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。     

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.     

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。     

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.     

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.     

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.     

泌乳与妊娠奶牛营养需要模型及其Basic程序编制

本文以《中国奶牛营养需要和饲养标准》(2000年,修订第二版)为重要文献,详细介绍了泌乳与妊娠奶牛营养需要模型的基本常数、数学模型和计算方法,编制出了实用的计算泌乳与妊娠奶牛营养需要的Basic程序。     

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.     

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。     

Experimental Infection of Cows with Bovine Viral Diarrhoea Virus in Early Pregnancy – Findings in Serum and Foetal Fluids

Nineteen pregnant cows were experimentally infected with bovine viral diarrhoea virus (BVDV) between day 74 and 81 of pregnancy. All cows became infected and developed serum antibodies. Sixteen of the cows delivered persistently infected (PI) offspring, whereas the remaining three gave birth to calves with detectable serum antibodies and free from BVDV. The 16 cows with PI foetuses developed higher levels of antibodies in serum during pregnancy than did their three peers carrying non‐PI calves. Multivariate analysis showed that the antibody levels in these two groups of cows were significantly different from day 135 of pregnancy. Foetal fluid was successfully collected from 18 of the 19 infected cows and from five uninfected control cows between 10 and 24 days before delivery by use of a percutaneous, blind puncture technique. No negative effects were observed in the cows or their offspring. BVDV was isolated and detected with an immunoperoxidase test in foetal fluid from 13 of the 16 cows carrying PI foetuses, and from 15 of the cows when a quantitative fluorescent polymerase chain reaction (PCR) technique was used. The negative sample in the PCR assay was positive for BVDV antibodies. The number of viral copies per microlitre in foetal fluids varied between 103 and 1080 in the positive samples. All samples taken from the cows carrying non‐PI foetuses were negative for BVDV in both assays. In this experiment, examination of either serum or foetal fluids could identify the cows carrying a PI foetus. Examination of serum for BVDV antibodies was a reliable indicator of a PI foetus if the serum was collected during the last 2 months of pregnancy. For examination of foetal fluids, both viral and serological analyses should be performed. For viral analysis, PCR should be the test of choice. High levels of BVDV antibodies in conjunction with a negative result in the PCR may be indicative of a false‐negative virus result. Further experience with the method of collection of foetal fluids is necessary for evaluation of its safety. Investigation of pregnant cows in order to discover a PI offspring before it is born could be a useful tool in control and eradication of BVDV.     

母海狸鼠早期怀孕的诊断

采用MCD Ⅰ型检侧仪对 16 2 0只海狸鼠进行了早期怀孕诊断 ,并与分娩记录相比较 ,其符合率高 ,最早探测宫血音、胎心音、胎血音时间分别为 8,12 ,16d。宫血音诊断与分娩记录符合率在配种后 10～ 15d ,16～ 2 5d分别为 97 7% ,10 0 % ;未怀孕的符合率为 10 0 %。本试验还采用AlokaSSD 2 10DXⅡ型超声诊断装置 (超声断层扫描、B超 )鉴定了已用MCD Ⅰ型检测仪诊断为怀孕的母海狸鼠 2 3只 ,这两种检测结果一致。超声断层扫描最早可在配种后 8～ 13d对怀孕作出诊断 ,故证明MCD Ⅱ型检测仪检测宫血音的效果是可靠的 ,因此 ,母海狸鼠的早期怀孕诊断可用宫血音阳性和超声断层扫描作为其判定怀孕的主要依据     

泌乳早期乳中尿素氮含量对奶牛繁殖性能的影响

为探索奶牛泌乳早期乳中尿素氮含量对产后繁殖性能的影响,本研究选取海丰奶牛场的头胎荷斯坦牛1932头,于产犊后30d内采集乳样,测定乳中尿素氮含量,并收集试验牛只产后繁殖资料,用多因素方差分析法分析乳中尿素氮对产后繁殖性能的影响。结果表明,试验牛只产犊后1个月内乳中尿素氮含量为9．22±2．78mg／dL;乳中尿素氮含量对个体情期受胎率有显著影响（P〈0．05）,而对产后首次配种天数有极显著影响（P〈0．01）,乳中尿素氮含量大于15mg／dL的奶牛产后第一次配种天数显著高于低于此数值的奶牛,且个体情期受胎率显著低于此数值低的奶牛。同时乳中尿素氮含量与个体情期受胎率呈极显著负相关（P〈0．01）。据此认为,作为反映能氮平衡的关键指标,泌乳早期乳中尿素氮含量可作为预测奶牛产后繁殖性能的指标加以利用。     

Termination of Pregnancy with Cloprostenol and Dexamethasone in Intact or Ovariectomized Cows

Termination of pregnancy in cows was investigated using sham-operated (SH) or ovariectomized (OV) cows treated with either a saline vehicle (V), cloprostenol (PG), dexamethasone (DEX) or dexamethasone and cloprostenol (DEX+PG). Surgery was done at 210 days of pregnancy and treatment was administered 72 hours later.

Days (mean±S.E.) from treatment to termination of pregnancy for the treatment groups were: sham-operated +vehicle (SH+V): 61.5±11.3; ovariectomized+vehicle (OV+V): 53.4±15.7; sham-operated+cloprostenol (SH+PG): 61.8±1.7; ovariectomized+cloprostenol (OV+PG): 54.5±13.1; shamoperated+dexamethasone (SH+DEX): 74.8±4.8; ovariectomized+dexamethasone (OV+DEX): 2.8±0.4; shamoperated+dexamethasone+cloprostenol (SH+DEX+PG) 26.0±23.0; ovariectomized+dexamethasone+cloprostenol (OV+DEX+PG): 7.2±4.9. Pregnancies in the OV+DEX and OV+DEX+PG groups were terminated significantly earlier than in all other groups (P<0.05) except the SH+DEX+PG group. These findings suggest that dexamethasone will terminate pregnancy in cows near seven months of gestation after the ovarian source of progesterone has been removed by either an injection of prostaglandin or by ovariectomy.