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Age-Related Influence of a Cocktail of Xylanase, Amylase, and Protease or Phytase Individually or in Combination in Broilers¹

O. A. Olukosi^*, A. J. Cowieson and O. Adeola^*,2

^* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907; and Danisco Animal Nutrition, Marlborough, Wiltshire, SN8 1XN, UK

² Corresponding author: ladeola{at}purdue.edu

	ABSTRACT

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This 21-d experiment was conducted to determine if the response of chicks to a cocktail of xylanase, amylase, and protease (XAP) or Escherichia coli-derived phytase individually or in combination when fed a nutritionally marginal corn-soybean meal diet is age-dependent. Six hundred 1-d-old chicks were allocated to 5 dietary treatments in a randomized complete block design. The treatments were as follows: 1) positive control with supplemental inorganic P; 2) negative control (NC) marginal in P and ME; 3) NC plus XAP to provide (per kg of diet) 650, 1,650, and 4,000 U of xylanase, amylase, and protease, respectively; 4) NC plus phytase added to provide 1,000 phytase units/kg; and 5) NC plus a combination of XAP and phytase. Low ME and P in the NC diet depressed weight gain and gain:feed (P < 0.001). A cocktail of XAP alone did not improve performance, but phytase supplementation improved (P < 0.001) weight gain. The enzymes were additive in their effects on growth performance. The enzymes had no effect on ileal digestible energy. Ileal N digestibility was higher (P < 0.05) in diet with XAP or phytase individually compared with NC. Both phytase and XAP individually and in combination improved (P < 0.01) ileal P digestibility compared with NC. Total tract nutrient retention and ME increased (P < 0.01) as the birds grew older. There were age x diet interactions (P < 0.001) on total tract retention of P and Ca; improvement in P retention due to phytase use decreased by 50% as the chicks matured. The current study shows that a combination of XAP and phytase improved performance, but the enhancement in performance appears to be mainly from phytase. Both XAP and phytase were effective in improving P digestibility and retention of chicks receiving nutritionally marginal corn-soybean meal. The data also shows that the chicks benefited more from the enzyme addition at a younger age and that the contribution of the enzymes to nutrient retention decreased with age in chickens.

Key Words: age • broiler • carbohydrase • performance • phytase

	INTRODUCTION

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Corn and soybean meal (SBM) are widely used in diets for pigs and chickens because of their perceived high nutritional value. However, Malathi and Devegowda (2001) reported that the level of nonstarch polysaccharides (NSP) is up to 29% in SBM and 9% in corn. Exogenous enzymes have been used to enhance the feeding value of rye and wheat-based diets, because these feed-stuffs are high in soluble NSP that induce viscosity, but it is thought that feedstuffs like corn and SBM that induce less viscosity may not benefit from exogenous enzymes. However, Hong et al. (2002) found that the use of an enzyme cocktail that has xylanase, amylase, and protease (XAP) activities improved the digestibility of a corn–SBM-based diet for ducks. Marsman et al. (1997) similarly reported improved ileal digestibility of nutrients in broilers fed a corn–SBM diet supplemented with exogenous protease and carbohydrases. Others (Ritz et al., 1995; Zanella et al., 1999; Gracia et al., 2003) have similarly reported improved performance when carbohydrases are used. The positive effects of the enzymes are suggested to be due to enhancement of nutrient digestibility in young chicks that are <1 wk old as well as digestion of soluble and insoluble NSP in corn and SBM.

Phytase has been shown to improve performance of chicks fed diets marginally deficient in P (yla et al., 2000; Onyango et al., 2005). Phytase and xylanase, when used separately, have been shown to improve the performance of broilers (Ravindran et al., 1999; Kocher et al., 2000; Boling-Frankenbach et al., 2001; Meng et al., 2005). Association between phytase and carbohydrases has been categorized as additive, subadditive, or synergistic (Cowieson and Adeola, 2005; Juanpere et al., 2005). The interaction between phytase and carbohydrases may be considered from 2 perspectives. On one hand, when feed-stuffs high in NSP are used in the absence of carbohydrases, the cell wall shields nutrients, including phytate P, and, hence, makes phytate inaccessible to phytase, thus reducing the efficiency of the enzyme. In a similar fashion, insufficiency of phytase will prevent carbohydrases from liberating other nutrients that may be bound with the phytate molecule.

From another perspective, the limitation imposed by a nutrient may attenuate the response that another enzyme may produce (Cowieson and Adeola, 2005). For example, when ME is limiting, the presence of phytase may fail to produce improvement in performance, even though P is liberated from phytate. In contrast, limitation imposed by insufficient P in the absence of phytase may limit the response to additional ME made available by the use of carbohydrases. The kind of interactions described above may require simultaneous use of enzymes with many different activities that are able to target different components of feedstuffs used. When an enzyme cocktail containing several activities is used in a broiler diet, it is more likely to have greater effect than when they are used separately. yla et al. (1996) demonstrated in vitro that complete dephosphorylation of phytate will only occur if a cocktail of enzymes is used.

The posthatch growth period in poultry is a critical time during which the bird must regulate nutrient supply to support growth and other metabolic functions. At hatching and through the first week of life, the gut represents a small portion of the total body mass, and compared with the next 2 wk, this period is the time for maximal body growth along with corresponding demand for energy and protein (Obst and Diamond, 1992). During this time, the ability of the bird to extract nutrients may be limited and thus presents a bottleneck to development. Mahagna and Nir (1996) observed that intestinal brush border disaccharidases and alkaline phosphatase activities increased with age in chicks. In view of this, it could be expected that enzymes that improve availability of ME and P may be especially beneficial to chickens at a very young age.

With these in mind, this experiment was designed to study efficacy of a cocktail of XAP or an Escherichia coli-derived phytase individually or in combination on performance, total tract nutrient retention, and ileal digestibility of broilers fed nutritionally marginal corn–SBM diets. Specifically, the study examined whether nutrient retention response to these enzymes is dependent on age of the birds.

	MATERIALS AND METHODS

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Six hundred male broiler chicks (Ross 308) were used for the 21-d growth performance study. The chicks were randomly allocated to 5 dietary treatments, and each treatment had 12 replicates with 10 birds per replicate cage. The birds were raised in a temperature-controlled house with a light regimen of 23L:1D; room temperature was maintained at 32°C for wk 1 and reduced by 2°C every 1 wk after. Diets were randomly assigned to cages in each block. The experimental design was a randomized complete block design, with the chickens being blocked by initial BW. The diets were corn–SBM fed in mash form. The 5 dietary treatments were as follows: 1) a positive control (PC) treatment adequate in all nutrients, with dicalcium phosphate as the source of inorganic P; 2) a negative control (NC) diet formulated so that it was marginally deficient in Ca, P, and ME; 3) an NC diet supplemented with XAP (A; Avizyme 1505, Danisco Animal Nutrition), added to provide 650, 1,650, and 4,000 U of XAP per kilogram of diet (as-fed basis), respectively; 4) NC plus supplemental phytase (B; Phyzyme XP 5000, Danisco Animal Nutrition) added to provide 1,000 phytase units (FTU)/kg of feed. One FTU is defined as the quantity of enzyme required to liberate 1 µmol of inorganic P per minute, at pH 5.5, from an excess of 15 µM of sodium phytate at 37°C; and 5) NC plus a combination of A and B (C). All the enzymes were added as granulates. The compositions of the PC and NC diets are presented in Table 1.

Chemical Analyses
Excreta, diets, and ileal contents were dried and ground to pass through a 0.5-mm screen using a mill grinder (Retsch ZM 100, Retsch GmbH and Co., K.G., Haan, Germany). Samples were dried at 105°C in a drying oven (Precision Scientific Co., Chicago, IL) for 24 h for DM determination. Gross energy was determined in a bomb calorimeter (Parr 1261 bomb calorimeter, Parr Instrument Co., Moline, IL) using benzoic acid as a calibration standard. Chromium concentration in the feed, excreta, and ileal digesta samples was determined using the method of Fenton and Fenton (1979). Nitrogen was determined using the combustion method (Leco FP analyzer model 602600, Leco Corp., St. Joseph, MI), using EDTA as an internal standard. Samples were digested in concentrated nitric acid and 70% perchloric acid to solubilize Ca and P. The concentration of P in the supernatant was measured using a kit (Sigma kit no. 670, Sigma Diagnostics Inc., St. Louis, MO) as described by Onyango et al. (2004). Briefly, ammonium molybdate was added to the supernatant to form phosphomolybdate, which was then reduced to form a blue phosphomolybdenum complex. The color intensity of the complex is proportional to P concentration and was determined at 620 nm. The Ca content of the supernatant was determined using flame atomic absorption spectrometry (Varian FS240 AA, Varian Inc., Palo Alto, CA).

Calculations
Apparent nutrient ileal digestibility or total tract retention was calculated using the following relation:

where ND = nutrient digestibility or retention; C_i and C_o = concentration of Cr in the diet and excreta or digesta, respectively; N_i and N_o = concentration of nutrient in the diet and excreta or digesta, respectively.

Metabolizable energy (kcal/g) was calculated using this relation:

where GE_i = gross energy (kcal/g) in feed; GE_o = the gross energy (kcal/g) in excreta; C_i and C_o = concentration of Cr in the diet and excreta or digesta, respectively.

Percentage increase in nutrient retention from one week to another was calculated when necessary using the following relation:

where R_inc = percentage increase in nutrient retention from wk Y to wk X; R_X and R_Y = retention values in weeks X and Y, respectively, with the value for wk X being for older chickens.

Statistical Analysis
Data were analyzed as a randomized complete block design using the GLM procedure of SAS Institute (2004). Initial BW was used as the blocking criteria in the experiment. Means were separated using specific orthogonal contrasts to compare NC with PC or elucidate the effect of phytase, XAP, and their combination on growth performance and apparent nutrient digestibility. Because excreta samples were collected 3 times during the study, the main effect of age and interaction between age and diet were also tested. When there were no interactions, the main effects means of age and dietary treatments were presented. When interactions were present, the simple effect means within each week were also presented. Both linear and quadratic effects of age on total tract nutrient retention were tested.

Additivity for the effects of phytase and XAP on the response criteria was tested as follows. For each response criterion of interest, the response observed in the NC treatment was used as a baseline to which other treatments were compared. The difference between the response to other treatments and the response to NC was taken as the effect of enzyme addition. The sum of the individual effects of XAP (A) and phytase (B) would not be different from the effects of the combination of XAP and phytase (C) if the effects of individual enzymes were additive. The values were then analyzed using the GLM procedure of SAS, and the sum of the means for effects of enzymes A and B were compared with mean of the effect of enzyme C using orthogonal contrasts. Statistical significance was determined at a 5% probability level.

	RESULTS

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The analyzed (enzyme analyses were done by Danisco Animal Nutrition) levels of the individual enzyme activities were 645 U/kg and 1,660 U/kg for xylanase and amylase, respectively, in A; 1,200 FTU/kg for phytase activity in B; and 689, 1,809, and 1,344 FTU/kg for xylanase, amylase, and phytase activities, respectively, in C. There were no analyses for the protease activity in the enzyme mixtures.

The result of the growth performance is shown in Table 2. The final BW on d 21 of chicks receiving diet that is marginally deficient in energy and P was the lowest; the chicks on the PC diet had higher BW than NC (P < 0.001) and were 15% heavier than birds on the NC diet. The addition of XAP alone did not affect BW, but when phytase alone or in combination with XAP were added, there was an increase (P < 0.001) in BW above the NC diet. The total weight gain followed the same pattern as the final BW. Gain per feed was higher (P < 0.001) in the chicks on the PC diet than those on the NC diet. Although XAP and phytase produced numerical step-wise increases in gain:feed above the NC diet, neither of the enzymes individually had significant effect on feed efficiency; however, the combination of the 2 enzymes improved feed efficiency (P < 0.05) above the NC treatment.

	DISCUSSION

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Phytase is able to liberate phytate-bound P and make more P available to the animals. Studies have reported improvements in performance when phytase was used in chickens (yla et al., 2000; Onyango et al., 2005), layers (Silverside et al., 2006), and pigs (Young et al., 1993; Yi et al., 1996; Adeola et al., 1998; Matsui et al., 2000; Jendza et al., 2005). Onyango et al. (2004) compared 3 phytases produced using different yeast production systems and reported that only phytase produced in Pichia pastoris was able to outperform low-P diets in terms of weight gain, feed intake, and feed efficiency. Ravindran et al. (1999) reported no beneficial effect of phytase or glycanase (principally, xylanase activity) alone on the weight gain of chickens, but reported a linear improvement as higher concentrations of the combination of the 2 enzymes were used.

In this study, both phytase and XAP individually or in combination were used in corn–SBM diets that were marginally deficient in both energy and P. Because phytase acts on the phosphate groups associated with the inositol ring of phytic acid backbone and thus releases P, it is expected that the use of phytase would result in improved performance of the animal if P is the nutrient limiting for growth. In the current study, addition of phytase alone produced a significant improvement in performance above the NC, demonstrating that P was a limiting nutrient in the current study. The XAP cocktail is expected to act on resistant starch and improve accessibility and solubility of nutrients, thus improving ME and protein digestibility. This is expected to improve performance above the NC diet when ME is limiting.

The NC diet was formulated to be marginally deficient in ME, the analyzed ME was 3,030 kcal/kg, which is lower than the 3,200 kcal/kg requirement for male broiler chicks of that age (NRC, 1994). However, improvement in performance due to XAP addition alone was not observed in this current study. It should be noted that P is still limiting in the NC diet plus XAP only; therefore, the effect of P limitation likely limited the response to XAP in this study. The calculated ME in the NC diet before analysis was 3,073 kcal/kg, whereas the analyzed level was 3,030 kcal/kg; these values show that XAP did not improve ME availability as was expected. This lack of response to XAP may explain why there was no improvement in performance when XAP alone was used in the diet. Cowieson and Adeola (2005) reported a linear increase in the weight gain of chicks in response to XAP addition, and the combination of phytase and XAP gave about a 2-fold increase in weight gain than when either of the enzymes alone was used. Kocher et al. (2003) reported that using an enzyme cocktail containing pectinase, amylase, and protease in corn–SBM-based diets for chicks resulted in improved performance.

If the enzymes were additive in their effect, it would be expected that the sum of the effect attributed to each enzyme individually should not be different from the effect attributed to the use of the enzymes in combination. Indeed, our data for weight gain and gain:feed in the present study indicate additivity of the effect of the 2 enzymes on these response criteria. Neither of the enzymes alone had any significant effect on gain:feed; however, the combination of the 2 enzymes produced a significant improvement in this response above the NC diet. Furthermore, the current data shows that the chicks that received a combination of the 2 enzymes had higher final BW, weight gain, and gain:feed than the chicks that received only XAP. However, there was no difference in these response criteria between the treatments that received only phytase and those that received the combination of the 2 enzymes. This observation suggested that the effect seen when the 2 enzymes were used was probably more from phytase. However, this is likely due to the diet being formulated to be deficient in ME and P rather than a failure of XAP to beneficially modify its substrate.

Cowieson and Adeola (2005) reported a response to XAP and phytase enzymes, demonstrating that both ME and P were limiting in their study. Although the current study and that reported by Cowieson and Adeola (2005) used corn–SBM-based diets, the addition of rye to the diet used in the Cowieson and Adeola (2005) study may limit availability of energy in the absence of XAP, whereas the effect of rye NSP may be lacking in the current study. Clearly the response obtained in studies like these would depend on both the limiting nutrients as well as the feed-stuff being used, as other studies have shown (Kidd et al., 2001; Café et al., 2002).

The period immediately following hatch is a critical time of development during which the development of the intestinal tract may present a bottleneck to development. The absorptive capacity of young chicks is limited and so is the level of pancreatic and intestinal enzymes. Mahagna and Nir (1996) reported low levels of plasma glucose, protein, and P at hatch in broiler-type chickens. The study also reported the dynamics of intestinal disaccharidases and alkaline phosphates, among others, showing that they attained peak at different times, usually increasing from a hatch-time low level to a peak by the middle of the second week of life. These changes no doubt reflect the change in the diet supply of the chicken from lipid-based hydrophobic substances to hydrophilic ones (Noy and Sklan, 2001; Sklan, 2003). Mahagna et al. (1995) reported a decrease in pancreatic chymotrypsin activity in a chick diet supplemented with an enzyme containing amylase and protease. With these challenges, exogenous enzymes used in the diets of young chicks would be very beneficial in improving nutrient digestibility in at least 2 ways: 1) by supplying enzymes that the chick cannot produce in sufficient quantity by itself, or 2) even though the chick can produce enough of an enzyme by itself, exogenous enzyme may reduce the requirement for the enzyme, thus making more nutrients and energy available for growth of the chick at that critical stage.

There were both linear and quadratic effects of age on the total tract retention of DM, N, and ME. This is similar to reports by Leeson et al. (1996) and Palander et al. (2005) and is probably related to the development of digestive and absorptive capacities of the intestine, as has been demonstrated for pigs (Adeola and King, 2006) and domestic fowl (Obst and Diamond, 1992; Gonzalez and Vinardell, 1996). There were effects of both age and diets and an interaction of the 2 factors on P and Ca retention. Because the response to phytase is consistent in this study, the difference in the benefit that chickens derive from dietary phytase as they grow older can be demonstrated using this response criterion. In wk 1, P retention of the chicks fed the diet with phytase was 2.4 times as much as chicks fed the NC diet. In wk 2 and 3, P retention in the same treatment was 1.5 and 1.2 times, respectively, compared with chicks fed the NC diet. Using the P retention data of NC treatment, it is calculated that the ability of the chicks to extract P from the NC diet without phytase increased by 120% from wk 1 to 2 but only increased by 40% from wk 2 to 3; a similar trend is also noticed for ME. These data show that very young chickens will likely derive greater benefit from the enzymes used to target specific antinutrients in their diet than older chickens would. Therefore, it is important to consider whether nutritional intervention in terms of enzyme use should target such critical times as the first week of life, using higher enzyme activity at that age, while decreasing the enzyme activity in subsequent weeks. Further studies are required to establish the practical and nutritional advantages of such intervention.

Total tract retention of DM and energy was higher in PC than NC, and there was no effect of any of the enzymes alone or in combination on DM retention or ME. Nitrogen retention was higher in the PC than NC diet, and both phytase and XAP individually and in combination improved N retention. Nitrogen retention in the treatments with phytase and XAP individually and in combination was similar and was about 5 percentage points lower than for the PC diet. Marsman et al. (1997) showed that carbohydrases and proteases individually or in combination improved the digestibility of protein and that the carbohydrases improved the digestibility of the NSP fraction of SBM, although no improvement in the performance of broilers was observed. Cowieson and Adeola (2005) similarly reported that XAP linearly improved the digestibility of N and DM of chickens in their study. Harper et al. (1997) found no effect of phytase on digestibility of DM.

It is suggested that phytate is able to reduce the efficacy of digestive enzymes by its ability to form indigestible complexes with the enzymes or their cofactors, especially Ca (Singh and Krikorian, 1982). Because phytase is able to hydrolyze phytate, it could be expected that phytase use would improve digestibility of nutrients both by releasing enzymes that are likely bound with phytate as well as by releasing phytate-bound nutrients. Results from different studies have not been consistent on the effect of phytase on protein and amino acid digestibility. Onyango et al. (2005), using E. coli-derived phytase, reported improved N retention and some amino acids over the low-P diet in broiler chicks receiving corn–SBM-based diets. Traylor et al. (2001) found little to no effect of addition of Natuphos phytase on digestibility of CP and amino acids in pigs fed semipurified diets with dehulled SBM as the sole protein source. Liao et al. (2005) suggested that amino acid response to phytase supplementation may be dependent on diet composition. In the current study, both XAP and phytase individually and in combination improved N retention. It would be logical that the enzymes exerted their effect via different mechanisms. The cocktail of XAP contained protease, and this activity might be responsible, at least in part, for the increase in N retention observed when it was used, whereas the release of N and other nutrients may be responsible for part of the response observed when phytase alone was used.

Metabolizable energy was higher in the PC compared with the NC diet, and there were no effects of any of the enzymes alone or in combination on ME. Wu et al. (2004a) reported that the combination of phytase and xylanase, but neither of the enzymes alone, improved ME in wheat-based diets to chickens. Juanpere et al. (2005) tested the effect of glycosidases and phytase alone or in combination on 3 cereals (corn, wheat, and barley) and reported that corn-based diets with phytase had higher ME than diets based on barley and wheat and that addition of glycosidases had positive effects on ME availability from barley, but not corn or wheat. Zanella et al. (1999) similarly reported improved nutrient digestibility when XAP was added to corn–SBM-based diets. Palander et al. (2005) reported that the use of enzyme containing ß-glucanase-xylanase activities improved AME_n in wheat-, barley-, and oat-based diets for turkey.

Ileal digestible energy presents the amount of energy available to chickens in a way that minimizes the effect of microorganisms on energy measured in the excreta. In the current study, neither of the enzymes individually or in combination improved IDE above the NC diet. There was a trend toward improved IDE when phytase alone was used in the diet. Cowieson and Adeola (2005) reported that XAP linearly improved IDE in chickens fed a corn–SBM-based diet laced with rye to increase the quantity of NSP in the diet. Kocher et al. (2003), using an enzyme cocktail containing pectinase, protease, and amylase, found that this enzyme cocktail was able to improve the ME available from a low-energy corn–SBM-based diet but not high-energy corn–SBM diet. Café et al. (2002) compared the effect of XAP on low- and high-digestible energy SBM fed in a corn–SBM-based diet and observed that XAP increased ME to a greater degree in SBM that had lower IDE than SBM that had higher IDE. Xylanase alone improved TME more in high-viscosity than low-viscosity wheat when fed to ducks (Adeola and Bedford, 2004). Comparison of the composition of the diets used in the studies cited above and the current study along with observation of the effects of carbohydrases on ME and IDE in the studies compared with the current study suggest that the ability of XAP to improve the ME may depend on the feedstuff being fed. Although the basal diet in the present study was marginally deficient in energy, the energy in the diets was likely very digestible, as could be seen from the similarity in ME and IDE values across all the diets, with the exception of the PC diet, so that the enzymes were not able to improve ME.

Phytase has consistently been shown to improve P digestibility and retention in chickens (Simon et al., 1990; Liu et al., 1998; yla et al., 2000; Dilger et al., 2004; Onyango et al., 2004; Juanpere et al., 2005; Onyango et al., 2005) and pigs (Cromwell et al., 1993; Adeola et al., 1995; Yi et al., 1996; Harper et al., 1997; Dilger et al., 2004; Jendza et al., 2005). The total tract retention and ileal digestibility data in the current study show that both XAP and phytase individually improved P liberation above the NC diet. It is known that phytase improved P digestibility by catalyzing the phosphate monoester hydrolysis of phytic acid, resulting in the stepwise formation of myo-inositol and orthophosphates via inositol pentakisphosphate, up to monophosphates as intermediary or end products resulting in liberation of P (Liu et al., 1998). The cocktail of XAP is not known to act in this manner, and its ability to enhance P digestibility and retention probably occurs via a different mechanism.

One possible mechanism of how XAP improves P digestibility could be deduced by taking note of the following. Although a phytate molecule is a strong chelating agent, it is not the only molecule that is responsible for chelation of nutrients. There is evidence that some soluble fibers, including soluble NSP, are responsible as well (Frølich, 1990). The author pointed out that the soluble NSP and phytic acid are in the aleurone cell layer. Parkkonen et al. (1997), using in vitro digestion techniques, reported that xylanase increased the permeability of the aleurone cell wall layer, which is the site of phytate storage. It is possible that XAP, by improving aleurone layer permeability, enhances the access of endogenous phytase to phytate molecules, hence improving P digestibility and retention.

yla et al. (1996) found in their study that to obtain complete dephosphorylation of a corn–SBM diet fed to poults, it was necessary to use an enzyme cocktail rather than just 1 enzyme that had single activity. An enzyme cocktail that contained phytase, pectinase, acid phosphatase, and protease activities was more effective than phytase alone at promoting the performance of turkey poults. It could be expected that an enzyme that is capable of breaking the NSP layer will also provide easier access to phytate. If phytase alone is used, the ability of the enzyme to act on phytate will be limited by its lack of access to its substrate if phytase is within the NSP matrix. Hence, glycosidases (xylanase and amylase) that are able to break down the NSP fraction can facilitate the contact between phytase and phytate. Additionally, some soluble fiber-bound P may be released in the presence of glycosidases, and this may explain how XAP alone is able to increase P digestibility.

Juanpere et al. (2005) reported interaction between phytase and glycosidases in wheat- and barley-based diets but not for corn-based diets for P retention. Wu et al. (2004b) reported that a combination of xylanase and phytase in a wheat-based diet adequate in nonphytate P did not produce better response in performance than was obtained with the enzymes used individually. In the current study, interactions were observed between phytase and XAP for Ca and P digestibility, which probably indicates that 1 of the enzymes in the cocktail affected the activity of the other. It is not clear whether phytase could have affected the activity of any of the enzymes in XAP cocktail. However, because in the 2 cases (ileal digestibility and total tract retention), the interaction was significant for P, and taking into consideration that phytase is the principal enzyme that improved P release, it is reasoned that the interaction observed was very likely an attenuation of phytase activity.

In conclusion, the addition of phytase and a cocktail of enzymes that contains XAP improved the performance of chickens fed a corn–SBM-based diet that was marginally deficient in energy and P. Improvement in performance observed when a combination of phytase and XAP was used was more likely from phytase, because phytase alone was able to improve daily gain, but XAP alone did not. We recognize the effect that the limiting nutrients have on chickens’ response to the enzymes used in this study. The current study also helps to confirm that chickens derive greater benefit from enzyme use in their diet when they are juvenile and that the benefit of such intervention as well as the magnitude of such benefit becomes smaller as the bird matures. It is likely that older birds would require different doses of enzymes, because their physiological needs and capacity are different from newly hatched chicks; this aspect requires further study to establish the practical benefit of such dietary intervention.

	ACKNOWLEDGMENTS

 
We acknowledge the assistance of the staff of Purdue Animal Sciences Small Animal House for set-up and maintenance of animal holding equipment and Pat Jaynes for technical assistance.

	FOOTNOTES

 
¹ Journal paper no. 17925 of the Purdue University Agricultural Research Program.

Received for publication May 1, 2006. Accepted for publication August 22, 2006.

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