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Effects of Enzyme Combinations on Apparent Metabolizable Energy of Corn-Soybean Meal-Based Diets in Broilers
2003 Poultry Science Association, Inc.
Effects of Enzyme Combinations on Apparent Metabolizable Energy of Corn–Soybean Meal-Based Diets in Broilers A. Kocher,*,1 M. Choct,* G. Ross,† J. Broz,‡ and T. K. Chung†
Primary Audience: Nutritionists, Researchers, Feed Manufacturers SUMMARY Corn and soybean meal (SBM) are high-quality feed ingredients for broiler diets. Antinutritive factors in SBM, such as protease inhibitors and lectins, are successfully inactivated by heat treatment. However, the nutritive value of these ingredients also depends on the amount of indigestible carbohydrates, in particular the amount of oligosaccharides and nonstarch polysaccharides (NSP). Despite the fact that such diets are low in indigestible carbohydrates, it has been suggested that the inclusion of exogenous feed enzymes to such diets could improve nutrient availability and, subsequently, improve energy digestibility. Four consecutive AME bioassays were conducted to investigate the effects of three singleactivity enzyme products, two multi-activity enzyme preparations, and a commercially available enzyme product added to a corn–SBM-based broiler diet. None of the evaluated enzyme combinations successfully improved the performance of 3- to 4wk-old broiler chickens. However, in experiments 1 and 2, when enzymes were included in a lowerenergy corn–SBM basal diet, the combination of pectinase, protease, and amylase significantly improved AMEn in comparison to the unsupplemented basal diet. Subsequent experiments with a higher energy and protein basal diet failed to show the same improvement when enzymes were added. Results of this study indicated that although enzyme addition to corn–SBM-based basal diets can significantly improve AMEn, the success of such improvement depended greatly on the raw ingredients available at the time. Key words: apparent metabolizable energy, broiler, corn, enzyme, soybean meal 2003 J. Appl. Poult. Res. 12:275–283
DESCRIPTION OF PROBLEM High levels of soluble nonstarch polysaccharides (NSP) in wheat and barley exhibit antinutritive effects. Endogenous enzymes of broilers cannot adequately digest these carbo1
hydrates, and subsequently the ingestion of high levels of soluble NSP leads to increased digesta viscosity and reduced nutrient digestibility and absorption [1]. In many parts of the world exogenous glycanases are added to broiler diets to improve performance and re-
To whom correspondence should be addressed: [email protected].
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*School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia; †Roche Vitamins Asia Pacific Pte. Ltd., Singapore 079120; and ‡Roche Vitamins Ltd., Animal Nutrition and Health R&D, Basel 4070, Switzerland
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It seems that the response to enzyme addition in corn–SBM diets varies greatly between studies and may depend heavily on the enzyme product and its activities. Therefore, the objective of the current study was to evaluate the effects of different enzymes with known activities and their combination on AME of a corn– SBM-based diet and the performance of 3-wkold broiler chickens.
MATERIALS AND METHODS Bird Husbandry Four consecutive AME bioassays were conducted to investigate the effects of three single-activity enzyme products, two multi-activity enzyme preparations, and a commercially available enzyme product added to a corn–SBM-based broiler diet. Enzyme activities are listed in Table 1 and were determined by the supplier according to the Nelson-Somogyi method for the determination of reducing sugar content [12]. All four assays were conducted over a 6-mo period (December 2000 through May 2001) at the University of New England, Armidale, Australia. Single-sex (male) Ross broiler chickens were raised from hatch to 24 d of age in brooders on enzyme-free commercial starter crumbles. At d 24, chickens were weighed in groups of four and transferred to metabolism cages located in three rooms kept at 22 to 25°C. A 3-d period enabled the chickens to adapt to the new environment. During the following 4 d, feed intake was measured and all excreta collected and dried. Moisture content of excreta collected was measured. At the end of the 7d period, birds were weighed in groups (n = 4). The excreta were dried in a fan-forced oven at 80°C for 36 h. The DM content of the diets was determined by drying the diets at 105°C for 16 h. Gross energy contents of diets and excreta were determined via an isoperibol bomb calorimeter. The nitrogen content of diets and excreta samples was determined with a LECO FP-2000 automatic analyzer. The protein contents were calculated using a multiplication factor of 6.25. Experimental Design and Diet Composition The experimental basal diet was formulated using a commercially available feed formula-
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duce digesta viscosity by depolymerizing the soluble NSP of cereal grains. In regions such as the United States or Asia, the inclusion of these feed enzymes is less common simply because the basis of a common broiler diet are low-NSP cereals, for example, corn or sorghum in combination with soybean meal (SBM). The nutrients contained in corn and SBM are generally considered to be highly digestible, and heat treatment is commonly used to inactivate antinutritive factors (ANF) such as protease inhibitors and lectins in SBM [2]. However, the energy utilization in corn and SBM also depends on the amount of indigestible carbohydrates present in particular oligosaccharides and NSP. Corn contains approximately 0.9% soluble NSP and 6% insoluble NSP, whereas SBM contains approximately 6% soluble NSP and 18 to 21% insoluble NSP [3]. The nutritive value of SBM oligosaccharides (OS) in broiler diets remains unclear. Two studies by Leske et al. [4, 5] showed that the removal of OS via ethanol extraction resulted in increased true metabolizable energy (TMEN) in SBM. In contrast, the depolimerization of OS by the addition of α-galactosidase to cornSBM diets failed to show any beneficial effects on the nutritive value of SBM [6]. In order to improve the nutritive value of corn–SBM diets, several studies have investigated the effect of carbohydrase supplementation to corn and SBM diets for broilers. Marsman et al. [7] and Zanella et al. [8] showed that commercial carbohydrase products improved weight gain and feed conversion ratio (FCR) in broilers fed a corn–soy diet as a result of increased ileal digestibility of protein and NSP. Kocher et al. [9] found that the inclusion of an experimental galactanase product improved AMEn of the diet but not the growth or the FCR of the broilers. Pack et al. [10] reported that the addition of a commercial enzyme preparation to broiler and layer diets improved bird performance as a result of the action of this enzyme preparation on the arabinoxylan present in corn and SBM. However, an extensive evaluation of three different enzyme products designed to hydrolyze NSP in vegetable proteins, such as SBM, failed to show any effects of such enzyme products on AME, FCR, or digestibility of NSP [11].
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TABLE 1. Activities of enzyme products Enzyme
Commercial name
Activity
Single-activity enzymes A B C
Ronozyme Pro (CT) Ronozyme WX (CT) Celluclast
Endo-protease (1,500,000 HUT/g) Xylanase (1,000 FXU/g) Cellulase (concentrated β-glucanse)
Multiple-activity enzymes D EA
Ronozyme A (CT) Ronozyme VP (CT)
α-Amylase (75 KNU/g), β-glucanase (250 BGU/g) β-Glucanase (54.7 FBG/g), hemicellulase (15,000 VHCH/g), pectinase (3,017 U/g)
Commercial enzyme preparation F
Avizyme 1500
Xylanase (800 U/g), protease (6,000 U/g), amylase (2,000 U/g)
A
Two separate batches of Enzyme E were examined. Enzyme activities were determined by the supplier according to the Nelson-Somogyi method for determination of reducing sugar content [12].
B
TABLE 2. Composition of experimental basal diets (g/ kg) Ingredient
(g/kg)
Corn Wheat millrun (²⁄₃ bran, ¹⁄₃ pollard) SBM (48%) Canola meal Meat and bone meal Dicalcium phosphate Limestone Salt Methionine Lysine Choline chloride Premix1
600 70 250 30 15 17 9 2 3 1 1 2
Calculated composition CP ME MJ/kg Methionine + cystine Lysine Calcium Nonphytate phosphorus
prior to pelleting, as outlined in Table 3. All diets were cold-pelleted (60°C) using a Templewood provender press. Experimental Design and Statistical Analysis The experimental design used was a randomized block design with six (experiments 1 and 2) or eight replicates (experiments 3 and 4) per diet. SAS [14] was used to perform the statistical analyses for all four studies, and data were analyzed according to the GLM procedure. A randomized design was used with the experimental unit consisting of the mean value of a cage. Data were analyzed by GLM to determine the significance of the main effect (enzyme inclusion). Duncan’s multiple-range test was used to separate means when significant effects (P < 0.05) were detected by multifactorial analysis of variance [15]. Animal Ethics
(g/kg of DM) 204.0 12.30 9.3 10.0 9.8 5.0
The Animal Ethics Committees of the University of New England approved this study. Health and animal husbandry practices complied with the “Code of Practice for the Use of Animals for Scientific Purposes” issued by the Australian Bureau of Animal Health [16].
1
Provided per kilogram of diet: vitamin A (as all-trans retinol), 12,000 IU; cholecalciferol, 3,500 IU; vitamin E (as D-α-tocopherol), 44.7 IU; vitamin K3, 2 mg; thiamine, 2 mg; riboflavin, 6 mg; pyridoxine hydrochloride, 5 mg; vitamin B12, 0.2 mg; biotin, 0.1 mg; niacin, 50 mg; D-calcium pantothenate, 12 mg; folic acid, 2 mg; monesin, 500 mg; Mn, 80 mg; Fe, 60 mg; Cu, 8 mg; I, 1 mg; Co, 0.3 mg; Se, 0.1 mg; and Mo, 1 mg.
RESULTS AND DISCUSSION The aim of the four experiments was to examine whether a combination of currently available commercial enzyme products in diets based on corn and SBM would have any effects
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tion program [13; Table 2]. First, the minor ingredients were mixed in a small rotary mixer and then were pooled with the major ingredients for thorough mixing. All experimental diets were identical in composition. All enzymes were added directly to other ingredients
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Inclusion rate (ppm) Diet/enzyme
Enzyme A
Enzyme B
Enzyme C
— — — — 50 50 50 100
— — — 50 50 50 50 100
— — — — — — 5 —
— — — 50 100 — — 50 —
— — — 50 100 — — — 50
— — — — — — — — —
— — — — 50 50 50 50 100 100
— — — — — — — — — 100
— — — — — — — — — —
Experiment 1 Corn control (low ME corn) Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme
control combination combination combination combination combination combination combination
1-1 1-2 1-3 1-4 1-5 1-6 1-7
2-1 2-2 2-4 2-7 2-8 2-9 2-10 2-11
control combination combination combination combination combination combination combination combination combination
3-2 3-8 3-9 3-10 3-12 3-13 3-15 3-16 3-17
control combination combination combination combination combination combination combination combination
4-5 4-5B 4-10 4-10B 4-13 4-13B 4-14 4-14B
— 300A 500A 300A 300A 300A 300A 200A
1,000 — — — — — — —
— — — — 100 — 50 50 50
— 300A 500A 300A 200A 400A 300A 300A 300A
1,000 — — — — — — — —
— — — 50 50 — 50 50 100 100
— 500B 400B 300B 300B 300B 250B 200B — —
1,000 — — — — — — — — —
— 50 50 50 50 50 50 50 50
— 300A 300B 300A 300B 250A 250B 250A 250B
1,000 — — — — — — — —
No enzyme
Experiment 4 Corn control (high ME corn) Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme
— — — — — 50 50 100 No enzyme
Experiment 3 Corn control (high ME corn) Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme
Enzyme F
No enzyme — 50 50 50 50 50 50 50 50
— 50 50 — — — — 50 50
— — — — — — — — —
A
Enzyme E batch 1. Enzyme E batch 2.
B
on excreta moisture, broiler performance, or AMEn. The enzyme products tested in the current study contained activities to depolymerize cell walls in corn and SBM and assist endogenous enzymes to digest starch and protein. En-
zymes used in diets containing high levels of SBM are mostly multiglycanase products containing polygalacturonase (pectinase) to hydrolyze pectins present in vegetable products, as well as a range of other enzymes such as
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control combination combination combination combination combination combination combination combination
Enzyme E
No enzyme
Experiment 2 Corn control (low ME corn) Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme
Enzyme D
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TABLE 4. Feed conversion ratio (FCR) of broiler chickens fed diets containing corn–soybean meal (SBM) with or without enzymes FCR Corn
Mean
Low ME Corn
Experiment 1
1 2 3 4 5 6 7 8 9 10 11
High ME corn Corn control Enzyme control Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination
SD
2 5 5A 8A 9A 10 10A 12 13 13A 14 14A 15 16 17
1.842 1.921 1.845 1.844 1.857 2.029 1.909 1.852 1.853
± ± ± ± ± ± ± ± ±
B
0.06 0.10 0.13 0.10 0.10 0.06 0.12 0.07 0.08
Mean
SD
Experiment 2B 1.837 1.789 1.767 1.797
± ± ± ±
0.09 0.12 0.06 0.06
1.797 ± 0.12
1.812 1.720 1.842 1.909 1.793
± ± ± ± ±
0.08 0.09 0.01 0.03 0.07
Experiment 3C
Experiment 4C
1.930 ± 0.05 1.950 ± 0.11 1.968 ± 0.13
1.961 ± 0.12 19.50 ± 0.10
2.049 ± 0.17 2.025 ± 0.12 2.053 ± 0.12 2.027 ± 0.12 2.002 ± 0.10 1.965 ± 0.10 2.046 ± 0.25 2.010 ± 0.14
1.973 ± 0.06 1.910 ± 0.10 2.009 ± 0.16 2.038 ± 0.11 2.004 2.008 1.947 1.935
± ± ± ±
0.12 0.12 0.10 0.13
A
Enzyme E batch 2. Mean value of six replicates. C Mean value of eight replicates. B
cellulase and hemicellulases targeting neutral NSP. Reports in the literature indicate that the enzyme products selected for these experiments should have some effects on broiler performance or AME. Addition of Enzyme E to a corn–SBM broiler diet resulted in a significant improvement in weight gain and FCR as a result of increased ileal digestibility of CP, starch, and fat [7]. Similarly, Zanella et al. [8] reported a significant improvement in ileal digestibility of CP and NSP in corn–soy broiler diets. It was concluded from both studies that the added enzyme products not only had cell
wall degrading activities but also exhibited protease activity, which explained the improved nutrient digestibility. Our results do not confirm these findings. Enzyme E alone or in combination with protease, xylanase, or amylase had no effect on bird performance (Table 4). However, these performance data were gathered over a 1-wk period only in the current study. In order to accurately predict changes in performance as a result of different enzyme combinations, FCR needs to be measured over a longer period. In the current experimental setup, these data are used as an overall indication about the trial, for example, bird welfare,
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Control corn Enzyme control Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination
FCR
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TABLE 5. AMEn of broiler diets containing corn–soybean (SBM) with or without enzymes (low ME corn) Experiment 1 (AMEn MJ/kg DMA) Diet
1 2 3 4 5 6 7 8 9 10 11
STD
12.72 12.79 12.76 13.04 12.90 12.96 13.07 13.03 13.12
Mean
± ± ± ± ± ± ± ± ±
0.18 0.21cd 0.18cd 0.21ab 0.08bcd 0.15abc 0.17ab 0.12ab 0.12a
STD
12.71 12.74 12.95 12.99
± ± ± ±
0.17e 0.09de 0.09bcd 0.22bc
13.19 ± 0.17b
12.90 13.01 13.00 13.42 13.09
12.93
Source of variance Diet
Mean
d
± ± ± ± ±
0.22cde 0.16bc 0.24bc 0.15a 0.17bc
12.99 Probability of greater F value
***
***
Values within a column with unlike superscripts differ significantly (P < 0.05). Mean value of six replicates. ***P < 0.001. a–c A
rather than a precise reflection on the performance. Such a setup only targets large differences between experimental diets and is not designed to find subtle differences. A more accurate tool to determine the effects of different enzyme combinations on the nutritive value of a corn–SBM basal diet in a brief experiment is the classical AME assay. In experiments 1 and 2, Enzyme E included at a higher dosage or Enzyme E in combinations with Enzymes D, A, or B significantly improved AMEn of the diet in comparison with the unsupplemented basal diet (Table 5). The mechanisms by which these enzyme combinations have improved AMEn remain speculative. Kocher et al. [9] reported that it is most likely that Enzyme E solubilized pectic NSP such as rhamnogalacturonan, arabinogalactan, and galactan found in SBM and released nutrients entrapped in the cell walls, which could be successfully utilized by the bird. However, these effects were only significant when Enzyme E was added at a higher dosage. Minor activities of other enzymes present in Enzyme E might have further improved nutrient availability. There is evidence in the literature that the addition of exogenous protease and amy-
lase can improve the utilization of protein in SBM and assist the digestion of starch encapsulated in the endosperm in the upper gastrointestinal tract [8, 10]. The fact that Enzyme E at a lower dosage in combination with protease (Enzyme A) or amylase (Enzyme D) had a similar effect in improving AMEn supports this hypothesis. Although it is evident that soluble NSP in SBM or corn do not exhibit strong antinutritive activities and can be utilized to some extent throughout the intestine by microbial digestion [9], results from experiments 1 and 2 clearly show that AMEn of a corn–SBM basal diet can be improved by adding pectinase (Enzyme E) in combination with amylase (Enzyme D) and protease (Enzyme A). The benefits of enzyme addition lies in the increased access to intracellular entrapped nutrients as well as in an improved energy utilization in comparison with the microbial fermentation. In order to verify the findings in the first two experiments and to find a commercially beneficial enzyme combination, two further experiments were conducted (experiments 3 and 4; Table 6). Surprisingly, the results from the previous studies could not be repeated despite the fact that the same experimental conditions
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Corn control Enzyme control Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination
Mean
Experiment 2 (AMEn MJ/kg DMA)
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TABLE 6. AMEn of broiler diets containing corn–soybean meal (SBM) with or without enzymes (high ME corn) Experiment 3 (AMEn MJ/kg DMB) Mean
2 5 5A 8A 9A 10 10A 12 13 13A 14 14A 15 16 17
SD
Mean
12.89 ± 0.23 12.69 ± 0.13 12.79 ± 0.16
13.04 ± 0.14a 12.65 ± 0.14de 12.67 ± 0.05de 12.77 ± 0.10cd
12.70 ± 0.30 12.70 ± 0.11
13.01 ± 0.05ab 12.88 ± 0.15bc
12.84 ± 0.20 12.72 ± 0.14
12.94 12.49 12.95 12.62
12.81 ± 0.16 12.61 ± 0.14 12.64 ± 0.11 12.77 ± 0.22
Mean
SD
12.73
± ± ± ±
0.10ab 0.14f 0.11ab 0.16e
12.80
Source of variance
Probability of greater F value
Diet
NS
***
Values within a column with unlike superscripts differ significantly (P < 0.05). Enzyme E batch 2. B Mean value of eight replicates. *** P < 0.001. a–f A
and protocols were used. Results in Table 7 show that there are indeed significant differences between the batch of Enzyme E used in experiments 1 and 2 and that used in Experiment 3. However, the addition of either batch of Enzyme E in Experiment 4 failed to show improvement in AMEn, as previously seen in Experiments 1 and 2.
All major ingredients, including corn and SBM, used in the four experiments were purchased from a local supplier approximately 3 wk prior to the start of the experiment. Because the four experiments were conducted over a 9mo period, it has to be assumed that corn and SBM source varied between experiments. It is well-known that the quality and amount of CP
TABLE 7. Comparison of two different batches of Enzyme E (experiment 4), mean values of eight replicates AMEn (MJ/kg DM)
FCRA Batch 1 2 Mean
Mean
SD
1.983 ± 0.11 1.973 ± 0.12
12.79 Probability of greater F value
NS
Values within a column with unlike superscripts differ significantly (P < 0.05). Feed conversion ratio. ***P < 0.001. a,b A
SD
12.90 ± 0.15a 12.69 ± 0.20b
1.978
Source of variance Batch
Mean
***
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Corn control Enzyme control Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination
Experiment 4 (AMEn MJ/kg DMB)
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TABLE 8. Comparison of control diets between four consecutive AME bioassays Experiment 1 2 3 4
(low ME corn) (low ME corn) (high ME corn) (high ME corn)
Gross energyA (MJ/kg DM)
CPA (%)
FCRB (g feed/g gain)
AMEnB (MJ/kg DM)
AMEn:CPA (kJ/g)
18.15 18.28 18.60 18.88
21.0 21.9 23.7 23.4
1.842b 1.837b 1.930ab 1.961a
12.72b 12.71b 12.89ab 13.04a
60.6 58.0 54.4 55.7
Source of variance
Probability of greater F value
Experiment
*
*
Values within a column with unlike superscripts differ significantly (P < 0.05). Measured as single value per diet. B Mean value of six or eight replicates. *P < 0.05. a,b A
speculated that the release of entrapped cell wall proteins resulted in an increase in the total protein turnover. The energy loss associated with the increased protein turnover resulted in lower AMEn [22]. On the other hand, Irish and Balnave [18] found that the performance of broilers is highly correlated with the concentration of oligosaccharides in the digesta. Although corn is generally regarded as a low-NSP cereal, the actual levels of NSP can vary between 5 and 8% [3, 23]. It has been shown in vitro that the highly branched glucurono-arabinoxylans in corn can be degraded by exogenous enzymes releasing a series of oligomers [23]. Hence, it is possible that the enzymes depolymerized part of the NSP of corn, as well as the rhamnogalacturonan and arabinogalactan of SBM, resulting in an increase of oligosaccharides in the digesta. These lowmolecular-weight carbohydrates are not only osmotically active but also highly fermentative. There are two possible effects of their action on the ME value. Firstly, loss of water in the lumen through osmotic up-regulation is an energy-expensive process, and secondly, proliferation of microorganisms in the gut leads to increased organic mass of the excreta, lowering AME value in a balance trial.
CONCLUSIONS AND APPLICATIONS 1. Addition of any enzyme combination to a corn–SBM basal diet had no effects on performance measured over a short period. 2. Combined addition of pectinase, protease, and amylase significantly improved AMEn when added to a corn–SBM basal diet low in energy and protein. 3. Addition of the same enzyme combination to a corn–SBM basal diet with increased energy and protein content results in a significant reduction of AMEn.
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and NSP in SBM can vary widely as a result of processing methods and conditions [16, 17]. Similarly, it has been reported that oil and fiber contents in corn can significantly differ between commercial hybrids [19]. This was the case in the current study, as the basal diets diet used in experiments 3 and 4 had a higher energy and protein contents than in those used in experiments 1 and 2. Furthermore, the energy:protein ratio (E:P) in experiments 1 and 2 was larger in comparison to experiments 3 and 4 (Table 8). Douglas et al. [17] found that the addition of enzyme to corn–SBM diets is most effective in corn–SBM diets containing a low digestible energy level. Wiseman [20] reported that in diets with a low E:P ratio, excess protein will be catabolized rather than deposited as meat. Thus, the lack of response to enzyme addition in experiments 3 and 4 may be due to the increased AMEn of the basal diet. In experiment 4, the enzymes resulted in a significant reduction of AMEn of the diet. A previous study by Kocher et al. [21] found a similar reduction in AMEn when a commercial multi-activity enzyme product was added to a corn–canola meal-based diet. These authors
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REFERENCES AND NOTES 1. Annison, G. 1991. Relationship between the levels of soluble nonstarch polysaccharides and the apparent metabolizable energy of wheat assayed in broiler chickens. J. Agric. Food Chem. 39:1252– 1256. 2. Campbell, G. L., and A. F. B. van der Poel. 1998. Use of enzymes and process technology to inactivate antinutritional factors in legume seeds and rapeseed. Pages 377–386 in Proc. Recent Advances of Research in Antinutritional Factors in Legume Seeds and Rapeseed.Wageningen Press, Wageningen, The Netherlands. 3. Bach Knudsen, K. E. 1997. Carbohydrate and lignin contents of plant materials used in animal feeding. Anim. Feed Sci. Technol. 67:319–338.
5. Leske, K. L., C. J. Jevne, and C. N. Coon. 1993. Effect of oligosaccharide additions on nitrogen-corrected true metabolizable energy of soy protein concentrate. Poult. Sci. 72:664–668. 6. Irish, G. G., G. W. Barbour, H. L. Classen, R. T. Tyler, and M. R. Bedford. 1995. Removal of the alpha-galactosides of sucrose from soybean meal using either ethanol extraction or exogenous alpha-galactosidase and broiler performance. Poult. Sci. 74:1484– 1494. 7. Marsman, G. J., H. Gruppen, A. F. van der Poel, R. R. Kwakkel, M. W. Verstegen, and A. G. Voragen. 1997. The effect of thermal processing and enzyme treatments of soybean meal on growth performance, ileal nutrient digetibilities, and chyme characteristics in broiler chicks. Poult. Sci. 76:864–872. 8. Zanella, I., N. K. Sakomura, F. G. Silversides, A. Fiqueirdo, and M. Pack. 1999. Effect of enzyme supplementation of broiler diets based on corn and soybeans. Poult. Sci. 78:561–568. 9. Kocher, A., M. Choct, M. D. Porter, and J. J. Broz. 2002. Effects of feed enzymes on nutritive value of soybean meal fed to broilers. Br. Poult. Sci. 43:54–63. 10. Pack, M., D. Creswell, and H. Graham. 1996. Applying enzymes to sorghum and maize based broiler diets. Pages 252–258 in Proc. of 10th Australian Poultry and Feed Convention, Melbourne, Australia. 11. Kocher, A. 2000. Enzymatic degradation of non-starch polysaccharides in vegetable proteins in poulty diets. PhD Thesis. University of New England, Armidale. 12. Somogyi, M. 1960. Modification of two methods for the assay of amylase. Clin. Chem. 6:23–35.
14. SAS Institute Inc. 2001. The SAS System for Windows, Version 8.02 SAS Institute Inc., Cary, NC. 15. Duncan, D. B. 1955. Multiple range test and multiple F-tests. Biometrics 11:1–42. 16. National Health and Medical Research Council. 1990. Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. National Health and Medical Research Council, Commonwealth Scientific and Industrial, Research Organisation, Australian Agricultural Council. Australian Govt. Pub. Service, Canberra, Australia. 17. Douglas, M. W., C. M. Parsons, and M. R. Bedford. 2000. Effect of various soybean meal sources and Avizyme on chick growth performance and ileal digestible energy. J. Appl. Poult. Res. 9:74–80. 18. Irish, G. G., and D. Balnave. 1993. Non-starch polysaccharides and broiler performance on diets containing soyabean meal as the sole protein concentrate. Aust. J. Agric. Res. 44:1483–1499. 19. Singh, V., R. A. Moreau, A. E. Haken, S. R. Eckhoff, K. B. Hicks, and V. Singh. 2000. Hybrid variability and effect of growth location on corn fiber yields and corn fiber oil composition. Cereal Chem. 77:692–695. 20. Wiseman, J. 2001. High energy diets for poultry: Effects of diet composition on performance and carcass quality. Pages 87–106 in Recent Advances in Animal Nutrition. P. C. Gransworthy and J. Wiseman, ed. Nottingham University Press, Nottingham, U.K. 21. Kocher, A., M. Choct, M. D. Porter, and J. Broz. 2000. The effects of enzyme addition to broiler diets containing high levels of canola or sunflower meal. Poult. Sci. 79:1767–1774. 22. Nieto, R., C. Prieto, I. Fernandez-Figares, and J. F. Aguilera. 1995. Effect of dietary protein quality on energy metabolism in growing chickens. Br. J. Nut. 74:163–173. 23. Huisman, M. M. H., H. A. Schols, and A. G. J. Voragen. 2000. Glucuronoarabinoxylans from maize kernel cell walls are more complex than those from sorghum kernel cell walls. Carbohydrate Polymers 43:269–279.
Acknowledgments Roche Vitamins Ltd, Basel, Switzerland, supported this study. The authors are grateful to Geoff Ross, Roche Ltd, for his efforts in arranging the supply of the enzyme products. We gratefully acknowledge the efforts of the staff of the Division of Animal Science, University of New England.
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4. Leske, K. L., O. Akavanichan, T. K. Cheng, and C. N. Coon. 1991. Effect of ethanol extract on nitrogen-corrected true metabolizable energy for soybean meal with broilers and roosters. Poult. Sci. 70:892–895.
13. Feedmania, Saltbush Enterprise, Armidale, Australia.
Effects of Enzyme Combinations on Apparent Metabolizable Energy of Corn–Soybean Meal-Based Diets in Broilers A. Kocher,*,1 M. Choct,* G. Ross,† J. Broz,‡ and T. K. Chung†
Primary Audience: Nutritionists, Researchers, Feed Manufacturers SUMMARY Corn and soybean meal (SBM) are high-quality feed ingredients for broiler diets. Antinutritive factors in SBM, such as protease inhibitors and lectins, are successfully inactivated by heat treatment. However, the nutritive value of these ingredients also depends on the amount of indigestible carbohydrates, in particular the amount of oligosaccharides and nonstarch polysaccharides (NSP). Despite the fact that such diets are low in indigestible carbohydrates, it has been suggested that the inclusion of exogenous feed enzymes to such diets could improve nutrient availability and, subsequently, improve energy digestibility. Four consecutive AME bioassays were conducted to investigate the effects of three singleactivity enzyme products, two multi-activity enzyme preparations, and a commercially available enzyme product added to a corn–SBM-based broiler diet. None of the evaluated enzyme combinations successfully improved the performance of 3- to 4wk-old broiler chickens. However, in experiments 1 and 2, when enzymes were included in a lowerenergy corn–SBM basal diet, the combination of pectinase, protease, and amylase significantly improved AMEn in comparison to the unsupplemented basal diet. Subsequent experiments with a higher energy and protein basal diet failed to show the same improvement when enzymes were added. Results of this study indicated that although enzyme addition to corn–SBM-based basal diets can significantly improve AMEn, the success of such improvement depended greatly on the raw ingredients available at the time. Key words: apparent metabolizable energy, broiler, corn, enzyme, soybean meal 2003 J. Appl. Poult. Res. 12:275–283
DESCRIPTION OF PROBLEM High levels of soluble nonstarch polysaccharides (NSP) in wheat and barley exhibit antinutritive effects. Endogenous enzymes of broilers cannot adequately digest these carbo1
hydrates, and subsequently the ingestion of high levels of soluble NSP leads to increased digesta viscosity and reduced nutrient digestibility and absorption [1]. In many parts of the world exogenous glycanases are added to broiler diets to improve performance and re-
To whom correspondence should be addressed: [email protected].
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*School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia; †Roche Vitamins Asia Pacific Pte. Ltd., Singapore 079120; and ‡Roche Vitamins Ltd., Animal Nutrition and Health R&D, Basel 4070, Switzerland
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It seems that the response to enzyme addition in corn–SBM diets varies greatly between studies and may depend heavily on the enzyme product and its activities. Therefore, the objective of the current study was to evaluate the effects of different enzymes with known activities and their combination on AME of a corn– SBM-based diet and the performance of 3-wkold broiler chickens.
MATERIALS AND METHODS Bird Husbandry Four consecutive AME bioassays were conducted to investigate the effects of three single-activity enzyme products, two multi-activity enzyme preparations, and a commercially available enzyme product added to a corn–SBM-based broiler diet. Enzyme activities are listed in Table 1 and were determined by the supplier according to the Nelson-Somogyi method for the determination of reducing sugar content [12]. All four assays were conducted over a 6-mo period (December 2000 through May 2001) at the University of New England, Armidale, Australia. Single-sex (male) Ross broiler chickens were raised from hatch to 24 d of age in brooders on enzyme-free commercial starter crumbles. At d 24, chickens were weighed in groups of four and transferred to metabolism cages located in three rooms kept at 22 to 25°C. A 3-d period enabled the chickens to adapt to the new environment. During the following 4 d, feed intake was measured and all excreta collected and dried. Moisture content of excreta collected was measured. At the end of the 7d period, birds were weighed in groups (n = 4). The excreta were dried in a fan-forced oven at 80°C for 36 h. The DM content of the diets was determined by drying the diets at 105°C for 16 h. Gross energy contents of diets and excreta were determined via an isoperibol bomb calorimeter. The nitrogen content of diets and excreta samples was determined with a LECO FP-2000 automatic analyzer. The protein contents were calculated using a multiplication factor of 6.25. Experimental Design and Diet Composition The experimental basal diet was formulated using a commercially available feed formula-
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duce digesta viscosity by depolymerizing the soluble NSP of cereal grains. In regions such as the United States or Asia, the inclusion of these feed enzymes is less common simply because the basis of a common broiler diet are low-NSP cereals, for example, corn or sorghum in combination with soybean meal (SBM). The nutrients contained in corn and SBM are generally considered to be highly digestible, and heat treatment is commonly used to inactivate antinutritive factors (ANF) such as protease inhibitors and lectins in SBM [2]. However, the energy utilization in corn and SBM also depends on the amount of indigestible carbohydrates present in particular oligosaccharides and NSP. Corn contains approximately 0.9% soluble NSP and 6% insoluble NSP, whereas SBM contains approximately 6% soluble NSP and 18 to 21% insoluble NSP [3]. The nutritive value of SBM oligosaccharides (OS) in broiler diets remains unclear. Two studies by Leske et al. [4, 5] showed that the removal of OS via ethanol extraction resulted in increased true metabolizable energy (TMEN) in SBM. In contrast, the depolimerization of OS by the addition of α-galactosidase to cornSBM diets failed to show any beneficial effects on the nutritive value of SBM [6]. In order to improve the nutritive value of corn–SBM diets, several studies have investigated the effect of carbohydrase supplementation to corn and SBM diets for broilers. Marsman et al. [7] and Zanella et al. [8] showed that commercial carbohydrase products improved weight gain and feed conversion ratio (FCR) in broilers fed a corn–soy diet as a result of increased ileal digestibility of protein and NSP. Kocher et al. [9] found that the inclusion of an experimental galactanase product improved AMEn of the diet but not the growth or the FCR of the broilers. Pack et al. [10] reported that the addition of a commercial enzyme preparation to broiler and layer diets improved bird performance as a result of the action of this enzyme preparation on the arabinoxylan present in corn and SBM. However, an extensive evaluation of three different enzyme products designed to hydrolyze NSP in vegetable proteins, such as SBM, failed to show any effects of such enzyme products on AME, FCR, or digestibility of NSP [11].
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TABLE 1. Activities of enzyme products Enzyme
Commercial name
Activity
Single-activity enzymes A B C
Ronozyme Pro (CT) Ronozyme WX (CT) Celluclast
Endo-protease (1,500,000 HUT/g) Xylanase (1,000 FXU/g) Cellulase (concentrated β-glucanse)
Multiple-activity enzymes D EA
Ronozyme A (CT) Ronozyme VP (CT)
α-Amylase (75 KNU/g), β-glucanase (250 BGU/g) β-Glucanase (54.7 FBG/g), hemicellulase (15,000 VHCH/g), pectinase (3,017 U/g)
Commercial enzyme preparation F
Avizyme 1500
Xylanase (800 U/g), protease (6,000 U/g), amylase (2,000 U/g)
A
Two separate batches of Enzyme E were examined. Enzyme activities were determined by the supplier according to the Nelson-Somogyi method for determination of reducing sugar content [12].
B
TABLE 2. Composition of experimental basal diets (g/ kg) Ingredient
(g/kg)
Corn Wheat millrun (²⁄₃ bran, ¹⁄₃ pollard) SBM (48%) Canola meal Meat and bone meal Dicalcium phosphate Limestone Salt Methionine Lysine Choline chloride Premix1
600 70 250 30 15 17 9 2 3 1 1 2
Calculated composition CP ME MJ/kg Methionine + cystine Lysine Calcium Nonphytate phosphorus
prior to pelleting, as outlined in Table 3. All diets were cold-pelleted (60°C) using a Templewood provender press. Experimental Design and Statistical Analysis The experimental design used was a randomized block design with six (experiments 1 and 2) or eight replicates (experiments 3 and 4) per diet. SAS [14] was used to perform the statistical analyses for all four studies, and data were analyzed according to the GLM procedure. A randomized design was used with the experimental unit consisting of the mean value of a cage. Data were analyzed by GLM to determine the significance of the main effect (enzyme inclusion). Duncan’s multiple-range test was used to separate means when significant effects (P < 0.05) were detected by multifactorial analysis of variance [15]. Animal Ethics
(g/kg of DM) 204.0 12.30 9.3 10.0 9.8 5.0
The Animal Ethics Committees of the University of New England approved this study. Health and animal husbandry practices complied with the “Code of Practice for the Use of Animals for Scientific Purposes” issued by the Australian Bureau of Animal Health [16].
1
Provided per kilogram of diet: vitamin A (as all-trans retinol), 12,000 IU; cholecalciferol, 3,500 IU; vitamin E (as D-α-tocopherol), 44.7 IU; vitamin K3, 2 mg; thiamine, 2 mg; riboflavin, 6 mg; pyridoxine hydrochloride, 5 mg; vitamin B12, 0.2 mg; biotin, 0.1 mg; niacin, 50 mg; D-calcium pantothenate, 12 mg; folic acid, 2 mg; monesin, 500 mg; Mn, 80 mg; Fe, 60 mg; Cu, 8 mg; I, 1 mg; Co, 0.3 mg; Se, 0.1 mg; and Mo, 1 mg.
RESULTS AND DISCUSSION The aim of the four experiments was to examine whether a combination of currently available commercial enzyme products in diets based on corn and SBM would have any effects
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tion program [13; Table 2]. First, the minor ingredients were mixed in a small rotary mixer and then were pooled with the major ingredients for thorough mixing. All experimental diets were identical in composition. All enzymes were added directly to other ingredients
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Inclusion rate (ppm) Diet/enzyme
Enzyme A
Enzyme B
Enzyme C
— — — — 50 50 50 100
— — — 50 50 50 50 100
— — — — — — 5 —
— — — 50 100 — — 50 —
— — — 50 100 — — — 50
— — — — — — — — —
— — — — 50 50 50 50 100 100
— — — — — — — — — 100
— — — — — — — — — —
Experiment 1 Corn control (low ME corn) Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme
control combination combination combination combination combination combination combination
1-1 1-2 1-3 1-4 1-5 1-6 1-7
2-1 2-2 2-4 2-7 2-8 2-9 2-10 2-11
control combination combination combination combination combination combination combination combination combination
3-2 3-8 3-9 3-10 3-12 3-13 3-15 3-16 3-17
control combination combination combination combination combination combination combination combination
4-5 4-5B 4-10 4-10B 4-13 4-13B 4-14 4-14B
— 300A 500A 300A 300A 300A 300A 200A
1,000 — — — — — — —
— — — — 100 — 50 50 50
— 300A 500A 300A 200A 400A 300A 300A 300A
1,000 — — — — — — — —
— — — 50 50 — 50 50 100 100
— 500B 400B 300B 300B 300B 250B 200B — —
1,000 — — — — — — — — —
— 50 50 50 50 50 50 50 50
— 300A 300B 300A 300B 250A 250B 250A 250B
1,000 — — — — — — — —
No enzyme
Experiment 4 Corn control (high ME corn) Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme
— — — — — 50 50 100 No enzyme
Experiment 3 Corn control (high ME corn) Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme
Enzyme F
No enzyme — 50 50 50 50 50 50 50 50
— 50 50 — — — — 50 50
— — — — — — — — —
A
Enzyme E batch 1. Enzyme E batch 2.
B
on excreta moisture, broiler performance, or AMEn. The enzyme products tested in the current study contained activities to depolymerize cell walls in corn and SBM and assist endogenous enzymes to digest starch and protein. En-
zymes used in diets containing high levels of SBM are mostly multiglycanase products containing polygalacturonase (pectinase) to hydrolyze pectins present in vegetable products, as well as a range of other enzymes such as
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control combination combination combination combination combination combination combination combination
Enzyme E
No enzyme
Experiment 2 Corn control (low ME corn) Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme
Enzyme D
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TABLE 4. Feed conversion ratio (FCR) of broiler chickens fed diets containing corn–soybean meal (SBM) with or without enzymes FCR Corn
Mean
Low ME Corn
Experiment 1
1 2 3 4 5 6 7 8 9 10 11
High ME corn Corn control Enzyme control Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination
SD
2 5 5A 8A 9A 10 10A 12 13 13A 14 14A 15 16 17
1.842 1.921 1.845 1.844 1.857 2.029 1.909 1.852 1.853
± ± ± ± ± ± ± ± ±
B
0.06 0.10 0.13 0.10 0.10 0.06 0.12 0.07 0.08
Mean
SD
Experiment 2B 1.837 1.789 1.767 1.797
± ± ± ±
0.09 0.12 0.06 0.06
1.797 ± 0.12
1.812 1.720 1.842 1.909 1.793
± ± ± ± ±
0.08 0.09 0.01 0.03 0.07
Experiment 3C
Experiment 4C
1.930 ± 0.05 1.950 ± 0.11 1.968 ± 0.13
1.961 ± 0.12 19.50 ± 0.10
2.049 ± 0.17 2.025 ± 0.12 2.053 ± 0.12 2.027 ± 0.12 2.002 ± 0.10 1.965 ± 0.10 2.046 ± 0.25 2.010 ± 0.14
1.973 ± 0.06 1.910 ± 0.10 2.009 ± 0.16 2.038 ± 0.11 2.004 2.008 1.947 1.935
± ± ± ±
0.12 0.12 0.10 0.13
A
Enzyme E batch 2. Mean value of six replicates. C Mean value of eight replicates. B
cellulase and hemicellulases targeting neutral NSP. Reports in the literature indicate that the enzyme products selected for these experiments should have some effects on broiler performance or AME. Addition of Enzyme E to a corn–SBM broiler diet resulted in a significant improvement in weight gain and FCR as a result of increased ileal digestibility of CP, starch, and fat [7]. Similarly, Zanella et al. [8] reported a significant improvement in ileal digestibility of CP and NSP in corn–soy broiler diets. It was concluded from both studies that the added enzyme products not only had cell
wall degrading activities but also exhibited protease activity, which explained the improved nutrient digestibility. Our results do not confirm these findings. Enzyme E alone or in combination with protease, xylanase, or amylase had no effect on bird performance (Table 4). However, these performance data were gathered over a 1-wk period only in the current study. In order to accurately predict changes in performance as a result of different enzyme combinations, FCR needs to be measured over a longer period. In the current experimental setup, these data are used as an overall indication about the trial, for example, bird welfare,
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Control corn Enzyme control Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination
FCR
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TABLE 5. AMEn of broiler diets containing corn–soybean (SBM) with or without enzymes (low ME corn) Experiment 1 (AMEn MJ/kg DMA) Diet
1 2 3 4 5 6 7 8 9 10 11
STD
12.72 12.79 12.76 13.04 12.90 12.96 13.07 13.03 13.12
Mean
± ± ± ± ± ± ± ± ±
0.18 0.21cd 0.18cd 0.21ab 0.08bcd 0.15abc 0.17ab 0.12ab 0.12a
STD
12.71 12.74 12.95 12.99
± ± ± ±
0.17e 0.09de 0.09bcd 0.22bc
13.19 ± 0.17b
12.90 13.01 13.00 13.42 13.09
12.93
Source of variance Diet
Mean
d
± ± ± ± ±
0.22cde 0.16bc 0.24bc 0.15a 0.17bc
12.99 Probability of greater F value
***
***
Values within a column with unlike superscripts differ significantly (P < 0.05). Mean value of six replicates. ***P < 0.001. a–c A
rather than a precise reflection on the performance. Such a setup only targets large differences between experimental diets and is not designed to find subtle differences. A more accurate tool to determine the effects of different enzyme combinations on the nutritive value of a corn–SBM basal diet in a brief experiment is the classical AME assay. In experiments 1 and 2, Enzyme E included at a higher dosage or Enzyme E in combinations with Enzymes D, A, or B significantly improved AMEn of the diet in comparison with the unsupplemented basal diet (Table 5). The mechanisms by which these enzyme combinations have improved AMEn remain speculative. Kocher et al. [9] reported that it is most likely that Enzyme E solubilized pectic NSP such as rhamnogalacturonan, arabinogalactan, and galactan found in SBM and released nutrients entrapped in the cell walls, which could be successfully utilized by the bird. However, these effects were only significant when Enzyme E was added at a higher dosage. Minor activities of other enzymes present in Enzyme E might have further improved nutrient availability. There is evidence in the literature that the addition of exogenous protease and amy-
lase can improve the utilization of protein in SBM and assist the digestion of starch encapsulated in the endosperm in the upper gastrointestinal tract [8, 10]. The fact that Enzyme E at a lower dosage in combination with protease (Enzyme A) or amylase (Enzyme D) had a similar effect in improving AMEn supports this hypothesis. Although it is evident that soluble NSP in SBM or corn do not exhibit strong antinutritive activities and can be utilized to some extent throughout the intestine by microbial digestion [9], results from experiments 1 and 2 clearly show that AMEn of a corn–SBM basal diet can be improved by adding pectinase (Enzyme E) in combination with amylase (Enzyme D) and protease (Enzyme A). The benefits of enzyme addition lies in the increased access to intracellular entrapped nutrients as well as in an improved energy utilization in comparison with the microbial fermentation. In order to verify the findings in the first two experiments and to find a commercially beneficial enzyme combination, two further experiments were conducted (experiments 3 and 4; Table 6). Surprisingly, the results from the previous studies could not be repeated despite the fact that the same experimental conditions
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Corn control Enzyme control Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination
Mean
Experiment 2 (AMEn MJ/kg DMA)
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TABLE 6. AMEn of broiler diets containing corn–soybean meal (SBM) with or without enzymes (high ME corn) Experiment 3 (AMEn MJ/kg DMB) Mean
2 5 5A 8A 9A 10 10A 12 13 13A 14 14A 15 16 17
SD
Mean
12.89 ± 0.23 12.69 ± 0.13 12.79 ± 0.16
13.04 ± 0.14a 12.65 ± 0.14de 12.67 ± 0.05de 12.77 ± 0.10cd
12.70 ± 0.30 12.70 ± 0.11
13.01 ± 0.05ab 12.88 ± 0.15bc
12.84 ± 0.20 12.72 ± 0.14
12.94 12.49 12.95 12.62
12.81 ± 0.16 12.61 ± 0.14 12.64 ± 0.11 12.77 ± 0.22
Mean
SD
12.73
± ± ± ±
0.10ab 0.14f 0.11ab 0.16e
12.80
Source of variance
Probability of greater F value
Diet
NS
***
Values within a column with unlike superscripts differ significantly (P < 0.05). Enzyme E batch 2. B Mean value of eight replicates. *** P < 0.001. a–f A
and protocols were used. Results in Table 7 show that there are indeed significant differences between the batch of Enzyme E used in experiments 1 and 2 and that used in Experiment 3. However, the addition of either batch of Enzyme E in Experiment 4 failed to show improvement in AMEn, as previously seen in Experiments 1 and 2.
All major ingredients, including corn and SBM, used in the four experiments were purchased from a local supplier approximately 3 wk prior to the start of the experiment. Because the four experiments were conducted over a 9mo period, it has to be assumed that corn and SBM source varied between experiments. It is well-known that the quality and amount of CP
TABLE 7. Comparison of two different batches of Enzyme E (experiment 4), mean values of eight replicates AMEn (MJ/kg DM)
FCRA Batch 1 2 Mean
Mean
SD
1.983 ± 0.11 1.973 ± 0.12
12.79 Probability of greater F value
NS
Values within a column with unlike superscripts differ significantly (P < 0.05). Feed conversion ratio. ***P < 0.001. a,b A
SD
12.90 ± 0.15a 12.69 ± 0.20b
1.978
Source of variance Batch
Mean
***
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Corn control Enzyme control Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination Enzyme combination
Experiment 4 (AMEn MJ/kg DMB)
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TABLE 8. Comparison of control diets between four consecutive AME bioassays Experiment 1 2 3 4
(low ME corn) (low ME corn) (high ME corn) (high ME corn)
Gross energyA (MJ/kg DM)
CPA (%)
FCRB (g feed/g gain)
AMEnB (MJ/kg DM)
AMEn:CPA (kJ/g)
18.15 18.28 18.60 18.88
21.0 21.9 23.7 23.4
1.842b 1.837b 1.930ab 1.961a
12.72b 12.71b 12.89ab 13.04a
60.6 58.0 54.4 55.7
Source of variance
Probability of greater F value
Experiment
*
*
Values within a column with unlike superscripts differ significantly (P < 0.05). Measured as single value per diet. B Mean value of six or eight replicates. *P < 0.05. a,b A
speculated that the release of entrapped cell wall proteins resulted in an increase in the total protein turnover. The energy loss associated with the increased protein turnover resulted in lower AMEn [22]. On the other hand, Irish and Balnave [18] found that the performance of broilers is highly correlated with the concentration of oligosaccharides in the digesta. Although corn is generally regarded as a low-NSP cereal, the actual levels of NSP can vary between 5 and 8% [3, 23]. It has been shown in vitro that the highly branched glucurono-arabinoxylans in corn can be degraded by exogenous enzymes releasing a series of oligomers [23]. Hence, it is possible that the enzymes depolymerized part of the NSP of corn, as well as the rhamnogalacturonan and arabinogalactan of SBM, resulting in an increase of oligosaccharides in the digesta. These lowmolecular-weight carbohydrates are not only osmotically active but also highly fermentative. There are two possible effects of their action on the ME value. Firstly, loss of water in the lumen through osmotic up-regulation is an energy-expensive process, and secondly, proliferation of microorganisms in the gut leads to increased organic mass of the excreta, lowering AME value in a balance trial.
CONCLUSIONS AND APPLICATIONS 1. Addition of any enzyme combination to a corn–SBM basal diet had no effects on performance measured over a short period. 2. Combined addition of pectinase, protease, and amylase significantly improved AMEn when added to a corn–SBM basal diet low in energy and protein. 3. Addition of the same enzyme combination to a corn–SBM basal diet with increased energy and protein content results in a significant reduction of AMEn.
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and NSP in SBM can vary widely as a result of processing methods and conditions [16, 17]. Similarly, it has been reported that oil and fiber contents in corn can significantly differ between commercial hybrids [19]. This was the case in the current study, as the basal diets diet used in experiments 3 and 4 had a higher energy and protein contents than in those used in experiments 1 and 2. Furthermore, the energy:protein ratio (E:P) in experiments 1 and 2 was larger in comparison to experiments 3 and 4 (Table 8). Douglas et al. [17] found that the addition of enzyme to corn–SBM diets is most effective in corn–SBM diets containing a low digestible energy level. Wiseman [20] reported that in diets with a low E:P ratio, excess protein will be catabolized rather than deposited as meat. Thus, the lack of response to enzyme addition in experiments 3 and 4 may be due to the increased AMEn of the basal diet. In experiment 4, the enzymes resulted in a significant reduction of AMEn of the diet. A previous study by Kocher et al. [21] found a similar reduction in AMEn when a commercial multi-activity enzyme product was added to a corn–canola meal-based diet. These authors
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REFERENCES AND NOTES 1. Annison, G. 1991. Relationship between the levels of soluble nonstarch polysaccharides and the apparent metabolizable energy of wheat assayed in broiler chickens. J. Agric. Food Chem. 39:1252– 1256. 2. Campbell, G. L., and A. F. B. van der Poel. 1998. Use of enzymes and process technology to inactivate antinutritional factors in legume seeds and rapeseed. Pages 377–386 in Proc. Recent Advances of Research in Antinutritional Factors in Legume Seeds and Rapeseed.Wageningen Press, Wageningen, The Netherlands. 3. Bach Knudsen, K. E. 1997. Carbohydrate and lignin contents of plant materials used in animal feeding. Anim. Feed Sci. Technol. 67:319–338.
5. Leske, K. L., C. J. Jevne, and C. N. Coon. 1993. Effect of oligosaccharide additions on nitrogen-corrected true metabolizable energy of soy protein concentrate. Poult. Sci. 72:664–668. 6. Irish, G. G., G. W. Barbour, H. L. Classen, R. T. Tyler, and M. R. Bedford. 1995. Removal of the alpha-galactosides of sucrose from soybean meal using either ethanol extraction or exogenous alpha-galactosidase and broiler performance. Poult. Sci. 74:1484– 1494. 7. Marsman, G. J., H. Gruppen, A. F. van der Poel, R. R. Kwakkel, M. W. Verstegen, and A. G. Voragen. 1997. The effect of thermal processing and enzyme treatments of soybean meal on growth performance, ileal nutrient digetibilities, and chyme characteristics in broiler chicks. Poult. Sci. 76:864–872. 8. Zanella, I., N. K. Sakomura, F. G. Silversides, A. Fiqueirdo, and M. Pack. 1999. Effect of enzyme supplementation of broiler diets based on corn and soybeans. Poult. Sci. 78:561–568. 9. Kocher, A., M. Choct, M. D. Porter, and J. J. Broz. 2002. Effects of feed enzymes on nutritive value of soybean meal fed to broilers. Br. Poult. Sci. 43:54–63. 10. Pack, M., D. Creswell, and H. Graham. 1996. Applying enzymes to sorghum and maize based broiler diets. Pages 252–258 in Proc. of 10th Australian Poultry and Feed Convention, Melbourne, Australia. 11. Kocher, A. 2000. Enzymatic degradation of non-starch polysaccharides in vegetable proteins in poulty diets. PhD Thesis. University of New England, Armidale. 12. Somogyi, M. 1960. Modification of two methods for the assay of amylase. Clin. Chem. 6:23–35.
14. SAS Institute Inc. 2001. The SAS System for Windows, Version 8.02 SAS Institute Inc., Cary, NC. 15. Duncan, D. B. 1955. Multiple range test and multiple F-tests. Biometrics 11:1–42. 16. National Health and Medical Research Council. 1990. Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. National Health and Medical Research Council, Commonwealth Scientific and Industrial, Research Organisation, Australian Agricultural Council. Australian Govt. Pub. Service, Canberra, Australia. 17. Douglas, M. W., C. M. Parsons, and M. R. Bedford. 2000. Effect of various soybean meal sources and Avizyme on chick growth performance and ileal digestible energy. J. Appl. Poult. Res. 9:74–80. 18. Irish, G. G., and D. Balnave. 1993. Non-starch polysaccharides and broiler performance on diets containing soyabean meal as the sole protein concentrate. Aust. J. Agric. Res. 44:1483–1499. 19. Singh, V., R. A. Moreau, A. E. Haken, S. R. Eckhoff, K. B. Hicks, and V. Singh. 2000. Hybrid variability and effect of growth location on corn fiber yields and corn fiber oil composition. Cereal Chem. 77:692–695. 20. Wiseman, J. 2001. High energy diets for poultry: Effects of diet composition on performance and carcass quality. Pages 87–106 in Recent Advances in Animal Nutrition. P. C. Gransworthy and J. Wiseman, ed. Nottingham University Press, Nottingham, U.K. 21. Kocher, A., M. Choct, M. D. Porter, and J. Broz. 2000. The effects of enzyme addition to broiler diets containing high levels of canola or sunflower meal. Poult. Sci. 79:1767–1774. 22. Nieto, R., C. Prieto, I. Fernandez-Figares, and J. F. Aguilera. 1995. Effect of dietary protein quality on energy metabolism in growing chickens. Br. J. Nut. 74:163–173. 23. Huisman, M. M. H., H. A. Schols, and A. G. J. Voragen. 2000. Glucuronoarabinoxylans from maize kernel cell walls are more complex than those from sorghum kernel cell walls. Carbohydrate Polymers 43:269–279.
Acknowledgments Roche Vitamins Ltd, Basel, Switzerland, supported this study. The authors are grateful to Geoff Ross, Roche Ltd, for his efforts in arranging the supply of the enzyme products. We gratefully acknowledge the efforts of the staff of the Division of Animal Science, University of New England.
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4. Leske, K. L., O. Akavanichan, T. K. Cheng, and C. N. Coon. 1991. Effect of ethanol extract on nitrogen-corrected true metabolizable energy for soybean meal with broilers and roosters. Poult. Sci. 70:892–895.
13. Feedmania, Saltbush Enterprise, Armidale, Australia.