Stabilwax da

The nutritional characteristics (ADF, NDF, starch, crude protein, soluble protein, and ethanol soluble carbohydrates) of samples of fresh forage were analyzed at Cumberland Valley Analytical Services (Waynesboro, PA, USA) using wet chemistry methods (https://www.foragelab.com/Resources/Lab‐Procedures). Volatile fatty acids (VFA), lactic acid, ethanol, and pH were also analyzed at Cumberland Valley Analytical Services. Briefly, each fermented silage sample was mixed, and a 25 g wet sample was taken and diluted with 200 ml deionized water. After incubation for 2 h in the refrigerator, the sample mixture was then blended for 2 min and filtered through a coarse (20–25 µm particle retention) filter paper. For VFA quantification and ethanol, 3 ml of extract was filtered through a 0.2 µm filter membrane, and a 1.0 µl subsample was injected into a Perkin Elmer AutoSystem gas chromatograph (Model 710, Perkin Elmer, USA) equipped with a Restek column packed with Stabilwax‐DA (Restek, USA). For lactic acid quantification, a 1:1 ratio of extract to deionized water was placed in a YSI 2700 Select Biochemistry Analyzer to determine lactic acid. pH was measured with a Mettler DL12 Titrator (Mettler‐Toledo, USA) using 0.1 N NaOH to a pH of 6.5.

Fecal acetate, propionate and n-butyrate were extracted using ether by the method described previously [18 (link)]. The fecal samples were injected onto a gas chromatography (GC-2014, Shimadzu, Tokyo, Japan) fitted with a glass capillary column (0.25 mm, 30 m Stabilwax-DA, Restek Corp., Bellefonte, PA, USA) and a flame ionization detector by the method described previously [18 (link)].

Fecal short-chain fatty acid concentrations were analyzed using gas chromatography at the Agricultural, Food and Nutritional Science chromatography core facility as previously described [10 (link)]. Briefly, 800 μL of 0.1 N hydrochloric acid and 200 μL of 25% phosphoric acid were added to approximately 0.2 g of stool. The contents were vortexed until fully homogenized and centrifuged at 5000× g for 15 min or until a clear supernatant was obtained. An internal standard solution (150 mg of 4-methyl-valeric acid, S381810, Sigma-Aldrich), 5% phosphoric acid, and supernatant was then added to glass chromatography tubes and stored at −80 °C prior to analysis. Samples were analyzed with a gas chromatograph (Bruker SCION 456-GC, Bruker Corporation, Billerica, MA, USA) using a 30 m × 0.53 mm inner diameter × 0.5 μm film thickness capillary column (Stabilwax-DA, Restek Corporation, Belefonte, PA, USA).

We based the profiles of fatty acids (FAs) in the diets of the experimental period on a previous analysis (Table 1) [14 (link)], which evaluated the total lipidic content using a modification [26 ] of the method proposed by Hartman and Lago [27 (link)]. The FA profile was determined using a gas chromatograph (GC) (Shimadzu GC-2010, Nishinokyo, Japan) with an AOC-5000 auto-injector and flame ionization detector (FID). We used a Restek Stabilwax-DA (Bellefonte, PA, USA) column (fused silica) (30 m × 0.25 mm; 0.25 µm), and both injector and FID operated at 250 °C. The peaks of methyl esters were identified by comparison of their retention times in the column with the retention time of the standards of FA methyl esters, and the quantification was determined according to the American Oil Chemists′ Society (AOCS) Ce 1e-91 method and using an area correction factor [28 (link)].
Based on the composition of free FAs, we assessed the nutritional quality using three different indices: the atherogenic index (AI) (Equation (1)), the thrombogenic index (TI) (Equation (2)), [29 (link)] and the hypocholesterolemic:hypercholesterolemic (HH) ratio (Equation (3)) [30 (link)].
$\mathrm{AI} = \frac{{\mathrm{C}}_{12:0}+ 4 \times { \mathrm{C}}_{14:0} +{ \mathrm{C}}_{16:0}}{{{\displaystyle \sum }}^{\text{}}\mathrm{MUFA} + {{\displaystyle \sum }}^{\text{}}\mathrm{n}6 + {{\displaystyle \sum }}^{\text{}}\mathrm{n}3}$
$\mathrm{TI} = \frac{{\mathrm{C}}_{14:0}+{ \mathrm{C}}_{16:0} +{ \mathrm{C}}_{18:0}}{0.5 \times {{\displaystyle \sum }}^{\text{}}\mathrm{MUFA}+0.5 \times {{\displaystyle \sum }}^{\text{}}\mathrm{n}6+3 \times {{\displaystyle \sum }}^{\text{}}\mathrm{n}3+\left(\mathrm{n}3/\mathrm{n}6\right)}$
$\mathrm{HH} = \frac{{\mathrm{C}}_{18:1\mathrm{cis}9}+{ \mathrm{C}}_{18:2\mathrm{n}6} +{ \mathrm{C}}_{20:4\mathrm{n}6}+{ \mathrm{C}}_{18:3\mathrm{n}3}+{ \mathrm{C}}_{20:5\mathrm{n}3}+{ \mathrm{C}}_{22:5\mathrm{n}3}+{ \mathrm{C}}_{22:6\mathrm{n}3}}{{\mathrm{C}}_{14:0}+{ \mathrm{C}}_{16:0}}$

The VFA analysis was carried out as previously described [15 ]. The VFA profile was analyzed by using gas chromatography (Trace 1300, Thermo Scientific, Waltham, MA) equipped with a flame ionization detector (FID), AS 1310 series automatic liquid sampler, and a 30 m × 0.53 mm internal diameter column (Stabilwax-DA, Restek, Restek Corporation, Bellefonte, PA). Helium was supplied as a carrier gas at a flow rate of 14.5 mL/min, and run time was set for 15 min. The temperature of the injector port and detector port was set at 200°C and 240°C, respectively. Trimethyl acetate (TMA) was used as an internal standard. Data handling and processing were performed on ChromeleonTM 7.2 software (Thermo Scientific, Waltham, MA).

Human stool samples analysed for short-chain fatty acid (SCFA) content will be freeze-dried and subsequently analysed using a gas chromatograph. The samples will be weighed (~ 200 mg of dry matter) and a 10-fold dilution with physiological NaCl saline (1.8 mL) will be produced. Following vortexing (2 min, Vortexer MS 3 basic, IKA-Werke GmbH & Co. KG, Staufen, Germany), an aliquot of 200 μL will be mixed with 0.36 M HClO₄ (280 μL) and 1 M NaOH (270 μL). The solution will be lyophilised at − 35 °C (alpha 1–4 LSC, CHRIST, Osterode am Harz, Germany). The obtained lyophilisate will be dissolved in 100 μL 5 M HCOOH and 400 μL acetone and centrifuged (5 min at 4000×g, RT; Pico 17, Thermo Electron LED GmbH, Langenselbold, Germany). Concentrations of the SCFA will be determined in the supernatant using a GC-2010 Plus gas chromatograph (Shimadzu Deutschland GmbH, Duisburg, Germany) equipped with flame ionisation detection and a thin-film capillary column Stabilwax®-DA 30 m × 0.25 mm × 0.5 μm (Restek, Bad Homburg, Germany). The samples will be spread out by split injection using the auto-sampler AOC-20s/I (Shimadzu Deutschland GmbH). GCsolution Chromatography Data System (Shimadzu Deutschland GmbH) will be used for data processing. For the determination of the SCFA, an external standard (Supelco™ WSFA-1 Mix, Supelco Sigma-Aldrich Co., Bellefonte PA) will be used.