Esta publicação foi financiada pela pesquisa de Horizonte 2020 da União Europeia e o programa de inovação no âmbito do acordo de concessão de Marie Sklodowska-Curie não 642893 (IMPRESS), o escritório de custo (custo de ação FA 1205, AQUAGAMETE) e o centro de excelência em pesquisa- 1476-4/2016/FEKUT.

Todos os procedimentos para trabalhar com enguia europeia descrita neste protocolo foram aprovados pelo Comitê de ética da experimentação do Animal na Universitat Politécnica de València, seguindo as leis espanholas e regulamentos controlando as experiências e procedimentos em animais vivos.
1. peixe de manutenção
<ol><li>Trazer as enguias europeias para uma instalação de pesquisa e colocá-los em um aquário de 200 L com um sistema de recirculação. Uso de termostatos e refrigeradores para manter uma temperatura constante de 20 ° C.</li>
	<li>Mantenha o peixe no escuro para evitar stress22 e sem comida durante o experimento.</li>
	<li>Manter o peixe em água doce durante os 3 primeiros dias. Então mudar 1/3 da água e encha novamente com água do mar todos os dias até atingir uma salinidade de 37,0 ± 0,3 g/L.</li>
</ol>2. tratamento hormonal
Nota: O tratamento hormonal consiste em injeções semanais de gonadotrofina coriônica humana (hCG) durante toda a duração inteira (nove semanas) do experimento.
<ol><li>Preparar o hormônio hCG antecipadamente em uma concentração de 1 IU / µ l diluindo o hormônio em solução salina (0,9% NaCl). Nota: O hormônio pode ser preservado diluído nessa concentração para mais de uma semana a-18 ° C.</li>
	<li>Para anestesiar o peixe, prepare um balde de flexível de 40 L com 5 L de água do sistema.<ol>
<li>Em um balão de 250 mL, dilua 300 mg de benzocaína em 100 mL de etanol a 70%.</li>
		<li>Despeje a benzocaína diluída na caçamba (concentração final 0,36 mM) e misture bem. Isto é para uma concentração final de benzocaína de 60 mg/L.</li>
		<li>Transfira o peixe, individualmente, para a água com benzocaína e aguarde alguns segundos até que a benzocaína entra em vigor e o peixe está devidamente anestesiado. Nota: Para confirmar que o peixe é anestesiado, coloque o peixe em posição supina e verificar que ela permaneça ainda nessa posição.</li>
	</ol>
</li>
	<li>Para administrar o hormônio, pesar o peixe e preparar o hormônio hCG na dose de 1,5 UI/g de peixe, um tubo de plástico de 1,5 mL.<ol>
<li>Encha uma seringa de 1 mL com o hormônio hCG do tubo de plástico.</li>
		<li>Coloque o peixe anestesiado, em posição supina e com a assistência ou a seringa, injetar o hormônio cuidadosamente na área intraperitoneal.</li>
	</ol>
</li>
	<li>Devolver o peixe para o aquário e monitorá-lo até que se recuperou totalmente. Nota: A administração hormonal tem que ser realizados semanalmente durante o experimento. Normalmente, as enguias europeus começam spermating após 6-7 semanas de tratamento hormonal.</li>
</ol>3. esperma amostragem
<ol><li>Para obter as melhores amostras de qualidade, extrair esperma 24 h após a administração hormonal6,23.</li>
	<li>Anestesia o peixe com benzocaína, conforme descrito no passo 2.2.</li>
	<li>Coloque o peixe anestesiado em posição supina, limpe a área genital com um esguicho de água destilada e seque cuidadosamente com papel, para evitar a contaminação de fezes presente na área genital ou ativação acidental esperma pelo contato com água do mar vem as aquários.</li>
	<li>Colocando os dedos de ambos os lados laterais do peixe, massagem cuidadosamente pressionando lateralmente das barbatanas peitorais para a área genital para forçar o esperma para fora.<ol>
<li>Repita a massagem até que não mais esperma sai da abertura genital.</li>
	</ol>
</li>
	<li>Colete o esperma em tubos de plástico de 15 mL usando uma bomba de vácuo.</li>
	<li>Dilua o esperma extraído 01:10 em modificados Tanaka´s extensor solução24 (137 mM NaCl, NaHCO3, em água destilada de 76,2 mM) a 4 ° c. Nota: O esperma na solução extensor deve ser mantido a 4 ° c.</li>
</ol>4. avaliação da qualidade de esperma
<ol><li>Preparar a água do mar artificial13 (em mM: NaCl 354.7, MgCl2 52,4, CaCl2 9.9, Na2SO4 28,2, 9,4 de KCl, em água destilada) com 2% albumina de soro bovino (BSA) (w:v), ajustar o pH a 8.2, osmolalidade mOsm/kg de 1100 e manter é a 4 ° c para evitar o crescimento bacteriano.</li>
	<li>Abra o software para análise de esperma assistida por computador (CASA) e selecione o módulo do esperma de peixe.<ol>
<li>Clique em Propriedades para abrir a configuração do software do sistema. Lá, defina as opções de captura em 60 imagens por segundo. Selecione fase negativa óptica, câmara de Makler e escala de X 10.</li>
		<li>Os valores de análise, selecione peixes como espécie e área de partículas maiores que 2 µm2 e menor que 20 µm2.</li>
		<li>Sobre os parâmetros de velocidade, definida como lenta quando as células mover-se entre 10 e 45 µm/s, definido como médio , quando se deslocam entre 45 e 100 µm/s e definida como rápida quando se movendo mais rápido do que 100 µm/s. Nota: Espermatozoides com mais lento do que 10 µm/s de velocidade foram considerados fixa.</li>
		<li>Selecione os valores progressivos como 80% da retidão (STR) e salvar as propriedades.</li>
		<li>Clique no Campo de captura (uma janela com as imagens ao vivo da câmera será aberta). Nota: O sistema de análise de esperma assistida por computador é formado por um microscópio, uma câmera e um software de análise de imagem.</li>
	</ol>
</li>
	<li>Na câmara de contagem, coloque 4 µ l de água do mar artificial e adicione 0,5 µ l de solução de esperma (esperma diluída em solução de extensor).<ol>
<li>Focar a imagem com o microscópio com ampliação de 10x na fase negativa. 15 s após a ativação, clique em capturar - de vídeo do software de análise de esperma assistida por computador.</li>
		<li>Analise cada amostra em triplicado e selecionadas amostras com uma mobilidade superior a 70% para criopreservação. Nota: É muito importante selecionar apenas espermatozoides de alta qualidade para o sucesso do presente protocolo de criopreservação.</li>
	</ol>
</li>
</ol>5. método de congelamento de esperma
<ol><li>Prepare antecipadamente o nitrogénio (aproximadamente 2,5 L) em um 34 x 34 cm x 30 cm e grossa caixa de isopor de 5 cm. Nota: Manter um nível de nitrogênio líquido de 4-5 cm de altura em todos os momentos.<ol>
<li>Construa uma estrutura flutuante para pre-congelar o esperma. Use 2 pedaços de isopor (20 cm x 5 cm), ligá-los com 2 tubos de plástico e coloque a estrutura sobre o nitrogênio líquido. Essa estrutura precisa flutuar sobre o nitrogênio líquido a uma altura aproximada de 3 cm sobre a superfície (Figura 1).</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56835/56835fig1.jpg"> Figura 1 . Esquemático, desenho da estrutura flutuante usada para pré-congelamento com nitrogênio líquido. A estrutura é composta de duas peças de baixa densidade de isopor de 20 cm x 4 cm x 5 cm conectado com tubos de plástico de 14 cm. As palhetas são colocadas sobre os tubos de plástico a 3 cm sobre o nitrogênio líquido. <a href="//cloudflare.jove.com/files/ftp_upload/56835/56835fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<ol start="2"><li>Preparar a solução de criopreservação misturando esperma, solução de extensor e metanol na proporção de 1:8:1 em plástico de 1,5 mL a tubos e mexa delicadamente.</li>
	<li>Com a ajuda de uma pipeta, adicione 480 µ l da solução de criopreservação nos 500 canudos µ l e se necessário, marcá-los, fechando uma extremidade com argila de modelagem.</li>
	<li>Colocar a palha no dispositivo flutuante a uma altura de 3 cm sobre o nitrogênio líquido por 3 min. Em seguida, coloque a palha para o nitrogênio líquido.</li>
	<li>Após 10-15 min, transfira os canudos em um tanque de armazenamento de nitrogênio líquido usando pinça longa e mantê-los submersos em nitrogênio líquido em todos os momentos. Aqui, o esperma pode ser armazenado indefinidamente.</li>
</ol>6. método de descongelamento
<ol><li>Preparar uma caixa de isopor com nitrogênio líquido, conforme descrito na etapa 5.1 e preparar um banho de água usando um copo de 3 L com água da torneira a 40 ° c.</li>
	<li>Transferi o palitinho com esperma congelado do tanque de armazenamento na caixa de isopor com nitrogênio líquido, usando pinça longa.<ol>
<li>Coloque cada palha em um banho de água a 40 ° c para 13 s.</li>
		<li>Despeje o esperma em um tubo de plástico de 1,5 mL cortando as extremidades fechadas da palha com uma tesoura.</li>
	</ol>
</li>
	<li>Analise a mobilidade do esperma, usando o sistema de análise de esperma assistida por computador, como explicado no passo 4.1.</li>
	<li>Manter 100 µ l de esperma para analisar a viabilidade de espermatozoides com um citômetro de fluxo.</li>
</ol>7. fluxo Cytometry
<ol><li>Use um kit fluorescente contendo iodeto de propidium (PI), que é um composto fluorescente vermelho que mancha os núcleos das células mortas e um membrana-permeant nuclear composto fluorescente, que verde manchas os núcleos das células vivas.<ol>
<li>Prepare a solução de coloração verde fluorescente, diluindo-a partir da solução-mãe (1mm) 01:10 Tanaka´s médio para uma solução de trabalho de 100 µM.</li>
		<li>Não dilua a solução de PI. A solução é a 2,4 mM.</li>
	</ol>
</li>
	<li>Para cada amostra, tomar 50 µ l de esperma fresca ou descongelada e adicione 0,5 µ l de verde fluorescente, manchando a solução de trabalho (concentração final de 1 µM) e 2 µ l da solução de PI (concentração final de 100 µM).</li>
	<li>Incube as amostras contendo os corantes (PI e a coloração verde fluorescente) no escuro por 5 min.<ol>
<li>Diluir as amostras em 500 µ l de solução de Tanaka´s extensor e analisar com o citômetro de fluxo.</li>
	</ol>
</li>
	<li>Ligue o citômetro de fluxo e criar um novo protocolo que contém pelo menos 2 terrenos: SS log log de vs FS e vs FL1 FL3. Nota: Ambos, verde fluorescente iodeto de coloração e propidium pode ser excitado com comprimento de onda visível luz. Quando ligado ao DNA, a emissão de fluorescência máxima destes corantes são 516 nm e 617 nm, respectivamente.<ol>
<li>Ajustar as tensões dos diferentes lasers: SS = 199; FS = 199; FL1 = 377; FL3 = 372</li>
		<li>Configurar o acordo de aquisição configurações para eventos máximos = 5000 ou 15 s (a concentração final da amostra deve ser de aproximadamente 1 milhão de células/mL). Leia a amostra usando um fluxo baixo .</li>
		<li>Selecione o modo de leitura único tubo fixada modo posição.</li>
		<li>Colocar a amostra no número certo de carrossel</li>
		<li>Leia a amostra (pressionando F9) e salvar os dados coletados para um arquivo do excel (pressionando F7) para posterior análise.</li>
	</ol>
</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+sperm+motility+and+factors+affecting+sperm+quality+in+cultured+fish.">The measurement of sperm motility and factors affecting sperm quality in cultured fish.</a> Aquaculture. 234 (1-4), 1-28 (2004).</li><li>Verstegen, J., Iguer-Ouada, M., Onclin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+assisted+semen+analyzers+in+andrology+research+and+veterinary+practice.">Computer assisted semen analyzers in andrology research and veterinary practice.</a> Theriogenology. 57 (1), 149-179 (2002).</li><li>Magnotti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+vitrification+of+fish+semen:+a+review+with+special+emphasis+on+marine+species.">Cryopreservation and vitrification of fish semen: a review with special emphasis on marine species.</a> Rev. Aquacult. , (2016).</li><li>Dziewulska, K., Rzemieniecki, A., Czerniawski, R., Domaga&#322;a, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-thawed+motility+and+fertility+from+Atlantic+salmon+(Salmo+salar+L.)+sperm+frozen+with+four+cryodiluents+in+straws+or+pellets.">Post-thawed motility and fertility from Atlantic salmon (Salmo salar L.) sperm frozen with four cryodiluents in straws or pellets.</a> Theriogenology. 76 (2), 300-311 (2011).</li><li>Babiak, I., Bolla, S., Ottesen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suitable+methods+for+cryopreservation+of+semen+from+Atlantic+halibut,+Hippoglossus+hippoglossus+L.">Suitable methods for cryopreservation of semen from Atlantic halibut, Hippoglossus hippoglossus L.</a> Aquac. Int. 16 (6), 561-572 (2008).</li><li>M&#252;ller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Japanese+eel+(Anguilla+japonica+Temminck+&amp;+Schlegel,+1846)+propagation+using+cryopreserved+sperm+samples.">Japanese eel (Anguilla japonica Temminck &amp; Schlegel, 1846) propagation using cryopreserved sperm samples.</a> J. Appl. Ichthyol. 33 (3), 550-552 (2017).</li></ol>

Os autores declaram que eles têm não tem interesses financeiro concorrente.

Este protocolo descreve o processo completo para criopreservação de esperma, a manipulação e a maturação de enguia europeia. As condições de criação descritas aqui são ideais para rápida maturação e produção de grandes volumes de espermatozoides de alta qualidade nesta espécie 6,7,25. O sucesso do presente protocolo de criopreservação e seu uso potencial para a fertilização pós-descongelação dependem gran...

Nos últimos 25 anos, o número da enguia europeia (Anguilla anguilla) chegando no litoral europeu diminuíram progressivamente 90% 1,2,3. Existem vários fatores que explicam essa queda drástica, incluindo a poluição, infecções, destruição de sobrepesca e habitat. Tudo isso teve um efeito profundo sobre esta espécie, levando à inclusão da enguia europeia na União Internacional para a lista de conservação da natureza (IUCN) como em perigo crítico 4. Consequentemente, o desenvolvimento de técnicas e protocolos para a reprodução em cativeiro são necessárias.
A maturação da enguia europeia em cativeiro é alcançada pelo tratamento hormonal 5,6,7 , mas a produção de gametas em ambos os sexos é difícil sincronizar a 8. Mesmo que o desenvolvimento de novos implantes de andrógeno tem mostrado para acelerar a ovogênese em enguias 9,10, o tempo de maturação final nas fêmeas ainda é altamente variável e difícil de controlar 11. Portanto, a curto prazo armazenamento de esperma 12,13,14 e criopreservação técnicas são necessárias para a gestão da reprodução, tornando o gameta sincronização desnecessária 8.
Criopreservação de esperma de enguia europeia tem sido desenvolvida desde 2003 15,16. Vários pesquisadores projetado protocolos bem sucedidos usando dimetilsulfóxido (DMSO) ou metanol como crioprotectores 16,17,18,19,20. Embora ambos os protocolos têm sido utilizados com sucesso, a viabilidade de células obtidas de espermatozoides descongelados criopreservadas com DMSO é inferior com metanol 20,21. Além disso, a enguia esperma é ativada em contato com DMSO e requer mais tedioso esperma manipulação 19, portanto o metanol é um crioprotetor mais adequado para que o esperma de enguia europeia que DMSO.
Aqui, o protocolo de tratamento ideal e tratamento hormonal da enguia europeia será descrito abaixo. Além disso, o melhor protocolo de criopreservação de esperma de enguia europeia utilizando metanol como um crioprotetor e um protocolo para a avaliação da qualidade do esperma nesta espécie também será descrito.

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

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

Durante os últimos anos, diversos grupos de pesquisa têm trabalhado no desenvolvimento e aperfeiçoamento de novos protocolos para o tratamento de enguia europeia e maturação. A partir de ainda, injeções semanais de gonadotrofina coriônica humana (hCG) demonstraram maturate machos após apenas 5-6 semanas de tratamento, produzindo grandes volumes de espermatozoides de alta qualidade durante várias semanas. Além disso, protocolos de criopreservação de esperma usando diferentes extensores, crioprotectores e refrigeração e descongelamento vezes foram anteriormente descritas para a enguia europeia. Aqui, nós mostramos que solução de extensor de Tanaka´s pode ser usada diretamente para a fertilização ou para criopreservação, tornando desnecessária a utilização de diferentes tipos de soluções e diluições. Além disso, o uso do metanol como um crioprotetor faz este protocolo fácil de usar como metanol tem baixa toxicidade e não ativa o esperma. O esperma não precisa ser criopreservado imediatamente após a adição do crioprotetor, e pode ser usado muito tempo depois de ser descongelado. Além disso, é ainda alta motilidade espermática pós-descongelação embora seja menor do que de esperma fresca. O objetivo deste trabalho é mostrar o melhor protocolo disponível para a enguia europeia, manipulação, maturação e criopreservação de esperma.

Esperma de 18 enguias com uma mobilidade do esperma de 70% ou superior, foi selecionado para este estudo. Os resultados mostraram uma redução em todos os parâmetros de qualidade após descongelamento comparados de esperma fresca (tabela 1 e Figura 2). Os resultados de motilidade (média ± no MEV mostrou, n = 18) mostrou uma maior motilidade total e uma motilidade progressiva no esperma fresca do que o total mobilidade e motilidade progres...

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

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

Protocolos de criopreservação de maturação e esperma de enguia Europeia foram melhorados nos últimos anos. Este artigo descreve o melhor protocolo disponível usando gonadotrofina coriônica humana (hCG) para induzir a maturação e metanol como crioprotetor.

Manipulação e tratamento de macho Europeu enguias (Anguilla anguilla) para maturação Hormonal e criopreservação de esperma

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

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

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

Research

JoVE Journal

Developmental Biology

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

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

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

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

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

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

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

Manipulação e<em&gt; In Vitro</em&gt; Maturação<em&gt; Xenopus laevis</em&gt; Oócitos, seguido de injeção intracitoplasmática de espermatozóide, para estudar o desenvolvimento embrionário

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

Biologia do Desenvolvimento

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

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

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

O isolamento e criopreservação de Neonatais Rato Cardiomyocytes

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

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

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

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

Modificada Vitrificação MicroSecure: Um Altamente procedimento seguro, simples e eficaz para Criopreservação blastócitos humanos

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

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

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

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

Maturação de cardiomiócitos estaminais humanas derivadas de células em Biowires Usando Electrical Stimulation

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

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Uma abordagem padronizada para a purificação multiespecífica de células germinais masculinas de mamíferos por dissociação mecânica do tecido e citometria de fluxo

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

Induction and Validation of Cellular Senescence in Primary Human Cells

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

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

Indução e validação da senescência celular em células humanas primárias

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

Fluorimetric Techniques for the Assessment of Sperm Membranes

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

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

Fluorimétrica técnicas para a avaliação das membranas de esperma

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

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

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

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

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

Medindo a orientação e a motilidade do esperma dentro do intervalo reprodutivo hermafrodita dos elegans de Caenorhabditis

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

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

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

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

Definindo o Programa de Tradução materna mRNA durante a maturação in vitro usando um ensaio de repórter oócito único

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

Combinando imagens com um sistema de entrega de estimulantes para estudar espermatozoides vivos de camundongos

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

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

Autor em destaque: uma técnica de imagem de células vivas para estudar a sinalização de cálcio e exocitose acrossômica em espermatozoides de camundongos 

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

Imagem em Tempo Real da Dinâmica do Cálcio Acrossomal e Exocitose em Espermatozoides Vivos de Camundongos

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