Effects of wheat cultivar, metabolizable energy level, and xylanase supplementation to laying hens diet on performance, egg quality traits, and selected blood parameters

A 2 × 2 × 2 factorial arrangement of treatments was conducted to evaluate the effects of two dietary apparent metabolizable energy (AME) levels (2,720 and 2,580 kcal kg diet) and enzyme (0 and 0.3 g kg diet, Grindazym GP 15,000 with mostly xylanase activity) supplementation on the performance of laying hens fed diets based on two wheat cultivars (Marvdasht and Sardari). Experimental diets were formulated to have a constant energy to protein ratio and were fed to 65-wk-old Lohmann LSL-Lite laying hens for 7 wk. The lower level of AME reduced egg production and egg mass (p < 0.05) and increased feed conversion ratio (p < 0.05). Enzyme addition increased feed intake of the birds fed a diet with Sardari cultivar (p < 0.05) but had no effect on feed intake of the birds fed a diet with Marvdasht cultivar (p > 0.05). Nevertheless, birds receiving diets based on Marvdasht cultivar had higher feed intake and egg mass than that of those receiving diets based on Sardari cultivar (p < 0.05). The birds fed diets based on Marvdasht cultivar produced less undesired eggs and had better yolk color as compared with the birds fed diets based on Sardari cultivar (p < 0.05). The serum concentration of glucose increased by enzyme supplementation when birds receiving lower AME level (p < 0.05). These results indicate that enzyme supplementation may have a positive effect on the feed intake of laying hens when fed on wheat-based diets; however, this effect is cultivar dependent and does not necessarily mean that enzyme supplementation always benefit production. Additional key words: Triticum aestivum; feed intake; egg production; serum glucose; leukocyte profile. * Corresponding author: torki@razi.ac.ir Received: 17-03-14. Accepted: 06-10-14. Abbreviations used: AME (apparent metabolizable energy); BGU (β-glucanase unit); CF (crude fiber); CP (crude protein); DM (dry matter); EE (ether extract); FCR (feed conversion ratio); FXU (fungal xylanase unit); HDL (high density lipoprotein cholesterol); HU (Haugh unit); LDL (low density lipoprotein cholesterol); NSP (non-starch polysaccharides); T3 (triiodothyronine); T4 (thyroxine). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2014 12(4): 1071-1081 http://dx.doi.org/10.5424/sjar/2014124-5929 ISSN: 1695-971X eISSN: 2171-9292 RESEARCH ARTICLE OPEN ACCESS (Choct & Hughes, 1999). However, several other physical and chemical factors can influence energy available from wheat and as a result animal performance. Among them hardness (Carré et al., 2002), pelleting (Ghobadi & Karimi, 2012), starch, crude protein (CP) and ether extract contents (Steenfeldt, 2001; Svihus & Gullord, 2002) can be underlined. Parsaie et al. (2006) reported that Iranian wheat cultivars are variable in their apparent metabolizable energy (AME, 1,893 to 3,062 kcal kg), NSP (9.6 to 14.9%), CP (9.5 to 14.0%) and other chemicals content. Rafuse et al. (2005) reported that CP of five varieties of Canadian wheat varies between 11.4 and 15.5%. In a survey of 18 wheat cultivars, Kim et al. (2003) reported that starch content ranged between 58.5 and 73.7%, CP between 9.7 and 19.1%, and NSP between 7.8 and 11.0%. In addition, Mollah et al. (1983) found AME ranging between 2,670 and 3,860 kcal kg of dry matter (DM) when 22 wheat samples were fed to growing broiler chickens. Moreover, some wheat cultivars considered to be of high quality have produced broilers with unexpectedly low performance (Gutiérrez-Álamo et al., 2008b). The exogenous xylanase is used to reduce or to eliminate the negative effects of the soluble NSP on performance of young chickens (Bedford & Schultz, 1998; Choct et al., 2004; Gutiérrez-Álamo et al., 2008b; Zhang et al., 2012; Zou et al., 2013), but the effects are limited and inconclusive for laying hens. It has been reported that addition of xylanase to wheatbased layer diets improves egg production rate (Pan et al., 1998). Additionally, Pan et al. (1998) reported significant improvements in AME and feed conversion ratio (FCR) with supplementation of a commercial enzyme preparation containing β-glucanase, cellulase, and xylanase, when diets containing 80% wheat or 65% rye were fed to 22-wk-old hens. Mathlouthi et al. (2002) found that xylanase supplementation improved egg mass of laying hens fed diets containing 70% wheat and wheat-barley (49% wheat and 20% barley). As mentioned above, enzyme supplementation can change the nutritional status and improve productive performance of birds fed wheat-included diets, but which are also closely related to the regulation of metabolism and functioning of the growth-related endocrine system. For example, triiodothyronine (T3) and thyroxine (T4) in peripheral blood of laying hens played their physiological functions in many ways such as facilitating the differentiation, growth and development of tissue, promoting the formation of protein and enzymes, increasing the utilization of carbohydrate, and enhancing the disintegration of fats (Ooi et al., 2004). Nutritional status is an important factor in the regulation of plasma hormones and intermediary metabolism in laying hens (Swennen et al., 2005; Xiao-Ying et al., 2010). So we hypothesized that the effects of enzyme supplementation on productive performance may be associated with changes in the concentration of metabolic hormones and metabolites in laying hens fed wheat-based diets. The objective of the present study was to examine the effects of two levels of AME and enzyme supplementation on the performance, egg quality traits and selected blood parameters of laying hens fed diets based on two wheat cultivars. Material and methods Birds and experimental design All procedures used in this 7-wk experiment were approved by the Animal Ethics Committee of Razi University and complied with the guidelines for the care and use of animals in research (Federation of Animal Science Societies, 2010). A total number of 240 65wk-old Lohmann LSL-Lite laying hens (after production peak) with similar body weight (1,450 ± 14 g) and egg production rate (80.3 ± 3.8%) were randomly distributed in 40 cages (6 hens/cage) in eight experimental groups. The hens were placed in wire-floored cages arranged in a single tier within a conventional open-sided house. The cages were located in an environmentally controlled room with the room temperature kept at 21-23°C and the photoperiod set at 16 h of light (incandescent lighting, 10-lx) and 8 h dark. As is presented in Table 1, eight experimental diets including two wheat cultivars (Marvdasht and Sardari) and two levels of AME (2,720 and 2,580 kcal kg) with or without a commercial enzyme (0.0 and 0.3 g kg Grindazym GP 15,000: 36,000 FXU g endo-1, 4-β-xylanase EC 3.2.1.8., 15,000 BGU g endo-1, 4-β-glucanase EC 3.2.1.4. per g) fed to hens with 5 replicates per diet during 7-wk trial period. One unit of xylanase (FXU) is defined as the amount of enzyme that liberates 1 μmol reducing sugars from xylan, measured as xylose equivalents, under the conditions of the assay. One unit of β-glucanase (BGU) is defined as the amount of enzyme that liberates 0.27 μmol reducing sugars from β-glucan, measured as glucose equiva1072 M. Mirzaee et al. / Span J Agric Res (2014) 12(4): 1071-1081

. However, several other physical and chemical factors can influence energy available from wheat and as a result animal performance. Among them hardness (Carré et al., 2002), pelleting (Ghobadi & Karimi, 2012), starch, crude protein (CP) and ether extract contents (Steenfeldt, 2001;Svihus & Gullord, 2002) can be underlined. Parsaie et al. (2006) reported that Iranian wheat cultivars are variable in their apparent metabolizable energy (AME, 1,893 to 3,062 kcal kg -1 ), NSP (9.6 to 14.9%), CP (9.5 to 14.0%) and other chemicals content. Rafuse et al. (2005) reported that CP of five varieties of Canadian wheat varies between 11.4 and 15.5%. In a survey of 18 wheat cultivars, Kim et al. (2003) reported that starch content ranged between 58.5 and 73.7%, CP between 9.7 and 19.1%, and NSP between 7.8 and 11.0%. In addition, Mollah et al. (1983) found AME ranging between 2,670 and 3,860 kcal kg -1 of dry matter (DM) when 22 wheat samples were fed to growing broiler chickens. Moreover, some wheat cultivars considered to be of high quality have produced broilers with unexpectedly low performance (Gutiérrez-Álamo et al., 2008b).
The exogenous xylanase is used to reduce or to eliminate the negative effects of the soluble NSP on performance of young chickens (Bedford & Schultz, 1998;Choct et al., 2004;Gutiérrez-Álamo et al., 2008b;Zhang et al., 2012;Zou et al., 2013), but the effects are limited and inconclusive for laying hens. It has been reported that addition of xylanase to wheatbased layer diets improves egg production rate (Pan et al., 1998). Additionally, Pan et al. (1998) reported significant improvements in AME and feed conversion ratio (FCR) with supplementation of a commercial enzyme preparation containing β-glucanase, cellulase, and xylanase, when diets containing 80% wheat or 65% rye were fed to 22-wk-old hens. Mathlouthi et al. (2002) found that xylanase supplementation improved egg mass of laying hens fed diets containing 70% wheat and wheat-barley (49% wheat and 20% barley).
As mentioned above, enzyme supplementation can change the nutritional status and improve productive performance of birds fed wheat-included diets, but which are also closely related to the regulation of metabolism and functioning of the growth-related endocrine system. For example, triiodothyronine (T3) and thyroxine (T4) in peripheral blood of laying hens played their physiological functions in many ways such as facilitating the differentiation, growth and development of tissue, promoting the formation of protein and enzymes, increasing the utilization of carbohydrate, and enhancing the disintegration of fats (Ooi et al., 2004). Nutritional status is an important factor in the regulation of plasma hormones and intermediary metabolism in laying hens (Swennen et al., 2005;Xiao-Ying et al., 2010). So we hypothesized that the effects of enzyme supplementation on productive performance may be associated with changes in the concentration of metabolic hormones and metabolites in laying hens fed wheat-based diets.
The objective of the present study was to examine the effects of two levels of AME and enzyme supplementation on the performance, egg quality traits and selected blood parameters of laying hens fed diets based on two wheat cultivars.

Birds and experimental design
All procedures used in this 7-wk experiment were approved by the Animal Ethics Committee of Razi University and complied with the guidelines for the care and use of animals in research (Federation of Animal Science Societies, 2010). A total number of 240 65wk-old Lohmann LSL-Lite laying hens (after production peak) with similar body weight (1,450 ± 14 g) and egg production rate (80.3 ± 3.8%) were randomly distributed in 40 cages (6 hens/cage) in eight experimental groups. The hens were placed in wire-floored cages arranged in a single tier within a conventional open-sided house. The cages were located in an environmentally controlled room with the room temperature kept at 21-23°C and the photoperiod set at 16 h of light (incandescent lighting, 10-lx) and 8 h dark. As is presented in Table 1, eight experimental diets including two wheat cultivars (Marvdasht and Sardari) and two levels of AME (2,720 and 2,580 kcal kg -1 ) with or without a commercial enzyme (0.0 and 0.3 g kg -1 Grindazym ® GP 15,000: 36,000 FXU g -1 endo-1, 4-β-xylanase EC 3.2.1.8., 15,000 BGU g -1 endo-1, 4-β-glucanase EC 3.2.1.4. per g) fed to hens with 5 replicates per diet during 7-wk trial period. One unit of xylanase (FXU) is defined as the amount of enzyme that liberates 1 µmol reducing sugars from xylan, measured as xylose equivalents, under the conditions of the assay. One unit of β-glucanase (BGU) is defined as the amount of enzyme that liberates 0.27 µmol reducing sugars from β-glucan, measured as glucose equiva-lents, under the conditions of the assay. Diets were formulated to have a constant energy to protein ratio; however, the reduced-AME diets were supplemented with DL-methionine and L-lysine in a way that the ratio of total sulfur amino acids to lysine was similar to that in the normal-AME diets (Khajali et al., 2008). All the experimental diets were given in mash form, and the birds had free access to diets and water. The two wheat cultivars used in the study came from the agriculture and natural resources center of Kermanshah province, in 2009. They were selected to have relatively similar starch and CP contents, and to obtain a large range in NSP content according to previously published data (Parsaie et al., 2006). The nutrient analysis of the two wheat cultivars (Table 2) was carried out according to the standard methods of analysis (AOAC, 1995) in order to determine dry matter (DM, method 934.01), crude protein (CP, method 954.01), crude fiber (CF, method 962.09), ether extract (EE, method

Performance production and egg quality traits
Production performance of the laying hens was measured from 65 to 72 wk of age. Daily egg production per replicate cage was recorded, and at the end of the experiment, the total number of eggs laid per bird was calculated. Similarly, eggs laid per replicate cage were weighed daily and at the end of the experiment, the average egg weight per bird was calculated. Undesired eggs such as soft-shelled, cracked, and broken were also recorded daily. The generated data (number of eggs and egg weight) were used to calculate egg mass per bird (egg number in replicate × average egg weight). Feed intake and estimated AME intake were measured on a weekly basis. Data on feed intake and egg mass were used to calculate FCR (feed intake/egg mass, g g -1 ). Body weights were recorded at the beginning and the end of the experiment to determine body weight changes.
For measuring the egg quality characteristics, 3-d eggs from each replicate were collected at the end of the experimental period (wk-7) and weighed. Eggshell weight, eggshell thickness, egg specific gravity, albumen height, yolk index and yolk color were measured on 10 eggs from each treatment (2 eggs per replicate). Egg specific gravity was determined using 11 gradient saline solutions varying in specific gravity from 1.060 to 1.100 at 0.005-unit increments (Holder & Bradford, 1979). The eggshell thickness was measured using a FHK eggshell thickness gauge (Fujihira Co. Ltd., Tokyo, Japan). Haugh units were calculated as an indicator of interior egg quality. Albumen height was do-cumented at three different sites by using a spherometer, and Haugh units (Eisen et al., 1962) were calculated as follows: HU = 100 log (H + 7.57 -1.7 W 0.37 ) where H = albumin height (mm) and W = egg weight (g). Yolks were separated using an egg separator and weighed. Albumen weight was calculated by subtracting the yolk and eggshell weight from the total egg weight. The yolk index was determined as the ratio of the yolk height to the yolk width and yolk color was compared to the Roche yolk color fan, which ranges from a pale yellow at score 1 to a dark orange at score 15 (Vuilleumier, 1969).

Blood parameters
Blood samples were collected from the wing vein of six randomly selected birds per treatment (one hen per replicate) at d-35 of the experiment. The blood samples for differential counts of white blood cells (leukocytes profile) were collected into bottles pretreated with heparin, as anti-coagulant. After providing the blood smear and staining by May-Grünwald-Giemsa stain, differential counting of white blood cells (leukocyte profile) was done using light microscope (Gross & Siegel, 1983) and heterophil to lymphocyte (H/L) ratio was calculated. Blood samples for serum metabolites were collected into sample bottles containing no anti-coagulant and then centrifuged (15 min, 3,000 rpm). The sera were removed and stored at -20°C until further analysis. Serum glucose, triglycerides, total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) were analyzed using the diagnostic kit (Pars Azmun, Tehran, Iran), and enzymatic methods. Serum cortisol was analyzed with the RIA kit (Pars Azmun, Tehran, Iran) and serum T3 and T4 were measured by an ELISA kit (Pishtaz Teb, Tehran, Iran).

Statistical analysis
Data were subjected to ANOVA in a completely randomized design with 2 × 2 × 2 factorial arrangements of treatments using GLM procedure of SAS (v.9.1, SAS Inst. Inc., Cary, NC, USA). All statements of significance are based on a probability of < 0.05. The mean values were compared by least significant difference (LSD) test. The following model was considered for analysis: where Y ijkl is the characteristic that was measured; µ is the overall mean; A i is main effect of the wheat cultivar; B j is the main effect of AME; C k is the main effect of enzyme; (AB) ij is the effect of the interaction between wheat cultivar and AME; (AC) ik is the effect of the interaction between wheat cultivar and enzyme; (BC) jk is the effect of the interaction between AME and enzyme; (ABC) ijk is the three-way interaction of the wheat cultivar, AME, and enzyme; and ε ijkl is the random error term. Where the interaction effect was significant, the effects of the main factors were not discussed.

Performance production
The effects of dietary treatments on production performance of laying hens during the 7-wk period of the study are presented in Table 3. The results indicated that a decrease in the energy content of the diet had no effect on feed intake (p > 0.05), but resulting in a decrease in AME intake (p < 0.05). The AME reduction reduced egg production and egg mass (p < 0.05). However, the AME concentration of the diet did not affect egg weight. Moreover, the birds receiving a diet with a low AME content had lower body weight and higher FCR as compared with birds receiving a diet with a normal AME content (p < 0.05). Enzyme addition had no significant effect on production performance and there was no interaction between AME and enzyme on the measured performance criteria. An interaction was detected between wheat cultivar and enzyme on feed intake and AME intake (p < 0.05). Dietary enzyme addition caused an increase in feed intake and AME intake of the birds fed the diet with Sardari cultivar (p < 0.05) but had no significant effect on the feed intake or AME intake of the birds receiving Marvdasht cultivar. Nevertheless, birds receiving a diet with Marvdasht cultivar had higher feed intake and Wheat nutritive value and laying hens' performance 1075 AME intake (p < 0.05) and exhibited higher egg mass than that of those receiving a diet with Sardari cultivar (p < 0.05).

Egg quality traits
Egg quality traits of the experimental groups are summarized in Table 4. The birds receiving a diet with Marvdasht cultivar had better yolk color compared to those receiving a diet with Sardari cultivar (p<0.05). Moreover, birds receiving a diet with Marvdasht cultivar produced lower undesired eggs (p<0.05). No significant effect of dietary treatments was found on egg index, yolk index, Haugh unit, eggshell weight and eggshell thickness.

Blood parameters
As shown in Table 5, among the serum biochemical parameters (glucose, triglycerides, total cholesterol, HDL and LDL) only serum level of glucose was affected by two-way interaction between AME level and enzyme (p < 0.05). Enzyme supplementation increased serum level of glucose when birds receiving a diet with low AME level (p < 0.05) but had no significant effect on serum glucose concentration when birds receiving a diet with normal AME level. No significant effect of dietary treatments was found on serum levels of T3, T4 and cortisol hormones (Table 5). Similarly, differential counts of white blood cells did not differ among birds receiving different dietary treatments (Table 6).

Discussion
The results of the present study indicated that a decrease in the energy content of the diet from 2,720 to 2,580 kcal kg -1 had no significant effect on feed intake, but resulting in a net decrease in AME intake of 5%. This was unexpected as others (Stilborn & Waldroup, 1990;De Lange & Birkett, 2005)   ported that laying hens will increase feed intake when fed low AME diets. However, similar findings have been cited (Jalal et al., 2007). Likewise, Jalal et al. (2006) reported no significant effect of dietary AME level (2,800, 2,850 and 2,900 kcal kg -1 of diet) on feed intake in laying hens from 20 to 35 wk of age. On the other hand, they found no effect of dietary AME level on the egg production and egg mass, whereas in the present study, the AME reduction significantly reduced egg production and egg mass in the overall experimental period. Similar results have been reported by Mathlouthi et al. (2002) comparing diets with 2,650 and 2,750 kcal of AME kg -1 . In contrast, Harms et al. (2000), in Single Comb White Leghorn (SCWL) hens fed diets varying from 2,500 to 3,100 kcal of AME kg -1 , did not detect any significant difference in egg production with changes in the energy content of the diet. Moreover, in the present study the birds receiving a diet with a low AME content had significantly lower body weight as compared with birds receiving a diet with a normal AME content. The AME concentration of the diet did not affect egg weight, which is consistent with the results repor-ted by Çiftci et al. (2003b), Pérez-Bonilla et al. (2012) and Li et al. (2013). On the other hand, Bouvarel et al. (2010) analyzed data from 11 experiments conducted for the last 20 years and reported that egg weight increased 0.96 g per each 10 kcal of extra energy intake per day. The reasons for the inconsistencies in relation to the effects of an increase in AME content of the diet on egg weight are not apparent but might depend on the fat and the linoleic acid content of the diets. When the AME concentration of the diet increases, there is usually a concomitant increase in both fat and linoleic acid contents. If the linoleic acid content of the control diet is below hen requirements, an increase in AME will result in higher intake of this nutrient and increase in egg weight (Grobas et al., 2001). In the current study, the level of dietary added fat increased from 0.50 to 1.74% as the energy content of the diet increased. Grobas et al. (1999Grobas et al. ( , 2001 reported that an increase in fat content of the diet resulted in increase in egg weight. However, they suggested that laying hens require no more than 1.15% linoleic acid in the diet to maximize egg weight and that when this minimal amount of linoleic acid was met, an increase in Wheat nutritive value and laying hens' performance 1077 supplemental fat resulted in further increases in egg weight, irrespective of its linoleic acid content. The AME reduction significantly increased FCR, in agreement with most previous reports (Grobas et al., 1999;Pérez-Bonilla et al., 2012;Li et al., 2013). In contrast, Keshavarz (1998) reported no difference in FCR in SCWL hens from 18 to 66 wk of age fed diets with 2,820 or 3,040 kcal of AME kg -1 . Pérez-Bonilla et al. (2012) noted that hens given the high-energy diet may have lower feed intake but have higher energy intake than hens fed the normal or low-energy diets, but the excess of energy was used for increases in body weight rather than for improvements in egg mass production. Consequently, the efficiency of converting feed energy to egg mass was hindered when the very high-energy diet was used.
Enzyme addition had no significant effect on laying hens' performance and there was no interaction between AME and enzyme on the measured performance criteria. However, Mathlouthi et al. (2002) found that xylanase addition in laying hens fed with low-AME wheat-based diets was equivalent to an increase of at least 100 kcal of AME kg -1 . They also reported significant improvement of egg production, egg mass and FCR as a result of dietary xylanase supplementation. The experimental period could be considered among the factors responsible for differences obtained in the present study. Mathlouthi et al. (2003) reported no significant improvement in egg production, egg weight or egg mass when a commercial enzyme preparation containing xylanase and β-glucanase were added to a cornsoybean meal diet fed to 45-wk-old laying hens for 9 wk (from 45 to 54 wk of age). They concluded that enzyme supplementation might be beneficial during production peak because laying hens need high levels of nutrients to maintain body growth and high egg production. To support this view, Mirzaie et al. (2012) found that xylanase supplementation increased egg production and egg mass and improved FCR per kg of eggs throughout the experimental period (25 to 47 wk of age), with the benefits being more pronounced during the first stage of the laying period (25 to 33 wk of age).
An interesting interaction was detected between wheat cultivar and enzyme on feed intake and AME intake. Dietary enzyme addition caused an increase in feed intake and AME intake of the birds fed the diet with Sardari cultivar but had no significant effect on the feed intake or AME intake of the birds receiving Marvdasht cultivar. The greater response of Sardari cultivar to enzyme may be partly related to the NSP content of it (13.90 vs. 9.60% for Sardari and Marvdasht cultivars, respectively). According to the range provided by Parsaie et al. (2006), poor quality wheats tend to have greater responses to enzyme. Nevertheless, birds receiving a diet with Marvdasht cultivar had significantly higher feed intake and AME intake and exhibited significantly higher egg mass than that of those receiving a diet with Sardari cultivar. Pirgozliev et al. (2010) examined the effect of dietary xylanase (400, 800, 1,200, or 1,600 FXU kg -1 diet) on the availability of nutrients for laying hens when fed on wheat-rye-soybased diets and reported that the AME and nitrogen metabolizability coefficients of xylanase-supplemented diets were greater than the control diet. In addition, they reported that xylanase supplementation significantly improved the coefficients of metabolizability of indispensable, dispensable and total amino acids. The wheat cultivar significantly affected yolk color and percentage of undesired eggs. The birds receiving a diet with Marvdasht cultivar had better yolk color compared to those receiving a diet with Sardari cultivar. The higher soluble carotenoid content of the Marvdasht cultivar might be involved, which were not analyzed in the present study.
No significant effect of dietary treatments was found on egg index, yolk index, Haugh unit, eggshell weight and eggshell thickness. Typically, xylanase addition to layer feed appears to have little effects on egg quality traits. Mirzaie et al. (2012) found no effect of dietary xylanase on any of the egg quality traits, except eggshell thickness at 47 wk of age that was improved by xylanase supplementation in laying hens fed diets containing 23 to 69% wheat. Roberts & Choct (2006) reported that enzyme supplementation improved eggshell breaking strength in hens fed wheat-based diets. In contrast, Rafuse et al. (2004) observed that diets based on wheat with a mixture of xylanase and protease did not affect albumen height, or weight of yolk, eggshell and albumen when measured 3 wk (32 wk of age) after the hens began consuming the experimental diets. Likewise, Çiftci et al. (2003a) reported no effect on eggshell thickness when 30% corn was substituted by wheat in diets for SCWL hens from 27 to 43 wk of age.
The information available on the effects of AME level of wheat-based diets on egg-quality traits is scarce. Çiftci et al. (2003b) reported no effect on egg shape index, breaking strength and shell thickness when Hisex Brown laying hens fed wheat-based diets con-taining two levels of AME (2,680 and 2,790 kcal kg -1 ). Similar results were reported by other researchers (Jalal et al., 2007;Novak et al., 2008;Pérez-Bonilla et al., 2012;Li et al., 2013) who evaluated the effect of different dietary AME levels on egg quality traits in laying hens fed corn-based diets.
Thyroid hormones play an important role on the regulation of general metabolism, growth and tissue differentiation as well as gene expression (Ooi et al., 2004). According to Collin et al. (2003), enzyme addition can directly or indirectly promote the activity of deiodinase in liver and kidney tissues, and, thus, promoting the transformation of T4 into T3. However, in the present study, dietary treatments did not have any significant influence on the serum concentration of T4. Similarly, serum levels of triglycerides, total cholesterol, HDL, LDL, and cortisol hormone as well as differential counts of white blood cells did not differ among birds receiving different dietary treatments. However, serum level of glucose was affected by two-way interaction between AME level and enzyme. Enzyme supplementation increased serum level of glucose when birds receiving a diet with low AME level but had no significant effect on serum glucose concentration when birds receiving a diet with normal AME level. Enzyme addition can increase intestinal starch digestibility  and sugar absorption; however, blood glucose concentration depends upon many factors such as blood levels of insulin and glucagon.
In conclusion, the results of the present study suggest that enzyme supplementation may have a positive effect on the feed intake of laying hens when fed on wheat-based diets; however, its effect is cultivar dependent and does not necessarily mean that enzyme supplementation always benefit production. Further, the results do not support the idea that laying hens tend to increases feed intake when fed low-AME diets.