このプロジェクトは、コストの協会から資金を受けている (食品と農業コスト アクション FA1205: インプレス (ジョージア州 No 642893) プロジェクト AQUAGAMETE と欧州連合のホライゾン 2020年研究と技術革新プログラムの下でマリー マリアスクウォドフスカ キュリー。具体的にはこのプロジェクトの記録ビデオで彼の積極的な参加の学生アルベルト ・ ヴェンドレル ベルナベウ、PROiSER の科学的なチームに感謝したいと思います。

含む動物対象としている手順は、農業生産や家畜、大学パラオレイ バレンシアでの一般的な方向性によって (2015/VSC/エンドウ/00064) を承認しました。
1. 捕われの身で精子を成熟したヨーロッパのウナギから収集します。
注: 使用ヨーロッパうなぎ男性海水と一定した温度 (20 ° C) での再循環システム タンクに保持されます。毎週の腹腔内注射 (ひと絨毛性ゴナドトロピン (hCG); 魚体重の g あたり 1.5 IU) を通してホルモンを扱います。慣らす徐々 に始まる 3 日間淡水魚に海水交換が続く (1/3 水タンク内の) 37.0 g/mL の塩分濃度に到達するまで 2 日おき。
<ol><li>良質の精子のサンプルを取得するホルモン注射後 24 h ウナギを麻酔します。<ol>
<li>あらかじめ、麻酔の準備: 70% エタノール 100 mL にベンゾカインの 300 mg を追加。よく混合し、4 ° C で保存</li>
		<li>60 mg/L の最終的な集中を取得し、正しくミックス システム水の 5 L の柔軟なバケツにベンゾカインを希釈します。</li>
		<li>ベンゾカイン システム水とバケツに魚を転送します。魚が落ち着くまで、数分を待ってください。</li>
	</ol>
</li>
	<li>水で陰部をクリアし、糞便、尿や海水によって精子サンプルの汚染を避けるために吸水紙を乾燥します。</li>
	<li>腹部に穏やかな圧力を適用し、真空ポンプを使った 15 mL プラスチック チューブに精子を採取します。精子の量まで 6-7 mL、男性によって。</li>
	<li>運動解析を実施するまで少なくとも 1 時間のための 4 ° C で精子サンプルを保ちます。</li>
</ol>2. 冷蔵庫で冷やし、精子サンプルを希釈
<ol><li>原因の解決策を事前に準備します。 注: それぞれの種に固有のエクステンダー ・ ソリューションで精子サンプルを希釈する必要があります。<ol>
<li>うなぎ精子サンプル (P1 中) の非活性化メディアを準備します。重炭酸ナトリウム、塩化ナトリウム、塩化マグネシウム、塩化カリウムと 250 mL の蒸留水に塩化カルシウム 0.037 g 0.56 g の 0.127 g の 1.828 g 0.42 g を追加します。ミックスも解散します。4 ° C でのストア</li>
	</ol>
</li>
	<li>一定温度でサンプルを維持するために 4 ° c のクーラーのブロックを設定します。温度が安定するまで待ちます。</li>
	<li>それぞれの種にソリューション固有の攪拌機構を用いた精子サンプルを希釈します。 注: 希釈倍率固有種であり、プロトコルの標準化を定義する必要があります。まず、手順 3 と 4 で説明したように、ソフトウェアを使用して運動の事前の分析で得られた濃度に基づく精子濃度を予測します。この分析とは、運動の割合に基づいて最高の精子のサンプルを選択することが可能ですも。この後、研究者は正しい濃度で最高のサンプルを用いた実験のためのデータ収集を開始できます。<ol>
<li>1:50 の比率で P1 中うなぎ精子サンプルを希釈します。500 μ L の希釈うなぎ精子を準備するには、新鮮な精子サンプルを 50 μ l 添加し、P1 の 450 μ L を追加します。よく混ぜます。</li>
	</ol>
</li>
</ol>3. 精子の運動パラメーターを評価します。
<ol><li>
セットアップ ソフトウェアの運動モジュール
	<ol>
<li>4 ° C のクーラーの段階を設定し、それが安定するまでを待ちます。</li>
		<li>ソフトウェアを開き、運動モジュールを選択します。必要な場合は、ユーザー名とパスワードを作成します。</li>
		<li>プロパティと精子分析を開始する前に必要なパラメーターを選択します。<ol>
<li>種をクリックして |魚。</li>
			<li>それぞれの種の最高の技術的な条件によって1 秒あたりのフレームとイメージの数を選択します。1 秒あたり 120 の画像の両方のオプションを設定します。</li>
			<li>否定的なコントラストを選択します。精子の軌跡16の正確な再生のために必須です。</li>
			<li>対応する商工会議所のカウントとビデオカメラのスケールを選択します。実験で使用される倍率レンズのカメラを調整します。カウント部屋として SpermTrack10 と 10 倍の規模を設定します。 注: 一般的に、カウントの商工会議所で 10 μ m の深さと 10 倍の倍率のレンズが推奨条件以来、彼らはすべての精子のよりよい焦点を提供し、セルの最大数をキャプチャします。ただし、それぞれの種の運動解析のための最適な条件の以前の研究をすることをお勧めします。</li>
			<li>それぞれの種の粒子領域と接続を調整します。2 の2 μ m と 7 μ m で接続で最小の粒子領域を設定します。 注: 魚のため、粒子面積の範囲は 2-5 μ m と接続との間の最小値は精子の速度とフレーム レートに依存します。</li>
			<li>設定を保存します。</li>
		</ol>
</li>
		<li>キャプチャを選択します。</li>
	</ol>
</li>
	<li>
ソフトウェアでのヨーロッパウナギ精子を分析します。
	<ol>
<li>2 %bsa の蒸留水 25 mL に市販塩の 0.946 g を追加することによってソリューションの活性化 (人工海水) を準備します。</li>
		<li>500 mL の活性剤溶液 (人工海水) を取り出して、クーラーのブロックにします。 注: 一般に、魚の精子をアクティブに浸透衝撃イベント、いくつかの種のイオン濃度も重要かもしれませんが。海洋生物の培地は、高張べき間淡水種のため活性化中低張性浸透圧が必要です。</li>
		<li>冷却ステージの下で数える商工会議所を置き、4 ° C に温度が安定するまで待つ</li>
		<li>ミックス活性化と希薄化後精子精子をアクティブにし、アクティブ化後 5-10 秒間録画運動を開始します。<ol>
<li>カウントの商工会議所の活性化ソリューションの場所 4 μ L を取る。</li>
			<li>優しく精子細胞の損傷を防ぐため、遠心の 3 回を揺することによって希釈した精子をホモジナイズしてください。</li>
			<li>0.5 μ L の希釈した精子を取り出して活性剤混ぜてカウントの商工会議所ですぐにカバーをかけます。 注: この手順は、5 以上を取る必要がありますいない s 分析をできるだけ早く開始します。</li>
			<li>精子細胞に焦点を当てるし、セル (補足図 1 a) 間の遮断を避けるために精子 (150 に 200 細胞) の低い数字によって定義された最高の視覚領域を見つけます。速度に従って分かれている精子のトラックを取得するビデオのキャプチャを選択します。</li>
			<li>キャプチャを取得、結果の変動が少なく進み以前のように最適な間隔である 7 に 3 フィールドを取得するを選択します。120 の s ライセンス認証後カウント チャンバーのカバーの結露を避けるためにまでビデオを録画できます。</li>
			<li>出口の一般的なビューのすべてのフィールドを選択します。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 運動データを取得します。
<ol><li>フィールドを選択 |部分的な |保存とファイル名を選択します。保存をクリックします。 注: スプレッドシートは、部分的なデータ、グラフおよび精子トラックの画像の平均値を提供します。</li>
	<li>一般的なデータを選択 |ファイル名 |保存。 注: データは、すべてのフィールドの個々 の精子の運動の値を提供します。</li>
</ol>

<ol><li>Kime, D. E., et al. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/S0166445X96008065">Use of computer-assisted sperm analysis (CASA) for monitoring the effects of pollution on sperm quality of fish; application to the effects of heavy metals.</a> Aquatic Toxicology. 36, 223-237 (1996).</li>
<li>Kime, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+sperm+analysis+%28CASA%29+as+a+tool+for+monitoring+sperm+quality+in+fish%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+-+Part+C%3A+Toxicology+%26+Pharmacology%22%5BJournal%5D)">Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish.</a> Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology. 130, 425-433 (2001).</li>
<li>Bobe, J., Labbé, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Egg+and+sperm+quality+in+fish%5BTitle%5D)+AND+%22General+and+Comparative+Endocrinology%22%5BJournal%5D)">Egg and sperm quality in fish.</a> General and Comparative Endocrinology. 165, 535-548 (2010).</li>
<li>Rurangwa, E., Kime, D. E., Ollevier, F., Nash, J. P. <a target="_blank" href="https://www.sciencedirect.com/science/article/abs/pii/S0044848603008330">The measurement of sperm motility and factors affecting sperm quality in cultured fish.</a> Aquaculture. 234, 1-28 (2004).</li>
<li>Bekkevold, D., Hansen, M. M., Loeschcke, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Male+reproductive+competition+in+spawning+aggregations+of+cod+%28Gadus+morhua%2C+L.%29%5BTitle%5D)+AND+%22Molecular+Ecology%22%5BJournal%5D)">Male reproductive competition in spawning aggregations of cod (Gadus morhua, L.).</a> Molecular Ecology. 11, 91-102 (2002).</li>
<li>Chong, A. P., Walters, C. A., Weinrieb, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neglected+laboratory+test%3A+the+semen+analysis%5BTitle%5D)+AND+%22Journal+of+Andrology%22%5BJournal%5D)">The neglected laboratory test: the semen analysis.</a> Journal of Andrology. 4, 280-282 (1983).</li>
<li>Overstreet, J. W., Katz, D. F., Hanson, F. W., Fonesca, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laboratory+tests+for+human+male+reproductive+risk+assessment%5BTitle%5D)+AND+%22Teratogenesis%2C+Carcinogenesis%2C+and+Mutagenesis%22%5BJournal%5D)">Laboratory tests for human male reproductive risk assessment.</a> Teratogenesis, Carcinogenesis, and Mutagenesis. 4, 67-82 (1984).</li>
<li>Gallego, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardization+of+European+eel+%28Anguilla+anguilla%29+sperm+motility+evaluation+by+CASA+software%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software.</a> Theriogenology. 79, 1034-1040 (2013).</li>
<li>Fauvel, C., Suquet, M., Cosson, J. <a target="_blank" href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1439-0426.2010.01529.x">Evaluation of fish sperm quality.</a> Journal of Applied Ichthyology. 26, 636-643 (2010).</li>
<li>Mortimer, S. T., Schoëvaërt, D., Swan, M. A., Mortimer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+observations+of+flagellar+motility+of+capacitating+human+spermatozoa%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Quantitative observations of flagellar motility of capacitating human spermatozoa.</a> Human Reproduction. 12, 1006-1012 (1997).</li>
<li>Bompart, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CASA-Mot+technology%3A+How+results+are+affected+by+the+frame+rate+and+counting+chamber%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">CASA-Mot technology: How results are affected by the frame rate and counting chamber.</a> Reproduction, Fertility and Development. , (2018).</li>
<li>Vladić, T., Järvi, T. <a target="_blank" href="https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1095-8649.1997.tb01632.x">Sperm motility and fertilization time span in Atlantic salmon and brown trout - the effect of water temperature.</a> Journal of Fish Biology. 50, 1088-1093 (1997).</li>
<li>Rurangwa, E., Volckaert, F. A. M., Huyskens, G., Kime, D. E., Ollevier, F. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/11245263">Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardizes fertilisation in African catfish (Clarias gariepinus).</a> Theriogenology. 55, 751-769 (2001).</li>
<li>Cosson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marine+fish+spermatozoa%3A+racing+ephemeral+swimmers%5BTitle%5D)+AND+%22Reproduction%22%5BJournal%5D)">Marine fish spermatozoa: racing ephemeral swimmers.</a> Reproduction. 136, 277-294 (2008).</li>
<li>Castellini, C., Dal Bosco, A., Ruggeri, S., Collodel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+is+the+best+frame+rate+for+evaluation+of+sperm+motility+in+different+species+by+computer-assisted+sperm+analysis%3F%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">What is the best frame rate for evaluation of sperm motility in different species by computer-assisted sperm analysis?</a> Fertility and Sterility. 96, 24-27 (2011).</li>
<li>Soler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+holographic+solution+for+sperm+motility+analysis+in+boar+samples.+Effect+of+counting+chamber+depth%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">A holographic solution for sperm motility analysis in boar samples. Effect of counting chamber depth.</a> Reproduction, Fertility and Development. , (2018).</li>
<li>Elliot, F. I., Sherman, J. K., Elliot, E. J., Sullivan, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+photo+method+of+measuring+sperm+motility%5BTitle%5D)+AND+%22Journal+of+Animal+Science%22%5BJournal%5D)">A photo method of measuring sperm motility.</a> Journal of Animal Science. 37, 310(1973).</li>
<li>Katz, D. F., Dott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+of+measuring+swimming+speed+of+spermatozoa%5BTitle%5D)+AND+%22Journal+of+Reproduction+and+Fertility%22%5BJournal%5D)">Methods of measuring swimming speed of spermatozoa.</a> Journal of Reproduction and Fertility. 45, 263-272 (1975).</li>
<li>Liu, Y. T., Warme, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computerized+evaluation+of+sperm+cell+motility%5BTitle%5D)+AND+%22Computers+and+Biomedical+Research%22%5BJournal%5D)">Computerized evaluation of sperm cell motility.</a> Computers and Biomedical Research. 10, 127-138 (1977).</li>
<li>Jecht, E. W., Russo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+system+for+the+quantitative+analysis+of+human+sperm+motility%5BTitle%5D)+AND+%22Andrologia%22%5BJournal%5D)">A system for the quantitative analysis of human sperm motility.</a> Andrologia. 5, 215-221 (1973).</li>
<li>Holt, W. V., Palomo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+a+continuous+real-time+computerized+semen+analysis+system+for+ram+sperm+motility+assessment%2C+and+evaluation+of+four+methods+of+semen+preparation%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">Optimization of a continuous real-time computerized semen analysis system for ram sperm motility assessment, and evaluation of four methods of semen preparation.</a> Reproduction, Fertility and Development. 8, 219-230 (1996).</li>
<li>Stephens, D. T., Hickman, R., Hoskins, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Description%2C+validation%2C+and+performance+characteristics+of+a+new+computer-automated+sperm+motility+analysis+system%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Description, validation, and performance characteristics of a new computer-automated sperm motility analysis system.</a> Biology of Reproduction. 38, 577-586 (1988).</li>
<li>Mortimer, D., Goel, N., Shu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+CellSoft+automated+semen+analysis+system+in+a+routine+laboratory+setting%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Evaluation of the CellSoft automated semen analysis system in a routine laboratory setting.</a> Fertility and Sterility. 50, 960-968 (1988).</li>
<li>Mortimer, S. T., Swan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinematics+of+capacitating+human+spermatozoa+analysed+at+60+Hz%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Kinematics of capacitating human spermatozoa analysed at 60 Hz.</a> Human Reproduction. 10, 873-879 (1995).</li>
<li>Holt, W. V., O'Brien, J., Abaigar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+and+interpretation+of+computer-assisted+sperm+analyses+and+sperm+sorting+methods+in+assisted+breeding+and+comparative+research%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">Applications and interpretation of computer-assisted sperm analyses and sperm sorting methods in assisted breeding and comparative research.</a> Reproduction, Fertility and Development. 19, 709-718 (2007).</li>
<li>Gill, H. Y., Van Arsdalen, K., Hypolote, J., Levin, R., Ruzich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+study+of+two+computerized+semen+motility+analyzers%5BTitle%5D)+AND+%22Andrologia%22%5BJournal%5D)">Comparative study of two computerized semen motility analyzers.</a> Andrologia. 20, 433-440 (1988).</li>
<li>Jasko, D. J., Lein, D. H., Foote, R. H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/2254179">A comparison of two computer-assisted semen analysis instruments for the evaluation of sperm motion characteristics in the stallion.</a> Journal of Andrology. 11, 453-459 (1990).</li>
<li>Vantman, D., Koukoulis, G., Dennison, L., Zinaman, M., Sherins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+semen+analysis%3A+Evaluation+of+method+and+assessment+of+the+influence+of+sperm+concentration+on+linear+velocity+determination%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Computer-assisted semen analysis: Evaluation of method and assessment of the influence of sperm concentration on linear velocity determination.</a> Fertility and Sterility. 49, 510-515 (1988).</li>
<li>Kime, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+sperm+analysis+%28CASA%29+as+a+tool+for+monitoring+sperm+quality+in+fish%5BTitle%5D)+AND+%22Comparative+Biochemistry+and+Physiology+-+Part+C%3A+Toxicology+%26+Pharmacology%22%5BJournal%5D)">Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish.</a> Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology. 130, 425-433 (2001).</li>
<li>Scherr, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidics+and+numerical+simulation+as+methods+for+standardization+of+zebrafish+sperm+cell+activation%5BTitle%5D)+AND+%22Biomedical+Microdevices%22%5BJournal%5D)">Microfluidics and numerical simulation as methods for standardization of zebrafish sperm cell activation.</a> Biomedical Microdevices. 17, 65-75 (2015).</li>
<li>Mortimer, S. T., Swan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+image+sampling+frequency+on+established+and+smoothing-independent+kinematic+values+of+capacitating+human+spermatozoa%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Effect of image sampling frequency on established and smoothing-independent kinematic values of capacitating human spermatozoa.</a> Human Reproduction. 14, 997-1004 (1999).</li>
<li>Hoogewijs, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+counting+chamber+type+on+CASA+outcomes+of+equine+semen+analysis%5BTitle%5D)+AND+%22Equine+Veterinary+Journal%22%5BJournal%5D)">Influence of counting chamber type on CASA outcomes of equine semen analysis.</a> Equine Veterinary Journal. 44, 542-549 (2012).</li>
<li>Soler, C., et al. <a target="_blank" href="https://www.researchgate.net/publication/289987642_Effect_of_counting_chamber_on_seminal_parameters_analyzing_with_the_ISASv1_R">Effect of counting chamber on seminal parameters, analyzing with the ISASv1®.</a> Revista Internacional de Andrología. 10, 132-138 (2012).</li>
<li>Didion, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+semen+analysis+and+its+utility+for+profiling+boar+semen+samples%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">Computer-assisted semen analysis and its utility for profiling boar semen samples.</a> Theriogenology. 70, 1374-1376 (2008).</li>
<li>David, G., Serres, C., Jouannet, P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/2384341">Kinematics of human spermatozoa.</a> Gamete Research. 4, 83-95 (1981).</li>
<li>Björndahl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+is+normal+semen+quality%3F+On+the+use+and+abuse+of+reference+limits+for+the+interpretation+of+semen+results%5BTitle%5D)+AND+%22Human+Fertility+%28Cambridge%29%22%5BJournal%5D)">What is normal semen quality? On the use and abuse of reference limits for the interpretation of semen results.</a> Human Fertility (Cambridge). 14, 179-186 (2011).</li>
<li>Verstegen, J., Iguer-ouada, M., Onclin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computer-assisted+semen+analyzers+in+andrology+research+and+veterinary+practice%5BTitle%5D)+AND+%22Theriogenology%22%5BJournal%5D)">Computer-assisted semen analyzers in andrology research and veterinary practice.</a> Theriogenology. 57, 149-179 (2002).</li>
<li>Alavia, S. M. H., Cosson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sperm+motility+in+fishes.+I.+Effects+of+temperature+and+pH%3A+a+review%5BTitle%5D)+AND+%22Cell+Biology+International%22%5BJournal%5D)">Sperm motility in fishes. I. Effects of temperature and pH: a review.</a> Cell Biology International. 29, 101-110 (2005).</li>
<li>Islam, M. S., Akhter, T. <a target="_blank" href="http://article.sapub.org/10.5923.j.als.20110101.03.html">Tale of Fish Sperm and Factors Affecting Sperm Motility: A Review.</a> Advancements in Life Sciences. 1, 11-19 (2011).</li>
<li>Dadras, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+common+carp+Cyprinus+carpio+sperm+motility+and+lipid+composition+using+different+in+vitro+temperatures%5BTitle%5D)+AND+%22Anim.+Reprod.+Sci%22%5BJournal%5D)">Analysis of common carp Cyprinus carpio sperm motility and lipid composition using different in vitro temperatures.</a> Anim. Reprod. Sci. 180, 37-43 (2017).</li>
<li>Soler, C., García, A., Contell, J., Segervall, J., Sancho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinematics+and+subpopulations%27+structure+definition+of+blue+fox+%28Alopex+lagopus%29+sperm+motility+using+the+ISASV1+CASA+system%5BTitle%5D)+AND+%22Reproduction+in+Domestic+Animals%22%5BJournal%5D)">Kinematics and subpopulations' structure definition of blue fox (Alopex lagopus) sperm motility using the ISASV1 CASA system.</a> Reproduction in Domestic Animals. 49, 560-567 (2014).</li>
<li>Vásquez, F., Soler, C., Camps, P., Valverde, A., GarcíaMolina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spermiogram+and+sperm+head+morphometry+assessed+by+multivariate+cluster+analysis+results+during+adolescence+%2812-18+years%29+and+the+effect+of+varicocele%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Spermiogram and sperm head morphometry assessed by multivariate cluster analysis results during adolescence (12-18 years) and the effect of varicocele.</a> Asian Journal of Andrology. 18, 824-830 (2016).</li>
<li>Soler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dog+sperm+head+morphometry%3A+its+diversity+and+evolution%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Dog sperm head morphometry: its diversity and evolution.</a> Asian Journal of Andrology. 19, 149-153 (2017).</li>
<li>Valverde, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphometry+and+subpopulation+structure+of+Holstein+bull+spermatozoa%3A+variations+in+ejaculates+and+cryopreservation+straws%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Morphometry and subpopulation structure of Holstein bull spermatozoa: variations in ejaculates and cryopreservation straws.</a> Asian Journal of Andrology. 18, 851-857 (2016).</li>
</ol>

著者が明らかに何もありません。

このプロトコルで使われる精子分析ソフトウェア使用されています研究者によって世界的な種、魚など。しかし、魚は精子の評価に影響を与えることができるいくつかの特定の機能にあります。魚の精子はすぐに低下し、活性化した後短時間の運動につながる活性化の瞬間に高速を示した。その上、再現の温度は種に依存し、いくつかのケースでは約 4 ° C2,<sup class=...

再現性は、両方の配偶子 (卵と精子)1,2の品質に依存します。これは、正常な受精の可能な子孫3,4の開発が可能に貢献する主要な要因です。配偶子の便利な評価は、標本の不妊の可能性を定義するための最良のツールです。
多くの水生商業種4の生産に一般的な方法は、複数の男性から精子を混合します。ただし、男性の精子変動が精子競争につながることができますそして、したがって、すべての男性は、同じように遺伝子プール5に貢献しています。この意味で、運動などの個々 の射精/精子機能の適切な評価は潜在的な個々 の男性不妊に関する差別的な情報を取得に不可欠です。それは時間と一貫性の欠如、結果6、7の非互換性につながる経験を必要と、精子の運動の直接観察は不正確で、主観的なデータを生成できます。ただし、信頼性の高い精子の品質分析2,4を提供することができます多くの革新的な迅速かつ定量的手法があります。
精子のコンピューター支援型分析は、精子品質8に関する正確なデータを提供する開発されました。この技術には、精子の運動性の評価をできる位相差顕微鏡に関連するソフトウェアの開発が含まれています。しかし、運動パラメーターの制限要因はビデオ カメラのフレーム レートです。軌道が精子に基づいている個々 の精子頭部重心位置の連続したフレームでビデオ録画の鞭毛運動パターン3,9,10,と相関している11. 測定するメインの速度論的パラメーターは直線速度 (VSL)、曲線速度 (VCL) 平均パス速度 (VAP)。VSL は、開始と精子を時間で割った値で撮影したエンドポイントとの間の距離です。VCL は、精子で撮影した正確な軌道に沿って実際の速度です。VAP は、軌道の派生平滑化されたパスに沿って速度です。これらのパラメーターは、直線性 (LIN)、真直度 (STR)、揺れ (一) 横方向のヘッドの動き (ALH) とビート クロス周波数 (BCF)4,10の振幅のような暴行測定など追加の運動情報を許可します。
哺乳類の精子分析システムがもともと、ドナー (約 37 ° C) の体温で動作するシステムの要件の 1 つです。このソフトウェアは魚種にも使えるとはいえ、精子分析結果の誤差を減らすためにいくつかの適応を作るべきです。サケやウナギの8,12など、いくつかの魚種で受精は低温 (およそ 4 ° C)2,4で発生します。したがって、不快な労働条件を避けるためには、冷却装置を開発する必要があります。また、魚類精子のいない精液の運動性、運動を有効に浸透衝撃を必要です。海洋生物の培地は、高張べき間淡水種のため活性化中低張性浸透圧が必要です。しかし、いくつかの種は、サケ科魚類、としてイオン濃度もかもしれない重要な3,4,9。活性化後の魚の精子は運動 (2 分以内)13,14高速で信頼性の高いデータ15を取得する最適なフレーム レートを決定することが重要されているの急速な減少によって特徴付けられます。
本研究の目的は、デザインし、魚精子サンプルの冷凍システムを適用です。さらに、このプロトコルは、種に応じて標準プロトコルの確立のための最適なフレーム レートを決定する方法を定義します。このプロトコルの使用は、モデルとしてヨーロッパのウナギを用いた魚精評価のコンテキストで新しい扉を開きます。

<table><tbody><tr><td>Human Chorionic Gonadotropin</td><td>Argent Chemical Laboratories</td><td>hCG</td><td>Hormone</td></tr><tr><td>Benzocaine</td><td>Merck</td><td>E1501 Sigma</td><td>Anesthesia</td></tr><tr><td>sodium bicarbonate</td><td>Merck</td><td>S5761 Sigma&amp;nbsp;</td><td>P1 medium</td></tr><tr><td>sodium chloride</td><td>Merck</td><td>1.06406 EMD Millipore</td><td>P1 medium</td></tr><tr><td>magnesium chloride</td><td>Merck</td><td>1374248 USP</td><td>P1 medium</td></tr><tr><td>potassium chloride</td><td>Merck</td><td>P3911-500G</td><td>P1 medium</td></tr><tr><td>calcium chloride</td><td>Merck</td><td>C7902-500G</td><td>P1 medium</td></tr><tr><td>commercial salt</td><td>Aqua Medic&amp;nbsp;</td><td>Meersalz</td><td>Activator solution&amp;nbsp;</td></tr><tr><td>BSA</td><td>Merck</td><td>05470 Sigma</td><td>Activator solution&amp;nbsp;</td></tr><tr><td>Falcon tubes 15 ml</td><td>Merck</td><td>T1943-1000EA</td><td></td></tr><tr><td>Falcon tubes support</td><td>Merck</td><td>R5651-5EA</td><td></td></tr><tr><td>Eppendorfs</td><td>Merck</td><td>T9661-1000EA</td><td></td></tr><tr><td>Micropipet 20 &amp;micro;l</td><td>Gilson</td><td>PIPETMAN&amp;reg; Classic</td><td></td></tr><tr><td>Micropipet 10 &amp;micro;l</td><td>Merck</td><td>Z683787-1EA</td><td></td></tr><tr><td>Tips for micropipets 20 &amp;micro;l</td><td>Merck</td><td>Z740030-1000EA</td><td></td></tr><tr><td>Tips for micropipets 10 &amp;micro;l</td><td>Merck</td><td>Z740028-2000EA</td><td></td></tr><tr><td>Spermtrack</td><td>PROiSER</td><td>Counting chamber</td><td></td></tr><tr><td>TruMorph</td><td>PROiSER</td><td>TruMorph</td><td></td></tr><tr><td>Microscope UB 200i Serie</td><td>PROiSER</td><td>Microscope</td><td></td></tr><tr><td>Cooler plate</td><td>PROiSER</td><td></td><td>Prototype</td></tr><tr><td>Cooler block</td><td>PROiSER</td><td></td><td>Prototype</td></tr><tr><td>ISAS v1</td><td>PROiSER</td><td>ISAS</td><td>Software</td></tr></tbody></table>

fish sperm assessment using software and cooling devices

配偶子品質評価のため養殖のための有用なデータを提供できる革新的な迅速かつ定量的な手法があります。精子分析のためのコンピューター化されたシステムは、いくつかのパラメーターを測定するため開発された測定する最も一般的の 1 つ、精子の運動です。
当初は、魚の精子分析のため使用することもできますが、このコンピューター技術は哺乳類種のため設計されました。魚は、精子評価など短い運動時間活性化した後と、場合によっては、温度を下げるための適応に影響を与えることができる特定の機能を持っています。したがって、運動解析を魚の精子分析のためより効率的にソフトウェアとハードウェアのコンポーネントを変更する必要は。哺乳動物の精子、精子の最適温度を維持するために加熱プレートを使用します。しかし、いくつかの魚種のため、以来、精子は 2 分以内にアクティブなまま、運動の持続時間を延ばすために低温を使用する便利です。したがって、冷却装置が分析、光学顕微鏡などの時間の経過とともに一定の温度でサンプルを冷蔵する必要です。このプロトコルでは、魚の精子の運動性の精子分析のためのソフトウェアを使用して、新しい結果を最適化するデバイスを冷却の分析について説明します。

精子の運動の時間効果の解析
15 から静的な精子の割合増加するヨーロッパのウナギの場合 120 s (36.9% 20.9%) から s (24.4% から 40.7%)、活性化とモバイルの進歩的な精子の割合が減少しました。(図 1 aと1 b)。速度に基づいて、精子細胞は時間 (図 1と1 D</...

Watch this Scientific Journal Video about Fish Sperm Assessment Using Software and Cooling Devices at JoVE.com

Fish Sperm Assessment Using Software and Cooling Devices

この議定書は、精子のコンピューター支援型分析を使用して、デバイスを冷却魚精子評価の手順を説明します。ソフトウェアは、迅速、正確なと養殖業の再生の成功を改善するために便利なツールをすることができます、精子運動に基づく魚の精子の質の定量分析を与えます。

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 ...

fish-sperm-assessment-using-software-and-cooling-devices

Research

JoVE Journal

Biology

8.7K ビュー.  PROiSER R+D S.L.. この議定書は、精子のコンピューター支援型分析を使用して、デバイスを冷却魚精子評価の手順を説明します。ソフトウェアは、迅速、正確なと養殖業の再生の成功を改善するために便利なツールをすることができます、精子運動に基づく魚の精子の質の定量分析を与えます。

ビデオ: Fish Sperm Assessment Using Software and Cooling Devices

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

遺伝子編集アメリカナマズ、 Ictalurus イシガキダイ胚に CRISPR/Cas9 蛋白質のマイクロインジェクション

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

遺伝学

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, ...

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 ...

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

ゼブラフィッシュの生殖細胞系列編集の迅速な分離のための精子のスクリーニング

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...

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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

蛍光その場で</emヒト精子の>ハイブリダイゼーション（FISH）プロトコル

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

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, ...

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 ...

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 ...

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 ...

魚の精子の評価ソフトウェアを使用して、冷却装置

要約

さらに動画を探す

遺伝子編集アメリカナマズ、 Ictalurus イシガキダイ胚に CRISPR/Cas9 蛋白質のマイクロインジェクション

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

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

ゼブラフィッシュの生殖細胞系列編集の迅速な分離のための精子のスクリーニング

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

蛍光その場でハイブリダイゼーション（FISH）プロトコル

蛍光光度法による精子膜評価技術

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

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

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

魚の精子の評価ソフトウェアを使用して、冷却装置

要約

さらに動画を探す

遺伝子編集アメリカナマズ、 Ictalurus イシガキダイ胚に CRISPR/Cas9 蛋白質のマイクロインジェクション

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

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

ゼブラフィッシュの生殖細胞系列編集の迅速な分離のための精子のスクリーニング

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

蛍光その場でハイブリダイゼーション（FISH）プロトコル

蛍光光度法による精子膜評価技術

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

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

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

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