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Supplementation of Corn–Soy-Based Diets with an Eschericia coli-Derived Phytase: Effects on Broiler Chick Performance and the Digestibility of Amino Acids and Metabolizability of Minerals and Energy

A. J. Cowieson^*, T. Acamovic^*,1 and M. R. Bedford

^* Scottish Agricultural College, West Mains Road, Edinburgh, EH9 3JG, UK; and Syngenta Animal Nutrition, Chestnut House, Beckhampton, Wiltshire, SN8 1QJ, UK

¹ Corresponding author: thomas.acamovic{at}sac.ac.uk

	ABSTRACT

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The effect of the supplementation of diets containing low available P concentrations with low and supra-activities of an Eschericia coli 6-phytase was assessed using growing broiler chicks. A total of 384 female Ross broiler chicks were weighed at d 1 of life and assigned to 1 of 8 experimental treatments. There were 12 replicate cages with 4 chicks per cage, and the diets were fed from d 1 of life for a period of 16 d. A positive control diet (5 g/kg of available P) and a negative control diet (3 g/kg of available P) were used, and 6 more diets were manufactured by supplementing the negative control diet with 150, 300, 600, 1,200, 2,400, and 24,000 U/kg of exogenous phytase. Body weight gain and feed conversion ratios were determined, as were nutrient digestibility coefficients and toe ash values. Birds fed the negative control diet had lower (P < 0.05) weight gains than those fed the positive control diet. The addition of exogenous phytase above 150 U/kg improved (P < 0.05) weight gain, toe ash percentage, and nutrient utilization of the birds fed the negative control diet. Furthermore, the 24,000 U/kg of diet improved (P < 0.05) toe ash percentage and the utilization of several nutrients beyond that of the lower doses of phytase. It can be concluded that the supplementation of diets containing 3 g/kg of available P with exogenous phytase can improve the performance of chicks to that of birds fed a diet containing 5 g/kg of available P. In addition, the use of high doses of phytase (>1,000 U/kg of diet) can improve nutrient availability in poultry diets beyond that of diets containing lower (<1,000 U/kg) phytase activities. These results may be mediated partially by reduced endogenous loss as well as an increase in the availability of dietary nutrients as indicated by improvements in digestibility coefficients.

Key Words: broiler • phytase • mineral • amino acid • energy

	INTRODUCTION

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Feeds of intensively reared poultry typically contain a high proportion of cereals, grain legumes, and oilseed meals (McDonald et al., 1990). These feed ingredients contain variable concentrations of P, which ranges from around 19 g/kg in extracted rice bran to less than 1 g/kg in some tubers (Eeckhout and De Paepe, 1994). However, approximately two-thirds of the P is present as phytate P (Eeckhout and De Paepe, 1994; Harland and Oberleas, 1999). Phytate is the term for salts of phytic acid (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and is ubiquitous in many types of plant material (Harland and Morris, 1995). The use (frequently measured as loss in the gastrointestinal tract) of phytate P by single-stomached animals has been reported to vary from less than 10 to over 50% (Ravindran et al., 1999; Selle et al., 2000; Ravindran et al., 2001; Cowieson et al., 2006a). The discrepancies in the availability of phytate P reported by others are likely to be due to differences in the design of the diets offered, the concentration of endogenous phytases present in the feedstuffs offered, and the age and species of livestock. Regardless of differences in reported values, the availability of P from phytate for chickens is poor because they do not possess endogenous enzymes for the effective hydrolysis of phytic acid (Hu et al., 1996; Miyazawa et al., 1996; Bedford and Schulze, 1998; Bedford, 2000; Maenz, 2001).

The structure of phytic acid has been well described by others (Johnson and Tate, 1969; Blank et al., 1971; Costello et al., 1976), and although differing conclusions have been reached regarding the alignment of the phosphate groups on the inositol nucleus, the conformation is well elucidated. Phytic acid contains 12 dissociable protons with pK_a values that range from 1.5 to around 10 (Costello et al., 1976). Because phytic acid is a polyanionic molecule, it can chelate di- and trivalent cations and interact with proteins and carbohydrates, reducing the availability of these compounds for poultry (Lonnerdal et al., 1999; Angel et al., 2002; Sandberg, 2002). Furthermore, the ingestion of phytic acid by poultry can increase the excretion of endogenous compounds, further impairing the performance of the animal (Cowieson et al., 2004b). However, some of the detrimental effects of phytic acid can be ameliorated by the addition of exogenous phytase to the diet (Ravindran et al., 1999, 2001; Selle et al., 2000; Cowieson et al., 2006a).

The supplementation of poultry diets with exogenous phytases can dramatically improve the use of phytate P (Leske and Coon, 1999; Augspurger et al., 2003; Baidoo et al., 2003; Cowieson et al., 2006a). However, the effects of the addition of exogenous phytases to the diets of poultry are not confined to improvements in P retention but include improved gain, feed conversion ratio (FCR), nutrient use, and bone mineralization. These improvements are often above that which can be explained by improved P retention only (Ravindran et al., 1999; Newkirk and Classen, 2001). Although many studies have reported improvements in performance parameters associated with the addition of phytases to the diet, few studies have added exogenous phytase at concentrations exceeding 1,000 phytase units (FTU)/kg of diet. It may be that the supplementation of poultry diets with phytase at concentrations exceeding 1,000 FTU/kg of diet would have further beneficial effects on nutrient use, health, and on the environment compared with lower phytase activities. However, the economics of adding high levels of phytases, or other enzymes, need to be considered carefully. It was the purpose of the study to assess the effect of supplemental Eschericia coli 6-phytase (Quantum, Syngenta Animal Nutrition Inc., Raleigh, NC) in a diet designed to contain 3 g/kg of available P at doses from 150 to 24,000 FTU/kg of diet.

	MATERIALS AND METHODS

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Animal Husbandry and Toe Ash Determination

The study was conducted with the approval of the Animal Ethics Committee of the Scottish Agricultural College (SAC). The formulation and calculated nutrient provision of the experimental diets is presented in Table 1. The positive control (PC) diet was prepared in a single batch in a horizontal mixer and fed without supplemental phytase. The negative control (NC) diet was prepared as a single batch in a horizontal mixer. Appropriate quantities of the basal diet were selected, and phytase was added prior to remixing with and without 150, 300, 600, 1,200, 2,400, and 24,000 FTU/kg of supplemental phytase. The phytase is an evolved E. coli 6-phytase (Syngenta Animal Nutrition Inc.) optimized for improved thermal and gastric tolerance and expressed in Pichia pastoris (Garrett et al., 2004). The diets were provided as a mash to 384, 1-d-old female Ross broiler chicks, which were obtained from a local hatchery, weighed and assigned to cages, and stratified by d 1 BW such that each block had minimal variation in BW. There were 12 replicate cages, each containing a total of 4 chicks, giving a total of 48 chicks on each experimental diet. Temperature was approximately 31°C on d 1 of the study and was gradually reduced to around 24°C on d 16. Water and diet were provided for ad libitum consumption via a suspended nipple drinker line at the rear of each cage and a trough at the front of each cage, respectively. Lighting was set at 23 h per day. On d 7 and 14, feed intake and BW gain were determined. On d 16, 2 birds per cage were killed by overdose of sodium pentobarbitone introduced into the wing vein, and the middle toe on the right foot was removed. The toes were oven dried for 24 h at 80°C, weighed, ashed for 16 h in a muffle furnace at 390°C, and then reweighed to determine the concentration of ash per gram of DM. The mean ash content of the toe from both birds per cage was used in the final statistical analysis.

On d 14, trays were placed beneath each cage, and a total collection of excreta was made over a 48-h period. On d 16, the excreta were quantitatively collected, and feed consumption was determined. The excreta were dried to a constant weight in a forced-draft oven at <80°C, and digestibility coefficients of nutrients were calculated. The choice of oven drying as opposed to freeze drying was based on evidence that moderate oven drying does not compromise the recovery of labile nutrients in feed or excreta (Wallis and Balnave, 1983; Dale et al., 1985). The concentration of N in the excreta was determined using a Leco FP-2000 analyzer (St. Joseph, MI). Amino acid concentrations in the excreta were determined by HPLC (Roth, 1971; Jones et al., 1981; Alltech data sheet, D28062 [GenBank] , Alltech Associates, Carnforth, UK) following acid hydrolysis and precolumn derivatization using orthophthaldialdehyde. The HPLC system comprised a Dionex ASI-100 autosampler fitted with a Dionex P580 pump and a Dionex RF-2000 detector (Sunnyvale, CA). The flow rate used was 1.5 mL/min and the column used was an Adsorbosphere OPA-HR (150 x 4.6mm; Alltech Associates). Because this method of hydrolysis destroys methionine, cystine, and tryptophan, data on these amino acids are not reported. However, the results for the analyses of S may be interpreted as an indicator of the methionine and cystine content and effects. The mineral content of the excreta, after digestion with nitric acid in a microwave digestion unit, was determined using an Optima 4300 DV Dual View ICP-OE spectrometer (PerkinElmer, Beaconsfield, UK). The concentration of phytate P in the excreta was determined colorimetrically by the method of Haug and Lantzsch (1983). Phytase activity in the feed was determined (Enzyme Services and Consultancy, Gwent, UK) independently without knowledge of the treatments by measuring the release of inorganic phosphate from sodium phytate at pH 5.5 and 37°C. One unit of phytase activity was defined as the quantity of enzyme that liberated one micromole of inorganic P per minute from sodium phytate at pH 5.5 and 37°C. The quantity of liberated inorganic P was measured using an acid molybdate/vanadate reagent, which terminated the reaction and produced a colored complex with the liberated phosphate. The color of the complex is a measure of the quantity of phosphate present and was compared with a standard curve to calculate phosphate release (Engelen et al., 2001). Gross energy of samples was determined using an adiabatic bomb calorimer using benzoic acid as the reference material (Gallenkamp, Loughborough, UK).

Statistical Analyses

Data were analyzed (JMP Software, SAS Institute Inc., Cary, NC) by standard least squares to assess the effect of treatment and block. A further analysis was conducted whereby the PC was excluded from the analysis, and the dose of the enzyme was investigated as a continuous variable with log-determined dose (log₁₀) response as the x variable. In addition, specific contrasts were carried out to assess the overall effects of diet and phytase. In all instances, differences were significant at P 0.05 (Snedecor and Cochran, 1980).

	RESULTS

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Performance

All animals survived and remained healthy until the end of the study. The compositions of the NC and PC diets are shown in Table 1. The analyzed chemical composition agreed fairly closely with the calculated values. The main differences between the NC and PC were the P and Ca. The recovery of phytase was approximately in agreement with anticipated activities (Table 2). The concentration of endogenous phytase in the corn and soybean meal was low (<25 U/kg of feed).

The effect of phytase on metabolizability of the NC is presented in Tables 4 through 7. In most instances, birds that were fed the NC were as efficient at retaining nutrients from the diet as those fed the PC. Exceptions to this were for coefficients of P, Na, Ca, Cu, Fe, and Mn retention, which were higher (P < 0.05) for the NC than for the PC. Toe ash percentage was significantly lower from birds fed the NC than for those fed the PC. However, for AME_n and the retention of amino acids and N, there was little difference between chicks fed the PC and those fed the NC.

The effect of phytase on amino acid digestibility coefficients is presented in Tables 6 and 7. The coefficients of digestibility of all amino acids were improved by the addition of phytase, and, in most instances, there was little to be gained from adding phytase above 600 FTU/ kg, with significant beneficial effects often attained at only 150 or 300 FTU/kg. The digestibility coefficients of amino acids were not all affected to the same degree by the addition of phytase, and improvements ranged from 2% for arginine to almost 6% for threonine. The average improvement in coefficients of digestibility of amino acids was approximately 3% compared with the NC.

The highly significant effects of phytase on BW gain and FCR can be explained by the effect on the intake of retainable nutrients (Table 8). Birds that received the NC supplemented with exogenous phytase had higher (P < 0.01) intakes of retainable DM, N, energy, P, Ca, K, Na, S, and amino acids compared with birds fed the unsupplemented NC. However, in some cases, phytase supplementation failed to achieve retainable nutrient intakes equivalent to that of the PC.

	DISCUSSION

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Recent work has shown that the inclusion of phytase, with xylanase, amylase, and protease, to nutritionally deficient and nutritionally rich diets can improve performance and uniformity of BW. The improvements may be due to an improvement in the net energy value of the diet (Cowieson and Adeola, 2005; Cowieson et al., 2006b,c). It may be that to realize the full potential of phytase on P retention, diets should be designed to be adequately nutrient dense and balanced with regard to their supply of amino acids and energy, in the presence of phytase. Furthermore, it is possible that to maximize the response to phytase it should be added to the diet in combination with accessory enzymes such as xylanase or other enzymes that are capable of improving access to dietary phytate.

Dephosphorylation of inositol hexaphosphate by phytase takes place in a stepwise manner, which, for the phytase used in the current experiment, starts with the phosphate group at position 6 (Greiner et al., 2000). This dephosphorylation forms lower-molecular weight inositol (Ins) isomers [D/L-Ins(1,2,3,4,5)P₅, D/L-Ins(2,3,4,5)P₄, D/L-Ins(2,4,5)P₃, D/L-Ins(2,5)P₂, and, Ins(2)P (6/1/3/4/5)]. It seems that most phytases are unable to cleave all 6 phosphate groups from the inositol ring within the time and environmental constraints in the gut of the chicken (Greiner et al., 2000), although they have been shown to produce the monophosphate (Venekamp et al., 1995). As phytase sequentially dephosphorylates the inositol nucleus, the rate of hydrolysis decreases with each catalytic event (Greiner et al., 1993). This is likely to be linked to both product inhibition (perhaps related to pH changes as phosphoric acid is produced) and an inherently lower rate of hydrolysis of the lower molecular weight inositol polyphosphate esters (Greiner at al, 1993). As the K_m of the enzyme for the lower molecular weight intermediates increases, it is likely that substrate concentration plays a role in restricting further dephosphorylation. This results in phytases degrading the "pool" of higher molecular weight isopropyl esters prior to dephosphorylation of lower molecular weight isopropyl esters (Kerovuo et al., 2000; Shin et al., 2001). Given the kinetics of complete dephosphorylation of inositol hexaphosphate, it may be that the addition of higher concentrations of phytase allows dephosphorylation to occur more effectively in the relatively restricted conditions within the gastrointestinal tract of the chicken. The results of the present study tended to support this theory, with higher toe ash, dephosphorylation of phyate (about 60% at the highest concentration of phytase), and retention coefficients of P associated with the addition of ‘supra’ doses of phytase to the NC compared with more normal (250 to 500 FTU) concentrations. However, although phytase improved the retention of amino acids and energy, the additional beneficial effects of high doses of phytase were not as apparent for these nutrients as for the coefficient of P retention, toe ash percentage, and phytate P hydrolysis. This may be explained by the reduced capacity of lower molecular weight isopropyl esters to interact with starches, proteins, and an ability to stimulate an increase in endogenous losses (Selle et al., 2000; Angel et al., 2002; Cowieson et al., 2004b; Cowieson and Adeola, 2005). The consequence of this is that most of the improvements in amino acid and energy retention associated with phytase addition may be expected to be achieved by the removal of 1 or 2 phosphate groups, with further improvements increasingly unlikely as each subsequent phosphate is cleaved. This means that the addition of relatively low doses of phytase has the capacity to improve the retention of nonmineral nutrients and that the addition of higher doses may only have a significant effect on P retention and not on energy or amino acids. However, these effects are likely to be linked to the phytate concentration in the diet and also to the Ca:P ratio and the ratios among available nutrients. Diets containing rice bran or canola meal may benefit more from higher doses of phytase than for those based on corn and soybean meal, because rice bran and canola contain higher concentrations of phytate P (Eeckhout and De Paepe, 1994).

It is interesting that although the coefficients of digestibility of all amino acids were improved by the addition of phytase, there was a large variation in response that was dependent on the amino acid. The digestibility coefficient of arginine was improved by only 2% by the addition of phytase, whereas for valine, threonine, isoleucine, aspartic acid, and alanine the improvements were between 4.5 and 5.6%. This is in agreement with findings by Kornegay (1996), Ravindran et al. (1999), and Namkung and Leeson (1999), who found that the digestibility of valine, threonine, and isoleucine in particular was improved by phytase. Furthermore, Rutherfurd et al. (2002) found that phytase improved the digestibility coefficients of aspartic acid, valine, and threonine compared with unsupplemented corn. Although published results on the effect of phytase on amino acid digestibilities vary (Selle et al., 2000; Adeola and Sands, 2003), it is clear that, when phytase influences amino acid digestibility coefficients, it does not do so to the same extent for all amino acids. This may be linked to differential interactions between amino groups and phytate or it may be associated with the ability of phytate to increase the loss of endogenous compounds, such as mucins, that are rich in certain amino acids (Forstner and Forstner, 1994; Mansoori and Acamovic, 1998; Cowieson et al., 2004b).

Consistent with the effect of phytase on amino acid digestibility, the effect on ME can be variable. Newkirk and Classen (2001) reported that phytase improved the AME of canola meal and concluded that phytate was responsible for decreasing the energy availability in canola for chickens. This is supported by data showing that phytate has the ability to interfere with starch digestion, perhaps associated with complexing of Ca, a required cofactor for -amylase or due to the complexing of starch with phytate, reducing the solubility and digestibility of starch (Knuckles and Betschart, 1987; Cowieson et al., 2004a). In the present study, phytase improved (P < 0.01) AME_n by 50 to 150 kcal/kg (about 5%) depending on the dose of phytase. This improvement is somewhat higher than has been reported for phytase, and whether phytase can improve ME consistently is still open for debate (Adeola and Sands, 2003; Cowieson and Adeola, 2005). However, there was no indication that high doses of phytase improved the AME_n of the NC more than could be achieved with 150 to 300 FTU/kg.

The intake of digestible nutrients (Table 8) may explain the significant effects of phytase on performance of the birds. Supplementation of the NC improved (P < 0.001) the intake of retainable DM, N, ME, P, Ca, K, Na, S, and amino acids compared with the unsupplemented NC. These data are a function of the improved coefficients of retention and the higher feed intakes, caused by supplementation with phytase. Nevertheless, intake of P, the limiting nutrient in the NC diet compared with the PC, never reached that of the PC, whereas the performance of birds fed 300 U/kg or greater equaled or exceeded that of the PC. This suggests that P intake in this study was not limiting when it exceeded ~0.18 g/d, and the interaction with Ca may have also impaired further improvement. There is also an indication that there was little substrate available for the enzyme when a 0.18-g intake had been achieved. If substrate had not become limiting, then it is possible that the higher doses of phytase used in this study may have yielded greater benefits in animal performance. Indeed, if the NC had been formulated to contain a lower concentration of digestible amino acids and ME, the effect of phytase on performance may have been greater. The effect of phytase on the intake of retainable K and Na is particularly interesting as anecdotal evidence (T. Acamovic, unpublished data) suggests that phytase may have adverse consequences on litter quality associated with an increase in the consumption and excretion of water. Data presented in Table 8 suggest that a reason for an increase in water consumption with phytase supplementation of poultry diets may be linked to changes in the retention and balance of mineral ions. This would affect the osmotic balance (increased osmolality) within the gastrointestinal tract of the birds, thereby increasing the requirement for water to maintain homeostasis.

It can be concluded that supplemental 6-phytase is effective in improving the nutritional value of corn- and soy-based diets for young broiler chickens fed a diet that is formulated to be suboptimal in terms of available P and Ca. Furthermore, phytase can improve amino acid digestibility coefficients, AME, and the retention coefficients of many minerals. The benefits on amino acids and energy are maximized at relatively low doses, whereas with minerals the benefits seem to continue to increase with incremental dose, which is in accord with the increased loss of phytate P as phytase activity increased. It is unclear, although likely, that the acid-base balance and osmotic stressors influence the effects of phytases. It is also possible that the effects of phytase on water intake are linked to liberation of ions from inositol hexaphosphate complexes. Both of these topics warrant further investigation.

	ACKNOWLEDGMENTS

 
The Scottish Executive Environment and Rural Affairs Department financially support SAC. The assistance of technical staff at the Avian Science Research Centre, SAC, is gratefully acknowledged.

Received for publication November 22, 2005. Accepted for publication March 16, 2006.

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