A total of 162, one-day old broiler chicks (ROSS 308) purchased from a commercial company were maintained in a wire battery cage in a room equipped with forced air ventilation system and a light/dark cycle (light on 04:00–24:00). Immediately after a day acclimation, 3-d-old birds were randomly divided into three groups; each group had six replicate cages with 54 birds. Birds were fed the soy-corn-wheat basal diet (Table 1) supplemented with none (CON), 200 mg/kg of vitamin C (ascorbic acid, purity 99%, VIC) or 100 mg/kg of vitamin E (α-tocopheryl acetate, purity 50%, VIE) until 35 days of age. A thermostat was installed in the room to monitor temperature fluctuation for the entire experimental periods. The weather in summer during experimental period showed a typical East Asian climate, with an average air temperature of 32°C to 34°C during the day and 27°C to 29°C during the night especially at the months of July and August.

All birds received the respective starter (3 to 21 days of age) and finisher (22 to 35 days of age) diets ad libitum and had free access to water nipples. Body weight and feed consumption were monitored on days 3, 21 and 35 to determine growth performance and feed to gain ratio. The animal handling procedures were followed according to the Institutional Animal Care and Use Committee (IACUC) at the Gyeongnam National University of Science and Technology, Korea.

After 35 days of age, six birds with the average body weight per group were sacrificed by cervical dislocation. Immediately after bleeding from the jugular vein, several organs including the liver, spleen and thymus were harvested and weighed. The liver tissue and serum were rapidly frozen in liquid nitrogen and kept at −70°C until further assay.

A quantification of mRNA using Real Time-PCR was carried out to assess mRNA expression of pro-inflammatory cytokines including IL-1β, IL-6, IL-18, interferon (IFN)-γ and toll like receptor (TLR)-4, and HSP70. The cDNA primers to amplify each gene are presented in Table 2. Hepatic tissue from each individual was used for the extraction of total RNA using method of RNAzol B (TRIzol Reagent, Invitron, Eugene, OR, USA). In brief, 2 mL of RNAzol solution was added to 100 mg of hepatic tissues. The tissues were thoroughly homogenized using a glass-glass homogenizer. The lysate was transferred to an Eppendorf-tube and 100 μL of chloroform added and incubated for 5 min on ice. And then, the aqueous phase was obtained by centrifugation for 15 min at 15,000×g. Isolated RNA was precipitated with the same volume of isopropanol and centrifuged for 15 min at 15,000×g. The isolated total RNA was washed with 75% ethyl alcohol, dried, and diluted with diethylpyrocarbonate-treated H₂O. The isolated total mRNA was quantified by a spectrophotometer (GeneQuant pro, Amersham, Piscataway, NJ, USA). To synthesize the first strand cDNA, 5.0 μg of total RNA were incubated with 1.0 μg of oligo dT (Invitrogen Co., Carlsbad, CA, USA) at 70°C for 5 min and 4°C for 5 min. The reaction solution was incubated at 42°C for 50 min, 90°C for 10 min and 42°C for 50 min in a reaction mixture containing 5X 1st strand buffer, 2.5 mM dNTP, 0.1 M DTT, superscript III and RT-mixture. After that, 0.5 μL of RNase H was added to RT-products to remove RNA hybridized with cDNA for 20 min at 37°C. The quantification of the target genes and internal control (RPL27) was examined using real-time quantitative PCR with SYBR green supermix (Bio-Rad, Hercules, CA, USA). Real-time PCR assay was performed under the following conditions: 5 min at 95°C, 40 cycles of denaturation at 95°C for 15 s, and annealing at 60°C for 30 s and extension 72°C for 30 s. The PCR amplification cycle at which dye fluorescence passed the selected baseline (Ct) was determined by real-time monitoring and the expression of all mRNAs were normalized by 2[deltadelta] method (Livak and Schmittgen, 2001) to calculate relative changes in gene expression.

Serum total antioxidant status (TAS) was assayed using a Randox reagent set (Randox, Antrim, UK), which was adapted to an enzyme-linked immunosorbent assay (ELISA). The determination is based on the reaction of 2, 29-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) with a peroxidase and H₂O₂ to produce the radical cation ABTS. The radical cation has a relatively stable blue-green color, which can be detected at 600 nm. Antioxidants contained in the sample give rise to the suppression of this color development based on the trolox equivalent antioxidant capacity.

Serum and hepatic microsome were used to analyze lipid peroxidation. To harvest microsomal fractions from the liver, the homogenized tissues were centrifuged at 10,000×g, and the resulting supernatant was centrifuged at 105,000×g in a Centrikon T-2080 (Kontron Instruments, Zürich, Switzerland) ultracentrifuge. The resulting pellet (microsomes) was suspended in a phosphate buffer containing 150 mM KCl (pH 7.4) to produce a protein concentration of 20mg/ml. After then, the microsomes were frozen in liquid nitrogen and kept at −80°C for further assay of lipid perodixation. The lipid peroxidation in serum and microsomes was measured by the amount of 2-thiobarbituric acid (TBA) reactive substances using a spectrophotometer at 532 nm (Bidlack and Tappel, 1973). TBA reaction is expressed as nanomoles of malondialdehyde (MDA) per milligram of protein. Protein was assayed by the BCA method (Thermo Scientific, Rockford, IL, USA) using ELISA plate reader (V_Max, Molecular Devices, Sunnyvale, CA, USA).

The effect of the dietary vitamin C or E on data was analyzed by the ANOVA Procedure of SAS (SAS Institute Inc., Cary, NC, USA). When the treatment effect was significant at p<0.05, Tukey test was applied to identify significant differences among dietary groups. The level of probability indicating a statistical difference was determined as p<0.05. Data are presented as means±SD.

Growth performance and the relative organ weights are shown in Table 3 and 4, respectively. Dietary supplementation of vitamin C or E did not affect body weight, feed intake and feed conversion in birds during summer conditions. However, the VIE group was shown to have a significant (p<0.05) increase in the relative thymus weight by 77% compared to the CON group without change in the weights of the liver and spleen. The relative weights of organs did not differ between the VIC and VIE groups.

The mRNA expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-18, and IFN-γ) and biomarker proteins (TLR-4 and HSP70) in the liver of birds fed diets supplemented with vitamin C and E are presented in Table 5. The mRNA expression level of IL-1β, IL-6, IFN-γ and TLR-4 in the VIC group was significantly (p<0.05) lower than that in the CON group. Compared with the VIE group, the VIC group resulted in significant (p<0.05) up-regulation of IL-1β, IFN-γ and TLR-4 (Table 5). The VIE group also significantly (p<0.05) increased in mRNA expression of IL-6 compared with the CON group. Furthermore, mRNA expression of HSP70 was markedly (p<0.05) lower in birds fed diet supplemented with vitamin C and E than in those fed a basal diet. Therefore, mRNA expression level of some pro-inflammatory cytokines and HSP70 in response to summer diurnal HS were significantly (p<0.05) attenuated by dietary antioxidant vitamins, particularly vitamin C.

Total antioxidant status (TAS) and lipid peroxidation (MDA) in birds fed diets supplemented with vitamin C and E during summer HS are shown in Figure 1 and 2, respectively. TAS in serum was significantly (p<0.05) higher in the VIC group compared with the CON group (Figure 1). Moreover, dietary supplementation with vitamin C or E to birds subjected to summer HS markedly (p<0.05) decreased lipid peroxidation (MDA) in serum or liver compared with the CON group (Figure 2). There was no difference in TAS and MDA between the VIC and VIE groups.