Chloroform methanol

At the end of the experiment, two broilers from each pen were slaughtered and the meat was trimmed and prepared for fatty acid profile and peroxide value measurement. The breast muscle was trimmed and stored at − 20 °C. Next, the meat sample (50 g) was mixed in a blender with 150 ml of Chloroform-methanol (Merck) in a 1:2 ratio for a minute. Then, 50 mL of sodium chloride (0.88%) was added to the mixture. Potassium chloride was used for dewatering, the aqueous phase was collected and methanol- potassium chloride 0.88% (v/v: 1/1) was mixed with the aqueous phase. The final solution was dried at 35 °C, after that, the residual solvent was evaporated under nitrogen gas pressure to obtain pure oil. Eventually, the fat was obtained for the measurement of peroxidation. To measure meat peroxidation, chloroform–acetic acid (25 ml) was added to one g of extracted fat in a ratio of 2:3. Then, one mL of potassium iodide was added to the mixture, which was stored for five min in the dark. The mixture was tittered with normal 1% potassium thiosulfate^{6 (link)}. Peroxidation value was calculated based on the amount of peroxide (meq/kg extracted fat), according to the American Oil Chemists Society²² . The fatty acid profile of the meat was analyzed by gas chromatography of the method as described by Ghasemi-Sadabadi^{6 (link),27 (link)}.

The total lipids were extracted from the 10 mg of lyophilized biomass with a chloroform-methanol (2:1 v/v) solvent mixture (Merck, Darmstadt, Germany) using a procedure similar to the Folch’s method [34 (link)]. Fatty acid methyl esters (FAMEs) were produced from the extracted lipid by a transesterification reaction. Briefly, methanol was added to the extracted lipid with sulfuric acid as a catalyst and a transesterification reaction was allowed to occur at 100°C for 10 minutes. After the reaction, 1 ml of deionized water was added and the organic phase was separated from water phase by centrifugation at 4000 rpm for 10 minutes. A total of 1 ml of chloroform containing 0.5 mg of heptadecanoic acid (C17:0; Sigma Aldrich, St. Louis, MO, USA) was added to each tube as an internal standard. The FAMEs in organic phase were analyzed by gas chromatography (HP5890, Agilent, Santa Clara, CA, USA) with a flame ionized detector (FID) and INNOWAX capillary column (Agilent, Santa Clara, United States, 30 m × 0.32 mm × 0.5 μm).

Total lipids were extracted from liver samples with chloroform/methanol (2:1, Merck, Darmstadt, Germany) with roughly 20 times the sample weight and frozen overnight at −20 °C. Neutral and polar lipids of the sample extracts were separated by solid phase extraction before analysis of the FA composition, as described by Sissener et al. [75 (link)]. Nonadecanoic acid (19:0) was added to all samples as an internal standard for quantitative determination. Briefly, extracts were filtered and evaporated, then saponified and methylated with BF₃ in methanol. FA separation was conducted using the AutoGC (Autosystem XL, Perkin Elmer Inc., Waltham, MA, USA) fitted with a flame ionisation detector. For integration, Chromeleon^® version 7.2 (Thermo Scientific, Waltham, MA, USA) was used.

We used the modified method of Moilanen [31 (link)], that is itself a modification of the method described by Folch [32 (link),33 (link)]. Fatty acids extraction and preparation of fatty acid methyl esters(FAME) from red blood cells (RBC) and tissue samples were carried out as previously described [16 (link),34 (link)]. Briefly, total lipids from phospholipids of RBC membranes were extracted by adding 0.9 mL of an acidified salt solution (H₂SO₄ 2 × 10⁻⁴ M, NaCl 0.1%). For fatty acids extraction from tissues, about 20 mg wet tissues were homogenized with 0.8 mL of ice cold 0.9% NaCl. All samples received 5.0 mL of chloroform:methanol (2:1, v/v) (Sigma-Aldrich, Milan, Italy) and the samples were mixed thoroughly and centrifuged at 1000× g for 10 min. The lower layer, containing fatty acids, was removed with care, replaced in a new tube and dried by a centrifugal evaporator (Bio-Rad, Milan, Italy). The FAME were obtained by adding toluene and BF₃·MeOH 14% (Sigma-Aldrich, Milan, Italy) and incubating for 2 h at 80 °C. After the addition of toluene and 5% aqueous sodium chloride solution, the samples were centrifuged at 470× g for 10 min. Fatty acid methyl esters contained in the upper layer of the tubes, were collected and transferred into a vial and analyzed.

Liver lipids were extracted by following the protocol developed previously [46 (link)]. Liver tissues were homogenized in 0.15 M NaCl (Sigma-Aldrich, St. Louis, MO, USA), and the tissue pulps were mixed with chloroform/methanol (2:1, v/v) (Sigma-Aldrich). After centrifugation at 400× g for 5 min, the supernatants containing liver lipids were collected. Immediately, the supernatants were measured with an enzymatic colorimetric assay (Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) to examine the levels of hepatic cholesterol and triacylglycerol.