The soft tissue was removed and then homogenized with a grinder (Retsch Grindomix GM200, Düsseldorf, Germany; 5,000 rpm; material: PP cup and stainless steel knifes), vacuum packed and frozen at −80 °C.
The frozen samples were freeze-dried for 48 h at −50 °C under low pressure (approximately 10To extract total amino acids (protein bound free), 14.8–15.8 mg of sample were placed in 10-ml ampoules with 3 ml of 6 M HCl (Merck) containing 0.1 % phenol (Merck), according to the method described by AOAC ().
The moisture content of the upper phase was removed using anhydrous sodium sulfate (Panreac).
A 2 µL aliquot from the upper phase was then injected onto a gas chromatograph (Varian Star 3800 Cp, Walnut Creek, CA, USA) equipped with an autosampler and fitted with a flame ionization detector at 250 °C for FAME analysis.
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FA levels are expected to increase with greater food availability and throughout gametogenesis and decrease with spawning.