α-amylase Supplementation of Broiler Diets Based on Corn 1

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1 α-amylase Supplementation of Broiler Diets Based on Corn 1 M. I. Gracia, M. J. Araníbar, 2 R. Lázaro, P. Medel, 3 and G. G. Mateos 4 Departamento de Produccion Animal, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, Madrid, Spain ABSTRACT A 42-d trial was conducted to study the for CP or fat digestibility. Nutrient digestibility and AME n influence of exogenous α-amylase on digestive and performance traits in broilers fed a corn-soybean meal diet. There were two treatments (control and α-amylase supplemented diet) and six replicates (14 Cobb male chicks caged together) per treatment. At 7 d of age, α-amylase supplementation improved daily gain by 9.4% (P 0.05) and feed conversion by 4.2% (P 0.01). At the end of the trial, birds fed the α-amylase-supplemented diet ate more and grew faster (P 0.05) and had better feed conversion (P 0.10) than broilers fed the control diet. Also, α-amylase supplementation improved apparent fecal digestibility of organic matter and starch (P 0.01) and AME n of the diet (P 0.001). However, no effects were detected of the diet increased with age (P 0.001); however, no interactions of α-amylase age were observed for any trait. Coefficients of apparent ileal and fecal digestibility of starch at 28 d of age were similar, which indicated that most of the undigested starch was not fermented in the hindgut of the chick. α-amylase supplementation reduced relative pancreas weight (P 0.001) but did not affect the weight of the remaining organs. consistently reduced intestinal viscosity and relative weights of all the organs (P 0.001). The data indicated that α- amylase supplementation of a corn-soybean meal diet improved digestibility of nutrients and performance of broilers. (Key words: amylase, corn, digestibility, digestive organ size, broiler) 2003 Poultry Science 82: INTRODUCTION Enzyme supplementation improves performance and nutrient digestibility of broilers fed diets containing high levels of grains rich in nonstarch polysaccharides (NSP). The reason is not totally clear but has been related to a decrease in intestinal viscosity, which may improve nutrient digestibility and increase feed intake (Petterson et al., 1991; Salih et al., 1991; Lázaro et al., 2003a,b). Corn and sorghum grains are low in NSP content and therefore do not present problems of viscosity. As a consequence, a less response to addition of enzymes targeted for viscous grains should be expected in diets based on these cereals. However, the insoluble components of the NSP present in corn may be encapsulating nutrients and as such could be responsive to exogenous xylanase. Gastrointestinal tract (GIT) development after hatching has been widely investigated in birds (Dror et al., 1977; Nitsan et al., 1991a,b; Nir et al., 1993). All these studies emphasize the major role of the pattern of growth of the 2003 Poultry Science Association, Inc. Received for publication September 4, Accepted for publication November 27, Supported by funding through Project AGF , MCYT (Spain). 2 Present address: Universidad Nacional del Altiplano, Puno, Peru. 3 Present address: Imasde Agropecuaria, S. L. Isabel Colbrand 10 4a, Madrid, Spain. 4 To whom correspondence should be addressed: ggmateos@pan. etsia.upm.es. GIT during the first week of life, a period in which the digestive organs increase in size more rapidly than the remaining body. Oral intake of nutrients stimulates the development of the GIT in the chick, but limited synthesis of some pancreatic enzymes during the first days after hatching may limit early growth. Noy and Sklan (1995) reported that daily net secretion of amylase into the duodenum was low at d 4 and steadily increased up to d 21. Uni et al. (1995) also reported that the secretion of amylase per gram of feed intake was low on d 4, increased up to d 7, and then stabilized. Therefore, an exogenous supply of amylase might be needed to match the requirement of birds and improve performance early in life. Early studies have shown beneficial effects of amylase and protease preparations on growth and feed efficiency of chicks when added to diets (Jensen et al., 1957; Fry et al., 1958; Burnett, 1966). Mahagna et al. (1995), however, did not find any beneficial effect of enzyme complexes, including amylase and protease, on performance or digestibility of nutrients of broilers from 1 to 14 d of age fed a sorghum-soybean meal diet. Amylase supplementation to corn-soybean meal diets significantly improves performance of turkey poults (Ritz et al., 1995). Recent studies have indicated that addition of a mixture of amylase, protease, and xylanase benefits birds fed diets based Abbreviation Key: cp = centipoise; EE = ether extract; GIT = gastrointestinal tract; NSP = nonstarch polysaccharides; OM = organic matter. 436

2 AMYLASE SUPPLEMENTATION OF CORN 437 on corn and soybean meal (Zanella et al., 1999; Douglas et al., 2000; Café et al., 2002), which indicates that young birds might be deficient in certain endogenous enzymes. However, information on the influence of amylase as a single exogenous enzyme added to broiler diets is scarce. Therefore, we conducted a trial to study the effects of supplementing a corn-soybean meal diet with pure α- amylase on performance, viscosity, nutrient digestibility, and development of the GIT of broilers. Diets MATERIALS AND METHODS There were two dietary treatments: a corn-soybean diet and the same diet supplemented with 40 ppm of an enzyme preparation. 5 The preparation contained, as confirmed by duplicate analysis, 43,000 units of α-amylase (EC ) per gram from Bacillus amyloliquefaciens. The premixed enzyme preparation was added as dry material to the mixer. The experimental diet included 1,720 units of α-amylase per kilogram of diet. Chicks were fed a starter diet from 1 to 21 d of age and a grower diet from 21 to 42 d, and both diets were formulated to meet or exceed the minimum nutrient requirements recommended by the National Research Council (1994). The ingredient composition, estimated nutrient value according to FEDNA (1999), and determined chemical analyses of the experimental diets are given in Table 1. Birds A total of d-old male Cobb 500 chicks was obtained from a commercial hatchery 6 and randomly allotted in groups of 14 chicks to 12 battery cages ( cm) within a windowless, environmentally controlled facility. The initial temperature of 32 C was reduced sequentially according to the age of the birds until reaching 26 C at 21 d. The chicks were kept on a 23-h light program, with free access to mash feed and water throughout the experiment. At 21 d, two birds per replicate were removed and housed together in metabolism cages ( m) for measuring digestibility of nutrients. The remaining birds were moved to 12 battery cages ( cm) where they were kept for 21 additional d. Therefore, the experimental unit was formed by nine birds from 21 to 42 d of age. Experimental procedures followed the principles for care of animals in experimentation (Spanish Royal Decree 223/88, 1988). Performance Body weight and feed consumption by pen were recorded weekly, and the data were used to determine 5 Natustarch, BASF, Ludwigshafen, Germany. 6 Cobb Expaña, Alcalá de Henares, Madrid, Spain. 7 Model DV III, Brookfield Engineering Laboratories Inc., Soughton, MA. 8 Food Chemicals Codex Grade, Celite Corp., Lompar, CA. TABLE 1. Composition, and nutritive value of the experimental diets Ingredients 0 21 d d 1 (%, as fed) Dent corn Soybean meal, 44% CP Lard Dicalcium phosphate Limestone NaCl Vitamin and mineral premix DL-Methionine Celite Estimated analysis 4 AME n, kcal/kg 2,980 3,184 Crude protein, % Lysine, % Methionine, % Methionine + cystine, % Ether extract, % Starch, % Ash, % Calcium, % Phosphorus, % Available phosphorus, % Determined analysis Gross energy, kcal/kg 4,699 4,788 Dry matter, % Crude protein, % Ether extract, % Starch, % Ash, % Acid-insoluble ash, % Calcium, % Phosphorus, % With 0 or 40 ppm of α-amylase added. 2 Provided per kilogram of diet: vitamin A, 7,500 IU (retinyl acetate); cholecalciferol, 37.5 µg; vitamin E, 10 IU (DL-α-tocopherol); menadione, 3 mg; thiamin, 1.8 mg; riboflavin, 5.3 mg; pyridoxine, 3.5 mg; vitamin B 12, 12.5 µg, folic acid, 0.5 mg; biotin, 0.1 mg, choline, 350 mg; calcium pantothenate, 10 mg; niacin, 35 mg; Mn, 80 mg; Zn, 50 mg; I, 2 mg; Fe, 80 mg; Cu, 8 mg; Co, 0.2 mg; Se, 0.2 mg; ethoxyquin, 100 mg. 3 Acid-washed diatomaceous earth. Food Chemicals Codex Grade, Celite Corp., Lompar, CA. 4 According to FEDNA (1999). performance parameters on a cumulative basis at 7, 21, and 42 d of age. Intestinal Viscosity To measure intestinal viscosity, three (7 d of age) or two (28 d of age) broilers chosen at random from each replicate were euthanatized by cervical dislocation, and the intestinal content was collected from the jejunum, defined as the region from the pancreas tail to the Meckel s diverticulum as described by Bedford et al. (1991). The viscosity (centipoise, cp) of a 0.5-mL aliquot obtained from the supernatant solution was determined by using a Brookfield digital viscometer 7 at 24 C. Each sample was read twice and the average value was used for statistical analysis. Digestibility of Nutrients Apparent fecal digestibilities of DM, organic matter (OM), CP, ether extract (EE), starch, and gross energy

3 438 GRACIA ET AL. and the AME n of the diets were measured at 7 d with a digesta marker and at 28 d of age by the total collection method (European reference method; Bourdillon et al., 1990). At 7 d, samples of the excreta produced for the previous 24 h were collected from each pen, frozen ( 20 C), and stored. The AME n of the diets was calculated according to the method described by Hill and Anderson (1958). The fecal digestibility coefficients at 7 d were estimated using 4 N-HCl insoluble ash as indicator. Celite 8 (acid-washed diatomaceous earth) was added at 0.5% to the starter diets as an additional source of acid-insoluble ash. At 28 d, fecal digestibility was measured by the total collection method, using the birds housed at 21 d in the metabolism cages. After a 3-d adaptation period, excreta samples were collected daily from each cage for 4 consecutive d, frozen, and stored in plastic bags ( 20 C) until analysis. Prior to analysis, excreta samples were thawed overnight, homogenized, dried (72 h; 60 C), and ground (1-mm screen). Ileal apparent digestibility of DM and starch was measured at 28 d of age. Two broilers from each replicate were euthanatized by cervical dislocation, and the intestinal content was collected from the ileum, defined as the region from the Meckel s diverticulum to the ileocecal junction, pooled, frozen ( 20 C), and stored. Prior to analysis, ileal samples were freeze-dried and ground. The ileal digestibility coefficients at 28 d were estimated using 4 N-HCl insoluble ash as indicator, but no additional acidinsoluble ash was added to the diet. Organ Size Proventriculus, gizzard, pancreas, liver, and small intestine were removed from a chick per replicate pen used for measuring viscosity on d 7 and 28. The weights of the empty organs relative to BW were calculated in these chicks. Chemical Analyses Feeds and feces were analyzed for moisture by oven drying method (930.15), ash by muffle furnace (942.05), CP by Kjeldahl method (984.13), EE by Soxhlet fat analysis (920.39), and starch by α-amylase glucosidase method (996.11) as described by the Association of Official Analytical Chemists (2000). The percentage of OM was calculated as 100 minus ash content. Gross energy was determined by an adiabatic bomb calorimeter, 9 and acid-insoluble ash was determined by the method described by Vogtmann et al. (1975). Ileal samples were analyzed for moisture, starch, and acid-insoluble ash by the same methods used for feeds and feces. Statistical Analysis The general lineal models (GLM) procedure of SAS software (SAS Institute, 1990) was used to analyze the 9 Parr 1356, Parr Instrument Company, Moline, IL. data as a completely randomized design with α-amylase supplementation as main effect. For intestinal viscosity, apparent digestibility of nutrients, and relative weight of selected organs, age was also included in the model. Pen means served as the experimental unit. RESULTS In general, α-amylase supplementation improved productive traits throughout the trial (Table 2). At7dofage, α-amylase supplementation improved daily gain (14.0 vs g/d; P 0.05) and feed conversion (1.13 vs g/ g; P 0.01). Moreover, at the end of the trial, birds fed the α-amylase-supplemented diet ate more (90.5 vs g/d; P 0.05), grew faster (57.7 vs g/d; P 0.05), and tended to have better feed conversion (1.57 vs g/g; P 0.10) than broilers fed the control diet. Mortality was very low (1.2%) and independent of dietary treatment (data not shown). Intestinal viscosity was not affected by α-amylase supplementation but decreased with age from 2.34 cp at 7 d to 1.69 cp at 28 d (P 0.001) (Table 3). At 7 d of age, α- amylase supplementation improved apparent fecal digestibility of DM (72.0 vs. 70.2%; P 0.01), OM (74.6 vs. 72.8%; P 0.01), starch (96.2 vs. 93.5%; P 0.01), gross energy (75.2 vs. 73.9%; P 0.05), and AME n of the diet (2,976 vs. 2,927 kcal/kg; P 0.05), and the beneficial effects on digestibility persisted at 28 d of age (Table 3). However, no effects of α-amylase supplementation were detected for CP or EE digestibility at any age (P > 0.10). Also, apparent ileal digestibility of DM (78.5 vs. 76.8%; P 0.01) and starch (97.9 vs. 96.4%; P 0.01) were improved by α-amylase at 28 d of age (Table 3). Apparent fecal digestibility of all nutrients studied and the AME n of the diets increased with age of birds (P 0.001). α-amylase supplementation reduced the weight of pancreas at 7 d (0.485 vs % BW; P 0.05) and at 28 d (0.323 vs % BW, P 0.01) but did not affect any of the other digestive organs studied (Table 4). Relative weights of all the organs were lower at 28 d than at 7 d of age (P 0.001). DISCUSSION α-amylase supplementation improved broiler performance throughout the trial, results that agree with Ritz et al. (1995) who observed improvements of 3% in daily gain and 4% in feed consumption in 21-d-old poults fed a corn-soybean meal diet supplemented with an enzyme complex containing predominantly amylase. However, Mahagna et al. (1995) reported a 4% depression in feed utilization from 1 to 7 d in broilers fed a sorghum-soybean meal diet supplemented with amylase and protease. The reasons for these discrepancies are unknown but the enzyme preparation used by Mahagna et al. (1995) was produced by Bacillus subtilis and Penicillium emersonii, whereas ours was obtained from Bacillus amyloliquefaciens. Also, the activity of amylase per kilogram of feed, the

4 AMYLASE SUPPLEMENTATION OF CORN 439 TABLE 2. Productive performance of broilers SEM Treatment Control α-amylase 1 (n = 6) P Daily gain 0 7 d * 0 21 d ** d * 0 42 d * Feed intake (g) 0 7 d NS 0 21 d * d d * Feed conversion (g/g) 0 7 d ** 0 21 d NS d d ppm of α-amylase. P *P **P (g) main cereal (sorghum vs. corn), and the form of the feed (crumbles vs. mash) were different in these trials. As expected, intestinal viscosity was low and not affected by α-amylase supplementation. Similar results have been found by Zanella et al. (1999) when they supplemented a corn-soybean meal diet with a mixture of amylase, protease, and xylanase. Cereals such as corn do not present viscosity values as high as barley or wheat because of the negligible amounts of β-glucans and the low content of soluble pentosans of the seed. Choct and Annison (1990) have found that the content of pentosans + β-glucans on DM basis is 4.3% for corn but 11.0% for barley. Intestinal viscosity decreased with age of birds, which agrees with previous research (Salih et al., 1991; Petersen et al., 1999). Apparent fecal digestibility of starch was improved by α-amylase supplementation that also increased digestibility of DM, OM, gross energy, and AME n of the diet. Noy and Sklan (1995) reported that the daily net secretion of amylase into the duodenum was low at d 4 and steadily increased with age. Thus, supplementation with α-amylase might improve starch digestibility and consequently DM, OM, and energy digestibility. Apparent ileal digestibility of starch and DM were also improved by exogenous α-amylase. Zanella et al. (1999) found that the ileal and fecal digestibility of starch in 37-d-old broilers increases from 91.2 to 93.0% and from 98.2 to 98.5% when a corn-soybean meal diet is supplemented with an enzyme complex containing amylase, protease, and xylanase. Also, these authors found that ileal ME of the diet increases from 3,076 to 3,153 kcal/kg with enzyme supplementation. Douglas et al. (2000) reported improvements in ileal digestibility of energy at 21 d of age when amylase, protease, and xylanase are used in a corn-soybean meal diet for broilers. The increased digestibility of starch and the higher AME n value with α-amylase may explain the improvement in feed efficiency observed in our work. Also, the increase in feed intake justified, in part, the improvements in broiler growth observed. In the present study, the improvement in digestibility observed with α-amylase supplementation at d 7 was also evident at d 28, indicating that supplementation with α-amylase might be beneficial even in the grower period when the digestive system of broilers is assumed to be totally developed. Yuste et al. (1991), however, observed that the digestibility of starch of a variety of ingredients is lower in 21-d-old broilers than in adult cockerels, which indicates that the GIT of 21-d-old broilers is not fully developed and able to digest starch. Similar data have been reported for turkeys fed a corn diet (Persia et al., 2002). They observed that starch disappearance at the Meckle s region is 79% in 6-wk-old hens and 86% in 12-wk-old hens and that the AME n of the diet also increases from 2,280 to 2,470 kcal/kg with age. Ritz et al. (1995) reported that α-amylase supplementation increases the length of the villi within the jejunal and ileal sections of 3-wk-old turkey poults fed corn-soybean meal diets. The increase in surface area suggested by the increased villus length might enhance nutrient absorption and improve nutrient digestibility (Caspary, 1992). In our study, fecal and ileal digestibility coefficients of starch at 28 d were similar, indicating that the undigested starch fraction was not fermented to a great extent in the hindgut of the chicken. Similar data have been reported by Yuste et al. (1991) working with semipurified starches of a variety of origins in 28-d-old broilers and by Weurding et al. (2001) in 28-d-old broilers fed diets containing different starch sources. These observations are consistent with the report of Kussaibati et al. (1982) who indicated that the undigested starch fraction is similar for conventional and germ-free chicks. The digestibility of all nutrients studied increased with age. The most striking effect was observed for EE that increased from 76.1% at 7 d to 85.2% at 28 d. Several

5 440 GRACIA ET AL. TABLE 3. Intestinal viscosity (centipoise; cp) and apparent digestibility of DM, organic matter, CP, ether extract, starch, and gross energy, and AME n of the diet SEM Treatment Control α-amylase 1 (n = 6) α-amylase Interaction 2 Viscosity α-amylase d NS *** NS 28 d Fecal digestibility (%) Dry matter α-amylase d ** *** NS 28 d Organic matter α-amylase d ** *** NS 28 d Ether extract α-amylase d NS *** NS 28 d Starch α-amylase d ** *** NS 28 d Crude protein α-amylase d NS *** NS 28 d Gross energy α-amylase d *** *** NS 28 d (AME n, kcal/kg) α-amylase 3,030 3,102 7 d 2,927 2,976 2, *** *** NS 28 d 3,132 3,227 3, Ileal digestibility (%) Dry matter 28 d ** Starch 28 d ** 1 40 ppm of α-amylase. 2 α-amylase age. **P ***P authors (Nitsan et al., 1991a; Dunnington and Siegel, 1995) have found that endogenous lipase activity is limited in young birds. Also, Carew et al. (1972) reported that the percentage of excretal fat of chicks supplemented with 20% of different fat sources is greater at 7 d than at 11 d of age. Therefore, a reduction in lipase excretion or activity may have contributed to the low values of fat digestibility observed at 7 d. Also, the grower diet had 3% higher content of fat, as lard than the starter diet and supplemental fat is better digested than fat contained in raw materials of vegetable origin. The apparent fecal digestibility of starch increased from 94.9% at 7 d to 97.1% at 28 d of age. Few and discrepant data are available in the literature. Magahna et al. (1995) reported a decrease in fecal starch digestibility from 96.7% at 7 d to 93.7% at 21 d of age in broilers fed a sorghum-soybean meal diet. However, Plavnik and Sklan (1995) with 21-d-old broilers and Weurding et al (2001) with 28-d-old broilers have found coefficients of starch digestibility of 97.3 and 97.4%, respectively, results that are close to our data. Also, Batal

6 AMYLASE SUPPLEMENTATION OF CORN 441 TABLE 4. Relative weight (% BW) of selected digestive organs of chicks SEM Treatment Control α-amylase 1 (n = 6) α-amylase Interaction 2 Proventriculus α-amylase d NS *** NS 28 d Gizzard α-amylase d NS *** NS 28 d Pancreas α-amylase d *** *** NS 28 d Liver α-amylase d NS *** NS 28 d Small intestine α-amylase NS *** NS 7 d d ppm of α-amylase. 2 α-amylase age. ***P and Parsons (2002a) have found that starch digestibility increases in chicks from 97% at 7 d of age to 99% at 21 d of age, values that are slightly higher than ours. The percentage of CP digestibility increased by 4.0% from 7 to 28 d, an increase that is lower than the 15% found by Noy and Sklan (1995) for broilers of similar ages. The AME n of the diets were determined by different methods at 7 and 28 d. However, Vogtmann et al (1975) found that 4 N-HCl insoluble ash method had similar accuracy to the total collection method; therefore, we have studied the effects of age on this parameter. The AME n of the diet increased from 2,952 kcal/kg at 7 d to 3,180 kcal/kg at 28 d, an increase which is consistent with the improvement observed for most nutrients and with Batal and Parsons (2002a,b) who found improvements in fat digestibility and in the ME n of the diet with age. The weight of pancreas as a percentage of BW decreased with α-amylase supplementation, which indicates that secretion of pancreatic enzymes might be affected by the concentration of enzymes and substrates or products of their hydrolysis in the lumen of the small intestine (Twombly-Snook and Meyer, 1964a,b; Moran, 1985). Mahagna et al. (1995) found that secretion of amylase and proteases (trypsin and chymotripsin) by the pancreas was reduced when chicks were fed diets supplemented with amylase and protease. Therefore, the reduction in pancreas weight observed in our trial might have been related to less secretion of endogenous amylase due to the presence of exogenous amylase in the intestine. Relative weights of all the organs studied decreased with age, data that are consistent with Nitsan et al. (1991a,b) and Iji et al. (2001) who indicated that the maximum weights of these organs are reached before 9 d of life. The experimental data indicate that α-amylase supplementation to corn-soybean meal diets increases digestibility of starch and the AME n of the diet and can be used to improve performance of broilers at any age. REFERENCES Association of Official Analytical Chemists Official Methods of Analysis. 16th ed. AOAC, Arlington, VA. Batal, A. B., and C. M. Parsons. 2002a. Effects of age on nutrient digestibility in chicks fed different diets. Poult. Sci. 81: Batal, A. B., and C. M. Parsons. 2002b. Effect of fasting versus feeding oasis after hatching on nutrient utilization in chicks. Poult. Sci. 81: Bedford, M. R., H. L. Classen, and G. L. Campbell The effect of pelleting, salt, and pentosanase on the viscosity of intestinal contents and the performance of broilers fed rye. Poult. Sci. 70: Bourdillon, A., B. Carré, L. Conan, J. Duperray, G. Huyghebaert, B. Leclercq, M. Lessire, J. McNab, and J. Wiseman European reference method for the in vivo determination of metabolisable energy with adult cockerels: Reproducibility, effect of food intake and comparison with individual laboratory methods. Br. Poult. Sci. 31: Burnett, G. S The effect of damaged starch, amylolytic enzymes, and proteolytic enzymes on the utilization of cereals by chickens. Br. Poult. Sci. 3: Café, M. B., C. A. Borges, C. A. Fritts, and P. W. Waldroup Avizyme improves performance of broilers fed cornsoybean meal-based diets. J. Appl. Poult. Res. 11: Carew, L. B., R. H. Machemer, R. W. Sharp, and D. C. Foss Fat absorption by very young chick. Poult. Sci. 51:

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