O microscópio confocal foi comprado com fundos de o US National Science Foundation (NSF), grant #DBI-0722569. A pesquisa foi apoiada por os E.U. institutos de saúde nacional/Instituto Nacional de General Medical Ciências (NIH/NIGMS) concessão #GM085290 e E.U. departamento de defesa (DOD) grant #W81XWH-16-1-0466 atribuído a R.M. Brewster. E. Vital foi apoiada por uma concessão a UMBC do Howard Hughes Medical Institute, através da pre-faculdade e programa de educação científica de graduação, conceder #52008090. S.P. Brown foi apoiado por um departamento E.U. de educação GAANN Fellowship, uma bolsa de pós-graduação Meyerhoff financiado pela concessão de NIH/NIGMS, #GM055036 e um trabalho de assistente de investigação financiado pelos E.U. DOD grant #W81XWH-16-1-0466.

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+chemical+modulation+of+spastin-dependent+axon+outgrowth+in+zebrafish+embryos+indicates+a+role+for+impaired+microtubule+dynamics+in+hereditary+spastic+paraplegia.">Genetic and chemical modulation of spastin-dependent axon outgrowth in zebrafish embryos indicates a role for impaired microtubule dynamics in hereditary spastic paraplegia.</a> Dis Model Mech. 3 (11-12), 743-751 (2010).</li><li>Yoo, S. K., Lam, P. -. Y., Eichelberg, M. R., Zasadil, L., Bement, W. M., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microtubules+in+neutrophil+polarity+and+migration+in+live+zebrafish.">The role of microtubules in neutrophil polarity and migration in live zebrafish.</a> J Cell Sci. 125 (23), 5702-5710 (2012).</li><li>Andersen, E. F., Halloran, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+movements+in+vivo+correlate+with+specific+neurite+formation+downstream+of+LIM+homeodomain+transcription+factor+activity.">Centrosome movements in vivo correlate with specific neurite formation downstream of LIM homeodomain transcription factor activity.</a> Development. 139 (19), 3590-3599 (2012).</li><li>Lee, S. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+microtubule+and+actin+cytoskeleton+in+zebrafish+epiboly.">Dynamic regulation of the microtubule and actin cytoskeleton in zebrafish epiboly.</a> Biochem Biophys Res Commun. 452 (1), 1-7 (2014).</li><li>Bulinski, J. C., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+and+post-translational+modification+of+microtubules+during+cellular+morphogenesis.">Stabilization and post-translational modification of microtubules during cellular morphogenesis.</a> BioEssays. 13 (6), 285-293 (1991).</li><li>Magiera, M. M., Janke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+16+-+Investigating+Tubulin+Posttranslational+Modifications+with+Specific+Antibodies.">Chapter 16 - Investigating Tubulin Posttranslational Modifications with Specific Antibodies.</a> Methods Cell Biol. 115, 247-267 (2013).</li><li>Hong, E., Jayachandran, P., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+polarity+protein+Pard3+is+required+for+centrosome+positioning+during+neurulation.">The polarity protein Pard3 is required for centrosome positioning during neurulation.</a> Dev Biol. 341 (2), 335-345 (2010).</li><li>Westermann, S., Weber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+regulate+microtubule+function.">Post-translational modifications regulate microtubule function.</a> Nat Rev Mol Cell Biol. 4 (12), 938-948 (2003).</li><li>Jayachandran, P., Olmo, V. N., 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=Microtubule-associated+protein+1b+is+required+for+shaping+the+neural+tube.">Microtubule-associated protein 1b is required for shaping the neural tube.</a> Neural Develop. 11, 1 (2016).</li><li>Nam, S. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Tau,+a+microtubule+associated+protein,+in+Drosophila+photoreceptor+morphogenesis.">Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis.</a> Genes N Y N 2000. 54 (11), 553-561 (2016).</li><li>Abal, M., Piel, M., Bouckson-Castaing, V., Mogensen, M., Sibarita, J. -. B., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+release+from+the+centrosome+in+migrating+cells.">Microtubule release from the centrosome in migrating cells.</a> J Cell Biol. 159 (5), 731-737 (2002).</li><li>Delgehyr, N., Sillibourne, J., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+nucleation+and+anchoring+at+the+centrosome+are+independent+processes+linked+by+ninein+function.">Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function.</a> J Cell Sci. 118 (8), 1565-1575 (2005).</li><li>Manning, J. A., Lewis, M., Koblar, S. A., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+function+for+the+centrosomal+protein+NEDD1+in+zebrafish+development.">An essential function for the centrosomal protein NEDD1 in zebrafish development.</a> Cell Death Differ. 17 (8), 1302-1314 (2010).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn Off Publ Am Assoc Anat. 203 (3), 253-310 (1995).</li><li>Beck, A. P., Watt, R. 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Z-functions - ImageJ Available from: <a target="_blank" href="https://imagej.net/Z-functions">https://imagej.net/Z-functions</a> (2017)</li></ol>

Os autores não têm nada para divulgar.

Atualmente existem muitos métodos de imagem dinâmica de MT no início do desenvolvimento do zebrafish, variando de imagens ao vivo de moléculas marcou a immunolabeling de fixa tecido11,12,13,14. Embora MTs em uma única célula podem existir nos Estados dinâmicos ou estáveis, epitelização é um processo no qual o MTs são estabilizadas progressivamente ao longo do tempo. Usando marcadore...

Microtúbulos (MTs) são polímeros de α - e β-tubulina que reúne numa protofilamentos lineares, vários dos quais se combinam para formar um tubo oco1,2. MTs são estruturas polarizadas, com rápido crescimento além de extremidades e crescimento lento menos extremidades ancorados na centrossoma ou outros microtubule-organização centro (MTOC)3. De novo Formação de MT é iniciada pela nucleação no complexo anel γ-tubulina (γ-TURC), que fornece um modelo para montagem de MT4. Em qualquer determinada célula, duas populações de MTs distinguem-se a curva sobre a taxas diferentes. MTs dinâmicos explorar seu ambiente celular alternando entre fases de crescimento e retração em um processo conhecido como instabilidade dinâmica5. Ao contrário de dinâmicas MTs, MTs estáveis são não-crescimento e tem uma meia-vida mais longa que a dinâmica MTs6.

Décadas de pesquisa em biologia celular tem fornecido um sofisticado conjunto de ferramentas para estudar a função e estrutura de MT e resultou em um grande corpo de conhecimento sobre esses elementos do citoesqueleto. Por exemplo, MTs desempenham um papel central no estabelecimento e na manutenção da polaridade da célula, que é atribuível não só a sua polaridade intrínseca, mas também para a distribuição diferencial subcellular de estábulo versus dinâmico MTs7, 8. em contraste, muito menos é compreendido sobre arquitetura de MT e função em ambientes (3D) tridimensionais mais complexos, tais como os embriões vertebrados, em parte devido ao desafio de citoesqueleto MT em alta resolução de imagem. Apesar desta limitação, a geração recente de transgênico GFP-expressando as linhas esse rótulo MTs ou expressão transiente de marcadores de MT marcados fluorescentemente aumentou nossa compreensão das mudanças dinâmicas que se submetem a MTs e seus celulares e papel do desenvolvimento no embrião zebrafish. Toda a rede de MT pode ser fotografada em linhas transgénicas na qual tubulina ou é diretamente rotulados polímeros9 ou tubulina indiretamente são etiquetados usando proteínas associadas MT Doublecortin-como-quinase (Dclk) ou Ensconsin (EMTB)10, 11. Outras linhas (e construções) foram geradas que permitem avaliação de polaridade intrínseca de MT por rotulagem especificamente MT além termina ou centrossoma ancorada menos termina11,12,13, 14. o poder dessas ferramentas encontra-se na capacidade de estudar a dinâmica de MT em ao vivo, organismos em desenvolvimento. Tais estudos têm revelado, por exemplo, a distribuição espacial e dinâmica de MTs em populações de células específicas, a orientação de mitótica fusos em tecidos submetidos a morfogênese (um indicador do plano de divisão celular), a polaridade do polímero MT no que se refere aos processos, tais como alongamento celular e migração e a taxa de crescimento de MT determinada pelo cometa velocidade9,13,15. A limitação dessas ferramentas é que eles não prontamente qualquer discriminação entre as populações de MT estáveis e dinâmicas.

Desenho da literatura biologia celular rico, immunolabeling métodos de imagem estáveis e dinâmicos MTs no zebrafish embrião são descritos aqui, que são complementares para o uso de linhas transgénicas. O uso generalizado de tais métodos immunolabeling no zebrafish tem sido um pouco dificultado pela dificuldade em preservar a integridade de MT durante o procedimento de fixação. 1 protocolo descreve um método otimizado para immunolabeling total, dinâmico, e MTs estáveis em secções do rombencéfalo zebrafish desenvolvimento transversais. Além disso, um método simples usando comercialmente software disponível é descrita para quantificar estas populações de MT. MTs estáveis são distintos dos MTs dinâmicos com base em várias modificações borne-translational do α-tubulina, tais como a acetilação e detyrosination, que se acumulam na MTs estáveis ao longo do tempo16,17. No embrião zebrafish, acetilação ocorre em ciliares e axonal MTs mas não em interfase estável MTs18, limitando a utilidade deste marcador a um subconjunto de MTs estabilizados. Em contraste, detyrosination parece ocorrer em todos os MTs estáveis em zebrafish embrião18. Esta modificação pós-traducional expõe o ácido glutâmico carboxy-terminal de α-tubulina (detyrosinated tubulina)18 e pode ser detectada usando anti-Glu-tubulina19. Embora detyrosination ocorre preferencialmente na MTs estáveis, evidência experimental indica que esta modificação pós-traducional é um resultado de, ao invés de uma causa de, estabilidade de MT16. Distingue-se a população MT recíproca, composta por MTs dinâmicos, usando um anticorpo, anti-Tyr-tubulina, que reconhece especificamente a forma tyrosinated de α-tubulina19. Após immunolabeling com estes marcadores e imagem latente confocal, análise quantitativa de MTs (comprimento, número e abundância relativa) pode ser realizada em regiões definidas do tubo neural em desenvolvimento. Aqui, é fornecido um método simplificado para realizar esta análise, utilizando o software de processamento de imagem em 3D. Esse método pode ser aplicado para responder a perguntas sobre morfogênese e o estabelecimento ou a maturação de células polaridade20. Com efeito, a elaboração de matrizes polarizadas de estáveis MTs acompanha muitos eventos do desenvolvimento, incluindo fotorreceptoras morfogênese21, epitelização das células no desenvolvimento do tubo neural18 e axônio formação8.

2 protocolo descreve uma adaptação na vivo de um ensaio de biologia de pilha para analisar MTs durante sua montagem fase (nucleação/fixação e crescimento)22,23. MTs nascentes são nucleated na centrossoma e posteriormente ancoradas apêndices subdistal da mãe centriole23. É descrito um método para analisar a nascente MT rebrota após despolimerização. Este protocolo fornece detalhes sobre o tratamento de nocodazole para depolymerize MTs, o processo de esmaecimento de droga e o período de recuperação pós-tratamento. Re-crescimento de MT é monitorado em intervalos regularesesmaecimento de post s por immunolabeling com marcadores para MTs totais (anti β-tubulina) ao lado de marcadores para a centrossoma (anti γ-tubulina) e núcleo (4', 6-diamidino-2-phenylindole (DAPI)), de acordo com os procedimentos gerais descritos no protocolo 1. A etapa de despolimerização MT do presente protocolo é essencial, como permite a avaliação do crescimento de MT de novo , em vez de extensão de MTs preexistentes. Esta técnica é, portanto, distinta de outros procedimentos publicados para medir as taxas de crescimento de MT (na ausência de despolimerização) usando um marcador de ponta mais como fim proteína 3 fundido a proteína verde fluorescente (EB3-GFP), conforme mostrado no Tran et al., 201211. Além disso, este ensaio é particularmente útil para a análise de embriões com defeito na montagem de novo MT, tais como os mutantes NEDD1 relatados anteriormente, em que o recrutamento de γ-tubulina para a centrossoma é prejudicado, resultando em incompleta formação do tubo neural e defeitos neuronal24.

<table border="0" cellpadding="0" cellspacing="0" width="916">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="140">
			<td height="140" style="height:140px;width:207px;">Low Melting Point Agarose</td>
			<td style="width:177px;">IBI scientific</td>
			<td style="width:159px;">IB70050</td>
			<td style="width:373px;">Only used for embedding. 
			 
			Prepare 4% LMP agarose 
			by heating a solution of&#160; 4 grams LMP agarose per 100 ml 1X TBS in a microwave 
			until polymerized. Keep warm on a 50 &#730;C hot plate with the cap secured</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Agarose</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">Used to treat petridishes.&#160;&#160; Prepare 1% agarose 
			by heating a solution of&#160; 1 gram agarose per 100 ml 1X embryo medium in a microwave 
			until polymerized.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Nocodazole</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">487928-10MG</td>
			<td style="width:373px;">Make stock by dissolving entire bottle of powder in distilled water and aliquoting into 500ul aliquots.&#160; Store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">8% Formaldehyde</td>
			<td style="width:177px;">Electron Microscopy Sciences</td>
			<td style="width:159px;">157-8-100</td>
			<td style="width:373px;">Aliquot and store at -20 &#730;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Kpipes</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P7643</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">NaCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">S7653</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Tris-HCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">T3253-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">KCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P9333-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">CaCl2&#183;2H2O</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">C5080</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">NP-40</td>
			<td style="width:177px;">American Bioanalyticals</td>
			<td style="width:159px;">AB01424</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">EGTA</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">E3889-25G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">MgCl2</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">M2670-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Normal Goat Serum</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">S26-100ml</td>
			<td style="width:373px;">Aliquot and store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Bovine serum albumin (BSA)</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">BP1605</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Triton-x</td>
			<td style="width:177px;">American Bioanalyticals</td>
			<td style="width:159px;">AB02025</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Pronase E (non-specific Protease from Streptomyces griseus)</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P5147-1G</td>
			<td style="width:373px;">make stock solution by diluting 10mg per ml of ddH2O. Aliquot in 1ml aliquots and store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Anti-Fade mounting medium</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:159px;">P10144</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">DAPI</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:159px;">D1306</td>
			<td style="width:373px;">Prepare 50 ml DAPI solution by first combining 1 &#181;l DAPI stock&#160; with 1 ml sterile water and then adding the DAPI/water mix to 49 ml 1X TBS; store in foil at 4 &#730;C for months.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:207px;">Mouse anti-&#946;-tubulin</td>
			<td style="width:177px;">Developmental studies Hybridoma Bank</td>
			<td style="width:159px;">E7</td>
			<td style="width:373px;">1/200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Rabbit anti-&#947;-tubulin</td>
			<td style="width:177px;">Genetex</td>
			<td style="width:159px;">GTX113286</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Rabbit anti-&#945;-tubulin</td>
			<td style="width:177px;">Genetex</td>
			<td style="width:159px;">GTX108784</td>
			<td style="width:373px;">1/1000*</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Rabbit anti-detyrosinated-tubulin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">AB3201</td>
			<td style="width:373px;">1/200-1/1000*&#160; Titrate antibody with first use of new lot.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Rabbit anti-tyrosinated-tubulin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">ABT171</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Mouse anti-centrin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">04-1624</td>
			<td style="width:373px;">1/1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 488 anti-rabbit</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11008</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 594 anti-rabbit</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11012</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 594 anti-mouse</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11005</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 488 anti-mouse</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11001</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Vibratome</td>
			<td style="width:177px;">Vibratome</td>
			<td style="width:159px;">1500</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Forceps</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td style="width:159px;">555227F</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">100 mm petri dish</td>
			<td style="width:177px;">Cell treat</td>
			<td style="width:159px;">229693</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">35 mm petri dish</td>
			<td style="width:177px;">Cell treat</td>
			<td style="width:159px;">229638</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">50 ml falcon tube</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-432-22</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Woven nylon mesh 70 um</td>
			<td style="width:177px;">Amazon.com</td>
			<td style="width:159px;">B0043D1SZG&#160;</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Micropipette</td>
			<td style="width:177px;">Gilson</td>
			<td style="width:159px;">F123602</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Glass pipette</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">NC-999363-9</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Aquarium sealant</td>
			<td style="width:177px;">Amazon.com, by MarineLand</td>
			<td style="width:159px;">Silicone Sealer 1 oz (Tube)&#160;</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Ring stand</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-675BO</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Microbore PTFE Tubing, 0.022&#34;ID</td>
			<td style="width:177px;">Cole-Parmer</td>
			<td style="width:159px;">WU-06417-21</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Modeling clay</td>
			<td style="width:177px;">Amazon.com</td>
			<td style="width:159px;">Sargent Art 22-4000</td>
			<td style="width:373px;">Any wax or oil based non-toxic modeling clay will suffice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Clamp</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">02-215-466</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">60ml syringe</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-820-11</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:207px;">Embryo medium (E3)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">34.8 g NaCl 
			1.6 g KCl 
			5.8 g CaCl2&#183;2H2O 
			9.78 g MgCl2&#183;6H2O 
			 
			To prepare a 60X stock, dissolve the ingredients in H2O, to a final volume of 2 L. Adjust the pH to 7.2 with NaOH. Autoclave. To prepare 1X medium, dilute 16.5 mL of the 60X stock to 1 L.&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Blocking Solution</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">50 ml TBS-NP-40 
			2.5 ml normal goat serum 
			1 g BSA 
			625 &#181;l Triton-X</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">TBS-NP-40 (pH 7.6)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">155 mM NaCl 
			10 mM Tris HCl 
			0.1% NP-40</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">2x MAB (pH6.4)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">160 mM KPIPES 
			10 mM EGTA 
			2 mM MgCl2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Commercial 3-D Image processing Software</td>
			<td style="width:177px;">PerkinElmer</td>
			<td style="width:159px;">Volocity (V 6.2)</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Dry block heater&#160;</td>
			<td style="width:177px;">VWR</td>
			<td style="width:159px;">12621-108</td>
			<td style="width:373px;">Used as a hot plate to melt agarose in Protocol 1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Dissecting Microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:159px;">MZ12</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Confocal Microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:159px;">SP5</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Flat embedding mold</td>
			<td style="width:177px;">emsdiasum.com</td>
			<td style="width:159px;">BEEM 70904-01</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Public domain image processing software</td>
			<td style="width:177px;">NIH</td>
			<td style="width:159px;">ImageJ (V 1.5)</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:532px;">* Success varies by lot number</td>
		</tr>
	</tbody>
</table>

use of immunolabeling to analyze stable, dynamic, and nascent microtubules in the zebrafish embryo

Microtúbulos (MTs) são estruturas dinâmicas e frágeis que são desafiadores para imagem na vivo, particularmente em embriões de vertebrados. Immunolabeling métodos são descritos aqui para analisar populações distintas de MTs em tubo neural em desenvolvimento do embrião do zebrafish. Enquanto o foco estiver em tecido neural, esta metodologia é amplamente aplicável para outros tecidos. Os procedimentos são otimizados para cedo aos embriões de meados-somitogenesis-estágio (1 somite para 12 somitas), no entanto podem ser adaptadas a uma gama de outras fases com relativamente pequenos ajustes. O primeiro protocolo fornece um método para avaliar a distribuição espacial da MTs estáveis e dinâmicos e realizar uma análise quantitativa dessas populações com software de processamento de imagem. Essa abordagem complementa as ferramentas existentes para imagem microtubule dinâmica e distribuição em tempo real, usando linhas transgénicas ou expressão transiente de construções etiquetadas. Com efeito, tais ferramentas são muito úteis, porém eles não facilmente distinguir entre MTs dinâmicos e estáveis. A capacidade de imagem e analisar essas populações distintas do microtubule tem implicações importantes para os mecanismos de compreensão subjacente a polarização celular e morfogênese. O segundo protocolo descreve uma técnica para analisar especificamente o nascentes MTs. Isso é realizado através da captura as propriedades de crescimento de novo do MTs ao longo do tempo, seguindo microtubule despolimerização com a drogas nocodazole e um período de recuperação após o fracasso de drogas. Esta técnica ainda não tiver sido aplicada ao estudo da MTs em embriões de peixe-zebra, mas é um ensaio valioso para investigar a função no vivo das proteínas implicado na montagem de microtúbulos.

Análise de MTs estáveis e dinâmicos usando immunolabeling No protocolo n º 1, a distribuição das subpopulações de MT durante o início (quilha neural) e os estágios finais (haste neural) do desenvolvimento do tubo neural são revelados, usando Glu-tubulina e Tyr-tubulina como marcadores para MTs estáveis e dinâmicos, respectivamente. MTs dinâmicas predominam o rombencéfalo na fase neural da quilha (4-5 somitas) (Figura...

Watch this Scientific Journal Video about Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo at JoVE.com

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Immunolabeling métodos para analisar populações distintas de microtúbulos no cérebro em desenvolvimento zebrafish são descritos aqui, que são amplamente aplicáveis a outros tecidos. O primeiro protocolo descreve um método otimizado para immunolabeling estável e dinâmica de microtúbulos. O segundo protocolo fornece um método para a imagem e quantificar microtúbulos nascentes especificamente.

Uso de Immunolabeling para analisar os microtúbulos estáveis, dinâmicos e emergentes no embrião Zebrafish

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

use-immunolabeling-to-analyze-stable-dynamic-nascent-microtubules

Research

JoVE Journal

Developmental Biology

8.2K visualizações.  University of Maryland, Baltimore County. Immunolabeling métodos para analisar populações distintas de microtúbulos no cérebro em desenvolvimento zebrafish são descritos aqui, que são amplamente aplicáveis a outros tecidos. O primeiro protocolo descreve um método otimizado para immunolabeling estável e dinâmica de microtúbulos. O segundo protocolo fornece um método para a imagem e quantificar microtúbulos nascentes especificamente.

Vídeo: Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Produção e Uso de Lentivírus a seletivamente transduzem Primária Oligodendrocyte células precursoras para<em&gt; In Vitro</em&gt; Os ensaios mielinização

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

Biologia do Desenvolvimento

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

Uso do Sistema de Teton para estudar os mecanismos moleculares de Regeneração Zebrafish

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microbead Implantação no embrião de peixe-zebra

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análise de Zebrafish larvas Músculo Esquelético Integrity com Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Imagiologia estruturas celulares no embrião vivo Zebrafish

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Observando mitótico Division e Dinâmica em um embrião de peixe-zebra ao vivo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

Microinjeção de embriões e Eletroporação na cordados<em&gt; Intestinalis Ciona</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

AFM e Microrheology na célula de embrião de Zebrafish gema

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Imobilização de Caenorhabditis elegans , para analisar o transporte intracelular nos neurônios

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

Eficácia do ar despojamento em dois peixes salmonídeos, truta arco-íris (Oncorhynchus Mykiss) e truta (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...