*2.3. Formulas*

Infant formulas from SF and EF were based on cow's milk and were provided by ORDESA Laboratories, S.L., Barcelona, Spain. The experimental formula was characterized by the presence of LCPUFAs AA (*Mortierella alpine*) and DHA (fish oil), milk fat globule membrane (MFGM) components {10% of total protein content (wt:wt}, symbiotics, gangliosides, nucleotides and sialic acid (Nutriexpert® factor). Fat blend, OMEGA FATS (palm, palm-kemel, rapeseed, sunflower, oleic sunflower fatty acids) and BETAPOL (Palm, palm-kemel, sunflower and rapeseed oils) were present in both formulas. For more information regarding the lipid profile, consult the Supplementary Materials (Table S2). Additionally, both formulas followed the guidelines of the Committee on Nutrition of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN), and the international and national recommendations for the composition of infant formulas. Nutritional composition of infant formulas is shown in Table 1.

**Table 1.** Standard and Experimental Infant Formula Nutrition Facts per 100 mL.


Powder diluted in water (13.5%) \* With alpha-lactoalbumin (15% of total protein) and with Immunoglobulins. # With 22% of palmitic acid in beta position.

#### *2.4. Cheek Cell Sample Collection*

Cheek cell samples were collected at 3 months of age to analyze FAs and genotype FADS SNPs. Samples were collected 1 h after feeding by scraping the inside of the cheeks with a Rovers® EndoCervex-Brush®. The tip of the brush was transferred and jolted in a cryotube with distilled water, shaking the tip before removing the brush. After centrifugation, the supernatant was carefully discarded. The cell pellets were stored at −80 ◦C until analysis.

#### *2.5. Cheek Cell Fatty Acid Analysis*

A modified version of the method described by de la Garza et al. [30] was used to analyze FAs from the glycerophospholipid fraction. Methanol with butylated hydroxytoluene (BHT) was used for lipid extraction. FA reactions with sodium methylate in methanol (25 wt% in methanol) and boron trifluoride methanol solution (14% *v*/*v*) were used to obtain FA methyl esters (FAMEs). Next, the FAs were separated by rapid gas chromatography following the method developed by Bondia et al. [31]. The system consisted of a Shimadzu GC-2010 gas chromatograph (Kyoto, Japan) equipped with a "split-splitless" injector, an automatic injector with AOC-20i-AOC-20s sampler and a flame ionization detector (FID). The separation of the methyl esters from the FAs was carried out with a fast capillary column of fused silica VF-23ms (10 m × 0,10 mm internal diameter, 0,10 μm film thickness) coated with a stationary phase 100% cyanopropyl-phenyl-methyl-polysiloxane of varian (Palo Alto, CA, USA). The methyl esters of the FAs were identified by comparison with the retention times of standards, FAME-37 and PUFA-2 animal. Quantification was done by normalization, expressing the results in relative amounts (percentage). Enzyme activities were estimated as product:precursor indexes of individual FAs as follows: GLA:LA and DGLA:LA indexes for D6D enzyme activity, and the AA:DGLA index for D5D enzyme activity. Additionally, AA:LA and eicosapentaenoic acid (EPA):ALA indexes were analyzed.

#### *2.6. SNP Selection and Genotyping*

SNPs within the FADS gene were selected if they were documented in previous studies for comparison purposes [19,27,32–38] and if their minor allele frequency (MAF) was higher than 10%. DNA material was extracted from infant cheek cells. FADS1 (rs174537, rs174545, rs174546, rs174548, and rs174553) and FADS2 (rs1535, rs174570, and rs2072114) SNPs were genotyped from 2.5 μl of DNA mixed with 2.5 μL of 2X TaqMan® OpenArray® Genotyping Master Mix. Analysis was then performed with 4 μL of the mixture in a microplate using the TaqMan® OpenArray® genotyping technology. Analyses were carried out at the *Autonomous University of Barcelona* (UAB) using the QuantStudio 12 k Flex® instrument (ThermoFisher) and the corresponding OpenArray® SNP Genotyping Analysis software.
