After semen collection and analysis, blood samples were drawn from the cephalic vein, transferred into serum gel tubes with clot activator (Greiner Bio-one, Kremsmünster, Austria), and placed at 4 °C for 30 min to allow clot formation. Tubes were then centrifuged at 2000× g for 5 min and sera were immediately stored at −80 °C until analysis in an external specialized laboratory (Algemeen Medisch Laboratorium, Sonic Healthcare Benelux, Antwerp, Belgium). Serum AMH levels were quantified by electrochemiluminescence immunoassay (ECLIA) using the Elecsys AMH Plus immunoassay on the cobas e411 analyzer (Roche Diagnostics International Ltd., Rotkreuz, Switzerland). Results were determined via a calibration curve generated by 2-point calibration and a master curve provided by the manufacturer. The analyzer automatically provided the AMH concentration and controls were performed for each analysis. Samples with AMH concentrations above the measuring range (>23 mg/L) were diluted to obtain a definite value. The intra- and inter-assay precision were ≤1.3 and ≤4.1%, respectively. The limits of blank, quantitation, and detection were 0.007 ng/mL, 0.030 µg/L, and 0.010 µg/L, respectively. The method was standardized against the Beckman Coulter AMH Gen II ELISA assay.
Amh gen 2 elisa assay
Manufactured by Beckman Coulter
Sourced in Germany
The AMH Gen II ELISA assay is a quantitative immunoassay for the measurement of Anti-Müllerian Hormone (AMH) in human serum and plasma. AMH is a glycoprotein produced by the granulosa cells of the ovary and has been shown to be a useful marker for ovarian reserve.
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3 protocols using amh gen 2 elisa assay
1
AMH Quantification by Electrochemiluminescence
2
Plasma AMH Level Quantification
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Plasma anti-Müllerian hormone (AMH) concentrations were analysed using the AMH Gen II ELISA assay according to the manufacturers instructions (Beckman Coulter, Sinsheim, Germany). The detection limit of the assay was 0.16 ng/ml; the intra-assay coefficient of variation was 5.3%.
3
Plasma AMH and Metabolic Assays in Cows
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Plasma anti-Müllerian hormone (AMH) was determined using the AMH Gen II ELISA assay (Beckman Coulter) using 50 μL of undiluted plasma, as previously described (Rico et al. 2009) (link), from samples collected the first day of the diet supplementation and stored at -20°C. AMH, an indicator of the ovarian response after hormonal stimulation, was assessed for any differences in plasma AMH between the groups.
For metabolic assays, plasma samples were collected once per week from caudal venipuncture before feeding, during the supplementation period. Plasma samples were stored at -20°C until the assays were conducted. Plasma NEFA, urea and glucose were determined using enzymatic colorimetry with a multi-parameter analyser (KONE Instruments Corporation, Espoo, Finland).
Plasma and follicular fluid fatty acid compositions were determined using gas chromatography, as previously described (Lefils et al. 2010) (link). Plasma fatty acid composition was determined at the beginning of the supplementation period and in samples corresponding to OPU leading to in vitro embryo production (at 2, 5 and 7 weeks postpartum, Fig. 1). Follicular fluid fatty acid composition was determined at 2, 5 and 7 weeks postpartum.
For metabolic assays, plasma samples were collected once per week from caudal venipuncture before feeding, during the supplementation period. Plasma samples were stored at -20°C until the assays were conducted. Plasma NEFA, urea and glucose were determined using enzymatic colorimetry with a multi-parameter analyser (KONE Instruments Corporation, Espoo, Finland).
Plasma and follicular fluid fatty acid compositions were determined using gas chromatography, as previously described (Lefils et al. 2010) (link). Plasma fatty acid composition was determined at the beginning of the supplementation period and in samples corresponding to OPU leading to in vitro embryo production (at 2, 5 and 7 weeks postpartum, Fig. 1). Follicular fluid fatty acid composition was determined at 2, 5 and 7 weeks postpartum.
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