作者要感谢珍妮特·海斯-克鲁格和凯利·安德森管理蒙特雅克殖民地，艾伦·杨博士向我们介绍了蒙特雅克和Cervid解决方案公司的克莱夫·特德福德先生为这个项目提供种子蒙贾克。这项工作得到了NIH赠款R01AI093634的支持。

1. 程序准备
所述所有动物程序都已由科罗拉多州立大学动物护理和使用机构委员会（实验室动物护理评估和认证协会）审查和批准，该协会是国际认可机构。
<ol><li>在检查之前，使用适当的静剂或麻醉方案固定动物，以提供放松，并允许适当的定位。一个处理者限制鹿，而另一个人给动物施用安定。 注： 作者已经找到以下组合，以提供有效的静剂在这个物种：丁醇（0.45毫克/千克），阿扎佩龙（0.035毫克/千克）和甲基胺（0.04毫克/千克），肌肉内在臀部肌肉管理。</li>
	<li>一旦脚趾达到允许合规处理的沉着状态，将她定位在复明或横向重新任职的检查台上或 V 槽内。稍微提升头部、颈部和胸腔（约 20-30°），以降低反流和渴望的神经内含物的风险，并便于在手术过程中进行适当的通风。 注： 在大多数腹腔或其他反胃动物中，腹部超声波在站立动物中进行：然而，这个物种的体积小和行为考虑需要大量的沉淀检查和胸骨定位因此更加困难，并可能排除有效的成像技术。将动物置于胸骨和横向重新任职之间的中途，可能是降低再叛乱和低气流风险的理想之选，如果可能，应予以考虑。在任何卧存位置，必须仔细监测动物的呼吸，并立即停止检查，如果发现动物出现回流或通风过低，则将其置于严重重新监禁状态。</li>
	<li>如有必要，从腹部区域剪发，以便实现传感器的脚印和皮肤之间的适当接触。 注： 在妊娠早期，可以在乳腺附近的腹腔腹部检测到怀孕的证据。蒙特雅克的这个区域通常是无毛的，因此此时可能不会指示剃须。随着胎儿的生长和子宫的衰减，在腹部占据更颅骨的位置，在成像之前需要剪头发。应小心避免对皮肤造成任何机械创伤，包括剃须刀刀片的撕裂或电剪的热损伤。</li>
	<li>将超声波装置放置在一个位置，使技术人员能够在用占主导地位的手进行检查时轻松可视化监视器。将动物识别信息（名称、识别号 等）输入系统，以自动标记保存的图像和视频循环。</li>
</ol>2. 跨性别生殖超声检查
<ol><li>将大量的声学耦合凝胶涂抹在腹腔上。</li>
	<li>将 5 - 10 MHz 弯曲的线性传感器放在腹腔皮肤上，立即颅到，然后缓慢地朝颅面方向移动，以评估骨盆腔内的结构。扫描胆汁腹部的左右象限，以评估蒙贾克双角子宫的两个角。 注： 全面的腹部超声波检查包括所有腹部内脏，包括肝脏、肾脏、脾脏、胃肠道、尿囊和生殖道。为了本议定书的目的，将只详细讨论对怀孕的检查：然而，其他腹腔器官的正常外观应被识别为这些结构可能会在研究过程中遇到。在这个位置最常见的器官包括胃肠道、尿囊和生殖道的片段。大肠和小肠通过肠道壁的特征分层外观来识别。流明可能发音，也可能不发音，具体取决于内容的类型和数量，其中可能包括液体、气体、粘液或食物颗粒。除非尿液分离，否则膀胱可能难以识别，膀胱壁的层明显不如肠道的层明显不同。除了子宫外，卵巢也可能在雌性蒙贾克的生殖道的超声波上找到。卵巢将以卵巢结构出现，靠近肾脏的卵巢极，其外观可能会在骨循环过程中发生变化。皮层内存在多个成熟、充满液体的卵泡，这暗示着雌激素。 注： 通过旋转和/或"扇动"传感器的扫描头，结构可以用横向或下垂平面进行评估。<ol>
<li>如果需要区分子宫与尿囊，通过对腹腔腹部施加温和压力或放置尿导管并重复扫描，手动排空膀胱。 注： 只有在动物处于胸骨或直立位置时，才应尝试手动表达膀胱，这有利于腹部内脏更自然地方向，并降低对其他结构造成创伤的风险。子宫也可以通过评估器官壁的厚度来区分尿囊。中度放开的膀胱的典型厚度约为1.4毫米24，而作者发现，在蒙贾克，大子宫的肌肉壁通常尺寸在7-8毫米之间。 注：由于缺乏可识别的胎儿结构，早孕很难确定，事实上，怀孕的第一个迹象可能只是腹腔内的一个小液体口袋。如果此时看到，发育中的胚胎将呈超选择性，卵形到子宫内的无定形结构（图1）。</li>
	</ol></li>
	<li>根据需要对深度、频率和增益进行调整，以改善对比度和图像细节。 注意：更高的频率与较短的波长相关联，这是提高图像分辨率所必需的。大多数转导器能够在频率范围内工作（例如，6-10 MHz）24。频率可以通过超声波单元上的扫描仪控制进行调整，增加频率将导致图像更清晰。增加频率同时降低声波渗透深度：因此，减少频率可能是必要的可视化更深的结构，但分辨率可能会成为妥协24。因此，选择尽可能高的频率以达到所需的组织深度非常重要。增益控制的调整将改变声波从组织返回到传感器的放大，并影响图像24的对比度。增益应设置为实现高选择组织和低氧组织之间的最佳对比的点，这些设置可能需要根据正在成像的结构进行调整。</li>
</ol>3. 获得胎儿测量
注意：许多家畜物种的妊娠年龄可以使用从超声检查获得的胎儿测量的既定参考范围来估算。最常用的测量包括直冠凸长（SCRL）、胸部深度（CD）和头部长度（HL）17。虽然尚未收集到足够的数据，以制定可靠的妊娠年龄指南在里维斯的蒙贾克鹿，测量可以收集，以开始建立这些参考。胎儿心率 （FHR） 已在其他类型的子宫颈和反胃动物中显示，随着妊娠进展到某一点（通常在妊娠的第 60 天左右），线性增加，此时心率开始下降17。FHR可用于与其他超声波观察一起估计妊娠年龄，但更常用于测量胎儿存活率17，24。FHR早在观察后35天就计算在里维斯的蒙贾克。
<ol><li>要测量 SCRL，将子宫定位在监视器上，并对传感器的位置进行微调，直到获得整个胎儿的下垂视图（图 2和图 3）。冻结图像并测量头骨最后部和囊基之间的距离（图 2和3）。</li>
	<li>要测量 CD，获取胎儿的下垂视图（可用于测量 SCRL 的相同图像），并测量胸腔的支部和腹腔方面之间的距离（图 2和图 3）。</li>
	<li>要测量 HL，使用与 SCRL 和 CD 测量中描述的胎儿相同的下垂视图，测量从腹股顶到鼻底的距离（图 2和图 3）。</li>
	<li>要计算胎儿心率，通过可视化胎儿的有节奏收缩来识别胎儿胸腔内的心脏。通过计算在 1 分钟内发生的收缩（心跳）数来计算心率。 注： 如果使用的超声波装置具有多普勒功能，则此功能可能用于进一步帮助区分心脏和其他非特定胎儿运动。</li>
	<li>验证所有保存的图像和视频循环包括所有必要的识别信息，包括动物名称或数量、日期、测量和适当的结构标签。</li>
</ol>4. 程序结论和恢复
<ol><li>使用软布或纸巾从动物腹部取出所有残留的超声波接触凝胶。在存储前从传感器中清洁凝胶。</li>
	<li>如果使用步骤 1.1 中描述的沉静协议，以 0.25 毫克/kg 的剂量在皮下施用阿提帕梅索莱，以扭转甲基霉素的影响。如果使用其他可用拮抗剂的缓解协议，则在程序结束时根据已公布的剂量将这种剂给动物管理，以加快恢复速度。</li>
	<li>将 muntjac 放在软床表面的胸骨重新任职，头部稍微抬起，以减少反胃和渴望的鲁梅纳尔内容的风险。密切监视动物，直到完全恢复，并能够在没有帮助的情况下在围栏周围流动。</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chapter 2. Small Animal Diagnostic Ultrasound. , 19-29 (2002).</li><li>Huang, F., Cockrell, D. C., Stephenson, T. R., Noyes, J. H., Sasser, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serum+pregnancy+test+with+a+specific+radioimmunoassay+for+moose+and+elk+pregnancy-specific+protein+B.">A serum pregnancy test with a specific radioimmunoassay for moose and elk pregnancy-specific protein B.</a> J. Wildl. Manag. 64, 492-499 (2000).</li></ol>

作者没有什么可透露的。

该协议描述了在里维斯的蒙特雅克鹿怀孕诊断中应用跨性别超声波。与其他超声波研究一致，适当的设备和技术熟练程度对于成功进行蒙贾克多伊的生殖检查至关重要。仔细审查哺乳动物的基本解剖学、诊断超声波的物理原理以及腹部内脏典型的超声学外观将有助于减少误诊。此外，选择适当的转导器和熟悉仪器和扫描仪控制对于获得高质量的图像和准确的解释至关重要。
对于许多缺乏经验的超声学家来说，成像文物是图像分辨率差和结构评估不准确的常见原因。皮肤传感器接口的回响是兽医腹部超声波中最常见的人工制品之一，通过消除两个表面之间的气体存在，可以显著减少。足够的脱毛和声胶在皮肤上的慷慨应用应该足以尽量减少这种神器的影响。其他成像文物的讨论可以在人类和兽医超声学的各种文本中找到，因此这里不会进一步详细讨论：然而，对这些现象的一般了解将使超声学家能够更有效地控制它们。
如果进行生殖检查，应该明白，子宫可能很难在正常、非怀孕的动物中显像。在早孕时，子宫的流明可能会随着妊娠囊的形成而成为缺氧空间的低音。在蒙贾克的这个阶段，子宫通常可以通过扫描乳腺的水平，或稍微小到乳腺来找到。在骨骼矿化或胎儿心跳出现之前，在出现其他生殖条件导致子宫内积液时，可能会误诊怀孕，这可能包括病理学，如皮奥梅特拉、水经或粘膜。超声波诊断应与动物在治疗前进行完整的临床评估相结合。在早期妊娠期，概念将以球形出现，在羊膜囊内出现卵巢、超选择结构。特定的发展时间点在这个物种中没有很好的特征：然而，骨骼矿化和胎儿心脏运动似乎在交配后约8周始终可以检测到。
在理想条件下，跨位超声波可以成为评估许多物种的生殖结构的非常有用的工具，包括里维斯的蒙特雅克。然而，这项技术的价值在很大程度上取决于动物住房设施的具体能力。与更被动的妊娠检测机制不同，超声波需要动物在5-10分钟或更长时间内进行物理和/或化学约束。在许多设施中，这是不可能的或不实际的，应根据研究的具体目标考虑审查的必要性。此外，某些生理或病理状态可能表明，由于捕获和镇静相关的痛苦的潜在有害影响，某些动物的超声波是禁忌症。在开始研究之前，必须评估每种动物是否适合进行超声波检查，在这些项目的规划阶段应咨询兽医。
目前在里维斯的muntjac和其他野生动物物种中发现怀孕的做法在性质上通常是被动的（观察交配行为和腹部衰减）。血清孕酮或妊娠相关糖蛋白水平可以测量，以确认诊断18，25，但这些检测结果不提供任何有关妊娠阶段或胎儿生存能力的信息。超声波的好处是允许实时评估生殖状态的准确性类似于或大于血清激素检测18，同时保持最小的侵入性和痛苦动物。在育种日期难以或无法确定的操作中，超声波可能提供一个更准确的机制，通过该机制来估计妊娠阶段，而不是荷尔蒙检测，即使没有公布妊娠表。此外，超声检查的物理和化学约束似乎对健康的蒙贾克造成最小的痛苦，并提供了一个理想的机会，进行彻底的体格检查，并收集额外的样本（血液，尿液 等），以进行一般健康评估。
通过超声波测量，在许多物种中建立了妊娠年龄估计，包括牛1、绵羊2、3、山羊4和马5，在数量有限的种群中也描述了8-19种。在没有确切的繁殖日期或既定的妊娠表的情况下，里维斯的胎儿年龄只能根据其他物种概述的胎儿发育参数粗略估计。这里描述的工作的一个具体目标是建立更正式的指南，在这个物种的胎儿老化。有了足够的规范性数据，未来在这个殖民地的超声学应用将包括识别胎儿发育中的异常，在存在不同的实验变量的情况下，并评估胎儿的生存能力。此外，可以确定跨多面超声波的敏感性和特异性，并与其他常用的检测子宫颈物种怀孕的机制进行比较。

超声检查是牛1、绵羊2、3、山羊4、马5、伴侣动物6、7等家畜种孕期诊断方法。这种成像方式也被确定为一个有价值的工具，以怀孕检测和胎儿年龄估计在几个品种的圈养颈椎，包括休养鹿8-11，红鹿12-16，北海道西卡鹿17，驯鹿18，19。在许多情况下，超声学产生的准确率高于其他妊娠检测机制，如血清孕酮或妊娠相关糖蛋白分析9，18。此外，生殖道的超声学可视化可以提供有关胎儿生存能力、妊娠年龄和发育异常的识别的其他信息，否则将难以评估。超声学用于评估生殖状态表明，这种方法应适应其他使用活性繁殖程序的物种。
里维斯的蒙特雅克鹿（蒙蒂亚库斯里韦西）是原产于东南亚20岁的小种。虽然这些动物的繁殖周期在它们的本领地并不明显，但对英格兰南部部分地区的野性种群的研究已经加深了对它们繁殖模式的了解。与大多数认证不同，里维斯的蒙特雅克似乎全年繁殖，没有明显的季节性讨好高峰20-22。雌性小鹿通常在怀孕210天后生下一只小鹿，并在24小时20-22小时内可靠地进入产后雌性。这是否不设想在这个产后期间将返回到埃斯特鲁斯约24-25天后20。在俘虏的情况下，繁殖通常是通过住房与一个或多个完整的男性实现的。该系统产生令人满意的受孕率，对人员要求很少的技术技能，但确切的育种日期很难确定，如果不是不可能的话。
蒙特雅克是细胞遗传学研究中常用的，因为不同物种23的双体染色体数量广泛，卡约蒂奇多样化率高。这些动物目前正在作为普里昂病发病机理和传播的模型进行调查，特别感兴趣的是研究慢性浪费病（CWD）在子宫颈的垂直传播。蒙古人在若干不同研究应用中的重要性表明，应建立改进的生殖技术，以补充目前的调查程序。具体来说，评估妊娠期的非侵入性机制有可能阐明不同实验条件下生殖性能和胎儿生长发育的关键变化。此外，在无法确定繁殖日期的情况下，开发实时胎儿测量参考值对于估计妊娠年龄具有潜在的价值。先前的研究已经描述了使用胎儿测量的机制，如冠冠长度（CRL）、胸部深度（CD）和头部长度（HL），以估计11-14、16，17等物种妊娠的大致阶段。此外，其中一些研究还建立了指导方针，描述何时可以识别特定的发展里程碑，如骨骼矿化或胎儿心跳。虽然这些数据可能有助于指导muntjac的生殖超声波分析，但大多数其他被研究的认证物种的体型要大得多，妊娠期更长，降低了这些研究对muntjac应用的转化价值。
此处概述的协议描述了女性蒙贾克的跨性别超声波扫描，用于诊断和监测怀孕。此协议的成功执行可促进胎儿早期妊娠检测和胎儿生长发育评估。该技术在研究繁殖、 子宫发育 或以小型子宫颈模型垂直传播传染病的研究方面具有宝贵的应用，也可用于圈养繁殖手术的临床目的。

<table><tbody><tr><td>Butorphanol tartrate (Torbugesic) (0.45 mg/kg)</td><td>Fort Dodge Animal Health, Fort Dodge, Iowa</td><td>NADA 135-780</td><td></td></tr><tr><td>Azaperone tartrate (0.035 mg/kg)</td><td>ZooPharm</td><td></td><td></td></tr><tr><td>Medetomidine HCl (0.04 mg/kg)</td><td>ZooPharm</td><td></td><td></td></tr><tr><td>Ultrasound contact gel</td><td>Medline, Mundelein, Illinois</td><td>MDS092005</td><td></td></tr><tr><td>Atipamezole hydrochloride (Antisedan) (0.25 mg/kg)</td><td>Orion Corporation, Espoo, Finland</td><td>NADA 141-033</td><td></td></tr><tr><td>V-trough (optional)</td><td>N/A</td><td>N/A</td><td>Animal may be supported in dorsal recumbency on a standard examination table or other surface without the use of a V-trough</td></tr><tr><td>Electrical clippers or razor blades for hair removal</td><td>Oster, McMinnville, Tennessee</td><td>78005-140</td><td></td></tr><tr><td>Ultrasound system: Ibex Pro portable ultrasound</td><td>E.I. Medical Imaging, Loveland, CO</td><td>Ibex Pro</td><td></td></tr><tr><td>5-10 MHz transducer: CL3.8 5-2.5 MHz 60 mm curved linear array</td><td>E.I. Medical Imaging, Loveland, CO</td><td>290420</td><td></td></tr><tr><td>3 ml syringes</td><td>Covidien</td><td>1.18E+09</td><td></td></tr><tr><td>22 G needles</td><td>Covidien</td><td>8.88E+09</td><td></td></tr></tbody></table>

transabdominal ultrasound for pregnancy diagnosis in reeves' muntjac deer

里维斯的蒙特雅克鹿（蒙蒂亚库斯·里韦西）是原产于东南亚的小型认证物种，目前正在被调查为普里昂病传播和发病机理的潜在模型。垂直传播是研究传染病（包括普里昂病）的研究人员感兴趣的一个领域，这些调查需要有效的方法来评估母体感染对生殖性能的影响。超声检查是诊断妊娠和评估胎儿健康的一个成熟的工具，在许多动物物种1-7，包括几个品种的养殖证明8-19，但这种技术还没有在里维斯的蒙贾克鹿描述。在这里，我们描述了跨位超声波的应用，以检测怀孕在蒙贾克做，并评估胎儿生长和发展在整个妊娠期。使用此程序，怀孕的动物早在多巴克配对后 35 天就被识别出来，这是定期安全监测怀孕的有效方法。这项工作的未来目标将包括建立正常的胎儿测量参考，以估计妊娠年龄，确定诊断妊娠不同阶段怀孕的技术的敏感性和特殊性，以及确定不同实验条件下胎儿生长发育的变化。

使用此处描述的协议，可以使用微创技术在里夫的蒙特雅克鹿中诊断和监测怀孕。作者可以检测到怀孕的最早时间点是在观察到的交配行为之后大约35天。
在妊娠的早期阶段，通过可视化一个被确定为胆汁腹部缺氧空间的充满液体的子宫来诊断怀孕。在子宫内，发育中的胚胎以超选择卵巢结构出现，几乎没有可识别的特征（图1）。随着妊娠的进展，胎儿结构变得明显，在对骨骼矿化和胎儿心跳（图2 和 3）进行观察后，可以做出决定性的诊断。
胎儿测量可以获得和用于建立参考指南，以估计妊娠年龄的情况下，确切的繁殖日期不知道，虽然我们还没有完全开发这个系统在muntjac。直冠隆长（SCRL）、HL和CD是标准胎儿测量，通常用于估计许多物种17的胎儿年龄，这些测量结果是利用此处描述的协议从蒙贾克怀孕的超声波图像中获得的。所有这些测量都可以从相同的正确定位的下垂图像（图2 和 图3）获得，但如果不能在单个下垂视图中捕获整个胎儿，则可以使用单独的图像。将来，这些技术还可用于识别不同实验条件下观察到的生长发育变化，并确定胎儿的存活率。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50855/50855fig1highres.jpg" src="/files/ftp_upload/50855/50855fig1.jpg"> 图1。超声波图像显示里维斯的蒙特雅克鹿早孕。这项研究中的多伊被安置了46天完整的雄性蒙贾克，但具体的繁殖日期尚不清楚。胎儿特征在妊娠的这个阶段是无法区分的。
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50855/50855fig2highres.jpg" src="/files/ftp_upload/50855/50855fig2.jpg"> 图2。图1中描述的同一怀孕的超声波图像，在将多伊引入降压后的91天。（CD-胸部深度，HL-头部长度，P-胎盘，SCRL-直冠凸长）。
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50855/50855fig3highres.jpg" src="/files/ftp_upload/50855/50855fig3.jpg"> 图3。第二次怀孕的超声波图像，显示获得胎儿测量的适当定位。图像拍摄于介绍多伊到降压 91 天后。（CD-胸部深度，HL-头部长度，P-胎盘，SCRL-直冠凸长）。

Watch this Scientific Journal Video about Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer at JoVE.com

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

跨地超声波被描述为评估里维斯蒙贾克鹿生殖状态的有效、非侵入性手段。这些方法可用于实现妊娠早期诊断和评估胎儿存活率。这项技术的未来应用包括估计妊娠年龄和母体疾病对胎儿发育的影响。

里维斯的蒙特雅克鹿怀孕诊断的跨性别超声波

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of ...

transabdominal-ultrasound-for-pregnancy-diagnosis-reeves-muntjac

Nanyang Technological University

Research

JoVE Journal

Medicine

10.3K 次观看.  Colorado State University. 跨地超声波被描述为评估里维斯蒙贾克鹿生殖状态的有效、非侵入性手段。这些方法可用于实现妊娠早期诊断和评估胎儿存活率。这项技术的未来应用包括估计妊娠年龄和母体疾病对胎儿发育的影响。

视频: Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

小鼠胎儿超声心动图

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

科研

医学

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

发育生物学

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

在家兔模型宫内生长受限胎儿超声心动图和多普勒超声脉冲波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

高频超声在胎儿和胎盘发育分析中的影响

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

小鼠中Pax8定向缺失肿瘤抑制因子 Arid1a 和 Pten 的子宫内膜癌进展的非侵入性超声评估

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

癌症研究

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

我的老鼠怀孕了吗？高频超声波评估

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

在胚胎第7.5天通过子宫内纳米注射（NEPTUNE）靶向小鼠神经板

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

大鼠实验性生殖研究中的超声检查

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

牛奶收集方法小鼠和里夫斯&#39;麂鹿

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

生物学

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

两栖动物卵巢监测和控制的生殖技术

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...