Plasma concentrations of NEFA and β-hydroxybutyrate were determined by enzymatic analyses using the Wako NEFA-HR (2) (Wako Chemicals GmbH, Neuss, Germany) and the β-Hydroxybutyrate LiquiColor® kit (Stanbio Laboratory, Boerne, TX, USA), respectively. Spectrophotometric measurements were performed for both NEFA and β-hydroxybutyrate, using a Cobas Mira S Chemistry Analyzer (Roche, Basel, Switzerland).
Wako nefa hr 2
Manufactured by Fujifilm
Sourced in Spain
The Wako NEFA-HR (2) is a lab equipment product for the measurement of non-esterified fatty acids (NEFA) in biological samples. It is a quantitative, enzymatic colorimetric assay used for the determination of NEFA concentrations.
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Lab products found in correlation
5 protocols using wako nefa hr 2
1
NEFA and β-Hydroxybutyrate Quantification
2
Postpartum Blood Sampling Protocol
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Blood samples were collected from the coccygeal vessels (8.5-mL clot accelerator tubes, Becton Dickson, Franklin Lakes, NJ). Immediately, samples were centrifuged at 3000 x g for 20 min and serum was stored frozen (-20°C) until further analysis. Serum was analyzed for NEFA concentration at the Animal Endocrine and Metabolism laboratory, Veterinary Faculty, Montevideo, Uruguay. Non-esterified fatty acid (NEFA) concentrations were measured by colorimetric assays on an A25 autoanalyzer (© Biosystems S.A., Barcelona, Spain) using commercial kits: Wako NEFA-HR (2), Wako Pure Chemical Industries Ltd., Osaka, Japan. The inter-assay coefficient of variation (CV) for commercial quality controls was less than 10%.
At approximately 1 week postpartum (5 to 8 DIM), a second blood sample was taken in K2- EDTA 6.5-mL tubes (Becton Dickson, Franklin Lakes, NJ) from the coccygeal vessels and sent immediately to the laboratory for total and differential WBC count. The total WBC count was determined using an automated haemocytometer (Sysmex XT1000, Roche Diagnostic, CA, USA) and cell morphology was assessed by microscopic examination of blood smears. The inter-assay CV for commercial quality controls was less than 5%.
At approximately 1 week postpartum (5 to 8 DIM), a second blood sample was taken in K2- EDTA 6.5-mL tubes (Becton Dickson, Franklin Lakes, NJ) from the coccygeal vessels and sent immediately to the laboratory for total and differential WBC count. The total WBC count was determined using an automated haemocytometer (Sysmex XT1000, Roche Diagnostic, CA, USA) and cell morphology was assessed by microscopic examination of blood smears. The inter-assay CV for commercial quality controls was less than 5%.
3
Colorimetric Assay for NEFA
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Non-esterified fatty acid concentrations were determined at the Animal Endocrine and Metabolism Laboratory, Veterinary Faculty, Montevideo, Uruguay. Colorimetric assays were performed on an A25 autoanalyzer (© Biosystems S.A., Barcelona, Spain) using commercial kits: Wako NEFA-HR (2), Wako Pure Chemical Industries Ltd., Osaka, Japan. The inter-assay coefficient of variation (CV) for commercial quality controls was less than 10%.
4
Prepartum Body Condition Assessment and NEFA Analysis
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At d -10 to -7 from the expected calving date (enrollment), BCS was assessed (Ferguson et al., 1994) (link) and recorded by each of the veterinary technicians. At the same time, blood samples were collected from the coccygeal vessel (8.5-mL clot accelerator tubes, Becton Dickson). Control and PEG animals were blood sampled again within 24 h after calving (i.e., when PEG cows also received the second treatment), for further determinations beyond the aim of this report. Blood samples were centrifuged at 3,000 × g for 20 min and serum was stored frozen (-20°C) until further analysis for NEFA concentrations at the Animal Endocrine and Metabolism Laboratory, Veterinary Faculty, Montevideo, Uruguay. Nonesterified fatty acid concentrations were measured by colorimetric assays on an A25 autoanalyzer (Biosystems S.A.) using commercial kits: Wako NEFA-HR (2) (Wako Pure Chemical Industries Ltd.), as reported before (Barca et al., 2021a,b) . Laboratory personnel were blinded to treatment status.
5
Plasma Metabolite Assay Protocol
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The plasma FFA levels were measured using the enzymatic colorimetric method assay (Wako NEFA-HR2, Wako Diagnostics Inc. Richmond, U.S.A.) on an automatic analyzer (BML Inc., Tokyo, Japan) [13] . The minimum detectable level was 0.0014 mEq/l and the mean intra-assay and inter-assay coefficients of variation (CV) were 0.64% and 1.27%, respectively. The plasma levels of lactate and pyruvate were measured by respectively by means of the oxidase system on an auto-analyzer (Hitachi Inc. Tokyo, Japan). Intra and inter CV of plasma levels of lactate and pyruvate were 1.2%, 2.1% and 0.8%, 0.8% respectively [14] . The levels of total ketone bodies and β-hydroxybutyrate (βOHB) were measured by enzyme circling methods produced by Kinos Inc. Tokyo Japan. The CV of plasma levels of these substances were under 3.0% [15] . Acetoacetate was calculated by total ketone bodies minus βOHB. The other biochemical analyses were done using standard laboratory procedures.
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