J. Anim. Sci. 2003. 81:2751-2757
© 2003 American Society of Animal Science
Effectiveness of an experimental consensus phytase in improving dietary phytate-phosphorus utilization by weanling pigs1
J. M. Gentile,
K. R. Roneker,
S. E. Crowe,
W. G. Pond and
X. G. Lei2
Department of Animal Science, Cornell University, Ithaca, NY 14853
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Abstract
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Consensus phytase is a new biosynthetic, heat-stable enzyme derived from the sequences of multiple homologous phytases. Two experiments were conducted to determine its effectiveness, relative to inorganic P and a mutant enzyme of Escherichia coli phytase (Mutant-EP), in improving dietary phytate-P availability to pigs. In Exp. 1, 36 pigs (3 wk old, 7.00 ± 0.24 kg of BW) were fed a low-P corn-soybean meal basal diet plus consensus phytase at 0, 250, 500, 750, 1,000, or 1,250 U/kg of feed for 5 wk. Plasma inorganic P concentration, plasma alkaline phosphatase activity, bone strength, and overall ADG and gain:feed ratio of pigs were improved (P < 0.05) by consensus phytase in both linear (R2 = 0.20 to 0.70) and quadratic (R2 = 0.30 to 0.70) dose-dependent fashions. In Exp. 2, 36 pigs (4 wk old, 9.61 ± 0.52 kg BW) were fed the basal diet + inorganic P at 0.1 or 0.2%, consensus phytase at 750 or 450 U/kg of feed, Mutant-EP at 450 U/kg of feed, or 225 U consensus + 225 U Mutant-EP/kg of feed. Pigs fed 750 U of consensus phytase or 450 U of Mutant-EP/kg feed had plasma inorganic concentrations and bone strength that fell between those of pigs fed 0.1 or 0.2% inorganic P. These two measures were 16 to 29% lower (P < 0.05) in pigs fed 450 U of consensus phytase/kg of feed than those of pigs fed 0.2% inorganic P. Plasma inorganic P concentrations were 14 to 29% higher (P < 0.05) in pigs fed Mutant-EP vs. consensus phytase at 450 U/kg at wk 2 and 3. In conclusion, the experimental consensus phytase effectively releases phytate P from the corn–soy diet for weanling pigs. The inorganic P equivalent of 750 U of consensus phytase/kg of feed may fall between 0.1 and 0.2%, but this requires further determination.
Key Words: Alkaline Phosphatase • Bone Strength • Consensus • Escherchia coli Phytase • Phosphorus • Pigs
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Introduction
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Effectiveness of supplemental microbial phytases in diets for swine in improving phytate P bioavailability and reducing manure P excretion has been demonstrated by many research groups around the world (Simons et al., 1990
; Cromwell et al., 1993; Young et al., 1993). One major limitation of currently available commercial phytases in animal feed is their inability to withstand the heat denaturation (60 to 80°C) involved in feed pelleting. To overcome this limitation, Lehmann et al. (2000) have used a novel consensus approach and have developed a synthetic phytase named "experimental consensus phytase," based on the homologous sequences of 13 known fungal phytases. Whereas the naturally occurring phytases have temperature optima between 45 and 55°C, the consensus phytase has a temperature optimum at 71°C. The unfolding temperature, at which the enzyme melts and becomes denatured, is between 56 to 63°C for all known phytases, but is 78°C for the consensus phytase (Lehmann et al., 2000). In addition, the catalytic activity and pH optimum of consensus phytase are comparable with those of Aspergillus fumigatis and Emericella nidulans phytases at 37°C (Lehmann et al., 2000). Apparently, consensus phytase has tremendous potential to become a heat-stable and catalytically efficient enzyme for use in heat-processed (e.g., pelleted) animal feed. Therefore, its effectiveness in releasing phytate P from typical diets for swine needs to be assessed. Our objectives of this study were to determine the dose-dependent effectiveness of consensus phytase in a corn-soybean meal diet for weanling pigs and its efficacy relative to that of inorganic P or a mutant enzyme of the Escherichia coli phytase (Mutant-EP) (Rodriguez et al., 2000) that was developed in our laboratory, as well as possible synergistic effects between these two phytases.
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Materials and Methods
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Phytases
Consensus phytase was used in granular form and was provided by Roche Vitamins Ltd. (Basel, Switzerland). The Mutant-EP was produced in a yeast system from an E. coli phytase variant gene prepared in our laboratory (Rodriguez et al., 2000). The actual activities of consensus phytase and Mutant-EP phytase used for this study were 3,250 and 4,500 U/g by analysis, respectively. To make their activity comparable and practically relevant, we determined phytase activity of both enzymes by the release of inorganic P from sodium phytate in 0.2 M citrate buffer, pH 5.5, at 37°C (Han et al., 1998). One unit of phytase was defined as the amount of enzyme that releases 1 µmol of inorganic P per minute from sodium phytate under the assay conditions. The released inorganic P was measured based on the formation and reduction of a phosphomolybdate complex colorimetrically at 820 nm (DU 640 Spectrophotometer, Beckman Instruments Inc., Fullerton, CA).
Animals, Diets, and Measures
Our protocols were approved by the Institutional Animal Care and Use Committee of Cornell University. A total of 72 weanling gilts (Duroc-Landrace-Yorkshire cross) was selected from the Cornell University Swine Farm, and placed into treatment groups based on litter, BW, and plasma inorganic P concentrations. In Exp. 1, 36 weanling pigs (3 wk old, 7.00 ± 0.24 kg of BW) were allotted into six treatment groups (n = 6) to determine the dose effects of dietary consensus phytase. Each group was assigned to a diet containing 0; 250; 500; 750; 1,000; or 1,250 U/kg of feed. In Exp. 2, 36 weanling pigs (4 wk old, 9.61 ± 0.52 kg of BW) were allotted into six treatment groups (n = 6) to compare relative efficacy of consensus phytase to inorganic P and Mutant-EP, and to check for a possible synergistic effect between the two phytases. Each group was assigned to a diet containing inorganic P at 0.1 or 0.2% (designated as 0.1% P and 0.2% P, respectively), consensus phytase at 450 or 750 U/kg of feed, Mutant-EP at 450 U/kg of feed or the two phytases each at 225 U/kg of feed. The duration for Exp. 1 and 2 was 5 and 4 wk, respectively. Prior to the beginning of both experiments, all pigs were fed the basal diet (BD, Table 1) to adjust for 3 d. The BD contained adequate levels of all nutrients (NRC, 1998), except that no inorganic P was supplemented and Ca level was reduced (Lei et al., 1994). Within each experiment, all phytase-supplemented diets were prepared from the same batch of BD. In Exp. 2, the two inorganic P supplemented diets were made from the same BD, with minor adjustments in corn and soybean meal contents for the elevated inorganic P and Ca levels. The ratios of Ca:Ptotal in all experimental diets were close to 1.2:1 (Lei et al., 1994; Liu et al., 1998). All pigs were housed in individual pens in an environmentally controlled barn (23 to 25°C; 12-light:12-dark cycle) and allowed free access to feed and water. Feed waste was collected daily, and BW of pigs was measured weekly for calculation of ADG, ADFI, and gain:feed ratio. Blood samples of all individual pigs (fasted for 8 h, from 2200 to 0600) were collected weekly from the anterior vena cava into heparinized syringes for the assay of plasma inorganic P concentrations and alkaline phosphatase activity. At the end of both studies, the pigs were killed to collect the third and fourth metacarpals and metatarsals to determine bone strength.
Biochemical Analysis
Plasma was prepared by spinning chilled whole blood samples for 10 min at 4°C and 3,000 x g (GS-6KR Centrifuge, Beckman Instruments Inc.). After being deproteinated with 12.5% tricholoacetic acid, plasma inorganic P concentration was determined using Elon (p-methylaminophenol sulfate) solution (Gomori, 1942). Plasma alkaline phosphatase activity was assayed by the hydrolysis of p-nitrophenol phosphate to p-nitrophenol (Bowers and McComb, 1966). The enzyme unit was defined as 1 µmol of p-nitrophenol released from p-nitrophenol phosphate per minute at 30°C.
Bone Strength
The collected third and fourth metacarpals and metatarsals were prepared by removing the skin, muscle, connective tissue, tendons, and the other nearby bones and stored in closed plastic bags at 4°C (<7 d) before the strength analysis. The bone strength was determined at room temperature (23°C) by subjecting each bone to a three-point bending test (Turner and Burr, 1993) using the Instron machine (model 4502, Instron, Canton, MA). During the testing, the crosshead speed was set at 50.0 mm/min, and the two supports were spaced 2 cm apart. From each test, a load-displacement curve was generated, which indicated the maximum load at the breaking point of each bone. Actual values were expressed as the means of the third and fourth metacarpals or metatarsals or the overall averages of all four bones.
Statistical Analyses
All data were processed using SAS (SAS Inst., Inc., Cary, NC). In Exp. 1, dose effects of dietary consensus phytase activity on weekly and/or overall growth performance, plasma inorganic P concentration, and alkaline phosphatase activity, and bone strength were evaluated by polynomial evaluations. In Exp. 2, growth performance and plasma data were analyzed using ANOVA with time-repeated measurements (Gill, 1986). The bone data were analyzed by ANOVA. The individually penned pig was used as the experimental unit. The Bonferroni t-test was used to compare treatment means. The significance level was set at P < 0.05.
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Results
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Experiment 1
The overall ADG of pigs responded to dietary consensus phytase doses in both linear (P < 0.001, R2 = 0.29) and quadratic fashions (P < 0.001, R2 = 0.49; Table 2). Likewise, effects of the phytase doses on the overall gain:feed ratio of pigs fit linear (P < 0.001, R2 = 0.29) and quadratic (P < 0.001, R2 = 0.40) polynomial equations. However, the linear response of the overall ADFI of pigs to the dietary phytase doses was fairly weak (P = 0.06, R2 = 0.10), and the quadratic response was moderate (P < 0.03, R2 = 0.20). Based on the quadratic polynomial equations, the predicted dietary consensus phytase activities for the maximal ADG, ADFI, and gain/feed ratio were 875, 868, and 913 U/kg of feed, respectively. On the weekly basis, the linear or quadratic effects of the consensus phytase doses on these three growth performance measures were similar to those with the aforementioned overall averages (data not shown).
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Table 2. Effects of dietary supplemental consensus phytase on overall growth performance of pigs in Experiment 1a
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Initial plasma inorganic P concentrations were similar among the six treatment groups (Figure 1). At the end of the trial (wk 5), plasma inorganic P concentrations were a linear (P < 0.001, R2 = 0.69) function of dietary consensus phytase doses. Their relationship also fit a quadratic polynomial (P < 0.001, R2 = 0.70). The linear and quadratic effects (P < 0.05) of the phytase doses were relatively stronger at wk 3 and 4 (R2 = 0.50 to 0.70) than at wk 1 and 2 (R2 = 0.3 to 0.4) (data not shown). Based on the weekly quadratic polynomial equations, the average dietary consensus phytase activity predicated for the maximal plasma inorganic P concentration was 1,497 U/kg of feed. In contrast, plasma alkaline phosphatase activity was inversely correlated with dietary phytase doses, and the relationship (P < 0.001) fit linear (R2 = 0.40) and quadratic (R2 = 0.50) polynomial equations at wk 5 (Figure 2). The dietary treatment had no (P = 0.24 to 0.46) linear or quadratic (R2 = 0.02 to 0.08) effect on plasma alkaline phosphatase activity at wk 1 or 2, but showed weak or moderate (P < 0.03, R2 = 0.20 to 0.41) effects at wk 3 and 4 (data not shown). The estimated average dietary consensus phytase activity for the lowest plasma alkaline phosphatase activity was 1,094 U/kg of feed. The average breaking strength of the third and the fourth metacarpals or metatarsals as well as the mean strength of both bones responded to dietary phytase virtually in a nearly identical linear fashion (P < 0.001, R2 = 0.53 to 0.57, Figure 3). The responses could also be described by quadratic polynomial equations (P < 0.001, R2 = 0.55 to 0.59), and the maximal mean strength of both bones was predicated to occur at 1,559 U/kg of feed.
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Figure 1. Effects of supplemental dietary consensus phytase activity on plasma inorganic P concentrations of pigs in Exp. 1. Values are means of six individually penned pigs. The overall dose response at wk 5 fit linear (P < 0.001, R2 = 0.70) and quadratic (P < 0.001, R2 = 0.70) polynomial equations.
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Figure 2. Effects of supplemental dietary consensus phytase activity on plasma alkaline phosphatase activity of pigs in Exp. 1. Values are means of six individually penned pigs. The overall dose response at wk 5 fit linear (P < 0.001, R2 = 0.41) and quadratic (P < 0.001, R2 = 0.46) polynomial equations.
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Figure 3. Effects of supplemental dietary consensus phytase activity on the average breaking strength of the third and the fourth metacarpals or metatarsals and the overall average strength of the four bones of pigs in Exp. 1. Values are means of six individually penned pigs for the 0 U/kg feed group, and four pigs for the other groups. The overall dose responses of both bones and their average fit linear and quadratic polynomial equations (P < 0.001; R2 = 0.53 to 0.59).
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Experiment 2
There was no significant main effect of dietary treatments on ADG (P = 0.92), ADFI (P = 0.91), or gain:feed ratio (P = 0.44) (Table 3). Pigs fed 0.1% P and 450 U of consensus phytase/kg feed had 25 and 26% lower (P < 0.05) average bone strength of the third and the fourth metacarpals and metatarsals than that of pig fed 0.2% P, respectively. The other three groups of pigs had intermediate bone strengths that were not different (P = 0.10 to 0.84) from those of pigs fed 0.1 or 0.2% P. Separate analyses of metacarpals or metatarsals gave results similar to the means of both bones (data not shown).
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Table 3. Effects of dietary supplemental inorganic P, consensus phytase, and Mutant-EP alone or in combination on overall growth performance and bone strength of pigs in Experiment 2a,b
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At initial, plasma inorganic P concentrations of all six groups of pigs were similar (Figure 4). Throughout the experiment, pigs fed 450 U of consensus phytase/kg feed had 16 to 29% lower (P < 0.05) plasma inorganic P concentrations than those of pigs fed 0.2% P. The concentrations in pigs fed 750 U consensus phytase or 450 U of Mutant-EP/kg feed were not different from those fed 0.1 or 0.2% P (P = 0.10 to 0.99). At wk 2 and 3, plasma inorganic P concentrations were 14 to 29% higher (P < 0.05) in pigs fed 450 U of Mutant-EP/kg feed than those fed 450 U of consensus phytase/kg of feed. The latter also had lower (P < 0.05) plasma inorganic P concentrations than those of pigs fed 0.1% P at wk 2. Plasma inorganic P concentrations in pigs fed the two phytases in combination did not differ (P = 0.16 to 0.70) from those of pigs fed each enzyme alone. There was no main effect of dietary treatments (P = 0.13) on plasma alkaline phosphatase activity (data not shown).
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Figure 4. Effects of supplemental 0.1 or 0.2% inorganic P (as 0.1 or 0.2% P), 750 or 450 U of consensus phytase/kg of feed (750 or 450 Con), 450 U of Mutant-EP/kg of feed (450 Mut), and 225 U of consensus + 225 U of Mutant-EP/kg of feed (450 Combo) on plasma inorganic P concentrations of pigs in Exp. 2. Values are means of six individually penned pigs. Within each week, means not sharing a common superscript letter differ (P < 0.05). The SEM were: wk 0, 0.23; wk 1, 0.25; wk 2, 0.23; wk 3, 0.39; wk 4, 0.40.
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Discussion
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Our results from Exp. 1 have clearly demonstrated the effectiveness of this experimental consensus phytase in improving dietary phytate P utilization by weanling pigs. When it was supplemented up to 1,250 U/kg of feed into the corn-soybean meal diet without inorganic P added, the enzyme resulted in linear and quadratic improvements in growth performance, bone strength, and plasma inorganic P concentrations in pigs. The phytase dose-dependent (linear and quadratic at wk 3, 4, and 5) decrease in plasma activity of alkaline phosphatase, an enzyme that catalyzes phosphorus release from bone, indicates that consensus phytase indeed facilitated digestion of dietary phytate-P, preventing excessive bone resorption or demineralization. The effectiveness of consensus phytase, along with its excellent thermostability (Lehmann et al., 2000), has at least two implications. First, a practically effective consensus phytase offers great potential in overcoming the constraint of the currently available phytases in withstanding heat denaturation by feed pelleting. Since its melting temperature of 78°C is 15 to 22°C higher than those of all other known phytases (Lehmann et al., 2000), consensus phytase may be resistant to heat inactivation by the standard feed pelleting (60 to 80°C) that causes up to 52% activity loss of other phytases (Simons et al., 1990). Second, the good efficacy of the consensus phytase in the practical diets supports the notion that thermostable enzymes do not necessarily compromise their catalytic activity for stability (Lehmann et al., 2000). Because of its high optimal temperature (71°C), it is indeed a real concern that the enzyme may be inefficient at the physiological temperature of the swine gastrointestinal tracts (37°C). Apparently, its structure stability or rigidity does not substantially reduce its ability to catalyze the hydrolysis of phytate P from digesta in pigs. Similarly, another heat-stable A. fumigatus fungal phytase has also been shown to be effective at 500 U/kg in improving phytate P bioavailability in diets for growing-finishing pigs (Simoes and Guggenbuhl, 1998). Subsequently, more studies should be conducted to determine the actual tolerance of consensus phytase to heat denaturation by feed pelleting and its efficacy in the pellet feed.
Based on the quadratic polynomial equations derived from Exp. 1, the average dietary consensus phytase activity predicted for the maximal growth performance is 885 U/kg of feed, whereas the activity for the maximal plasma inorganic P concentration and bone strength is 1,497 and 1,559 U/kg of feed, respectively. These differences can be explained by the higher phosphorus requirement of pigs for the maximal bone strength than for the maximal growth (NRC, 1998). Apparently, the actual effects of these doses need testing in future studies. In Exp. 2, pigs fed 750 U of consensus phytase/kg of feed had responses in all measures that fell between those of pigs fed 0.1 and 0.2% P. Meanwhile, 450 U of consensus phytase/kg of feed produced bone strength nearly identical to that of pigs fed 0.1% P. However, the precise inorganic P equivalence of these consensus phytase activities needs to be assayed with multiple levels of inorganic P (Biehl and Baker, 1996; Traylor et al., 2001; Augspurger et al., 2003).
Compared with pigs fed 0.2% P, pigs fed 450 U consensus phytase/kg of feed had 16 to 29% lower (P < 0.05) plasma inorganic P concentrations or bone strength. In contrast, these two measures in pigs fed 450 U of Mutant-EP phytase/kg of feed did not differ from those of pigs fed 0.2% P. More directly, 450 U of Mutant-EP phytase/kg of feed yielded 14 to 29% higher (P < 0.05) plasma inorganic P concentrations at wk 2 and 3 than 450 U of consensus phytase/kg feed. Seemingly, Mutant-EP phytase was more efficacious than consensus phytase in releasing dietary phytate P for maintaining plasma inorganic P concentrations in weanling pigs. Despite this, caution should be given in generalizing an overall efficacy comparison between these two enzymes, due to the lack of statistically significant differences in the final plasma inorganic P concentrations or bone strength between the two groups of pigs in Exp. 2. However, efficacy of E. coli phytases has been determined in chicks by Leeson et al. (2000) and in pigs by Stahl et al. (2000). An improved effectiveness of this Mutant-EP phytase over that of other two fungal phytases in both swine and poultry diets has been reported (Applegate et al., 2003; Augspurger et al., 2003). Any efficacy difference between Mutant-EP phytase and consensus phytase or other fungal phytases might be partially related to their pH optima. Although the optimal pH for consensus phytase is 6.5 (Lehmann et al., 2000), the Mutant-EP phytase is most active at a more acidic pH (Rodriguez et al., 2000). It is understandable that 450 U of Mutant-EP phytase, measured at pH 5.5, bears more functioning activity at the stomach pH of pigs (Yi and Kornegy, 1996) than that of consensus phytase. In addition, E. coli phytase has a much greater turnover rate than the fungal enzyme (Greiner et al., 1993), and is resistant to proteolysis (Rodriguez et al., 1999).
A combination of these two rather different phytases resulted in no apparent synergistic effect on the measures in Exp. 2. Although pigs fed 225 U of consensus phytase + 225 U of Mutant-EP phytase/kg had numerically higher plasma inorganic P concentrations and bone strength than the calculated averages of pigs fed each enzyme alone at 450 U/kg, there was no significant difference among these three groups. Future research should examine if there is any synergistic effect of these two enzymes under dietary conditions different from those in the present study. Because properties of phytases vary, it is possible to maximize dietary phytate P bioavailability to pigs by using multiple phytases in combination, along with low-phytate grain (Spencer et al., 2000; Sands et al., 2001), organic acids (Han et al., 1998; Radcliffe et al., 1998), intrinsic phytases in feeds (Han et al., 1997), and an effective feeding regimen (Mroz et al., 1994).
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Implications
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A newly developed experimental heat-stable consensus phytase was effective in improving phytate phosphorus bioavailability in corn-soybean meal diets for weanling pigs. Linear and quadratic responses in growth performance and body nutritional status of phosphorus were observed in these pigs fed this enzyme at 0 to 1,250 U/kg of corn-soybean meal diet, without supplemental inorganic phosphorus. Future research will be needed to determine the exact inorganic phosphorus equivalents of consensus phytase and any possible synergistic effects between the enzyme and other phytases.
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Footnotes
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1 This research was partially supported by the Cornell Biotechnology Program and Roche Vitamins Ltd. (Basel, Switzerland). We thank J. A. Bartsch for his help in bone strength analysis, and J. Broz, N. E. Ward, and M. Wyss for their useful discussions on the research. 
2 Correspondence: Morrison Hall 252 (phone: 607-254-4703; fax: 607-255-9829; E-mail: XL20{at}cornell.edu).
Received for publication February 10, 2003.
Accepted for publication July 17, 2003.
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A. K. Kies, P. A. Kemme, L. B. J. Sebek, J. Th. M. van Diepen, and A. W. Jongbloed
Effect of graded doses and a high dose of microbial phytase on the digestibility of various minerals in weaner pigs
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May 1, 2006;
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J. A. Jendza, R. N. Dilger, S. A. Adedokun, J. S. Sands, and O. Adeola
Escherichia coli phytase improves growth performance of starter, grower, and finisher pigs fed phosphorus-deficient diets
J Anim Sci,
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83(8):
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[Abstract]
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C. H. Stahl, K. R. Roneker, W. G. Pond, and X. G. Lei
Effects of combining three fungal phytases with a bacterial phytase on plasma phosphorus status of weanling pigs fed a corn-soy diet
J Anim Sci,
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[Abstract]
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Top
Abstract
Introduction
