The effect of β-mannanase on nutrient utilization and blood parameters in chicks fed diets containing soybean meal and guar gum

The effect of β-mannanase on nutrient utilization and blood parameters in chicks fed diets... Abstract The present study was conducted to determine whether the addition of β-mannanase in broiler feed changes hormonal profiles in the blood and broiler performance and nutrient availability. Five hundred and four Cobb male chickens were studied during d 7 to 21. Three corn–soybean meal (SBM) based diets 1) Low SBM (18% SBM); 2) High SBM (31% SBM); and 3) High SBM+GG (31% SBM + Guar Gum (GG) 0.5%) with 3 levels of β-mannanase (0, 200, and 400 ppm) were mixed to produce 9 diets. A factorial design 3 × 3 was performed with JMP pro 13 (SAS, 2017). Analysis of variance and contrast analysis were used to test significance level at P < 0.05. Glucose (190 and 188 mg/dL) was increased with 200 and 400 ppm of β-mannanase, respectively, compared to control (182 mg/dL) in the fasted state (P < 0.037). Glucose was higher in chicks fed with the High SBM and High SBM + GG diets but lower in the fasted re-fed state (P < 0.01). Insulin was higher with 200 and 400 ppm added β-mannanase in the fed state (P < 0.021). Insulin-like growth factor-1 was higher with 400 ppm added to High SBM+GG. β-mannanase improved feed conversion ratio (FCR) 9 points with 400 ppm in High SBM diet (P < 0.01) and 16 and 18 points with 200 and 400 ppm, respectively, added to the High SBM+GG diet (P < 0.01). Viscosity decreased from 19.2 to 7 cps with both enzyme doses in the High SBM + GG diet (P < 0.01). Digestible energy was +152 kcal/kg with 400 ppm β-mannanase in the High SBM diet and +200 kcal/kg with both levels of enzyme in High SBM+GG diet. Digestibility of amino acids was improved from 0.8 to 3.6% with β-mannanase in High SBM+GG diet (P < 0.05). In conclusion, chicks fed with High SBM and High SBM+GG diets with added β-mannanase significantly improved blood glucose and anabolic hormone homeostasis, FCR, digestible energy, and digestible amino acids compared to chicks fed with same diets without β-mannanase. INTRODUCTION Common cereals and protein crops used as feedstuffs in poultry diets have high nonstarch polysaccharides (NSP). Nonstarch polysaccharides, predominantly the soluble form, are considered detrimental for nutrient utilization in broilers, but the soluble NSP also have the greatest potential to be degraded by exogenous enzymes (Bach Knudsen, 2014). The purpose of adding exogenous enzymes to animal feeds is to increase the availability of nutrients while reducing poultry production cost. Processed soybean meal (SBM) is the world's largest source of plant protein used in the feed, but SBM contains various NSP that diminish nutrient utilization by poultry. One of the NSP in SBM is β-mannan that has a concentration of 1.26 ± 0.14% in dehulled and 1.61±0.20% in nondehulled SBM (Hsiao, et al., 2006). The exogenous enzyme β-mannanase represents a potential NSP enzyme capable of reducing the negative effects of β-mannan. Several researchers (Jackson et al., 1999; Daskiran, et al., 2004; Mohammad and Kazem, 2012; Hussain et al., 2012) reported negative effects of β-mannan from guar and soybean meal on poultry performance and the potential to overcome its effects with mannanase. The structure of β-mannan in guar is almost identical to that of β-mannan in SBM (Daskiran, et al., 2004; and Hsiao et al., 2006). Because SBM is the major protein source in poultry diets, the degradation of β-mannan in SBM could lead to improved performance of poultry. β-mannan in the feed has been reported to lower body weight gain and increased feed:gain in d 1 to 14 broilers with guar gum (GG) (Daskiran et al., 2004); increase feed:gain in d 1 to 42 broilers with guar meal (Kamran et al., 2002); increase feed:gain and produce higher plasma lipids for d 1 to 42 broilers fed guar meal (Lee et al., 2005; Mohammad and Kazem, 2012); however, there is still a limited information available on the metabolic effects of β-mannan on glucose metabolism and hormonal change. Rainbird and Low (1986) and Nunes and Malmlof (1992) have reported a reduction in postprandial blood glucose and insulin when GG was fed to pigs. An attempt to understand the β-mannanase effect at the metabolic level was conducted by Jozefiak et al. (2010) when the group added a multicarbohydrase enzyme with mannanase to a wheat–soybean and full-fat rapeseed meal diet and showed increased insulin receptors in the liver of chickens. Arsenault et al. (2017) found that the addition of β-galactomannan impacted the immune response in the jejunum of d 42 broiler; and adding β-mannanase eliminated most of the immune signaling effect. More information is needed to determine whether blood glucose and anabolic hormones such as insulin and insulin-like growth factor-1 (IGF-1) in broilers fed corn-SBM-based diets are influenced by the supplementation of mannanase. The present experiment is intended to test the negative effects of β-mannan from SBM and GG in diets on energy metabolism in broilers and to determine whether an added β-mannanase enzyme will reverse this effect. MATERIALS AND METHODS All management practices and procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee (IACUC) #12,041. Birds and Diets Male broiler chicks (504) of a commercial strain (Cobb-Vantress, Inc.) were obtained from a local hatchery. The chicks were maintained on floor pens for 7 d post hatching on an environmentally controlled room and fed a standard starter diet (CP 22.2%; 3035 kcal/kg) that meet the breeder recommendation levels. On d 7, all chicks were weighed individually, allocated to 72 wire metabolic cages (dimensions: 91 cm × 30 cm each), and randomly assigned to 9 dietary treatments with 7 chicks per metabolic cage with 8 replications for a 2 wk experimental period. Each cage started with similar coefficient of variance (CV) on body weight (BW) (Range: 12.1 to 13.7%). Birds had free access to mash feed and water during the experimental period. Individual BW and feed consumption per cage were recorded at d 21 post hatching. All broilers received 1 of the 9 experimental diets structured in a 3 × 3 factorial arrangement from d 7 to 21. Three diets × three levels of the enzyme β-mannanase (0, 200, and 400 ppm). Legumes such as SBM contain higher β-mannan (1.2% DM) compared to corn (0.3% DM) (Bach Knudsen, 1997). SBM and GG were used as the main mannan source and meat and bone meal was added to reduce the SBM level in basal diet 1. GG used in the present study had 80% β-mannans and it was used to increase the β-mannans in the third diet. The experimental diets evaluated were 1) Low SBM, 2) High SBM, and 3) High SBM+ GG. Diet 1, low SBM had 18% SBM, diet 2 had 31% SBM, and diet 3 was diet 2 plus 0.5% GG. Total calculated mannans were 4,677; 5,335; and 9,330 ppm in diets 1, 2, and 3, respectively. These values were calculated from corn, corn dried distillers grain solubles (DDGS), and SBM from recent publications (Bach Knudsen, 2014; Jaworski et al., 2015), and GG was purchased with 80% mannan (Polypro International, Minnesota, US) contributing with 4,000 ppm of the 9,330 ppm of mannan in diet 3. The enzyme β-mannanase was added to the 3 basal diets (Table 1) at 3 inclusion rates: 0, 200, and 400 ppm generating 9 diets. The β-mannanase enzyme was produced from Bacillus lentus. One unit of mannanase activity was defined as the amount of enzyme which generates 0.72 micrograms of reducing sugars per minute from a mannose-containing substrate at pH 6.6 and temperature of 104°F. The assay detects a value <15 × 106 activity units of mannanase/MT in the basal diet; therefore, activity values in the basal diets of the present study were acceptable. Feed production started by making 3 basal feeds, and splitting each basal into 3 separate containers. The enzyme was premixed with 4.5 kg of the corresponding diet before mixing the diet for the experimental treatment. Control diets with no enzyme addition (Low SBM-0, High SBM-0, and High SBM+GG-0) were mixed again to avoid differences of mixing due to other feeds being mixed with the corresponding enzyme level. Samples of each diet were sent for enzyme analysis verification to a commercial laboratory (Elanco, IN, USA) (Table 2). The results confirmed that the enzyme activities were ±30% within the guaranteed values. Table 1. Composition and nutrient calculations (g/100 g as fed) of the basal diets for d 7 to 21.   Basal Low  Basal High  Basal High  Ingredients  SBM  SBM  SBM + GG1  Corn 8.8% CP  65.1  60.8  59.8  Soybean meal 46.4% CP  18.4  31.4  31.6  Meat & Bone Meal, 54% CP2  7.8      Corn DDGS  5.0      Guar Gum (80% mannan)      0.50  Poultry fat  0.40  2.40  2.74  DL-Methionine 98.5%  0.30  0.31  0.31  L-Lysine HCl, 78%  0.47  0.29  0.29  L-Threonine, 98%  0.18  0.15  0.15  Limestone  0.47  1.05  1.05  Dicalcium phosphate  0.41  2.15  2.16  Salt  0.41  0.38  0.38  Vitamin and mineral premix3  0.52  0.52  0.52  Titanium dioxide  0.50  0.50  0.50  Calculated Nutrients        β-Mannan, ppm4  4,677  5,335  9,330  AME, kcal/kg  3,000  3,000  3,000  Crude protein, %  21.0  21.0  21.0  Calcium, %  1.00  1.00  1.00  Digestible phosphorus, %  0.50  0.50  0.50  Digestible lysine, %  1.10  1.10  1.10  Digestible methionine + cysteine, %  0.83  0.83  0.83  Digestible threonine, %  0.73  0.73  0.73  Analyzed Nutrients5        Crude protein, %  20.6  20.9  20.5  Digestible lysine, %  1.04  1.18  1.15  Digestible methionine + cysteine, %  0.77  0.79  0.79  Digestible threonine, %  0.70  0.72  0.69  Digestible phosphorus, %  0.50  0.48  0.45    Basal Low  Basal High  Basal High  Ingredients  SBM  SBM  SBM + GG1  Corn 8.8% CP  65.1  60.8  59.8  Soybean meal 46.4% CP  18.4  31.4  31.6  Meat & Bone Meal, 54% CP2  7.8      Corn DDGS  5.0      Guar Gum (80% mannan)      0.50  Poultry fat  0.40  2.40  2.74  DL-Methionine 98.5%  0.30  0.31  0.31  L-Lysine HCl, 78%  0.47  0.29  0.29  L-Threonine, 98%  0.18  0.15  0.15  Limestone  0.47  1.05  1.05  Dicalcium phosphate  0.41  2.15  2.16  Salt  0.41  0.38  0.38  Vitamin and mineral premix3  0.52  0.52  0.52  Titanium dioxide  0.50  0.50  0.50  Calculated Nutrients        β-Mannan, ppm4  4,677  5,335  9,330  AME, kcal/kg  3,000  3,000  3,000  Crude protein, %  21.0  21.0  21.0  Calcium, %  1.00  1.00  1.00  Digestible phosphorus, %  0.50  0.50  0.50  Digestible lysine, %  1.10  1.10  1.10  Digestible methionine + cysteine, %  0.83  0.83  0.83  Digestible threonine, %  0.73  0.73  0.73  Analyzed Nutrients5        Crude protein, %  20.6  20.9  20.5  Digestible lysine, %  1.04  1.18  1.15  Digestible methionine + cysteine, %  0.77  0.79  0.79  Digestible threonine, %  0.70  0.72  0.69  Digestible phosphorus, %  0.50  0.48  0.45  The β-mannanase was added to each of the 3 basal diets at 3 inclusion rates: 0, 200, and 400 ppm generating 9 diets. 1Inclusion: 0.5% Guar Gum. 2Meat and Bone meal was sourced from H. J. Baker as Proplus, 54%. 3Supplied per kilogram of diet: 200 mg antioxidant, 15,432 UI vitamin A, 11,023 IU vitamin D3, 110 UI vitamin E, 3 mg menadione, 13 mg riboflavin, 20 mg pantothenic acid, 77 mg niacin, 2 mg folic acid, 0.03 mg vitamin B12, 6 mg pyridoxine, 0.20 mg biotin, 3 mg thiamine, 1,200 mg of choline chlorine, 100 mg Mn, 27 mg Mg, 100 mg Zn, 50 mg Fe, 10 mg Cu, 1 mg I, and 0.20 mg Se. 4Adapted from Bach Knudsen, 2014; and Jaworski et al., 2015. 5The analyzed digestible AA were calculated with the total AA value multiplied by the digestibility value. View Large Table 1. Composition and nutrient calculations (g/100 g as fed) of the basal diets for d 7 to 21.   Basal Low  Basal High  Basal High  Ingredients  SBM  SBM  SBM + GG1  Corn 8.8% CP  65.1  60.8  59.8  Soybean meal 46.4% CP  18.4  31.4  31.6  Meat & Bone Meal, 54% CP2  7.8      Corn DDGS  5.0      Guar Gum (80% mannan)      0.50  Poultry fat  0.40  2.40  2.74  DL-Methionine 98.5%  0.30  0.31  0.31  L-Lysine HCl, 78%  0.47  0.29  0.29  L-Threonine, 98%  0.18  0.15  0.15  Limestone  0.47  1.05  1.05  Dicalcium phosphate  0.41  2.15  2.16  Salt  0.41  0.38  0.38  Vitamin and mineral premix3  0.52  0.52  0.52  Titanium dioxide  0.50  0.50  0.50  Calculated Nutrients        β-Mannan, ppm4  4,677  5,335  9,330  AME, kcal/kg  3,000  3,000  3,000  Crude protein, %  21.0  21.0  21.0  Calcium, %  1.00  1.00  1.00  Digestible phosphorus, %  0.50  0.50  0.50  Digestible lysine, %  1.10  1.10  1.10  Digestible methionine + cysteine, %  0.83  0.83  0.83  Digestible threonine, %  0.73  0.73  0.73  Analyzed Nutrients5        Crude protein, %  20.6  20.9  20.5  Digestible lysine, %  1.04  1.18  1.15  Digestible methionine + cysteine, %  0.77  0.79  0.79  Digestible threonine, %  0.70  0.72  0.69  Digestible phosphorus, %  0.50  0.48  0.45    Basal Low  Basal High  Basal High  Ingredients  SBM  SBM  SBM + GG1  Corn 8.8% CP  65.1  60.8  59.8  Soybean meal 46.4% CP  18.4  31.4  31.6  Meat & Bone Meal, 54% CP2  7.8      Corn DDGS  5.0      Guar Gum (80% mannan)      0.50  Poultry fat  0.40  2.40  2.74  DL-Methionine 98.5%  0.30  0.31  0.31  L-Lysine HCl, 78%  0.47  0.29  0.29  L-Threonine, 98%  0.18  0.15  0.15  Limestone  0.47  1.05  1.05  Dicalcium phosphate  0.41  2.15  2.16  Salt  0.41  0.38  0.38  Vitamin and mineral premix3  0.52  0.52  0.52  Titanium dioxide  0.50  0.50  0.50  Calculated Nutrients        β-Mannan, ppm4  4,677  5,335  9,330  AME, kcal/kg  3,000  3,000  3,000  Crude protein, %  21.0  21.0  21.0  Calcium, %  1.00  1.00  1.00  Digestible phosphorus, %  0.50  0.50  0.50  Digestible lysine, %  1.10  1.10  1.10  Digestible methionine + cysteine, %  0.83  0.83  0.83  Digestible threonine, %  0.73  0.73  0.73  Analyzed Nutrients5        Crude protein, %  20.6  20.9  20.5  Digestible lysine, %  1.04  1.18  1.15  Digestible methionine + cysteine, %  0.77  0.79  0.79  Digestible threonine, %  0.70  0.72  0.69  Digestible phosphorus, %  0.50  0.48  0.45  The β-mannanase was added to each of the 3 basal diets at 3 inclusion rates: 0, 200, and 400 ppm generating 9 diets. 1Inclusion: 0.5% Guar Gum. 2Meat and Bone meal was sourced from H. J. Baker as Proplus, 54%. 3Supplied per kilogram of diet: 200 mg antioxidant, 15,432 UI vitamin A, 11,023 IU vitamin D3, 110 UI vitamin E, 3 mg menadione, 13 mg riboflavin, 20 mg pantothenic acid, 77 mg niacin, 2 mg folic acid, 0.03 mg vitamin B12, 6 mg pyridoxine, 0.20 mg biotin, 3 mg thiamine, 1,200 mg of choline chlorine, 100 mg Mn, 27 mg Mg, 100 mg Zn, 50 mg Fe, 10 mg Cu, 1 mg I, and 0.20 mg Se. 4Adapted from Bach Knudsen, 2014; and Jaworski et al., 2015. 5The analyzed digestible AA were calculated with the total AA value multiplied by the digestibility value. View Large Table 2. Enzyme activity analysis in feed (enzyme × 106 units of β-mannanase/ton). Type of  Enzyme,  Analyzed Enzyme  Guaranteed Enzyme  Analyzed:  diet  ppm  Activity  Activity  Guaranteed,1%  Low SBM  Dose 0  9.9      Low SBM  Dose 200  34.2  29.0  83.8  Low SBM  Dose 400  81.7  58.0  123.8  High SBM  Dose 0  6.3      High SBM  Dose 200  41.7  29.0  122.1  High SBM  Dose 400  74.0  58.0  116.7  High SBM + GG  Dose 0  9.8      High SBM + GG  Dose 200  46.2  29.0  125.5  High SBM + GG  Dose 400  86.4  58.0  132.1  Type of  Enzyme,  Analyzed Enzyme  Guaranteed Enzyme  Analyzed:  diet  ppm  Activity  Activity  Guaranteed,1%  Low SBM  Dose 0  9.9      Low SBM  Dose 200  34.2  29.0  83.8  Low SBM  Dose 400  81.7  58.0  123.8  High SBM  Dose 0  6.3      High SBM  Dose 200  41.7  29.0  122.1  High SBM  Dose 400  74.0  58.0  116.7  High SBM + GG  Dose 0  9.8      High SBM + GG  Dose 200  46.2  29.0  125.5  High SBM + GG  Dose 400  86.4  58.0  132.1  1Analyzed enzyme values for 200 and 400 ppm dose levels were deducted from the basal before they were divided over their corresponding guaranteed enzyme activity values × 100. View Large Table 2. Enzyme activity analysis in feed (enzyme × 106 units of β-mannanase/ton). Type of  Enzyme,  Analyzed Enzyme  Guaranteed Enzyme  Analyzed:  diet  ppm  Activity  Activity  Guaranteed,1%  Low SBM  Dose 0  9.9      Low SBM  Dose 200  34.2  29.0  83.8  Low SBM  Dose 400  81.7  58.0  123.8  High SBM  Dose 0  6.3      High SBM  Dose 200  41.7  29.0  122.1  High SBM  Dose 400  74.0  58.0  116.7  High SBM + GG  Dose 0  9.8      High SBM + GG  Dose 200  46.2  29.0  125.5  High SBM + GG  Dose 400  86.4  58.0  132.1  Type of  Enzyme,  Analyzed Enzyme  Guaranteed Enzyme  Analyzed:  diet  ppm  Activity  Activity  Guaranteed,1%  Low SBM  Dose 0  9.9      Low SBM  Dose 200  34.2  29.0  83.8  Low SBM  Dose 400  81.7  58.0  123.8  High SBM  Dose 0  6.3      High SBM  Dose 200  41.7  29.0  122.1  High SBM  Dose 400  74.0  58.0  116.7  High SBM + GG  Dose 0  9.8      High SBM + GG  Dose 200  46.2  29.0  125.5  High SBM + GG  Dose 400  86.4  58.0  132.1  1Analyzed enzyme values for 200 and 400 ppm dose levels were deducted from the basal before they were divided over their corresponding guaranteed enzyme activity values × 100. View Large Sample Collection Blood samples were taken at three different feeding conditions (fed, fasted, and re-fed). On d 21 at 7 AM (fed), 1 chick per cage (8 chicks per treatment) were randomly selected as soon as the weight was recorded, it was also when the time of fasting started. The second blood collection occurred after 24 h of fasting on a second set of 8 chicks per treatment starting at 7 AM on d 22 (fasted). Finally, the last set of blood samples were taken on a third set of 8 chicks per treatment, 2 h after re-feeding starting at 9 AM on d 22 (re-fed). One chick per cage was taken for blood analysis resulting in 8 chicks per treatment, and 72 chicks for the study. Chicks were sampled in blocks, so every block had all 9 treatments to reduce the bias on the analytics due to time of sampling. Bleeding for each block took about 20 min, so the bleeding was finished in approximately 3 h for the study. Blood was collected via jugular to avoid hemolysis. Blood from each chick was collected into one serum separator tube. Chicks were euthanized using gas CO2 inhalation soon after the blood collection. All blood samples were centrifuged at 3,000 rpm for 15 min and the serum portion was transferred into 3 separate tubes and stored at −20°C for further glucose and hormone analysis. On d 23, after 9 h of fasting, the remaining birds were provided feed for 2 h and then 4 chicks per cage were humanely euthanized using CO2 gas inhalation, and the contents of the jejunum and ileum collected. The jejunum was defined from the end of the duodenal loop to the vitelline diverticulum (Meckel's diverticulum). The ileum was defined as the portion of the small intestine extending from vitelline diverticulum to 4 cm proximal to the ileocecal junction. Jejunum contents were processed and analyzed for viscosity within 2 h after the last collection. The ileal contents from 4 chicks were stored in 100 mL cups at −20°C for 48 h, lyophilized for 7 d, and finely ground for further nutrient analysis. Blood Analysis Serum was analyzed for glucose, insulin, and IGF-1. Glucose concentration was analyzed using 200 μL of serum sample with Ciba-Corning Glucose hexokinase (HK) reagents. This method assays serum glucose concentration enzymatically. Glucose is phosphorylated with adenosine triphosphate in the reaction catalyzed by HK. The product, glucose-6-phosphate, is then oxidized with the concomitant reduction of nicotinamide adenine dinucleotide (NDA+) to NADH in the reaction catalyzed by glucose-6-phosphate dehydrogenase. The formation of NADH causes an increase in absorbance at 340 nm, which is directly proportional to the amount of glucose in the sample. The absorbance was measured using the analyzer Express plus 550 (Ciba Corning, NY, USA). Serum insulin and IGF-1 were detected using a chicken insulin ELISA kit (Cusabio, San Diego, CA, USA), which has higher sensitivities for chicken insulin than regular insulin kits made for rodents or humans. Same method was used by Shi et al. (2014). The absorbance was measured using the ELISA multi detection microplate reader Synergy HT (Biotek, Winooski, VT, USA). Results were analyzed against a standard curve. Viscosity and Chemical Analysis The jejunum contents from 4 chicks per cage were filled into 50-mL tubes and centrifuged at 3,000 rpm for 10 min. The supernatant was decanted and the viscosity of a 0.5 ml aliquot measured in centipoise (cps) using Brookfield Digital Viscometer (Model DV-II, Brookfield Engineering Laboratories, Stoughton, MA). Diets and ileal contents were analyzed for dry matter, gross energy (GE), amino acids (AA), phosphorus (P), and titanium dioxide (TiO2). Samples were analyzed following AOAC official methods (AOAC, 1990). Dry matter was analyzed with AOAC 934.01. Gross energy was determined in a bomb calorimeter (Parr 6200 bomb calorimeter, Parr Instruments Co., Moline, IL.). Amino acids were determined with AOAC 982.30 and AOAC 985.28 methods using high-performance (high-pressure) liquid chromatography, P was analyzed with AOAC, 968.08 using inductively-coupled plasma. TiO2 was measured on a UV spectrophotometer (Shimadzu model UV-2101 PC, Scientific Instruments Inc., Hawthorne, NY) utilizing the method of Myers et al. (2004). The apparent ileal nutrient digestibility (AIND) of energy, phosphorus, and amino acids were calculated using the equation AIND = [((NT/Ti)d − (NT/Ti)i)/(NT/Ti)d] × 100; where (NT/Ti)d = ratio of nutrient and Ti in diet and (NT/Ti)i = ratio of nutrient and Ti in ileal digesta (Amerah and Ravindran, 2015). Apparent ileal digestible energy (AIDE) was calculated by multiplying the diet GE content by the apparent ileal energy digestibility. Statistical Analysis All data analyses were performed using JMP pro13 statistical analysis software (SAS institute, 2017). Growth and nutrient digestibility data were analyzed as a complete randomized design with 3 × 3 factorial arrangement with 1 cage as the experimental unit. Data analyzed by analysis of variance was presented as mean ± SEM (standard error of the mean). Significant differences of means were separated using Tukey HSD test at P ≤ 0.05. Blood data were analyzed as a completely block design because the time of termination of bleeding was considered as a block to avoid bias on the results due to timing instead of effect on the treatments. One chick was the experimental unit in the blood analysis data. The arrangement was a 3 × 3 factorial arrangement (type of diet and enzyme inclusion) within nutritional state. Data are presented as means ± SE (standard error). Differences among treatments were determined using single degree of freedom orthogonal contrasts. Six comparisons performed were: 1) Low SBM – 0 ppm vs. Low SBM – 200 ppm; 2) Low SBM – 0 ppm vs. Low SBM – 400 ppm, 3) High SBM – 0 ppm vs. High SBM – 200 ppm, 4) High SBM – 0 ppm vs. High SBM – 400, 5) High SBM+GG – 0 ppm vs. High SBM+GG – 200 ppm, 6) High SBM+GG – 0 ppm vs. High SBM+GG – 400 ppm. Mortality data was transformed to Arcsine before analysis. Significance was considered at P < 0.05. RESULTS Performance Final BW at 21 d after 14 d experimental period was different between types of diets. Low SBM, High SBM, and High SBM+GG produced 733, 755, and 695 g/bird, respectively (Table 3). All three diets differed from each other (P ≤ 0.01). Enzyme dose levels and interaction effects tended to be significant (P < 0.076) for final BW; therefore, a contrast analysis showed that the addition of both 200 and 400 ppm β-mannanase to diets with high SBM + GG improved 21 d BW compared to their respective control, High SBM+GG-200: 702 g vs. High SBM+GG-0: 669 g (P ≤ 0.01), and High SBM+GG-400: 715 g vs. High SBM+GG-0: 669 g (P ≤ 0.01). BW gain, feed intake, feed:gain, and mortality are presented in Table 3. BW gain on d 21 followed the same trend as BW because the initial BW was similar for the treatments. Feed intake was lower for chicks fed with the diet containing 400 ppm of β-mannanase compared to the control chicks (0 ppm enzyme): 889 vs. 918 g/bird, respectively (P ≤ 0.01). The feed intake for chicks fed the diet containing 200 ppm of the enzyme (908 g/bird) was not different compared to either treatment. No differences were shown in feed intake between type of diets; however, there was a trend (P < 0.086) for the interaction effect. Chicks fed with High SBM-400 ppm diet had lower feed intake compared to chicks fed with High SBM-0 ppm diet: 878 g vs. 937 g/bird, respectively (P ≤ 0.01). Differences in BW and feed intake established differences in feed: gain. The interaction effects of feed type and enzyme levels were significant for feed:gain (P < 0.01), where High SBM-400 ppm produced the best feed: gain (1.406) and High SBM+GG-0 ppm produced the poorest feed: gain (1.693). The contrast analysis showed feed: gain was improved by 0.162 and 0.182 when 200 ppm (1.531) and 400 ppm (1.511) of β-mannanase was added to High SBM+GG-0 ppm diets (1.693), respectively. Feed:gain was improved by 0.09 when 400 ppm was added to High SBM-0 ppm diet. No effect was found in feed:gain when enzyme was added to Low SBM or when only 200 ppm enzyme was added to the High SBM diet. Mortality was the highest with Low SBM (P ≤ 0.034) compared only to High SBM+GG diet. Table 3. Body weight, body weight gain, feed intake, feed conversion ratio, and mortality of broilers from 7 to 21 d. Item  Initial BW  Final BW  Gain,  Feed Intake,  Feed:  Mortality,    d 7, g/bird  d 21, g/bird  g/bird  g/bird  Gain1 g/g  %  Type of diet              Low SBM    127.9  732.5b  604.7b  907.2  1.513b  3.57a  High SBM    128.2  755.3a  627.2a  915.1  1.461c  1.27a,b  High SBM+GG    127.5  695.2c  567.4c  893.2  1.578a  0.00b  Enzyme β-D-mannanase, ppm              Dose: 0 ppm    128.2  718.3  590.0  918.4a  1.566a  0.60  Dose: 200 ppm    127.5  729.9  602.7  908.3a,b  1.515b  2.38  Dose: 400 ppm    127.9  734.9  606.6  888.7b  1.471c  2.38  Type of diet  Enzyme              Low SBM  Dose: 0 ppm  128.6  730.2  601.6  903.6  1.511b  1.79  Low SBM  Dose: 200 ppm  126.8  734.0  607.3  915.7  1.531b  3.57  Low SBM  Dose: 400 ppm  128.3  733.4  605.1  902.2  1.496b  5.36  High SBM  Dose: 0 ppm  128.4  755.8  627.2  936.7  1.494b  0.00  High SBM  Dose: 200 ppm  128.6  754.1  626.5  930.6  1.481b  3.57  High SBM  Dose: 400 ppm  127.6  756.1  627.9  878.0  1.406c  1.79  High SBM+GG  Dose: 0 ppm  127.5  669.0  541.3  914.9  1.693a  0.00  High SBM+GG  Dose: 200 ppm  127.2  701.5  574.3  878.6  1.531b  0.00  High SBM+GG  Dose: 400 ppm  127.6  715.2  586.7  886.0  1.511b  0.00  SEM    1.56  8.80  8.45  13.63  0.02  1.63  P-value for main effects and interaction            Type of diet    0.965  <0.01**  <0.01**  0.147  <0.01**  0.034*  Enzyme    0.647  0.071  0.051  0.033*  <0.01**  0.311  Type of diet × Enzyme  0.987  0.076  0.070  0.086  <0.01**  0.668  P-value for contrasts of enzyme level              Low SBM-0 vs. Low SBM-200  0.420  0.759  0.609  0.523  0.496  0.444  Low SBM-0 vs. Low SBM-400  0.864  0.798  0.764  0.945  0.618  0.128  High SBM-0 vs. High SBM-200  0.685  0.895  0.954  0.755  0.686  0.128  High SBM-0 vs. High SBM-400  0.853  0.983  0.953  <0.01**  <0.01**  0.444  High SBM+GG-0 vs. High SBM+GG-200  0.862  0.010*  <0.01**  0.061  <0.01**  1.000  High SBM+GG-0 vs. High SBM+GG-400  0.725  <0.01**  <0.01**  0.146  <0.01**  1.000  Item  Initial BW  Final BW  Gain,  Feed Intake,  Feed:  Mortality,    d 7, g/bird  d 21, g/bird  g/bird  g/bird  Gain1 g/g  %  Type of diet              Low SBM    127.9  732.5b  604.7b  907.2  1.513b  3.57a  High SBM    128.2  755.3a  627.2a  915.1  1.461c  1.27a,b  High SBM+GG    127.5  695.2c  567.4c  893.2  1.578a  0.00b  Enzyme β-D-mannanase, ppm              Dose: 0 ppm    128.2  718.3  590.0  918.4a  1.566a  0.60  Dose: 200 ppm    127.5  729.9  602.7  908.3a,b  1.515b  2.38  Dose: 400 ppm    127.9  734.9  606.6  888.7b  1.471c  2.38  Type of diet  Enzyme              Low SBM  Dose: 0 ppm  128.6  730.2  601.6  903.6  1.511b  1.79  Low SBM  Dose: 200 ppm  126.8  734.0  607.3  915.7  1.531b  3.57  Low SBM  Dose: 400 ppm  128.3  733.4  605.1  902.2  1.496b  5.36  High SBM  Dose: 0 ppm  128.4  755.8  627.2  936.7  1.494b  0.00  High SBM  Dose: 200 ppm  128.6  754.1  626.5  930.6  1.481b  3.57  High SBM  Dose: 400 ppm  127.6  756.1  627.9  878.0  1.406c  1.79  High SBM+GG  Dose: 0 ppm  127.5  669.0  541.3  914.9  1.693a  0.00  High SBM+GG  Dose: 200 ppm  127.2  701.5  574.3  878.6  1.531b  0.00  High SBM+GG  Dose: 400 ppm  127.6  715.2  586.7  886.0  1.511b  0.00  SEM    1.56  8.80  8.45  13.63  0.02  1.63  P-value for main effects and interaction            Type of diet    0.965  <0.01**  <0.01**  0.147  <0.01**  0.034*  Enzyme    0.647  0.071  0.051  0.033*  <0.01**  0.311  Type of diet × Enzyme  0.987  0.076  0.070  0.086  <0.01**  0.668  P-value for contrasts of enzyme level              Low SBM-0 vs. Low SBM-200  0.420  0.759  0.609  0.523  0.496  0.444  Low SBM-0 vs. Low SBM-400  0.864  0.798  0.764  0.945  0.618  0.128  High SBM-0 vs. High SBM-200  0.685  0.895  0.954  0.755  0.686  0.128  High SBM-0 vs. High SBM-400  0.853  0.983  0.953  <0.01**  <0.01**  0.444  High SBM+GG-0 vs. High SBM+GG-200  0.862  0.010*  <0.01**  0.061  <0.01**  1.000  High SBM+GG-0 vs. High SBM+GG-400  0.725  <0.01**  <0.01**  0.146  <0.01**  1.000  Means with no common superscripts (a, b, c) within a column are different at *P ≤ 0.05, or highly significant **P ≤ 0.01 for Tukey-HSD test. 1Feed:gain, adjusted for mortality. View Large Table 3. Body weight, body weight gain, feed intake, feed conversion ratio, and mortality of broilers from 7 to 21 d. Item  Initial BW  Final BW  Gain,  Feed Intake,  Feed:  Mortality,    d 7, g/bird  d 21, g/bird  g/bird  g/bird  Gain1 g/g  %  Type of diet              Low SBM    127.9  732.5b  604.7b  907.2  1.513b  3.57a  High SBM    128.2  755.3a  627.2a  915.1  1.461c  1.27a,b  High SBM+GG    127.5  695.2c  567.4c  893.2  1.578a  0.00b  Enzyme β-D-mannanase, ppm              Dose: 0 ppm    128.2  718.3  590.0  918.4a  1.566a  0.60  Dose: 200 ppm    127.5  729.9  602.7  908.3a,b  1.515b  2.38  Dose: 400 ppm    127.9  734.9  606.6  888.7b  1.471c  2.38  Type of diet  Enzyme              Low SBM  Dose: 0 ppm  128.6  730.2  601.6  903.6  1.511b  1.79  Low SBM  Dose: 200 ppm  126.8  734.0  607.3  915.7  1.531b  3.57  Low SBM  Dose: 400 ppm  128.3  733.4  605.1  902.2  1.496b  5.36  High SBM  Dose: 0 ppm  128.4  755.8  627.2  936.7  1.494b  0.00  High SBM  Dose: 200 ppm  128.6  754.1  626.5  930.6  1.481b  3.57  High SBM  Dose: 400 ppm  127.6  756.1  627.9  878.0  1.406c  1.79  High SBM+GG  Dose: 0 ppm  127.5  669.0  541.3  914.9  1.693a  0.00  High SBM+GG  Dose: 200 ppm  127.2  701.5  574.3  878.6  1.531b  0.00  High SBM+GG  Dose: 400 ppm  127.6  715.2  586.7  886.0  1.511b  0.00  SEM    1.56  8.80  8.45  13.63  0.02  1.63  P-value for main effects and interaction            Type of diet    0.965  <0.01**  <0.01**  0.147  <0.01**  0.034*  Enzyme    0.647  0.071  0.051  0.033*  <0.01**  0.311  Type of diet × Enzyme  0.987  0.076  0.070  0.086  <0.01**  0.668  P-value for contrasts of enzyme level              Low SBM-0 vs. Low SBM-200  0.420  0.759  0.609  0.523  0.496  0.444  Low SBM-0 vs. Low SBM-400  0.864  0.798  0.764  0.945  0.618  0.128  High SBM-0 vs. High SBM-200  0.685  0.895  0.954  0.755  0.686  0.128  High SBM-0 vs. High SBM-400  0.853  0.983  0.953  <0.01**  <0.01**  0.444  High SBM+GG-0 vs. High SBM+GG-200  0.862  0.010*  <0.01**  0.061  <0.01**  1.000  High SBM+GG-0 vs. High SBM+GG-400  0.725  <0.01**  <0.01**  0.146  <0.01**  1.000  Item  Initial BW  Final BW  Gain,  Feed Intake,  Feed:  Mortality,    d 7, g/bird  d 21, g/bird  g/bird  g/bird  Gain1 g/g  %  Type of diet              Low SBM    127.9  732.5b  604.7b  907.2  1.513b  3.57a  High SBM    128.2  755.3a  627.2a  915.1  1.461c  1.27a,b  High SBM+GG    127.5  695.2c  567.4c  893.2  1.578a  0.00b  Enzyme β-D-mannanase, ppm              Dose: 0 ppm    128.2  718.3  590.0  918.4a  1.566a  0.60  Dose: 200 ppm    127.5  729.9  602.7  908.3a,b  1.515b  2.38  Dose: 400 ppm    127.9  734.9  606.6  888.7b  1.471c  2.38  Type of diet  Enzyme              Low SBM  Dose: 0 ppm  128.6  730.2  601.6  903.6  1.511b  1.79  Low SBM  Dose: 200 ppm  126.8  734.0  607.3  915.7  1.531b  3.57  Low SBM  Dose: 400 ppm  128.3  733.4  605.1  902.2  1.496b  5.36  High SBM  Dose: 0 ppm  128.4  755.8  627.2  936.7  1.494b  0.00  High SBM  Dose: 200 ppm  128.6  754.1  626.5  930.6  1.481b  3.57  High SBM  Dose: 400 ppm  127.6  756.1  627.9  878.0  1.406c  1.79  High SBM+GG  Dose: 0 ppm  127.5  669.0  541.3  914.9  1.693a  0.00  High SBM+GG  Dose: 200 ppm  127.2  701.5  574.3  878.6  1.531b  0.00  High SBM+GG  Dose: 400 ppm  127.6  715.2  586.7  886.0  1.511b  0.00  SEM    1.56  8.80  8.45  13.63  0.02  1.63  P-value for main effects and interaction            Type of diet    0.965  <0.01**  <0.01**  0.147  <0.01**  0.034*  Enzyme    0.647  0.071  0.051  0.033*  <0.01**  0.311  Type of diet × Enzyme  0.987  0.076  0.070  0.086  <0.01**  0.668  P-value for contrasts of enzyme level              Low SBM-0 vs. Low SBM-200  0.420  0.759  0.609  0.523  0.496  0.444  Low SBM-0 vs. Low SBM-400  0.864  0.798  0.764  0.945  0.618  0.128  High SBM-0 vs. High SBM-200  0.685  0.895  0.954  0.755  0.686  0.128  High SBM-0 vs. High SBM-400  0.853  0.983  0.953  <0.01**  <0.01**  0.444  High SBM+GG-0 vs. High SBM+GG-200  0.862  0.010*  <0.01**  0.061  <0.01**  1.000  High SBM+GG-0 vs. High SBM+GG-400  0.725  <0.01**  <0.01**  0.146  <0.01**  1.000  Means with no common superscripts (a, b, c) within a column are different at *P ≤ 0.05, or highly significant **P ≤ 0.01 for Tukey-HSD test. 1Feed:gain, adjusted for mortality. View Large Apparent Ileal Digestible Energy, Phosphorus Digestibility, and Viscosity The interaction effect between type of diet × enzyme level was significant for AIDE where High SBM-400 was 3,165 kcal/kg compared to High SBM-200: 2,819 kcal/kg, and High SBM+GG-0: 2,777 kcal/kg (P ≤ 0.01) (Table 4). Phosphorus digestibility (PD) and viscosity are also presented in Table 4. PD showed no differences for the main effects (type of diets and enzyme level). The contrast analysis showed higher PD for Low SBM-200 compared to its respective control Low SBM-0. Viscosity was the highest with High SBM+GG-0 when no enzyme was added (19.2 cps) compared to birds fed with diets supplemented with β-mannanase: High SBM+GG-200 (7.40 cps) and High SBM+GG-400 (6.83 cps) (P ≤ 0.01). The β-mannanase enzyme did not lower the jejunum viscosity in birds fed with diets with Low SBM-0 and High SBM-0 ppm. Table 4. Digestible energy (kcal/kg), phosphorus digestibility (%), and jejunum viscosity (cps) at 23 d of age. Item  Apparent Digestible Energy, Kcal/kg  Phosphorus Digestibility, %  Jejunum Viscosity, cps  Type of diet    Low SBM    3,071a  63.98  2.55b  High SBM    2,999a,b  62.50  2.27b  High SBM+GG    2,920b  61.50  11.14a  Enzyme β-D-mannanase, ppm        Dose: 0 ppm    2,957b  63.04  8.02a  Dose: 200 ppm    2,958b  63.40  4.08b  Dose: 400 ppm    3,074a  61.50  3.86b  SEM    34.76  0.89  0.14  Type of diet  Enzyme  ±       Low SBM  Dose: 0 ppm  3,085a,b  62.66  2.53c  Low SBM  Dose: 200 ppm  3,072a,b  67.73  2.54c  Low SBM  Dose: 400 ppm  3,057a,b  61.54  2.57c  High SBM  Dose: 0 ppm  3,013a,b,c  65.49  2.33c  High SBM  Dose: 200 ppm  2,819c  61.09  2.30c  High SBM  Dose: 400 ppm  3,165a  62.66  2.18c  High SBM+GG  Dose: 0 ppm  2,777c  60.98  19.20a  High SBM+GG  Dose: 200 ppm  2,982a,b,c  63.16  7.40b  High SBM+GG  Dose: 400 ppm  3,001a,b,c  60.23  6.83b  SEM    58.83  1.78  0.25  P-value for main effects and interaction      Type of diet    0.015*  0.252  <0.01**  Enzyme    0.018*  0.399  <0.01**  Type of diet × Enzyme  <0.01**  0.103  <0.01**  P-value for contrasts of enzyme level        LowSBM-0 vs. LowSBM-200  0.878  0.047*  0.986  LowSBM-0 vs. LowSBM-400  0.728  0.655  0.905  HighSBM-0 vs. HighSBM-200  0.113  0.094  0.930  HighSBM-0 vs. HighSBM-400  0.013*  0.263  0.668  HighSBM+GG-0 vs. HighSBM+GG-200  0.015*  0.386  <0.01**  HighSBM+GG-0 vs. HighSBM+GG-400  <0.01**  0.765  <0.01**  Item  Apparent Digestible Energy, Kcal/kg  Phosphorus Digestibility, %  Jejunum Viscosity, cps  Type of diet    Low SBM    3,071a  63.98  2.55b  High SBM    2,999a,b  62.50  2.27b  High SBM+GG    2,920b  61.50  11.14a  Enzyme β-D-mannanase, ppm        Dose: 0 ppm    2,957b  63.04  8.02a  Dose: 200 ppm    2,958b  63.40  4.08b  Dose: 400 ppm    3,074a  61.50  3.86b  SEM    34.76  0.89  0.14  Type of diet  Enzyme  ±       Low SBM  Dose: 0 ppm  3,085a,b  62.66  2.53c  Low SBM  Dose: 200 ppm  3,072a,b  67.73  2.54c  Low SBM  Dose: 400 ppm  3,057a,b  61.54  2.57c  High SBM  Dose: 0 ppm  3,013a,b,c  65.49  2.33c  High SBM  Dose: 200 ppm  2,819c  61.09  2.30c  High SBM  Dose: 400 ppm  3,165a  62.66  2.18c  High SBM+GG  Dose: 0 ppm  2,777c  60.98  19.20a  High SBM+GG  Dose: 200 ppm  2,982a,b,c  63.16  7.40b  High SBM+GG  Dose: 400 ppm  3,001a,b,c  60.23  6.83b  SEM    58.83  1.78  0.25  P-value for main effects and interaction      Type of diet    0.015*  0.252  <0.01**  Enzyme    0.018*  0.399  <0.01**  Type of diet × Enzyme  <0.01**  0.103  <0.01**  P-value for contrasts of enzyme level        LowSBM-0 vs. LowSBM-200  0.878  0.047*  0.986  LowSBM-0 vs. LowSBM-400  0.728  0.655  0.905  HighSBM-0 vs. HighSBM-200  0.113  0.094  0.930  HighSBM-0 vs. HighSBM-400  0.013*  0.263  0.668  HighSBM+GG-0 vs. HighSBM+GG-200  0.015*  0.386  <0.01**  HighSBM+GG-0 vs. HighSBM+GG-400  <0.01**  0.765  <0.01**  Means with no common superscripts (a, b, c) within a column are different at P ≤ 0.05 for Tukey-HSD test. *P-value < 0.05. **P-value < 0.01. View Large Table 4. Digestible energy (kcal/kg), phosphorus digestibility (%), and jejunum viscosity (cps) at 23 d of age. Item  Apparent Digestible Energy, Kcal/kg  Phosphorus Digestibility, %  Jejunum Viscosity, cps  Type of diet    Low SBM    3,071a  63.98  2.55b  High SBM    2,999a,b  62.50  2.27b  High SBM+GG    2,920b  61.50  11.14a  Enzyme β-D-mannanase, ppm        Dose: 0 ppm    2,957b  63.04  8.02a  Dose: 200 ppm    2,958b  63.40  4.08b  Dose: 400 ppm    3,074a  61.50  3.86b  SEM    34.76  0.89  0.14  Type of diet  Enzyme  ±       Low SBM  Dose: 0 ppm  3,085a,b  62.66  2.53c  Low SBM  Dose: 200 ppm  3,072a,b  67.73  2.54c  Low SBM  Dose: 400 ppm  3,057a,b  61.54  2.57c  High SBM  Dose: 0 ppm  3,013a,b,c  65.49  2.33c  High SBM  Dose: 200 ppm  2,819c  61.09  2.30c  High SBM  Dose: 400 ppm  3,165a  62.66  2.18c  High SBM+GG  Dose: 0 ppm  2,777c  60.98  19.20a  High SBM+GG  Dose: 200 ppm  2,982a,b,c  63.16  7.40b  High SBM+GG  Dose: 400 ppm  3,001a,b,c  60.23  6.83b  SEM    58.83  1.78  0.25  P-value for main effects and interaction      Type of diet    0.015*  0.252  <0.01**  Enzyme    0.018*  0.399  <0.01**  Type of diet × Enzyme  <0.01**  0.103  <0.01**  P-value for contrasts of enzyme level        LowSBM-0 vs. LowSBM-200  0.878  0.047*  0.986  LowSBM-0 vs. LowSBM-400  0.728  0.655  0.905  HighSBM-0 vs. HighSBM-200  0.113  0.094  0.930  HighSBM-0 vs. HighSBM-400  0.013*  0.263  0.668  HighSBM+GG-0 vs. HighSBM+GG-200  0.015*  0.386  <0.01**  HighSBM+GG-0 vs. HighSBM+GG-400  <0.01**  0.765  <0.01**  Item  Apparent Digestible Energy, Kcal/kg  Phosphorus Digestibility, %  Jejunum Viscosity, cps  Type of diet    Low SBM    3,071a  63.98  2.55b  High SBM    2,999a,b  62.50  2.27b  High SBM+GG    2,920b  61.50  11.14a  Enzyme β-D-mannanase, ppm        Dose: 0 ppm    2,957b  63.04  8.02a  Dose: 200 ppm    2,958b  63.40  4.08b  Dose: 400 ppm    3,074a  61.50  3.86b  SEM    34.76  0.89  0.14  Type of diet  Enzyme  ±       Low SBM  Dose: 0 ppm  3,085a,b  62.66  2.53c  Low SBM  Dose: 200 ppm  3,072a,b  67.73  2.54c  Low SBM  Dose: 400 ppm  3,057a,b  61.54  2.57c  High SBM  Dose: 0 ppm  3,013a,b,c  65.49  2.33c  High SBM  Dose: 200 ppm  2,819c  61.09  2.30c  High SBM  Dose: 400 ppm  3,165a  62.66  2.18c  High SBM+GG  Dose: 0 ppm  2,777c  60.98  19.20a  High SBM+GG  Dose: 200 ppm  2,982a,b,c  63.16  7.40b  High SBM+GG  Dose: 400 ppm  3,001a,b,c  60.23  6.83b  SEM    58.83  1.78  0.25  P-value for main effects and interaction      Type of diet    0.015*  0.252  <0.01**  Enzyme    0.018*  0.399  <0.01**  Type of diet × Enzyme  <0.01**  0.103  <0.01**  P-value for contrasts of enzyme level        LowSBM-0 vs. LowSBM-200  0.878  0.047*  0.986  LowSBM-0 vs. LowSBM-400  0.728  0.655  0.905  HighSBM-0 vs. HighSBM-200  0.113  0.094  0.930  HighSBM-0 vs. HighSBM-400  0.013*  0.263  0.668  HighSBM+GG-0 vs. HighSBM+GG-200  0.015*  0.386  <0.01**  HighSBM+GG-0 vs. HighSBM+GG-400  <0.01**  0.765  <0.01**  Means with no common superscripts (a, b, c) within a column are different at P ≤ 0.05 for Tukey-HSD test. *P-value < 0.05. **P-value < 0.01. View Large Apparent Amino Acid Digestibility The present study evaluated the digestibility of 16 amino acids (AA) (Table 5). Type of diets and enzyme levels affected apparent amino acid digestibility (AAAD). There was a diet × AA interaction for AAAD for 5 AA (Gly, His, Ala, Asp, and Glu). The AAAD was significantly higher for 10 AA (Lys, Met, Cys, Val, Iso, Tyr, Ser, His, Asp, and Glu) for diets containing High SBM and High SMB+GG compared to Low SBM (P ≤ 0.01). As for the contrast analysis, 10 out of 16 AAAD (Lys, Thr, Arg, Leu, Phe, Gly, Ser, His, Asp, and Glu) were improved when 200 ppm of β-mannanase was added to High SBM+GG (P ≤ 0.01) and 6 out of 16 AAAD (Thr, Leu, Phe, Gly, His, and Glu) were improved when 400 ppm of β-mannanase was added to High SBM+GG (P ≤ 0.01). Four AAAD (Val, Iso, His, and Ala) were lower when 200 ppm of β-mannanase was added to High SBM (P ≤ 0.01). Only Lys digestibility was improved when 200 ppm of β-mannanase was added to diet containing Low SBM (P ≤ 0.02). Table 5. Apparent ileal amino acid digestibility (%) at 23 d of age. Item  Lys  Met  Cys  Thr  Arg  Val  Leu  Iso  Phe  Tyr  Gly  Ser  His  Ala  Asp  Glu  Type of diet                                  Low SBM    81.7b  90.2b  67.4b  76.5b  93.0b  75.0b  80.6b  75.8b  79.4b  79.1b  75.4b  76.5b  80.8c  80.8  75.7b  83.4b  High SBM    84.9a  91.6a  73.2a  78.8a  93.8a  79.5a  82.7a  79.8a  81.9a  81.3a  78.4a  79.8a  84.1b  82.4  80.6a  86.6a  High SBM+GG    84.1a  92.5a  71.2a  77.9a,b  93.4a,b  78.7a  81.8a,b  79.0a  80.9a,b  81.3a  77.1a,b  79.5a  87.5a  81.8  79.8a  85.8a  Enzyme β-D-mannanase, ppm                                  Dose: 0 ppm    82.5  91.4  70.2  77.2  93.2  77.7  81.4  78.0  80.3  80.2  76.6  78.1  83.8  81.7  78.3  85.0  Dose: 200 ppm    84.5  91.1  70.4  78.0  93.6  77.7  81.9  78.3  81.1  80.6  77.3  78.8  84.1  81.5  79.1  85.6  Dose: 400 ppm    83.8  91.7  71.3  78.0  93.5  77.7  81.8  78.3  80.9  80.9  77.0  78.8  84.6  81.8  78.7  85.3  Type of diet  Enzyme                                  Low SBM  Dose: 0 ppm  79.7  90.5  67.4  76.4  92.9  74.7  80.5  75.5  79.2  78.7  75.3a,b,c  76.3  80.0d  80.8a,b  75.2b  83.3b,c  Low SBM  Dose: 200 ppm  83.6  90.5  68.3  77.5  93.4  76.3  81.6  77.0  80.6  80.0  77.2a,b,c  77.6  82.4b,c,d  82.2a,b  77.3a,b  84.3a,b,c  Low SBM  Dose: 400 ppm  81.7  90.4  66.6  75.6  92.9  74.0  79.8  74.9  78.6  78.5  73.6c  75.4  80.1d  79.4b  74.6b  82.6c  High SBM  Dose: 0 ppm  85.2  92.3  74.0  79.5  94.0  80.7  83.6  81.1  82.8  81.9  79.7a  80.7  85.6a,b,c  83.7a  81.8a  87.4a  High SBM  Dose: 200 ppm  84.1  90.7  71.3  77.5  93.5  77.6  81.5  78.0  80.7  79.8  76.5a,b,c  78.1  81.6c,d  80.3a,b  79.1a,b  85.7a,b,c  High SBM  Dose: 400 ppm  85.6  92.3  74.6  79.4  94.0  80.1  82.9  80.3  82.2  82.2  79.2a,b  80.5  85.1a,b,c  83.3a,b  80.9a  86.8a  High SBM+GG  Dose: 0 ppm  82.5  91.5  69.3  75.6  92.8  77.6  80.0  77.3  79.1  79.9  74.7b,c  77.4  87.5a,b  80.5a,b  77.8a,b  84.2a,b,c  High SBM+GG  Dose: 200 ppm  85.7  92.6  71.7  79.1  93.9  79.2  82.7  80.0  81.9  82.1  78.3a,b,c  80.7  88.4a  82.1a,b  80.9a  86.7a,b  High SBM+GG  Dose: 400 ppm  84.3  93.3  72.7  79.1  93.6  79.1  82.8  79.7  81.9  82.1  78.3a,b,c  80.4  88.5a  82.8a,b  80.5a  86.6a,b  SEM    1.15  0.65  1.55  1.09  0.29  1.05  0.93  1.11  0.99  0.98  1.09  1.17  0.88  0.91  1.04  0.78  P-value for main effects and interaction                                  Type of diet    <0.01  <0.01  <0.01  0.04*  <0.01  <0.01  0.03*  <0.01  0.01*  <0.01  <0.01  <0.01  <0.01  0.106  <0.01  <0.01  Enzyme    0.10  0.41  0.17  0.54  0.25  1.00  0.74  0.90  0.65  0.64  0.71  0.70  0.55  0.93  0.62  0.62  Type of diet × Enzyme    0.24  0.23  0.37  0.07  0.09  0.08  0.06  0.06  0.07  0.13  <0.01  0.076  <0.01  <0.01  0.03*  0.04*  P-value for contrasts of enzyme level                                  Low SBM-0 vs. Low SBM-200    0.02*  0.97  0.69  0.46  0.18  0.28  0.38  0.31  0.30  0.37  0.23  0.42  0.07  0.29  0.15  0.32  Low SBM-0 vs Low SBM-400    0.26  0.87  0.73  0.61  0.99  0.62  0.60  0.71  0.69  0.86  0.28  0.58  0.98  0.28  0.66  0.56  High SBM-0 vs. High SBM-200    0.51  0.09  0.23  0.21  0.28  0.04*  0.12  0.05*  0.14  0.13  0.05  0.14  <0.01  <0.01  0.08  0.14  High SBM-0 vs. High SBM-400    0.80  1.00  0.79  0.94  0.93  0.65  0.60  0.60  0.66  0.83  0.74  0.93  0.70  0.70  0.56  0.56  High SBM+GG-0 vs. High SBM+GG-200    0.05*  0.21  0.26  0.03*  <0.01  0.29  0.05*  0.09  0.04*  0.12  0.02*  0.04*  0.04*  0.22  0.04*  0.03*  High SBM+GG-0 vs. High SBM+GG-400    0.26  0.06  0.12  0.02*  0.06  0.33  0.04*  0.13  0.05*  0.11  0.02*  0.07  0.03*  0.10  0.07  0.03*  Item  Lys  Met  Cys  Thr  Arg  Val  Leu  Iso  Phe  Tyr  Gly  Ser  His  Ala  Asp  Glu  Type of diet                                  Low SBM    81.7b  90.2b  67.4b  76.5b  93.0b  75.0b  80.6b  75.8b  79.4b  79.1b  75.4b  76.5b  80.8c  80.8  75.7b  83.4b  High SBM    84.9a  91.6a  73.2a  78.8a  93.8a  79.5a  82.7a  79.8a  81.9a  81.3a  78.4a  79.8a  84.1b  82.4  80.6a  86.6a  High SBM+GG    84.1a  92.5a  71.2a  77.9a,b  93.4a,b  78.7a  81.8a,b  79.0a  80.9a,b  81.3a  77.1a,b  79.5a  87.5a  81.8  79.8a  85.8a  Enzyme β-D-mannanase, ppm                                  Dose: 0 ppm    82.5  91.4  70.2  77.2  93.2  77.7  81.4  78.0  80.3  80.2  76.6  78.1  83.8  81.7  78.3  85.0  Dose: 200 ppm    84.5  91.1  70.4  78.0  93.6  77.7  81.9  78.3  81.1  80.6  77.3  78.8  84.1  81.5  79.1  85.6  Dose: 400 ppm    83.8  91.7  71.3  78.0  93.5  77.7  81.8  78.3  80.9  80.9  77.0  78.8  84.6  81.8  78.7  85.3  Type of diet  Enzyme                                  Low SBM  Dose: 0 ppm  79.7  90.5  67.4  76.4  92.9  74.7  80.5  75.5  79.2  78.7  75.3a,b,c  76.3  80.0d  80.8a,b  75.2b  83.3b,c  Low SBM  Dose: 200 ppm  83.6  90.5  68.3  77.5  93.4  76.3  81.6  77.0  80.6  80.0  77.2a,b,c  77.6  82.4b,c,d  82.2a,b  77.3a,b  84.3a,b,c  Low SBM  Dose: 400 ppm  81.7  90.4  66.6  75.6  92.9  74.0  79.8  74.9  78.6  78.5  73.6c  75.4  80.1d  79.4b  74.6b  82.6c  High SBM  Dose: 0 ppm  85.2  92.3  74.0  79.5  94.0  80.7  83.6  81.1  82.8  81.9  79.7a  80.7  85.6a,b,c  83.7a  81.8a  87.4a  High SBM  Dose: 200 ppm  84.1  90.7  71.3  77.5  93.5  77.6  81.5  78.0  80.7  79.8  76.5a,b,c  78.1  81.6c,d  80.3a,b  79.1a,b  85.7a,b,c  High SBM  Dose: 400 ppm  85.6  92.3  74.6  79.4  94.0  80.1  82.9  80.3  82.2  82.2  79.2a,b  80.5  85.1a,b,c  83.3a,b  80.9a  86.8a  High SBM+GG  Dose: 0 ppm  82.5  91.5  69.3  75.6  92.8  77.6  80.0  77.3  79.1  79.9  74.7b,c  77.4  87.5a,b  80.5a,b  77.8a,b  84.2a,b,c  High SBM+GG  Dose: 200 ppm  85.7  92.6  71.7  79.1  93.9  79.2  82.7  80.0  81.9  82.1  78.3a,b,c  80.7  88.4a  82.1a,b  80.9a  86.7a,b  High SBM+GG  Dose: 400 ppm  84.3  93.3  72.7  79.1  93.6  79.1  82.8  79.7  81.9  82.1  78.3a,b,c  80.4  88.5a  82.8a,b  80.5a  86.6a,b  SEM    1.15  0.65  1.55  1.09  0.29  1.05  0.93  1.11  0.99  0.98  1.09  1.17  0.88  0.91  1.04  0.78  P-value for main effects and interaction                                  Type of diet    <0.01  <0.01  <0.01  0.04*  <0.01  <0.01  0.03*  <0.01  0.01*  <0.01  <0.01  <0.01  <0.01  0.106  <0.01  <0.01  Enzyme    0.10  0.41  0.17  0.54  0.25  1.00  0.74  0.90  0.65  0.64  0.71  0.70  0.55  0.93  0.62  0.62  Type of diet × Enzyme    0.24  0.23  0.37  0.07  0.09  0.08  0.06  0.06  0.07  0.13  <0.01  0.076  <0.01  <0.01  0.03*  0.04*  P-value for contrasts of enzyme level                                  Low SBM-0 vs. Low SBM-200    0.02*  0.97  0.69  0.46  0.18  0.28  0.38  0.31  0.30  0.37  0.23  0.42  0.07  0.29  0.15  0.32  Low SBM-0 vs Low SBM-400    0.26  0.87  0.73  0.61  0.99  0.62  0.60  0.71  0.69  0.86  0.28  0.58  0.98  0.28  0.66  0.56  High SBM-0 vs. High SBM-200    0.51  0.09  0.23  0.21  0.28  0.04*  0.12  0.05*  0.14  0.13  0.05  0.14  <0.01  <0.01  0.08  0.14  High SBM-0 vs. High SBM-400    0.80  1.00  0.79  0.94  0.93  0.65  0.60  0.60  0.66  0.83  0.74  0.93  0.70  0.70  0.56  0.56  High SBM+GG-0 vs. High SBM+GG-200    0.05*  0.21  0.26  0.03*  <0.01  0.29  0.05*  0.09  0.04*  0.12  0.02*  0.04*  0.04*  0.22  0.04*  0.03*  High SBM+GG-0 vs. High SBM+GG-400    0.26  0.06  0.12  0.02*  0.06  0.33  0.04*  0.13  0.05*  0.11  0.02*  0.07  0.03*  0.10  0.07  0.03*  Means with no common superscripts (a, b, c, d) within a column are different at P ≤ 0.05 for Tukey-HSD test. *Means P-value < 0.05. View Large Table 5. Apparent ileal amino acid digestibility (%) at 23 d of age. Item  Lys  Met  Cys  Thr  Arg  Val  Leu  Iso  Phe  Tyr  Gly  Ser  His  Ala  Asp  Glu  Type of diet                                  Low SBM    81.7b  90.2b  67.4b  76.5b  93.0b  75.0b  80.6b  75.8b  79.4b  79.1b  75.4b  76.5b  80.8c  80.8  75.7b  83.4b  High SBM    84.9a  91.6a  73.2a  78.8a  93.8a  79.5a  82.7a  79.8a  81.9a  81.3a  78.4a  79.8a  84.1b  82.4  80.6a  86.6a  High SBM+GG    84.1a  92.5a  71.2a  77.9a,b  93.4a,b  78.7a  81.8a,b  79.0a  80.9a,b  81.3a  77.1a,b  79.5a  87.5a  81.8  79.8a  85.8a  Enzyme β-D-mannanase, ppm                                  Dose: 0 ppm    82.5  91.4  70.2  77.2  93.2  77.7  81.4  78.0  80.3  80.2  76.6  78.1  83.8  81.7  78.3  85.0  Dose: 200 ppm    84.5  91.1  70.4  78.0  93.6  77.7  81.9  78.3  81.1  80.6  77.3  78.8  84.1  81.5  79.1  85.6  Dose: 400 ppm    83.8  91.7  71.3  78.0  93.5  77.7  81.8  78.3  80.9  80.9  77.0  78.8  84.6  81.8  78.7  85.3  Type of diet  Enzyme                                  Low SBM  Dose: 0 ppm  79.7  90.5  67.4  76.4  92.9  74.7  80.5  75.5  79.2  78.7  75.3a,b,c  76.3  80.0d  80.8a,b  75.2b  83.3b,c  Low SBM  Dose: 200 ppm  83.6  90.5  68.3  77.5  93.4  76.3  81.6  77.0  80.6  80.0  77.2a,b,c  77.6  82.4b,c,d  82.2a,b  77.3a,b  84.3a,b,c  Low SBM  Dose: 400 ppm  81.7  90.4  66.6  75.6  92.9  74.0  79.8  74.9  78.6  78.5  73.6c  75.4  80.1d  79.4b  74.6b  82.6c  High SBM  Dose: 0 ppm  85.2  92.3  74.0  79.5  94.0  80.7  83.6  81.1  82.8  81.9  79.7a  80.7  85.6a,b,c  83.7a  81.8a  87.4a  High SBM  Dose: 200 ppm  84.1  90.7  71.3  77.5  93.5  77.6  81.5  78.0  80.7  79.8  76.5a,b,c  78.1  81.6c,d  80.3a,b  79.1a,b  85.7a,b,c  High SBM  Dose: 400 ppm  85.6  92.3  74.6  79.4  94.0  80.1  82.9  80.3  82.2  82.2  79.2a,b  80.5  85.1a,b,c  83.3a,b  80.9a  86.8a  High SBM+GG  Dose: 0 ppm  82.5  91.5  69.3  75.6  92.8  77.6  80.0  77.3  79.1  79.9  74.7b,c  77.4  87.5a,b  80.5a,b  77.8a,b  84.2a,b,c  High SBM+GG  Dose: 200 ppm  85.7  92.6  71.7  79.1  93.9  79.2  82.7  80.0  81.9  82.1  78.3a,b,c  80.7  88.4a  82.1a,b  80.9a  86.7a,b  High SBM+GG  Dose: 400 ppm  84.3  93.3  72.7  79.1  93.6  79.1  82.8  79.7  81.9  82.1  78.3a,b,c  80.4  88.5a  82.8a,b  80.5a  86.6a,b  SEM    1.15  0.65  1.55  1.09  0.29  1.05  0.93  1.11  0.99  0.98  1.09  1.17  0.88  0.91  1.04  0.78  P-value for main effects and interaction                                  Type of diet    <0.01  <0.01  <0.01  0.04*  <0.01  <0.01  0.03*  <0.01  0.01*  <0.01  <0.01  <0.01  <0.01  0.106  <0.01  <0.01  Enzyme    0.10  0.41  0.17  0.54  0.25  1.00  0.74  0.90  0.65  0.64  0.71  0.70  0.55  0.93  0.62  0.62  Type of diet × Enzyme    0.24  0.23  0.37  0.07  0.09  0.08  0.06  0.06  0.07  0.13  <0.01  0.076  <0.01  <0.01  0.03*  0.04*  P-value for contrasts of enzyme level                                  Low SBM-0 vs. Low SBM-200    0.02*  0.97  0.69  0.46  0.18  0.28  0.38  0.31  0.30  0.37  0.23  0.42  0.07  0.29  0.15  0.32  Low SBM-0 vs Low SBM-400    0.26  0.87  0.73  0.61  0.99  0.62  0.60  0.71  0.69  0.86  0.28  0.58  0.98  0.28  0.66  0.56  High SBM-0 vs. High SBM-200    0.51  0.09  0.23  0.21  0.28  0.04*  0.12  0.05*  0.14  0.13  0.05  0.14  <0.01  <0.01  0.08  0.14  High SBM-0 vs. High SBM-400    0.80  1.00  0.79  0.94  0.93  0.65  0.60  0.60  0.66  0.83  0.74  0.93  0.70  0.70  0.56  0.56  High SBM+GG-0 vs. High SBM+GG-200    0.05*  0.21  0.26  0.03*  <0.01  0.29  0.05*  0.09  0.04*  0.12  0.02*  0.04*  0.04*  0.22  0.04*  0.03*  High SBM+GG-0 vs. High SBM+GG-400    0.26  0.06  0.12  0.02*  0.06  0.33  0.04*  0.13  0.05*  0.11  0.02*  0.07  0.03*  0.10  0.07  0.03*  Item  Lys  Met  Cys  Thr  Arg  Val  Leu  Iso  Phe  Tyr  Gly  Ser  His  Ala  Asp  Glu  Type of diet                                  Low SBM    81.7b  90.2b  67.4b  76.5b  93.0b  75.0b  80.6b  75.8b  79.4b  79.1b  75.4b  76.5b  80.8c  80.8  75.7b  83.4b  High SBM    84.9a  91.6a  73.2a  78.8a  93.8a  79.5a  82.7a  79.8a  81.9a  81.3a  78.4a  79.8a  84.1b  82.4  80.6a  86.6a  High SBM+GG    84.1a  92.5a  71.2a  77.9a,b  93.4a,b  78.7a  81.8a,b  79.0a  80.9a,b  81.3a  77.1a,b  79.5a  87.5a  81.8  79.8a  85.8a  Enzyme β-D-mannanase, ppm                                  Dose: 0 ppm    82.5  91.4  70.2  77.2  93.2  77.7  81.4  78.0  80.3  80.2  76.6  78.1  83.8  81.7  78.3  85.0  Dose: 200 ppm    84.5  91.1  70.4  78.0  93.6  77.7  81.9  78.3  81.1  80.6  77.3  78.8  84.1  81.5  79.1  85.6  Dose: 400 ppm    83.8  91.7  71.3  78.0  93.5  77.7  81.8  78.3  80.9  80.9  77.0  78.8  84.6  81.8  78.7  85.3  Type of diet  Enzyme                                  Low SBM  Dose: 0 ppm  79.7  90.5  67.4  76.4  92.9  74.7  80.5  75.5  79.2  78.7  75.3a,b,c  76.3  80.0d  80.8a,b  75.2b  83.3b,c  Low SBM  Dose: 200 ppm  83.6  90.5  68.3  77.5  93.4  76.3  81.6  77.0  80.6  80.0  77.2a,b,c  77.6  82.4b,c,d  82.2a,b  77.3a,b  84.3a,b,c  Low SBM  Dose: 400 ppm  81.7  90.4  66.6  75.6  92.9  74.0  79.8  74.9  78.6  78.5  73.6c  75.4  80.1d  79.4b  74.6b  82.6c  High SBM  Dose: 0 ppm  85.2  92.3  74.0  79.5  94.0  80.7  83.6  81.1  82.8  81.9  79.7a  80.7  85.6a,b,c  83.7a  81.8a  87.4a  High SBM  Dose: 200 ppm  84.1  90.7  71.3  77.5  93.5  77.6  81.5  78.0  80.7  79.8  76.5a,b,c  78.1  81.6c,d  80.3a,b  79.1a,b  85.7a,b,c  High SBM  Dose: 400 ppm  85.6  92.3  74.6  79.4  94.0  80.1  82.9  80.3  82.2  82.2  79.2a,b  80.5  85.1a,b,c  83.3a,b  80.9a  86.8a  High SBM+GG  Dose: 0 ppm  82.5  91.5  69.3  75.6  92.8  77.6  80.0  77.3  79.1  79.9  74.7b,c  77.4  87.5a,b  80.5a,b  77.8a,b  84.2a,b,c  High SBM+GG  Dose: 200 ppm  85.7  92.6  71.7  79.1  93.9  79.2  82.7  80.0  81.9  82.1  78.3a,b,c  80.7  88.4a  82.1a,b  80.9a  86.7a,b  High SBM+GG  Dose: 400 ppm  84.3  93.3  72.7  79.1  93.6  79.1  82.8  79.7  81.9  82.1  78.3a,b,c  80.4  88.5a  82.8a,b  80.5a  86.6a,b  SEM    1.15  0.65  1.55  1.09  0.29  1.05  0.93  1.11  0.99  0.98  1.09  1.17  0.88  0.91  1.04  0.78  P-value for main effects and interaction                                  Type of diet    <0.01  <0.01  <0.01  0.04*  <0.01  <0.01  0.03*  <0.01  0.01*  <0.01  <0.01  <0.01  <0.01  0.106  <0.01  <0.01  Enzyme    0.10  0.41  0.17  0.54  0.25  1.00  0.74  0.90  0.65  0.64  0.71  0.70  0.55  0.93  0.62  0.62  Type of diet × Enzyme    0.24  0.23  0.37  0.07  0.09  0.08  0.06  0.06  0.07  0.13  <0.01  0.076  <0.01  <0.01  0.03*  0.04*  P-value for contrasts of enzyme level                                  Low SBM-0 vs. Low SBM-200    0.02*  0.97  0.69  0.46  0.18  0.28  0.38  0.31  0.30  0.37  0.23  0.42  0.07  0.29  0.15  0.32  Low SBM-0 vs Low SBM-400    0.26  0.87  0.73  0.61  0.99  0.62  0.60  0.71  0.69  0.86  0.28  0.58  0.98  0.28  0.66  0.56  High SBM-0 vs. High SBM-200    0.51  0.09  0.23  0.21  0.28  0.04*  0.12  0.05*  0.14  0.13  0.05  0.14  <0.01  <0.01  0.08  0.14  High SBM-0 vs. High SBM-400    0.80  1.00  0.79  0.94  0.93  0.65  0.60  0.60  0.66  0.83  0.74  0.93  0.70  0.70  0.56  0.56  High SBM+GG-0 vs. High SBM+GG-200    0.05*  0.21  0.26  0.03*  <0.01  0.29  0.05*  0.09  0.04*  0.12  0.02*  0.04*  0.04*  0.22  0.04*  0.03*  High SBM+GG-0 vs. High SBM+GG-400    0.26  0.06  0.12  0.02*  0.06  0.33  0.04*  0.13  0.05*  0.11  0.02*  0.07  0.03*  0.10  0.07  0.03*  Means with no common superscripts (a, b, c, d) within a column are different at P ≤ 0.05 for Tukey-HSD test. *Means P-value < 0.05. View Large Table 6. Nutritional states, effect of type of diet, and mannanase on serum glucose (mg/dL), insulin (μIU/mL) and IGF-1 (pg/mL) at 21 to 22 d broilers.     Glucose HK (mg/dL)  Insulin (μIU/mL)  IGF-1 (pg/mL)  Item  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Type of diet                    Low SBM    216 ± 3.17b  185 ± 2.15  277 ± 5.21a  10.72 ± 0.35  11.39 ± 0.47  9.73 ± 0.27  543 ± 25.70  779 ± 25.56  668 ± 23.60  High SBM    223 ± 3.17a  187 ± 2.20  244 ± 5.07b  10.64 ± 0.37  10.03 ± 0.44  9.66 ± 0.28  507 ± 23.01  787 ± 32.98  730 ± 24.16  High SBM+GG    225 ± 3.09a  188 ± 2.15  252 ± 5.21b  10.47 ± 0.37  10.88 ± 0.45  9.33 ± 0.28  505 ± 29.06  778 ± 29.21  674 ± 24.15  Enzyme β-D-mannanase, ppm                    Dose: 0 ppm    220 ± 3.17  182 ± 2.2b  263 ± 5.07  9.73 ± 0.41b  10.89 ± 0.46  9.60 ± 0.28  472 ± 28.79  816 ± 27.89  684 ± 24.70  Dose: 200 ppm    222 ± 3.17  190 ± 2.15a  256 ± 5.33  11.23 ± 0.34a  10.65 ± 0.47  9.24 ± 0.27  557 ± 23.19  738 ± 28.74  691 ± 23.60  Dose: 400 ppm    220 ± 3.09  188 ± 2.15a  255 ± 5.07  10.87 ± 0.35a  10.76 ± 0.43  9.87 ± 0.28  526 ± 23.76  790 ± 29.04  697 ± 23.60  Type of diet   Enzyme                    Low SBM   Dose: 0 ppm  213 ± 5.35  182 ± 3.91  269 ± 8.79  10.18 ± 0.58  12.65 ± 0.80  9.72 ± 0.46  490 ± 34.82  806 ± 43.34  700 ± 40.88  Low SBM   Dose: 200 ppm  216 ± 5.77  187 ± 3.63  284 ± 9.47  11.58 ± 0.58  9.71 ± 0.87  9.31 ± 0.46  568 ± 50.09  738 ± 42.86  690 ± 40.88  Low SBM   Dose: 400 ppm  218 ± 5.35  185 ± 3.63  279 ± 8.79  10.38 ± 0.63  11.82 ± 0.74  10.16 ± 0.47  571 ± 49.91  792 ± 47.60  613 ± 40.88  High SBM   Dose: 0 ppm  218 ± 5.77  183 ± 3.91  253 ± 8.79  9.97 ± 0.76  9.99 ± 0.80  9.52 ± 0.51  493 ± 41.77  811 ± 59.75  748 ± 43.70  High SBM   Dose: 200 ppm  234 ± 5.35  191 ± 3.91  238 ± 8.79  10.77 ± 0.58  10.36 ± 0.74  9.57 ± 0.46  536 ± 31.24  746 ± 67.52  698 ± 40.88  High SBM   Dose: 400 ppm  218 ± 5.35  187 ± 3.63  243 ± 8.79  11.18 ± 0.58  9.72 ± 0.74  9.88 ± 0.47  494 ± 45.14  802 ± 39.94  744 ± 40.88  High SBM+GG   Dose: 0 ppm  229 ± 5.35  181 ± 3.63  266 ± 8.79  9.05 ± 0.76  10.03 ± 0.80  9.56 ± 0.46  433 ± 74.62  829 ± 46.11  604 ± 43.70  High SBM+GG   Dose: 200 ppm  222 ± 5.35  193 ± 3.63  247 ± 9.47  11.32 ± 0.58  11.87 ± 0.80  8.85 ± 0.47  567 ± 40.20  731 ± 40.67  684 ± 40.88  High SBM+GG   Dose: 400 ppm  224 ± 5.35  191 ± 3.92  244 ± 8.79  11.04 ± 0.58  10.74 ± 0.74  9.58 ± 0.51  514 ± 30.45  774 ± 65.26  732 ± 40.88  P-value for main effects and interaction                  Type of diet    0.048*  0.509  <0.01**  0.884  0.108  0.560  0.529  0.977  0.138  Enzyme    0.882  0.037*  0.543  0.021*  0.933  0.269  0.082  0.152  0.931  Type of diet × Enzyme  0.131  0.863  0.301  0.517  0.066  0.937  0.837  0.993  0.099  Block    <0.01**  0.731  0.045*  <0.01**  0.965  0.022*  0.139  0.580  0.297  P-value for contrasts of enzyme level                    LowSBM-0 vs. LowSBM-200  0.735  0.431  0.268  0.092  0.017*  0.536  0.195  0.267  0.810  LowSBM-0 vs. LowSBM-400  0.441  0.611  0.454  0.817  0.449  0.511  0.197  0.827  0.137  HighSBM-0 vs. HighSBM-200  0.031*  0.152  0.233  0.404  0.740  0.952  0.409  0.450  0.408  HighSBM-0 vs. HighSBM-400  0.955  0.417  0.413  0.209  0.801  0.604  0.982  0.901  0.963  HighSBM+GG-0 vs. HighSBM+GG-200  0.359  0.026*  0.158  0.021*  0.111  0.289  0.118  0.109  0.186  HighSBM+GG-0 vs. HighSBM+GG-400  0.461  0.084  0.091  0.042*  0.516  0.977  0.343  0.504  0.036*      Glucose HK (mg/dL)  Insulin (μIU/mL)  IGF-1 (pg/mL)  Item  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Type of diet                    Low SBM    216 ± 3.17b  185 ± 2.15  277 ± 5.21a  10.72 ± 0.35  11.39 ± 0.47  9.73 ± 0.27  543 ± 25.70  779 ± 25.56  668 ± 23.60  High SBM    223 ± 3.17a  187 ± 2.20  244 ± 5.07b  10.64 ± 0.37  10.03 ± 0.44  9.66 ± 0.28  507 ± 23.01  787 ± 32.98  730 ± 24.16  High SBM+GG    225 ± 3.09a  188 ± 2.15  252 ± 5.21b  10.47 ± 0.37  10.88 ± 0.45  9.33 ± 0.28  505 ± 29.06  778 ± 29.21  674 ± 24.15  Enzyme β-D-mannanase, ppm                    Dose: 0 ppm    220 ± 3.17  182 ± 2.2b  263 ± 5.07  9.73 ± 0.41b  10.89 ± 0.46  9.60 ± 0.28  472 ± 28.79  816 ± 27.89  684 ± 24.70  Dose: 200 ppm    222 ± 3.17  190 ± 2.15a  256 ± 5.33  11.23 ± 0.34a  10.65 ± 0.47  9.24 ± 0.27  557 ± 23.19  738 ± 28.74  691 ± 23.60  Dose: 400 ppm    220 ± 3.09  188 ± 2.15a  255 ± 5.07  10.87 ± 0.35a  10.76 ± 0.43  9.87 ± 0.28  526 ± 23.76  790 ± 29.04  697 ± 23.60  Type of diet   Enzyme                    Low SBM   Dose: 0 ppm  213 ± 5.35  182 ± 3.91  269 ± 8.79  10.18 ± 0.58  12.65 ± 0.80  9.72 ± 0.46  490 ± 34.82  806 ± 43.34  700 ± 40.88  Low SBM   Dose: 200 ppm  216 ± 5.77  187 ± 3.63  284 ± 9.47  11.58 ± 0.58  9.71 ± 0.87  9.31 ± 0.46  568 ± 50.09  738 ± 42.86  690 ± 40.88  Low SBM   Dose: 400 ppm  218 ± 5.35  185 ± 3.63  279 ± 8.79  10.38 ± 0.63  11.82 ± 0.74  10.16 ± 0.47  571 ± 49.91  792 ± 47.60  613 ± 40.88  High SBM   Dose: 0 ppm  218 ± 5.77  183 ± 3.91  253 ± 8.79  9.97 ± 0.76  9.99 ± 0.80  9.52 ± 0.51  493 ± 41.77  811 ± 59.75  748 ± 43.70  High SBM   Dose: 200 ppm  234 ± 5.35  191 ± 3.91  238 ± 8.79  10.77 ± 0.58  10.36 ± 0.74  9.57 ± 0.46  536 ± 31.24  746 ± 67.52  698 ± 40.88  High SBM   Dose: 400 ppm  218 ± 5.35  187 ± 3.63  243 ± 8.79  11.18 ± 0.58  9.72 ± 0.74  9.88 ± 0.47  494 ± 45.14  802 ± 39.94  744 ± 40.88  High SBM+GG   Dose: 0 ppm  229 ± 5.35  181 ± 3.63  266 ± 8.79  9.05 ± 0.76  10.03 ± 0.80  9.56 ± 0.46  433 ± 74.62  829 ± 46.11  604 ± 43.70  High SBM+GG   Dose: 200 ppm  222 ± 5.35  193 ± 3.63  247 ± 9.47  11.32 ± 0.58  11.87 ± 0.80  8.85 ± 0.47  567 ± 40.20  731 ± 40.67  684 ± 40.88  High SBM+GG   Dose: 400 ppm  224 ± 5.35  191 ± 3.92  244 ± 8.79  11.04 ± 0.58  10.74 ± 0.74  9.58 ± 0.51  514 ± 30.45  774 ± 65.26  732 ± 40.88  P-value for main effects and interaction                  Type of diet    0.048*  0.509  <0.01**  0.884  0.108  0.560  0.529  0.977  0.138  Enzyme    0.882  0.037*  0.543  0.021*  0.933  0.269  0.082  0.152  0.931  Type of diet × Enzyme  0.131  0.863  0.301  0.517  0.066  0.937  0.837  0.993  0.099  Block    <0.01**  0.731  0.045*  <0.01**  0.965  0.022*  0.139  0.580  0.297  P-value for contrasts of enzyme level                    LowSBM-0 vs. LowSBM-200  0.735  0.431  0.268  0.092  0.017*  0.536  0.195  0.267  0.810  LowSBM-0 vs. LowSBM-400  0.441  0.611  0.454  0.817  0.449  0.511  0.197  0.827  0.137  HighSBM-0 vs. HighSBM-200  0.031*  0.152  0.233  0.404  0.740  0.952  0.409  0.450  0.408  HighSBM-0 vs. HighSBM-400  0.955  0.417  0.413  0.209  0.801  0.604  0.982  0.901  0.963  HighSBM+GG-0 vs. HighSBM+GG-200  0.359  0.026*  0.158  0.021*  0.111  0.289  0.118  0.109  0.186  HighSBM+GG-0 vs. HighSBM+GG-400  0.461  0.084  0.091  0.042*  0.516  0.977  0.343  0.504  0.036*  Mean ± SE. Means with no common superscripts (a, b) within a column are different at P ≤ 0.05 for Tukey-HSD test. *P-value < 0.05. **P-value < 0.01. View Large Table 6. Nutritional states, effect of type of diet, and mannanase on serum glucose (mg/dL), insulin (μIU/mL) and IGF-1 (pg/mL) at 21 to 22 d broilers.     Glucose HK (mg/dL)  Insulin (μIU/mL)  IGF-1 (pg/mL)  Item  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Type of diet                    Low SBM    216 ± 3.17b  185 ± 2.15  277 ± 5.21a  10.72 ± 0.35  11.39 ± 0.47  9.73 ± 0.27  543 ± 25.70  779 ± 25.56  668 ± 23.60  High SBM    223 ± 3.17a  187 ± 2.20  244 ± 5.07b  10.64 ± 0.37  10.03 ± 0.44  9.66 ± 0.28  507 ± 23.01  787 ± 32.98  730 ± 24.16  High SBM+GG    225 ± 3.09a  188 ± 2.15  252 ± 5.21b  10.47 ± 0.37  10.88 ± 0.45  9.33 ± 0.28  505 ± 29.06  778 ± 29.21  674 ± 24.15  Enzyme β-D-mannanase, ppm                    Dose: 0 ppm    220 ± 3.17  182 ± 2.2b  263 ± 5.07  9.73 ± 0.41b  10.89 ± 0.46  9.60 ± 0.28  472 ± 28.79  816 ± 27.89  684 ± 24.70  Dose: 200 ppm    222 ± 3.17  190 ± 2.15a  256 ± 5.33  11.23 ± 0.34a  10.65 ± 0.47  9.24 ± 0.27  557 ± 23.19  738 ± 28.74  691 ± 23.60  Dose: 400 ppm    220 ± 3.09  188 ± 2.15a  255 ± 5.07  10.87 ± 0.35a  10.76 ± 0.43  9.87 ± 0.28  526 ± 23.76  790 ± 29.04  697 ± 23.60  Type of diet   Enzyme                    Low SBM   Dose: 0 ppm  213 ± 5.35  182 ± 3.91  269 ± 8.79  10.18 ± 0.58  12.65 ± 0.80  9.72 ± 0.46  490 ± 34.82  806 ± 43.34  700 ± 40.88  Low SBM   Dose: 200 ppm  216 ± 5.77  187 ± 3.63  284 ± 9.47  11.58 ± 0.58  9.71 ± 0.87  9.31 ± 0.46  568 ± 50.09  738 ± 42.86  690 ± 40.88  Low SBM   Dose: 400 ppm  218 ± 5.35  185 ± 3.63  279 ± 8.79  10.38 ± 0.63  11.82 ± 0.74  10.16 ± 0.47  571 ± 49.91  792 ± 47.60  613 ± 40.88  High SBM   Dose: 0 ppm  218 ± 5.77  183 ± 3.91  253 ± 8.79  9.97 ± 0.76  9.99 ± 0.80  9.52 ± 0.51  493 ± 41.77  811 ± 59.75  748 ± 43.70  High SBM   Dose: 200 ppm  234 ± 5.35  191 ± 3.91  238 ± 8.79  10.77 ± 0.58  10.36 ± 0.74  9.57 ± 0.46  536 ± 31.24  746 ± 67.52  698 ± 40.88  High SBM   Dose: 400 ppm  218 ± 5.35  187 ± 3.63  243 ± 8.79  11.18 ± 0.58  9.72 ± 0.74  9.88 ± 0.47  494 ± 45.14  802 ± 39.94  744 ± 40.88  High SBM+GG   Dose: 0 ppm  229 ± 5.35  181 ± 3.63  266 ± 8.79  9.05 ± 0.76  10.03 ± 0.80  9.56 ± 0.46  433 ± 74.62  829 ± 46.11  604 ± 43.70  High SBM+GG   Dose: 200 ppm  222 ± 5.35  193 ± 3.63  247 ± 9.47  11.32 ± 0.58  11.87 ± 0.80  8.85 ± 0.47  567 ± 40.20  731 ± 40.67  684 ± 40.88  High SBM+GG   Dose: 400 ppm  224 ± 5.35  191 ± 3.92  244 ± 8.79  11.04 ± 0.58  10.74 ± 0.74  9.58 ± 0.51  514 ± 30.45  774 ± 65.26  732 ± 40.88  P-value for main effects and interaction                  Type of diet    0.048*  0.509  <0.01**  0.884  0.108  0.560  0.529  0.977  0.138  Enzyme    0.882  0.037*  0.543  0.021*  0.933  0.269  0.082  0.152  0.931  Type of diet × Enzyme  0.131  0.863  0.301  0.517  0.066  0.937  0.837  0.993  0.099  Block    <0.01**  0.731  0.045*  <0.01**  0.965  0.022*  0.139  0.580  0.297  P-value for contrasts of enzyme level                    LowSBM-0 vs. LowSBM-200  0.735  0.431  0.268  0.092  0.017*  0.536  0.195  0.267  0.810  LowSBM-0 vs. LowSBM-400  0.441  0.611  0.454  0.817  0.449  0.511  0.197  0.827  0.137  HighSBM-0 vs. HighSBM-200  0.031*  0.152  0.233  0.404  0.740  0.952  0.409  0.450  0.408  HighSBM-0 vs. HighSBM-400  0.955  0.417  0.413  0.209  0.801  0.604  0.982  0.901  0.963  HighSBM+GG-0 vs. HighSBM+GG-200  0.359  0.026*  0.158  0.021*  0.111  0.289  0.118  0.109  0.186  HighSBM+GG-0 vs. HighSBM+GG-400  0.461  0.084  0.091  0.042*  0.516  0.977  0.343  0.504  0.036*      Glucose HK (mg/dL)  Insulin (μIU/mL)  IGF-1 (pg/mL)  Item  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Type of diet                    Low SBM    216 ± 3.17b  185 ± 2.15  277 ± 5.21a  10.72 ± 0.35  11.39 ± 0.47  9.73 ± 0.27  543 ± 25.70  779 ± 25.56  668 ± 23.60  High SBM    223 ± 3.17a  187 ± 2.20  244 ± 5.07b  10.64 ± 0.37  10.03 ± 0.44  9.66 ± 0.28  507 ± 23.01  787 ± 32.98  730 ± 24.16  High SBM+GG    225 ± 3.09a  188 ± 2.15  252 ± 5.21b  10.47 ± 0.37  10.88 ± 0.45  9.33 ± 0.28  505 ± 29.06  778 ± 29.21  674 ± 24.15  Enzyme β-D-mannanase, ppm                    Dose: 0 ppm    220 ± 3.17  182 ± 2.2b  263 ± 5.07  9.73 ± 0.41b  10.89 ± 0.46  9.60 ± 0.28  472 ± 28.79  816 ± 27.89  684 ± 24.70  Dose: 200 ppm    222 ± 3.17  190 ± 2.15a  256 ± 5.33  11.23 ± 0.34a  10.65 ± 0.47  9.24 ± 0.27  557 ± 23.19  738 ± 28.74  691 ± 23.60  Dose: 400 ppm    220 ± 3.09  188 ± 2.15a  255 ± 5.07  10.87 ± 0.35a  10.76 ± 0.43  9.87 ± 0.28  526 ± 23.76  790 ± 29.04  697 ± 23.60  Type of diet   Enzyme                    Low SBM   Dose: 0 ppm  213 ± 5.35  182 ± 3.91  269 ± 8.79  10.18 ± 0.58  12.65 ± 0.80  9.72 ± 0.46  490 ± 34.82  806 ± 43.34  700 ± 40.88  Low SBM   Dose: 200 ppm  216 ± 5.77  187 ± 3.63  284 ± 9.47  11.58 ± 0.58  9.71 ± 0.87  9.31 ± 0.46  568 ± 50.09  738 ± 42.86  690 ± 40.88  Low SBM   Dose: 400 ppm  218 ± 5.35  185 ± 3.63  279 ± 8.79  10.38 ± 0.63  11.82 ± 0.74  10.16 ± 0.47  571 ± 49.91  792 ± 47.60  613 ± 40.88  High SBM   Dose: 0 ppm  218 ± 5.77  183 ± 3.91  253 ± 8.79  9.97 ± 0.76  9.99 ± 0.80  9.52 ± 0.51  493 ± 41.77  811 ± 59.75  748 ± 43.70  High SBM   Dose: 200 ppm  234 ± 5.35  191 ± 3.91  238 ± 8.79  10.77 ± 0.58  10.36 ± 0.74  9.57 ± 0.46  536 ± 31.24  746 ± 67.52  698 ± 40.88  High SBM   Dose: 400 ppm  218 ± 5.35  187 ± 3.63  243 ± 8.79  11.18 ± 0.58  9.72 ± 0.74  9.88 ± 0.47  494 ± 45.14  802 ± 39.94  744 ± 40.88  High SBM+GG   Dose: 0 ppm  229 ± 5.35  181 ± 3.63  266 ± 8.79  9.05 ± 0.76  10.03 ± 0.80  9.56 ± 0.46  433 ± 74.62  829 ± 46.11  604 ± 43.70  High SBM+GG   Dose: 200 ppm  222 ± 5.35  193 ± 3.63  247 ± 9.47  11.32 ± 0.58  11.87 ± 0.80  8.85 ± 0.47  567 ± 40.20  731 ± 40.67  684 ± 40.88  High SBM+GG   Dose: 400 ppm  224 ± 5.35  191 ± 3.92  244 ± 8.79  11.04 ± 0.58  10.74 ± 0.74  9.58 ± 0.51  514 ± 30.45  774 ± 65.26  732 ± 40.88  P-value for main effects and interaction                  Type of diet    0.048*  0.509  <0.01**  0.884  0.108  0.560  0.529  0.977  0.138  Enzyme    0.882  0.037*  0.543  0.021*  0.933  0.269  0.082  0.152  0.931  Type of diet × Enzyme  0.131  0.863  0.301  0.517  0.066  0.937  0.837  0.993  0.099  Block    <0.01**  0.731  0.045*  <0.01**  0.965  0.022*  0.139  0.580  0.297  P-value for contrasts of enzyme level                    LowSBM-0 vs. LowSBM-200  0.735  0.431  0.268  0.092  0.017*  0.536  0.195  0.267  0.810  LowSBM-0 vs. LowSBM-400  0.441  0.611  0.454  0.817  0.449  0.511  0.197  0.827  0.137  HighSBM-0 vs. HighSBM-200  0.031*  0.152  0.233  0.404  0.740  0.952  0.409  0.450  0.408  HighSBM-0 vs. HighSBM-400  0.955  0.417  0.413  0.209  0.801  0.604  0.982  0.901  0.963  HighSBM+GG-0 vs. HighSBM+GG-200  0.359  0.026*  0.158  0.021*  0.111  0.289  0.118  0.109  0.186  HighSBM+GG-0 vs. HighSBM+GG-400  0.461  0.084  0.091  0.042*  0.516  0.977  0.343  0.504  0.036*  Mean ± SE. Means with no common superscripts (a, b) within a column are different at P ≤ 0.05 for Tukey-HSD test. *P-value < 0.05. **P-value < 0.01. View Large Blood Glucose, Insulin, and IGF-1 Serum glucose concentrations in 21 d chicks fed with High SBM (223 mg/dL) and High SBM+GG (225 mg/dL) were significantly higher than glucose in chicks fed with Low SBM diet (216 mg/dL) (P ≤ 0.048), see Table 6. Low SBM diets in re-fed chicks produced an opposite effect on serum glucose (277 mg/dL) compared to High SBM (244 mg/dL), and High SBM+GG (252 mg/dL) for chicks under re-fed conditions. In the fasted state, chicks previously fed diets with 200 and 400 β-mannanase had increased serum glucose concentrations of 190 and 188 mg/dL, respectively, compared to fasted glucose concentrations (182 mg/dL) for chicks fed control diets containing 0 ppm mannanase (P ≤ 0.01). Insulin was not affected by type of feed under any nutritional state, but was higher when 200 and 400 ppm of mannanase was added: 11.23 and 10.97 μIU/mL, respectively, compared to 9.73 μIU/mL (P ≤ 0.01) for control chicks under fed conditions. Contrast analysis showed that both 200 and 400 ppm mannanase added to High SBM diets produced higher insulin values in chicks than the control diet. Regarding IGF-1, there were no main effect differences, only contrast of High SBM+GG-400 diet (732 pg/mL) higher compared to 604 pg/mL in High SMB+GG-0 (P ≤ 0.036). DISCUSSION The present study had the purpose of evaluating performance, nutrient utilization, and metabolic changes when β-mannanase was added to 3 types of different diets. The enzyme β-mannanase affected variables differently with different dose levels and different types of diets. Performance Only 2 wk of evaluation showed that the addition of β-mannanase improved the feed:gain ratio in some but not all feeds. When β-mannanase was added by 200 and 400 ppm to High SBM+GG feeds, the feed:gain improved in 0.16 and 0.18, respectively. These results are expected due to the high β-mannan concentration in the feed (see Table 1). Daskiran et al. (2004) also found statistical differences when 500 ppm of β-mannanase was added to a feed with 0.5% of GG; however, the differences in feed:gain ratio between using the enzyme or not were lower than the values found in this study. The present experiment showed an opportunity for the enzyme β-mannanase to improve performance when High SBM and High SBM+GG diets are provided to broilers. It was expected that chicks fed diets with Low SBM would have equal performance to chicks fed with High SBM diets; however, AA analysis of the Low SBM diets (Table 1) were lower. Meat and bone meal or DDGS could have had lower nutrient content or nutrient digestibility in the Low SBM diet, although the individual ingredients were evaluated by near infrared prior to formulation. The higher mortality in Low SBM was unexpected and the authors have no explanation. The enzyme showed different response to different types of diets as expected. The enzyme will provide a different value when different ingredients are used in poultry diets because it is expected to produce a larger response with more substrate in the diet. Β-mannanase has been shown to improve broiler (Zou et al., 2006) and laying hen (Wu et al., 2005) performance when fed corn-soybean based diets. Broiler nutrition is about economics and producers around the world search for quality feedstuffs that are less expensive and available in quantities to support intense poultry production. Often, alternative ingredients that become available are byproducts of key ingredients needed for the human food industry. Many of the grain and legume byproducts contain increased levels of nonstarch polysaccharides including β-mannans. GG diets with high levels of mannan created an opportunity that shows the importance of the use of β-mannanase in broiler diets. High SBM diets with β-mannanase showed partial improvements, in a lower degree than the feeds with GG; however, it can be noted that this study was carried only for 2 wk. A longer period of evaluation may give higher differences or better understanding on the activity of the β-mannanase. There are ingredients in many parts of the work with high mannan content, and β-mannanase can be suitable to improve performance and/or reduce the feed cost without compromising broiler performance. Nutrient Utilization and Viscosity Improvement in feed:gain ratio for chicks fed with High SBM+GG diets with added β-mannanase in the present experiment can be explained by increased digestible energy, AA digestibility, and lower viscosity when 200 and 400 ppm β-mannanase were added to this diet. Chicks fed the High SBM diet with 400 ppm added β-mannanase also had an improved feed:gain ratio because energy digestibility was shown to be increased. In the present experiment, β-mannanase (200 and 400 ppm) added to High SBM+GG diets improved the digestible energy (DE) by 205 and 224 kcal/kg, respectively. The dose level of 400 ppm β-mannanase added to the High SBM diet without added GG improved DE by 152 kcal. These DE improvements with the additions of β-mannanase are significantly higher and probably the main reason of improvements in feed:gain ratio in the present study. Other authors have shown 59 kcal/kg energy improvements for MEn with 500 ppm of β-mannanase (same enzyme as the present study) added to High SBM+GG 0.5% broiler diets with broilers 1–14 d (Daskiran et al., 2004). Mannanase added to broiler diets as part of a carbohydrase composite enzyme has been reported to increase the AMEn by 144 kcal AMEn in broilers (Meng et al., 2005). β-mannanase may be releasing mannans to be used as source of energy in the metabolism. AAAD increased for chicks fed diets with β-mannanase added to High SBM-GG diets. The AAAD improvement ranged from +0.90% for histidine digestibility to +3.5 and +3.6 for threonine and glycine digestibility, respectively. Threonine and glycine are both indirectly involved in the gastrointestinal health of broilers by supporting mucin production. β-mannanase may be decreasing the substrate β-mannan and allowing better AA digestibility. Researchers have previously reported an improvement in AA digestibility in broilers fed corn-SBM diets when mannanase was included in a carbohydrase composite enzyme (Meng et al., 2005; Rutherfurd et al., 2007). Kong et al. (2011) reported no improvements in AA digestibility with added β-mannanase for broiler diets. Kong et al. (2011) were using a mannanase from Bacillus subtilis that is different from the Bacillus lentus used in the present experiment. Other publications showed that carbohydrases such as β-mannanase may improve the AA digestibility of feeds by releasing the protein that is caged within the polysaccharides (Cowieson and Ravindran, 2008). The improvement of digestibility of major AA but not body gain brings an important topic of discussion. There is likely a situation where increasing viscosity slows the transit and therefore the digestibility is better; however, because the transit is slower, the feed intake is decreased and no improvement in body gain. Further studies need to be accomplished to understand this paradigm because transit time was not measured in the present study. Glucose, Insulin, and IGF-1 changes Glucose is the primary nutrient source of energy for vital cells in an organism (Clarenburg, 1992) and insulin and IGF-1 are anabolic hormones that respond to glucose metabolism and other nutrients. Chickens have much higher plasma glucose levels than mammals but similar levels of insulin (chicken glucose: 200 mg/dL; mammals 100 mg/dL) (Hazelwood et al., 1968; Simon and Rosselin 1978). Modern high yielding broilers have been reported to contain even higher levels of glucose compared to previous broiler glucose levels: 234–309 mg/dL (Barcellos et al., 2012). Previous experiments with swine using high mannan feeds from GG produced lower glucose and insulin post-prandial (Rainbird and Low, 1986; Nunes and Malmlof, 1992). The importance of present study was to evaluate the negative impact of mannan from GG and SBM on plasma glucose, insulin, and IGF-1 and determine whether adding β-mannanase to the feed will ameliorate the negative effects. Rainbird and Low (1986) and Nunes and Malmlof (1992) reported the lower blood glucose values in pigs was because GG slowed glucose digestion and absorption. Fiber types such as GG have been tested in swine mainly as a means to help human obesity with the objective of reducing digestion and absorption of glucose. In the present experiment, the objective was to improve the negative effects of GG using the enzyme β-mannanase that reduced the viscosity of the jejunum and lowering viscosity helped increase the plasma insulin levels in fed conditions. There were no effects on insulin levels for chicks fed with β-mannanase during fasting and re-fed conditions. The question arises on which nutrient is causing the chick insulin levels to increase because glucose did not change with the enzyme treatments for chicks in the fed state. It is known that insulin is secreted not only in response to the rise of glucose but also fatty acids, and AA in the blood (Gropper and Smith, 2012). Fatty acids were not measured but the digestibility of several AA was increased with β-mannanase for chicks fed with High SBM+GG feeds. β-mannanase may have released more nutrients that increased insulin response because the feed intake was lower with the enzyme. Insulin response due to changes in glucose in broilers is not well understood. Broilers are hyperglycemic, but have the same amount of insulin compared to mammals and show low sensitivity to injections of insulin (Dupont et al., 2008). Dakovic et al. (2014) recently reported that chickens have lost some genes involved in insulin metabolism and the response to glucose metabolism is uncertain. Glucose metabolism was clearly affected in fasting chicks (24 h of feed deprivation) that were being fed diets containing both 200 and 400 ppm concentrations of β-mannanase. During fasting, glucose levels diminished to a certain level but there is a need to maintain a certain level of glucose during fasting because it is the only fuel for the brain and the nervous system (Clarenburg, 1992). In a human, carbohydrate reserves are exhausted in 1 d (Berg et al., 2012) and the process of gluconeogenesis begins to reload glucose in the blood and cells. A hypothesis on the mechanism of how β-mannanase elevates blood insulin and glucose in fasting chicks may be offered from the present study. First, the enzyme diminished the viscosity of the feed and this may have increased glucose metabolism due to better digestion and absorption. Second, mannose is a monosaccharide that can be absorbed passively and converted to glucose from fructose 6 PO4 (Clarenburg, 1992). It is possible that this pathway may have been stimulated to allow more glucose in the system in a fasting scenario. Third, gluconeogenesis increases glucose from noncarbohydrate sources such as AA that may have been stimulated. It is usually believed that carbohydrates are used as a fuel first, followed by fat, and finally proteins; however, the specific timing of the physiological transitions is difficult to consistently identify, because animals oxidize a mixture of carbohydrates, lipids, and proteins (McCue and Pollock, 2013). The results in the present study showed that the enzyme increased the glucose levels during fasting only. Glucose was not changed with the enzyme in other nutritional conditions; however, glucose changed due to the type of diet being fed. In the fed state, feeds with the higher β-mannan content showed higher glucose, and the opposite effect was found in the re-fed state. The re-fed state resembles the swine study where glucose was found to be lower right after a meal that was high in GG (Rainbird and Low, 1986; Nunes and Malmlof, 1992). Two hours may not be enough time for the enzyme to reduce the negative effects of β-mannans for the chick at the cellular level but the enzyme may have adequate time to reduce the viscosity in a 2 wk feeding study (fed state). This argument is supported by increased AA digestibility caused by additional β-mannanase with higher β-mannan feeds. The hormone IGF-1 is another anabolic hormone besides insulin playing an important role in cell proliferation and metabolism after hatch. Lu et al. (2007) reported IGF-1 increased at 14 d during embryo development and kept increasing after hatch through the end of study at 3 wk of age. IGF-1 was significantly increased in chicks in the present study only when 400 ppm of β-mannanase was added to High SBM+GG feed compared to their respective control. The High SBM+GG feed had the highest concentration of mannan, thereby providing more substrate and opportunity for the enzyme to work. Serum glucose was affected by block when samples were collected in the feeding state, but not during the fasting state, probably due to lower standard error between the birds in the fasting state. Serum glucose decreased with time of sampling (block) but at different rate between the treatments, which is the reason for the block to be statistically significant. This same blocking effect was seen with insulin in the feeding state. This may be due to diurnal patterns in insulin (Ekmay et al., 2010). Because the sampling was done in about 3 h during the morning, this could have affected the glucose and insulin status. No blocking differences were seen when evaluating IGF-1. In conclusion, β-mannanase enzyme added to feed showed higher nutritional benefits (performance and physiological) when used with High SBM and High SBM+GG diets. 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The effect of β-mannanase on nutrient utilization and blood parameters in chicks fed diets containing soybean meal and guar gum

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey099
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Abstract

Abstract The present study was conducted to determine whether the addition of β-mannanase in broiler feed changes hormonal profiles in the blood and broiler performance and nutrient availability. Five hundred and four Cobb male chickens were studied during d 7 to 21. Three corn–soybean meal (SBM) based diets 1) Low SBM (18% SBM); 2) High SBM (31% SBM); and 3) High SBM+GG (31% SBM + Guar Gum (GG) 0.5%) with 3 levels of β-mannanase (0, 200, and 400 ppm) were mixed to produce 9 diets. A factorial design 3 × 3 was performed with JMP pro 13 (SAS, 2017). Analysis of variance and contrast analysis were used to test significance level at P < 0.05. Glucose (190 and 188 mg/dL) was increased with 200 and 400 ppm of β-mannanase, respectively, compared to control (182 mg/dL) in the fasted state (P < 0.037). Glucose was higher in chicks fed with the High SBM and High SBM + GG diets but lower in the fasted re-fed state (P < 0.01). Insulin was higher with 200 and 400 ppm added β-mannanase in the fed state (P < 0.021). Insulin-like growth factor-1 was higher with 400 ppm added to High SBM+GG. β-mannanase improved feed conversion ratio (FCR) 9 points with 400 ppm in High SBM diet (P < 0.01) and 16 and 18 points with 200 and 400 ppm, respectively, added to the High SBM+GG diet (P < 0.01). Viscosity decreased from 19.2 to 7 cps with both enzyme doses in the High SBM + GG diet (P < 0.01). Digestible energy was +152 kcal/kg with 400 ppm β-mannanase in the High SBM diet and +200 kcal/kg with both levels of enzyme in High SBM+GG diet. Digestibility of amino acids was improved from 0.8 to 3.6% with β-mannanase in High SBM+GG diet (P < 0.05). In conclusion, chicks fed with High SBM and High SBM+GG diets with added β-mannanase significantly improved blood glucose and anabolic hormone homeostasis, FCR, digestible energy, and digestible amino acids compared to chicks fed with same diets without β-mannanase. INTRODUCTION Common cereals and protein crops used as feedstuffs in poultry diets have high nonstarch polysaccharides (NSP). Nonstarch polysaccharides, predominantly the soluble form, are considered detrimental for nutrient utilization in broilers, but the soluble NSP also have the greatest potential to be degraded by exogenous enzymes (Bach Knudsen, 2014). The purpose of adding exogenous enzymes to animal feeds is to increase the availability of nutrients while reducing poultry production cost. Processed soybean meal (SBM) is the world's largest source of plant protein used in the feed, but SBM contains various NSP that diminish nutrient utilization by poultry. One of the NSP in SBM is β-mannan that has a concentration of 1.26 ± 0.14% in dehulled and 1.61±0.20% in nondehulled SBM (Hsiao, et al., 2006). The exogenous enzyme β-mannanase represents a potential NSP enzyme capable of reducing the negative effects of β-mannan. Several researchers (Jackson et al., 1999; Daskiran, et al., 2004; Mohammad and Kazem, 2012; Hussain et al., 2012) reported negative effects of β-mannan from guar and soybean meal on poultry performance and the potential to overcome its effects with mannanase. The structure of β-mannan in guar is almost identical to that of β-mannan in SBM (Daskiran, et al., 2004; and Hsiao et al., 2006). Because SBM is the major protein source in poultry diets, the degradation of β-mannan in SBM could lead to improved performance of poultry. β-mannan in the feed has been reported to lower body weight gain and increased feed:gain in d 1 to 14 broilers with guar gum (GG) (Daskiran et al., 2004); increase feed:gain in d 1 to 42 broilers with guar meal (Kamran et al., 2002); increase feed:gain and produce higher plasma lipids for d 1 to 42 broilers fed guar meal (Lee et al., 2005; Mohammad and Kazem, 2012); however, there is still a limited information available on the metabolic effects of β-mannan on glucose metabolism and hormonal change. Rainbird and Low (1986) and Nunes and Malmlof (1992) have reported a reduction in postprandial blood glucose and insulin when GG was fed to pigs. An attempt to understand the β-mannanase effect at the metabolic level was conducted by Jozefiak et al. (2010) when the group added a multicarbohydrase enzyme with mannanase to a wheat–soybean and full-fat rapeseed meal diet and showed increased insulin receptors in the liver of chickens. Arsenault et al. (2017) found that the addition of β-galactomannan impacted the immune response in the jejunum of d 42 broiler; and adding β-mannanase eliminated most of the immune signaling effect. More information is needed to determine whether blood glucose and anabolic hormones such as insulin and insulin-like growth factor-1 (IGF-1) in broilers fed corn-SBM-based diets are influenced by the supplementation of mannanase. The present experiment is intended to test the negative effects of β-mannan from SBM and GG in diets on energy metabolism in broilers and to determine whether an added β-mannanase enzyme will reverse this effect. MATERIALS AND METHODS All management practices and procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee (IACUC) #12,041. Birds and Diets Male broiler chicks (504) of a commercial strain (Cobb-Vantress, Inc.) were obtained from a local hatchery. The chicks were maintained on floor pens for 7 d post hatching on an environmentally controlled room and fed a standard starter diet (CP 22.2%; 3035 kcal/kg) that meet the breeder recommendation levels. On d 7, all chicks were weighed individually, allocated to 72 wire metabolic cages (dimensions: 91 cm × 30 cm each), and randomly assigned to 9 dietary treatments with 7 chicks per metabolic cage with 8 replications for a 2 wk experimental period. Each cage started with similar coefficient of variance (CV) on body weight (BW) (Range: 12.1 to 13.7%). Birds had free access to mash feed and water during the experimental period. Individual BW and feed consumption per cage were recorded at d 21 post hatching. All broilers received 1 of the 9 experimental diets structured in a 3 × 3 factorial arrangement from d 7 to 21. Three diets × three levels of the enzyme β-mannanase (0, 200, and 400 ppm). Legumes such as SBM contain higher β-mannan (1.2% DM) compared to corn (0.3% DM) (Bach Knudsen, 1997). SBM and GG were used as the main mannan source and meat and bone meal was added to reduce the SBM level in basal diet 1. GG used in the present study had 80% β-mannans and it was used to increase the β-mannans in the third diet. The experimental diets evaluated were 1) Low SBM, 2) High SBM, and 3) High SBM+ GG. Diet 1, low SBM had 18% SBM, diet 2 had 31% SBM, and diet 3 was diet 2 plus 0.5% GG. Total calculated mannans were 4,677; 5,335; and 9,330 ppm in diets 1, 2, and 3, respectively. These values were calculated from corn, corn dried distillers grain solubles (DDGS), and SBM from recent publications (Bach Knudsen, 2014; Jaworski et al., 2015), and GG was purchased with 80% mannan (Polypro International, Minnesota, US) contributing with 4,000 ppm of the 9,330 ppm of mannan in diet 3. The enzyme β-mannanase was added to the 3 basal diets (Table 1) at 3 inclusion rates: 0, 200, and 400 ppm generating 9 diets. The β-mannanase enzyme was produced from Bacillus lentus. One unit of mannanase activity was defined as the amount of enzyme which generates 0.72 micrograms of reducing sugars per minute from a mannose-containing substrate at pH 6.6 and temperature of 104°F. The assay detects a value <15 × 106 activity units of mannanase/MT in the basal diet; therefore, activity values in the basal diets of the present study were acceptable. Feed production started by making 3 basal feeds, and splitting each basal into 3 separate containers. The enzyme was premixed with 4.5 kg of the corresponding diet before mixing the diet for the experimental treatment. Control diets with no enzyme addition (Low SBM-0, High SBM-0, and High SBM+GG-0) were mixed again to avoid differences of mixing due to other feeds being mixed with the corresponding enzyme level. Samples of each diet were sent for enzyme analysis verification to a commercial laboratory (Elanco, IN, USA) (Table 2). The results confirmed that the enzyme activities were ±30% within the guaranteed values. Table 1. Composition and nutrient calculations (g/100 g as fed) of the basal diets for d 7 to 21.   Basal Low  Basal High  Basal High  Ingredients  SBM  SBM  SBM + GG1  Corn 8.8% CP  65.1  60.8  59.8  Soybean meal 46.4% CP  18.4  31.4  31.6  Meat & Bone Meal, 54% CP2  7.8      Corn DDGS  5.0      Guar Gum (80% mannan)      0.50  Poultry fat  0.40  2.40  2.74  DL-Methionine 98.5%  0.30  0.31  0.31  L-Lysine HCl, 78%  0.47  0.29  0.29  L-Threonine, 98%  0.18  0.15  0.15  Limestone  0.47  1.05  1.05  Dicalcium phosphate  0.41  2.15  2.16  Salt  0.41  0.38  0.38  Vitamin and mineral premix3  0.52  0.52  0.52  Titanium dioxide  0.50  0.50  0.50  Calculated Nutrients        β-Mannan, ppm4  4,677  5,335  9,330  AME, kcal/kg  3,000  3,000  3,000  Crude protein, %  21.0  21.0  21.0  Calcium, %  1.00  1.00  1.00  Digestible phosphorus, %  0.50  0.50  0.50  Digestible lysine, %  1.10  1.10  1.10  Digestible methionine + cysteine, %  0.83  0.83  0.83  Digestible threonine, %  0.73  0.73  0.73  Analyzed Nutrients5        Crude protein, %  20.6  20.9  20.5  Digestible lysine, %  1.04  1.18  1.15  Digestible methionine + cysteine, %  0.77  0.79  0.79  Digestible threonine, %  0.70  0.72  0.69  Digestible phosphorus, %  0.50  0.48  0.45    Basal Low  Basal High  Basal High  Ingredients  SBM  SBM  SBM + GG1  Corn 8.8% CP  65.1  60.8  59.8  Soybean meal 46.4% CP  18.4  31.4  31.6  Meat & Bone Meal, 54% CP2  7.8      Corn DDGS  5.0      Guar Gum (80% mannan)      0.50  Poultry fat  0.40  2.40  2.74  DL-Methionine 98.5%  0.30  0.31  0.31  L-Lysine HCl, 78%  0.47  0.29  0.29  L-Threonine, 98%  0.18  0.15  0.15  Limestone  0.47  1.05  1.05  Dicalcium phosphate  0.41  2.15  2.16  Salt  0.41  0.38  0.38  Vitamin and mineral premix3  0.52  0.52  0.52  Titanium dioxide  0.50  0.50  0.50  Calculated Nutrients        β-Mannan, ppm4  4,677  5,335  9,330  AME, kcal/kg  3,000  3,000  3,000  Crude protein, %  21.0  21.0  21.0  Calcium, %  1.00  1.00  1.00  Digestible phosphorus, %  0.50  0.50  0.50  Digestible lysine, %  1.10  1.10  1.10  Digestible methionine + cysteine, %  0.83  0.83  0.83  Digestible threonine, %  0.73  0.73  0.73  Analyzed Nutrients5        Crude protein, %  20.6  20.9  20.5  Digestible lysine, %  1.04  1.18  1.15  Digestible methionine + cysteine, %  0.77  0.79  0.79  Digestible threonine, %  0.70  0.72  0.69  Digestible phosphorus, %  0.50  0.48  0.45  The β-mannanase was added to each of the 3 basal diets at 3 inclusion rates: 0, 200, and 400 ppm generating 9 diets. 1Inclusion: 0.5% Guar Gum. 2Meat and Bone meal was sourced from H. J. Baker as Proplus, 54%. 3Supplied per kilogram of diet: 200 mg antioxidant, 15,432 UI vitamin A, 11,023 IU vitamin D3, 110 UI vitamin E, 3 mg menadione, 13 mg riboflavin, 20 mg pantothenic acid, 77 mg niacin, 2 mg folic acid, 0.03 mg vitamin B12, 6 mg pyridoxine, 0.20 mg biotin, 3 mg thiamine, 1,200 mg of choline chlorine, 100 mg Mn, 27 mg Mg, 100 mg Zn, 50 mg Fe, 10 mg Cu, 1 mg I, and 0.20 mg Se. 4Adapted from Bach Knudsen, 2014; and Jaworski et al., 2015. 5The analyzed digestible AA were calculated with the total AA value multiplied by the digestibility value. View Large Table 1. Composition and nutrient calculations (g/100 g as fed) of the basal diets for d 7 to 21.   Basal Low  Basal High  Basal High  Ingredients  SBM  SBM  SBM + GG1  Corn 8.8% CP  65.1  60.8  59.8  Soybean meal 46.4% CP  18.4  31.4  31.6  Meat & Bone Meal, 54% CP2  7.8      Corn DDGS  5.0      Guar Gum (80% mannan)      0.50  Poultry fat  0.40  2.40  2.74  DL-Methionine 98.5%  0.30  0.31  0.31  L-Lysine HCl, 78%  0.47  0.29  0.29  L-Threonine, 98%  0.18  0.15  0.15  Limestone  0.47  1.05  1.05  Dicalcium phosphate  0.41  2.15  2.16  Salt  0.41  0.38  0.38  Vitamin and mineral premix3  0.52  0.52  0.52  Titanium dioxide  0.50  0.50  0.50  Calculated Nutrients        β-Mannan, ppm4  4,677  5,335  9,330  AME, kcal/kg  3,000  3,000  3,000  Crude protein, %  21.0  21.0  21.0  Calcium, %  1.00  1.00  1.00  Digestible phosphorus, %  0.50  0.50  0.50  Digestible lysine, %  1.10  1.10  1.10  Digestible methionine + cysteine, %  0.83  0.83  0.83  Digestible threonine, %  0.73  0.73  0.73  Analyzed Nutrients5        Crude protein, %  20.6  20.9  20.5  Digestible lysine, %  1.04  1.18  1.15  Digestible methionine + cysteine, %  0.77  0.79  0.79  Digestible threonine, %  0.70  0.72  0.69  Digestible phosphorus, %  0.50  0.48  0.45    Basal Low  Basal High  Basal High  Ingredients  SBM  SBM  SBM + GG1  Corn 8.8% CP  65.1  60.8  59.8  Soybean meal 46.4% CP  18.4  31.4  31.6  Meat & Bone Meal, 54% CP2  7.8      Corn DDGS  5.0      Guar Gum (80% mannan)      0.50  Poultry fat  0.40  2.40  2.74  DL-Methionine 98.5%  0.30  0.31  0.31  L-Lysine HCl, 78%  0.47  0.29  0.29  L-Threonine, 98%  0.18  0.15  0.15  Limestone  0.47  1.05  1.05  Dicalcium phosphate  0.41  2.15  2.16  Salt  0.41  0.38  0.38  Vitamin and mineral premix3  0.52  0.52  0.52  Titanium dioxide  0.50  0.50  0.50  Calculated Nutrients        β-Mannan, ppm4  4,677  5,335  9,330  AME, kcal/kg  3,000  3,000  3,000  Crude protein, %  21.0  21.0  21.0  Calcium, %  1.00  1.00  1.00  Digestible phosphorus, %  0.50  0.50  0.50  Digestible lysine, %  1.10  1.10  1.10  Digestible methionine + cysteine, %  0.83  0.83  0.83  Digestible threonine, %  0.73  0.73  0.73  Analyzed Nutrients5        Crude protein, %  20.6  20.9  20.5  Digestible lysine, %  1.04  1.18  1.15  Digestible methionine + cysteine, %  0.77  0.79  0.79  Digestible threonine, %  0.70  0.72  0.69  Digestible phosphorus, %  0.50  0.48  0.45  The β-mannanase was added to each of the 3 basal diets at 3 inclusion rates: 0, 200, and 400 ppm generating 9 diets. 1Inclusion: 0.5% Guar Gum. 2Meat and Bone meal was sourced from H. J. Baker as Proplus, 54%. 3Supplied per kilogram of diet: 200 mg antioxidant, 15,432 UI vitamin A, 11,023 IU vitamin D3, 110 UI vitamin E, 3 mg menadione, 13 mg riboflavin, 20 mg pantothenic acid, 77 mg niacin, 2 mg folic acid, 0.03 mg vitamin B12, 6 mg pyridoxine, 0.20 mg biotin, 3 mg thiamine, 1,200 mg of choline chlorine, 100 mg Mn, 27 mg Mg, 100 mg Zn, 50 mg Fe, 10 mg Cu, 1 mg I, and 0.20 mg Se. 4Adapted from Bach Knudsen, 2014; and Jaworski et al., 2015. 5The analyzed digestible AA were calculated with the total AA value multiplied by the digestibility value. View Large Table 2. Enzyme activity analysis in feed (enzyme × 106 units of β-mannanase/ton). Type of  Enzyme,  Analyzed Enzyme  Guaranteed Enzyme  Analyzed:  diet  ppm  Activity  Activity  Guaranteed,1%  Low SBM  Dose 0  9.9      Low SBM  Dose 200  34.2  29.0  83.8  Low SBM  Dose 400  81.7  58.0  123.8  High SBM  Dose 0  6.3      High SBM  Dose 200  41.7  29.0  122.1  High SBM  Dose 400  74.0  58.0  116.7  High SBM + GG  Dose 0  9.8      High SBM + GG  Dose 200  46.2  29.0  125.5  High SBM + GG  Dose 400  86.4  58.0  132.1  Type of  Enzyme,  Analyzed Enzyme  Guaranteed Enzyme  Analyzed:  diet  ppm  Activity  Activity  Guaranteed,1%  Low SBM  Dose 0  9.9      Low SBM  Dose 200  34.2  29.0  83.8  Low SBM  Dose 400  81.7  58.0  123.8  High SBM  Dose 0  6.3      High SBM  Dose 200  41.7  29.0  122.1  High SBM  Dose 400  74.0  58.0  116.7  High SBM + GG  Dose 0  9.8      High SBM + GG  Dose 200  46.2  29.0  125.5  High SBM + GG  Dose 400  86.4  58.0  132.1  1Analyzed enzyme values for 200 and 400 ppm dose levels were deducted from the basal before they were divided over their corresponding guaranteed enzyme activity values × 100. View Large Table 2. Enzyme activity analysis in feed (enzyme × 106 units of β-mannanase/ton). Type of  Enzyme,  Analyzed Enzyme  Guaranteed Enzyme  Analyzed:  diet  ppm  Activity  Activity  Guaranteed,1%  Low SBM  Dose 0  9.9      Low SBM  Dose 200  34.2  29.0  83.8  Low SBM  Dose 400  81.7  58.0  123.8  High SBM  Dose 0  6.3      High SBM  Dose 200  41.7  29.0  122.1  High SBM  Dose 400  74.0  58.0  116.7  High SBM + GG  Dose 0  9.8      High SBM + GG  Dose 200  46.2  29.0  125.5  High SBM + GG  Dose 400  86.4  58.0  132.1  Type of  Enzyme,  Analyzed Enzyme  Guaranteed Enzyme  Analyzed:  diet  ppm  Activity  Activity  Guaranteed,1%  Low SBM  Dose 0  9.9      Low SBM  Dose 200  34.2  29.0  83.8  Low SBM  Dose 400  81.7  58.0  123.8  High SBM  Dose 0  6.3      High SBM  Dose 200  41.7  29.0  122.1  High SBM  Dose 400  74.0  58.0  116.7  High SBM + GG  Dose 0  9.8      High SBM + GG  Dose 200  46.2  29.0  125.5  High SBM + GG  Dose 400  86.4  58.0  132.1  1Analyzed enzyme values for 200 and 400 ppm dose levels were deducted from the basal before they were divided over their corresponding guaranteed enzyme activity values × 100. View Large Sample Collection Blood samples were taken at three different feeding conditions (fed, fasted, and re-fed). On d 21 at 7 AM (fed), 1 chick per cage (8 chicks per treatment) were randomly selected as soon as the weight was recorded, it was also when the time of fasting started. The second blood collection occurred after 24 h of fasting on a second set of 8 chicks per treatment starting at 7 AM on d 22 (fasted). Finally, the last set of blood samples were taken on a third set of 8 chicks per treatment, 2 h after re-feeding starting at 9 AM on d 22 (re-fed). One chick per cage was taken for blood analysis resulting in 8 chicks per treatment, and 72 chicks for the study. Chicks were sampled in blocks, so every block had all 9 treatments to reduce the bias on the analytics due to time of sampling. Bleeding for each block took about 20 min, so the bleeding was finished in approximately 3 h for the study. Blood was collected via jugular to avoid hemolysis. Blood from each chick was collected into one serum separator tube. Chicks were euthanized using gas CO2 inhalation soon after the blood collection. All blood samples were centrifuged at 3,000 rpm for 15 min and the serum portion was transferred into 3 separate tubes and stored at −20°C for further glucose and hormone analysis. On d 23, after 9 h of fasting, the remaining birds were provided feed for 2 h and then 4 chicks per cage were humanely euthanized using CO2 gas inhalation, and the contents of the jejunum and ileum collected. The jejunum was defined from the end of the duodenal loop to the vitelline diverticulum (Meckel's diverticulum). The ileum was defined as the portion of the small intestine extending from vitelline diverticulum to 4 cm proximal to the ileocecal junction. Jejunum contents were processed and analyzed for viscosity within 2 h after the last collection. The ileal contents from 4 chicks were stored in 100 mL cups at −20°C for 48 h, lyophilized for 7 d, and finely ground for further nutrient analysis. Blood Analysis Serum was analyzed for glucose, insulin, and IGF-1. Glucose concentration was analyzed using 200 μL of serum sample with Ciba-Corning Glucose hexokinase (HK) reagents. This method assays serum glucose concentration enzymatically. Glucose is phosphorylated with adenosine triphosphate in the reaction catalyzed by HK. The product, glucose-6-phosphate, is then oxidized with the concomitant reduction of nicotinamide adenine dinucleotide (NDA+) to NADH in the reaction catalyzed by glucose-6-phosphate dehydrogenase. The formation of NADH causes an increase in absorbance at 340 nm, which is directly proportional to the amount of glucose in the sample. The absorbance was measured using the analyzer Express plus 550 (Ciba Corning, NY, USA). Serum insulin and IGF-1 were detected using a chicken insulin ELISA kit (Cusabio, San Diego, CA, USA), which has higher sensitivities for chicken insulin than regular insulin kits made for rodents or humans. Same method was used by Shi et al. (2014). The absorbance was measured using the ELISA multi detection microplate reader Synergy HT (Biotek, Winooski, VT, USA). Results were analyzed against a standard curve. Viscosity and Chemical Analysis The jejunum contents from 4 chicks per cage were filled into 50-mL tubes and centrifuged at 3,000 rpm for 10 min. The supernatant was decanted and the viscosity of a 0.5 ml aliquot measured in centipoise (cps) using Brookfield Digital Viscometer (Model DV-II, Brookfield Engineering Laboratories, Stoughton, MA). Diets and ileal contents were analyzed for dry matter, gross energy (GE), amino acids (AA), phosphorus (P), and titanium dioxide (TiO2). Samples were analyzed following AOAC official methods (AOAC, 1990). Dry matter was analyzed with AOAC 934.01. Gross energy was determined in a bomb calorimeter (Parr 6200 bomb calorimeter, Parr Instruments Co., Moline, IL.). Amino acids were determined with AOAC 982.30 and AOAC 985.28 methods using high-performance (high-pressure) liquid chromatography, P was analyzed with AOAC, 968.08 using inductively-coupled plasma. TiO2 was measured on a UV spectrophotometer (Shimadzu model UV-2101 PC, Scientific Instruments Inc., Hawthorne, NY) utilizing the method of Myers et al. (2004). The apparent ileal nutrient digestibility (AIND) of energy, phosphorus, and amino acids were calculated using the equation AIND = [((NT/Ti)d − (NT/Ti)i)/(NT/Ti)d] × 100; where (NT/Ti)d = ratio of nutrient and Ti in diet and (NT/Ti)i = ratio of nutrient and Ti in ileal digesta (Amerah and Ravindran, 2015). Apparent ileal digestible energy (AIDE) was calculated by multiplying the diet GE content by the apparent ileal energy digestibility. Statistical Analysis All data analyses were performed using JMP pro13 statistical analysis software (SAS institute, 2017). Growth and nutrient digestibility data were analyzed as a complete randomized design with 3 × 3 factorial arrangement with 1 cage as the experimental unit. Data analyzed by analysis of variance was presented as mean ± SEM (standard error of the mean). Significant differences of means were separated using Tukey HSD test at P ≤ 0.05. Blood data were analyzed as a completely block design because the time of termination of bleeding was considered as a block to avoid bias on the results due to timing instead of effect on the treatments. One chick was the experimental unit in the blood analysis data. The arrangement was a 3 × 3 factorial arrangement (type of diet and enzyme inclusion) within nutritional state. Data are presented as means ± SE (standard error). Differences among treatments were determined using single degree of freedom orthogonal contrasts. Six comparisons performed were: 1) Low SBM – 0 ppm vs. Low SBM – 200 ppm; 2) Low SBM – 0 ppm vs. Low SBM – 400 ppm, 3) High SBM – 0 ppm vs. High SBM – 200 ppm, 4) High SBM – 0 ppm vs. High SBM – 400, 5) High SBM+GG – 0 ppm vs. High SBM+GG – 200 ppm, 6) High SBM+GG – 0 ppm vs. High SBM+GG – 400 ppm. Mortality data was transformed to Arcsine before analysis. Significance was considered at P < 0.05. RESULTS Performance Final BW at 21 d after 14 d experimental period was different between types of diets. Low SBM, High SBM, and High SBM+GG produced 733, 755, and 695 g/bird, respectively (Table 3). All three diets differed from each other (P ≤ 0.01). Enzyme dose levels and interaction effects tended to be significant (P < 0.076) for final BW; therefore, a contrast analysis showed that the addition of both 200 and 400 ppm β-mannanase to diets with high SBM + GG improved 21 d BW compared to their respective control, High SBM+GG-200: 702 g vs. High SBM+GG-0: 669 g (P ≤ 0.01), and High SBM+GG-400: 715 g vs. High SBM+GG-0: 669 g (P ≤ 0.01). BW gain, feed intake, feed:gain, and mortality are presented in Table 3. BW gain on d 21 followed the same trend as BW because the initial BW was similar for the treatments. Feed intake was lower for chicks fed with the diet containing 400 ppm of β-mannanase compared to the control chicks (0 ppm enzyme): 889 vs. 918 g/bird, respectively (P ≤ 0.01). The feed intake for chicks fed the diet containing 200 ppm of the enzyme (908 g/bird) was not different compared to either treatment. No differences were shown in feed intake between type of diets; however, there was a trend (P < 0.086) for the interaction effect. Chicks fed with High SBM-400 ppm diet had lower feed intake compared to chicks fed with High SBM-0 ppm diet: 878 g vs. 937 g/bird, respectively (P ≤ 0.01). Differences in BW and feed intake established differences in feed: gain. The interaction effects of feed type and enzyme levels were significant for feed:gain (P < 0.01), where High SBM-400 ppm produced the best feed: gain (1.406) and High SBM+GG-0 ppm produced the poorest feed: gain (1.693). The contrast analysis showed feed: gain was improved by 0.162 and 0.182 when 200 ppm (1.531) and 400 ppm (1.511) of β-mannanase was added to High SBM+GG-0 ppm diets (1.693), respectively. Feed:gain was improved by 0.09 when 400 ppm was added to High SBM-0 ppm diet. No effect was found in feed:gain when enzyme was added to Low SBM or when only 200 ppm enzyme was added to the High SBM diet. Mortality was the highest with Low SBM (P ≤ 0.034) compared only to High SBM+GG diet. Table 3. Body weight, body weight gain, feed intake, feed conversion ratio, and mortality of broilers from 7 to 21 d. Item  Initial BW  Final BW  Gain,  Feed Intake,  Feed:  Mortality,    d 7, g/bird  d 21, g/bird  g/bird  g/bird  Gain1 g/g  %  Type of diet              Low SBM    127.9  732.5b  604.7b  907.2  1.513b  3.57a  High SBM    128.2  755.3a  627.2a  915.1  1.461c  1.27a,b  High SBM+GG    127.5  695.2c  567.4c  893.2  1.578a  0.00b  Enzyme β-D-mannanase, ppm              Dose: 0 ppm    128.2  718.3  590.0  918.4a  1.566a  0.60  Dose: 200 ppm    127.5  729.9  602.7  908.3a,b  1.515b  2.38  Dose: 400 ppm    127.9  734.9  606.6  888.7b  1.471c  2.38  Type of diet  Enzyme              Low SBM  Dose: 0 ppm  128.6  730.2  601.6  903.6  1.511b  1.79  Low SBM  Dose: 200 ppm  126.8  734.0  607.3  915.7  1.531b  3.57  Low SBM  Dose: 400 ppm  128.3  733.4  605.1  902.2  1.496b  5.36  High SBM  Dose: 0 ppm  128.4  755.8  627.2  936.7  1.494b  0.00  High SBM  Dose: 200 ppm  128.6  754.1  626.5  930.6  1.481b  3.57  High SBM  Dose: 400 ppm  127.6  756.1  627.9  878.0  1.406c  1.79  High SBM+GG  Dose: 0 ppm  127.5  669.0  541.3  914.9  1.693a  0.00  High SBM+GG  Dose: 200 ppm  127.2  701.5  574.3  878.6  1.531b  0.00  High SBM+GG  Dose: 400 ppm  127.6  715.2  586.7  886.0  1.511b  0.00  SEM    1.56  8.80  8.45  13.63  0.02  1.63  P-value for main effects and interaction            Type of diet    0.965  <0.01**  <0.01**  0.147  <0.01**  0.034*  Enzyme    0.647  0.071  0.051  0.033*  <0.01**  0.311  Type of diet × Enzyme  0.987  0.076  0.070  0.086  <0.01**  0.668  P-value for contrasts of enzyme level              Low SBM-0 vs. Low SBM-200  0.420  0.759  0.609  0.523  0.496  0.444  Low SBM-0 vs. Low SBM-400  0.864  0.798  0.764  0.945  0.618  0.128  High SBM-0 vs. High SBM-200  0.685  0.895  0.954  0.755  0.686  0.128  High SBM-0 vs. High SBM-400  0.853  0.983  0.953  <0.01**  <0.01**  0.444  High SBM+GG-0 vs. High SBM+GG-200  0.862  0.010*  <0.01**  0.061  <0.01**  1.000  High SBM+GG-0 vs. High SBM+GG-400  0.725  <0.01**  <0.01**  0.146  <0.01**  1.000  Item  Initial BW  Final BW  Gain,  Feed Intake,  Feed:  Mortality,    d 7, g/bird  d 21, g/bird  g/bird  g/bird  Gain1 g/g  %  Type of diet              Low SBM    127.9  732.5b  604.7b  907.2  1.513b  3.57a  High SBM    128.2  755.3a  627.2a  915.1  1.461c  1.27a,b  High SBM+GG    127.5  695.2c  567.4c  893.2  1.578a  0.00b  Enzyme β-D-mannanase, ppm              Dose: 0 ppm    128.2  718.3  590.0  918.4a  1.566a  0.60  Dose: 200 ppm    127.5  729.9  602.7  908.3a,b  1.515b  2.38  Dose: 400 ppm    127.9  734.9  606.6  888.7b  1.471c  2.38  Type of diet  Enzyme              Low SBM  Dose: 0 ppm  128.6  730.2  601.6  903.6  1.511b  1.79  Low SBM  Dose: 200 ppm  126.8  734.0  607.3  915.7  1.531b  3.57  Low SBM  Dose: 400 ppm  128.3  733.4  605.1  902.2  1.496b  5.36  High SBM  Dose: 0 ppm  128.4  755.8  627.2  936.7  1.494b  0.00  High SBM  Dose: 200 ppm  128.6  754.1  626.5  930.6  1.481b  3.57  High SBM  Dose: 400 ppm  127.6  756.1  627.9  878.0  1.406c  1.79  High SBM+GG  Dose: 0 ppm  127.5  669.0  541.3  914.9  1.693a  0.00  High SBM+GG  Dose: 200 ppm  127.2  701.5  574.3  878.6  1.531b  0.00  High SBM+GG  Dose: 400 ppm  127.6  715.2  586.7  886.0  1.511b  0.00  SEM    1.56  8.80  8.45  13.63  0.02  1.63  P-value for main effects and interaction            Type of diet    0.965  <0.01**  <0.01**  0.147  <0.01**  0.034*  Enzyme    0.647  0.071  0.051  0.033*  <0.01**  0.311  Type of diet × Enzyme  0.987  0.076  0.070  0.086  <0.01**  0.668  P-value for contrasts of enzyme level              Low SBM-0 vs. Low SBM-200  0.420  0.759  0.609  0.523  0.496  0.444  Low SBM-0 vs. Low SBM-400  0.864  0.798  0.764  0.945  0.618  0.128  High SBM-0 vs. High SBM-200  0.685  0.895  0.954  0.755  0.686  0.128  High SBM-0 vs. High SBM-400  0.853  0.983  0.953  <0.01**  <0.01**  0.444  High SBM+GG-0 vs. High SBM+GG-200  0.862  0.010*  <0.01**  0.061  <0.01**  1.000  High SBM+GG-0 vs. High SBM+GG-400  0.725  <0.01**  <0.01**  0.146  <0.01**  1.000  Means with no common superscripts (a, b, c) within a column are different at *P ≤ 0.05, or highly significant **P ≤ 0.01 for Tukey-HSD test. 1Feed:gain, adjusted for mortality. View Large Table 3. Body weight, body weight gain, feed intake, feed conversion ratio, and mortality of broilers from 7 to 21 d. Item  Initial BW  Final BW  Gain,  Feed Intake,  Feed:  Mortality,    d 7, g/bird  d 21, g/bird  g/bird  g/bird  Gain1 g/g  %  Type of diet              Low SBM    127.9  732.5b  604.7b  907.2  1.513b  3.57a  High SBM    128.2  755.3a  627.2a  915.1  1.461c  1.27a,b  High SBM+GG    127.5  695.2c  567.4c  893.2  1.578a  0.00b  Enzyme β-D-mannanase, ppm              Dose: 0 ppm    128.2  718.3  590.0  918.4a  1.566a  0.60  Dose: 200 ppm    127.5  729.9  602.7  908.3a,b  1.515b  2.38  Dose: 400 ppm    127.9  734.9  606.6  888.7b  1.471c  2.38  Type of diet  Enzyme              Low SBM  Dose: 0 ppm  128.6  730.2  601.6  903.6  1.511b  1.79  Low SBM  Dose: 200 ppm  126.8  734.0  607.3  915.7  1.531b  3.57  Low SBM  Dose: 400 ppm  128.3  733.4  605.1  902.2  1.496b  5.36  High SBM  Dose: 0 ppm  128.4  755.8  627.2  936.7  1.494b  0.00  High SBM  Dose: 200 ppm  128.6  754.1  626.5  930.6  1.481b  3.57  High SBM  Dose: 400 ppm  127.6  756.1  627.9  878.0  1.406c  1.79  High SBM+GG  Dose: 0 ppm  127.5  669.0  541.3  914.9  1.693a  0.00  High SBM+GG  Dose: 200 ppm  127.2  701.5  574.3  878.6  1.531b  0.00  High SBM+GG  Dose: 400 ppm  127.6  715.2  586.7  886.0  1.511b  0.00  SEM    1.56  8.80  8.45  13.63  0.02  1.63  P-value for main effects and interaction            Type of diet    0.965  <0.01**  <0.01**  0.147  <0.01**  0.034*  Enzyme    0.647  0.071  0.051  0.033*  <0.01**  0.311  Type of diet × Enzyme  0.987  0.076  0.070  0.086  <0.01**  0.668  P-value for contrasts of enzyme level              Low SBM-0 vs. Low SBM-200  0.420  0.759  0.609  0.523  0.496  0.444  Low SBM-0 vs. Low SBM-400  0.864  0.798  0.764  0.945  0.618  0.128  High SBM-0 vs. High SBM-200  0.685  0.895  0.954  0.755  0.686  0.128  High SBM-0 vs. High SBM-400  0.853  0.983  0.953  <0.01**  <0.01**  0.444  High SBM+GG-0 vs. High SBM+GG-200  0.862  0.010*  <0.01**  0.061  <0.01**  1.000  High SBM+GG-0 vs. High SBM+GG-400  0.725  <0.01**  <0.01**  0.146  <0.01**  1.000  Item  Initial BW  Final BW  Gain,  Feed Intake,  Feed:  Mortality,    d 7, g/bird  d 21, g/bird  g/bird  g/bird  Gain1 g/g  %  Type of diet              Low SBM    127.9  732.5b  604.7b  907.2  1.513b  3.57a  High SBM    128.2  755.3a  627.2a  915.1  1.461c  1.27a,b  High SBM+GG    127.5  695.2c  567.4c  893.2  1.578a  0.00b  Enzyme β-D-mannanase, ppm              Dose: 0 ppm    128.2  718.3  590.0  918.4a  1.566a  0.60  Dose: 200 ppm    127.5  729.9  602.7  908.3a,b  1.515b  2.38  Dose: 400 ppm    127.9  734.9  606.6  888.7b  1.471c  2.38  Type of diet  Enzyme              Low SBM  Dose: 0 ppm  128.6  730.2  601.6  903.6  1.511b  1.79  Low SBM  Dose: 200 ppm  126.8  734.0  607.3  915.7  1.531b  3.57  Low SBM  Dose: 400 ppm  128.3  733.4  605.1  902.2  1.496b  5.36  High SBM  Dose: 0 ppm  128.4  755.8  627.2  936.7  1.494b  0.00  High SBM  Dose: 200 ppm  128.6  754.1  626.5  930.6  1.481b  3.57  High SBM  Dose: 400 ppm  127.6  756.1  627.9  878.0  1.406c  1.79  High SBM+GG  Dose: 0 ppm  127.5  669.0  541.3  914.9  1.693a  0.00  High SBM+GG  Dose: 200 ppm  127.2  701.5  574.3  878.6  1.531b  0.00  High SBM+GG  Dose: 400 ppm  127.6  715.2  586.7  886.0  1.511b  0.00  SEM    1.56  8.80  8.45  13.63  0.02  1.63  P-value for main effects and interaction            Type of diet    0.965  <0.01**  <0.01**  0.147  <0.01**  0.034*  Enzyme    0.647  0.071  0.051  0.033*  <0.01**  0.311  Type of diet × Enzyme  0.987  0.076  0.070  0.086  <0.01**  0.668  P-value for contrasts of enzyme level              Low SBM-0 vs. Low SBM-200  0.420  0.759  0.609  0.523  0.496  0.444  Low SBM-0 vs. Low SBM-400  0.864  0.798  0.764  0.945  0.618  0.128  High SBM-0 vs. High SBM-200  0.685  0.895  0.954  0.755  0.686  0.128  High SBM-0 vs. High SBM-400  0.853  0.983  0.953  <0.01**  <0.01**  0.444  High SBM+GG-0 vs. High SBM+GG-200  0.862  0.010*  <0.01**  0.061  <0.01**  1.000  High SBM+GG-0 vs. High SBM+GG-400  0.725  <0.01**  <0.01**  0.146  <0.01**  1.000  Means with no common superscripts (a, b, c) within a column are different at *P ≤ 0.05, or highly significant **P ≤ 0.01 for Tukey-HSD test. 1Feed:gain, adjusted for mortality. View Large Apparent Ileal Digestible Energy, Phosphorus Digestibility, and Viscosity The interaction effect between type of diet × enzyme level was significant for AIDE where High SBM-400 was 3,165 kcal/kg compared to High SBM-200: 2,819 kcal/kg, and High SBM+GG-0: 2,777 kcal/kg (P ≤ 0.01) (Table 4). Phosphorus digestibility (PD) and viscosity are also presented in Table 4. PD showed no differences for the main effects (type of diets and enzyme level). The contrast analysis showed higher PD for Low SBM-200 compared to its respective control Low SBM-0. Viscosity was the highest with High SBM+GG-0 when no enzyme was added (19.2 cps) compared to birds fed with diets supplemented with β-mannanase: High SBM+GG-200 (7.40 cps) and High SBM+GG-400 (6.83 cps) (P ≤ 0.01). The β-mannanase enzyme did not lower the jejunum viscosity in birds fed with diets with Low SBM-0 and High SBM-0 ppm. Table 4. Digestible energy (kcal/kg), phosphorus digestibility (%), and jejunum viscosity (cps) at 23 d of age. Item  Apparent Digestible Energy, Kcal/kg  Phosphorus Digestibility, %  Jejunum Viscosity, cps  Type of diet    Low SBM    3,071a  63.98  2.55b  High SBM    2,999a,b  62.50  2.27b  High SBM+GG    2,920b  61.50  11.14a  Enzyme β-D-mannanase, ppm        Dose: 0 ppm    2,957b  63.04  8.02a  Dose: 200 ppm    2,958b  63.40  4.08b  Dose: 400 ppm    3,074a  61.50  3.86b  SEM    34.76  0.89  0.14  Type of diet  Enzyme  ±       Low SBM  Dose: 0 ppm  3,085a,b  62.66  2.53c  Low SBM  Dose: 200 ppm  3,072a,b  67.73  2.54c  Low SBM  Dose: 400 ppm  3,057a,b  61.54  2.57c  High SBM  Dose: 0 ppm  3,013a,b,c  65.49  2.33c  High SBM  Dose: 200 ppm  2,819c  61.09  2.30c  High SBM  Dose: 400 ppm  3,165a  62.66  2.18c  High SBM+GG  Dose: 0 ppm  2,777c  60.98  19.20a  High SBM+GG  Dose: 200 ppm  2,982a,b,c  63.16  7.40b  High SBM+GG  Dose: 400 ppm  3,001a,b,c  60.23  6.83b  SEM    58.83  1.78  0.25  P-value for main effects and interaction      Type of diet    0.015*  0.252  <0.01**  Enzyme    0.018*  0.399  <0.01**  Type of diet × Enzyme  <0.01**  0.103  <0.01**  P-value for contrasts of enzyme level        LowSBM-0 vs. LowSBM-200  0.878  0.047*  0.986  LowSBM-0 vs. LowSBM-400  0.728  0.655  0.905  HighSBM-0 vs. HighSBM-200  0.113  0.094  0.930  HighSBM-0 vs. HighSBM-400  0.013*  0.263  0.668  HighSBM+GG-0 vs. HighSBM+GG-200  0.015*  0.386  <0.01**  HighSBM+GG-0 vs. HighSBM+GG-400  <0.01**  0.765  <0.01**  Item  Apparent Digestible Energy, Kcal/kg  Phosphorus Digestibility, %  Jejunum Viscosity, cps  Type of diet    Low SBM    3,071a  63.98  2.55b  High SBM    2,999a,b  62.50  2.27b  High SBM+GG    2,920b  61.50  11.14a  Enzyme β-D-mannanase, ppm        Dose: 0 ppm    2,957b  63.04  8.02a  Dose: 200 ppm    2,958b  63.40  4.08b  Dose: 400 ppm    3,074a  61.50  3.86b  SEM    34.76  0.89  0.14  Type of diet  Enzyme  ±       Low SBM  Dose: 0 ppm  3,085a,b  62.66  2.53c  Low SBM  Dose: 200 ppm  3,072a,b  67.73  2.54c  Low SBM  Dose: 400 ppm  3,057a,b  61.54  2.57c  High SBM  Dose: 0 ppm  3,013a,b,c  65.49  2.33c  High SBM  Dose: 200 ppm  2,819c  61.09  2.30c  High SBM  Dose: 400 ppm  3,165a  62.66  2.18c  High SBM+GG  Dose: 0 ppm  2,777c  60.98  19.20a  High SBM+GG  Dose: 200 ppm  2,982a,b,c  63.16  7.40b  High SBM+GG  Dose: 400 ppm  3,001a,b,c  60.23  6.83b  SEM    58.83  1.78  0.25  P-value for main effects and interaction      Type of diet    0.015*  0.252  <0.01**  Enzyme    0.018*  0.399  <0.01**  Type of diet × Enzyme  <0.01**  0.103  <0.01**  P-value for contrasts of enzyme level        LowSBM-0 vs. LowSBM-200  0.878  0.047*  0.986  LowSBM-0 vs. LowSBM-400  0.728  0.655  0.905  HighSBM-0 vs. HighSBM-200  0.113  0.094  0.930  HighSBM-0 vs. HighSBM-400  0.013*  0.263  0.668  HighSBM+GG-0 vs. HighSBM+GG-200  0.015*  0.386  <0.01**  HighSBM+GG-0 vs. HighSBM+GG-400  <0.01**  0.765  <0.01**  Means with no common superscripts (a, b, c) within a column are different at P ≤ 0.05 for Tukey-HSD test. *P-value < 0.05. **P-value < 0.01. View Large Table 4. Digestible energy (kcal/kg), phosphorus digestibility (%), and jejunum viscosity (cps) at 23 d of age. Item  Apparent Digestible Energy, Kcal/kg  Phosphorus Digestibility, %  Jejunum Viscosity, cps  Type of diet    Low SBM    3,071a  63.98  2.55b  High SBM    2,999a,b  62.50  2.27b  High SBM+GG    2,920b  61.50  11.14a  Enzyme β-D-mannanase, ppm        Dose: 0 ppm    2,957b  63.04  8.02a  Dose: 200 ppm    2,958b  63.40  4.08b  Dose: 400 ppm    3,074a  61.50  3.86b  SEM    34.76  0.89  0.14  Type of diet  Enzyme  ±       Low SBM  Dose: 0 ppm  3,085a,b  62.66  2.53c  Low SBM  Dose: 200 ppm  3,072a,b  67.73  2.54c  Low SBM  Dose: 400 ppm  3,057a,b  61.54  2.57c  High SBM  Dose: 0 ppm  3,013a,b,c  65.49  2.33c  High SBM  Dose: 200 ppm  2,819c  61.09  2.30c  High SBM  Dose: 400 ppm  3,165a  62.66  2.18c  High SBM+GG  Dose: 0 ppm  2,777c  60.98  19.20a  High SBM+GG  Dose: 200 ppm  2,982a,b,c  63.16  7.40b  High SBM+GG  Dose: 400 ppm  3,001a,b,c  60.23  6.83b  SEM    58.83  1.78  0.25  P-value for main effects and interaction      Type of diet    0.015*  0.252  <0.01**  Enzyme    0.018*  0.399  <0.01**  Type of diet × Enzyme  <0.01**  0.103  <0.01**  P-value for contrasts of enzyme level        LowSBM-0 vs. LowSBM-200  0.878  0.047*  0.986  LowSBM-0 vs. LowSBM-400  0.728  0.655  0.905  HighSBM-0 vs. HighSBM-200  0.113  0.094  0.930  HighSBM-0 vs. HighSBM-400  0.013*  0.263  0.668  HighSBM+GG-0 vs. HighSBM+GG-200  0.015*  0.386  <0.01**  HighSBM+GG-0 vs. HighSBM+GG-400  <0.01**  0.765  <0.01**  Item  Apparent Digestible Energy, Kcal/kg  Phosphorus Digestibility, %  Jejunum Viscosity, cps  Type of diet    Low SBM    3,071a  63.98  2.55b  High SBM    2,999a,b  62.50  2.27b  High SBM+GG    2,920b  61.50  11.14a  Enzyme β-D-mannanase, ppm        Dose: 0 ppm    2,957b  63.04  8.02a  Dose: 200 ppm    2,958b  63.40  4.08b  Dose: 400 ppm    3,074a  61.50  3.86b  SEM    34.76  0.89  0.14  Type of diet  Enzyme  ±       Low SBM  Dose: 0 ppm  3,085a,b  62.66  2.53c  Low SBM  Dose: 200 ppm  3,072a,b  67.73  2.54c  Low SBM  Dose: 400 ppm  3,057a,b  61.54  2.57c  High SBM  Dose: 0 ppm  3,013a,b,c  65.49  2.33c  High SBM  Dose: 200 ppm  2,819c  61.09  2.30c  High SBM  Dose: 400 ppm  3,165a  62.66  2.18c  High SBM+GG  Dose: 0 ppm  2,777c  60.98  19.20a  High SBM+GG  Dose: 200 ppm  2,982a,b,c  63.16  7.40b  High SBM+GG  Dose: 400 ppm  3,001a,b,c  60.23  6.83b  SEM    58.83  1.78  0.25  P-value for main effects and interaction      Type of diet    0.015*  0.252  <0.01**  Enzyme    0.018*  0.399  <0.01**  Type of diet × Enzyme  <0.01**  0.103  <0.01**  P-value for contrasts of enzyme level        LowSBM-0 vs. LowSBM-200  0.878  0.047*  0.986  LowSBM-0 vs. LowSBM-400  0.728  0.655  0.905  HighSBM-0 vs. HighSBM-200  0.113  0.094  0.930  HighSBM-0 vs. HighSBM-400  0.013*  0.263  0.668  HighSBM+GG-0 vs. HighSBM+GG-200  0.015*  0.386  <0.01**  HighSBM+GG-0 vs. HighSBM+GG-400  <0.01**  0.765  <0.01**  Means with no common superscripts (a, b, c) within a column are different at P ≤ 0.05 for Tukey-HSD test. *P-value < 0.05. **P-value < 0.01. View Large Apparent Amino Acid Digestibility The present study evaluated the digestibility of 16 amino acids (AA) (Table 5). Type of diets and enzyme levels affected apparent amino acid digestibility (AAAD). There was a diet × AA interaction for AAAD for 5 AA (Gly, His, Ala, Asp, and Glu). The AAAD was significantly higher for 10 AA (Lys, Met, Cys, Val, Iso, Tyr, Ser, His, Asp, and Glu) for diets containing High SBM and High SMB+GG compared to Low SBM (P ≤ 0.01). As for the contrast analysis, 10 out of 16 AAAD (Lys, Thr, Arg, Leu, Phe, Gly, Ser, His, Asp, and Glu) were improved when 200 ppm of β-mannanase was added to High SBM+GG (P ≤ 0.01) and 6 out of 16 AAAD (Thr, Leu, Phe, Gly, His, and Glu) were improved when 400 ppm of β-mannanase was added to High SBM+GG (P ≤ 0.01). Four AAAD (Val, Iso, His, and Ala) were lower when 200 ppm of β-mannanase was added to High SBM (P ≤ 0.01). Only Lys digestibility was improved when 200 ppm of β-mannanase was added to diet containing Low SBM (P ≤ 0.02). Table 5. Apparent ileal amino acid digestibility (%) at 23 d of age. Item  Lys  Met  Cys  Thr  Arg  Val  Leu  Iso  Phe  Tyr  Gly  Ser  His  Ala  Asp  Glu  Type of diet                                  Low SBM    81.7b  90.2b  67.4b  76.5b  93.0b  75.0b  80.6b  75.8b  79.4b  79.1b  75.4b  76.5b  80.8c  80.8  75.7b  83.4b  High SBM    84.9a  91.6a  73.2a  78.8a  93.8a  79.5a  82.7a  79.8a  81.9a  81.3a  78.4a  79.8a  84.1b  82.4  80.6a  86.6a  High SBM+GG    84.1a  92.5a  71.2a  77.9a,b  93.4a,b  78.7a  81.8a,b  79.0a  80.9a,b  81.3a  77.1a,b  79.5a  87.5a  81.8  79.8a  85.8a  Enzyme β-D-mannanase, ppm                                  Dose: 0 ppm    82.5  91.4  70.2  77.2  93.2  77.7  81.4  78.0  80.3  80.2  76.6  78.1  83.8  81.7  78.3  85.0  Dose: 200 ppm    84.5  91.1  70.4  78.0  93.6  77.7  81.9  78.3  81.1  80.6  77.3  78.8  84.1  81.5  79.1  85.6  Dose: 400 ppm    83.8  91.7  71.3  78.0  93.5  77.7  81.8  78.3  80.9  80.9  77.0  78.8  84.6  81.8  78.7  85.3  Type of diet  Enzyme                                  Low SBM  Dose: 0 ppm  79.7  90.5  67.4  76.4  92.9  74.7  80.5  75.5  79.2  78.7  75.3a,b,c  76.3  80.0d  80.8a,b  75.2b  83.3b,c  Low SBM  Dose: 200 ppm  83.6  90.5  68.3  77.5  93.4  76.3  81.6  77.0  80.6  80.0  77.2a,b,c  77.6  82.4b,c,d  82.2a,b  77.3a,b  84.3a,b,c  Low SBM  Dose: 400 ppm  81.7  90.4  66.6  75.6  92.9  74.0  79.8  74.9  78.6  78.5  73.6c  75.4  80.1d  79.4b  74.6b  82.6c  High SBM  Dose: 0 ppm  85.2  92.3  74.0  79.5  94.0  80.7  83.6  81.1  82.8  81.9  79.7a  80.7  85.6a,b,c  83.7a  81.8a  87.4a  High SBM  Dose: 200 ppm  84.1  90.7  71.3  77.5  93.5  77.6  81.5  78.0  80.7  79.8  76.5a,b,c  78.1  81.6c,d  80.3a,b  79.1a,b  85.7a,b,c  High SBM  Dose: 400 ppm  85.6  92.3  74.6  79.4  94.0  80.1  82.9  80.3  82.2  82.2  79.2a,b  80.5  85.1a,b,c  83.3a,b  80.9a  86.8a  High SBM+GG  Dose: 0 ppm  82.5  91.5  69.3  75.6  92.8  77.6  80.0  77.3  79.1  79.9  74.7b,c  77.4  87.5a,b  80.5a,b  77.8a,b  84.2a,b,c  High SBM+GG  Dose: 200 ppm  85.7  92.6  71.7  79.1  93.9  79.2  82.7  80.0  81.9  82.1  78.3a,b,c  80.7  88.4a  82.1a,b  80.9a  86.7a,b  High SBM+GG  Dose: 400 ppm  84.3  93.3  72.7  79.1  93.6  79.1  82.8  79.7  81.9  82.1  78.3a,b,c  80.4  88.5a  82.8a,b  80.5a  86.6a,b  SEM    1.15  0.65  1.55  1.09  0.29  1.05  0.93  1.11  0.99  0.98  1.09  1.17  0.88  0.91  1.04  0.78  P-value for main effects and interaction                                  Type of diet    <0.01  <0.01  <0.01  0.04*  <0.01  <0.01  0.03*  <0.01  0.01*  <0.01  <0.01  <0.01  <0.01  0.106  <0.01  <0.01  Enzyme    0.10  0.41  0.17  0.54  0.25  1.00  0.74  0.90  0.65  0.64  0.71  0.70  0.55  0.93  0.62  0.62  Type of diet × Enzyme    0.24  0.23  0.37  0.07  0.09  0.08  0.06  0.06  0.07  0.13  <0.01  0.076  <0.01  <0.01  0.03*  0.04*  P-value for contrasts of enzyme level                                  Low SBM-0 vs. Low SBM-200    0.02*  0.97  0.69  0.46  0.18  0.28  0.38  0.31  0.30  0.37  0.23  0.42  0.07  0.29  0.15  0.32  Low SBM-0 vs Low SBM-400    0.26  0.87  0.73  0.61  0.99  0.62  0.60  0.71  0.69  0.86  0.28  0.58  0.98  0.28  0.66  0.56  High SBM-0 vs. High SBM-200    0.51  0.09  0.23  0.21  0.28  0.04*  0.12  0.05*  0.14  0.13  0.05  0.14  <0.01  <0.01  0.08  0.14  High SBM-0 vs. High SBM-400    0.80  1.00  0.79  0.94  0.93  0.65  0.60  0.60  0.66  0.83  0.74  0.93  0.70  0.70  0.56  0.56  High SBM+GG-0 vs. High SBM+GG-200    0.05*  0.21  0.26  0.03*  <0.01  0.29  0.05*  0.09  0.04*  0.12  0.02*  0.04*  0.04*  0.22  0.04*  0.03*  High SBM+GG-0 vs. High SBM+GG-400    0.26  0.06  0.12  0.02*  0.06  0.33  0.04*  0.13  0.05*  0.11  0.02*  0.07  0.03*  0.10  0.07  0.03*  Item  Lys  Met  Cys  Thr  Arg  Val  Leu  Iso  Phe  Tyr  Gly  Ser  His  Ala  Asp  Glu  Type of diet                                  Low SBM    81.7b  90.2b  67.4b  76.5b  93.0b  75.0b  80.6b  75.8b  79.4b  79.1b  75.4b  76.5b  80.8c  80.8  75.7b  83.4b  High SBM    84.9a  91.6a  73.2a  78.8a  93.8a  79.5a  82.7a  79.8a  81.9a  81.3a  78.4a  79.8a  84.1b  82.4  80.6a  86.6a  High SBM+GG    84.1a  92.5a  71.2a  77.9a,b  93.4a,b  78.7a  81.8a,b  79.0a  80.9a,b  81.3a  77.1a,b  79.5a  87.5a  81.8  79.8a  85.8a  Enzyme β-D-mannanase, ppm                                  Dose: 0 ppm    82.5  91.4  70.2  77.2  93.2  77.7  81.4  78.0  80.3  80.2  76.6  78.1  83.8  81.7  78.3  85.0  Dose: 200 ppm    84.5  91.1  70.4  78.0  93.6  77.7  81.9  78.3  81.1  80.6  77.3  78.8  84.1  81.5  79.1  85.6  Dose: 400 ppm    83.8  91.7  71.3  78.0  93.5  77.7  81.8  78.3  80.9  80.9  77.0  78.8  84.6  81.8  78.7  85.3  Type of diet  Enzyme                                  Low SBM  Dose: 0 ppm  79.7  90.5  67.4  76.4  92.9  74.7  80.5  75.5  79.2  78.7  75.3a,b,c  76.3  80.0d  80.8a,b  75.2b  83.3b,c  Low SBM  Dose: 200 ppm  83.6  90.5  68.3  77.5  93.4  76.3  81.6  77.0  80.6  80.0  77.2a,b,c  77.6  82.4b,c,d  82.2a,b  77.3a,b  84.3a,b,c  Low SBM  Dose: 400 ppm  81.7  90.4  66.6  75.6  92.9  74.0  79.8  74.9  78.6  78.5  73.6c  75.4  80.1d  79.4b  74.6b  82.6c  High SBM  Dose: 0 ppm  85.2  92.3  74.0  79.5  94.0  80.7  83.6  81.1  82.8  81.9  79.7a  80.7  85.6a,b,c  83.7a  81.8a  87.4a  High SBM  Dose: 200 ppm  84.1  90.7  71.3  77.5  93.5  77.6  81.5  78.0  80.7  79.8  76.5a,b,c  78.1  81.6c,d  80.3a,b  79.1a,b  85.7a,b,c  High SBM  Dose: 400 ppm  85.6  92.3  74.6  79.4  94.0  80.1  82.9  80.3  82.2  82.2  79.2a,b  80.5  85.1a,b,c  83.3a,b  80.9a  86.8a  High SBM+GG  Dose: 0 ppm  82.5  91.5  69.3  75.6  92.8  77.6  80.0  77.3  79.1  79.9  74.7b,c  77.4  87.5a,b  80.5a,b  77.8a,b  84.2a,b,c  High SBM+GG  Dose: 200 ppm  85.7  92.6  71.7  79.1  93.9  79.2  82.7  80.0  81.9  82.1  78.3a,b,c  80.7  88.4a  82.1a,b  80.9a  86.7a,b  High SBM+GG  Dose: 400 ppm  84.3  93.3  72.7  79.1  93.6  79.1  82.8  79.7  81.9  82.1  78.3a,b,c  80.4  88.5a  82.8a,b  80.5a  86.6a,b  SEM    1.15  0.65  1.55  1.09  0.29  1.05  0.93  1.11  0.99  0.98  1.09  1.17  0.88  0.91  1.04  0.78  P-value for main effects and interaction                                  Type of diet    <0.01  <0.01  <0.01  0.04*  <0.01  <0.01  0.03*  <0.01  0.01*  <0.01  <0.01  <0.01  <0.01  0.106  <0.01  <0.01  Enzyme    0.10  0.41  0.17  0.54  0.25  1.00  0.74  0.90  0.65  0.64  0.71  0.70  0.55  0.93  0.62  0.62  Type of diet × Enzyme    0.24  0.23  0.37  0.07  0.09  0.08  0.06  0.06  0.07  0.13  <0.01  0.076  <0.01  <0.01  0.03*  0.04*  P-value for contrasts of enzyme level                                  Low SBM-0 vs. Low SBM-200    0.02*  0.97  0.69  0.46  0.18  0.28  0.38  0.31  0.30  0.37  0.23  0.42  0.07  0.29  0.15  0.32  Low SBM-0 vs Low SBM-400    0.26  0.87  0.73  0.61  0.99  0.62  0.60  0.71  0.69  0.86  0.28  0.58  0.98  0.28  0.66  0.56  High SBM-0 vs. High SBM-200    0.51  0.09  0.23  0.21  0.28  0.04*  0.12  0.05*  0.14  0.13  0.05  0.14  <0.01  <0.01  0.08  0.14  High SBM-0 vs. High SBM-400    0.80  1.00  0.79  0.94  0.93  0.65  0.60  0.60  0.66  0.83  0.74  0.93  0.70  0.70  0.56  0.56  High SBM+GG-0 vs. High SBM+GG-200    0.05*  0.21  0.26  0.03*  <0.01  0.29  0.05*  0.09  0.04*  0.12  0.02*  0.04*  0.04*  0.22  0.04*  0.03*  High SBM+GG-0 vs. High SBM+GG-400    0.26  0.06  0.12  0.02*  0.06  0.33  0.04*  0.13  0.05*  0.11  0.02*  0.07  0.03*  0.10  0.07  0.03*  Means with no common superscripts (a, b, c, d) within a column are different at P ≤ 0.05 for Tukey-HSD test. *Means P-value < 0.05. View Large Table 5. Apparent ileal amino acid digestibility (%) at 23 d of age. Item  Lys  Met  Cys  Thr  Arg  Val  Leu  Iso  Phe  Tyr  Gly  Ser  His  Ala  Asp  Glu  Type of diet                                  Low SBM    81.7b  90.2b  67.4b  76.5b  93.0b  75.0b  80.6b  75.8b  79.4b  79.1b  75.4b  76.5b  80.8c  80.8  75.7b  83.4b  High SBM    84.9a  91.6a  73.2a  78.8a  93.8a  79.5a  82.7a  79.8a  81.9a  81.3a  78.4a  79.8a  84.1b  82.4  80.6a  86.6a  High SBM+GG    84.1a  92.5a  71.2a  77.9a,b  93.4a,b  78.7a  81.8a,b  79.0a  80.9a,b  81.3a  77.1a,b  79.5a  87.5a  81.8  79.8a  85.8a  Enzyme β-D-mannanase, ppm                                  Dose: 0 ppm    82.5  91.4  70.2  77.2  93.2  77.7  81.4  78.0  80.3  80.2  76.6  78.1  83.8  81.7  78.3  85.0  Dose: 200 ppm    84.5  91.1  70.4  78.0  93.6  77.7  81.9  78.3  81.1  80.6  77.3  78.8  84.1  81.5  79.1  85.6  Dose: 400 ppm    83.8  91.7  71.3  78.0  93.5  77.7  81.8  78.3  80.9  80.9  77.0  78.8  84.6  81.8  78.7  85.3  Type of diet  Enzyme                                  Low SBM  Dose: 0 ppm  79.7  90.5  67.4  76.4  92.9  74.7  80.5  75.5  79.2  78.7  75.3a,b,c  76.3  80.0d  80.8a,b  75.2b  83.3b,c  Low SBM  Dose: 200 ppm  83.6  90.5  68.3  77.5  93.4  76.3  81.6  77.0  80.6  80.0  77.2a,b,c  77.6  82.4b,c,d  82.2a,b  77.3a,b  84.3a,b,c  Low SBM  Dose: 400 ppm  81.7  90.4  66.6  75.6  92.9  74.0  79.8  74.9  78.6  78.5  73.6c  75.4  80.1d  79.4b  74.6b  82.6c  High SBM  Dose: 0 ppm  85.2  92.3  74.0  79.5  94.0  80.7  83.6  81.1  82.8  81.9  79.7a  80.7  85.6a,b,c  83.7a  81.8a  87.4a  High SBM  Dose: 200 ppm  84.1  90.7  71.3  77.5  93.5  77.6  81.5  78.0  80.7  79.8  76.5a,b,c  78.1  81.6c,d  80.3a,b  79.1a,b  85.7a,b,c  High SBM  Dose: 400 ppm  85.6  92.3  74.6  79.4  94.0  80.1  82.9  80.3  82.2  82.2  79.2a,b  80.5  85.1a,b,c  83.3a,b  80.9a  86.8a  High SBM+GG  Dose: 0 ppm  82.5  91.5  69.3  75.6  92.8  77.6  80.0  77.3  79.1  79.9  74.7b,c  77.4  87.5a,b  80.5a,b  77.8a,b  84.2a,b,c  High SBM+GG  Dose: 200 ppm  85.7  92.6  71.7  79.1  93.9  79.2  82.7  80.0  81.9  82.1  78.3a,b,c  80.7  88.4a  82.1a,b  80.9a  86.7a,b  High SBM+GG  Dose: 400 ppm  84.3  93.3  72.7  79.1  93.6  79.1  82.8  79.7  81.9  82.1  78.3a,b,c  80.4  88.5a  82.8a,b  80.5a  86.6a,b  SEM    1.15  0.65  1.55  1.09  0.29  1.05  0.93  1.11  0.99  0.98  1.09  1.17  0.88  0.91  1.04  0.78  P-value for main effects and interaction                                  Type of diet    <0.01  <0.01  <0.01  0.04*  <0.01  <0.01  0.03*  <0.01  0.01*  <0.01  <0.01  <0.01  <0.01  0.106  <0.01  <0.01  Enzyme    0.10  0.41  0.17  0.54  0.25  1.00  0.74  0.90  0.65  0.64  0.71  0.70  0.55  0.93  0.62  0.62  Type of diet × Enzyme    0.24  0.23  0.37  0.07  0.09  0.08  0.06  0.06  0.07  0.13  <0.01  0.076  <0.01  <0.01  0.03*  0.04*  P-value for contrasts of enzyme level                                  Low SBM-0 vs. Low SBM-200    0.02*  0.97  0.69  0.46  0.18  0.28  0.38  0.31  0.30  0.37  0.23  0.42  0.07  0.29  0.15  0.32  Low SBM-0 vs Low SBM-400    0.26  0.87  0.73  0.61  0.99  0.62  0.60  0.71  0.69  0.86  0.28  0.58  0.98  0.28  0.66  0.56  High SBM-0 vs. High SBM-200    0.51  0.09  0.23  0.21  0.28  0.04*  0.12  0.05*  0.14  0.13  0.05  0.14  <0.01  <0.01  0.08  0.14  High SBM-0 vs. High SBM-400    0.80  1.00  0.79  0.94  0.93  0.65  0.60  0.60  0.66  0.83  0.74  0.93  0.70  0.70  0.56  0.56  High SBM+GG-0 vs. High SBM+GG-200    0.05*  0.21  0.26  0.03*  <0.01  0.29  0.05*  0.09  0.04*  0.12  0.02*  0.04*  0.04*  0.22  0.04*  0.03*  High SBM+GG-0 vs. High SBM+GG-400    0.26  0.06  0.12  0.02*  0.06  0.33  0.04*  0.13  0.05*  0.11  0.02*  0.07  0.03*  0.10  0.07  0.03*  Item  Lys  Met  Cys  Thr  Arg  Val  Leu  Iso  Phe  Tyr  Gly  Ser  His  Ala  Asp  Glu  Type of diet                                  Low SBM    81.7b  90.2b  67.4b  76.5b  93.0b  75.0b  80.6b  75.8b  79.4b  79.1b  75.4b  76.5b  80.8c  80.8  75.7b  83.4b  High SBM    84.9a  91.6a  73.2a  78.8a  93.8a  79.5a  82.7a  79.8a  81.9a  81.3a  78.4a  79.8a  84.1b  82.4  80.6a  86.6a  High SBM+GG    84.1a  92.5a  71.2a  77.9a,b  93.4a,b  78.7a  81.8a,b  79.0a  80.9a,b  81.3a  77.1a,b  79.5a  87.5a  81.8  79.8a  85.8a  Enzyme β-D-mannanase, ppm                                  Dose: 0 ppm    82.5  91.4  70.2  77.2  93.2  77.7  81.4  78.0  80.3  80.2  76.6  78.1  83.8  81.7  78.3  85.0  Dose: 200 ppm    84.5  91.1  70.4  78.0  93.6  77.7  81.9  78.3  81.1  80.6  77.3  78.8  84.1  81.5  79.1  85.6  Dose: 400 ppm    83.8  91.7  71.3  78.0  93.5  77.7  81.8  78.3  80.9  80.9  77.0  78.8  84.6  81.8  78.7  85.3  Type of diet  Enzyme                                  Low SBM  Dose: 0 ppm  79.7  90.5  67.4  76.4  92.9  74.7  80.5  75.5  79.2  78.7  75.3a,b,c  76.3  80.0d  80.8a,b  75.2b  83.3b,c  Low SBM  Dose: 200 ppm  83.6  90.5  68.3  77.5  93.4  76.3  81.6  77.0  80.6  80.0  77.2a,b,c  77.6  82.4b,c,d  82.2a,b  77.3a,b  84.3a,b,c  Low SBM  Dose: 400 ppm  81.7  90.4  66.6  75.6  92.9  74.0  79.8  74.9  78.6  78.5  73.6c  75.4  80.1d  79.4b  74.6b  82.6c  High SBM  Dose: 0 ppm  85.2  92.3  74.0  79.5  94.0  80.7  83.6  81.1  82.8  81.9  79.7a  80.7  85.6a,b,c  83.7a  81.8a  87.4a  High SBM  Dose: 200 ppm  84.1  90.7  71.3  77.5  93.5  77.6  81.5  78.0  80.7  79.8  76.5a,b,c  78.1  81.6c,d  80.3a,b  79.1a,b  85.7a,b,c  High SBM  Dose: 400 ppm  85.6  92.3  74.6  79.4  94.0  80.1  82.9  80.3  82.2  82.2  79.2a,b  80.5  85.1a,b,c  83.3a,b  80.9a  86.8a  High SBM+GG  Dose: 0 ppm  82.5  91.5  69.3  75.6  92.8  77.6  80.0  77.3  79.1  79.9  74.7b,c  77.4  87.5a,b  80.5a,b  77.8a,b  84.2a,b,c  High SBM+GG  Dose: 200 ppm  85.7  92.6  71.7  79.1  93.9  79.2  82.7  80.0  81.9  82.1  78.3a,b,c  80.7  88.4a  82.1a,b  80.9a  86.7a,b  High SBM+GG  Dose: 400 ppm  84.3  93.3  72.7  79.1  93.6  79.1  82.8  79.7  81.9  82.1  78.3a,b,c  80.4  88.5a  82.8a,b  80.5a  86.6a,b  SEM    1.15  0.65  1.55  1.09  0.29  1.05  0.93  1.11  0.99  0.98  1.09  1.17  0.88  0.91  1.04  0.78  P-value for main effects and interaction                                  Type of diet    <0.01  <0.01  <0.01  0.04*  <0.01  <0.01  0.03*  <0.01  0.01*  <0.01  <0.01  <0.01  <0.01  0.106  <0.01  <0.01  Enzyme    0.10  0.41  0.17  0.54  0.25  1.00  0.74  0.90  0.65  0.64  0.71  0.70  0.55  0.93  0.62  0.62  Type of diet × Enzyme    0.24  0.23  0.37  0.07  0.09  0.08  0.06  0.06  0.07  0.13  <0.01  0.076  <0.01  <0.01  0.03*  0.04*  P-value for contrasts of enzyme level                                  Low SBM-0 vs. Low SBM-200    0.02*  0.97  0.69  0.46  0.18  0.28  0.38  0.31  0.30  0.37  0.23  0.42  0.07  0.29  0.15  0.32  Low SBM-0 vs Low SBM-400    0.26  0.87  0.73  0.61  0.99  0.62  0.60  0.71  0.69  0.86  0.28  0.58  0.98  0.28  0.66  0.56  High SBM-0 vs. High SBM-200    0.51  0.09  0.23  0.21  0.28  0.04*  0.12  0.05*  0.14  0.13  0.05  0.14  <0.01  <0.01  0.08  0.14  High SBM-0 vs. High SBM-400    0.80  1.00  0.79  0.94  0.93  0.65  0.60  0.60  0.66  0.83  0.74  0.93  0.70  0.70  0.56  0.56  High SBM+GG-0 vs. High SBM+GG-200    0.05*  0.21  0.26  0.03*  <0.01  0.29  0.05*  0.09  0.04*  0.12  0.02*  0.04*  0.04*  0.22  0.04*  0.03*  High SBM+GG-0 vs. High SBM+GG-400    0.26  0.06  0.12  0.02*  0.06  0.33  0.04*  0.13  0.05*  0.11  0.02*  0.07  0.03*  0.10  0.07  0.03*  Means with no common superscripts (a, b, c, d) within a column are different at P ≤ 0.05 for Tukey-HSD test. *Means P-value < 0.05. View Large Table 6. Nutritional states, effect of type of diet, and mannanase on serum glucose (mg/dL), insulin (μIU/mL) and IGF-1 (pg/mL) at 21 to 22 d broilers.     Glucose HK (mg/dL)  Insulin (μIU/mL)  IGF-1 (pg/mL)  Item  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Type of diet                    Low SBM    216 ± 3.17b  185 ± 2.15  277 ± 5.21a  10.72 ± 0.35  11.39 ± 0.47  9.73 ± 0.27  543 ± 25.70  779 ± 25.56  668 ± 23.60  High SBM    223 ± 3.17a  187 ± 2.20  244 ± 5.07b  10.64 ± 0.37  10.03 ± 0.44  9.66 ± 0.28  507 ± 23.01  787 ± 32.98  730 ± 24.16  High SBM+GG    225 ± 3.09a  188 ± 2.15  252 ± 5.21b  10.47 ± 0.37  10.88 ± 0.45  9.33 ± 0.28  505 ± 29.06  778 ± 29.21  674 ± 24.15  Enzyme β-D-mannanase, ppm                    Dose: 0 ppm    220 ± 3.17  182 ± 2.2b  263 ± 5.07  9.73 ± 0.41b  10.89 ± 0.46  9.60 ± 0.28  472 ± 28.79  816 ± 27.89  684 ± 24.70  Dose: 200 ppm    222 ± 3.17  190 ± 2.15a  256 ± 5.33  11.23 ± 0.34a  10.65 ± 0.47  9.24 ± 0.27  557 ± 23.19  738 ± 28.74  691 ± 23.60  Dose: 400 ppm    220 ± 3.09  188 ± 2.15a  255 ± 5.07  10.87 ± 0.35a  10.76 ± 0.43  9.87 ± 0.28  526 ± 23.76  790 ± 29.04  697 ± 23.60  Type of diet   Enzyme                    Low SBM   Dose: 0 ppm  213 ± 5.35  182 ± 3.91  269 ± 8.79  10.18 ± 0.58  12.65 ± 0.80  9.72 ± 0.46  490 ± 34.82  806 ± 43.34  700 ± 40.88  Low SBM   Dose: 200 ppm  216 ± 5.77  187 ± 3.63  284 ± 9.47  11.58 ± 0.58  9.71 ± 0.87  9.31 ± 0.46  568 ± 50.09  738 ± 42.86  690 ± 40.88  Low SBM   Dose: 400 ppm  218 ± 5.35  185 ± 3.63  279 ± 8.79  10.38 ± 0.63  11.82 ± 0.74  10.16 ± 0.47  571 ± 49.91  792 ± 47.60  613 ± 40.88  High SBM   Dose: 0 ppm  218 ± 5.77  183 ± 3.91  253 ± 8.79  9.97 ± 0.76  9.99 ± 0.80  9.52 ± 0.51  493 ± 41.77  811 ± 59.75  748 ± 43.70  High SBM   Dose: 200 ppm  234 ± 5.35  191 ± 3.91  238 ± 8.79  10.77 ± 0.58  10.36 ± 0.74  9.57 ± 0.46  536 ± 31.24  746 ± 67.52  698 ± 40.88  High SBM   Dose: 400 ppm  218 ± 5.35  187 ± 3.63  243 ± 8.79  11.18 ± 0.58  9.72 ± 0.74  9.88 ± 0.47  494 ± 45.14  802 ± 39.94  744 ± 40.88  High SBM+GG   Dose: 0 ppm  229 ± 5.35  181 ± 3.63  266 ± 8.79  9.05 ± 0.76  10.03 ± 0.80  9.56 ± 0.46  433 ± 74.62  829 ± 46.11  604 ± 43.70  High SBM+GG   Dose: 200 ppm  222 ± 5.35  193 ± 3.63  247 ± 9.47  11.32 ± 0.58  11.87 ± 0.80  8.85 ± 0.47  567 ± 40.20  731 ± 40.67  684 ± 40.88  High SBM+GG   Dose: 400 ppm  224 ± 5.35  191 ± 3.92  244 ± 8.79  11.04 ± 0.58  10.74 ± 0.74  9.58 ± 0.51  514 ± 30.45  774 ± 65.26  732 ± 40.88  P-value for main effects and interaction                  Type of diet    0.048*  0.509  <0.01**  0.884  0.108  0.560  0.529  0.977  0.138  Enzyme    0.882  0.037*  0.543  0.021*  0.933  0.269  0.082  0.152  0.931  Type of diet × Enzyme  0.131  0.863  0.301  0.517  0.066  0.937  0.837  0.993  0.099  Block    <0.01**  0.731  0.045*  <0.01**  0.965  0.022*  0.139  0.580  0.297  P-value for contrasts of enzyme level                    LowSBM-0 vs. LowSBM-200  0.735  0.431  0.268  0.092  0.017*  0.536  0.195  0.267  0.810  LowSBM-0 vs. LowSBM-400  0.441  0.611  0.454  0.817  0.449  0.511  0.197  0.827  0.137  HighSBM-0 vs. HighSBM-200  0.031*  0.152  0.233  0.404  0.740  0.952  0.409  0.450  0.408  HighSBM-0 vs. HighSBM-400  0.955  0.417  0.413  0.209  0.801  0.604  0.982  0.901  0.963  HighSBM+GG-0 vs. HighSBM+GG-200  0.359  0.026*  0.158  0.021*  0.111  0.289  0.118  0.109  0.186  HighSBM+GG-0 vs. HighSBM+GG-400  0.461  0.084  0.091  0.042*  0.516  0.977  0.343  0.504  0.036*      Glucose HK (mg/dL)  Insulin (μIU/mL)  IGF-1 (pg/mL)  Item  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Type of diet                    Low SBM    216 ± 3.17b  185 ± 2.15  277 ± 5.21a  10.72 ± 0.35  11.39 ± 0.47  9.73 ± 0.27  543 ± 25.70  779 ± 25.56  668 ± 23.60  High SBM    223 ± 3.17a  187 ± 2.20  244 ± 5.07b  10.64 ± 0.37  10.03 ± 0.44  9.66 ± 0.28  507 ± 23.01  787 ± 32.98  730 ± 24.16  High SBM+GG    225 ± 3.09a  188 ± 2.15  252 ± 5.21b  10.47 ± 0.37  10.88 ± 0.45  9.33 ± 0.28  505 ± 29.06  778 ± 29.21  674 ± 24.15  Enzyme β-D-mannanase, ppm                    Dose: 0 ppm    220 ± 3.17  182 ± 2.2b  263 ± 5.07  9.73 ± 0.41b  10.89 ± 0.46  9.60 ± 0.28  472 ± 28.79  816 ± 27.89  684 ± 24.70  Dose: 200 ppm    222 ± 3.17  190 ± 2.15a  256 ± 5.33  11.23 ± 0.34a  10.65 ± 0.47  9.24 ± 0.27  557 ± 23.19  738 ± 28.74  691 ± 23.60  Dose: 400 ppm    220 ± 3.09  188 ± 2.15a  255 ± 5.07  10.87 ± 0.35a  10.76 ± 0.43  9.87 ± 0.28  526 ± 23.76  790 ± 29.04  697 ± 23.60  Type of diet   Enzyme                    Low SBM   Dose: 0 ppm  213 ± 5.35  182 ± 3.91  269 ± 8.79  10.18 ± 0.58  12.65 ± 0.80  9.72 ± 0.46  490 ± 34.82  806 ± 43.34  700 ± 40.88  Low SBM   Dose: 200 ppm  216 ± 5.77  187 ± 3.63  284 ± 9.47  11.58 ± 0.58  9.71 ± 0.87  9.31 ± 0.46  568 ± 50.09  738 ± 42.86  690 ± 40.88  Low SBM   Dose: 400 ppm  218 ± 5.35  185 ± 3.63  279 ± 8.79  10.38 ± 0.63  11.82 ± 0.74  10.16 ± 0.47  571 ± 49.91  792 ± 47.60  613 ± 40.88  High SBM   Dose: 0 ppm  218 ± 5.77  183 ± 3.91  253 ± 8.79  9.97 ± 0.76  9.99 ± 0.80  9.52 ± 0.51  493 ± 41.77  811 ± 59.75  748 ± 43.70  High SBM   Dose: 200 ppm  234 ± 5.35  191 ± 3.91  238 ± 8.79  10.77 ± 0.58  10.36 ± 0.74  9.57 ± 0.46  536 ± 31.24  746 ± 67.52  698 ± 40.88  High SBM   Dose: 400 ppm  218 ± 5.35  187 ± 3.63  243 ± 8.79  11.18 ± 0.58  9.72 ± 0.74  9.88 ± 0.47  494 ± 45.14  802 ± 39.94  744 ± 40.88  High SBM+GG   Dose: 0 ppm  229 ± 5.35  181 ± 3.63  266 ± 8.79  9.05 ± 0.76  10.03 ± 0.80  9.56 ± 0.46  433 ± 74.62  829 ± 46.11  604 ± 43.70  High SBM+GG   Dose: 200 ppm  222 ± 5.35  193 ± 3.63  247 ± 9.47  11.32 ± 0.58  11.87 ± 0.80  8.85 ± 0.47  567 ± 40.20  731 ± 40.67  684 ± 40.88  High SBM+GG   Dose: 400 ppm  224 ± 5.35  191 ± 3.92  244 ± 8.79  11.04 ± 0.58  10.74 ± 0.74  9.58 ± 0.51  514 ± 30.45  774 ± 65.26  732 ± 40.88  P-value for main effects and interaction                  Type of diet    0.048*  0.509  <0.01**  0.884  0.108  0.560  0.529  0.977  0.138  Enzyme    0.882  0.037*  0.543  0.021*  0.933  0.269  0.082  0.152  0.931  Type of diet × Enzyme  0.131  0.863  0.301  0.517  0.066  0.937  0.837  0.993  0.099  Block    <0.01**  0.731  0.045*  <0.01**  0.965  0.022*  0.139  0.580  0.297  P-value for contrasts of enzyme level                    LowSBM-0 vs. LowSBM-200  0.735  0.431  0.268  0.092  0.017*  0.536  0.195  0.267  0.810  LowSBM-0 vs. LowSBM-400  0.441  0.611  0.454  0.817  0.449  0.511  0.197  0.827  0.137  HighSBM-0 vs. HighSBM-200  0.031*  0.152  0.233  0.404  0.740  0.952  0.409  0.450  0.408  HighSBM-0 vs. HighSBM-400  0.955  0.417  0.413  0.209  0.801  0.604  0.982  0.901  0.963  HighSBM+GG-0 vs. HighSBM+GG-200  0.359  0.026*  0.158  0.021*  0.111  0.289  0.118  0.109  0.186  HighSBM+GG-0 vs. HighSBM+GG-400  0.461  0.084  0.091  0.042*  0.516  0.977  0.343  0.504  0.036*  Mean ± SE. Means with no common superscripts (a, b) within a column are different at P ≤ 0.05 for Tukey-HSD test. *P-value < 0.05. **P-value < 0.01. View Large Table 6. Nutritional states, effect of type of diet, and mannanase on serum glucose (mg/dL), insulin (μIU/mL) and IGF-1 (pg/mL) at 21 to 22 d broilers.     Glucose HK (mg/dL)  Insulin (μIU/mL)  IGF-1 (pg/mL)  Item  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Type of diet                    Low SBM    216 ± 3.17b  185 ± 2.15  277 ± 5.21a  10.72 ± 0.35  11.39 ± 0.47  9.73 ± 0.27  543 ± 25.70  779 ± 25.56  668 ± 23.60  High SBM    223 ± 3.17a  187 ± 2.20  244 ± 5.07b  10.64 ± 0.37  10.03 ± 0.44  9.66 ± 0.28  507 ± 23.01  787 ± 32.98  730 ± 24.16  High SBM+GG    225 ± 3.09a  188 ± 2.15  252 ± 5.21b  10.47 ± 0.37  10.88 ± 0.45  9.33 ± 0.28  505 ± 29.06  778 ± 29.21  674 ± 24.15  Enzyme β-D-mannanase, ppm                    Dose: 0 ppm    220 ± 3.17  182 ± 2.2b  263 ± 5.07  9.73 ± 0.41b  10.89 ± 0.46  9.60 ± 0.28  472 ± 28.79  816 ± 27.89  684 ± 24.70  Dose: 200 ppm    222 ± 3.17  190 ± 2.15a  256 ± 5.33  11.23 ± 0.34a  10.65 ± 0.47  9.24 ± 0.27  557 ± 23.19  738 ± 28.74  691 ± 23.60  Dose: 400 ppm    220 ± 3.09  188 ± 2.15a  255 ± 5.07  10.87 ± 0.35a  10.76 ± 0.43  9.87 ± 0.28  526 ± 23.76  790 ± 29.04  697 ± 23.60  Type of diet   Enzyme                    Low SBM   Dose: 0 ppm  213 ± 5.35  182 ± 3.91  269 ± 8.79  10.18 ± 0.58  12.65 ± 0.80  9.72 ± 0.46  490 ± 34.82  806 ± 43.34  700 ± 40.88  Low SBM   Dose: 200 ppm  216 ± 5.77  187 ± 3.63  284 ± 9.47  11.58 ± 0.58  9.71 ± 0.87  9.31 ± 0.46  568 ± 50.09  738 ± 42.86  690 ± 40.88  Low SBM   Dose: 400 ppm  218 ± 5.35  185 ± 3.63  279 ± 8.79  10.38 ± 0.63  11.82 ± 0.74  10.16 ± 0.47  571 ± 49.91  792 ± 47.60  613 ± 40.88  High SBM   Dose: 0 ppm  218 ± 5.77  183 ± 3.91  253 ± 8.79  9.97 ± 0.76  9.99 ± 0.80  9.52 ± 0.51  493 ± 41.77  811 ± 59.75  748 ± 43.70  High SBM   Dose: 200 ppm  234 ± 5.35  191 ± 3.91  238 ± 8.79  10.77 ± 0.58  10.36 ± 0.74  9.57 ± 0.46  536 ± 31.24  746 ± 67.52  698 ± 40.88  High SBM   Dose: 400 ppm  218 ± 5.35  187 ± 3.63  243 ± 8.79  11.18 ± 0.58  9.72 ± 0.74  9.88 ± 0.47  494 ± 45.14  802 ± 39.94  744 ± 40.88  High SBM+GG   Dose: 0 ppm  229 ± 5.35  181 ± 3.63  266 ± 8.79  9.05 ± 0.76  10.03 ± 0.80  9.56 ± 0.46  433 ± 74.62  829 ± 46.11  604 ± 43.70  High SBM+GG   Dose: 200 ppm  222 ± 5.35  193 ± 3.63  247 ± 9.47  11.32 ± 0.58  11.87 ± 0.80  8.85 ± 0.47  567 ± 40.20  731 ± 40.67  684 ± 40.88  High SBM+GG   Dose: 400 ppm  224 ± 5.35  191 ± 3.92  244 ± 8.79  11.04 ± 0.58  10.74 ± 0.74  9.58 ± 0.51  514 ± 30.45  774 ± 65.26  732 ± 40.88  P-value for main effects and interaction                  Type of diet    0.048*  0.509  <0.01**  0.884  0.108  0.560  0.529  0.977  0.138  Enzyme    0.882  0.037*  0.543  0.021*  0.933  0.269  0.082  0.152  0.931  Type of diet × Enzyme  0.131  0.863  0.301  0.517  0.066  0.937  0.837  0.993  0.099  Block    <0.01**  0.731  0.045*  <0.01**  0.965  0.022*  0.139  0.580  0.297  P-value for contrasts of enzyme level                    LowSBM-0 vs. LowSBM-200  0.735  0.431  0.268  0.092  0.017*  0.536  0.195  0.267  0.810  LowSBM-0 vs. LowSBM-400  0.441  0.611  0.454  0.817  0.449  0.511  0.197  0.827  0.137  HighSBM-0 vs. HighSBM-200  0.031*  0.152  0.233  0.404  0.740  0.952  0.409  0.450  0.408  HighSBM-0 vs. HighSBM-400  0.955  0.417  0.413  0.209  0.801  0.604  0.982  0.901  0.963  HighSBM+GG-0 vs. HighSBM+GG-200  0.359  0.026*  0.158  0.021*  0.111  0.289  0.118  0.109  0.186  HighSBM+GG-0 vs. HighSBM+GG-400  0.461  0.084  0.091  0.042*  0.516  0.977  0.343  0.504  0.036*      Glucose HK (mg/dL)  Insulin (μIU/mL)  IGF-1 (pg/mL)  Item  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Fed  Fasted  Re-fed  Type of diet                    Low SBM    216 ± 3.17b  185 ± 2.15  277 ± 5.21a  10.72 ± 0.35  11.39 ± 0.47  9.73 ± 0.27  543 ± 25.70  779 ± 25.56  668 ± 23.60  High SBM    223 ± 3.17a  187 ± 2.20  244 ± 5.07b  10.64 ± 0.37  10.03 ± 0.44  9.66 ± 0.28  507 ± 23.01  787 ± 32.98  730 ± 24.16  High SBM+GG    225 ± 3.09a  188 ± 2.15  252 ± 5.21b  10.47 ± 0.37  10.88 ± 0.45  9.33 ± 0.28  505 ± 29.06  778 ± 29.21  674 ± 24.15  Enzyme β-D-mannanase, ppm                    Dose: 0 ppm    220 ± 3.17  182 ± 2.2b  263 ± 5.07  9.73 ± 0.41b  10.89 ± 0.46  9.60 ± 0.28  472 ± 28.79  816 ± 27.89  684 ± 24.70  Dose: 200 ppm    222 ± 3.17  190 ± 2.15a  256 ± 5.33  11.23 ± 0.34a  10.65 ± 0.47  9.24 ± 0.27  557 ± 23.19  738 ± 28.74  691 ± 23.60  Dose: 400 ppm    220 ± 3.09  188 ± 2.15a  255 ± 5.07  10.87 ± 0.35a  10.76 ± 0.43  9.87 ± 0.28  526 ± 23.76  790 ± 29.04  697 ± 23.60  Type of diet   Enzyme                    Low SBM   Dose: 0 ppm  213 ± 5.35  182 ± 3.91  269 ± 8.79  10.18 ± 0.58  12.65 ± 0.80  9.72 ± 0.46  490 ± 34.82  806 ± 43.34  700 ± 40.88  Low SBM   Dose: 200 ppm  216 ± 5.77  187 ± 3.63  284 ± 9.47  11.58 ± 0.58  9.71 ± 0.87  9.31 ± 0.46  568 ± 50.09  738 ± 42.86  690 ± 40.88  Low SBM   Dose: 400 ppm  218 ± 5.35  185 ± 3.63  279 ± 8.79  10.38 ± 0.63  11.82 ± 0.74  10.16 ± 0.47  571 ± 49.91  792 ± 47.60  613 ± 40.88  High SBM   Dose: 0 ppm  218 ± 5.77  183 ± 3.91  253 ± 8.79  9.97 ± 0.76  9.99 ± 0.80  9.52 ± 0.51  493 ± 41.77  811 ± 59.75  748 ± 43.70  High SBM   Dose: 200 ppm  234 ± 5.35  191 ± 3.91  238 ± 8.79  10.77 ± 0.58  10.36 ± 0.74  9.57 ± 0.46  536 ± 31.24  746 ± 67.52  698 ± 40.88  High SBM   Dose: 400 ppm  218 ± 5.35  187 ± 3.63  243 ± 8.79  11.18 ± 0.58  9.72 ± 0.74  9.88 ± 0.47  494 ± 45.14  802 ± 39.94  744 ± 40.88  High SBM+GG   Dose: 0 ppm  229 ± 5.35  181 ± 3.63  266 ± 8.79  9.05 ± 0.76  10.03 ± 0.80  9.56 ± 0.46  433 ± 74.62  829 ± 46.11  604 ± 43.70  High SBM+GG   Dose: 200 ppm  222 ± 5.35  193 ± 3.63  247 ± 9.47  11.32 ± 0.58  11.87 ± 0.80  8.85 ± 0.47  567 ± 40.20  731 ± 40.67  684 ± 40.88  High SBM+GG   Dose: 400 ppm  224 ± 5.35  191 ± 3.92  244 ± 8.79  11.04 ± 0.58  10.74 ± 0.74  9.58 ± 0.51  514 ± 30.45  774 ± 65.26  732 ± 40.88  P-value for main effects and interaction                  Type of diet    0.048*  0.509  <0.01**  0.884  0.108  0.560  0.529  0.977  0.138  Enzyme    0.882  0.037*  0.543  0.021*  0.933  0.269  0.082  0.152  0.931  Type of diet × Enzyme  0.131  0.863  0.301  0.517  0.066  0.937  0.837  0.993  0.099  Block    <0.01**  0.731  0.045*  <0.01**  0.965  0.022*  0.139  0.580  0.297  P-value for contrasts of enzyme level                    LowSBM-0 vs. LowSBM-200  0.735  0.431  0.268  0.092  0.017*  0.536  0.195  0.267  0.810  LowSBM-0 vs. LowSBM-400  0.441  0.611  0.454  0.817  0.449  0.511  0.197  0.827  0.137  HighSBM-0 vs. HighSBM-200  0.031*  0.152  0.233  0.404  0.740  0.952  0.409  0.450  0.408  HighSBM-0 vs. HighSBM-400  0.955  0.417  0.413  0.209  0.801  0.604  0.982  0.901  0.963  HighSBM+GG-0 vs. HighSBM+GG-200  0.359  0.026*  0.158  0.021*  0.111  0.289  0.118  0.109  0.186  HighSBM+GG-0 vs. HighSBM+GG-400  0.461  0.084  0.091  0.042*  0.516  0.977  0.343  0.504  0.036*  Mean ± SE. Means with no common superscripts (a, b) within a column are different at P ≤ 0.05 for Tukey-HSD test. *P-value < 0.05. **P-value < 0.01. View Large Blood Glucose, Insulin, and IGF-1 Serum glucose concentrations in 21 d chicks fed with High SBM (223 mg/dL) and High SBM+GG (225 mg/dL) were significantly higher than glucose in chicks fed with Low SBM diet (216 mg/dL) (P ≤ 0.048), see Table 6. Low SBM diets in re-fed chicks produced an opposite effect on serum glucose (277 mg/dL) compared to High SBM (244 mg/dL), and High SBM+GG (252 mg/dL) for chicks under re-fed conditions. In the fasted state, chicks previously fed diets with 200 and 400 β-mannanase had increased serum glucose concentrations of 190 and 188 mg/dL, respectively, compared to fasted glucose concentrations (182 mg/dL) for chicks fed control diets containing 0 ppm mannanase (P ≤ 0.01). Insulin was not affected by type of feed under any nutritional state, but was higher when 200 and 400 ppm of mannanase was added: 11.23 and 10.97 μIU/mL, respectively, compared to 9.73 μIU/mL (P ≤ 0.01) for control chicks under fed conditions. Contrast analysis showed that both 200 and 400 ppm mannanase added to High SBM diets produced higher insulin values in chicks than the control diet. Regarding IGF-1, there were no main effect differences, only contrast of High SBM+GG-400 diet (732 pg/mL) higher compared to 604 pg/mL in High SMB+GG-0 (P ≤ 0.036). DISCUSSION The present study had the purpose of evaluating performance, nutrient utilization, and metabolic changes when β-mannanase was added to 3 types of different diets. The enzyme β-mannanase affected variables differently with different dose levels and different types of diets. Performance Only 2 wk of evaluation showed that the addition of β-mannanase improved the feed:gain ratio in some but not all feeds. When β-mannanase was added by 200 and 400 ppm to High SBM+GG feeds, the feed:gain improved in 0.16 and 0.18, respectively. These results are expected due to the high β-mannan concentration in the feed (see Table 1). Daskiran et al. (2004) also found statistical differences when 500 ppm of β-mannanase was added to a feed with 0.5% of GG; however, the differences in feed:gain ratio between using the enzyme or not were lower than the values found in this study. The present experiment showed an opportunity for the enzyme β-mannanase to improve performance when High SBM and High SBM+GG diets are provided to broilers. It was expected that chicks fed diets with Low SBM would have equal performance to chicks fed with High SBM diets; however, AA analysis of the Low SBM diets (Table 1) were lower. Meat and bone meal or DDGS could have had lower nutrient content or nutrient digestibility in the Low SBM diet, although the individual ingredients were evaluated by near infrared prior to formulation. The higher mortality in Low SBM was unexpected and the authors have no explanation. The enzyme showed different response to different types of diets as expected. The enzyme will provide a different value when different ingredients are used in poultry diets because it is expected to produce a larger response with more substrate in the diet. Β-mannanase has been shown to improve broiler (Zou et al., 2006) and laying hen (Wu et al., 2005) performance when fed corn-soybean based diets. Broiler nutrition is about economics and producers around the world search for quality feedstuffs that are less expensive and available in quantities to support intense poultry production. Often, alternative ingredients that become available are byproducts of key ingredients needed for the human food industry. Many of the grain and legume byproducts contain increased levels of nonstarch polysaccharides including β-mannans. GG diets with high levels of mannan created an opportunity that shows the importance of the use of β-mannanase in broiler diets. High SBM diets with β-mannanase showed partial improvements, in a lower degree than the feeds with GG; however, it can be noted that this study was carried only for 2 wk. A longer period of evaluation may give higher differences or better understanding on the activity of the β-mannanase. There are ingredients in many parts of the work with high mannan content, and β-mannanase can be suitable to improve performance and/or reduce the feed cost without compromising broiler performance. Nutrient Utilization and Viscosity Improvement in feed:gain ratio for chicks fed with High SBM+GG diets with added β-mannanase in the present experiment can be explained by increased digestible energy, AA digestibility, and lower viscosity when 200 and 400 ppm β-mannanase were added to this diet. Chicks fed the High SBM diet with 400 ppm added β-mannanase also had an improved feed:gain ratio because energy digestibility was shown to be increased. In the present experiment, β-mannanase (200 and 400 ppm) added to High SBM+GG diets improved the digestible energy (DE) by 205 and 224 kcal/kg, respectively. The dose level of 400 ppm β-mannanase added to the High SBM diet without added GG improved DE by 152 kcal. These DE improvements with the additions of β-mannanase are significantly higher and probably the main reason of improvements in feed:gain ratio in the present study. Other authors have shown 59 kcal/kg energy improvements for MEn with 500 ppm of β-mannanase (same enzyme as the present study) added to High SBM+GG 0.5% broiler diets with broilers 1–14 d (Daskiran et al., 2004). Mannanase added to broiler diets as part of a carbohydrase composite enzyme has been reported to increase the AMEn by 144 kcal AMEn in broilers (Meng et al., 2005). β-mannanase may be releasing mannans to be used as source of energy in the metabolism. AAAD increased for chicks fed diets with β-mannanase added to High SBM-GG diets. The AAAD improvement ranged from +0.90% for histidine digestibility to +3.5 and +3.6 for threonine and glycine digestibility, respectively. Threonine and glycine are both indirectly involved in the gastrointestinal health of broilers by supporting mucin production. β-mannanase may be decreasing the substrate β-mannan and allowing better AA digestibility. Researchers have previously reported an improvement in AA digestibility in broilers fed corn-SBM diets when mannanase was included in a carbohydrase composite enzyme (Meng et al., 2005; Rutherfurd et al., 2007). Kong et al. (2011) reported no improvements in AA digestibility with added β-mannanase for broiler diets. Kong et al. (2011) were using a mannanase from Bacillus subtilis that is different from the Bacillus lentus used in the present experiment. Other publications showed that carbohydrases such as β-mannanase may improve the AA digestibility of feeds by releasing the protein that is caged within the polysaccharides (Cowieson and Ravindran, 2008). The improvement of digestibility of major AA but not body gain brings an important topic of discussion. There is likely a situation where increasing viscosity slows the transit and therefore the digestibility is better; however, because the transit is slower, the feed intake is decreased and no improvement in body gain. Further studies need to be accomplished to understand this paradigm because transit time was not measured in the present study. Glucose, Insulin, and IGF-1 changes Glucose is the primary nutrient source of energy for vital cells in an organism (Clarenburg, 1992) and insulin and IGF-1 are anabolic hormones that respond to glucose metabolism and other nutrients. Chickens have much higher plasma glucose levels than mammals but similar levels of insulin (chicken glucose: 200 mg/dL; mammals 100 mg/dL) (Hazelwood et al., 1968; Simon and Rosselin 1978). Modern high yielding broilers have been reported to contain even higher levels of glucose compared to previous broiler glucose levels: 234–309 mg/dL (Barcellos et al., 2012). Previous experiments with swine using high mannan feeds from GG produced lower glucose and insulin post-prandial (Rainbird and Low, 1986; Nunes and Malmlof, 1992). The importance of present study was to evaluate the negative impact of mannan from GG and SBM on plasma glucose, insulin, and IGF-1 and determine whether adding β-mannanase to the feed will ameliorate the negative effects. Rainbird and Low (1986) and Nunes and Malmlof (1992) reported the lower blood glucose values in pigs was because GG slowed glucose digestion and absorption. Fiber types such as GG have been tested in swine mainly as a means to help human obesity with the objective of reducing digestion and absorption of glucose. In the present experiment, the objective was to improve the negative effects of GG using the enzyme β-mannanase that reduced the viscosity of the jejunum and lowering viscosity helped increase the plasma insulin levels in fed conditions. There were no effects on insulin levels for chicks fed with β-mannanase during fasting and re-fed conditions. The question arises on which nutrient is causing the chick insulin levels to increase because glucose did not change with the enzyme treatments for chicks in the fed state. It is known that insulin is secreted not only in response to the rise of glucose but also fatty acids, and AA in the blood (Gropper and Smith, 2012). Fatty acids were not measured but the digestibility of several AA was increased with β-mannanase for chicks fed with High SBM+GG feeds. β-mannanase may have released more nutrients that increased insulin response because the feed intake was lower with the enzyme. Insulin response due to changes in glucose in broilers is not well understood. Broilers are hyperglycemic, but have the same amount of insulin compared to mammals and show low sensitivity to injections of insulin (Dupont et al., 2008). Dakovic et al. (2014) recently reported that chickens have lost some genes involved in insulin metabolism and the response to glucose metabolism is uncertain. Glucose metabolism was clearly affected in fasting chicks (24 h of feed deprivation) that were being fed diets containing both 200 and 400 ppm concentrations of β-mannanase. During fasting, glucose levels diminished to a certain level but there is a need to maintain a certain level of glucose during fasting because it is the only fuel for the brain and the nervous system (Clarenburg, 1992). In a human, carbohydrate reserves are exhausted in 1 d (Berg et al., 2012) and the process of gluconeogenesis begins to reload glucose in the blood and cells. A hypothesis on the mechanism of how β-mannanase elevates blood insulin and glucose in fasting chicks may be offered from the present study. First, the enzyme diminished the viscosity of the feed and this may have increased glucose metabolism due to better digestion and absorption. Second, mannose is a monosaccharide that can be absorbed passively and converted to glucose from fructose 6 PO4 (Clarenburg, 1992). It is possible that this pathway may have been stimulated to allow more glucose in the system in a fasting scenario. Third, gluconeogenesis increases glucose from noncarbohydrate sources such as AA that may have been stimulated. It is usually believed that carbohydrates are used as a fuel first, followed by fat, and finally proteins; however, the specific timing of the physiological transitions is difficult to consistently identify, because animals oxidize a mixture of carbohydrates, lipids, and proteins (McCue and Pollock, 2013). The results in the present study showed that the enzyme increased the glucose levels during fasting only. Glucose was not changed with the enzyme in other nutritional conditions; however, glucose changed due to the type of diet being fed. In the fed state, feeds with the higher β-mannan content showed higher glucose, and the opposite effect was found in the re-fed state. The re-fed state resembles the swine study where glucose was found to be lower right after a meal that was high in GG (Rainbird and Low, 1986; Nunes and Malmlof, 1992). Two hours may not be enough time for the enzyme to reduce the negative effects of β-mannans for the chick at the cellular level but the enzyme may have adequate time to reduce the viscosity in a 2 wk feeding study (fed state). This argument is supported by increased AA digestibility caused by additional β-mannanase with higher β-mannan feeds. The hormone IGF-1 is another anabolic hormone besides insulin playing an important role in cell proliferation and metabolism after hatch. Lu et al. (2007) reported IGF-1 increased at 14 d during embryo development and kept increasing after hatch through the end of study at 3 wk of age. IGF-1 was significantly increased in chicks in the present study only when 400 ppm of β-mannanase was added to High SBM+GG feed compared to their respective control. The High SBM+GG feed had the highest concentration of mannan, thereby providing more substrate and opportunity for the enzyme to work. Serum glucose was affected by block when samples were collected in the feeding state, but not during the fasting state, probably due to lower standard error between the birds in the fasting state. Serum glucose decreased with time of sampling (block) but at different rate between the treatments, which is the reason for the block to be statistically significant. This same blocking effect was seen with insulin in the feeding state. This may be due to diurnal patterns in insulin (Ekmay et al., 2010). Because the sampling was done in about 3 h during the morning, this could have affected the glucose and insulin status. No blocking differences were seen when evaluating IGF-1. In conclusion, β-mannanase enzyme added to feed showed higher nutritional benefits (performance and physiological) when used with High SBM and High SBM+GG diets. 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Poultry ScienceOxford University Press

Published: May 11, 2018

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