Research Progress on Early Diagnosis of Pregnancy in Cattle and Sheep
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摘要:
规模化养殖中，母畜空怀将使养殖场遭受严重的经济损失，应用早期妊娠诊断技术，可有效降低空怀母畜数量，缩短产犊（羔）间隔，提高养殖效益。本文从诊断方法、诊断时间、诊断准确率等方面对牛羊早期妊娠临床诊断法和妊娠相关标志物诊断法进行综述。在临床诊断中，B超妊娠诊断较普遍，妊娠28～35 d超声检查可以获得比较可靠的结果，但其可靠性很大程度上取决于所用设备频率、操作者技能。在妊娠相关标志物诊断中，孕酮（Progesterone, P4）浓度检测法操作繁琐，对检测环境要求高，难以在生产中大面积推广。早期妊娠因子是受精后最早出现的特异性指标，但目前其检测完全依赖于使用玫瑰花环抑制试验，后续需开发简便的检测方法。干扰素刺激基因和外泌体miRNAs可能有助于牛羊早期妊娠诊断，但这些技术尚处于研究开发阶段。商业化的妊娠相关糖蛋白（pregnancy-associated glycoproteins, PAGs）检测试剂盒可作为B超妊娠诊断的替代方法，用于确定牛羊的早期妊娠或晚期胚胎损失。未来需要研发国产的商业化PAG检测试剂盒，以降低检测成本。本文通过总结不同检测方法的优缺点及实际应用效果，为生产者选择早期妊娠诊断方法提供参考，为牛羊早期妊娠诊断方法后期研究方向提供参考。
Abstract:In a commercial livestock or dairy farm, the so-called “empty sows” of those female animals failed to conceive in impregnation means economic loss for the business. Being able to accurately detect a pregnancy early, therefore, is indispensable for a timely determination allowing a short calving interval to enhance operational efficiency and profitability. This article reviewed the availability and advancements in the techniques and markers for the diagnosis on cattle and sheep. Published reports on the methodologies, examination time, and test result accuracy on the diagnosis are summarized with comments. For instance, B-ultrasound is commonly applied in clinical practices for 28–35 d pregnancy in the animals. Although relatively reliable results can be expected from the tests, they depend on the frequency and equipment selected as well as the skill of the operator. As a marker, progesterone (P4) is an indicator whose concentration in the animal is measured for the diagnosis. The method required specific testing environment, and consequently, is not popularly employed in the field. The initial fertilization indicator or physiochemical pregnancy signs in impregnated animals are currently detected by using the complex erythrocyte rosette test. Hence, not until a simple method is developed can a wide application based on the approach be realized. In theory, the interferon-stimulated genes and exosomal miRNAs may be useful for the diagnosis, but no testing technology has yet been established. And the commercially available PAG test kits for early-stage pregnancy or late-stage embryo loss are cost prohibitive for average farmers at present. By briefly describing the basics and presenting the pros and cons of various available devices and methods, this article provides a concise reference for the livestock ranchers in their decision-making and for the animal husbandry scientists in directing their efforts to develop a reliable and affordable means of fertilization detection in cattle and sheep.
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Keywords:
- cattle /
- sheep /
- pregnancy diagnosis /
- miRNAs /
- PAGs
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0. 引言
随着畜牧业的快速发展，集约化、标准化的养殖模式已经成为养殖业发展的大趋势[1]。目前，定时输精技术的应用为牛羊集约化养殖带来了可观的经济效益。然而，妊娠早期胚胎丢失造成的母畜空怀给养殖场带来较大的经济损失[2]。同时，妊娠失败显著增加产间距和淘汰率[3,4]，是造成养殖场经济损失的主要原因。因此，尽早对母畜进行妊娠诊断[5]，可有效提高母畜繁殖效率，缩短产间距，增加养殖场经济效益[6]。
早期妊娠诊断是在动物配种后一段时间，通过外部观察法、直肠触诊法（牛）、超声探查法等临床诊断方法[7]，或孕酮浓度检测法[8]、早孕因子检测法[9]、妊娠相关糖蛋白检测法[10]、干扰素刺激基因检测[11]等来判断母畜是否妊娠，对妊娠母畜进行精细化管理，未妊娠母畜及时补配或者进行淘汰，有利于提高母畜繁殖效率，促进集约化养殖。然而，这些方法均存在一定的局限性，在生产应用中还有待进一步完善。本文主要综述了近年来早期妊娠诊断技术的优点、局限性和目前较为前沿的实验室早期妊娠诊断方法的研究进展，为进一步深入研究牛羊早孕诊断方法和推广牛羊早孕诊断技术提供参考。
1. 临床诊断方法
1.1 直肠触诊
直肠触诊诊断妊娠发展至今已有近两百年的历史，该方法在牛上应用较普遍，并且需要操作人员大量实践，才能逐渐提高诊断的准确率。操作者通过触诊羊膜囊、胎膜滑动、子叶和胎儿这4种妊娠诊断标志来判断母畜是否妊娠；还可以通过触诊生殖道的其他变化辅助确诊，这些变化包括妊娠黄体、子宫中动脉扩张等。值得注意的是，如果没有触摸到4种基本的妊娠诊断标志中的任何一种，则不能给出明确的诊断结果[12]。 经验丰富的技术人员，在人工授精（artificial insemination, AI）35 d之后，通过触摸牛卵巢黄体和子宫角形状，就能得出较为准确的判断[13]。由于直肠触诊仅适用于大型动物，在操作过程中，一些生殖疾病可能导致误诊，如子宫炎或子宫积水，如果操作不当还可能会造成胚胎损失，增加医源性流产的风险，所以该项技术需要操作者具有较高的直肠触诊经验。
1.2 直肠超声波探查
超声波探查是与声学结合的一种物理诊断方法。自1966年将超声诊断技术引入兽医学以来，超声诊断技术得到了广泛的应用。20世纪80~90年代，A超、D超和B超在我国畜牧生产中得到推广应用，其中A超价格低廉，但准确度不高，D超准确率高，但价格昂贵，所以没有得到广泛应用。在牛羊等家畜中，B超成为主要的妊娠诊断工具，并且经直肠超声检查是一种无创、准确、有效的早期妊娠诊断方法[14]。
在B超妊娠诊断中，当子宫腔内发现一个形状不规则、无回声的黑点（或斑点）时，表明存在绒毛膜尿囊液，并在卵巢同侧的子宫角内有正常数量的绒毛膜尿囊液和黄体，即可证明已经妊娠[15]。需要注意的是，妊娠早期绒毛膜尿囊中的液体积累很容易在发情前和发情期间与子宫内的子宫液相混淆。研究发现，把识别心脏的跳动作为胎儿超声诊断阳性的标志时，与识别绒毛膜尿囊液相比，假阴性明显减少（P＜0.001）[16]，因此，检测胎儿心跳被业界认为是妊娠诊断的金标准。超声诊断试验证明，在AI后24 d就可以检测到胎儿心跳[17]。
但在生产中，很难在早期阶段检测到胎儿心跳。在生产中利用B超进行妊娠诊断主要是看母畜的羊水状态，必要时可以查看卵巢上的妊娠黄体或胎儿的脐带，妊娠黄体在母畜妊娠后1～2月内大小一般不会发生变化[18]，胎儿的脐带在妊娠后32 d就可以被观测到[19]。在规模化奶牛场运用B超进行妊娠诊断，在AI后28～34 d 诊断准确率为100%[20]；刘宗玲等[21]发现，湖羊在配种后20 d直肠B超的妊娠准确率仅为30.8%，随着时间的推移，30 d时准确率可达到100%，但熊琪等[22]在28 d的妊娠诊断准确率就可达到100%，这可能是操作者不同造成的差异。大部分操作者认为，经直肠超声妊娠诊断最有效的时间是妊娠后28～35 d[23,24]。使用不同的设备对诊断结果也有一定的影响。Silva等[15]报道，在母牛妊娠第27 天使用5～10 MHz线阵探头检测情况，子宫腔内存在绒毛膜尿囊液可能导致误诊。兽用B超探头发射的超声波频率一般在2～10 MHz，频率越高，分辨率就越好，能够探测到更小的物体，但是穿透深度就会相应降低，在生产中，根据牛羊体型差异，牛一般使用6.5 MHz线阵探头，羊一般使用3.5 MHz凸阵探头。
B超在牛羊早期妊娠诊断中具有准确率高、速度快等特点，但诊断结果易受操作者主观判断影响，如母畜个体差异和一些生殖系统疾病（如持久黄体、卵巢囊肿等）可能会导致操作者误判，且设备价格昂贵，很难在中小型养殖场推广。
2. 妊娠相关标志物诊断法
2.1 孕酮（progesterone, P4）
P4通过促进子宫内膜血管增生，从而支持早期胚胎发育和附植。研究发现，牛羊在整个妊娠期都能维持较高的P4水平，妊娠初期，P4主要由黄体分泌，为维持妊娠、胚胎着床和胚胎发育提供必要条件[25,26]。研究表明，血液和乳汁中的P4浓度可以作为妊娠检测的重要指标。在奶牛上，以乳汁中P4含量高于4.0 ng·mL−1判定为已妊娠、P4含量低于2.0 ng·mL−1判定为未妊娠为标准，妊娠诊断准确率为84.31%[27]；在山羊上，以P4含量低于8.3 ng·mL−1判定为空怀，妊娠诊断准确率为 96.5% [28]。
此外，血液中P4浓度不仅影响卵母细胞质量，还与流产密切相关。Martins等[29]发现，在奶牛妊娠35～56 d，血液中P4含量低的流产率显著高于P4含量高的流产率。类似地，在AI后10、21、30 d，流产奶牛血液中P4含量显著低于妊娠奶牛[30]。赵梦圆[31]发现，绵羊在AI后14～30 d，妊娠组血液中P4含量显著高于未妊娠组。因此，牛羊AI后血液中P4含量较低，可作为妊娠失败判别的重要依据。但是，P4浓度的测定要经过采血、离心、加样、温育、洗涤、显色、终止反应、检测等程序，且对检测环境要求严格，无法在生产实践中大面积推广。
2.2 早期妊娠因子（early pregnancy factor, EPF）
EPF最初是在小鼠中被发现的，并随后被证明对早期胚胎存活具有重要作用，不久之后，在羊、牛等家畜中也发现了EPF的存在。EPF作为胚胎发育中的免疫耐受因子，抑制母体对胎儿的免疫排斥，从而完成胚胎着床并妊娠。EPF最早可在交配或受精后数小时内出现，但妊娠失败或流产后便会迅速消失，EPF的变化可能与妊娠早期母体免疫调节有关[32]。在生产中，牛常用玫瑰花环抑制试验测定EPF，羊主要用血清滴定测定EPF。贺加双等[33]用玫瑰花环抑制试验对配种后1个月的母牛进行妊娠诊断，妊娠与空怀母牛EPF差异显著（P＜0.05）；郑毛亮等[34]采用血清滴定法对绵羊进行早孕诊断，准确率为90.81%。然而， EPF不是胎盘特异性蛋白，容易出现假阳性[35]。
在EPF之后，伴侣蛋白10被鉴定出具有免疫抑制和生长因子的特性，它属于热休克蛋白家族，与该家族其他成员不同，它可以在细胞外检测到EPF，但是目前还需要进一步试验来验证其准确性[16]。
2.3 干扰素（interferon tau, IFN-τ）及干扰素刺激基因（interferon-stimulated genes, ISGs）
胚胎在子宫定殖的过程中，滋养层细胞分泌IFN-τ进入子宫腔，通过抑制前列腺素F2α（PGF2α），从而阻止黄体的溶解，确保卵巢黄体继续产生孕酮维持妊娠[36]，同时，调节母体免疫，保障胚胎着床[37,38]。由于IFN-τ在子宫外组织和外周血中含量极低，因此，可以把IFN-τ浓度变化当作母体妊娠的识别信号。然而，目前尚未建立基于IFN-τ浓度检测来准确诊断牛羊早期妊娠的方法。
ISGs被认为是可以替代IFN-τ的检测方法。研究发现，IFN-τ通过诱导ISG15、MX1、MX2、OAS1基因表达，从而激活JAK激酶和转录激活因子信号通路调节胚胎植入[39]。奶牛在AI后14～24 d，外周血中性粒细胞中ISG15、MX1、MX2、OAS1的表达均上升[40]；此外，Yoshino等[41]观察到，在妊娠第21 天，妊娠牛中ISGs的表达显著高于未妊娠牛和晚期胚胎死亡牛；同样地，Domingues等[42]发现，AI后第22 天流产奶牛中ISG15比未妊娠奶牛高，但与妊娠奶牛相比，其表达较低；这些研究表明ISGs可以作为流产奶牛的妊娠标志物。在绵羊上，AI后的15 d内，受体转运蛋白4（receptor transporter 4, RTP4）基因和ISG15的表达均上调，在第18 天对妊娠母羊注射PGF2α后，RTP4和ISG15 的mRNA水平在第23 天和25 天下降（P＜0.001） [43]；Mauffré等[44]利用妊娠早期免疫循环细胞中ISG表达水平变化，检测绵羊妊娠情况，在检测未怀孕的母羊中发现有17%的母羊是妊娠母羊，说明妊娠早期免疫循环细胞中ISG存在高度变异，因此，通过检测ISG不能准确预测母羊是否妊娠。
2.4 妊娠相关糖蛋白（pregnancy-associated glycoproteins, PAGs）
在奶牛中，配种后15～22 d [45]、第24天[46]就可以在母体血液中检测到PAGs。 Filho等[47]在奶牛妊娠30～100 d中，发生晚期胚胎死亡的奶牛在妊娠24 d时PAG浓度低于维持妊娠至100 d的奶牛； Nanas等[48]研究表明，奶牛在人工授精后31 d或32 d低水平的PAG与胚胎丢失呈正相关。此外，在AI后第28天，经历流产的母牛循环PAGs浓度低于维持妊娠的母牛，而且PAG每减少1 ng·mL−1，流产的概率相应增加20%[49]。Domingues等[50]发现，AI后22 d流产的奶牛PAG水平显著低于维持妊娠的奶牛。PAG作为牛妊娠标志物在妊娠后短时间内就可以被检测到，并根据其浓度的变化预测胚胎损失，但预测胚胎损失的PAG浓度阈值还需要进一步研究。
在羊上，AI后18～22 d就能在母体血浆中检测到PAGs的存在 [51,52]，且随妊娠的进展呈线性增加[53]。Roberts等[54]发现，单胎母羊在分娩前后PAGs浓度均低于双胎母羊，同样的，PAG1可能是决定扎莱比山羊是否产双羔的关键因素[55]，但具体预测多胎的PAG浓度阈值并没有相关报道。在Lacaune羊和Sarda羊中，妊娠前60 d PAG的浓度持续增加，60 d后开始下降，持续到120 d 后又显著上升，在分娩时达到高峰[56]，这为通过PAG浓度预测母羊妊娠提供了时间参考。在羊的早孕检测中，由于PAG家族具有相似的抗原表位，大多使用牛的PAG-ELISA进行检测，但结果参差不齐，因此，亟需开发针对羊的PAG-ELISA来提供更加稳定的检测结果。
在实际生产应用中，妊娠相关糖蛋白的检测试剂盒已投入使用。Lamglait等[57]使用商用ELISA对妊娠期Barbary羊的PAGs进行测定，敏感性为100.0%，特异性为95.8%；De等[58]也有类似的报道。李艳艳[59]使用由北京爱德士元亨生物科技有限公司提供的 PAG ELISA 早孕检测试剂盒对牛进行早孕检测，结果未出现假阳性；薄小辉[60]使用美国爱德士（IDEXX）可视怀孕检测试剂盒（PAG检测试剂盒）检测荷斯坦奶牛妊娠情况，准确率可达93.9%，检测成本约50～60 元·头−1。Moussafir等[61]在牛AI后30～40 d内，使用商业化的ELISA-PAG试剂盒进行早期妊娠诊断，诊断准确率为94%。目前，我国尚无成熟的PAG检测技术，商业化的PAG早孕检测试剂盒均由美国爱德士公司生产，尽管其具有检测速度快、准确性高的特点，但检测成本高且无法预测母畜是否存在多胎妊娠，这使大多数养殖场望而却步，限制了其在国内大规模推广应用。
2.5 外泌体miRNAs
外泌体是由各种细胞释放到细胞外液中的微囊泡[62]，直径为30～150 nm，并广泛存在于所有的体液中。外泌体通过调节包括母体免疫反应和代谢适应在内的几个过程，在妊娠期内循环外泌体呈增加趋势，并在胚胎-子宫内膜的信息交流中发挥重要作用[63]。研究发现，成熟的miRNAs是外泌体中含量最多的RNA，并且循环miRNAs表达谱比循环mRNA表达谱具有更高的准确性[64]，可能是因为血液中的miRNAs不受内源性RNA酶的影响。此外，外泌体中的miRNAs很容易从体液中获取，实现无创伤检测，满足研究者对理想分子标志物的要求。
在哺乳动物中，miRNAs广泛存在于生殖器官中，并与繁殖性状高度相关[65]，研究人员猜测，妊娠期血浆中的特异性循环miRNAs可能与妊娠状态有关。Pohler等[66]观察到奶牛妊娠的第24天，血清中外泌体来源的miRNAs就可以区分其妊娠状态；同样的，Zhao等[67]发现，妊娠奶牛和未怀孕奶牛的血液外泌体miRNAs存在差异，可以作为判断奶牛妊娠状态的生物标志物。Ioannidis等[68]确认miR-26a是一种潜在妊娠诊断生物标志物，并在奶牛妊娠第8天 时，血浆中的miR-26a水平升高。Markkandan等[69]对妊娠30 d的奶牛进行血浆外泌体miRNA测序，发现有29个差异miRNAs，它们的靶基因与代谢过程和信号传导有关。此外，在妊娠失败和维持妊娠的奶牛之间也存在一些差异表达的外泌体miRNAs。Pohler等[66]发现，在妊娠第17天的胚胎死亡和妊娠奶牛中，有3种miRNAs（miR-25、miR-16b和miR-3596）在胚胎死亡奶牛中的表达显著升高。
在羊上，胚胎和子宫内膜分泌的外泌体参与了胚胎植入母体子宫内膜的生理过程[70]，Klisch等[71]认为双核滋养层分泌的外泌体是一种母胎交流的新类型。Cleys等[72]采集了绵羊妊娠30 d和90 d的血液和胎盘组织，发现只有miR-379在血清外泌体和胎盘组织样中差异表达，血清差异miRNAs靶向细胞生长、增殖和器官发育途径。Burns等[73]对绵羊子宫腔液中的外泌体进行测序，发现81个保守成熟的miRNAs来自子宫内膜上皮细胞或者滋养外胚层，并且认为外泌体在建立和维持妊娠中至关重要。目前，外泌体对牛羊妊娠的影响还处在研究阶段，筛选到差异表达的miRNA，还需深入研究其功能，为开发相应的检测产品奠定基础。
妊娠相关标志物诊断法具备各自的优点，但同时也存在一定的局限性（表1），导致不能大规模推广应用。
表 1 不同妊娠相关标志物诊断法在牛羊生产中的应用Table 1. Various methods for pregnancy diagnosis on cattle and sheep based on pregnancy-related markers妊娠诊断方法
Pregnancy diagnostic生产中应用效果
Application effect in production优点
Advantage局限性
Disadvantage孕酮浓度检测
P4 detectionAI后10、21和30 d，流产奶牛P4显著低于妊娠奶牛[29]；
羊在AI后14～30 d，妊娠组P4显著高于未妊娠组[30]；方便、可批量操作 程序复杂、对检测环境要求严格 早孕因子检测
EPF detection牛配种后1个月，妊娠与空怀差异显著 [32]；
绵羊进行早孕诊断，准确率90.81%[33]；诊断时间早，妊娠24 h后即可检出 肿瘤 EPF可能造成假阳性结果 IFN-τ及ISGs
IFN-τ and ISGsAI后22 d流产奶牛ISG15比妊娠奶牛低[41]；
羊在AI后15 d内 ISG15的表达均上调[41]；诊断时间早 判别标准需要进一步研究 妊娠相关糖蛋白检测
PAG detection牛PAG检测试剂盒准确率可达93.9%[60]；
羊PAG检测试剂盒敏感性100.0%，特异性95.8%[57]；诊断时间早 检测成本和技术要求高 外泌体miRNAs 牛：miR-26a可能是潜在妊娠诊断标志物[65]；
羊：miR-379可能是潜在妊娠诊断标志物[69]；诊断时间早 在试验研究阶段，无相应检测产品 3. 总结与展望
在牛羊早期妊娠诊断领域，多种检测方法各具特点与局限。直肠超声波探查在早期妊娠诊断方面具有较高的准确性，但它价格昂贵，而且操作者的主观判断可能导致假阳性结果； P4、早期妊娠因子检测法因操作繁琐，且对检测环境要求高，还需进一步优化检测方法。干扰素刺激基因和miRNAs，虽为牛羊早期妊娠诊断带来新的可能性，但目前这些技术尚不成熟，还需深入研究，才有可能在生产中应用。商业化的PAG检测试剂盒可以作为B超妊娠诊断的替代方法，用于确定牛羊的早期妊娠或晚期胚胎损失。但当前市场上的此类试剂盒依赖进口，使用成本较高，未来有必要研发国产的商业化PAG检测试剂盒，降低检测成本，提高其在生产中的应用推广。
近年来，电化学免疫传感器凭借灵敏度高、选择性好、低成本、快速响应、易操作等优势，被用于分析和检测疾病生物标志物。随着纳米技术的快速发展，研究人员将纳米材料和电化学免疫传感器相结合，使检测在可见光照射下呈现肉眼可见的不同颜色，这为早期妊娠诊断标志物的检测开辟了新路径。此外，在胚胎着床和胎儿发育过程中，母体免疫系统发生巨大变化，而这种变化被认为与母体肠道微生物和代谢物有关[74]。因此，肠道微生物、代谢物和免疫之间可能存在潜在生物学联系，且与妊娠相关。然而，这种潜在的生物学联系仍需在未来的研究中进一步验证。
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表 1 不同妊娠相关标志物诊断法在牛羊生产中的应用
Table 1 Various methods for pregnancy diagnosis on cattle and sheep based on pregnancy-related markers
妊娠诊断方法
Pregnancy diagnostic生产中应用效果
Application effect in production优点
Advantage局限性
Disadvantage孕酮浓度检测
P4 detectionAI后10、21和30 d，流产奶牛P4显著低于妊娠奶牛[29]；
羊在AI后14～30 d，妊娠组P4显著高于未妊娠组[30]；方便、可批量操作 程序复杂、对检测环境要求严格 早孕因子检测
EPF detection牛配种后1个月，妊娠与空怀差异显著 [32]；
绵羊进行早孕诊断，准确率90.81%[33]；诊断时间早，妊娠24 h后即可检出 肿瘤 EPF可能造成假阳性结果 IFN-τ及ISGs
IFN-τ and ISGsAI后22 d流产奶牛ISG15比妊娠奶牛低[41]；
羊在AI后15 d内 ISG15的表达均上调[41]；诊断时间早 判别标准需要进一步研究 妊娠相关糖蛋白检测
PAG detection牛PAG检测试剂盒准确率可达93.9%[60]；
羊PAG检测试剂盒敏感性100.0%，特异性95.8%[57]；诊断时间早 检测成本和技术要求高 外泌体miRNAs 牛：miR-26a可能是潜在妊娠诊断标志物[65]；
羊：miR-379可能是潜在妊娠诊断标志物[69]；诊断时间早 在试验研究阶段，无相应检测产品 
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