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><tbody><tr><td>AQUACEN BENZOCA&amp;Iacute;NA +D2A2:D24</td><td>AQUACEN</td><td>3394ESP</td><td>Benzocaine</td></tr><tr><td>NaCl</td><td>Avantor&amp;nbsp;</td><td>3624-19</td><td></td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>305501</td><td>Syringe 1 mL</td></tr><tr><td>FC500</td><td>Beckman Coulter</td><td></td><td>Flow cytometer</td></tr><tr><td>Air pump 100</td><td>EHEIM</td><td>4011708370032</td><td>Modified for suction</td></tr><tr><td>Eppendorf 1.5</td><td>Eppendorf</td><td>175508</td><td>Plastic tubes 1.5 mL</td></tr><tr><td>Falcon tubes</td><td>Falcon</td><td>175747</td><td>Plastic tubes 15 mL</td></tr><tr><td>Flexible bucket</td><td>Fiel</td><td>1040101</td><td>40 L, Black color.</td></tr><tr><td>Ethanol</td><td>Guinama SL</td><td>Mg96270</td><td></td></tr><tr><td>Cyopreservation straws&amp;nbsp;</td><td>IMV Technologies</td><td>14550</td><td>500 &amp;micro;L straws</td></tr><tr><td>Live/Dead Sperm Viability Kit</td><td>Invitrogen</td><td>L7011</td><td>Cytometer Kit</td></tr><tr><td>Modelling clay</td><td>JOVI</td><td>Art 30</td><td>4 different colors</td></tr><tr><td>Precision scale PCB</td><td>KERN &amp;amp; Sohn GmbH</td><td>PCB35002</td><td>Digital scale</td></tr><tr><td>Saline solution Vitulia 0.9%</td><td>Laboratorios Ern, S.A.</td><td>C.N. 999790.8</td><td>Saline solution</td></tr><tr><td>Forceps</td><td>Levantina de Lab. SL</td><td>3710025</td><td>30 cm metal forceps</td></tr><tr><td>Scissors</td><td>Levantina de Lab. SL</td><td>3700014</td><td>Scissors 14cm</td></tr><tr><td>Ovitrelle (r-hCG)</td><td>Merck SL</td><td>EMEA/H/C/000320</td><td>Human chorionic gonadotropin</td></tr><tr><td>Nikon Eclipse 80i</td><td>Nikon</td><td></td><td>Microscope</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>2112211210</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>3257091611</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>1416381210</td><td></td></tr><tr><td>782M Camera</td><td>Proiser</td><td>782M</td><td>Camera for CASA</td></tr><tr><td>ISAS v1</td><td>Proiser</td><td>ISAS v1</td><td>CASA software</td></tr><tr><td>SpermTrack-10</td><td>Proiser</td><td>SpermTrack-10</td><td>Countig chamber for CASA</td></tr><tr><td>Beaker Pyrex 3L</td><td>PYREX</td><td>2110668</td><td>Glass beaker 3L</td></tr><tr><td>Flask Pyrex 250 GL-45</td><td>PYREX</td><td>21801365</td><td>Glass flask 250 mL</td></tr><tr><td>KCl</td><td>Scharlab SL</td><td>PO02000500</td><td></td></tr><tr><td>Methanol</td><td>Scharlab SL</td><td>ME03061000</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Scharlab SL</td><td>MA00370500</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>05482</td><td>Bovine serum albumina</td></tr><tr><td>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。
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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 ...

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

Universidad de Cartagena UDEC

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. 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。

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

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

排水処理プロセスの有効性の評価のための化学的および生態毒性メソッドのバッテリーの使用は、エストロゲン効力を削除します

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

環境学

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

熱ショック2処理とゼブラフィッシュ胚の倍数性マニピュレーション

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology has been identified as a way to obtain more detailed information on movement and behavior across a broader spatial scale than is possible with other known technology. The purpose of this method is to provide a detailed step by step instruction on tag implantation for both lampreys and eel. For laboratory studies using actively migrating juvenile Pacific Lamprey (120-160 mm), we determined that the presence of the tag did not alter the swimming ability between tagged and untagged Individuals or have any significant tag loss (&lt;3%). Similar results were determined during laboratory testing of Yellow phase American Eels (113-175 mm). No mortality occurred during a 38-day holding period and there was minimal tag loss (3.8%). The presence of the tag did not have any significant effect on the swimming ability or survival of tagged eels compared to untagged controls and there was minimal tag loss.

This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

少年太平洋ヤツメウナギやアメリカのウナギの新しいマイクロ音響タグの注入

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

生物学

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高スループット<em&gt;ゼブラフィッシュ</em&gt;エネルギー消費アッセイ

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

全体の睾丸針によるゼブラフィッシュを精原細胞の凍結保存に超急速冷却浸漬

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

魚の精子の評価ソフトウェアを使用して、冷却装置

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

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 ...

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (Oryzias latipes)

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka is also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and, thus, reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including teleost fish. Collecting sperm is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article, therefore, describes two methods of sperm collection in the small model fish, Japanese medaka: testes dissection and abdominal massage. This paper demonstrates that both approaches are feasible for medaka and shows that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. This article also describes a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). These procedures, specified for this useful small teleost model, will greatly enhance understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka Oryzias latipes

This article describes two quick and efficient methods for collecting sperm from the small model fish medaka (Oryzias latipes), as well as a protocol for reliably assessing sperm quality using computer-assisted sperm analysis (CASA).

硬骨動物モデルにおける精子採取とコンピュータ支援精子解析 メダカ

Japanese medaka (Oryzias latipes) is a teleost fish and&#160;an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology ...

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

要約

さらに動画を探す

操作と

排水処理プロセスの有効性の評価のための化学的および生態毒性メソッドのバッテリーの使用は、エストロゲン効力を削除します

熱ショック2処理とゼブラフィッシュ胚の倍数性マニピュレーション

少年太平洋ヤツメウナギやアメリカのウナギの新しいマイクロ音響タグの注入

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

高スループット

全体の睾丸針によるゼブラフィッシュを精原細胞の凍結保存に超急速冷却浸漬

魚の精子の評価ソフトウェアを使用して、冷却装置

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

硬骨動物モデルにおける精子採取とコンピュータ支援精子解析メダカ

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

要約

さらに動画を探す

操作と

排水処理プロセスの有効性の評価のための化学的および生態毒性メソッドのバッテリーの使用は、エストロゲン効力を削除します

熱ショック2処理とゼブラフィッシュ胚の倍数性マニピュレーション

少年太平洋ヤツメウナギやアメリカのウナギの新しいマイクロ音響タグの注入

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

高スループット

全体の睾丸針によるゼブラフィッシュを精原細胞の凍結保存に超急速冷却浸漬

魚の精子の評価ソフトウェアを使用して、冷却装置

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

硬骨動物モデルにおける精子採取とコンピュータ支援精子解析 メダカ

硬骨動物モデルにおける精子採取とコンピュータ支援精子解析メダカ