Metabolizable energy values of diets supplemented with xylanase determined with laying hens

The objective of this study was to evaluate the effect of the supplementation of xylanase in diets with reduced energy level on the apparent metabolizable energy corrected for nitrogen, determined with laying hens at 14, 36, 60 and 80 weeks of age. Four digestibility trials were conducted, using 80 Hy-line W36 laying hens aged 14, 36, 60 and 80 weeks of age. Birds were distributed in a completely randomized design in 2 × 2 factorial arrangement (energy level × inclusion of xylanase), totaling four treatments with 10 replicates of two birds each. Treatments were: positive control (balanced diet for their age); positive control + xylanase; negative control (diet with reduction of 100 kcal/kg in the level of metabolizable energy); and negative control + xylanase. Xylanase, produced by microorganism Trichoderma reesei, was added to the diets at 100 g/t (16,000 BXU/kg) for diets fed at 14 weeks and 75 g/t for diets of 36, 60 and 80 weeks (12,000 BXU/kg). The data obtained were subjected to analysis of variance at 5% probability. Supplementation of xylanase promoted higher values for AME (apparent metabolizable energy) and AMEn (apparent metabolizable energy corrected for nitrogen) determined with 80-week-old laying hens, subjected to diet with energy level according to the nutritional requirements for their age. Supplementation of xylanase increases the matabolizability coefficient of the dietary crude protein and improves the nitrogen retention of laying hens at 14 weeks. In addition, xylanase associated with adequate levels of dietary energy promotes higher values for AME and AMEn determined with laying hens at 80 weeks of age.


Introduction
It is known that, in laying hens, approximately 20% of energy is used for production; therefore, the decision of the amount to be provided is fundamental, since the bird will preferentially direct the energy for maintenance and decrease the production if the input is insufficient.A diet deficient in energy can cause a reduction in the growth of the bird, as well as decrease in the laying rate and weight loss (Leeson & Summers, 1997).
Non-starch polysaccharides (NSP) stand out as one of the anti-nutritional factors.Corn, which is added to poultry feed as the main source of energy, contains only 1% of soluble NSP, especially arabinoxylans, compared with 24, 45 and 46% for wheat, barley and rye, respectively (Choct, 2010).However, although maize is an easily-digested ingredient, there is evidence suggesting the presence of starch resistant to digestion, which may limit the value of corn energy and consequently of the dietary energy (Slominski, 2001).
Exogenous enzymes improve the nutritional value of corn and consequently of diets based on corn and soybean meal by several potential mechanisms (Cowieson, 2005).Among them, it is known that enzymes can hydrolyze polysaccharides that prevent the digestion of starch grains and protein, making these compounds available for digestion by the action of endogenous enzymes (Bedford, 1996).
Xylanase has been successfully used in the wheatbased diets, having a high content of arabinoxylans and reported success reducing the viscosity of the chyme and degradation of the cell wall.Moreover, it presents great tolerance to pH, being effective in the range of 3.5 to 6.5, so it can act from the beginning of digestion to the ileum (Costa et al., 2004).
In this context, although the presence of NSP in corn is not a major problem, the use of xylanase may be beneficial in diets based on corn and soybean meal for poultry, probably by improving the coefficients of nutrient digestibility, mediated by changes in the cell wall structure, once xylans and arabinoxylans undergoing hydrolisys, under the action of exogenous enzyme, can release encapsulated nutrients (Cowieson, 2005).
The objective of this study was to evaluate de effect of supplementation of xylanase in diets with reduced energy level on the apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen, determined with laying hens at 14, 36, 60 and 80 weeks of age.

Material and Methods
Four digestibility trials were conducted with 80 laying hens of the Hy-line strain variety W36 at the ages of 14, 36, 60 and 80 weeks of age, housed in metabolism cages with dimensions 1.00 × 0.50 × 0.60 m arranged in battery, provided with front internal feeder, nipple drinking system and trays previously coated with plastic for the collection of excreta.
The trials were developed in two phases of rearing: growth (14 weeks) and laying (36, 60 and 80 weeks), and each trial consisted of five days of adaptation to diets and facilities and five days for collection of excreta.The birds were distributed in a completely randomized design in 2 × 2 factorial arrangement (energy level × inclusion of xylanase), totaling four treatments with 10 replicates of two birds each.
The treatments were: positive control (diet balanced according to age); positive control + xylanase; negative control (diet with reduction of 100 kcal/kg in metabolizable energy); and negative control + xylanase.The diets were based on corn and soybean meal and formulated to make up the requirements recommended by Rostagno et al. (2005), for periods of 13 to 17 weeks (Rearing II) and 33 to 80 weeks (Laying II), except for the energy level of the diets of negative control treatments (with or without inclusion of xylanase) (Tables 1 and 2).Water and feed were given ad libitum throughout the trial.
The method of total excreta collection was adopted, conducted in the four digestibility trials.The feed were weighed before and after collection periods for determining the feed intake, and marked with 1% ferric oxide to determine the beginning and end of the collections (Table 3).
During the collection period, the excreta were collected twice daily (8h00 and 16h00) to avoid loss of material, packed in plastic bags identified per experimental unit, and frozen for subsequent analysis.
At the end of the collection period, the excreta were joined per experimental unit, thawed, weighed and homogenized.From the homogeneous mass, a sample was collected and placed in a forced-ventilation oven, at a temperature of 55 ºC for 72 hours, in order to carry out the pre-drying.Subsequently, the pre-dried samples were exposed to air so that there was balance with the environment temperature and humidity.They were then weighed, ground and packed in plastic containers for laboratory analysis.
The contents of humidity, dry matter, nitrogen and ether extract of the excreta and feed were determined according to Silva & Queiroz (2002), and gross energy by bomb calorimeter.From the results of laboratory analysis information of dry matter intake, metabolizability coefficients of dry matter, crude protein, ether extract, gross energy, value of apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention of the feed and nitrogen retention per day were obtained.To calculate the apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention and metabolizability coefficients, the formulas described by Matterson et al. (1965) apud Sakomura & Rostagno (2007) were used.The data were subjected to analysis of variance by the procedure PROC GLM of SAS (Statistical Analysis System,version 9.1), according to statistical model: Yijk = µ + Ei+ Zj+ (E x Z)ij + eijk in which Yijk = observation of the characteristic in birds of the k experimental unit, of the energy level i and of the form of enzymatic supplementation j; µ = constant common to all experimental units; Ei = effect of the i-th level of dietary energy (i = 1.2);Zj = effect of the j-th form of enzymatic supplementation (j=1.2);EZij = effect of the interaction of the i-th energy level and j-th form of enzymatic supplementation; eijk = error associated with the observation Yijk; eijk ~ N (0, σ 2 ).

Results and Discussion
There was interaction (P<0.05) between the factors for apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention determined at 14 weeks of age (Table 4).The supplementation with the enzyme xylanase showed higher (P<0.05)percentage of metabolizability coefficient of the dietary crude protein; likewise, there was greater nitrogen retention per day.
It can be inferred that the xylanase supplementation was effective in reducing the viscosity of the digesta, improving the digestibility and protein adsorption, and probably lowering the amount of available substrate for bacterial fermentation in the growth stage of the poultry.Starch and protein in the diet that were not used by the animal can favor the migration of microorganisms, which would typically be found in large quantities in the poultry caecum, to the small intestine, which is the place of greatest absorption of nutrients (Campbell & Bedford, 1992).
Although not observed in this study, the high bacterial load would possibly irritate the intestinal epithelium, promoting a thickened mucosa, damaged microvilli and reduction in nutrient absorption.This could have implications for the economy of poultry protein, in other words, the synthesis of more protein given the growth of organs, enzyme secretion and repair of intestinal mucosa could promote a reduction in the availability of protein for tissue growth or egg production in chickens (Jaroni et al., 1999).
The highest values (P<0.05) of apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention were obtained for diets balanced according to nutritional recommendation for their age without enzyme supplementation determined with laying hens at 14 weeks (Table 5).
Probably, the values of apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention, determined with laying hens at 14 weeks of age, were results of availability in the diet with 100 more kcal of ME/kg (ME = metabolizable energy) than in the diets formulated with the recommended energy level.However, it was expected that xylanase supplementation, in diets with reduced energy level, could promote similar or greater effects than those found in diets without supplementation and with the recommended energy level.Novak et al. (2007) observed greater energy retention (kcal/bird/day) for birds subjected to diets with reduction in the energy level and supplementation of enzyme complex (amylase + protease + xylanase) during the growth period (10 to 15 weeks).
Interaction (P<0.05) was observed between the factors for metabolizability coefficient of dry matter, crude protein, metabolizable energy corrected for nitrogen retention and nitrogen retention (Table 6).
The values of dry matter intake and metabolizability coefficient of ether extract at 36 weeks were higher (P<0.05) for the birds subjected to diets with reduction in the energy level at 100 kcal/kg.Likewise, the factor energy influenced the metabolizability coefficient of the gross energy and apparent metabolizable energy; however, the highest values were obtained for diets with adequate energy level for their age.The increase in dry matter intake observed was expected, since poultry increase the feed intake in an attempt to supplement their demand for energy.
Xylanase, associated with the reduction of dietary energy, reduced the metabolizability coefficient of dry matter at 36 weeks of age, but promoted better results (P<0.05) for metabolizability coefficient of crude protein and nitrogen retention and less apparent metabolizable energy corrected for nitrogen retention when the enzyme was added to the diet with recommended energy level for the age of laying hens (Table 7).
The highest values for metabolizability coefficient of crude protein and nitrogen retention indicated that xylanase supplementation could improve the digestibility of nutrients such as protein and amino acids by reduction of intestinal viscosity.Choct et al. (1996) report that the digestibility of starch, protein and lipid can be significantly improved by enzyme supplementation in diets containing NSP. Novak et al. (2008) found that the interaction between enzyme supplementation and level of dietary energy  resulted in differences in the percentage of energy retention at 38 weeks of age.The same authors reported that the absence of enzyme supplementation (amylase + protease + xylanase) promoted greater percentage of energy retention in laying hens fed diets formulated with recommended energy level for their age compared with those with a 3% reduction in the energy level; however, associated with enzymatic supplementation, the level of dietary energy did not influence the percentage of retained energy.Furthermore, laying hens fed diets with recommended energy level for their age and supplementation of enzymatic complex (amylase + protease + xylanase) showed reduction in the percentage of protein retention at the age of 38 weeks (Novak et al., 2008).
It was found that only apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention, determined at 60 weeks of age, were affected by treatments, with interaction (P<0.05)observed between the factors level of dietary energy and xylanase supplementation (Table 8).
It was found that diets formulated to meet the energy requirement and without xylanase presented apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention superior (P<0.05) to those formulated with reduced energetic level (Table 9).It was observed that the xylanase supplementation to the diet with reduced energetic level resulted in apparent metabolizable energy and apparent metabolizable energy corrected for  nitrogen similar (P<0.05) to those of the diet formulated according to nutritional requirement.Interaction (P<0.05) was observed between the factors energy and xylanase for metabolizability coefficient of crude protein, apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention determined with 80-week-old laying hens (Table 10).The energy factor influenced (P<0.05)dry matter intake, metabolizability coefficient of dry matter and of crude protein, and the diets formulated according to the recommendations of Rostagno et al. (2005) promoted lower (P<0.05)intake of crude protein and higher (P<0.05)percentage for metabolizability coefficient of dry matter and crude protein.
It was also found that xylanase supplementation reduced (P<0.05) the nitrogen retention.However, the xylanase supplementation promoted higher values (P<0.05) for metabolizability coefficient of gross energy, apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention determined with 80-weekold laying hens, subjected to diet with energy level according to the nutritional requirement for their age (Table 11).
In an experiment with broilers, Mathlouthi et al. (2003) observed better digestibility of nutrients and of apparent metabolizable energy in diets composed of corn, rice meal and barley, supplemented with xylanase + β-glucanase.Cowieson et al. (2006), working with reduction in the level of dietary energy and addition of enzymatic complex (xylanase + protease + amylase + phytase) in diets for broilers observed intake values of digestible fractions (dry matter, nitrogen, energy) greater than or similar to those resulting from supply of diets formulated with adequate nutritional levels without inclusion of enzyme.
On the other hand, Viana (2009), studying the comparison of productive parameters from the supply of diets based on corn and soybean meal, with adequate nutritional levels or reduction in the percentage of the apparent metabolizable energy and xylanase supplementation, reported that the mean value of apparent    for metabolizability coefficient between the two levels of dietary energy.The values of apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen retention determined with 80-week-old laying hens could enable formulations with inclusion of xylanase to provide superior performance compared with those birds fed diets nutritionally adequate without enzyme supplementation.However, information about the digestion and nutritional value of diets with the exogenous enzymes supplementation are important for nutritionists, once they probably enable low-cost formulations and may reduce the risk of having unbalanced nutrients (Cowieson et al., 2006).

Conclusions
The supplementation of xylanase increases the metabolizability coefficient of the dietary crude protein and improves the nitrogen retention of laying hens at 14 weeks of age.Xylanase, associated with adequate energy levels in the diet promotes higher values of apparent metabolizable energy and apparent metabolizable energy corrected for nitrogen determined with 80-week-old laying hens.

Table 1 -
Percentage and calculated composition of experimental diets for the trial of 14 weeks of age

Table 2 -
Percentage and calculated composition of experimental diets for the trials of 36, 60 and 80 weeks of age

Table 3 -
Body weight at the beginning of the period of collection of excreta from laying hens fed diets containing xylanase R.Bras.Zootec., v.41, n.12, p.2433-24, 201212

Table 4 -
Metabolizability coefficients, metabolizable energy and nitrogen retention of 14-week-old laying hens fed diets containing xylanase 1 EN, XL, EN×XL = effect of the energy level of diet, supplementation of xylanase and their interaction, respectively.
Means followed by different letters in the column are significantly different by the F test. 1 EN -energy; XL -xylanase; REQ -diet formulated as recommended by Rostagno et al. (2005) for their age; RED -diet with reduction of 100 kcal/kg metabolizable energy in relation to REQ; DMI -dry matter intake; MCDM -metabolizability coefficient of dry matter; MCCP -metabolizability coefficient of crude protein; MCEE -metabolizability coefficient of ether extract; MCCE -metabolizability coefficient of crude energy; AME -apparent metabolizable energy; AMEn -apparent metabolizable energy corrected for nitrogen; NR -nitrogen retention; CV -coefficient of variation. 2Data expressed on a dry matter basis. 3

Table 5 -
Deployment of metabolizable energy determined with laying hens fed diets containing xylanase at 14 weeks of age

Table 6 -
Metabolizability coefficients, metabolizable energy and nitrogen retention of 36-week-old laying hens fed diets containing xylanase Rostagno et al. (2005)erent letters in the same column differ significantly by the F test.EN -energy; XL -xylanase; REQ -diet formulated as recommended byRostagno et al. (2005)for their age; RED -diet with reduction of 100 kcal/kg metabolizable energy in relation to REQ; DMI -dry matter intake; MCDM -metabolizability coefficient of dry matter; MCCP -metabolizability coefficient of crude protein; MCEE -metabolizability coefficient of ether extract; MCCE -metabolizability coefficient of crude energy; AME -apparent metabolizable energy; AMEn -apparent metabolizable energy corrected for nitrogen; NR -nitrogen retention; CV -coefficient of variation.1Dataexpressed on a dry matter basis. 2 EN, XL, EN×XL -effect of the energy level of diet, supplementation of xylanase and their interaction, respectively.

Table 7 -
Deployment for metabolizability of dry matter, crude protein, metabolizable energy and nitrogen retention determined with laying hens fed diets containing xylanase at 36 weeks of age1 Rostagno et al. (2005)erent uppercase letters in the same column and different lowercase letters in the same row differ significantly by the F test (P<0.05).MCDM -metabolizability coefficient of dry matter; MCCP -metabolizability coefficient of crude protein; AMEn -apparent metabolizable energy corrected for nitrogen; NR -nitrogen retention; CV -coefficient of variation.1EnergyrequirementaccordingtoRostagno et al. (2005); Reduced in 100 kcal/kg in relation to the energy requirement.
1Data expressed on a dry matter basis. 2 EN, XL, EN×XL -effect of the energy level of diet, supplementation of xylanase and their interaction, respectively.

Table 9 -
Deployment for metabolizable energy determined with laying hens fed diets containing xylanase at 60 weeks of age

Table 10 -
Rostagno et al. (2005)icients, metabolizable energy and nitrogen retention of laying hens at 80 weeks of age fed diets containing xylanase Means followed by different letters in the same column differ significantly by the F test.EN -energy; XL -xylanase; REQ -diet formulated as recommended byRostagno et al. (2005)for their age; RED -diet with reduction of 100 kcal/kg metabolizable energy in relation to REQ; DMI -dry matter intake; MCDM -metabolizability coefficient of dry matter; MCCP -metabolizability coefficient of crude protein; MCEE -metabolizability coefficient of ether extract; MCCE -metabolizability coefficient of crude energy; AME -apparent metabolizable energy; AMEn -apparent metabolizable energy corrected for nitrogen; NR -nitrogen retention; CV -coefficient of variation.1Dataexpressed on a dry matter basis. 2 EN, XL, EN×XL -effect of the energy level of diet, supplementation of xylanase and their interaction, respectively.

Table 11 -
Deployment for metabolizability coefficients of gross energy and apparent metabolizable energy determined with laying hens fed diets containing xylanase at 80 weeks of age Rostagno et al. (2005)erent uppercase letters in the same column and different lowercase letters in the same row differ significantly by the F test (P<0.05).MCCE -metabolizability coefficient of crude energy; AME -apparent metabolizable energy; AMEn -apparent metabolizable energy corrected for nitrogen; NR -nitrogen retention; CV -coefficient of variation.1EnergyrequirementaccordingtoRostagno et al. (2005); Reduced in 100 kcal/kg in relation to the energy requirement.