Db fatwax ui

The composition of fatty acids in murine epididymal adipose tissue and serum was measured by gas chromatography–mass spectrometry (GC/MS), Agilent 7890B/5977B (Agilent Technologies, Santa Clara, CA, USA). A total of 15 µg of epididymal adipose tissue and 25 µl of serum were methylated with a fatty acid methylation kit (nacalai tesque, Kyoto, Japan). The final product was loaded onto a Varian capillary column (DB-FATWAX UI; Agilent Technologies). The capillary column used for fatty acid separation was CP-Sil 88 for FAME (100 m × an inner diameter of 0.25 mm × membrane thickness of 0.20 μm, Agilent Technologies). The column temperature was maintained at 100°C for 4 min and then increased gradually by 3°C/min to 240°C and held there for 7 min. The sample was injected in split mode with split ratio 5:1. Each fatty acid methyl ester was detected in selected ion monitoring mode. All results were normalized to the peak height of the C17:0 internal standard (24 (link)).

The soleus muscle composition of free fatty acids was determined by using the same gas chromatography-mass spectrometry (GC/MS) system as that used for SCFA and amino acid quantification. and an Agilent 7890B/7000D (Agilent Technologies, Santa Clara, CA, USA). Soleus muscle (15 µg) was methylated with a fatty acid methylation kit (Nacalai Tesque, Kyoto, Japan). The final product was loaded onto a Varian capillary column (DB-FATWAX UI; Agilent Technologies). The capillary column used was CP-Sil 88 for FAME (length, 100 mm; membrane thickness, 0.20 μm; inner diameter, 0.25 mm; Agilent Technologies) for fatty acid separation. The column temperature was maintained at 100 °C for four minutes, increased gradually at a rate of 3 °C/min to 240 °C, and maintained for seven minutes. The samples were injected in the split mode at a ratio of 5:1. The fatty acid methyl esters were detected in the selected ion monitoring mode. Each result was normalized to the peak height of the C17:0 internal standard.

The fatty acid composition of the murine liver and serum samples was analyzed using gas chromatography-mass spectrometry on an Agilent 7890B/5977B System (Agilent Technologies, Santa Clara, CA, USA). Liver tissue (15 µg) and serum (25 µL) samples were methylated using a Fatty Acid Methylation Kit (Nacalai Tesque, Kyoto, Japan). The final product was loaded onto a Varian capillary column (DB-FATWAX UI; Agilent Technologies). The capillary column used for fatty acid separation was CP-Sil 88 for FAME (100 m × an inner diameter of 0.25 mm × membrane thickness of 0.20 μm, Agilent Technologies). The column temperature was maintained at 100°C for 4 min, increased gradually by 3°C/min to 240°C, and then held there for 7 min. The sample was injected in split mode with a split ratio of 5:1. Each fatty acid methyl ester was detected in the select ion-monitoring mode. All results were normalized to the peak height of the C17:0 internal standard (14 (link)).

Basal culture media and CFSs were assessed for SCFAs content after a liquid–liquid extraction method with methyl tert-butyl ether (MTBE). SCFAs were then analyzed using a gas chromatography-mass spectrometer GC-TOFMS (BT, Leco Corp., St. Josef, MI, USA), as previously described^{24 (link)}. Briefly, the column adopted was a 30 m DB-FATWAX-UI (Agilent Technologies, Santa Clara, CA), while high-purity helium (99,9999%) was used as the carrier gas. One μL of each sample was injected in splitless mode at 250 °C. The program was as follows: the initial temperature was 40 °C for 2 min, then ramped 7 °C/min up to 165 °C, 25 °C/min up to 240 °C, and maintained for 5 min. The electron impact ionization was applied at 70 eV. The ion source temperature was set at 250 °C, the mass range at 40 to 300 m/z with an extraction frequency of 32 kHz and an acquisition rate of 200 spectra/s.
The lactic acid content was measured by using the Megazyme Lactic Acid Assay Kit (NEOGEN Europe Ltd), following the manufacturer’s instructions.

In order to determine the fatty acid profile of the extracted oil, a trans-esterification for converting fatty acids to fatty acid methyl esters (FAME) was carried out using the methylation reagent trimethylsulfonium hydroxide (TMSH). For this, between 0.02 g and 0.03 g of the extracted oil was weighed into a flask and filled up to 1 mL with hexane. Aliquots of 100 µL of this solution were pipetted into a GC vial, adding 50 µL of TMSH and subsequently 50 µL of ethyl acetate. The sample was then mixed and allowed to equilibrate for 30 min before proceeding with the GC analysis. A total of 1μL of each sample solution was injected into the GC System, an Agilent 7890B equipped with a DB-FATWAX UI (Agilent Technologies Inc. G3903-63008) column (30 m × 0.25 mm × 0.25 μm). Fatty acids were eluted under a programme with a starting temperature of 100 °C, heating at 15 °C min⁻¹ to 200 °C (held for 50 min), followed by a second heating step at 50 °C min⁻¹ to 240 °C (held for 10 min), a flow rate of 1.2 mL/min (32 cm/s), injector temperature of 240 °C, and FID-detector temperature set to 245 °C. The extraction was conducted from two biological replicates, each in technical duplicates.