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An Escherichia coli phytase expressed in yeast effectively replaces inorganic phosphorus for finishing pigs and laying hens¹

N. R. Augspurger^,2, D. M. Webel and D. H. Baker^*

JBS United Inc., Sheridan, IN 46069; and ^* Department of Animal Sciences, University of Illinois, Urbana 61801

	Abstract

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Two experiments determined the efficacy of an Escherichia coli phytase (ECP) added to P-deficient, corn-soybean meal diets fed to finishing pigs and second-cycle laying hens. Sixty finishing pigs (49 ± 0.9 kg) were formed into blocks within sex based on weight and ancestry and allotted to a P-deficient diet unsupplemented or supplemented with 0.10% inorganic P (iP) from KH₂PO₄ or ECP at 250, 500, 1,000, or 10,000 phytase units (FTU)/kg. Individually fed pigs were allowed ad libitum access to the experimental diets until a BW of 120 ± 3 kg was achieved, at which time the pigs were euthanized and the left fibula and fourth metatarsal were excised for determination of bone ash. Pigs were fed a 2-phase diet program for early- and late-finishing pigs; available P in the basal diets was set 0.10% below the requirement. Dietary supplementation of iP or ECP increased weight gain (P < 0.10) and G:F (P < 0.01); performance was not different (P > 0.13) among the phytase-supplemented groups. Fibula ash was greatest (P < 0.01) for pigs fed diets containing 10,000 FTU of ECP/kg. Two hundred forty second-cycle hens were allotted to a P-deficient diet or a P-deficient diet supplemented with 0.10% iP or ECP at 150, 300, or 10,000 FTU/kg for a 12-wk experiment. The basal diet was a corn-soybean meal diet with no added iP (17% CP, 3.8% Ca, 0.10% available P). Hens fed the P-deficient diet were removed from the experiment after 4 wk due to poor egg production. Supplementation of iP or ECP resulted in increased (P < 0.01) feed intake, egg weight, and egg production during the first 4 wk. During the entire 12-wk period, there were no differences (P > 0.28) between the iP- and ECP-supplemented groups in feed intake, egg weight, or egg production. These experiments reveal that ECP was as efficacious as supplemental iP and that supplementation of an excess dose of ECP was efficacious and without negative effects in finishing pigs and laying hens.

Key Words: hen • phosphorus • phytase • pig

	INTRODUCTION

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Phytase increases the bioavailability of P from plant ingredients for pigs (Cromwell et al., 1993; Kemme et al., 1997) and chickens (Nelson et al., 1971; Perney et al., 1993; Boling et al., 2000a) by cleaving phosphate groups from the phytate complex. Utilization of this technology can lower the amount of P excreted, which can leach into water systems and stimulate eutrophication of surface water areas through land application of animal wastes (Ryden et al., 1973). Additionally, phytase supplementation reduces the need for inorganic P (iP), which is the third-most expensive component of nonruminant diets, after energy and protein. Recoverable mineral P is also becoming a scarce resource and will last only for an estimated 80 yr at the current rate of extraction (Smil, 2000).

Recent work with pigs and chicks from our laboratory has shown greater P-releasing efficacy for an E. coli-derived phytase (ECP) than that for fungal phytases at similar phytase concentrations. The ECP released approximately 0.20% P at 1,000 phytase units (FTU)/kg (Augspurger et al., 2003, 2004; Augspurger and Baker, 2004), which would be able to eliminate the need for iP in some situations in pig and poultry nutrition. The pH range for optimum activity of this phytase is 2.5 to 3.5 (Rodriguez et al., 1999a). These pH values are similar to the pH of the pig stomach and the chicken proventriculus and gizzard (Riley and Austic, 1984; Yi and Kornegay, 1996; Radcliffe et al., 1998), which has been shown to be the primary site of phytase action (Yi and Kornegay, 1996; Pagano et al., 2005). This E. coli-derived phytase has also been shown to be more resistant than an Aspergillus niger-derived phytase to the gastric protease, pepsin (Rodriguez et al., 1999b).

The objective of the research presented herein was to investigate the efficacy of increasing activities of ECP to improve performance and bone mineralization in finishing pigs and laying hens, as well as to test the efficacy and safety of very high activities of ECP in pigs and hens. We hypothesized that high dietary activities of phytase would not adversely affect growth performance or bone mineralization relative to lower activity concentrations of phytase in pigs and laying hens.

	MATERIALS AND METHODS

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Phytase

The E. coli phytase (ECP; OptiPhos, JBS United Inc., Sheridan, IN) was isolated from a strain of E. coli found in the colon of pigs (Rodriguez et al., 1999a). The enzyme was expressed and produced through a yeast expression system that has been described previously (Rodriguez et al., 1999a). The purified phytase was assayed for activity before use as previously described (Han et al., 1999). One unit of phytase activity (FTU) was defined as the amount of enzyme required to release 1 µmol of iP per min from sodium phytate at 37°C.

Pig Experiment

The University of Illinois Institutional Animal Care and Use Committee approved all experimental procedures involving animals. This experiment utilized 60 AusGene pigs (30 barrows and 30 gilts) with a BW of 49.0 ± 0.9 (SD) kg. The pigs were selected from a larger group and were moved into a facility equipped with individual pens (1.1 x 1.9 m) and given a 1-wk period to acclimate to the facility. The pigs were then fasted for 12 h, weighed, and allocated to blocks within sex based on weight and ancestry, from which they were randomly allotted to dietary treatments. Pigs were given ad libitum access to their experimental diet and water until the average BW of the pigs within a block was 120 ± 3 (SD) kg. At that point, the pigs were fasted overnight before their final BW was recorded. The pigs were euthanized by intravenous injection of an overdose of a euthanasia solution (86 mg of sodium pentobarbital/kg of BW and 11 mg of sodium phytoin/kg of BW) for collection of the fibula and fourth metatarsal bones from the left leg. These bones were cleaned of adhering tissue by autoclaving for 20 min, dried at 105°C for 24 h, and then ashed in a muffle furnace at 600°C for 24 h. Bone ash is presented as grams per pig and as percent of dried bone (Peter et al., 2001; Snow et al., 2004; Jendza et al., 2005).

The pigs were fed a 2-phase dietary program based on BW ranges and amino acid requirements for early-and late-finishing pigs (NRC, 1998). The diets were switched when the average BW of the pigs within a block within sex reached 80 ± 3 (SD) kg. The basal diets for each phase were based on corn and soybean meal and were formulated to be adequate in all nutrients except available P, which was set at 0.10% available P below the NRC (1998) requirement (Table 1). Dietary treatments were the P-deficient basal diet, or the basal diet supplemented with 0.10% iP from reagent-grade KH₂PO₄ (Sigma Aldrich, St. Louis, MO), or with ECP at 250, 500, 1,000, or 10,000 FTU/kg. Supplementation of ECP at 10,000 FTU/kg was included to examine the efficacy of a dose of phytase that exceeded 10 to 20 times that of the dose levels of 1,000 and 500 FTU/kg, respectively. This treatment was included as part of the United States Food & Drug Administration requirements for product approval.

The University of Illinois Institutional Animal Care and Use Committee approved all experimental procedures involving animals. Two hundred forty second-cycle Single-Comb White Leghorn hens (Dekalb Sigma) were randomly allotted to 1 of 5 dietary treatments in a 12-wk feeding trial. The hens were previously fed a corn-soybean meal, layer diet that met or exceeded the NRC (1994) requirements and contained 17% CP, 3.8% Ca, and 0.45% available (nonphytate) P; as-fed basis. The hens were housed in a completely enclosed, mechanically ventilated, cage-layer building, where they were exposed to a daily lighting schedule of 16 h light:8 h dark. The experiment was begun in late November and ended in late February. At the beginning of the experiment, the hens were weighed and assigned to treatment according to a completely randomized design. Four groups of 12 hens per group were each allowed ad libitum access to their experimental diet and water for 12 wk. One experimental unit consisted of 4 adjacent cages (30 x 46 cm) of 3 hens per cage, all fed from a common feeder; there were a total of 48 hens (16 cages) per treatment group. These experimental procedures are similar to previous work by Boling et al. (2000a, b) and Wu et al. (2006).

The basal diet was a corn-soybean meal diet with no supplemental iP (Table 1) and was calculated to contain an estimated 0.10% available (nonphytate) P (0.28% analyzed total P) and 3.8% Ca (Boling et al., 2000a,b; Snow et al., 2004). Dietary treatments consisted of the P-deficient basal diet or the basal diet supplemented with 0.10% iP from reagent-grade KH₂PO₄, or ECP at 150, 300, or 10,000 FTU/kg. The P-supplemented diet was calculated to provide 240 mg of available P per day for laying hens consuming 120 g of feed per day, a surfeit level relative to the requirement of 209 mg estimated by Snow et al. (2004) for second-cycle hens. As in the pig experiment, the treatment diet containing 10,000 FTU of ECP/kg of BW was included to examine the efficacy of a 33- to 66-fold excess dose of phytase relative to standard doses of 300 and 150 FTU/kg as part of United States Food & Drug Administration requirements for product approval. The diets were mixed every 4 wk.

Diet Analysis

Crude protein was determined in triplicate for all basal diets by the macro-Kjeldahl method (AOAC, 1995), and amino acids were quantified chromatographically (Beckman model 6300; Beckman Instruments, Palo Alto, CA) after a 24-h hydrolysis in HCl (Spackman et al., 1958). Basal diet samples were dry-ashed in a 600°C muffle furnace for 4 h and then wet-ashed using HCl and HNO₃ (AOAC, 1995; Augspurger et al., 2003). Total P was quantified colorimetrically in each basal diet according to AOAC (1995) procedures.

Statistical Analysis

Data from the pig experiment were analyzed according to ANOVA procedures appropriate for a randomized, complete block design with a split-plot arrangement of treatments (Hahn et al., 1995; Steel et al., 1997) using the MIXED procedure (SAS Inst. Inc., Cary, NC). Sex (1 df) was used as the main plot, and diet (5 df) was the subplot. The sex x diet interaction was not significant (P > 0.05) for any of the criteria evaluated. Therefore, the replicate x diet interaction (40 df) was used as the appropriate error term to evaluate diet main effects. Data from the laying hen experiment were analyzed according to ANOVA procedures appropriate for a completely randomized design using the GLM procedure of SAS.

Orthogonal, single degree-of-freedom comparisons were used to evaluate treatment differences in both experiments; namely, 1) P-deficient vs. supplemented diets; 2) iP- vs. phytase-supplemented diets; 3) 250 vs. 500, 1,000, and 10,000 FTU/kg phytase; 4) 500 vs. 1,000 and 10,000 FTU/kg; and 5) 1,000 vs. 10,000 FTU/kg phytase. For the laying hen experiment, egg production (%) during wk 1 was included as a covariable for analysis of weekly and cumulative egg production (%). An alpha level of 0.05 was used to denote significance.

	RESULTS

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Pig Experiment

Barrows grew 16% faster (P = 0.003) and consumed 17% more (P = 0.003) feed than the gilts (data not shown), but there were no differences (P > 0.15) between them for gain/feed ratio or any of the bone ash measurements. Dietary supplementation of iP or ECP increased (basal vs. supplemented diets, P < 0.01) weight gain and increased (basal vs. supplemented diets, P < 0.01) gain/feed (Table 2). Growth performance (weight gain and gain/feed) of pigs fed diets containing supplemental ECP at 250 FTU/kg was not different (250 vs. higher FTU/kg, P = 0.407 for weight gain, P = 0.136 for gain/feed) from those fed the higher concentrations. All bone ash variables responded (basal vs. supplemented diets, P < 0.001) positively to supplemental iP and phytase (Table 2). Fibula ash (g) was greatest (1,000 vs. 10,000 FTU/kg, P < 0.01) for pigs fed diets containing 10,000 FTU of ECP/kg. Metatarsal ash (g) was greater (250 vs. higher FTU/kg, P < 0.05) for pigs fed diets containing ECP at 500, 1,000, and 10,000 FTU/kg than for those pigs fed diets containing 250 FTU/kg.

Hens on the P-deficient diet were removed from the experiment after 4 wk due to poor feed intake and egg production. During the first 4 wk, feed intake, egg production, and egg weight were all lowest (P-deficient vs. supplemented diets, P < 0.001) for hens fed the P-deficient basal diet (Table 3). Feed intake of the hens on the iP- or phytase-supplemented treatments was similar to expected levels for second-cycle hens in this production system (Boling et al., 2000a; Snow et al., 2004). There were no differences in feed intake (iP vs. ECP-supplemented diets, P = 0.33) or egg production (iP vs. ECP-supplemented diets, P = 0.77) among the supplemented diets during the last 8 wk (Table 3). Over the course of the entire 12 wk, feed intake, egg production, and egg weight were similar (iP vs. ECP-supplemented diets, P > 0.28) for hens fed iP- or ECP-supplemented diets (Table 3).

	DISCUSSION

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The production responses of the laying hens to dietary supplementation of iP or phytase to a P-deficient diet were similar to responses reported by van der Klis et al. (1997) and Boling et al. (2000b), who reported egg production of first-cycle hens fed diets containing 250 and 100 FTU/kg of phytase, respectively, were similar to 0.10% iP supplementation. Increases in dietary phytase activity, however, did not elicit further increases in egg production or in any other response criteria. A similar pattern of response was reported for tibia weight, in that no further increase in tibia weight was seen above the lowest phytase concentration (van der Klis et al., 1997). Additionally, dietary supplementation of phytase to P-deficient laying hen diets restored egg production performance to levels similar to the P-adequate diets in first-cycle hens (Francesch et al., 2005).

Both egg production and egg weight in hens fed diets with 0.10% supplemental iP or phytase were similar to previous data from second-cycle layers fed nutritionally adequate diets (Boling et al., 2000a; Snow et al., 2004). Snow et al. (2004), however, reported that second-cycle laying hens have a higher available P requirement than younger first-cycle hens. Thus, first-cycle hens required about 0.18% available P (198 mg/d), whereas second-cycle hens required in excess of 0.20% available P (209 mg/d). Our diet with 0.10% supplemental iP contained an estimated 0.20% available P, which was somewhat below the requirement estimated by Snow et al. (2004) for second-cycle layers, but feed intake was such that the diet provided 242 mg of available P per day, which was greater than the daily P requirement of 209 mg. Hence, even though our egg production results appeared normal, we cannot conclude that our iP or phytase-supplemented diets had, in fact, met the P requirement. Indeed, this was not the intent of our study. Instead, our objective was to ascertain whether phytase-supplemented diets would allow the same egg laying performance as that occurring with 0.10% iP addition. The data in Table 3 suggest that 150 to 300 FTU/kg of ECP was as effective as 0.10% iP in providing P for egg production.

The poor feed intake and egg production of these second-cycle hens fed the basal diet (0.10% estimated available P) has been shown previously in laying hens. Punna and Roland (1999) reported a 17% reduction in feed intake and a 44% reduction in egg production in first-cycle hens fed a diet containing 0.10% available P compared with hens consuming the 0.10% available P diet with 300 FTU/kg phytase (Natuphos) from 21 to 36 wk of age. Egg production declined further to below 20% at 39 wk of age. Boling et al. (2000b) also showed reduced production in first-cycle hens fed diets containing only 0.10% available P, so much so that those hens were removed from the experiment and switched to a diet containing 0.45% available P. Interestingly, egg production of those hens previously fed the low-P diet increased to a level similar to that of the hens receiving the other treatments within 2 wk after returning them to the high-P diet. Snow et al. (2004) also showed poor egg production in hens fed diets containing 0.10% available P, but Scott et al. (1999) and Jalal and Scheideler (2001) both reported little effect of low dietary available P on feed intake or egg production.

The data from these 2 experiments are qualitative and do not reveal a specific efficacy estimate; indeed, the experiments were not designed to determine a quantitative efficacy estimate. The small difference between the P concentrations in the P-deficient and P-adequate diets in these experiments [0.10% P separates the deficient and a P-adequate diet in the pig experiment (NRC, 1998)] precludes drawing an objective conclusion as to the efficacy of a particular dose of phytase. Just because the responses to a certain activity concentration of phytase are similar to those of 0.10% iP supplementation does not mean that 0.10% P was released from the diet. The close proximity of the basal dietary available P concentration and the requirement of finishing pigs (NRC, 1998) and laying hens (NRC, 1994) prevents one from assuming that the response of any given criterion is linear between the available P concentrations of the basal diet and the iP-supplemented diet. This is due to the curvilinear nature of the response as the supply of any nutrient nears the requirement and evidence that the requirement for available P to support growth performance is less than that required to support or maximize bone mineralization (NRC, 1998). In the laying hen, this issue is further complicated due to recent evidence (Boling et al., 2000a; Snow et al., 2004) that the available P requirement may be substantially lower than previous information has suggested (NRC, 1994).

An ECP supplemented to a corn-soybean meal diet at 10,000 FTU/kg elicited responses that were similar to lower concentrations of phytase in both pigs and hens. There is a paucity of data in the literature concerning the effectiveness of an excess dose of phytase. Recent data from our laboratory, however, suggest that concentrations of ECP up to 5,000 and 10,000 FTU/kg were efficacious for improving weight gain and bone ash of young chicks fed a P-deficient corn-soybean meal diet (Augspurger and Baker, 2004). Moreover, tibia ash was not further increased when concentrations above 1,000 FTU/kg were fed, indicating that 1,000 FTU of ECP/kg elicited the maximum release of P from the diet. Shirley and Edwards (2003) showed that 12,000 FTU/kg of a fungal phytase added to a P-deficient diet produced bone ash and performance responses that were similar to a positive control, indicating near complete digestion of dietary phytate-phosphorus.

Our work herein shows that an E. coli-derived phytase can safely replace 0.10% inorganic P supplementation in finishing pigs and laying hens without sacrifice of any growth or production responses. Phytase concentrations as high as 10,000 FTU/kg were observed to be both efficacious and safe. Supplementation of an ECP to diets for finishing pigs and laying hens that contain no supplemental inorganic P resulted in performance and egg production that were equal to that occurring with P-supplemented diets.

	Footnotes

 
¹ Appreciation is expressed to Eric Parr and Lindsey Wilson of JBS United Inc. for daily management of the pigs.

² Corresponding author: nathan.augspurger{at}jbsunited.com

Received for publication May 25, 2006. Accepted for publication January 12, 2007.

	LITERATURE CITED

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