Animals, diet, and experimental design
The experiment was conducted following animal welfare regulations at the Veterinary Control and Research Institute of Elazig, Turkey, with all procedures approved by the Institutional Animal Care and Use Committee. A total of 360 ten-day-old Japanese quail (Coturnix coturnix japonica) were randomly divided into 12 groups in a 2x2x3 factorial design (2 environmental temperatures x 2 levels of dietary PUFA x 3 levels of dietary genistein), with each group containing 30 birds subdivided into five replicates with six birds each. The birds were kept in a temperature-controlled room at either 22±2°C for 24 h/d (thermoneutral, TN) or 34±2°C for 8 h/d (08:00 to 17:00 h) followed by 22°C for 16 h (heat stress, HS) during the experimental period (from d 10 to d 45). Animals were fed a basal diet (Table 1) or basal diet supplemented with two PUFA levels at 15 or 45% of total fat (g/100 gr fat) or three genistein levels at 0, 400, or 800 mg/kg. The basal diet contained 9% added fat source as either tallow only or as a combination of tallow (ELET Inc. Elazig, Turkey), linseed oil, and fish oil (Oz-Gida Inc. Elazig, Turkey). The PUFA levels as 15 or 45% (g/g fat) for PUFA15 and PUFA45 in the diets were formulated as reported by Cortinas et al. (2004). The fatty acid compositions of the PUFA15 and PUFA45 of the diets are presented in Table 2. Genistein contained 98% aglycone form genistein and 2% starches as a carrier (Bonistein, DSM Nutritional Products, Istanbul, Turkey). Feed and water were offered ad libitum throughout the experiment.
Samples and data collection
Cumulative feed intakes and body weight gains were recorded weekly. Weight gains and feed conversion ratio (FCR: feed intake, g: weighed gained, g) was then calculated.
At the end of the study, a total of 120 animals (10 from each group and 2 from each subgroup) were randomly chosen and slaughtered to take blood and left thigh meat samples. Blood samples were taken into gel biochemistry tubes (Standard plus & Medical Co. Ltd. Germany) and centrifuged in a cooled centrifuge (Universal 320R, Hettich, Tuttlingen, Germany) at 5000 rpm for 10 minutes at 4 °C, and serum samples were obtained. All samples were stored at -70°C in the deep freezer (Hettich, Tuttlingen, Germany) until analyzed.
Analysis of fatty acids by gas chromatography
For the analysis of fatty acids, samples were extracted according to the method reported by Hara and Radin (1978). 1 g feed and left thigh samples were homogenized with 5 ml of hexane-isopropanol (3:2,v:v), centrifuged and the supernatant was taken. Fatty acid methyl esters were prepared in the hexane/isopropanol phase, and 5 ml of 2% methanolic sulfuric acid was added, mixed, and left to methylated in a 50 °C oven (Memmert, Germany). Then 5 ml of 5% sodium chloride (NaCl, Merck, Darmstadt, Germany) was added. Fatty acid methyl esters were extracted with hexane, and the hexane phase was treated with 5 ml of 2% potassium bicarbonate (KHCO3, Merck, Darmstadt, Germany). The supernatant was taken and evaporated. The residue was dissolved with 1 ml of hexane and analyzed by Gas Chromatography (Shimadzu GC 17, Japan). The column temperature program was set from 120 °C to 220 °C. Fatty acid peaks were recorded and integrated using a Hewlett-Packard 3396 integrator. Calculations were made using the GC Solution (LabSolution GC solution 2.3) package program. Fatty acids of muscle were reported as a percentage (%).
Analysis of malondialdehyde, vitamins, and genistein by HPLC
Tissue and serum MDA, vitamin and genistein levels were performed by using an HPLC (Shimadzu, Japan) with ultraviolet (UV) detector (SPD-20A), pump (LC-20AD), automatic sampling device (SIL-20A), column oven (CTO-10ASVP) units and C18 column (ODS-3, 5µm, 4.6 x 250mm, Inertsil, GL Sciences, Japan). LC Solution (LabSolution LCL solution Release 1.21) package program was used.
The malondialdehyde (MDA) and vitamin C levels of serum and thigh meat samples were determined as reported previously (Sahin et al. 2016). Briefly, 300 µl 0.5 M perchloric acid (HClO4, Riedel, Seelze, Germany) was added to the 400 µl serum sample, centrifuged, and the supernatant was taken. For tissue analyses, 0.5 g of thigh meat samples were homogenized with 1 ml ultra-pure water, 100 µl butylhydroxytoluene (500 µg/ml; 2,6-di t-butyl-p-cresol, BHT) and 1 ml 0.5 M HClO4. Then, samples were centrifuged at 5000 rpm and 4 °C for 10 minutes, and the supernatant was taken for HPLC analyses. HPLC analysis conditions included column oven temperature as 30 °C, mobile phase, 30 mM KH2PO4 and methanol (82.5: 17.5, v/v, pH 3.6), flow rate 1 ml/min, and wavelength 250 nm.
To detect serum and tissue fat-soluble vitamins A and E levels, samples were analyzed as described by Barim and Karatepe (2010). Briefly, 500 µl of extraction solution was added to the 300 µl serum sample to precipitate its proteins and extracted with 300 µl of n-hexane (Riedel, Seelze, Germany) three times and hexane phases were evaporated. The residue was dissolved with methanol. For tissue analyses, 2 ml of ultrapure water and 200 µl of BHT (500 µg / ml) were added to the 0.5 gr sample and homogenized. Then, 3 ml of extraction solution was added, mixed, and 500 µl of n-hexane was added and centrifuged at 5000 rpm at 4 ° C for 10 minutes, and the upper hexane phase was removed. The extraction with hexane was repeated three times. The residue was dissolved with 200 µl of methanol. Analysis conditions; column oven temperature was set as 40 °C, mobile phase flow rate 1 ml/min, and wavelengths 326 nm for vitamin A, and 296 nm for vitamin E.
Genistein concentrations in serum and tissue samples were determined as defined previously with minor modifications (D’Souza et al. 2005; Tacyildiz et al. 2010). 300 µl of 0.2M sodium acetate (pH 5.0, C2H3NaO2, Sigma-Aldrich, St. Louis, MO) containing 3500 units of β-glucorinidase enzyme (Sigma Chemical, St. Louis, MO) was added to 300 µl serum sample and 0.5 g tissue sample and incubated for 6 h. Then, 4400 µl of 80% methanol was added, mixed, and sonicated. The samples were centrifuged and supernatant was taken and evaporated. The residue was dissolved in methanol. Genistein levels were measured by a linear gradient using the reverse-phase separation technique on the HPLC device. Mobile phases forming the linear gradient; A mixture of 0.1% acetic acid (Merck, Germany), 5% acetonitrile and 94.9% ultrapure water, A mobile phase, 0.1% acetic acid, and 99.9% acetonitrile mixture was prepared as mobile phase B. Linear gradient applied during measurement; Initially, it was 84% A mobile phase and 16% B mobile phase, and until the 15th minute, the A mobile phase was adjusted to 30% and the B mobile phase was 70%, and continued at these rates until the 18th minute. The analysis was continued until 25 minutes by setting the mobile phase ratios to be A 84% and B 16% at 20 minutes. Analysis conditions; column oven temperature was set as 40 °C, mobile phase flow rate 1 ml/min, and wavelength 260 nm.
Statistical analysis
Quail were assigned randomly to one of 12 treatments in a 2 x 3 x 2 factorial arrangement of 2 environmental temperatures (TN vs. HS), 2 dietary PUFA levels (15 vs. 45% of total fat), and 3 genistein levels (0, 400, and 800 mg/kg). Analysis of variance was conducted using the PROC MIXED (SAS, 2004) in a complete randomized design. The linear model to test the effects of treatments on response variables was as follows:
Yijkl = m + ETi + GNj + PUFAk + (ET*GN)ij + (ET*PUFA)ik + (GN*PUFA)jk + (ET*GN*PUFA)ijk + eijkl,
where Yijkl = response variable, m = population mean, ETi = environmental temperature (i = TN and HS), PUFAk = PUFA level (i = 15 and 45%), GNj = genistein level (0, 400, and 800 mg/kg), and eijkl = residual error, N (0, 1).
Linear and quadratic responses to genistein levels were attained using the polynomial contrast option. The linear model included the time effect and its relevant interaction as repeated measures for the performance variable. Data were reported as mean ± SEM. A probability value less than 0.05 was considered significant.