Effect of Nongenetically Modified Phytase Supplementation on ...

2003 Poultry Science Association, Inc.

Effect of Nongenetically Modified Phytase Supplementation on Commercial Leghorns D. A. Roland, Sr.,*,1 H. A. Ahmad,† S. S. Yadalam,† and T. Sefton‡ *341 Poultry Science Department, Auburn University, Auburn, Alabama 36849-5416; †107 Food Animal Production Center, Tuskegee University, Tuskegee, Alabama 36088; and Biotechnology Center, 3031 Catnip Hill Pike, Nicholasville, Kentucky 40356

Primary Audience: Poultry Nutritionists, Poultry Extension Personnel, Researchers, Commercial Producers SUMMARY Efficient phosphorus utilization and reduced excretion from poultry has been a matter of great concern due mainly to the detrimental effect on the environment. This research was conducted to analyze the effect of Allzyme2x, a nongenetically modified phytase produced by solid-state fermentation on performance of commercial Leghorns. The experiment was designed using a factorial analysis with two lysine levels of 0.83 and 0.92% and three available phosphorus (aP) levels of 0.4% (control), 0.1% (without phytase), and 0.1% (with phytase). There were eight replicates of 20 hens (40 wk old) for each treatment, and the diets were fed for 8 wk. Results indicated that reducing the lysine level from 0.92 to 0.83% significantly reduced egg weight and feed consumption but had no influence on egg production. Reducing the aP level from 0.4 to 0.1% reduced egg production, egg weight and feed consumption. Supplementing 11,400 phytase units (PTU)/kg Allzyme2x (phytase) to the phosphorus-deficient diet alleviated the drop in egg production and egg weight. These results indicate the nongenetically modified phytase completely reversed the negative effects of aP deficiency in commercial layers. Key words: phytase, phosphorus, layer, performance criteria 2003 J. Appl. Poult. Res. 12:257–263

DESCRIPTION OF PROBLEM Microbial phytase supplementation in animal feeds, particularly in poultry, results in hydrolysis of the plant-phytate molecule [1, 2]. Gordon and Roland [3] observed the benefits of phytase on phosphorus utilization in layers, whereas other studies noted similar effects in swine [4], broilers [5], and turkeys [6]. Published literature indicates that phytase (Natuphos) [7] also improves the utilization of minerals other than phosphorus [3, 8] and amino acids [9, 10]. 1

Commercially available phytase has the ability to release approximately 40% of phytic phosphorus in commercial layer rations [11]. Because of a high turnover of phosphorus in the poultry production cycle from feed to birds to environment, it is imperative that producers be proactive in managing poultry wastes. For example 0.1% reduction in nonphytate phosphorus results in a 25% reduction in phosphorus excretion by layers [12]. Given over 270 million layers in the United States this reduction translates into a substantial

To whom correspondence should be addressed: [email protected].

JAPR: Research Report

258 decline in phosphorus excretion, which could have significant positive effects on the environment. Considerable research [2, 3, 4, 5, 6, 9] has been conducted with Natuphos [7]. Natuphos is a phytase obtained by liquid fermentation using a genetically modified Aspergillus strain. The genetic information originated from Aspergillus ficuum and was transferred to Aspergillus niger, which is commonly used as a highly efficient producer in fermentation processes. After fermentation, the production strain organism is killed by increasing the temperature and lowering the pH and is then removed in several filtration steps. The resulting enzyme solution, called ultrafiltrate, is free of any production organism. More recently, another commercial phytase Allzyme2x [13] (a nongenetically modified enzyme) produced by solid-state fermentation has been introduced to the market. Very little research has been conducted with nongenetically modified phytase and none between commercial egg strains in the US. Differences in utilization of phytate phosphorus have been reported within and between strains [8, 14]. The objective of this study was to determine the influence of Allzyme2x on the reproductive performance of commercial Leghorns during the second phase of their first laying cycle. The experiment was conducted using two different protein (lysine) levels commonly used by the industry to increase the sensitivity of the trial.

MATERIALS AND METHODS A randomized complete block design, with two levels of lysine (0.83 and 0.92%) and two levels of available phosphorus (aP) were used. Both lysine levels and 0.4% aP were equal or greater than NRC [15] recommendations. Each lysine level contained three treatments. The control diets of both lysine levels contained 0.4% aP with no added phytase. The other two treatments within each lysine level supplied 0.1% aP with and without phytase [11,400 phytase units (PTU)/ kg). One phytase unit is the amount of enzyme that will liberate 1 µg of inorganic phosphate/ min under the conditions of the assay [16]. All diets were formulated to contain the same quantity of energy (isocaloric). A detailed description of the diets is presented in Table 1.

Bovans laying hens (n = 960, 40 wk old) were randomly assigned to six dietary treatments for 8 wk. Each treatment contained eight replicates of 20 hens, with four hens housed per cage (40.6 cm × 45.7 cm) in five adjacent cages. Hens in each replicate shared a feed trough and had access to drinking cups. Replicates were equally distributed into upper and lower cage levels to minimize cage level effect. Feed and water were provided ad libitum. A photoperiod of 8D:16L was provided. An average daily temperature of approximately 78°F was maintained (84°F during the day and 70°F during the night). Response criteria were egg production, egg weights, feed consumption, and egg specific gravity. Feed consumption, egg production, and egg weight (2 d of eggs/wk) were recorded on a weekly basis. Egg specific gravity was determined biweekly for all eggs laid on 2 consecutive d. Egg specific gravity was determined using 11 gradient saline solutions varying in specific gravity from 1.060 to 1.100 in 0.005-unit increments [17]. Data was subjected to ANOVA [18] using general linear model procedure, and means were separated by Student-Newman-Keuls test whenever P ≤ 0.05 was detected [19].

RESULTS AND DISCUSSION Egg Production Lysine had no significant effect on egg production. Overall egg production average for hens fed diets containing 0.83% lysine was 81.4 vs. 82.8% for hens fed diets containing 0.92% lysine (Table 2). Reducing dietary aP from 0.4% (positive control) to 0.1% (negative control) significantly reduced egg production (Table 2) as early as wk 3, and the effect continued throughout the experiment. Reducing the dietary phosphorus level to 0.1% significantly reduced the 8-wk average egg production from 89.6 to 69.5%. Within 7 wk, egg production of hens fed the diet containing 0.l% aP was reduced from 93.7 to 48.4% production, clearly indicating the hens were severely phosphorus deficient. When 11,400 PTU/g phytase was added to the phosphorus deficient diet, no decrease in production was observed, and production remained equal to the control diet containing 0.4% aP. No interactions were observed between phytase and lysine levels, except during wk 3.

ROLAND ET AL.: PHYTASE SUPPLEMENTATION

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TABLE 1. Composition of experimental diets Diet (%) 0.92% Lysine IngredientA % Corn Soybean meal (48% CP) CaCO3 (%) Hardshell (%)B Dicalcium phosphate (%) Poultry oil (%) Salt (%) Vitamin premix (%)C Mineral premix (%)D DL-Methionine (%) Phytase Calculated analysisE Protein ME (kcal/lb) Calcium (%) TP (%) aP (%) Na (%) Methionine (%) TSAA (%) Lysine (%) Tryptophan (%)

0.83% Lysine 0.1% aP + phytase

0.4% aP

0.1% aP

61.47 25.14 4.54 4.50 1.63 1.59 0.46 0.25 0.25 0.19

62.83 25.02 5.45 4.50

62.74 25.03 5.45 4.50

1.06 0.45 0.25 0.25 0.18

1.10 0.45 0.25 0.25 0.18 0.05

17.35 1,282 4.00 0.61 0.40 0.20 0.48 0.76 0.92 0.21

17.41 1,282 4.00 0.31 0.10 0.20 0.48 0.76 0.92 0.21

17.41 1,282 4.00 0.31 0.10 0.20 0.48 0.76 0.92 0.21

0.1% aP + phytase

0.4% aP

0.1% aP

65.54 21.81 4.55 4.50 1.64 0.88 0.44 0.25 0.25 0.14

66.92 21.69 5.47 4.50

66.82 21.70 5.47 4.50

0.35 0.44 0.25 0.25 0.14

0.38 0.44 0.25 0.25 0.14 0.05

16.11 1,283 4.00 0.60 0.40 0.20 0.43 0.69 0.83 0.21

16.17 1,283 4.00 0.30 0.10 0.20 0.43 0.69 0.83 0.21

16.16 1,283 4.00 0.30 0.10 0.20 0.43 0.69 0.83 0.21

aP = available phosphorus; TP = total phosphorus. Hardshell = large particle (passing US mesh #4 and retained by US mesh #6) CaCo3 supplied by Franklin Industrial Minerals, Lowell, Florida. C Amount provided per kilogram of diet : vitamin A, 8,000 IU; cholecalceferol, 2,200 ICU; vitamin E, 8 IU; vitamin B 12, 0.02 mg; riboflavin, 5.5 mg; D-calcium pantothenate acid, 13 mg; niacin, 13 mg; choline, 500 mg; folic acid, 0.5 mg; thiamin, 1 mg; pyridoxin, 2.2 mg; biotin, 0.05 mg; menadione sodium bisulfite, 2 mg. D Amount provided per kilogram of diet: manganese, 65 mg; iodine, 1 mg; iron, 55 mg; copper, 6 mg; zinc, 55 mg; selenium, 0.3 mg. E Amino acid analyses of corn and soybean meal were determined by chemical analysis. A B

These results are in agreement with those of Carlos and Edwards [20] who observed a significant increase in egg production when 600 PTU/kg phytase (Natuphos) was added to an aP-deficient diet. Gordon and Roland [3] observed a decline in production with 0.1% low nonphytate phosphorus diet, and supplementing the phosphorus-deficient diet with phytase (Natuphos) completely corrected the decline. Similarly, Peter [21] reported that after 12 wk of consuming a low-nonphytate phosphorus diet (0.12%) hens without access to phytase (Natuphos) had production rates of 70%, whereas those consuming phytase-supplemented diets maintained production rates of 86%. Egg Weight Lysine had a significant positive effect on egg weights starting from wk 1. The egg weight

average for birds fed diets containing 0.83% lysine was 59.8 vs. 60.7 g for hens fed diets containing 0.92% lysine (Table 3). Reducing dietary phosphorus from 0.4 to 0.1% significantly decreased egg weights by the second week. Addition of 11,400 PTU/kg phytase to the phosphorusdeficient diet prevented any decrease in egg weight from occurring. There was no significant difference in egg weight between hens fed the positive control diet (0.4% aP) and the phosphorus-deficient diet (0.1% aP) containing 11,400 PTU/kg phytase. No interaction between phytase and lysine was observed. Other researchers [2, 20] have also reported that egg weights associated with nonphytate phosphorus-deficient diets were improved by the inclusion of genetically modified phytase in the diet.


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TABLE 2. Influence of lysine, phosphorus, and phytase on egg production of commercial Leghorns Average weekly egg production (%) Treatment

A

1

2

3

4

5

6

7

8

Average (1–8)

Lysine 0.83% 0.92% SEM

NS 92.6 92.7 1.21

NS 90.0 90.8 1.39

NS 87.0 88.6 1.54

NS 82.4 85.8 2.08

NS 79.6 80.3 2.34

NS 75.2 75.1 2.68

NS 71.8 75.0 2.58

NS 72.1 74.8 2.60

NS 81.4 82.8 1.38

Phytase 0.1%, aP 0.1%, aP + phytase 0.4% aP SEM

NS 93.7 91.6 92.7 1.92

NS 90.6 89.8 90.7 2.20

** 83.8b 88.8a 91.0a 2.44

*** 73.1b 88.2a 90.9a 3.29

*** 63.3b 87.9a 88.8a 3.70

*** 52.4b 84.7a 88.5a 4.23

*** 48.4b 83.7a 87.7a 4.08

*** 50.9b 83.9a 86.3a 4.12

*** 69.5b 87.3a 89.6a 2.19

Lysine × phytase 0.83 0.1% aP 0.1% aP + phytaseB 0.4% aP 0.92 0.1% aP 0.1% aP + phytaseB 0.4% aP SEM

NS 94.1 91.7 92.0 93.3 91.5 93.2 2.72

NS 90.4 89.8 89.8 90.8 89.9 91.7 3.11

** 80.8b 91.0a 89.6a 86.8b 86.9b 92.5a 3.46

NS 68.7 88.9 89.7 77.5 87.6 92.0 4.65

NS 60.8 89.0 84.0 65.4 86.8 88.7 5.23

NS 50.0 86.3 89.6 55.0 83.0 87.4 5.99

NS 46.0 82.0 87.3 51.0 85.2 88.1 5.77

NS 48.2 82.0 87.9 54.0 85.8 84.9 5.82

NS 67.3 88.0 89.3 71.7 87.0 89.0 3.10

Means in a column with different superscripts are significantly different, P ≤ 0.05. aP = available phosphorus. Phytase enzyme (11,400 phytase units/kg) was added to the negative control diet containing 0.1% aP. **P ≤ 0.01; *** ≤ 0.001.

a,b A B

TABLE 3. Influence of lysine, phosphorus and phytase on egg weights of commercial Leghorns Average weekly egg weights (g) Treatment

A

1

2

3

4

5

6

7

8

Average (1–8)


** 59.4 60.1 0.2

** 59.2 60.3 0.2

** 59.5 60.3 0.2

** 59.7 60.7 0.2

*** 59.5 60.8 0.3

** 60.0 61.1 0.2

** 60.7 61.5 0.2

*** 60.1 61.3 0.2

*** 59.8 60.7 0.1


NS 59.5 59.8 59.9 0.3

** 59.2b 60.1a 60.0a 0.3

** 59.0b 60.2a 60.6a 0.3

*** 59.1b 60.5a 61.9a 2.7

*** 58.2b 61.0a 60.4a 0.3

*** 58.6b 61.4a 61.7a 0.4

*** 59.2b 61.7a 62.4a 0.3

*** 59.0b 61.3a 61.8a 0.2

*** 59.0b 60.8a 61.1a 0.2


NS 59.2 59.4 59.5 59.8 60.2 60.4 0.3

NS 58.5 59.6 59.5 59.8 60.6 60.6 0.4

NS 58.5 59.7 60.4 59.4 60.7 60.7 0.4

NS 58.4 60.1 60.6 59.9 60.9 61.2 0.4

NS 57.4 60.5 60.7 59.1 61.4 62.0 0.4

NS 57.8 60.9 61.2 59.4 62.0 62.2 0.5

NS 59.0 61.0 62.0 59.5 62.3 62.7 0.5

NS 58.6 60.6 61.1 59.3 62.0 62.4 0.3

NS 58.4 60.2 60.6 59.5 61.3 61.5 0.3

Means in a column with different superscripts are significantly different, P ≤ 0.05. aP = available phosphorus. B Phytase enzyme (11,400 phytase units/kg) were added to the negative control diet containing 0.1% aP. **P ≤ 0.01; ***P ≤ 0.001. a,b A


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TABLE 4. Influence of lysine, phosphorus, and phytase on feed consumption of commercial Leghorns Average weekly feed consumption (g/h per d) Treatment

A

1

2

3

4

5

6

7

8

Average

NS 98.7 99.7 0.55

NS 95.0 96.3 0.47

** 96.3 99.3 0.43

NS 95.3 97.2 0.43

** 95.4 98.6 0.37

NS 96.1 98.3 0.52

** 97.0 99.9 0.50

NS 94.1 96.8 0.65

** 95.9 98.3 0.35


NS 96.8 99.7 101.2 0.88

** 92.2c 95.5b 99.1a 0.75

*** 89.4c 98.9b 105.2a 0.68

*** 85.8c 98.4b 104.8a 0.68

*** 83.1c 100.8b 107.1a 0.58

*** 81.7c 102.2b 107.8a 0.82

*** 84.2c 102.5b 108.0a 0.79

*** 80.4c 102.6b 106.0a 1.03

*** 86.8c 99.8b 104.8a 0.56


NS 95.7 98.8 110.2 98.2 100.3 100.3 1.25

NS 90.9 95.3 98.5 93.7 95.8 99.6 1.06

NS 86.0 98.8 104.3 93.0 99.1 106.0 0.97

NS 83.0 99.1 103.8 88.9 97.7 105.2 1.03

* 78.7 100.4 107.2 87.6 101.2 107.1 0.82

NS 79.2 101.8 107.5 84.2 102.7 108.0 1.16

NS 81.2 101.6 107.0 87.1 103.6 108.9 1.12

NS 77.3 99.9 105.2 83.5 100.1 106.8 1.46

NS 94.0 99.4 104.4 89.5 100.1 105.2 0.80


Means within a column with different superscripts are significantly different, P ≤ 0.05. aP = available phosphorus. Phytase enzyme (11,400 phytase units/kg) were added to the negative control diet containing 0.1% aP. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

a–c A B

Feed Consumption Lysine had a significant effect on feed consumption. Hens fed 0.92% lysine consumed 98.3 g of feed vs. 95.9 g for the hens fed the diet containing 0.83% lysine (Table 4). By the second week, and in subsequent weeks, hens fed the phosphorus-deficient diet (0.1% aP) consumed significantly less feed than all other groups. The decline in feed intake preceded a decrease in egg production. Average feed consumption of hens fed the phosphorus-deficient diet was 86.8 vs. 104.8 g for the positive control diet containing 0.4% aP. Addition of 11,400 PTU/kg to the phosphorus-deficient diet prevented (P ≤ 0.05) much of the decline in feed consumption (99.8 g). During wk 5 a significant interaction occurred between lysine and phytase. The beneficial effect of phytase could be clearly observed. At 0.83% lysine, hens fed the phosphorus-deficient diet (0.1% aP) consumed 78.7 vs. 107.2 g for the hens fed the positive control diet with 0.4%. Addition of 11,400 PTU/kg phytase increased feed consumption to 100.4 g. At the higher level of lysine (0.92%), hens fed the phosphorus-deficient diet consumed 87.6 vs. 107.1 g for the hens fed the positive control diet with 0.4% aP. Addition of

11,400 PTU/kg enzyme improved feed consumption to 101.2 g. At both levels of lysine (0.83 and 0.92%) feed consumption was statistically different for all three phosphorus groups. This trend was observed for the rest of the experiment; however, no other significant interactions were observed. Egg Specific Gravity Dietary lysine had no influence on average egg specific gravity. However, during wk 4, significant differences in egg specific gravity were observed between the two lysine levels of 0.83 and 0.92% (Table 5). The effect of lysine on egg specific gravity was probably related to egg weight. Hens fed the 0.83% lysine had a higher egg specific gravity than hens fed the 0.92% lysine (1.084 vs. 1.083). Phytase had no significant influence on egg specific gravity. No interactions among lysine, phosphorus, and phytase were observed. There are two distinct phytases on the market; one is derived from a genetically modified organism, and the other is from a nongenetically modified organism. Both are marketed for their ability to release phytate phosphorus. In this experiment,


262

TABLE 5. Influence of lysine, phosphorus, and phytase on egg specific gravity of commercial Leghorns Weekly egg specific gravity Treatment

A

2

4

8

Average


NS 1.0833 1.0835 0.0003

** 1.0847 1.0832 0.0004

NS 1.0846 1.0846 0.0004

NS 1.0842 1.0838 0.0003


NS 1.0831 1.0846 1.0831 0.0004

NS 1.0849 1.0834 1.0835 0.0006

NS 1.0845 1.0848 1.0845 0.0005

NS 1.0842 1.0841 1.0837 0.0003

NS 1.0828 1.0842 1.0830 1.0833 1.0838 1.0833 0.0005

NS 1.0853 1.0845 1.0843 1.0846 1.0823 1.0828 0.0008

NS 1.0843 1.0850 1.0844 1.0847 1.0847 1.0845 0.0007

NS 1.0841 1.0846 1.0839 1.0842 1.0836 1.0835 0.0004

0.83

0.92

SEM

Lysine × phytase 0.1% 0.1% 0.4% 0.1% 0.1% 0.4%

aP aP + phytase1 aP aP aP + phytase1 aP

aP = available phosphorus. Phytase enzyme (11,400 phytase units/kg) were added to the negative control diet containing 0.1% aP. **P ≤ 0.01.

A B

when the dietary phosphorus level was reduced, significant declines in feed consumption, egg production, and egg weight occurred. When 11,400 PTU/kg of the nongenetically modified organism phytase was added to the phosphorus-deficient diet, no decrease in egg production and egg weights occurred. Feed consumption of hens fed the diet containing phytase was significantly less than that from hens fed the control diet. However, no differences in the egg weight or egg production occurred even though less feed was consumed resulting in a feed conversion (lb of feed/dozen) of 3.1 vs. 3.0 (data not shown). This result suggests that phytase might have improved the availability of nutrients other than phosphorus, per-

haps amino acids and energy [3, 8, 9]. Filler [22] has demonstrated that phytase produced by solidstate fermentation (Allzyme2x) also contains some enzyme activities not found in enzyme preparation from submerged culture systems. The complex nature of feedstuffs may make these side activities beneficial to the animal industry [22]. In vitro comparisons have shown increased rates of reducing sugar and amino nitrogen and an associated increase in phosphate release by the solid-state fermentation enzyme. Results of the current study and another [11] clearly indicate that the nongenetically modified organism phytase (Allzyme2x) is effective in improving phytate phosphorus availability.

CONCLUSIONS AND APPLICATIONS 1. Reducing the lysine level from 0.92 to 0.83% significantly reduced egg weight and feed consumption but not egg production. 2. Reducing aP from 0.4 to 0.1% reduced egg production, egg weight, and feed consumption. 3. Supplementation of 11,400 PTU/kg of nongenetically modified phytase to the phosphorusdeficient diet of 0.1% aP alleviated the adverse effects of phosphorus deficiency on egg production and egg weight. The improvements in egg production and egg weight were further enhanced because phytase supplementation did so at reduced feed consumption level compared to the 0.4% aP diet.


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