2.2. Egg Fatty Acid Analysis. Three

2.2. Egg Fatty Acid Analysis. Three samples of eggs fromeach
dietary group were obtained randomly for fatty acid analysis
at the end of the sixth week of the feeding study. Fatty acid
methyl esters (FAMEs) were prepared using methanol/2N
NaOH and extracted with n-hexane and then analyzed by
gas chromatography (GC). For this purpose, samples were
injected into a Supelcowax 10 column (60 m-0.25mm i.d.,
0.25 lm film thickness; Supelco, Bellefonte, PA, USA) coated
with polyethylene glycol. The column was connected to a
Hewlett-Packard 5890 Series II (Little Falls,Wilmington, DE,
USA) GC equipped with a flame-ionization detector. The
oven temperature was programmed as follows: 180∘C for
2min,which increased to 200∘Cat 2∘C/min, held at 200∘C for
a further 10min, and then increased to 215∘C at 2∘C/min and
kept there for 10 min. The injector and detector temperatures
were 200∘C and 250∘C, respectively. Helium was used as the
carrier gas at a flow rate of 1.5 mL/min. FAME identification
was based on retention times compared with thoseof standard
FAMEs. C17:0 was used as an internal standard.
2.3. Statistical Analysis. One-way analysis of variance
(ANOVA) was carried out to determine the effects of marigold
flour on egg production, egg weight, feed conversion
ratio, egg yolk fatty acid composition, and egg quality
parameters in laying chickens. Significance between individual
means was identified using the Tukey’s multiple
range test. Mean differences were considered significant at