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ANIMAL NUTRITION |
* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054;
and
Danisco Animal Nutrition, Marlborough SN8 1XN, U.K.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Escherichia coli • Nutrient Balance • Phosphorus • Phytase • Pigs
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Phytate consists of an inositol ring binding up to six phosphate groups that, unless cleaved from the inositol ring, are unavailable for absorption (Maga, 1982). Exogenous supplementation of feeds with phytase of microbial origin has demonstrated the ability to increase P bioavailability and thus growth rates in pigs (Cromwell et al., 1993; Young et al., 1993; Adeola and Sands, 2003) by the cleavage of P molecules from the phytate (Maga, 1982).
Considerable variation exists in the efficacy of phytase enzymes of different origins (Gentile et al., 2003) due to resulting differences in pH optima, temperature optima, and resistance to pepsin (Liu et al., 1998). As a result, phytases with similar activity in one species or stage of production may differ significantly in a different species or stage of production. The phytase of interest in this paper is derived from Escherichia coli and expressed in Schizosaccharomyces pombe. Commensal bacteria like E. coli are capable of colonizing the gut of a pig because they have evolved mechanisms and enzymes well suited to compete and thrive in this environment, whereas fungal species such as Aspergillus niger are not found colonizing the gut because they lack the adaptations necessary to thrive in this particular environment. It is hypothesized that selecting a species of origin that is well suited to the pig gut will result in a highly efficacious enzyme.
The objective of this experiment was to assess the effect of an E. coli phytase on growth performance, bone ash, and blood plasma concentrations of P in starter, grower, and finisher pigs, as well as apparent nutrient intake and absorption in starter pigs fed no supplemental inorganic P.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Growth Performance: Starter (Exp. 1)
A total of 48 pigs (24 barrows, 24 gilts) with an average initial BW of 10.6 kg were assigned to four treatments, resulting in six barrows and six gilts per treatment. The basal diet (B) was formulated to contain 7.1 and 4.0 g/kg of Ca and P, respectively, no added phytase, and no additional inorganic P but adequate in all other nutrients (Table 1). The high-P control (HPC) was formulated to supply 1.7 g/kg of additional total P and raise the available P from 0.8 to 2.4 g/kg of diet. The third and fourth diets consisted of the basal diet plus 500 and 1,000 phytase units (FTU)/kg of an E. coli phytase (Phyzyme XP, Danisco Animal Nutrition, Marlborough, U.K.). All four treatments were in mash form and formulated to be identical in all but total and available P; however, as a result of using cornstarch as a filler, the calculated DE and ME values were lower for the HPC diet at 3,541 and 3,371 kcal/kg than the other three diets at 3,557 and 3,387 kcal/kg. Treatments were assigned randomly to 12 replicate blocks of four pens per block in a randomized complete block design. Pigs were blocked by weight and sex, and assigned to pens, and thus treatments, such that the average weight across treatments was similar.
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Growth Performance: Grower (Exp. 2)
A total of 128 pigs (64 barrows and 64 gilts) with an average initial BW of 22.8 kg were assigned to four treatments. Four replicate pens (1.7 x 3.0 m) of barrows and four replicate pens of gilts consisting of four pigs per pen were used in a randomized complete block design. The HPC diet was formulated to be adequate in all nutrients with Ca and P concentrations of 6.4 and 5.3 g/kg (Table 1
). The basal diet was formulated to be adequate in all nutrients except total and available P by removing all inorganic P supplementation and thereby decreasing the total P to 3.6 g/kg and the available P from 2.3 to 0.7 g/kg. The third and fourth diets consisted of the basal diet plus 500 and 1,000 FTU/kg respectively. All four treatments were in mash form and formulated to be identical in all but total and available P, but as a result of using cornstarch as a filler, the calculated DE and ME were lower for the HPC diet at 3,529 and 3,379 kcal/kg than the other three at 3,545 and 3,395 kcal/kg. Treatments were assigned randomly to pens in eight blocks of four diets. Pigs were blocked by weight and sex, and assigned to pens such that the average weight across treatments is similar.
Body weight and feed consumption were recorded every 2 wk, and ad libitum access to feed and water was provided for 42 d. On d 43, one pig was randomly selected from each pen and euthanized. The third and fourth metacarpal bones were excised from the left leg for use in the determination of bone ash, and blood was collected during exsanguination for analysis of plasma P concentration in a heparinized tube.
Growth Performance: Finisher (Exp. 3)
A total of 160 pigs (80 barrows and 80 gilts) with an average initial BW of 52.7 kg were assigned to four treatments. Four replicate pens (1.7 x 3.0 m) of barrows and four replicate pens of gilts consisting of five pigs per pen were used in a randomized complete block design. Treatments consisted of a HPC diet formulated to be adequate in all nutrients with Ca and P concentrations of 6.2 and 5.1 g/kg. The basal diet was formulated to be adequate in all but total and available P by removing all inorganic P supplementation, decreasing the total P to 3.4 g/kg, and the available P from 2.3 to 0.6 g/kg. The third and fourth diets consisted of the basal diet plus 500 and 1,000 FTU/kg, respectively. All four treatments were in mash form and formulated to be identical in all but total and available P, but, once again, as a result of using cornstarch as filler, the calculated DE and ME were lower for the HPC diet at 3,515 and 3,374 kcal/kg than the other three diets at 3,529 and 3,387 kcal/kg. Treatments were assigned randomly to pens in eight blocks of four diets. Pigs were blocked by weight and sex and assigned to pens, and thus treatments, such that the average weight across treatments was similar.
Procedures for the 42-d period were as described for Exp. 2. As in Exp. 2, one pig was selected randomly from each pen and euthanized for collection of the third and fourth metacarpal bones and the determination of bone ash. Blood collection also was done as described for Exp. 2.
Apparent Total Tract Digestibility (Exp. 4)
Barrows with an average initial BW of 14.5 kg were assigned to stainless steel metabolism crates (0.83 x 0.71 m) that allowed for separate collection of feces and urine. The metabolism crates were located in an environmentally controlled room with a temperature of 21 ± 2°C. Pigs had ad libitum access to water, and were fed in two equal feedings daily (0900 and 1500) in a mash form. The study consisted of a 5-d adjustment period followed by a 5-d period of total, but separate, collection of feces and urine. During the 5-d period of adjustment to metabolism crates and diets, daily feed intake averaging 700 g/d (on an as-fed basis) was achieved and maintained throughout the collection period. Feces were collected twice daily, weighed, mixed for each pig, and stored at –18°C until subsequent analysis.
Treatments consisted of a HPC diet formulated to be adequate in all nutrients with Ca and P concentrations of 7.1 and 4.6 g/kg. The basal diet was formulated to be adequate in all but total and available P by removing all inorganic P supplementation, decreasing the total P to 3.3 g/kg, and the available P from 2.3 to 0.6 g/kg. The third and fourth diets consisted of the basal diet plus 500 and 1,000 FTU/kg respectively. Chromic oxide was added to the diets at 0.3% as an indigestible marker. All four treatments were formulated to be identical in all but total and available P, but as in previous experiments, the calculated DE and ME were lower for the HPC diet at 3,515 and 3,366 kcal/kg than the other three diets at 3,531 and 3,382 kcal/kg. Treatments were assigned randomly to metabolism crates in six blocks of four diets in a randomized complete block design. The 24 barrows were blocked by weight and assigned to crates such that the average weight across treatments was similar.
Chemical Analyses
Fecal samples were dried at 55°C in a forced-draft oven for 5 d. Fecal and diet samples were then ground to pass a 1-mm screen. Samples were then used to determine DM content by oven drying in a 100°C drying oven for 24 h. Nitrogen content of diets was determined by the combustion method (Method 990.03, AOAC, 2000; model FP2000, Leco Corp., St. Joseph, MI) and GE by adiabatic bomb calorimetry (Adeola and Bajjalieh, 1997; model 1261, Parr Instrument Co., Moline, IL). Feed and fecal samples were prepared using a nitric/perchloric wet ash (AOAC, 2000; Method 968.08D[b]), and Cr (Spectronic 21D; Milton Roy Co., Rochester, NY) was determined. Calcium was determined in digests by atomic absorption spectrophotometry (AAnalyst 300, Perkin Elmer, Norwalk, CT). Phosphorus concentration was determined using a colorimetric assay. Acid molybdate and Fiske’s SubbaRow reducer solution were added to the supernatant/digest to form a phospho-molybdenum complex. Color intensity was proportional to P concentration and was determined with a spectrophotometer using absorbance at 620 nm (AOAC, 2000; Method 965.17; Packard SpectraCount, model No. AS1000, Meriden, CT). Phytase activity was determined by the method of Engelen et al. (1994). One phytase unit (FTU) is defined as the quantity of enzyme required to hydrolyze 1 µmol of inorganic P/min, at pH 5.5, from an excess of 15 µM sodium phytate at 37°C (IUB, 1979). The E. coli-derived phytase used has a pH optimum of 4.5, resulting in potentially greater actual activity compared with a phytase having pH optima of 5.5, with similar calculated FTU/kg.
Left metacarpal bones were excised at slaughter and defleshed by making a longitudinal slit on opposite sides of the bone using a knife, and then autoclaved at 121°C for 3 min, with the autoclave set for fast exhaust. Bones were ashed for 16 h at 600°C in a muffle furnace to determine bone ash.
Statistical Analyses
Data from all four experiments were analyzed as a randomized complete block design using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC). Individual pig was the experimental unit for Exp. 1 and 4, and pen was the experimental unit for Exp. 2 and 3. A contrast of the HPC diet and basal diet was used to determine the effect of removing inorganic P, and orthogonal polynomial contrasts of the diets containing no supplemental P were used to determine linear and quadratic effects of supplemental phytase on the measures of growth performance and nutrient digestibility.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). This linear response was also noted in the final plasma concentrations (P < 0.01) and final BW (P < 0.05). The magnitude of overall improvements in performance of starter pigs fed 500 and 1,000 FTU/kg of E. coli phytase compared with the basal diet were 12 and 12% for ADG, and 14 and 20% for G:F, respectively.
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The Ca:P ratio in the finisher diets (Exp. 3) was determined to be 2.7 and 1.7 for the basal and HPC diets, respectively. The mean ± SD of the analyzed phytase activities in diets formulated to contain 500 or 1,000 FTU/kg were 670 ± 144 and 1,388 ± 91 FTU/kg. In finishing pigs, phytase supplementation of the basal diet resulted in a linear increase (P < 0.01) in ADG, G:F, and final BW. Phytase supplementation of the basal diet did not affect ADFI or plasma P concentration at slaughter. The magnitude of improvements in the performance of finisher pigs fed 500 and 1,000 FTU/kg E. coli phytase compared with the basal diet were 5 and 13% for ADG, and 6 and 15% for G:F, respectively. Bone ash showed a significant decrease from the HPC diet to basal diet (P < 0.01), as well as a quadratic trend in response to dietary phytase (P < 0.06).
Apparent Total-Tract Digestibility
The Ca:P ratio in the starter diets used in Exp. 4 were determined to be 2.2 and 1.5 for the basal and HPC diets, respectively, and the analyzed phytase activities in diets formulated to contain 500 or 1,000 FTU/kg were 611 or 1,055 FTU/kg. Removal of supplemental inorganic P resulted in a decrease in P intake, Ca (P < 0.05) and P absorption (Table 3), apparent digestibility of Ca and P, as well as an increase in apparent digestibility of DM, energy, and N (P < 0.01). Exogenous phytase supplementation of starter diets resulted in a linear increase (P < 0.01) in P absorption and digestibility of Ca and P in the basal diets, and it also evoked a quadratic response in apparent digestibility of energy and P (P < 0.05). The magnitude of the increases in P absorption of starter pigs fed 500 and 1,000 FTU/kg of E. coli phytase compared with the basal diet was 0.18 and 0.62 g/d, or 32 and 111%. Phosphorus absorbed from the HPC diet and basal diet plus 1,000 FTU/kg were numerically the same, at 1.18 g/d.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Kemme et al., 1999; Traylor et al., 2001), results have recently been published concerning alternative phytase enzymes (Rutherfurd et al., 2002; Gentile et al., 2003; Augspurger and Baker, 2004) of different biological origins, expression vectors, and efficacies. In the current study, growth performance and nutrient digestibility increased with phytase supplementation of P-deficient diets. These improvements in performance are attributed to the liberation of phytate-bound P by the supplemental phytase as previously documented with other phytase products (Lei et al., 1993a; Kemme et al., 1999). Plasma P concentrations increased in starter pigs. Gentile et al. (2003) with 750 FTU/kg of consensus phytase, Adeola et al. (1995) with 1,500 FTU/kg of A. niger phytase, and Lei et al. (1993) with A. niger phytase, all demonstrated a similar plasma P response with supplemental phytase in weanling pigs fed otherwise P deficient diets. Increased plasma P is interpreted as the result of increase P bioavailability resulting from supplementation of P-deficient diets with phytase. The increased bioavailability enables the pig to maintain more normal plasma P concentrations needed for maintenance, as well as growth and development, compared with diets supplying inadequate P and no supplemental phytase. This effect on plasma P was not seen in growing or finishing pigs, possibly as a result of greater body stores of P from which to draw over the course of the trial. As a result, plasma P concentration may not be a useful measure for describing the P status of grower and finisher pigs.
Average daily gain, G:F, and BW at the end of the starter, grower, and finisher phases, as well as ADFI during the grower phase, were all improved by supplementation of the P-deficient diet with phytase. This result is attributed to the ability of phytase to increase P availability (Harper et al., 1997; Gentile et al., 2003; Johnston et al., 2004). It would seem that the E. coli phytase used is more efficacious in the grower phase than in the starter and finisher phases. The percent increases in feed efficiency and ADG were larger, and increases in feed intake were noted exclusively in the grower phase. Despite the disparity between formulated and analyzed dietary phytase and Ca concentrations in the current studies, growth performance improvements were similar to some results in the literature. Adeola et al. (2004) reported that including an E. coli-derived phytase at 500 and 1,000 FTU/kg improved ADG by 20 and 30%, ADFI by 1 and 10%, and G:F by 18 and 17%, respectively, in 20-kg pigs fed a P-deficient corn–soybean meal diet.
Gentile et al. (2003) demonstrated in weanling pigs an ADG increase of 42 and 44% for a P-deficient diet supplemented with a consensus phytase at 500 and 1,000 FTU/kg, respectively, but the greater increase in ADG was accompanied by a feed intake increase of 20%. Our results were slightly less than the 13 and 23% ADG increases Harper et al. (1997) reported in the grower and finisher phases, respectively, at 500 FTU/kg. Both Gentile et al. (2003) and Harper et al. (1997) used diets with lower Ca, P, and Ca:P ratios than was determined in the current study, which would result in greater responses to phytase supplementation as demonstrated by Lei et al. (1994) and Liu et al. (2000). Adeola et al. (2004) demonstrated a 30% increase in the ADG of grower pigs by supplementing 1,000 FTU of an E. coli-derived phytase expressed in Bacillus subtilis per kilogram of diet, which is similar to the 28% increase we observed at the same inclusion level. The percent increase in ADG Harper et al. (1997) demonstrated was greater for the finisher than the grower phase, which is in contrast with our results. However, Harper et al. (1997) fed P-deficient diets for 16 wk, as opposed to our 6-wk period, resulting in a more chronic deficiency with more pronounced effects due to P deficiency.
Our results for G:F compare favorably with increases in feed efficiency of 19 and 16% for weanling pigs at 500 and 1,000 FTU of consensus phytase per kilogram, respectively (Gentile et al., 2003). Using an A. niger phytase at 500 FTU/kg of diet, Harper et al. (1997) demonstrated 4 and 3% increases in G:F in grower and finisher pigs, and O’Quinn et al. (1997) demonstrated a 10% improvement for 50- to 80-kg pigs, which is comparable to observations in the current study. O’Quinn et al. (1997) had much lower determined Ca and P concentrations and a Ca:P ratio of approximately 1:1, which would require greater efficiency to meet metabolic requirements and would avoid the negative effects of a wide Ca:P ratio demonstrated by Lei et al. (1994) and Liu et al. (2000).
The current thought is that if phytase has an effect on AA digestibility, it is most likely the result of the phytase hydrolyzing the phosphate groups off of the inositol ring, thereby destroying or preventing the formation of protein-phytate complexes (Selle et al., 2000). Unfortunately, this effect has been inconsistently demonstrated (Selle et al., 2000; Adeola and Sands, 2003). In this study, we were unable to detect any change in N absorption or digestibility that could be linked to a change in AA digestibility as a result of phytase supplementation. The only change we observed in N balance was a decrease in digestibility with the addition of inorganic P.
Lei et al. (1993) realized 23% greater apparent digestibility of P in weanling pigs fed a P-deficient diet supplemented with 750 FTU/kg of phytase from A. niger. Augspurger et al. (2003) compared two commercial, fungal-derived phytases and an experimental E. coli-derived phytase in young chicks and pigs at 500 FTU/kg. The P-release value of E. coli-derived experimental phytase of 0.125% was greater than the P-release values for A. niger and Peniophora lycii phytase, with 0.032 and 0.028%, respectively, in poultry (Augspurger et al., 2003). The differences between sources in swine were far less dramatic, however, with 0.081, 0.043, and 0.108% for A. niger phytase, P. lycii, and the E. coli-derived phytase, respectively. In Exp. 4, supplementation of the basal diet with 1,000 FTU/kg of E. coli phytase was able to restore the P absorption to the level of the HPC diet in weanling pigs. Augspurger et al. (2003) attributed the superior efficacy of E. coli-derived phytase to a greater resistance to pepsin, and lower, wider pH optima, which Adeola et al. (2004) determined to be between pH 2 and 4.5 based on the greatest inorganic P release in vitro. These combined enabled the E. coli-derived phytase to remain active longer in the stomach, which Yi and Kornegay (1996) determined as the primary site of phytase action in the young pig. As a result, E. coli-derived phytases have the potential to be more efficacious at similar inclusion levels than currently available, fungal-derived commercial phytase preparations because phytase activity is frequently determined using the procedure of Engelen et al. (1994) and pH 5.5, at which Rodriguez et al. (1999) showed a loss of activity of 35% compared with pH 3.5.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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2 Correspondence: 915 W. State St. (phone: 765-494-4848; fax: 765-494-9346; e-mail: ladeola{at}purdue.edu).
Received for publication June 21, 2004. Accepted for publication April 28, 2005.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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 O. A. Olukosi, A. J. Cowieson, and O. Adeola Age-Related Influence of a Cocktail of Xylanase, Amylase, and Protease or Phytase Individually or in Combination in Broilers Poult. Sci., January 1, 2007; 86(1): 77 - 86. [Abstract] [Full Text] [PDF]
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T. L. Veum, D. W. Bollinger, C. E. Buff, and M. R. Bedford A genetically engineered Escherichia coli phytase improves nutrient utilization, growth performance, and bone strength of young swine fed diets deficient in available phosphorus J Anim Sci, May 1, 2006; 84(5): 1147 - 1158. [Abstract] [Full Text] [PDF]
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