A total of three-hundred twenty 33-week-old Hy-Line Brown laying hens (Gallus domesticus) were evenly assigned to four experimental diets of 2,750, 2,850, 2,950, and 3,050 kcal AME_n/kg in pens with floors covered with deep litter of rice hulls. There were four replicates of each treatment, each consisting of 20 birds in a pen. A commercial type basal diet was formulated to meet or exceed the nutrient recommendations of the National Research Council [8] for layers (Table 1). The diets were analysed in duplicate to determine the crude protein (CP), crude ash, ether extract, and the gross energy (GE) content. Nitrogen for CP analysis was measured using a nitrogen analyzer (NS-2000, Leco Corp., St. Joseph, MI, USA). Dry matter was determined according to the method of Association of Official Analytical Chemists [9], and GE was determined using a Parr adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, IL, USA). The diet samples were subjected to analyses of ether extract [9]. The diets were offered daily and were stored at 4°C during the course of the trial. During the 10-week experimental period, hens were provided with feed and water ad libitum and were exposed to a 16 h:8 h (light:dark) schedule. The temperature and humidity of the laying house was maintained at 20°C±3°C and 65% to 70% respectively.

Hen-day egg production rate, floor eggs, broken egg production rate and egg weight were recorded daily, whereas feed intake and the feed conversion ratio were recorded weekly. Egg mass was calculated as per Hayat et al [10].

Ten eggs per replicate were randomly collected at the end of the each week. Eggshell strength, eggshell thickness, egg yolk color, and Haugh units (HU) were measured. Eggshell strength was measured by the Texture Systems Compression Test Cell (model T2100C, Food Technology Co., Ltd., Rockville, MD, USA) and expressed as units of compression force exposed to units of eggshell surface area (kg/cm²). Eggshell thickness is defined as the mean value of measurements at 3 different locations on the egg (air cell, equator, and sharp end) and was measured with a dial pipe gauge (model 7360, Mitutoyo Co. Ltd., Kawasaki, Japan) and calculated using the following formula [11]:

Egg yolk colour was evaluated by the Roche Yolk Color Fan (Hoffman-La Roche Ltd., Basel, Switzerland; 15 = dark orange; 1 = light pale). Haugh unit values were calculated using a micrometer (model S-8400, Ames, Walthman, MA, USA) with the following formula described by Eisen et al [12]:

Where W is egg weight, and H is albumen height.

At the end of the 10-week feeding trial, 2 birds/replicate (i.e., 8 birds per treatment) with a body weight near the mean were selected to be euthanized by cervical dislocation. Immediately after death, a 5 mL blood sample was collected from the jugular vein of each bird using ethylenediaminetetraacetic acid (EDTA)-treated and non-EDTA treated vacutainer tubes (Becton Dicknson, Franklin Lakes, NJ, USA). The whole blood samples were kept on ice and used immediately for hematological analysis. Leukocytes (white blood cells, heterophils, lymphocytes, monocytes, eosinophils, and basophils) were analyzed using the Hemavet Multi-species Hematology System (Drew Scientific Inc., Oxford, CT, USA). The H/L ratios were determined by dividing the number of heterophils by the number of lymphocytes.

Serum samples were obtained by centrifuging the samples for 20 min at 25,000×g and 4°C and were stored at −15°C. Total cholesterol, triglyceride, asparate aminotransferase (AST), alanine transferase (ALT), and calcium in the serum were quantified using an ADVIA 1650 Chemistry System (Bayer Diagnostic, Puteaux, France).

Birds were fed the experimental diets for 8 d with a 5-d adaptation period to the diet and following 3-d excreta collection period. The marker to marker method was used to ensure total collection of excreta voided from the birds [13]. Chromic oxide (0.3%) and ferric oxide (0.3%) were added to the diet at the start of collection period and at the conclusion of the collection period, respectively. Excreta collection was started when chromic oxide was appeared in excreta, and the collection was finished when ferric oxide was appeared in excreta. Full caution was paid to prevent disturbing the birds whenever the feces were monitored. Feed intake was recorded and excreta were collected on a daily basis, and stored at −4°C be for analysis. Excreta sample were dried in a force-air oven at 60°C for 72 h and finely ground for the subsequent analysis. Diets and excreta samples were analyzed for GE using bomb calorimeter (Parr Instrument, USA). The diets and excreta samples were analyzed for dry matter (Method 934.01), CP (Method 990.93), ether extract (Method 920.39), crude ash (Method 942.05) using standard procedures of Association of Official Analytical Chemists [9]. The ATTD of GE and nutrients in the diet was calculated as: 100 − (total GE or nutrient excretion/total GE or nutrient intake)×100 [14,15].

Morphometric analysis of the ileum (from Meckel’s diverticulum to the ileo-caeco-colic junction) was evaluated according to Giannenas et al [16]. One-centimetre-long segments were taken from the centre of each part and fixed in 10% buffered formalin for morphometrical assays under light microscopy. Formalin-fixed intestinal tissues were processed, embedded in paraffin wax, sectioned at 3 μm and stained the haematoxylin-eosin method. Histological sections were examined with a Nikon phase contrast microscope coupled with a Microcomp integrated digital imaging analysis system (Nikon Eclipse 80i; Nikon, Tokyo, Japan). Images were viewed using a 4× Eplan objective (40×) to measure morphometric parameters of intestinal architecture. For this purpose, three favourably orientated sections cut perpendicularly from villus enterocytes to the muscularis mucosae were selected from each animal and measurements were carried as follows: villus height was estimated by measuring the vertical distance from the villus tip to villus-crypt junction level for 10 villi per section; crypt depth (the vertical distance from the villus-crypt junction to the lower limit of the crypt) was estimated for 10 corresponding crypts per section.

All data were analyzed by analysis of variance according to completely randomized design using the Proc MIXED procedure of SAS [17]. Outlier data were identified by the UNIVARIATE procedure of SAS, but no outliers were found. The experimental unit for all data was replicate. The LSMEANS procedure was used to calculate mean values. The orthogonal polynomial contrast test was performed to determine linear and quadratic effects of increasing AME_n concentrations in diets.

AME_n intake increased (linear, p<0.05) as the inclusion level of AME_n in diets increased. Feed intake decreased (linear, p< 0.01) as the inclusion level of AME_n in diets increased. Increasing inclusion level of AME_n in diets tended to improve hen-day egg production. Hen-day egg production of the 2,850, 2950, and 3,050 kcal/kg treatments compared to the 2,750 kcal/kg treatment trended toward a non-significant increase of 1.09%, 2.54%, and 3.51%, respectively. Feed conversion ratio was improved (linear, p<0.05) with inclusion level of AME_n in diets increased. The feed conversion ratio of the other treatments improved by 1.59%, 4.76%, and 7.16%, respectively, compared to the 2,750 kcal/kg treatment (Table 2). Floor eggs, broken egg production, egg weight, and egg mass were not influenced by an increase in the inclusion level of AME_n in diets (Table 3).

Hens eat to satisfy their energy requirements, and therefore, an increase in the energy content of the diet should decrease feed intake [18]. However, an increase in the current energy content of the diet from 2,750 to 3,050 kcal of AME_n/kg (an 11% increase) resulted in a decreased feed intake of 4.7%, resulting in a net increase in energy intake of 5.8%. Bouvarel et al [19] reviewed experiments conducted in laying hens during the last 20 yr and reported that as an average, a 10% increase in AME_n content of the diet resulted in a reduction in feed intake of only 5.5%, in agreement with the results of the current experiment. An increase in energy content of the diet is generally attained by increasing the amount of fat and supplemental fat results often in higher energy intake, probably because of less dust formation and improved palatability of the diet [19,20]. Harms et al [2] reported in Brown hens and Single Comb White Leghorns (SCWL) fed diets varying in AME_n from 2,500 to 3,100 kcal/kg, there was no significant difference in hen-day egg production with changes in the energy content of the diet. These data support the hypothesis that an excess in energy intake caused by changes in diet composition results primarily in increases in body weight gain rather than in further increases in egg mass production. Egg weight was not affected by energy concentration of the diet, consistent with data of Grobas et al [1] and Bouvarel et al [19]. Bouvarel et al [19] 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 discrepancies in relation to the effects of an increase in energy content of the diet on egg weight are not apparent but might depend on the fat and the linoleic acid content of the diet. When the energy concentration of the diet increase, there is usually an increase in both fat and linoleic acid content. Feed conversion ratio improved as the energy content of the diet increased, in agreement with most published reports [1,4,5]. Hens eat feed to satisfy their energy requirements, and therefore, high AME_n diets results in improved feed conversion ratio. Moreover, supplemental fat has been shown to reduce rate of feed passage, facilitating the contact between digesta and enzymes and improving digestibility and utilization of other nutrients such as the lipid and carbohydrate fractions of the diet. The results suggest that modern brown-egg-laying hens might not regulate accurately feed intake according to energy requirements when very high or low energy diets are used. Hens fed high AME_n diet (i.e., 3,050 kcal/kg in the current experiment) tended to overconsume energy with a positive effect on feed intake and feed conversion ratio but not in egg production and egg mass. Energy concentration of the diet did not affect the percentage of dirty, broken or shell-less egg production rate throughout the laying period, consistent with data of Grobas et al [1].

During the experiment, leukocyte concentration and blood biochemistry (total cholesterol, triglyceride, glucose, total protein, calcium, AST, and ALT) were not influenced by increasing level of AME_n in diets (Tables 4, 5). Blood parameters are good indicators of general health status, reflecting any physiological, nutritional and pathological changes occurring in the organism; however, it is important to establish appropriate reference values for these parameters [21]. Leukocyte count has also been used as a measure of immune function in birds [22]. Many factors, such as exposure to various microbes and chemicals, can cause changes in both granulocytic white blood cells [23]. The lack of adequate data on the role of increasing level of AME_n in altering blood parameters in poultry requires further research.

Nutrient digestibility from the present experiment is summarized in Table 6. Dry matter, CP, crude fiber, and crude ash were not influenced by increasing level of AME_n in diets. However, GE and ether extract were increased (linear, p<0.01) with inclusion level of AME_n in diets increased (Table 6).

Mateos et al [24] reported that supplemental fat delays the intestinal transit time of the digesta, allowing a better contact between the components of the digesta and the endogenous enzymes present in the gastrointestinal tract, which in turn could improve nutrient utilization. The fat is almost completely absorbed in the small intestine of the bird, and therefore, GE ileal digestibility was expected to be higher for the supplemental fat containing diets [25].

Intestinal morphology from the present experiment is summarized in Table 7. Villus height increased (linear, p<0.05) as the inclusion level of AME_n in diets increased. Crypth depth and villus height:crypth depth ratio was not influenced by increasing level of AME_n in diets. Development of intestinal morphology could reflect the health status of the gastrointestinal tract of an animal. New epithelial cells are produced in the intestinal mucosal crypts and migrate along with the villus to the top [26]. As the intestine is the major site of the enzyme digestion and absorption of nutrients, the efficiency of absorption and hence feed conversion ratio largely depends on the morphology of intestine [27]. A deeper crypt may indicate faster tissue turnover to permit renewal of the villus, which suggests that the host’s intestinal response mechanism is trying to compensate for normal sloughing or atrophy of villus due to inflammation from pathogens and their toxins [28]. Previous research has shown that the intestinal morphology, specifically structures such as villus, crypts and the thickness of mucosa, were altered by the composition of diet [29]. The increase in villus height and crypt depth is associated with healthy turnover of epithelial cell and active cell mitosis [30].