Fp 428

Proximate composition analysis of the diets and whole fish was performed by the following methods: dry matter, by drying at 105 °C for 24 h; ash, by combustion at 550 °C for 12 h; crude protein (N × 6.25), by a flash combustion technique followed by gas chromatographic separation and thermal conductivity detection (LECO FP428); fat, after petroleum ether extraction, by the Soxhlet method; total phosphorus, according to the ISO/DIS 6491 method, using the vanado-molybdate reagent; gross energy, in an adiabatic bomb calorimeter (IKA).

Chemical analyses were run in duplicate, following the AOAC⁵² procedures. Fresh gonads were analysed for dry matter after drying at 105 °C for 24 h, and samples of freeze-dried gonads were analysed for: ash content by incineration in a muffle furnace at 500 °C for 6 h (Nabertherm L9/11/B170, Germany); crude protein (N x 6.25) by a flash combustion technique followed by a gas chromatographic separation and thermal conductivity detection (LECO FP428, USA) and gross energy in an adiabatic bomb calorimeter (IKA C2000, Germany). Total lipids were determined following the method described by Folch, et al.^{53 (link)} with dichloromethane-methanol (2:1) and gravimetric determination.
The fatty acid methyl esters (FAME) contained in total lipid extracts were transesterified by acidic methylation^{54 (link)}, as described by Campos, et al.^{55 (link)}. To each sample was added 1 mL of internal standard solution (1 mg C23:0/1 mL hexane; C23:0, Matreya LLC, USA). FAME were analysed in duplicate, using a Shimadzu GC-2010 Plus gas chromatograph (Shimadzu Europe GmbH, Germany), equipped with a flame-ionization detector and an Omegawax 250 capillary column (30 m x 0.25 mm i.d. x 0.25 µm film thickness; Supelco, Bellefonte, USA). FAME were identified by comparing their retention times with known standards and quantified as mg.g⁻¹ of dry biomass, using the internal standard C23:0.

Total nitrogen was determined in the samples based on the Dumas Method (Dumas, 1831 ). Samples were dried, and ground or sieved prior to analysis. The samples were combusted in a sealed system. Nitrogen compounds released were reduced to N₂ gas, which was measured by a thermal conductivity cell using the LECO FP428.