Influence of β-Mannase on Broiler Performance, Digestibility, and Intestinal Fermentation

2002 Poultry Science Association, Inc.

Influence of β-Mannase on Broiler Performance, Digestibility, and Intestinal Fermentation Departament de Cie`ncia Animal i dels Aliments, Facultat de Veterina`ria. Universitat Auto`noma de Barcelona, Bellaterra E-08-193, Barcelona, Spain

Primary Audience: Nutritionists, Broiler Producers SUMMARY Despite the increasing availability of feed enzymes and a growing interest in soybean meal (SBM) carbohydrates, there is little information on the potential beneficial effects of dietary supplements of enzymes to increase nutritive value. Here we hypothesize that a combination of mannase (polysaccharidase) and protease enzymes could reduce the anti-nutritive effect described in soybean β-manans and consequently increase the degradability of carbohydrates and proteins in diets based on SBM. We assessed the effect of adding mannase and protease to a corn-SBM diet fed to broiler chicks in batteries, particularly on productive performance and nutrient digestibility. Our results showed that neither BW gain nor feed intake was affected. No consistent effects were observed with the individual enzymes (mannase or protease) on the ileal or fecal digestibility of crude protein and crude fat. However, organic matter digestibility and apparent metabolizable energy of diets tended to increase with mannase supplementation. Addition of this enzyme to the diet also reduced ileal microbial flora (as measured by purine base concentration), which indicated that feed enzymes might contribute to the prevention of digestive diseases. Key words: mannase, protease, digestibility, carbohydrate, fermentation, broiler 2002 J. Appl. Poult. Res. 11:244–249

DESCRIPTION OF PROBLEM Soybean meal (SBM) has high protein content and a well-balanced amino acid pattern, which make it the most important protein source in livestock feed (World trade of soybean ≈101 millions tonnes [1]). However, the nutritional value of SBM is not exploited, and because of anti-nutritional factors and thermal over- or under-processing, the digestibility of carbohydrates by poultry is low, and 10 to 15% of amino acids are indigestible [2]. Moreover, some carbohydrates in SBM, such as oligosac1

charides (±6%, [3]) and β-mannans (±2%, [4]), are associated with decreases in the efficiency of feed utilization by broilers [5], probably because of changes in intestinal microflora. The nutritive value of SBM can be improved by supplementation with enzymes. However, to our knowledge, few studies have addressed the response of broiler chicks to cereal-soy diets supplemented with mannase [6, 7]. Here we evaluate the effects of including mannase and protease in broiler diets based on corn and SBM. Weight gain, digestibility, and

To whom correspondence should be addressed: [email protected].

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I. Ouhida, J. F. Perez,1 M. Anguita, and J. Gasa

OUHIDA ET AL.: MANNASE FOR BROILERS TABLE 1. Ingredient composition (g/kg) and analysis of the basal diet g/kg

Corn grain Soybean meal (44% CP) Full-fat soybean meal Sunflower oil DL-Methionine Dicalcium phosphate Calcium carbonate PremixA

593.3 258.1 100.1 10.0 2.3 17.7 10.7 7.8

Calculated analysis CP AMEn (kcal/kg) Total Lys Total Met + Cys Calcium Nonphytate phosphorus

195 3,150 11.0 8.5 10.0 4.5

A Premix contained per kilogram of diet: NaCl, 4.8 g; vitamin A, 10,000 IU; vitamin D3, 3,000 IU; vitamin E (αtocopherol), 15.3 mg; vitamin K3; 15 µg; vitamin B1, 2 mg; vitamin B2, 4.5 mg; vitamin B6, 3 mg; vitamin B12, 15 µg; pantothenic acid, 8 mg; choline chloride, 230 mg; folic acid, 0.5 mg; nicotinic acid, 25 mg; biotin, 30 µg. Minerals: Co, 0.25 mg; Se, 0.2 mg; Fe, 40 mg; Cu, 8 mg; Mn, 150 mg; Zn, 80 mg; I, 1 mg; ethoxiquin, 0.9 mg.

intestinal microbial fermentations were evaluated in a mannase dose-response study, with and without protease supplements.

MATERIALS AND METHODS Chick Assays One-day-old commercial male broiler chicks of the Ross strain were placed in a restricted area and fed on a corn-SBM starter diet for the first 6 d after hatching. On Day 6, the chicks were weighed (85.9 ± 2.4 g/bird) and housed in wire cages. Six chicks were placed in each of 36 cages (50 × 60 cm2) in a temperaturecontrolled room. A cage was considered a replicate experimental unit. Replicates were allotted to the dietary treatments, which were provided feed ad libitum. The experiment comprised six treatments in a 3 × 2 factorial design (six replicates per treatment). Basal diet consisted of corn-SBM (Table 1), following NRC [2] requirements to provide 19.5% CP, 3,150 kcal AMEn/kg, 0.85% Met + Cys, 1.0% Ca, and 0.45% nonphytate P. The protein content of the diet was intentionally formulated to be lower than the NRC [2] reference value of 22.5% in order to make the animals more sensitive to the

likely improvement in protein use by enzyme supplementation. The basal diet was supplemented with one of three levels (0, 0.35, and 0.875 g/kg) of Mannase (1,400,000 mannase U/g) and two amounts (0 and 0.35 g/kg) of Bioprotease N120P (120,000 protease U/g, produced from a selected strain of Bacillus subtilis). Both enzyme complexes were provided by the Quest International Company (Ireland). The highest mannase supplementation was fixed at 0.87 g/ kg, because we previously observed that higher doses (1.74 g/kg) promoted detrimental effects on feed intake and feed efficiency. Chromium sexquioxide was added (1 g Cr2O3/Kg) as an indigestible marker. BW and feed intake per replicate were recorded at 21 and 42 d posthatch. Digestibility Analysis and Whole-Tract Nutrient Retention On Days 22 and 42, feces were collected, sampled, and dried in a forced-air oven (105°C). After collection of feces, three chicks from each cage were slaughtered by an intracardiac injection of 1 mL sodium pentobarbital. Ileum (15 cm starting from the ileocaecal junction) and ceca digesta were collected by gentle manipulation and pooled on a replicate basis. Feed, feces, and digesta were analyzed for organic matter (OM), CP, crude fat (CF) and gross energy (GE) using the procedures of the Association of Official Analytical Chemists [8]. Chromium was analyzed using the method described by Williams et al. [9]. Whole-tract nutrient retention and ileal digestibility were estimated using the marker (Cr) ratio method. AMEn (kcal/kg) corrected for nitrogen was calculated as described by Sibbald [10]. Microbial Proliferation and Fermentation Parameters Bacteria proliferation and fermentation in ileum and ceca digesta were estimated using the concentration in digesta of purine bases (PB) as microbial markers and short-chain volatile fatty acids (SCVFA) as the main fermentation end products. PB in digesta were determined by HPLC after acid hydrolysis with 2 mL 2N perchloric acid at 100°C for 1 h, with

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Ingredients and analysis

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TABLE 2. Average valuesA for BW, feed intake, and feed:gain ratio of broiler chickens fed on corn-soybean meal diets with or without enzyme supplementation Enzyme combinations Mannase (g/kg)

6–42 d old

Protease (g/kg)

BW (g)

Feed intake (g)

Feed:gain

BW (g)

Feed intake (g)

Feed:gain

0 0 0 0.35 0.35 0.35

704 703 713 727 712 713 712 15.4

968 1,000 984 1,000 967 979 983 29.4

1.57 1.62 1.57 1.56 1.54 1.56 1.57 0.024

2,035 2,014 2,048 2,098 2,027 2,060 2,046 45.5

3,377 3,422 3,408 3,451 3,386 3,415 3,409 85.3

1.73 1.77 1.74 1.72 1.74 1.73 1.74 0.017

Probability Protease Mannase Protease × mannase

0.40 0.86 0.75

0.94 0.99 0.55

0.07 0.76 0.18

0.43 0.58 0.81

0.83 0.99 0.80

0.18 0.16 0.75

A

Means represent six pens of six birds per treatment.

an addition of 0.75 µmol of allopurinol and neutralization immediately with 4.5 M KOH [11]. For the SCVFA analysis, a fraction of ceca digesta was immediately acidified with H3PO4 [approximately 2 g fresh weight digesta/ 1 mL of 5% (wt/wt) H3PO4, 1% (wt/wt) mercuric chloride acid, and 50 mM 3-methyl valerate as internal standard] during digestive collection. SCVFA concentrations in cecal digesta were measured by gas liquid chromatography, following the method proposed by Jouany [12]. Statistical Analyses Data were studied by analysis of variance using the general linear models procedure of SAS software (SAS Institute, [13]), with cage as the experimental unit. Statistical significance of differences among treatments was assessed using the least significant difference test [14].

RESULTS AND DISCUSSION Supplementation with mannase or protease did not affect feed intake, BW gain, or the feed/ gain ratios (Table 2). Ward and Fodge [6] and McNaughton et al. [7] reported a significant increase in the average daily gain and feed efficiency of broilers fed SBM diets supplemented with β-mannase. Moreover, these authors observed significant increases in the energy digestibility, as broilers fed low-energy feeds treated with enzymes showed similar BW and

feed:gain ratios compared to those fed higherenergy feeds. In the present experiment, we deliberately reduced the dietary content of protein (19.5%) to focus the study on the effects of enzymes on amino acid availability. This restriction could have precluded productive changes if energy digestibility was affected. Protease and mannase supplementation did not significantly modify the ileum digestibility of OM, CP, or CF (Table 3). However, mannase supplementation tended to increase (P = 0.06) the apparent whole-tract digestibility of OM in 21-d-old birds and in 42-d-old birds supplemented simultaneously with protease (P = 0.07). Changes were not observed in CP or CF, which may indicate a more specific effect of the enzymes on the carbohydrate fraction. Detrimental effects have been reported [15] in poultry fed corn-SBM diets supplemented (1 to 3 g/kg food) with guar gum (known as βmannan chains to which D-galactose units are attached in a 2:3 ratio). This gum increases intestinal viscosity and simultaneously decreases nutrient absorption. On the other hand, Marsman et al. [16] observed an increase in the digestibility of the nonstarch polysacharides in broilers fed a corn-SBM diet supplemented with carbohydrase (Energex). However, in contrast to Ward and Fodge [6] and McNaughton et al. [7], Marsman et al. [16] did not observe any effect on the broiler weight gain or feed

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0 0.35 0.87 0 0.35 0.87 Mean SE

6–21 d old

0 0 0 0.35 0.35 0.35

Protease (g/kg)

0.49 0.98 0.79

76.8 1.26

76.8 77.4 76.1 75.5 76.2 78.5

OM

Ileum

0.18 0.96 0.96

82.7 1.05

82.0 82.2 82.1 83.2 83.1 83.6

CP

Means represent six pens of three birds per treatment.

A

Protease Mannase Protease × mannase

Mean SE

0 0.35 0.87 0 0.35 0.87

Mannase (g/kg)

0.24 0.06 0.49

79.8 0.47

79.6 79.0 80.3 79.6 80.0 80.8

OM

0.27 0.37 0.88

0.59 0.93 0.98

84.3 0.68

84.1 84.4 84.0 84.4 84.5 84.4

CF

Whole-tract

79.5 0.91

79.0 78.7 79.5 80.2 78.9 80.6

CP

21 d old

79.6 0.59

79.7 79.4 80.7 79.6 79.5 78.8

OM

0.16 0.69 0.12

0.19 0.86 0.22

Probability

3,317 21.8

3,312 3,326 3,277 3,306 3,329 3,356

AMEn

Ileum

0.72 0.29 0.15

84.6 0.64

84.3 84.0 85.7 83.8 84.4 85.2

CP

0.98 0.38 0.07

80.7 0.73

80.7 81.1 80.2 79.6 80.2 82.2

OM

CF 82.7 82.2 82.8 81.7 82.1 83.3 82.4 0.89 0.63 0.46 0.50

78.8 80.9 80.6 80.6 80.4 80.9 80.4 1.02 0.53 0.53 0.52

Whole-tract CP

42 d old

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Enzyme combinations

0.74 0.32 0.07

3,374 34.4

3,382 3,394 3,359 3,318 3,346 3,445

AMEn

TABLE 3. Ileal and whole-tract organic matter (OM), CP, and crude fat (CF) digestibilitiesA (%) and AMEn1 of 21- and 42-d-old broilers fed on corn-soybean meal diets with or without enzyme supplementation

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TABLE 4. Short-chains volatile fatty acid (SCVFA) concentrationsA in cecal digesta and purine base contentsA (µmol/g) in ileum and cecum digesta of 21- and 42-d-old broilers fed on corn-soybean meal diets with or without enzymes Enzyme combinations Mannase (g/kg)

Mean SE

Protease (g/kg) 0 0 0 0.35 0.35 0.35

SCVFA 21 d

Ileum 42 d

Ceca

21 d

42 d

21 d

42 d

107 102 113 128 125 136

118 127 132 164 101 95

5.8 5.5 4.8 6.9 5.9 4.9

7.1 6.9 6.7 9.0 9.5 5.6

80.5 81.5 75.1 74.5 78.3 79.1

76.3 75.8 72.6 73.4 69.8 69.2

118 28.2

123 19.7

5.6 0.42

7.6 0.74

78.2 3.51

72.9 2.81

0.16 0.06 0.70

0.87 0.27 0.73

0.08 0.38 0.84

Probability Protease Mannase Protease × mannase

0.40 0.89 0.66

0.41 0.27 0.12

0.11 0.04 0.48

A

Means represent six pens of three birds per treatment.

efficiency. This finding is also supported by our results. In addition, we evaluated other effects of feed enzymes on the digestive tract that may impact on the microbial environment and nutrient digestibility. β-Mannans are readily fermentable viscous substrates and may be involved in changes in the microflora colonization of the digestive tract [17, 18]. Average SCVFA concentration in cecal digesta (Table 4) was 118 µmol/g, but a large variability (59% CV) was observed within treatments. Therefore, SCVFA is not suitable to show the microbial effect of experimental treatments. On the other hand, PB concentrations in ileum and ceca digesta (µmol/g DM) presented a lower variability (CV of 20 and 10%, respectively). Average PB concentrations increased from 6.9 to 82 µmol/g DM from the ileum to ceca, respectively. Mannase supplementation promoted significant decreases in the PB content in ileum digesta. Average values were 6.35 vs. 5.7 and 4.85 µmol/g (P < 0.05) in 21-d-old broilers for

0, 0.35, and 0.875 g/kg of mannase, respectively. In 42-d-old birds values were 8.05 and 8.2 vs. 6.15 µmol/g (P = 0.06) for 0, 0.35, and 0.875 g/kg of mannase, respectively. Microflora in ileum and ceca obtain their energy mainly from dietary sugars that escape foregut digestion, which indicates that mannase could indirectly affect intestinal flora through hydrolysis and removal of polysaccharides. An overgrowth of bacteria in the small intestine has been associated with significant decreases in the digestion of saturated fatty acids by deconjugation of bile acids by enterococci [19] and could increase the incidence of disease in practical conditions. Our experimental conditions in batteries probably differed greatly from on-floor microbial challenges. However, given that removal of antibiotic growth promoters from animal rations exacerbates the relevance of small intestinal diseases in poultry production, we propose that supplementation of poultry diets with mannase may prevent bacterial growth in the digestive tract.

CONCLUSIONS AND APPLICATIONS 1. Supplementation with mannase or protease does not affect productive or digestibility coefficients in broiler chick grown in batteries and fed on soybean-corn diets.

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0 0.35 0.87 0 0.35 0.87

Purine bases

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2. Mannase supplementation reduces microbial flora (as measured by PB concentration) in ileal digesta, which may be of considerable relevance in the prevention of digestive disease in poultry.

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Acknowledgments This work was supported by the FEDER project 2FD 1997-1470 and Loders Croklaan, Wormerveer, The Netherlands.

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