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The objective of the research reported here was to establish the possibility of using cellulases and xylanases to improve the nutritive value of alfalfa for poultry. The influence oF introducing plant cell wall hydrolases in diets containing moderate to high levels of alfalfa on the growth and feed conversion rates of broiler chicks was assessed. Finally, we measured the impact of alfalfa consumption on the levels of pigmentation of broiler skin.

MATERIALS AND METHODS

Chickens and Husbandry

Two experiments were conducted to determine the effect ofcellulase and xylanase supplementation on the nutritional value of alfalra. Commercial broiler chick males (Ross 308) were housed in 20 battery brooders exposed to constant light for the duration of the trial. Each cage was provided with an individual feeder and 2 automatic pipette drinkers unless otherwise stated. Water was available for ad libitum consumption throughout the experiments. The brooders were located in a temperature-controlled room, which was adjusted daily to the recommended values, according to standard brooding practice. In both experiments, birds were individually weighed at the commencement of the experiment, marked, and assigned with an experimental number. Birds were divided into 20 experimental replicates, equalizing both the mean and the variance of BW and allocated randomly into the 20 cages. Peed consumption and individual BW were recorded weekly. Gain-to-feed ratios were calculated by dividing the weight gain per pen, including the weight gain of any dead birds, by the weight of feed consumed. At the end of the experiments, 5 birds from each treatment, 1 per cage, were killed by cervical dislocation, and digesta were collected from the various gastrointestinal compartments. The samples were frozen at -20°C for later analysis.

Experiment 1

The objective of this study was to examine the effects on broiler performance of supplementing diets containing 20% alfalfa with cellulases and xylanases, using animals between 35 to 63 d old. The 4 treatments under analysis consisted of a basal diet, containing 20% of alfalfa, not supplemented, or supplemented with the commercial cellulase-xylanase enzyme cocktail Roxazyme G, with a recombinant xylanase or with 2 recombinant enzymes, consisting of cellulase plus xylanase. The composition of the basal diet is displayed in Table 1, while enzyme activities presented on Roxazyme G and enzyme preparation are displayed elsewhere [16]. Five replicates of 4 birds were randomly assigned to each treatment in a total of 80 animals. During the first week of the experiment, all animals were fed the basal diet for adaptation (28 to 35 d old). Broilers were provided feed ad libitum on the experimental diets for 4 wk (35 to 63 d old).

Experiment 2

The main aim of this study was to evaluate the impact of polysaccharidase supplementation on the nutritive value of alfalfa for broilers until 8 wk of age. An extra feeder was introduced into the cages, since the birds were fed on 2 feeds: a high-energy commercial feed and dehydrated alfalfa. Consumption of the commercial feed was restricted to 50 and 75% of the estimated values, while alfalfa was offered ad libitum. Using this strategy, alfalfa consumption was stimulated. Therefore, the 4 treatments under analysis consisted of 2 levels of high-energy feed restriction (50 and 75%) and 2 levels of alfalfa polysaccharidase supplementation (0 and /kg of Roxazyme G). Five replicates of 5 birds, for a total of 100 birds, were randomly assigned to each of the 4 treatments, which were C50+0, high-energy feed restricted to 50% of the estimated values and alfalfa not supplemented with enzymes; C50+E, high-energy feed restricted to 50% of the estimated values and alfalfa supplemented with enzymes; C75+0, high-energy feed restricted to 75% of the estimated values and alfalfa not supplemented with enzymes; and C75+E, high-energy feed restricted to 75% of the estimated values and alfalfa supplemented with enzymes. The determined composition of the high-energy diets and alfalfa are displayed in Table 2. High-energy feed was distributed once per day. During the first week, all animals were provided both alfalfa and the high-energy feed for ad libitum consumption. At d 7 animals were individually weighed, marked, and assigned to each replicate as described previously. Experimental diets were given from 7 to 56 d. At the end of the experiment, 3 animals per cage were slaughtered at a commercial processing plant.

Analytical Procedures

Analyses for dry matter, ether extract, CP, and dietary fiber were performed according to the AOAC methods [17]. Cellulase and xylanase assays were performed with soluble xylan and barley [beta]-glucan, according to the methods described by Fontes et al. [20]. Analysis of cellulase and xylanase activity in situ was assessed, using the referred polysaccharides at % final concentration, in 10 mM Tris-HCl, pH , agar plates. Activity was detected after 16 h incubation at 37°C through the Congo red assay plate, as described by Ferreira et al. [21].

Skin Color Evaluation

The carcasses were refrigerated for 24 h, and color of breasts and backs was evaluated using a Minolta chroma meter [22]. The readings were taken on equivalent positions of the carcasses. For each reading 3 measurements were performed, and the final value for each animal is the average of those readings. Skin color was expressed in the CIELAB dimensions of lightness (L*), redness (a*), and yellowness (b*). Meat pH and water holding capacity were determined as described in Sierra [23]. After these measurements were taken, carcasses were frozen at -20°C until the consumer preference tests were performed.

Statistical Analyses

Statistical treatment of data related to bird performance and meat quality was conducted by ANOVA using SAS software [24] for data concerning consumer preferences using Statistical Package for the Social Sciences [25]. Unless otherwise stated, differences were considered significant when P

RESULTS AND DISCUSSION

In the first experiment, addition of cellulases and xylanases, from commercial and recombinant sources, did not result in significant improvements on daily weight gain and efficiency of feed use of poultry (Table 3). The statistical analysis demonstrated that the recombinant xylanase, individually, had a considerably negative impact on the final weight, daily weight gain, and feed intake, while no significant differences were apparent for feed conversion ratio (FCR). In addition, the recombinant xylanase, combined with the recombinant cellulase, had a similar impact in the performance of the broiler chicks when compared with the commercial enzyme mixture or the control diets. Although not significant, the nonsupplemented animals reached higher final weights and had higher daily weight gains when compared with the birds receiving the exogenous hydrolases. Feed consumption was lower for animals supplemented with the recombinant enzymes. These data suggest that birds receiving exogenous enzymes had lower daily growth rates (on average less 4 g/d) and that this was a consequence of poorer feed conversion efficiencies and of lower feed intakes, particularly in the animals receiving the recombinant xylanase. Digesta samples collected from the various gastrointestinal compartments were tested for cellulase and xylanase activity, using the Congo red plate assay. The data (not shown) demonstrated that low exogenous polysaccharidase activity was present in the crop and duodenum of birds fed on diets supplemented with enzymes. Under the same conditions, no enzyme was detected on the corresponding compartments of birds fed on the control no-enzyme diet. Chick mortality was low and was not related to treatment. However, in the final days of the experiments some animals were affected by mobility problems most probably due to their heavy weights. Taken together, these results suggest that at low levels polysaccharidases were functional in the gastrointestinal tract of the enzymesupplemented birds and that, although nonsignificant, the enzymes negatively affected the performance of broiler chicks.

In a second experiment, 1-d-old male broilers were used and kept under the experimental conditions until d 56. Considering results from experiment 1, which show that supplementation with recombinant enzymes had some negative impacts on broiler performance, only the commercially cellulase-xylanase mixture was used in this second experiment. In addition, in vitro enzyme assays against alfalfa NSP (not shown), and neutral detergent fiber preparations demonstrated that the commercial enzyme mixture was more effective in releasing reducing sugars from alfalfa than the recombinant enzymes (Figure 1). Alfalfa consumption was stimulated by restricting the intake of the high-energy commercial feed mixture to 50 and 75% of the dailyestimated values for these birds. The data, presented in Table 4, demonstrates that restricting the consumption of the high-energy feed led to a significant reduction on final BW and daily weight gains. Moreover, alfalfa consumption was increased (on average 250%) and FCR were deteriorated by points. The addition of the cellulase-xylanase enzyme mixture had no effect on the final BW and on the daily weight gains during the entire period of the experiment. Although there were no significant differences in total consumption of alfalfa among treatments with identical high energy feed restriction, it is apparent that during early periods of the experiment enzyme-supplemented birds had significantly lower levels of alfalfa intake (not shown). Resulting from this observation, the FCR of animals supplemented with plant cell wall hydrolases were consequently slightly better, during that period, than that of the animals fed on the basal diet. The differences, although not significant, were reflected in the final FCR of the animals from the 50% restriction in which higher consumption of alfalfa was observed. At the end of the experiment on d 56, digesta samples were collected from various gastrointestinal compartments of 5 animals of each treatment. The samples were assayed for cellulase and xylanase activity. The data, not shown, demonstrated that considerable levels of polysaccharidase activity could be detected in the crops and small intestine of the animals receiving exogenous enzymes, while no activity was present in nonsupplemented animals. Mortality was low and was not related to treatments. Taken together, these results suggest that although the animals were consuming a significant amount of dehydrated alfalfa, exogenous enzymes selected for these experiments were unable to significantly increase its nutritive value.

The data presented here questions the effectiveness of using the chosen cellulases and xylanase for improving the nutritive value of alfalfa for broilers. The addition of exogenous enzymes was unable to improve final weights, daily weight gains, and FCR of broiler chicks fed on diets containing medium and high levels of alfalfa. It is possible that exogenous enzymes were inhibited or degraded during passage through the gastrointestinal tract. However, the recombinant polysaccharidases used in these experiments were previously shown to he completely resistant to proteolysis [19]. Alternatively, xylanase-inhibitors, which have been discovered in wheat [28, 29], could have affected the function of the exogenous plant cell wall polysaccharidases in vivo. At least in the first experiment, in which the diets contained high levels of wheat, it is possible that the low levels of duodenal xylanase activity recovered could have resulted from the action of endogenous xylanase inhibitors. Considering the high levels of cellulase and xylanase activity detected in the crop and small intestine of animals receiving the exogenous microbial enzymes from the second experiment, it is unlikely that such inhibitor proteins exist, at least in high concentrations, in alfalfa.

Daily weight gains and Iced intakes were slightly deteriorated as a consequence of enzyme supplementation. Moreover, in the first experiment supplementation with a recombinant xylanase significantly decreased the daily weight gains and feed intakes. Consistently, in the second experiment animals supplemented with exogenous polysaccharidases and with higher high-energy feed restrictions had smaller but nonsignificant alfalfa feed intakes, although growth rates were comparable to the nonsupplemented birds. We had anticipated that enzymes could contribute for a considerable breakdown of plant cell wall polysaccharides during digestion resulting in improved animal performances. Il is possible that as a consequence of plant cell wall polysaccharide hydrolysis other nonbeneficial factors are released from the feed. It is well known that alfalfa contains significant levels of saponins, which are antinutritional factors for poultry [30]. The action of polysaccharidases, especially xylanases considering the result of experiment 1, can contribute to release a higher percentage of saponins that were otherwise trapped, therefore, potentiating the negative impacts of these bioactive compounds. It remains to be established whether the lower feed intakes and growth rates expressed in the 2 experiments resulted from the release of higher percentages of antinutritional factors from the feed. In addition, the carbohydrates produced from cellulase and xylanase hydrolysis could affect the endogenous microflora of the animals, which were not fed from d 1 on diets supplemented with polysaccharidases [5], This could explain the more drastic results verified in experiment 1, which used older animals. Under these conditions the microflora, which colonize the last compartments of the gastrointestinal tract, was not able to adapt to the change in carbohydrate profile resulting from the action of polysaccharidases.

Although exogenous polysaccharidases were detected in the digesta, it is possible that the enzyme mixture used in these experiments was not the most appropriate for the targeted substrates or that time for action was short. Considering the short transit periods verified in poultry, it is possible that enzymes, such as polysaccharidase that act at considerably slower rates due to the recalcitrance of their substrates, did not have the required time to act. One alternative to maximize the effect of the exogenous enzymes would be to supplement the diets with the biocatalysts and promote some polysaccharidase hydrolysis before consumption by animals. A similar phenomenon occurs in nature. It is believed that the impact of enzyme supplementation on wheat- and rye-based diets decreases with storage time, and this is a consequence of endogenous polysaccharidase activity, leading to a considerable reduction on the degree of polymerization of polysaccharides [3 IJ. A more controlled but similar mechanism could be attempted for the hydrolysis of the complex polysaccharides of alfalfa prior to consumption by animals.

Our data also imply that there is work to be done on the identification of the most efficient enzyme mixtures to be used for the substrate under analysis. Our preliminary in vitro experiments, presented in this report in Figure 1, demonstrated that different enzyme mixtures had very different capacities to release reducing sugars from alfalfa fiber preparations. Clearly, the data suggest that commercial enzyme cocktails performed better than individual recombinant enzymes and that cellulases might be more important than xylanases. This is not completely unexpected considering the complexity of the plant cell wall. It is well recognized that the hydrolysis of plant cell wall polysaccharides requires the synergistic interaction of a large consortium of enzymes [33]. Commercial enzyme mixtures, containing a large variety of enzymes and substrate activities, therefore, are more promising when hydrolysis of plant cell wall polysaccharides is required. In addition, the data also suggest that xylanases released smaller percentages of reducing sugars from the alfalfa cell walls and that these enzymes can have a negative impact in the release of detrimental factors and that enzyme mixtures for alfalfa supplementation should contain high levels of cellulase activity. Therefore, in the future more work should be developed in the identification of enzyme mixtures more suited for the hydrolysis of alfalfa polysaccharides.

At the end of the animal trial (d 56), 15 animals from treatments C50+0 and C75+0 were slaughtered at a commercial processing plant. After 24 h, carcasses were evaluated for skin color using a color meter, over the most homogeneous portion of the samples. For color comparison purposes, 15 male Cobb broilers grown conventionally in a commercial producing unit, were acquired at slaughter day. Although from a different strain and having different ages, these animals were grown under nutritional and management programs that aim at producing the deep yellow carcasses appreciated by Portuguese consumers. Results of the colorimetric evaluation of breast and back skin are presented as the CIELAB values of L*, a*, and b* in Table 5. Broilers fed diets containing alfalfa had lower L* scores, both in breast and backs, than the commercially grown animals, indicating a more deeply pigmented skin. However, there was no significant difference among the treatments receiving alfalfa. Interestingly, the addition of alfalfa at the higher levels of inclusion caused a significant decrease in the a* of broiler carcasses, both in the breast and backs, showing that the usually undesirable pink and red tones in the skin were less developed. This effect was particularly marked on the breast skin. Finally, as expected all broilers fed on alfalfa had higher b* values, particularly on the breast, compared with the commercial broilers. Moreover, this parameter was significantly higher in the animals consuming higher percentages of alfalfa. However, b* values of the back were only significantly higher for animals with higher alfalfa feed intakes, with no apparent difference between the commercial and C75+0 animals. Other parameters, such as pH and water-holding capacity, were not significantly different between the treatments and commercial carcasses (not shown). These findings parallel those described in the literature [11, 33] but suggest that higher levels of alfalfa consumption considerably reduce the redness usually associated with broiler carcasses. This is particularly interesting, considering that red tonalities in poultry carcasses are usually not appreciated [34] and may result from the extremely low levels of red pigments found in dehydrated alfalfa [35|.

CONCLUSIONS AND APPLICATIONS

1. Roxazyme G enzymes, cellulase 5a from Cellvibrio mixtus and xylanase GH11-CBM6 from Clostridium thermocellum were not effective in improving the performance of broiler chicks fed alfalfa-containing diets.

2. The results suggest that ingestion of high levels of alfalfa meal by broiler chicks do not cause any apparent health problems in the animals.

3. Alfalfa considerably contributes to the pigmentation of broiler skin, particularly with yellow pigments, while red and pink undesirable tones were significantly reduced.

REFERENCES AND NOTES

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16. Roxazyme G contains a minimum of 1,600 U/g of cellulase, 3,600 U/g of endo-1,3(4)-[beta]-glucanase, and 5,200 U/g of endo-1,4-[beta]-xylanase. The recombinant enzymes used in this experiment were the single domain ccllulase 5a (Cel5a) from Cellvibrio mixius [18] and a truncated derivative of xylanase 11a (Xynl 1a) from Clostridiun thennocellum, termed GH11-CBM6 [19]. The bacterial xylanase is a modular enzyme containing a catalytic domain and a noncatalytic xylan-binding module separated by a short linker sequence [19], Plasmids containing the DNA encoding regions of both proteins, under the control of a T7 promoter in the prokaryotic expression vector pET21a (Novagen), were transformed in Escherichia coli BL21 cells. Recombinant E. coli strains were grown on Luria Bertani media to mid-exponential phase (A^sub 600nm^ of ) and polysaccharidase gene expression induced by adding isopropyl [beta]-D-thiogalactoside to a final concentration of 1 mM. Cells were collected after 5 h induction at 37°C, and protein extracts prepared by ultrasonication as described by Fernandas et al. 119]. Extracts were incubated at 50°C for 20 min and centrifuged for 30 min at 10,000 × g to remove much of the E. coli proteins (both recombinant enzymes are thermostable at the referred temperature). Total enzyme used in each treatment was commercial polysaceharidase mixture, g/kg of Roxazyme G; recombinant xylanase, 4,000 U/kg of GH11-CBM6; and recombinant cellulase plus a xylanase, 4,000 U/kg of GH11-CBM6 plus 4,000 U/kg of Cel5a (1 U of enzyme activity released 1 (mol of product/ min at 37°C).

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Acknowledgments

The authors express their gratitude to Sociedade Agricola da Quinta da Freiria SA for supplying the 1-d-old broiler chicks. This work was supported by Instituto Nacional de Investigação Agrária (Programa Agros, projecto 57).

P. I. P. tonteD,* L. M. A. Ferreira,* M. A. C. Soares,[dagger] M. A. N. M. Aguiar,* J. P. C. Lemos,* I. Mendes,:): and C. M. G. A. Fontes*'1

*CIISA - Faculdade de Medicina Veterinaria, Polo Universitario do Alto da Ajuda, Rua Prof. Cid dos Santos, 1300-477 Lisboa, Portugal; [dagger]lnstituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, Portugal; and [double dagger]Estaçâo Zootécnica National, Fonte Boa, 2005-048 VaIe de Santarém, Portugal

1 To whom correspondence should be addressed: .pt.