Omegawax 250

We analyzed a pool of feed and supplemental FO to evaluate the FA profile (Table 2). Semen samples for lipid analysis were collected on days 120 of each period and stored at -80ºC until analysis.
Samples for lipid extraction were analyzed following the method described by Folch et al. (20 (link)). Once lipids were obtained, they were saponified with potassium hydroxide dissolved in ethanol for 45 minutes at 80ºC, then acidified with a 0.5 ml hydrochloric acid concentrate. The acids were esterified with boron trifluoride at 64ºC for 1.5 hours. FA composition was determined by gas liquid chromatography with a 30 mm capillary column (Omega Wax 250, Supelco, Bellefonte, PA, USA). Temperature was programmed for a linear increase of 3°C per minute from 175 to 230°C. The chromatographed peaks were identified by comparing their retention times with standards.

Blood sample were centrifuged at 3000 rpm for 10 min at 4°C and then separated into plasma or serum. Alpha-tocopherol was measured using high-performance liquid chromatography. Total cholesterol was determined by the cholesterol dehydrogenase (UV-End) method. LDL-cholesterol and HDL-cholesterol were determined by a direct method. We calculated non-HDL cholesterol as the difference between total and HDL cholesterol. TG was determined by an enzyme method. The remnant like particles (RLP) -cholesterol was determined by the immune adherence method. Cholesterol ester transfer protein activity (CETP) was measured using a commercially available ELISA kit (Daiichi-kagaku, Tokyo Japan). Apolipoproteins (apo A-1, B, C-3 and E) were measured by the turbidimetric immunoassay (TIA) system. Apo B-48 was measured by the chemiluminescent enzyme immunoassay (CLEIA). Serum fatty acids composition was measured by gas–liquid chromatography. Briefly, total lipids in the serum were extracted using the Folch procedure and fatty acids were then methylated with BF₃/methanol. Transesterified fatty acids was then analyzed using a gas chromatograph (GC-17A; Shimadzu, Kyoto, Japan) with a capillary column Omegawax 250 (Supelco, Bellefonte, PA). Measurements of sd-LDL-cholesterol were performed using sd-LDL-EX “SEIKEN” (Denka Seiken, Tokyo, Japan).

On enrollment in the baseline study, venous blood was collected early in the morning after fasting for at least 12 h. Blood samples were centrifuged at 3500 × g for 15 min. The serum was refrigerated before analysis for fatty acid content. Serum DHA, EPA, and arachidonic acid (ARA) concentrations were measured by gas-liquid chromatography at a clinical laboratory (SRL, Tokyo, Japan). Briefly, total lipids in the serum were extracted using the Folch procedure, and fatty acids were then methylated with BF3/methanol. Transesterified fatty acids were then analyzed using a gas chromatograph (GC-17A; Shimadzu, Kyoto, Japan) with a capillary column (Omegawax 250; Supelco, Bellefonte, PA, USA). The weights of DHA and EPA (g/mL) as fatty acid concentrations were identified by comparison with known standards. Intra- and inter-assay precision and accuracy values (coefficient of variation [CV]) were 3.2 and 5.8 CV% for ARA, 2.7 and 6.9 CV% for EPA, and 1.9 and 6.9 CV% for DHA, respectively [18 (link)]. In the statistical analyses, serum EPA, DHA, ARA concentrations, and the EPA/ARA ratio, which predict future cardiovascular events [19 (link)], were selected as serum fatty acid factors.

The profile of volatile organic compounds (VOCs) produced by L. paracasei 85 and L. buchneri 93 were determined using an Agilent 7890bsystem (Santa Clara, CA, USA) coupled with Agilent 5977 Inert XL MSD apparatus. The system was equipped with a fused-silica capillary column coated with a polar stationary phase (OMEGAWAX™250, 30 m × 0.25 mm × 0.25 μm, Supelco, Bellefonte, PA, USA). The bacterial culture with or without ZEN treatment was inoculated with Tryptic Soy Agar (TSA) medium (Sigma-Aldrich, Steinheim, Germany). The vials were placed at an angle into the incubator. The VOCs were extracted with 50 μm polydimethylsiloxane (PDMS)/divinylbenzene (DVB) fiber (Supelco, Bellefonte, PA, USA) at 37 °C for 50 min. The absorbed molecules were desorbed into the injection port for 5 min at 250 °C. VOCs were separated with the following program: held 40 °C for 5 min, heated at 5 °C/min to 185 °C, held for 1 min, heated at 5 °C/min to 200 °C, held for 10 min, and then heated at 10 °C/min to 240 °C. Helium was used as the carrier gas. The MS interface and the ion source were maintained at 250 and 230 °C, respectively. Acquisition was performed in electron impact mode (70 eV) with the mass range of 30–300 m/z. Compounds were identified by matching mass spectra with the NIST14.1 library [2 (link)]. The analyses for each sample were carried out in triplicate.

The total lipids were quantified following the method of Folch et al. (60 (link)), modified by using dichloromethane:methanol (2:1) instead of trichloromethane:methanol (2:1). The fatty acids were transesterified to fatty acid methyl esters by acid-catalyzed methylation (61 (link)). Non-adecanoic acid (C19:0, Matreya LLC, State College, PA, USA) was added as an internal standard. Fatty acid methyl esters were analyzed by gas chromatography, using a Shimadzu GC-2010 Plus gas chromatograph (Shimadzu Europe GmbH, Duisburg, Germany) equipped with a capillary column (Omegawax 250, 30 m × 0.25 mm × 0.25 μm; Supelco, Bellefonte, PA, USA) and a flame-ionization detector. The carrier gas was helium at 1.30 ml min⁻¹, with a split ratio of 1:100, and the injection volume was 1.0 μl. The initial column temperature of 150°C was held for 7 min, increased at 3°C min⁻¹ to 170°C and held for 25 min, and then increased at 3°C min⁻¹ to 220°C and held for 30 min. The injector and detector temperatures were 250 and 260°C, respectively. Fatty acids were identified by comparing retention times with those of commercially available standards (Supelco 37 Component FAME Mix, BAME Mix, PUFA No.1, PUFA No.2, PUFA No.3, Sigma-Aldrich Co. LLC; GLC-110 Mixture, Matreya LLC) and quantified by using the internal standard (C19:0). Analyses were run in duplicate.