TY - JOUR
AU - Adeola, O.
AB - ABSTRACT Two experiments were conducted to investigate the concept that the addition of corn expressing an Escherichia coli-derived gene (corn-based phytase; CBP) to a P-deficient diet would improve growth performance and P utilization in pigs. An E. coli-derived microbial phytase (expressed in Pichia pastoris) sprayed onto a wheat carrier (Quantum) was included for comparison. In Exp. 1, forty-eight 10-kg pigs were blocked by BW into 6 blocks and allotted to 8 dietary treatments such that the BW among dietary treatments was similar and given free access to feed for 28 d. The dietary treatments were a negative control (NC) with no inorganic P supplementation; NC + 2, 4, or 6 g of monosodium phosphate/kg; NC + 16,500, 33,000, or 49,500 phytase units (FTU) of CBP/kg; and NC + 16,500 FTU of Quantum/kg. In Exp. 2, twenty-four 13-kg barrows were assigned to the NC, NC + 16,500 or 33,000 FTU of CBP/kg, or NC + 16,500 FTU of Quantum/kg, in a nutrient- and energy-balance study consisting of 5 d of adjustment and 5-d collection periods. The total collection method was used to determine nutrient and energy balance. Addition of CBP to the low-P NC diet linearly increased (P < 0.01) ADG, G:F, and plasma P concentration of pigs during the 28-d study. There was no difference in ADG, G:F, or plasma P concentration between pigs fed the CBP or Quantum phytase at 16,500 FTU/kg. Weight gain, G:F, and plasma P concentration of pigs increased (P < 0.01) with monosodium phosphate supplementation, confirming P deficiency of the NC diet. Linear improvements (P < 0.05) in DM digestibility and energy retention were observed with CBP supplementation of the NC diet. Although there were linear (P < 0.01) and quadratic (P < 0.05) increases in N digestibility, N retention was unaffected by CBP supplementation of the NC diet in growing pigs. Phosphorus and Ca digestibilities and retentions improved linearly and quadratically (P < 0.01) with the addition of CBP to the NC diet. There was no difference in digestive utilization of P or Ca between pigs fed CBP and Quantum phytase at 16,500 FTU/kg. The data showed that the addition of a corn expressing an E. coli-derived gene to a P-deficient diet improved growth performance and indices of P utilization in pigs, and corn expressing phytase was as efficacious as Quantum phytase when supplemented in P-deficient diets for weanling pigs. INTRODUCTION The nominal intestinal phytase activity of nonruminant animals limits the utilization of phytate P, leading to its subsequent excretion in manure, which can cause environmental pollution problems (Adeola, 1999). Microbial phytase supplementation of nonruminant diets is an effective means of improving the utilization of phytate-bound P (Jongbloed et al., 1992; Cromwell et al., 1995; Adeola et al., 2004). Advances in recombinant DNA technology have enabled the production of transgenic plants expressing microbial-derived phytase in tobacco seeds (Pen et al., 1993), soybean (Li et al., 1997), and canola (Zhang et al., 2000). However, there is no record of a microbial phytase gene expressed in corn. Corn constitutes the bulk of nonruminant diets. Moreover, unlike canola and soybean, corn is not subjected to the extreme heat employed in processing soybean and canola meals. Corn is therefore an attractive system for the production of transgenic plants expressing microbial-derived phytase. It can be directly incorporated into diets with minimal risk of losing phytase activity through postharvest processing of corn. Herein we report the results of 2 studies to demonstrate that up to 49,500 units of an Escherichia coli-derived phytase expressed in corn (corn-based phytase; CBP) could be directly incorporated in a P- and Ca-deficient weanling pig diet without any deleterious effects. Phytase activity of 16,500 units/kg from CBP was also compared with E. coli-derived microbial phytase (expressed in Pichia pastoris, a species of methylo-trophic yeast) sprayed onto a wheat carrier (Quantum, Syngenta Animal Nutrition, Research Triangle Park, NC). It was hypothesized that the addition of CBP to P-deficient, low-Ca, corn-soybean diets would improve growth performance and indices of P utilization, and weanling pigs fed CBP-supplemented diets would respond in a manner similar to those pigs fed Quantum phytase-supplemented diets. MATERIALS AND METHODS All animal procedures were approved by the Purdue Animal Care and Use Committee. Experiment 1 Dietary Treatments. The corn-based phytase was determined to have a phytase activity of 660 phytase units (FTU)/g. One phytase unit is defined as the quantity of enzyme required to hydrolyze 1 μmol of inorganic P/min, at pH 5.5, from an excess of 1.5 mM sodium phytate at 37°C (International Union of Biochemistry, 1979). The 8 dietary treatments (Table 1), presented as a mash, were a negative control (NC) formulated to be adequate (NRC, 1998) in all nutrients and ME except P and Ca [nonphytate P (nPP) = 1.41 g/kg and Ca = 5.3 g/kg]; NC + 2, 4, or 6 g/kg of monosodium phosphate (MSP1, MSP2, or MSP3); NC + 16,500 (CBP1; analyzed to contain 15,329 FTU/kg), 33,000 (CBP2; analyzed to contain 29,766 FTU/kg), or 49,500 (CBP3; analyzed to contain 44,303 FTU/kg) CBP FTU/kg; or NC + 16,500 (analyzed to contain 17,162 FTU/kg) Quantum phytase FTU/kg. The gene source of the phytase enzyme in the corn and the Quantum phytase sample was identical, which is E. coli-derived, 6-phytase mutated for greater thermostability. The phytase gene was mutated from the wild type E. coli phytase using Gene Site Saturation Mutagenesis technology (Diversa Corporation; San Diego, CA), with the selection pressure being that of thermotolerance. The mutant gene was then inserted into Pichia pastoris (Syngenta Biotechnology Inc., Research Triangle Park, NC) as a fermentation host or into corn as a production host. Eight pigs in each of 6 blocks were randomly allotted to 8 diets. Table 1. Composition of the negative control diet (as-fed basis) Ingredient, g/kg    Corn1  621.0  Soybean meal (48.0% CP)  331.0  Corn oil  10.0  Salt  3.0  Limestone (38% Ca)  11.0  Trace-mineral premix2  1.0  Vitamin premix3  1.5  Selenium4  0.5  L-Lysine·HCl  1.0  Corn5  20.0  Calculated nutrients and energy      Protein, g/kg  210      DE, kcal/kg  3,567      ME, kcal/kg  3,395      Ca, g/kg  5.3      Total P, g/kg  4.08      Nonphytate P, g/kg  1.41      Ca:P  1.29  Ingredient, g/kg    Corn1  621.0  Soybean meal (48.0% CP)  331.0  Corn oil  10.0  Salt  3.0  Limestone (38% Ca)  11.0  Trace-mineral premix2  1.0  Vitamin premix3  1.5  Selenium4  0.5  L-Lysine·HCl  1.0  Corn5  20.0  Calculated nutrients and energy      Protein, g/kg  210      DE, kcal/kg  3,567      ME, kcal/kg  3,395      Ca, g/kg  5.3      Total P, g/kg  4.08      Nonphytate P, g/kg  1.41      Ca:P  1.29  1 Normal corn was replaced with an equal portion of 25, 50, or 75 g/kg of corn-based phytase to supply phytase activity of 16,500, 33,000, or 49,500 phytase units (FTU)/kg of diet, respectively. One phytase unit is defined as the quantity of enzyme required to liberate 1 μmol of inorganic P/min, at pH 5.5, from an excess of 1.5 mM sodium phytate at 37°C (International Union of Biochemistry, 1979). 2 Provided per kilogram of diet: 97 mg of Fe (ferrous sulfate); 12 mg of Mn (manganese oxide); 97 mg of Zn (zinc sulfate); 9 mg of Cu (copper sulfate); 0.34 mg of I (potassium iodate). 3 Provided per kilogram of diet: 2,423 IU of vitamin A; 242 IU of vitamin D3; 17.6 IU of vitamin E; 2.4 mg of vitamin K activity; 804 μg of menadione; 14.1 μg of vitamin B12; 2.8 mg of riboflavin; 9 mg of D-pantothenic acid; and 13 mg of niacin. 4 Provided 300 μg of Se per kilogram of diet. 5 Fine ground corn used as a monosodium phosphate carrier to supply 0.5, 1.0, or 1.5 g of inorganic P/kg, or mixed with Quantum phytase to obtain a premix with a phytase activity of 825 FTU/g. View Large Table 1. Composition of the negative control diet (as-fed basis) Ingredient, g/kg    Corn1  621.0  Soybean meal (48.0% CP)  331.0  Corn oil  10.0  Salt  3.0  Limestone (38% Ca)  11.0  Trace-mineral premix2  1.0  Vitamin premix3  1.5  Selenium4  0.5  L-Lysine·HCl  1.0  Corn5  20.0  Calculated nutrients and energy      Protein, g/kg  210      DE, kcal/kg  3,567      ME, kcal/kg  3,395      Ca, g/kg  5.3      Total P, g/kg  4.08      Nonphytate P, g/kg  1.41      Ca:P  1.29  Ingredient, g/kg    Corn1  621.0  Soybean meal (48.0% CP)  331.0  Corn oil  10.0  Salt  3.0  Limestone (38% Ca)  11.0  Trace-mineral premix2  1.0  Vitamin premix3  1.5  Selenium4  0.5  L-Lysine·HCl  1.0  Corn5  20.0  Calculated nutrients and energy      Protein, g/kg  210      DE, kcal/kg  3,567      ME, kcal/kg  3,395      Ca, g/kg  5.3      Total P, g/kg  4.08      Nonphytate P, g/kg  1.41      Ca:P  1.29  1 Normal corn was replaced with an equal portion of 25, 50, or 75 g/kg of corn-based phytase to supply phytase activity of 16,500, 33,000, or 49,500 phytase units (FTU)/kg of diet, respectively. One phytase unit is defined as the quantity of enzyme required to liberate 1 μmol of inorganic P/min, at pH 5.5, from an excess of 1.5 mM sodium phytate at 37°C (International Union of Biochemistry, 1979). 2 Provided per kilogram of diet: 97 mg of Fe (ferrous sulfate); 12 mg of Mn (manganese oxide); 97 mg of Zn (zinc sulfate); 9 mg of Cu (copper sulfate); 0.34 mg of I (potassium iodate). 3 Provided per kilogram of diet: 2,423 IU of vitamin A; 242 IU of vitamin D3; 17.6 IU of vitamin E; 2.4 mg of vitamin K activity; 804 μg of menadione; 14.1 μg of vitamin B12; 2.8 mg of riboflavin; 9 mg of D-pantothenic acid; and 13 mg of niacin. 4 Provided 300 μg of Se per kilogram of diet. 5 Fine ground corn used as a monosodium phosphate carrier to supply 0.5, 1.0, or 1.5 g of inorganic P/kg, or mixed with Quantum phytase to obtain a premix with a phytase activity of 825 FTU/g. View Large Animals and Blood Collection. A total of 48 (24 barrows and 24 gilts), 10-kg pigs housed individually in stainless-steel pens (0.76 × 0.89 m) equipped with nipple drinkers, stainless-steel feeders, and plastic-coated, expanded metal floors were given free access to water and feed for 28 d. The pens were located in environmentally regulated rooms with a temperature of 23°C and a light/dark cycle of 12 h. The pigs were allotted to diets such that the average initial BW among dietary treatments was similar. On d 14 and 28, BW and feed intake were recorded, but after the pigs were weighed on d 28, feed was withdrawn for 16 h, after which the pigs were fed 500 g of their respective diets, and blood was collected 2 h later from the anterior vena cava into 6-mL heparinized syringes (spray coated with 86 heparin units as sodium heparin per 6-mL tube) for analysis of plasma P concentration. Experiment 2 Four of the diets (NC, CBP1, CBP2, or NC + 16,500 Quantum phytase FTU/kg) used in Exp. 1 were chosen for the nutrient balance study consisting of 5-d adjustment and 5-d collection periods. Diets CBP1 and Quantum phytase are NC + 16,500 FTU/kg from E. coli-derived phytase expressed in corn and E. coli-derived microbial phytase (expressed in Pichia pastoris) sprayed on to a wheat carrier, respectively. Twenty-four 13-kg barrows were randomly assigned to the 4 diets in a randomized complete block design, such that there were 6 pigs per dietary treatment. Pigs were housed in stainless-steel metabolism crates (0.83 × 0.71 m) that allowed for separate collection of feces and urine using the protocols of Adeola and Bajjalieh (1997). Barrows were fed a total 50 g/kg of BW in 2 equal portions, which were offered at 0600 and 1800. Chemical Analyses Frozen feces and an aliquot of strained urine were dried in a forced-draft oven at 55°C for 5 d. The dried fecal samples and diets were ground to pass through a 0.5-mm screen and mixed thoroughly before analysis. For DM determination, feces and diets were oven-dried at 105°C for 24 h until there was no change in weight. Gross energy of diets, feces, and urine was determined using adiabatic bomb calorimetry. The N content of diets, urine, and fecal samples was determined by the combustion method (Method 990.03; AOAC, 2000) using a combustion analyzer (Leco Model FP 2000, Leco Corp., St. Joseph, MI). Diets and fecal samples were wet-ashed in nitric/ perchloric acids (Method 968.08D[b]; AOAC, 2000). Protein in reconstituted urine (prepared by adding urine to deionized water) samples was precipitated with 10% (wt/vol) trichloroacetic acid. The supernatant was used for the determination of P and Ca. Blood in heparinized tubes was centrifuged at 1,000 × g for 10 min at 4°C and the supernatant treated with 10% trichloroacetic acid and centrifuged at 3,500 × g for 15 min at room temperature on a table-top microcentrifuge to precipitate plasma proteins. The concentration of P in the supernatant was determined using a colorimetric assay. Briefly, acid molyb-date and Fiske's SubbaRow reducer solution was added to the wet-ash, acid digest to form a phospho-molybdenum complex. The blue color intensity, measured with a spectrophotometer at 620 nm (Method 965.17, AOAC, 2000; Packard SpectraCount, Model No. AS 1000, Meriden, CT), is proportional to P concentration. Calcium content was determined by flame atomic absorption spectrophotometry (Varian FS 240 AA, Varian Inc., Palo Alto, CA). Phytase activity was determined according to the method of Engelen et al. (2001), with modifications, which are optimized for the E. coli phytase, involving the use of 250 mM acetate buffer, 0.1% Tween, and ammonium heptamolybdate and ammonium vanadate as yellow-color reagents. Statistical Analysis Data collected from the trials were analyzed using the GLM procedure (SAS Inst. Inc., Cary, NC) appropriate for a randomized complete block design. Individual pigs served as experimental units, and an alpha value of less than 0.05 was considered significant. Means were separated using the following contrasts: NC vs. Quantum phytase (Exp. 1 and 2); CBP1 vs. Quantum phytase (Exp. 1 and 2); MSP3 vs. CBP1 (Exp. 1); linear and quadratic contrasts of NC, CBP1, CBP2, and CBP3 (Exp. 1); and linear and quadratic contrasts of NC, CBP1, and CBP2 (Exp. 2). RESULTS Growth Performance and Plasma P The analyzed composition of experimental diets on an as-fed basis is presented in Table 2. Table 3 shows the growth performance and plasma P for weanling pigs fed P-deficient, low-Ca diets supplemented with MSP, CBP, or Quantum phytase in Exp. 1. Feed intake was unaffected by dietary treatment. Addition of Quantum phytase to NC at phytase activity of 16,500 FTU/kg of diet improved (P < 0.01) ADG, G:F, and plasma P concentration of pigs. There was no difference between pigs fed Quantum phytase and CBP1 in G:F, ADG, or plasma P or between pigs fed MSP3 and CBP1. There were linear increases (P < 0.001) in ADG and G:F of pigs in response to CBP or MSP supplementation of the NC diet. Furthermore, there were linear and quadratic increases (P < 0.001) in plasma P concentration of pigs due to CBP supplementation of the NC diet. There was a linear (P < 0.001) relationship between plasma P and the supplementation of the NC diet with inorganic P from monosodium phosphate (R2 = 0.86). Because the plasma P concentrations of pigs fed diets containing CBP were numerically greater than those fed inorganic P from monosodium phosphate, it was inappropriate to generate P equivalency values. Table 2. Analyzed components of the diets on an as-fed basis   Diet1  Item  NC  MSP1  MSP2  MSP3  CBP1  CBP2  CBP3  Quantum phytase  DM, g/kg  891.8  893.4  895.2  894.7  892.8  900.3  900.7  902.0  GE, kcal/kg  3,798  3,792  3,804  3,791  3,830  3,856  3,862  3,871  N, g/kg  31.33  31.41  31.32  31.51  31.61  31.39  31.39  30.84  Ca, g/kg  4.98  5.0  5.14  5.32  5.45  4.98  5.23  5.31  P, g/kg  3.78  4.18  4.63  5.07  3.79  3.72  3.82  3.79  Phytase, FTU/kg  <50  <50  <50  <50  15,329  29,766  44,303  17,162    Diet1  Item  NC  MSP1  MSP2  MSP3  CBP1  CBP2  CBP3  Quantum phytase  DM, g/kg  891.8  893.4  895.2  894.7  892.8  900.3  900.7  902.0  GE, kcal/kg  3,798  3,792  3,804  3,791  3,830  3,856  3,862  3,871  N, g/kg  31.33  31.41  31.32  31.51  31.61  31.39  31.39  30.84  Ca, g/kg  4.98  5.0  5.14  5.32  5.45  4.98  5.23  5.31  P, g/kg  3.78  4.18  4.63  5.07  3.79  3.72  3.82  3.79  Phytase, FTU/kg  <50  <50  <50  <50  15,329  29,766  44,303  17,162  1 NC = negative control; MSP1 = NC + 2 g/kg of monosodium phosphate; MSP2 = NC + 4 g/kg of monosodium phosphate; MSP3 = NC + 6 g/kg of monosodium phosphate; CBP1 = NC + corn-based phytase at a phytase activity of 16,500 FTU/kg of diet; CBP2 = NC + corn-based phytase at a phytase activity of 33,000 FTU/kg of diet; CBP3 = NC + corn-based phytase at a phytase activity of 49,500 FTU/kg of diet; Quantum phytase = NC + Quantum phytase at a phytase activity of 16,500 FTU/kg of diet. View Large Table 2. Analyzed components of the diets on an as-fed basis   Diet1  Item  NC  MSP1  MSP2  MSP3  CBP1  CBP2  CBP3  Quantum phytase  DM, g/kg  891.8  893.4  895.2  894.7  892.8  900.3  900.7  902.0  GE, kcal/kg  3,798  3,792  3,804  3,791  3,830  3,856  3,862  3,871  N, g/kg  31.33  31.41  31.32  31.51  31.61  31.39  31.39  30.84  Ca, g/kg  4.98  5.0  5.14  5.32  5.45  4.98  5.23  5.31  P, g/kg  3.78  4.18  4.63  5.07  3.79  3.72  3.82  3.79  Phytase, FTU/kg  <50  <50  <50  <50  15,329  29,766  44,303  17,162    Diet1  Item  NC  MSP1  MSP2  MSP3  CBP1  CBP2  CBP3  Quantum phytase  DM, g/kg  891.8  893.4  895.2  894.7  892.8  900.3  900.7  902.0  GE, kcal/kg  3,798  3,792  3,804  3,791  3,830  3,856  3,862  3,871  N, g/kg  31.33  31.41  31.32  31.51  31.61  31.39  31.39  30.84  Ca, g/kg  4.98  5.0  5.14  5.32  5.45  4.98  5.23  5.31  P, g/kg  3.78  4.18  4.63  5.07  3.79  3.72  3.82  3.79  Phytase, FTU/kg  <50  <50  <50  <50  15,329  29,766  44,303  17,162  1 NC = negative control; MSP1 = NC + 2 g/kg of monosodium phosphate; MSP2 = NC + 4 g/kg of monosodium phosphate; MSP3 = NC + 6 g/kg of monosodium phosphate; CBP1 = NC + corn-based phytase at a phytase activity of 16,500 FTU/kg of diet; CBP2 = NC + corn-based phytase at a phytase activity of 33,000 FTU/kg of diet; CBP3 = NC + corn-based phytase at a phytase activity of 49,500 FTU/kg of diet; Quantum phytase = NC + Quantum phytase at a phytase activity of 16,500 FTU/kg of diet. View Large Table 3. Growth performance and plasma P concentration of pigs fed diets containing phytase in Exp. 1   Diet2    P-value of contrasts  Item  NC  MSP1  MSP2  MSP3  CBP1  CBP2  CBP3  Quantum Phytase  SD3  NC vs. Quantum phytase  CBP1 vs. Quantum phytase  MSP3 vs. CBP1  Linear4  Quadratic5  Linear6  Quadratic7  No. of observations1  6  6  6  6  6  6  6  6                  Initial BW, kg  9.84  9.82  9.92  9.85  9.86  9.89  9.86  9.89  0.282                Final BW, kg  20.92  21.16  23.08  23.39  24.84  24.07  24.97  24.85  1.798                ADG, g  396  405  470  483  535  506  540  534  60.5  <0.001  0.97  0.15  <0.001  0.029  0.008  0.61  ADFI, g  1,066  1,051  1,054  997  1,084  1,060  1,030  1,027  113.1  0.46  0.28  0.19  0.49  0.58  0.39  0.70  G:F, g/kg  384  401  462  502  496  483  538  529  65.6  <0.001  0.38  0.88  0.004  0.35  0.002  0.66  Plasma P, mg/L  33.8  41.8  62.6  74.1  78.4  76.2  76.6  74.5  6.08  <0.001  0.24  0.25  <0.001  <0.001  <0.001  0.60    Diet2    P-value of contrasts  Item  NC  MSP1  MSP2  MSP3  CBP1  CBP2  CBP3  Quantum Phytase  SD3  NC vs. Quantum phytase  CBP1 vs. Quantum phytase  MSP3 vs. CBP1  Linear4  Quadratic5  Linear6  Quadratic7  No. of observations1  6  6  6  6  6  6  6  6                  Initial BW, kg  9.84  9.82  9.92  9.85  9.86  9.89  9.86  9.89  0.282                Final BW, kg  20.92  21.16  23.08  23.39  24.84  24.07  24.97  24.85  1.798                ADG, g  396  405  470  483  535  506  540  534  60.5  <0.001  0.97  0.15  <0.001  0.029  0.008  0.61  ADFI, g  1,066  1,051  1,054  997  1,084  1,060  1,030  1,027  113.1  0.46  0.28  0.19  0.49  0.58  0.39  0.70  G:F, g/kg  384  401  462  502  496  483  538  529  65.6  <0.001  0.38  0.88  0.004  0.35  0.002  0.66  Plasma P, mg/L  33.8  41.8  62.6  74.1  78.4  76.2  76.6  74.5  6.08  <0.001  0.24  0.25  <0.001  <0.001  <0.001  0.60  1 Number of observations is 5 for plasma P concentration of pigs on diet CBP2. 2 NC = negative control; MSP1 = NC + 2 g/kg of monosodium phosphate; MSP2 = NC + 4 g/kg of monosodium phosphate; MSP3 = NC + 6 g/kg of monosodium phosphate; CBP1 = NC + corn-based phytase at a phytase activity of 16,500 FTU/kg of diet; CBP2 = NC + corn-based phytase at a phytase activity of 33,000 FTU/kg of diet; CBP3 = NC + corn-based phytase at a phytase activity of 49,500 FTU/kg of diet; Quantum phytase = NC + Quantum phytase at a phytase activity of 16,500 FTU/kg of diet. 3 SD = pooled SD. 4 Linear (NC, CBP1, CBP2, and CBP3). 5 Quadratic (NC, CBP1, CBP2, and CBP3). 6 Linear (NC, MSP1, MSP2, and MSP3). 7 Quadratic (NC, MSP1, MSP2, and MSP3). View Large Table 3. Growth performance and plasma P concentration of pigs fed diets containing phytase in Exp. 1   Diet2    P-value of contrasts  Item  NC  MSP1  MSP2  MSP3  CBP1  CBP2  CBP3  Quantum Phytase  SD3  NC vs. Quantum phytase  CBP1 vs. Quantum phytase  MSP3 vs. CBP1  Linear4  Quadratic5  Linear6  Quadratic7  No. of observations1  6  6  6  6  6  6  6  6                  Initial BW, kg  9.84  9.82  9.92  9.85  9.86  9.89  9.86  9.89  0.282                Final BW, kg  20.92  21.16  23.08  23.39  24.84  24.07  24.97  24.85  1.798                ADG, g  396  405  470  483  535  506  540  534  60.5  <0.001  0.97  0.15  <0.001  0.029  0.008  0.61  ADFI, g  1,066  1,051  1,054  997  1,084  1,060  1,030  1,027  113.1  0.46  0.28  0.19  0.49  0.58  0.39  0.70  G:F, g/kg  384  401  462  502  496  483  538  529  65.6  <0.001  0.38  0.88  0.004  0.35  0.002  0.66  Plasma P, mg/L  33.8  41.8  62.6  74.1  78.4  76.2  76.6  74.5  6.08  <0.001  0.24  0.25  <0.001  <0.001  <0.001  0.60    Diet2    P-value of contrasts  Item  NC  MSP1  MSP2  MSP3  CBP1  CBP2  CBP3  Quantum Phytase  SD3  NC vs. Quantum phytase  CBP1 vs. Quantum phytase  MSP3 vs. CBP1  Linear4  Quadratic5  Linear6  Quadratic7  No. of observations1  6  6  6  6  6  6  6  6                  Initial BW, kg  9.84  9.82  9.92  9.85  9.86  9.89  9.86  9.89  0.282                Final BW, kg  20.92  21.16  23.08  23.39  24.84  24.07  24.97  24.85  1.798                ADG, g  396  405  470  483  535  506  540  534  60.5  <0.001  0.97  0.15  <0.001  0.029  0.008  0.61  ADFI, g  1,066  1,051  1,054  997  1,084  1,060  1,030  1,027  113.1  0.46  0.28  0.19  0.49  0.58  0.39  0.70  G:F, g/kg  384  401  462  502  496  483  538  529  65.6  <0.001  0.38  0.88  0.004  0.35  0.002  0.66  Plasma P, mg/L  33.8  41.8  62.6  74.1  78.4  76.2  76.6  74.5  6.08  <0.001  0.24  0.25  <0.001  <0.001  <0.001  0.60  1 Number of observations is 5 for plasma P concentration of pigs on diet CBP2. 2 NC = negative control; MSP1 = NC + 2 g/kg of monosodium phosphate; MSP2 = NC + 4 g/kg of monosodium phosphate; MSP3 = NC + 6 g/kg of monosodium phosphate; CBP1 = NC + corn-based phytase at a phytase activity of 16,500 FTU/kg of diet; CBP2 = NC + corn-based phytase at a phytase activity of 33,000 FTU/kg of diet; CBP3 = NC + corn-based phytase at a phytase activity of 49,500 FTU/kg of diet; Quantum phytase = NC + Quantum phytase at a phytase activity of 16,500 FTU/kg of diet. 3 SD = pooled SD. 4 Linear (NC, CBP1, CBP2, and CBP3). 5 Quadratic (NC, CBP1, CBP2, and CBP3). 6 Linear (NC, MSP1, MSP2, and MSP3). 7 Quadratic (NC, MSP1, MSP2, and MSP3). View Large Apparent Digestibility and Retention of Energy and Nutrients The data from energy and nutrient balance study (Exp. 2) are summarized in Table 4. Dry matter intake and digestibility linearly increased (P < 0.05) with the addition of CBP to the NC diet. There were linear increases (P < 0.01) in GE intake and energy retention as phytase added from CBP to the NC diet increased. Total tract N digestibility, but not N retention, increased linearly (P < 0.01) when the NC diet was supplemented with CBP. Table 4. Nutrient and energy balance in weanling pigs fed supplemental phytase in Exp. 2   Diet1    P-value of contrasts  Item  NC  CBP1  CBP2  Quantum phytase  SD2  CBPI vs. Quantum phytase  Linear3  Quadratic3  No. of barrows  6  6  6  6          BW, kg  13.3  13.3  13.3  13.4          DMI, g/d  554.86  574.63  575.35  580.00  13.707  0.51  0.04  0.22  DM digestibility, %  88.38  90.16  90.29  90.74  0.82  0.24  0.03  0.08  Energy balance      Intake, kcal/d  2,365  2,465  2,464  2,489  58.80  0.49  0.01  0.11      Digestibility, %  87.55  88.55  88.67  89.27  0.960  0.21  0.08  0.41      Retention, kcal/d  2,005  2,114  2,109  2,164  40.48  0.046  0.001  0.014      Retention, %  84.78  85.74  85.63  87.02  1.040  0.05  0.18  0.32  N balance      Intake, g/d  19.51  20.35  20.06  19.83  0.475  0.08  0.06  0.03      Digestibility, %  86.67  89.73  89.08  89.67  1.280  0.93  0.01  0.02      Retention, g/d  13.04  14.05  13.44  14.45  0.733  0.36  0.36  0.04      Retention, %  66.83  69.10  67.03  73.07  4.090  0.11  0.94  0.32  Ca balance      Intake, g/d  3.10  3.51  3.18  3.42  0.080  0.0755  0.105  <0.001      Digestibility, %  49.56  82.32  79.74  78.60  7.230  0.39  <0.001  <0.001      Retention, g/d  1.53  2.89  2.53  2.67  0.242  0.1437  <0.001  <0.001      Retention, %  49.47  82.17  79.65  78.41  7.230  0.38  <0.001  <0.001  P balance      Intake, g/d  2.35  2.44  2.38  2.44  0.057  0.96  0.41  0.02      Digestibility, %  57.85  83.42  83.35  84.26  4.230  0.74  <0.001  <0.001      Retention, g/d  1.36  2.04  1.98  2.06  0.109  0.76  <0.001  <0.001      Retention, %  57.76  83.37  83.28  84.19  4.220  0.74  <0.001  <0.001    Diet1    P-value of contrasts  Item  NC  CBP1  CBP2  Quantum phytase  SD2  CBPI vs. Quantum phytase  Linear3  Quadratic3  No. of barrows  6  6  6  6          BW, kg  13.3  13.3  13.3  13.4          DMI, g/d  554.86  574.63  575.35  580.00  13.707  0.51  0.04  0.22  DM digestibility, %  88.38  90.16  90.29  90.74  0.82  0.24  0.03  0.08  Energy balance      Intake, kcal/d  2,365  2,465  2,464  2,489  58.80  0.49  0.01  0.11      Digestibility, %  87.55  88.55  88.67  89.27  0.960  0.21  0.08  0.41      Retention, kcal/d  2,005  2,114  2,109  2,164  40.48  0.046  0.001  0.014      Retention, %  84.78  85.74  85.63  87.02  1.040  0.05  0.18  0.32  N balance      Intake, g/d  19.51  20.35  20.06  19.83  0.475  0.08  0.06  0.03      Digestibility, %  86.67  89.73  89.08  89.67  1.280  0.93  0.01  0.02      Retention, g/d  13.04  14.05  13.44  14.45  0.733  0.36  0.36  0.04      Retention, %  66.83  69.10  67.03  73.07  4.090  0.11  0.94  0.32  Ca balance      Intake, g/d  3.10  3.51  3.18  3.42  0.080  0.0755  0.105  <0.001      Digestibility, %  49.56  82.32  79.74  78.60  7.230  0.39  <0.001  <0.001      Retention, g/d  1.53  2.89  2.53  2.67  0.242  0.1437  <0.001  <0.001      Retention, %  49.47  82.17  79.65  78.41  7.230  0.38  <0.001  <0.001  P balance      Intake, g/d  2.35  2.44  2.38  2.44  0.057  0.96  0.41  0.02      Digestibility, %  57.85  83.42  83.35  84.26  4.230  0.74  <0.001  <0.001      Retention, g/d  1.36  2.04  1.98  2.06  0.109  0.76  <0.001  <0.001      Retention, %  57.76  83.37  83.28  84.19  4.220  0.74  <0.001  <0.001  1 NC = negative control; CBP1 = NC + corn-based phytase at a phytase activity of 16,500 FTU/kg of diet; CBP2 = NC + corn-based phytase at a phytase activity of 33,000 FTU/kg of diet; Quantum phytase = NC + Quantum phytase at a phytase activity of 16,500 FTU/kg of diet. 2 SD = pooled SD. 3 Linear and quadratic (NC, CBP1, and CBP2). View Large Table 4. Nutrient and energy balance in weanling pigs fed supplemental phytase in Exp. 2   Diet1    P-value of contrasts  Item  NC  CBP1  CBP2  Quantum phytase  SD2  CBPI vs. Quantum phytase  Linear3  Quadratic3  No. of barrows  6  6  6  6          BW, kg  13.3  13.3  13.3  13.4          DMI, g/d  554.86  574.63  575.35  580.00  13.707  0.51  0.04  0.22  DM digestibility, %  88.38  90.16  90.29  90.74  0.82  0.24  0.03  0.08  Energy balance      Intake, kcal/d  2,365  2,465  2,464  2,489  58.80  0.49  0.01  0.11      Digestibility, %  87.55  88.55  88.67  89.27  0.960  0.21  0.08  0.41      Retention, kcal/d  2,005  2,114  2,109  2,164  40.48  0.046  0.001  0.014      Retention, %  84.78  85.74  85.63  87.02  1.040  0.05  0.18  0.32  N balance      Intake, g/d  19.51  20.35  20.06  19.83  0.475  0.08  0.06  0.03      Digestibility, %  86.67  89.73  89.08  89.67  1.280  0.93  0.01  0.02      Retention, g/d  13.04  14.05  13.44  14.45  0.733  0.36  0.36  0.04      Retention, %  66.83  69.10  67.03  73.07  4.090  0.11  0.94  0.32  Ca balance      Intake, g/d  3.10  3.51  3.18  3.42  0.080  0.0755  0.105  <0.001      Digestibility, %  49.56  82.32  79.74  78.60  7.230  0.39  <0.001  <0.001      Retention, g/d  1.53  2.89  2.53  2.67  0.242  0.1437  <0.001  <0.001      Retention, %  49.47  82.17  79.65  78.41  7.230  0.38  <0.001  <0.001  P balance      Intake, g/d  2.35  2.44  2.38  2.44  0.057  0.96  0.41  0.02      Digestibility, %  57.85  83.42  83.35  84.26  4.230  0.74  <0.001  <0.001      Retention, g/d  1.36  2.04  1.98  2.06  0.109  0.76  <0.001  <0.001      Retention, %  57.76  83.37  83.28  84.19  4.220  0.74  <0.001  <0.001    Diet1    P-value of contrasts  Item  NC  CBP1  CBP2  Quantum phytase  SD2  CBPI vs. Quantum phytase  Linear3  Quadratic3  No. of barrows  6  6  6  6          BW, kg  13.3  13.3  13.3  13.4          DMI, g/d  554.86  574.63  575.35  580.00  13.707  0.51  0.04  0.22  DM digestibility, %  88.38  90.16  90.29  90.74  0.82  0.24  0.03  0.08  Energy balance      Intake, kcal/d  2,365  2,465  2,464  2,489  58.80  0.49  0.01  0.11      Digestibility, %  87.55  88.55  88.67  89.27  0.960  0.21  0.08  0.41      Retention, kcal/d  2,005  2,114  2,109  2,164  40.48  0.046  0.001  0.014      Retention, %  84.78  85.74  85.63  87.02  1.040  0.05  0.18  0.32  N balance      Intake, g/d  19.51  20.35  20.06  19.83  0.475  0.08  0.06  0.03      Digestibility, %  86.67  89.73  89.08  89.67  1.280  0.93  0.01  0.02      Retention, g/d  13.04  14.05  13.44  14.45  0.733  0.36  0.36  0.04      Retention, %  66.83  69.10  67.03  73.07  4.090  0.11  0.94  0.32  Ca balance      Intake, g/d  3.10  3.51  3.18  3.42  0.080  0.0755  0.105  <0.001      Digestibility, %  49.56  82.32  79.74  78.60  7.230  0.39  <0.001  <0.001      Retention, g/d  1.53  2.89  2.53  2.67  0.242  0.1437  <0.001  <0.001      Retention, %  49.47  82.17  79.65  78.41  7.230  0.38  <0.001  <0.001  P balance      Intake, g/d  2.35  2.44  2.38  2.44  0.057  0.96  0.41  0.02      Digestibility, %  57.85  83.42  83.35  84.26  4.230  0.74  <0.001  <0.001      Retention, g/d  1.36  2.04  1.98  2.06  0.109  0.76  <0.001  <0.001      Retention, %  57.76  83.37  83.28  84.19  4.220  0.74  <0.001  <0.001  1 NC = negative control; CBP1 = NC + corn-based phytase at a phytase activity of 16,500 FTU/kg of diet; CBP2 = NC + corn-based phytase at a phytase activity of 33,000 FTU/kg of diet; Quantum phytase = NC + Quantum phytase at a phytase activity of 16,500 FTU/kg of diet. 2 SD = pooled SD. 3 Linear and quadratic (NC, CBP1, and CBP2). View Large There was no difference in digestibility or retention of N, Ca, and P in pigs fed CBP1 or Quantum phytase. Calcium digestibility and retention linearly increased (P < 0.01) with the addition of CBP to the NC diet. Supplementation of the NC diet with phytase from CBP linearly increased (P < 0.01) the digestibility and retention of P. DISCUSSION Phosphorus has been identified as the most important factor in accelerating eutrophication of water bodies (Sharpley et al., 1994). This is especially important in animal production where phytate P is poorly utilized as a result of limited inherent phytase activity in the gastrointestinal tract of nonruminants (Adeola et al., 2004). One of the most effective means of reducing P output from pig production units has been the use of exogenous phytase supplementation to low-P diets (Jongbloed et al., 1992; Sands et al., 2001; Johnston et al., 2004). The efficacy of an E. coli-derived phytase expressed in the endosperm of corn (CBP) or in a microbial expression system and sprayed onto wheat carrier (Quantum phytase) was compared in their P releasing ability, growth performance, and nutrient balance in weanling pigs in the current study. Phytase supplementation, as CBP or Quantum, of the NC diet improved ADG, feed efficiency, and plasma P in weanling pigs. The addition of CBP to the NC diet improved P and Ca digestibility. The digestibility, but not retention, of N was improved in the NC diet supplemented with CBP. There were no differences in growth performance, plasma P concentration, and nutrient balance in pigs fed the NC diet supplemented with Quantum phytase or CBP. The beneficial effects of microbial phytase supplementation of low-P diets in pigs have been well documented (Cromwell et al., 1995; Stahl et al., 1999; Adeola et al., 2004). Improvements in BW gain, feed efficiency, and plasma P were reported by Stahl et al. (2000) in weanling pigs fed supplemental microbial phytase. The G:F, feed intake, and ADG values reported herein are consistent with those reported for pigs of similar BW by Sands et al. (2001) and Adeola et al. (2004). The P requirement of pigs fed corn-soybean meal diets could be met without inorganic P supplementation, provided that a significant portion of phytate P in corn and soybean are degraded by phytase (Cromwell et al., 1995). The linear increase in plasma P with MSP supplementation of the NC diet is indicative of the increase in available P in those diets. Supplementation of the NC diet with phytase numerically increased plasma P in excess of the increase observed with supplementation of the NC diet with 6 g of MSP/kg. This clearly underscores the central role that exogenous phytase supplementation of nonruminant diets can play in degrading phytate-bound minerals. The increase in available P with microbial phytase supplementation is therefore regarded as the reason for the improvement in growth performance usually reported with phytase supplementation of low-P diets (Cromwell et al., 1993; Lei et al., 1993). Other reports indicate that the improvements in growth performance with microbial phytase supplementation of swine diets are multidimensional. Stahl et al. (1999) elegantly demonstrated that microbial phytase supplementation improved blood Fe concentration and hemoglobin concentration in anemic weanling pigs similar to pigs fed supplemental Fe. Earlier, Adeola et al. (1995) had reported an increase in plasma Zn concentration with microbial phytase supplementation and attributed the growth-promoting effect of phytase supplementation to an overall increase in mineral availability. In pigs fed diets with added phytase, improvements in growth rate, feed efficiency, and plasma P content were numerically greater than those pigs fed the highest level of MSP. Raboy (2003) reviewed the numerous physiological and biochemical roles that products of myo-inositol-1,2,3,4,5,6-hexakisphosphate play in the body, including serving as regulators in signal transduction pathways and functioning in RNA export, DNA repair, and ATP regeneration. Therefore, phytase supplementation of the P-deficient, low-Ca diets not only increased available P, but also provided metabolic benefits consistent with the aforementioned effects of increasing available P. It has been estimated that supplying microbial phytase in excess of 1,050 FTU/kg of diet would further improve P utilization only marginally in weanling pigs (Kornegay, 2001). In broiler chickens, it has been shown that greater doses of phytase activity than current industrial recommended doses improve P digestibility in low-P diets (Shirley and Edwards, 2003; Augspurger and Baker, 2004). Recently, Kies et al. (2006) reported improved mineral utilization in weanling pigs supplied with 15,000 FTU of Natuphos/kg of diet. The nutrient balance study reported herein is consistent with the report of Kies et al. (2006). Phosphorus digestibility was improved in weanling pigs fed 16,500 FTU/kg CBP-or Quantum phytase-supplemented diets compared with those pigs fed the NC diet. The product of P digestibility (57.8%) and the analyzed P content in the NC diet (4.24 g/kg) implies that 2.45 g of total P/kg of the NC diet was digested, leaving 1.79 g of undigested P/ kg available for hydrolysis by phytase. The addition of CBP to the the NC diet thus released approximately 61% of this otherwise unavailable P. Doubling the phytase activity in CBP2 to 33,000 FTU/kg of diet, however, did not seem to further improve P digestibility. In general, the linear and quadratic effects attributed to CBP (Exp. 2) are associated with the response to phytase vs. NC and not a specific dose response. The improvement in P digestibility nearly mirrors the increase in plasma P of pigs fed CBP-supplemented diets compared with those pigs fed the NC diet. This clearly demonstrates that phytase supplementation ameliorates the negative effect of P deficiency through hydrolysis of phytate-bound P by CBP, thereby increasing available P. Urinary P excretion was negligible, with pigs retaining near 100% of the digestible P. This observation is consistent with those reported by Lei et al. (1993) and Adeola et al. (1995). Thus, not only is available P content improved in low-P diets, but also P excretion is considerably reduced. Pigs fed phytase-supplemented diets excreted less than one-half as much P as pigs fed diets with no phytase supplementation, thereby reducing the impact of P-related environmental pollution. The release of phytate-bound minerals other than P with microbial phytase supplementation of low P and Ca diets is reflected in the high Ca digestibility and retention observed in the phytase-supplemented diets compared with pigs fed the NC diet. The Ca digestibility values reported herein are consistent with high dosage microbial phytase supplementation of low P and Ca diets (Kies et al., 2006). Contrary to the improvements in Ca digestibility and retention observed in the present research, Adeola et al. (2004) reported no improvement in Ca digestibility and utilization with microbial phytase supplementation of P-deficient diet fed to 13-kg barrows, but showed linear increases in 19-kg barrows fed P-deficient diets. These contrasting results were attributed to possible age effects and differences in the Ca:P ratio in the diets. It is important to recognize that unlike phosphate, circulating Ca concentration in the body is tightly regulated and only minimal fluctuations occur because of the body's ability to selectively increase or decrease intestinal absorption of Ca in the ingesta depending on Ca status (Guyton and Hall, 2000). Diet formulation, therefore, would play a crucial role in investigating the effect of phytase on Ca digestibility. Diets formulated to be adequate in Ca would be most unlikely to yield improvements in Ca digestibility and retention because although phytate degradation could have led to the release of chelated Ca ions, intestinal absorption would be downregulated to reflect increased supply, and as a result, there would not be a concomitant increase in digestibility or retention. Digestibility and retention values of Ca were proportionately lower compared with values for P. The absorption of Ca from the small intestine is usually not as efficient as P partly due to the divalent nature of Ca (Guyton and Hall, 2000). The effect of microbial supplementation on protein and amino acid utilization remains a debated issue (Adeola and Sands, 2003). Although N digestibility was improved with CBP supplementation of the NC diet, there was no improvement in N retention of pigs fed these N-adequate diets because increased N digestibility led to an increase in urinary N output. The increased N digestibility notwithstanding, improvement in N utilization was therefore not realized. Improvements in DMI in pigs fed CBP-supplemented diets could account for the higher P and Ca intake in those pigs compared with pigs fed the NC diet. There was no difference in the amount of feed allotted to barrows in a block. Although there was no feed refusal, differences in feeding behavior led to some feed wastage, which resulted in differences in feed intake that could not be attributed to dietary effect, even though 2 pigs on the NC diet were the most culpable. The expression of microbial phytase in the endosperm of corn is an innovative means of delivering microbial phytase to nonruminants to enhance the utilization of phytate P. The results reported herein clearly demonstrate that CBP compares favorably with Quantum phytase expressed in a microbial system at 16,500 FTU/ kg. This observation is consistent with the production of other transgenic phytase-containing seeds (Pen et al., 1993; Li et al., 1997; Zhang et al., 2000). Corn-based phytase improved growth performance, plasma P, nutrient, and energy balance as much as Quantum phytase in weanling pigs. It can be stated that the expression of the E. coli-derived gene in corn will increase the available P, and therefore the improvement observed in pigs fed CBP is a result of increase in available P in the corn. In pigs fed CBP1, CBP constituted only 25 g/ kg of the diet, but the extent of improvement exceeded, albeit numerically, the improvements recorded for pigs fed 1.5 g of inorganic P/kg from MSP. These improvements in pigs fed CBP1 could therefore be attributable to the phytase activity. It can be concluded that the E. coli-derived microbial phytase expressed in corn is as efficacious as that expressed in a microbial expression system at 16,500 FTU/kg when weanling pigs were fed P-deficient low-Ca diet over a 28-d period. LITERATURE CITED Adeola, O. 1999. Nutrient management procedures to enhance environmental conditions: An introduction J. Anim. Sci.  77: 427– 429. Google Scholar CrossRef Search ADS   Adeola, O., and N. L. Bajjalieh 1997. 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TI - Corn expressing an Escherichia coli-derived phytase gene: A proof-of-concept nutritional study in pigs
JF - Journal of Animal Science
DO - 10.2527/jas.2007-0037
DA - 2007-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/corn-expressing-an-escherichia-coli-derived-phytase-gene-a-proof-of-U7Uh2P06vd
SP - 1946
EP - 1952
VL - 85
IS - 8
DP - DeepDyve
ER -