An SCFA profile in ruminal liquid was performed by a gas chromatography (GC-2014; Shimadzu, Kyoto, Japan) split-injector, a flame ionization detector and a capillary column (Stabilwax®, Restek, Bellefonte, PA, USA) at 145 °C (isothermal), as described by Bueno et al. in 2020 [15 (link)]. Acetic, propionic, isobutyric, butyric, isovaleric and valeric acid (99.5% purity, Chem Service, West Chester, PA, USA) were used as quantitative external standards. The operational conditions were: injector and detector temperatures at 250 °C; helium as the carrier gas at 8.01 mL/min; hydrogen flow to the flame jet at 60 kPa; and synthetic air at 40 kPa. The samples were thawed at room temperature and centrifuged at 14,500× g for 10 min. The supernatant (800 µL) was transferred to a dry and clean flask with 200 µL formic acid (98–100%) and 100 µL of the internal standard (100 mM 2-ethyl butyric acid, Chem service, West Chester, PA, USA).
Stabilwax
Manufactured by Restek
Sourced in United States
Stabilwax is a high-performance capillary column designed for the analysis of polar compounds. It features a bonded, stabilized polyethylene glycol stationary phase that provides excellent peak shape and reproducibility. Stabilwax columns are suitable for a wide range of applications, including the analysis of alcohols, aldehydes, ketones, esters, and other polar analytes.
Automatically generated - may contain errors
Lab products found in correlation
Gc 2014, by Shimadzu (8 mentions)
Pegasus 4d, by Leco (3 mentions)
2 ethyl butyric acid, by Chem Service (2 mentions)
Rxi 624sil, by Restek (2 mentions)
Gcms tq8030, by Shimadzu (1 mentions)
Crossbond carbowax polyethylene glycol, by Restek (1 mentions)
Rtx 1, by Restek (1 mentions)
Gc 2010 plus, by Shimadzu (1 mentions)
Qp2010 ultra, by Shimadzu (1 mentions)
Ankom a 200i fiber analyzer, by ANKOM Technology (1 mentions)
Dvb carboxen pdms stableflex, by Merck Group (1 mentions)
Bicinchoninic acid protein quantification kit, by Yeasen (1 mentions)
Chromatof software version 4, by Leco (1 mentions)
Agilent 7890 gc, by Agilent Technologies (1 mentions)
Mps autosampler, by Gerstel (1 mentions)
Isovaleric acid, by Merck Group (1 mentions)
Gc 2025, by Shimadzu (1 mentions)
Formic acid, by Merck Group (1 mentions)
Carboxen 1000, by Merck Group (1 mentions)
Tedlar bag, by SKC (1 mentions)
24 protocols using stabilwax
1
SCFA Profiling by Gas Chromatography
2
GC-MS Analysis of Volatile Compounds
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The GC-MS analysis was performed using a Shimadzu GC-MS TQ8030. In order to characterize both non-polar and polar volatile compounds, two different columns have been used: a nonpolar capillary column [Crossbond 100% dimethyl polysiloxane, 30 m, 0.25 mm ID, 0.25 (jm df, RTX-1 (Restek, USA)] and a polar capillary column [Crossbond carbowax polyethylene glycol, 30 m, 0.32 mm ID, 0.25 μηι df, Stabilwax (Restek, USA)]. Injection mode was splitless at a split ratio: 40.0. Helium was used as a carrier gas at a flow rate of 1.0 mL/min. The GC oven was initially at 50 C and was held for 5 min after injection for both columns, followed by temperature ramping at 10 °C /min up to a final temperature of 230 °C and 250 °C respectively for the polar and non-polar column. The fragmentation was carried out by electron impact under a field of 70 eV. The scanning range was from 40 Da to 600 Da. The total run time was approximately 23 and 28 min for the polar and nonpolar column, respectively. Peak identifications were tentatively performed by comparison with the NIST11 library (NIST 11-MassSpectral Library, 2011 version).
3
Profiling Scent Compounds in Rose Petals
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Petals from R. hybrida 'Penny Lane' and 'Vital' at each of the five developmental stages were immersed for 24 hours in 2 mL of hexane per gram (FW) of petals at room temperature with no light to extract the scent compounds (Bergougnoux et al., 2007) (link).
The composition of the scent compounds was detected by gas chromatography (GC2010plus, Shimadzu, Japan) with a flame ionization detector and a wax column, 30 m × 0.32 mm inner diameter, 0.25-µm film thickness (Stabilwax; Restek, Bellafonte, PA, USA). The column condition was programmed for 3 min at 100°C, with an increase of 5°C min -1 to 220°C and a 10-min hold. Helium (99.999% purity) was used as the carrier gas at a flow rate of 1.0 mL•min -1 in linear mode. The sample was injected in split mode with a 5:1 ratio at 280°C. The detector was set at 280°C. To quantify scent compounds of the five developmental stages of the two cultivars, 13 standards (Sigma, USA) as the main scent compounds in roses (Baldermann et al., 2009) were analyzed (phenolics; 2-phenylethanol, 3,5-dimethoxytoluene, eugenol, and methyl eugenol;
terpenes: β-citronellol, β-ionone, geranial, geraniol, linalool, neral, nerol, (-)rose oxide, and trans-caryophyllene).
The composition of the scent compounds was detected by gas chromatography (GC2010plus, Shimadzu, Japan) with a flame ionization detector and a wax column, 30 m × 0.32 mm inner diameter, 0.25-µm film thickness (Stabilwax; Restek, Bellafonte, PA, USA). The column condition was programmed for 3 min at 100°C, with an increase of 5°C min -1 to 220°C and a 10-min hold. Helium (99.999% purity) was used as the carrier gas at a flow rate of 1.0 mL•min -1 in linear mode. The sample was injected in split mode with a 5:1 ratio at 280°C. The detector was set at 280°C. To quantify scent compounds of the five developmental stages of the two cultivars, 13 standards (Sigma, USA) as the main scent compounds in roses (Baldermann et al., 2009) were analyzed (phenolics; 2-phenylethanol, 3,5-dimethoxytoluene, eugenol, and methyl eugenol;
terpenes: β-citronellol, β-ionone, geranial, geraniol, linalool, neral, nerol, (-)rose oxide, and trans-caryophyllene).
4
Aroma Compound Extraction and GC-MS Analysis
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Extraction of aroma compounds. It was performed using adsorption and separate elution from an Isolute (IST Ltd, Mid Glamorgan, UK) ENV1 cartridge packed with 1 g of a highly cross-linked styrene-divinyl benzene (SDVB) polymer. Treatment of samples and GC-MS analysis were performed as described previously 6 in a Shimadzu-QP 2010 ULTRA (Tokyo, Japan) mass spectrometer equipped with a Stabilwax (30 m × 0.25 mm i.d., 0.25 µm film thickness, Restek) capillary column.
5
Analyzing Rumen Fluid Composition and Microbial Protein Production
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The VFA concentrations in the rumen fluid samples were determined using gas chromatography (Shimadzu GC-2014, Japan) equipped with a capillary column (Stabilwax, Restek, Bellefonte, PA, United States). The NH3-N concentration was measured using a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., China) according to a method described by Broderick and Kang (Broderick and Kang, 1980 (link)). The MCP production was determined according to the method of Makkar and Becker (1999) (link).
The feed and feces samples were dried at 60°C and ground through a 1-mm stainless steel screen. The dry matter, ash, and crude fat of the feed and fecal samples were determined according to methods 967.03, 924.05, and 920.39 of the AOAC (Cunniff, 1995 ), respectively. The neutral detergent fiber and acid detergent fiber were analyzed using an Ankom A200i Fiber Analyzer (ANKOM Technology Co., New York, NY, United States) according to the methods of Van Soest et al. (1991) (link). The total nitrogen contents of the feed and feces samples were determined according to procedure 984.13 of the AOAC (Cunniff, 1995 ).
The feed and feces samples were dried at 60°C and ground through a 1-mm stainless steel screen. The dry matter, ash, and crude fat of the feed and fecal samples were determined according to methods 967.03, 924.05, and 920.39 of the AOAC (Cunniff, 1995 ), respectively. The neutral detergent fiber and acid detergent fiber were analyzed using an Ankom A200i Fiber Analyzer (ANKOM Technology Co., New York, NY, United States) according to the methods of Van Soest et al. (1991) (link). The total nitrogen contents of the feed and feces samples were determined according to procedure 984.13 of the AOAC (Cunniff, 1995 ).
6
Rumen and Serum Biomarker Analysis Protocol
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The VFA concentrations in the rumen fluid samples were determined using gas chromatography (Shimadzu GC-2014, Japan) equipped with a capillary column (Stabilwax, Restek, Bellefonte, PA, USA). The NH3-N concentration was measured using a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., China) according to a method described by Broderick and Kang [47 (link)]. MCP production was determined according to the method of Coomassie Brilliant Blue [48 ].
Serum cortisol, triiodothyronine (T3), thyroxine (T4), IgG, IgA, IgM, tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1 (IL-1β), IL-6, lipopolysaccharide (LPS) and rumen fluid LPS were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The level of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (T-AOC) in the serum samples were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute). Serum and rumen fluid eDNA content analysis was assessed by a dsDNA HS Assay Kit for Qubit® (Kit: Shanghai Yisheng Bioengineering Institute, Shanghai, China).
Serum cortisol, triiodothyronine (T3), thyroxine (T4), IgG, IgA, IgM, tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1 (IL-1β), IL-6, lipopolysaccharide (LPS) and rumen fluid LPS were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The level of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total antioxidant capacity (T-AOC) in the serum samples were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute). Serum and rumen fluid eDNA content analysis was assessed by a dsDNA HS Assay Kit for Qubit® (Kit: Shanghai Yisheng Bioengineering Institute, Shanghai, China).
7
Ruminal Fluid Analysis: pH, VFA, and NH3-N
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Ruminal fluid samples (straining digesta from 5 different sites of the rumen) were collected on d 18 of each experimental period, before (time 0), and 2, 4, 6, 8, 10, and 12 h relative to the morning feeding. After sampling, pH of ruminal fluid was measured using a digital pH meter (MB-10, Marte Cientifica, Santa Rita do Sapucai, Brazil). Aliquots from ruminal fluid samples (1.6 mL) were mixed with methanoic acid (98-100% H 2 CO 2 , 400 µL), centrifuged at 7,000 × g for 15 min at 4°C, and the supernatant of each sample was frozen for subsequent VFA analysis. Other aliquots from ruminal fluid samples (2 mL) were mixed with 1 mL of sulfuric acid (0.5 M) and frozen for NH 3 -N analysis using the colorimetric phenol-hypochlorite method (Broderick and Kang, 1980) . Ruminal concentration of VFA was determined according to Erwin et al. (1961) using a gas chromatograph (GC-2014, Shimadzu Corporation) equipped with a capillary column (Stabilwax, Restek, Bellefonte, PA). The gases used in the analyses were helium (8.01 mL/min flow) as the carrier gas, hydrogen (pressure of 60 kPa) as the fuel gas, and synthetic air (pressure of 40 kPa) as the oxidizer gas. The steamer temperature was set at 220°C, the ionization detector flames at 250°C, and the separation column at 145°C for 3 min, which was raised by 10°C/min until it reached 200°C.
8
Analysis of Fir Oil Composition
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We examined the composition of fir oil. One microliter of fir oil was deposited into 40-mL glass vials that were sealed with tin foil. A triphase 50/30 µm DVB/Carboxen/PDMS StableFlex solid-phase microextraction (SPME) fiber for volatiles and semivolatiles with molecular weight between 40 and 275 (Supelco, Bellefonte, PA, USA) was inserted through the tin foil lid and exposed to the fir oil odor for 5 s. The SPME fiber was desorbed for 5 min at 240 °C under splitless conditions and the odor constituents were separated over 37 min on a Stabilwax (Restek, Bellefonte, PA, USA) capillary column (60 m × 0.25 mm inner diameter; 0.5 µm film thickness) using a temperature gradient from 40 to 240 °C at 7 °C/min, and hold at 240 °C for 8 min. Helium was used as a carrier gas at 2 mL/min. Identification of the compounds was performed using a Clarus 500 quadrupole mass spectrometer and turbo mass software (Perkin Elmer, Shelton, CT, USA). Linear retention times of authentic standards, when available, and mass spectra from the NIST database, were used to identify compounds.
9
Rumen Fluid Analysis and Immune Response
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The VFA concentrations in the rumen fluid samples were determined using gas chromatography (Shimadzu GC-2014, Japan) equipped with a capillary column (Stabilwax, Restek, Bellefonte, PA, USA). The NH3-N concentration was measured using a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., China) according to a method described by Broderick and Kang [89 (link)]. MCP production was determined according to the method of Coomassie Brilliant Blue [90 ]. Serum lactate dehydrogenase (LDH), creatine kinase (CK), cortisol (COR), adreno cortico tropic hormone (ACTH), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), malondialdehyde (MDA), IgA, IgG, IgM, interleukin-1β (IL-1β), IL-4, IL-6, and tumor necrosis factor-α (TNF-α) were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
10
Fatty Acid Profiling of Longissimus Thoracis
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Samples of Longissimus thoracis muscle were taken from the 10th to 13th rib [4 (link)]. Muscle samples were packed into polyethylene bags and stored at −30 °C until analysis. The fatty acid (FA) profile of the fat extracted from the internal part of the muscle was analyzed by gas chromatography (GC) of the methyl-esters derived from the fatty acids of the samples. The fat methylation process was performed by mixing 10 mg of sample with 0.25 mL of a sodium methoxide solution 5% (w/v) in methanol and heating it at 60 °C for 30 min [24 (link)]. The methyl-esters were analyzed and quantified by GC using a Stabilwax (Restek Corp., Bellefonte, PA, USA) capillary column (60 m × 0.25 mm × 0.25 µm; Varian 3400, Palo Alto, CA, USA). The FA peaks were identified through comparison with known reference methyl-esters (Supelco 37 Component FAME Mix, 47885-U, Sigma-Aldrich Co., Bellefonte, PA, USA).
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