Effects of dietary supplementation of an enzyme blend on the ileal and fecal digestibility of nutrients in growing pigs F. Ji, D. P. Casper, P. K. Brown, D. A. Spangler, K. D. Haydon and J. E. Pettigrew J Anim Sci 2008.86:1533-1543. doi: 10.2527/jas.2007-0262 originally published online Mar 14, 2008; The online version of this article, along with updated information and services, is located on the World Wide Web at: http://jas.fass.org/cgi/content/full/86/7/1533 www.asas.org Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. Effects of dietary supplementation of an enzyme blend on the ileal and fecal digestibility of nutrients in growing pigs1 F. Ji,* D. P. Casper,† P. K. Brown,† D. A. Spangler,† K. D. Haydon,‡ and J. E. Pettigrew*2 *University of Illinois, Department of Animal Sciences, Urbana 61801; †Agri-King Inc., Fulton, IL 61252; and ‡Prince Agri Products, Quincy, IL 62306 ABSTRACT: The objective of this experiment was to determine the effect of a β-glucanase-protease enzyme blend product (EBP) on fecal digestibility (FD), apparent ileal digestibility (AID), standardized ileal digestibility, and digestibility in the hindgut of growing pigs. Twelve ileal-cannulated, growing barrows (38.2 ± 0.5 kg) were housed in individual metabolism crates, blocked by previous feed intake into 3 groups with 4 pigs each, and randomly assigned to 1 of 4 treatments within a square (group) of 3 replications of 4 × 4 Latin square design. Treatments were basal diet (Basal), Basal + 0.05% of EBP (0.05% EBP), Basal + 0.10% of EBP (0.10% EBP), and hydrolyzed casein for measurement of endogenous amino acids. The Basal diet consisted of corn and soybean meal and was calculated to have 3.36 Mcal of ME/kg and 1.1% of total lysine, asfed basis. Feed intake of each replicate of the Latin square during the first period was 85% of the minimum feed intake of the 4 pigs during the preliminary period and was equalized within each square. The feeding level was increased by 100 g/d in each subsequent period. Each of the experimental periods was 14 d, including 4 d of dietary adaptation, 5 d of fecal collection, 3 d of transition period, and 2 d of ileal collection. Ileal efflu- ents were collected continuously for the same 12-h interval each day. Pigs fed the EBP demonstrated increased (P < 0.05) FD of DM, OM, energy, CP, nonfiber carbohydrate, total dietary fiber, insoluble dietary fiber, acid-hydrolyzed fat, ash, Ca, and P compared with pigs fed Basal. The AID of NDF and hemicellulose was increased (P < 0.05) by supplying the EBP either at 0.05 or 0.10% in the diets, but AID of DM and energy was not increased. The AID of acid-hydrolyzed fat tended to be greater (P = 0.051) for the pigs fed the EBP than for those fed Basal. Ileal digestibility of most amino acids was not affected by treatment, but the EBP reduced the apparent and standardized digestibility of methionine, alanine, and serine (P < 0.05). The difference between FD and AID of hemicellulose was lower (P < 0.05) for the pigs fed the EBP than for those fed Basal. These results demonstrated that the EBP fed to growing pigs improved the FD of DM, OM, energy, CP, nonfiber carbohydrate, total dietary fiber, acid-hydrolyzed fat, Ca, and P, and the AID of NDF and hemicellulose, but the standardized ileal digestibility of amino acids was not improved by supplying the EBP in corn-soybean meal-based diets of growing pigs. Key words: digestibility, enzyme, growing pig, nutrient ©2008 American Society of Animal Science. All rights reserved. INTRODUCTION The effects of enzymes on the nutrient digestion by the pig may vary by enzyme products, age of the pigs, and feed ingredients. Baas and Thacker (1996) showed that commercial products of β-glucanase varied in their effects on digestibility of DM, energy, and CP. It seems the most likely application would be in weanling pig 1 We wish to acknowledge the financial support of Agri-King Inc. (Fulton, IL). 2 Corresponding author: jepettig@uiuc.edu Received May 10, 2007. Accepted March 3, 2008. J. Anim. Sci. 2008. 86:1533–1543 doi:10.2527/jas.2007-0262 diets in which pancreatic enzyme production may be limiting (Lindemann et al., 1986). Exogenous enzymes seem less effective in older pigs (Thacker et al., 1988, 1989, 1992; Baas and Thacker, 1996; Nyachoti et al., 2006), because older pigs are more able to digest fiber than younger pigs. Exogenous enzymes were also more effective in chick diets based on poorly digested cereals (Classen et al., 1995; Scott et al., 1998a,b), which contain more fiber (Fernandez and Jørgenson, 1986; Noblet and Bach-Knudsen, 1997; Le Goff et al., 2002). Previous studies (Thacker et al., 1990; Li et al., 1996; Zijlstra et al., 2004) on supplying carbohydrases in pig diets were mainly focused on barley, wheat, or ryebased diets. Gaining an advantage from the use of exogenous enzymes has been more challenging for corn- 1533 Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1534 Ji et al. soybean meal (SBM) diets due to decreased fiber concentrations and high nutrient digestibility. Corn and SBM are known to contain 10 and 22% of nonstarch polysaccharides, respectively (CVB, 1998). In conjunction with the variation in nutrient composition of corn (Sullivan et al., 1989; Burgoon et al., 1992; Summers, 2001) and SBM (Grieshop et al., 2003), there may be potential to improve digestibility of corn-SBM diets with exogenous enzymes. A novel β-glucanase-protease enzyme blend product (EBP) has been developed to improve performance of grower-finisher pigs fed a cornSBM-based diet, in which exogenous enzymes have not often been shown to be effective. The objective of this experiment was to investigate the effect of EBP on digestibility of energy and nutrients in the small intestine, hindgut, and overall gastrointestinal tract. MATERIALS AND METHODS Animals and Facilities Experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Illinois. Fifteen growing barrows (22.5 ± 0.5 kg of BW, PIC337 sire × Camborough-22, PIC dam) were housed in galvanized metal metabolism crates (0.86 × 1.60 m) with mesh floors in a power ventilated, environmentally controlled room. Pigs were able to move about freely, turn around, and lie down in the crates. Each crate was equipped with a low-pressure drinking nipple, a feeder, and a screen for collecting feces. Room lights were illuminated for 24 h/d, and the room temperature was controlled to approximately 22°C. Surgical Procedures and Animal Care The pigs had 7 d to adapt to their surroundings before the surgery. Pigs had ad libitum access to feed during this period. Feed was withheld from each animal 12 h before surgery, but the pigs had continuous access to water. Pigs were surgically fitted with a simple T-cannula at approximately 10 cm cranial to the ileo-cecal junction, according to procedures adapted from Sauer et al. (1983). The cannulation site was cleaned daily with soft detergent and warm water during the entire experiment. Also, a zinc oxide-lanolin-based cream (Desitin, Pfizer Inc., New York, NY) was applied daily to the flank area to minimize irritation and to maintain skin integrity during the entire experiment. The pigs were fed 2 meals daily until full feeding was achieved 4 d after surgery. A period of 10 d was allowed for surgical recovery before the beginning of the experiment. Experimental Design, Enzyme Product, and Diets Twelve of the 15 cannulated pigs (38.2 ± 1.7 kg of BW at the beginning of period 1) were blocked by previ- Table 1. Composition of the experimental diets, as-fed basis Item Ingredient, % Corn Soybean meal Animal-vegetable fat blend Dicalcium phosphate Limestone Salt-trace mineral premix1 Vitamin premix2 Lysine HCl Threonine Methionine Corn starch Sucrose Cellulose3 Hydrolyzed casein4 MgO (58% Mg) K2CO3 (55% K) Total Calculated composition ME,5 Mcal/kg CP, % Lysine, % True ileal digestible lysine, % Ca, % Available P, % Basal diet Hydrolyzed casein diet 70.386 25.80 1.00 1.23 0.90 0.35 0.10 0.175 0.04 0.019 — — — — — — 100.00 — — 3.00 1.61 0.77 0.35 0.10 — — — 57.62 20.00 5.00 10.00 0.15 1.40 100.00 3.361 18.29 1.10 0.98 0.70 0.29 3.622 9.04 0.74 0.74 0.70 0.29 1 Supplied the following per kilogram of complete diet: Fe, 90 mg (FeSO4ⴢH2O); Zn, 100 mg (ZnO); Mn, 20 mg (MnO); Cu, 8 mg (CuSO4ⴢH2O); I, 0.35 mg (CaI2); Se, 0.3 mg (Na2SeO3); and NaCl, 3 g. 2 Supplied the following per kilogram of complete diet: 6,608 IU of vitamin A as retinyl acetate; 680 IU of vitamin D as cholecalciferol; DL-α-tocopheryl acetate, 88 mg; menadione sodium bisulfite complex, 4 mg; niacin, 33 mg; D-Ca-pantothenate, 24 mg; riboflavin, 9 mg; vitamin B12, 35 g; and choline chloride, 324 mg. 3 Solka-Floc 40 FCC (powdered cellulose, International Fiber Corp., North Tonawanda, NY). 4 American Casein Company, Burlington, NJ. 5 The assumed ME values of the ingredients were from the NRC (1998); ME of Solka-floc was assumed to be zero. ous feed intake into 3 groups with 4 pigs each. Three extra pigs were available to be used in the event that pigs were removed from the study, but none was used. Within each group, pigs were randomly assigned to 1 of 4 treatments in a 4 × 4 Latin square design, with pigs and periods as the main factors. The 4 treatments were basal diet (Basal), Basal + 0.05% of EBP (0.05% EBP), Basal + 0.10% of EBP (0.10% EBP), and a hydrolyzed casein diet (Casein). The Casein contained hydrolyzed casein at a 10% level (as-fed basis) as the sole protein source and was included to enable the calculation of standardized ileal digestibility of CP and amino acids. The Basal consisted mainly of corn and SBM and was formulated to provide 3.36 Mcal of ME/kg and 1.1% total lysine (Table 1) to meet or exceed the nutrient requirements (NRC, 1998). The EBP (Agri-King Inc., Fulton, IL) contained β-glucanase activity (guaranteed not less than 660,000 β-glucanase units/kg) and protease activity (guaranteed not less than 22,000 hemoglobin units/kg). One β-glucanase unit liberates 1 mM reducing sugar (glucose equivalence) per minute. One he- Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1535 Effect of exogenous enzymes on nutrient digestibility Table 2. Analyzed composition of the experimental diets (DM basis) Item Casein1 Basal2 0.05% EBP3 0.10% EBP4 DM, % OM, % GE, Mcal/kg DE, Mcal/kg CP, % Starch, % Nonfiber carbohydrate, % ADF, % NDF, % Hemicellulose, % Total dietary fiber, % Insoluble dietary fiber, % Soluble dietary fiber, % Ether extract, % Acid-hydrolyzed fat, % Arginine, % Histidine, % Isoleucine, % Leucine, % Lysine, % Methionine, % Phenylalanine, % Threonine, % Tryptophan, % Valine, % Alanine, % Aspartic acid, % Cysteine, % Glutamate, % Glycine, % Serine, % Tyrosine, % Ash, % Ca, % P, % Mg, % K, % Na, % S, % Cl, % Fe, ppm Cu, ppm Zn, ppm Mn, ppm 92.0 96.5 4.148 — 9.5 56.5 78.2 4.4 5.4 1.0 — — — 3.5 2.4 0.39 0.34 0.57 0.99 0.78 0.24 0.49 0.41 0.13 0.74 0.29 0.70 0.05 2.52 0.19 0.55 0.50 3.48 0.50 0.29 0.03 0.72 0.31 0.10 0.09 131 3 116 30 87.3 95.0 4.367 3.588 20.5 54.0 61.9 4.0 8.9 4.9 12.9 10.8 2.1 4.2 3.8 1.43 0.53 0.95 1.74 1.22 0.30 0.98 0.82 0.28 1.06 1.01 2.18 0.28 4.05 0.91 1.11 0.72 5.02 0.86 0.65 0.16 0.92 0.13 0.22 0.25 321 14 137 40 87.2 95.4 4.386 3.818 20.3 54.1 63.3 3.5 8.5 5.0 13.1 10.6 2.5 4.0 3.7 1.42 0.51 0.94 1.72 1.18 0.29 0.96 0.80 0.27 1.05 0.99 2.10 0.28 3.94 0.84 1.03 0.71 4.59 0.86 0.65 0.16 0.90 0.14 0.23 0.23 332 13 135 46 87.2 95.2 4.362 3.806 20.1 54.8 63.2 3.9 8.6 4.7 13.0 11.2 1.8 4.0 3.9 1.42 0.50 0.94 1.74 1.23 0.30 0.97 0.80 0.27 1.06 1.00 2.13 0.26 3.97 0.89 1.08 0.71 4.80 0.81 0.62 0.16 0.89 0.14 0.23 0.23 338 15 145 43 1 Hydrolyzed casein diet. Basal diet. Basal + 0.05% of the enzyme blend product (EBP). 4 Basal + 0.10% of the EBP. 2 3 moglobin unit of protease produces, in 1 min, a hydrolysate, in which absorbance at 275 nm is equal to that of a solution containing 1.1 mg/mL of tyrosine in 0.006 N hydrochloric acid. The manufacturer’s suggested usage of the EBP in growing pig diets is 0.05%. The 0.05% EBP and 0.10% EBP were made by mixing 0.05 and 0.10% of the EBP into the Basal, respectively, on an as-fed basis. The 4 chromic oxide diets were made by mixing 0.35% of chromic oxide (as-fed basis) into the 4 regular diets (Basal, 0.05% EBP, 0.10% EBP, and Casein). Both the regular and chromic oxide diets were sampled at the feed mill when the diets were mixed and at the feeders during period 4 of the experiment. Because the analyzed nutrient contents of the diets sampled at both the feed mill and the feeder were similar, the nutrient contents of the diets sampled at the feed mill were used in calculation of digestibility. The analyzed nutrient contents of the 4 regular diets sampled at the feed mill are listed in Table 2. Animal Feeding and Sample Collection The daily feed intake of pigs allowed ad libitum consumption before the beginning of the experiment ranged from 1,725 to 2,400 g/d. To ensure that all pigs could consume the feed allowance, the feed intake of Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1536 Ji et al. all 4 pigs within each square during the first period was restricted to 85% of the previous intake of the pig with the lowest intake (i.e., 1,460, 1,820, and 1,980 g/ d, respectively). The feeding level was increased by 100 g/d in each subsequent period to accommodate the increased nutrient needs due to growth of the pigs. The feeding levels were 3.3 to 4.3 times the maintenance energy requirement (106 kcal of ME per kg of BW0.75; NRC, 1998) in the first period and 2.7 to 3.1 times maintenance in the last period. Pigs were fed 2 equal meals daily, at 12-h intervals. Water was available free choice. Spilled feed was collected and, unless it was heavily contaminated with feces, was returned to the feeder. There were 4 periods in the experiment, and each pig received different treatment diets in each period. Each period was 14 d in duration. The first 4 d of each period were the diet adaptation phase. The chromic oxide diets were fed at the beginning and end of fecal collection to identify when to begin and end the collection of feces. Pigs were fed the diets containing chromic oxide in the morning meal of d 5. After that, the pigs were still fed the regular diets. Approximately 24 to 36 h later, the chromic oxide appeared in the feces, after which the feces were collected twice daily at 12-h intervals. Fecal collections were conducted from d 6 to 11. In the morning meal on d 10 of each period (120 h after the first feeding of chromic oxide), pigs were fed the diets containing chromic oxide until the end of the period. Again, approximately 24 to 36 h later, chromic oxide appeared in the feces, and collection of feces was stopped. The feces of the 5-d collection were pooled by pig. Fresh feces were frozen at −20°C. The collection phase of ileal digesta was conducted on d 13 and 14. Ileal effluents were collected continuously for the same 12-h interval. Ileal digesta were collected by attaching polyethylene tubing (5 × 25 cm; Rand Materials Handling Equipment Co. Inc., Pawtucket, RI) to the cannula barrel with a cable tie. There was no chemical preservative in the tubing. The tubing was changed at least once every hour. As soon as ileal digesta was dumped into a container, it was placed in a freezer at −20°C for storage. Ileal digesta samples were pooled by pig and frozen at −20°C until the end of each collection period. Chemical Analyses Fresh feces of each pig were weighed. All fecal and ileal samples were thawed and mixed for homogeneity. A subsample was taken for chemical analyses. Fecal, ileal, and feed samples were dried at 60°C and ground to an 800-m particle size before analyses. The contents of DM, ADF, NDF, total dietary fiber, CP, ether extract, ash, Ca, P, Mg, K, Na, Fe, Cu, Zn, Mn, S, Cl, and pH were determined by the methods of the AOAC (2002). Hemicellulose was calculated as the difference between NDF and ADF. Soluble dietary fiber and insoluble dietary fiber were analyzed according to the method de- scribed by Prosky et al. (1992). Amino acids were determined by the method of the AOAC (2002), but using an Adsorbosphere Opa HPLC precolumn (Alltech, 2000). Tryptophan was determined by an HPLC method using 7.5 N NaOH at 103 kPa for 16 h rather than barium hydroxide. Energy was measured using a bomb calorimeter (Model 1108, Parr Instrument Company, Moline, IL). Starch was measured by the method of Trinder (1969). Acid-hydrolyzed fat was determined by the method of Oser (1965). Chromium was analyzed by the method developed by Williams and Iismaa (1962) and Binnerts et al. (1968). Ammonia-nitrogen was determined by the method of Peters et al. (2003). The VFA were analyzed by capillary electrophoresis using a method adapted from that of Cancalon (1993). Calculations of Digestibility The fecal digestibility was determined by the equation described by Adeola (2001). The apparent ileal digestibility, endogenous amino acid loss, and standardized ileal digestibility were determined by the equations described by Smiricky et al. (2002). The endogenous amino acid loss used to calculate the standardized ileal digestibility in a square (group) was the average of the 4 pigs in the square. The digestibility of nutrients in the hindgut was calculated by using the following equations: difference between fecal and ileal digestibility (%) = fecal digestibility − apparent ileal digestibility; and digestibility based on the nutrients entering the hindgut (%) = ⎡ ⎢1 ⎣ − ⎤ FW × NCf ⎥ × 100, DMI × NCF × (1 − AID/100)⎦ where FW = the dried fecal weight (kg of DM); NCf = the nutrient content in feces (% of DM); DMI = the DM intake (kg); NCF = the nutrient content in feed (% of DM); and AID = apparent ileal digestibility (%). Statistical Analyses Data were analyzed by the GLM and UNIVARIATE procedures (SAS Inst. Inc., Cary, NC) to test for homogeneity, normality, and outliers. Data were then analyzed by the MIXED procedure of SAS with groups, pigs, periods, and diets in the model. Means were reported as least squares means. Treatment comparisons were made using the following orthogonal contrasts: 1) effect of EBP [(0.05% EBP + 0.10% EBP)/2 vs. Basal] and 2) effect of level of EBP (0.05 vs. 0.10% EBP), with an α level of 0.05 used to determine statistical significance. RESULTS Enzyme Activities and Diet Components The calculated β-glucanase activities were 330 and 660 β-glucanase units/kg of diet, and the calculated Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1537 Effect of exogenous enzymes on nutrient digestibility Table 3. Feed intake and characteristics of feces and ileal digesta (DM basis) Contrast5 Treatment Basal1 (n6 = 10) Item Feed intake, g as fed Fecal weight, g of DM In feces DM content, % pH NH3, ppm Acetic acid, % Propionic acid, % Butyric acid, % Valeric acid, % In ileal digesta DM content, % pH NH3, ppm Acetic acid, % Propionic acid, % Butyric acid, % Valeric acid, % 0.05% EBP2 (n6 = 11) 0.10% EBP3 (n6 = 11) 8,287 1,043 8,236 935 8,189 936 44.81 6.91 3,434 0.987 0.51 0.36 0.14 45.29 6.79 2,863 1.27 0.55 0.39 0.14 41.68 6.85 3,140 1.26 0.65 0.41 0.17 n8 = 11 n8 = 12 n8 = 12 10.46 7.39 86.85 0.014 0.004 0.003 ND9 9.60 7.19 89.31 0.015 0.004 0.002 ND 8.53 7.29 88.36 0.014 0.003 0.001 0.001 SD4 98 44 3.67 0.21 562 0.15 0.15 0.10 0.04 3.65 0.30 14.57 0.010 0.000 0.000 0.000 1 2 0.065 <0.001 0.275 0.965 0.360 0.360 0.064 <0.001 0.130 0.242 0.375 0.032 0.507 0.264 0.906 0.106 0.576 0.068 0.318 0.204 0.719 0.854 0.704 0.502 0.412 0.479 0.412 0.875 0.940 0.188 0.315 0.133 1 Basal diet. Basal + 0.05% of the enzyme blend product (EBP). 3 Basal + 0.10% of the EBP. 4 Pooled SD. 5 Contrasts were as follows: 1) effect of EBP [(0.05% EBP + 0.10% EBP)/2 vs. Basal] and 2) effect of level of EBP (0.05 vs. 0.10%). 6 One outlier was removed from 0.05% EBP and 0.10% EBP; 2 outliers were removed from Basal. 7 One more outlier was removed from Basal. 8 One outlier was removed from Basal. 9 ND = not detected. 2 protease activities were 11 and 22 hemoglobin units/ kg of diet for 0.05% EBP and 0.10% EBP, respectively. The contents of DM, GE, CP, total dietary fiber, and indispensable amino acids among Basal, 0.05% EBP, and 0.10% EBP were similar after manufacturing (Table 2), indicating that the EBP did not change the main nutrient composition of the diets. < 0.001) in the pigs fed the EBP than in the pigs fed the Basal. Fecal pH and concentrations of DM, ammonia, propionic acid, butyric acid, and valeric acid did not differ between the pigs fed the EBP and the Basal. Acetic acid was greater (P < 0.001) in feces from the pigs fed the EBP than from those fed the Basal. Periods Fecal Digestibility of Nutrients The fecal digestibility and apparent ileal digestibility of DM, CP, and energy, as well as apparent ileal digestibility of amino acids, were decreased (P < 0.001) in period 1 compared with periods 2, 3, and 4 (data not shown). Although pigs were allowed 10 d for recovery after surgery with 4 additional days for dietary adaptation, this may not have been sufficient time for recovery from surgery. However, digestibility values did not vary among periods 2, 3, and 4, indicating the effect of age of grower-finisher pigs on nutrient digestibility was limited. Feed Intake and Fecal Characteristics All pigs consumed the allotted feed, gained weight, had no diarrhea, and appeared healthy during the experiment. Feed intake did not differ among the treatments (Table 3). Dried fecal weight was decreased (P The fecal digestibility of DM, OM, energy, CP, and ash was greater (P < 0.05) for the pigs fed the EBP than the pigs fed the Basal (Table 4), whereas the fecal digestibility of these items did not differ between the pigs fed 0.05% EBP and 0.10% EBP. Pigs fed the EBP had greater fecal digestibility (P < 0.05) of total dietary fiber and acid-hydrolyzed fat than pigs fed the Basal. Within total dietary fiber, the fecal digestibility of insoluble dietary fiber was increased (P < 0.05) by the EBP, whereas the fecal digestibility of soluble dietary fiber was not affected by the EBP. Almost all starch was digested over the whole digestive tract, and there was no difference in fecal digestibility of starch among treatments. The fecal digestibility of Ca, P, Mg, S, Fe, and Cu also was increased (P < 0.05) when pigs were fed the EBP. Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1538 Ji et al. Table 4. Effect of β-glucanase-protease enzyme blend product on fecal digestibility (%) Treatment Contrast5 Item Basal1 (n6 = 10) 0.05% EBP2 (n6 = 11) 0.10% EBP3 (n6 = 11) SD4 1 2 DM OM Energy CP Starch Nonfiber carbohydrate ADF NDF Hemicellulose Total dietary fiber Insoluble dietary fiber Soluble dietary fiber Ether extract Acid-hydrolyzed fat Ash Ca P Mg K Na S Cl Fe Cu Zn Mn 87.42 88.99 86.51 86.47 99.24 94.83 64.84 54.62 46.33 60.61 53.81 95.59 80.14 44.97 57.79 57.33 53.80 26.33 75.05 86.317 81.70 95.50 10.13 5.07 −34.337 −5.01 88.61 89.89 87.42 88.08 99.26 95.22 61.40 55.62 51.62 65.36 57.79 96.80 80.51 48.27 60.77 65.31 61.73 35.20 77.41 87.62 84.76 95.40 21.07 10.46 −22.67 18.63 88.50 89.87 87.26 87.39 99.31 95.63 65.92 56.05 47.99 65.61 60.97 94.68 78.24 51.80 60.95 61.20 57.83 33.36 76.50 88.42 84.33 95.28 19.03 20.89 −7.52 12.07 0.47 0.45 0.56 0.94 0.15 0.48 3.54 3.33 5.06 3.33 3.79 5.64 2.95 2.58 2.34 2.89 2.41 2.89 2.14 3.51 1.16 1.30 3.42 3.56 10.77 3.77 <0.001 <0.001 0.002 0.003 0.527 0.006 0.400 0.357 0.096 0.002 0.002 0.947 0.508 <0.001 0.004 <0.001 <0.001 <0.001 0.036 0.252 <0.001 0.754 <0.001 <0.001 <0.001 <0.001 0.617 0.885 0.511 0.101 0.436 0.065 0.009 0.765 0.112 0.861 0.066 0.390 0.090 0.005 0.857 0.004 0.002 0.156 0.334 0.601 0.389 0.842 0.180 <0.001 0.005 0.001 1 Basal diet. Basal + 0.05% of the enzyme blend product (EBP). Basal + 0.10% of the EBP. 4 Pooled SD. 5 Contrasts were as follows: 1) effect of EBP [(0.05% EBP + 0.10% EBP)/2 vs. Basal] and 2) effect of level of EBP (0.05 vs. 0.10%). 6 One outlier was removed from 0.05% EBP and 0.10% EBP; 2 outliers were removed from Basal. 7 One outlier was removed from Basal. 2 3 Apparent and Standardized Ileal Digestibility of Nutrients The apparent ileal digestibility of DM, energy, starch, and nonfiber carbohydrate did not differ among treatments (Table 5). The pigs fed EBP had decreased (P < 0.05) apparent ileal digestibility of CP than the pigs fed the Basal. The apparent ileal digestibility of NDF and hemicellulose of the pigs fed the EBP was greater (P < 0.05) compared with that of the pigs fed the Basal. The apparent ileal digestibility of acid-hydrolyzed fat in the pigs fed the EBP tended to be greater (P = 0.051) than in those fed the Basal. Ileal digestibility of most amino acids was not affected by treatment, but the EBP reduced the apparent (Table 5) and standardized (Table 6) digestibility of methionine, alanine, and serine (P < 0.05) and arginine, glycine, threonine, and valine (P < 0.05 for apparent digestibility only). Digestion of Nutrients in the Hindgut There was no difference among treatments in the difference between fecal and ileal digestibility except for hemicellulose, which was decreased (P < 0.05) for the pigs fed the EBP compared with pigs fed the Basal (Table 7). The difference between fecal and ileal digestibility of NDF for the pigs fed the EBP tended to be decreased (P = 0.050) compared with the pigs fed the Basal. For the digestibility based on the nutrients entering the hindgut (Table 8), the pigs fed the EBP had greater values (P < 0.05) for CP and DM digestibility compared with pigs fed the Basal treatment. DISCUSSION The EBP containing β-glucanase and protease was developed to improve performance of growing pigs. The current results showed that the EBP supplied at either 0.05 or 0.10% of diet increased fecal digestibility of DM, energy, CP, nonfiber carbohydrate, total dietary fiber, acid-hydrolyzed fat, Ca, and P. Digestion of NDF and hemicellulose in the small intestine of growing pigs was also improved by supplying EBP. However, the EBP did not improve the standardized ileal digestibility of amino acids. This indicates that β-glucanase in the EBP Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1539 Effect of exogenous enzymes on nutrient digestibility Table 5. Effect of β-glucanase-protease enzyme blend product on apparent ileal digestibility (%) Treatment Contrast5 Item Basal1 (n6 = 11) 0.05% EBP2 (n6 = 12) 0.10% EBP3 (n6 = 12) SD4 1 2 DM Energy CP Starch Nonfiber carbohydrate ADF NDF Hemicellulose Ether extract Acid-hydrolyzed fat Ash Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Alanine Aspartic acid Cystine Glutamate Glycine Serine Tyrosine Ca P Mg K Na S Cl Fe Cu Zn Mn 70.86 70.93 78.29 97.95 82.11 4.33 1.21 −1.19 61.40 52.06 17.62 94.58 86.89 81.58 83.78 87.04 82.76 83.90 67.10 74.12 81.29 73.03 80.13 76.06 84.90 68.43 81.83 86.72 54.71 49.62 6.12 74.47 −540.83 70.84 −20.89 9.24 −12.61 −7.76 −4.23 69.13 69.48 75.51 98.01 80.68 4.36 9.52 13.11 62.94 53.84 8.19 93.68 85.16 79.62 82.55 85.01 80.35 82.64 62.27 72.35 78.95 69.04 77.85 72.72 82.37 61.94 78.83 85.03 57.51 49.54 13.23 71.18 −567.11 67.62 −59.48 12.39 20.88 −1.97 5.03 70.50 70.71 76.54 98.12 82.04 5.22 10.05 13.42 62.18 57.73 16.41 93.39 85.24 80.63 83.13 85.91 79.07 83.53 63.99 73.76 79.71 70.37 79.33 75.087 84.89 64.81 79.18 84.39 56.31 49.00 11.81 72.64 −513.68 71.16 −16.857 3.56 12.597 −10.58 12.19 2.81 2.84 2.76 0.49 2.17 10.26 8.61 8.56 4.48 4.80 7.94 1.28 3.49 2.18 1.79 2.06 2.82 2.14 4.37 2.98 2.31 3.84 2.62 3.47 2.48 5.67 2.56 3.65 7.74 5.18 9.47 5.27 94.28 3.88 28.52 10.88 23.26 15.32 9.75 0.329 0.441 0.041 0.514 0.362 0.905 0.015 <0.001 0.493 0.051 0.088 0.041 0.210 0.092 0.174 0.054 0.009 0.320 0.025 0.347 0.035 0.032 0.130 0.107 0.188 0.028 0.008 0.154 0.455 0.859 0.086 0.208 0.990 0.326 0.114 0.758 0.003 0.797 0.002 0.246 0.305 0.371 0.588 0.145 0.840 0.881 0.931 0.683 0.062 0.021 0.576 0.955 0.272 0.437 0.295 0.281 0.322 0.348 0.262 0.433 0.409 0.183 0.117 0.023 0.231 0.739 0.670 0.708 0.803 0.717 0.505 0.182 0.038 0.002 0.062 0.399 0.185 0.089 1 Basal diet. Basal + 0.05% of the enzyme blend product (EBP). 3 Basal + 0.10% of the EBP. 4 Pooled SD. 5 Contrasts were as follows: 1) effect of EBP [(0.05% EBP + 0.10% EBP)/2 vs. Basal] and 2) effect of level of EBP (0.05 vs. 0.10%). 6 One outlier was removed from Basal. 7 One outlier was removed. 2 is effective even under the difficult conditions of a lowfiber corn-SBM diet and limit fed to older pigs, but the protease may be ineffective. The carbohydrate of cereal grains includes sugars, starch, and cell wall polysaccharides. The endosperm cell walls of barley, oat, wheat, rye, sorghum, corn, and triticale are composed mainly of arabinoxylans and βglucans (Henry, 1985; Bedford, 1995; de Lange, 2000). The current results showed that the fecal digestibility of total dietary fiber and insoluble dietary fiber was improved by supplying the EBP. The total dietary fiber consists of soluble materials such as β-glucans, arabinoxylans, pectins, and gums, and the insoluble galactoand glucomannans, cellulose and lignin (Asp et al., 1983; Bedford, 1995; Souffrant, 2001). It is unclear why the EBP increased digestibility of insoluble rather than soluble dietary fiber, because the substrate of the βglucanase is soluble. The increase in ileal digestibility of NDF and hemicellulose when the EBP was added to the diet, with no change in fecal digestibility, indicates that the enzyme product may have shifted some of the digestion of these Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1540 Ji et al. Table 6. Effect of β-glucanase-protease enzyme blend product on standardized ileal digestibility (%) Treatment Item CP Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Alanine Aspartic acid Cystine Glutamate Glycine Serine Tyrosine Contrast5 Basal1 (n6 = 11) 0.05% EBP2 (n6 = 12) 0.10% EBP3 (n6 = 12) SD4 1 2 88.47 101.29 91.01 87.01 87.52 90.91 87.83 87.23 84.66 81.78 87.29 82.33 85.49 80.98 91.17 95.38 91.31 90.55 86.09 100.64 89.48 85.28 86.39 89.00 85.50 86.07 80.45 79.95 85.15 78.59 83.46 77.84 88.93 89.88 88.76 89.02 87.35 100.55 89.64 86.12 86.95 89.83 84.69 86.89 82.28 81.36 85.89 80.05 84.86 79.68 91.00 92.59 89.15 88.47 2.76 1.28 3.49 2.19 1.79 2.06 2.82 2.14 4.36 2.98 2.31 3.85 2.62 3.41 2.49 5.67 2.55 3.65 0.105 0.156 0.279 0.124 0.215 0.068 0.018 0.362 0.057 0.323 0.055 0.049 0.191 0.097 0.208 0.065 0.023 0.200 0.277 0.867 0.916 0.358 0.455 0.337 0.489 0.361 0.316 0.262 0.443 0.364 0.209 0.203 0.056 0.258 0.717 0.718 1 Basal diet. Basal + 0.05% of the enzyme blend product (EBP). Basal + 0.10% of the EBP. 4 Pooled SD. 5 Contrasts were as follows: 1) effect of EBP [(0.05% EBP + 0.10% EBP)/2 vs. Basal] and 2) effect of level of EBP (0.05 vs. 0.10%). 6 One outlier was removed from Basal. 2 3 nutrients from hindgut fermentation to small intestinal enzymatic digestion. Such a change would avoid the fermentative loss of energy as heat and methane and presumably increase the energetic efficiency of fiber use. The cell walls enclose starch, protein, and lipids. Supplying enzymes to enhance breakdown of the components of these cell walls can increase exposure of the nutrients in cells to the digestive enzymes. However, because of the difference of ingredients, age of pigs, and enzyme products, the effectiveness of supplying enzyme in pig diets may be different. Some studies showed that supplying β-glucanase increased ileal digestibility of DM, energy, CP or fibers (Taverner and Campbell, 1988; Inborr and van der Meulen, 1993; Yin et al., 2001a,b), and fecal digestibility of fibers (Yin et al., 2001a), whereas other studies showed supplying β-glucanase did not improve ileal digestibility of DM, energy, or CP (Graham et al., 1989; Nyachoti et al., 2006) and fecal digestibility of DM, energy, CP, or fiber (Taverner and Campbell, 1988; Graham et al., 1989; Nyachoti et al., 2006). These previous studies focused on the enzyme effect on diets based on fibrous cereals such as barley and wheat. The effect of enzyme on corn-SBM-based diet has not previously been well addressed. The current results showed that apparent ileal digestibility of NDF and hemicellulose and the fecal digestibility of DM, energy, CP, and total dietary fiber were improved by the EBP. This indicates that even under the difficult conditions of corn-SBM diet and pigs several months old, supplying exogenous enzymes still have the potential to improve digestibility of DM, energy, and fibers. However, because the capacity of degrading fiber is different for the enzymes obtained from different microbes (Nevins et al., 1978), the effectiveness of various commercial enzyme products was different (Baas and Thacker, 1996). The enzyme product used in the current study was an effective fiber-degrading enzyme product. The effectiveness of an enzyme may be affected by many factors, such as the activity of the enzyme, the type and concentration of substrate, particle size, pH, temperature, and time of the feed staying in the small intestine. The calculated β-glucanase activities were 330 and 660 units/kg of diet for 0.05% EBP and 0.10% EBP, respectively. The levels of β-glucanase in previous studies varied from 10 (Graham et al., 1986) and 100 (Graham et al., 1989) units per kilogram of diet to 600 β-glucanase units/kg of diet (Yin et al., 2001a,b). In this case, the lower level of EBP tested (0.05%) seemed to be sufficient to achieve maximum effect. The apparent ileal digestibility of acid-hydrolyzed fat was increased by supplying EBP, whereas Graham et al. (1989) reported no effect. The increase in fecal digestibility of acid-hydrolyzed fat by supplying EBP was caused by increased digestibility in the small intestine. Because the fecal digestibility and apparent ileal digestibility of ether extract did not differ, the increase of apparent ileal digestibility of acid-hydrolyzed fat may Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1541 Effect of exogenous enzymes on nutrient digestibility Table 7. Effect of β-glucanase-protease enzyme blend product on the digestibility in the hindgut (%, the difference between fecal and ileal digestibility) Treatment Item DM Energy CP Starch Nonfiber carbohydrate ADF NDF Hemicellulose Ether extract Acid-hydrolyzed fat Ash Ca P Mg K Na S Cl Fe Cu Zn Mn Contrast5 Basal1 (n6 = 11) 0.05% EBP2 (n6 = 12) 0.10% EBP3 (n6 = 12) SD4 1 2 17.25 16.00 9.13 1.46 13.00 61.86 55.93 50.94 19.31 −4.83 42.24 4.30 6.16 24.03 3.74 618.30 11.78 118.44 4.94 27.51 −19.46 4.31 19.68 18.19 12.86 1.34 14.87 56.17 45.36 37.87 17.52 −5.30 52.78 6.92 12.37 23.38 8.00 651.58 17.39 155.58 8.88 −10.37 −20.80 14.28 18.32 16.96 11.15 1.25 13.73 61.06 45.93 34.19 16.69 −5.60 44.99 4.02 8.38 21.48 6.24 603.99 13.45 128.33 14.88 4.34 0.66 −1.28 3.79 3.86 4.01 0.62 2.58 12.97 12.95 14.41 5.95 8.95 10.71 9.61 7.50 12.56 7.19 104.61 5.50 39.74 15.81 23.42 28.57 14.85 0.236 0.294 0.075 0.478 0.167 0.514 0.050 0.014 0.338 0.855 0.118 0.749 0.153 0.739 0.228 0.812 0.097 0.134 0.259 0.003 0.394 0.700 0.392 0.444 0.310 0.713 0.384 0.368 0.916 0.540 0.737 0.935 0.093 0.471 0.210 0.716 0.557 0.281 0.098 0.112 0.366 0.143 0.084 0.020 1 Basal diet. Basal + 0.05% of the enzyme blend product (EBP). Basal + 0.10% of the EBP. 4 Pooled SD. 5 Contrasts were as follows: 1) effect of EBP [(0.05% EBP + 0.10% EBP)/2 vs. Basal] and 2) effect of level of EBP (0.05 vs. 0.10%). 6 One outlier was removed from Basal. 2 3 be because EBP digested cell wall materials and released the lipids from the cell membranes, which constitute most of the difference between ether extract and acid-hydrolyzed fat. The fecal digestibility of minerals including Ca, P, Mg, S, Cl, Fe, Cu, and Mn was improved by the EBP and may result from the EBP releasing minerals bound to or in the cell wall. The EBP did not increase ileal digestibility of amino acids. We cannot explain why the standardized ileal digestibility of methionine, alanine, and serine was reduced by the EBP. In previous studies, the effect of supplying exogenous enzymes on ileal digestibility of protein was contradictory in pigs. Protease treatment of SBM had no effect on ileal digestibility of CP and amino acids in newly weaned pigs (Caine et al., 1997a,b). Li et al. (1996) demonstrated that β-glucanase supplementation increased the ileal digestibility of βglucans, CP, and the majority of amino acids in a hulless barley-SBM diet. Yin et al. (2001b) showed that supplying β-glucanase and xylanase in weaned pig diets increased the apparent ileal digestibility of lysine, glycine, threonine, histidine, and valine. Yin et al. (2001a) obtained similar results from another study, in which β-glucanase improved apparent ileal digestibility of CP and of most amino acids. The current study showed that the supplementation of protease in the corn-SBM- based growing pig diets was ineffective. It may be because the protease activities in the EBP (11 and 22 hemoglobin units/kg of diet for 0.05% EBP and 0.10% EBP, respectively) were decreased compared with that in a previous study (500 units/kg in pig diets; Yin et al., 2001a). The feeding levels were 3.3, 4.1, and 4.3 times maintenance in the first period and 2.7, 3.0, and 3.1 times maintenance in the fourth period. The degree of restriction increased as the pigs grew. The feeding levels did not affect the fecal digestibility or apparent ileal digestibility of DM, CP, or energy, as well as apparent ileal digestibility of all amino acids, which agrees with the previous results of Haydon et al. (1984), Albin et al. (2001), Gómez et al. (2002), and Moter and Stein (2004). Other studies showed an inverse relationship between feeding levels and fecal digestibility of DM and CP (Rao and McCracken, 1991) and ileal digestibility of CP and most amino acids (Moter and Stein, 2004). Because the comparisons were at different levels of restriction rather than restriction to ad libitum, it is possible the response to the EBP may be greater in ad libitum pigs. In conclusion, the fecal digestibility of DM, OM, energy, CP, total dietary fiber, acid-hydrolyzed fat, Ca, and P and the apparent ileal digestibility of hemicellulose and NDF were improved, but standardized ileal Downloaded from jas.fass.org at Serials/Acq. Dept., Library on May 1, 2009. 1542 Ji et al. Table 8. Digestibility (%) based on the nutrients entering the hindgut Treatment Item DM Energy CP Starch Nonfiber carbohydrate ADF NDF Hemicellulose Ether extract Acid-hydrolyzed fat Ash Ca P Mg K Na S Cl Fe Cu Zn Mn Contrast5 Basal1 (n6 = 11) 0.05% EBP2 (n6 = 12) 0.10% EBP3 (n6 = 12) SD4 1 2 57.78 54.41 39.20 63.00 71.85 63.71 54.38 47.00 48.86 −13.29 49.73 5.13 9.86 24.02 −5.94 97.58 37.99 96.39 2.45 29.54 −24.34 1.38 62.70 58.53 50.71 61.11 75.08 58.70 49.78 42.92 46.03 −13.43 56.39 13.35 23.21 24.47 1.66 97.92 51.95 96.78 7.23 −53.30 −25.56 12.62 61.14 56.83 46.67 63.81 75.81 63.69 50.76 39.47 42.10 −14.63 52.84 8.04 15.85 23.33 4.26 98.01 45.85 95.97 14.74 −21.13 −1.66 −2.19 4.56 6.42 7.68 12.00 4.13 5.53 6.47 8.22 11.09 18.82 5.69 21.20 10.62 11.52 19.64 0.89 9.27 0.86 15.51 99.31 30.34 12.95 0.026 0.188 0.004 0.906 0.032 0.238 0.105 0.075 0.288 0.916 0.034 0.489 0.026 0.978 0.239 0.262 0.006 0.953 0.157 0.088 0.351 0.436 0.413 0.527 0.216 0.589 0.673 0.042 0.714 0.318 0.334 0.878 0.145 0.548 0.109 0.812 0.750 0.788 0.126 0.033 0.253 0.439 0.072 0.013 1 Basal diet. 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