INTRODUCTION Most cereals contain a significant proportion of

soluble nonstarch polysaccharides (NSP), which are known to display a variety of antinutritive properties for monogastric animals, particularly for poultry (Hes-selman and Aman, 1986). Incorporation of exogenous β-1,3–1,4-glucanases, in barley, and β-1,4-xylanases, in wheat or rye-based diets, improves the efficiency of feed utilization, enhances growth, and contributes to a better use of low cost feed ingredients (Chesson, 1993; Bedford, 2000). Plant cell wall-degrading enzymes con-tribute to reduce digesta viscosity that is associated with the intake of soluble indigestible carbohydrates, therefore improving the rate of diffusion of substrates,

endogenous digestive enzymes, and nutrients (White et al., 1981; Fengler and Marquardt, 1988; Bedford et al., 1991; Bedford and Classen, 1992). A reduction in digesta viscosity also increases the velocity of feed pas-sage, thus decreasing the proliferation of fermenting mi-crobes in the upper regions of the gastrointestinal (GI) tract (van der Klis et al., 1993) while improving feed intake. However, it is well known that in certain cases the inclusion of exogenous enzymes in diets containing a high percentage of wheat, barley, or rye fails to have any effect in animal performance (Bedford, 2000). Nu-merous hypotheses have been advanced to explain this observation. Strong evidence exists that the availability of energy from cereal grains in poultry is inversely re-lated to the content of soluble NSP (Villamide et al., 1997). Thus, levels of NSP may vary between cereal lots, resulting in cereals expressing different nutritive values. It is well established that a multitude of factors may affect cereal content in NSP, which include cereal genotype and growing conditions, length of the cereal

Levels of endogenous β-glucanase activity in barley affect the efficacy of exogenous enzymes used to supplement barley-based diets for poultry

T. Ribeiro,* M. M. S. Lordelo,† P. I. P. Ponte,* B. Maçãs,‡ J. A. M. Prates,* M. Aguiar Fontes,* L. Falcão,† J. P. B. Freire,† L. M. A. Ferreira,* and C. M. G. A. Fontes*1

*CIISA–Faculdade de Medicina Veterinária, Pólo Universitário do Alto da Ajuda, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; †Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa,

Portugal; and ‡INRB/INIA, Estrada Gil Vaz, 7350-228 Elvas, Portugal

ABSTRACT To improve the nutritive value of barley-based diet for broilers, 2 experiments using 2 different barley lots were performed to evaluate the capacity of a mesophilic cellulase when fused to a β-glucan spe-cific family 11 carbohydrate-binding module. The data revealed that the recombinant β-glucanase derivatives were not appropriate for feed supplementation because of a lack of stability at acidic pH levels. However, un-der the same experimental conditions, a commercial enzyme mixture improved the nutritive value of 1 of the cereal lots used. Analysis of the nutritive value of the 2 barleys revealed intrinsic differences in the lev-els of endogenous β-glucanase activity. These differ-ences were extensively evident when the studies were expanded to a range of 64 barley lots. Thus, to clarify the effect of endogenous cellulases on the efficacy of ex-ogenous β-glucanases used to supplement barley-based

diets for poultry, 2 barley lots presenting low and high levels of endogenous plant cell wall-degrading enzymes were selected. These lots were used to prepare 2 barley-based diets, which were supplemented with or without a commercial enzyme product and fed to broiler chicks. The data revealed that the exogenous enzymes were effective when the basal diet presented low levels of endogenous β-glucanases but were unable to improve the nutritive value of the barley lot displaying higher β-glucanase activity. Thus, these studies suggest that levels of endogenous β-glucanases may affect the ef-ficacy of exogenous enzymes used to improve the nu-tritive value of barley-based diets for broilers. The de-velopment of a quick β-glucanase assay that could be applied for cereal-based feeds may help identify those barley-based diets that are more responsive to the ac-tion of feed enzymes.

Key words: feed enzyme , barley-based diet , β-glucanase , nonstarch polysaccharide , broiler

2011 Poultry Science 90 :1245–1256doi: 10.3382/ps.2010-01218

1245

Received November 3, 2010. Accepted February 3, 2011. 1 Corresponding author: cafontes@fmv.utl.pt

© 2011 Poultry Science Association Inc.

storage period, grain cultivar, growing season, or soil type, among others (Villamide et al., 1997). Although other factors may explain the unpredictable response to enzyme supplementation, such as the levels of endog-enous cellulase and hemicellulase activities within the grain, these remain relatively uncharacterized.

Glycoside hydrolases (EC 3.2.1) that participate in the hydrolysis of plant cell wall carbohydrates display re-markably elaborate molecular architectures comprising both catalytic and noncatalytic carbohydrate-binding modules (CBM). These CBM contribute to establish a close interaction between enzymes and plant polysac-charides allowing the appended catalytic domain to in-timately contact its target substrates, thus potentiating catalysis (Boraston et al., 2004). Carbohydrate-binding modules have been classified into sequence-based fami-lies in the CAZy database (www.cazy.org; Boraston et al., 2004). The role of CBM in the function of feed en-zymes has been investigated. A family 6 xylan-binding domain was shown to improve the efficacy of a micro-bial recombinant xylanase in vivo when the enzyme was used to supplement wheat or rye-based diets for poul-try (Fontes et al., 2004). Thus, animals supplemented with a bimodular xylanase containing both catalytic and xylan-binding domains grew significantly faster than animals fed diets containing exclusively the xyla-nase catalytic domain. More recently, a family 11 CBM that is highly specific for β-1,3–1,4-glucans was shown to improve the efficacy of the associated catalytic do-main used to supplement a barley-based diet (Ribeiro et al., 2008). The higher efficacy of the modular enzyme allowed for a significant reduction in enzyme dosage.

The primary objective of this work was to evaluate, in 2 replicated experiments, the capacity of a family 11 CBM that binds specifically to β-1,3–1,4-mixed linked glucans to improve the efficacy of a single-domain cel-lulase from Cellvibrio mixtus. A different response to enzyme supplementation in these 2 initial experiments motivated the measurement of the levels of β-glucan, β-glucanase activity, and viscosity in the 2 barley lots used. The experiments were extended to a range of more than 60 barleys. Because levels of endogenous plant β-glucanases were found to vary widely in barley, a third experiment was conducted in which the capac-ity of exogenous enzymes to improve the nutritive value of barley-based diets containing different levels of plant β-glucanases was evaluated.

MATERIALS AND METHODS

Enzyme PreparationThe gene encoding the family 11 CBM of Clos-

tridium thermocellum CtLic26A-Cel5E was amplified through PCR from genomic DNA using the thermo-stable polymerase NZYPremium (NZYTech Ltd., Lis-boa, Portugal) and the following primers: forward, 5′-CTCGTCGACCCAACTCCAAGACCGACC; re-

verse, 5′-CACCTCGAGAGCACCAATCAGCTTGAT. The PCR product was cloned into pNZY28, sequenced to ensure that no mutations accumulated during PCR, and subsequently subcloned into the XhoI site of pCF1 (Fontes et al., 1997) generating pTR1. Plasmid pCF1 encoded the single-domain β-glucanase Cel5A and pTR1 encoded the unimodular cellulase fused to the clostridial CBM11, which was termed Cel5A-CBM11 (Figure 1). The 2 plasmids were used to transform BL21 Escherichia coli cells. Recombinant E. coli strains were grown on Luria-Bertani media to mid exponential phase (absorbance at 600 nm = 0.5) and recombinant gene expression was induced by adding isopropyl β-d-thiogalactoside to a final concentration of 1 mM. Cells were collected after 5 h of induction at 37°C and pro-tein extracts were prepared by ultrasonication followed by centrifugation. The recombinant proteins were puri-fied by metal-affinity chromatography as described by Fontes et al. (2004). Both recombinant proteins retain considerable catalytic activity at 40°C and are resistant to proteolytic degradation (Fontes et al., 1997).

Birds, Diets, and ManagementThe composition of the barley-based diets used in this

study, which were formulated to contain adequate nu-trient levels as defined by the NRC (1994), is presented in Table 1. In the first studies (experiments 1 and 2), 2 different barley lots were used to produce basal diets that were supplemented with no enzyme or with 15 U/kg of Cel5A (referred to as treatment Cel5A) or Cel5A-CBM11 (referred to as treatment Cel5A-CBM11). This level of supplementation corresponds to the calculated level of supplementation of the positive control enzyme (see below). In addition, a fourth treatment included a basal diet supplemented with a calculated 15 U/kg of the commercial enzyme cocktail Rovabio Excel AP (Adisseo, Antony, France; referred to as treat-ment C+), which corresponds to an incorporation ratio of 50 g of enzyme/tonne of feed as recommended by the manufacturer. In experiment 3, 2 basal diets were produced using barley batches that contained high or low levels of endogenous β-glucanases. The diets were supplemented with or without the commercial enzyme product Rovabio Excel AP as described above. Thus, the effect of β-glucanase supplementation was tested in barley lots presenting high and low levels of endogenous

Figure 1. Domain organization of Cellvibrio mixtus and its recom-binant derivative used in this study. The cellulase (GH5) and β-glucan binding domain (CBM11) are indicated. The gray and the black boxes represent the signal peptide and linker sequences, respectively.

1246 RIBEIRO ET AL.

β-glucanase. Basal diets were provided in the pelleted form and enzyme preparations were mixed with the feed just before administration to the animals. Animal experiments were conducted in accordance with the principles and specific guidelines presented in Euro-pean Union (1986), reviewed by the Ethics Committee of CIISA Faculdade de Medicina Veterinária, and ap-proved by the Animal Care Committee of the National Veterinary Authority (Direcção-Geral de Veterinária, Lisboa, Portugal).

For each experiment, 1-d-old chicks (Ross 308; n = 160) were divided into 40 battery brooders (capacity of 4 animals/pen) exposed to constant light for the du-ration of the trial. Water and diets were available ad libitum throughout the experiment and were provided from automatic drinking nipples and a hanging feeder, respectively. The brooders were located in an environ-mentally controlled room, which was adjusted daily to the recommended temperatures according to standard brooding practice. Birds were individually weighed at the beginning of the experiment and were randomly as-signed to one of the 4 treatments, with 10 replicates/treatment. Feed consumption and individual BW were recorded weekly. Feed conversion ratios were calculated by dividing the weight gain per pen per week and at the end of the experiment, including the weight gain of any dead birds, by the total feed consumed during the respective period. Bird mortality was recorded daily. At the end of the experiment, on d 28, 1 bird/pen was slaughtered by an intravenous injection of an aqueous isotonic solution of 125 mg of Tiopental Braun (Braun, Barcelona, Spain). The size of the various GI compart-ments was measured or weighted and digesta samples were collected and stored at −20°C for later analysis. In experiments 1 and 2 the weight of the GI compart-ments was determined full. In contrast, in experiment

3 the various portions of the GI tract were emptied before weighing. Levels of β-glucanase activity in the GI tract were measured as described below.

Analytical Procedures

To standardize the number of enzyme units used to supplement the basal diets, the catalytic activity of the various exogenous enzymes, including the commercial mixture, was determined under identical experimental conditions. Catalytic activity was determined at 40°C by measuring reducing sugar released, following the method described by Fontes et al. (2000), using barley β-glucan (Megazyme, Bray, UK) as the substrate. One unit of catalytic activity is defined as the amount of enzyme required to release 1 μmol of product/min. The extract containing Rovabio Excel AP enzymes was pre-pared by ressuspending 75 mg of the enzyme mixture in 10 mL of 50 mM Na-HEPES buffer (pH 7.5) followed by incubation overnight at 4°C with gentle agitation and a centrifugation at 13,000 × g for 5 min. Previous to detection of β-glucanase activity, digesta samples were centrifuged and the supernatant was recovered for analysis. Initially, qualitative analysis of β-glucanase activity in the digesta samples recovered from the vari-ous GI compartments was assessed in agar plates, us-ing barley β-glucan (Megazyme) at 0.1% (wt/vol) final concentration, in 10 mM Tris-HCl (pH 7.0). Catalytic activity was detected after 16 h of incubation at 37°C through the Congo red assay plate, as described in Pon-te et al. (2004) and Mourão et al. (2006). For measuring the viscosity of small intestine contents, samples were centrifuged for 10 min at 7,500 × g and the viscosity of the supernatant was measured using a Brookfield viscometer (model LVDVCP-II, Brookfield Engineering Laboratories, Middleboro, MA) with a cup maintained at 24°C. Analyses for DM (method 934.01), crude fat (method 920.39), CP (method 954.01), NDF (method 2002.04), and ADF and acid detergent lignin (method 973.18) were performed according to the methods of AOAC (1980).

A large range of barley lots from different varieties was selected in September and October 2009, and levels of viscosity and contents in β-glucan and β-glucanase were measured. All barleys were harvested in 2009 and thus could be considered as young barleys because the assays were performed in the autumn of 2009. Barley content in β-glucan and levels of cereal β-glucanase ac-tivities were determined with a β-glucan assay kit and a β-glucanase assay kit (Megazyme), respectively. The method for determining β-glucanase activity followed the manufacturer’s protocol with a modification on the incubation period that was extended from 10 min to 3 h. The method used for measuring the levels of viscos-ity followed the steps described above, although the barley samples were milled at 0.5 mm and mixed with 15 mL of phosphate citrate buffer (pH 6.5) by vigorous shaking for 5 min before the start of the experiment.

Table 1. Ingredient composition and calculated analysis of the cereal-based feed

Item (% unless noted) Value

Ingredient Barley 61.48 Soybean meal (47%) 29.40 Soybean oil 5.90 Sodium chloride 0.25 Calcium carbonate 0.80 Dicalcium phosphate (18%) 1.80 dl-Methionine 0.17 Mineral and vitamin premix1 0.20Calculated nutrient content Energy (MJ of ME/kg of DM) 12.1 CP 20.8 Ether extract 7.5 Crude cellulose 5.1 Ash 5.7 Methionine 0.47

1Mineral and vitamin premix provided the following per kilogram of diet: vitamin A, 9,000 IU; vitamin D3, 2,100 IU; vitamin E, 20 mg; nico-tinic acid, 30 mg; vitamin B12, 0.12 mg; calcium pantothenate, 10 mg; vitamin K3, 2 mg; thiamin, 1 mg; riboflavin, 4.2 mg; vitamin B6, 1.7 mg; folic acid, 0.5 mg; biotin, 0.5 mg; Fe, 80 mg; Cu, 10 mg; Mn, 100 mg; Zn, 80 mg; Co, 0.2 mg; I, 1.0 mg; Se, 0.3 mg; monensin, 100 mg.

1247ENDOGENOUS β-GLUCANASE AND FEED ENZYME EFFICACY

Statistical AnalysisStatistical analysis of data related to bird perfor-

mance was conducted by ANOVA using the GLM pro-cedure of SAS (SAS Inst. Inc., Cary, NC). Means with a significant F-ratio were separated by the least signifi-cant difference test. The experimental unit was a cage of 4 birds. Unless otherwise stated, differences were considered significant at P < 0.05. Regression analy-ses were conducted to test for linearity between level of β-glucans and viscosity, between level of β-glucans and β-glucanase activity, and between viscosity and β-glucanase activity in barley lots.

RESULTS AND DISCUSSION

Recombinant Derivatives of Cel5A from C. mixtus Were Unable to Improve the Nutritive Value of Barley-Based Diets for Poultry

In contrast to the majority of plant cell wall-degrad-ing enzymes, Cel5A from C. mixtus is a unimodular enzyme which, despite being produced by a mesophilic bacterium, presents resistance to proteolytic inacti-vation and significant thermostability (Fontes et al., 1997). Previously, it was shown that a family 11 CBM has the capacity to increase the catalytic activity of the associated β-glucanase and cellulase catalytic domains in vivo by improving the efficacy of the catalytic mod-ules to enhance the nutritive value of barley-based diets

for poultry (Guerreiro et al., 2008; Ribeiro et al., 2008). Here, we investigated the capacity of a recombinant form of Cel5A from C. mixtus to decrease the levels of β-glucans found in barley-based diets for poultry. In ad-dition, the capacity of the family 11 CBM to enhance the efficacy of Cel5A was evaluated by engineering a recombinant fusion enzyme combining the 2 modules (Figure 1).

In experiment 1, BW of birds fed barley-based diets supplemented with or without the exogenous polysac-charidases was not significantly different on d 7, 14, 21, or 28 (final BW; bird mortality was 3.1%; Table 2). No differences were found in weight gain among the dif-ferent groups during the entire trial. In addition, feed intake and feed conversion ratios did not differ among the groups. Taken together, the results suggest that the exogenous enzymes of microbial (CelA and CelA-CBM11) or fungal (Rovabio Excel AP; treatment C+) origin were unable to improve the nutritive value of the barley-based diet used in experiment 1, which con-tained more than 60% (wt/wt) of barley.

The exact same experiment was repeated (experi-ment 2) using a basal diet including the same propor-tion of ingredients (as described in Table 1) but prepar-ing it using different barley, soybean meal, and soybean oil; all other remaining components of the diet had the same origin as in experiment 1. Bird BW, weight gain, feed intake, and feed conversion ratio of experiment 2 are summarized in Table 3 (bird mortality was 1.9%). The data revealed that final BW of birds fed the basal diet supplemented with the commercial enzyme cock-

Table 2. Performance of broilers fed a barley-based diet supplemented with different β-glucanase preparations studied in experiment 1

Item

Treatment1

SEM p(F)2C− C+ CelA CelA-CBM11

BW (g) 0 d 42.9 43 42.9 42.9 0.150 0.945 7 d 135.9 137.3 130.4 131.4 2.570 0.182 14 d 310.5 331.4 310.2 307.4 7.330 0.095 21 d 594.5 627.8 591.3 589.1 11.590 0.077 28 d 1,028.7 1,068.5 1,015.1 1,040.3 22.350 0.391Weight gain (g) 0–7 d 93 94.3 87.5 88.5 2.57 0.186 7–14 d 174.6 194.1 179.8 176.1 5.56 0.070 14–21 d 284.1 296.5 281.1 281.7 6.16 0.268 21–28 d 434.2 440.7 423.9 451.3 15.59 0.655 0–28 d 985.8 1,025.5 972.2 997.4 22.32 0.391Feed intake (g) 0–7 d 122.0 127.1 123.2 120.6 3.66 0.636 7–14 d 261.4 288.6 274.3 272.7 6.64 0.052 14–21 d 438.6 455.8 444.4 432.3 14.65 0.709 21–28 d 695 698.7 716.7 714.4 16.58 0.729 0–28 d 1,516.9 1,570.2 1,558.6 1,540.0 32.81 0.683Feed conversion ratio 0–7 d 1.320 1.360 1.410 1.360 0.044 0.545 7–14 d 1.500 1.490 1.540 1.580 0.044 0.520 14–21 d 1.550 1.540 1.590 1.560 0.033 0.708 21–28 d 1.650 1.590 1.700 1.630 0.042 0.381 0–28 d 1.570 1.540 1.600 1.630 0.036 0.257

1C−: birds not supplemented with an exogenous enzyme; C+: birds supplemented with an enzymatic commercial mixture; CelA: birds supplemented with Cellvibrio mixtus; CelA-CBM11: birds supplemented with the protein CelA-CBM11.

2p(F) < 0.05 is statistically significant.

1248 RIBEIRO ET AL.

tail (Rovabio Excel AP; treatment C+) was significant-ly higher than birds of the other groups. Differences in BW were visible as soon as d 7 and remained signifi-cant for the duration of the experiment. However, dif-ferences in weight gain were particularly acute on d 7, revealing that the exogenous enzymes were particularly effective in earlier stages of growth. In addition, no dif-ferences in feed intake were found between treatments, although feed conversion ratio in animals receiving the fungal enzymes was smaller when compared with the other groups (0–28 d). Thus, these data suggest that an improvement in BW in animals receiving the com-mercial feed enzymes resulted from a better use of the feed ingredients rather from an increase in feed intake. Taken together the results revealed that, in contrast to the commercial enzyme mixture, the recombinant en-zymes were unable to improve the nutritive value of the barley-based diet of experiment 2. In addition, broilers fed the basal diets of experiments 1 and 2 responded differently to the addition of the fungal exogenous en-zymes that were effective only in experiment 2.

It is well known that diets presenting high levels of soluble NSP induce considerable enlargement of some portions of the GI tract (Brenes et al., 1993) and stimu-late an increase in protein turnover rates (Dänicke et al., 2000). Enzyme supplementation decreases digesta viscosity and therefore improves the feed passage rate and nutrient absorption. In addition, the relative weight of the digestive tract decreases, leading to an increase in carcass yield (Petterson and Aman, 1989). Fuente et al. (1998) found an equation relating the empty weight

of the digestive tract to digesta viscosity. Therefore, the effects of dietary treatments in the relative length and weight of GI tract compartments of broiler chickens of experiments 1 and 2 were evaluated. Enzyme supple-mentation had no effect on crop, gizzard, and liver rela-tive weights, on digesta viscosity, or on the duodenum, jejunum, and cecum relative lengths in experiment 1 (data not shown). In contrast, in experiment 2 jejunum and ileum relative lengths were significantly reduced (P < 0.05) in birds receiving the commercial enzyme mix-ture when compared with animals of other treatments (Table 4). Thus, exogenous enzymes in treatment C+ contributed to reduce the levels of digestive antinutri-tive β-glucans, which through an increase in viscosity contribute to increase the size of the digestive com-partments. Although it significantly contributed to im-prove broiler performance, the commercial β-glucanase (treatment C+) had no effect on the viscosity of the intestinal contents (Table 4). Digesta viscosity was de-termined exclusively at the end of the experimental period, on d 28. At later stages of animal growth, the endogenous microflora might contribute to consider-ably reduce the chain length of soluble glucans, thus reducing digesta viscosity.

In contrast with previous reports (Philip et al., 1995; Guerreiro et al., 2008; Ribeiro et al., 2008) that con-firmed the capacity of recombinant cellulases Cel5E (Philip et al., 1995) and CtLic26A-Cel5E (Guerreiro et al., 2008; Ribeiro et al., 2008) from C. thermocellum to improve the nutritive value of barley-based diets for poultry, data presented here suggest that Cel5A from

Table 3. Performance of broilers fed a barley-based diet supplemented with different β-glucanases studied in experiment 2

Item

Treatment1

SEM p(F)2C− C+ CelA CelA-CBM11

BW (g) 0 d 45.3 44.7 45.2 45.3 0.450 0.759 7 d 141.2b 157.3a 132.3b 133.6b 4.730 0.002 14 d 365.0b 397.3a 344.9b 351.5b 9.020 0.001 21 d 699.2b 750.8a 664.3b 677.1b 16.100 0.003 28 d 1,171.9b 1,230.4a 1,112.6b 1,154.0b 23.210 0.009Weight gain (g) 0–7 d 96.0b 112.6a 87.1b 88.3b 4.67 0.002 7–14 d 223.8 240.1 212.6 218.0 8.60 0.146 14–21 d 334.3ac 353.5a 319.4bc 325.6bc 8.10 0.029 21–28 d 472.6 479.8 458.4 477.0 11.06 0.538 0–28 d 1,126.6ac 1,185.9a 1,077.5bc 1,108.7bc 23.90 0.022Feed intake (g) 0–7 d 140.8 148.4 136.0 142.0 6.07 0.551 7–14 d 327.1 338.5 322.7 335.2 9.65 0.640 14–21 d 591.9 595.6 587.5 612.4 16.17 0.716 21–28 d 917.7 877.5 910.1 917.6 21.69 0.513 0–28 d 1,977.5 1,960.0 1,956.2 2,007.5 38.98 0.783Feed conversion ratio 0–7 d 1.460 1.370 1.560 1.630 0.079 0.117 7–14 d 1.480 1.440 1.520 1.550 0.058 0.535 14–21 d 1.780 1.690 1.840 1.890 0.058 0.091 21–28 d 1.940 1.840 2.000 1.930 0.049 0.158 0–28 d 1.760ab 1.660b 1.820a 1.820a 0.038 0.014

a–cMeans within the same row with different superscripts are significantly different (P < 0.05).1C−: birds not supplemented with an exogenous enzyme; C+: birds supplemented with an enzymatic commercial mixture; CelA: birds supplemented

with Cellvibrio mixtus; CelA-CBM11: birds supplemented with the protein CelA-CBM11.2p(F) < 0.05 is statistically significant.

1249ENDOGENOUS β-GLUCANASE AND FEED ENZYME EFFICACY

C. mixtus is unable to reduce the antinutritive effects of soluble glucans present in barley-based diets. One of the major actions of feed β-glucanases is to decrease the degree of polymerization of β-1,3–1,4-glucans pres-ent in barley through the random cleavage of glycosydic bonds of the polysaccharide backbone. The reduction in carbohydrate chain length contributes to decrease the levels of digesta viscosity (Fengler and Mar-quardt, 1988; Bedford and Morgan, 1996; Apajalahti and Bedford, 1999). Data presented here suggest that the exogenous recombinant CelA derivatives were not catalytically active. Thus, to evaluate the stability of exogenous glycoside hydrolases during passage through the GI tract, β-glucanase activity was qualitatively de-termined in digesta samples collected in the various digestive compartments of 10 animals/treatment. The data, presented in Table 5, revealed that the major-ity of digesta samples collected from birds receiving the commercial enzyme mixture expressed β-glucanase activity. However, the levels of enzyme activity pres-ent in the GI tract of birds supplemented with the re-combinant enzymes were very small or below the assay detection limit, particularly after the crop. The lower activity of the recombinant enzymes in the crop when compared with animals supplemented with the fungal β-glucanases is difficult to explain considering that the

crop presents a milder environment in terms of pH and proteases. It is possible that some degree of enzyme in-hibition may have occurred as a result of the presence of specific CelA inhibitors in the diet, although this possibility was unexplored in the current work. As was anticipated, only birds not receiving exogenous enzymes expressed significant levels of β-glucanase activity in the cecum. Thus, the data suggest that although a sim-ilar level of exogenous enzymes was added to the basal diets of treatments C+, Cel5A, and Cel5A-CBM11, the activity of the recombinant enzymes was significantly reduced after the passage through the proventriculus. This suggests that CelA and its recombinant deriva-tives are particularly sensitive to denaturation under acidic conditions. Assays performed in vitro confirmed this hypothesis; the 2 recombinant derivatives were shown to retain only approximately 10% of their initial activity after incubation for 10 min at pH 3.

Taken together the data presented here revealed that the nutritive value of the barley lots used in the 2 ex-periments differed because the diets reacted differently to the supplementation with exogenous cellulases. In addition, the recombinant enzymes derived from C. mixtus CelA are not appropriate for feed supplemen-tation because of their limited stability at lower pH levels.

Table 4. Relative weight and length of the gastrointestinal tract and viscosity of digesta samples of broilers fed a barley-based feed supplemented with different exogenous β-glucanases (data from experiment 2)

Item

Treatment1

SEM p(F)2C− C+ CelA CelA-CBM11

Relative weight (g/100 g of BW) Crop 3.790a 2.940b 3.130b 3.460ab 0.221 0.051 Gizzard 13.710 12.600 13.420 13.260 0.888 0.840Relative length (cm/kg of BW) Duodenum 22.06 21.82 23.37 21.54 0.686 0.261 Jejunum 60.28b 53.35a 63.83b 60.10b 1.813 0.002 Ileum 64.04ab 59.39a 68.67b 61.42ab 2.196 0.030 Cecum 14.70 13.32 15.07 14.34 0.542 0.140Content viscosity (cP) Duodenum + jejunum 6.753 5.983 6.632 6.937 0.483 0.919 Ileum 12.529 9.553 12.485 12.128 0.974 0.670

a,bMeans within the same row with different superscripts are significantly different (P < 0.05).1C−: birds not supplemented with an exogenous enzyme; C+: birds supplemented with an enzymatic commercial mixture; CelA: birds supplemented

with Cellvibrio mixtus; CelA-CBM11: birds supplemented with the protein CelA-CBM11.2p(F) < 0.05 is statistically significant.

Table 5. Qualitative detection1 of cellulase activity in digesta collected from the gastrointestinal compartments of 40 broilers fed a barley-based feed supplemented with recombinant cellulases displaying different molecular architectures2 (data from experiment 2)

Item C− C+ CelA CelA-CBM11

Crop +/–/–/+/–/–/–/–/–/– +/+/+/+/+/+/+/+/+/+ +/+/+/–/–/+/+/–/–/– –/–/+/–/+/–/+/–/+/+Gizzard –/–/–/–/+/–/–/–/–/+ +/+/+/+/–/+/+/+/–/+ –/–/–/–/–/–/–/–/–/– –/–/–/–/–/–/–/–/+/–Duodenum –/–/–/+/–/–/–/–/–/– +/+/–/+/–/–/+/+/+/– –/–/–/–/–/–/–/–/–/– –/–/–/–/–/–/–/–/–/–Jejunum –/–/–/–/–/–/–/–/–/– +/+/–/–/–/–/+/–/+/+ –/–/–/–/–/–/–/–/–/– –/–/–/–/–/–/–/–/+/–Ileum –/–/–/–/+/+/–/–/–/– +/–/–/–/+/–/–/–/+/+ –/–/–/–/–/–/–/–/+/– –/–/+/–/–/–/–/–/–/–Cecum +/+/+/+/+/+/+/+/+/+ +/+/+/+/+/+/+/+/+/+ +/+/+/+/+/+/+/+/+/+ +/+/+/+/+/+/+/+/+/+

1Symbols refer to no (–) or detectable (+) β-glucanase activity.2C−: birds not supplemented with an exogenous enzyme; C+: birds supplemented with an enzymatic commercial mixture; CelA: birds supplemented

with Cellvibrio mixtus; CelA-CBM11: birds supplemented with the protein CelA-CBM11.

1250 RIBEIRO ET AL.

Different Barley Lots Expressed Different Levels of Endogenous β-Glucanase Activity

Data revealed that the exogenous enzymes of Rov-abio Excel AP were unable to improve the nutritive value of the basal diet of experiment 1. Because diets of experiments 1 and 2 contained more than 60% of barley but presented the same formulation, it is sug-gested that the observed differential response to en-zyme supplementation resulted from the different composition of the 2 barley lots used in experiments 1 and 2. To test this possibility, viscosity, β-glucan, and β-glucanase contents of the 2 barley lots used in the above-described experiments were determined. The data revealed that levels of β-glucan and viscosity were higher in the barley lot used in experiment 2 (barley of experiment 2 contained 3.44% of β-glucan and 1.36 cP viscosity, whereas the barley lot of experiment 1 presented levels of β-glucan and viscosity of 2.26% and 1.25 cP). In contrast, the barley lot used in experiment 1 presented almost 5 times more β-glucanase activity than the barley lot of experiment 2 (589 and 134 U/kg of β-glucanase activity in barleys of experiments 1 and 2, respectively). Thus, the data suggest that a variation exists in the levels of β-glucan and digesta viscosity but particularly of endogenous β-glucanase activity in the barleys used in experiments 1 and 2. These observed variations may explain the differential response to en-zyme supplementation, as will be explored below.

Levels of viscosity, β-glucan, and β-glucanase activ-ity were determined in a large range of barley lots to assess the variation in barley composition in relation to those factors (see Materials and Methods section). The data, depicted in Table 6, revealed that consider-able variation exists in the levels of β-glucanase activ-ity expressed by the different barley lots, which ranged from >1,300 U/kg to <60 U/kg (in Table 6, barleys B9 and B11 correspond to the cereals used in experi-ments 1 and 2, respectively). A regression analysis to test the existence of linear effects between the level of β-glucan and viscosity, between the level of β-glucan and β-glucanase activity, and between viscosity and β-glucanase activity was performed. However, the coefficients of determination were 0.019, 0.097, and 0.331, respectively, which indicates a low correlation between the variables. However, β-glucanase activity varies much more widely than the levels of viscosity and β-glucans. Taken together, these observations sug-gest that endogenous levels of β-glucanase activity and not exclusively the content in β-glucans may affect the nutritive value of barley for poultry.

Levels of Endogenous β-Glucanase Activity Affect the Nutritive Value of Barley-Based Diets for Poultry

Data presented above suggest that levels of endog-enous plant β-glucanases in barley may affect the effi-

cacy of exogenous cellulases used to supplement barley-based diets for poultry. To test this possibility 2 barley lots were selected for a comparative study aiming to evaluate the capacity of an exogenous cellulase mix-ture to improve the nutritive value of barley-based diets with different levels of endogenous plant enzymes for broilers. The 2 barley lots selected were B13 (present-ing high β-glucanase activity; HA) and B23 (presenting low β-glucanase activity; LA), with barley B13 display-ing approximately 4 times more β-glucanase activity than barley B23. In contrast, the viscosity and levels of β-glucan were similar in barleys B13 and B23 (Table 6). The chemical composition of the 2 selected barley lots was similar (Table 7), although levels of NDF were higher in barley HA (Table 7). Thus, the formula of Table 1 was used to produce 2 different barley-based diets using either barley B13 or barley B23. The 2 bas-al diets, alone or supplemented with the commercial enzyme mixture Rovabio Excel AP, were used to feed broiler chicks until d 28 as described in the Materi-als and Methods section (experiment 3). The results of experiment 3, expressed as final BW, weight gain, feed intake, and feed conversion ratios are summarized in Table 8 (bird mortality was 3.6%). Final BW of birds fed the HA basal diet were significantly higher than those of animals fed the LA diet. In contrast with what was observed in the 2 preceding experiments, final BW of birds fed HA diets was similar to what is expected in standard commercial conditions, confirming that bar-ley B13 displayed a higher nutritive value when com-pared with the other barleys used in this study. The addition of exogenous β-glucanases had no effect on the weight gain of animals receiving the HA diet. In contrast, addition of exogenous enzymes to diet LA sig-nificantly improved bird final BW, although birds did not reach the final weight of birds receiving the HA di-ets. No variations were found in feed intake among the 4 groups, suggesting that barley source and exogenous enzymes lead to different efficiencies of nutrient utiliza-tion rather to an increase or decrease of feed intake. This was confirmed by analyzing the data on feed con-version ratios, which were substantially smaller for the groups receiving the HA diet and the group receiving the LA diet supplemented with the exogenous enzymes when compared with the animals receiving the LA basal diet not supplemented with microbial enzymes. Taken together, the data suggest that levels of endog-enous β-glucanase activity may have a major effect on the nutritive value of barley-based diets. Hence, addi-tion of exogenous β-glucanases may be effective only in the case of barley-based diets containing lower levels of endogenous plant cell wall-degrading activity, such as barley B23. Notwithstanding the suggested implica-tions of endogenous plant enzymes in the effectiveness of exogenous enzymes used to supplement barley diets, it is clear that diets used in experiment 3 may have pre-sented unaccounted variations that may have affected the nutritive value of the 2 barley lots. Clear proof of the concept would be possible by comparing the nutri-

1251ENDOGENOUS β-GLUCANASE AND FEED ENZYME EFFICACY

tive value of a barley batch similar to B13 before and after specifically inactivating the endogenous enzymes (for example, through heating). However, although it

can be anticipated that endogenous plant β-glucanases are thermolabile, it is clear that a relative thermo-stability exists because diets used in this study were

Table 6. Viscosity and levels of β-glucan and β-glucanase activity in different barley lots1,2

Barley no. Variety β-Glucan (%) Viscosity (cP) β-Glucanase activity (U/kg)

B1 Scarlett 2.96 1.97 554B2 Braeman 2.83 1.23 930B3 Braeman 3.52 1.86 569B4 Braeman 3.14 1.71 1,209B5 Unk 3.43 1.41 1,010B6 Unk 2.62 2.12 541B7 Unk 3.01 1.97 221B8 Unk 2.89 1.53 339B9 Scarlett 2.26 1.25 589B10 Unk 2.35 1.23 672B11 Unk 3.44 1.36 134B12 Unk 2.50 1.23 1,381B13 Unk 2.80 1.46 1,047B14 Unk 3.02 1.41 1,125B15 Unk 2.50 1.46 1,304B16 Unk 3.24 2.05 174B17 Unk 2.40 1.20 1,202B18 Unk 3.40 1.61 254B19 Unk 3.20 1.43 1,024B20 Unk 3.24 1.46 843B21 Unk 3.08 1.20 1,064B22 Scarlett 2.44 1.43 113B23 Unk 2.58 1.49 287B24 Braeman 2.68 1.23 407W1 Esterel 1.65 2.02 108W2 Regina 2.75 1.69 97W3 Wintmalt 3.94 1.46 315W4 Cartel 3.47 1.69 126W5 Malice 3.41 1.77 95W6 Metaxa 4.12 1.71 128W7 Malicorne 3.57 1.84 123W8 Manureva 3.87 1.89 113W9 Melodica 3.62 1.64 60W10 Violetta 3.91 2.00 66W11 Vanessa 2.95 2.12 111CS1 Scarlett 3.73 2.04 61CS2 Prestige 2.99 2.10 78CS3 Vivaldi 3.11 2.71 234CS4 Maltasia 3.27 2.25 150CS5 Kangoo 3.79 2.27 241CS6 Jolika 3.06 2.12 1,038CS7 Belgravia 3.34 2.28 157CS8 Thorgal 3.22 1.99 98CS9 Cropton 3.07 1.99 296CS10 Jennifer 2.84 1.82 436CS11 Signora 2.98 2.22 177CS12 Calcule 4.38 1.56 188CS13 Concerto 3.08 2.07 173CS14 Azalea 3.10 1.64 84CS15 Primadonna 2.93 1.64 93CS16 Pewter 3.40 2.04 100CS17 Margret 3.97 2.33 57IB1 Quench 3.65 1.84 52IB2 Clairion 3.60 1.74 267IB3 Concerto 3.78 2.15 77IB4 Erlina 3.62 2.27 67IB5 NFC106–119 3.28 1.87 58IB6 180/02 5B 3.78 1.77 115IB7 302/02-G 3.75 2.05 203IB8 IN0616 3.86 2.68 67IB9 SYN407–143 1.12 2.50 117IB10 Charmay 2.39 2.25 133IB11 Chogun 3.26 1.66 151IB12 Parigus 3.67 1.38 111

1Barley B9 was used in experiment 1; barley B11 was used in experiment 2; barleys B13 and B23 were used in experiment 3.2The Unk varieties are mixtures of several barley varieties. Barley identification numbers with the letter B refer to Portuguese barleys; barley

identification numbers with the letter W refer to European winter barleys; barley identification numbers with the letters CS refer to European spring barleys; and barley identification numbers with the letters IB refer to Iberian spring barleys.

1252 RIBEIRO ET AL.

subjected to pelleting, where temperatures can reach temperatures of 80°C. However, it is also expected that a considerable degree of enzyme substrate protection exists, which contributes to enhance the stability of the endogenous enzyme (Fontes et al., 1995).

The effects of the different dietary treatments on the relative length or weight of different organs and GI tract compartments of broiler chickens of experiment 3 were evaluated; the data are presented in Table 9. The data revealed lower relative lengths and weights for the duodenum, jejunum, and ileum of birds receiving the HA diet when compared with birds receiving the LA treatment. In addition, supplementation of the LA diet with β-glucanase activity reduced organ sizes. As dis-cussed above, these data are in agreement with results reported by several other authors who have shown that

exogenous enzymes decrease digestive viscosity affect-ing the digestive tract weight, or length, or both, when expressed as a percentage of live weight (Brenes et al., 1993; Peterson et al., 1993; Viveros et al., 1994). Diges-ta viscosity in the hindgut and foregut of birds fed the different dietary treatments was measured; the data are presented in Table 9. Duodenum and jejunum viscosi-ties were higher in digesta samples from birds receiving the LA diet not supplemented with exogenous enzymes when compared with the 3 other groups. Therefore, the data suggest that endogenous and exogenous enzymes have a similar capacity to reduce the degree of polym-erization of soluble glucans, thus contributing to a con-siderable reduction in digesta viscosity. Because only 1 animal/pen was slaughtered for sample collection, as a result of logistic limitations, these values and those

Table 7. Chemical composition of the 2 barleys used in experiment 31

Chemical composition (% unless noted) Barley HA (B13) Barley LA (B23)

DM 11.65 13.66Crude energy (MJ/kg of DM) 16.30 16.05CP 9.15 9.00Ash 2.32 1.90NDF 25.73 20.35ADF 4.99 5.70Acid detergent lignin 1.56 1.57

1Barley HA = barley with high β-glucanase activity. Barley LA = barley with low β-glucanase activity. See Table 6 for explanation of B13 and B23.

Table 8. Performance of broilers fed 2 barley-based diets supplemented or not with a commercial β-glucanase preparation (data from experiment 3)

Item

Treatment1

SEM p(F)2HA− HA+ LA− LA+

BW (g) 0 d 48.06 47.72 47.71 48.19 0.248 0.42266 7 d 154.46ab 159.71a 131.19c 144.21b 4.934 0.00130 14 d 404.16a 421.30a 293.93c 361.25b 11.457 <0.0001 21 d 784.67a 811.52a 576.74c 693.33b 21.923 <0.0001 28 d 1,323.00a 1,304.57a 992.65c 1,163.93b 30.847 <0.0001Weight gain (g) 0–7 d 106.38ab 111.93b 83.95c 96.14ac 5.006 0.0019 7–14 d 249.68a 261.16a 162.29b 217.27c 7.735 <0.0001 14–21 d 378.88c 390.24c 282.85a 332.08b 12.826 <0.0001 21–28 d 533.58c 493.08c 415.92a 470.62b 14.134 <0.0001 0–28 d 1,274.86c 1,256.85c 944.95a 1,115.86b 30.922 <0.0001Feed intake (g) 0–7 d 141.74 144.29 152.95 132.9 10.202 0.5854 7–14 d 412.14 394.43 348.51 373.27 24.524 0.3051 14–21 d 721.13b 681.82ab 596.38a 662.38ab 37.420 0.1406 21–28 d 986.53 925.78 854.71 867.86 55.615 0.3319 0–28 d 1,957.88 1,888.00 1,952.54 1,875.27 79.271 0.8307Feed conversion ratio 0–7 d 1.27a 1.25a 1.83b 1.40a 0.063 <0.0001 7–14 d 1.50a 1.47a 2.23b 1.72a 0.095 <0.0001 14–21 d 1.75a 1.70a 2.15b 2.01b 0.086 0.0016 21–28 d 1.69a 1.82a 2.08b 1.86ab 0.090 0.0333 0–28 d 1.67a 1.66a 2.09b 1.84a 0.068 0.0002

a–cMeans within the same row with different superscripts are significantly different (P < 0.05).1HA−: barley-based diet displaying high endogenous β-glucanase activity not supplemented with an enzymatic commercial mixture of cellulases;

HA+: barley-based diet displaying high endogenous β-glucanase activity supplemented with an enzymatic commercial mixture of cellulases; LA−: barley-based diet displaying low endogenous β-glucanase activity not supplemented with an enzymatic commercial mixture of cellulases; LA+: barley-based diet displaying low endogenous β-glucanase activity supplemented with an enzymatic commercial mixture of cellulases.

2p(F) < 0.05 is statistically significant.

1253ENDOGENOUS β-GLUCANASE AND FEED ENZYME EFFICACY

reported for experiments 1 and 2 should be viewed with some caution considering that variation among animals for the measured variables can be significantly high.

Various authors have suggested that a range of fac-tors such as animal age, microbial challenge, cereal genotype, cereal growing conditions, or cereal storage length (Fuente et al., 1998; Svihus and Gullord, 2002) may contribute to the reduction of effectiveness of ex-ogenous β-1,3–1,4-glucanases used to supplement poul-try diets. However, the molecular mechanisms underly-ing this lack of response, in a variety of circumstances, remain unknown. It is clear that if levels of β-glucans are smaller the resulting digesta viscosity will be lower and consequently enzymes have no margin to function (Campbell et al., 1989; Choct, 2006). In addition, it is known that cereal genotype, growing conditions, and storage length affect the levels of barley β-glucan. Data presented here revealed that barley may also express significant but variable levels of endogenous β-glucanases. The plant enzymes seem to be active un-der the conditions of the GI tract and thus contribute to a significant depolymerization of the antinutritive β-glucans. For example, plant β-glucanases of barley B13 were sufficient to decrease duodenal viscosity of nonsupplemented birds to the levels observed in ani-mals supplemented with the exogenous enzyme. Thus, endogenous enzymes may contribute to affect microbial enzyme effectiveness by reducing substrate availabil-ity to the exogenous enzymes. The role of endogenous β-glucanases in plant metabolism remains relatively unexplored. During germination of barley grains, the cell walls of the starchy endosperm are degraded by β-1,3–1,4-glucanases. This allows the amylases and proteases secreted from the surrounding aleurone or scutellar tissues to reach their starch and storage pro-tein substrates inside the endosperm cells (Fincher et al., 1986). The activation of β-glucanase activity most possibly contributes to improve the nutritive value of

soaked or germinated barley (Svihus et al., 1997). In these examples, the activity of the endogenous enzymes reduces the degree of polymerization of the antinutri-tive β-glucans, resulting in an improvement of the diet nutritive value. However, data presented here suggest that the basal levels of expression of barley cellulases and β-1,3–1,4-glucanases before the germination pro-cess starts may vary and can reach significant values in a range of barley lots.

The molecular structure of barley cellulases remains to be identified. In addition, the barley genome se-quence, which could reveal the complete set of plant cell wall hydrolase genes encoded by this cereal, is pres-ently unknown. However, work developed in the last 20 yr led to the cloning of 2 β-1,3–1,4-glucanase encoding genes (Fincher et al., 1986). In addition, 12 expressed sequence tags, which contain plant cell wall hydrolase genes, were identified in various plant tissues (data ob-tained from GenBank; http://www.ncbi.nlm.nih.gov/genbank/). Cellulase and β-1,3–1,4-glucanase activities are of utmost importance during the germination of cereal grains to allow the degradation of cell walls of the nonliving starchy endosperm (Fincher et al., 1986). However, levels of expression of genes encoding plant cell wall hydrolases in barley grains are presently un-known because most studies have been performed in germinating seeds. Therefore, more work is required to characterize the expression profiles of genes encoding plant cell wall-degrading enzymes in grains, particu-larly of β-1,3–1,4-glucanases and cellulases. Differences in the expression pattern of the different genes should be evaluated with cereal lots from different origins. The collected data may contribute to the development of novel targets for barley breed improvement. Clearly the development of barley varieties expressing higher levels of endogenous cellulase activity would contrib-ute to improve the nutritive value of the corresponding barley-based diets for monogastric animals. The devel-

Table 9. Relative weight and length of the gastrointestinal tract and viscosity of digesta samples of broilers fed 2 barley-based diets supplemented or not with exogenous β-glucanases (data from experiment 3)

Item

Treatment1

SEM p(F)2HA− HA+ LA− LA+

Relative weight (g/100 g of BW)             Crop 3.51 3.07 3.91 3.53 0.231 0.1062 Gizzard 15.98ab 17.09ac 14.71bcd 13.03d 0.866 0.0141Relative length (cm/kg of BW)              Duodenum 20.85ab 19.91a 27.16c 22.07b 0.749 <0.0001 Jejunum 55.04a 54.50a 68.82b 56.15a 2.302 0.0002 Ileum 55.34a 54.63a 75.28b 57.68a 2.266 <0.0001 Cecum 12.72a 12.62a 16.01b 13.90a 0.530 0.0002Content viscosity (cP)             Duodenum + jejunum 3.463b 2.813b 8.612a 3.596b 0.437 <0.0001 Ileum 6.509b 7.836b 4.998b 12.678a 0.853 0.0019

a–cMeans within the same row with different superscripts are significantly different (P < 0.05).1HA−: barley-based diet displaying high endogenous β-glucanase activity not supplemented with an enzymatic commercial mixture of cellulases;

HA+: barley-based diet displaying high endogenous β-glucanase activity supplemented with an enzymatic commercial mixture of cellulases; LA−: barley-based diet displaying low endogenous β-glucanase activity not supplemented with an enzymatic commercial mixture of cellulases; LA+: barley-based diet displaying low endogenous β-glucanase activity supplemented with an enzymatic commercial mixture of cellulases.

2p(F) < 0.05 is statistically significant.

1254 RIBEIRO ET AL.

opment of novel barley varieties with increased β-1,3–1,4-glucanase activity could eventually contribute to preclude the requirement for feed supplementation of barley-based diets in monogastric nutrition.

ConclusionsData presented here revealed that endogenous

β-glucanases may be present in moderate levels in bar-ley seeds and may remain active throughout the GI tract, thus contributing to reduce digesta viscosity and consequently the effectiveness of exogenous cellulases used to supplement poultry feed. In addition, the re-sults revealed that levels of endogenous β-glucanases vary largely between different barley lots, a variation that is more pronounced than the levels of β-glucans. Therefore, a variable response to enzyme supplementa-tion may result, among other factors, from the variable levels of β-glucanase activity present in barley grains. The implementation of a quick assay to detect the lev-els of both barley β-glucan and β-glucanase may help rationalize the supplementation of poultry feed with exogenous enzymes.

ACKNOWLEDGMENTS

We thank Sociedade Agrícola da Quinta da Freiria SA (Bombarral, Portugal) for supplying the 1-d-old broilers used in these experiments and Central de Cervejas (Vialonga, Portugal) for kindly donating the barley used in experiment 3. This work was supported by Fundação para a Ciência e a Tecnologia (Lisboa, Portugal; grants PTDC/CVT/69329/2006 and PTDC/CVT/103942/2008). Patrícia I. P. Ponte and Teresa Ribeiro were supported by Fundação para a Ciência e a Tecnologia through individual fellowships SFRH/BD/17969/2004 and SFRH/BD/32321/2006, respec-tively.

REFERENCESApajalahti, J., and M. R. Bedford. 1999. Improve bird performance

by feeding its microflora. World’s Poult. Sci. J. 15:20–23.AOAC. 1980. Official Methods of Analysis. 3rd ed. Association of

Analytical Chemists, Washington, DC.Bedford, M. R. 2000. Exogenous enzymes in monogastric nutrition—

Their current value and future benefits. Anim. Feed Sci. Tech-nol. 86:1–13.

Bedford, M. R., G. L. Campbell, and H. L. Classen. 1991. The ef-fect of pelleting, salt and pentosanase on the viscosity of intesti-nal contents and the performance of broiler fed rye. Poult. Sci. 70:1571–1577.

Bedford, M. R., and A. Classen. 1992. Reduction of intestinal vis-cosity through manipulation of dietary rye and pentosanase con-centration is effected throughout changes in carbohydrate com-position of the intestinal aqueous phase and results in improved growth rate and food conversion efficiency of broiler chicks. J. Nutr. 122:560–569.

Bedford, M. R., and A. J. Morgan. 1996. The use of enzymes in poultry diets. World’s Poult. Sci. J. 52:61–68.

Boraston, A. B., D. N. Bolam, H. J. Gilbert, and G. J. Davies. 2004. Carbohydrate-binding modules: Fine-tuning polysaccharide rec-ognition. Biochem. J. 382:769–781.

Brenes, A., W. Guener, and R. R. Marquardt. 1993. Effect of en-zyme supplementation on the performance and digestive tract size of broiler chickens fed wheat- and barley-based diets. Poult. Sci. 72:1731–1739.

Campbell, G. L., B. G. Rossnagel, H. L. Classen, and P. A. Thacker. 1989. Genotypic and environmental differences in extract vis-cosity of barley and their relationship to its nutritive value for broiler chickens. Anim. Feed Sci. Technol. 26:221–230.

Chesson, A. 1993. Feed enzymes. Anim. Feed Sci. Technol. 45:65–69.

Choct, M. 2006. Enzymes for the feed industry: Past, present and future. World’s Poult. Sci. J. 62:5–16.

Dänicke, S., H. Jeroch, W. Bottcher, and O. Simon. 2000. Inter-actions between dietary fat type and enzyme supplementation in broiler diets with high pentosan contents: Effects on precae-cal and total tract digestibility of fatty acids, metabolizability of gross energy, digesta viscosity and weights of small intestine. Anim. Feed Sci. Technol. 84:279–294.

European Union. 1986. Council Directive of 24 November 1986 on the approximation of laws, regulations and administrative provi-sions of the Member States regarding the protection of animals used for experimental and other scientific purposes (86/609/EEC). European Union, Brussels, Belgium.

Fengler, A. I., and R. R. Marquardt. 1988. Water-soluble arabinox-ylans from rye. II. Effects of rate of dialysis and on the retention of nutrient by the chick. Cereal Chem. 65:298–302.

Fincher, G. B., P. A. Lock, M. M. Morgan, K. Lingelbach, R. E. Wettenhall, J. F. Mercer, A. Brandt, and K. K. Thomsen. 1986. Primary structure of the (1–3,1–4)-β-d-glucan 4-glucohydrolase from barley aleurone. Proc. Natl. Acad. Sci. USA 83:2081–2085.

Fontes, C. M. G. A., J. H. Clarke, G. P. Hazlewood, T. H. Fer-nandes, H. J. Gilbert, and L. M. A. Ferreira. 1997. Possible roles for a non-modular, thermostable and proteinase-resistant cellu-lase from the mesophilic aerobic soil bacterium Cellvibrio mixtus. Appl. Microbiol. Biotechnol. 48:473–479.

Fontes, C. M. G. A., C. M. Fontes, J. Hall, B. H. Hirst, G. P. Hazle-wood, and H. J. Gilbert. 1995. The resistance of cellulases and xylanases to proteolytic inactivation. Appl. Microbiol. Biotech-nol. 43:52–57.

Fontes, C. M. G. A., H. J. Gilbert, G. P. Hazelwood, J. H. Clarke, J. A. M. Prates, V. A. McKie, T. Nagy, T. H. Fernandes, and L. M. A. Ferreira. 2000. A novel Cellvibrio mixtus family 10 xyla-nase that is both intracellular and expressed under non-inducing conditions. Microbiology 146:1959–1967.

Fontes, C. M. G. A., P. I. P. Ponte, T. C. Reis, M. C. Soares, L. T. Gama, F. M. V. Dias, and L. M. A. Ferreira. 2004. A family 6 carbohydrate-binding module potentiates the efficiency of a recombinant xylanase used to supplement cereal-based diets for poultry. Br. Poult. Sci. 45:648–656.

Fuente, J. M., P. Perez de Ayala, A. Flores, and M. J. Villamide. 1998. Effect of storage time and dietary enzyme on the metabo-lizable energy and digesta viscosity of barley-based diets for poul-try. Poult. Sci. 77:90–97.

Guerreiro, C. I. P. D., T. Ribeiro, P. I. P. Ponte, M. M. S. Lordelo, L. Falcão, J. P. B. Freire, L. M. A. Ferreira, J. A. M. Prates, and C. M. G. A. Fontes. 2008. Probing the role of a family 11 car-bohydrate binding module (CBM) in the function of a recombi-nant cellulase used to supplement a barley-based diet for broiler chicken. Br. Poult. Sci. 49:446–454.

Hesselman, K., and P. Aman. 1986. The effect of β-glucanase on the utilisation of starch and nitrogen by broiler chickens fed on barley of low and high viscosity. Anim. Feed Sci. Technol. 15:83–93.

Mourão, J. L., P. I. P. Ponte, J. A. M. Prates, M. S. J. Centeno, L. M. A. Ferreira, M. A. C. Soares, and C. M. G. A. Fontes. 2006. Use of β-glucanases and β-1,4-xylanases to supplement diets con-taining alfalfa and rye for laying hens: Effects on bird perfor-mance and egg quality. J. Appl. Poult. Res. 15:256–265.

National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Nat. Acad. Press, Washington, DC.

Petterson, D., and P. Aman. 1989. Enzyme supplementation of a poultry diet containing rye and wheat. Brit. J. Nutr. 62:139–149.

Petersen, S. T., J. Wiseman, and M. R. Bedford. 1993. The effect of age and diet on the viscosity of intestinal contents in broiler chicks. Proc. Br. Soc. Anim. Prod. 56:434.

1255ENDOGENOUS β-GLUCANASE AND FEED ENZYME EFFICACY

Philip, J. S., H. J. Gilbert, and R. R. Smithard. 1995. Growth, viscosity and β-glucanase activity of intestinal fluid in broiler chickens fed on barley-based diets with or without exogenous β-glucanase. Br. Poult. Sci. 36:599–603.

Ponte, P. I. P., L. M. A. Ferreira, M. A. C. Soares, L. T. Gama, and C. M. G. A. Fontes. 2004. Xylanase inhibitors affect the action of exogenous enzymes used to supplement Triticum durum-based diets for broiler chicks. J. Appl. Poult. Res. 13:660–666.

Ribeiro, T., P. I. P. Ponte, C. I. P. D. Guerreiro, H. M. Santos, L. Falcão, J. P. B. Freire, L. M. A. Ferreira, J. A. M. Prates, C. M. G. A. Fontes, and M. M. Lordelo. 2008. A family 11 carbohydrate binding module (CBM) improves the efficacy of a recombinant cellulase used to supplement a barley based diet for broiler chick-ens. Br. Poult. Sci. 49:600–608.

Svihus, B., and M. Gullord. 2002. Effect of chemical content and physical characteristics on nutritional value of wheat, barley and oats for poultry. Anim. Feed Sci. Technol. 102:71–92.

Svihus, B., R. K. Newman, and C. W. Newman. 1997. Effect of soaking, germination, and enzyme treatment of whole barley on

nutritional value and digestive tract parameters of broiler chick-ens. Br. Poult. Sci. 38:390–396.

van der Klis, J. D., A. Van Voorst, and C. Van Cruyningen. 1993. Effect of a soluble polysaccharide (carboxymethyl cellulose) on the physico-chemical conditions in the gastrointestinal tract of broilers. Br. Poult. Sci. 34:971–983.

Villamide, M. J., J. M. Fuente, P. Perez de Ayala, and A. Flores. 1997. Energy evaluation of eight barley cultivars for poultry: Ef-fect of dietary enzyme addition. Poult. Sci. 76:834–840.

Viveros, A., A. Brenes, M. Pizarro, and M. Castaño. 1994. Effect of enzyme supplementation of a diet based on barley, and auto-clave treatment, on apparent digestibility, growth performance and gut morphology of broiler diets. Anim. Feed Sci. Technol. 48:237–251.

White, W. B., H. R. Bird, M. L. Sunde, W. C. Burger, and J. A. Marlett. 1981. The viscosity interaction of barley β-glucan with Trichoderma viride cellulose in the chick intestine. Poult. Sci. 60:1043–1048.

1256 RIBEIRO ET AL.