Questa pubblicazione è stata finanziata dalla ricerca Orizzonte 2020 dell'Unione europea e l'innovazione del programma nell'ambito dell'accordo di sovvenzione Marie Sklodowska-Curie No 642893 (IMPRESS), l'ufficio di costo (COST Action FA 1205, AQUAGAMETE) e il centro di ricerca di eccellenza- 1476-4/2016/FEKUT.

Tutte le procedure per l'utilizzo di anguilla europea descritta in questo protocollo è sono approvate dal comitato di etica della sperimentazione animale presso la Universitat Politècnica de València, seguendo le leggi spagnole e regolamenti gli esperimenti di controllo e procedure sugli animali vivi.
1. pesce manutenzione
<ol><li>Portare le anguille europee per una struttura di ricerca e metterli in un acquario di 200L con un sistema di ricircolo. Utilizzare termostati e refrigeratori per mantenere una temperatura costante di 20 ° C.</li>
	<li>Tenere i pesci in condizioni di oscurità per evitare lo stress22 e senza cibo durante l'esperimento.</li>
	<li>Tenere i pesci in acqua dolce durante i primi 3 giorni. Poi cambiare 1/3 dell'acqua e riempire con acqua di mare ogni giorno fino a raggiungere una salinità di 37.0 ± 0,3 g/L.</li>
</ol>2. trattamento ormonale
Nota: Il trattamento ormonale consiste di iniezioni settimanali di gonadotropina corionica umana (hCG) in tutta l'intera durata (nove settimane) dell'esperimento.
<ol><li>Preparare l'ormone hCG in anticipo ad una concentrazione di 1 IU / µ l diluendo l'ormone in soluzione fisiologica (0,9% NaCl). Nota: L'ormone possa essere conservato diluiti a questa concentrazione per più di una settimana a-18 ° C.</li>
	<li>Per anestetizzare il pesce, preparare un secchio flessibile 40 L con 5 L di acqua dell'impianto.<ol>
<li>In un pallone da 250 mL, diluire 300 mg di benzocaina in 100 mL di etanolo al 70%.</li>
		<li>Versare la benzocaina diluito nel secchio (concentrazione finale 0,36 mM) e mescolare bene. Questo è per una concentrazione finale di benzocaina di 60 mg/L.</li>
		<li>Trasferire il pesce, individualmente, in acqua con benzocaina e attendere alcuni secondi fino a quando la benzocaina ha effetto e il pesce è adeguatamente anestetizzato. Nota: Per confermare che il pesce è anestetizzato, mettere il pesce in una posizione supina e controllare che rimanga ancora in quella posizione.</li>
	</ol>
</li>
	<li>Per amministrare l'ormone, pesare il pesce e preparare l'ormone hCG alla dose di 1,5 UI/g di pesce, in un tubo di plastica da 1,5 mL.<ol>
<li>Riempire una siringa da 1 mL con l'ormone hCG dal tubo di plastica.</li>
		<li>Mettere il pesce anestetizzato in posizione supina e con l'assistenza o la siringa, iniettare l'ormone attentamente in zona intraperitoneale.</li>
	</ol>
</li>
	<li>Restituire il pesce per acquari e monitorare fino a che completamente ha recuperato. Nota: L'amministrazione ormonale deve essere condotta settimanale in tutto l'esperimento. Normalmente, anguille europee iniziano spermating dopo 6-7 settimane di trattamento ormonale.</li>
</ol>3. prelievi di sperma
<ol><li>Per ottenere i migliori campioni di qualità, estrarre spermatozoi 24 h dopo la somministrazione ormonale6,23.</li>
	<li>Anestetizzare il pesce con benzocaina come descritto al punto 2.2.</li>
	<li>Mettere il pesce anestetizzato in posizione supina, pulire la zona genitale con uno spruzzo di acqua distillata e asciugare accuratamente con carta, per evitare la contaminazione di feci presenti nella zona genitale o attivazione accidentale dello sperma dal contatto con acqua di mare dal aquaria.</li>
	<li>Posizionando le dita su entrambi i lati laterali del pesce, massaggiare accuratamente premendo lateralmente da pinne pettorali alla zona genitale per forzare lo sperma fuori.<ol>
<li>Ripetere il massaggio fino a quando non più sperma esce dall'apertura genitale.</li>
	</ol>
</li>
	<li>Raccogliere lo sperma in tubi di plastica da 15 mL utilizzando una pompa a vuoto.</li>
	<li>Diluire lo spermatozoi estratti 01:10 in modificato Tanaka´s extender soluzione24 (137 mM NaCl, 76,2 mM NaHCO3, in acqua distillata) a 4 ° c. Nota: Lo sperma nella soluzione extender deve essere mantenuto a 4 ° c.</li>
</ol>4. valutazione della qualità sperma
<ol><li>Preparare acqua di mare artificiale13 (in mM: NaCl 354,7, MgCl2 52,4, CaCl2 9,9, Na2SO4 28.2, KCl 9.4, in acqua distillata) con 2% albumina di siero bovino (BSA) (corrispondente), regolare il pH a 8.2, osmolalità a 1100 mOsm/kg e mantenere e ' a 4 ° c per evitare la crescita batterica.</li>
	<li>Aprire il software per l'analisi computerizzata dello sperma (CASA) e selezionare il modulo di sperma di pesce.<ol>
<li>Fare clic su Proprietà per aprire la configurazione di sistema del software. Lì, è possibile impostare le opzioni di cattura 60 immagini al secondo. Ottica di selezionare fase negativa, camera di Makler e scala X 10.</li>
		<li>Quindi i valori di analisi, selezionare pesce come specie e zona di particelle più grandi di 2 µm2 e più piccoli di 20 µm2.</li>
		<li>Ai parametri di velocità, impostata su lento quando le cellule spostare tra 10 e 45 µm/s, impostato su medio , quando si spostano tra 45 e 100 µm/s e impostare rapidamente quando si muove più velocemente di 100 µm/s. Nota: Gli spermatozoi con velocità più lenta di 10 µm/s sono stati considerati immotile.</li>
		<li>Selezionare i valori progressivi come 80% di rettilineità (STR) e salvare le proprietà.</li>
		<li>Fare clic sul Campo Capture (si aprirà una finestra con le immagini live dalla fotocamera). Nota: Il sistema di analisi computerizzata dello sperma è formato da un microscopio, una telecamera e un software di analisi di immagine.</li>
	</ol>
</li>
	<li>Sulla camera di conteggio, mettere 4 µ l di acqua di mare artificiale e 0,5 µ l di soluzione di sperma (sperma diluito in soluzione extender).<ol>
<li>Mettere a fuoco l'immagine con il microscopio a ingrandimento 10x in fase negativa. 15 s dopo l'attivazione, fare clic su Capture - dal video del software di analisi computerizzata dello sperma.</li>
		<li>Analizzare ogni campione in triplice copia e selezionare campioni con una motilità superiore al 70% per la crioconservazione. Nota: È molto importante selezionare solo alta qualità sperma per il successo di questo protocollo di crioconservazione.</li>
	</ol>
</li>
</ol>5. metodo di congelamento di spermatozoi
<ol><li>Preparare in anticipo liquido azoto (circa 2,5 litri) in un 34 cm x 34 cm x 30 cm e scatola di polistirolo spessa 5 cm. Nota: Mantenere un livello di azoto liquido di 4-5 cm di altezza in ogni momento.<ol>
<li>Costruire una struttura galleggiante per pre-congelare lo sperma. Utilizzare 2 pezzi di polistirolo (20 x 5 cm), li legano con 2 tubi di plastica e posizionare la struttura sull'azoto liquido. Questa struttura deve galleggiare sopra l'azoto liquido ad un'altezza approssimativa di 3 cm sopra la superficie (Figura 1).</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56835/56835fig1.jpg"> Figura 1 . Disegno della struttura galleggiante utilizzata per pre-congelamento sopra azoto liquido schematico. La struttura è composta da due pezzi di bassa densità polistirolo di 20 cm x 4 cm x 5 cm collegato con tubi di plastica di 14 cm. Le cannucce sono disposti sopra i tubi in plastica a 3 cm sopra l'azoto liquido. <a href="//cloudflare.jove.com/files/ftp_upload/56835/56835fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<ol start="2"><li>Preparare la soluzione di crioconservazione dello sperma, la soluzione di estensione e metanolo al rapporto di 1:8:1 a 1,5 mL in plastica tubi e mescolare delicatamente.</li>
	<li>Con l'aiuto di una pipetta, aggiungere 480 µ l della soluzione crioconservazione in 500 cannucce µ l e se necessario, li segnano chiudendo un'estremità con argilla da modellare.</li>
	<li>Mettere la paglia sul dispositivo mobile ad un'altezza di 3 cm sopra l'azoto liquido per 3 min. Quindi, inserire la cannuccia nell'azoto liquido.</li>
	<li>Dopo 10-15 minuti, trasferire le cannucce in un serbatoio di stoccaggio di azoto liquido utilizzando pinze lunghe e tenerli immersi in azoto liquido a tutte le ore. Qui, gli spermatozoi possono essere conservati a tempo indeterminato.</li>
</ol>6. metodo di scongelamento
<ol><li>Preparare una scatola di polistirolo con azoto liquido, come descritto al punto 5.1 e preparare un bagno di acqua utilizzando un becher da 3 litri con rubinetto acqua a 40 ° c.</li>
	<li>Trasferire le cannucce con sperma congelato dal serbatoio di deposito nella scatola di polistirolo con azoto liquido utilizzando pinze lunghe.<ol>
<li>Mettere ogni paglia in un bagno di acqua a 40 ° c per 13 s.</li>
		<li>Versare lo sperma in un tubo di plastica da 1,5 mL tagliando le estremità chiuse della paglia con le forbici.</li>
	</ol>
</li>
	<li>Analizzare la motilità dello sperma usando il sistema di analisi computerizzata dello sperma come spiegato al punto 4.1.</li>
	<li>Tenere 100 µ l di sperma per analizzare la vitalità degli spermatozoi con un citometro a flusso.</li>
</ol>7. flusso Cytometry
<ol><li>Utilizzare un kit fluorescente contenente ioduro di propidio (PI), che è un composto fluorescente rosso che colora i nuclei delle cellule morte e un composto fluorescente nucleare membrana-permeante, che verdi macchie i nuclei delle cellule viventi.<ol>
<li>Preparare la soluzione di colorante fluorescente verde diluendo dalla soluzione di riserva (1 mM) 01:10 in mezzo Tanaka´s a una soluzione di lavoro di 100 µM.</li>
		<li>Non diluire la soluzione PI. La soluzione di riserva è a 2,4 mM.</li>
	</ol>
</li>
	<li>Per ogni campione, prendere 50 µ l di spermatozoi freschi o scongelati e 0,5 µ l di verde fluorescente macchiatura soluzione di lavoro (concentrazione finale di 1 µM) e 2 µ l di soluzione di PI (concentrazione finale 100 µM).</li>
	<li>Incubare i campioni contenenti i coloranti (PI e colorazione fluorescente verde) al buio per 5 min.<ol>
<li>Diluire i campioni in 500 µ l di soluzione di estensione Tanaka´s e analizzare con il citometro a flusso.</li>
	</ol>
</li>
	<li>Accendere il citometro a flusso e creare un nuovo protocollo contenente almeno 2 appezzamenti: SS log log vs FS e FL1 vs FL3. Nota: Entrambi, verde fluorescente macchiatura e propidio ioduro di possa essere eccitato con luce di lunghezza d'onda visibile. Quando è associato al DNA, l'emissione di fluorescenza massima di questi coloranti sono 516 nm e 617 nm, rispettivamente.<ol>
<li>Regolare le tensioni dei laser diversi: SS = 199; FS = 199; FL1 = 377; FL3 = 372</li>
		<li>Impostare l'accordo di impostazioni di acquisizione per numero massimo di eventi = 5000 o 15 s (la concentrazione finale del campione dovrebbe essere di circa 1 milione di cellule/mL). Leggere l'esempio utilizzando un flusso basso .</li>
		<li>Selezionare la modalità di lettura singolo tubo posizione modalità fissata.</li>
		<li>Mettere il campione del numero giusto del carosello</li>
		<li>Leggere il campione (premendo F9) e salvare i dati raccolti in un file di excel (premendo F7) per ulteriori analisi.</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>

Gli autori dichiarano di non avere nessun concorrenti interessi finanziari.

Questo protocollo descrive il processo completo per la crioconservazione di spermatozoi, la manipolazione e la maturazione di anguilla europea. Le condizioni di allevamento descritte qui sono ottimale per la produzione di elevati volumi di alta qualità dello sperma in questo specie 6,7,25e veloce maturazione. Il successo di questo protocollo di crioconservazione e suo uso potenziale per fertilizzazione dopo lo scongelamento dip...

Negli ultimi 25 anni, il numero di anguilla europea (Anguilla anguilla) che arrivano al litorale europeo sono diminuiti costantemente dal 90% 1,2,3. Ci sono diversi fattori che spiegano questo drastico calo compreso le infezioni, inquinamento, distruzione di sovrasfruttamento e habitat. Tutto questo ha avuto un effetto profondo su questa specie, che l'inclusione dell'anguilla europea su Unione internazionale per la lista di conservazione della natura (IUCN) come in pericolo critico 4. Di conseguenza, lo sviluppo di tecniche e protocolli per la riproduzione in cattività sono necessarie.
La maturazione delle uova dell'anguilla europea in cattività avviene mediante trattamento ormonale 5,6,7 , ma la produzione di gameti in entrambi i sessi è difficile sincronizzare 8. Anche se lo sviluppo di nuovi impianti di androgeni è indicata per accelerare oogenesi anguille 9,10, i tempi di maturazione finale nelle femmine è ancora altamente variabili e difficili da controllare 11. Pertanto, deposito a breve termine delle tecniche di sperma 12,13,14 e crioconservazione sono necessarie per la gestione della riproduzione, rendendo gamete sincronizzazione inutili 8.
Crioconservazione di spermatozoi di anguilla europea è stato sviluppato dal 2003 15,16. Diversi ricercatori progettato protocolli successo utilizzando solfossido dimetilico (DMSO) o metanolo come crioprotettori 16,17,18,19,20. Sebbene entrambi i protocolli sono stati utilizzati con successo, l'attuabilità delle cellule ottenuti di spermatozoi scongelati crioconservati con DMSO è inferiore con metanolo 20,21. Inoltre, sperma di Anguilla è attivato il contatto con DMSO e richiede per la manipolazione di sperma più noioso 19, pertanto il metanolo è un crioprotettore più adatto per gli spermatozoi di anguilla europea di DMSO.
Qui, il protocollo per la gestione ottima e trattamento ormonale dell'anguilla europea sarà descritto di seguito. Oltre a questo, il miglior protocollo di crioconservazione di spermatozoi anguilla europea usando il metanolo come crioprotettore e un protocollo per la valutazione della qualità dello sperma in questa specie sarà anche descritto.

<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

Negli ultimi anni, diversi gruppi di ricerca hanno lavorato sullo sviluppo e miglioramento di nuovi protocolli per la gestione di anguilla europea e maturazione. Come di ancora, iniezioni settimanali di gonadotropina corionica umana (hCG) hanno dimostrato di maturate maschi dopo appena 5-6 settimane di trattamento, produzione di elevati volumi di alta qualità dello sperma durante parecchie settimane. Inoltre, protocolli di crioconservazione di spermatozoi utilizzando diversi dispositivi Extender, crioprotettori e raffreddamento e scongelamento volte precedentemente sono stati descritti per anguilla europea. Qui, indichiamo che Tanaka´s la soluzione di estensione può essere direttamente utilizzata per la fecondazione o per la crioconservazione, rendendo inutile l'utilizzo di diversi tipi di soluzioni e diluizioni. Inoltre, l'uso del metanolo come crioprotettore rende questo protocollo facile da usare come metanolo ha una bassa tossicità e non attivare lo sperma. Lo sperma non deve necessariamente essere crioconservati immediatamente dopo l'aggiunta del crioprotettore, e può essere utilizzato a lungo dopo essere scongelati. Inoltre, la motilità dello sperma è ancora alta dopo lo scongelamento anche se è più basso di quello di sperma fresco. Lo scopo di questo lavoro è quello di mostrare il miglior protocollo disponibile per anguilla europea la gestione, la maturazione e la crioconservazione degli spermatozoi.

Sperma da 18 anguille con una motilità dello sperma del 70% o superiore, è stato selezionato per questo studio. I risultati hanno mostrato una riduzione in tutti i parametri di qualità dopo lo scongelamento rispetto a quelli da sperma fresco (tabella 1 e Figura 2). I risultati di motilità (media ± s.e.m., n = 18) hanno mostrato una maggiore motilità totale e una motilità progressiva nello sperma fresco rispetto alla motilità totale e ...

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

Protocolli per la crioconservazione di maturazione e lo sperma di anguilla europea sono state migliorate negli ultimi anni. Questo articolo viene descritto il protocollo migliore disponibile con gonadotropina corionica umana (hCG) per indurre la maturazione e metanolo come crioprotettore.

Gestione e trattamento di maschio anguilla europea (Anguilla anguilla) per maturazione ormonale e crioconservazione degli spermatozoi

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 Views.  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

Video: 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.

Manipolazione e<em&gt; In Vitro</em&gt; Maturazione di<em&gt; Xenopus laevis</em&gt; ovociti, seguito da Iniezione intracitoplasmatica di spermatozoi, per studiare lo sviluppo embrionale

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

Ricerca

Biologia dello sviluppo

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.

Isolamento e Crioconservazione di neonatali Rat 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.

Modificato vetrificazione MicroSecure: Un altamente Procedura Crioconservazione sicuro ed efficace, semplice e per blastocisti umane

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.

Maturazione di staminali umane cardiomiociti derivati ​​dalle cellule in Biowires Utilizzando Stimolazione Elettrica

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.

Un approccio standardizzato per la depurazione multispecie di cellule germinali maschili di mammiferi mediante dissociazione tissutale meccanica e citometria del flusso

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.

Induzione e convalida della senescenza cellulare in cellule umane primarie

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.

Fluorimetrica tecniche per la valutazione delle membrane degli spermatozoi

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.

Misurare la guida dello sperma e la motilità all'interno del tratto riproduttivo ermafrodita di Caenorhabditis elegans

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.

Definizione del programma di traduzione dell'mRNA materno durante la maturazione in vitro utilizzando un saggio single oocyte reporter

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

Combinazione dell'imaging con un sistema di somministrazione di stimolanti per studiare lo sperma di topo vivo

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.

Autore in primo piano: una tecnica di imaging di cellule vive per studiare la segnalazione del calcio e l'esocitosi dell'acrosomia nello sperma di topo 

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.

Imaging in tempo reale della dinamica del calcio acrosomiale e dell'esocitosi in spermatozoi di topo vivi

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