妊娠相关糖蛋白及其在反刍动物早期妊娠诊断中的应用

妊娠相关糖蛋白(pregnancy-associated glycoprotein,PAG)是由胎盘组织产生并释放到母体外周血液中的大分子蛋白,在妊娠过程中发挥着重要作用.目前,母畜妊娠早期诊断的研究多集中在对奶牛伤害小、准确性高、特异性强的间接检测方法上,如检测血液或乳汁中孕酮等激素,而近年来发展起来的对血液中PAG的检测就是一种主流的方法.作者现就PAG的功能、生化特性、母畜怀孕期间PAG的浓度变化及其在反刍动物早期妊娠诊断中的应用情况进行综述.     

牛妊娠相关糖蛋白ELISA对早期妊娠诊断效果的影响

选取273头荷斯坦奶牛,在人工输精后的28d和75d分别采用牛妊娠相关糖蛋白(PAG)ELISA和直肠检查的方法进行妊娠诊断,比较PAG ELISA和直肠检查法的结果,旨在评价PAG ELISA对配种28d的奶牛进行早期妊娠诊断的准确性。结果表明,PAG ELISA法妊娠诊断的敏感性、特异性、阳性预测值、阴性预测值和准确性分别为100%、75.5%、86.5%、100%和90.5%,与75d直肠检查结果相同,可用于母牛的早期妊娠诊断。妊娠诊断对于母牛保胎、分群管理及提高繁殖效率等具有非常重要的意义。     

奶牛早期妊娠诊断及妊娠相关糖蛋白(PAG)ELISA检测技术进展

奶牛配种后,及时且准确了解奶牛的妊娠状况至关重要。奶牛早期妊娠诊断可显著缩短平均育种间隔,提高奶牛养殖效率。随着早期妊娠诊断技术不断发展,检测牛妊娠相关糖蛋白(PAG)逐渐被人们重视,尤其是酶联免疫吸附试验(ELISA)得到更多的开发与利用。目前国外已有PAG ELISA牛妊娠检测试剂盒用于检测怀孕早期PAGs,该方法快速、准确、灵敏,而国内现存的试纸条准确率低。本文综述了传统的奶牛早期妊娠诊断方法和PAG ELISA方法的发展现状,对ELISA检测方法在动物繁殖领域的应用进行展望。     

牛妊娠相关糖蛋白的研究进展

妊娠相关糖蛋白(pregnancy associated glycoprotein, PAG)是由反刍动物胎盘表达的一种天冬氨酸蛋白酶家族,在反刍动物滋养外胚层的单核和双核细胞中合成。在牛科动物中,PAG家族蛋白种类较多。PAG家族各蛋白成员在分子质量、氨基酸序列和生理功能等方面有一定差别,还需要大量的科学研究来揭示这些蛋白质的功能和作用。PAGs在奶牛妊娠过程中发挥了重要作用,可以作为检测奶牛妊娠及诊断妊娠丢失的标志物,同时还可以作为监测胎盘功能的指标。笔者综述了PAG家族的起源、在牛体内的生物学功能及在牛妊娠监测中的应用,旨在为奶牛早期妊娠诊断研究提供理论参考。     

妊娠相关糖蛋白检测试剂盒在奶牛早期妊娠诊断上的应用

为了研究妊娠相关糖蛋白(PAG)检测试剂盒在人工授精(AI)后28~32 d奶牛早期妊娠诊断上的应用效果,试验采集49头AI后28~32 d奶牛血液样品,分离血清并应用PAG检测试剂盒进行检测;采血当天利用兽用B型超声诊断仪(B超)进行妊娠检查,并于AI后第35天用B超复检。结果表明:AI后28~32 d,PAG检测试剂盒检测奶牛早期妊娠的灵敏度、特异性、阳性预测值、阴性预测值、准确性分别为92.0%、95.8%、95.8%、92.0%、93.9%,与B超检测方法结果的一致性(Kappa值)为0.632。说明PAG检测试剂盒可以高效应用于奶牛的早期妊娠诊断中。     

绵羊妊娠相关糖蛋白PAG3的制备

妊娠相关糖蛋白(pregnancy-associated glycoprotein,PAG)是一类动物妊娠后胎盘产生的特异性蛋白质,是判断妊娠的重要信号物质。为了获得绵羊妊娠相关糖蛋白PAG3的制备方法,以期通过检测绵羊体内的妊娠相关糖蛋白的含量来进行早期妊娠诊断。本试验通过原核表达生物工程合成的方法,将绵羊妊娠相关糖蛋白编码基因序列插入到表达载体pET20b中,利用原核表达转化大肠杆菌获得了融合His标签的目的蛋白PAG3。经过超声波裂解、His标签亲和层析的方法纯化获得了妊娠相关糖蛋白PAG3。     

奶牛早期妊娠诊断研究进展

奶牛早期妊娠诊断一直以来是奶牛养殖亟待解决的问题,特别是近年来奶牛养殖业飞速发展,奶牛早期妊娠诊断更加影响着奶牛的发展。家畜早期妊娠诊断方法研究历史悠久,从早期观察配种后母畜周期发情停止和使用最为直接的直肠检查法,到20世纪50年代开始至今相继发展起来的妊娠诊断方法很多。本文仅将在生产中具有实际应用和发展前景的早期妊娠诊断技术应用现状综述如下。     

干扰素刺激基因ISG15、OAS1和RSAD2在奶牛早期妊娠诊断中的应用

研究旨在探讨干扰素刺激基因15(Interferon stimulated gene 15,ISG15)、2′-5′寡腺苷酸合成酶1(2′-5′-oligoadenylate Synthetase 1,OAS1)和S-腺苷甲硫氨酸基区域蛋白2(Radical S-adenosyl methionine domain containing 2,RSAD2)对奶牛早期妊娠诊断的价值,为其在奶牛早期妊娠诊断中的应用提供理论依据。36头青年母牛分别于人工授精(artificial insemination,AI)后0d、18d、21d、28d采集外周血,利用荧光定量PCR技术对ISG15、OAS1和RSAD2在18d外周血总白细胞中的相对表达量(相对于0d)进行了分析;分别利用化学发光法和ELISA技术对血清孕酮(18d、21d)和牛妊娠相关糖蛋白(pregnancy-associated glycoprotein,PAG),(28d)进行了检测。通过建立受试者工作特征曲线(ROC曲线)综合评估了三个候选生物标志物单独或联合检测在奶牛早期妊娠诊断中的统计学指标,并与孕酮检测和PAG检测的诊断效能进行了同步比较分析。对ISG15、OAS1和RSAD2三者的分析结果表明,ISG15与RSAD2联合检测的敏感性和特异性分别为94.7%和100%,具有最优的诊断价值;以血清中孕酮含量作为参考指标时,AI后21d妊娠诊断的敏感性和特异性与18d相比较优,分别为88.9%和94.1%,但诊断效能不及ISG15、RSAD2二者联用;PAG ELISA最早于AI后28d可做出诊断,且相对于ISG15-RSAD2联用其诊断的特异性较低,为94.12%。综上所述,利用ISG15和RSAD2基因不仅可望在AI后18d成功实现妊娠诊断而且具有最高的灵敏度和特异性,研究结果为下一步建立奶牛新型早期妊娠诊断技术奠定了基础。     

奶牛早期妊娠诊断研究综述

奶牛早期妊娠诊断研究综述西昌农业专科学校（６１５０１３）陶莉，张学舜（审校）这个研究主要是就我们所采用的奶牛早期妊娠诊断方法及国外方法的简要综述，并论证其可行性。奶牛和其它繁殖母牛的早期妊娠诊断是养牛业生产中亟待解决的问题。奶牛饲养管理的目标除了提高...     

妊娠奶牛PAPPA和PAG mRNA表达规律的研究

[目的]分析PAPPA(妊娠相关血浆蛋白A)和PAG(妊娠相关糖蛋白)mRNA在不同妊娠时期奶牛外周血的表达规律.[方法]提取奶牛空怀期和妊娠30 d、60 d、90 d、150 d和240 d血浆中游离RNA,RT-PCR扩增PAPPA和PAG的基因,分析其在妊娠期的相对表达水平,并研究其表达规律.[结果]PAG基因...     

Application of an ELISA Milk Pregnancy Test in Beef Cows

Pregnancy‐associated glycoproteins (PAG) are secreted by the binucleate giant cells of the ruminant placenta and enter maternal circulation at the time of placental attachment. The IDEXX Milk Pregnancy Test (IDEXX, Westbrook, ME) detects a subset of PAG in milk. Although designed as a management tool for dairy cows, there is potential for using the milk PAG test in beef cows. Our objective was to compare the performance of the milk PAG ELISA with a gold standard method for pregnancy diagnosis and determine the agreement between milk and serum PAG analysis in lactating beef cows. Angus and Angus‐crossed cows (n = 332) from two Michigan beef herds were enrolled in this study. Cows were subjected either to timed artificial insemination followed by exposure to a bull or exclusively exposed to a bull. The bulls and cows were separated 30 days prior to examination. Serum and milk samples were collected and submitted within 24 h of collection to a commercial laboratory for PAG analysis using the IDEXX Milk Pregnancy Assay (milk) and the IDEXX Bovine Pregnancy Assay (serum). Concurrently with milk and serum collection, each cow was examined transrectally by palpation or ultrasonography. When compared to transrectal examination, the performance (and 95% confidence intervals) of the milk PAG ELISA was sensitivity of 99.7% (99.0–100.0%) and specificity of 80.8% (65.6–95.9%). The lower specificity is likely due to the low prevalence (9.9%) of open cows (n = 30) in the herds examined. Of the 332 cows examined, 1.8% (n = 6) were classified as rechecks using the milk PAG ELISA. Results of the milk and serum PAG ELISA were in high agreement (kappa coefficient = 0.91). The milk PAG ELISA was accurate in predicting pregnancy status using milk collected from beef cattle between days 37 and 125 post‐insemination and may be useful for aiding management decisions in beef herds.     

Establishment of an ELISA for Measuring Bovine Pregnancy-Associated Glycoprotein in Serum or Milk and Its Application for Early Pregnancy Detection

It has been shown in several mammalian species that during pregnancy, trophoblast cells express a range of pregnancy-associated glycoproteins (PAG). The presence of PAG in the maternal serum of cows may serve as an indicator of pregnancy from day 28 after AI onward. The present study addresses (1) conversion of an existing PAG-RIA to a competitive double antibody ELISA using a polyclonal anti-bPAG-IgG and an anti-rabbit-IgG raised in sheep for coating and (2) application of newly established ELISA to test its suitability for pregnancy detection by measuring PAG in serum or milk. The intraassay coefficients of variation (CV) for the PAG-ELISA were 2–14% for serum and 10–12% for milk; the corresponding interassay CVs were 8–22% and 12–22%, respectively. Pregnancy-associated glycoprotein profiles established in milk and serum of 12 pregnant cows showed a characteristic pattern with measurable amounts from approximately day 20 onwards in serum and from day 60 onwards in milk. In a field trial, serum PAG was determined in 397 cows sampled between 20 and 50 days after insemination. The outcome was that, pregnancy could reliably be diagnosed from day 28 onwards in serum and from day 150 onwards in milk. In conclusion, it may be stated that the established ELISA provides an efficient and reliable means of pregnancy diagnosis that will, in our judgement, gain in popularity with cattle breeding. The ELISA proved to be an adequate and efficient way of measuring PAG in maternal serum or milk and will be a useful means of pregnancy detection in cows.     

Secretory Profiles of Pregnancy-Associated Glycoproteins at Different Stages of Pregnancy in the Goat

Plasma concentrations of pregnancy‐associated glycoproteins (PAG) were determined in goats during pregnancy by two homologous radioimmunoassays that employed caprine PAG₅₅₊₆₂ (caPAG₅₅₊₆₂) and caprine PAG₅₅₊₅₉ (caPAG₅₅₊₅₉) and their specific antisera. The effects of fetal number on PAG concentrations were analysed. The concentrations of caPAG₅₅₊₆₂ were higher than that of caPAG₅₅₊₅₉ throughout pregnancy (p <0.05). Both caPAG₅₅₊₆₂ and caPAG₅₅₊₅₉ reached maximal levels in week 8 (48.6 ± 5.0 and 30.4 ± 4.3 ng/ml, respectively), decreased between weeks 12 and 14 (45.5 ± 2.5 to 31.9 ± 2.7 ng/ml and 30.6 ± 2.1 to 15.8 ± 2.8 ng/ml, respectively, p <0.01) and remained relatively constant until parturition. Twin‐bearing goats had higher PAG concentrations than single‐bearing animals, but the difference was only significant in week 4 (52.3 ± 3.7 versus 30.9 ± 3.9 ng/ml and 18.9 ± 1.7 versus 12.6 ± 2.0 ng/ml, for caPAG₅₅₊₆₂ and caPAG₅₅₊₅₉, respectively, p <.,0.05). This is the first study of PAG concentrations in goat throughout pregnancy by a homologous radioimmunoassay system. The profiles of caPAG₅₅₊₆₂ and caPAG₅₅₊₅₉ were closely parallel. Concentrations of PAG were lower than those obtained by a heterologous radioimmunoassay and their patterns were also different, due to a different specificity of the antisera used in heterologous system. Goats that delivered twins had higher PAG concentrations at the time of implantation than those that delivered a single fetus, indicating that PAG concentrations provide a useful measure of the trophoblast secretory activity.     

Pregnancy-associated Glycoprotein Profile during the First Trimester of Pregnancy in Egyptian Buffalo Cows

Pregnancy-associated glycoprotein (PAG) concentrations were measured in buffalo cows starting from day 28 after breeding. Oestrus was synchronized in 10 buffaloes using two injections of 25 mg prostraglandin (PG)F_{2 α} (Lutalyse^®) at a 11-day interval. Blood sampling was conducted nearly twice weekly. Results indicated that plasma PAG concentrations in non-pregnant buffaloes were low (<0.20 ng/ml) during the whole experimental period (day 28 to 103), while in pregnant animals plasma PAG levels increased from day 28 (4.48 ± 0.92 ng/ml) until day 41 (27.27 ± 6.74 ng/ml), remaining high (20.71 ± 9.20 ng/ml) until day 103. Progesterone levels were significantly (p < 0.0001) higher in pregnant (3.51–4.80 ng/ml) than in non-pregnant buffaloes (0.28–1.52 ng/ml). A significant difference (p < 0.0001) in plasma PAG concentrations between pregnant and non-pregnant animals starting at day 28 after breeding suggests that PAG-radioimmunoassay could be suitable for pregnancy diagnosis in buffaloes during this period. In conclusion, PAG test offers the advantages that it requires a single plasma sample for early pregnancy diagnosis as well as the accuracy of the test for the detection of pregnancy as early as day 28.     

Comparison of Five Radioimmunoassay Systems for PAG Measurement: Ability to Detect Early Pregnancy in Cows

This study was conducted to describe the minimum detection limit, reproducibility, accuracy, specificity and parallelism of different pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) systems: RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool. Their ability to distinguish between non-pregnant and pregnant females at day 30 after artificial insemination (AI) was investigated. The antisera were raised in rabbits against different PAG preparations. All RIA systems proved to be sensitive, repeatable and accurate for measuring PAG concentrations. The dilutions of plasma samples taken at an early stage of pregnancy were found to be parallel to the standard curves. No cross-reaction was observed with different carbohydrates, either with Pregnant Mare Serum Gonadotropin (PMSG) or human Chorionic Gonadotropin (hCG). The concentrations of PAG in pregnant females at day 30 after AI were shown to be higher with the use of antisera R#706, R#780, R#809 and Pool when compared with antiserum R#497. All the RIA systems gave 100% sensitivity and negative predictive values. On the other hand, the use of antisera R#780 and R#809 resulted in lower specificity and positive predictive values. The present study clearly shows that the ability of PAG-RIA systems to diagnose pregnancy specifically at day 30 after AI can be improved by using a combination of antisera raised against different forms of PAG.     

Pregnancy‐Associated Glycoprotein Secretion in North Moroccan Goats

The plasma profile of pregnancy‐associated glycoprotein (PAG) and its relationship with fetal number were studied in 14 North Moroccan goats using a semi‐heterologous radioimmunoassay (RIA). Peripheral blood was collected every day during the first month of pregnancy, afterward the blood samples were collected trice a week. The PAG were first detected at day 20 of pregnancy, their levels increase to week 8 of gestation then decrease slightly until parturition. Statistical differences between goats carrying one or two foetuses are observed from week 5 of pregnancy until parturition. Factorial Discriminant Analysis provides mathematical models for the discrimination between the litter size using the PAG level and the week of pregnancy. Using only one blood sample per week, high sensitivity, specificity and overall accuracy (74%, 88% and 81%) were obtained using these models. It is concluded that the PAG RIA is an effective tool for early diagnosis of pregnancy and for discrimination between the litter sizes in North Moroccan goats.     

Storage strategies and processing of bovine milk samples for pregnancy-associated glycoproteins detection test

Three experiments were performed to test if the time and storage conditions of milk samples, the preheating of samples in a water bath, as well as the carryover effect in laboratory analysis equipment could affect the pregnancy-associated glycoproteins (PAG) levels, and consequently the results of a pregnancy test. The pregnancy test used in both experiments uses the enzyme-linked immunosorbent assay method to measure the concentrations of PAG and classify the samples as pregnant, nonpregnant or suspicious. As a result, PAG levels showed no variation when the samples were analyzed up to 9 days after collection, whether stored in ambient temperature or refrigerated. The preheating of the samples in a water bath and prior to the analysis of SCC and composition also did not affect the levels of PAG, allowing the same sample used in the quality analysis to be used for the pregnancy test.