The field evaluation of the diagnostic performance of a commercial pregnancy-associated glycoprotein-based lateral flow assay for early pregnancy detection in ewes.

The present study aimed to determine the diagnostic accuracy of the commercial Alertys OnFarm Pregnancy Test (AOPT) based on pregnancy-associated glycoprotein (PAG) for early pregnancy diagnosis in ewes. A total of 100 multiparous Awassi ewes were synchronised for estrus and mated during the non-breeding season. Whole blood samples were collected from the jugular vein of all ewes on days 21, 28, and 35 after mating. The AOPT was first performed on the collected whole blood samples. Plasma PAG concentrations were then determined by Enzyme-Linked Immunosorbent Assay (ELISA). Pregnancy diagnosis was made on the same days by transrectal ultrasonography (USG), which was accepted as the reference standard. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of AOPT compared to USG on days 21, 28, and 35 after mating were calculated. In addition, the diagnostic performance of plasma PAG concentrations for predicting pregnancy status was evaluated by determining cut-off values and conducting receiver operating characteristic (ROC) curve analysis. When the results were examined, the sensitivity of AOPT was calculated as 77.55 %, specificity as 92.15 %, PPV as 90.47 %, NPV as 81.03 % and accuracy as 85.00 % at 21 days after mating and showed strong agreement with transrectal USG (Kappa= 0.699; P < 0.001). In contrast, the sensitivity (95.55 %) and accuracy (93.00 %) of AOPT on day 28 improved significantly, showing almost perfect agreement with USG (Kappa= 0.859; P < 0.001). On day 35, AOPT achieved the highest diagnostic performance compared to other pregnancy days with 100 % sensitivity, 96.49 % specificity, and 98.00 % accuracy (Kappa= 0.959; P < 0.001). Plasma PAG levels (OD) were significantly higher in pregnant ewes than in non-pregnant ewes at all time points (P < 0.001). The optimal cut-off values for pregnancy detection were determined as > 0.28 on day 21 (AUC= 0.941), > 0.48 on day 28 (AUC= 0.986), and > 0.43 on day 35 (AUC= 1.000). In conclusion, AOPT can be considered a practical, rapid, and reliable method for early pregnancy detection in ewes, particularly from day 28 post-mating. However, possible false-negative results that may occur very early in pregnancy and false-positive results due to embryonic loss later in pregnancy necessitate careful interpretation and, if necessary, repeat testing or confirmation with USG.