Fatty acid composition determination
Total fat from the diets and egg yolks were extracted using the cold extraction using the chloroform-method cold extraction method for total lipid according to AOAC method 983.23(AoAC 2005) as modified by Phillips and others(1997) and similar to the methods by Coorey and others(2012).
Samples(5 g) were weighed into a 500 mL centrifuge bottle and 0.5 mol sodium acetate(32 ml), methanol(80 mL), and chloroform(40 ml) were added.
Centrifuge bottles were then placed in a Ratek swB20 shaking water bath(Ratek Instruments, Boronia, VIC, Australia) at 25 oC and shaken for dded to the centrifuge 30 min.
Chloroform(40 mL) was then added to the centrifuge bottles and shaken again for a further 30 min.
Deionized water(40 mL) was added and samples were shaken for another 30 min.
Subsequently samples were centrifuged at a speed of 2300 rpm for 10 min.
The bottom organic layer containing the fat was carefully siphoned off, placed in a beaker, and left overnight in a fume hood to evaporate the chloroform.
Eggs collected on the last day of each week(week 1 to week 4) were used to determine fatty acids composition.
Yokes were separated using a hand held yoke separator before lipids extraction using a gram of sample, thus, the sample to solvents ratio adjusted accordingly.