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METABOLISM AND NUTRITION |
Center of Excellence for Poultry Science, University of Arkansas, Fayetteville 72701
2 Corresponding author: ccoon{at}uark.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: phytate P hydrolysis • phytase concentration • broiler • Ca and nonphytate phosphate levels • P physiological threshold
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There have been several published research reports using graded concentrations of phytase to hydrolyze PP but there have been few studies to determine the maximum amount of PP hydrolysis that could be obtained with mega doses of dietary phytase. Shirley and Edwards (2003) reported 94.8% PP hydrolysis in broilers fed 12,000 units of phytase (Natuphos 5000) per kilogram of diet.
Dietary phytase can be supplied to hydrolyze PP but the amount of NPP that can be spared (phosphorus equivalency) with phytase depends on the quantity of PP hydrolyzed. The retention of released P from PP hydrolysis has been shown to be dependent upon the optimum ratio of Ca:NPP or Ca:TP. In calculating P equivalency values for phytase, researchers (Schöner et al., 1991; Denbow et al., 1995; Mitchell and Edwards, 1996a,b; Kornegay et al., 1996) have only used 1 to 3 concentrations of NPP, thus limiting the ability to develop regression curves showing optimum PP hydrolysis. These reports do not show the effect of phytase on optimum PP hydrolysis in relation to dietary Ca using a broad range of graded concentrations of dietary NPP with fixed concentrations of Ca. In addition to formulating feed for the correct ratio of Ca to NPP, adequate concentrations of Ca and NPP are important for promoting bone deposition instead of bone resorption. A decrease in dietary NPP content increases percentage P retention but has been shown to also decrease Ca retention (Viveros et al., 2002). The decrease in Ca retention caused by greater Ca to NPP ratios could be attributed to increased intestinal pH and a reduced soluble fraction of minerals available for absorption (Shafey, 1993). An imbalance caused by an increased Ca to P ratio may result in bone resorption to maintain serum P while enhancing Ca excretion, thus causing lower Ca retention. Phytase supplementation increases both P and Ca retention (Sebastian et al., 1996; Qian et al., 1997). Retention of TP can be increased for poultry by maintaining a constant ratio of Ca:TP (2:1) with a fixed concentration of PP either by increasing dietary NPP concentrations or with graded concentrations of dietary phytase supplementation (Kornegay et al., 1996). Although the ratio of bone Ca to P is 2:1, the optimum ratio of 2.57:1 (0.9% Ca:0.35% NPP) has been recommended for broilers fed for the 3- to 6-wk period (NRC, 1994). The ability to hydrolyze PP by phytase may be reduced at greater concentrations of Ca (Taylor, 1965) because of increased pH in the intestine and an increase in Ca-PP salt formation. Reducing dietary Ca concentrations from 1 to 0.5% helps keep the Ca balanced with the lower concentrations of P and has been shown to increase the PP utilization by 15% in chicks (Mohammed et al., 1991).
The main objectives of the present study were to 1) determine the optimum dietary does of microbial phytase enzyme (Phyzyme XP 5000G, Danisco Animal Nutrition, Marlborough, UK) necessary for maximal PP utilization by broilers fed a corn-soybean meal-based diet from 21 to 26 d of age; 2) determine percentage PP hydrolysis in broilers fed corn-soybean meal-based diets with or without 1,000 FTU phytase/kg in combination with 8 different concentrations of dietary NPP, 2 Ca concentrations, and a low (fixed) concentration of PP from 21 to 26 d of age; and 3) evaluate P equivalency values based on PP hydrolysis with phytase at different dietary Ca:P concentrations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A 5-d chick bioassay was used to determine PP hydrolysis, TP retention, and phytase equivalency similar to 5-d bioassay previously reported by Leske and Coon (1999). One-day-old male broiler chicks (Cobb 500) were offered a standard starter diet (based on NRC, 1994 recommendation) in floor pens until 21 d of age. Chicks of a uniform body weight were placed in individual metabolic cages (38 cm wide x 51 cm long x 38 cm high) and offered corn-soybean test diets (Table 1
) with 8 concentrations of microbial phytase (Phyzyme XP 5000G, Danisco Animal Nutrition) added on top. Phyzyme XP 5000G, a phytase product derived from Escherichia coli and expressed in Schizosaccharomyces pombe is a 6-phytase (EC 3.1.3.2
[EC]
6) with acid phosphatase activity (pH optimum of 2 to 5.5). The 8 concentrations of the phytase tested were 0, 250, 500, 750, 1,000, 1,500, 2,000, and 5,000 FTU of phytase (based on analyzed product premix concentration). The broiler grower test diets were formulated to contain 0.70% Ca, 0.12% NPP, 0.28% PP on an as-is basis. The analyzed values of experimental diets are shown in Table 2. Ten individual broiler chicks were fed each test diet and subjected to a 23L:1D lighting schedule. Environmental housing temperatures from d 1 to 7, 8 to 14, and 15 to 25 were kept at 35, 32, and 27°C, respectively. Two percent acid insoluble ash (Celite, Celite Corp., Lompoc, CA) was added to the feed and used as a digestive marker. Chicks were acclimated to cages and diets, and excreta samples were collected for the last 24-h period during the experiment. The excreta were collected on trays, frozen (–20°C), and freeze-dried before analysis. Birds had unlimited access to feed and water for the duration of the experiment. Feed consumption and weight gain were recorded during the entire 5-d period of the experiment.
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One-day-old male broiler chicks (Cobb 500) were housed in floor pens and fed a crumbled corn-soybean meal-based starter diet that was formulated to meet or exceed all of their known nutrient requirements (NRC, 1994). On d 22, chicks of uniform body weight were then placed in individual metabolic cages and provided test diets consisting of 8 concentrations (0.08, 0.13, 0.18, 0.23, 0.28, 0.33, 0.38, and 0.45%) of NPP combined with a fixed concentration (0.5%) of Ca (Table 3). The Ca added to the basal diets consisted of ground limestone and Ca from reagent-grade calcium phosphate, dibasic, monohydrate (product C129-12; Fisher Scientific, Fairlawn, NJ). Sixteen diets were fed and consisted of 8 concentrations of NPP (0.08, 0.13, 0.18, 0.23, 0.28, 0.33, 0.38, and 0.45%) with or without 1,000 FTU/kg of phytase (Table 3). Ten broiler chicks each were fed 1 of the 16 test diets. Two percent acid insoluble ash (Celite) was added to the feed and used as a digestive marker. Husbandry practices and excreta collections were similar to those of Experiment 1. Feed consumption and weight gains were recorded during the entire 5-d period of the experiment.
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Experiment 3 was similar to Experiment 2 except that the Ca concentration was maintained at 0.9% (NRC, 1994 for 3- to 6 wk-old broilers) by adding ground limestone and Ca from reagent-grade calcium phosphate, dibasic, monohydrate (product C129-12; Fisher Scientific; Table 3
). The analyzed values for experimental diets used in Experiments 2 and 3 are shown in Table 4. Animal use protocols for the 3 experiments were approved by the University of Arkansas Institutional Animal Care and Use Committee.
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In all 3 experiments, diets and excreta were analyzed for TP and Ca by inductively coupled plasma emission spectroscopy as described by Leske and Coon (2002). Acid insoluble ash was determined in experimental diets and excreta samples using dry ash and the hydrochloric acid digestion technique of Scott and Balnave (1991). Diet and excreta PP were measured as inositol hexaphosphate by using ion-exchange chromatography as described by Bos et al. (1991). The N in feed and moisture in feed and excreta were determined by methods 990.03 (AOAC International, 1995) and 934.01 (AOAC, 1990) respectively. Retention of TP, apparent PP hydrolysis, and retention of NPP were determined using the diet and excreta P, PP, and acid insoluble ash concentrations according to Scott and Balnave (1991). The experimental diets were assayed for phytase by Danisco Animal Nutrition (Marlborough, UK) using the method reported by Engelen et al. (1993). Retainable P (RP) was determined by the method reported by Leske and Coon (2002). The P equivalency values for phytase were calculated based on the differences between the amount (%) of dietary PP that is hydrolyzed by birds fed diets with and without inclusion of phytase using the following equation: [(% excreta PP hydrolysis with dietary phytase inclusion x % PP in the diet)/100] – [(% excreta PP disappearance with no dietary phytase inclusion x % PP in the diet)/100].
The P equivalency values obtained for different levels of dietary phytase inclusion were expressed based on the DM content of the experimental diets.
Data from Experiment 1 were subjected to one-way ANOVA to determine the overall effect of phytase, and data from Experiments 2 and 3 were subjected to 2-way ANOVA to determine the main effects of NPP and phytase and their interaction using the GLM procedure (SAS Institute, 1999). The significance was tested at P 
0.05. Orthogonal polynomial contrast coefficients were used to determine the linear and quadratic responses of increasing dietary phytase concentration (Experiment 1) and increasing dietary NPP concentration with and without phytase supplementation and interaction of phytase and NPP (Experiments 2 and 3). Data on TP retention and PP hydrolysis from Experiment 1 were subjected to nonlinear regression analysis (Fox and Shotton, 1995). Data on retention of TP and NPP from Experiments 2 and 3 were subjected to 1-slope broken-line and 2-slope broken-line regression analysis (Coon et al., 2007).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body weight gain (BWG) and feed:gain ratio (F:G) improved quadratically (P < 0.05) whereas feed intake (FI) increased linearly with phytase supplementation from 0 to 5,000 FTU/kg (Table 5). The intake of TP, PP, and NPP increased linearly (P < 0.05) with the increase in dietary phytase supplementation. The apparent TP retention and PP hydrolysis increased quadratically. Chicks fed the diets with added phytase at 250, 500, 750, 1,000, 1,500, 2,000, and 5,000 FTU/kg of feed contributed P equivalency (based on PP hydrolysis) of 0.08, 0.1, 0.13, 0.13, 0.15, 0.17, and 0.17%, respectively. The RP values determined for all 8 experimental diets based on TP retention indicate that the maximum RP was obtained for chicks fed 1,000 FTU phytase/kg of diet (Table 2).
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Broiler chicks fed diets containing 0.5% Ca and increasing concentrations of dietary NPP from 0.08 to 0.45% without dietary phytase supplementation exhibited a linear increase in BWG, FI, TP intake, PP intake, and NPP intake (Table 6). The F:G ratio decreased linearly and TP and NPP retention decreased quadratically for chicks fed increasing amounts of NPP. The addition of 1,000 FTU of phytase/kg of diet combined with increasing concentrations of dietary NPP resulted in a linear increase in BWG, TP intake, and NPP intake and a linear decrease in F:G, TP retention, and apparent PP hydrolysis. The broiler chicks fed phytase and additional NPP produced a quadratic decrease in NPP retention. The orthogonal polynomial contrasts revealed a significant linear interaction of NPP and phytase on NPP intake, TP retention, apparent PP hydrolysis, and NPP retention.
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Broiler chicks fed diets containing 0.9% Ca and increasing concentrations of dietary NPP from 0.08 to 0.45% without dietary phytase supplementation exhibited linear increases in BWG, TP intake, and NPP intake (Table 7). The F:G ratio decreased linearly with increasing dietary NPP, whereas TP retention, apparent PP hydrolysis, and NPP retention decreased quadratically. The addition of 1,000 FTU of phytase/kg of diet to increasing concentrations of dietary NPP resulted in a linear increase in intake of TP and NPP. The apparent PP hydrolysis and retention of TP and NPP decreased quadratically. The orthogonal polynomial contrasts showed a significant linear interaction of NPP and phytase on the retention of TP and for apparent PP hydrolysis.
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). Broilers fed increasing concentrations of NPP above 0.08% in combination with 0.5% Ca exhibited a decline in the P equivalency values for phytase. The P equivalency value was lowest (0.06%) for phytase when broilers were fed 0.90% Ca along with the lowest inclusion level of dietary NPP (0.08%). The maximum P equivalency (0.12%) for phytase was obtained for broilers fed 0.23% dietary NPP in combination with 0.90% Ca. Dietary increases above 0.23% NPP with 1,000 FTU of phytase/kg of diet (with 0.9% Ca) produced a plateau response for P equivalency values.
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). The estimates for feed TP retention and feed percentage TP (Experiment 2) with no added phytase were 59.33 ± 2.79% and 0.37 ± 0.02%, and with added phytase were 68.85 ± 3.14% and 0.34 ± 0.04%, respectively (Figure 2). Similarly, the estimates for NPP retention and feed percentage NPP at breakpoint for birds fed 0.9% Ca (Experiment 3) diet with no added phytase were 87.47 ± 5.52% and 0.31 ± 0.03%, and with added phytase were 63.34 ± 5.58% and 0.18 ± 0.03%, respectively (Figure 3). The estimates for TP retention and feed percentage TP (Experiment 3) with no added phytase were 52.45 ± 3.04% and 0.54 ± 0.03%, and with added phytase were 64.83 ± 2.10% and 0.40 ± 0.05%, respectively (Figure 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The possibility of microbial degradation of excreta PP during a 24-h excreta collection in the present bioassays was ruled out because the hydrolysis of PP (disappearance of PP in excreta) was greater with increasing concentrations of enzyme (Experiment 1) compared with hydrolysis of PP from broilers fed diets without added phytase. Previous research by Sooncharernying and Edwards (1993) could not detect microbial or enzymatic degradation of PP in excreta that was collected from broilers on dropping trays for a 32-h collection period.
The RP calculated from breakpoints for percentage TP retention indicated that RP would be approximately the same for broilers fed diets with or without phytase when the broilers were fed the same dietary concentration of Ca. Broilers fed diets with 0.5% Ca (Experiment 2) had RP of 0.23 and 0.22% with and without dietary phytase, respectively. Broilers fed diets with 0.9% Ca had RP values of 0.26 and 0.28% with and without dietary phytase, respectively. The data indicate a diminishing return for RP when broilers consumed additional biologically available P above the breakpoint for maximum P retention. The biological available P concentration above the breakpoint that caused a reduction in RP could be from added dietary NPP, phytase supplementation, or a combination of phytase and NPP.
A key observation from the present research was the innate ability of broilers to utilize NPP more efficiently at the lowest dietary NPP concentrations with Ca:NPP ratios of 6.25:1 (Experiment 2) compared with 11.25:1 (Experiment 3). The efficiency of TP retention was maximal with dietary phytase supplementation compared with broilers fed no added phytase. The percentage TP retention began declining when broilers consumed dietary TP above a set amount (Figures 2 and 4) irrespective of phytase addition. A similar observation was also seen for broilers fed increasing levels of phytase in Experiment 1. Leske and Coon (2002) reported that the inorganic P from reagent or food-grade monocalcium phosphate, dicalcium phosphate, and defluorinated phosphate was retained at 85 to 90% until the physiological steady-state concentration for P was reached. The researchers showed that P retention from a reagent-grade monocalcium phosphate declined from 94 to 58.5% when NPP was increased from 0.21% to the NRC (1994) suggested level of 0.45%. Manangi and Coon (2006) reported that colostomized broilers fed increasing amounts of dietary P showed an increase in plasma P that eventually reached a steady state; with additional P intake, the broilers increased P excretion in urine and feces. The researchers showed that the biological P threshold was substantially below the suggested NRC (1994) requirement for available P. Coon et al. (2007) recently reported a lower physiological P threshold than the suggested NRC (1994) requirement for available P with broilers fed 10 levels of NPP (from 0.15 to 0.85%) and a fixed level of PP (0.18%). The increased P retention in the present study for broilers fed diets with less NPP compared with greater NPP concentrations was similar to findings of Um and Paik (1999), Ravindran et al. (2000), Keshavarz (2000), and Viveros et al. (2002).
Previous research has shown that the P equivalency value for phytase of different origins may be from 500 to 900 U/kg of diet to replace 0.1% of inorganic P in broiler diets (Schöner et al., 1991; Mitchell and Edwards, 1996a,b; Kornegay et al., 1996). It may be inappropriate to compare previous reported P equivalency values for phytase with those of the present study based on actual quantification of PP (IP6) hydrolysis or disappearance because previous reports on phytase P equivalency were based on comparing bird performance or bone ash with birds fed monocalcium or defluorinated phosphate. The discrepancy or variation in phytase P equivalency in previous reports compared with the present work may be attributed to a) source of microbial phytase and its efficacy in the biological system, b) source and amount of dietary phytate, c) amount of dietary Ca and available P, d) age of birds or period of study, and e) phytase overage in the product.
In summary, nearly complete hydrolysis (99.45%) of PP was obtained for broilers fed high concentrations of dietary phytase (5,000 FTU/kg) but maximum TP retention was obtained with only 1,000 FTU of added phytase. Maximum PP hydrolysis occurred for broilers fed diets with 1,000 FTU added phytase when the diets contained the lowest concentration (0.08%) of dietary NPP with either 0.5 or 0.9% dietary Ca concentrations. Broilers fed 0.9% dietary Ca have a greater P physiological threshold before a loss in percentage RP compared with broilers fed 0.5% dietary Ca. Dietary NPP, P released from dietary phytase, or a combination of these dietary P sources will decrease the percentage RP when fed at concentrations above the P physiological threshold. The average P equivalency provided from phytase at 1,000 FTU/kg was approximately the same in Experiment 2 (0.094%) and Experiment 3 (0.099%); however, the main difference in P equivalency when using phytase with 0.5 and 0.9% dietary Ca diets is that when the diet contains increasing TP, the equivalency of P from phytase will increase with 0.9% dietary Ca and decrease with 0.5% dietary Ca.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 13, 2007. Accepted for publication April 19, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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