Comparison of the Ability of Three Radioimmunoassay to Detect Pregnancy-associated Glycoproteins in Bovine Plasma

Pregnancy‐associated glycoproteins (PAGs) constitute a large family of glycoproteins that are synthesized in the superficial layer of the ruminant placenta according to a spatial and temporal expression pattern. When PAGs are released in the maternal blood they can be used for pregnancy diagnosis, pregnancy follow‐up and for the monitoring of the trophoblastic function. Three different radioimmunoassay systems (RIA 1, RIA 2 and RIA 3) using antisera produced against PAG I₆₇ (RIA 1), PAG₅₅₊₆₂ (RIA 2) and PAG₅₅₊₅₉ (RIA 3) were used in this investigation in order to measure the PAG concentration in plasma samples withdrawn from pregnant cows and heifers during different periods following artificial insemination (AI). These systems were able to detect PAG molecules in the maternal blood as early as 21 days after AI in different concentrations (RIA 1: 0.43 ± 0.24 ng/ml, mean ± SD; RIA 2: 0.48 ± 0.24 ng/ml; RIA 3: 0.64 ± 0.37 ng/ml). On days 32 and 42 RIA 2 (4.30 ± 1.32 ng/ml and 5.56 ± 1.95 ng/ml) and RIA 3 (4.17 ± 1.15 ng/ml and 5.60 ± 1.89 ng/ml) presented significantly (p < 0.0001) higher PAG concentrations than those of RIA 1 (2.43 ± 0.81 ng/ml and 4.01 ± 1.48 ng/ml), respectively. After day 21, significant correlations (p < 0.0001; r ≥ 0.929) were determined between the three systems. Additionally the three individual PAG profiles presented in this study showed that PAG molecules secreted in the maternal blood between 21 and 50 days after AI were better recognized by the RIA 2 and RIA 3 systems. This study clearly indicated that the ability of a RIA test to recognize PAG molecules in the maternal blood can be improved by carefully selecting the antiserum.     

Measurement of Ovine Pregnancy-Associated Glycoprotein (PAG) During Early Pregnancy in Lacaune Sheep

This study describes ovine pregnancy‐associated glycoprotein (ovPAG) concentrations in 20 Lacaune sheep during early pregnancy. Measurements were performed by using semi‐purified ovPAG as standard, tracer and immunogens for antibody production in rabbits. Antisera R780 (against ovPAG_57+59kDa) and R805 (against ovPAG5_58+61kDa) were used respectively in RIA‐780 and RIA‐805. Blood samples were collected at days 0, 18, 20, 22 and 25 after artificial insemination. From day 18 after breeding onward, the mean ovPAG concentration was significantly higher (p < 0.001) in plasma samples from pregnant ewes (n = 17) than in non‐pregnant ones (n = 3). The specific activity of the tracer was 11 760 Ci/mmol in RIA‐780 and 14 900 Ci/mmol in RIA‐805. The minimal detection limits for RIA‐780 and RIA‐805 were 0.2 ng/ml and 0.3 ng/ml, respectively. The intra‐assay CV of samples with low (1.0 ng/ml), medium (2.5 ng/ml) and high (4.0 ng/ml) PAG concentrations were 3%, 6% and 9% for RIA‐780 and 8%, 9% and 5% for RIA‐805. The inter‐assay CV in the same samples were 13%, 12% and 7% for RIA‐780 and 13%, 11% and 5% for RIA‐805. The recovery was higher than 95% in both assays. No cross‐reaction was observed with members of aspartic proteinase family as well as with other tested proteins. In both RIA‐780 and RIA‐805, inhibition of the binding of the tracer by antisera was parallel between standard curve and serial dilutions of pregnant ewe samples. In conclusion, the two homologous RIA systems are suitable for early quantification of ovPAG concentrations in ewe plasma samples from day 18 after breeding.     

奶牛早孕诊断技术研究进展

高繁殖率是提高奶牛场经济效益的重要保障,而早孕诊断又是提高奶牛繁殖率的重要手段之一。传统的妊娠诊断方法较难在配种后21 d内确定奶牛妊娠状态,而近年研发的新型妊娠诊断技术可以对奶牛进行有效早孕诊断。本文综述了多普勒彩超技术,中红外线光谱技术及妊娠相关蛋白、循环核苷酸、干扰素τ诱导基因检测等技术在奶牛早孕诊断中的研究进展,比较分析了这些技术的实际应用效果及优缺点,并对新的奶牛早孕诊断技术进行了展望,以期为开发和应用新型高效奶牛早孕诊断技术提供思路和借鉴。     

奶牛早期妊娠诊断技术的研究进展

高效准确的早期妊娠诊断,可以缩短母牛的产犊间隔,从而达到提高母牛繁殖效率的目的。家畜早期妊娠诊断方法的研究具有悠久的历史,从原始的外部观察法到目前应用最广泛且最直接的直肠检查法,再到后期发展起来的超声波检查法以及近年来研究比较多的易于商业化的间接检测法。本文对在生产中具有实际应用效果且具有一定发展前景的早期妊娠诊断技术应用现状进行了综述。     

奶牛早期妊娠诊断技术研究

该文应用直检和B超对奶牛配后16~21 d进行卵巢检测,根据可参配母牛发情时的卵泡和配后黄体发育的状态与质量,对是否妊娠做出综合判断,进而减少和缩短母牛空怀期,提高奶牛生产效益。     

奶牛早孕诊断试纸条的研制及临床试验

用抗孕酮单克隆抗体S3B2D4A6和羊抗鼠IgG分别固定于硝酸纤维膜，并与胶体金标记的抗孕酮单克隆抗体S3B3E1G1及其它试剂，应用层析式双抗体夹心法原理研制奶牛早孕诊断试纸，在北京10家奶牛场进行临床检测试验，对1559头荷斯坦奶牛乳汁中孕酮含量进行临床符合验证。结果表明。该试纸条灵敏度高，最低检出量达5～8ng／mL。与无关抗原均无交叉，且批内、批间变异小；检测1559头奶牛。孕牛1316头，其中试纸检测结果阳性1274头。可疑32头。复测阳性22头，孕牛阳性临床符合率96．8％；复测后阳性总符合率达98．5％。阴性对照243头，其中试纸检测结果阴性236头。可疑6头。复测阴性2头。阴性临床符合率为97．1％。复测后阴性总符合率97．9％。结论：北京万华生物工程有限公司研制的奶牛早孕诊断试纸能够快速、准确地进行奶牛早期妊娠诊断。     

现场诊断奶牛早孕与发情的酶免疫分析法的改进

报道了一种建立并经改进的现场检测乳汁孕酮诊断奶牛早 和发情的酶免疫分析法，有准、早、易、安全等优点。     

应用EPF抗血清进行奶牛超早期妊娠诊断的研究

为了建立一种快速、高效的奶牛早期和超早期妊娠诊断的方法,采用多肽合成仪合成了早孕因子(EPF)的部分肽段(MC29),将MC29与载体蛋白BSA连接后免疫Balb/C小鼠,制备EPF抗血清,用EPF抗血清阻断ELISA对6头奶牛进行超早期妊娠诊断。结果表明:妊娠10～21d的4头奶牛血清检测均为阴性,2头未妊娠奶牛血清均为阴性,该结果与输精2个月后的直肠检查和B超鉴定的结果相一致。说明应用EPF抗血清进行阻断ELISA可以准确的进行奶牛超早期妊娠诊断。     

用血清酸滴定法对牛进行早期妊娠诊断的研究

采用血清酸滴定法对配种后16 d～45 d的1 008头不同品种的母牛进行了早期妊娠诊断研究,检出妊娠牛764头,未妊娠牛244头;与直肠检查结果对比,妊娠符合率为90.31%(690/764),未孕符合率为93.03%(227/244),总符合率为90.97%(917/1008).对3种不同品种牛试验结果经3组间及两组间卡方检验统计分析,无显著差异(P＞0.05).表明血清酸滴定法用于牛的早孕诊断具有诊断时间早、方法简便、结果准确等优点,宜于在基层推广.     

Failure to Detect Soluble Tumor Associated Antigen by Radioimmunoassay in Bovine Lymphoma

The detection of circulating soluble tumor-associated antigen by radioimmunoassay was attempted in cows with lymphoma. The assay system did detect PAGE-purified tumor-associated antigen but did not detect immunoreactive substances in the sera of 20 affected animals and 20 normal cattle. Similarly, ultrafiltration of low pH-dissociated serum to separate immune complexes did not aid in the detection of antigen.     

3种食蟹猴早孕诊断方法的比较

本试验旨在对3种食蟹猴早孕诊断方法进行比较,找出比较准确、可靠的诊断方法。在食蟹猴怀孕第20天时用直肠触诊、B超检查和孕酮值检测3种方法进行早孕诊断,在怀孕第30天时B超确诊是否怀孕。结果表明,B超检查的准确率为96.4%;直肠触诊的准确率为92.9%;孕酮值检测的准确率为64.3%。因此B超检查准确率高,是食蟹猴怀孕诊断的金标法;直肠触诊准确率较高且方便、经济,适合基层生产中进行大批量诊断;孕酮值检测确诊率较低,但可作为早孕诊断的辅助方法。     

Impact of Probing the Reproductive Tract During Early Pregnancy on Fertility of Beef Cows

This short communication reports the impact of endometrial biopsies, uterine flushings and follicular fluid aspiration procedures at day 6 post artificial insemination (AI) on pregnancy rates. In Experiment 1, cows were timed AI (TAI) and assigned to the following treatment groups: control (n = 37), uterine flushing (n = 35) and endometrial biopsy (n = 38). On day 30 post AI, pregnancy rates were 40.5%, 33% and 28.5%, respectively (p > 0.1). Pregnancy rate on day 60 was lower (p < 0.004) in flushed cows than in the controls. In Experiment 2, oestrus was detected and cows were assigned to flushing (n = 32) or biopsy (n = 33) treatments 6 days after AI, which resulted in pregnancy rates of 31% and 36%, respectively (p > 0.1). In Experiment 3, cows were, 6 days after TAI, randomly assigned to the following treatments: control (n = 84) or aspiration of the largest follicle (n = 73). Pregnancy rates on day 30 post AI were 63.5% for the control group and 53% for the aspirated group (p > 0.1). In conclusion, uterine flushing and endometrial biopsy negatively affect pregnancy rates, but neither procedure can be considered to be incompatible with pregnancy maintenance. Follicular aspiration during pregnancy does not interact with pregnancy success. The amount and quality of samples obtained are compatible with the use of cellular and molecular analysis of uterine variables from cows that failed or succeeded on maintaining pregnancy.     

赐力健对奶牛生产性能、免疫力、抗热应激能力和受胎率的影响

采用对照试验设计,在北京一奶牛场选择40头奶牛,研究赐力健对奶牛生产性能、免疫力、抗热应激能力和受胎率的影响.结果表明:赐力健可以提高产奶量、增强抗热应激能力、降低体细胞数,明显提高受胎率,对提高免疫球蛋白含量没有作用.     

Measurement of Faecal Progesterone Metabolites and its Application for Early Screening of Open Cows Post-insemination

The present study investigated the changes of serum progesterone (P4) and its faecal metabolites in pregnant and non-pregnant cows (Expt 1) and the feasibilty of using faecal P4 metabolites for early screening of open cows post-insemination (Expt 2). In Expt 1, seven crossbred Holstein-Friesian (HF) cows were studied. Serum and faecal samples were collected once daily from the day of artificial insemination (AI) until 25 days after AI. In Expt 2, 27 crossbred HF inseminated cows were employed. Serum and faecal samples were obtained on the day of AI (day 0) and on days 19-22 post-insemination. Enzyme immunoassay measurements of serum P4 and faecal P4 metabolites were established. The low detection limit of the assay was 0.01 ng/ml and the amount of P4, resulting in a 50% reduction in the initial binding value, was 1.07 ng/ml. The intra- and inter-assay coefficients of variation were <8% and <14%, respectively. A positive correlation between the levels of serum P4 and faecal P4 metabolites was found in every single cow (r = 0.73-0.88, p < 0.001) and pooled data (r = 0.78, p < 0.001). The estimated value of faecal P4 metabolites at 100 ng/g of faeces was equal to the serum P4 levels of 1 ng/ml. The accuracies of pregnancy and non-pregnancy diagnosis based on the analyses of faecal P4 metabolites between day 0 and days 19-22 post-insemination, were 67% and 100%, respectively. In conclusion, the measurement of faecal P4 metabolites can be a potentially alternative method for early screening of open cows post-insemination with the same accuracy and precision, as measured by serum P4 assay.     

Aspartic Proteinase Members Secreted by the Ruminant Placenta: Specificity of Three Radioimmunoassay Systems for the Measurement of Pregnancy-associated Glycoproteins

Pregnancy‐associated glycoproteins (PAGs) isolated from the placenta of various ruminant species are enzymatically inactive members of the aspartic proteinase family. The measurement of these proteins in the maternal blood can be a good indicator of the presence of a live embryo. As certain aspartic proteinases are present in biological fluids in physiological and pathological conditions at various concentrations, it was necessary to determine the specificity of three radioimmunoassay (RIA) systems currently used for the detection of PAG molecules. Commercially available members of the aspartic proteinase family like pepsinogen, pepsin, chymosin, rennet, cathepsin D and renin were tested in a wide concentration range (10 ng/ml – 1 mg/ml). Pepsinogen cross‐reacted in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 50 μg/ml and 500 μg/ml concentrations, respectively. In the presence of pepsin, cross‐reaction was observed in RIA 1, RIA 2 and RIA 3 over 1 mg/ml, 500 μg/ml and 1 mg/ml concentrations, respectively. Chymosin and rennet could cross‐react in RIA 2 and RIA 3, while renin and cathepsin D did not decrease the binding of the tracer to antisera more, than that of the minimal detection limit. As the plasma/serum concentrations of the examined aspartic proteinases reported in the literature were outside the concentration range where cross‐reaction was observed, it can be concluded that these RIA systems were specific for the detection of PAGs in biological fluids.     

皮毛中5种病毒基因芯片检测方法的研究

根据GenBank上蓝舌病病毒(BTV)、O型口蹄疫病毒(FMDV)、山羊痘病毒(GPV)、绵羊痘病毒(SPPV)和牛病毒性腹泻病毒(BVDV)等5种病毒的特异性保守序列,分别设计多重荧光标记引物和相应寡核苷酸探针.使用芯片点样液稀释各探针至终浓度30μmol/L,点样制备11×11阵列芯片.核酸杂交后,建立并优化基因芯片检测方法.结果显示,使用470 mL/L甲酰胺杂交液,42℃摇转杂交4h为最佳基因芯片杂交条件.建立的基因芯片检测方法与伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、禽流感病毒(AIV)和新城疫病毒(NDV)等无交叉反应,检测敏感性可达20拷贝病毒核酸.制备的基因芯片稳定,保存6个月可用.对151份临床样品进行基因芯片和商业化PCR试剂盒平行检测,两者的符合率为100％.     

Effect of Complex Vertebral Malformation on Luteal Function in Holstein Cows During Oestrous Cycle and Early Pregnancy

The reason why cows carrying the mutation of complex vertebral malformation (CVM) show poor reproductive capability although they carry only one mutant allele is still not fully understood. Monitoring the progesterone profiles during oestrous cycle and early pregnancy in carrier cows might help explain their lowered reproductive capability. Progesterone concentration was measured in 19 CVM carrier cows and 21 control cows during oestrous cycle and early pregnancy. Milk samples were collected from all cows starting on the day of artificial insemination until day 45 post‐AI. Progesterone was measured in skim milk using enzyme‐linked immunosorbent assay (ELISA). Progesterone concentration was significantly reduced on day 7 (p < 0.05) and day 9 (p < 0.01) post‐insemination in conceived CVM carrier cows when compared with that in control conceived cows. The mean progesterone concentration during early pregnancy was significantly lower (p < 0.05) in conceived cows with CVM than that of control cows in the same period. However, the mean progesterone concentration did not differ significantly (p = 0.072) in CVM cows that showed fertilization failure or embryonic death than that of control cows. Additionally, of 13 conceived control cows, eight cows (61.5%) showed normal luteal function. In contrast, of nine conceived CVM cows, only four cows (44.4%) showed normal luteal function. The conception rate was 47.4% in CVM carrier cows and 61.9% in control cows, but this difference did not reach significance. In conclusion, progesterone concentration might be lowered during early pregnancy in conceived CVM cows compared with that in control cows.     

Pregnancy‐Associated Glycoprotein (PAG) Profile of Holstein–Friesian Cows as Compared to Dual‐Purpose and Beef Cows

Pregnancy‐associated glycoproteins (PAGs) are produced by mono‐ and binucleate trophoblast cells in the ruminant placenta. PAG appears in maternal blood and, from approximately 4 weeks after fertilization onward, may serve as a reliable means of diagnosing pregnancy. A range of factors are said to affect plasma PAG concentrations, such as number and sex of foetus, mass of calf and placenta, level of milk production and genetic constitution. In this study, PAG pregnancy profiles of a dual‐purpose (Simmental) and two beef breeds (Uckermark and Aubrac) are compared with the profile of the specialized dairy breed Holstein–Friesian. Holstein–Friesian cows were sampled weekly; the levels of the other breeds were presented at 3‐week intervals. The overall significant breed difference (p = 0.013) was founded on deviations during the initial 3 weeks of pregnancy and from 23 weeks onward. During the period critical for the detection of pregnancy, between four and 22 weeks, agreement between PAG levels of various breeds was close (p > 0.05). No significant effect of body mass of cow or calf (relative to mass of dam) was detected. These findings imply that the PAG pregnancy test may be executed uniformly irrespective of breed or type of cow, affirming the suitability of the test as a valuable asset for the cattle industry.