## 2 Materials and Methods
### Nutritional Composition and Fatty Acid Profile of N. oceanica, Hen Diets, and Eggs
#### 2.3.4 Fatty Acid Profile  
Fatty acid methyl esters (FAME) of eggs, microalgae, standard and supplemented feeds were obtained by direct acid transmethylation of samples [30]. Briefly, samples (50 mg) were mixed with 2 mL of H\({}_{2}\)S\({}_{0}\) (2%, \(v/v\)) in methanol and heated during 2 h at 80 \({}^{\circ}\)C. After the addition of 1 mL of MilliQ water and 2 mL of hexane, mixtures were vortex (1 min) and centrifuged (Eppendorf, Centrifuge 5810 R) at 123\(\times\)_g_ during 5 min. Finally, the hexane layer was collected and analyzed by gas chromatography (GC). GC analysis were carried out in a Finnigan Ultra Trace gas chromatograph, equipped with a Thermo Tr-FAME capillary column (60 m \(\times\) 0.25 mm ID, 0.25 \(\upmu\)m film thickness); an auto sampler AS 3000 from Thermo Electron Corporation and a flame ionization detector (FID). The temperatures of the detector and injector (splitless) were 280 \({}^{\circ}\)C and 250 \({}^{\circ}\)C, respectively. Owen temperature was set at 100 \({}^{\circ}\)C for 1 min, followed by an increase at 10 \({}^{\circ}\)C/min to 160 \({}^{\circ}\)C (maintaining for 10 min) and a second increase at 4 \({}^{\circ}\)C/min to 235 \({}^{\circ}\)C (during 10 min). Helium was used as carrier gas at a constant flow of 1.2 mL/min. Air and hydrogen were supplied to the detector at flow rates of 350 mL/min and 35 mL/min, respectively.  
Fatty acids were identified by comparison of its retention time with those of Supelco 37 standard mixture. Eicosapentaenoic (EPA), alpha-linolenic (ALA) and docosahexaenoicenoic (DHA) fatty acids were quantified by the external standard method, using a calibration curve (linear regression) constructed with a serial dilution of Supelco 37 mix: Area = 4 \(\times\) 10\({}^{8}\)\(\times\) [EPA] (\(\upmu\)g/mL)-8 \(\times\) 10\({}^{8}\), R = 0.990; Area = 2 \(\times\) 10\({}^{8}\)\(\times\) [ALA] (\(\upmu\)g/mL)-7 \(\times\) 10\({}^{8}\), R = 0.989; Area = 4 \(\times\) 10\({}^{8}\)\(\times\) [DHA] (\(\upmu\)g/mL)-9 \(\times\) 10\({}^{8}\), R = 0.981. Results were expressed as mg of FA per 100 g of sample (mg/100 g), taking into account the FAME concentration, the volume of hexane used to extract the FAME and the samples mass.  
Aiming to evaluate if eggs could be labeled as a source of n-3 (40 mg EPA + DHA per 100 Kcal) or as high in n-3 (80 mg EPA + DHA per 100 Kcal) (CE n\({}^{\circ}\) 1924/2006), Equation (1) was applied, based in the amount of Kcal supplied per gram of protein, lipids, and carbohydrates. The maximum value of carbohydrate content (1%) was considered for the calculation [31].  
\[EPA+DHA=\frac{(EPA+DHA)}{(4\times protein+9\times lipids+4\times carbohydrates)} \tag{1}\]  
where \(EPA\) + \(DHA\) is expressed as mg/100 g; protein, lipids and carbohydrates content expressed as %.  
The efficiency of n-3 PUFA incorporation in eggs, was estimated by Equation (2) as described by [32]:  
\[Efficiency\left(\%\right)=\frac{(ALA_{Wx}+EPA_{Wx}+DHA_{Wx})-(ALA_{C}+EPA_{C}+DHA _{C})}{(ALA_{SDF}+EPA_{SDF}+DHA_{SDF})-(ALA_{SPF}+EPA_{SPF}+DHA_{SPF})}\times 100 \tag{2}\]  
where fatty acids content is expressed as mg/100 g; \(Wx\)--eggs from hens fed with supplemented diet for \(x\) weeks; \(C\)--control eggs; \(SDF\)--standard feed; \(SPF\)--supplemented feed.