Abstract：
The objective of the current study was to evaluate pregnancy-associated glycoproteins (PAGs) based enzyme-linked immunosorbent assay (ELISA) kits utilizing whole blood, serum, or milk samples for diagnosis of early pregnancy status in lactating Nili-Ravi buffaloes. Dairy buffaloes (n = 174) of mixed parity, 4-6 years of age, having mean (± standard deviation) days in milk 165 ± 87, and body condition score of 3.26 ± 0.34 were randomly enrolled in this study. Buffaloes were exposed to penile deviated bulls with 12 h interval for estrus detection during peak breeding season and eventually bred naturally at their respective standing estrus (day 0). Blood and milk samples were collected at days 24, 28, and 35 post-breeding to run a rapid visual pregnancy test® (RVPT) as a buffalo-side test or ELISA-based assay in the laboratory to detect early pregnancy status. Transrectal B-mode ultrasonography was performed to diagnose pregnancy at day 35 post-breeding and used as a gold standard to validate results of RVPT or ELISA-based tests. The RVPT is a visual readout test for pregnancy detection and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (77.9 vs. 89.7 vs. 93.3%), and accuracy (84.5 vs. 90.1 vs. 94.2%) at days 24, 28, and 35 post-breeding, respectively. The PAGs were assayed using ELISA kits in serum and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (84.2 vs. 87.7 vs. 93.9%), and accuracy (82.1 vs. 88.4 vs. 93.7%) at days 24, 28, and 35 post-breeding, respectively. Similarly, PAGs were also analysed using ELISA kits in milk samples and had sensitivity (77.6 vs 89.5 vs 95.0%), specificity (89.1 vs 91.9, vs 93.9%), and accuracy (85.1 vs 91.1 vs 94.3%) at days 24, 28, and 35 post-breeding, respectively. Overall, the Kappa values in this study exceeded 0.85 at day 35 post-breeding using RVPT or ELISA-based test kits in serum or milk samples, indicating a high level of agreement between PAGs detection method and gold standard for pregnancy diagnosis. The pregnancy outcomes based on ELISA-based PAGs detection at day ≥28 post-breeding had a high negative predictive value, indicating that the probability of incorrectly administering prostaglandins to pregnant buffaloes would be low if these tests were implemented on a commercial dairy herd. Taken together, it is concluded that PAGs-based determination of pregnancy using RVPT or ELISA in blood, serum, or milk samples can be used effectively for pregnancy diagnosis at ≥28 days post-breeding with more than 90% accuracy in Nili-Ravi buffaloes.
摘要：
本研究的目的是利用全血评估基于妊娠相关糖蛋白（PAG）的酶联免疫吸附测定（ELISA）试剂盒，血清,或用于诊断泌乳Nili-Ravi水牛早期妊娠状态的牛奶样品。混合平价的水牛(n=174)，4-6岁,平均(±标准差)日奶165±87天,身体状况评分3.26±0.34被随机纳入本研究.在繁殖高峰期，水牛以12小时的间隔暴露于阴茎偏斜的公牛，以进行发情检测，并最终在各自的站立发情期（第0天）自然繁殖。在繁殖后第24、28和35天收集血液和牛奶样品，以进行快速视觉妊娠测试（RVPT），作为实验室中的水牛侧测试或基于ELISA的测定，以检测早期妊娠状态。在繁殖后第35天进行经直肠B超检查以诊断妊娠，并用作金标准以验证RVPT或基于ELISA的测试的结果。RVPT是用于妊娠检测的视觉读出测试，具有敏感性（77.9vs.89.7vs.93.3％），特异性（77.9vs.89.7vs.93.3％），和准确性(84.5vs.90.1vs.94.2%)在繁殖后第24、28和35天，分别。使用ELISA试剂盒在血清中测定PAG，并具有敏感性（77.9vs.89.7vs.93.3％），特异性（84.2vs.87.7vs.93.9%），和准确性（82.1与88.4vs.93.7%)在繁殖后第24、28和35天，分别。同样，还使用ELISA试剂盒对牛奶样品中的PAG进行了分析，并具有敏感性（77.6vs89.5vs95.0％），特异性（89.1vs91.9，vs93.9%），育种后第24、28和35天的准确性（85.1vs91.1vs94.3％），分别。总的来说,本研究中的Kappa值在育种后第35天使用RVPT或基于ELISA的检测试剂盒在血清或牛奶样品中超过0.85，表明PAGs检测方法与妊娠诊断的金标准之间的高度一致性。基于ELISA的PAGs检测在繁殖后第28天的妊娠结局具有较高的阴性预测值，这表明，如果对商业乳牛群实施这些测试，对怀孕的水牛不正确施用前列腺素的可能性将很低。一起来看,结论是使用血液中的RVPT或ELISA基于PAGs的妊娠测定，血清,在Nili-Ravi水牛中，或牛奶样品可在繁殖后≥28天有效地用于妊娠诊断，准确率超过90％。
