INTRODUCTION. S. A. Kaczmarek,* A. Rogiewicz,* M. Mogielnicka,* A. Rutkowski, R. O. Jones, and B. A. Slominski * 1

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1 The effect of protease, amylase, and nonstarch polysaccharide-degrading enzyme supplementation on nutrient utilization and growth performance of broiler chickens fed corn-soybean meal-based diets S. A. Kaczmarek,* A. Rogiewicz,* M. Mogielnicka,* A. Rutkowski, R. O. Jones, and B. A. Slominski * 1 * Department of Animal Science, University of Manitoba, Winnipeg, Canada R3T 2N2; Department of Animal Nutrition and Feed Management, University of Life Sciences, Poznan, Poland ; and Canadian Bio-Systems Inc., Calgary, Canada T2C 0J7 ABSTRACT A study was conducted to determine if amylase and protease addition would improve nutrient digestion during the first 2 wk of growth. The experimental treatments included a control corn-soybean meal-based diet and diets supplemented with either amylase or amylase plus protease. No effect of enzyme supplementation was observed on BW gain and feed conversion ratio. This was corroborated by similar ileal starch and protein digestibility values, which averaged 96.8, 96.8, and 96.9% and 83.9, 80.1, and 79.6%, respectively, for the control and for the amylase or amylase plus protease supplemented diets. Total tract digestibility of starch averaged 97.8, 97.7 and 97.7% for the 3 diets and was followed by a similar diet with AME n values of 3,129, 3,129, and 3,106 kcal/kg. In another study, a 2 3 factorial arrangement of 8 dietary treatments was used to evaluate the effect of corn particle size (conventional or coarse vs. fine) and the addition of a nonstarch polysaccharide enzyme, amylase, or both on growth performance and nutrient utilization of broiler chickens from 1 to 21 d of age. Chickens fed a diet containing a INTRODUCTION conventionally ground corn (geometric mean diameter of 736 µm) showed higher (P < 0.001) BW gain (808 vs. 750 g/bird) and lower feed conversion ratio (1.27 vs. 1.32) than those consuming a fine corn-containing diet (geometric mean diameter of 482 µm). This was further substantiated by a lower AME n content (2,852 vs. 2,972 kcal/kg). Addition of amylase had no effect on growth performance of chickens fed a conventional corn-containing diet, but improved BW gain, feed conversion ratio, and diet AME n in those fed the finely ground corn, possibly due to increased starch digestion in the upper gut. Addition of nonstarch polysaccharide enzymes was effective for both diets, with the most pronounced effects observed in feed converstion ratio for the conventional corn-containing diet (1.27 vs. 1.23) and BW gain (750 vs. 789 g/bird) for the fine corn-containing diet. This was followed by the same magnitude of difference in diet AME n content, which increased from 2,972 to 3,042 and 2,852 to 3,009 kcal/ kg following enzyme addition. Key words: corn particle size, amylase, protease, nonstarch polysaccharide enzyme, broiler chicken 2014 Poultry Science 93 : Poultry Science Association Inc. Received November 6, Accepted March 19, Corresponding author: Bogdan.Slominski@umanitoba.ca A concerted effort to improve the nutritive value of poultry diets with enzyme supplementation has been ongoing in the poultry industry. In addition to the use of the conventional xylanase, glucanase, phytase, and, more recently, multicarbohydrase preparations, the application of normal digestive tract enzymes (i.e., protease, amylase, lipase) have also been proposed (Jiang et al., 2008; Angel et al., 2011; Kalmendal and Tauson, 2012; Yegani and Korver, 2013). The application of these enzymes has been driven by the idea that newly hatched chicks may be deficient in key digestive enzymes; this has been corroborated by Nitsan et al. (1991), Noy and Sklan (1995) and Jin et al. (1998), who showed that specific activities of lipase, amylase, and trypsin rapidly increase up to 2 to 3 wk posthatch. Consequently, it has been suggested that the immaturity of the digestive system of young chicks may result in the poor utilization of dietary nutrients (Jin et al., 1998), and nutrient digestion rather than the ability to absorb nutrients has been indicated to be the primary limiting factor (Parsons, 2004). These findings led to studies of the need to supplement diets of young broiler 1745

2 1746 Kaczmarek et al. chickens with normal endogenous enzymes to enhance growth performance. The current study concentrated on the use of protease and amylase microbial enzymes, as earlier work in our laboratory (Meng et al., 2004) demonstrated no effect of lipase supplementation on fat digestibility, diet AME n, or growth performance of young broiler chickens fed diets containing either tallow or vegetable oil. In addition, we hypothesized that the differences in growth response from amylase and protease supplementation reported by different laboratories could be due to factors other than the endogenous enzyme deficiency. Therefore, particle size of the corn component of the diet was investigated in an attempt to elucidate the variability in research data on the effect of dietary amylase supplementation on growth performance and starch utilization in broiler chickens. This was driven by the fact that, in studies examining the effect of feed particle size on growth performance of broiler chickens (Amerah et al., 2008), coarse particles decreased feed intake (FI) and improved feed conversion ratio (FCR), suggesting that coarse grinding of corn, as opposed to fine grinding, has beneficial effects on broiler performance. In contrast, Lott et al. (1992) found that coarsely ground corn significantly decreased chickens BW gain (BWG) and FCR at 21 d of age compared with a diet containing finely ground corn. Kilburn and Edwards (2001) also reported improvements in FCR and true ME content when the diet contained medium corn particles compared with diets containing very coarse particles. However, Reece et al. (1985, 1986) reported that corn particle size (i.e., 0.68 vs mm) had no effect on growth performance of broilers fed crumbled or pelleted diets. In addition, Péron et al. (2005) and Lentle et al. (2006) reported that wheat particle size distribution did not change after pelleting, whereas Engberg et al. (2002) and Svihus et al. (2004) reported that pelleting minimized the differences in particle size distribution in wheat-based diets. Thus, it has been suggested that even though cornbased diets are not viscous, some amounts of starch and protein escape digestion, reach the hindgut, and undergo fermentation with a relatively low energy yield (Zanella et al., 1999). As recently reviewed (Slominski, 2011), some studies have shown that multicarbohydrase preparation could be effective for the improvement of the nutritive value of low-viscosity, corn and soybean meal (SBM) diets, and in this regard research data on the possible interactions between corn particle size and dietary amylase, protease, and nonstarch polysaccharide (NSP) enzyme supplementation in pelleted cornbased diets is not available. In the current study, the objective of experiment 1 was to investigate the effect of amylase alone or in combination with protease on growth performance and nutrient utilization in broiler chickens fed a mash corn-sbm diet. The effect of corn particle size and NSP-degrading enzyme or starch-hydrolyzing enzyme addition was evaluated in a growth performance and nutrient digestibility study with broiler chickens in experiment 2. MATERIALS AND METHODS Experimental Design and Diets In experiment 1, 9 replicate cages of 5 birds each were randomly assigned to 3 dietary treatments: control, control plus amylase (10,000 U/kg) and amyloglucosidase (10 U/kg), and control plus amylase, amyloglucosidase, and protease (4,000 U/kg). All diets were fed in a mash form throughout the 2-wk experimental period. In experiment 2, 9 replicate cages of 5 birds each were randomly assigned to a factorial arrangement of 8 dietary treatments to investigate the effect of corn particle size (conventional vs. fine) without or with supplementation with an NSP-degrading enzyme, starchhydrolyzing enzyme, or both on nutrient utilization and growth performance of broiler chickens. All diets were fed in a pelleted form throughout the 3-wk period. The NSP enzyme supplied 600 U of xylanase, 100 U of glucanase, 300 U of cellulase, 600 U of pectinase, 30 U of mannanase, and 30 U of galactanase per kilogram of diet. The amylase supplement supplied 10,000 U of amylase and 10 U of amyloglucosidase per kilogram of diet. The protease supplement supplied 4,000 U of protease per kilogram of diet. Enzyme premixes were provided by Canadian Bio-Systems Inc. (Calgary, Alberta, Canada) and were added to the diets at the expense of corn. Due to the detection limits (i.e., amylase at 40 U/g, protease at 80 U/g) and the interference from the carbohydrate components of feed ingredients in many enzyme assays based on the dinitrosalicylic acid reagent, enzyme activities were determined in the enzyme premixes rather than in complete feeds. The exception to this was xylanase activity, which was determined in both enzyme premixes and diets used in experiment 2 using Xylazyme AX tablets (Megazyme International Ireland Ltd., Bray, Ireland). The determined activities were 34, 715, 26, and 743 U/kg for the control diet containing coarse corn as well as the diets supplemented with NSP enzyme, amylase, and NSP enzyme + amylase, respectively, and 27, 732, 34, and 756 U/kg for the control diet containing fine corn as well as the diets supplemented with NSP enzyme, amylase, and NSP enzyme + amylase, respectively. The composition of experimental diets is presented in Table 1. In experiment 1, corn-sbm diets were suboptimal in nutrient composition and were formulated to meet 95% of the NRC (1994) requirement for AME n and 92% for CP, Met, Ca, and available P. Other nutrients met or exceeded the NRC specifications for broiler chickens. All diets were provided in a mash form ad libitum. Each diet contained chromic oxide (0.3%) as an indigestible marker for the calculation of nutrient digestibility coefficients and AME n content.

3 Table 1. Composition and calculated analysis of experimental diets Item Experiment 1 In experiment 2, diets were formulated to meet 95% of the NRC (1994) requirement for AME n and CP. Titanium dioxide (0.3%) was used as an internal marker to determine starch and NSP digestibility and AME n content. Diets were pelleted using the conditioning temperature of 65 C. Diets and water were provided ad libitum. The 2 corn particle geometric mean diameters (GMD) of 736 µm (conventional) and 482 µm (fine) were manufactured using a Wiley hammer mill standard model no. 3 grinder (Arthur H. Thomas Company, Philadelphia, PA) with a screen size of 3.18 mm; ground corn came from a single grinding (conventional) or multiple grindings (fine). The GMD was determined according to the American Society of Agricultural Engineers (2003). Bird Management and Sample Collection EXOGENOUS ENZYMES IN BROILER CHICKEN NUTRITION Experiment 2 Diet 1 Diet 2 Ingredient (g/kg) Corn (conventional, 736 µm) Corn (fine, 482 µm) Soybean meal (46% CP) Canola meal (37% CP) Canola oil Dicalcium P Limestone dl-met Mineral premix Vitamin premix Chromic oxide 3.0 Titanium dioxide Total 1, , ,000.0 Calculated analysis 5 CP (%) AME n (kcal/kg) 3,052 3,008 3,008 Lys (%) Met (%) Met + Cys (%) Ca (%) Available P (%) Analyzed composition CP (%) AME n (kcal/kg) 3,129 2,972 2,852 1 Contained 21% Ca and 18% P. Contained 38% Ca. 3 Mineral premix provided per kilogram of diet: Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se, 0.1 mg; I, 0.36 mg; Na, 1.6 g. 4 Vitamin premix provided per kilogram of diet: vitamin A, 8,250 IU; vitamin D 3, 1,000 IU; vitamin E, 11 IU; vitamin B 12, mg; vitamin K, 1.1 mg; niacin, 53 mg; choline, 1,020 mg; folic acid, 0.75 mg; biotin, 0.25 mg; riboflavin, 5.5 mg. 5 Calculated based on analyzed values of feed ingredients. One-day-old male Ross 308 broiler chicks were obtained from a local hatchery. The birds were individually weighed and sorted into 5 weight classes. Groups of 5 birds, 1 from each weight class, were then randomly assigned to cages such that the average initial BW of birds was similar across cages. The birds were held in electrically heated battery cages, had free access to water and feed and were provided with continuous light for the duration of experiment. Body weight and FI were monitored weekly, with the cage serving as the experimental unit. Before weighing, the birds were fasted for 4 h. Mean weight gain, FI, and feed-to-gain ratio were used to determine growth performance following any necessary corrections for mortality. Mortality in both experiments was very low and averaged 1.1% for experiment 1 and 0.9% for experiment 2. At the termination of each experiment (d 14 for experiment 1 and d 21 for experiment 2), excreta samples were collected from each cage over a 3-h period and subsequently frozen, freeze-dried, and finely ground. On d 15 (experiment 1) or 22 (experiment 2), 12 birds from each treatment (9 birds from each cage plus 3 birds selected from 3 different cages) were randomly selected and euthanized by cervical dislocation. The contents of the terminal ileum (15 cm, adjacent to the ileo-cecal junction) were collected. The digesta samples from 3 birds within a treatment were pooled to provide sufficient material for chemical analysis. The digesta samples were frozen, freeze-dried, and ground before further analyses. All animal procedures were conducted according to the guidelines of the Canadian Council on Animal Care, and the protocol for this study was approved by the Local Animal Care Committee of the University of Manitoba. Chemical Analyses Diet, digesta, and excreta samples were analyzed in duplicate. Chromic oxide was determined using the procedure described by Williams et al. (1962). Titanium dioxide was determined according to the procedure described by Lomer et al. (2000) and was read on a Varian inductively coupled plasma mass spectrometer (Varian Canada Inc., Mississauga, Ontario, Canada). Nitrogen content was analyzed by the combustion method with a Leco Model FP 2000 combustion analyzer (Leco Corp., St. Joseph, MI); protein contents were calculated using a multiplication factor of Gross energy was determined using a Parr 1261 adiabatic bomb calorimeter (Parr Instrument Co., Moline, IL) standardized with benzoic acid. Nonstarch polysaccharide content was determined by gas-liquid chromatography (component-neutral sugars) and by colorimetry (uronic acids). The procedure for neutral sugars was as described by Englyst and Cummings (1984, 1988) with some modifications (Slominski and Campbell, 1990). Uronic acids were determined using the procedure described by Scott (1979). Starch was determined colorimetrically using a Megazyme total starch determination kit and the AOAC Method (AOAC International, 2012). Calculations and Statistical Analysis 1747 Using starch digestibility calculation as an example, the following equations were used to calculate the di-

4 1748 Kaczmarek et al. Table 2. Growth performance of broiler chickens (1 14 d) fed corn-soybean meal-based diets supplemented with amylase or amylase and protease enzymes (experiment 1) Feed intake (g/bird) BW gain (g/bird) Feed/gain (g/g) Treatment 1 7 d 7 14 d 1 14 d 1 7 d 7 14 d 1 14 d 1 7 d 7 14 d 1 14 d Control (no enzyme) Amylase Amylase 2 + protease Pooled SEM Probability Means of 9 replicate cages of 5 birds each. 2 Supplied 10,000 U of amylase and 10 U of amyloglucosidase per kilogram of diet. 3 Supplied 4,000 U of protease per kilogram of diet. gestibility of various dietary components and AME n content of experimental diets: Digestibility (%) = {1 [(IM %diet /IM %digesta/excreta ) (starch %digesta/excreta /starch %diet )]}, and AME n (kcal/kg) = GE kcal/kg of diet [GE kcal/kg of excreta (IM %diet /IM %excreta )] 8.22 {N %diet [N %excreta (IM %diet /IM %excreta )]}, where GE = gross energy; N = nitrogen; IM = internal marker (chromic oxide in experiment 1 or titanium oxide in experiment 2); and 8.22 = the energy equivalent of uric acid nitrogen (i.e., 8.22 kcal/kg of uric acid nitrogen). Statistical differences between treatments in experiment 1 were calculated by one-way ANOVA using the GLM procedure of SAS (SAS Institute Inc., Cary, NC). Experiment 2 was a factorial experiment applied to a completely randomized design. All data were subjected to 3-way ANOVA using the GLM procedure of SAS (SAS Institute Inc.) to determine the effect of particle size, NSP enzyme addition, amylase addition, and their interactions. Differences among diet means were determined using a Duncan s means comparison when the significance of the factor was P < Experiment 1 RESULTS AND DISCUSSION Growth performance results for broiler chickens fed corn-sbm diets supplemented with either amylase and amyloglucosidase or amylase, amyloglucosidase, and protease are summarized in Table 2. No effect of enzyme supplementation (P > 0.05) on growth performance was observed. Body weight gain and FCR values were similar for the control and the amylase or amylase plus protease supplemented diets in both wk 1 and 2 of the experiment. Although similar apparent ileal starch digestibility values were observed (Table 3), protein digestibility decreased (P < 0.05) slightly for birds fed the amylase or amylase plus protease supplemented diets. Total tract digestibility of starch for the control and the enzymesupplemented diets were similar and were followed by nearly identical AME n values. The use of amylase and protease in poultry diets to improve nutrient digestibility and growth performance has been proposed (Zanella et al., 1999; Cafe et al., 2002; Gracia et al., 2003; Iji et al., 2003; Cowieson and Adeola, 2005; Kaczmarek et al., 2009). Some authors (Noy and Sklan, 1995; Uni et al., 1995) suggest that enzymes, such as amylase and protease, may improve digestibility and growth performance because of limited Table 3. Apparent ileal, total tract starch and protein digestibility, and diet AME n content of cornsoybean meal-based diets supplemented with amylase or amylase and protease (experiment 1) Treatment Starch digestibility (%) Ileal Total tract Ileal protein digestibility (%) AME n (kcal/kg) Control (no enzyme) a 3,129 Amylase b 3,129 Amylase 3 + protease b 3,106 Pooled SEM Probability a,b Means within columns with no common superscripts differ significantly (P < 0.05). 1 Means of 4 pooled samples of 3 birds each. 2 Means of 9 samples of 5 birds each. 3 Supplied 10,000 U of amylase and 10 U of amyloglucosidase per kilogram of diet. 4 Supplied 4,000 U of protease per kilogram of diet.

5 pancreatic enzyme synthesis during the first days posthatch. In disagreement with earlier research (Noy and Sklan, 1995; Zanella et al., 1999; Gracia et al., 2003; Meng and Slominski, 2005), the present study demonstrated that starch digestibility in birds fed a corn-sbm diet was as high at the ileal level as when measured by excreta collection, and no effect of amylase supplementation was observed. Earlier research (Zanella et al., 1999) showed that digestion of starch in the small intestine is incomplete and continues in the lower gut as a result of microbial fermentation. However, the current study, as well as those by Weurding et al. (2001) and Gracia et al. (2003), showed no difference between ileal and total tract starch digestibility. In addition, the total tract starch digestibility value determined in the current study agreed well with those reported earlier for a corn-based diet fed to broiler chickens (Weurding et al., 2001; Parsons, 2004; Meng and Slominski, 2005). It could be hypothesized that the undigested starch fraction is not fermented to any significant extent in the hindgut of the chicken, which is consistent with the data of Kussaibati et al. (1982), who reported similar amounts of undigested starch in conventional and germ-free birds. As starch is the most important dietary energy source, its high digestibility resulted in high AME n values (Table 3), which only slightly increased the expected values when formulating the diets (Table 1). Contrary to earlier research indicating that very young chicks have reduced ability to digest soybean protein (Parsons, 2004), no effect of protease addition on protein utilization was noted in the current study, with birds fed the control treatment showing the highest protein digestibility values at the ileal level. Depressed protein digestibility in the presence of amylase with or without protease addition observed in the current study does not agree with the data of some research (Zanella et al., 1999; Gracia et al., 2003). However, the results of the current study agree with those of Mahagna et al. (1995), who observed some depression in feed utilization from 1 to 7 d of age in birds fed sorghum-sbm diets supplemented with protease and amylase. Those authors suggested that the depression in nutrient digestibility may be a consequence of reduced pancreatic amylase and protease secretions. In this regard, the reduction in protein digestibility noted in the current study may have been influenced by lower trypsin and chymotrypsin secretions due to exogenous enzymes presence at higher concentrations in the enzyme-supplemented birds than those of the birds in the control treatment (Snook and Meyer, 1964). Experiment 2 EXOGENOUS ENZYMES IN BROILER CHICKEN NUTRITION As illustrated in Table 4, the diets containing fine corn particle sizes (i.e., GMD of 482 µm) had no effect on FI from 1 to 21 d of age, except for the first 7 d when birds consumed more feed (P < 0.5). Birds fed 1749 diets containing fine corn particle sizes had lower BWG (P < 0.05) than those fed diets containing conventional ground corn (i.e., GMD of 736 µm). Feed conversion ratio results demonstrated a particle size effect, with birds fed diets containing fine corn having higher FCR than those fed the conventionally ground corn. Amerah et al. (2008) demonstrated that coarse grinding of corn may improve broiler chicken performance, which agrees with the negative effect of fine corn particle size observed in the current study. In contrast, Amerah et al. (2007) and Reece et al. (1986) showed that pelleting minimized the differences in particle size distribution of diets consisting of wheat or corn, with particle sizes having no effect on growth performance. Conversely, Lentle et al. (2006) and Péron et al. (2005) suggested that differences in particle size distribution existed even after pelleting and inferred that this difference may affect broiler chicken performance. In general, it is believed that diet particle size may influence broiler chicken performance as a consequence of a longer residence time of coarse matter within the gizzard. Slower passage rate through the gizzard may increase the exposure time of nutrients to digestive enzymes, leading to improved nutrient digestibility and energy utilization (Nir et al., 1994; Svihus et al., 2002). Ferket (2000) showed that a well-developed gizzard in birds improved gut motility due to higher levels of cholecystokinin release (Svihus et al., 2004), which, in turn, stimulated the secretion of pancreatic enzymes. In this regard, Jacobs et al. (2010) clearly showed that gizzard weight increased as dietary corn particle size increased. The overall FCR values for birds from 1 to 21 d of age showed that NSP enzyme supplementation increased feed utilization, but only when conventional corn (i.e., GMD of 736 µm) was used (P < 0.05), although an increase in BWG with NSP enzyme supplementation to the finely ground corn diet was noted. Amylase supplementation influenced FI (P < 0.05), but BWG at 21d of age was unaffected when using a conventionally ground corn and increased when fine corn particles were used. From 1 to 21 d of age, the use of amylase tended (P = 0.09) to affect FCR, but the effect was only significant when finely ground corn was used. It is possible that fine grinding of corn lead to better amylase access to starch granules and, as a consequence, resulted in higher starch utilization. However, ileal starch digestibility observed in the current study does not confirm this hypothesis (Table 5), as only a trend (P = 0.133) in improved ileal starch digestibility was observed with amylase addition. However, the use of NSP enzyme in concert with amylase improved (P = 0.025) ileal starch digestibility and AME n contents of diets containing both conventional and finely ground corn. Some authors (Mahagna et al., 1995; Gracia et al., 2003; Cowieson et al., 2004) have suggested that the use of exogenous enzymes could improve bird performance via limiting endogenous amino acid loss. Cowieson et al. (2004) suggested that endogenous losses were

6 1750 Kaczmarek et al. Table 4. Growth performance of broiler chickens (1 to 21 d) fed corn-soybean meal-based diets containing corn of different particle size without or with enzyme supplementation (experiment 2) Feed intake (g/bird) BW gain (g/bird) Feed/gain (g/g) Treatment 1 7 d 7 14 d d 1 21 d 1 7 d 7 14 d d 1 21 d 1 7 d 7 14 d d 1 21 d Corn (conventional, 736 µm) Control (no enzyme) 140 c,1 371 c 507 a 1,022 ab cde 397 ab 808 bc 1.12 c 1.30 cde 1.28 bc 1.27 cd Nonstarch polysaccharide (NSP) enzyme bc 379 bc 494 ab 1,020 ab ab 404 ab 830 ab 1.15 bc 1.25 e 1.23 d 1.23 e Amylase abc 376 bc 500 ab 1,023 ab abc 397 ab 822 bc 1.17 abc 1.25 e 1.26 dc 1.25 de NSP enzyme + amylase 142 bc 395 a 517 a 1,056 a a 413 a 854 a 1.11 c 1.26 de 1.25 dc 1.24 de Corn (fine, 482 µm) Control (no enzyme) 143 bc 374 c 474 b 992 b e 351 c 750 d 1.15 bc 1.37 a 1.35 a 1.32 a NSP enzyme 155 a 378 bc 494 ab 1,025 ab de 383 b 789 c 1.23 a 1.35 ab 1.30 bc 1.30 ab Amylase 154 ab 391 ab 512 a 1,054 a bcd 394 ab 818 bc 1.18 abc 1.34 abc 1.31 abc 1.29 bc NSP enzyme + amylase 153 ab 382 abc 504 a 1,039 a bcd 382 b 802 bc 1.21 ab 1.31 bcd 1.31 ab 1.30 ab Pooled SEM Source of variation Corn particle size < <0.001 <0.001 < <0.001 <0.001 <0.001 NSP enzyme Amylase < < Particle size NSP enzyme Particle size amylase NSP enzyme amylase Particle size NSP enzyme amylase a e Means within a columns with no common superscripts differ significantly (P < 0.05). 1 Means of 9 replicate cages of 5 birds each. 2 Supplied 600 U of xylanase, 100 U of glucanase, 300 U of cellulase, 600 U of pectinase, 30 U of mannanase, and 30 U of galactanase per kilogram of diet. 3 Supplied 10,000 U of amylase and 10 U of amyloglucosidase per kilogram of diet.

7 EXOGENOUS ENZYMES IN BROILER CHICKEN NUTRITION 1751 Table 5. Apparent ileal and total tract starch, nonstarch polysaccharides (NSP) digestibility, and AME n content of diets containing corn of different particle sizes without or with amylase and NSPdegrading enzyme supplementation (experiment 2) Treatment Ileal NSP (%) Total tract Ileal starch (%) AME n (kcal/kg of diet) Corn (conventional, 736 µm) Control (no enzyme) 0.3 b, cd, ,972 c NSP enzyme ab 16.7 b ,042 ab Amylase b 11.7 bcd ,005 bc NSP enzyme + amylase 14.9 a 29.3 a ,070 a Corn (fine, 482 µm) Control (no enzyme) 2.5 bc 6.3 d ,852 d NSP enzyme 0.2 c 9.9 cd ,009 abc Amylase 0.9 c 8.5 d ,950 c NSP enzyme + amylase 9.4 abc 14.4 bc ,977 c Pooled SEM Source of variation Particle size < <0.001 NSP enzyme < <0.001 Amylase < Particle size NSP enzyme Particle size amylase NSP enzyme amylase Particle size NSP enzyme amylase a d Means within columns with no common superscripts differ significantly (P < 0.05). 1 Means of 4 pooled samples of 3 birds each. 2 Means of 9 samples of 5 birds each. 3 Supplied 600 U of xylanase, 100 U of glucanase, 300 U of cellulase, 600 U of pectinase, 30 U of mannanase, and 30 U of galactanase per kilogram of diet. 4 Supplied 10,000 U of amylase and 10 U of amyloglucosidase per kilogram of diet. decreased by enzymatic degradation of antinutritional factors, whereas Gracia et al. (2003) proposed that exogenous amylase and protease may supplement endogenous production of enzymes. In the current study, diet particle size affected NSP degradation (P < 0.038). In addition, the use of NSP-degrading enzymes had a positive effect on the ileal utilization of NSP, resulting in a significant particle size by NSP enzyme interaction. Nonstarch polysaccharides and starch digestibilities, as well as energy utilization, were indicative of a slower digesta passage rate when coarse corn was used. A lower total tract NSP digestibility when fine material was used may be related to the increase in passage rate and a limited exposure of the cell wall material to microbial fermentation. In the current study, when a combination of enzymes was used, improved growth performance and energy utilization were noted. These results confirm that the addition of NSP-degrading enzymes to corn-sbm diets improves NSP degradation, as documented by earlier studies (Kocher et al., 2003; Meng and Slominski, 2005). The mode of action of NSP enzymes, based on elimination of the physical barrier of endosperm cell walls and nutrient encapsulation, provides an explanation as to why the enzymes applied in the current study improved FCR in birds fed diets containing conventional corn particle size, whereas no such effect was observed when finely ground corn was used. The NSPdegrading enzymes may release portions of starch and protein encapsulated by the cell wall structure, thus facilitating additional energy utilization. Wu and Ravindran (2004) found that the efficacy of supplemental xylanase was enhanced in the presence of whole wheat in broiler diets. Conversely, Kim et al. (2005) failed to show any effect of wheat particle size or xylanase supplementation on the growth performance of pigs. In the present study, enzyme supplementation increased AME n content to a greater extent in diets containing conventionally ground corn as opposed to finely ground corn. Regardless of amylase addition, NSP-degrading enzymes improved AME n level of diets containing both conventionally and finely ground corn. It is believed that NSP hydrolysis products can be fermented by the resident microflora into short-chain fatty acids, and thus contribute to the increase in energy utilization and diet AME n content. However, Engberg et al. (2002) found only a small difference in short-chain fatty acid concentration when broiler chickens were fed fine or coarse ground feed either as mash or pellets. Kilburn and Edwards (2001) also reported improvements in FCR and TME when diets included medium ground corn rather than very coarse corn. Similar findings were noted in the current study, as a consistent effect of corn particle size was noted for improved NSP degradation due to the use of NSP enzyme with or without amylase addition. The greatest improvement, however, was observed when NSP enzymes were used in combination with amylase, which resulted in significant NSP enzyme by amylase and particle size by NSP enzyme by amylase interactions. It can be concluded from the current study that the digestive enzyme deficiency in young chickens may not

8 1752 Kaczmarek et al. be as pronounced as originally thought. Consequently, supplementation of diets for young poultry with starchand protein-hydrolyzing enzymes may result in little or no response. No synergistic effect of NSP enzyme and amylase was noted. Different corn particle sizes may contribute to the variability in growth performance observed with amylase supplementation. It can also be suggested that NSP enzymes would improve the nutritive value of corn-based diets when coarse grinding of corn (i.e., GMD of ~700 µm) is employed. ACKNOWLEDGMENTS The authors express many thanks to Thomas Davie and Harry Muc of the Department of Animal Science, University of Manitoba, for their technical assistance. Funding for this study was provided by the Natural Science and Engineering Research Council of Canada and Canadian Bio-Systems Inc., Calgary, Alberta, Canada. REFERENCES Amerah, A. M., R. G. Lentle, and V. 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