2.6. Chemical Analysis of Diets

The nutritional levels of diets were analysed in duplicate according to the rules of the Official Association of Analytical Chemists [22]. Analyses of DM (method 930.15), ashes (method 942.05), crude energy (EB), total phosphorus (P, method 946.05), calcium (Ca, method 978.02) and nitrogen (N) was made using the Kjeldahl digestion and distillation procedure (TE-036/1, Tecnal, Brazil) and the crude protein was calculated as N × 6.25 (method 990.03), and crude fibre (CF) [23]. The ether extract (EE) was analysed in duplicate according to the rules of the Official Association of Analytical Chemists [24].

Lipids were extracted from samples of the diets according to the Folch method using chloroform/methanol at 2:1 v/v [25]. A sample of approximately 1 g was used to determine the FA composition of the diets offered. A frozen sample was homogenised in 20 mL of a solution of chloroform/methanol (2:1) using a Turrax homogeniser, disintegrator and emulsifier. After that, an aliquot of lipid extract was methylated according to [26]. The quantification of lipids used was by GC 2010 gas chromatograph (Shimadzu Corp., Kyoto, Japan) with a SP-2560 capillary column (100 mL × 0.20 mm D with 0.02 μm film thickness; Supelco, Bellefonte, PA, USA). Initially the temperature was adjusted to 70 °C for 4 min and then increased by 13 °C/min until reaching 175 °C and then maintained for 27 min. The temperature was then increased by 4 °C/min to 215 °C and then maintained for 31 min. Hydrogen was used as carrier gas at a flow rate of 40 cm³/s.