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Efficacy of an Escherichia coli Phytase in Broilers Fed Adequate or Reduced Phosphorus Diets and Its Effect on Carcass Characteristics
Efficacy of an Escherichia coli Phytase in Broilers Fed Adequate or Reduced Phosphorus Diets and Its Effect on Carcass Characteristics P. B. Pillai, T. O’Connor-Dennie, C. M. Owens, and J. L. Emmert1 Department of Poultry Science, University of Arkansas, Fayetteville 72701 tibia ash of chicks fed E. coli phytase (250, 500, or 1,000 phytase units/kg) did not differ (P > 0.05) from that of chicks fed the P-adequate diet. In addition, carcass yield of broilers fed E. coli phytase was not reduced (P > 0.05). In EXP 4, E. coli phytase effectively supported weight gain, tibia ash, breast yield, and leg yield compared with birds fed the P-adequate diet, but clavicle breakage during processing was increased in birds fed E. coli phytase. In EXP 5, E. coli phytase again effectively supported weight gain, and no differences (P > 0.05; compared with the P-adequate diet) were noted for clavicle ash, diameter, or breaking strength. No differences (P > 0.05) in bone breakage during processing were noted among treatments. These results indicate that the addition of E. coli phytase to P-deficient broiler diets improves growth, bone, and carcass performance and is more effective at releasing phytate-bound P than the other phytase products that were tested.
Key words: phosphorus, broiler chick, bioavailability, phytase 2006 Poultry Science 85:1737–1745
INTRODUCTION A major issue facing the broiler industry is maintaining bone integrity while reducing feed cost and reducing environmental pollution. Bone breakage in broilers is not only an economic issue for processors but a welfare issue as well (Rath et al., 2000), with quality of nutrition playing an important role. The use of phytase enzymes may prove to be a method to address both issues. The effectiveness of phytase in releasing bound Ca, P, and other divalent minerals important in bone development has been well established (Biehl and Baker, 1997; Harper et al., 1997; Zanini and Sazzad, 1999; Boling-Frankenbach et al., 2001; Augspurger et al., 2003; Augspurger and Baker, 2004a,b; Shelton et al., 2004; Onyango et al., 2005). It has been estimated that phytase releases at least 0.09% bound Ca and P (Augspurger et al., 2003; Augspurger and Baker, 2004b; Shelton et al., 2004) and improves digestibility of
2006 Poultry Science Association Inc. Received March 23, 2006. Accepted June 11, 2006. 1 Corresponding author: [email protected]
these nutrients (Harper et al., 1997; Zanini and Sazzad, 1999; Jalal and Scheideler, 2001; Dilger et al., 2004). With the importance of P in the environment, phytase source and efficacy has become an important issue to the poultry industry. Dephosphorylation of the phytate molecule occurs at different reaction sites, depending on the origin of the phytase enzyme that is catalyzing the reaction (Rodriguez et al., 1999b; Tamim et al., 2004). Furthermore, phytase enzyme origin also affects the pH at which the enzyme is most effective and its ability to resist breakdown in the stomach and small intestine (Rodriguez et al., 1999a; Augspurger et al., 2003), hence the differences in the efficacy of different phytase enzymes. Recent research has indicated that a new Escherichia coli phytase (OptiPhos, JBS United Inc., Sheridan, IN) is more efficacious than other commercially available phytases in releasing at least 0.10% P (Augspurger et al., 2003). Although information provided in these research findings is invaluable, there exists a lack of information about the effect of phytase enzymes on carcass characteristics such as the incidence of bone breakage during processing. Breakage of bones, such as the tibia, clavicle, and coracoid, affects both product condemnation rates and food safety,
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ABSTRACT Five experiments (EXP) were conducted to assess the efficacy of an Escherichia coli phytase compared with 2 commercially available fungal phytases. In EXP 1 and 2, male broiler chicks were fed experimental diets that included a P-deficient control (0.13% available P; 0.88% Ca) alone or with graded levels of KH2PO4 (0, 0.05, 0.10, or 0.15%) or phytase at levels of 250, 500, 1,000, 2,000, or 4,000 phytase units/kg of E. coli phytase (EXP 1 and 2), fungal phytase 1 (EXP 2), or fungal phytase 2 (EXP 2). In EXP 1 and 2, weight gain and tibia ash (mg/ chick and %) responded linearly (P < 0.05) to inorganic P addition. In EXP 2, each level of E. coli phytase released more P than either fungal phytases 1 or 2, whether based on tibia ash weight (mg/chick) or percentage. In EXP 3, 4, and 5, dietary treatments containing adequate or deficient levels of P were fed with or without supplemental E. coli phytase. In EXP 3, weight gain and tibia ash were reduced (P < 0.05) by P deficiency, but gain and
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which are extremely important for the broiler industry. Five experiments (EXP) were conducted with the objectives of assessing the relative P-releasing ability of E. coli phytase and comparing its efficacy to 2 commerciallyavailable fungal phytase products [fungal phytase 1 (FP1; Natuphos, BASF, Mount Olive, NJ) and fungal phytase 2 (FP2; Ronozyme, DSM Nutritional Products Inc., Parsippany, NJ)]. The effect of E. coli phytase on growth performance, tibia and clavicle ash, and bone breakage during processing in commercial broilers was measured.
MATERIALS AND METHODS
EXP 1 and 2 On d 9 or 8 posthatching (EXP 1 and 2, respectively), chicks were weighed, wing-banded, and randomly allotted to dietary treatments such that each pen within an EXP would have a similar average initial weight and weight range. Each EXP consisted of 5 replicates of 5 chicks per replicate housed in batteries with raised wire floors, and a 24-h photoperiod was used. Experimental diets (Table 1) were fed until d 23 or 22 posthatching (EXP 1 and 2, respectively), at which time chicks and feed were weighed for determination of weight gain, feed intake, and feed efficiency. At the termination of each EXP, chicks were killed by cervical dislocation, and right tibias were collected for subsequent analysis of ash weight per chick (mg/chick) and percentage of ash. In EXP 1 and 2, standard-curve methodology (Biehl et al., 1995) was used, with KH2PO4 (22.8% P) serving as the standard. Dietary additions were made to a 23.1% CP corn–soybean meal diet that contained no supplemental inorganic P (iP) and was analyzed to contain 0.40% total P and 0.88% Ca (calculated to contain 0.13% iP; Table 1).
EXP 3 Experiment 3 consisted of 5 dietary treatments, replicated 8 times, with each replicate containing 20 birds. All diets were based on corn and soybean meal and met or exceeded NRC (1994) recommendations for all nutrients, with the exception of P, when appropriate (Table 1). Experimental diets (Table 1) were fed from 3 to 21 d and 21 to 42 d, and treatments consisted of the following diets: 1) a positive control containing adequate levels of Ca (1.0 and 0.9% during the starter and grower periods, respectively) and iP (0.47 and 0.46% during the starter and grower periods, respectively); 2) a negative control containing an adequate level of Ca (1.0 and 0.9% during the starter and grower periods, respectively) and a deficient level of iP (0.31 and 0.28% during the starter and grower periods, respectively); and 3), 4), and 5) diet 2 with graded levels of E. coli phytase (250, 500, and 1,000 FTU/kg). Birds and feed were weighed at EXP initiation, during phase changes, and at assay termination (d 42) to calculate weight gain, feed intake, feed efficiency, and livability for each period and overall. Feeders were removed from experimental pens 10 h before EXP termination. Following weighing, 5 birds per pen were randomly selected for processing at the University of Arkansas Poultry Processing Plant.
EXP 4 and 5 In EXP 4, there were 6 dietary treatments with 7 replicate pens (20 birds per pen); in EXP 5, there were 4 dietary treatments with 4 replicate pens (15 birds per pen). All diets were based on corn and soybean meal and met or exceed NRC (1994) recommendations for all nutrients, with the exception of Ca and P, when appropriate (Table 1). Birds and feed were weighed at EXP initiation, during phase changes, and at assay termination (d 50 or 56 in EXP 4 and 5, respectively) to calculate weight gain, feed intake, feed efficiency, and livability for each period and overall. Feeders were removed from experimental pens 10 h before EXP termination. Following weighing, 5 birds per pen (EXP 4) were randomly selected for processing at the University of Arkansas Poultry Processing Plant; all birds from EXP 5 were processed. In EXP 4, chicks were weighed and placed on treatment diets on d 1 posthatch. Experimental diets (Table 1) were fed as follows: starter (1 to 18 d), grower (18 to 32 d), finisher (32 to 40 d), and withdrawal (40 to 50 d). Dietary treatments consisted of the following diets: 1) a positive control adequate in all essential nutrients for the respective phases; 2) diet 1 with Ca reduced by 0.05% and P
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All procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee. Before the beginning of EXP 1 and 2 and throughout EXP 3, 4, and 5, male Cobb × Cobb (Cobb-Vantress Inc., Siloam Springs, AR) broilers were housed in floor pens with pine shavings, hanging tube feeders, and Plasson waterers. Before the initiation of EXP 1, 2, and 3, chicks were fed a common corn–soybean meal starter diet that contained 24% CP and met or exceeded all NRC (1994) nutrient recommendations. Water and feed were freely available. A review of the differences among the different phytase enzymes used in these EXP is given by Rodriguez et al. (1999b), Applegate et al. (2003b), Augspurger et al. (2003), and Onyango et al. (2005). Dietary additions of phytase were made according to the experimental procedures of Augspurger et al. (2003) and Augspurger and Baker (2004a), who assayed the same phytases used herein and defined phytase activity as the quantity of the enzyme that will liberate 1 mol of inorganic P per min from 5.1 mM Na phytate at a pH of 5.5 and temperature of 37°C. Thus, dietary additions in each EXP were made to accomplish dietary phytase activity levels as defined under the aforementioned conditions.
In EXP 1 and 2, graded levels (0, 0.05, 0.10, 0.15%) of KH2PO4 were used to construct the standard curve. In EXP 1, 5 levels of E. coli phytase (250, 500, 750, 1,000, or 10,000 phytase units (FTU)/kg) were tested; in EXP 2, 5 levels of E. coli phytase, FP1, or FP2 (250, 500, 1,000, 2,000, 4,000) were added to the basal diet to allow quantification of phytate-bound P release.
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EFFECT OF PHYTASE ON CARCASS CHARACTERISTICS Table 1. Percentage composition of experimental diets EXP1 3
3
4 and 52
4 and 5
4 and 5
4 and 52
Ingredient
Starter3
3 to 21 d
21 to 42 d
0 to 18 d
18 to 32 d
32 to 40 d
40 to 56 d
Corn Soybean meal Soybean oil Poultry fat Dicalcium phosphate Limestone NaCl Vitamin mix4 Mineral mix4 Choline Cl (60%) DL-Met L-Thr Sacox salinomycin5 Cornstarch Composition6 ME, kcal/kg CP, % Total P, %7 Total Ca, %
51.69 39.69 5.00 — — 1.67 0.40 0.20 0.15 0.20 0.20 — — to 100
50.93 39.69 5.00 — 1.20 1.59 0.40 0.20 0.10 0.20 0.25 — — to 100
57.16 34.00 5.00 — 0.70 1.65 0.40 0.20 0.10 0.20 0.15 — — to 100
55.95 37.18 — 3.05 0.58 0.93 0.49 0.20 0.10 0.15 0.18 0.03 0.05 to 100
61.14 32.33 — 2.99 0.36 0.90 0.49 0.20 0.10 0.15 0.17 0.01 0.05 to 100
68.71 25.25 — 2.64 0.87 0.27 0.51 0.20 0.10 0.15 0.11 0.03 0.05 to 100
70.63 23.39 — 2.99 0.08 0.85 0.49 0.20 0.10 0.15 0.02 — — to 100
3,100.00 23.1 0.40 0.88
3,115.00 23.2 0.578 1.00
3,185.00 21.0 0.538 0.90
3,050.00 23.0 0.63/0.589 1.00/0.829
3,100.00 21.1 0.53/0.489 0.80/0.809
3,050.00 18.4 0.45/0.439 0.76/0.739
3,200.00 17.7 0.41/0.419 0.72/0.659
1
EXP = experiment. The starter feed was fed from 8 to 18 d in EXP 5; the withdrawal feed was fed from 40 to 50 d in EXP 4. 3 Diets fed from 9 to 23 d in EXP 1 and from 8 to 22 d in EXP 2. 4 Vitamin and mineral mix in EXP 1 and 2 from Han and Baker (1993); vitamin and mineral mix in EXP 3 and 4 from Emmert et al. (1999). 5 Sacox 60, Hoechst-Roussel Agri-Vet. Co., Somerville, NJ; provided 66 mg/kg of salinomycin activity. 6 Metabolizable energy and CP values were calculated; diets were analyzed for total P and Ca. 7 Diets were estimated to contain the following inorganic P levels: 0.13% for EXP 1 and 2; 0.31 and 0.28% for the starter and grower periods, respectively, in EXP 3; 0.28, 0.22, 0.18, and 0.14% for the starter, grower, finisher, and withdrawal periods, respectively, in EXP 4; 0.26, 0.20, 0.17, and 0.14% for the starter, grower, finisher, and withdrawal periods, respectively, in EXP 5. 8 Positive control diets (diet 1) were analyzed to contain 0.73% total P (0.47% estimated available P) and 0.61% total P (0.46% estimated available P) for the starter and grower periods, respectively. 9 Analytical values for dietary Ca and P are shown for EXP 4 and 5 (EXP 4 values are to the left of the slash). 2
reduced by 0.10%; 3) diet 1 with 300 FTU/kg of E. coli phytase; 4) diet 2 with 300 FTU/kg of E. coli phytase; 5) diet 1 with dietary Ca reduced by 0.05% and dietary P reduced by 0.15% P and 600 FTU/kg of E. coli phytase; and 6) diet 1 with dietary Ca reduced by 0.05% and dietary P reduced by 0.20% and 1,000 FTU/kg of E. coli phytase. Treatment additions to the basal diet were made at the expense of cornstarch. Due to concerns over the detrimental effects of feeding birds diets that were too deficient in Ca and iP for the whole duration of the project, corresponding negative controls for treatments 5 and 6 were not formulated. Experiment 5 was similar to EXP 4, with the exception of the EXP initiation (d 8) and termination (d 56) and the dietary treatments, which included only treatments 1, 2, 4, and 6 from EXP 4.
Processing Variables After arrival at the processing plant, birds were hung on a shackle line and commercially processed to evaluate carcass and parts yields, and the incidence of bones that were broken or disjointed. Birds were electrically stunned (11 V, 11 mA, 11 s), manually bled by severing the left carotid artery and jugular vein, bled out (1.5 min), softscalded (129°F, 2 min), and feathers were picked with the use of inline commercial defeathering equipment. Evis-
cerating and rinsing followed, after which carcasses were placed in a prechill tank at 12 C for 15 min. Carcasses were then moved to an immersion chiller (1°C) for 45 min, after which carcasses were removed, packed in ice, and aged at 4°C until time of deboning (EXP 4 and 5) at 4 h postmortem. Carcasses were separated into breast (pectoralis major and pectoralis minor), wings, legs, and frame, followed by weighing and removal of the tibia from the legs for subsequent ash analysis.
Bone Response Variables Tibias were autoclaved for 45 min to remove adhering muscle and cartilage, dried at 110°C for 24 h, and subsequently weighed. Dry tibias were ashed in a muffle furnace at 600°C for 18 h. After cooling, tibia ash was weighed for determination of ash weight and bone ash percentage. In EXP 4 and 5, the incidence of broken bones (tibia, coracoid, clavicle, and radius and ulna) and disjointed wings that had occurred during processing were noted at the time that birds were separated into parts. The incidence of broken or disjointed bones was expressed as a percentage of the number of birds selected for processing. For clavicles, the incidence rate reflects the sum of clavicles broken on the shaft or at the point of fusion. In EXP 5, clavicles were collected at the time of deboning
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1 and 2
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Statistical Analysis Data were subjected to ANOVA (SAS Institute, 2004) appropriate for a completely randomized design; treatment means were separated using the least significant difference multiple-comparison procedure or Duncan’s multiple range test (in the case of missing replicate values). Single df contrasts were used to test overall effects of phytase (when appropriate). Standard curves were established (SAS Institute, 2004), with tibia ash (mg/chick or %) as the dependent variable and consumption (g) of supplemental P as the independent variable for EXP 1 and 2. By insertion of replicate values for tibia ash (mg/ chick or %) into the standard curve equation, the amount of P released by the enzyme was calculated for treatments 6 to 10 (EXP 1). Pens served as the experimental unit for all data analyzed, and means were considered significant at P < 0.05.
RESULTS EXP 1 Growth and bone response variables are represented in Table 2. Increasing iP resulted in a linear increase in weight gain and feed intake (P < 0.01); adding E. coli phytase resulted in a linear and quadratic increase in weight gain (P < 0.05). Feed efficiency increased with increasing iP and E. coli phytase levels (P < 0.05). Increasing iP resulted in a linear increase in tibia weight and
tibia ash (P < 0.05). Standard curve regression equations (Table 2) were as follows: for percentage of tibia ash, Y = 38.95 + 8.48X (r2 = 0.72) and for milligrams of tibia ash, Y = 272.98 + 163.62X (r2 = 0.65). Bioavailable P release estimates ranged from 0.11 to 0.19% for 250 to 10,000 FTU/kg of supplemented E. coli phytase when based on tibia ash weight (mg/chick) and 0.09 to 0.19% for 250 to 10,000 FTU/kg of supplemented E. coli phytase when based on tibia ash expressed as a percentage (Table 2). It should be noted that the tibia ash values for birds fed diets with 750 FTU of E. coli phytase/kg or greater exceeded the tibia ash value for birds fed 0.15% supplemental iP (diet 4).
EXP 2 Growth and bone response variables are presented in Table 3. Increasing the level of supplemental iP and phytase increased weight gain and feed intake (P < 0.01), regardless of phytase source. Feed efficiency also increased (P < 0.01) with increasing iP or phytase levels. Overall, weight gain and feed efficiency were greater (P < 0.05) for chicks fed E. coli phytase than for chicks fed FP1 or FP2. Whether expressed as milligrams per chick or a percentage, tibia ash increased linearly (P < 0.01) with increasing iP or phytase levels, regardless of phytase source. Overall, tibia ash (mg/chick or %) was greater (P < 0.05) for chicks fed E. coli phytase than for chicks fed FP1 or FP2. Standard curve regression equations (Table 3) were Y = 34.53 + 11.46X; r2 = 0.87 for percentage of tibia ash, and Y = 340.58 + 249.43X; r2 = 0.85 for milligrams of tibia. The P-releasing ability of E. coli phytase exceeded that of the other phytase sources; estimates for E. coli phytase ranged from 0.12 to 0.24% for 250 to 4,000 FTU of E. coli phytase/kg, compared with 0.07 to 0.18% for FP1 and 0.03 to 0.11% for FP2 (at the same inclusion levels) when based on tibia ash weight (Table 3). When based on tibia ash percentage, P-releasing ability estimates were 0.12 to 0.15%, 0.04 to 0.12, and 0.02 to 0.07% for E. coli phytase, FP1, and FP2, respectively. Similar to EXP 1, tibia ash values for birds fed the higher levels of E. coli phytase exceeded the tibia ash value for birds fed 0.15% iP.
EXP 3 Growth performance data for EXP 3 is shown in Table 4. Birds fed the negative control (diet 2) had reduced (P < 0.05) feed intake, tibia ash, and live weight compared with birds fed the positive control (diet 1), but no differences (P > 0.05) in weight gain, feed efficiency, and carcass yield were detected. The addition of 500 FTU/kg of E. coli phytase reversed the negative effects of the low-iP diet on weight gain and tibia ash (P < 0.05); 250 FTU/kg of E. coli phytase reversed the negative effects on tibia ash; and 1,000 FTU/kg of E. coli phytase reversed all the negative effects of the low-iP diet. In addition, birds fed diets supplemented with 1,000 FTU/kg of E. coli phytase
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for measurement (on the same d) of diameter, tension force, and break force. Intact clavicles (at time of collection) were subjected to a tension test, in which tension force (the force required to separate the clavicles at the point of fusion) was measured in newtons on a texture analyzer (TA-XT2i, Texture Technologies Corp., Scarsdale, NY) equipped with a 5-kg load cell and 2 attached adjustable grips (TA-96, Texture Technologies Corp.) placed opposite each other on the machine. The ends of the bone (opposite of midpoint) were trimmed so that the bone could be mounted in adjustable grips. The clavicle was placed in the grip 15 mm away from the midpoint of the clavicle on both sides of the bone. The clavicle bone was then pulled apart until broken at a speed of 5 mm/sec with tension strength (N) measurements being recorded. Following the tension test, these bones, in addition to the bones that were previously broken at the point of fusion during processing, were broken by using a 3-point bendbreaking method on 1 side of the clavicle. The 3-point bend test was conducted using the texture analyzer (TAXT2i, Texture Technologies Corp.) fitted with a 1.5 × 11 mm incisor knife blade (TA-45, Texture Technologies Corp.) attachment at a speed of 5 mm/sec. Force (N) required to break bones was recorded and represented break force. Any clavicles which were broken along the shaft before testing were not used in bone strength analysis; all clavicles were subjected to ash analysis (following strength measurements) as described for tibias.
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EFFECT OF PHYTASE ON CARCASS CHARACTERISTICS 1
Table 2. Effect of phytase on growth performance and tibia ash in Experiment 1 Weight gain3 (g)
Diet 6
1. Basal diet 2. Diet 1 + 0.05% iP (KH2PO4) 3. Diet 1 + 0.10% iP 4. Diet 1 + 0.15% iP 5. Diet 1 + 250 FTU/kg of Escherichia coli phytase 6. Diet 1 + 500 FTU/kg of E. coli phytase 7. Diet 1 + 750 FTU/kg of E. coli phytase 8. Diet 1 + 1,000 FTU/kg of E. coli phytase 9. Diet 1 + 10,000 FTU/kg of E. coli phytase Pooled SEM
289 332 357 374 365 359 387 352 388 18.8
Gain:feed (g/kg) b
561 565b 593ab 577ab 624a 588ab 600ab 568ab 589ab 20.9
Supplemental P intake (mg) 0 295 601 968 0 0 0 0 0 —
Tibia ash3,4,5
Bioavailable P release from tibia ash2
(%)
(mg)
(%)
(mg)
39.5 41.7 45.3 46.5 44.4 47.0 48.6 47.5 49.7 0.78
281 314 375 427 356 382 479 404 476 27.9
— — — — 0.11 0.16 0.18 0.16 0.19 0.012
— — — — 0.09 0.13 0.19 0.13 0.19 0.022
Means within a column lacking a common superscript differ (P < 0.05). Values are means of 5 pens of 5 male chicks fed the experimental diets from 9 to 23 d. 2 Diet 5 vs. diets 6, 7, 8, and 9 (P < 0.05). 3 Weight gain and tibia ash (% and mg) responded linearly to supplemental inorganic P (iP; P < 0.05); linear and quadratic responses were significant for weight gain and tibia ash (% and mg) regressed against E. coli phytase levels including 250, 500, 750, and 1,000 phytase units (FTU)/ kg (P < 0.05). 4 The linear regression equation of tibia ash (%) for diets 1 to 4 as a function of supplemental iP intake (g) was Y = 38.95 (±0.77) + 8.48 (±1.27)X (r2 = 0.72). 5 The linear regression equation of tibia ash (mg) for diets 1 to 4 as a function of supplemental iP intake (g) was Y = 272.98 (±16.81) + 163.62 (±28.51)X (r2 = 0.65). 6 The basal diet was analyzed to contain 0.40% total P and estimated to contain 0.13% inorganic P (iP). a,b 1
Diet
Weight Supplemental gain2 Gain:feed3 P intake (g) (g/kg) (mg)
1. Basal diet7 331 2. Diet 1 + 0.05% iP (KH2PO4) 424 3. Diet 1 + 0.10% iP 455 4. Diet 1 + 0.15% iP 486 5. Diet 1 + 250 FTU8/kg of Escherichia coli phytase 472 6. Diet 1 + 500 FTU/kg of E. coli phytase 510 7. Diet 1 + 1,000 FTU/kg of E. coli phytase 542 8. Diet 1 + 2,000 FTU/kg of E. coli phytase 548 9. Diet 1 + 4,000 FTU/kg of E. coli phytase 584 10. Diet 1 + 250 FTU/kg of FP1 425 11. Diet 1 + 500 FTU/kg of FP1 455 12. Diet 1 + 1,000 FTU/kg of FP1 451 13. Diet 1 + 2,000 FTU/kg of FP1 499 14. Diet 1 + 4,000 FTU/kg of FP1 518 15. Diet 1 + 250 FTU/kg of FP2 385 16. Diet 1 + 500 FTU/kg of FP2 413 17. Diet 1 + 1,000 FTU/kg of FP2 457 18. Diet 1 + 2,000 FTU/kg of FP2 428 19. Diet 1 + 4,000 FTU/kg of FP2 481 Pooled SEM 19.5 Contrasts E. coli phytase vs. FP1 P < 0.05 E. coli phytase vs. FP2 P < 0.05 FP1 vs. FP2 P < 0.05 1
629 668 687 684 665 703 748 732 747 667 670 647 687 694 636 655 702 650 685 19.1
0 323 664 1,067 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
P < 0.05 P < 0.05 —
— — —
Tibia ash4,5,6
Bioavailable P release from tibia ash
(%)
(mg)
(%)
(mg)
33.4 39.8 42.2 46.2 44.3 47.8 47.5 49.0 47.7 37.2 38.8 44.6 42.8 44.7 35.0 35.7 35.7 39.4 40.5 0.69
334 421 520 599 550 661 759 807 806 424 464 513 559 673 384 391 463 475 539 19.8
— — — — 0.12 0.16 0.16 0.17 0.15 0.04 0.06 0.13 0.10 0.12 0.02 0.05 0.02 0.07 0.07 0.009
— — — — 0.12 0.18 0.23 0.25 0.24 0.07 0.07 0.10 0.12 0.18 0.03 0.05 0.08 0.08 0.11 0.009
P < 0.05 P < 0.05 P < 0.05
P < 0.05 P < 0.05 P < 0.05
P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05
Values are means of 5 pens of 5 male chicks fed the experimental diets from 8 to 22 d. Linear response of weight gain to supplemental inorganic P (iP), E. coli phytase, fungal phytase 1 (FP1), and fungal phytase 2 (FP2; P < 0.05); quadratic response to iP (P < 0.05). 3 Linear response of feed efficiency to supplemental iP and E. coli phytase. 4 Linear response of tibia ash (% and mg) to supplemental iP, E. coli phytase, FP1, and FP2 (P < 0.05); quadratic response of tibia ash (%) to iP, E. coli phytase, and FP1 (P < 0.05); quadratic response of tibia ash (mg) to E. coli phytase (P < 0.05). 5 The linear regression equation of tibia ash (%) for diets 1 to 4 as a function of supplemental iP intake (g) was Y = 34.53 (±0.67) + 11.46 (±1.03)X (r2 = 0.87). 6 The linear regression equation of tibia ash (mg) for diets 1 to 4 as a function of supplemental iP intake (g) was Y = 340.58 (±16.07) + 249.43 (±24.78)X (r2 = 0.85). 7 The basal diet was analyzed to contain 0.40% total P and calculated to contain 0.13% inorganic P. 8 FTU = phytase unit. 2
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Table 3. Effect of phytase on growth performance and tibia ash in Experiment 21
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Table 4. Growth, bone, and carcass response variables in Experiment 31 Diet2 1. Basal diet (adequate Ca and P) 2. Diet 1 − 0.10% iP 3. Diet 2 + 250 FTU5/kg of Escherichia coli phytase 4. Diet 2 + 500 FTU/kg of E. coli phytase 5. Diet 2 + 1,000 FTU/kg of E. coli phytase Pooled SD
Weight gain (g)
Feed intake (g)
Gain:feed (g/kg)
Tibia ash (%)
Live weight3 (kg)
Carcass yield4 (%)
2,469ab 2,371b 2,450ab 2,479a 2,493a 86.7
4,303a 4,105b 4,196ab 4,234ab 4,293a 158.6
574 579 584 586 581 16.9
44.4a 41.8b 44.3a 44.7a 45.3a 1.26
2.59a 2.47b 2.55ab 2.55ab 2.60a 0.10a
67.0c 67.3bc 68.0ab 68.0ab 68.9a 1.18
Means within a column lacking a common superscript differ (P < 0.05). Values are means of 8 pens of 20 male chicks fed the starter diets from 3 to 21 d and the grower diets from 21 to 42 d. 2 Diet 1 was calculated to contain 0.47 and 0.36% inorganic P (iP) during the starter and grower periods, respectively; diets 2, 3, 4, and 5 were calculated to contain 0.31 and 0.28% iP during the starter and grower periods, respectively. 3 Represents average live weight of the 5 birds per pen that were randomly chosen for processing. 4 Calculated as hot eviscerated carcass as a percentage of live BW. 5 FTU = phytase unit. a–c 1
EXP 4 Growth performance and bone response variables for EXP 4 are shown in Tables 5 and 6. Slightly decreasing dietary iP and Ca (diet 2) did not reduce (P > 0.05) growth performance and carcass yield compared with birds fed the positive control diet (diet 1), but tibia ash was reduced (P < 0.05). Addition of E. coli phytase to diets containing reduced Ca and iP did not affect (P > 0.05) growth performance or carcass yield compared with the corresponding control diets, with the exception of wing yield, which was slightly higher (P < 0.05) for birds fed diet 4 compared with diet 2. Addition of 300 FTU of E. coli phytase/kg to diets containing reduced Ca and iP improved (P < 0.05) tibia ash compared with birds fed the negative control diet (diet 2), and adding 600 or 1,000 FTU of E. coli phytase/kg (diets 5 and 6, respectively) prevented a reduction in growth performance, yield, and tibia ash relative to birds fed the positive control diet (diet 1).
The incidence rate for bone breakage (Table 6) during processing was variable, with diet having little effect (P > 0.05) on the incidence of broken coracoids or tibias or on the incidence of disjointed wings. However, the incidence of broken clavicles was increased (P < 0.05; compared with diet 1) in birds fed the negative control diet (diet 2) and diets containing E. coli phytase. The incidence of broken wings did not appear to follow a logical pattern; however, variability within this measurement was high, so none of the treatments differed (P > 0.05) from the positive control (diet 1).
EXP 5 Growth performance and bone response variables for EXP 5 are shown in Table 7. In this EXP, slightly decreasing dietary iP and Ca (diet 2) reduced (P < 0.05) weight gain compared with birds fed the other diets. No differences (P > 0.05) among treatments were noted for clavicle ash (%), but clavicle diameter and tension force (force required to separate the clavicles at the point of fusion) was reduced (P < 0.05) in birds fed diet 2. No differences
Table 5. Growth and carcass response variables in Experiment 41
Diet2 1. 2. 3. 4. 5.
Basal diet (adequate Ca and P) Diet 1 − 0.10% iP and 0.05% Ca Diet 1 + 300 FTU5/kg of Escherichia coli phytase Diet 2 + 300 FTU/kg of E. coli phytase Diet 1 − 0.15% iP and 0.05% Ca + 600 FTU/kg of E. coli phytase 6. Diet 1 − 0.20% iP and 0.05% Ca + 1,000 FTU/kg of E. coli phytase Pooled SEM
Weight gain (g)
Feed intake (g)
Gain:feed (g/kg)
Carcass yield3 (%)
Breast yield4 (%)
Wing yield4 (%)
Leg yield4 (%)
2,699c 2,709bc 2,782abc 2,781abc
5,125b 5,108b 5,193ab 5,156b
527b 530ab 536ab 539a
70.7 70.7 70.6 70.4
25.5b 26.0ab 25.6ab 25.9ab
11.7bc 11.6c 11.9ab 11.8ab
34.3ab 34.0b 34.0b 34.5ab
2,859a
5,335a
536ab
70.1
26.3a
11.7abc
34.7a
2,803ab 32.8
5,237ab 61.7
535ab 3.3
70.6 0.45
25.9ab 0.24
12.0a 0.09
34.7a 0.20
Means within a column lacking a common superscript differ (P < 0.05). Growth values are means of 7 pens of 20 male chicks; yield values are means of 7 pens of 5 randomly-selected chicks per pen; birds were fed the starter diets from 0 to 18 d, grower diets from 18 to 32 d, finisher diets from 32 to 40 d, and withdrawal diets from 40 to 50 d. 2 Diet 1 was calculated to contain 0.48, 0.42, 0.38, and 0.34% inorganic P (iP) during the starter, grower, finisher, and withdrawal periods, respectively. 3 Represents chilled, ready-to-cook carcass as a percentage of live BW. 4 Calculated as a percentage of chilled, ready-to-cook carcass weight. 5 FTU = phytase unit. a–c 1
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had a higher carcass yield than birds fed diets 1 and 2 (P < 0.003).
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EFFECT OF PHYTASE ON CARCASS CHARACTERISTICS 1
Table 6. Bone response variables in Experiment 4
Incidence rate2 Broken Broken Broken Tibia ash clavicle coracoid tibia (%) (%) (%) (%)
Diet3 1. 2. 3. 4. 5.
Basal diet (adequate Ca and P) Diet 1 − 0.10% iP and 0.05% Ca Diet 1 + 300 FTU5/kg of Escherichia coli phytase Diet 2 + 300 FTU/kg of E. coli phytase Diet 1 − 0.15% iP and 0.05% Ca + 600 FTU/kg of E. coli phytase 6. Diet 1 − 0.20% iP and 0.05% Ca + 1,000 FTU/kg of E. coli phytase Pooled SEM
Broken wing4 (%)
Wing disjoint (%)
42.0ab 39.9c 42.1a 41.2ab
17.1b 28.6ab 45.7a 45.7a
0.0 2.9 2.9 5.7
17.1 20.0 22.9 14.3
17.1abc 5.7c 25.7ab 2.9c
2.9 0.0 0.0 8.6
40.8bc
31.4ab
2.9
11.4
34.3a
8.6
ab
ab
42.0 0.4
37.1 7.4
5.7 2.9
5.7 6.3
bc
14.3 6.4
2.9 3.0
Means within a column lacking a common superscript differ (P < 0.05). Values are means of 8 pens of 5 randomly selected male chicks per pen that were fed the starter diets from 0 to 18 d, grower diets from 18 to 32 d, finisher diets from 32 to 40 d, and withdrawal diets from 40 to 50 d. 2 Incidence rates represent the percentage of birds per treatment that exhibited the noted defect. 3 Diet 1 was calculated to contain 0.48, 0.42, 0.38, and 0.34% inorganic P (iP) during the starter, grower, finisher, and withdrawal periods, respectively. 4 A broken wing was designated as one in which the radius, ulna, or both were broken. 5 FTU = phytase unit. a–c 1
DISCUSSION Previous EXP utilizing E. coli phytase products investigated the Ca- and P-releasing ability of these microbial phytases in broilers during the starter phase of production
(Augspurger et al., 2003; Augspurger and Baker, 2004a,b). Although the information provided in these papers contributes to the elucidation of the mode of action of this particular enzyme and its efficacy, information about the effects of this enzyme on key broiler carcass characteristics, such as leg and breast yield and the incidence of broken legs, wings, and clavicles, is not available. There exists, therefore, a paucity of data detailing the effect of the level of iP and phytase supplementation during the finisher and withdrawal stages of broiler production on commercial processing parameters. The addition of phytase to diets deficient in available P at various production phases of broilers has been shown to increase growth and improve bone (usually tibia) response variables (Zyla et al., 2000a,b; Applegate et al., 2003a; Augspurger et al., 2003; Augspurger and Baker,
Table 7. Growth and bone response variables in Experiment 51 Incidence rate2
Clavicle attributes
Diet3 1. Basal diet (adequate Ca and P) 2. Diet 1 − 0.10% iP and 0.05% Ca 3. Diet 2 + 300 FTU6/kg of Escherichia coli phytase 4. Diet 1 − 0.20% iP and 0.05% Ca + 1,000 FTU/kg of E. coli phytase Pooled SD
Weight gain (g)
Gain:feed (g/kg)
Ash (%)
Diameter (mm)
Tension force4 (N)
Break force4 (N)
Broken clavicle (%)
Broken tibia (%)
Broken wing5 (%)
Wing disjoint (%)
3,865a 3,612b
458b 454b
54.2 54.3
2.75a 2.63b
31.0a 19.0b
100.4 89.5
24.5 38.1
5.7 2.4
1.9 2.4
3.8 4.8
3,826a
483a
55.6
2.71ab
28.6ab
98.6
32.1
7.5
1.9
5.7
3,839a 109
463ab 7
54.4 4.6
2.75a 0.27
33.6a 6.7
98.3 24.7
27.9 46.1
4.7 22.3
0.0 12.5
7.0 22.3
Means within a column lacking a common superscript differ (P < 0.05). Growth values are means of 4 pens of 15 male broilers; yield values are means of 4 pens of 15 birds per pen; birds were fed the starter diets from 8 to 18 d, grower diets from 18 to 32 d, finisher diets from 32 to 40 d, and withdrawal diets from 40 to 56 d. 2 Incidence rates represent the percentage of birds per treatment that exhibited the noted defect. 3 Diet 1 was calculated to contain 0.43, 0.40, 0.37, and 0.35% inorganic P (iP) during the starter, grower, finisher, and withdrawal periods, respectively. 4 Tension force is defined as the force required to separate the clavicles at the point of fusion; break force is the force required to break the bone. 5 A broken wing was designated as one in which the radius, ulna, or both were broken. 6 FTU = phytase unit. a,b 1
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(P < 0.05) in clavicle diameter or tension force were noted in birds fed diet 1 or the diets containing E. coli phytase. Break force (force required to break the clavicle) did not differ (P > 0.05) among treatments, although break force for clavicles from birds fed diet 2 was numerically lower. As for EXP 4, bone breakage rates in EXP 5 were variable, and no differences (P < 0.05) in the incidence of bone breakage during processing were observed. It should be noted that, for clavicles, the majority (>80%; data not shown) of breakage occurred at the point of fusion.
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yield in pigs appears to be variable (O’Quinn et al., 1997; Liu et al., 1998), and Walz and Pallauf (2003) reported that although phytase increased apparent digestibility of P, Ca, and Zn, it had no effect on carcass and meat characteristics in barrows. Similar results have been obtained in drakes supplemented with phytase, Lys, or both, with no difference in carcass yield and meat quality reported by Attia (2003). However, Shelton et al. (2004) reported that the inclusion of phytase to diets deficient in Ca, P, and trace minerals reversed the negative effects of these deficient diets on carcass lean content, weight, dressing percentage, and bone associated with the removal of these minerals in growing-finishing pigs. The effect of phytase on carcass characteristics in broilers is also important, but it is limited. In the current EXP, the inclusion of the E. coli phytase enzyme not only returned bone ash percentage to that of the control in broilers fed P-deficient diets (Tables 2, 3, 4, and 6), but it also prevented negative effects on carcass and breast yield in birds fed diets containing substantially reduced P in EXP 4. It should be noted that corresponding negative control diets were not fed in EXP 4 and 5 because of welfare concerns associated with feeding such drastic reductions in Ca and P over the duration of the trials, but in a separate trial, short-term feeding of negative control diets (diets 5 and 6 in EXP 4 and diet 4 in EXP 5 fed without E. coli phytase) verified that these diets substantially reduce growth and tibia ash in the absence of E. coli phytase (data not shown). Bone integrity has an important role to play in total yield of the carcasses intended for the whole-bird market and the further-processing market (Rath et al., 2000) and in the rate of product loss due to bone breakage. Chen and Moran (1994) reported that reducing the level of P in the withdrawal phase of production resulted in increased defects and decreased production of grade-“A” carcasses. We were interested in determining whether phytase would affect the incidence of bone breakage during processing. In EXP 4, bone breakage results were highly variable, and we were surprised by the high incidence of broken clavicles. We have also noted high rates of clavicle breakage (approaching 50%) in other research projects involving processing of broilers raised on nutritionallyadequate diets. The departmental processing plant in which birds were processed must accommodate a wide range of bird sizes, and it is possible that evisceration equipment was not optimally adjusted for birds in our trial, resulting in a higher clavicle breakage rate. Because of this, and because the rate of wing breakage in EXP 4 was so variable (and seemingly illogical), portions of EXP 4 were repeated in EXP 5. Although variability associated with bone breakage was still high in EXP 5, clavicle and wing breakage rates were lower overall, and results indicated that bone breakage should not be increased in broilers fed diets containing low levels of iP combined with supplemental phytase. In conclusion, based on the data obtained from these EXP, E. coli phytase releases more P than the fungal phytases that were tested. When based on tibia ash weight
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2004a,b; Dilger et al., 2004; Shirley and Edwards, 2003). The degree of response has been the subject of much investigation, and researchers have estimated that fungal phytases release an amount of available P ranging from less than 0.05% (Angel et al., 2001) to as much as 0.113% (Biehl et al., 1995). The level of improved P utilization and growth seems largely dependent on the source of phytase supplementation, as some of the major constraints to the catalytic properties of phytase enzyme are pH and time limitations with regard to the unique nature of the gastrointestinal tract of the broiler (Zyla et al., 2004). Augspurger et al. (2003) reported greater P-release values from phytase produced by E. coli expressed in yeast when compared with phytase derived from FP1 and FP2 for young chicks and pigs during the starter phases of production. Applegate et al. (2003b) reported that turkeys fed diets supplemented with an E. coli-derived phytase had a consistently higher iP-sparing effect than FP1 and FP2. Augspurger et al. (2003) attributed the greater P release by E. coli phytase to the different activation levels of FP1 (pH 2.5 and 5.5), FP2 (pH 4.0 to 4.5), and the E. coliderived phytase (pH 2.5 to 3.5). Rodriguez et al. (1999a) reported that the E. coli phytase enzyme expressed in Pichia pastoris released more P from phytate in soybean meal than Aspergillus niger phyA at each enzyme’s optimum pH. Moreover, E. coli phytase appears to be more resistant to pepsin breakdown; Rodriguez et al. (1999b) reported that another E. coli phytase had a 30% increase in phytase activity after being incubated in pepsin at a pH of 2, whereas FP1 had a 58 to 77% decrease in activity under the same experimental conditions. However, FP1 was more resistant to trypsin degradation at a pH of 7 than E. coli phytase enzyme. Our results confirm the efficacy of E. coli phytase (Table 2) and indicate an increased ability of E. coli phytase to release bound P, compared with the fungal phytases that were used (Table 3). As described previously (Rodriguez et al., 1999a,b; Applegate et al., 2003b; Augspurger et al., 2003), attributes of E. coli phytase (resistance to pepsin degradation, optimum pH) appear to enhance its P-releasing ability. Based on tibia ash, we noted improvements in estimated iP release ranging from 23 to 200% (depending on phytase level) for E. coli phytase, compared with FP1, and 118 to 700% (depending on phytase level) for E. coli phytase, compared with FP2. As previously noted, at the higher levels of E. coli phytase supplementation in EXP 1 and 2, tibia ash values exceeded those of the standard curve; although this brings into question the accuracy of E. coli phytase efficacy estimates at the higher levels of E. coli phytase supplementation, it does suggest that E. coli phytase releases >0.15% iP. Our iP-sparing values for the various phytase enzymes are similar to those reported by Augspurger et al. (2003). In other farm species of economic importance, investigators have largely focused on the effect of phytase in conjunction with varying amino acid and energy levels on carcass quality of pigs and drakes (Harper et al., 1997; Attia, 2003; Walz and Pallauf, 2003; Shelton et al., 2004). The effect of phytase enzyme supplementation on carcass
EFFECT OF PHYTASE ON CARCASS CHARACTERISTICS
(mg/chick or %), the amount of available P released ranged from 0.119 to 0.239% for E. coli phytase, compared with 0.07 to 0.18% from FP1 and 0.03 to 0.11% from FP2 at phytase supplementation levels of 250 and 4,000 FTU/ kg, respectively. When based on tibia ash weight (mg/ chick), P release appeared to be maximized at a level of 1,000 FTU/kg of E. coli phytase. Results also indicate that E. coli phytase is effective at maintaining carcass yield characteristics without increasing the incidence of bone breakage (based on EXP 5).
REFERENCES
diets improves performance, phosphorus digestibility, and bone mineralization and reduces phosphorus excretion. J. Anim. Sci. 75:3174–3186. Jalal, M. A., and S. E. Scheideler. 2001. Effect of supplementation of two different sources of phytase on egg production parameters in laying hens and nutrient digestiblity. Poult. Sci. 80:1463–1471. Liu, J., D. W. Bollinger, D. R. Ledoux, and T. L. Veum. 1998. Lowering the dietary calcium to total phosphorus ratio increases phosphorus utilization in low-phosphorus corn–soybean meal diets supplemented with microbial phytase for growing-finishing pigs. J. Anim. Sci. 76:808–813. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Onyango, E. M., M. R. Bedford, and O. Adeola. 2005. Efficacy of an evolved Escherichia coli phytase in diets of broiler chicks. Poult. Sci. 84:248–255. O’Quinn, P. R., D. A. Knabe, and E. J. Gregg. 1997. Efficacy of Natuphos in sorghum-based diets of finishing swine. J. Anim. Sci. 75:1299–1307. Rath, N. C., G. R. Huff, W. E. Huff, and J. M. Balog. 2000. Factors regulating bone maturity and strength in poultry. Poult. Sci. 79:1024–1032. Rodriguez, E., Y. Han, and X. G. Lei. 1999a. Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon. Biochem. Biophys. Res. Commun. 257:117–123. Rodriguez, E., J. M. Porres, Y. Han, and X. G. Lei. 1999b. Different sensitivity of recombinant Aspergillus niger phytase (rPhyA) and Escherichia coli pH 2.5 acid phosphatase (r-AppA) to trypsin and pepsin in vitro. Arch. Biochem. Biophys. 365:262–267. SAS Institute. 2004. SAS/STAT User’s Guide: Statistics. Version 9.2 Edition. SAS Inst. Inc., Cary, NC. Shelton, J. L., L. L. Southern, F. M. LeMieux, T. D. Bidner, and T. G. Page. 2004. Effects of microbial phytase, low calcium and phosphorus, and removing the dietary trace mineral premix on carcass traits, pork quality, plasma metabolites, and tissue mineral content in growing-finishing pigs. J. Anim. Sci. 82:2630–2639. Shirley, R. B., and H. M. Edwards Jr. 2003. Graded levels of phytase past industry standards improves broiler performance. Poult. Sci. 82:671–680. Tamim, N. M., R. Angel, and M. Christman. 2004. Influence of dietary calcium and phytase on phytate phosphorus hydrolysis in broiler chickens. Poult. Sci. 83:1358–1367. Walz, O. P., and J. Pallauf. 2003. The effect of the combination of microbial phytase and amino acid supplementation of diets for finishing pigs on P and N excretion and carcass quality. Arch. Tierernahr. 57:413–428. Zanini, S. F., and M. H. Sazzad. 1999. Effects of microbial phytase on growth and mineral utilisation in broilers fed on maize soyabean-based diets. Br. Poult. Sci. 40:348–352. Zyla, K., J. Koreleski, S. Swiatkiewicz, A. Wikiera, M. Kujawski, J. Piironen, and D. R. Ledoux. 2000a. Effects of phosphorolytic and cell wall-degrading enzymes on the performance of growing broilers fed wheat-based diets containing different calcium levels. Poult. Sci. 79:66–76. Zyla, K., M. Mika, B. Stodolak, A. Wikiera, J. Koreleski, and S. Swiatkiewicz. 2004. Towards complete dephosphorylation and total conversion of phytates in poultry feeds. Poult. Sci. 83:1175–1186. Zyla, K., A. Wikiera, J. Koreleski, S. Swiatkiewicz, J. Piironen, and D. R. Ledoux. 2000b. Comparison of the efficacies of a novel Aspergillus niger mycelium with separate and combined effectiveness of phytase, acid phosphatase, and pectinase in dephosphorylation of wheat-based feeds fed to growing broilers. Poult. Sci. 79:1434–1443.
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Angel, R., A. S. Dhandu, T. J. Applegate, and M. Christman. 2001. Phosphorus sparing effect of phytase, 25-hydroxycholecalciferol, and citric acid when fed to broiler chicks. Poult. Sci. 80(Suppl. 1):133. (Abstr.) Applegate, T. J., R. Angel, and H. L. Classen. 2003a. Effects of dietary calcium, 25-hydroxycholecalciferol, or bird strain on small intestinal phytase activity in broiler chickens. Poult. Sci. 82:1140–1148. Applegate, T. J., D. M. Webel, and X. G. Lei. 2003b. Efficacy of a phytase derived from Escherichia coli and expressed in yeast on phosphorus utilization and bone mineralization in turkey poults. Poult. Sci. 82:1726–1732. Attia, Y. A. 2003. Performance, carcass characteristics, meat quality and plasma constituents of meat type drakes fed diets containing different levels of lysine with or without a microbial phytase. Arch. Tierernahr. 57:39–48. Augspurger, N. R., and D. H. Baker. 2004a. High dietary phytase levels maximize phytate-phosphorus utilization but do not affect protein utilization in chicks fed phosphorus- or amino acid-deficient diets. J. Anim. Sci. 82:1100–1107. Augspurger, N. R., and D. H. Baker. 2004b. Phytase improves dietary calcium utilization in chicks, and oyster shell, carbonate, citrate, and citrate-malate forms of calcium are equally bioavailable. Nutr. Res. 24:293–301. Augspurger, N. I., D. M. Webel, X. G. Lei, and D. H. Baker. 2003. Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs. J. Anim. Sci. 81:474–483. Biehl, R. R., and D. H. Baker. 1997. Utilization of phytate and nonphytate phosphorus in chicks as affected by source and amount of vitamin D3. J. Anim. Sci. 75:2986–2993. Biehl, R. R., D. H. Baker, and H. F. DeLuca. 1995. 1 α-Hydroxylated cholecalciferol compounds act additively with microbial phytase to improve phosphorus, zinc and manganese utilization in chicks fed soy-based diets. J. Nutr. 125:2407–2416. Boling-Frankenbach, S. D., C. M. Peter, M. W. Douglas, J. L. Snow, C. M. Parsons, and D. H. Baker. 2001. Efficacy of phytase for increasing protein efficiency ratio values of feed ingredients. Poult. Sci. 80:1578–1584. Chen, X., and E. T. Moran Jr. 1994. Response of broilers to omitting dicalcium phosphate from withdrawal feed: Live performance, carcass downgrading and further-processing yields. J. Appl. Poult. Res. 3:74–79. Dilger, R. N., E. M. Onyango, J. S. Sands, and O. Adeola. 2004. Evaluation of microbial phytase in broiler diets. Poult. Sci. 83:962–970. Emmert, J. L., M. W. Douglas, S. D. Boling, C. M. Parsons, and D. H. Baker. 1999. Bioavailability of lysine from a liquid lysine source in chicks. Poult. Sci. 78:383–386. Han, Y., and D. H. Baker. 1993. Effects of sex, heat stress, body weight, and genetic strain on the dietary lysine requirement of broiler chicks. Poult. Sci. 72:701–708. Harper, A. F., E. T. Kornegay, and T. C. Schell. 1997. Phytase supplementation of low-phosphorus growing-finishing pig
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Key words: phosphorus, broiler chick, bioavailability, phytase 2006 Poultry Science 85:1737–1745
INTRODUCTION A major issue facing the broiler industry is maintaining bone integrity while reducing feed cost and reducing environmental pollution. Bone breakage in broilers is not only an economic issue for processors but a welfare issue as well (Rath et al., 2000), with quality of nutrition playing an important role. The use of phytase enzymes may prove to be a method to address both issues. The effectiveness of phytase in releasing bound Ca, P, and other divalent minerals important in bone development has been well established (Biehl and Baker, 1997; Harper et al., 1997; Zanini and Sazzad, 1999; Boling-Frankenbach et al., 2001; Augspurger et al., 2003; Augspurger and Baker, 2004a,b; Shelton et al., 2004; Onyango et al., 2005). It has been estimated that phytase releases at least 0.09% bound Ca and P (Augspurger et al., 2003; Augspurger and Baker, 2004b; Shelton et al., 2004) and improves digestibility of
2006 Poultry Science Association Inc. Received March 23, 2006. Accepted June 11, 2006. 1 Corresponding author: [email protected]
these nutrients (Harper et al., 1997; Zanini and Sazzad, 1999; Jalal and Scheideler, 2001; Dilger et al., 2004). With the importance of P in the environment, phytase source and efficacy has become an important issue to the poultry industry. Dephosphorylation of the phytate molecule occurs at different reaction sites, depending on the origin of the phytase enzyme that is catalyzing the reaction (Rodriguez et al., 1999b; Tamim et al., 2004). Furthermore, phytase enzyme origin also affects the pH at which the enzyme is most effective and its ability to resist breakdown in the stomach and small intestine (Rodriguez et al., 1999a; Augspurger et al., 2003), hence the differences in the efficacy of different phytase enzymes. Recent research has indicated that a new Escherichia coli phytase (OptiPhos, JBS United Inc., Sheridan, IN) is more efficacious than other commercially available phytases in releasing at least 0.10% P (Augspurger et al., 2003). Although information provided in these research findings is invaluable, there exists a lack of information about the effect of phytase enzymes on carcass characteristics such as the incidence of bone breakage during processing. Breakage of bones, such as the tibia, clavicle, and coracoid, affects both product condemnation rates and food safety,
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ABSTRACT Five experiments (EXP) were conducted to assess the efficacy of an Escherichia coli phytase compared with 2 commercially available fungal phytases. In EXP 1 and 2, male broiler chicks were fed experimental diets that included a P-deficient control (0.13% available P; 0.88% Ca) alone or with graded levels of KH2PO4 (0, 0.05, 0.10, or 0.15%) or phytase at levels of 250, 500, 1,000, 2,000, or 4,000 phytase units/kg of E. coli phytase (EXP 1 and 2), fungal phytase 1 (EXP 2), or fungal phytase 2 (EXP 2). In EXP 1 and 2, weight gain and tibia ash (mg/ chick and %) responded linearly (P < 0.05) to inorganic P addition. In EXP 2, each level of E. coli phytase released more P than either fungal phytases 1 or 2, whether based on tibia ash weight (mg/chick) or percentage. In EXP 3, 4, and 5, dietary treatments containing adequate or deficient levels of P were fed with or without supplemental E. coli phytase. In EXP 3, weight gain and tibia ash were reduced (P < 0.05) by P deficiency, but gain and
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which are extremely important for the broiler industry. Five experiments (EXP) were conducted with the objectives of assessing the relative P-releasing ability of E. coli phytase and comparing its efficacy to 2 commerciallyavailable fungal phytase products [fungal phytase 1 (FP1; Natuphos, BASF, Mount Olive, NJ) and fungal phytase 2 (FP2; Ronozyme, DSM Nutritional Products Inc., Parsippany, NJ)]. The effect of E. coli phytase on growth performance, tibia and clavicle ash, and bone breakage during processing in commercial broilers was measured.
MATERIALS AND METHODS
EXP 1 and 2 On d 9 or 8 posthatching (EXP 1 and 2, respectively), chicks were weighed, wing-banded, and randomly allotted to dietary treatments such that each pen within an EXP would have a similar average initial weight and weight range. Each EXP consisted of 5 replicates of 5 chicks per replicate housed in batteries with raised wire floors, and a 24-h photoperiod was used. Experimental diets (Table 1) were fed until d 23 or 22 posthatching (EXP 1 and 2, respectively), at which time chicks and feed were weighed for determination of weight gain, feed intake, and feed efficiency. At the termination of each EXP, chicks were killed by cervical dislocation, and right tibias were collected for subsequent analysis of ash weight per chick (mg/chick) and percentage of ash. In EXP 1 and 2, standard-curve methodology (Biehl et al., 1995) was used, with KH2PO4 (22.8% P) serving as the standard. Dietary additions were made to a 23.1% CP corn–soybean meal diet that contained no supplemental inorganic P (iP) and was analyzed to contain 0.40% total P and 0.88% Ca (calculated to contain 0.13% iP; Table 1).
EXP 3 Experiment 3 consisted of 5 dietary treatments, replicated 8 times, with each replicate containing 20 birds. All diets were based on corn and soybean meal and met or exceeded NRC (1994) recommendations for all nutrients, with the exception of P, when appropriate (Table 1). Experimental diets (Table 1) were fed from 3 to 21 d and 21 to 42 d, and treatments consisted of the following diets: 1) a positive control containing adequate levels of Ca (1.0 and 0.9% during the starter and grower periods, respectively) and iP (0.47 and 0.46% during the starter and grower periods, respectively); 2) a negative control containing an adequate level of Ca (1.0 and 0.9% during the starter and grower periods, respectively) and a deficient level of iP (0.31 and 0.28% during the starter and grower periods, respectively); and 3), 4), and 5) diet 2 with graded levels of E. coli phytase (250, 500, and 1,000 FTU/kg). Birds and feed were weighed at EXP initiation, during phase changes, and at assay termination (d 42) to calculate weight gain, feed intake, feed efficiency, and livability for each period and overall. Feeders were removed from experimental pens 10 h before EXP termination. Following weighing, 5 birds per pen were randomly selected for processing at the University of Arkansas Poultry Processing Plant.
EXP 4 and 5 In EXP 4, there were 6 dietary treatments with 7 replicate pens (20 birds per pen); in EXP 5, there were 4 dietary treatments with 4 replicate pens (15 birds per pen). All diets were based on corn and soybean meal and met or exceed NRC (1994) recommendations for all nutrients, with the exception of Ca and P, when appropriate (Table 1). Birds and feed were weighed at EXP initiation, during phase changes, and at assay termination (d 50 or 56 in EXP 4 and 5, respectively) to calculate weight gain, feed intake, feed efficiency, and livability for each period and overall. Feeders were removed from experimental pens 10 h before EXP termination. Following weighing, 5 birds per pen (EXP 4) were randomly selected for processing at the University of Arkansas Poultry Processing Plant; all birds from EXP 5 were processed. In EXP 4, chicks were weighed and placed on treatment diets on d 1 posthatch. Experimental diets (Table 1) were fed as follows: starter (1 to 18 d), grower (18 to 32 d), finisher (32 to 40 d), and withdrawal (40 to 50 d). Dietary treatments consisted of the following diets: 1) a positive control adequate in all essential nutrients for the respective phases; 2) diet 1 with Ca reduced by 0.05% and P
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All procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee. Before the beginning of EXP 1 and 2 and throughout EXP 3, 4, and 5, male Cobb × Cobb (Cobb-Vantress Inc., Siloam Springs, AR) broilers were housed in floor pens with pine shavings, hanging tube feeders, and Plasson waterers. Before the initiation of EXP 1, 2, and 3, chicks were fed a common corn–soybean meal starter diet that contained 24% CP and met or exceeded all NRC (1994) nutrient recommendations. Water and feed were freely available. A review of the differences among the different phytase enzymes used in these EXP is given by Rodriguez et al. (1999b), Applegate et al. (2003b), Augspurger et al. (2003), and Onyango et al. (2005). Dietary additions of phytase were made according to the experimental procedures of Augspurger et al. (2003) and Augspurger and Baker (2004a), who assayed the same phytases used herein and defined phytase activity as the quantity of the enzyme that will liberate 1 mol of inorganic P per min from 5.1 mM Na phytate at a pH of 5.5 and temperature of 37°C. Thus, dietary additions in each EXP were made to accomplish dietary phytase activity levels as defined under the aforementioned conditions.
In EXP 1 and 2, graded levels (0, 0.05, 0.10, 0.15%) of KH2PO4 were used to construct the standard curve. In EXP 1, 5 levels of E. coli phytase (250, 500, 750, 1,000, or 10,000 phytase units (FTU)/kg) were tested; in EXP 2, 5 levels of E. coli phytase, FP1, or FP2 (250, 500, 1,000, 2,000, 4,000) were added to the basal diet to allow quantification of phytate-bound P release.
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EFFECT OF PHYTASE ON CARCASS CHARACTERISTICS Table 1. Percentage composition of experimental diets EXP1 3
3
4 and 52
4 and 5
4 and 5
4 and 52
Ingredient
Starter3
3 to 21 d
21 to 42 d
0 to 18 d
18 to 32 d
32 to 40 d
40 to 56 d
Corn Soybean meal Soybean oil Poultry fat Dicalcium phosphate Limestone NaCl Vitamin mix4 Mineral mix4 Choline Cl (60%) DL-Met L-Thr Sacox salinomycin5 Cornstarch Composition6 ME, kcal/kg CP, % Total P, %7 Total Ca, %
51.69 39.69 5.00 — — 1.67 0.40 0.20 0.15 0.20 0.20 — — to 100
50.93 39.69 5.00 — 1.20 1.59 0.40 0.20 0.10 0.20 0.25 — — to 100
57.16 34.00 5.00 — 0.70 1.65 0.40 0.20 0.10 0.20 0.15 — — to 100
55.95 37.18 — 3.05 0.58 0.93 0.49 0.20 0.10 0.15 0.18 0.03 0.05 to 100
61.14 32.33 — 2.99 0.36 0.90 0.49 0.20 0.10 0.15 0.17 0.01 0.05 to 100
68.71 25.25 — 2.64 0.87 0.27 0.51 0.20 0.10 0.15 0.11 0.03 0.05 to 100
70.63 23.39 — 2.99 0.08 0.85 0.49 0.20 0.10 0.15 0.02 — — to 100
3,100.00 23.1 0.40 0.88
3,115.00 23.2 0.578 1.00
3,185.00 21.0 0.538 0.90
3,050.00 23.0 0.63/0.589 1.00/0.829
3,100.00 21.1 0.53/0.489 0.80/0.809
3,050.00 18.4 0.45/0.439 0.76/0.739
3,200.00 17.7 0.41/0.419 0.72/0.659
1
EXP = experiment. The starter feed was fed from 8 to 18 d in EXP 5; the withdrawal feed was fed from 40 to 50 d in EXP 4. 3 Diets fed from 9 to 23 d in EXP 1 and from 8 to 22 d in EXP 2. 4 Vitamin and mineral mix in EXP 1 and 2 from Han and Baker (1993); vitamin and mineral mix in EXP 3 and 4 from Emmert et al. (1999). 5 Sacox 60, Hoechst-Roussel Agri-Vet. Co., Somerville, NJ; provided 66 mg/kg of salinomycin activity. 6 Metabolizable energy and CP values were calculated; diets were analyzed for total P and Ca. 7 Diets were estimated to contain the following inorganic P levels: 0.13% for EXP 1 and 2; 0.31 and 0.28% for the starter and grower periods, respectively, in EXP 3; 0.28, 0.22, 0.18, and 0.14% for the starter, grower, finisher, and withdrawal periods, respectively, in EXP 4; 0.26, 0.20, 0.17, and 0.14% for the starter, grower, finisher, and withdrawal periods, respectively, in EXP 5. 8 Positive control diets (diet 1) were analyzed to contain 0.73% total P (0.47% estimated available P) and 0.61% total P (0.46% estimated available P) for the starter and grower periods, respectively. 9 Analytical values for dietary Ca and P are shown for EXP 4 and 5 (EXP 4 values are to the left of the slash). 2
reduced by 0.10%; 3) diet 1 with 300 FTU/kg of E. coli phytase; 4) diet 2 with 300 FTU/kg of E. coli phytase; 5) diet 1 with dietary Ca reduced by 0.05% and dietary P reduced by 0.15% P and 600 FTU/kg of E. coli phytase; and 6) diet 1 with dietary Ca reduced by 0.05% and dietary P reduced by 0.20% and 1,000 FTU/kg of E. coli phytase. Treatment additions to the basal diet were made at the expense of cornstarch. Due to concerns over the detrimental effects of feeding birds diets that were too deficient in Ca and iP for the whole duration of the project, corresponding negative controls for treatments 5 and 6 were not formulated. Experiment 5 was similar to EXP 4, with the exception of the EXP initiation (d 8) and termination (d 56) and the dietary treatments, which included only treatments 1, 2, 4, and 6 from EXP 4.
Processing Variables After arrival at the processing plant, birds were hung on a shackle line and commercially processed to evaluate carcass and parts yields, and the incidence of bones that were broken or disjointed. Birds were electrically stunned (11 V, 11 mA, 11 s), manually bled by severing the left carotid artery and jugular vein, bled out (1.5 min), softscalded (129°F, 2 min), and feathers were picked with the use of inline commercial defeathering equipment. Evis-
cerating and rinsing followed, after which carcasses were placed in a prechill tank at 12 C for 15 min. Carcasses were then moved to an immersion chiller (1°C) for 45 min, after which carcasses were removed, packed in ice, and aged at 4°C until time of deboning (EXP 4 and 5) at 4 h postmortem. Carcasses were separated into breast (pectoralis major and pectoralis minor), wings, legs, and frame, followed by weighing and removal of the tibia from the legs for subsequent ash analysis.
Bone Response Variables Tibias were autoclaved for 45 min to remove adhering muscle and cartilage, dried at 110°C for 24 h, and subsequently weighed. Dry tibias were ashed in a muffle furnace at 600°C for 18 h. After cooling, tibia ash was weighed for determination of ash weight and bone ash percentage. In EXP 4 and 5, the incidence of broken bones (tibia, coracoid, clavicle, and radius and ulna) and disjointed wings that had occurred during processing were noted at the time that birds were separated into parts. The incidence of broken or disjointed bones was expressed as a percentage of the number of birds selected for processing. For clavicles, the incidence rate reflects the sum of clavicles broken on the shaft or at the point of fusion. In EXP 5, clavicles were collected at the time of deboning
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1 and 2
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PILLAI ET AL.
Statistical Analysis Data were subjected to ANOVA (SAS Institute, 2004) appropriate for a completely randomized design; treatment means were separated using the least significant difference multiple-comparison procedure or Duncan’s multiple range test (in the case of missing replicate values). Single df contrasts were used to test overall effects of phytase (when appropriate). Standard curves were established (SAS Institute, 2004), with tibia ash (mg/chick or %) as the dependent variable and consumption (g) of supplemental P as the independent variable for EXP 1 and 2. By insertion of replicate values for tibia ash (mg/ chick or %) into the standard curve equation, the amount of P released by the enzyme was calculated for treatments 6 to 10 (EXP 1). Pens served as the experimental unit for all data analyzed, and means were considered significant at P < 0.05.
RESULTS EXP 1 Growth and bone response variables are represented in Table 2. Increasing iP resulted in a linear increase in weight gain and feed intake (P < 0.01); adding E. coli phytase resulted in a linear and quadratic increase in weight gain (P < 0.05). Feed efficiency increased with increasing iP and E. coli phytase levels (P < 0.05). Increasing iP resulted in a linear increase in tibia weight and
tibia ash (P < 0.05). Standard curve regression equations (Table 2) were as follows: for percentage of tibia ash, Y = 38.95 + 8.48X (r2 = 0.72) and for milligrams of tibia ash, Y = 272.98 + 163.62X (r2 = 0.65). Bioavailable P release estimates ranged from 0.11 to 0.19% for 250 to 10,000 FTU/kg of supplemented E. coli phytase when based on tibia ash weight (mg/chick) and 0.09 to 0.19% for 250 to 10,000 FTU/kg of supplemented E. coli phytase when based on tibia ash expressed as a percentage (Table 2). It should be noted that the tibia ash values for birds fed diets with 750 FTU of E. coli phytase/kg or greater exceeded the tibia ash value for birds fed 0.15% supplemental iP (diet 4).
EXP 2 Growth and bone response variables are presented in Table 3. Increasing the level of supplemental iP and phytase increased weight gain and feed intake (P < 0.01), regardless of phytase source. Feed efficiency also increased (P < 0.01) with increasing iP or phytase levels. Overall, weight gain and feed efficiency were greater (P < 0.05) for chicks fed E. coli phytase than for chicks fed FP1 or FP2. Whether expressed as milligrams per chick or a percentage, tibia ash increased linearly (P < 0.01) with increasing iP or phytase levels, regardless of phytase source. Overall, tibia ash (mg/chick or %) was greater (P < 0.05) for chicks fed E. coli phytase than for chicks fed FP1 or FP2. Standard curve regression equations (Table 3) were Y = 34.53 + 11.46X; r2 = 0.87 for percentage of tibia ash, and Y = 340.58 + 249.43X; r2 = 0.85 for milligrams of tibia. The P-releasing ability of E. coli phytase exceeded that of the other phytase sources; estimates for E. coli phytase ranged from 0.12 to 0.24% for 250 to 4,000 FTU of E. coli phytase/kg, compared with 0.07 to 0.18% for FP1 and 0.03 to 0.11% for FP2 (at the same inclusion levels) when based on tibia ash weight (Table 3). When based on tibia ash percentage, P-releasing ability estimates were 0.12 to 0.15%, 0.04 to 0.12, and 0.02 to 0.07% for E. coli phytase, FP1, and FP2, respectively. Similar to EXP 1, tibia ash values for birds fed the higher levels of E. coli phytase exceeded the tibia ash value for birds fed 0.15% iP.
EXP 3 Growth performance data for EXP 3 is shown in Table 4. Birds fed the negative control (diet 2) had reduced (P < 0.05) feed intake, tibia ash, and live weight compared with birds fed the positive control (diet 1), but no differences (P > 0.05) in weight gain, feed efficiency, and carcass yield were detected. The addition of 500 FTU/kg of E. coli phytase reversed the negative effects of the low-iP diet on weight gain and tibia ash (P < 0.05); 250 FTU/kg of E. coli phytase reversed the negative effects on tibia ash; and 1,000 FTU/kg of E. coli phytase reversed all the negative effects of the low-iP diet. In addition, birds fed diets supplemented with 1,000 FTU/kg of E. coli phytase
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for measurement (on the same d) of diameter, tension force, and break force. Intact clavicles (at time of collection) were subjected to a tension test, in which tension force (the force required to separate the clavicles at the point of fusion) was measured in newtons on a texture analyzer (TA-XT2i, Texture Technologies Corp., Scarsdale, NY) equipped with a 5-kg load cell and 2 attached adjustable grips (TA-96, Texture Technologies Corp.) placed opposite each other on the machine. The ends of the bone (opposite of midpoint) were trimmed so that the bone could be mounted in adjustable grips. The clavicle was placed in the grip 15 mm away from the midpoint of the clavicle on both sides of the bone. The clavicle bone was then pulled apart until broken at a speed of 5 mm/sec with tension strength (N) measurements being recorded. Following the tension test, these bones, in addition to the bones that were previously broken at the point of fusion during processing, were broken by using a 3-point bendbreaking method on 1 side of the clavicle. The 3-point bend test was conducted using the texture analyzer (TAXT2i, Texture Technologies Corp.) fitted with a 1.5 × 11 mm incisor knife blade (TA-45, Texture Technologies Corp.) attachment at a speed of 5 mm/sec. Force (N) required to break bones was recorded and represented break force. Any clavicles which were broken along the shaft before testing were not used in bone strength analysis; all clavicles were subjected to ash analysis (following strength measurements) as described for tibias.
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EFFECT OF PHYTASE ON CARCASS CHARACTERISTICS 1
Table 2. Effect of phytase on growth performance and tibia ash in Experiment 1 Weight gain3 (g)
Diet 6
1. Basal diet 2. Diet 1 + 0.05% iP (KH2PO4) 3. Diet 1 + 0.10% iP 4. Diet 1 + 0.15% iP 5. Diet 1 + 250 FTU/kg of Escherichia coli phytase 6. Diet 1 + 500 FTU/kg of E. coli phytase 7. Diet 1 + 750 FTU/kg of E. coli phytase 8. Diet 1 + 1,000 FTU/kg of E. coli phytase 9. Diet 1 + 10,000 FTU/kg of E. coli phytase Pooled SEM
289 332 357 374 365 359 387 352 388 18.8
Gain:feed (g/kg) b
561 565b 593ab 577ab 624a 588ab 600ab 568ab 589ab 20.9
Supplemental P intake (mg) 0 295 601 968 0 0 0 0 0 —
Tibia ash3,4,5
Bioavailable P release from tibia ash2
(%)
(mg)
(%)
(mg)
39.5 41.7 45.3 46.5 44.4 47.0 48.6 47.5 49.7 0.78
281 314 375 427 356 382 479 404 476 27.9
— — — — 0.11 0.16 0.18 0.16 0.19 0.012
— — — — 0.09 0.13 0.19 0.13 0.19 0.022
Means within a column lacking a common superscript differ (P < 0.05). Values are means of 5 pens of 5 male chicks fed the experimental diets from 9 to 23 d. 2 Diet 5 vs. diets 6, 7, 8, and 9 (P < 0.05). 3 Weight gain and tibia ash (% and mg) responded linearly to supplemental inorganic P (iP; P < 0.05); linear and quadratic responses were significant for weight gain and tibia ash (% and mg) regressed against E. coli phytase levels including 250, 500, 750, and 1,000 phytase units (FTU)/ kg (P < 0.05). 4 The linear regression equation of tibia ash (%) for diets 1 to 4 as a function of supplemental iP intake (g) was Y = 38.95 (±0.77) + 8.48 (±1.27)X (r2 = 0.72). 5 The linear regression equation of tibia ash (mg) for diets 1 to 4 as a function of supplemental iP intake (g) was Y = 272.98 (±16.81) + 163.62 (±28.51)X (r2 = 0.65). 6 The basal diet was analyzed to contain 0.40% total P and estimated to contain 0.13% inorganic P (iP). a,b 1
Diet
Weight Supplemental gain2 Gain:feed3 P intake (g) (g/kg) (mg)
1. Basal diet7 331 2. Diet 1 + 0.05% iP (KH2PO4) 424 3. Diet 1 + 0.10% iP 455 4. Diet 1 + 0.15% iP 486 5. Diet 1 + 250 FTU8/kg of Escherichia coli phytase 472 6. Diet 1 + 500 FTU/kg of E. coli phytase 510 7. Diet 1 + 1,000 FTU/kg of E. coli phytase 542 8. Diet 1 + 2,000 FTU/kg of E. coli phytase 548 9. Diet 1 + 4,000 FTU/kg of E. coli phytase 584 10. Diet 1 + 250 FTU/kg of FP1 425 11. Diet 1 + 500 FTU/kg of FP1 455 12. Diet 1 + 1,000 FTU/kg of FP1 451 13. Diet 1 + 2,000 FTU/kg of FP1 499 14. Diet 1 + 4,000 FTU/kg of FP1 518 15. Diet 1 + 250 FTU/kg of FP2 385 16. Diet 1 + 500 FTU/kg of FP2 413 17. Diet 1 + 1,000 FTU/kg of FP2 457 18. Diet 1 + 2,000 FTU/kg of FP2 428 19. Diet 1 + 4,000 FTU/kg of FP2 481 Pooled SEM 19.5 Contrasts E. coli phytase vs. FP1 P < 0.05 E. coli phytase vs. FP2 P < 0.05 FP1 vs. FP2 P < 0.05 1
629 668 687 684 665 703 748 732 747 667 670 647 687 694 636 655 702 650 685 19.1
0 323 664 1,067 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
P < 0.05 P < 0.05 —
— — —
Tibia ash4,5,6
Bioavailable P release from tibia ash
(%)
(mg)
(%)
(mg)
33.4 39.8 42.2 46.2 44.3 47.8 47.5 49.0 47.7 37.2 38.8 44.6 42.8 44.7 35.0 35.7 35.7 39.4 40.5 0.69
334 421 520 599 550 661 759 807 806 424 464 513 559 673 384 391 463 475 539 19.8
— — — — 0.12 0.16 0.16 0.17 0.15 0.04 0.06 0.13 0.10 0.12 0.02 0.05 0.02 0.07 0.07 0.009
— — — — 0.12 0.18 0.23 0.25 0.24 0.07 0.07 0.10 0.12 0.18 0.03 0.05 0.08 0.08 0.11 0.009
P < 0.05 P < 0.05 P < 0.05
P < 0.05 P < 0.05 P < 0.05
P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05
Values are means of 5 pens of 5 male chicks fed the experimental diets from 8 to 22 d. Linear response of weight gain to supplemental inorganic P (iP), E. coli phytase, fungal phytase 1 (FP1), and fungal phytase 2 (FP2; P < 0.05); quadratic response to iP (P < 0.05). 3 Linear response of feed efficiency to supplemental iP and E. coli phytase. 4 Linear response of tibia ash (% and mg) to supplemental iP, E. coli phytase, FP1, and FP2 (P < 0.05); quadratic response of tibia ash (%) to iP, E. coli phytase, and FP1 (P < 0.05); quadratic response of tibia ash (mg) to E. coli phytase (P < 0.05). 5 The linear regression equation of tibia ash (%) for diets 1 to 4 as a function of supplemental iP intake (g) was Y = 34.53 (±0.67) + 11.46 (±1.03)X (r2 = 0.87). 6 The linear regression equation of tibia ash (mg) for diets 1 to 4 as a function of supplemental iP intake (g) was Y = 340.58 (±16.07) + 249.43 (±24.78)X (r2 = 0.85). 7 The basal diet was analyzed to contain 0.40% total P and calculated to contain 0.13% inorganic P. 8 FTU = phytase unit. 2
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Table 3. Effect of phytase on growth performance and tibia ash in Experiment 21
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PILLAI ET AL.
Table 4. Growth, bone, and carcass response variables in Experiment 31 Diet2 1. Basal diet (adequate Ca and P) 2. Diet 1 − 0.10% iP 3. Diet 2 + 250 FTU5/kg of Escherichia coli phytase 4. Diet 2 + 500 FTU/kg of E. coli phytase 5. Diet 2 + 1,000 FTU/kg of E. coli phytase Pooled SD
Weight gain (g)
Feed intake (g)
Gain:feed (g/kg)
Tibia ash (%)
Live weight3 (kg)
Carcass yield4 (%)
2,469ab 2,371b 2,450ab 2,479a 2,493a 86.7
4,303a 4,105b 4,196ab 4,234ab 4,293a 158.6
574 579 584 586 581 16.9
44.4a 41.8b 44.3a 44.7a 45.3a 1.26
2.59a 2.47b 2.55ab 2.55ab 2.60a 0.10a
67.0c 67.3bc 68.0ab 68.0ab 68.9a 1.18
Means within a column lacking a common superscript differ (P < 0.05). Values are means of 8 pens of 20 male chicks fed the starter diets from 3 to 21 d and the grower diets from 21 to 42 d. 2 Diet 1 was calculated to contain 0.47 and 0.36% inorganic P (iP) during the starter and grower periods, respectively; diets 2, 3, 4, and 5 were calculated to contain 0.31 and 0.28% iP during the starter and grower periods, respectively. 3 Represents average live weight of the 5 birds per pen that were randomly chosen for processing. 4 Calculated as hot eviscerated carcass as a percentage of live BW. 5 FTU = phytase unit. a–c 1
EXP 4 Growth performance and bone response variables for EXP 4 are shown in Tables 5 and 6. Slightly decreasing dietary iP and Ca (diet 2) did not reduce (P > 0.05) growth performance and carcass yield compared with birds fed the positive control diet (diet 1), but tibia ash was reduced (P < 0.05). Addition of E. coli phytase to diets containing reduced Ca and iP did not affect (P > 0.05) growth performance or carcass yield compared with the corresponding control diets, with the exception of wing yield, which was slightly higher (P < 0.05) for birds fed diet 4 compared with diet 2. Addition of 300 FTU of E. coli phytase/kg to diets containing reduced Ca and iP improved (P < 0.05) tibia ash compared with birds fed the negative control diet (diet 2), and adding 600 or 1,000 FTU of E. coli phytase/kg (diets 5 and 6, respectively) prevented a reduction in growth performance, yield, and tibia ash relative to birds fed the positive control diet (diet 1).
The incidence rate for bone breakage (Table 6) during processing was variable, with diet having little effect (P > 0.05) on the incidence of broken coracoids or tibias or on the incidence of disjointed wings. However, the incidence of broken clavicles was increased (P < 0.05; compared with diet 1) in birds fed the negative control diet (diet 2) and diets containing E. coli phytase. The incidence of broken wings did not appear to follow a logical pattern; however, variability within this measurement was high, so none of the treatments differed (P > 0.05) from the positive control (diet 1).
EXP 5 Growth performance and bone response variables for EXP 5 are shown in Table 7. In this EXP, slightly decreasing dietary iP and Ca (diet 2) reduced (P < 0.05) weight gain compared with birds fed the other diets. No differences (P > 0.05) among treatments were noted for clavicle ash (%), but clavicle diameter and tension force (force required to separate the clavicles at the point of fusion) was reduced (P < 0.05) in birds fed diet 2. No differences
Table 5. Growth and carcass response variables in Experiment 41
Diet2 1. 2. 3. 4. 5.
Basal diet (adequate Ca and P) Diet 1 − 0.10% iP and 0.05% Ca Diet 1 + 300 FTU5/kg of Escherichia coli phytase Diet 2 + 300 FTU/kg of E. coli phytase Diet 1 − 0.15% iP and 0.05% Ca + 600 FTU/kg of E. coli phytase 6. Diet 1 − 0.20% iP and 0.05% Ca + 1,000 FTU/kg of E. coli phytase Pooled SEM
Weight gain (g)
Feed intake (g)
Gain:feed (g/kg)
Carcass yield3 (%)
Breast yield4 (%)
Wing yield4 (%)
Leg yield4 (%)
2,699c 2,709bc 2,782abc 2,781abc
5,125b 5,108b 5,193ab 5,156b
527b 530ab 536ab 539a
70.7 70.7 70.6 70.4
25.5b 26.0ab 25.6ab 25.9ab
11.7bc 11.6c 11.9ab 11.8ab
34.3ab 34.0b 34.0b 34.5ab
2,859a
5,335a
536ab
70.1
26.3a
11.7abc
34.7a
2,803ab 32.8
5,237ab 61.7
535ab 3.3
70.6 0.45
25.9ab 0.24
12.0a 0.09
34.7a 0.20
Means within a column lacking a common superscript differ (P < 0.05). Growth values are means of 7 pens of 20 male chicks; yield values are means of 7 pens of 5 randomly-selected chicks per pen; birds were fed the starter diets from 0 to 18 d, grower diets from 18 to 32 d, finisher diets from 32 to 40 d, and withdrawal diets from 40 to 50 d. 2 Diet 1 was calculated to contain 0.48, 0.42, 0.38, and 0.34% inorganic P (iP) during the starter, grower, finisher, and withdrawal periods, respectively. 3 Represents chilled, ready-to-cook carcass as a percentage of live BW. 4 Calculated as a percentage of chilled, ready-to-cook carcass weight. 5 FTU = phytase unit. a–c 1
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had a higher carcass yield than birds fed diets 1 and 2 (P < 0.003).
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EFFECT OF PHYTASE ON CARCASS CHARACTERISTICS 1
Table 6. Bone response variables in Experiment 4
Incidence rate2 Broken Broken Broken Tibia ash clavicle coracoid tibia (%) (%) (%) (%)
Diet3 1. 2. 3. 4. 5.
Basal diet (adequate Ca and P) Diet 1 − 0.10% iP and 0.05% Ca Diet 1 + 300 FTU5/kg of Escherichia coli phytase Diet 2 + 300 FTU/kg of E. coli phytase Diet 1 − 0.15% iP and 0.05% Ca + 600 FTU/kg of E. coli phytase 6. Diet 1 − 0.20% iP and 0.05% Ca + 1,000 FTU/kg of E. coli phytase Pooled SEM
Broken wing4 (%)
Wing disjoint (%)
42.0ab 39.9c 42.1a 41.2ab
17.1b 28.6ab 45.7a 45.7a
0.0 2.9 2.9 5.7
17.1 20.0 22.9 14.3
17.1abc 5.7c 25.7ab 2.9c
2.9 0.0 0.0 8.6
40.8bc
31.4ab
2.9
11.4
34.3a
8.6
ab
ab
42.0 0.4
37.1 7.4
5.7 2.9
5.7 6.3
bc
14.3 6.4
2.9 3.0
Means within a column lacking a common superscript differ (P < 0.05). Values are means of 8 pens of 5 randomly selected male chicks per pen that were fed the starter diets from 0 to 18 d, grower diets from 18 to 32 d, finisher diets from 32 to 40 d, and withdrawal diets from 40 to 50 d. 2 Incidence rates represent the percentage of birds per treatment that exhibited the noted defect. 3 Diet 1 was calculated to contain 0.48, 0.42, 0.38, and 0.34% inorganic P (iP) during the starter, grower, finisher, and withdrawal periods, respectively. 4 A broken wing was designated as one in which the radius, ulna, or both were broken. 5 FTU = phytase unit. a–c 1
DISCUSSION Previous EXP utilizing E. coli phytase products investigated the Ca- and P-releasing ability of these microbial phytases in broilers during the starter phase of production
(Augspurger et al., 2003; Augspurger and Baker, 2004a,b). Although the information provided in these papers contributes to the elucidation of the mode of action of this particular enzyme and its efficacy, information about the effects of this enzyme on key broiler carcass characteristics, such as leg and breast yield and the incidence of broken legs, wings, and clavicles, is not available. There exists, therefore, a paucity of data detailing the effect of the level of iP and phytase supplementation during the finisher and withdrawal stages of broiler production on commercial processing parameters. The addition of phytase to diets deficient in available P at various production phases of broilers has been shown to increase growth and improve bone (usually tibia) response variables (Zyla et al., 2000a,b; Applegate et al., 2003a; Augspurger et al., 2003; Augspurger and Baker,
Table 7. Growth and bone response variables in Experiment 51 Incidence rate2
Clavicle attributes
Diet3 1. Basal diet (adequate Ca and P) 2. Diet 1 − 0.10% iP and 0.05% Ca 3. Diet 2 + 300 FTU6/kg of Escherichia coli phytase 4. Diet 1 − 0.20% iP and 0.05% Ca + 1,000 FTU/kg of E. coli phytase Pooled SD
Weight gain (g)
Gain:feed (g/kg)
Ash (%)
Diameter (mm)
Tension force4 (N)
Break force4 (N)
Broken clavicle (%)
Broken tibia (%)
Broken wing5 (%)
Wing disjoint (%)
3,865a 3,612b
458b 454b
54.2 54.3
2.75a 2.63b
31.0a 19.0b
100.4 89.5
24.5 38.1
5.7 2.4
1.9 2.4
3.8 4.8
3,826a
483a
55.6
2.71ab
28.6ab
98.6
32.1
7.5
1.9
5.7
3,839a 109
463ab 7
54.4 4.6
2.75a 0.27
33.6a 6.7
98.3 24.7
27.9 46.1
4.7 22.3
0.0 12.5
7.0 22.3
Means within a column lacking a common superscript differ (P < 0.05). Growth values are means of 4 pens of 15 male broilers; yield values are means of 4 pens of 15 birds per pen; birds were fed the starter diets from 8 to 18 d, grower diets from 18 to 32 d, finisher diets from 32 to 40 d, and withdrawal diets from 40 to 56 d. 2 Incidence rates represent the percentage of birds per treatment that exhibited the noted defect. 3 Diet 1 was calculated to contain 0.43, 0.40, 0.37, and 0.35% inorganic P (iP) during the starter, grower, finisher, and withdrawal periods, respectively. 4 Tension force is defined as the force required to separate the clavicles at the point of fusion; break force is the force required to break the bone. 5 A broken wing was designated as one in which the radius, ulna, or both were broken. 6 FTU = phytase unit. a,b 1
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(P < 0.05) in clavicle diameter or tension force were noted in birds fed diet 1 or the diets containing E. coli phytase. Break force (force required to break the clavicle) did not differ (P > 0.05) among treatments, although break force for clavicles from birds fed diet 2 was numerically lower. As for EXP 4, bone breakage rates in EXP 5 were variable, and no differences (P < 0.05) in the incidence of bone breakage during processing were observed. It should be noted that, for clavicles, the majority (>80%; data not shown) of breakage occurred at the point of fusion.
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PILLAI ET AL.
yield in pigs appears to be variable (O’Quinn et al., 1997; Liu et al., 1998), and Walz and Pallauf (2003) reported that although phytase increased apparent digestibility of P, Ca, and Zn, it had no effect on carcass and meat characteristics in barrows. Similar results have been obtained in drakes supplemented with phytase, Lys, or both, with no difference in carcass yield and meat quality reported by Attia (2003). However, Shelton et al. (2004) reported that the inclusion of phytase to diets deficient in Ca, P, and trace minerals reversed the negative effects of these deficient diets on carcass lean content, weight, dressing percentage, and bone associated with the removal of these minerals in growing-finishing pigs. The effect of phytase on carcass characteristics in broilers is also important, but it is limited. In the current EXP, the inclusion of the E. coli phytase enzyme not only returned bone ash percentage to that of the control in broilers fed P-deficient diets (Tables 2, 3, 4, and 6), but it also prevented negative effects on carcass and breast yield in birds fed diets containing substantially reduced P in EXP 4. It should be noted that corresponding negative control diets were not fed in EXP 4 and 5 because of welfare concerns associated with feeding such drastic reductions in Ca and P over the duration of the trials, but in a separate trial, short-term feeding of negative control diets (diets 5 and 6 in EXP 4 and diet 4 in EXP 5 fed without E. coli phytase) verified that these diets substantially reduce growth and tibia ash in the absence of E. coli phytase (data not shown). Bone integrity has an important role to play in total yield of the carcasses intended for the whole-bird market and the further-processing market (Rath et al., 2000) and in the rate of product loss due to bone breakage. Chen and Moran (1994) reported that reducing the level of P in the withdrawal phase of production resulted in increased defects and decreased production of grade-“A” carcasses. We were interested in determining whether phytase would affect the incidence of bone breakage during processing. In EXP 4, bone breakage results were highly variable, and we were surprised by the high incidence of broken clavicles. We have also noted high rates of clavicle breakage (approaching 50%) in other research projects involving processing of broilers raised on nutritionallyadequate diets. The departmental processing plant in which birds were processed must accommodate a wide range of bird sizes, and it is possible that evisceration equipment was not optimally adjusted for birds in our trial, resulting in a higher clavicle breakage rate. Because of this, and because the rate of wing breakage in EXP 4 was so variable (and seemingly illogical), portions of EXP 4 were repeated in EXP 5. Although variability associated with bone breakage was still high in EXP 5, clavicle and wing breakage rates were lower overall, and results indicated that bone breakage should not be increased in broilers fed diets containing low levels of iP combined with supplemental phytase. In conclusion, based on the data obtained from these EXP, E. coli phytase releases more P than the fungal phytases that were tested. When based on tibia ash weight
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2004a,b; Dilger et al., 2004; Shirley and Edwards, 2003). The degree of response has been the subject of much investigation, and researchers have estimated that fungal phytases release an amount of available P ranging from less than 0.05% (Angel et al., 2001) to as much as 0.113% (Biehl et al., 1995). The level of improved P utilization and growth seems largely dependent on the source of phytase supplementation, as some of the major constraints to the catalytic properties of phytase enzyme are pH and time limitations with regard to the unique nature of the gastrointestinal tract of the broiler (Zyla et al., 2004). Augspurger et al. (2003) reported greater P-release values from phytase produced by E. coli expressed in yeast when compared with phytase derived from FP1 and FP2 for young chicks and pigs during the starter phases of production. Applegate et al. (2003b) reported that turkeys fed diets supplemented with an E. coli-derived phytase had a consistently higher iP-sparing effect than FP1 and FP2. Augspurger et al. (2003) attributed the greater P release by E. coli phytase to the different activation levels of FP1 (pH 2.5 and 5.5), FP2 (pH 4.0 to 4.5), and the E. coliderived phytase (pH 2.5 to 3.5). Rodriguez et al. (1999a) reported that the E. coli phytase enzyme expressed in Pichia pastoris released more P from phytate in soybean meal than Aspergillus niger phyA at each enzyme’s optimum pH. Moreover, E. coli phytase appears to be more resistant to pepsin breakdown; Rodriguez et al. (1999b) reported that another E. coli phytase had a 30% increase in phytase activity after being incubated in pepsin at a pH of 2, whereas FP1 had a 58 to 77% decrease in activity under the same experimental conditions. However, FP1 was more resistant to trypsin degradation at a pH of 7 than E. coli phytase enzyme. Our results confirm the efficacy of E. coli phytase (Table 2) and indicate an increased ability of E. coli phytase to release bound P, compared with the fungal phytases that were used (Table 3). As described previously (Rodriguez et al., 1999a,b; Applegate et al., 2003b; Augspurger et al., 2003), attributes of E. coli phytase (resistance to pepsin degradation, optimum pH) appear to enhance its P-releasing ability. Based on tibia ash, we noted improvements in estimated iP release ranging from 23 to 200% (depending on phytase level) for E. coli phytase, compared with FP1, and 118 to 700% (depending on phytase level) for E. coli phytase, compared with FP2. As previously noted, at the higher levels of E. coli phytase supplementation in EXP 1 and 2, tibia ash values exceeded those of the standard curve; although this brings into question the accuracy of E. coli phytase efficacy estimates at the higher levels of E. coli phytase supplementation, it does suggest that E. coli phytase releases >0.15% iP. Our iP-sparing values for the various phytase enzymes are similar to those reported by Augspurger et al. (2003). In other farm species of economic importance, investigators have largely focused on the effect of phytase in conjunction with varying amino acid and energy levels on carcass quality of pigs and drakes (Harper et al., 1997; Attia, 2003; Walz and Pallauf, 2003; Shelton et al., 2004). The effect of phytase enzyme supplementation on carcass
EFFECT OF PHYTASE ON CARCASS CHARACTERISTICS
(mg/chick or %), the amount of available P released ranged from 0.119 to 0.239% for E. coli phytase, compared with 0.07 to 0.18% from FP1 and 0.03 to 0.11% from FP2 at phytase supplementation levels of 250 and 4,000 FTU/ kg, respectively. When based on tibia ash weight (mg/ chick), P release appeared to be maximized at a level of 1,000 FTU/kg of E. coli phytase. Results also indicate that E. coli phytase is effective at maintaining carcass yield characteristics without increasing the incidence of bone breakage (based on EXP 5).
REFERENCES
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