Name: avian semen collection by cloacal massage and isolation of dna from sperm
Uploaded: 2018-02-05T12:30:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm at JoVE.com

排せつのマッサージと早期支援ありがとうサンノゼ施設と研究所のジェフリー ・ Kittilson をサポートします。この作品は、BJH に ND EPSCoR FAR0022429 賞によって支えられました。出版費用は寛大清心生物学科によってをサポートされていました。

このプロトコルは脊椎動物の動物科目を含み、機関動物ケアおよび使用委員会 (IACUC) ノースダコタ州立大学でによって承認されています。
1. 精液採取排せつマッサージを使用してスズメ目の鳥から
注: 排せつマッサージ繁殖アクティブな鳥のみ、効果的な精液収集手法ですが、適切な飼育状況で繁殖シーズン以外正常に実行することができます。排せつのマッサージを使用する前にターゲット種の生殖活動を決定してください。キャプチャ時に野生の鳥や鳥が飼育下で開催、この手法を使用することができます。
<ol><li>(繁殖条件) で、雄の鳥から始まる非利き手でのバンダのグリップは、腹側 (図 1) の手のひらに触れると支配的な手に鳥を転送します。<ol>
<li>鳥の頭は手のひらの外側の縁に最も近いことを確認します。軽く頭と小指、リングおよび中指を使用してボディを保護します。これは、鳥の口と尾公開を残します。</li>
		<li>緩んでいるか、軽く手のひらを使用して拘束された鳥の足を残します。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55324/55324fig1.jpg"> 図 1: 排せつマッサージ グリップします。鳥はその腹側の手のひらと小指担保の頭に触れると、軽く利き手でセキュリティ保護されます。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol start="2"><li>腔の優れた終わりのどちら側でも支配的な人差し指と親指を置き、軽く尾 (図 2) の方の非常にわずかな圧力を適用するときに一緒に指を挟んだり。排泄腔が片脚、排せつの粘膜 (ピンク インテリア) を公開します。 注: 鳥がない場合排せつの突起が小さいことができる現在、精液を生産と排泄腔が片脚にくい。経験を持つ、繁殖アクティブおよび非アクティブの男性5,11cloacas 区別するために簡単です。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55324/55324fig2.jpg"> 図 2: 位置や排せつ突起を指します。優れたエンド (A) にわずかな圧力を適用することによって、総排出腔を片脚します。繁殖アクティブな男性のスズメ目、排せつの突起は明白と (B) の会社。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol start="3"><li>非支配的な手で手を安定させるために尾のベースの背側に指のパッドを配置し、すぼまり腔下親指の先端。 注: 短く、滑らかなサムネイルは排せつマッサージを実行するため有利であり傷害から鳥を保護します。</li>
	<li>中型圧力、クロアカ以下の滞在の反復運動に深いと頭側非利き手親指を移動します。鳥が射精するまで続行します。個人によって通常 1 といくつかのダース ストローク間かかります。</li>
	<li>鳥が射精すると、すぐに microhematocrit チューブで精液を収集します。複数射精を取得する排せつマッサージを繰り返します。 注: スズメ目精液は明るい茶色で、通常やや濃厚。白や暗い物質排泄腔から排出、精液は、精液のサンプルを汚染する可能性があります。腔に少量の血液の外観または混合される精液は、通常のマッサージ中にあまりにも多くの圧力によって引き起こされます。</li>
	<li>DNA の抽出の 1x PBS (pH 7.4) の 20 μ L の微量遠心チューブに精液を配置します。注: 1 × PBS で保存精液人工授精には適していませんが、後日 DNA 抽出のため使用することができます-80 ° C で保存する場合PBS 室温または冷蔵しますが、精液のサンプルが到着したとき、液体の状態にする必要があります。<ol>
<li>サンプルで精子の存在を確認するには、複合顕微鏡 (図 3) の下で希釈サンプルの少量を表示します。</li>
	</ol>
</li>
	<li>抽出まで-80 ° C で希釈精液を格納します。</li>
</ol>2. 鳥の精液からの DNA の抽出
注: このプロトコルは、複数の商業 DNA 抽出キットでテストされていますが、のみ 1 つの DNA キット12で使用する場合は成功しています。キットのプロトコルからの変更手順で示されます *。
<ol><li>ジチオトレイトール (DTT) 水での 1 M 溶液を準備し、vortex.* を使用してミックス<ol>
<li>因数 DTT ソリューション使用直前まで-20 ° C で保存します。抽出する各サンプルは 1 M の DTT の solution.* の 10 μ L を必要とします。</li>
	</ol>
</li>
	<li>20 ミリメートル pH 8.0; トリス-の Cl を含むバッファーを準備します。20 mM EDTA;200 mM の NaCL;4 %sds。各サンプルを抽出するこの buffer.* の 90 μ L が必要です。<ol>
<li>室温でバッファーを保存します。それは沈殿させた場合 dissolve.* 56 ° C に温めてください。</li>
	</ol>
</li>
	<li>ロッカーまたは 65 にシェーカーを含むインキュベーターを予熱 ° C.*</li>
	<li>すぐに抽出を開始、する前にバッファーの場合は 10 試料を抽出手順 2.2 各サンプル抽出する、例えば900 μ L バッファーに加え 100 μ L DTT ソリューションで準備の 90 μ L で 10 μ L DTT ソリューションをミックスします。この混合物は DTT buffer.* と呼ばれる、</li>
	<li>全体の希釈精液サンプルを 70 μ L 1 × PBS を追加し、vortex.* でよく混ぜ</li>
	<li>100 μ L を追加 DTT バッファー、サンプルに 10 μ L プロティナーゼ K ソリューション。20 秒間、渦、予熱したインキュベーターで 1 時間インキュベート ロッカーまたは shaker.*</li>
	<li>200 μ L バッファー アルと 200 μ L を追加新鮮なエタノール (100%)、20 seconds.* の渦</li>
	<li>スピン列にサンプル全体を転送し、6,000 x g. 破棄で 1 分コレクション チューブを遠心分離します。</li>
	<li>新しいチューブに列を配置し、500 μ L 準備バッファー AW1 を追加します。6,000 x g. 破棄で 1 分コレクション チューブを遠心分離機します。</li>
	<li>新しいチューブに列を配置し、500 μ L 準備バッファー AW2 を追加します。6,000 x g に 1 分間遠心は、フロースルーと列がチューブに戻って配置を破棄します。</li>
	<li>20,000 x g 破棄に 3 分コレクション チューブを遠心し、1.5 mL 遠心チューブに列を配置します。</li>
	<li>ピペット 35 μ L AE の列膜に直接バッファーし、5 分ではあ、室温でインキュベート</li>
	<li>スピン列 14,000 x g 破棄に 1 分を遠心し、-80 ° C で抽出されたサンプルを格納</li>
</ol>

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著者は、彼らは競合する金銭的な利益があることを宣言します。

シンプルで信頼性の高い繁殖アクティブ状態でない中小鳥から精液を収集し、鳥の精子から DNA を抽出法について述べる.
記述されていた精液の DNA の抽出のプロトコルは便宜上、キットからは変更が、鳥ザーメン使用の洗練されました。精子が標準抽出化学9,13、溶解耐性と産スズメ目鳥から収集した精液のサンプルが非常に小さい (通常数マイクロリットル)。これらの要因は、DTT の適切な濃度を使用し、65 ° c. で孵化によって正常に克服するための課題を提示します。このメソッドを使用して、我々 は一貫して精子テロメアの長さ (通常 40 250 ng/μ L、射精量に応じて) 間の qPCR の分析のための使用可能な量の DNA を分離しています。
DTT は、フェノール/クロロホルム反応13との組み合わせで鳥の精子から DNA を抽出する以前使用されています。フェノール/クロロホルム反応は、DNA を隔離するため効果がより時間がかかり、危険なし、ヒューム フードを必要とします。私たちの抽出のプロトコルの主な制限は、それだけ成功させるには、1 つのブランドとタイプの少量サンプルを開始のための抽出キットで使用されているです。(未満 1 μ L) の非常に小さい精液サンプルまたは密度の低い精子サンプルは成功の十分な DNA のサンプルでは、現在のための DNA の抽出を防ぐことができますが、条件の繁殖の鳥に排せつマッサージが実行されるときにこの問題を発生していない私たち.この問題を回避するには、は、排せつマッサージを抽出するためにプールすることができます複数の射精を収集するためにすぐに繰り返すことが。
排せつマッサージ、多く種1,4,5,6,7,15,16,17、何十年も使用されているし、は、多くの場合鳥から精液を収集の迅速かつ効果的な手段。捕獲できる野生または未熟な鳥を含む研究項目に交尾し、彼の精液を喜んで入金に雄の鳥を説得力に依存するコレクションの方法よりもより効果的です4。別の精液コレクション メソッドは精巣の精 glomera 郭清が、これは繰り返しサンプリング4,14または保全目的のためのオプションではありません。排せつマッサージの主な制限は、デモなし学習の難しさです。書かれた説明多いを理解し、学ぶプロセスを実装するための十分なないです。排せつマッサージの詳細を示すは、我々 は技術を学びたい人のための学習時間と難易度を減らすために願っています。

鳥が生物学の研究に幅広く使用されている精子は排せつを使用して簡単にサンプリングすることができますと精子の質と競争1、精子テロメア ダイナミクス2エピジェネティクス3と似たようなトピックを含む研究のための理想的な科目マッサージ。排せつマッサージは鳥4,5,6から精液を収集するための穏やかな、非侵襲的な手動技術です。繰り返しサンプルを簡単に得ることができる、特別な工具も不要、簡単にフィールドまたはラボで実行できるように。排せつのマッサージは、何十年も使用されている、利用可能な説明が書かれてから学ぶことは困難です。このパブリケーションは、排せつマッサージを学習に関わる不確実性を低減するものです。
排せつマッサージを使用して鳥から収集した精液は、運動評価4,7または人工授精8、すぐに使用または高度なイメージングおよび DNA の抽出等の用途として冷凍することができます。スズメ目の鳥から精液サンプルは小さいが密集精子を含まれています。精子9の特殊な保護機能を克服するための変更を簡単にするため商業抽出キットを使用して DNA を抽出します。抽出後、qPCR10を使用して精子テロメアを測定できます。

<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

精液の採取は、精子の質、精子テロメア ダイナミクス、エピジェネティクス研究を含むアプリケーションの広い範囲のために役立ちます。鳥は広く使用されている科目は、生物学的研究、繰り返し精子サンプルを含む研究に最適です。ただし、いくつかのリソースは現在利用可能な収集および鳥の精子から DNA を抽出する方法を学びたい人のためです。ここで排せつマッサージ、鳥精子を収集するための穏やかな、非侵襲的な手動技術について述べる。文献でこの手法を確立すると、使用可能な記述から学ぶことは困難ことはできます。また、変更と商業抽出キットを使用して鳥の精液から DNA を抽出するための情報を提供いたします。排せつマッサージは、生殖状態で中規模男性鳥で容易に使用することができます。次のコレクション、精液は運動アッセイのためすぐに使用または記載プロトコルを次の DNA の抽出のために冷凍できます。この抽出プロトコル鳥精子の洗練された、いくつかのスズメ目種 (通行人国立公園、 Spizella passerina、 Haemorhous メキシコ、およびつぐみ migratorius から収集されたサンプルで正常に使用されています。) と 1 つの columbid (コルンバ ・ リヴィア)。

排せつマッサージと DNA 抽出を使用していくつかのスズメ目種と 1 つの columbid (表 1) で行われた記述のプロトコル。排せつマッサージによって収集された精液に精子の存在は、400 倍の倍率 (図 3) で少量の化合物顕微鏡下で希釈した試料を表示することによって確認されました。ここで説明されているプロトコルを使用して家すずめから収集された精液のサンプルから DNA を抽出後、8 個のサンプルはゲル電気泳動法 (図 4の 3 つのサンプルを見ることができる) DNA の梯子で実行しました。高品質 DNA の分離を成功させるに示すサンプル レーンのすべての 8 で塗りつけなし明瞭なバンドを見た。このプロトコルを使用して抽出した他のすべてのサンプルは、分光光度計 (テーブル 1を参照) を用いた DNA 濃度の評価されました。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55324/55324fig3.jpg"> 図 3: 2 つの小さな産スズメ目鳥から精子。左、メキシコマシコ (Haemorhous メキシコ) から排せつマッサージと右、家すずめ (通行人国立公園)、採取したこれらの精子は、2016 年にファーゴ、ノースダコタに閉じ込められました。サンプルそれぞれ 20 μ L の PBS で希釈され、それぞれ 1,000 倍と 400 倍の倍率での顕微鏡の下に表示。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55324/55324fig4.jpg"> 図 4: 精子 DNA の電気泳動のゲルの DNA します。3 つのサンプルの家すずめの精液から抽出した DNA は DNA の梯子が付いているゲルで実行します。高品質の DNA を正しく抽出を示す明瞭なバンドの存在に注意してください。<a href="//cloudflare.jove.com/files/ftp_upload/55324/55324fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>家すずめ (通行人国立公園)</td>
			<td>チャガシラヒメドリ (Spizella passerina)</td>
			<td>家フィンチ (Haemorhous メキシコ)</td>
			<td>アメリカのロビン (つぐみ migratorius)</td>
			<td>カワラバト (コルンバ ・ リヴィア)</td>
		</tr>
<tr>
<td>収集されたサンプルの数</td>
			<td>156</td>
			<td>3</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
		</tr>
<tr>
<td>抽出サンプル数</td>
			<td>90</td>
			<td>3</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
		</tr>
<tr>
<td>精液回収成功率</td>
			<td>98%</td>
			<td>75%</td>
			<td>100%</td>
			<td>67%</td>
			<td>67%</td>
		</tr>
</tbody></table>表 1: 種およびサンプルの収集まで抽出します。出版物の時に、サンプルは、スズメ目 4 種と 1 columbid から記述されているプロトコルを使用して処理されています。精液回収成功率は試行の合計数のうち正常に取得されましたサンプルをそれぞれの割合を指します。

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

このプロトコルでは、排せつマッサージ、鳥、および変更と商業抽出キットを使用して鳥の精子から DNA の抽出から精液を収集するため非侵襲的な手動技術について説明します。

排せつマッサージと精子から DNA の隔離による鳥の精液採取

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

Nanyang Technological University

Research

JoVE Journal

Biology

15.9K ビュー.  North Dakota State University. このプロトコルでは、排せつマッサージ、鳥、および変更と商業抽出キットを使用して鳥の精子から DNA の抽出から精液を収集するため非侵襲的な手動技術について説明します。

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

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

ポスト交配精液女性の生殖器官とマウスの精液の液状化測定からのコレクション

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

発生生物学

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.

処理と治療の男性欧州ウナギ (アンギラ) ホルモンの成熟、精子凍結保存

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.

標準的な実験装置を用いてマウス精巣からパチテン精子細胞と精子を濃縮

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.

バイオレット励起細胞透過性DNA結合色素を用いたマウス精子細胞の分離

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.

アイソレーションと試験管内で</emの>活性化線虫（Caenorhabditis elegans）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.

トランスジェニック齧歯類アッセイ男性生殖細胞変異頻度を定量化するための

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.

密度勾配遠心法を用いた差動品質の精液採取

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.

アスティアナックスメキシコのガメテコレクションと体外受精

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.

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.

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for ...

U-Shaped Horizontal Swimming Technique for Preparing High-Quality Sperm with Low DNA Fragmentation Index

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and viscous seminal plasma. High-quality sperm refers to progressively motile spermatozoa with normal morphology, which often exhibit a lower DNA fragmentation index (DFI) and higher fertilization potential. Preparing high-quality sperm is a critical step in human-assisted reproductive technology. The traditional sperm preparation method, discontinuous density gradient centrifugation (DGC), is time-consuming and labor-intensive. Repeated centrifugation can damage sperm DNA, thereby affecting subsequent fertilization and embryo development. This study introduces a U-shaped horizontal swimming (UHS) method for preparing intracytoplasmic sperm injection (ICSI) sperm, which significantly eliminates the detrimental effects of&#160;centrifugation on sperm DNA.&#160;The UHS method involves creating a UHS lane using a fertilization medium within an ICSI operating dish. A 10 &#181;L fertilization medium microdroplet is placed at the starting point on the left side of the UHS lane to hold the semen. Two additional 10 &#181;L fertilization medium buffer droplets are positioned at intervals along the left middle section of the lane, with all droplets connected by fertilization medium. The dish is then covered with culture oil and incubated overnight at 37 &#176;C with 6% CO2 to equilibrate. Subsequently, 3 &#181;L of semen is added to the microdroplet at the starting point. High-quality spermatozoa swim to the right side of the UHS lane, facilitating their suction into the ICSI injection needle. Dead sperm, cell debris, and other viscous impurities largely remain at the initial point or in the buffer droplets. We simultaneously processed 21 semen samples using both UHS and DGC techniques and compared their DFI. The results demonstrated that the DFI in the DGC group was 5.5% &#177; 3.2%, whereas the DFI in the UHS group was 1.7% &#177; 1.1%. The difference between the two groups was statistically significant (P &#60; 0.05).

This protocol describes a method for screening high-quality sperm with a low DNA fragmentation index by utilizing sperm motility characteristics and thigmotaxis. The method employs a U-shaped horizontal swimming lane, enabling high-quality sperm to reach the opposite side through buffer droplets, while dead sperm, cell debris, and impurities are excluded.

低DNA断片化指数で高品質の精子を調製するためのU字型水平遊泳技術

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and ...