This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

全ての動物手順は、三重大学の倫理委員会によって承認された、と「動物の愛護及び管理に関する法律「日本動物福祉の規制（日本の環境省）に従って行われ、国際的なガイドラインを遵守しました。 ニードルの作製注：全ての実験は、麻酔下で行い、すべての努力が苦痛を最小限にするために行われました。安楽死のために、魚を≥20分間（≤4℃で5部の氷/水1部）を氷水浴中に浸漬しました。 <ol><li>針プラー（ 図1A）と1.0 mmの外径のガラスキャピラリーを引いて、ガラスマイクロキャピラリー針を準備します。 </li><li>斜めに細かいハサミ（ 図1B）を使用して、針の先端をカットします。 200ミクロン（ 図1C） -理想的な先端の直径は約100である必要があります。先端diamete場合rが狭すぎる、血液が針に入ることはありません。 </li><li> 5mg / mlの濃度に生理食塩水でヘパリンを溶解します。 </li><li>吸引管アセンブリのノーズピース端でのプレカット針を置き、口の中にマウスピースを保持する、または電球ディスペンサーに針を接続します。ヘパリン溶液に針の先端を浸し、吸引により針をヘパリンで治療すると、溶液（ 図1Dおよび1E）を吹き抜けます。 注：吸引管アセンブリは、ゼブラフィッシュ精子の凍結保存の16のために使用されてきました。ロングゴムチューブは、口の中に飛んで任意の血液を停止することができます。チューブの途中にフィルタを設定すると、危険を避けることができます。 </li><li>少なくとも1時間、10-cmのシャーレと空気乾燥でヘパリン処理針を保管してください。針の多くは、事前に製造することができます。 </li></ol> 2.麻酔<ol><li> 2（2-フェノキシエタノール、100μlの魚の水200mlを混合することにより、小さなプラスチックケースで麻酔液を準備します-PE）。麻酔薬の最終濃度は500ppmです。 注意！ 2-PEは、迅速な効果があります。 </li><li>循環系からの魚（AB株）の所望の数を削除します。 </li><li> 2分（ 図1F） - 1のための麻酔薬に魚を転送するためにネットを使用してください。 1分以内に急速な蓋の動きを徐々に、泳ぐ、水平にあえぎを胸ひれを広げ、持っている魚を観察します。 </li><li>時が経つにつれ、魚がケースの底に横たわっ観察し、最終的に水泳を停止します。魚が息をのむために停止し、蓋の動きが遅いとき麻酔の外科的平面に到達します。この時点で、魚は採血のための準備ができています。 </li><li>スキマーを使用すると、2-PEから麻酔した魚を持ち上げ、ゆっくりと麻酔薬（ 図1G）に浸したペーパータオルの上に置きます。目の乾燥を防ぎ、やさしくするために別の乾いた柔らかいティッシュペーパーを使用することも2-PE液に浸した柔らかいティッシュペーパーで魚の頭をカバー体表面を乾燥。 </li></ol> 3.採血<ol><li>吸引チューブアセンブリ（または電球ディスペンサー）のノーズピース端でのヘパリン処理針を置き、口の中に吸引管アセンブリのマウスピース端部を保持します。 </li><li>ノーズピース端と一緒に針をつかみ、慎重に針の先端に干渉スケールを除去。採血部位へ45°の角度 - 30に針を挿入します。消化管（ 図1H）の穿刺を避けます。バルブディスペンサーを用いた場合には、親指と中指とバルブを押して、上記のように針を挿入し、最初の指で電球の先端の穴をブロックします。 注：採血用サイトでは、背側大動脈の領域で肛門に体軸と後部に沿っています。背側大動脈（DA）と後部主静脈（PCV）は、脊椎に腹だけ（ 図2）です。 </li><li>吸うために開始針が脊椎に接触したときに感じられる吸引管アセンブリの端部がマウスピース。血液が上昇しない場合、血液の流れを促進するために手で微妙に針の先端を移動させます。血液が針に上昇されると、すぐに揺れ停止し（ 図1I）優しく吸うことに注意してください。バルブディスペンサーを用いた場合には、血液を吸引するためのバルブの圧力を解放します。 </li><li>血液がゆっくりため動脈圧の可能性が高い、吸引することなく、拍動性の方法で針に上昇します確認します。したがって、針が正しく動脈を貫通した場合吸引する必要はありません。 </li><li>血液の適切な量を収集した後、吸引を停止します。魚から針を外し、任意の出血を止めるために、柔らかいティッシュペーパーを使用して穿刺部位を押してください。指の圧力の20秒（ 図1J） -約10後の出血の停止を確認します。 </li><li>出血が停止した後、直ちにCLEに戻し魚を転送します暖かい水（〜28℃）のタンク。それは泳ぐことを始めるまで優しく鰓に向かって水を旋回することによって回復する魚を助けます。 注意：2リットルタンクで5 postsampling魚まで保ち、魚に酸素する循環システムにタンクを接続します。魚の水への抗生物質の添加は不要です。通常の住宅と給紙に魚を維持します。 </li><li>パラフィルムの一部（ 図1K）のクリーン領域に針から血液を追放。 </li><li>すべての市販のハンドヘルドグルコメーター（ 図1L）を使用して、血糖値を測定します。 注：血糖計は、グルコースデヒドロゲナーゼ、フラビンアデニンジヌクレオチドの電極を使用し、0.6μlのサンプル量が必要です。 <ol><li>メーター内に完全テストストリップを挿入し、直接血液滴をタッチします。自動的にテストストリップに採血を確認し、表示領域の血糖結果5秒を取得します。結果を記録し、テストストリップを廃棄します。使用各測定のために新しいテストストリップ。 </li></ol></li><li> （オプションのステップ）は、ピペッティングにより血液の正確な量を取り、さらなる分析（ヘモグロビン、トリアシルグリセロール（TG）、総コレステロールなど ）のためのマイクロ遠心チューブに血液を移します。必要に応じて、生理食塩水で1魚から全血を希釈します。室温で680×gで3分間、血液サンプルを遠心分離し、血漿を採取します。新しいチューブにプラズマを転送します。この時点で、それは、生化学的分析に使用する準備ができています。 </li></ol>

<ol><li>Lieschke, G., Currie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+human+disease%3A+zebrafish+swim+into+view%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li>
<li>Penberthy, W. T., Shafizadeh, E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+as+a+model+for+human+disease%5BTitle%5D)+AND+%22Front+Biosci%22%5BJournal%5D)">The zebrafish as a model for human disease.</a> Front Biosci. 7, d1439-d1453 (2002).</li>
<li>Stachura, D. L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+dissection+of+zebrafish+hematopoiesis%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 101, 75-110 (2011).</li>
<li>Jagadeeswaran, P., Sheehan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+blood+coagulation+in+the+zebrafish%5BTitle%5D)+AND+%22Blood+Cells+Mol+Dis%22%5BJournal%5D)">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol Dis. 25 (3-4), 239-249 (1999).</li>
<li>Jagadeeswaran, P., Sheehan, J. P., Craig, F. E., Troyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+zebrafish+thrombocytes%5BTitle%5D)+AND+%22Br+J+Haematol%22%5BJournal%5D)">Identification and characterization of zebrafish thrombocytes.</a> Br J Haematol. 107 (4), 731-738 (1999).</li>
<li>Lieschke, G. J., Oates, A. C., Crowhurst, M. O., Ward, A. C., Layton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphologic+and+functional+characterization+of+granulocytes+and+macrophages+in+embryonic+and+adult%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult.</a> Blood. 98 (10), 3087-3096 (2001).</li>
<li>Iwanami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+as+a+model+for+understanding+the+evolution+of+the+vertebrate+immune+system+and+human+primary+immunodeficiency%5BTitle%5D)+AND+%22Exp+Hematol%22%5BJournal%5D)">Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency.</a> Exp Hematol. 42 (8), 697-706 (2014).</li>
<li>Babin, P. J., Goizet, C., Raldua, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+models+of+human+motor+neuron+diseases%3A+advantages+and+limitations%5BTitle%5D)+AND+%22Prog+Neurobiol%22%5BJournal%5D)">Zebrafish models of human motor neuron diseases: advantages and limitations.</a> Prog Neurobiol. 118, 36-58 (2014).</li>
<li>Seth, A., Stemple, D. L., Barroso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+emerging+use+of+zebrafish+to+model+metabolic+disease%5BTitle%5D)+AND+%22Dis+Mod+Mech%22%5BJournal%5D)">The emerging use of zebrafish to model metabolic disease.</a> Dis Mod Mech. 6 (5), 1080-1088 (2013).</li>
<li>Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostasis%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis.</a> Zebrafish. 7 (2), 205-213 (2010).</li>
<li>Moss, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+the+pancreas+in+adult+zebrafish%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Regeneration of the pancreas in adult zebrafish.</a> Diabetes. 58 (8), 1844-1851 (2009).</li>
<li>Oka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity%5BTitle%5D)+AND+%22BMC+Physiol%22%5BJournal%5D)">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC Physiol. 10 (21), (2010).</li>
<li>Chu, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overexpression+of+Akt1+enhances+adipogenesis+and+leads+to+lipoma+formation+in+zebrafish%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.</a> PLoS One. 7 (5), e36474(2012).</li>
<li>Velasco-Santamaría, Y. M., Korsgaard, B., Madsen, S. S., Bjerregaard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bezafibrate%2C+a+lipid-lowering+pharmaceutical%2C+as+a+potential+endocrine+disruptor+in+male+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22Aquat+Toxicol%22%5BJournal%5D)">Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio).</a> Aquat Toxicol. 105 (1-2), 107-118 (2011).</li>
<li>Zang, L., Shimada, Y., Nishimura, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel%2C+reliable+method+for+repeated+blood+collection+from+aquarium+fish%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">A novel, reliable method for repeated blood collection from aquarium fish.</a> Zebrafish. 10 (3), 425-432 (2013).</li>
<li>Carmichael, C., Westerfield, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryopreservation+and+in+vitro+fertilization+at+the+zebrafish+international+resource+center%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Cryopreservation and in vitro fertilization at the zebrafish international resource center.</a> Methods Mol Biol. 546, 45-65 (2009).</li>
<li>Thorson, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+partitioning+of+body+water+in+Osteichthyes%3A+phylogenetic+and+ecological+implications+in+aquatic+vertebrates%5BTitle%5D)+AND+%22Biol+Bull-US%22%5BJournal%5D)">The partitioning of body water in Osteichthyes: phylogenetic and ecological implications in aquatic vertebrates.</a> Biol Bull-US. 120, 238-254 (1961).</li>
<li>Conte, F. P., Wagner, H. H., Harris, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+blood+volume+in+the+fish+%28Salmo+gairdneri+gairdneri%29%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Measurement of blood volume in the fish (Salmo gairdneri gairdneri).</a> Am J Physiol. 205, 533-540 (1963).</li>
<li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood%2C+including+routes+and+volumes%5BTitle%5D)+AND+%22J+Appl+Toxicol%22%5BJournal%5D)">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J Appl Toxicol. 21 (1), 15-23 (2001).</li>
<li>Nahas, K., Provost, J. -P., Baneux, P. H., Rabemampianina, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+acute+blood+removal+via+the+sublingual+vein+on+haematological+and+clinical+parameters+in+Sprague-Dawley+rats%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Effects of acute blood removal via the sublingual vein on haematological and clinical parameters in Sprague-Dawley rats.</a> Lab Anim. 34 (4), 362-371 (2000).</li>
<li>Curado, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+targeted+cell+ablation+in+zebrafish%3A+a+new+tool+for+regeneration+studies%5BTitle%5D)+AND+%22DevDyn%22%5BJournal%5D)">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> DevDyn. 236 (4), 1025-1035 (2007).</li>
<li>Andersson, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenosine+signaling+promotes+regeneration+of+pancreatic+beta+cells+in+vivo%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">Adenosine signaling promotes regeneration of pancreatic beta cells in vivo.</a> Cell Metab. 15 (6), 885-894 (2012).</li>
<li>Hiramitsu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eriocitrin+ameliorates+diet-induced+hepatic+steatosis+with+activation+of+mitochondrial+biogenesis%5BTitle%5D)+AND+%22Sci+Rep-UK%22%5BJournal%5D)">Eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.</a> Sci Rep-UK. 4, 3708(2014).</li>
<li>Zang, L., Shimada, Y., Kawajiri, J., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Yuzu+%28Citrus+junos+Siebold+ex+Tanaka%29+peel+on+the+diet-induced+obesity+in+a+zebrafish+model%5BTitle%5D)+AND+%22J+Funct+Foods%22%5BJournal%5D)">Effects of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet-induced obesity in a zebrafish model.</a> J Funct Foods. 10, 499-510 (2014).</li>
<li>Schlegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+non-alcoholic+fatty+liver+disease+with+zebrafish%3A+a+confluence+of+optics%2C+genetics%2C+and+physiology%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">Studying non-alcoholic fatty liver disease with zebrafish: a confluence of optics, genetics, and physiology.</a> Cell Mol Life Sci. 69 (23), 3953-3961 (2012).</li>
<li>Stoletov, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vascular+lipid+accumulation%2C+lipoprotein+oxidation%2C+and+macrophage+lipid+uptake+in+hypercholesterolemic+zebrafish%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.</a> Circ Res. 104 (8), 952-960 (2009).</li>
<li>Thomas, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nutrient+balance+and+energy+expenditure+during+ad+libitum+feeding+of+high-fat+and+high-carbohydrate+diets+in+humans%5BTitle%5D)+AND+%22Am+J+Clin+Nutr%22%5BJournal%5D)">Nutrient balance and energy expenditure during ad libitum feeding of high-fat and high-carbohydrate diets in humans.</a> Am J Clin Nutr. 55 (5), 934-942 (1992).</li>
</ol>

The authors have nothing to disclose.

ここでは、直列大人のゼブラフィッシュから血液を取得するための詳細なプロトコールを提示します。この方法は、実施するのは簡単であると私たちは日常的に研究室で使用します。この採血方法は、ゼブラフィッシュの背側大動脈にガラスキャピラリ針の挿入に基づいています。この手順の間、それは背側大動脈を検索するための基準であるため、脊椎を切除しないように注意することが?...

その臓器や遺伝学は人間1,2と同様であるため、ゼブラフィッシュは、ヒト疾患の貴重なモデルとして増加し人気を集めています。発生生物学の分野では、多くの研究は、ゼブラフィッシュとヒトのショーは造血3、止血4,5、および骨髄造血6の類似性をマークしていることを実証しました。大人のゼブラフィッシュはまた、このモデル生物を共有ので、ヒトの疾患において破壊と共通の経路を免疫学7、神経変性8および肥満関連疾患9を研究するために使用されています。肥満及び肥満関連疾患（糖尿病、脂肪肝と非アルコール性脂肪肝炎およびアテローム性動脈硬化症）のために、ゼブラフィッシュ血糖および脂質のレベルは完全にいくつかのトランスジェニックおよび食餌性肥満モデル10-13に研究されてきました。 個々の動物からの反復採血は動物の使用量を削減し、12月になります個人間の違いをrease。しかし、繰り返しサンプルの収集は、その比較的少量の血液量と簡単にアクセス血管の欠如のようなゼブラフィッシュのような小動物では技術的に困難です。これらの方法は、自分の欠点を持っているが、ゼブラフィッシュからの1回の血液採取のためのいくつかの方法は、組織損傷および制限された血液量に関連する、致死性を含む、開発されています。例えば、1〜5μlの血液は背側大動脈5の領域の長さは0.3程度センチ横切開から収穫することができます。胸ガードルを切断することにより、はさみで断頭は、5〜10μlの血液10を収集することができます 。別の便利な採血方法は尾切除14です。心臓穿刺は、同じ魚から繰り返し採血のための1つの潜在的な代替方法ですが、この手順で取得した、非常に少量（約50 NL）はperforすることができます分析の数を制限11のmed。したがって、新しいプロトコルは、この生物はヒト疾患のための標準的なモデル生物であることをするために必要な重要な進歩となるであろう繰り返さ非致死性の血液サンプリングを、有効にするために必要とされています。この技術は、薬理学的応答、分子診断のためのバイオマーカー、予後の決意の発見、ならびに代謝性疾患、変性疾患および悪性疾患のいくつかの種類のような様々な疾患の監視を試験することを可能にします。 したがって、我々は、ゼブラフィッシュシリアル15から血液を得るための低侵襲的方法を開発しました。ここでは、視覚的に手順を示し、この技術の詳細なプロトコルを提供します。空腹時血糖、ヘモグロビンを含む様々なパラメータに基づいて、正常値をこの方法を用いて、そして健康な成人のゼブラフィッシュの血液中の脂質を評価しました。さらに、我々はまた、この方法は、mで一連の試料を必要とする研究に適しているかどうかを評価しましたオーバーフィード実験中血糖値の時間変化をonitoring。

<table><tbody><tr><td>Glass capillaries with filament</td><td>Narishige</td><td>GD-1</td><td>1.0 mm outer diameter.</td></tr><tr><td>Needle puller</td><td>Narishige</td><td>PC-10</td><td>To produce the needles</td></tr><tr><td>Heparin</td><td>Wako Pure Chemical Industries</td><td>081-00136</td><td>For heparinization</td></tr><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td>For blood collection</td></tr><tr><td>Bulb dispenser</td><td>Drummond</td><td>1-000-9000</td><td>For blood collection</td></tr><tr><td>2-phenoxyethanol</td><td>Wako Pure Chemical Industries</td><td>163-12075</td><td>For anesthetizing the fish</td></tr><tr><td>DRI-CHEM3500V</td><td>Fujifilm</td><td>-</td><td>For hemoglobin measurement</td></tr><tr><td>DRI-CHEM Slides</td><td>Fujifilm</td><td>Hb-WII</td><td>For hemoglobin measurement</td></tr><tr><td>Glutest Neo Super</td><td>Sanwa Kagaku Kenkyusho</td><td>-</td><td>For bood glucose measurement</td></tr><tr><td>Wako L-type TG kit</td><td>Wako Pure Chemical Industries</td><td>464-44201</td><td>For TG measurement</td></tr><tr><td>Wako L-type CHO kit</td><td>Wako Pure Chemical Industries</td><td>460-44301</td><td>For total cholesterol measurement</td></tr><tr><td>Parafilm M</td><td>Alcan Packaging</td><td>PM996</td><td>To expel the blood on</td></tr></tbody></table>

repeated blood collection for blood tests in adult zebrafish

反復採血は、動物実験で実行される最も一般的な技術の一つです。しかしながら、ゼブラフィッシュからの血液採取のための非致死プロトコルが確立されていません。ゼブラフィッシュからの採血のための従来の方法は、横切開、断頭とテールアブレーションとして、致死的です。したがって、私たちはここで小説「繰り返し」採血方法、および現在のこの手順を概説し、詳細なプロトコルを開発しました。この方法は、低侵襲性であるため、同じ個体から繰り返し採血を可能にする、ゼブラフィッシュのための非常に低い死亡率（2.3％）が得られます。採血の最大量は、魚の体重に依存します。間隔で繰り返し採血用のボリュームが血中ヘモグロビンの測定により評価した2週間毎に、体重の≤0.4％毎週または≤1％であるべきです。さらに、ヘモグロビン、空腹時血糖、血漿トリアシルグリセロール（TG）、総C男性と女性の大人のゼブラフィッシュにおけるholesterolレベルを測定しました。また、食餌性肥満におけるグルコース代謝の調節不全を調査するためにこの方法を適用しました。この血液採取法は、グルコースおよび脂質代謝およびヒト疾患モデル生物としてゼブラフィッシュの使用を増加させる血液学の研究を含む多くのアプリケーションを可能にします。

この血液採取法は、ゼブラフィッシュ（&lt;1 mmの穿刺;図1J）に最小の損傷を引き起こし、2.3％の非常に低い死亡率をもたらします。我々は、単一の魚から収集し、その体重（ 図3）への関係を評価することができた血の最大音量を検討しました。我々は、収集した最大血液量が直線的に体重（R = 0.813）と相関することを見出しました。個々の魚（体重= 1.07...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

アダルトゼブラフィッシュにおける血液検査のための反復採血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

35.0K ビュー.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

ビデオ: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

In Vivoモデル システムとしてゼブラフィッシュを用いた DNA 複製タイミングのプロファイリング

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

遺伝学

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 ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

アダルトゼブラフィッシュにおける眼窩インジェクション

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

生物学

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

発達中のゼブラフィッシュにおける変化した血液形成を検出し定量するためのフローサイトメトリーの使用

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

発生生物学

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

ゼブラフィッシュモデルの筋肉再生を調べる

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

アダルトゼブラフィッシュへの腹腔内注射

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

成体ゼブラフィッシュにおける静脈内注射の最適化

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

がん研究

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

大人のゼブラフィッシュの生化学的解析のための採血

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavagingアダルトゼブラフィッシュ

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

アダルトゼブラフィッシュにおける血液検査のための反復採血

要約

さらに動画を探す

In Vivoモデルシステムとしてゼブラフィッシュを用いた DNA 複製タイミングのプロファイリング

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

アダルトゼブラフィッシュにおける眼窩インジェクション

発達中のゼブラフィッシュにおける変化した血液形成を検出し定量するためのフローサイトメトリーの使用

ゼブラフィッシュモデルの筋肉再生を調べる

アダルトゼブラフィッシュへの腹腔内注射

成体ゼブラフィッシュにおける静脈内注射の最適化

大人のゼブラフィッシュの生化学的解析のための採血

Gavagingアダルトゼブラフィッシュ

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

アダルトゼブラフィッシュにおける血液検査のための反復採血

要約

さらに動画を探す

In Vivoモデル システムとしてゼブラフィッシュを用いた DNA 複製タイミングのプロファイリング

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

アダルトゼブラフィッシュにおける眼窩インジェクション

発達中のゼブラフィッシュにおける変化した血液形成を検出し定量するためのフローサイトメトリーの使用

ゼブラフィッシュモデルの筋肉再生を調べる

アダルトゼブラフィッシュへの腹腔内注射

成体ゼブラフィッシュにおける静脈内注射の最適化

大人のゼブラフィッシュの生化学的解析のための採血

Gavagingアダルトゼブラフィッシュ

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

In Vivoモデルシステムとしてゼブラフィッシュを用いた DNA 複製タイミングのプロファイリング