89 Comparative Quantification of Plasma Progesterone Through Radioimmumoassay and Enzyme-Linked Fluorescent Assay Techniques in Cattle

Abstract

Progesterone (P4) is an important component of oestrous cyclicity and is critical to fertility. A concentration >1 ng mL⁻¹ reflects the function of the corpus luteum (CL) and is considered indicative of a cyclic cow. Recent publications have shown that P4 at the onset of synchronization programs is critical to pregnancy outcomes in primiparous cows (Stevenson et al. 2015 J. Anim. Sci. 93, 2111-2123) and cows with P4 <5 ng mL⁻¹ on Day 14 could predict pregnancy loss (Kenyon et al. 2013 Anim. Reprod. Sci. 136, 223-230). Currently, the gold standard technique to quantify P4 is radioimmunoassay (RIA). However, new techniques are emerging. The objective of this study was to evaluate a new commercial in vitro diagnostic assay to quantify P4 based on enzyme immunoassay by competition with detection of final fluorescence (enzyme-linked fluorescence assay, ELFA). A total of 30 cows were synchronized on Day 0 with an intravaginal device (IVD) containing 500 mg of P4 (Cronipres, Biogénesis Bagó, Argentina). On Day 7 and Day 8 all cows received 150 mg of prostaglandin F_2α (Enzaprost, Biogénesis Bagó). All IVD were removed on Day 8. A total of 95 blood samples were taken at Days 0, 9, 9.5, and 10 using BD Vacutainer (Becton-Dickinson, Franklin Lakes, NJ, USA) with sodium heparin by jugular venipuncture and centrifuged at 3000 × g for 30 min for plasma separation, which was frozen at –20°C until analysis. Samples were measured in duplicate by IM1188-Progesterone-RIA (Beckman Coulter, Brea, CA, USA) and VIDAS-PRG-ELFA (Biomerieux, Marcy l’Étoile, France). Concentrations of P4 obtained by RIA were classified in 2 groups: (A) P4 <1 ng mL⁻¹, and (B) P4 ≥1 ng mL⁻¹ and matched with P4 concentrations obtained by ELFA. Kappa (κ) test was used to determine agreement between both techniques, intra-assay coefficients of variation was determined for RIA and ELFA; and sensitivity (SE), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) were determined for ELFA. There was very good agreement between the RIA and ELFA techniques, κ = 0.95, to determine P4 concentrations for group A (62 and 62 samples, respectively) and group B (33 and 33 samples, respectively). The intra-assay coefficients of variation were 5% (RIA) and 2.9% (ELFA). Values for SE = 0.97, SP = 0.98, PPV = 0.97, and NPV = 0.98 were obtained for ELFA. We were able to quantify P4 in bovine plasma in all samples using the ELFA technique with similar reliability to the RIA technique. Only 2 samples (2.1%) differed in their concentrations and clinical interpretation. There was a slight discrepancy between the results found for both techniques with an excellent SE and SP in ELFA compared with RIA. Based on the analytical results, we believe that this in vitro diagnostic assay developed for use with an autoanalyzer could be useful for routine bovine reproduction programs. Further studies should be carried out to strengthen these conclusions.