Variation in levels of non-starch polysaccharides and endogenous endo-1,4-ß-xylanases affects the nutritive value of wheat for poultry.

1. Endo-1,4-ß-xylanase is known to improve the nutritive value of wheat-based diets for poultry by degrading dietary arabinoxylans. However, broilers' response to supplementation of wheat-based diets with exogenous endo-1,4-ß-xylanase is not always observed. 2. In this study, 108 different wheat lots were analysed for levels of extract viscosity as well as for endogenous endo-1,4-ß-xylanase activity, and the impact of these two variables in animal performance was tested. 3. Results revealed that endogenous endo-1,4-ß-xylanase activity and extract viscosity content varied widely among different wheat lots. Thus, a trial was conducted to evaluate the efficacy of exogenous enzyme supplementation in broiler diets using wheats with different levels of extract viscosity and endogenous endo-1,4-ß-xylanase activity. 4. The data revealed that exogenous enzyme supplementation was only effective when the wheat present in the diet had high levels of extract viscosity (14.8 cP) with low endogenous endo-1,4-ß-xylanase activity (347.0 U/kg). Nevertheless, it is apparent that exogenous microbial xylanases reduce digesta extract viscosity and feed conversion ratio independently of the endogenous properties presented by different wheat lots. 5. The data suggest that extract viscosity and/or endogenous endo-1,4-ß-xylanase activity affect the response to enzyme supplementation by poultry fed on wheat-based diets.

The prion-related protein (testis-specific) gene (PRNT) is highly polymorphic in Portuguese sheep.

The objective of this study was to search for polymorphisms in the ovine prion-related protein (testis-specific) gene (PRNT). Sampling included 567 sheep from eight Portuguese breeds. The PRNT gene-coding region was analyzed by single-strand conformation polymorphism and sequencing, allowing the identification of the first ovine PRNT polymorphisms, in codons 6, 38, 43 and 48: c.17C>T (p.Ser6Phe, which disrupts a consensus arginine-X-X-serine/threonine motif); c.112G>C (p.Gly38>Arg); c.129T>C and c.144A>G (synonymous) respectively. Polymorphisms in codons 6, 38 and 48 occur simultaneously in 50.6% of the animals, 38.8% presenting as heterozygous. To study the distribution of the polymorphism in codon 43, a restriction fragment length polymorphism analysis was performed. Polymorphic variant c.129C, identified in 89.8% of the animals with 32.8% presented as heterozygous, was considered the wild genotype in Portuguese sheep. Eight different haplotypes which have comparable distribution in all breeds were identified for the PRNT gene. In conclusion, the PRNT coding region is highly polymorphic in sheep, unlike the prion protein 2 dublet gene (PRND), in which we previously found only one synonymous substitution (c.78G>A), in codon 26. The absence or reduced number of PRND heterozygotes (c.78G>A) was significantly associated with three PRNT haplotypes (17C-112G-129T-144A,17CT-112GC-129CT-144AG and 17T-112C-129C-144G), and the only three animals found homozygous at c.78A had the 17C-112G-129C-144A PRNT haplotype. These results constitute evidence of an association between polymorphic variation in PRND and PRNT genes, as has already been observed for PRND and prion protein gene (PRNP).

Effect of reduced dietary protein and supplementation with a docosahexaenoic acid product on broiler performance and meat quality.

1. Chicken breast meat is a lean meat due to its low content of intramuscular fat (IMF) resulting in an overall lower acceptability by consumers due to a decrease in juiciness, flavour and increased chewiness. Recently, studies performed in pigs suggested the possibility of increasing IMF by decreasing dietary crude protein (CP) content, an effect possibly mediated through an increased lipogenesis. 2. Dietary supplementation with lipids rich in omega 3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) may modulate an increase in the content of these fatty acids in meat from monogastric animals and, thus, promote the daily intake of n-3 LC-PUFA by humans. 3. LC-PUFA are very susceptible to oxidation, resulting in off-flavours that affect meat quality and consumers' acceptability. 4. This trial was conducted to assess the effect of reducing dietary CP, from 21% to 17%, on chicken's meat IMF content and, simultaneously, to evaluate if a complementary supplementation with a proprietary n-3 LC-PUFA source (DHA Gold™) could improve meat quality. These effects were assessed by measuring productive performance and meat quality, oxidative stability, sensory traits and fatty acid profile. 5. A reduction in CP content of broiler diets, from 21% to 17%, balanced for lysine, improved performance while it was not sufficient to increase IMF content in chicken meat. In contrast, DHA Gold™ supplementation had a positive impact both in broiler productive parameters and in meat fatty acid profile. 6. In addition, incorporation of 7.4% of DHA Gold™ in the diet promoted carcass yield but negatively affected chicken meat acceptability by consumers, due to a decrease of meat oxidative stability. 7. Overall the data suggest that neither a dietary supplementation with DHA Gold™ nor a reduction in CP have a direct positive effect in the levels of IMF present in broiler meat.

Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-Arabinofuranosidase.

The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) from Clostridium thermocellum, cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (Ka of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (Ka of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant Ka of 230 M(-1) and binding capacity (N0) of 11 µmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca2+, indicating that Ca2+ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic ß-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.

Direct supplementation of diet is the most efficient way of enriching broiler meat with n-3 long-chain polyunsaturated fatty acids.

1. Concentrations of beneficial omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) in poultry meat can be improved by increasing the concentration of n-3 PUFA in poultry diets. 2. A decrease in flavour quality is, however, usually associated with the dietary supplementation with n-3 PUFA, which is due to the susceptibility of PUFA to oxidation. 3. This experiment was conducted to study the effects of introducing two different n-3 fatty acid sources (extruded linseed and DHA Gold™, a proprietary algal product rich in docosahexaenoic acid), either separately or together, on broiler productive performance, and meat quality, oxidative stability, sensory traits and LC-PUFA profile. 4. Birds given the algal product displayed better productive performances than animals from other groups. 5. The data revealed an improvement in the fatty acid nutritional value of meat from birds receiving the algal product and an inefficient conversion of α-linolenic acid (LNA) into LC-PUFA. 6. Metabolisation of LNA in vivo is not sufficient to improve meat quality in n-3 LC-PUFA and direct supplementation of the diet with n-3 LC-PUFA is a better alternative to modulate an increase in beneficial fatty acids of broiler meat. 7. The overall acceptability of meat was negatively affected by the dietary supplementation with 7.4% of DHA, in contrast to the supplementation with 3.7% of DHA, which showed to be efficient in improving LC-PUFA meat content without affecting its sensory properties.

The prion-like protein Doppel enhances ovine spermatozoa fertilizing ability.

The function of prion-like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post-thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim-up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post-swim-up spermatozoa incubated as above was tested through heterologous fertilization of bovine in vitro matured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhance in vitro spermatozoa fertilizing ability.

The thermostable ß-1,3-1,4-glucanase from Clostridium thermocellum improves the nutritive value of highly viscous barley-based diets for broilers.

1. Microbial ß-1,3-1,4-glucanases improve the nutritive value of barley-based diets for poultry by effectively decreasing the degree of polymerisation of the anti-nutritive soluble ß-glucans. Glycoside hydrolases (GHs) acting on recalcitrant polysaccharides display a modular architecture comprising a catalytic domain linked to one or more non-catalytic Carbohydrate-Binding Modules (CBMs). 2. GHs and CBMs have been classified in different families based on primary structure similarity (see CAZy webpage at http://www.cazy.org ). The role of CBMs is to anchor the appended GHs into their target substrates, therefore eliciting the efficient hydrolysis of structural carbohydrates. 3. Here we describe the biochemical properties of the family 16 GH from Clostridium thermocellum, termed CtGlc16A. CtGlc16A is a thermostable enzyme that specifically acts on ß-1,3-1,4-glucans with a remarkable catalytic activity (38000 U/mg protein). 4. CtGlc16A, individually or fused to the family 11 ß-glucan-binding domain of cellulase CtLic26A-Cel5E of C. thermocellum, was used to supplement a highly viscous barley-based diet for broilers. 5. The data showed that birds fed on diets supplemented with the recombinant enzymes displayed an improved performance when compared with birds given diets not supplemented with exogenous enzymes. However, inclusion of the non-catalytic CBMs had no influence on the capacity of CtGlc16A to reduce the anti-nutritive effects of soluble ß-1,3-1,4-glucans. 6. The data suggest that at elevated dosage rates, CBMs might be unable to potentiate the catalytic activity of appended catalytic domains; this effect may only be revealed when feed enzymes are incorporated at low levels.

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

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 ß-glucan specific family 11 carbohydrate-binding module. The data revealed that the recombinant ß-glucanase derivatives were not appropriate for feed supplementation because of a lack of stability at acidic pH levels. However, under 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 levels of endogenous ß-glucanase activity. These differences 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 exogenous ß-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 ß-glucanases but were unable to improve the nutritive value of the barley lot displaying higher ß-glucanase activity. Thus, these studies suggest that levels of endogenous ß-glucanases may affect the efficacy of exogenous enzymes used to improve the nutritive value of barley-based diets for broilers. The development of a quick ß-glucanase assay that could be applied for cereal-based feeds may help identify those barley-based diets that are more responsive to the action of feed enzymes.

Influence of dietary Chlorella vulgaris and carbohydrate-active enzymes on growth performance, meat quality and lipid composition of broiler chickens.

Herein, we investigated the effect of Chlorella vulgaris as ingredient (10% of incorporation) in broiler diets, supplemented or not with 2 formulations of Carbohydrate-Active enZymes (CAZymes; Rovabio Excel AP and a mixture of recombinant CAZymes, composed by an exo-ß-glucosaminidase, an alginate lyase, a peptidoglycan N-acetylmuramic acid deacetylase and a lysozyme), on growth performance, meat quality, fatty acid composition, oxidative stability, and sensory traits. One hundred twenty 1-day-old Ross 308 male birds were randomly assigned to one of the 4 experimental diets (n = 30): corn-soybean meal-basal diet (control), basal diet with 10% C. vulgaris (CV), CV supplemented with 0.005% of a commercial CAZyme cocktail (Rovabio Excel AP), (CV + R), and CV supplemented with 0.01% of a 4-CAZyme mixture previously selected (CV + M) during the experimental period lasted from day 21 to day 35. Body weight gain and feed conversion rate of broilers were not affected by C. vulgaris but digesta viscosity increased more than 2-fold (P < 0.001) relative to the control. In addition, neither cooking loss, shear force, juiciness, flavor nor off-flavor was impaired by dietary treatments (P > 0.05). By contrast, the dietary C. vulgaris increased tenderness, yellowness (b∗) and total carotenoids in breast and thigh meats. However, no additional protective effect against lipid oxidation was observed in meat with the inclusion of microalga. Chlorella vulgaris, independently of CAZymes, had a minor impact on meat fatty acid composition but improved the proportion of some beneficial fatty acids. In summary, our data indicate a slight improvement of broiler meat quality and lipid nutritional value, without impairment of broilers' growth performance, thus supporting the usefulness of this microalga in poultry diets, up to this high level of incorporation. By contrast, the selected CAZyme mixtures used do not significantly improve the release of microalga nutrients in poultry diets, through the disruption of microalga cell wall, which warrants further research.

Impact of dietary incorporation of Spirulina (Arthrospira platensis) and exogenous enzymes on broiler performance, carcass traits, and meat quality.

This study assessed the effect of Spirulina (Arthrospira platensis), individually and in combination with exogenous enzymes, on growth performance, carcass traits, and meat quality of broiler chickens. One hundred and twenty Ross 308 male chickens were allocated into 40 battery brooders, with 3 birds per cage, and fed ad libitum a corn-based diet during the first 21 D of the trial. The experimental period lasted from day 21 to 35, during which birds were fed 4 different diets: a corn-soybean basal diet, taken as the control group, a basal diet containing 15% Spirulina (MA), a basal diet containing 15% Spirulina plus 0.005% Rovabio Excel AP (MAR), and a basal diet containing 15% Spirulina plus 0.01% lysozyme (MAL). Body weight gain (P < 0.001) and feed conversion rate (P < 0.001) were improved in control chickens, when compared with those fed with Spirulina. In addition, Spirulina increased the length of duodenum plus jejunum in relation to the other treatment (P < 0.01). Chickens on the MAL diet showed a considerable increase in digesta viscosity (P < 0.05) compared with the control group. Breast and thigh meats from chickens fed with Spirulina, with or without the addition of exogenous enzymes, had higher values of yellowness (b*) (P < 0.001), total carotenoids (P < 0.001), and saturated fatty acids (P < 0.001), whereas n-3 polyunsaturated fatty acid (P < 0.01) and α-tocopherol (P < 0.001) decreased, when compared with the control. In conclusion, the incorporation of 15% Spirulina in broiler diets, individually or combined with exogenous enzymes, reduced birds' performance through a higher digesta viscosity, which is likely associated with the gelation of microalga indigestible proteins. In addition, cell wall of Spirulina was successfully broken by the addition of lysozyme, but not by Rovabio Excel AP. Therefore, we anticipate that the combination of lysozyme with an exogenous specific peptidase could improve the digestibility of proteins from this microalga and avoid their detrimental gelation.

Effect of dietary dehydrated pasture and citrus pulp on the performance and meat quality of broiler chickens.

Some feedstuffs containing significant levels of fiber may be a good source of bioactive compounds that may contribute to improving broiler meat quality. However, high fiber level can have a negative impact on broiler performance. A study was undertaken to investigate the impact of incorporating citrus pulp (5 or 10%) or dehydrated pasture (5 or 10%) on the performance, carcass yield, and characteristics of broiler chickens. A diet containing neither citrus pulp nor dehydrated pasture was used as control. The results on growth performances showed that daily weight gain was reduced by 26% in birds of the 10% citrus pulp treatment (P<0.05). Compared with the control treatment, increases in feed intake occurred in birds consuming diets with 5 or 10% citrus pulp, which resulted in significantly higher feed conversion rates with the 10% level. Under the same incorporation rate, dehydrated pasture had effects less evident on the performances of broiler chicken. In addition, diets containing citrus pulp, displaying higher percentages of soluble nonstarch polysaccharides, increased small intestine relative length, and reduced carcass yield. Inclusion of 10% dehydrated pasture in diets resulted in improved breast skin yellowness (P<0.05). Finally, the results revealed that incorporation of the nonstarch polysaccharide-rich feedstuffs had a major impact on the fatty acid profile (affected 16 of 21 fatty acids) of broiler meat. Polyunsaturated fatty acids content in meat was higher in birds consuming the highest levels of both citrus pulp and dehydrated pasture, leading to increased ratios of polyunsaturated to saturated fatty acids. Together, the results suggest that incorporation of moderate levels of dehydrated pastures in poultry diets has a minor impact on broiler performance and can contribute significantly to improve breast skin yellowness and fatty acid composition of meat.

Influence of pasture intake on the fatty acid composition, and cholesterol, tocopherols, and tocotrienols content in meat from free-range broilers.

Over the last centuries, Western diets acquired a dramatic imbalance in the ratio of polyunsaturated fatty acids (PUFA) to saturated fatty acids (SFA) with a concomitant reduction in the dietary proportion of n-3 PUFA. Pastures are a good source of n-3 fatty acids, although the effect of forage intake in the fatty acid profile of meat from free-range chicken remains to be evaluated. In addition, it is unknown if consumer interest in specialty poultry products derived from free-range or organic production systems is accompanied by a greater nutritional quality of these products. In this study, broilers of the RedBro Cou Nu x RedBro M genotype were fed on a cereal-based diet in portable floorless pens located either on subterranean clover (Trifolium subterraneum) or white clover (Trifolium repens) pastures. Control birds were maintained at the same site in identical pens but had no access to pasture. The capacity of ingested forage to modulate broiler meat fatty acid profiles and the meat content of total cholesterol, tocopherols, and tocotrienols was investigated in broiler chicks slaughtered at d 56. The results suggested that pasture intake (<5% DM) had a low impact on the fatty acid and vitamin E homologue profiles of meat from free-range broilers. However, breast meat from birds with free access to pasture presented lower levels of the n-6 and n-3 fatty acid precursors linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3), respectively. In spring the levels of eicosapentaenoic acid (20:5n-3) in breast meat were significantly greater in birds consuming pastures, which suggests greater conversion of alpha-linolenic acid into eicosapentaenoic acid in these birds. Finally, when compared with meat from slower-growing genotypes obtained under the conventional European free-range production systems with slaughtering at d 81, meat from birds of the Ross genotype raised intensively and slaughtered at d 35 seemed to have greater nutritional quality.

Improving the lipid nutritive value of poultry meat through the incorporation of a dehydrated leguminous-based forage in the diet for broiler chicks.

Dehydrated forages are assumed to be good sources of alpha-linolenic acid (ALA) and lipid-soluble antioxidant compounds (vitamin E homologs and beta-carotene). The effects of including a dehydrated leguminous-based forage in a typical diet for broiler chicken, on performance, meat quality, and fatty acid composition were evaluated. One hundred sixty 1-d-old male commercial broiler chicks (Ross 308) were housed in 20 battery brooders. During the 28-d growth period, the animals were fed ad libitum with a typical maize-soybean high-energy feed having access or not to a dehydrated leguminous-based forage provided in a separate feeder. The results revealed that dehydrated forage intake (which was 11.1% of the total intake) had no impact in broiler performance (P > 0.05). The capacity of ingested forage to modulate broiler meat fatty acid profile and the meat content in total cholesterol, tocopherols, tocotrienols, and beta-carotene was investigated in broiler chicks slaughtered at d 28. Dehydrated forage consumption had no effect on the lipid-soluble antioxidant compounds and cholesterol contents of broiler meat but had a significant effect on meat fatty acid profile. Although forage intake did not affect the linoleic acid and ALA contents in poultry meat, the levels of n-3 long-chain polyunsaturated fatty acids [eicosapentaenoic (P = 0.004), docosapentaenoic (P = 0.010), and docosahexaenoic (P = 0.007)] in breast meat were significantly higher in animals consuming leguminous biomass, which suggest a higher conversion of ALA into its derivatives in these birds. Overall, the data confirms that incorporation of a dehydrated leguminous-based forage in the diet for broiler chicks results in more favorable polyunsaturated fatty acids/saturated fatty acids and n-6/n-3 nutritional ratios for animals slaughtered at earlier stages of grow.

Pasture intake improves the performance and meat sensory attributes of free-range broilers.

Free-range chickens are assumed to consume low to moderate levels of pasture, although the effects of forage intake in broiler performance and poultry meat quality remain to be established. In addition, despite cellulases and hemicellulases being widely used as feed supplements to improve the nutritive value of cereal-based diets for fast-growing broilers, the potential interest of these biocatalysts in the production of free-range chicken is yet to be established. In this study, broilers of the RedBro Cou Nu x RedBro M genotype were fed a cereal-based diet in portable floorless pens located either on a rainfed subterranean clover (Trifolium subterraneum) pasture or on an irrigated white clover (Trifolium repens) pasture. Control birds were maintained at the same site in identical pens but with no access to pastures. The importance of pasture intake and enzyme supplementation in the performance and meat sensory properties of the free-range chicken from d 28 to 56 was investigated. The results revealed that although cellulase and hemicellulase supplementation had no impact on broiler performance (P > 0.05), birds foraging on legume-based pastures reached significantly greater final BW. The data suggest that the improvement in broiler performance results from increased intake of the cereal-based feed rather than from an improvement in the efficiency of nutrient utilization per se. Interestingly, although the intake of the subterranean clover pasture had no impact on the tenderness, juiciness, and flavor of broiler meat, members of a 30-person consumer panel classified the meat from grazing broilers with greater scores for overall appreciation. Together, the results suggest that pasture intake promotes bird performance while contributing to the production of broiler meat with preferred sensory attributes.

Restricting the intake of a cereal-based feed in free-range-pastured poultry: effects on performance and meat quality.

Pastures are assumed to be good sources of alpha-linolenic acid (ALA) and other bioactive compounds. In this study, we evaluated the effects of restricting the intake of a cereal-based feed on the consumption of a legume-based pasture, and consequently on poultry performance and meat quality. Broilers of the RedBro Cou Nu x RedBro M genotype were fed a cereal-based feed at different intake restriction levels (100, 75, or 50% of ad libitum intake) in portable floorless pens located on a subterranean clover (Trifolium subterraneum) pasture. Control birds were maintained at the same site in identical pens but had no access to pasture. The results revealed that, although the growth rate achieved was below the levels expected for the genotype, restriction of cereal-based feed intake had a significant impact on broiler weight gain and feed conversion while leading to an increase in relative leguminous pasture intake (from 1.6 to 4.9% of the total intake, on a DM basis). In addition, bird performance was positively influenced by pasture consumption. The capacity of ingested pasture to modulate carcass characteristics, broiler meat fatty acid profiles, and the meat content of total cholesterol, tocopherols, and to-cotrienols was investigated in broiler chickens slaughtered on d 64. Pasture intake decreased carcass yield (P < 0.05) and meat pH (P < 0.001) and improved breast skin pigmentation (P < 0.001). Consumption of the leguminous pasture had a marginal effect in the vitamin E profiles and cholesterol contents of broiler meat (P < 0.05), although it significantly affected the meat fatty acid profile. Although pasture intake did not influence the linoleic acid content of poultry meat, the levels of n-3 polyunsaturated fatty acids in breast meat [ALA (P < 0.001), eicosapentaenoic acid (P < 0.001), docosapentaenoic acid (P < 0.001), and docosahexaenoic acid (P < 0.001)] were significantly greater in birds consuming the leguminous biomass. Overall, the data suggest an important deposition of ALA and some conversion of ALA to its derivatives in pastured broilers subjected to a restriction of cereal-based feed.

Role of a family 11 carbohydrate-binding module in the function of a recombinant cellulase used to supplement a barley-based diet for broiler chickens.

1. Cellulases and xylanases display a modular architecture that comprises a catalytic module linked to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs have been classified into 52 different families, based on primary structure similarity. These non-catalytic modules mediate a prolonged and intimate contact of the enzyme with the target substrate eliciting efficient hydrolysis of the target polysaccharides. 2. A study was undertaken to investigate the importance of a family 11 CBM, displaying high affinities for barley beta-glucans, in the function of recombinant derivatives of cellulase CtLic26A-Cel5E of Clostridium thermocellum used to supplement a barley-based diet for broiler chicken. 3. The results showed that birds fed on diets containing the recombinant CtLic26A-Cel5E modular derivatives or the commercial enzyme mixture Rovabio Excel AP displayed improved performance when compared with birds fed on diets not supplemented with exogenous enzymes. 4. It is suggested that the enzyme dosage used in this study (30 U/kg of basal diet), was probably too high for the efficacy of the family 11 CBM to be noticed. It remains to be established if the targeting effect resulting from the incorporation of CBMs in plant cell wall hydrolases may be effective at lower exogenous enzyme dosages.

Crop beta-glucanase activity limits the effectiveness of a recombinant cellulase used to supplement a barley-based feed for free-range broilers.

1. The supplementation of diets rich in soluble polysaccharides with microbial cellulases and hemicellulases decreases digesta viscosity and promotes broiler performance. 2. In contrast, recent experiments suggest that polysaccharidases are ineffective for improving the nutritive value of pasture biomass used by free-range broilers. However, the feasibility of using cellulases and hemicellulases to improve the utilisation of cereal-based feeds by pastured poultry remains to be established. 3. A study was undertaken to investigate the capacity of a recombinant cellulase from Clostridium thermocellum to improve the nutritive value of a barley-based feed for free-range pastured broilers of the RedBro Cou Nu x RedBro M genotype. 4. The results show that supplementation of a barley-based diet with a recombinant beta-glucanase had no effect on the performance of free-range broilers, foraging in legume-based diets from d 28 to 56. In addition, the results confirm that the lack of effect of the recombinant enzyme in improving the nutritive value of the barley-based feed does not result from enzyme proteolysis or inhibition in the gastrointestinal tract. 5. Significantly, beta-glucanase activity was identified in the crop of non-supplemented animals. The data suggest that endogenous cellulases originated both from the barley-based feed and from the crop microflora. 6. The results presented here suggest that in older birds of slow-growing genotypes associated with free-range production systems, previously unknown sources of beta-glucanases, such as the feed and microbial symbiotic microflora, can affect the effectiveness of exogenous enzymes added to the feed.

A family 11 carbohydrate-binding module (CBM) improves the efficacy of a recombinant cellulase used to supplement barley-based diets for broilers at lower dosage rates.

1. Exogenous microbial beta-1,3-1,4-glucanases and hemicellulases contribute to improving the nutritive value of cereals rich in soluble non-starch polysaccharides for poultry. 2. In general, plant cell wall hydrolases display a modular structure comprising a catalytic module linked to one or more non-catalytic carbohydrate-binding modules (CBMs). Based on primary structure similarity, CBMs have been classified in 50 different families. CBMs anchor cellulases and hemicellulases into their target substrates, therefore eliciting efficient hydrolysis of recalcitrant polysaccharides. 3. A study was undertaken to investigate the effects of a family 11 beta-glucan-binding domain in the function of recombinant derivatives of cellulase CtLic26A-Cel5E of Clostridium thermocellum that were used to supplement a barley-based diet at lower dosage rates. 4. The results showed that birds fed on diets supplemented with the recombinant CtLic26A-Cel5E modular derivative containing the family 11 CBM or the commercial enzyme mixture Rovabio Excel AP tended to display improved performance when compared to birds fed diets not supplemented with exogenous enzymes. 5. It is suggested that at lower than previously reported enzyme dosage (10 U/kg vs 30 U/kg of basal diet), the beta-glucan-binding domain also elicits the function of the recombinant CtLic26A-Cel5E derivatives. 6. Finally, the data suggest that exogenous enzymes added to barley-based diets act primarily in the proximal section of the gastrointestinal tract.

Xylo-oligosaccharides display a prebiotic activity when used to supplement wheat or corn-based diets for broilers.

It is now well established that exogenous ß-1,4-xylanases improve the nutritive value of wheat-based diets for poultry. Among other factors, the mechanism of action of exogenous enzymes may involve a microbial route resulting from the generation of prebiotic xylo-oligosaccharides (XOS) in the birds' gastro-intestinal (GI) tract. In a series of three experiments, the effect of XOS on the performance of broilers fed wheat or corn-based diets was investigated. In experiment 1, birds receiving diets supplemented with XOS displayed an increased weight gain (P = 0.08). The capacity of XOS to improve the performance of animals during a longer trial (42 d) was investigated (Experiment 2). The data revealed that diet supplementation with XOS, tested at two incorporation rates (0.1 and 1 g/kg), or with an exogenous ß-1,4-xylanase resulted in an increased nutritive value of the wheat-based diet. An improvement in animal performance was accompanied by a shift in the microbial populations colonizing the upper portions of the GI tract. XOS were also able to improve the performance of broilers fed a corn-based diet, although the effects were not apparent at incorporation rates of 10 g/kg. Together these studies suggest that in some cases the capacity of ß-1,4-xylanases to improve the nutritive value of wheat-based diets is more related to their ability to produce prebiotic XOS than to their ability to degrade arabinoxylans. The extremely low quantities of XOS used in this study also challenge the depiction of a prebiotic being a quantitatively fermented substrate. These data also bring into question the validity of the "cell wall" mechanism, as XOS elicited an effect with clearly no action on endosperm cell wall integrity and yet the performance effects noted were equivalent or superior to the added enzymes.

Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (CtCBM11) of cellulosomal bifunctional cellulase from Clostridium thermocellum.

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with ß-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.