Xylanase and β-glucanase in diets for Japanese laying quails1

The rations of monogastric animals in Brazil are composed of corn and soybean meal. Among the obstacles to the use of cereal grains in diets is the presence of non-starch polysaccharides, constituents of low digestibility, undigested by poultry. Thus, it is important to study non-conventional ingredients and the use of additives, such as enzymes in animal feed. The objective of this study was to evaluate the productivity and metabolism of Japanese laying quails fed with corn or sorghum feeds containing or not xylanase and β-glucanase. The experimental design was completely randomized in a 2x2 factorial scheme, with four treatments, with five replicates and seven birds each, to 140 animals. The treatments were: 1) ration based on soybean meal and maize (RSMM); 2) RSMM with enzymes; 3) ration based on soybean meal and sorghum (RSMS) and 4) RSMS with enzymes. The evaluated variables were: zootechnical performance; metabolizable rations and excreta; quality and oxidative process of eggs; biometrics of organs and serum biochemistry. There was no significant interaction between the factors (ingredients x enzymes) for all evaluated characteristics. The rations did not cause changes in the productive variables, metabolizable, egg quality, biometry of the gastrointestinal tract organs and femur of Japanese laying quails. The total egg mass was higher in the different rations with enzymatic use. Serum calcium and phosphate concentrations were higher in the RSMM and RSMM with enzymes, however, the Ca:P ratios remained, indicating a metabolic normality condition in the Japanese quails from 26 to 35 weeks of age.


INTRODUCTION
The traditional rations of the monogastric animals are corn and soybean meal, and approximately 70% of the corn produced in the world is destined for animal feed, and only 15% for human consumption (OLIVEIRA; SANTOS; CUNHA, 2014).
Corn and soybeans are internationally traded products, their price of agricultural commodities, according to supply in response to the rhythm of demand, limited production in certain years, as well as high prices of inputs, causes an increase in the price of the costs of poultry farming. Such facts have led researchers to test unconventional ingredients in diets (NUNES; MAIER, 2012).
Among the numerous alternatives to substitute maize for poultry feeds, sorghum has a nutritional composition of 1.8 to 7.5% lower in metabolizable energy when compared to maize, however, similar crude protein content (ROSTAGNO et al., 2017).
However, when this food is included in the egg production chain, it is recommended to include pigmentants in the diets of laying hens, since the sorghum contains low levels of carotenoids and xanthophylls, responsible for the pigmentation of the yolk (FREITAS et al., 2014;MOURA;MELO;MIRANDA, 2016).
Grain-based diets have non-starch polysaccharides (PNAs) present in the cell wall of the seeds that forming the viscosity of the digesta (RESENDE et al., 2017). Birds do not secrete the endogenous enzymes necessary for the breakdown of β-glucans, arabinoxylans and other soluble or insoluble fibers present in cereals. Thus, the increase in viscosity results in impaired digestion and absorption of nutrients in the digestive tract of birds (VIANA et al., 2011). Campos et al. (2017), showed that the use of enzymes in bird feeding promotes a positive effect on cell membrane PNA degradation due to the reduction of the viscosity of the digesta, with the release of encapsulated nutrients in the cell wall structures (FERNANDES et al., 2017). The contact between these nutrients and the endogenous enzymes becomes favored, and the indigestible fraction can reach 27% with glucanases and 10% with xylanases. Thus, the study of conventional and/or alternative ingredients combined with different biotechnological inputs (enzymes) is a strategy to be evaluated.
The objective of this study was to evaluate the productive and physiological variables of Japanese laying quails fed with maize or sorghum-based diets containing or not xylanase and β-glucanase.

MATERIAL AND METHODS
The experiment was conducted in an aviary located in Rio Verde-Goiás, Brazil, with a duration of 63 days (three periods of 21 days). The Project was registered with the Committee of Ethics in Research with Animals n°: 3845300616.
A total of 140 quail (Coturnix coturnix japonica), 26 to 35 weeks of age, were used. The animal density was 117.85 cm 2 per bird. The experimental design was completely randomized (DCR), in a 2x2 factorial scheme (two rations, with and without enzymes and energy sources), in a total of four treatments, with five replicates of seven birds per experimental unit. The treatments corresponded to the following rations: 1) Ration based on soybean meal and maize (RSMM); 2) RSMM + enzymes; 3) Soybean meal and sorghum meal (RSMS); and 4) RSMS + enzymes.
The light supply was 16 hours a day and was controlled by an automatic timer which enabled the lights to be switched on and off during the night, according to the procedure adopted on commercial farms. Water and feed were provided at will during the trial period. Leftovers and feed wastes were weighed and discounted from the amount of heavy feed initially, and provided at different times. At the end of each 21-day period, the amount of feed consumed by the number of birds of each treatment was divided by the number of days, expressed in grams of feed consumed bird day -1 . In the case of poultry mortality, during the experimental period, mean consumption was corrected.
The eggs produced in each replicate were weighed to obtain the mean weight, which in turn was multiplied by the total number of eggs produced in the experimental period, thus obtaining the total egg mass. The feed conversion per dozen eggs was determined by the total feed consumption in grams divided by the dozen eggs produced (g dozen -1 ) and the conversion by egg mass by egg mass in kilograms (kg kg -1 ).
To evaluate the oxidative process of the eggs after 40 days of storage, peroxide (IP) and acidity (AI) were determined. The analyzes followed methodologies described in the Compendium of Animal Feeding (SINDICATO NACIONAL DA INDÚSTRIA DE ALIMENTAÇÃO ANIMAL, 2013).
The metabolizability coefficients of crude protein, ethereal extract and crude fiber were determined. Under the floor of the cages were placed trays of metal plates galvanized, coated with plastics, properly identified. The total excreta collection, twice a day, was carried out during the last five consecutive days of each experimental period. The marker used was ferric oxide (2%). Excreta and feed were analyzed following procedures described in Silva and Queiroz (2006).
At the end of the experimental period (63 rd day), a bird per treatment was weighed, sacrificed by cervical dislocation and had blood (serum), bone (femur) and organs of the digestive tract (liver, pancreas, proventriculus + gizzard and intestine) removed. The viscera were immediately weighed; the length of the intestine was measured using a metric ruler, measured from the beginning of the duodenum to the cloaca.
In the right femur in natura, all adherent tissue was removed, using scissors and tweezers, and then weighed in analytical balance, and measurements of the diameter (horizontal) and length (mm) were performed using a digital caliper. The Seedor index was obtained by dividing bone weight (mg) by bone length (mm), and taking into account measures bone density (SEEDOR; QUARRACCIO; THOMPSON, 1991), therefore the higher the index, the higher the density of the bone piece.
The collected blood was conditioned in a properly identified test tube; and centrifuged at 6000 rpm for 15 minutes to obtain serum (RAMOS et al., 2014), and then stored at -20 °C until the time of the evaluations. Analyzes of serum minerals, calcium and phosphorus were performed with commercial kits.
Data were submitted to analysis of variance in DCR, factorial scheme (2x2), considering the level of 5% of probability, and in case of significance the means were compared by means Tukey's test. The analyses were performed using SAEG (2007) software, version 9.5.

RESULT AND DISCUSSION
The use of maize or sorghum, with or without xylanase and β-glucanase in feed, did not influence the productive variables: feed consumption (total egg bird -1 day -1 ), egg weight (g), feed conversion by mass of eggs and per dozen produced and viability of birds ( Table 2). The results verified resemble those of Moura et al. (2010). The researchers did not find significant effects on the production characteristics of Japanese laying quails, from nine to 25 weeks of age, by replacing corn with sorghum (0; 25; 50; 75 and 100%) in the rations.
The total egg mass was altered by the inclusion of xylanase and beta-glucanase in the rations, presenting a higher amount when compared to the rations without the enzymes. The use of the enzymes was possibly efficient in the degradation of the non-starch polysaccharides (PNAs), improving the energetic utilization of the diets. According to Viana et al. (2011), xylanase supplementation in diets containing reduced energy (2,755 kcal ME kg -1 ) improved the egg production and egg mass of commercial laying hens from 24 to 48 weeks. Iwahashi et al. (2011), stated that the supplement with xylanase and β-glucanase enzyme complex was efficient in providing reduced dietary performance in ME and amino acids for the initial and growth stages of quails.
The sorghum has a similar bromatological profile to corn, with the exception of the amount of metabolizable energy -ME (ROSTAGNO et al., 2017) and carotenoids, which are smaller. Thus, the absence of significant difference between the rations evaluated, indicated the possibility of safe use of this feed in Japanese quail rations. However, the inclusion of synthetic pigments may be necessary to improve egg yolk color.
The accumulated mortality was 15.90% (1.77% week -1 ), and the viability was not influenced by the ingredients and/or enzymes; this result was due to the advancing age of birds. Togashi, Soares and Murakami (2008) reported that at the end of the batch life, mortality may reach 30%, and despite numerous reports of cloacal prolapse problems, many deaths have indeterminate causes.
There was no significant interaction between ingredients and enzymes (p>0.05) in these variables (Table 3). However, rations with soybean meal and maize (RSMM) showed higher crude protein metabolizability coefficient -CMCF (p=0.0396) when compared to soybean meal and sorghum (RSMS) rations. Also, with the enzymatic addition, the crude fiber metabolizability coefficient -CMCF verified were lower (p=0.0209).  Table 2 -Performance of Japanese laying quails fed with corn or sorghum feeds, with and without enzymes CV = coefficient of variation; ExR = Interaction Enzyme x Ration; ME = media rations with on without enzymes; MR = media rations; SEM = standard error of the mean Dissimilar to that found in this study, Iwahashi et al. (2011), verified an improvement in five percentage points in the metabolizability coefficient of neutral detergent fiber (CMNFD) due to the enzymatic addition (Xylanase + β-glucanase) in the feeds (negative controls) for quails. The explanation is the greater availability of intracellular nutrients contained in the vegetal wall.
Regarding egg quality, there was no significant interaction between feed and enzymes, as well as differences between treatments (Table 4). The internal characteristics of the egg: weight (g), pH, diameter (mm), height (mm) of albumen and yolk; were not influenced (p>0.05). Similarly, Viana et al. (2011), Oba et al. (2013), Geraldo et al. (2014), and Vargas et al. (2017), did not observe effects of the use of enzymatic complexes under the quality of the eggs, indicating that the requirements of the birds were met with the diets used.
In the study by Moura et al. (2010), the substitution of maize by sorghum in feed had no effect on yolk weight, eggshell, albumen and eggshell thickness. In the present study, eggshell thickness (mm) was altered as a function of the ingredient used, so corn rations (RSMM and RSMM + CV = coefficient of variation; ExR = Interaction Enzyme x Ration; ME = media rations with on without enzymes; MR = media rations; SEM = standard error of the mean  Murakami et al. (2007), also observed variations in eggshell thickness and Haugh Unit of eggs of commercial laying hens fed with the inclusion of multienzymatic complex in the rations. When evaluating the quality of the egg, some characteristics should be analyzed together, such as the thickness of the shell and the specific gravity. In general, the highest value of specific gravity is considered to be related to a thicker egg shell, a characteristic desirable by  Table 4 CV = coefficient of variation; ExR = Interaction Enzyme x Ration; ME = media rations with on without enzymes; MR = media rations; SEM = standard error of the mean the processing industries. Despite the modification of the thickness of the eggshell found, no change was observed in the specific gravity of the eggs, thus indicating the maintenance of quality.  In Table 5, the values of specific gravity (g cm -3 ), Haugh Unit and the indicators of egg oxidative process, peroxide index and acidity, are presented after 40 days of storage. There was no influence of the treatments in isolation, or even interaction between the ingredients and the enzymes. Oba et al. (2013), have pointed out that the addition of enzymes is able to reduce some antinutritional properties present in foods, such as PNAs, which have the capacity to increase the viscosity of the digesta, as it has high capacity to bind to water, forming a viscous gel, thus hindering the action of enzymes and the absorption of the digested nutrients. Despite the improvement in viscosity of the digesta, in general, the productive and egg quality variables were not influenced independently of the enzymatic use.
The biometry of the organs of the gastrointestinal tract (GIT) of quails was in accordance with those verified in the zootechnical performance, indicating that there was no physiological involvement of the birds (Table 6). It is important to consider the alterations of the digestive system, since these interrelate deeply with productivity, as a function of the influence on the utilization of all nutrients (ARTONI et al., 2014).
The intestinal length (cm), well the weights of each intestine (thin and thick) of the birds were not altered by receiving the different rations in an interactive and/ or isolated way. Saar et al. (2015), studied the intestinal morphometry of Japanese quails fed diets based on sorghum grain. These authors also did not find significant differences for the weight of the liver, gizzard and intestines, as well as intestinal size (cm). Also, because they did not compromise the gastrointestinal tract of the birds, they recommended the use of sorghum in the total or partial substitution of corn in the feed of quails.
The Table 7 shows the bone metric variables as well as the serum minerals (Ca and P) values. The femur weight, length (mm) and diameter (mm) were not Xylanase and β-glucanase in diets for Japanese laying quails   Serum calcium and phosphate levels were altered as a function of the feed ingredient. There was no significant interaction between the factors studied. The highest blood concentrations of Ca and P were observed in birds fed corn and soybean meal (RSMM and RSMS + enzymes), independent of the receipt of xylanase and β-glucanase.
The results of serum minerals found were within the normal range, similar to the values described by Silva et al. (2017), who evaluated turmeric in sorghumbased diets for Japanese laying quails. The investigators mentioned did not verify differences due to treatments for serum Ca and P, as well as performance, protein metabolism, ethereal extract, internal and external egg quality, biometry of the gastrointestinal tract, tibial and femoral morphometry.
Normal concentrations of Ca in birds may reach values much higher than those tolerated in mammalian species, reaching 30 g L -1 ; while the bands for P are 7 to 9 g L -1 (THRALL; BAKER; CAMPBELL, 2004). It should be noted that the changes in the mineral amounts of blood occur as a function of the physiological phase of laying hens, and in the reproductive stage the transport of Ca to the ovary, induced by estrogens, by the increase of Ca binding proteins such as vitellogenin and albumin (CAPITELLI;CROSTA, 2013;SILVA et al., 2017).
The Ca: P ratios were: 2.59:1; 2.40:1; 3.03:1 and 2.42:1 for RSMM, RSMM + enzymes; RSMS and RSMS + enzymes, respectively. The maintenance of these relationships results from the fact that laying hens maintain plasma Ca and phosphate proportional levels (CHOI; MILES;HARMS, 1979). In this study, the Ca and P values in diets provided were in accordance with the physiological needs of the laying quails under the conditions evaluated. Lecznieski (2006) explained that in diets with adequate or slightly deficient levels, the addition of enzymes can release nutrients that the animal does not Continuation Xylanase and β-glucanase in diets for Japanese laying quails need or even cannot convert into higher productive indexes. Such justification became plausible in the absence of additive effects of enzymatic use on the productive and physiological results found. Still, it is sometimes desired not to impair the productive and biochemical-physiological characteristics, and not only the improvement of these, especially in the case of the use of alternative foods.

CONCLUSIONS
1. In general, there were no significant differences in the main productive variables, metabolizability coefficient, egg quality, biometry of the gastrointestinal tract and organs of Japanese laying quails fed with rations containing maize or sorghum supplemented or not with xylanase + β-glucanase; 2. Based on the physiological responses obtained, an alternative is the optional use of the enzymes evaluated with maize or sorghum in Japanese quail rations, from 26 to 35 weeks of age.