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J. Anim. Sci. 2006. 84:1147-1158
© 2006 American Society of Animal Science

ANIMAL NUTRITION

A genetically engineered Escherichia coli phytase improves nutrient utilization, growth performance, and bone strength of young swine fed diets deficient in available phosphorus1

T. L. Veum*,2, D. W. Bollinger*, C. E. Buff* and M. R. Bedford{dagger}

* Department of Animal Sciences, University of Missouri, Columbia 65211; and and {dagger} Zymetrics, Inc., Golden Valley, MN 55427


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
A 28-d experiment was conducted using 126 crossbred barrows to evaluate the addition of a genetically engineered Escherichia coli phytase to diets that were 0.15% deficient in available P. Growth performance, bone strength, ash weight, and the apparent absorption of P, Ca, Mg, N, energy, DM, Zn, Fe, and Cu were the response criteria. The pigs (2 pigs/pen) averaged 7.61 kg of BW and 30 d of age initially. The low-P basal diet was supplemented with 0, 100, 500, 2,500, or 12,500 units (U) of E. coli phytase/kg of diet, or 500 U of Peniophora lycii phytase/kg of diet. The positive control (PC) diet was adequate in available P. Pigs were fed the diets in meal form. Fecal samples were collected from each pig from d 22 to 27 of the experiment. There were linear and quadratic increases (P < 0.001) in 28-d growth performance (ADFI, ADG, and G:F), bone breaking strength and ash weight, and the apparent absorption (g/d and %) of P, Ca, and Mg (P ≤ 0.01 for quadratic) with increasing concentrations of E. coli phytase. Pigs fed the low-P diets containing 2,500 or 12,500 U/kg of E. coli phytase had greater (P ≤ 0.01 or P < 0.001, respectively) values for growth performance, bone breaking strength and ash weight, and the apparent absorption (g/d and %) of P, Ca, and Mg than pigs fed the PC diet. The addition of E. coli phytase did not increase the apparent percentage absorption of N, GE, DM, Zn, Fe, or Cu. There were no differences in the efficacy of the E. coli or P. lycii phytase enzymes at 500 U/kg of low-P diet for any criterion measured. In conclusion, there were linear increases in growth performance, bone breaking strength and ash weight, and the apparent absorption of P, Ca, and Mg with increasing addition of E. coli phytase up to 12,500 U/kg of diet. Also, all of these criteria were greater for pigs fed the low-P diets containing 2,500 or 12,500 U of E. coli phytase/kg than for pigs fed the PC diet. The addition of 500, 2,500, or 12,500 U of E. coli phytase/kg of low-P diet reduced P excretion (g/d) in manure by 35, 42, and 61%, respectively, compared with pigs fed the PC diet.

Key Words: nutrient absorption • nutrient excretion • phosphorus • phytase • pig • swine


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
About 60 to 80% of the P in cereal grains and oil seeds is bound in the form of phytic acid or phytate (myo-inositol hexakis-dihydrogen phosphate; Maga, 1982Go; Lott et al., 2000Go). Phytate is poorly digested by swine because they produce little to no intestinal phytase, the enzyme required for phytate hydrolysis (Pointillart et al., 1984Go, 1987Go; Reddy et al., 1989Go). Consequently, most of the phytate P is excreted in the manure. However, P absorption is increased and P excretion reduced by the use of a commercial Aspergillus niger phytase (Natuphos; Harper et al., 1997Go; Liu et al., 1998Go; Stahl et al., 2000Go) or a Peniophora lycii (Ronozyme) phytase (Lassen et al., 2001Go; Augspurger et al., 2003Go; Stahl et al., 2004Go) in nonruminant diets. Soaking a swine diet containing A. niger phytase before feeding increased the phytase efficacy 2-fold (Liu et al., 1997Go). The potential development of low phytic acid grains (Raboy et al., 2001Go; Veum et al., 2001Go, 2002Go) and soybeans (Wilcox et al., 2000Go) may also increase P digestibility and reduce P excretion by nonruminants.

An Escherichia coli phytase with good thermostability, and expressed in Pichia pastoris yeast, effectively increased P bioavailability for weanling pigs (Stahl et al., 2000Go; Augspurger et al., 2003Go). Another genetically engineered E. coli phytase with enhanced thermostability, also expressed in P. pastoris yeast, was efficacious in increasing the bioavailability of P in broiler chicks (Onyango et al., 2004Go, 2005Go; Silversides et al., 2004Go). The objective of this experiment was to evaluate the efficacy of this new E. coli phytase at increasing concentrations in low-P diets fed to weanling swine with growth performance, bone strength and ash weight, and the apparent absorption and excretion of P, Ca, Mg, N, GE, DM, Zn, Fe, and Cu as the response criteria.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
This experiment was approved by the University of Missouri Animal Care and Use Committee.

Phytase Sources
The 2 phytase sources used in this experiment were a bacterial phytase from E. coli (Quantum phytase; Zymetrics, Inc., a division of Syngenta, Golden Valley, MN) and a fungal phytase from P. lycii (Ronozyme phytase; Roche Vitamins Inc., Parsippany, NJ). The E. coli phytase, with a pH optimum of 4.5, was genetically modified to enhance its thermal stability and cloned into a P. pastoris yeast host for production (Faber et al., 1995Go; Onyango et al., 2004Go, 2005Go). The P. pastoris yeast host also enhanced the thermal stability of the E. coli phytase at pelleting temperatures above 80°C (Silversides et al., 2004Go). The P. lycii phytase, with a pH optimum of 4.0 to 5.0, was cloned into a genetically modified Aspergillus oryzae host for production (Lassen et al., 2001Go). Both phytases initiate dephosphorylation of phytate at the 6-position of the inositol ring.

The phytase premixes were analyzed for phytase activity (Engelen et al., 1994Go, 2001Go) before diet mixing. The phytase activities (triplicate analyses) were 2,844 phytase units (U)/g for the E. coli phytase, and 3,000 phytase U/g for the P. lycii phytase. One phytase unit is defined as the amount of enzyme required to release 1.0 µmol of inorganic P per min from 5.1 mM sodium phytate at 37°C and pH 5.5.

Animals and Housing
High-health, isowean barrows (n = 126) from a multiplier herd (Pig Improvement Co., Franklin, KY) were weaned at an average BW of 4.93 ± 0.70 kg and age of 16 ± 1 d, ear-tagged, and transported to an enclosed nursery building at the University of Missouri. Pigs were placed in elevated pens (1.20 x 1.20 m) that had a nipple drinker, a stainless steel self-feeder, and woven wire flooring. There was a 14-d acclimation period before beginning the 28-d experiment that had a period 1 (d 14 to 28 postweaning) and a period 2 (d 28 to 42 postweaning). Pigs averaged 7.61 ± 0.02 kg of BW and 30 ± 1 d of age at the beginning of the experiment. There were 9 weight blocks (pens)/treatment, with 2 pigs in each pen. Room temperature was maintained at 32 ± 1°C during the 14-d acclimation period, and was lowered by 1.5°C each week during the 28-d experiment, with 12 h/d of light beginning at 0600. Pigs and feeders were inspected daily during the acclimation and experimental periods.

Dietary Treatments
At weaning, all pigs were fed the same diet during the 14-d acclimation period. The acclimation diet contained 38.0% ground yellow corn, 21.3% soybean meal (SBM, 48% CP), 20.0% spray-dried whey, 10.0% lactose, 6.0% spray-dried animal plasma, 2.0% corn oil, and mineral and vitamin supplementation to meet NRC (1998)Go nutrient requirements for 5- to 10-kg pigs. Published values (NRC, 1998Go) for nutrients were used to formulate the period 1 and 2 corn-SBM experimental diets (Table 1Go). However, analyzed nutrient values from the period 2 diets were used to determine nutrient absorption. The low-P basal diets were formulated to be 0.15% below the NRC (1998)Go requirement for available P (aP), whereas the positive control (PC) diets met the NRC (1998)Go aP requirement. For both the low-P basal and the PC diets, calcium concentrations were formulated to be approximately 0.05% below the NRC (1998)Go requirement.


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  		Table 1. Ingredient and chemical composition (%) of air-dry basal diets, as-fed basis1
 
Phytase treatments were chosen to examine the relationship between dose and response criteria with growing swine. An evaluation of 296 broiler chick experiments conducted worldwide indicated that microbial phytase at 2,500 U/kg of low-P diet tripled the improvement in feed efficiency compared with the commonly used (average) industry concentration of 634 U/kg of diet (Rosen, 2002Go). Therefore, we tested our E. coli phytase for linear and quadratic responses, beginning with 100 phytase U/kg of diet and sequentially increasing each concentration 5-fold up to 12,500 phytase U/kg of diet.

Seven dietary treatments were used. Six phytase treatments were made by subdividing batch mixes of the low-P period 1 and 2 basal diets and adding a premix containing 0 (negative control), 100, 500, 2,500, or 12,500 U of E. coli phytase/kg of diet (EC0, EC100, EC500, EC2500, or EC12500, respectively) or 500 U of P. lycii phytase/kg of diet (PL500). Our seventh dietary treatment was the PC. Period 2 diets contained 0.05% chromic oxide as an indigestible indicator to determine nutrient digestibilities. The diets were prepared in meal form and stored at 4 to 8°C before feeding.

Measurements
Pigs were weighed individually on d 0 (beginning of the 28-d experiment), 14, 21, and 28. Pen feed consumption was determined for periods 1 and 2, and for the 6-d fecal collection period. Fecal grab samples were collected from both pigs in each pen once daily from d 22 to 27 of the experiment and frozen in plastic freezer bags until analyzed. The fecal collections for each pen were thawed, pooled, and air-dried at 55°C. The dried fecal samples and samples of each diet were ground to pass through a 1.0-mm screen before analysis. Quadruplicate subsamples of the low-P basal batch mix and the PC diets, and duplicate subsamples of feces were digested using a wet ash procedure (AOAC, 1990Go). Digests were analyzed for the concentration of total P (tP) by the colorimetric molybdovanadate method (Spectra Rainbow Microplate Reader, Tecan, Inc., Durham, NC) and for the concentrations of Ca, Mg, Fe, Cu, Zn, and Cr by atomic absorption spectrophotometry (Spector AA-30, Varian Analytical Instruments, San Fernando, CA). Quadruplicate subsamples of diet and duplicate subsamples of feces were analyzed for N and DM (AOAC, 1990Go), and GE by oxygen bomb calorimetry (Parr Instrument Co., Moline, IL). Analyzed values for P, Ca, Mg, N, GE, DM, Zn, Fe, and Cu for the diets (Table 1Go) and fecal samples were used to determine nutrient digestibilities. There was good agreement between the calculated and analyzed dietary values for Ca and P.

On d 28 of the experiment, the pigs were killed (stunned by captive bolt followed by exsanguination). The right-front foot of each pig was removed and refrigerated at 2°C. The third metacarpal bone was excised and cleaned of all adhering tissue within 3 d for bone size and weight measurements, and the determination of breaking strength and ash weight. A caliper (Model CDS6, Mitutoyo Corp., Japan) was used to measure metacarpal bone length and the midshaft widths at the narrowest and widest points. Breaking strength of the fresh bones was determined using an Instron testing machine (Model TML, Instron Corp., Canton, MA), similar to the procedure described by Crenshaw (1986)Go. Force was applied to the center of the bone, which was held by 2 supports spaced 3.0 cm apart. After the determination of breaking strength, the bones were wrapped with cheesecloth, boiled in deionized water for 2 h, dried at 55°C for 24 h, and extracted with ethyl ether for 4 d. Ash weight was determined after the fat-free bones were dried at 55 and 100°C for 18 and 2 h, respectively, and ashed in a muffle furnace at 600°C for 16 h (AOAC, 1990Go).

Statistics
All data were analyzed by ANOVA as a randomized complete block design (Snedecor and Cochran, 1989Go) using SAS (SAS Inst., Inc., Cary, NC). Pens of pigs were the experimental units. The planned single df comparisons were the linear and quadratic responses for diets EC0 to EC12500, and the comparisons of PC vs. EC0, PC vs. EC500, PC vs. EC2500, PC vs. EC12500, and EC500 vs. PL500. The PC diet was not compared with EC100 because the concentration of phytase in EC100 was considered to be inadequate to enhance the response criteria compared with the other phytase treatments and the PC diet. Significance was designated as P ≤ 0.05 with a trend being between P ≥ 0.06 and P ≤ 0.10.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 
Growth Performance (Tables 2Go and 3Go)
One pig on diet EC500 died during the experiment, and that mortality was deemed unrelated to the experimental treatment. For period 1 (d 0 to 14 of the experiment), there were linear and quadratic increases (P < 0.01) in ADG and G:F with increasing dietary concentration of E. coli phytase from diets EC0 to EC12500. Pigs fed diets EC2500 or EC12500 had a greater (P = 0.03) period 1 ADG than pigs fed the PC diet. Pigs fed diet EC12500 also had a greater (P = 0.07) period 1 G:F than pigs fed the PC diet. However, pigs fed the PC diet had a greater (P = 0.03) period 1 G:F than pigs fed the negative control diet EC0. Pigs fed diets EC500 or PL500 did not differ from each other in period 1 growth performance response criteria.


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  		Table 2. Effect of a modified Escherichia coli phytase on pig growth performance1
 

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  		Table 3. Statistical significance for pig growth performance criteria1
 
For period 2 (d 14 to 28) and overall (d 0 to 28), there were linear (P < 0.001) and quadratic (P ≤ 0.01) increases in ADG, ADFI, and G:F with increasing concentration of E. coli phytase. Pigs fed diets EC2500 or EC12500 had greater (P ≤ 0.05) period 2 and overall ADG and ADFI than pigs fed the PC diet. Pigs fed diet EC12500 also had a greater (P < 0.001) period 2 and overall G:F than pigs fed the PC diet. However, pigs fed the PC diet had a greater (P ≤ 0.03) period 2 and overall ADG, ADFI, and G:F than pigs fed diet EC0, and a greater (P ≤ 0.05) period 2 ADG and ADFI, and overall ADG than pigs fed diet EC500. Pigs fed diets EC500 or PL500 did not differ in period 2 or overall growth performance criteria.

Metacarpal Bone Characteristics (Tables 4Go and 5Go)
There were linear and quadratic increases (P < 0.001) in metacarpal breaking strength, fresh and fat-free dry bone weight, bone ash weight, and most bone length and width measurements with increasing dietary concentration of E. coli phytase. Pigs fed diets EC2500 or EC12500 had greater (P ≤ 0.01 or P ≤ 0.001, respectively) metacarpal breaking strength, fat-free dry bone weight, and ash weight than pigs fed the PC diet. Metacarpal breaking strength and all other bone criteria were greater (P < 0.001) for pigs fed the PC diet than for pigs fed diet EC0. Metacarpal breaking strength and ash weight were also greater (P ≤ 0.01) for pigs fed the PC diet than for pigs fed diet EC500. Pigs fed diets EC500 or PL500 did not differ from each other for any metacarpal bone response criteria measured.


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  		Table 4. Effect of a modified Escherichia coli phytase on metacarpal bone characteristics
 

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  		Table 5. Statistical significance for metacarpal bone characteristics1
 
Apparent Absorption and Excretion of P, Ca, Mg, N, Energy, and DM (Tables 6Go and 7Go)
There were linear and quadratic increases (P < 0.001) in the daily intakes of P, Ca, Mg, N, and GE for diets EC0 to EC12500 during the fecal collections from d 22 to 27 because ADFI increased (P < 0.001) with increasing dietary concentration of E. coli phytase. Pigs fed diets EC2500 or EC12500 also had greater (P ≤ 0.01 or P < 0.001, respectively) ADFI than pigs fed the PC diet and consequently had greater daily intakes of Ca, Mg, N, and GE than pigs fed the PC diet. Pigs fed the PC diet, however, had a greater (P < 0.001) ADFI than pigs fed diet EC0 (P < 0.001) or diet EC500 (P = 0.03).


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  		Table 6. Effect of a modified Escherichia coli phytase on the apparent absorption and excretion of phosphorus, calcium, magnesium, nitrogen, energy, and dry matter digestibility1
 

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  		Table 7. Statistical significance for the apparent absorption and excretion of phosphorus, calcium, magnesium, nitrogen, energy, and dry matter digestibility1
 
There were linear and quadratic increases (P < 0.001) in the grams of P, Ca, and Mg absorbed per day and linear decreases (P < 0.001) in the grams of P excreted per day with increasing dietary concentration of E. coli phytase. Expressed as a percentage of intake, there were linear and quadratic increases (P < 0.001) in the absorption of P, Ca, and Mg (P = 0.01 for Mg quadratic) with equivalent decreases in excretion. Pigs fed diets EC2500 or EC12500 absorbed more (g/d, P = 0.01 or P < 0.001, respectively) P and more (g/d, P < 0.001) Ca and Mg than pigs fed the PC diet. Pigs fed diets EC2500 or EC12500 also excreted less (g/d, P < 0.001) P and Ca than pigs fed the PC diet. The percentages of P, Ca, and Mg absorbed and excreted, respectively, were also greater and lower (P < 0.001) for pigs fed diets EC2500 or EC12500 than for pigs fed the PC diet.

Pigs fed the PC diet absorbed and excreted more (g/d, P < 0.001) P than pigs fed diets EC0 or EC500. Pigs fed the PC diet also absorbed more (g/d, P ≤ 0.04) Ca and Mg than pigs fed diet EC0 and excreted more (g/d, P ≤ 0.02) Ca and Mg than pigs fed diets EC0 or EC500. However, expressed as a percentage of intake, pigs fed diet EC500 absorbed more and excreted less P (P = 0.06), Ca (P < 0.001), and Mg (P = 0.07) than pigs fed the PC diet. Pigs fed diets EC500 or PL500 did not differ in the absorption or excretion (g/d or % of intake) of P, Ca, or Mg.

For N and GE, there were linear and quadratic increases (g or Mcal/d, P < 0.001) in absorption and excretion with increasing concentration of E. coli phytase. Pigs fed diets EC2500 or EC12500 had greater (P = 0.01 or P < 0.001, respectively) intakes and absorption of N (g/d) and GE (Mcal/d), and a greater (P = 0.01) excretion of GE than pigs fed the PC diet. However, pigs fed the PC diet had greater intakes, absorption, and excretion of N and GE (g or Mcal/d) than pigs fed diet EC0 (P ≤ 0.001) or diet EC500 (P ≤ 0.10). Expressed as a percentage of intake, pigs fed the PC diet had lower and greater (P ≤ 0.01) percentages, respectively, of N absorbed and excreted than pigs fed diets EC0, EC500, or EC12500. For the percentages of GE absorbed and excreted, there were linear and quadratic responses (P = 0.02) with increasing concentration of E. coli phytase for diets EC0 to EC12500. There were no differences between pigs fed diets EC500 or PL500 in the absorption or excretion of N or GE. Dry matter digestibility (%) did not differ for any planned treatment comparison.

Apparent Absorption and Excretion of Zn, Fe, and Cu (Tables 8Go and 9Go)
There were linear and quadratic increases (mg/d, P < 0.001) in the intake and excretion of Zn, Fe, and Cu with increasing concentration of E. coli phytase. The increase in intake of these minerals is the result of an increase in ADFI with increasing E. coli phytase. There was a quadratic response (mg/d, P = 0.07) in Zn absorption, linear and quadratic increases (mg/d, P < 0.001) in Fe absorption, and a linear response (mg/d, P ≤ 0.03) in Cu absorption with increasing concentration of E. coli phytase. Pigs fed diets EC2500 or EC12500 had greater (mg/d, P ≤ 0.01) intakes of Zn and Cu, greater (mg/d, P ≤ 0.01) absorption of Zn and Fe, and greater (P ≤ 0.01) excretion of Cu and Zn (diet 5) than pigs fed the PC diet. However, pigs fed the PC diet had greater (mg/d, P ≤ 0.03) intakes and excretion of Zn, Fe, and Cu than pigs fed diets EC0 or EC500.


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  		Table 8. Effect of a modified Escherichia coli phytase on the apparent absorption and excretion of zinc, iron, and copper1
 

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  		Table 9. Statistical significance for the apparent absorption and excretion of zinc, iron, and copper1
 
Expressed as a percentage of intake, there were linear and quadratic increases (P < 0.01) in the percentages of Zn, Fe, and Cu excreted and corresponding decreases in the percentages absorbed with increasing concentrations of E. coli phytase. Pigs fed diets EC2500 or EC12500 had greater (P ≤ 0.001) percentages of Zn and Fe absorbed, and corresponding reductions in excretion (%), compared with pigs fed the PC diet. Pigs fed the PC diet had lower (P < 0.001) percentages of Zn and Fe absorption, and greater percentages of excretion, than pigs fed diets EC0 or EC500. Pigs fed the PC diet also had lower and greater (P < 0.001) percentages, respectively, of Cu absorption and excretion compared with pigs fed diets EC0 or EC500.


   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
IMPLICATIONS
 LITERATURE CITED
 
The E. coli phytase used in our experiment was very effective in hydrolyzing phytate in the low-P period 1 and 2 diets that were formulated to be 0.15% below the aP requirement for weanling swine NRC (1998)Go. There were linear increases (P < 0.001) in 28-d growth performance criteria, metacarpal strength and ash weight, and the absorption (g/d) of P, Ca, Mg, and N, and GE (kcal/d) with increasing concentrations of E. coli phytase up to our greatest level of 12,500 U/kg of diet. Because a plateau did not occur for these criteria at 12,500 U of E. coli phytase/kg of low-P diet, the maximum effective concentration of this E. coli phytase is unknown. Also, the responses for most of these criteria were greater (P ≤ 0.01) for pigs fed the low-P diets containing 2,500 or 12,500 U of E. coli phytase/kg than for pigs fed the PC diet that met the aP requirement. The results of our experiment confirm the efficacy of this E. coli phytase added at high dietary concentrations in low-P diets fed to weanling swine for 4 wk based on the response criteria reported.

Our results with young swine agree with the results of a chick experiment where chicks fed a low-P diet supplemented with 10,000 U/kg of a different E. coli-derived phytase had more (P < 0.05) absolute tibia ash than chicks fed a diet adequate in aP (Augspurger and Baker, 2004Go). Different E. coli-derived phytase products were also efficacious at concentrations from 250 to 1,200 U/kg of low-P diet fed to weanling swine, with increased growth performance, plasma inorganic P concentrations, and bone strength, and reduced plasma alkaline phosphatase activities (Stahl et al., 2000Go; Augspurger et al., 2003Go). Another E. coli-derived phytase added at 500 or 1,000 U/kg of low-P corn-SBM diet produced linear improvements in the apparent absorption of P in growing pigs, including the growth performance of starter, grower, and finishing pigs (Jendza et al., 2005Go). The apparent digestibilities of P, Ca, and Mg were also increased by adding an A. niger phytase to a low-P corn-SBM diet fed to growing-finishing swine (Kemme et al., 1999Go). An evaluation of different criteria used to estimate the bioavailability of P in feed phosphates for swine found that the apparent absorption of P was by far the best criterion, followed by bone and blood measurements, respectively (Dellaert et al., 1990Go).

Pigs fed the low-P diets in our experiment containing 2,500 or 12,500 U of E. coli phytase/kg consumed more feed (P ≤ 0.05), absorbed more (g/d and %, P ≤ 0.01) P, Ca, and Mg, and excreted less P and Ca than pigs fed the PC diet. Pigs fed the low-P diet containing 500 U of E. coli phytase/kg excreted less (g/d, P ≤ 0.02) P, Ca, Mg, and N, and had a greater (P ≤ 0.07) percentage absorption of those nutrients than pigs fed the PC diet. For P excretion, the addition of 500, 2,500, or 12,500 U of E. coli phytase/kg of diet reduced P excretion in manure by 35, 42, and 61%, respectively, compared with pigs fed the PC diet. Adding an A. niger phytase to low-P corn-SBM diets at 500 phytase U/kg reduced P excretion 30 to 40% at our station (Liu et al., 1997Go; 1998Go) and 31% at another station (Cromwell et al., 1995Go) compared with growing or growing-finishing swine fed PC diets. The solubility of P in swine manure may not be affected by adding phytase to low-P diets because almost all of the phytate in normal wild-type and low-phytate barley cultivars was hydrolyzed before excretion (Leytem et al., 2004Go), presumably by the intestinal microflora in the hindgut because swine have little to no production of phytase in the small intestine (Pointillart et al., 1984Go, 1987Go).

In our experiment, the addition of 500, 2,500, or 12,500 U of E. coli phytase/kg of low-P diet reduced Ca excretion 35, 34, and 37%, respectively, compared with pigs fed the PC diet, when dietary Ca concentration was standardized at 0.05% below NRC (1998)Go in our low-P and PC diets. High dietary Ca:tP ratios have a negative effect on P absorption (Adeola et al., 1998Go) and the efficiency of phytase to release P from phytate (Lantzsch et al., 1994Go). Lowering the Ca:tP ratio in low-P corn-SBM diets to 1.0:1 or 1.2:1 increased the efficacy of A. niger phytase for weaning and growing-finishing swine compared with greater Ca:tP ratios (Qian et al., 1996Go; Liu et al., 1998Go, 2000Go).

Pigs fed the PC diet in our experiment absorbed more (g/d, P < 0.001) P and had greater (P ≤ 0.05) values for overall ADG, bone strength, and bone ash weight than pigs fed the low-P diet containing 500 U of E. coli phytase/kg. The greater responses for pigs fed the PC diet may be explained by the fact that our low-P diets were 0.15% below the aP requirement for weanling swine. Augspurger et al. (2003)Go found that a different E. coli-derived phytase had a bioavailable P-release of 0.108% based on the linear regression of fibula ash on increasing inorganic P intake by weanling pigs. If the P-release efficiency of our E. coli phytase was equal to that of the E. coli-derived phytase tested in weanling pigs by Augspurger et al. (2003)Go, 500 U of our E. coli phytase/kg of diet would release 0.10 to 0.11% of aP from phytate (dietary basis), leaving a deficiency of 0.04 to 0.05% aP in our low-P diet compared with our PC diet. Stahl et al. (2000)Go found that the efficiency of P-release by an E. coli-derived phytase was equal to that of an A. niger phytase for weanling pigs based on growth performance, plasma inorganic P concentration, and pig mobility score. Harper et al. (1997)Go found that the P equivalency of 500 U of A. niger phytase/kg of diet relative to dicalcium phosphate was about 1.10 g of P/kg (0.11%) based on P digestibility and rib shear force of growing-finishing swine, similar to the P-release efficiency of the E. coli-derived phytase evaluated by Augspurger et al. (2003)Go.

In the current experiment the efficacy of our E. coli phytase at 500 U/kg was equal to that of the P. lycii phytase at 500 U/kg of low-P diet based on growth performance, bone strength and ash weight, and the apparent absorption of P, Ca, Mg, N, GE, and DM. Pigs fed our low-P diets containing 500 U of E. coli or P. lycii phytase/kg had growth performance and bone strength values similar to those reported in other experiments for weanling pigs fed low-P diets containing 500 U of either P. lycii or an E. coli-derived phytase (Gentile et al., 2003Go; Stahl et al., 2004Go).

The linear increases in the apparent absorption of N (g/d) and energy (Mcal/d) with increasing concentration of E. coli phytase in our experiment were a direct result of the increases in ADFI with increasing phytase concentration, because the percentages of N, GE, and DM absorbed were not increased by increasing concentration of E. coli phytase. The lack of an E. coli phytase effect on the percentage absorption of N and energy is in agreement with experiments in which A. niger phytase did not increase the apparent percentage absorption of N and energy in low-P corn-SBM diets fed to swine (Harper et al., 1997Go; Sands et al., 2001Go; Johnston et al., 2004Go) and in which A. niger phytase did not increase energy use of a corn-SBM diet by growing swine when carcass protein and fat accretion were the criteria (Shelton et al., 2003Go). Also, A. niger phytase did not increase the apparent ileal or total tract digestibilities of soybean protein or amino acids in semipurified diets fed to growing swine (Yi et al., 1996Go; Traylor et al., 2001Go), or in a high phytate diet containing rice bran fed to growing swine (Liao et al., 2005Go). Furthermore, an E. coli-derived phytase at dietary additions of 500 or 10,000 U/kg did not improve protein use from SBM or corn gluten meal by chicks (Augspurger and Baker, 2004Go).

In the current experiment, the linear increases (P ≤ 0.03) in the milligrams of Fe and Cu absorbed per day, and the quadratic increase (P = 0.07) in the milligrams of Zn absorbed per day are the result of increases in ADFI with increasing dietary concentration of E. coli phytase. Conversely, the percentages of Zn, Fe, and Cu absorption decreased (P < 0.001) with increasing dietary concentration of E. coli phytase. Our trace mineral premix provided these trace elements in excess of the requirement (2-fold for Zn and Fe, and 3-fold for Cu). The experimental test diet must be deficient in the nutrient that is being evaluated for bioavailability (Ammerman, 1995Go; Lewis and Bayley, 1995Go; Augspurger et al., 2003Go). In the current experiment, aP was deficient in our low-P test diets, Ca was 0.05% below NRC (1998)Go in all the diets, and all other nutrients were adequate or in excess. Therefore, we would not expect an increase in the percentage absorption of these trace elements by the addition of E. coli phytase because our test diets were not deficient in these nutrients. However, when corn-SBM test diets were deficient in 1 or more trace elements, A. niger phytase was effective in increasing trace element use by swine (Adeola, 1995Go; Stahl et al., 1999Go; Shelton et al., 2005Go).

In conclusion, the genetically engineered E. coli phytase evaluated in this experiment was very efficacious when fed at high concentrations, with linear increases in growth performance criteria, metacarpal strength and ash weight, and the absorption (g/d) of P, Ca, Mg, and N, and GE (Mcal/d) with increasing concentration of E. coli phytase up to 12,500 U/kg of diet. These criteria were also greater for swine fed the low-P diets containing 2,500 or 12,500 U of E. coli phytase/kg than for pigs fed the PC diet. The maximum effective concentration of this E. coli phytase is unknown because there was no plateau in the response criteria measured up to 12,500 U/kg of diet.


   IMPLICATIONS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
LITERATURE CITED
 
This genetically engineered Escherichia coli phytase was efficacious at concentrations up to 12,500 units per kilogram of low-phosphorus diet for growing swine, based on the response criteria of growth performance, bone strength and ash weight, and the apparent absorption of phosphorus, calcium, and magnesium. The maximum effective concentration of this Escherichia coli phytase is unknown. Response criteria for pigs fed the diets containing 2,500 or 12,500 units of Escherichia coli phytase per kilogram were greater than those of pigs fed the positive control diet, and reduced the excretion of phosphorus by 42 and 61%, respectively, compared with pigs fed the control diet. The efficacy of Escherichia coli phytase and that of Peniophora lycii phytase were equivalent at 500 phytase units per kilogram of diet. The use of Escherichia coli phytase in low-phosphorus diets fed to growing and finishing swine should greatly reduce phosphorus excretion in manure, and the environmental benefit will contribute to the sustainability of the swine industry worldwide.


   Footnotes
 
1 Supported in part by the Missouri Agric. Exp. Stn. and Zymetrics, Inc., Golden Valley, MN. We thank A. Kable, C. Kinnison, J. Lauer, R. Smiley, and L. Vanskike for assistance with animal care and data collection, and M. R. Ellersieck for assistance with statistical analysis. Back

2 Corresponding author: veumt{at}missouri.edu

Received for publication April 27, 2005. Accepted for publication December 7, 2005.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
 LITERATURE CITED
 


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