Este projeto recebeu financiamento da Associação de custo (alimentação e agricultura custo ação FA1205: AQUAGAMETE e Horizonte 2020 inovação programa de investigação e da União Europeia sob o Marie Sklodowska-Curie projeto IMPRESS (GA n 642893). Gostaríamos de agradecer a equipe científica de PROiSER, especificamente para o aluno Alberto Vendrell Bernabéu, por sua participação ativa na gravação do video deste projecto.

Procedimentos envolvendo assuntos animais tem sido aprovaram (2015/VSC/ervilha/00064) pela direção geral da produção agrícola e pecuária na Universitat Politécnica de València.
1. recolher o esperma maduro enguias europeias em cativeiro
Nota: Uso enguia europeia os machos mantidos em tanques com água do mar e um sistema de recirculação a uma temperatura constante (20 ° C). Tratar com hormônios através de injeção intraperitoneal semanal (gonadotrofina coriônica humana (hCG); 1,5 UI por g de peso corporal de peixes). Aclimatar gradualmente começando com 3 dias na água doce de peixe seguido de substituição com água do mar (1/3 da água total no tanque) cada 2 dias até atingir uma salinidade de 37,0 g/mL.
<ol><li>Anestesia a enguia 24h após a injeção de hormônio para obter amostras com melhor qualidade do esperma.<ol>
<li>Preparar a anestesia de antemão: Adicionar 300 mg de benzocaína para 100 mL de etanol a 70%. Misture bem e armazenar a 4 ° C.</li>
		<li>Dilua a benzocaína num balde flexível com 5 L de água do sistema para obter uma concentração final de 60 mg/L e misture corretamente.</li>
		<li>Transfira o peixe para o balde com água do sistema e benzocaína. Aguarde alguns minutos até que o peixe se acalme.</li>
	</ol>
</li>
	<li>Limpar a área genital com água e seque com papel absorvente para evitar a contaminação das amostras de esperma por fezes, urina ou água do mar.</li>
	<li>Aplique uma pressão suave na área abdominal e coletar as amostras de esperma em tubos de plástico de 15 mL usando a bomba de vácuo. Volume de esperma até 6-7 mL, dependendo do sexo masculino.</li>
	<li>Manter amostras de esperma a 4 ° C, durante pelo menos 1 h até análise de motilidade é realizado.</li>
</ol>2. leve à geladeira e diluir as amostras de esperma
<ol><li>Prepare a solução diluidor de antemão. Nota: As amostras de esperma devem ser diluídas na solução extensor específica para cada espécie.<ol>
<li>Prepare o suporte não-ativador para amostras de esperma de enguia (P1 médio). Adicione 0,42 g de bicarbonato de sódio, 1,828 g de cloreto de sódio, 0,127 g de cloreto de magnésio, 0,56 g de cloreto de potássio e 0,037 g de cloreto de cálcio para 250 mL de água destilada. Misture bem para dissolver. Loja a 4 ° C.</li>
	</ol>
</li>
	<li>Defina o bloco refrigerador a 4 ° C, para manter as amostras à temperatura constante. Espere até que a temperatura se estabilize.</li>
	<li>Dilua as amostras de esperma usando a diluidor solução específica para cada espécie. Nota: A razão de diluição é espécie específico e deve ser definida para padronizar o protocolo. Primeiro, estimar a concentração de espermatozoides, com base na concentração obtida na pré-análise da motilidade usando software, conforme explicado nas etapas 3 e 4. Com esta análise, também é possível selecionar as melhores amostras de esperma com base na porcentagem de motilidade. Depois disto, o pesquisador pode começar a coletar dados para o experimento usando as melhores amostras com a concentração correta.<ol>
<li>Dilua as amostras de esperma de enguia no meio de P1, com uma proporção de 01:50. Para preparar 500 µ l de esperma de enguia diluída, adicione 50 µ l de amostra de esperma fresco e 450 µ l de P1. Misture bem.</li>
	</ol>
</li>
</ol>3. avaliar os parâmetros de motilidade do esperma
<ol><li>
Instalação do módulo de motilidade do Software
	<ol>
<li>Definir o cenário refrigerador a 4 ° C e esperar até que estabiliza.</li>
		<li>Abra o software e selecione o Módulo de motilidade. Crie nome de usuário e senha, se necessário.</li>
		<li>Selecione Propriedades e escolha os parâmetros desejados antes de iniciar a análise do esperma.<ol>
<li>Clique sobre as espécies de | Peixe.</li>
			<li>Selecione os Quadros por segundo e o Número de imagens, dependendo das condições técnicas melhores para cada espécie. Defina as duas opções em 120 imagens por segundo.</li>
			<li>Escolha o contraste negativo. É obrigatório para reconstrução precisa dos espermatozoides trajetórias16.</li>
			<li>Selecione o correspondente Câmara de contagem e a escala da câmara. Calibre a câmera de vídeo para a lente de ampliação utilizada no experimento. Defina SpermTrack10 como câmara de contagem e escala de X 10. Nota: em geral, uma profundidade de 10 µm em câmara de contagem e uma lente de ampliação de 10x são condições recomendadas desde que eles fornecem a melhor focagem de todos os espermatozoides e capturar o maior número de células. No entanto, é aconselhável fazer um estudo prévio das condições ideais para a análise da mobilidade de cada espécie.</li>
			<li>Ajuste a Área de partícula e conectividade para cada espécie. Definir área de partícula mínimo 2 µm2 e conectividade em 7 µm. Nota: Para o peixe, o valor mínimo de intervalos de área de partículas entre 2-5 µm e conectividade depende a velocidade de espermatozoides e a taxa de quadros.</li>
			<li>Salve a configuração.</li>
		</ol>
</li>
		<li>Selecione capturar.</li>
	</ol>
</li>
	<li>
Analisar a enguia europeia esperma com Software
	<ol>
<li>Prepare a solução de ativador (água do mar artificial) adicionando 0,946 g de sal comercial para 25 mL de água destilada com 2% de BSA.</li>
		<li>Tomar 500 mL de solução ativador (água do mar artificial) e coloque no refrigerador bloco. Nota: em geral, esperma de peixe é ativada por um evento de choque osmotic, embora para algumas espécies a concentração do íon também pode ser importante. Para espécies de água doce, o meio de ativador terem osmolalidade hipotônica, enquanto para espécies marinhas a mídia deve ser hipertônica.</li>
		<li>Coloque a câmara de contagem sob a fase de arrefecimento e espere até a temperatura estabilizar a 4 ° C.</li>
		<li>Misture o ativador e esperma diluídas para ativar os espermatozoides e começar o motilidade de gravação de vídeo entre 5-10 s após a ativação.<ol>
<li>Tome 4 µ l de solução ativador e lugar na câmara de contagem.</li>
			<li>Homogeneizar delicadamente o esperma diluído agitando a microcentrifuga 3 vezes para evitar danos nas células de espermatozoides.</li>
			<li>Tomar 0,5 µ l de esperma diluídas, misture com o ativador e coloque a tampa na câmara contando rapidamente. Nota: Este passo não deve tomar mais de 5 s para iniciar a análise logo que possível.</li>
			<li>Centrar-se sobre as células de espermatozoides e encontrar a melhor área visual, que é definida por um baixo número de espermatozoides (150 a 200 células) para evitar a intercepção entre células (Supplemental figura 1A). Selecione Capturar vídeo para obter as faixas de espermatozoides, que são separadas de acordo com a velocidade.</li>
			<li>Selecione capturar para obter campos 3 a 7, que são o intervalo ideal para obter uma baixa variabilidade nos resultados e proceder como anteriormente. Gravar vídeos até 120 s pós da ativação para evitar condensação na tampa da câmara de contagem.</li>
			<li>Selecione a saída para ter uma visão geral de todos os campos.</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. obter dados de motilidade
<ol><li>Selecione os campos de | Parcial | Salve e escolher um nome de arquivo. Clique em salvar. Nota: A planilha fornece valores médios de dados parciais, gráficos e imagens de faixas de espermatozoides.</li>
	<li>Selecione dados gerais | Nome do arquivo | Salvar. Nota: Os dados fornecem valores cinéticas para espermatozoides individuais de todos os campos.</li>
</ol>

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<li>Islam, M. S., Akhter, T. <a target="_blank" href="http://article.sapub.org/10.5923.j.als.20110101.03.html">Tale of Fish Sperm and Factors Affecting Sperm Motility: A Review.</a> Advancements in Life Sciences. 1, 11-19 (2011).</li>
<li>Dadras, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+common+carp+Cyprinus+carpio+sperm+motility+and+lipid+composition+using+different+in+vitro+temperatures%5BTitle%5D)+AND+%22Anim.+Reprod.+Sci%22%5BJournal%5D)">Analysis of common carp Cyprinus carpio sperm motility and lipid composition using different in vitro temperatures.</a> Anim. Reprod. Sci. 180, 37-43 (2017).</li>
<li>Soler, C., García, A., Contell, J., Segervall, J., Sancho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinematics+and+subpopulations%27+structure+definition+of+blue+fox+%28Alopex+lagopus%29+sperm+motility+using+the+ISASV1+CASA+system%5BTitle%5D)+AND+%22Reproduction+in+Domestic+Animals%22%5BJournal%5D)">Kinematics and subpopulations' structure definition of blue fox (Alopex lagopus) sperm motility using the ISASV1 CASA system.</a> Reproduction in Domestic Animals. 49, 560-567 (2014).</li>
<li>Vásquez, F., Soler, C., Camps, P., Valverde, A., GarcíaMolina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spermiogram+and+sperm+head+morphometry+assessed+by+multivariate+cluster+analysis+results+during+adolescence+%2812-18+years%29+and+the+effect+of+varicocele%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Spermiogram and sperm head morphometry assessed by multivariate cluster analysis results during adolescence (12-18 years) and the effect of varicocele.</a> Asian Journal of Andrology. 18, 824-830 (2016).</li>
<li>Soler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dog+sperm+head+morphometry%3A+its+diversity+and+evolution%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Dog sperm head morphometry: its diversity and evolution.</a> Asian Journal of Andrology. 19, 149-153 (2017).</li>
<li>Valverde, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphometry+and+subpopulation+structure+of+Holstein+bull+spermatozoa%3A+variations+in+ejaculates+and+cryopreservation+straws%5BTitle%5D)+AND+%22Asian+Journal+of+Andrology%22%5BJournal%5D)">Morphometry and subpopulation structure of Holstein bull spermatozoa: variations in ejaculates and cryopreservation straws.</a> Asian Journal of Andrology. 18, 851-857 (2016).</li>
</ol>

Os autores não têm nada para divulgar.

O software de análise de esperma utilizado neste protocolo tem sido usado por pesquisadores em todo o mundo para diferentes espécies, incluindo peixe. No entanto, os peixes têm algumas características específicas que podem afetar a avaliação do esperma. Espermatozoides de peixes mostrou alta velocidade no momento da ativação que declina rapidamente e leva a um curto período de tempo de motilidade após a ativação. Além disso, a temperatura de reprodução é dependente da espécie e, em alguns casos, pode se...

A eficácia de reprodução depende da qualidade de ambos os gâmetas (óvulos e espermatozoides)1,2. Este é o principal fator que contribui para uma fecundação bem sucedida, permitindo o desenvolvimento da prole viável3,4. Conveniente avaliação da qualidade do material genético é a melhor ferramenta para definir o potencial de fertilidade de um espécime.
Mistura de esperma de vários machos é uma prática comum na produção de muitas espécies aquáticas comercial4. No entanto, a variabilidade de esperma entre machos pode levar à competição de esperma e, consequentemente, não todos os homens igualmente estão contribuindo para o pool de gene5. Neste sentido, a avaliação correta das características de ejaculação/espermatozoides individuais, tais como motilidade, é fundamental para obterem informações discriminatórias sobre potenciais individuais a fertilidade masculina. Observação directa da motilidade espermática pode produzir dados imprecisos e subjetivos como requer tempo e experiência, o que leva a uma falta de consistência e incompatibilidade dos resultados6,7. No entanto, existem muitas técnicas inovadoras, rápidas e quantitativas que podem fornecer um esperma confiável qualidade análise2,4.
Análise de esperma assistida por computador foi desenvolvido para oferecer dados precisos sobre o esperma de qualidade8. Esta tecnologia inclui o desenvolvimento de software associado com um microscópio de contraste de fase que permite a avaliação da motilidade espermática. No entanto, um fator limitante de parâmetro de motilidade é a taxa de quadros da câmara. Espermatozoides individuais trajetórias baseiam-se em espermatozoides cabeça posição de centroide em quadros consecutivos de gravações em vídeo, que está correlacionada com o movimento flagelar padrões3,9,10, 11. os principais parâmetros cinéticos medidos são a velocidade linear (VSI), velocidade curvilínea (VCL) e caminho de média velocidade (VAP). VSI é a distância entre o início e o ponto de extremidade tomadas por espermatozoides divididos pelo tempo. VCL é a velocidade real ao longo da trajetória precisa tomada por espermatozoides. VAP é a velocidade ao longo de um caminho suavizado derivada da trajetória. Estes parâmetros permitem que informações adicionais de cinéticas, incluindo linearidade (LIN), linearidade (STR), wobble (WOB) e medições de espancamento como amplitude de movimento lateral da cabeça (ALH) e frequência de batida-Cruz (BCF)4,10.
O sistema de análise de esperma foi originalmente utilizado para espécies de mamíferos, e um dos requisitos para o sistema está a funcionar a temperatura do corpo do doador (cerca de 37 ° C). Este software também pode ser utilizado para espécies de peixes; embora, seja necessário fazer algumas adaptações para reduzir o erro dos resultados de análise do esperma. Em algumas espécies de peixes, tais como os salmonídeos e enguia8,12, a fecundação ocorre em baixa temperatura (em torno de 4 ° C),2,4. Assim, dispositivos de arrefecimento deve ser desenvolvida para evitar condições de trabalho desconfortáveis. Além disso, peixes espermatozoides são fixa no fluido seminal e requerem um choque osmótica para ativar a motilidade. Para espécies de água doce, o meio de ativador terem osmolalidade hipotônica, enquanto para espécies marinhas a mídia deve ser hipertônica. No entanto, para algumas espécies, como os salmonídeos, a concentração do íon também poderia ser importante3,4,9. Após a ativação, esperma de peixe é caracterizada por uma rápida diminuição da motilidade (menos de 2 min)13,14 e alta velocidade, sendo vital para determinar a taxa de quadro ideal para obter dados fiáveis15.
Os objetivos deste estudo são projetar e aplicar sistemas de refrigeração para as amostras de esperma de peixe. Além disso, esse protocolo define como determinar as taxas de quadro ideal para o estabelecimento de protocolos padrão, dependendo da espécie. O uso deste protocolo abre novas portas no contexto da avaliação seminal de peixe, usando a enguia europeia como modelo.

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

fish sperm assessment using software and cooling devices

Para avaliação da qualidade de gametas, existem técnicas inovadoras, rápidas e quantitativas que podem fornecer dados úteis para a aquicultura. Sistemas informatizados para a análise de esperma foram desenvolvidos para medir vários parâmetros e dentre as mais comumente medido é a mobilidade do esperma.
Inicialmente, esta tecnologia de computador foi projetada para espécies de mamíferos, embora ele também pode ser usado para análise do esperma de peixe. Os peixes têm características específicas que podem afetar o esperma avaliação tais como um tempo curto de motilidade após a ativação e, em alguns casos, adaptação às temperaturas mais. Assim, é necessário modificar os componentes de software e hardware para fazer análise da motilidade mais eficiente para análise do esperma de peixe. Para espermatozoides de mamíferos, a placa de aquecimento é usada para manter a temperatura ideal de espermatozoides. No entanto, para algumas espécies de peixes, é vantajoso usar uma temperatura mais baixa para prolongar a duração da motilidade, uma vez que os espermatozoides permanecem ativos por menos de 2 min. Portanto, dispositivos de resfriamento são necessários para refrigerar amostras a temperatura constante ao longo do tempo de análise, incluindo no microscópio óptico. Este protocolo descreve a análise da mobilidade do esperma de peixe utilizando software para análise de esperma e resfriamento novos dispositivos para otimizar os resultados.

Análise do efeito do tempo na mobilidade do esperma
No caso da enguia europeia, a percentagem de espermatozoides estáticos aumentou de 15 s para 120 s após sua ativação (de 24,4% para 40,7%) e a porcentagem de espermatozoides móveis de progressivas diminuição (de 36,9% para 20,9%) (Figura 1A e 1B). Com base na velocidade, células de espermatozoides mostrara...

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

Fish Sperm Assessment Using Software and Cooling Devices

O presente protocolo descreve um procedimento de avaliação do esperma de peixe usando análise de esperma assistida por computador e dispositivos de refrigeração. O software dá uma rápida, precisa e a análise quantitativa da qualidade do esperma de peixe com base na mobilidade do espermatozoide, que pode ser uma ferramenta útil na aquicultura para melhorar o sucesso da reprodução.

Avaliação do esperma de peixe utilizando Software e dispositivos de refrigeração

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

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

Research

JoVE Journal

Biology

8.7K visualizações.  PROiSER R+D S.L.. O presente protocolo descreve um procedimento de avaliação do esperma de peixe usando análise de esperma assistida por computador e dispositivos de refrigeração. O software dá uma rápida, precisa e a análise quantitativa da qualidade do esperma de peixe com base na mobilidade do espermatozoide, que pode ser uma ferramenta útil na aquicultura para melhorar o sucesso da reprodução.

Vídeo: Fish Sperm Assessment Using Software and Cooling Devices

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

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

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

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

Microinjeção de proteína CRISPR/Cas9 em bagre do canal, Ictalurus punctatus, embriões para Gene Editing

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

Genética

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

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

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

Manipulação de ploidia de embriões Zebrafish com choque térmico 2 Tratamento

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

Biologia do Desenvolvimento

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

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

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

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

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 ...

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

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

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

Triagem de espermatozoides para o isolamento rápido de edições germinativas em peixes-zebra

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

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

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

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

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

Um método de alta taxa de transferência para Zebrafish criopreservação de espermatozóides e In Vitro Fertilização

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

Biologia

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

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

Fluorescence in situ hybridization FISH Protocol in Human Sperm

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

Fluorescência In situ Hybridization (FISH) Protocolo no esperma humano

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

Fluorimetric Techniques for the Assessment of Sperm Membranes

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

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

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, ...

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

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

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

Criopreservação de Zebrafish espermatogónias por toda agulha testículos imersos refrigeração ultra rápida

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

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

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

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

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

Coleta de espermatozoides e análise de espermatozoides assistida por computador no modelo Teleost japonês Medaka (Oryzias latipes)

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

U-Shaped Horizontal Swimming Technique for Preparing High-Quality Sperm with Low DNA Fragmentation Index

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and viscous seminal plasma. High-quality sperm refers to progressively motile spermatozoa with normal morphology, which often exhibit a lower DNA fragmentation index (DFI) and higher fertilization potential. Preparing high-quality sperm is a critical step in human-assisted reproductive technology. The traditional sperm preparation method, discontinuous density gradient centrifugation (DGC), is time-consuming and labor-intensive. Repeated centrifugation can damage sperm DNA, thereby affecting subsequent fertilization and embryo development. This study introduces a U-shaped horizontal swimming (UHS) method for preparing intracytoplasmic sperm injection (ICSI) sperm, which significantly eliminates the detrimental effects of&#160;centrifugation on sperm DNA.&#160;The UHS method involves creating a UHS lane using a fertilization medium within an ICSI operating dish. A 10 &#181;L fertilization medium microdroplet is placed at the starting point on the left side of the UHS lane to hold the semen. Two additional 10 &#181;L fertilization medium buffer droplets are positioned at intervals along the left middle section of the lane, with all droplets connected by fertilization medium. The dish is then covered with culture oil and incubated overnight at 37 &#176;C with 6% CO2 to equilibrate. Subsequently, 3 &#181;L of semen is added to the microdroplet at the starting point. High-quality spermatozoa swim to the right side of the UHS lane, facilitating their suction into the ICSI injection needle. Dead sperm, cell debris, and other viscous impurities largely remain at the initial point or in the buffer droplets. We simultaneously processed 21 semen samples using both UHS and DGC techniques and compared their DFI. The results demonstrated that the DFI in the DGC group was 5.5% &#177; 3.2%, whereas the DFI in the UHS group was 1.7% &#177; 1.1%. The difference between the two groups was statistically significant (P &#60; 0.05).

This protocol describes a method for screening high-quality sperm with a low DNA fragmentation index by utilizing sperm motility characteristics and thigmotaxis. The method employs a U-shaped horizontal swimming lane, enabling high-quality sperm to reach the opposite side through buffer droplets, while dead sperm, cell debris, and impurities are excluded.

Técnica de natação horizontal em forma de U para preparar espermatozóides de alta qualidade com baixo índice de fragmentação de DNA

Human semen is a complex mixture comprising progressively motile spermatozoa, non-progressively motile spermatozoa, immotile spermatozoa, cell debris, and ...