**3.4. Determination of hepatic antioxidant concentration**

The other two diets were the control diets supplemented with a standardised combination of PE (XTRACT 6930; Pancosma S.A., Geneva, Switzerland) including 5% carvacrol, 3% cinnamaldehyde and 2% capsicum oleoresin (100 grams per tonne, respectively, i.e. WC + PE; MC + PE). The PE was added in powder form to the diets and all diets were fed as mash. The diets did not contain any coccidiostat or antimicrobial growth promoters, prophylactic or

Day old male Ross 308 broiler chickens were purchased from a commercial hatchery and reared in floor pens littered with wood shavings. The temperatures were kept at 32°C during the first 2 days on birds arrival and were gradually reduced to 20°C by 21 days of age. A standard lighting programme following breeder's recommendations (Aviagen Ltd., Edinburgh, UK) for broilers was used. Access to the feed and the water was *ad libitum*. At 17 days of age, two birds from each pen were transferred to a pen with wire mesh floor and excreta samples were collected for four consecutive days from each pen, immediately dried at 60°C and then milled for further analyses. The birds were weighed on a per-pen basis and the average bird

In the experiments where dietary net energy (NE) was determined, at the end of the study, all chickens were killed by cervical dislocation and the carcass of the birds, including intestine, blood and feather, from each pen were frozen and then minced, thoroughly mixed and

In the experiments where the hepatic antioxidant content was determined, at 21 days of age, one bird from each pen was randomly selected, stunned/killed and the liver was collected and

In the experiments where the gene expression was measured, at 21 days of age one bird from each pen was randomly selected, stunned/killed and the left caecal tonsil was collected and stored in RNAlater® (Sigma-Aldrich, USA) at −70°C prior to analysis of the relative expres-

The experimental diets and the excreta were milled (0.75 mm mesh) and analysed further. Dry matter (DM) was determined by drying samples in a forced draft oven at 105°C to a constant weight. Crude protein (6.25 × N) in samples was determined by dry combustion method [33] using a LECO (FP-528 N, Leco Corp., St. Joseph, MI). Oil (as ether extract) was extracted with diethyl ether by the ether extraction method [33], using a Soxtec system (Foss UK Ltd.). The GE value of the samples was determined in a bomb calorimeter (model 6200; Parr Instrument

The N-corrected apparent metabolisable energy (AMEn) of the diets was calculated as

feed intake (FI), weight gain (WG) and gain: feed ratio (GF) were determined.

sampled, and used for following analysis and calculations.

108 Phytochemicals - Source of Antioxidants and Role in Disease Prevention

stored at –20°C prior to analysis of antioxidant contents.

**3.2. Chemical analysis of diets and excreta**

Co., Moline, IL), using benzoic acid as the standard.

**3.3. Dietary available energy determination**

other similar additives.

sion of selected genes.

described by [34].

Concentration of antioxidants in liver was determined by high-performance liquid chromatography (HPLC) [37, 38]. In brief: approximately 300 mg of liver samples were mixed in 0.7 ml 5% sodium chloride solution, then 1 ml ethanol was added and samples homogenised. During homogenisation, 2 ml hexane was added. Then samples were centrifuged and the hexane phase, containing the vitamin E and coenzyme Q10 were collected. Extraction with hexane was performed twice, and the combined phase was evaporated under nitrogen and re-dissolved in a mixture of dichloromethane–methanol (1:1, v/v).

Vitamin E (α-, γ- and ϭ-tocopherols) was determined as previously described [39] using an HPLC system (Shimadzu Liquid Chromatograph, LC-10 AD, Japan Spectroscopic Co Ltd., with a Jasco Intelligent Spectrofluorometer 821-FP) fitted with a Spherisorb, type S30DS2, 3 mm C18 reverse phase HPLC column, 15 cm × 4.6 mm (Phase Separations Limited, UK). Chromatography was performed using a mobile phase of methanol–water (97:3, v/v) at a flow rate of 1.05 ml/min. Fluorescence detection of tocopherols and tocotrienols involved excitation and emission wavelengths of 295 and 330 nm, respectively. Standard solutions of tocopherols in methanol were used for instrument calibration and tocol was used as an internal standard.

Coenzyme Q10 was analysed in the same extract by injecting 50 ml into the same HPLC system, but using a Vidac 201TP54 column (5 μm, 25 cm × 4.6 mm) and mobile phase ethanol– methanol–2-propanol (70:15:15, v/v) and flow rate of 1.5 ml/min with a diode array detection at 275 nm [40]. Coenzyme Q10 (Sigma, Poole, UK) standard was used for calibration.
