PAGmRNA在奶牛血液中表达的研究

本试验采用半定量RT-PCR的方法，检测和分析了牛PAG基因mRNA在妊娠牛及空怀牛血液中的表达规律。结果发现，PAG基因mRNA在妊娠21d的母牛及空怀牛血液中都有表达，而妊娠牛表达量显著高于空怀牛（P〈005）。这说明配种后21d奶牛血液中PAG基因的表达量可以作为奶牛早期妊娠诊断的依据。     

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。     

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。     

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。     

妊娠相关糖蛋白(PAG)在奶牛早期妊娠诊断上的应用现状

妊娠相关糖蛋白(pregnancy associated glycoproteins,PAG)是反刍动物妊娠后外周血液出现的特异性蛋白,在妊娠过程中发挥着重要的作用。目前,在生产上通过检测PAG在血清中浓度能够进行奶牛的妊娠诊断。作者综述了PAG的发现、分离纯化及其在奶牛体内的代谢水平和近年奶牛早期妊娠诊断上的应用情况。     

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。     

PAG ELISA早孕检测试剂盒对奶牛繁殖效率的影响

为了及时发现空怀牛并及时实施第2次配种,试验对配种后30~45 d未返情的奶牛采集血样,应用早孕因子(PAG)ELISA早孕检测试剂盒进行早期妊娠诊断。结果表明:应用PAG ELISA早孕检测试剂盒后,奶牛的平均空怀天数下降22.67 d,产犊间隔下降36.82 d。说明使用PAG ELISA早孕检测试剂盒进行奶牛早期妊娠诊断能够及时发现空怀牛并实施第2次配种,能够极显著降低奶牛空怀天数和产犊间隔(P0.01),提高奶牛繁殖效率。     

妊娠失败牛外周血中干扰素刺激基因和细胞因子的表达分析

为了探究牛人工授精后妊娠失败的原因,本研究分析了妊娠失败牛外周血干扰素刺激基因和细胞因子等的表达变化。试验选取11头健康状况良好、体况相近的未孕奶牛,采集人工授精后0d、18d和28d的血液样本,结合28d血清PAG检测和42d直肠触诊,对确定为妊娠失败牛的血液样本进行分析。结果表明,与0d相比,人工授精后18d血液RSAD2、MX1和RTP4 mRNA的表达均呈上调趋势,但无显著差异;18d时IL-2mRNA的表达显著高于0d,IL-10和CCL8 mRNA的表达显著低于0d,IFN-γ、IL-4和CXCL10 mRNA的表达均呈上调趋势,但无显著差异。说明妊娠失败与胚胎发育早期母体免疫失调及妊娠识别能力不足有关。     

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。     

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.     

母牛同期发情试验及受胎产犊效果观察

[目的]为了解决牛群分散,交通不变问题.[方法]本文利用同期发情处理方法,并为了观察牛同期发情及受胎产犊效果.在红塔、新平和元江分三批对102头母牛进行同期发情处理(放置阴道栓+注射氯前列烯醇),撤栓后根据发情情况用冷冻精液对97头母牛进行人工授精,[结果]表明同期发情率95.1%.150～160 d后对输精母牛进行妊...     

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.     

Estimation of sensitivity and specificity of pregnancy diagnosis using transrectal ultrasonography and ELISA for pregnancy-associated glycoprotein in dairy cows using a Bayesian latent class model

AIMS: To determine the sensitivity (Se) and specificity (Sp) of pregnancy diagnosis using transrectal ultrasonography and an ELISA for pregnancy-associated glycoprotein (PAG) in milk, in lactating dairy cows in seasonally calving herds approximately 85–100 days after the start of the herd’s breeding period.

METHODS: Paired results were used from pregnancy diagnosis using transrectal ultrasonography and ELISA for PAG in milk carried out approximately 85 and 100 days after the start of the breeding period, respectively, from 879 cows from four herds in Victoria, Australia. A Bayesian latent class model was used to estimate the proportion of cows pregnant, the Se and Sp of each test, and covariances between test results in pregnant and non-pregnant cows. Prior probability estimates were defined using beta distributions for the expected proportion of cows pregnant, Se and Sp for each test, and covariances between tests. Markov Chain Monte Carlo iterations identified posterior distributions for each of the unknown variables. Posterior distributions for each parameter were described using medians and 95% probability (i.e. credible) intervals (PrI). The posterior median estimates for Se and Sp for each test were used to estimate positive predictive and negative predictive values across a range of pregnancy proportions.

RESULTS: The estimate for proportion pregnant was 0.524 (95% PrI?=?0.485–0.562). For pregnancy diagnosis using transrectal ultrasonography, Se and Sp were 0.939 (95% PrI?=?0.890–0.974) and 0.943 (95% PrI?=?0.885–0.984), respectively; for ELISA, Se and Sp were 0.963 (95% PrI?=?0.919–0.990) and 0.870 (95% PrI?=?0.806–0.931), respectively. The estimated covariance between test results was 0.033 (95% PrI?=?0.008–0.046) and 0.035 (95% PrI?=?0.018–0.078) for pregnant and non-pregnant cows, respectively. Pregnancy diagnosis results using transrectal ultrasonography had a higher positive predictive value but lower negative predictive value than results from the ELISA across the range of pregnancy proportions assessed.

CONCLUSIONS AND CLINICAL RELEVANCE: Pregnancy diagnosis using transrectal ultrasonography and ELISA for PAG in milk had similar Se but differed in predictive values. Pregnancy diagnosis in seasonally calving herds around 85–100 days after the start of the breeding period using the ELISA is expected to result in a higher negative predictive value but lower positive predictive value than pregnancy diagnosis using transrectal ultrasonography. Thus, with the ELISA, a higher proportion of the cows with negative results will be non-pregnant, relative to results from transrectal ultrasonography, but a lower proportion of cows with positive results will be pregnant.     

Plasma Concentrations of Pregnancy‐Associated Glycoproteins Measured Using Anti‐Bovine PAG‐2 Antibodies on Day 120 of Gestation Predict Abortion in Dairy Cows Naturally Infected with Neospora caninum

The present study sought to determine: (i) the effects of Neospora caninum infection and twin pregnancy on plasma pregnancy‐associated glycoprotein‐2 (PAG‐2) concentrations throughout pregnancy and (ii) whether plasma PAG‐2 concentrations could predict abortion in N. caninum‐infected cows. The study was performed on a commercial Holstein‐Friesian dairy herd in northeastern Spain and the final data included those recorded in 53 non‐aborting and 19 aborting animals. Blood samples were collected immediately before pregnancy diagnosis (on Days 40, 90, 120, 150, 180 and 210 post‐insemination) in non‐aborting cows or until the time of abortion detection in aborting cows. General lineal models (GLM) repeated measures anova revealed the different behaviour of PAG‐1 and PAG‐2, and significant effects of Neospora seropositivity, cool season and twin pregnancy on plasma PAG‐2 concentrations throughout gestation (between‐subject effects). In addition, based on the odds ratios, the likelihood of abortion increased in Neospora‐seropositive cows (by a factor of 7.0) compared to seronegative animals and decreased in cows with a high plasma PAG‐2 concentration (>4.5 ng/ml) on Day 120 of pregnancy (by a factor of 0.24), compared to the remaining cows. In conclusion, there is a relationship between plasma PAG‐2 concentrations and the risk of abortion in Neospora‐infected dairy cows. Thus, plasma PAG concentrations measured using anti‐boPAG‐2 antiserum on Day 120 of gestation could serve as an indicator of the abortion risk in N. caninum infected animals; values <4.5 ng/ml indicating a high risk of abortion in chronically infected animals.     

Gamma Interferon Production and Plasma Concentrations of Pregnancy‐Associated Glycoproteins 1 and 2 in Gestating Dairy Cows Naturally Infected With Neospora caninum

Gamma interferon (IFN‐γ) production and cross‐breed pregnancy have been attributed a role in protecting dairy cows infected with Neospora caninum against abortion. Plasma levels of pregnancy‐associated glycoproteins‐1 (PAG‐1) are a marker of placental/foetal well‐being and of PAG‐2 is an abortion risk indicator in chronically N. caninum‐infected animals. The present study examines, in cross‐breed pregnancies, interactions between IFN‐γ production and levels of PAG‐1 and PAG‐2 in non‐aborting naturally Neospora‐infected dairy cows. Data were obtained from 60 pregnant Holstein‐Friesian cows: 44 Neospora‐seropositive and 16 Neospora‐seronegative; 12 became pregnant using Holstein‐Friesian semen and 48 using Limousin semen. Blood samples were collected on Days 40, 90, 120, 150, 180 and 210 of gestation. Gamma interferon was only detected in the plasma of nine of the 44 Neospora‐seropositive cows, all of them became pregnant using Limousin semen. Through GLM procedures, in cows inseminated with Limousin semen and Neospora‐seropositive cows showing no IFN‐γ production, PAG‐1 concentrations were high and increased throughout gestation compared to the levels detected in cows inseminated with Holstein‐Friesian semen and Neospora‐seropositive cows producing IFN‐γ, respectively. In Neospora‐seronegative cows and in Neospora‐seropositive cows showing no IFN‐γ production, significantly increased PAG‐2 concentrations were observed on gestation Day 120. Our findings indicate that IFN‐γ production correlates negatively and the production of antibodies against N. caninum is uncorrelated with plasma PAG concentrations during gestation in Neospora‐infected dairy cows. Accordingly, IFN‐γ production could be linked to the transplacental migration of tachyzoites, which may cause a reduction in PAG levels.     

玫瑰花环抑制实验检测肉牛早孕因子(EPF)活性

[目的]为了根据早孕因子活性的检测作为依据来判断肉牛妊娠状态.[方法]本实验采集不同孕期的肉牛的血样,同时进行了妊娠诊断,通过玫瑰花环抑制实验检测活性.[结果]实验表明,未怀孕牛和怀孕牛花环抑制滴度差异显著(P<0.05),不同孕期的早孕因子活性差异不显著.[结论]玫瑰花环抑制滴度高于8可以确认牛怀孕,低于4为未怀孕.     

Accuracy of transrectal palpation for early pregnancy diagnosis in Egyptian buffaloes

The present study was undertaken to evaluate the accuracy of transrectal palpation (TRP) for diagnosing early pregnancy in buffaloes and the false diagnoses of the TRP test by using the pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) test. Pregnancy was diagnosed in 168 buffalo–cows once by TRP and PAG-RIA test between days 31 and 55 after breeding. The sensitivity of TRP for detecting pregnant buffalo–cows was 37.5% at days 31–35, increased to 93.8% at days 46–50 and reached 100% at days 51–55 (P < 0.01). All cases of false negative diagnoses (n = 10) had PAG concentration higher than the threshold (≥1.8 ng/mL) for diagnosing pregnancy. The specificity of TRP for detecting non-pregnant buffalo cows ranged between 90.9%, and 100% between days 31 and 55. All cases of false positive diagnoses (n = 5) made by TRP had PAG concentrations lower than the threshold for diagnosing pregnancy. It could be concluded that TRP is an accurate method for diagnosing pregnant and non-pregnant buffalo cows from day 46 after breeding.     

Trächtigkeitsfeststellung beim Rind mit dem Echolot-Verfahren

Pregnancy detection in cows by pulse mode ultrasound
Two commercially available pulse mode ultrasound instruments for pregnancy detection in cows were examined by testing 50 pregnant Friesian cows twice weekly. Absolutely reliable results were obtained after d 181 ("Cow-Tone") and d 221 of pregnancy (Preg-Alert 7 respectively. A newly constructed instrument ("HK-Rheintechnik") operating with greater wavelength (0,7 MHz) was tested in a similar fashion on 62 pregnant cows. Zt produced absolutely reliable results from d 135 of pregnancy on, i.e. 46 days sooner than the best of the commercial instruments tested .     

南德温肉牛冻精应用效果调查

[目的]为了了解南德温牛冻精的受胎效果.[方法]选择607头母牛进行人工授精,并与西门塔尔牛受精效果(727头)进行对比.[结果]表明:南德温牛细管冻配受胎率达到92.3%,产子成活率达到95.6%,而西门塔尔牛细管冻配受胎率达到89.2%,产子成活率为90%.[结论]说明南德温牛的冻配在促进肉牛发展上具有较高的受胎率...