at 500° C. Percent ash was calculated as a percentage of the dry fat-free weight.
Mineral Analysis
The ashed MC III bones were prepared for mineral analysis by nitric acid wet digestion. Samples were transferred to Phillips beakers and the crucibles were rinsed with 20 mL of 14 M nitric acid (Omni Trace; EMD Chemicals, Inc., Gibbstown, NJ). Samples were digested in 30 mL of nitric acid with heat and then rehydrated to 100 mL with 0.5% nitric acid. Calcium concentrations were determined by flame atomic absorption spectrophotometry (Smith-Heiftje 4000; Thermo Jarrell Ash Corporation, Franklin, MA), and P concentrations were determined by the method of Gomori (1942) using a DU 7400 spectrophotometer (Beckman). Instrument accuracy for Ca and P analysis was maintained by simultaneous analysis of Bovine Liver Standard (NIST, Gaithersburg, MD). Bone mineral concentrations were calculated as percentages of dry fat-free weight.
Statistical Analysis
All data were analyzed by least squares ANOVA using the Proc Mixed procedures of SAS (SAS Inst. Inc., Cary, NC) for a randomized complete block design. Pig served as the experimental unit. The model included the fixed effects of the factorial treatments, their interaction, replication, and block by initial weight. Litter within replication was specified as a random effect. All means presented are least square means. Differences were considered significant at the level of P < 0.05.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Analysis of Diets
The full supplementation CSBM, full supplementation CSBM+WM, supplement withdrawal CSBM, and supplement withdrawal CSBM+WM diets were formulated to contain 0.58, 0.58, 0.29, and 0.45% Ca, and 0.49, 0.56, 0.38, and 0.52% total P, respectively (Table 3
). The analyzed mineral concentrations of the respective diets were 0.68, 0.67, 0.38, and 0.61% for Ca, and 0.45, 0.55, 0.34, and 0.52%, for total P. The resulting Ca to total P ratios were 1.51:1, 1.22:1, 1.12:1, and 1.17:1 in the respective diets. A suggested Ca to total P ratio for corn-soybean meal-based diets is between 1:1 and 1.25:1 (NRC, 1998).
As reported previously by Shaw et al. (2002), all late finishing diets provided Cu, Fe, Mn, and Zn in excess of requirements (NRC, 1998) with the exception of Zn in the CSBM withdrawal diet, which was approximately 30% below the stated requirement. Comparisons of these mineral contents to NRC (1998) were also made using analyzed values. The calculated Se concentration of the supplement withdrawal CSBM diet was 70% less than the NRC (1998) estimated requirement, but the supplement withdrawal CSBM+WM diet was calculated to be 187% of NRC (1998).
Bone Metabolism
Because initial osteocalcin and pyridinoline concentrations were statistically different between dietary treatments, the d 14 and 27 values were analyzed as deviations from the d 0 concentration. Other researchers also found that d 0 levels within groups varied significantly (Hoekstra et al., 1999; Hiney et al., 2004). Differences may be due to natural variation in bone metabolism within a population. By analyzing d 14 and 27 values as deviations from the d 0 concentration, we measured how each bone metabolism adapted to changes in dietary treatment. Supplement withdrawal, but not wheat middling inclusion, increased (P < 0.01) serum osteocalcin concentrations during the 27-d withdrawal period (Table 4). Similar responses have been noted in other swine studies. Carter et al. (1996) observed that serum osteocalcin concentrations decreased linearly as dietary Ca increased from 0.42 to 1.14% and dietary P increased from 0.35 to 0.95% over a 30-d period. In another 30-d trial, Eklou-Kalonji et al. (1999) found that reduction of dietary Ca from 0.90% to either 0.38 or 0.11% increased serum osteocalcin in growing and finishing pigs. Studies with rats also have concluded that serum osteocalcin concentrations are inversely correlated with dietary Ca, P content, or both (Lian et al., 1987; Tanimoto et al., 1991; Hämäläinen, 1994). A study done with young women on a short-term Ca-deficient diet also reported increased serum osteocalcin concentrations in the first 3 d of the diet (Akesson et al., 1998). Dietary deficiencies of Ca and P initiate bone resorption to meet the acute metabolic needs for those minerals. Thus, the pericellular matrix of many osteoblasts is weakened, and those cells experience increased mechanical loads. Osteoblasts respond by stimulating osteocalcin synthesis (Akhouayri et al., 1999). Additionally, osteoblast proliferation increases in rats during Ca repletion (Tanimoto et al., 1991). Thus, increased serum osteocalcin could be a reflection of increased cell numbers and not necessarily new bone formation (Akesson et al., 1998).
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found that decreasing dietary Ca from 0.86 to 0.39% for 8 wk did not alter serum osteocalcin in growing pigs. Also, Hillman et al. (1993) did not observe differences in serum osteocalcin when neonatal pigs were fed diets containing excess, adequate, or deficient Ca and P for 28 d. A major difference between these studies and ours is the age of the animals. Bone biological markers have been correlated with growth rate and age in humans (Spagnoli et al., 1996) and monkeys (Colman et al., 1999), respectively. Thus, younger animals have greater levels of bone bio-markers due to their rapid growth rate, which could make detecting dietary treatment differences more difficult (Bell et al., 2001). However, we were working with finishing pigs whose growth rates are slower relative to the neonatal and growing pigs. Thus, growth rate is not a confounding factor in our trials.
In our study, supplement withdrawal, but not wheat middling inclusion, had a greater effect (P < 0.05) on the change in serum pyridinoline concentration from d 0 to d 14 and 27 of the withdrawal period (Table 4). To our knowledge, we are one of the first groups to identify the relationship between serum pyridinoline and dietary mineral intake. However, studies with rats (Egger et al., 1994) and humans (Garnero et al., 1994; Shapses et al., 1995; Shen et al., 1995) reported that urinary pyridinium excretion was inversely proportional to dietary Ca concentration. Thus, our results are expected.
Bone Measurements
As shown in Table 5, supplement withdrawal decreased total bone mineral density, cortical bone mineral density, peak force, percent bone ash, and the Ca and P concentrations of the MC III (P < 0.04). These responses indicate that bone resorption exceeded bone formation. The increased bone resorption caused by supplement withdrawal is reflected by the change of serum pyridinoline from d 0 to 14 and from d 0 to 27 rather than the analyzed d 14 and 27 concentrations. Therefore, adjusting serum pyridinoline to reflect change over time seems to be an appropriate data modification.
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; Eklou-Kalonji et al., 1999) and P (Hall et al., 1991; Carter et al., 1996; O’Quinn et al., 1997) are moderately restricted. In addition to Ca and P, several other nutrients that contribute to bone quality were decreased in the supplement withdrawal diets. Dietary vitamin A (Zile et al., 1973), vitamin D (Sinha et al., 1988), and vitamin K (Knapen et al., 1993) are essential to maintain bone quality and were present in concentrations below the minimums suggested by the NRC (1998) in both of the supplement withdrawal diets. Dietary Zn is also essential (Miller et al., 1968) and was below the minimum suggested by the NRC (1998) in the supplement withdrawal CSBM diet but not in the supplement withdrawal CSBM+WM diet.
Supplement withdrawal decreased bone Ca and P concentrations when expressed as milligrams per gram of dry fat-free bone (Table 5). However, when bone Ca and P are calculated as concentration of bone ash, supplement withdrawal reduced percent Ca (43.70 ± 0.514 and 42.20 ± 0.514 for full supplementation and supplement withdrawal, respectively, P = 0.04) but did not affect percent P (17.43 ± 0.186 and 17.35 ± 0.186 for full supplementation and supplement withdrawal, respectively, P = 0.76). Crenshaw (2001) stated that Ca and P are deposited and reabsorbed in bone in a nearly constant 2.2:1 ratio. He further suggests that the Ca and P concentrations of bone ash do not change with extreme variation in dietary Ca and P. We do not have an explanation for why supplement withdrawal lowered Ca concentration of bone ash.
Bone Fractures
Although supplement withdrawal decreased metacarpal bone quality, no femoral or vertebral fractures were observed in this study. One pig, which received a fully supplemented CSBM diet, had a broken right tibia after hoisting by the chain.
Pond et al. (1969) reported that the vertebrae and the proximal and distal ends of the femur are depleted of mineral before the middiaphyseal region of the long bones in young pigs. Crenshaw et al. (1981) reported that decreasing bone quality in the metacarpals parallels decreasing bone quality of the vertebrae and femoral bones in growing and finishing pigs. Although no bone fractures were observed in the radiographic films, the decreased bone mineralization of the MC III indicates that the pigs were at an increased risk for vertebral and femoral fractures.
Dritz et al. (2000) described a case study in which a processor reported that the incidence of minor loin damage of pigs from a single farm was greater than twice that of pigs received from other producers. Most of the loin damage was reportedly caused by vertebral fractures that occurred during the stunning process. To correct the problem, the farm increased dietary available P from 4.4 to 5.7 g/d in pigs weighing 95 to 109 kg, and from 3.2 to 4.8 g/d in pigs weighing 109 kg to market weight. After a 2-mo transition period, the incidence of minor loin damage decreased to that of pigs from other producers.
In the current study, 31 pigs were fed supplement withdrawal CSBM and supplement withdrawal CSBM+WM diets providing 10.4 and 15.8 g of Ca daily, and 4.3 and 6.3 g of available P daily, respectively. Because wheat middlings are naturally high in P, only the 15 pigs fed CSBM supplement withdrawal diets received dietary Ca and P concentrations below the suggested NRC (1998) minimum requirements (13.8 and 4.6 g/d for Ca and available P, respectively). The available P concentration in the supplement withdrawal CSBM was only slightly less than the 4.8 g/d provided by Dritz et al. (2000), which as mentioned earlier, reduced the incidence of vertebral fractures. In a similar comparison, the supplement withdrawal CSBM+WM diet used in the current study provided about 30% more available P. Neither supplement withdrawal diet was preceded by diets containing less available P than suggested by NRC (1998). Assuming that the industry average for loin damage caused by vertebral fractures approaches the 0.58% reported by Dritz et al. (2000), it is not surprising that we did not observe vertebral and femoral fractures with supplement withdrawal in the current study. Furthermore, livestock handling practices at the university relative to commercial operations likely decreased the probability of observing bone fractures. Our animals were individually penned until slaughter, placed in pens with more than 60% solid concrete flooring, loaded into low elevation trailers without the use of ramps, allowed approximately 2 m2 space per animal during transport, transported for 15 min, and slaughtered at a rate of less than 5 animals per hour. In contrast, animals slaughtered in commercial operations are commonly housed in groups of 25 or more, placed in pens with 100% slotted flooring, loaded into elevated trucks using ramps, allowed about 0.35 m2 space per animal during transport, transported for several hours, and slaughtered at a rate of hundreds of pigs per hour.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Current address: Murphy-Brown LLC, Warsaw, NC 28466.
3 Corresponding author: rozeboom{at}msu.edu
Received for publication March 28, 2005. Accepted for publication December 6, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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