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© 2007 American Society for Nutrition J. Nutr. 137:1795-1801, July 2007

Nutrient Requirements and Optimal Nutrition

Supplemental Escherichia coli Phytase and Strontium Enhance Bone Strength of Young Pigs Fed a Phosphorus-Adequate Diet1,2

Angela R. Pagano3, Koji Yasuda3, Karl R. Roneker3, Thomas D. Crenshaw4 and Xin Gen Lei3,*

3 Department of Animal Science, Cornell University, Ithaca, NY 14853 and 4 Department of Animal Sciences, University of Wisconsin, Madison, WI 53706

* To whom correspondence should be addressed. E-mail: xl20{at}cornell.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Young pigs represent an excellent model of youth to assess potentials of dietary factors for improving bone structure and function. We conducted 2 experiments to determine whether adding microbial phytase (2,000 U/kg, OptiPhos, JBS United) and Sr (50 mg/kg, SrCO3 Alfa Aesar) into a P-adequate diet further improved bone strength of young pigs. In Expt. 1, 24 gilts (8.6 ± 0.1 kg body wt) were divided into 2 groups (n = 12), and fed a corn-soybean–meal basal diet (BD, 0.33% available P) or BD + phytase for 6 wk. In Expt. 2, 32 pigs (11.4 ± 0.2 kg) were divided into 4 groups (n = 8), and fed BD, BD + phytase, BD + Sr, or BD + phytase and Sr for 5 wk. Both supplemental phytase and Sr enhanced (P < 0.05) breaking strengths (11–20%), mineral content (6–15%), and mineral density (6–11%) of metatarsals and femurs. Supplemental phytase also resulted in larger total bone areas (P < 0.05) and a larger cross-sectional area of femur (P = 0.06). Concentrations of Sr were elevated 4-fold (P < 0.001) in both bones by Sr, and moderately increased (P = 0.05–0.07) in metatarsal by phytase. In conclusion, supplemental phytase at 2000 U/kg of P-adequate diets enhanced bone mechanical function of weanling pigs by modulating both geometrical and chemical properties of bone. The similar benefit of supplemental Sr was mainly due to an effect on bone chemical properties.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
As a primary concern for human health, bone fractures are associated with poor bone mineralization during growth spurts and increased risk of osteoporosis in elderly (1,2). Osteoporosis causes bone fractures by lowering bone mass and deteriorating bone architecture (3). Hip fractures alone are estimated to reach >6 million cases a year by 2050 (4). Although osteoporotic fractures occur mostly in people >70 y of age (5), enhancing peak bone mass early in life may postpone or prevent the incidents (6,7).

Mostly, only surrogates are available for in vivo bone property and function assessments in humans (7). Thus, animal models offer an advantage for the actual measurements of biophysical characteristics and chemical compositions of bones. Among several small and large animal models (8), canine and porcine bones resemble human bones in many features including density and stress-fracture properties (9). Because of the implications of estrogen in the occurrence of osteoporosis for women (10) and similarities of the pig estrus cycle to the human menstrual cycle (11), pigs seems to be a better model than dogs for human osteoporosis research.

Microbial phytase has been widely used during the past decade as a feed additive for swine to enhance utilization of phytate-P from plant feeds (12). Many studies have shown the effectiveness of the enzyme in replacing inorganic P supplementation to support normal growth performance and bone strength of pigs fed low-P diets (13,14). The enzyme releases P and other chelated elements including Ca, Fe, Zn, Mn, and Cu for absorption in the gastrointestinal tract, allowing for the possible incorporation of these elements into bone (15,16). A few experiments (17,18) have shown the potential benefits of dietary phytase to bone properties in pigs fed P-adequate diets. Because these experiments were conducted to optimize growth and production responses of pigs, data on bone responses of pigs from these studies offered limited implications for human bone health issues.

As young pigs represent an excellent model of bone mass and strength for humans (6,7,9,19), our initial objective was to determine whether a high level of phytase supplementation (2000 U/kg feed) in a P-adequate diet exerted any beneficial effect on bone strength of weanling pigs. The initial results showed a positive effect of supplemental phytase and also a possible involvement of strontium (Sr) accumulation in response to phytase. By uncoupling bone resorption and accretion, Sr was able to decrease the resorption rate and enhance the accretion rate (20). However, the element has been studied only in adults for osteoporosis treatment (21–23). Thus, another experiment was conducted with a 2 x 2 factorial arrangement of treatments [2 levels of phytase (0 and 2000 U/kg of feed) and 2 levels of Sr (0 and 50 mg/kg of feed)] to determine whether dietary phytase could be used additively with Sr to improve bone strength of young pigs fed P-adequate diets.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Animals, diets, and treatments. Our protocol was approved by the Institutional Animal Care and Use Committee of Cornell University. All pigs used in the study were weanling crossbreds (Landrace- Hampshire-Duroc) selected from the Cornell University Swine Farm. Pigs were weaned at 4 wk of age, and allotted into treatment groups based on body weight, litter, and sex. Two preliminary experiments using 56 gilts (5-wk–old) fed different levels of inorganic P concentration (0, 0.2, and 0.25%) and phytase activity (0, 1000, and 2000 U/kg), were conducted for 4 or 5 wk to determine the appropriate dietary inorganic P concentration and phytase activity for the experimental objectives. Based on results of the preliminary trials, Expt. 1 was conducted with 24 gilts (6-wk–old, 8.6 ± 0.1 kg body wt) to test the possible benefit of phytase to metatarsal breaking load of pigs fed the corn-soybean–meal basal diet (BD)5 (Table 1). The BD contained 0.33% available P and adequate concentrations of all other required nutrients (24). The selected pigs were divided into 2 groups (n = 12) and were fed BD or BD + phytase at 2000 U/kg for 6 wk. Subsequently, Expt. 2 was conducted with 32 pigs (8-wk–old, 11.4 ± 0.2 kg body wt) to determine the possible additive or synergistic effects of supplemental phytase and Sr on mechanical and chemical properties of metatarsal and femur bones. The pigs were divided into 4 groups (n = 8) and were fed BD, BD + phytase (2000 U/kg), BD + Sr (50 mg/kg) or BD + phytase (2000 U/kg) + Sr (50 mg/kg). The phytase used in both experiments was Escherichia coli AppA2 (OptiPhos, JBS United). After the actual activity was analyzed (25), the phytase enzyme was added to the diets at feed mixing. Strontium was added to the diet in the form of SrCO3 (Alfa Aesar). Pigs were penned in an environmentally controlled barn (20–25°C; 12 h light:12 h dark cycle), and were allowed free access to feed and water.


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  		TABLE 1 Composition of the basal diet1

 
    Growth performance and sample collection. In both experiments, the body weight of individual pigs was measured weekly. Blood samples of individual pigs were collected, at wk 0 and then weekly, from the anterior vena cava into heparinized tubes after an overnight fast (8 h). The collected whole blood samples were chilled on ice and centrifuged at 3000 x g for 10 min at 4°C (GS-6KR centrifuge, Beckman Instruments) to prepare plasma for assays of inorganic P concentrations and alkaline phosphatase activity. At the end of Expts. 1 and 2, 8 and 6 pigs from each treatment group were killed by electrical stunning and exsanguinations, respectively. Rear legs were amputated and stored on ice at 4°C until the femur and/or 3rd and 4th metatarsals were isolated for mechanical and/or image tests (see below). Following strength tests, bones were stored at –20°C for mineral analysis.

    Plasma biochemical analyses. After being deproteinated with 12.5% tricholoacetic acid, plasma samples were assayed for inorganic P concentrations using Elon (p-methylaminophenol sulfate) solution (26). The hydrolysis of p-nitrophenol phosphate to p-nitrophenol was used to measure plasma alkaline phosphatase activity (27). The enzyme unit was defined as 1 µmol of p-nitrophenol released per minute at 30°C.

    Bone geometrical and strength analyses. Third and 4th metatarsals in both experiments and femur (right leg) in Expt. 2 were prepared by manually removing surrounding skin, muscle, and other tissues. Bones were stored in closed plastic bags at 4°C until strength analysis. Maximal breaking load was measured using an Instron 4500 Machine at room temperature (23°C) by subjecting each bone to a 3-point bending test (28). During mechanical tests, force was applied to the center of the bone held by supports 2.0 cm apart for metatarsals and 3.3 cm apart for femur. The crosshead speed was set at 50 mm/min and the sample rate was 10 points/s. Final strength was determined from load-displacement curves indicating the maximum loads. The metatarsal breaking strength was expressed as the mean strength of 4 bones from both left and right feet of pigs in Expt. 1, and as the mean strength of the 2 bones from the right foot of pigs in Expt. 2.

In Expt. 2, bone mineral content (BMC, g) and bone mineral density (BMD, g/cm2) of the 3rd and 4th metatarsals from the left foot and the femur from the left leg of each pig were measured. After thawing to room temperature, entire bones were placed on a rice bag (to remove background effects) and scanned by dual energy X-ray absorptiometry using the GE Lunar Prodigy instrument (GE Lunar, Prodigy) in the small animal scan mode. Values of BMC and BMD were predicted by analysis of scans using Prodigy software (version 10.10.038).

Because the metatarsal bones do not have a clearly defined cortical bone wall, geometric and image measurements were made only in femur bones of the left leg in Expt. 2. A 3-point bending test was conducted on the dissected bones to generate load-deformation curves with an Instron Model 5566. Mechanical properties of bones were calculated using a formula as previously described (29). Bone cross sections were cut at the midpoint of loading and used to determine area moment of inertia. Cross sections were submersed for 5 min in a 0.4 mol/L sodium hypochlorite solution to remove periosteum and marrow tissue and then embedded in blue clay (Play-Doh, Hasbro) to prevent depth-of-field distortions and to enhance contrasts. The embedded sections were scanned with a flatbed scanner (Epson Perfection Model 3490) and analyzed using Image J software (30) to measure the x-y coordinates of bone pixels.

    Bone mineral concentration analyses. After the breaking strength analysis, samples (~100–200 mg) of metatarsals in both experiments, and the femur in Expt. 2 from the right legs, were used for mineral analysis. Cortical bones were isolated after removing attached connective tissue with a stainless steel scalpel and collecting individual shards using needle-nosed pliers with plastic-covered clamps. The samples were dried for 8 h at 105°C to measure dry weight. Concentrations of individual elements in the dried bone samples were measured using inductively coupled argon plasma spectrophotometer (ICAP 61E Trace Analyzer, Thermo Jarell Ash corporation) (31). Samples were digested in a mixture of HNO3 and HClO4 (9:1, v:v), and then diluted in 5% HNO3 before analysis. Standard reference materials (1573a, tomato leaves, and 1577b, bovine liver, National Institute of Standards and Technology) were used to validate the analytical procedures (32).

    Statistical analyses. Data were analyzed as a randomized block design using the General Linear Models procedure of SAS, version 6.12 (SAS Institute). Main effects of dietary treatments on various measures were analyzed using 1-way ANOVA in Expt. 1 and a 2 x 2 factorial ANOVA in Expt. 2. Each individual pig was used as the experimental unit. The Boneferroni t test was used to compare treatment means, and the significance level was set at P ≤ 0.05 (33). For the repeated-measured traits, including body weights, plasma inorganic P concentrations, and plasma alkaline phosphatase activity, only the data from the initial and final weeks were presented because of the similar trends at other times.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Expt. 1. Pigs fed BD + 2000 U/kg had 12% greater (P < 0.02) breaking strength of metatarsals (98.8 ± 3.2) than those fed only BD (112.0 ± 3.9 kg). These phytase-fed pigs had 7% (P < 0.05) higher Sr concentrations, but similar concentrations of other elements in the metatarsals, compared with the pigs fed BD (Table 2). These 2 groups of pigs had similar body weight (32.1 ± 0.6 vs. 33.4 ± 0.8 kg), plasma inorganic P concentration (81.7 ± 0.1 vs. 83.6 ± 0.1 g/L), and plasma alkaline phosphatase activity (161.7 ± 8.6 vs. 173.0 ± 6.8 U/L) at the end of the experiment.


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  		TABLE 2 Effect of dietary supplemental phytase on metatarsal mineral concentrations of pigs in Expt. 11

 
    Expt. 2. Although the final body weight, plasma inorganic P concentration, or plasma alkaline phosphatase activity of pigs did not differ among dietary treatment groups (Table 3), the breaking strengths of metatarsals (Fig. 1A) and femur (Fig. 1B) were elevated by supplemental phytase (P < 0.005) and Sr (P < 0.02), respectively. The improvement in metatarsal and femur strength was 16 and 20% by phytase and 11 and 10% by Sr, respectively. In both bones, BMC was enhanced 14–15% by phytase (P < 0.001), and 6–8% by Sr (P < 0.04) (Fig. 2). Meanwhile, BMD of the 2 bones was elevated 9–11% by phytase (P < 0.001) and 6% by Sr (P < 0.01). Total bone area (calculated by length x width, based on dual energy X-ray absorptiometry) of metatarsal (8.4 ± 0.1 vs. 9.0 ± 0.2 mm2) and femur (43.6 ± 0.6 vs. 45.8 ± 0.7 mm2) was increased (P < 0.05) 7 and 5%, respectively, by phytase. However, there was no interaction between phytase and Sr on the breaking strength, BMC, or BMD of either bone.


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  		TABLE 3 Effect of dietary supplemental phytase and strontium on growth performance and plasma measures of pigs in Expt. 21

 

Figure 1
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  		FIGURE 1  Effects of dietary supplemental phytase (Phy) and strontium (Sr) on metatarsal (A) and femur (B) breaking strength of pigs in Expt. 2. Values are means ± SE, n = 6. Means without a common letter differ, P < 0.05; *means tended to differ, P = 0.06.

 

Figure 2
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  		FIGURE 2  Effects of dietary supplemental phytase (Phy) and strontium (Sr) on bone mineral content (BMC, upper panel) and bone mineral density (BMD, lower panel) of metatarsal (A) and femur (B) of pigs in Expt. 2. Values are means ± SEM, n = 6. Means without a common letter differ, P < 0.05.

 
The cross-sectional area of femur from pigs fed phytase was 11% (P = 0.06) larger than that of pigs fed diets without phytase (Table 4). Supplemental phytase also tended to enhance cross-sectional area moment of inertia, particularly in pigs fed the diet without supplemental Sr, but the main effect of phytase or its interaction with Sr was not significant. The radius (distance from the neutral axis to maximum outer surface) of femur was not affected by supplemental phytase or Sr. The yield-bending moment of femur was enhanced 19% (P < 0.05) by supplemental phytase. No treatment differences were detected in the stress or strain values at either the yield or ultimate points on the load-deformation curves. There was no difference in modulus of elasticity (a measure of the rigidity of bone) among treatment groups. The interaction between supplemental phytase and Sr on maximal stress of femur was marginally significant (P = 0.09).


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  		TABLE 4 Effect of dietary supplemental phytase and strontium on femur geometrical and strength properties of pigs in Expt. 21

 
Metatarsal concentration of Sr was elevated 4-fold (P < 0.0001) by Sr and 11% (P < 0.07) by phytase (Table 5). Meanwhile, supplemental phytase reduced metatarsal concentrations of S (P < 0.01) and Cr (P < 0.05). Supplemental Sr tended (P = 0.09 to 0.10) to reduce metatarsal concentrations of Ca, P, and Fe, especially in pigs fed phytase. However, the interaction between Sr and phytase on either element was not significant (P = 0.13 to 0.65). There was a marginally significant interaction between phytase and Sr (P = 0.08) on metatarsal concentration of Na. Femur concentration of Sr was enhanced 3.9-fold (P < 0.0001) by Sr, but was not affected by phytase (Table 6). Femur concentration of S was decreased (P < 0.01) whereas that of Zn was increased (P < 0.05) by phytase. Supplemental Sr tended to reduce (P = 0.07) femur concentrations of Fe, Cr, and Mn.


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  		TABLE 5 Effect of dietary supplemental phytase and strontium on metatarsal mineral concentrations of pigs in Expt. 21

 

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  		TABLE 6 Effect of dietary supplemental phytase and strontium on femur mineral concentrations of pigs in Expt. 21

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The results of Expt. 1 clearly demonstrate that supplementing Escherichia coli AppA2 phytase at 2000 U/kg of diet enhanced breaking strength of metatarsals in P-adequate pigs. Although supplemental moderate levels of phytase activity were shown to effectively replace inorganic P in pigs fed the low-P diets (34–36), our findings illustrate a novel function of phytase when added at a relatively high level to diets with more than adequate concentrations of inorganic P. Because the final status of all assayed P-responsive measures (37,38), including metatarsal concentrations of P and Ca, was very similar between pigs fed only BD and BD + phytase diets, the benefit of supplemental phytase to metatarsal breaking strength was unlikely a response to further improvements in P availability.

The 7% elevation of metatarsal Sr concentration in pigs fed BD + phytase over those fed only BD in Expt. 1 prompted us to conduct Expt. 2 to examine a plausible synergistic interaction between phytase and Sr on bone metabolism. Indeed, the breaking strengths of metatarsals and femur were improved by both supplements. Because pig bones do not meet the length:diameter ratios needed for pure bending (compression and tension forces), the 3-point bending test, used to measure breaking strength of entire bones in both experiments, probably involved combined shear and tensile failure modes (28). Nevertheless, the results allow a relative comparison between dietary treatment groups. Further characterizations of entire femur mineral content by dual energy X-ray absorptiometry, along with geometrical analysis of cross sections and mechanical tests, helped reveal the mode of action for the 2 supplements. The enhancements of BMC and BMD in metatarsals and femur by both phytase and Sr indicate their common ability to shift bone chemical profiles. In contrast, only supplemental phytase, but not Sr, increased total bone surface areas of both bones and the cross-sectional area at the midshaft region of femur. Thus, there is a distinct difference between the 2 supplements in altering femur geometrical properties or anatomical structures. The larger cross-sectional area, along with the seemingly elevated cross-sectional area moment of inertia, allowed the femur of pigs fed phytase to withstand more force (greater yield-bending moment) than those of pigs fed diets without phytase. However, the stress and strain values at each point of bone, or the rigidity of bone, was not altered by either phytase or Sr.

The lack of significant interactions between phytase and Sr on bone geometrical or strength properties indicates fairly independent actions for the 2 supplements. In. Expt. 2, supplemental Sr resulted in a rather consistent elevation (4-fold) in Sr concentrations of cortical metatarsal and femur. In comparison, supplemental phytase caused a moderate elevation (11%) of Sr concentration in only metatarsals. Thus, the benefit of supplemental phytase to the properties of femur was not necessarily related to the enhanced Sr deposition in the cortical bone. Although mineral concentrations expressed on a dry bone basis may fluctuate with fat content, that source of variation unlikely exerted major impact on our results because Ca:P ratios in metatarsal or femur were essentially identical across the dietary treatment groups. Whereas supplemental phytase produced an inconsistent effect on Zn and Cr concentrations of metatarsals and femur, it consistently reduced S concentrations in both bones in Expt. 2. This raises the fascinating question of whether the enzyme improved bone metabolism of P-adequate pigs via modulating S incorporation and distribution. Copious amounts of S represent a structural component of proteoglycans in bone (39), and thus are involved in bone formation and repairing (40,41). The trend of decreasing femur concentrations of Fe, Cr, and Mn, as well as metatarsal concentrations of Ca, P, and Fe in Expt. 2, may not simply be explained by displacement of increased Sr deposition (42–44). In fact, the reduction in Ca and P concentrations was a magnitude greater than the increase in Sr concentration. Although the Sr-related reduction of metatarsal Ca and P concentrations was apparent in pigs fed phytase, which exhibited the best responses of bone properties, the observed enhancement of bone strength might be caused mainly by the changes of Sr per se. Because Sr can be enriched to high concentrations in cancellous bones (42) to stimulate bone remodeling (45) and cartilage matrix formation (46), we are now conducting active research in our laboratory to test whether a preferential accumulation of Sr in cancellous bones serves as the mechanism for the observed bone improvements by phytase or Sr (23,47).

The positive effect of low-level Sr supplementation on bone-breaking strength and material properties in Expt. 2 extends our knowledge of this element on bone metabolism. Because of its adverse effects on other minerals (48), Sr was abandoned as a therapeutic agent for osteoporosis. Interest in Sr as an osteopenic treatment was renewed with the realization that low doses of Sr [≤0.35 g Sr/(kg of body wt · d)] exerted no negative effects in the presence of adequate Ca intake (22). Overall, studies in rodents, monkeys, and humans indicate that low doses of Sr inhibit bone resorption and/or stimulate bone formation (42). In our study, supplemental Sr at 50 mg/kg of feed represented an mean intake of 3 mg Sr/(kg of body wt · d). Apparently, this dose of Sr was safe (22) and caused no obvious adverse responses. In summary, our research illustrates that high levels of bacterial phytase and low levels of inorganic Sr improve the bone-breaking strength of pigs fed adequate inorganic P. The former appeared to affect both geometrical and chemical properties of bone, whereas the latter mainly altered chemical properties of bones. Future research should attempt to elucidate the biochemical mechanisms for the benefits of these 2 supplements.


   ACKNOWLEDGMENTS
 
We thank Mike Rutzke of the USDA for help in mineral analysis; James Bartsch for instruction and the use of the Instron machine; and Taewan Kim, Taegu Ko, Carol A. Roneker, Michael S. Scimeca, Erin B. Peterson, and Jeremy D. Weaver for help in animal care and sample collection.


   FOOTNOTES
 
1 This project was supported by New York State and the Cornell University Center for Biotechnology, a NYSTAR Designated Center for Advanced Technology. Back

2 Author disclosures: A. R. Pagano, K. Yasuda, K. R. Roneker, T. D. Crenshaw, and X. G. Lei, no conflicts of interest. Back

5 Abbreviations used: BD, basal diet; BMC, bone mineral content; BMD, bone mineral density. Back

Manuscript received 1 March 2007. Initial review completed 9 April 2007. Revision accepted 23 April 2007.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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