Influence of supplemental phytase on performance of broilers four to six weeks of age

Influence of supplemental phytase on performance of broilers four to six weeks of age

METABOLISM AND NUTRITION Influence of Supplemental Phytase on Performance of Broilers Four to Six Weeks of Age1 S. S. SOHAIL and D. A. ROLAND, SR.2 De...

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METABOLISM AND NUTRITION Influence of Supplemental Phytase on Performance of Broilers Four to Six Weeks of Age1 S. S. SOHAIL and D. A. ROLAND, SR.2 Department of Poultry Science and Alabama Agricultural Experiment Station, Auburn University, Alabama 36849 ABSTRACT The influence of dietary phytase on phytate P availability was investigated using Ross × Hubbard male broiler chicks. A randomized complete block design with a factorial arrangement of 2 × 3 was used with eight replicates (n = 2,400; 50 chicks per replicate) per treatment. Diets were formulated to contain two levels of nonphytate P (NPP; 0.225 and 0.325%) and three levels of phytase [0, 300, and 600 phytase units (FTU)/kg] with 0.75% Ca. An additional diet with 0.425% NPP and 0.85% Ca was used as a positive control (n = 400). Prior to assigning treatments, all chicks were fed a commercial starter mash adequate in all nutrients until 3 wk of age. Neither performance nor bone strength was significantly influenced by a reduction of NPP to 0.325% and Ca to 0.75% as compared to the positive control. However, when NPP was reduced to 0.225% and Ca to 0.75%, significant

negative impacts on body weight, feed consumption, feed efficiency, and bone strength were observed. Phytase significantly increased BW at the lower NPP level but not at the higher NPP level. A significant NPP by phytase interaction occurred in bone criteria and livability. Phytase (300 FTU/kg) had greater influence on bone mineral content, bone density, bone breaking strength, and livability in broilers fed 0.225% NPP than in broilers fed 0.325% NPP. This study indicates that supplementing phytase in grower diets containing reduced levels of NPP and Ca significantly improved performance and bone strength of broilers. In diets containing marginal to deficient levels of either NPP or Ca or both, the addition of microbial phytase at 300 to 600 FTU/kg feed prevents P deficiency symptoms. Increasing phytase levels from 300 to 600 FTU/kg feed provided no additional benefit.

(Key words: bone strength, broiler, calcium, phytase, phosphorus) 1999 Poultry Science 78:550–555

Formation of insoluble phosphate makes Ca and P unavailable. Phytic acid has chelating potential and forms a wide variety of insoluble salts with di- and trivalent cations at neutral pH (Vohra et al., 1965; Oberleas, 1973). Zinc, Cu, Co, Mn, Fe, and Mg can complex, but Zn and Cu have the strongest binding affinity (Maddaiah et al., 1964; Vohra et al., 1965). Phytate complexes with metals and precipitates at the pH of the intestine (Oberleas, 1973), and potentially renders these minerals unavailable for intestinal absorption. Phytase (EC.3.1.3.8), myo-inositol hexakisphosphate phosphohydrolase, is the enzyme that releases P from phytate molecule (Gibson and Ullah, 1990). The efficacy of microbial phytase to improve dietary P bioavailability has been reported by several researchers (Simons et al., 1990; Denbow et al., 1995; Ravindran et al., 1995; Kornegay et al., 1996; Mitchell and Edwards, 1996; Gordon and Roland, 1997; Sohail and Roland, 1997). The results of trials in which the dose response to microbial

INTRODUCTION About two-thirds of the total P contained in feed ingredients of plant origin occurs as phytates. Phytate P is either unavailable or poorly utilized by monogastric animals due to insufficient quantities of endogenous phytase (Nelson, 1967). Thus, the availability of P in feedstuffs of plant origin is generally very low. Bioavailability estimates of P in corn and soybean meal for pigs and poultry range from 10 to 30% (Nelson, 1967; Calvert et al., 1978; Jongbloed and Kemme, 1990). This low availability of phytate P poses two problems for producers: 1) the need to add inorganic P supplements to diets, and 2) the excretion of large amounts of P in manure. In addition to low P availability, phytate limits availability of several other essential nutrients.

Received for publication July 10, 1998. Accepted for publication December 8, 1998. 1 Alabama Agriculture Experiment Journal Series Number 12-985940. 2To whom correspondence should be addressed: droland@acesag. auburn.edu

Abbreviation Key: 1,25-(OH)2D3 = 1,25-dihydroxycholecalciferol; D3 = cholecalciferol; FTU = phytase units; NPP = nonphytate phosphorus.

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BROILER RESPONSE TO PHYTASE TABLE 1. Ingredients and nutrient composition of experimental diets Diet Ingredients and composition

1

2

Corn 8.6% Soybean oil meal 48% Poultry oil Poultry meal Salt (NaCl) Calcium carbonate Defluorinated phosphate Meat and bone meal Vitamin premix1 Mineral premix2 DL-Methionine Natuphos 6003 Coban4 L-Lysine Nutrient composition5 Crude protein ME, kcal/kg Met + Cys Lysine Calcium Nonphytate P

63.80 26.12 3.85 2.00 0.283 0.194 0.93 2.00 0.25 0.25 0.189 . . . 0.084 0.049

64.42 26.00 3.62 2.00 0.348 0.439 0.335 2.00 0.25 0.25 0.188 . . . 0.084 0.052

20.05 3,212 0.84 1.10 0.85 0.43

20.05 3,212 0.84 1.10 0.75 0.33

3

4 64.34 26.02 3.65 2.00 0.346 0.429 0.335 2.00 0.25 0.25 0.188 0.05 0.084 0.051

20.05 3,212 0.84 1.10 0.75 0.32

5 (%) 64.26 26.04 3.68 2.00 0.343 0.418 0.335 2.00 0.25 0.25 0.188 0.10 0.084 0.051

20.05 3,212 0.84 1.10 0.75 0.32

63.93 27.09 3.76 2.00 0.400 0.953 0.055 1.00 0.25 0.25 0.186 . . . 0.083 0.042 20.05 3,212 0.84 1.10 0.75 0.22

6

7 63.85 27.11 3.78 2.00 0.397 0.943 0.055 1.00 0.25 0.25 0.186 0.05 0.083 0.041

20.05 3,212 0.84 1.10 0.75 0.22

63.77 27.12 3.81 2.00 0.395 0.932 0.055 1.00 0.25 0.25 0.186 0.10 0.084 0.041 20.05 3,212 0.84 1.10 0.75 0.22

1Provided per kilogram of diet: vitamin A (as retinyl acetate), 8,000 IU; cholecalciferol, 2,200 IU; vitamin E (as dl-a-tocopheryl acetate), 8 IU; vitamin B12, 0.02 mg; riboflavin, 5.5 mg; D-calcium pantothenic acid, 13 mg; niacin, 36 mg; choline, 500 mg; folic acid, 0.5 mg; thiamin mononitrate, 1 mg; pyridoxine hydrochloride, 2.2 mg; d-biotin, 0.05 mg; menadione sodium bisulfite complex, 2 mg. 2Provided per kilogram of diet: manganese, 65 mg; iodine, 1 mg; iron, 55 mg; copper, 6 mg; zinc, 55 mg; selenium, 0.3 mg. 3Natuphos 600 contains 600 phytase units/g. 4Coban contains monensin sodium 60 g/lb. A coccidiostat from Elanco Animal Health, Eli Lilly and Co., Indianapolis, IN 46285. 5Dietary Ca, total P, and nonphytate P values were determined by chemical analysis.

phytase was determined have shown a linear dependence on the dose up to 500 phytase units (FTU)/kg of feed. The response then tends to flatten to varying degrees at higher doses. In these trials, the diets were primarily corn-soybean meal diets containing negligible amounts of native phytase and similar to diets used commercially. Denbow et al. (1995) observed in soybean meal diets fed 0- to 3-wk-old male broilers, that phytase improved BW gains and feed intake at all the nonphytate phosphorus (NPP) levels. However, the magnitude of response was greatest at the low NPP level. Ravindran et al. (1995) found similar results in turkey poults, in which the response to phytase was highest at the lowest NPP level of 0.27%. Addition of phytase at 0.36 and 0.45% NPP levels completely corrected the gains equal to the positive control diet with 0.60% NPP. They suggested that 625 units of microbial phytase is equivalent to 1 g of P from defluorinated phosphate in soybean meal diets. Qian et al. (1997) reported that phytase linearly increased BW gain, feed intake, toe ash content, and P and Ca retention. Addition of cholecalciferol with phytase in the diets synergistically improved performance; however, widening the Ca:total P ratio, even with the inclusion of cholecalciferol and phytase, negatively influenced performance. Mitchell and Edwards (1996) demonstrated that 600 units of phytase could replace up to 0.1% inorganic P,

and a combination of 1-25-dyhydroxycholecalciferol [1,25-(OH)2D3] and phytase could replace 0.2% inorganic P in diets of young chicks. Qian et al. (1997) also reported that utilization of phytate P and Ca is influenced by Ca:total P ratio, and the level of cholecalciferol used. The above discussion clearly demonstrates the effectiveness of microbial phytase in improving the availability of P and other nutrients for simple stomached animals. Although a number of trials have been conducted using day-old chicks, few studies have investigated the efficacy of microbial phytase using 3-wk-old birds. This study was designed to determine the efficacy of phytase in broiler grower diets when fed from 3 through 6 wk of age.

MATERIALS AND METHODS Two thousand eight hundred day-old commercial broiler chicks (Ross × Hubbard) were randomly distributed in 56 pens with 50 chicks per pen. Commercial brooding and management procedures were followed, and all chicks were fed a typical commercial broiler starter ration for the first 3 wk. At 3 wk of age, they were randomly assigned to one of the seven dietary treatments with eight replicates per treatment. The seven diets (Table 1) were formulated to contain: 1) NPP and Ca levels of 0.425 and 0.85% Ca (positive control); 2) NPP reduced to 0.325% and Ca reduced to 0.75%; 3) as 2

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TABLE 2. Efficacy of microbial phytase in broiler grower and finisher diets on body weight from 4 to 6 wk of age Week Treatment

4

5

Control (0.425)2 NPP3 0.225 0.325 Phytase 0 FTU4/kg 300 FTU/kg 600 FTU/kg SEM

1,360 NS 1,368 1,375 NS 1,371 1,362 1,381 9.7

1,940 * 1,928 1,952 NS 1,937 1,937 1,946 5.4

6 (g) 2,430 *** 2,404 2,446 NS 2,423 2,412 2,439 13.9

x1 1,910 ** 1,900 1,924 NS 1,910 1,903 1,922 9.6

1Average

across Weeks 4 to 6 only. NPP diet (control) excluded from statistical analysis. 3NPP = nonphytate phosphorus. 4FTU = phytase units. *P < 0.05. **P < 0.01. ***P < 0.001. 20.425%

with 300 FTU3/kg diet; 4) as 2 with 600 FTU/kg diet; 5) NPP reduced to 0.225% and Ca reduced to 0.75%; 6) as 3 with 300 FTU/kg diet; and 7) as 3 with 600 FTU/kg diet. Criteria used to measure response were body weight gain, feed consumption, mortality, bone mineral content, bone density, and bone breaking strength. Body weight and feed consumption of chicks from all pens were measured weekly, and mortality of each pen was recorded on a daily basis. At the termination of the study, tibiae of 24 birds per treatment were removed by the direct excision method (Orban et al., 1993) to measure bone strength. Bone density and bone mineral content were measured with Norland bone densitometer (Model 2780),4 and bone breaking strength with an Instron Testing Instrument (Model 1011).5 Data were analyzed as a 2 × 3 factorial, and subjected to ANOVA (Steel and Torrie, 1980) using the General Linear Models procedure (SAS Institute, 1988), with the main effects of P and phytase, and P by phytase interaction being included in the model. Diet 1 was considered as a positive control, adequate in NPP and Ca; therefore phytase was not added. Because phytase was not included, data from Diet 1 were not included in the statistical analysis presented in Tables 2 to 5, but were included in Table 6.

RESULTS AND DISCUSSION After 1 wk on the experimental diets (4 wk of age), no significant impact of reducing NPP from 0.325 to 0.225% was evident nor was an effect of adding phytase found on the BW (Table 2). After 2 wk, birds receiving 0.325%

NPP were significantly (P < 0.05) heavier than birds receiving 0.225% NPP (1,952 vs 1,928 g). This lower BW was due to the deficiency of P in birds fed 0.225% NPP, which was much below the recommended level of 0.35% NPP for broilers 3 to 6 wk of age (National Research Council, 1994). Increasing NPP to 0.325% improved BW gain. This effect of P deficiency was also reported in broiler chicks 0 to 3 wk of age by Qian et al. (1996). A significant interaction (P < 0.05) was observed between NPP and phytase at 5 wk of age. This interaction was the result of steadily decreasing weights as phytase increased at NPP level of 0.325%, whereas weights increased (from 1,907 to 1,949 g) as phytase increased with the 0.225% NPP diets. This result was consistent with the results of Denbow et al. (1995), in which a NPP by phytase interaction was also due to a greater response of phytase at low NPP levels. At 6 wk of age, NPP levels had a highly significant (P < 0.001) impact on BW. Birds receiving 0.325% NPP weighed 2,446 g as compared to 2,404 g for birds receiving 0.225% NPP. The interaction between NPP and phytase approached significance at P < 0.06 (not shown). That is, when NPP levels were set at 0.325%, little effect from added phytase was observed as measured by BW. However, when NPP was reduced to 0.225%, phytase had a positive effect and resulted in weights increasing from 2,384 g at no added phytase to 2,392 g with 300 FTU/kg and 2,435 g with 600 FTU/kg. This result is in agreement with the previous findings in chicks 0 to 3 wk of age, that magnitude of response was greatest at low NPP levels, and supplementing low NPP diets with inorganic P or phytase improved performance of broilers (Denbow et al., 1995; Qian et al., 1996). Feed conversion data is presented in Table 3. After 1 wk on the experimental diets, birds receiving 0.325% NPP had a statistically (P < 0.05) lower feed conversion

TABLE 3. Efficacy of microbial phytase in broiler grower and finisher diets on the feed conversion ratio from 4 to 6 wk of age Week Treatment

4

5

Control (0.425)2 NPP3 0.225 0.325 Phytase 0 FTU4/kg 300 FTU/kg 600 FTU/kg SEM

1.84 * 1.77 1.74 NS 1.75 1.77 1.73 0.02

1.92 NS 1.95 1.93 NS 1.94 1.92 1.96 0.02

1Average

(g:g) 2.59 NS 2.60 2.58 NS 2.59 2.62 2.57 0.03

x1 2.11 * 2.11 2.08 NS 2.09 2.10 2.09 0.01

across Weeks 4 to 6 only. NPP diet (control) excluded from statistical analysis. 3NPP = nonphytate phosphorus. 4FTU = phytase units. *P < 0.05. 20.425%

3Natuphos, BASF Corp., Mt. Olive, NJ 07828-1234. 4Norland Corp., Fort Atkinson, WI 53538. 5Instron Corp., Canton, MA 02021.

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BROILER RESPONSE TO PHYTASE TABLE 4. Efficacy of microbial phytase in broiler grower and finisher diets on mortality from 4 to 6 wk of age Week Treatment

4

5

Control (0.425)2 NPP3 0.225 0.325 Phytase 0 FTU/kg4 300 FTU/kg 600 FTU/kg SEM

0.38 NS 0.42 0.33 NS 0.38 0.25 0.50 0.13

0.25 NS 0.29 0.42 ** 0.69 0.06 0.31 0.31

6 (%) 0.75 NS 0.88 0.58 NS 0.81 0.50 0.88 0.20

x1 0.46 NS 0.53 0.44 * 0.63 0.27 0.56 0.19

1Average

across Weeks 4 to 6 only. NPP diet excluded from statistical analysis. 3NPP = nonphytate phosphorus. 4FTU = phytase units. *P < 0.05. **P < 0.01. 20.425%

(1.74 vs 1.77) than birds receiving 0.225% NPP. No effect of adding phytase and no interaction between NPP and phytase were observed. The ANOVA (Diet 1 data excluded) for Weeks 5 and 6 revealed that no differences could be attributed to NPP, phytase, or to the interaction of NPP and phytase. This result was in agreement with the previous findings (Swick and Ivey, 1990; Denbow et al., 1995). Mortality data are presented in Table 4. During Week 5, birds not fed phytase had a significantly (P < 0.01) higher mortality than those fed phytase (300 FTU/kg), a finding consistent with the results and explanation of Simons et al. (1990) that not enough phytate P was available to the chicks to fulfill their requirement of P, which was much higher than the amount offered. Phosphorus requirement of chicks varies with the age, size, and strain of birds (Edwards, 1983; National Research Council, 1994; Punna and Roland, 1996a,b). Although live performance is an important measure of any dietary changes, when NPP and Ca levels or one of the two in the diet are changed, bone strength measures are generally more sensitive than performance factors. The effect of treatment on bone mineral content, bone density, and bone breaking strength are presented in Table 5. Again, Diet 1 was excluded from the statistical analysis because phytase was not included as a variable. A significant (P < 0.01) interaction between NPP and phytase as measured by bone mineral content was observed. When phytase was increased from 0 to 300 FTU/kg in diets containing 0.225% NPP, bone mineral content increased by 28.4%, from 0.141 to 0.181 g/cm. At 0.325% NPP, addition of phytase (300 FTU/kg) resulted in an increase in bone mineral content by 5.1%, from 0.178 to 0.187 g/cm. The effect of adding 300 FTU/kg was much more pronounced at 0.225% NPP than at 0.325% NPP.

For bone density, a significant interaction between NPP and phytase was also revealed. The inclusion of phytase at 300 FTU/kg diet and 600 FTU/kg diet resulted in an increase in bone density from 0.174 to 0.201 g/cm2 (15.5%) and to 0.206 g/cm2 (18.4%), respectively. With NPP set at 0.325%, a relatively small increase in bone density (6.4%) was observed with the addition of phytase, 300 FTU/kg diet (from 0.188 to 0.200 g/cm2). At 0.225% NPP, the effect of adding phytase (300 FTU/kg diet) was much more pronounced: density increased from 0.160 to 0.202 g/cm2. At 0.225 and 0.325% NPP, increasing phytase from 300 to 600 FTU/kg diet resulted in little change in bone density. From these data it may be concluded that bone density is maximized at both 0.225 and 0.325% NPP when the diet contains 300 FTU/kg. Bone breaking strength (kilograms) was also statistically impacted (P < 0.001) by phytase levels and the interaction (P < 0.01) between NPP and phytase. When the data were averaged across NPP levels, diets with added phytase had much higher breaking strength than did bones from birds not fed phytase. Only 34.35 kg pressure was required to break bones from birds not fed phytase, whereas 41.05 and 44.44 kg pressure was required for the birds fed phytase (Table 5). This result was consistent with the previous reports that inclusion of phytase in the diet improves bone strength (Perney et al., 1993; Qian et al., 1996). Clearly, birds fed phytase had stronger bones than birds not fed phytase. This significant interaction means that birds fed different levels of NPP responded differently to supplemental phytase. As

TABLE 5. Efficacy of microbial phytase in broiler grower and finisher diets on bone mineral content (BMC), bone density (BD), and bone breaking strength (BBS) Treatment

BMC

BD

BBS

Control (0.425)1 NPP2 0.225 0.325 Phytase 0 FTU3/kg 300 FTU/kg 600 FTU/kg NPP × Phytase 0.225 × 0 0.225 × 300 0.225 × 600 0.325 × 0 0.325 × 300 0.325 × 600 SEM

(g/cm) 0.180 ** 0.173 0.187 *** 0.160 0.184 0.195 ** 0.141 0.181 0.195 0.178 0.187 0.195 0.008

(g/cm2) 0.195 NS 0.189 0.198 *** 0.174 0.201 0.206 * 0.160 0.202 0.206 0.188 0.200 0.206 0.007

(kg) 39.37 NS 38.16 39.67 *** 34.35 41.05 41.44 ** 31.05 42.22 41.34 37.65 39.88 41.55 1.71

10.425%

NPP diet (control) excluded from statistical analysis. = nonphytate phosphorus. 3FTU = phytase units. *P < 0.05. **P < 0.01. ***P < 0.001. 2NPP

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SOHAIL AND ROLAND TABLE 6. Efficacy of microbial phytase in broiler grower and finisher diets; control vs treatment comparisons at 6 wk of age Treatment diets Variable

1 (Control)

2

3

5

6

Nonphytate P, % Calcium, % Phytase, FTU1/kg diet Body weight at 6 wk, g Feed conversion ratio, g:g Mortality, % Bone mineral content, g/cm Bone density, g/cm2 Bone breaking strength, kg

0.425 0.85 0 2,430 2.11 0.46 0.180 0.195 39.37

0.325 0.75 0 2,462 2.07 0.46 0.178 0.188 37.65

0.325 0.75 300 2,431 2.10 0.21 0.187 0.200 39.88

0.225 0.225 0.75 0.75 0 300 2,384 2,392 2.12 2.11 0.79 0.33 0.141*** 0.181 0.160*** 0.202 31.05*** 42.22

1FTU

= phytase unit. ***P < 0.001 compared to Diet 1.

might be expected, response to adding phytase was more pronounced on the lower dietary NPP treatments than on the higher NPP treatments. On the lower NPP diets, adding phytase at 300 FTU/kg diet resulted in increased bone breaking strength from 31.05 to 42.22 kg pressure, an increase of 36%. This 36% increase in bone breaking strength at the lower NPP level is contrasted with an increase of 5.84% (from 37.65 to 39.88 kg pressure) at the higher NPP level. However, at both NPP levels, little increase in bone breaking strength was seen as a result of increasing phytase from 300 to 600 FTU/kg diet. These results agree with the previous reports (Denbow et al., 1995; Mitchell and Edwards, 1996; Qian et al., 1996) in which supplemental phytase improved the quality of bone, and the effect of phytase on bone strength was greater at the lower levels of NPP. In this trial, Diet 1, containing 0.425% NPP and 0.85% Ca without phytase, was considered as a positive control and the test diets were compared to the control. Results of this statistical comparison are presented in Table 6. When Diet 1 was compared to Diet 2 or to Diet 3, reducing NPP and Ca by 0.1% (to 0.325% NPP and 0.75% Ca) without or with the addition of phytase had no effect on any of the parameters measured. However, when Diet 1 was compared to Diet 5, a highly significant effect on all measures of bone strength was recorded. Bone mineral content was reduced from 0.180 to 141 g/ cm, bone density was reduced from 0.195 to 0.160 g/ cm2, and breaking strength was reduced from 39.37 to 31.05 kg pressure. When Diet 1 was compared to Diet 6, no significant differences were observed. The addition of phytase (300 FTU/kg diet) completely reversed the negative effects associated with NPP and Ca reduction, and resulted in live performance and bone strength equal to the positive control diet. Based on the results of this study, the following conclusions were drawn: 1) Neither performance nor bone strength was significantly affected by a reduction of NPP to 0.325% and Ca to 0.75% as compared to diet containing 0.425% NPP and 0.85% Ca. However, when NPP was reduced to 0.225% and Ca to 0.75%, a highly significant negative impact on bone strength was

observed. 2) The negative effect on bone strength associated with a dietary level of 0.225% NPP and 0.75% Ca was completely reversed by the inclusion of phytase (300 FTU/kg diet). Addition of phytase resulted in improved bone mineral content, increased bone density and increased bone breaking strength. 3) In diets containing marginal to deficient levels of either NPP or Ca or both, the addition of microbial phytase (300 FTU/ kg diet) prevents phosphorus deficiency symptoms. Increasing phytase levels from 300 to 600 FTU/kg diet provided no additional benefit.

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