欧州連合のホライゾン 2020年研究によって資金が供給されたこの文書および革新プログラム マリアスクウォドフスカ キュリー付与契約の下で 642893 (インプレス) コスト オフィス (アクション コスト FA 1205, AQUAGAMETE)、および優れた研究センター-1476-4/2016/FEKUT。

このプロトコルで記述されているヨーロッパのウナギを操作するためのすべての手順は、次の実験を制御するスペイン法令、大学パラオレイ ・ デ ・ バレンシアで動物実験倫理委員会によって承認されたと生きている動物のプロシージャ。
1. 魚の維持
<ol><li>研究施設にヨーロッパのウナギをもたらすと循環システムの 200 L 水槽に入れてください。定数 20 ° C の温度を維持するためにサーモスタットとクーラーを使用します。</li>
	<li>実験中に食料もないとストレス22を避けるために暗い状態で魚を保ちます。</li>
	<li>最初の 3 日間は、新鮮な水で魚を保ちます。水の 1/3 に変更し、37.0 ± の塩分濃度に到達するまで毎日海水を補充 0.3 g/l.</li>
</ol>2. ホルモン治療
注: ホルモン治療毎週注射をひと絨毛性ゴナドトロピン (hCG) の実験の全期間 (9 週間) ではあります。
<ol><li>食塩でホルモンを希釈して 1 IU/μ L の濃度で事前に hCG ホルモンを準備 (0.9 %nacl)。 注: ホルモンは-18 ° C で一週間以上この濃度で希釈したを保存することができます。</li>
	<li>魚を麻酔するには、システム水量 5 L と 40 L 柔軟なバケットを準備します。<ol>
<li>250 mL のフラスコで 70% エタノール 100 mL にベンゾカインの 300 mg を希釈します。</li>
		<li>バケツで希釈ベンゾカインを注ぐ (最終濃度 0.36 mM) と適切にミックスします。これは、60 mg/L の最終ベンゾカイン濃度</li>
		<li>ベンゾカインと水に魚を個別に転送し、ベンゾカイン効力し、魚はきちんと麻酔まで数秒を待ちます。 注: 魚に麻酔がかかることを確認、仰臥位の魚を配置し、それはまだその位置にとどまることをチェックします。</li>
	</ol>
</li>
	<li>ホルモンを管理、魚の重量を量るし、1.5 mL プラスチック チューブに魚の 1.5 IU/グラムの用量で hCG ホルモンを準備します。<ol>
<li>プラスチック製のチューブから hCG ホルモンで 1 mL 注射器を満たしなさい。</li>
		<li>仰臥位と援助や注射器、麻酔下の魚を配置、腹腔内のエリアで慎重にホルモンを注入します。</li>
	</ol>
</li>
	<li>魚を水槽に戻るし、それを完全に回復するまでを監視します。 注: ホルモンの政権は、実験中は毎週実施しています。通常、ヨーロッパのウナギは、ホルモン治療の 6-7 週間後 spermating を開始します。</li>
</ol>3. 精子サンプリング
<ol><li>最高品質のサンプルを得るためには、ホルモン投与6,23後 24 h の精子を抽出します。</li>
	<li>手順 2.2 ベンゾカインで魚を麻酔します。</li>
	<li>仰臥位で麻酔をかけられた魚をプレイス、蒸留水の噴出と性器をきれいし、水槽から海水が付いている接触によって性器または偶発的な精子の活性化に糞便汚染を避けるために、紙で慎重に乾燥します。</li>
	<li>魚の両方の側面に指を置いて、慎重に押すと横方向に胸鰭から性器をうち精子を強制的にマッサージします。<ol>
<li>ないより多くの精子が生殖器の開口部から出るまでマッサージを繰り返します。</li>
	</ol>
</li>
	<li>真空ポンプを使用して 15 mL プラスチック チューブに精子を収集します。</li>
	<li>4 ° C で、採精された精子 1:10 修正 Tanaka´s エクステンダー ソリューション24 (137 mM NaCl、76.2 mM NaHCO3蒸留水) を希釈します。 メモ: エクステンダー ・ ソリューションで精子は 4 ° C で維持する必要があります。</li>
</ol>4. 精子品質評価
<ol><li>13人工海水を準備 (mm: NaCl 354.7、MgCl2 52.4、CaCl2 9.9、Na2SO4蒸留水 28.2、KCl 9.4) 2% ウシ血清アルブミン (BSA) (w:v)、8.2、1100 mOsm/kg に浸透するために pH を調整し、維持それは細菌の成長を避けるために 4 ° C で。</li>
	<li>精子のコンピューター支援型分析 (CASA) のソフトウェアを開き、魚精子モジュールを選択します。<ol>
<li>ソフトウェアのシステム セットアップを開きますのプロパティをクリックします。そこに、毎秒 60 枚のキャプチャ オプションを設定します。負位相光学、マークラー商工会議所、10 倍の規模。</li>
		<li>分析値の種と粒子領域より大きい 2 μ m2 20 μ m2よりも小さい魚を選択します。</li>
		<li>速度パラメーター セル 10、45 μ m/s の間移動、45 および 100 μ m/s の間を移動するときに中に設定し、100 μ m/s よりも速く動くとき急速に設定低速に設定します。 注: 10 μ m/秒よりも遅い速度で精子は immotile と考えられていた。</li>
		<li>真直度 (STR) 80% としてプログレッシブの値を選択し、プロパティを保存します。</li>
		<li>フィールドのキャプチャ(カメラからのライブ画像のウィンドウが開きます) をクリックします。 注: コンピューターによる精子分析システムは、顕微鏡、カメラ、画像解析ソフトによって形成されます。</li>
	</ol>
</li>
	<li>カウントのチャンバーの人工海水の 4 μ L を入れて精子ソリューション (エクステンダー ・ ソリューションで希釈した精子) の 0.5 μ L を追加します。<ol>
<li>イメージを集中させ、負のフェーズで 10 倍の倍率での顕微鏡を用いた。15 コンピューターによる精子分析ソフトウェアのビデオからのキャプチャ -をクリックして、アクティブ化後 s。</li>
		<li>凍結保存用 70% よりも高い運動と帳票サンプルを選択すべてのサンプルを分析します。 注: この凍結プロトコルの成功のため質の高い精子だけを選択する非常に重要です。</li>
	</ol>
</li>
</ol>5. 精子凍結法
<ol><li>34 cm × 34 cm × 30 cm、5 cm 厚の発泡スチロールの箱で事前に液体窒素 (約 2.5 L) を準備します。 注: は、すべての回で液体窒素の 4-5 cm の高さのレベルを保持します。<ol>
<li>あらかじめ精子を凍結するフローティング構造を構築します。ポリスチレン (20 cm × 5 cm) 2 個を使用して、2 のプラスチック チューブにそれらをバインドし、液体窒素で構造を配置します。この構造体は、表面 (図 1) に 3 cm のおおよその高さで液体窒素上にフロートする必要があります。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56835/56835fig1.jpg"> 図 1.あらかじめ液体窒素で凍結用フローティング構造模式。構造は低密度 5 x 20 cm × 4 cm のスタイロ フォームの 2 つの部分で構成されています 14 cm のプラスチック製の管と接続されている cm。ストローは、液体窒素の上 3 cm のプラスチック製のチューブ上に配置されます。<a href="//cloudflare.jove.com/files/ftp_upload/56835/56835fig1large.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>精子、エクステンダー ・ ソリューション 1 の比率でメタノールを混合して凍結保存ソリューションを準備: 8:1 1.5 mL プラスチック チューブし、軽くかき混ぜます。</li>
	<li>ピペットの助けを借りて、500 μ L ストローに凍結保存液の 480 μ l 添加し必要に応じて、粘土のモデリングの 1 つの端を閉じることによってそれらをマークします。</li>
	<li>3 分間液体窒素の上 3 cm の高さで浮遊装置にわらを置きます。その後、液体窒素にわらを配置します。</li>
	<li>10-15 分後、長い鉗子を使用して液体窒素貯蔵タンクにストローを転送し、すべての回で液体窒素に浸漬しておきます。ここでは、精子は無期限に保存できます。</li>
</ol>6. 解凍方法
<ol><li>5.1 の手順で説明されているように液体窒素で発泡スチロールの箱を準備し、40 ° C で、水道水を 3 L ビーカーを使用して水のお風呂を準備します。</li>
	<li>長い鉗子を使用して液体窒素貯蔵タンクから発泡スチロールの箱に冷凍精子をストローを転送します。<ol>
<li>13 の 40 ° C で水のお風呂にそれぞれ藁を入れて s。</li>
		<li>はさみでストローのクローズド エンドを切断することによって、1.5 mL プラスチック チューブに精子を注ぐ。</li>
	</ol>
</li>
	<li>4.1 の手順で説明したように、コンピューターによる精子解析システムを用いた精子の運動性を分析します。</li>
	<li>流れの cytometer で精子の生存率を分析する精子を 100 μ l 添加してください。</li>
</ol>7. フローサイトメトリー
<ol><li>Propidium ヨウ化 (PI)、死んだ細胞の核を汚れ赤蛍光化合物と膜側の透過物核蛍光化合物、細胞の核をグリーンに汚れを含む蛍光キットを使用します。<ol>
<li>100 μ M の実用的なソリューションにそれから、原液 (1 mM) 1:10 Tanaka´s 培地で希釈することによって緑の蛍光染色液を準備します。</li>
		<li>PI ソリューションを薄めないでください。原液は、2.4 mM です。</li>
	</ol>
</li>
	<li>各サンプルは、新鮮なまたは解凍精子の 50 μ L を取るし、緑の蛍光染色作業液 (最終濃度 1 μ M) の 0.5 μ L を追加し、PI ソリューション (最終濃度 100 μ M) の 2 μ L。</li>
	<li>5 分間暗い染料 (PI および緑蛍光染色) を含むサンプルをインキュベートします。<ol>
<li>Tanaka´s エクステンダー ・ ソリューションの 500 μ L のサンプルを希釈し、流れの cytometer で分析します。</li>
	</ol>
</li>
	<li>流れの cytometer をオンにし、少なくとも 2 のプロットを含む新しいプロトコルを作成する: SS ログ対 FS ログ、FL1 vs FL3。 注: 両方、緑蛍光染色と propidium ヨウ化は目に見える波長の光を励起できること。DNA にバインドされると、これらの染料の最大蛍光性の放出が 516 nm と 617 nm、それぞれ。<ol>
<li>異なるレーザーの電圧調整: SS = 199;FS = 199;FL1 = 377;FL3 = 372</li>
		<li>最大イベントに集録設定合意を設定 = 5000 または 15 秒 (サンプルの最終濃度は約 100 万セル/mL をする必要があります)。低フローを使用してサンプルをお読みください。</li>
		<li>読書モードシングル チューブ固定位置モードを選択します。</li>
		<li>カルーセルの適切な数のサンプルを置く</li>
		<li>(F9 キーを押して) サンプルを読むし、さらに分析 (f7 キーを押して) excel ファイルに収集されたデータを保存します。</li>
	</ol>
</li>
</ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sperm+cryopreservation+on+the+European+eel+sperm+viability+and+spermatozoa+morphology.">Effect of sperm cryopreservation on the European eel sperm viability and spermatozoa morphology.</a> Reprod. Domestic Anim. 42 (2), 162-166 (2007).</li><li>Mordenti, M., Di Biase, A., Sirri, R., Modugno, S., Tasselli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Sexual+Maturation+in+Wild+Female+European+Eels+(Anguilla+anguilla)+in+Darkness+and+Light.">Induction of Sexual Maturation in Wild Female European Eels (Anguilla anguilla) in Darkness and Light.</a> Isr. J. Aquac. 64, (2012).</li><li>Ohta, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+induction+of+maturation+and+fertilization+in+the+Japanese+eel,+Anguilla+japonica.">Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica.</a> Fish Physiol. Biochem. 17 (1), 163-169 (1997).</li><li>Tanaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cryopreservation+of+sperm+of+Japanese+eel.">Long-term cryopreservation of sperm of Japanese eel.</a> J. Fish Biol. 60 (1), 139-146 (2002).</li><li>Gallego, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subpopulation+pattern+of+eel+spermatozoa+is+affected+by+post-activation+time,+hormonal+treatment+and+the+thermal+regimen.">Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen.</a> Reprod. Fertil. Dev. 27 (3), 529-543 (2015).</li><li>Asturiano, J. F., Cabrita, E., Horv&#225;th, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress,+challenges+and+perspectives+on+fish+gamete+cryopreservation:+A+mini-review.">Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review.</a> Gen. Comp. Endocrinol. , (2016).</li><li>Kime, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-assisted+sperm+analysis+(CASA)+as+a+tool+for+monitoring+sperm+quality+in+fish.">Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish.</a> Comp. Biochem. Physiol. C Toxicol. Pharmacol. 130 (4), 425-433 (2001).</li><li>Rurangwa, E., Kime, D. E., Ollevier, F., Nash, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+sperm+motility+and+factors+affecting+sperm+quality+in+cultured+fish.">The measurement of sperm motility and factors affecting sperm quality in cultured fish.</a> Aquaculture. 234 (1-4), 1-28 (2004).</li><li>Verstegen, J., Iguer-Ouada, M., Onclin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+assisted+semen+analyzers+in+andrology+research+and+veterinary+practice.">Computer assisted semen analyzers in andrology research and veterinary practice.</a> Theriogenology. 57 (1), 149-179 (2002).</li><li>Magnotti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+vitrification+of+fish+semen:+a+review+with+special+emphasis+on+marine+species.">Cryopreservation and vitrification of fish semen: a review with special emphasis on marine species.</a> Rev. Aquacult. , (2016).</li><li>Dziewulska, K., Rzemieniecki, A., Czerniawski, R., Domaga&#322;a, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-thawed+motility+and+fertility+from+Atlantic+salmon+(Salmo+salar+L.)+sperm+frozen+with+four+cryodiluents+in+straws+or+pellets.">Post-thawed motility and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodiluents in straws or pellets.</a> Theriogenology. 76 (2), 300-311 (2011).</li><li>Babiak, I., Bolla, S., Ottesen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitable+methods+for+cryopreservation+of+semen+from+Atlantic+halibut,+Hippoglossus+hippoglossus+L.">Suitable methods for cryopreservation of semen from Atlantic halibut, Hippoglossus hippoglossus L.</a> Aquac. Int. 16 (6), 561-572 (2008).</li><li>M&#252;ller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Japanese+eel+(Anguilla+japonica+Temminck+&amp;+Schlegel,+1846)+propagation+using+cryopreserved+sperm+samples.">Japanese eel (Anguilla japonica Temminck &amp; Schlegel, 1846) propagation using cryopreserved sperm samples.</a> J. Appl. Ichthyol. 33 (3), 550-552 (2017).</li></ol>

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。

このプロトコルでは、ヨーロッパのウナギの成熟、処理および精子凍結保存の完全なプロセスについて説明します。ここで説明する飼育条件は早く成熟とこの種6,7,25で質の高い精子の大量生産のため最適です。この凍結プロトコルとその潜在的な解凍後受精の成功は新鮮な精子26の品質に大きく依存しま?...

過去 25 年間欧州ウナギ (アンギラ) ヨーロッパの海岸に到着の数は 90 1,2,3で着実に減少しています。汚染、感染症、乱獲や生息地の破壊を含む抜本的な低下を説明するいくつかの要因があります。これのすべてが重要な絶滅危惧種4としての性質 (IUCN) リストのための国際連合のヨーロッパのウナギを含めることにつながる、この種に大きな影響を与える。その結果、技術や飼育下で繁殖のためのプロトコルの開発が必要です。
捕われの身でヨーロッパのウナギの成熟はホルモン治療5,6,7によって達成されるが、雌雄の配偶子の生産は8を同期することは困難。ウナギ9,の形成を加速する新しいアンドロゲン インプラントの開発を示しているにもかかわらず10女性に最終的な成熟のタイミングはまだ変化して11を制御することは困難。したがって、精子12,13,14および凍結保存技術の短期的なストレージ、繁殖管理、配偶子同期不要な8を作るために必要です。
2003 15,16以来ヨーロッパうなぎ精子の凍結保存をしました。いくつかの研究では、凍結保護剤16,17,18,19,20としてジメチルスルホキシド (DMSO) やメタノールのいずれかを使用して成功したプロトコルを設計されています。両方のプロトコルが正常に使用されているが、DMSO を凍結保存後解凍精子の細胞生存率はメタノール20,21より低い。また、うなぎ精子は DMSO との接触で活性化されより退屈な精子操作19が必要です、そのためメタノールより適切な保護剤の検討欧州ウナギ精子の DMSO よりも。
ここでは、最適なハンドリングとヨーロッパのウナギのホルモン治療のプロトコルは、以下に記載します。これに加えて、この種の精子の質の評価のため、保護剤の検討とプロトコルとしてメタノールを使用して最高のヨーロッパウナギ精子凍結プロトコルは説明もします。

<table border="0" cellpadding="0" cellspacing="0" width="538">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:151px;">AQUACEN BENZOCA&#205;NA +D2A2:D24</td>
			<td style="width:128px;">AQUACEN</td>
			<td style="width:119px;">3394ESP</td>
			<td style="width:141px;">Benzocaine</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td>Avantor&#160;</td>
			<td>3624-19</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringe</td>
			<td>BD Plastipak</td>
			<td>305501</td>
			<td>Syringe 1 mL</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">FC500</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Air pump 100</td>
			<td>EHEIM</td>
			<td>4011708370032</td>
			<td>Modified for suction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Eppendorf 1.5</td>
			<td>Eppendorf</td>
			<td>175508</td>
			<td>Plastic tubes 1.5 mL</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Falcon tubes</td>
			<td>Falcon</td>
			<td>175747</td>
			<td>Plastic tubes 15 mL</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flexible bucket</td>
			<td>Fiel</td>
			<td>1040101</td>
			<td>40 L, Black color.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ethanol</td>
			<td>Guinama SL</td>
			<td>Mg96270</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cyopreservation straws&#160;</td>
			<td>IMV Technologies</td>
			<td>14550</td>
			<td>500 &#181;L straws</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Live/Dead Sperm Viability Kit</td>
			<td>Invitrogen</td>
			<td>L7011</td>
			<td>Cytometer Kit</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Modelling clay</td>
			<td>JOVI</td>
			<td>Art 30</td>
			<td>4 different colors</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Precision scale PCB</td>
			<td>KERN &#38; Sohn GmbH</td>
			<td>PCB35002</td>
			<td>Digital scale</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Saline solution Vitulia 0.9%</td>
			<td>Laboratorios Ern, S.A.</td>
			<td>C.N. 999790.8</td>
			<td>Saline solution</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Forceps</td>
			<td>Levantina de Lab. SL</td>
			<td>3710025</td>
			<td>30 cm metal forceps</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Scissors</td>
			<td>Levantina de Lab. SL</td>
			<td>3700014</td>
			<td>Scissors 14cm</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ovitrelle (r-hCG)</td>
			<td>Merck SL</td>
			<td>EMEA/H/C/000320</td>
			<td>Human chorionic gonadotropin</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Nikon Eclipse 80i</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CaCl2</td>
			<td>Panreac Quimica SAU</td>
			<td>2112211210</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Na2SO4</td>
			<td>Panreac Quimica SAU</td>
			<td>3257091611</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaHCO3</td>
			<td>Panreac Quimica SAU</td>
			<td>1416381210</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">782M Camera</td>
			<td>Proiser</td>
			<td>782M</td>
			<td>Camera for CASA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">ISAS v1</td>
			<td>Proiser</td>
			<td>ISAS v1</td>
			<td>CASA software</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SpermTrack-10</td>
			<td>Proiser</td>
			<td>SpermTrack-10</td>
			<td>Countig chamber for CASA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beaker Pyrex 3L</td>
			<td>PYREX</td>
			<td>2110668</td>
			<td>Glass beaker 3L</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flask Pyrex 250 GL-45</td>
			<td>PYREX</td>
			<td>21801365</td>
			<td>Glass flask 250 mL</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KCl</td>
			<td>Scharlab SL</td>
			<td>PO02000500</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Methanol</td>
			<td>Scharlab SL</td>
			<td>ME03061000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MgCl2</td>
			<td>Scharlab SL</td>
			<td>MA00370500</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>05482</td>
			<td>Bovine serum albumina</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Styrofoam box</td>
			<td></td>
			<td></td>
			<td>34x34x30 cm and 5cm thick</td>
		</tr>
	</tbody>
</table>

handling and treatment of male european eels (anguilla anguilla) for hormonal maturation and sperm cryopreservation

最後年の間にいくつかの研究グループは開発とヨーロッパのウナギ処理と成熟の新しいプロトコルの改善に取り組んでいます。としてまだ、ひと絨毛性ゴナドトロピン (hCG) の毎週の注射は治療の数週間中に質の高い精子の大量の生産わずか 5-6 週間後の男性の成熟に証明しています。さらに、精子凍結保存のプロトコルが異なるエクステンダーを使用して、凍結保護剤と冷却時間を解凍以前について記載されているヨーロッパのウナギ。ここで、紹介受精、凍結保存、Tanaka´s ・ エクステンダー ・ ソリューションが直接使用することができます不要な解決方法および希薄のさまざまな種類の使用法を作るします。さらに、このプロトコルにはメタノールは毒性が低いと、精子をアクティブにしません、保護剤の検討としてメタノールを使用では使いやすいのものとなります。精子は、保護剤の検討、添加後すぐに凍結保存する必要はありません、解凍されて後に長く使用できます。また、精子の運動性は新鮮な精子のそれより低いが融解後依然として高いです。この作業の目的は、ヨーロッパウナギの最高の利用可能なプロトコル処理、成熟、精子凍結保存を表示することです。

70% 以上精子の運動と 18 ウナギから精子はこの研究に選ばれました。新鮮な精子 (表 1および図 2) からのそれらと比較して融解した後、結果はすべての品質パラメーターの減少を示した。運動の結果 (意味 ± S.E.M.、n = 18) 総運動と解凍後精子サンプルは、進歩的な運動よりも新鮮な精子で高い総運動と進歩的な運動性を示した。
<p ...

Watch this Scientific Journal Video about Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation at JoVE.com

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

ヨーロッパのウナギの成熟と精子の凍結保存のためのプロトコルは、最後の年にわたって改善されています。この資料では、成熟と保護剤の検討としてメタノールを誘発するひと絨毛性ゴナドトロピン (hCG) で使用可能な最適なプロトコルについて説明します。

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 ...

Hi, my name is Juan Asturiano from the Polytechnic University of Valencia, and we are with some of our colleagues from the Szent Istv

handling-treatment-male-european-eels-anguilla-anguilla-for-hormonal

Research

JoVE Journal

Developmental Biology

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

7.8K ビュー.  Universitat Politècnica de València. Hi, my name is Juan Asturiano from the Polytechnic University of Valencia, and we are with some of our colleagues from the Szent Istv

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

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作と<em&gt;インビトロ</emの&gt;成熟<em&gt;アフリカツメガエル</em細胞質内精子注入し&gt;卵母細胞、胚発生を研究するために

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

発生生物学

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

新生児ラット心筋細胞の単離と凍結保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

修正されたMicroSecureガラス化：人間の胚盤胞のための、安全なシンプルで非常に効果的な凍結保存手順

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

電気刺激を使用してBiowires中のヒト幹細胞由来の心筋細胞の成熟

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

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.

機械的組織解離およびフローサイトメトリーによる哺乳動物雄胚細胞のマルチシード精製のための標準的アプローチ

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

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

ひと初代細胞における細胞老化の誘導性

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

蛍光光度法による精子膜評価技術

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Caenorhabditis elegansふたなり生殖管内の精子の誘導と運動性の測定

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

単一の卵母細胞レポーターアッセイを用いたインビトロ成熟時の母体母体翻訳プログラムの定義

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

イメージングと覚醒剤送達システムの組み合わせによるマウスの生精子の研究

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

著者スポットライト:マウス精子におけるカルシウムシグナル伝達と先端体エキソサイトーシスを研究するための生細胞イメージング技術 

The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

生きたマウス精子における先体カルシウム動態とエキソサイトーシスのリアルタイムイメージング

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...