Name: gonadectomy and blood sampling procedures in the small size teleost model japanese medaka (oryzias latipes)
Uploaded: 2020-12-11T15:00:00.000+00:00
Duration: 5 min 31 s
Description: Watch this Scientific Journal Video about Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes at JoVE.com

作者感谢卢尔德·卡伦·谭女士在养鱼方面给予的帮助。这项工作由NMBU、日本科学促进会（JSPS）的助学金（赠款编号18H04881和18K19323）资助，并资助住友基金会至S.K.的基础科学研究项目。

所有实验和动物处理都是根据挪威生命科学大学关于实验动物福利的建议进行的。挪威食品安全局（FOTS ID 24305）批准了使用淋病切除术的实验。
注:实验采用成年男性和女性（6-7个月大，体重约0.35克，长度约2.7厘米）日本美的进行。性别是通过区分次要性特征，如背鳍和肛鳍的大小和形状，如42，47描述确定。
1. 仪器和解决方案准备
<ol><li>准备麻醉库存溶液（0.6% 三卡因）。<ol>
<li>稀释 0.6 克三甲酸酯 （MS-222） 在 100 mL 的 10 倍磷酸盐缓冲盐水 （PBS）.</li>
		<li>将 1 mL 的 Tricaine 库存溶液分发到多个 1.5 mL 塑料管中，并储存在 -20 °C 下，直至使用。</li>
	</ol>
</li>
	<li>将 18 克 NaCl 加入 2 升水族馆水中，准备回收水（0.9% NaCl 溶液）。将溶液存放在室温下，直到使用。</li>
	<li>通过对角线打破剃须刀来准备切口工具，以获得一个锐点（图1A）。</li>
	<li>通过将 25 μL 肝素钠稀释到 1x PBS 的 500 μL 中，准备血液抗凝血溶液（0.05 U/μL 肝素钠）。将抗凝血液存储在 4 °C 下，直到使用。</li>
	<li>根据制造商的指示，用拔针器（图1B）拉玻璃毛细圈，从90毫米长的玻璃毛细圈中准备两根玻璃针。 注:玻璃针的外径为1毫米，内径为0.6毫米。</li>
	<li>通过切割盖子来准备一个1.5mL塑料管盖，并制作一个与针外径相符的孔（图1C）。要打孔，加热 9 毫米玻璃毛细孔的一端，并通过盖子刺穿加热的玻璃毛细圈。或者，使用针刺穿盖子，直到孔的直径与 9 毫米玻璃毛细孔相符。</li>
</ol>2. 妇科切除术
<ol><li>在30mL的水族馆水中稀释一管三甲酸酯（0.6%），准备0.02%的麻醉液。</li>
	<li>准备解剖工具，包括一个超细和两个细钳（一个有相对宽的尖端），小剪刀，尼龙线和剃须刀，如第1.3步所述。</li>
	<li>将鱼放入 0.02% 麻醉液中 30-60 秒，使鱼麻醉。 注:麻醉的持续时间取决于鱼的大小和重量，必须加以调整。为了确保鱼完全麻醉，鱼体可以用钳子轻轻捏捏。如果鱼没有反应，就可以开始做腺切除术。</li>
	<li>从麻醉液中取出鱼，将鱼水平放在其一侧，在解剖显微镜下出水。</li>
	<li>女性卵巢切除术 （OVX）<ol>
<li>取出卵卵（挂在女性身体外的卵子），如果有的话，刮切口区域的鳞片（图2A）。</li>
		<li>使用剃须刀刀片，在肋骨之间、骨盆和鼻鳍之间轻轻切口约2-2.5毫米长（图2A）。然后，轻轻捏鱼腹部，同时用宽尖的细钳一点一点地取出卵巢。</li>
		<li>使用细钳切开卵巢末端，将卵巢放在一边（图2B）。 注意:如果可能，请注意不要折断卵巢囊。如果卵巢囊破裂，尽可能完全去除任何腺体痕迹，而不留下任何未排卵的卵子。</li>
	</ol></li>
	<li>男性兰花切除术<ol>
<li>轻轻地在肛门上方的肋骨之间切开（图2A），然后用细钳慢慢打开切口。</li>
		<li>用细钳轻轻抓住睾试，慢慢取出睾试。之后，切开睾片的末端，以完全去除睾测试（图2B）。对于男性兰花切除术，所有制剂都类似于女性，直到切口部分。抓睾考试时，有时会获得类似于睾子的脂肪。然而，在恢复脂肪后，可以尝试再次找到睾试（图2B）。 注:对于男性和女性来说，重要的是尽量减少腹部的切口大小，以防止可能导致死亡的过度损伤。有时肠道也可能通过切口和淋头出现，因此请确保在关闭前将它们正确返回切口内。先前对卵巢和睾睾门在梅达卡腹部的位置的了解是必不可少的。</li>
	</ol></li>
	<li>缝合切口同样在男性和女性（图3）。<ol>
<li>将尼龙线放在切口区旁边，用超细钳子将切口右侧的皮肤刺入内侧腔内，用细钳将线带入（图3:1-2）。</li>
		<li>通过外体腔从切口左侧刺出皮肤以取出线（图3:3-4）。</li>
		<li>关闭切口开口，打两个结，切开过多的线（图3:4-6）。 注:缝合线必须足够紧，鱼的剩余线必须足够长，以防止缝合线松动。从麻醉到缝注的整个过程通常需要长达6分钟。更长的时间可能导致死亡。</li>
		<li>将鱼放入回收水中，并放置至少24小时，然后再将其转移到水族馆系统。 注:果纳切除鱼类通常在回收水中1-2小时后表现出正常行为。因此，根据实验目的，一个人可以在这个时间间隔后对鱼进行采样。</li>
	</ol></li>
</ol>3. 血液采样程序
<ol><li>准备工具:玻璃针、硅胶毛细管、带孔的塑料管、空的1.5毫升塑料管、迷你中心和胶带。</li>
	<li>使用第 2.1 步中描述的 0.02% 麻醉液对鱼进行麻醉，并将鱼置于垂直位置的解剖显微镜下（图 4A）。将鱼放在明亮的表面上，以方便眼花孔穿刺静脉的可视化。</li>
	<li>将玻璃针连接到硅胶毛细血管上（图4B），安装抽屉。用宽尖钳子打破针尖，通过吸气和吹动将针头内部涂上抗凝固液。 注意:建议使用吸盘和长度至少为 50 厘米的硅胶毛细血管，以采取安全措施，避免吸吸时直接接触血液。此外，确保针尖的开口足够大，以便抽取血液。</li>
	<li>将针头引向鱼的斑点区域，瞄准静脉（图5A），用嘴抽血，直到至少四分之一的针头总体积被填充（图5B）。 注意:在从鱼体中取出针头之前，必须停止吸气。</li>
	<li>松开针头，在针尖侧附近放一块胶带。将盖子与一个孔放在收集管上，将针头放在管内，通过孔，针尖放在外侧（图5C）。</li>
	<li>将鱼放入回收水中，并放置至少24小时，然后再将其转移到水族馆系统。 注:要从同一条鱼进行第二次血液取样，请在第一次血液采样一周后对血液进行取样。</li>
	<li>闪光旋转收集的血液1-2秒，在室温下1000克收集管内的血液。</li>
	<li>直接进入下游应用或将血液储存在 -20 °C 下，直到使用。 注: 参考以前的研究性类固醇提取从整个血液48.</li>
</ol>

作者没有什么可透露的。

如以往文献所报道，在其他模型物种中，长期以来一直使用造血术和血液采样来调查与性别类固醇在BPG轴调节中的作用有关的问题。然而，这些技术似乎只适合更大的动物。考虑到常用的远程模型（日本美田）体积小，我们为该物种的去结肠切除术和血液采样提供了详细的协议。
果子切除术鱼类的成活率达到近100%，表明在麦田上应用果子切除术是可行的。同样，血液采样过?...

在脊椎动物中，性类固醇，主要是由性腺动物产生的，通过各种反馈机制1，2，3，4，5在大脑-脑-脑-淋巴（BPG）轴的调节中起着重要的作用。此外，性类固醇影响大脑6，7，8和内分泌细胞，包括脑垂体9，10神经元的增殖和活动，从而在大脑和垂体可塑性的关键作用。 尽管在哺乳动物方面知识相对较好，但由性类固醇调停的BPG轴调节机制在非哺乳动物物种中还远远不为人所知，导致对进化保护原则的理解不充分。仍然有数量有限的研究记录性类固醇在大脑和垂体可塑性的作用， 从而提出了进一步研究性类固醇对各种脊椎动物物种的作用和影响的需要.
在脊椎动物中，远程运动已成为解决许多生物和生理问题的有力示范动物，包括应激反应12、13、生长14、15、营养生理16、17和繁殖2。Teleosts， 其中性别类固醇主要代表雌二 （E2） 和 11- 酮睾酮 （11-KT） 在男性18，19，长期以来一直是可靠的实验模型， 以调查跨物种繁殖的一般原理.Teleosts在下丘脑脑垂体连接20、21和独特的角细胞22中表现出独特性，这有时便于解释调节机制。此外，由于远程实验既适应实验室实验又进行实地实验，因此与其他生物体相比，远程运动具有许多优势。他们相对便宜购买和维持23，24。特别是小型远程模型，如斑马鱼（Danio rerio）和日本美达卡（Oryzias latipes），是具有非常高的活性，生命周期相对较短的物种，能够快速分析基因功能和疾病机制23，从而提供了更大的优势，解决大量的生物和生理问题，考虑到许多发达的协议和遗传工具包可用于这些物种25。
在许多研究中，去除淋巴（淋巴切除术）以及血液采样技术已被用作研究许多生理问题的方法，包括它对26、27、28、鸟类29和两栖动物30种哺乳动物的脊椎动物生殖生理学的影响。虽然腺切除术对生殖生理的影响可以替代模仿性类固醇拮抗剂，如塔莫西芬和氯米芬，药物的作用似乎是不一致的，由于双模效应31，32。长期接触性类固醇拮抗剂可能导致卵巢扩大33，34，这可能会禁用观察其影响的长期目的， 由于不健康的表型.此外， 不可能进行性类固醇拮抗剂治疗后的恢复实验， 以保证某些性类固醇的具体效果.与上述点， 性类固醇对抗剂使用的其他权衡已广泛审查31，32.因此， 结核切除术今天仍然作为一个强大的技术来研究性类固醇的作用.
虽然去角切除术和血液采样技术在更大的物种中相对容易执行， 如欧洲海贝（迪森特拉丘斯拉布拉克斯）35，蓝头瓦塞（塔拉索马双发）36，狗鱼（西利奥尔希努斯犬）37和猫鱼（异种化石和克拉里亚斯浴场）38，39，他们提出了挑战时，应用在小鱼作为梅达卡。例如，使用鱼麻醉输送系统（FADS）40不太可行，而且似乎容易对小鱼造成过度的身体伤害。此外，通常用于大鱼40的腺切除术不适合需要高精度以避免过度伤害的小鱼。最后，由于获得血管的机会有限，而且这些动物的血液量很小，血液采样具有挑战性。因此，在小手术中展示造血术和血液采样的每一步的明确协议非常重要。
该协议演示了腺切除术的分步程序，随后在日本产自东亚的小淡水鱼——日本美中进行血液取样。日本的美田有一个测序的基因组，几个分子和遗传工具可用25，和一个基因性别测定系统，允许调查性差异之前，二次性特征或性腺发育良好41。有趣的是，日本美达卡拥有融合的果子虫，这与许多其他的远程物种42相反。这两种技术加起来总共只需要8分钟，将完成该物种已经存在的视频协议列表，其中包括血管标签43，垂体部分44 和脑神经元45的补丁夹，和原细胞培养46。这些技术将使研究社区调查和更好地了解性类固醇在反馈机制中的作用，以及大脑和垂体可塑性在未来。

<table><tbody><tr><td>Glass capilary</td><td></td><td>GD1</td><td>Glass Capillary with Filament GD-1; Narishige</td></tr><tr><td>Heparin sodium salt</td><td></td><td>H4784-1G</td><td>Sigma-aldrich</td></tr><tr><td>Needle puller</td><td></td><td>P97</td><td>Flaming/Brown Micropipette puller Model P-97; Sutter Instrument</td></tr><tr><td>Nylon thread</td><td></td><td>N45VL</td><td>Polyamide suture, 0.2 metric; Crownjun</td></tr><tr><td>Plastic tube</td><td></td><td>T9661</td><td>Eppendorf Safe-lock microcentifuge tube 1.5 ml, Sigma-aldrich</td></tr><tr><td>Razor blade</td><td></td><td>-</td><td>Astra Superior Platinum Double Edge Razor Blades Green, salonwholesale.com</td></tr><tr><td>Silicone capillary</td><td></td><td>a16090800ux0403</td><td>Uxcell Silicone Tube 1 mm ID x 2 mm OD, amazon.com&amp;nbsp;</td></tr><tr><td>Tricaine</td><td></td><td>WXBC9102V</td><td>Aldrich chemistry</td></tr></tbody></table>

gonadectomy and blood sampling procedures in the small size teleost model japanese medaka (oryzias latipes)

性类固醇，由腺体产生，在大脑和垂体组织可塑性和神经内分泌控制所有脊椎动物通过提供大脑和垂体的反馈发挥关键作用。与哺乳动物相比，Teleost鱼在组织可塑性和生殖策略变化方面具有较高的水平，似乎是研究性类固醇的作用及其作用机制的有用模型。去除性类固醇生产的主要来源使用腺切除术与血液采样测量类固醇水平已经建立和相当可行的大鱼， 是一个强大的技术来调查性类固醇的作用和影响.但是，当在小型远程模型中实施时，这些技术会带来挑战。在这里，我们描述了日本母体的妇腺切除术的分步程序，然后是血液采样。这些协议被证明是非常可行的美达卡表明高存活率， 安全的寿命和表型的鱼， 并在性别类固醇清除方面可重复性.使用这些程序结合使用这种小远程模型的其他优势将大大提高对脊椎动物中性类固醇提供的生殖神经内分泌控制和组织可塑性的反馈机制的理解。

该协议描述了在小尺寸模型远程手术（日本医疗）中执行腺切除术和血液采样的每一步。雌性卵巢切除术（OVX）后鱼类的存活率为100%（10条鱼类中的10条），而94%（18条鱼类中的17条）的雄性在兰花切除术后存活下来。同时，在进行血液采样手术后，所有（38条鱼）都幸存了下来。
沙姆操作的雌性显示卵子（图6A?...

Watch this Scientific Journal Video about Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes at JoVE.com

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

本文描述了一个快速协议去纳切除术和采样血液从小远程鱼，使用日本美达卡（Oryzias拉蒂普斯）作为模型，以调查性类固醇在动物生理中的作用。

小尺寸远程模型日本美达卡（奥里齐亚斯拉蒂普斯）的果纳切除术和血液采样程序

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...

gonadectomy-blood-sampling-procedures-small-size-teleost-model

Research

JoVE Journal

Biology

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

4.3K 次观看.  Norwegian University of Life Sciences. 本文描述了一个快速协议去纳切除术和采样血液从小远程鱼，使用日本美达卡（Oryzias拉蒂普斯）作为模型，以调查性类固醇在动物生理中的作用。 

视频: Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

白银纳米胶体的盐度依赖性毒力测定使用青鳉鸡蛋

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

科研

发育生物学

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

作为一个型号的遗传生物鳉胚胎显微注射

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

生物学

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

鳉胚胎和细胞移植嵌合体生成Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射程序研究胚胎发育

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

小鼠圆形精子细胞注射

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

重复采血车在成年斑马鱼验血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

子宫内膜异位症异种小鼠病变大小的无创监测

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

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 ...

小尺寸远程模型日本美达卡（奥里齐亚斯拉蒂普斯）的果纳切除术和血液采样程序

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白银纳米胶体的盐度依赖性毒力测定使用青鳉鸡蛋

戒毒治疗和

作为一个型号的遗传生物鳉胚胎显微注射

鳉胚胎和细胞移植嵌合体生成Dechorionation

小鼠圆形精子细胞注射

小鼠圆形精子细胞注射

小鼠圆形精子细胞注射

重复采血车在成年斑马鱼验血

子宫内膜异位症异种小鼠病变大小的无创监测

两栖动物卵巢监测和控制的生殖技术