Agilent 6890 5973n

GC-MS study was conducted on an Agilent 6890-5973N USA (Agilent Technologies; Hewlett-Packard Company 2850 Centerville Road Wilmington, DE 19808-1610 USA), with the gas chromatograph set with an HP1 capillary column TG-5MS polydimethylsiloxane (30 m length × 250 µm diameter × 0.25 µm film thickness) interfaced with Hewlett Packard (5973N) mass selective detector. Parameters were; initial temperature was 70 °C (0 min) and last temperature increased to 200 °C with final time 10 °C min⁻¹ while, inlet temperature was 250 °C and split ratio was 10:1. MS quadruple and thermal aux temperatures were 150 and 285 °C, respectively. The MS scan range was 35–520 units and helium was used as the carrier gas with a flow rate of 1.0 mL min⁻¹. Compounds were identified and verified by comparing with gas chromatography mass spectrum literature or data provided by the National Institute of Standards and Technology Mass Spectral database Wiley/NIST.1998.1 [63 (link)]. To calculate the comparative yield of compounds raw data was followed based on gas chromatography (GC) areas with a FID correction factor.

Samples of feed and tissues were extracted using a mixture of chloroform and methanol (v/v 2:1) to obtain fatty acid methyl esters using the ISO 5509 method [15 ]. After phase separation, 2 mL of the upper layer was transferred to a sample injection bottle. The fatty acid composition of the samples was then measured by capillary gas chromatography (Agilent 6890-5973N; Agilent Technologies, Santa Clara, CA, USA). The chromatograph was equipped with a capillary column (30.0 m×0.25 mm i.d. and polyethylene glycol-film, thickness 0.25 μm; Chrompack, Palo Alto, CA, USA) and a flame ionization detector. To optimize separation, the initial oven temperature was set at 80°C, held for 1 min then increased to 220°C at 4°C/min up and held there for 14 min. The carrier gas (helium) flow rate was 0.9 mL/min. Both the injector and detector were set at 230°C and the split ratio was 50:1. The peaks were identified using purified standards and different fatty acids were quantified according the peak area, and expressed as percentage of total fatty acids detected.

The determination of fatty acid composition in muscle was performed according to a previously described method with some modifications (28 (link)). The muscle samples were dried by vacuum freeze-drying, and then, the total lipids of the liver were extracted from the dried muscle samples according to Folch’s procedure (29 (link)). Fatty acid methyl esters were prepared by referring to the method of Liu et al. (30 (link)) and then determined using GC-MS (Agilent 6890-5973N, Agilent Technologies, United States) equipped with a DB-WAX capillary column (60 m, 0.25 μm i.d., film thickness, Agilent Technologies, United States). The results were presented as the percentage of each fatty acid concerning the total fatty acids.