我们感谢何塞诺格拉的早期协助泄殖腔按摩, 和杰弗里 Kittilson 的实验室支持。这项工作得到了 EPSCoR FAR0022429 奖对 BJH 的支持。出版费用得到了 NDSU 生物科学部的慷慨支持。

该议定书涉及脊椎动物的主题, 并已批准的机构动物保育和使用委员会 (IACUC) 在北达科他州立大学。
1. 使用泄殖腔按摩的雀鸟的精子收集
注: 泄殖腔按摩是一种有效的生殖活禽的精子收集技术, 但可以在适当的圈养情况下成功地在繁殖季节之外进行。在使用泄殖腔按摩之前, 应确定目标物种的生殖活动。这种技术可以用于野生鸟类捕获或圈养鸟类。
<ol><li>从雄鸟开始, (在繁殖条件) 達的握在非主导的手, 转移鸟到支配的手与它的腹侧接触手掌 (图 1)。<ol>
<li>确保鸟的头部离手掌的侧边最近。用小指、环和中指轻轻地保护头部和身体。这就使得鸟的排气口和尾部露出来了。</li>
		<li>用手掌把鸟的腿松开或轻轻地克制。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> 图 1: 泄殖腔按摩手柄.这只鸟在占优势的手上被轻轻地固定着, 它的腹侧接触手掌, 头部被小指固定。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>将占主导地位的食指和拇指放在泄殖腔的任一侧, 轻轻地捏住手指, 对尾部施加非常轻微的压力 (图 2)。泄殖腔将埃弗特, 露出泄殖腔粘膜 (粉红色内部)。 注意: 如果鸟目前没有生产精子, 泄殖腔突起可以小, 泄殖腔很难埃弗特。有经验, 很容易区分 cloacas 的生殖活动和非活动的男性5,11。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> 图 2: 手指位置和泄殖腔突起.埃弗特泄殖腔通过施加轻微的压力到上级端 (A)。在生殖活跃的雄性雀中, 泄殖腔突起明显且牢固 (B)。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="3"><li>用非显性手, 将食指的垫放在尾巴底部的背侧以稳定手, 然后将拇指的尖端放在外翻泄殖腔的下方。 注意: 短而平滑的缩略图有利于进行泄殖腔按摩, 保护鸟免受意外伤害。</li>
	<li>移动非显性拇指深和 cranially 在一个重复的运动中压, 保持低于泄殖腔。继续, 直到鸟射精。根据个人, 这通常需要在1和几十个中风。</li>
	<li>鸟一射精, 就在 microhematocrit 管中收集精子。重复泄殖腔按摩以获得多射精。 注: 雀的精子呈浅褐色, 通常有较厚的稠度。从泄殖腔中排出的白色或深色物质不是精子, 可能会污染精子样本。在泄殖腔或与精子混合时出现少量的血液, 通常是由于按摩时压力太大造成的。</li>
	<li>对于 DNA 提取, 将精子放入离心管中, 20 µL 1x PBS (pH 7.4)。注: 1x PBS 中贮存的精子不适合人工受精, 但在-80 ° c 贮存时可用于 DNA 提取。PBS 可以是室温或冷冻的, 但在储存精子样本时应处于液态状态。<ol>
<li>通过在复合显微镜下观察少量的稀释样品 (图 3), 确认样品中是否存在精子。</li>
	</ol>
</li>
	<li>将稀释的精子贮存在-80 ° c 直至提取。</li>
</ol>2. 从禽精中提取 DNA
注: 本协议已通过多个商业 dna 提取试剂盒进行了测试, 但仅在使用一个 dna 套件12时才成功。从套件协议中修改的步骤由 * 表示。
<ol><li>在水中准备一个1米的糖 (德勤) 溶液, 并使用涡流混合。<ol>
<li>分的解决方案, 并存储在-20 ° c, 直到使用前立即。每个样品要提取需要10µL 1 M 的解决方案。</li>
	</ol>
</li>
	<li>准备一个包含20毫米三氯的缓冲液, pH 值 8.0;20毫米 EDTA;200毫米氯化钠;和 4% SDS。要提取的每个样本需要此缓冲区的90µL。<ol>
<li>在室温下存储缓冲区。如果它已经沉淀, 温暖它到56° c 溶化。</li>
	</ol>
</li>
	<li>将装有摇杆或振动筛的恒温箱预热至65° c。</li>
	<li>在开始提取之前, 在步骤2.2 中为每个抽取的样本准备10µL 的90µL 的缓冲液, 并在提取100样本时,例如900 µL 缓冲器加10µL 的解决方案。这种混合物将被称为 "数码缓冲"。</li>
	<li>添加70µL 1x PBS 到整个稀释的精子样本和混合良好使用涡流。</li>
	<li>添加100µL 的数码缓冲和10µL 蛋白酶 K 溶液的样品。漩涡20秒, 然后孵化在预热孵化器1小时的摇杆或振动筛。</li>
	<li>添加200µL 缓冲 AL 和200µL 新鲜乙醇 (100%) 和漩涡20秒。</li>
	<li>将整个样品转移到旋转柱, 离心机1分钟, 6000 x g. 丢弃收集管。</li>
	<li>将列放在新管上, 并添加500µL 准备的缓冲 AW1。离心机1分钟在 6000 x g. 丢弃收集管。</li>
	<li>将列放在新管上, 并添加500µL 准备的缓冲 AW2。离心机1分钟, 在 6000 x g. 丢弃流, 并将列放回管上。</li>
	<li>离心机3分钟在 2万 x g. 丢弃收集管, 将柱放在1.5 毫升离心管上。</li>
	<li>吸管35µL 将 AE 缓冲器直接放在柱膜上, 室温孵育5分钟。</li>
	<li>离心机1分钟在 1.4万 x g. 丢弃自旋柱, 并在-80 ° c 处储存提取的样品。</li>
</ol>

<ol><li>Briskie, J. V., Montgomerie, R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/1349186">Sperm size and sperm competition in birds.</a> Proc. R. Soc. B. 247, 89-95 (1992).</li>
<li>Monaghan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telomeres+and+life+histories%3A+the+long+and+the+short+of+it%5BTitle%5D)+AND+%22Ann.+N.Y.+Acad.+Sci%22%5BJournal%5D)">Telomeres and life histories: the long and the short of it.</a> Ann. N.Y. Acad. Sci. 1206, 130-142 (2010).</li>
<li>Carrell, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics+of+the+male+gamete%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Epigenetics of the male gamete.</a> Fertility and Sterility. 97 (2), 267-274 (2012).</li>
<li>Burrows, W. H., Quinn, J. P. <a target="_blank" href="https://academic.oup.com/ps/article-abstract/16/1/19/1564432?redirectedFrom=fulltext">The collection of spermatozoa from the domestic fowl and turkey.</a> Poultry Science. 16 (1), 19-24 (1937).</li>
<li>Wolfson, A. <a target="_blank" href="https://www.jstor.org/stable/4510381?seq=1#page_scan_tab_contents">The cloacal protuberance: a means for determining breeding condition in live male birds.</a> Bird-Banding. 23 (4), 159-165 (1952).</li>
<li>Kast, T. L., Ketterson, E. D., Nolan, V. <a target="_blank" href="https://www.jstor.org/stable/4089416?seq=1#page_scan_tab_contents">Variation in ejaculate quality in dark-eyed juncos according to season, stage of reproduction, and testosterone treatment.</a> The Auk. 115 (3), 684-693 (1998).</li>
<li>Helfenstein, F., Podevin, M., Richner, H. <a target="_blank" href="https://link.springer.com/article/10.1007/s00265-009-0871-x">Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus.</a> Behav. Ecol. Sociobiol. 64, 557-565 (2010).</li>
<li>Purchase, C. F., Earle, P. T. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1111/jai.12070/abstract">Modifications to the IMAGEJ computer assisted sperm analysis plugin greatly improve efficiency and fundamentally alter the scope of attainable data.</a> J. Appl. Icthyol. 28, 1013-1016 (2012).</li>
<li>Gill, P., Jeffreys, A. J., Werrett, D. J. <a target="_blank" href="https://www.nature.com/articles/318577a0">Forensic application of DNA 'fingerprints'.</a> Nature. 318 (12), 577-579 (1985).</li>
<li>Criscuolo, F., et al. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1111/j.1600-048X.2008.04623.x/abstract">Real-time quantitative PCR assay for measurement of avian telomeres.</a> J. Avian Biol. 40, 342-347 (2009).</li>
<li>Lovette, I. J., Fitzpatrick, J. W. <a target="_blank" href="http://www.birds.cornell.edu/homestudy/handbook-of-bird-biology">Cornell Lab of Ornithology Handbook of Bird Biology, 3rd Edition</a>. , Wiley-Blackwell, Princeton University Press. ISBN 9781118291047 (2016).</li>
<li><a target="_blank" href="https://www.scribd.com/document/54238221/QIAamp-DNA-Micro-Handbook">QIAamp DNA Micro Handbook</a>. , Third edition (2014).</li>
<li>Carter, K. L., Robertson, B. C., Kempenaers, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+differential+DNA+extraction+method+for+sperm+on+the+perivitelline+membrane+of+avian+eggs%5BTitle%5D)+AND+%22Mol.+Ecol%22%5BJournal%5D)">A differential DNA extraction method for sperm on the perivitelline membrane of avian eggs.</a> Mol. Ecol. 9, 2149-2154 (2000).</li>
<li>Birkhead, T. R., Buchanan, K. L., Devoogd, T. J., Pellatt, E. J., Catchpole, C. K. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0003347296904236">Song, sperm quality and testes asymmetry in the sedge warbler.</a> Anim. Behav. 53, 965-971 (1997).</li>
<li>Lüpold, S., Calhim, S., Immler, S., Birkhead, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sperm+morphology+and+sperm+velocity+in+passerine+birds%5BTitle%5D)+AND+%22Proc.+R.+Soc.+B%22%5BJournal%5D)">Sperm morphology and sperm velocity in passerine birds.</a> Proc. R. Soc. B. 276, 1175-1181 (2009).</li>
<li>Samour, J. H., Smith, C. A., Moore, H. D., Markham, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Semen+collection+and+spermatozoa+characteristics+in+budgerigars+%28Melopsittacus+undulates%29%5BTitle%5D)+AND+%22Vet.+Rec%22%5BJournal%5D)">Semen collection and spermatozoa characteristics in budgerigars (Melopsittacus undulates).</a> Vet. Rec. 118, 397-399 (1986).</li>
<li>Stelzer, G., Crosta, L., Bürkle, M., Krautwalt-Junghanns, M. E. <a target="_blank" href="https://www.researchgate.net/publication/232663050_Attempted_Semen_Collection_Using_the_Massage_Technique_and_Semen_Analysis_in_Various_Psittacine_Species">Attempted semen collection using the massage technique and semen analysis in various psittacine species.</a> J. Avian Med. Surg. 19 (1), 7-13 (2005).</li>
</ol>

作者声明他们没有竞争的金融利益。

本文介绍了一种简单可靠的生殖活性条件下从中小鸟类采集精子的方法, 并从禽精中提取 DNA。
所描述的精子 DNA 提取协议是从一个简单的套件修改, 但被提炼为使用在禽类的精子。精子抗裂解的标准萃取化学品9,13, 从形收集的精样本是相当小的 (通常是少数升)。这些因素提出了在65° c 的情况下使用适当浓度的德勤和孵化成功克服的?...

鸟类是研究精子质量和竞争的理想科目1, 精子端粒动力学2, 遗传学的3, 和类似的主题, 因为它们广泛用于生物研究和精子可以很容易地取样使用泄殖腔按摩.泄殖腔按摩是一种温和的, 非侵入性的手工收集精子的技术, 从鸟类4,5,6。重复样品可以很容易获得, 不需要特殊的工具, 使其易于在现场或实验室执行。虽然泄殖腔按摩已经使用了几十年, 很难从现有的书面说明。本刊物旨在减少学习泄殖腔按摩所涉及的时间和不确定因素。
从鸟类收集的精子使用泄殖腔按摩可立即用于运动评估4,7或人工授精8, 或冷冻其他用途, 如高级成像和 DNA 提取。来自雀鸟的精子样本很小, 但含有浓密的精子。DNA 的提取使用一个商业提取试剂盒简单, 与修改, 以克服专门的保护功能的精子9。提取后, 精子端粒可以使用 qPCR10进行测量。

<table><tbody><tr><td>Microhematocrit capillary tubes</td><td>Fisherbrand</td><td>22-362566</td><td>Any supplier can be used. Smaller tubes may be more effective for collection of small semen samples, such as from very small passerine birds like chipping sparrow.</td></tr><tr><td>Microcentrifuge tubes</td><td>Any</td><td></td><td></td></tr><tr><td>1x PBS</td><td>Any</td><td></td><td>Used for storing semen samples prior to DNA extraction.</td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>10708976001</td><td>Any supplier can be used.</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>1233508</td><td>Any supplier can be used.</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>793566</td><td>Any supplier can be used.</td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>1614363</td><td>Any supplier can be used.</td></tr><tr><td>QIAamp DNA micro kit</td><td>Qiagen</td><td>56304</td><td>Qiagen QIAamp DNA micro kit is recommended specifically. This DNA extraction protocol has been attempted with other commercial kits but with little success. The reasons for this are unknown to us.&amp;nbsp;</td></tr></tbody></table>

avian semen collection by cloacal massage and isolation of dna from sperm

收集的精子可能是有用的广泛的应用, 包括研究涉及精质, 精子端粒动力学和表观遗传学。鸟类是生物学研究中广泛使用的学科, 是涉及反复精子样本研究的理想选择。然而, 对于那些希望学习如何从禽类精子中收集和提取 DNA 的人来说, 目前还没有多少资源可用。在这里, 我们描述泄殖腔按摩, 一个温和的, 非侵入性的手工技术收集禽类精子。虽然这项技术是建立在文献, 它可能是很难从可用的描述学习。我们还提供信息, 从禽精提取 DNA 的商业提取试剂盒的修改。泄殖腔按摩可以很容易地用于任何小到中型雄鸟的生殖条件。在收集后, 可以立即使用精子进行运动化验, 或在本协议所述的 DNA 提取后冷冻。这一提取协议被提炼为禽精, 并已成功地用于收集的样本从几个雀物种 (过路人幼雏, Spizella passerina, Haemorhous mexicanus, 和鸫 migratorius) 和一个 columbid (鸽)。

泄殖腔按摩和 DNA 提取使用所描述的协议已经对几个雀物种和一个 columbid (表 1) 进行。在泄殖腔按摩收集的精子样本中, 通过在400X 放大倍数显微镜下观察少量稀释后的样品 (图 3), 证实了精的存在。从家庭麻雀收集的精子样本中提取的 dna 使用这里所描述的协议, 八样品在凝胶电泳和 dna 阶梯上运行 (三样本可以在图 4...

Watch this Scientific Journal Video about Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm at JoVE.com

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

本协议描述泄殖腔按摩, 一种非侵入性的手工技术, 用于从禽类中收集精子, 以及使用商业提取试剂盒进行 DNA 提取, 并进行修改。

泄殖腔按摩与精子 DNA 分离法采集禽精

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

avian-semen-collection-cloacal-massage-isolation-dna-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.0K 次观看.  North Dakota State University. 本协议描述泄殖腔按摩, 一种非侵入性的手工技术, 用于从禽类中收集精子, 以及使用商业提取试剂盒进行 DNA 提取, 并进行修改。

视频: Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

科研

发育生物学

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

使用标准实验室设备从小鼠测试中浓缩帕西滕苯细胞和精子

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

生物学

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic &#955;gt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental agents.

转基因检测鼠类量化男性生殖细胞突变频率

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

密度梯度离心法测定微分品质的精子

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

阿斯蒂亚纳斯墨西哥的Gamete收集与体外受精

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

<meta charset="utf8"></head><body>记录穿过哺乳动物精子细胞质膜的电流

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.