Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Hadrosaur diet wikipedia , lookup
Gluten-free diet wikipedia , lookup
Human nutrition wikipedia , lookup
Calorie restriction wikipedia , lookup
Vegetarianism wikipedia , lookup
Ketogenic diet wikipedia , lookup
Raw feeding wikipedia , lookup
Diet-induced obesity model wikipedia , lookup
Growth performance and nutrient utilization of broiler chickens fed diets supplemented with phytase alone or in combination with citric acid and multicarbohydrase T. A. Woyengo,* B. A. Slominski,*1 and R. O. Jones† *Department of Animal Science, University of Manitoba, Winnipeg, Canada R3T 2N2; and †Canadian Bio-Systems Inc., Calgary, Canada T2C 0J7 ABSTRACT An experiment was conducted to determine the effect of supplementing a corn-soybean meal-based diet with phytase alone or in combination with citric acid (CA) or multicarbohydrase, a preparation containing nonstarch polysaccharide-degrading enzymes, or both, on growth performance, nutrient utilization, and bone mineralization. A total of 360 one-day-old broiler chicks were assigned to 6 dietary treatments, consisting of 12 pens of 5 birds each, and were fed experimental diets from 1 to 21 d of age. The diets included a positive control (0.46% nonphytate P; 1.1% Ca) and a negative control (NC; 0.26% nonphytate P; 0.89% Ca) without or with phytase (600 U/kg) alone, phytase plus CA (5 g/kg), phytase plus multicarbohydrase (Superzyme OM; 0.6 g/kg), or phytase (Ronozyme P-CT) plus CA and multicarbohydrase. Birds fed the positive control diet had higher (P < 0.05) BW gain (764 vs. 594 g/21 d) and tibia ash content (50.0 vs. 38.3%) than those fed the NC diet. Phytase improved (P < 0.05) BW gain (632 g/21 d), which increased further (P < 0.05) to 673 g/21 d for the phytase plus multicarbohydrase diet. In contrast to phytase alone, phytase plus multicarbohydrase supplementation improved (P < 0.05) feed conversion ratio of the NC diet from 1.37 to 1.32. Tibia ash content for the NC diet increased (P < 0.05) from 38.3 to 42.4% due to phytase addition. Phytase improved (P < 0.05) ileal digestibility of P from 29.5 to 43%, and the addition of CA or multicarbohydrase, or both, to a phytase-supplemented diet further increased (P < 0.05) P digestibility to 51.5, 53.4, and 54.3%, respectively. Phytase addition improved (P < 0.05) diet AMEn content from 2,959 to 3,068 kcal/kg, which tended (P < 0.06) to increase further with CA (3,150 kcal/ kg) or multicarbohydrase (3,142 kcal/kg) addition. No beneficial interactions were detected between CA and multicarbohydrase for all response criteria measured. Results show that addition of multicarbohydrase to the phytase-supplemented broiler diets improved nutrient utilization and growth performance. Key words: broiler, phytase, multicarbohydrase, citric acid, nutrient utilization 2010 Poultry Science 89:2221–2229 doi:10.3382/ps.2010-00832 INTRODUCTION Approximately two-thirds of P in feedstuffs of plant origin is poorly digested by poultry because it is bound to phytic acid (PA), which is poorly hydrolyzed by endogenous enzymes of poultry (Ravindran et al., 1995). As a result, inorganic sources of P, which are expensive, are added to feeds to meet P requirements of poultry (Selle and Ravindran, 2007). In addition, the unabsorbed PA-bound P is discharged to environment, leading to environmental pollution (Selle and Ravindran, 2007). The negative effects that are associated with PA can be alleviated, in part, by the use of exogenous phytase, ©2010 Poultry Science Association Inc. Received April 7, 2010. Accepted June 21, 2010. 1 Corresponding author: [email protected] which degrades PA (Selle and Ravindran, 2007). Results from several studies have shown increased P digestibility and utilization and hence reduced P excretion into the environment due to phytase addition to poultry diets (Applegate et al., 2003; Penn et al., 2004; Angel et al., 2006; Leytem et al., 2007). The liberation of P from PA by phytase has, however, been far from complete (Olukosi et al., 2007; Woyengo et al., 2008) and averaged only 29% of phytate P present in poultry diets. This does not account for the 0.1% reduction in available P content commonly used for phytase-supplemented poultry diets. Several methods including dietary supplementation with nonstarch polysaccharide (NSP)-degrading carbohydrases and citric acid (CA), which have potential to improve the release of P from PA by phytase, have been investigated. The NSP-degrading enzymes have been shown to increase nutrient utilization in poultry 2221 2222 Woyengo et al. due to eliminating the nutrient-encapsulating effect of cell walls and reduction of digesta viscosity (Kim et al., 2005). The NSP-degrading enzymes may also increase the efficacy of phytase due to elimination of PA-chelating effects of NSP (Kim et al., 2005). This is because NSP have the capacity to bind multivalent cations (Debon and Tester, 2001), which associate with PA in both feedstuffs and in digesta. There is, however, limited and inconsistent information on the effect of adding NSP-degrading carbohydrases to phytasesupplemented diet on nutrient utilization by poultry. For instance, Ravindran et al. (1999) and Selle et al. (2003) observed greater improvement in nutrient digestibility and performance of broilers fed wheat-based diets when phytase and xylanase were supplemented in combination than when phytase was supplemented alone, whereas Wu et al. (2004) and Woyengo et al. (2008) did not observe any beneficial effect of adding xylanase to phytase-supplemented diets. In these studies, the NSP-degrading enzymes used were targeting the major NSP of cereal grains used in the diets (i.e., arabinoxylan). However, oilseed meals, which account for approximately 50% of PA in the practical poultry diets, contain NSP whose chemical structure is different from those found in cereal grains (Choct, 1997). Furthermore, cereal grains do not only contain one type of NSP; in addition to arabinoxylan, they contain some β-glucan and appreciative amounts of cellulose (Choct, 1997). Thus, supplementation of phytase plus a preparation of NSP-degrading enzymes to target all of the major NSP in the diet may result in better P and other nutrients utilization. Citric acid can improve the efficacy of phytase because it can chelate multivalent cations like Ca that form insoluble complexes with PA, thereby increasing PA solubility (Maenz et al., 1999; Boling et al., 2000). Furthermore, the organic acids like CA can reduce the pH of the digesta (Radcliffe et al., 1998), which can then result in increased dissociation between PA and minerals (Maenz et al., 1999) and increased activity of most phytases, which express their optimal activity at low pH (Simon and Igbasan, 2002). Snow et al. (2004) observed improved P utilization in broilers due to addition of CA to a phytase-supplemented diet at 30 or 40 g/kg. Therefore, supplemental phytase, NSP-degrading carbohydrases, and CA may additively improve performance and nutrient utilization by poultry due to NSP hydrolysis and thus an increase in PA availability, whose solubility could be increased further by CA supplementation. However, there is a lack of information on the effect of supplementing a combination of NSP-degrading carbohydrases and CA to a phytasesupplemented diet on nutrient utilization by poultry. Therefore, the objective of the current study was to determine the effect of supplementing a corn-soybean meal-based diet with phytase alone or in combination with CA or a preparation of NSP-degrading enzymes (multicarbohydrase), or both, on growth performance, bone mineralization, and nutrient digestibility and re- tention in broiler chickens. To optimize the response from these supplements, marginal levels of available (nonphytate) P (0.26%) and Ca (0.89%) were used for the enzyme- and CA-supplemented diet. MATERIALS AND METHODS Birds and Housing Three hundred sixty 1-d-old male broiler chicks of the Ross strain were obtained from a commercial hatchery and were used in this experiment, which lasted for 21 d. The chicks were individually weighed upon arrival and divided into 72 groups of 5 birds balanced for BW. They were then group-weighed, and each group was housed in a cage in electrically heated Petersime battery brooders (Petersime Incubator Company, Gettysburg, OH). The brooder and room temperatures were set at 32 and 29°C, respectively, during the first week. Light was provided for 24 h throughout the experiment. The birds were handled in accordance with guidelines described by the Canadian Council on Animal Care (CCAC, 1993). Experimental Diets Six corn-soybean meal-based diets were used in this study. The diets included a positive control (PC) and a negative control (NC) (Table 1) without or with phytase (Ronozyme P-CT, DSM Nutritional Products Inc., Parsippany, NJ; 600 phytase units/kg) alone, phytase plus CA (5 g/kg), phytase plus a multicarbohydrase (Superzyme OM, Canadian Bio-System Inc., Calgary, Alberta, Canada; 0.6 g/kg), or phytase plus a combination of CA and the multicarbohydrase. One phytase unit is defined as the amount of enzyme that liberates 1 µmol of inorganic P per minute from sodium phytate at a pH of 5.0 and temperature of 37°C. The multicarbohydrase enzyme supplement supplied 1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of amylase, and 120 U of protease per kilogram of diet. Both the phytase and the multicarbohydrase used in the current study are produced via a submerged fermentation process. The PC diet was formulated to meet or exceed the NRC (1994) nutrient requirements for broiler chickens. The NC diet was the same as the PC diet except that the Ca and nonphytate P levels were reduced by 0.20 percentage points. The phytase, CA, and multicarbohydrase were obtained from Canadian Bio-Systems Inc. All diets contained titanium oxide (0.3%) as an indigestible marker and were pelleted at 70°C and then crumbled. Experimental Procedure The 6 diets were randomly allocated to 72 cages to give 12 replicates per diet. The BW and feed consumption for each cage were determined weekly after with- 2223 ENZYMES AND CITRIC ACID IN BROILER DIETS drawing feed for 3 h before weighing on d 7, 14, and 21. On d 20, excreta samples from each cage were collected over a 3-h period and immediately frozen and stored at −20°C until needed for determination of apparent nutrient retention. Care was taken during the collection of excreta samples to avoid contamination from feathers and other foreign materials. On d 22, all of the birds were killed by cervical dislocation, and left tibiae and contents of the lower half of ileum (from the middle of the ileum to approximately 1 cm above the ileocecal junction) were obtained and stored at −20°C for determination of tibia ash and apparent ileal nutrient digestibility, respectively. Sample Preparation and Chemical Analyses The tibiae was defleshed after autoclaving at 121°C for 1 min and dried in an oven at 45°C for 3 d. They were then fat-extracted using hexane for 3 d, dried in a fume hood for 1 d to allow the hexane to evaporate, and ashed at 550°C in a muffle furnace for 12 h for the determination of tibia ash. Ileal and excreta samples were freeze-dried and finely ground in a grinder (CBG5 Smart Grind, Applica Consumer Products Inc., Shelton, CT) to pass through a 1-mm screen and were thoroughly mixed before analysis. Diet samples were similarly ground and thoroughly mixed before analysis. The samples were analyzed for gross energy (GE), total and phytate P, CP (N × 6.25), DM, N, Ca, and Ti contents. Dry matter was determined according to the method of AOAC (1990, method 925.09), and GE was determined using the Parr adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, IL). Nitrogen was determined using a N analyzer (model NS-2000, Leco Corporation, St. Joseph, MI). Titanium was determined according to the procedure described by Lomer et al. (2000) and read on a Varian inductively coupled plasma mass spectrometer (Varian Inc., Palo Alto, CA). Samples for Ca analysis were ashed for 12 h and digested according to AOAC (1990) procedures (method 990.08) and read on a Varian inductively coupled plasma mass spectrometer (Varian Inc.). Phytate P was determined by the method described by Haug and Lantzsch (1983). Total P was determined using the AOAC (1990) method 965.17. Nonphytate P was calculated as total P minus phytate P. Due to interference from dietary carbohydrates in many enzyme assays, only phytase, xylanase, and mannanase activities were determined in complete diets. Phytase activity was determined as described by Slominski et al. (2007), whereas xylanase and mannanase activities were assayed using Xylazyme AX and Mannazyme tablets, respectively (Megazyme International Ltd., Bray, Ireland). Calculations and Statistical Analysis Apparent ileal digestibility and apparent retention of nutrients in diets were calculated by the indicator method as described by Woyengo et al. (2008), whereas AMEn was determined as described by Meng and Slominski (2005). Data were analyzed using GLM procedure (SAS, 2002) in a completely randomized design. All of the treatment means were compared using Duncan’s multiple range test. The last 4 treatments were additionally analyzed as a 2 × 2 factorial to determine the main effects of CA and multicarbohydrase and interactions between the same (CA and multicarbohy- Table 1. Composition and analysis of basal diets Item Ingredient (% of diet) Corn Soybean meal (46% CP) Canola meal Canola oil Limestone Dicalcium phosphate Vitamin premix2 Mineral premix3 Methionine Titanium dioxide4 Total Calculated composition5 ME (kcal/kg) CP (%) Ca (%) Total P (%) Nonphytate P (%) Phytate P (%) Lys (%) Met (%) Met + Cys (%) Thr (%) Analyzed composition AMEn (kcal/kg) CP (%) Ca (%) Total P (%) Phytate P (%) Nonphytate P (%) Positive control 47.9 32.3 10.0 5.0 1.67 1.5 1.00 0.50 0.15 0.30 100.0 2,952 22.1 1.10 0.78 0.46 0.32 1.27 0.50 0.89 0.85 2,903 23.3 1.08 0.77 0.39 0.38 NC1 49.8 31.9 10.0 4.2 1.64 0.50 1.00 0.50 0.14 0.30 100.0 2,953 22.1 0.89 0.57 0.26 0.31 1.27 0.50 0.88 0.85 2,959 23.1 0.81 0.57 0.37 0.20 1The negative control (NC) diet was supplemented with either phytase (600 phytase units/kg of diet), phytase + citric acid (5 g/kg of diet), phytase + multicarbohydrase (Superzyme OM at 0.6 g/kg of diet, Canadian Bio-System Inc., Calgary, Alberta, Canada), or phytase + citric acid + multicarbohydrase. Enzyme and citric acid premixes were added at the expense of corn. The multicarbohydrase preparation supplied 1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of amylase, and 120 U of protease per kilogram of diet. The determined activities (U/kg of feed) of phytase, xylanase, and mannanase were, respectively, 28, 103, and 40 for the positive control diet; 26, 96, and 48 for the NC diet; 526, 105, and 52 for the NC diet + phytase; 506, 85, and 62 for the NC diet + phytase + citric acid; 488, 1,114, and 336 for the NC diet + phytase + multicarbohydrase; and 475, 1,220, and 346 for the NC diet + phytase + citric acid + multicarbohydrase. Due to interference from dietary carbohydrates in many enzyme assays, only phytase, xylanase, and mannanase were determined in complete diets. 2Vitamin premix provided the following per kilogram of diet: vitamin A, 8,250 IU; vitamin D3, 1,000 IU; vitamin E, 11 IU; vitamin B12, 0.012 mg; vitamin K, 1.1 mg; niacin, 53 mg; choline, 1,020 mg; folic acid, 0.75 mg; biotin, 0.25 mg; and riboflavin, 5.5 mg. 3Mineral premix provided the following per kilogram of diet: Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se, 0.1 mg; I, 0.36 mg; and Na, 1.6 g. 4Sigma T8141, Oakville, Ontario, Canada. 5Calculated nutrient content was based on ingredient composition data from NRC (1994). 2224 Woyengo et al. drase). All statements of significance are based on P ≤ 0.05. RESULTS Analyzed chemical composition of the PC and NC diets and enzyme activities of the 6 dietary treatments are presented in Table 1. The analyzed values of AMEn, CP, Ca, and total P were close to calculated values, whereas the analyzed nonphytate P values were slightly lower than the calculated values. Data on the effect of dietary treatments on broiler performance are presented in Table 2. Birds fed the PC diet had higher BW gain and feed intake than those fed the NC diet. The birds fed PC and NC diets had, however, a similar feed conversion ratio (FCR). Phytase supplementation to the NC diet improved BW gain. Also, the addition of multicarbohydrase to the phytase-supplemented diet resulted in a further increase in BW gain. There was, however, no effect of adding CA to the phytase-supplemented diet on BW gain. Although the BW gain increased after phytase and multicarbohydrase supplementations, the value did not reach that of the PC diet. Phytase supplementation to the NC diet tended to increase (P = 0.093) feed intake. However, supplementation of phytase together with CA or multicarbohydrase, or both, to the NC diet resulted in a significant increase in feed intake, but the values did not reach that of the PC diet. The differences between feed intake of birds fed the NC diet plus phytase alone and those fed the NC diet plus phytase with CA or multicarbohydrase, or both, were, however, not significant. Phytase supplementation to the NC diet did not affect FCR. However, supplementation of phytase together with multicarbo- hydrase resulted in improved FCR compared with the NC diet. No interactions were detected between CA and multicarbohydrase in phytase-supplemented diets on BW gain, feed intake, and FCR. Table 3 shows the tibia ash and apparent ileal P digestibility values. The tibia ash values were higher for the PC diet than for the NC diet. Phytase supplementation improved tibia ash values, but the addition of CA or multicarbohydrase, or both, to the phytasesupplemented diet did not result in a significant further increase in tibia ash. And like BW gain and feed intake, tibia ash values for the supplemented diets did not reach that of the PC diet. Phytase supplementation to the NC diet improved apparent ileal digestibility of total, nonphytate, and phytate P, and the addition of CA or multicarbohydrase, or both, to the phytasesupplemented diet further increased the digestibility of total and phytate P but not that of nonphytate P. No interactions were observed between CA and multicarbohydrase in phytase-supplemented diets for tibia ash and apparent ileal P digestibility. The effect of dietary treatment on AMEn and apparent retention of DM, GE, N, and total P are presented in Table 4. Phytase supplementation to the NC diet improved (P < 0.05) AMEn and apparent retention of DM, GE, N, and total P. The addition of CA to the phytase-supplemented diet increased the same response criteria. The addition of multicarbohydrase to the phytase-supplemented diet increased the apparent retention of DM and total P. There were interactions between CA and multicarbohydrase in phytasesupplemented diets on AMEn and apparent retention of DM, GE, N, and total P such that CA improved these response criteria only when it was supplemented Table 2. The effect of phytase alone or in combination with citric acid or multicarbohydrase enzyme, or both, on growth performance of broiler chickens from 1 to 21 d of age1 Item Diet Positive control Negative control (NC) NC + phytase2 NC + phytase + CA3 NC + phytase + MC4 NC + phytase + CA + MC SEM Contrasts5 CA MC CA × MC a–dMeans Feed intake (g/bird) BW gain (g/bird) Feed conversion ratio (g of feed:g of gain) 1,027.6a 811.9c 853.0bc 868.2b 890.5b 879.9b 17.1 764.4a 593.6d 631.7c 646.9bc 673.2b 662.4bc 12.1 1.34ab 1.37a 1.35ab 1.34ab 1.32b 1.33ab 0.01 0.870 0.091 0.370 0.850 0.018 0.272 0.966 0.077 0.459 within the same column with different superscripts differ (P < 0.05). are means of 12 replicates (cages) with 5 chicks per cage. 2Supplied 600 U of phytase per kilogram of diet. 3Supplied 5 g of citric acid per kilogram of diet. 4Supplied 1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of amylase, and 120 U of protease per kilogram of diet. 5CA = main effect of citric acid in phytase-supplemented diets; MC = main effect of multicarbohydrase in phytase-supplemented diets; CA × MC = interaction between citric acid and multicarbohydrase in phytasesupplemented diets. 1Data 2225 ENZYMES AND CITRIC ACID IN BROILER DIETS Table 3. The effect of phytase alone or in combination with citric acid or multicarbohydrase enzyme, or both, on tibia ash content and apparent ileal P digestibility in broiler chickens at 21 d of age1 Item Diet Positive control Negative control (NC) NC + phytase3 NC + phytase + CA4 NC + phytase + MC5 NC + phytase + CA + MC SEM Contrasts6 CA MC CA × MC Digestibility (%) Tibia ash (g/tibia) Tibia ash2 (%) Total P Nonphytate P Phytate P 1.21a 0.70c 0.79b 0.80b 0.84b 0.81b 0.03 50.0a 38.3c 42.4b 43.9b 44.0b 44.1b 0.8 47.0ab 29.5c 43.0b 51.6a 53.4a 54.3a 2.7 60.5a 39.9b 57.5a 63.0a 64.7a 65.0a 5.4 34.8b 24.9c 35.0b 45.8a 47.8a 47.6a 3.2 0.519 0.184 0.300 0.273 0.193 0.344 0.069 0.015 0.130 0.590 0.384 0.626 0.133 0.045 0.119 a–cMeans within the same column with different superscripts differ (P < 0.05). are means of 12 replicates (cages) with 5 chicks per cage. 2Percentage of fat-free tibia. 3Supplied 600 U of phytase per kilogram of diet. 4Supplied 5 g of citric acid per kilogram of diet. 5Supplied 1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of amylase, and 120 U of protease per kilogram of diet. 6CA = main effect of citric acid in phytase-supplemented diets; MC = main effect of multicarbohydrase in phytase-supplemented diets; CA × MC = interaction between citric acid and multicarbohydrase in phytase-supplemented diets. 1Data in absence of multicarbohydrase, whereas multicarbohydrase improved these response criteria only when it was supplemented in absence of CA. DISCUSSION The analyzed nonphytate P, but not total P, values for the PC and NC diets were slightly lower than the anticipated (calculated) values, which could be due to differences between the actual nonphytate P content in the ingredients used in the current study and the NRC (1994) nonphytate P values for the same ingredients, which were used in diet formulation. The nonphytate P as a proportion of total P is dependent on several factors, including the type and variety of ingredient and year of harvest (Steiner et al., 2007). Waldroup et al. (2000) and Powell et al. (2008) found a lower dietary concentration of nonphytate P (0.39%) than that recommended by NRC (1994; 0.45%) to be adequate for broilers in the starter phase. The nonphytate P content for the PC diet used in the current study was 0.38%, which may have not limited the performance of broilers. Table 4. The effect of phytase alone or in combination with citric acid or multicarbohydrase enzyme, or both, on AMEn content and apparent retention of DM, gross energy (GE), N, and P in broiler chickens at 21 d of age1 Retention (%) Item Diet Positive control Negative control (NC) NC + phytase2 NC + phytase + CA3 NC + phytase + MC4 NC + phytase + CA + MC SEM Contrasts5 CA MC CA × MC a–dMeans AMEn (kcal/kg) DM GE N Total P 2,903c 2,959c 3,068b 3,150a 3,142ab 3,091ab 27 61.8d 64.8c 67.8b 71.4a 70.7a 69.4ab 0.7 69.9d 71.9c 74.5b 76.5a 76.3ab 75.1ab 0.6 59.1c 61.4c 65.7b 68.9a 67.5ab 67.1ab 1.0 37.7c 38.6c 49.3b 56.3a 54.6a 52.7ab 1.7 0.575 0.782 0.026 0.138 0.518 0.003 0.567 0.783 0.026 0.118 0.975 0.046 0.045 0.484 0.002 within the same column with different superscripts differ (P < 0.05). are means of 12 replicates (cages) with 5 chicks per cage. 2Supplied 600 U of phytase per kilogram of diet. 3Supplied 5 g of citric acid per kilogram of diet. 4Supplied 1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of amylase, and 120 U of protease per kilogram of diet. 5CA = main effect of citric acid in phytase-supplemented diets; MC = main effect of multicarbohydrase in phytase-supplemented diets; CA × MC = interaction between citric acid and multicarbohydrase in phytase-supplemented diets. 1Data 2226 Woyengo et al. The growth performance and tibia ash (bone mineralization) of broilers fed the PC diet were indeed similar to what have been reported by Dilger et al. (2004), Onyango et al. (2004, 2005), Olukosi et al. (2007), and Woyengo et al. (2008) for broilers at 21 d of age fed nutrient-adequate diets. The growth performance and bone mineralization for the NC diet were lower than those for the PC (nutrientadequate) diet, confirming that the NC diet was indeed P-deficient. Phytase supplementation improved growth performance and tibia ash of broilers fed the NC diet, which was due to improved P digestibility. It is a wellknown fact that phytase supplementation results in improved performance and bone mineralization in broilers fed low-P diets (Dilger et al., 2004; Onyango et al., 2005; Angel et al., 2006). In the current study, the addition of CA to the phytase-supplemented diet did not result in significant improvement in growth performance and tibia ash of broilers despite increased ileal P digestibility. When supplemented alone, CA has been shown to improve performance and bone mineralization of broilers fed low-P diets (Boling et al., 2000; BolingFrankenbach et al., 2001; Snow et al., 2004; Liem et al., 2008), indicating improved utilization of P due to CA addition. However, the addition of CA to phytasesupplemented diets for broilers has yielded results that are inconsistent with regard to growth performance and bone mineralization. For instance, Brenes et al. (2003) did not observe improved growth performance and bone mineralization in broilers due to the addition of CA to a phytase-supplemented low-P diet, whereas Snow et al. (2004) reported improved growth performance and bone mineralization of broilers due to addition of CA to a phytase-supplemented low-P diet. It should, however, be noted that the concentration of the nonphytate P in the basal diet used in the study of Snow et al. (2004) was lower (0.13%) and that they observed improved growth performance and bone mineralization due to addition of CA to the phytase-supplemented diet only when vitamin D was also added to the diet or when the dietary Ca:nonphytate P ratio was widened (from 4.8:1 to 5.8:1 wt:wt) by increasing the dietary concentration of Ca. The concentration of the nonphytate P and the Ca:nonphytate P ratio in the basal diet used in the current study (0.26% and 4.1:1 wt:wt, respectively) and that of Brenes et al. (2003; 0.25% and 2.3:1 wt:wt, respectively) were, respectively, higher and narrower than those for the basal diet used in the study of Snow et al. (2004). An increase in the dietary concentration of vitamin D or Ca is known to result in increased absorption of Ca, which is required for incorporation of P into bones (Bronner, 1987). If Ca and P are absorbed in a smaller (narrower) Ca:P ratio than required, then the excess P is lost via urine. Because in the current study and in that of Brenes et al. (2003) the Ca:nonphytate P ratios were narrower and phytase and CA increases P digestibility, the lack of effect of adding CA to phytasesupplemented diets on broiler performance in these studies could be attributed to the fact that the dietary Ca was not adequate enough to allow for utilization of P that was made available by CA. It would thus be interesting to see the effect of combining phytase with CA in diets containing various ratios of Ca:nonphytate P on broiler performance, bone mineralization, and excretion of P via urine. Addition of multicarbohydrase to the phytase-supplemented diet for broilers resulted in further improvement in growth performance but not in tibia ash content. Because the addition of CA or multicarbohydrase to the phytase-supplemented diet resulted in improved P digestibility, but the improved P digestibility due to CA did not result in improved growth performance and tibia ash, the improved growth performance due to multicarbohydrase could have been due to improved availability of other nutrients by enzyme supplementation. The multicarbohydrase supplement used in the current study has been shown to improve the digestibility of several other nutrients including starch, protein, and oil in broilers (Meng and Slominski, 2005; Meng et al., 2006). Zyla et al. (1999), Selle et al. (2003), Cowieson and Adeola (2005) also observed improved growth performance of broilers when carbohydrases were added to phytase-supplemented diets. Citric acid and multicarbohydrase were not additive with regard to improving performance, which could be attributed to the lack of effect of combining these 2 supplements on nutrient digestibility. Growth performance and the tibia ash for the supplemented diets did not reach that of the PC diet, which is contrary to the findings of Onyango et al. (2004), Silversides et al. (2004), and Olukosi et al. (2007), who observed similar performance of broilers fed PC and phytase-supplemented low-P diets. It should, however, be noted that the nonphytate P content in the lowP diets used in the studies of Onyango et al. (2004), Silversides et al. (2004), and Olukosi et al. (2007) was either equal to the requirement or reduced by only 0.1 percentage point. In the current study, the analyzed nonphytate P content in the NC diet was 0.20%. Thus, the failure of growth performance and the tibia ash for the supplemented diets to reach that of the PC diet could have been due to the lower nonphytate P content in the basal diet than in that commonly used in phytase-supplemented diets. Zyla et al. (1999), Dilger et al. (2004), and Angel et al. (2006) have also observed inferior performance of broilers fed phytase-supplemented diets when the nonphytate P content in the diets was less than 0.23%. Phytase supplementation improved ileal phytate and total P digestibilities and retention of total P due to liberation of P from the PA molecule, which has also been reported by several other studies (Dilger et al., 2004; Onyango et al., 2004; Olukosi et al., 2007; Woyengo et al., 2008). The efficacy of phytase with regard to hydrolysis of PA is, however, limited, in part, by the presence of divalent cations like Ca in practical poultry diets, which form insoluble complexes with PA at pH found in the small intestine (Maenz, 2001; Selle and ENZYMES AND CITRIC ACID IN BROILER DIETS Ravindran, 2007). Addition of organic acids to phytasesupplemented diets can improve the efficacy of phytase because the organic acids chelate multivalent cations like Ca, thereby reducing the amount of the cations that are available for binding to PA (Maenz et al., 1999; Boling et al., 2000). Furthermore, the organic acids reduce the pH of the digesta (Radcliffe et al., 1998), which can result in increased dissociation between PA and minerals (Maenz et al., 1999) and thus increase phytase efficacy due to more acidic pH (Simon and Igbasan, 2002). Supplementation of low-P broiler diets with CA has been shown to result in increased bone mineralization (Boling et al., 2000; Snow et al., 2004; Liem et al., 2008), which indicates increased P digestibility. Results from the current study are in agreement with these findings. Addition of multicarbohydrase to the phytase-supplemented diet for broilers resulted in increased ileal digestibility of P. In feedstuffs of plant origin, PA is located within the cells (Prattley and Stanley, 1982). However, cell walls contain NSP, which are poorly digested by poultry and can reduce the availability of PA to phytase by encapsulation, leading to reduced PA degradation (Kim et al., 2005). In addition, NSP have capacity to chelate multivalent cations (Debon and Tester, 2001) and thus may reduce the availability of PA to phytase by binding the cations, which are associated with PA in both feedstuffs and in digesta. Therefore, improved P digestibility in broilers fed the phytase- and multicarbohydrase-supplemented diets could have been due to degradation of NSP by the multicarbohydrase, leading to increased accessibility of phytase to its PA substrate. Cowieson and Adeola (2005) and Olukosi et al. (2007) did not, however, observe increased digestibility of P and other nutrients due to addition of xylanase to a phytase-supplemented corn-soybean mealbased diet. In the current study, the carbohydrase used contained enzymes that can target several NSP in the diet including arabinoxylans, β-glucans, arabinogalactans, mannans, galactomannans, or pectic polysaccharides (Meng et al., 2005), whereas the carbohydrases used in the studies of Cowieson and Adeola (2005) and Olukosi et al. (2007) were most likely targeting only the major NSP (i.e., arabinoxylan) of cereal grains. However, oilseed meals, which account for approximately 50% of PA in the practical poultry diets, contain NSP, whose composition is different from those found in cereal grains. Also, cereal grains do not only contain one type of NSP. Thus, the difference between the current study and those of Cowieson and Adeola (2005) and Olukosi et al. (2007) with regard to the effect of combining phytase and carbohydrases on nutrient digestibility could be attributed to the differences in the carbohydrase supplements used in the studies. Phytase supplementation improved AMEn values of the NC diet, which may have been due to increased digestibility of energy-yielding nutrients such as protein, carbohydrates, and fats. Several other studies have also shown improved energy value for low-P broiler diets 2227 due to phytase supplementation (Ravindran et al., 2001; Onyango et al., 2004; Cowieson et al., 2006). Several mechanisms by which PA (phytase substrate) reduces the digestibility of energy-yielding nutrients have been proposed. They include the following: i) binding to protein in the stomach and small intestine (Prattley et al., 1982; Maenz, 2001; Selle et al., 2006), ii) binding to carbohydrates and lipids in small intestine (Thompson et al., 1987; Selle and Ravindran, 2007), iii) and binding to endogenous enzymes and metal cofactors of enzymes involved in hydrolysis of energy-yielding molecules (Thompson et al., 1987). Phytic acid has indeed been shown to reduce solubility of protein at stomach pH (Kies et al., 2006), solubility of protein in the presence of Ca at small intestinal pH (Prattley et al., 1982), gastric pepsin activity in piglets (Woyengo et al., 2010), trypsin activity in vitro (Singh and Krikorian, 1982), intestinal amylase activity in chickens (Liu et al., 2008), and intestinal lipase activity (Liu et al., 2009). These indicate that PA may reduce the digestibility of energyyielding nutrients by binding to such nutrients and affecting the enzymes involved in their digestion. The increased N retention due to phytase supplementation may also have been due to the liberation of amino acids and digestive enzymes from PA. The addition of CA or multicarbohydrase to the phytase-supplemented diet increased AMEn values of the NC diet, which may have been due to increased PA hydrolysis by CA or multicarbohydrase as evidenced by increased ileal phytate P digestibility by the same supplements. Both CA and multicarbohydrase may also improve energy value of diets by other mechanisms. For instance, CA can acidify the gastrointestinal contents, leading to increased activity of gastric enzymes (Biggs and Parsons, 2008), whereas the carbohydrases can increase the availability of nutrients other than P by hydrolyzing NSP (Bedford and Schuzle, 1998). There was no beneficial effect of combining CA and multicarbohydrase in the phytase-supplemented diet on all response criteria measured in the current study, which was contrary to the expectation. It had been assumed that the addition of a combination of CA and multicarbohydrase to the phytase-supplemented diet would result in increased P digestibility than when they are added individually because of the fact that the multicarbohydrase can hydrolyze NSP to increase the accessibility of phytase to PA and that the activity of phytase and the susceptibility of PA to phytase can be increased by CA. Therefore, the lack of a beneficial effect of combining CA and multicarbohydrase in the phytase-supplemented diet is not clear. It is concluded that under the conditions used in the current study, the addition of multicarbohydrase to the phytase-supplemented low-P and Ca broiler diets can result in improved growth performance and nutrient digestibility and retention, whereas the addition of CA to the phytase-supplemented low-P and Ca broiler diets can result in improved nutrient digestibility and retention. The addition of CA to phytase and multi- 2228 Woyengo et al. carbohydrase-supplemented diets for broilers would, however, not result in further improvement in growth performance and nutrient utilization. ACKNOWLEDGMENTS We thank Canadian Bio-Systems Inc. for providing the enzyme supplements. The assistance provided by A. Rogiewicz and T. Dave (University of Manitoba) with regard to taking care of birds and laboratory analyses, respectively, is highly appreciated. REFERENCES Angel, R., W. W. Saylor, A. D. Mitchell, W. Powers, and T. J. Applegate. 2006. Effect of dietary phosphorus, phytase, and 25-hydroxycholecalciferol on broiler chicken bone mineralization, litter phosphorus, and processing yields. Poult. Sci. 85:1200–1211. AOAC. 1990. Official Methods of Analysis. 15th ed. AOAC Int., Washington, DC. Applegate, T. J., B. C. Joern, D. L. Nussbaum-Wagler, and R. Angel. 2003. Water-soluble phosphorus in fresh broiler litter is dependent upon phosphorus concentration fed but not on fungal phytase supplementation. Poult. Sci. 82:1024–1029. Bedford, M. R., and H. Schuzle. 1998. Exogenous enzymes for pigs and poultry. Nutr. Res. Rev. 11:91–114. Biggs, P., and C. M. Parsons. 2008. The effects of several organic acids on growth performance, nutrient digestibilities, and cecal microbial populations in young chicks. Poult. Sci. 87:2581–2589. Boling, S. D., D. M. Webel, I. Mavromichalis, C. M. Parsons, and D. H. Baker. 2000. The effects of citric acid on phytate-phosphorus utilization in young chicks and pigs. J. Anim. Sci. 78:682–689. Boling-Frankenbach, S. D., J. L. Snow, C. M. Parsons, and D. H. Baker. 2001. The effect of citric acid on the calcium and phosphorus requirements of chicks fed corn-soybean meal diets. Poult. Sci. 80:783–788. Brenes, A., A. Viveros, I. Arija, C. Centeno, M. Pizarro, and C. Bravo. 2003. The effect of citric acid and microbial phytase on mineral utilization in broiler chicks. Anim. Feed Sci. Technol. 110:201–219. Bronner, F. 1987. Intestinal calcium absorption: Mechanisms and applications. J. Nutr. 117:1347–1352. CCAC. 1993. Guide to the Care and Use of Experimental Animals. 2nd ed. Vol.1. Can. Counc. Anim. Care, Ottawa, Ontario, Canada. Choct, M. 1997. Feed non-starch polysaccharides: Chemical structures and nutritional significance. Feed Mill. Int. June, 13–27. Cowieson, A. J., T. Acamovic, and M. R. Bedford. 2006. Supplementation of corn-soy-based diets with an Eschericia coli-derived phytase: Effects on broiler chick performance and the digestibility of amino acids and metabolizability of minerals and energy. Poult. Sci. 85:1389–1397. Cowieson, A. J., and O. Adeola. 2005. Carbohydrases, protease, and phytase have an additive beneficial effect in nutritionally marginal diets for broiler chicks. Poult. Sci. 84:1860–1867. Debon, S. J. J., and R. F. Tester. 2001. In vitro binding of calcium, iron and zinc by non-starch polysaccharides. Food Chem. 73:401–410. Dilger, R. N., E. M. Onyango, J. S. Sands, and O. Adeola. 2004. Evaluation of microbial phytase in broiler diets. Poult. Sci. 83:962–970. Haug, W., and H. J. Lantzsch. 1983. Sensitive method for the rapid determination of phytate in cereal and cereal products. J. Sci. Food Agric. 34:1423–1427. Kies, A. K., L. H. De Jonge, P. A. Kemme, and A. W. Jongbloed. 2006. Interaction between protein, phytate, and microbial phytase. In vitro studies. J. Agric. Food Chem. 54:1753–1758. Kim, J. C., P. H. Simmins, B. P. Mullan, and J. R. Pluske. 2005. The digestible energy value of wheat for pigs, with special ref- erence to the post-weaned animal. Anim. Feed Sci. Technol. 122:257–287. Leytem, A. B., P. W. Plumstead, R. O. Maguire, P. Kwanyuen, and J. Brake. 2007. What aspect of dietary modification in broilers controls litter water-soluble phosphorus: Dietary phosphorus, phytase, or calcium? J. Environ. Qual. 36:453–463. Liem, A., G. M. Pesti, and H. M. Edwards Jr.. 2008. The effect of several organic acids on phytate phosphorus hydrolysis in broiler chicks. Poult. Sci. 87:689–693. Liu, N., Y. Ru, J. Wang, and T. Xu. 2010. Effect of dietary sodium phytate and microbial phytase on the lipase activity and lipid metabolism of broiler chickens. Br. J. Nutr. 103:862–868. Liu, N., Y. J. Ru, F. D. Li, and A. J. Cowieson. 2008. Effect of diet containing phytate and phytase on the activity and messenger ribonucleic acid expression of carbohydrase and transporter in chickens. J. Anim. Sci. 86:3432–3439. Lomer, M. C. E., R. P. H. Thompson, J. Commisso, C. L. Keen, and J. J. Powell. 2000. Determination of titanium dioxide in foods usuing inductively coupled plasma optical emission spectrometry. Analyst (Lond.) 125:2339–2343. Maenz, D. D. 2001. Enzymatic and other characteristics of phytases as they relate to their use in animal feeds. Page 61–84 in Enzymes in Farm Animal Nutrition. M. R. Bedford and G.G. Partridge, ed. CABI Publ., Wallingford, Oxfordshire, UK. Maenz, D. D., C. M. Engele-Schaan, R. W. Newkirk, and H. L. Classen. 1999. The effect of minerals and mineral chelators on the formation of phytase-resistant and phytase-susceptible forms of phytic acid in solution and in a slurry of canola meal. Anim. Feed Sci. Technol. 81:177–192. Meng, X., and B. A. Slominski. 2005. Nutritive values of corn, soybean meal, canola meal, and peas for broiler chickens as affected by a multicarbohydrase preparation of cell wall degrading enzymes. Poult. Sci. 84:1242–1251. Meng, X., B. A. Slominski, L. D. Campbell, W. Guenter, and O. Jones. 2006. The use of enzyme technology for improved energy utilization from full-fat oilseeds. Part I. Canola seed. Poult. Sci. 85:1025–1030. Meng, X., B. A. Slominski, M. Nyachoti, L. D. Campbell, and W. Guenter. 2005. Degradation of cell wall polysaccharides by combinations of carbohydrase enzymes and their effect on nutrient utilization and broiler chicken performance. Poult. Sci. 84:37– 47. NRC. 1994. Nutrient Requirements of Poultry. 9th ed. Natl. Acad. Press, Washington, DC. Olukosi, O. A., A. J. Cowieson, and O. Adeola. 2007. Age-related influence of a cocktail of xylanase, amylase, and protease or phytase individually or in combination in broilers. Poult. Sci. 86:77–86. Onyango, E. M., M. R. Bedford, and O. Adeola. 2004. The yeast production system in which Escherichia coli phytase is expressed may affect growth performance, bone ash, and nutrient use in broiler chicks. Poult. Sci. 83:421–427. Onyango, E. M., M. R. Bedford, and O. Adeola. 2005. Efficacy of an evolved Escherichia coli phytase in diets of broiler chicks. Poult. Sci. 84:248–255. Penn, C. J., G. L. Mullins, L. W. Zelazny, J. G. Warren, and J. M. McGrath. 2004. Surface runoff losses of phosphorus from Virginia soils amended with turkey manure using phytase and high available phosphorus corn diets. J. Environ. Qual. 33:1431–1439. Powell, S., S. Johnston, L. Gaston, and L. L. Southern. 2008. The effect of dietary phosphorus level and phytase supplementation on growth performance, bone-breaking strength, and litter phosphorus concentration in broilers. Poult. Sci. 87:949–957. Prattley, C. A., and D. W. Stanley. 1982. Protein-phytate interactions in soybeans. I Localisation of phytate in protein bodies and globoids. J. Food Biochem. 6:243–253. Prattley, C. A., D. W. Stanley, and F. R. van de Voort. 1982. Protein-phytate interactions in soybeans. II. Mechanism of protein-phytate binding as affected by calcium. J. Food Biochem. 6:255–271. Radcliffe, J. S., Z. Zhang, and E. T. Kornegay. 1998. The effects of microbial phytase, citric acid, and their interaction in a corn-soybean meal-based diet for weanling pigs. J. Anim. Sci. 76:1880– 1886. ENZYMES AND CITRIC ACID IN BROILER DIETS Ravindran, V., W. L. Bryden, and E. T. Kornegay. 1995. Phytates: Occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol. Rev. 6:125–143. Ravindran, V., P. H. Selle, and W. L. Bryden. 1999. Effects of phytase supplementation, individually and in combination, with glycanase, on the nutritive value of wheat and barley. Poult. Sci. 78:1588–1595. Ravindran, V., P. H. Selle, G. Ravindran, P. C. H. Morel, A. K. Kies, and W. L. Bryden. 2001. Microbial phytase improves performance, apparent metabolizable energy, and ileal amino acid digestibility of broilers fed a lysine-deficient diet. Poult. Sci. 80:338–344. SAS. 2002. SAS User’s Guide: Statistics. SAS Inst. Inc., Cary, NC. Selle, P. H., and V. Ravindran. 2007. Microbial phytase in poultry nutrition. A review. Anim. Feed Sci. Technol. 35:1–41. Selle, P. H., V. Ravindran, W. L. Bryden, and T. Scott. 2006. Influence of dietary phytate and exogenous phytase on amino acid digestibility in poultry: A review. Jpn. Poult. Sci. 43:89–103. Selle, P. H., V. Ravindran, G. Ravindran, P. H. Pittolo, and W. L. Bryden. 2003. Influence of phytase and xylanase supplementation on growth performance and nutrient utilisation of broilers offered wheat-based diets. Asian-australas. J. Anim. Sci. 16:394–402. Silversides, F. G., T. A. Scott, and M. R. Bedford. 2004. The effect of phytase enzyme and level on nutrient extraction by broilers. Poult. Sci. 83:985–989. Simon, O., and F. Igbasan. 2002. In vitro properties of phytases from various microbial origins. Int. J. Food Sci. Technol. 37:813– 822. Singh, M., and A. D. Krikorian. 1982. Inhibition of trypsin activity in vitro by phytate. J. Agric. Food Chem. 30:799–800. Slominski, B. A., T. Davie, M. C. Nyachoti, and O. Jones. 2007. Heat stability of endogenous and microbial phytase during feed pelleting. Livest. Sci. 109:244–246. Snow, J. L., D. H. Baker, and C. M. Parsons. 2004. Phytase, citric acid, and 1α-hydroxycholecalciferol improve phytate phosphorus utilization in chicks fed a corn-soybean meal diet. Poult. Sci. 83:1187–1192. 2229 Steiner, T., R. Mosenthin, B. Zimmermann, R. Greiner, and S. Roth. 2007. Distribution of phytase activity, total phosphorus and phytate phosphorus in legume seeds, cereals and cereal byproducts as influenced by harvest year and cultivar. Anim. Feed Sci. Technol. 133:320–334. Thompson, L. U., C. L. Button, and D. J. A. Jenkins. 1987. Phytic acid and calcium affect the in vitro rate of navy bean starch digestion and blood glucose response in humans. Am. J. Clin. Nutr. 46:467–473. Waldroup, P. W., J. H. Kersey, E. A. Saleh, C. A. Fritts, F. Yan, H. L. Stilborn, R. C. Crum Jr., and V. Raboy. 2000. Nonphytate phosphorus requirement and phosphorus excretion of broiler chicks fed diets composed of normal or high available phosphate corn with and without microbial phytase. Poult. Sci. 79:1451– 1459. Woyengo, T. A., O. Adeola, and C. M. Nyachoti. 2010. Gastrointestinal digesta pH, pepsin activity and soluble mineral concentration responses to dietary phytic acid and phytase in piglets. Livest. Sci. doi:10.016/ j.livsci.2010.06.107 Woyengo, T. A., W. Guenter, J. S. Sands, C. M. Nyachoti, and M. A. Mirza. 2008. Nutrient utilisation and performance responses of broilers fed a wheat-based diet supplemented with phytase and xylanase alone or in combination. Anim. Feed Sci. Technol. 146:113–123. Wu, Y. B., V. Ravindran, D. G. Thomas, M. J. Birtles, and W. H. Hendriks. 2004. Influence of phytase and xylanase, individually or in combination, on performance of, apparent metabolisable energy, digestive tract measurements and gut morphology in broilers fed wheat-based diets containing adequate level of phosphorus. Br. Poult. Sci. 45:76–84. Zyla, K., D. Gogol, J. Koreleski, S. Swiatkiewicz, and D. R. Ledoux. 1999. Simultaneous application of phytase and xylanase to broiler feeds based on wheat: Feeding experiment with growing broilers. J. Sci. Food Agric. 79:1841–1848.