A total of 216 1-day-old Arbor Acres male broiler chicks (Gallus gallus domesticus L.) were obtained from a local hatchery (Da Bao Hatchery, Tai'an, China) and housed in cages in an environmentally controlled room. The brooding temperature was maintained at 35°C for the first 2 days and then gradually decreased by 2–3°C per week, according to the age of broilers (32, 33). The light regime was composed of 23 h of light and 1 h of darkness, and the ambient humidity was 40–50%. The composition and nutrient levels of the chicken diets used in the experiment are listed in Table 1. All birds received feed and water ad libitum during the rearing period. This study was approved by the Shandong Agricultural University and carried out in accordance with the Guidelines for Experimental Animals of the Ministry of Science and Technology (Beijing, China).

Broiler chickens were randomly divided into three groups, with 12 replicates per group and six chickens per replicate. Dietary treatment groups were as follows: (i) low energy diet (LED, ME = 2,900 kcal/kg), (ii) normal energy diet (control, ME = 3,200 kcal/kg), (iii) high energy diet (HED, ME = 3,500 kcal/kg). At 10 days of age, six cages of chickens in each dietary treatment were randomly assigned to receive subcutaneous injections of DEX (2 mg/kg BM/day for 3 days) or sham injection with saline.

At 10 days of age, one chick was selected from each cage for blood sample collection. At the end of the experiment (13 days of age), two chickens from each replicate were selected and sacrificed. Blood samples were obtained from the wing vein of each chicken using a heparinized syringe. Plasma was obtained after centrifugation at 400 g (4°C, 10 min) and stored at −20°C for further analysis (34, 35). After obtaining blood samples, the chickens were slaughtered through the intravenous injection of pentobarbital sodium (30 mg/kg body weight) and jugular exsanguination. The hypothalamus was collected, in accordance with the method described by Liu et al. (36), via a 4–5 mm deep incision, made parallel to the base of the brain (37). Hypothalamus samples were flash-frozen in liquid nitrogen and stored at −80°C.

The initial body weights of broilers were similar among the three groups (42.63 g in control group, 42.62 g in LED group and 42.52 g in HED group). At 10 and 13 days, broilers in each cage were weighed after 8 h of fasting, feed intake recorded, and replicate recorded values used to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed conversion rate (FCR) per group.

Plasma concentrations of various factors were measured spectrophotometrically using commercial diagnostic kits (Jiancheng Bioengineering Institute, Nanjing, P. R. China) as follows: total protein (TP; no. A045-2-2), albumin (ALB; no. A028-1-1), glucose (GLU; no. F006), uric acid (UA; no. C012-1-1), total cholesterol (TCHO; no. A111), triglyceride (TG; no. A110), high-density lipoprotein (HDL) cholesterol (no. A112), and low-density lipoprotein (LDL) cholesterol (no. A113). Plasma TP content was determined by Bradford method. Plasma ALB was measured using colorimetric method. Plasma glucose content was measured using the glucose oxidase method. Plasma UA content was determined using the colorimetric method. Plasma TCHO content was measured using the COD-PAP method. Plasma TG content was measured by the GPO-PAP enzymatic method. Plasma HDL-C and LDL were determined according to the kit instructions.

Gene expression in the hypothalamus was quantified by quantitative real-time PCR using total RNA extracted using the Trizol reagent (Invitrogen, San Diego, CA, USA). RNA integrity was assessed through agarose gel electrophoresis. The RNA was quantified by using a DeNovix spectrophotometer (DS-11; DeNovix Inc., Wilmington, DE, USA). RNA purity was verified by determining the absorbance ratio of 260 to 280 nm (OD260/280 = 1.8–2.0). mRNA was reverse transcribed into cDNA using a commercial kit (PrimeScript RT Reagent kit, TaKaRa, Dalian, P.R. China). Real-time PCR was carried out using an Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Foster, CA, USA). Each RT reaction served as a template in 20 μL PCR reaction mixtures containing 0.2 μmol/L of each primer and SYBR green master mix (Takara, Dalian, Liaoning, and PR China). Primer sequences (Table 2) were designed across exon-intron junctions using the Primer 5.0 software. Reaction conditions were as follows: pre-denaturation at 95°C for 30 s, then 40 cycles of denaturation at 95°C for 5 s, and annealing and extension at 60°C for 34 s. A standard curve was plotted to calculate the efficiency of the real-time PCR primers. Relative mRNA expression levels of genes were calculated using the 2^–ΔΔCt method and normalized to the value for β-actin expression. Results were verified using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and levels of the control + saline group were used as a calibrator. All processes were performed in accordance with previously described methods (38–40), and each sample was assayed in triplicate.

Differences between groups receiving different dietary treatments were analyzed using one-way ANOVA and the GLM procedure in Statistical Analysis Systems (SAS) software (version 8.02; SAS Institute Inc., Cary, NC). When differences among individual means were found in ANOVA tests (P < 0.05), means were compared using the Tukey's test. A two-way ANOVA model was used to analyze the main effects of diet energy, DEX treatment, and their interaction using SAS software. Results are presented as mean values and pooled standard error of the mean (SEM). For statistical analysis of ADG and ADFI, one cage was considered as one replicate. For measurement of plasma parameters and gene expression in the hypothalamus, one chick from each cage was sampled, and one replicate comprised one chick. P < 0.05 indicated statistical significance.

The effects of dietary energy levels on broiler performance are summarized in Table 3. From days 0 to 10, chicks fed with the HED had greater ADG and lower ADFI (P < 0.05) than those fed with the LED, while LED treatment resulted in higher ADFI and FCR (P < 0.05) than the control and HED treatments. The effects of DEX on the performance of broilers fed with different energy diets are presented in Table 4. ADG was decreased in response to DEX injection (P = 0.0002). The control group had a higher ADG and lower FCR than the control-DEX group (P = 0.0003). Further, ADFI in the LED group was lower than that in the control and HED groups (P = 0.0040), irrespective of DEX injection. No significant interaction of diet and DEX treatment on growth performance was detected.

The effects of dietary energy levels on the plasma parameters of broilers are presented in Table 5. HED treatment resulted in higher plasma TP, ALB, GLU, TCHO, HDL, and LDL (P < 0.05) relative to the control treatment group. UA concentration was increased in the LED group compared with that in the HED group (P < 0.05). No significant differences in plasma parameters were observed between the LED and control treatment groups. TG levels were not influenced by dietary treatment. The effects of DEX on the plasma parameters of broilers fed with different energy level diets are shown in Table 6; various plasma parameters were influenced by DEX injection. The contents of TP, ALB, TG, TCHO, HDL, and LDL in the control-DEX group were higher than those in the control group (P < 0.0001, P < 0.0001, P = 0.0740, P < 0.0001, P < 0.0001, and P < 0.0001, respectively). Further, UA content in both the LED and HED groups decreased in response to DEX (P = 0.0101), while the UA content of the LED group was higher than that of the HED group (P = 0.0036). In contrast GLU level was influenced by neither dietary energy level nor DEX injection. An interaction of dietary energy level and DEX injection was found to influence TCHO content (P = 0.0715), while no there was no evidence of an influence of such an interaction on TP, ALB, UA, GLU, TG, HDL, or LDL content.

The relative gene expression levels of AMPK and appetite-related genes are presented in Figures 1, 2, respectively. DEX treatment increased the gene expression levels of LKB1, AMPKα1, NPY, and glucocorticoid receptor (GR) in hypothalamus tissue from 13-day-old broilers (all P < 0.0001). LKB1 mRNA levels in the LED group were higher than those in the HED group (P = 0.0493). In addition, a trend of interaction was observed between DEX and the dietary energy treatments on LKB1 gene expression in the hypothalamus (P = 0.0663). Further, cholecystokinin (CCK) mRNA levels in the hypothalamus of 13-day-old broilers were not affected by either DEX or dietary energy level (P > 0.1).

Dietary energy in broiler nutrition plays a significant and central role in livestock maintenance and production (41). In this study, chicks fed a HED had higher ADG than those fed a LED, consistent with our previous findings (42). The increase in body weight induced by consumption of a HED has been widely verified in human and animal studies (21, 43–45). A LED reduces production performance, due to the low energy intake (46); however, Yuan et al. (1) has found that chicken body weight gain was not altered by dietary energy level. This controversial conclusion may be attributable to the age and the dietary energy level used in the study. Birds consume feed primarily to meet their energy requirements and increase their feed intake in response to dietary energy dilution (41, 47), consistent with our results. Chickens can control their feed intake according to changes in dietary ME concentration (48). The feed intake of broilers fed a low ME diet increased; however, this could not compensate for the poor weight gain and FCR, due to the physical limitations of feed consumption (47). In this study, as dietary energy levels increased, FCR decreased significantly. Several reports have revealed that increased dietary energy can reduce broiler FCR (45, 49, 50). Together, current data suggest that broilers can adjust their feed intake in response to dietary energy levels.

GC can evoke a particular appetite for foods rich in fat or energy (28). DEX had significant effects on ADG and FCR. This observation was consistent with the results of Lv et al. (51), who reported that DEX treatment decreases the body weight gain of broilers as FCR increases. Increased energy expenditure, protein oxidation, and reduced small intestine absorption are responsible for the suppressive effects of GC on growth rate (4, 52, 53). In the present study, the interaction between DEX and dietary energy level had no significant effect on ADG, ADFI, or FCR, similar to the findings of a previous study (4); however, Yuan et al. (1) found that chickens fed a HED had higher BW gain than those receiving a LED on corticosterone treatment, indicating the beneficial effects of a HED. Differences in dietary energy content or fat type may partially account for these discrepancies.

Serum GLU is an important energy source conducive to body tissue growth, whereas the serum ALB and TP reflect protein synthesis functions in the broiler liver, which may be associated with physiological status and growth (54). In birds, UA is the main product of nitrogen metabolism, and its content can reflect the direction of protein metabolism (55). In this study, birds fed with HED showed increased levels of plasma GLU, TP, and ALB at 10 days old. GLU in the blood can be oxidized to provide energy and channeled into pathways for fatty acid synthesis (56). The increased GLU concentration detected in the plasma of HED group chickens suggests a high rate of GLU use in fatty acid synthesis. Increased serum ALB and TP can be related to improved protein digestibility and increased availability of amino acid precursors for protein synthesis (57). In this study, at 13 days old, levels of TP and ALB were affected by treatment with DEX, consistent with the result of Lv et al. (51); however, dietary energy levels did not affect these parameters, in accordance with the findings of Yang et al. (4). The level of UA was affected by diet type and DEX treatment; however, no interaction between diet and DEX was evident. These results indicated that dietary energy level cannot alleviate the influence of stress on protein metabolism.

Lipid metabolism was also assessed in our study. Serum TG, TCHO, HDL-cholesterol, and LDL-cholesterol are key indicators of lipid metabolism balance (58–60). Increased levels of serum TCHO and HDL-cholesterol were observed in the HED group, consistent with the results of previous studies (50, 61, 62). HDL-cholesterol is responsible for the uptake of cholesterol from the peripheral tissues and blood and facilitates its transport back to the liver for catabolism (63), whereas LDL-cholesterol has the opposite function (63). Most fatty acids are synthesized in the liver and transported via LDL for storage as TG in adipose tissue (64). Therefore, in the HED group, cholesterol from the peripheral tissues was transported to the liver, as reported by Ge et al. (50), and TG was deposited in the adipose tissue. In the present experiment, DEX treatment significantly increased the levels of TG, TCHO, HDL-C, and LDL-C, regardless of the dietary energy level, as also revealed in previous studies (1, 51). A trend toward an effect of interaction of dietary energy level and DEX treatment on plasma TCHO levels was detected. This finding is consistent with previous research, revealing that the effect of GC on TG is minimal when the diet composition maintains a low lipid flux, but becomes highly significant when the dietary lipid flux increases (65).

In the CNS, the hypothalamus is vital in coordinating feed intake and regulating energy homeostasis in mammals and birds (66–68), and various orexigenic and anorexigenic neuropeptides have been identified in the hypothalamus of mammals and poultry (5). Hypothalamic NPY is a potently orexigenic neuropeptide, which can increase appetite in mammals and birds (23, 69, 70). Consistent with previous findings in rats (71) and chicks (28), our data demonstrate that GC treatment increased hypothalamic NPY levels, suggesting that appetite was stimulated. GR, which belongs to the steroid/sterol/thyroid/retinoid/orphan nuclear receptor superfamily, mediates the majority of the known actions of GC (72, 73) and regulates food intake and energy expenditure (22). In this study, GR mRNA levels were increased on treatment with DEX in both dietary energy level groups, indicating that the biological regulation of GC was strengthened. AMPK has emerged as a key molecular regulator with a pivotal role in cellular energy metabolism (74–76). In the CNS, AMPK participates in fasting, inflammation, stress, and other responses (77–80). Once activated, AMPK switches off anabolic pathways (such as fatty acid, TG, and cholesterol syntheses), in favor of catabolic pathways (such as glycolysis and fatty acid oxidation), to sustain energy homeostasis (75, 81). GC activate hypothalamic AMPK activity (82, 83), and this can lead to appetite stimulation (84). In this study, exogenous subcutaneous GC administration significantly increased hypothalamic NPY, AMPKα1, and GR gene expression, while the expression levels of these genes were not altered by dietary energy level; however, level of LKB1 were affected by diet and DEX. LKB1, a kinase upstream of AMPK, has the same function in poultry and mammals (27); LKB1 expression was reduced in DEX-treated chicks fed the HED, indicating that HED can partially reduce activation of AMPK signaling. In other words, high energy diet has the potential to inhibit the activation of central AMPK signaling by stress.