171 Blood sample type and storage conditions affect circulating anti-Müllerian hormone concentrations in cattle

Circulating anti-Müllerian hormone (AMH) varies greatly among individuals, is correlated with antral follicle count, and as such, is utilised as a predictor for embryo production in assisted reproductive technologies (ART) in cattle. There are, however, concerns that AMH is prone to preanalytical instability, which could hamper the ability to evaluate and select donors with superior embryo production potential. Thus, the objective of the present study was to investigate the effect of (1) sample type (plasma, serum, or whole blood), and (2) storage conditions (time and temperature) on circulating AMH concentration in cattle. Two experiments were performed using 20 Angus-cross suckled beef cows from which blood samples were collected at a random stage of the oestrous cycle. In experiment 1, blood samples were drawn into evacuated tubes containing either K₂ EDTA (#368589, BD Vacutainer®, Becton, Dickinson and Co.; 2 per cow) or a clot activator (#367820, BD Vacutainer®, 1 per cow) providing for three sample types: plasma, whole blood, and serum. Whole blood samples were directly transferred to microcentrifuge tubes and stored at −20°C, while plasma and serum samples were centrifuged, the plasma/serum separated, and stored at −20°C until assayed. In experiment 2, blood samples were drawn into evacuated tubes containing either K₂ EDTA (#368589, BD Vacutainer®, 10 per cow) or a clot activator (#367820, BD Vacutainer®, 10 per cow) providing for two sample types: plasma and serum. Both plasma and serum samples were incubated at 4°C or 20°C for either 0, 6, 12, 24, or 48 h. After incubation, samples were centrifuged, plasma/serum separated, and stored at −20°C until assayed. Samples were assayed for AMH using a commercially available ELISA (AnshLabs® Bovine AMH). Data were analysed using linear mixed models (SAS 9.4; SAS Institute Inc.) and presented as mean ± s.e.m. There were positive correlations in AMH concentrations (experiment 1) between plasma and serum (r = 0.65; P = 0.002), plasma and whole blood (r = 0.85; P < 0.001), and serum and whole blood (r = 0.78; P < 0.001). Mean circulating AMH concentration, however, was greater (P < 0.001) in plasma (793 ± 94 pg mL⁻¹) than in either whole blood (587 ± 68 pg mL⁻¹) or serum (542 ± 73 pg mL⁻¹), whereas there were no differences between serum and whole blood (P = 0.19). Plasma AMH concentration (experiment 2) was affected by storage time (P = 0.02) but not by temperature (P = 0.82), nor there was a temperature by time interaction (P = 0.28). Plasma AMH increased from 0 to 6 h (+ 8%) but remained constant thereafter. Serum AMH concentration was affected by storage time (P < 0.001) but was not affected by temperature (P = 0.91) or by temperature by time interaction (P = 0.23). Mean serum AMH increased with time such that mean serum AMH at 48 h was 37% greater than that at 0 h. In conclusion, plasma samples present the greatest circulating AMH concentration and have improved stability during storage at either 4°C or 20°C for up to 48 h. Thus, when assessing AMH for selection of cattle for embryo production, plasma should be the preferred sample type.