Star chromatography software

Total lipid content of female tissues, ova and swim-up fry was quantified gravimetrically after extraction by dichloromethane/methanol (2:1, v/v), containing 0.01% of butylated hydroxytoluene (BHT) as antioxidant, according to Folch et al. [31 (link)]. Neutral (NL) and polar lipid (PL) fractions were separated on silica cartridges (Sep-Pak, Waters, Ireland), according to Juaneda and Roquelin [32 (link)].
Fatty acid methyl esters (FAME) were prepared by acid-catalyzed transmethylation, using boron trifluoride according to Shantha & Ackman [33 ]. FAME were then analyzed in a Varian 3900 gas chromatograph equipped with a fused silica DB Wax capillary column (30m x 0.25 mm internal diameter, film thickness 0.25 μm; JW Alltech, France). Injection volume was 1 μl, using helium as carrier gas (1 ml/min). The temperatures of the injector and the flame ionization detector were 260°C and 250°C, respectively. The thermal gradient was as follows: 100–180°C at 8°C/min, 180–220°C at 4°C/ min and a constant temperature of 220°C for 20min. Fatty acids were identified with reference to a known standard mixture (Sigma, St Louis, MO, USA) and peaks were integrated using Varian Star Chromatography Software (Star Software, version 5). The results for individual FA were expressed as percentage of total identified FA methyl esters and as quantities (g/100g tissue) for ARA, EPA and DHA.

Fatty acid profiles of 6 individual spawns per diet and of a pool of sperm per diet were analyzed. Fatty acid methyl esters (FAME) were prepared from lipid extracts by acid-catalyzed transmethylation, using boron trifluoride according to Shantha and Ackman (1990) (link). FAME were then analyzed in a Varian 3900 gas chromatograph equipped with a fused silica DB Wax capillary column (30 m × 0.25 mm internal diameter, film thickness 0.25 μm; JW Alltech, France). Injection volume was 1 μl, using helium as carrier gas (1 ml/min). The temperatures of the injector and the flame ionization detector were 260°C and 250°C, respectively. The thermal gradient was as follows: 100–180°C at 8°C/min, 180–220°C at 4°C/min and a constant temperature of 220°C for 20 min. Fatty acids were identified with reference to a known standard mixture (Sigma, St. Louis, MO, United States) and peaks were integrated using Varian Star Chromatography Software (Star Software, version 5). The results for individual FA were expressed as percentage of total identified FAME.

Quantitative extraction of total fat was performed with chloroform and methanol in a ratio of 2:1 (v/v) [38 (link)]. Total extractable fat content was determined by weight and expressed as gram fat/100 g milk. Transesterification of fatty acids to form methyl esters (FAME) was performed with 0.5 N NaOH in methanol and 14% boron trifluoride in methanol [39 (link)]. The FAME were quantified with a Varian 430 GC, with a flame ionization detector and a fused silica capillary column, Chrompack CPSIL 88 (100 m length, 0.25 mm ID, 0.2 μm film thickness), and column temperature of 40–230 °C (hold 2 min; 4 °C/min; hold 10 min). The FAME in hexane (1 μL) was injected into the column by a Varian 4800 Autosampler, (Varian Inc. Walnut Creek, CA, USA) with a split ratio of 100:1. The injection port and detector were maintained at 250 °C. Hydrogen was used as carrier gas at 45 psi with nitrogen as makeup gas. Chromatograms were recorded by Varian Star Chromatography Software (Version 6.41). Hendecanoic acid (C11:0) was used as internal standard, after it was established that it was not detected in the samples under study. Identification of FAME was by comparison of the relative retention times of FAME peaks from samples with those of standards obtained from Supelco (Supelco 37 Component FAME Mix 47885-U with addition of C18:1c7, C18:2c9t11, C19:0, C22:5).