Bu yayın Avrupa Birliği'nin ufuk 2020 araştırma tarafından finanse edildi ve yenilik programı Marie Sklodowska-Curie hibe sözleşmesi kapsamında hiçbir 642893 (IMPRESS), maliyet Office (maliyet eylem SK 1205, AQUAGAMETE) ve mükemmellik Araştırma Merkezi- 1476-4/2016/FEKUT.

Bu protokol için açıklanan Avrupa yılan balığı ile çalışmak için tüm yordamları hayvan deney Etik Komitesi, İspanyol yasa ve yönetmeliklere deneyler kontrol takip Universitat Politècnica de València tarafından kabul edildi ve canlı hayvan işlemler.
1. balık bakım
<ol><li>Avrupa yılan balığı bir araştırma tesisine getir ve onları bir devridaim sistemi ile 200 L aquaria koyardım. Termostatlar ve soğutucular bir sabit 20 ° C sıcaklık sürdürmek için kullanır.</li>
	<li>Balık tutmak stres22 önlemek için karanlık koşullarda ve yiyecek olmayan deneme sırasında.</li>
	<li>Balık temiz suda ilk 3 gün boyunca tut. Sonra 1/3 su değiştirin ve her geçen gün bir tuzluluk 37,0 ± ulaşan kadar deniz suyu ile dolum 0.3 g/l</li>
</ol>2. hormonal tedavi
Not: Hormonal tedavi deneme tüm süresi (9 aylık) insan Korionik gonadotropin (hCG) haftalık enjeksiyonlari oluşur.
<ol><li>Tuzlu çözelti içinde hormon sulandrarak hCG hormonu 1 IU/µL bir konsantrasyon, önceden hazırlamak (% 0.9 NaCl). Not: Bu konsantrasyon için yere a hafta-18 ° C'de, seyreltilmiş hormon korunabilir</li>
	<li>Balık anestezi 5 L ile 40 L esnek kova sistemi su hazır olun.<ol>
<li>250 mL şişeye 300 mg 100 ml % 70 etanol benzokain sulandırmak.</li>
		<li>Kova içinde seyreltilmiş benzokain dökün (son konsantrasyonu 0,36 mM) ve düzgün karıştırın. 60 mg/l son benzokain konsantrasyon için bu</li>
		<li>Balık, tek tek, suya benzokain ile aktarmak ve benzokain etkin olur ve balık düzgün anestezi kadar birkaç saniye bekleyin. Not: balık anestezi onaylamak için balık sırtüstü pozisyonda yerleştirin ve hala o konumda kalır kontrol edin.</li>
	</ol>
</li>
	<li>Hormon yönetmek için balık tartmak ve 1,5 IU/g balık, 1,5 mL plastik tüp bir doz, hCG hormonu hazırlamak.<ol>
<li>HCG hormonu plastik tüp ile 1 mL şırıngaya doldur.</li>
		<li>Sırtüstü pozisyonda ve yardım veya şırınga ile imzalat balık yerleştirin, mayi bölgesinde özenle hormon enjekte.</li>
	</ol>
</li>
	<li>Balık akvaryumlar için dönün ve onu tamamen iyileşti kadar izlemek. Not: Haftalık deney boyunca yapılacak hormonal yönetim vardır. Normalde, Avrupa yılan balığı spermating hormonal tedavi 6-7 hafta sonra başlar.</li>
</ol>3. sperm örnekleme
<ol><li>En iyi kalite örneklerini almak için sperm 24 saat sonra hormonal yönetim6,23ayıklayın.</li>
	<li>Benzokain balıkla 2.2 adımda anlatıldığı gibi anestezi.</li>
	<li>İmzalat balık sırtüstü pozisyonda yerleştirin, bir su distile su ile genital bölge temiz ve dikkatli bir şekilde aquaria gelen deniz suyu ile temas dışkı kirlenmesini genital bölge veya kaza sonucu sperm harekete geçirmek bulunmasını önlemek için kağıt ile kuru.</li>
	<li>Parmakları balık her iki yan tarafta yerleştirerek, dikkatle yanal göğüs yüzgeçleri için genital bölge sperm dışarı zorlamak için tuşuna basarak masaj.<ol>
<li>Yok daha fazla sperm genital açılışa gelene masaj yineleyin.</li>
	</ol>
</li>
	<li>Sperm bir vakum pompası ile 15 mL plastik tüpler içine toplamak.</li>
	<li>Değiştirilmiş Tanaka´s extender çözüm24 (137 mM NaCl, 76.2 mM NaHCO3, distile su içinde) ayıklanan sperm 1:10 4 ºC sulandırmak. Not: 4 ºC extender çözüm sperm saklanması gerekebilir.</li>
</ol>4. sperm kalitesi değerlendirme
<ol><li>Yapay deniz suyu13 hazırlamak (mm: NaCl 354.7, MgCl2 52.4, CaCl2 9,9, Na2SO4 28.2, KCl 9.4, distile su içinde) % 2 sığır serum albümin ile (BSA) (w:v), pH 8.2, osmolalite 1100 mOsm/kg için ayarlamak ve sürdürmek Bakteriyel büyüme önlemek için 4 ºC bunda.</li>
	<li>Bilgisayar destekli sperm analizi (CASA) yazılımı açın ve balık sperm modülü seçin.<ol>
<li>Yazılım Sistem Kurulum açmak için Özellikler ' i tıklatın. Orada, saniyede 60 kare, yakalama seçeneklerini ayarlayın. Seçme negatif faz optik, Makler odası ve 10 X ölçeği.</li>
		<li>Sonra analiz değerleri üzerinde balık türleri ve parçacıklar alan 2 µm2 ' den daha büyük ve 20 µm2' den daha küçük seçin.</li>
		<li>Hücreleri 10 ila 45 µm/s arasında taşıdığınızda, 45 ila 100 µm/s arasında taşırken Orta olarak ayarlayın ve hızlı için 100 µm/s daha hızlı hareket ederken ayarla hız parametreleri üzerinde yavaş için ayarlayın. Not: Sperm hızı 10 µm/s yavaş immotile kabul edildi.</li>
		<li>Doğruluk (STR) % 80'i aşamalı değerleri seçin ve özelliklerini kaydedin.</li>
		<li>(Kameradan canlı görüntü içeren bir pencere açılacak) Yakalama alanı tıklatın. Not: Bilgisayar destekli sperm analizi sistemi mikroskop, bir kamera ve görüntü analiz yazılımı tarafından oluşturulur.</li>
	</ol>
</li>
	<li>Sayım odası, yapay deniz suyu 4 µL koymak ve sperm çözüm (sperm extender çözümde seyreltilmiş) 0.5 µL ekleyin.<ol>
<li>10 X büyütme negatif faz, mikroskop ile görüntü odak. 15 s harekete geçirmek sonra bilgisayar destekli sperm analiz yazılımı tıklatın - video yakalayabilir .</li>
		<li>Dondurma için % 70 daha yüksek bir hareketliliği ile her örnekte onaylatılacak ve seçkin örnekleri analiz. Not: Sadece yüksek kaliteli sperm bu dondurma Protokolü başarısı için seçmek çok önemlidir.</li>
	</ol>
</li>
</ol>5. sperm dondurma yöntemi
<ol><li>Önceden sıvı azot (yaklaşık 2.5 L) bir 34 cm x 34 cm x 30 cm ve 5 cm kalın strafor kutu hazırlayın. Not: bir sıvı azot 4-5 cm yüksekliği her zaman tutması.<ol>
<li>Önceden sperm dondurmak için kayan bir yapı inşa. Polistiren (20 cm x 5 cm) 2 adet kullanma, 2 plastik tüpler ile bağlamak ve yapısı üzerinde sıvı azot yer. Bu yapı bir yaklaşık yüksekliği 3 cm (şekil 1) yüzey üzerinde sıvı nitrojen içinde float gerekiyor.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56835/56835fig1.jpg"> Resim 1 . Şematik sıvı azot içinde önceden dondurma için kullanılan kayan yapısının çizim. Düşük yoğunluklu köpük 20 cm x 4 cm x 5 iki adet yapı oluşur cm 14 cm plastik tüpler ile bağlı. Payet sıvı azot içinde 3 cm'de plastik tüpler üzerine yerleştirilir. <a href="//cloudflare.jove.com/files/ftp_upload/56835/56835fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol start="2"><li>Dondurma çözüm sperm, extender çözüm ve metanol oranı 1, karıştırarak hazırlayın: 8:1 1,5 mL plastik tüpler ve hafifçe karıştırın.</li>
	<li>Bir pipet yardımıyla dondurma çözüm 480 µL 500 µL payet ekleyin ve gerekiyorsa onları kil modelleme ile bir ucunu kapatarak işaretleyin.</li>
	<li>3 min için sıvı azot içinde 3 cm yükseklikte aygıt kayan saman koymak. O zaman, saman sıvı azot yerleştirin.</li>
	<li>10-15 dk sonra payet uzun forseps kullanarak bir sıvı azot depolama tankı aktarmak ve sıvı azot içinde her zaman sular altında tutun. Burada, sperm süresiz olarak depolanabilir.</li>
</ol>6. çözme yöntemi
<ol><li>Sıvı nitrojen içeren bir Styrofoam kutusu 5.1 adımda anlatıldığı gibi ve musluk suyu 40 ºC, 3 L kabı kullanarak bir su banyosu hazırlayın.</li>
	<li>Dondurulmuş sperm ile çöp depolama tankından uzun forseps kullanarak sıvı azot ile Styrofoam kutusu aktarın.<ol>
<li>13 40 ºC, bir su banyosu içine her saman koymak s.</li>
		<li>Sperm saman kapalı uçlarını makasla keserek 1,5 mL plastik tüpün içine dökün.</li>
	</ol>
</li>
	<li>4.1. adımda açıklandığı gibi bilgisayar destekli sperm analizi sistemi kullanarak sperm motilitesi analiz.</li>
	<li>Sperm canlılığı sperm bir Akış Sitometresi ile analiz etmek için 100 µL tutun.</li>
</ol>7. Akış Sitometresi
<ol><li>Ölü hücreleri çekirdekleri lekeleri kırmızı bir floresan bileşik ve yeşil yaşam hücre çekirdekleri lekeleri bir membran permeant nükleer floresan bileşik propidium iyodür (PI), içeren bir floresan setini kullanın.<ol>
<li>Yeşil flüoresan boyama çözüm bu hisse senedi çözüm (1 mM) 1:10 Tanaka´s ortamda 100 µM çalışan bir çözüm için sulandrarak hazırlayın.</li>
		<li>PI çözüm sulandırmak değil. 2.4 mM hisse senedi çözümdür.</li>
	</ol>
</li>
	<li>Her örnek için 50 µL taze veya çözdürülen sperm alın ve yeşil floresan çalışma çözüm (son konsantrasyonu 1 µM) Boyama 0.5 µL ekleyin ve 2 µL PI çözüm (son toplama 100 µM).</li>
	<li>5 min için karanlıkta boyalar (PI ve yeşil flüoresan boyama) içeren örnekleri kuluçkaya.<ol>
<li>Tanaka´s extender çözeltinin 500 µL örneklerinde sulandırmak ve Akış Sitometresi ile analiz.</li>
	</ol>
</li>
	<li>Akış Sitometresi açmak ve en az 2 araziler içeren yeni bir iletişim kuralı oluşturun: SS günlük vs FS günlük ve FL1 vs FL3. Not: her iki, yeşil flüoresan boyama ve propidium iyodür dalga boyu görünür ışık ile heyecanlı. DNA'yı bağlandığında, bu boyalar en fazla Floresans emisyon vardır 516 nm ve 617 nm, anılan sıraya göre.<ol>
<li>Gerilimleri farklı lazer ayarlamak: SS 199; = FS = 199; FL1 377; = FL3 = 372</li>
		<li>Satın alma ayarları accord maksimum olaylara ayarlayın = 5000 ya da 15 s (örnek son konsantrasyonu yaklaşık 1 milyon hücre/ml olmalıdır). Düşük akış kullanarak örnek okuyun.</li>
		<li>Okuma modu tek tüp sabit pozisyon moduseçin.</li>
		<li>Atlıkarınca doğru sayıda örnek koymak</li>
		<li>(F9'a basarak) örnek okumak ve daha fazla analiz için (F7 tuşuna basarak) bir excel dosyası için toplanan verileri kaydetmek.</li>
	</ol>
</li>
</ol>

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E., 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=Computer-assisted+sperm+analysis+(CASA)+as+a+tool+for+monitoring+sperm+quality+in+fish.">Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish.</a> Comp. Biochem. Physiol. C Toxicol. Pharmacol. 130 (4), 425-433 (2001).</li><li>Rurangwa, E., Kime, D. E., Ollevier, F., Nash, J. 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>

Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Bu iletişim kuralı için Avrupa yılan balığı olgunlaşma, işleme ve sperm dondurma tam işlemini açıklar. Burada açıklanan yetiştirme koşulları hızlı olgunlaşma ve yüksek kaliteli sperm bu tür 6,7,25yüksek hacimli üretim için en iyi durumda. Başarısı bu dondurma Protokolü ve potansiyel kullanımı çözdürme sonra gübreleme için taze sperm 26kalitesini büyük ölçüde bağl?...

Son 25 yılda Avrupa sahilde gelen Avrupa yılan balığı (Anguilla anguilla) sayısı % 90 1,2,3tarafından giderek düştü. Kirliliği, enfeksiyonlar, aşırı avlanma ve habitat imha da dahil olmak üzere bu köklü damla açıklamak çeşitli faktörler vardır. Bunların hepsi Uluslararası Birliği için doğa koruma (IUCN) listesi olarak kritik tehlikedeki 4Tarih Avrupa yılan balığı eklenmesi için önde gelen bu tür üzerinde derin bir etkisi olmuştur. Sonuç olarak, geliştirme teknikleri ve protokolleri esaret üreme için gerekli.
Avrupa yılan balığı olgunlaşma tutsak hormonal tedavi 5,6,7 tarafından elde edilir ama her iki cinsiyette gamet üretim 8eşitlemek zordur. Yılan balıkları 9,oogenesis hızlandırmak için yeni androjen implantlar geliştirilmesi göstermiştir10, dişilerde son olgunlaşma zamanlaması hala son derece değişken ve 11kontrol etmek zor olsa bile. Bu nedenle, kısa süreli depolama sperm 12,13,14 ve dondurma teknikleri gamet eşitleme gereksiz 8yapma üreme yönetimi için gerekli.
Avrupa yılan balığı sperm dondurma 2003 15,16beri geliştirmiştir. Çeşitli araştırmacılar Dimetil sülfoksit (DMSO) veya metanol cryoprotectants 16,17,18,19,20kullanarak başarılı iletişim kuralları tasarlanmış. Her ne kadar her iki iletişim kurallarını başarıyla kullanmış, DMSO ile cryopreserved çözdürülen sperm elde edilen hücre canlılığı metanol 20,21ile daha düşüktür. Ayrıca, yılan balığı sperm DMSO ile temas harekete geçirmek ve daha sıkıcı sperm manipülasyon 19gerektirir, bu nedenle metanol DMSO daha Avrupa yılan balığı sperm için daha uygun bir cryoprotectant.
Burada, en uygun kullanım ve Avrupa yılan balığı hormonal tedavi için protokol aşağıda açıklanacaktır. Buna ek olarak, metanol bir cryoprotectant ve bir iletişim kuralı bu türler içinde sperm kalitesinin değerlendirilmesi için en iyi Avrupa yılan balığı sperm dondurma iletişim kuralı'nı da açıklanan olacaktır.

<table><tbody><tr><td>AQUACEN BENZOCA&amp;Iacute;NA +D2A2:D24</td><td>AQUACEN</td><td>3394ESP</td><td>Benzocaine</td></tr><tr><td>NaCl</td><td>Avantor&amp;nbsp;</td><td>3624-19</td><td></td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>305501</td><td>Syringe 1 mL</td></tr><tr><td>FC500</td><td>Beckman Coulter</td><td></td><td>Flow cytometer</td></tr><tr><td>Air pump 100</td><td>EHEIM</td><td>4011708370032</td><td>Modified for suction</td></tr><tr><td>Eppendorf 1.5</td><td>Eppendorf</td><td>175508</td><td>Plastic tubes 1.5 mL</td></tr><tr><td>Falcon tubes</td><td>Falcon</td><td>175747</td><td>Plastic tubes 15 mL</td></tr><tr><td>Flexible bucket</td><td>Fiel</td><td>1040101</td><td>40 L, Black color.</td></tr><tr><td>Ethanol</td><td>Guinama SL</td><td>Mg96270</td><td></td></tr><tr><td>Cyopreservation straws&amp;nbsp;</td><td>IMV Technologies</td><td>14550</td><td>500 &amp;micro;L straws</td></tr><tr><td>Live/Dead Sperm Viability Kit</td><td>Invitrogen</td><td>L7011</td><td>Cytometer Kit</td></tr><tr><td>Modelling clay</td><td>JOVI</td><td>Art 30</td><td>4 different colors</td></tr><tr><td>Precision scale PCB</td><td>KERN &amp;amp; Sohn GmbH</td><td>PCB35002</td><td>Digital scale</td></tr><tr><td>Saline solution Vitulia 0.9%</td><td>Laboratorios Ern, S.A.</td><td>C.N. 999790.8</td><td>Saline solution</td></tr><tr><td>Forceps</td><td>Levantina de Lab. SL</td><td>3710025</td><td>30 cm metal forceps</td></tr><tr><td>Scissors</td><td>Levantina de Lab. SL</td><td>3700014</td><td>Scissors 14cm</td></tr><tr><td>Ovitrelle (r-hCG)</td><td>Merck SL</td><td>EMEA/H/C/000320</td><td>Human chorionic gonadotropin</td></tr><tr><td>Nikon Eclipse 80i</td><td>Nikon</td><td></td><td>Microscope</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>2112211210</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>3257091611</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Panreac Quimica SAU</td><td>1416381210</td><td></td></tr><tr><td>782M Camera</td><td>Proiser</td><td>782M</td><td>Camera for CASA</td></tr><tr><td>ISAS v1</td><td>Proiser</td><td>ISAS v1</td><td>CASA software</td></tr><tr><td>SpermTrack-10</td><td>Proiser</td><td>SpermTrack-10</td><td>Countig chamber for CASA</td></tr><tr><td>Beaker Pyrex 3L</td><td>PYREX</td><td>2110668</td><td>Glass beaker 3L</td></tr><tr><td>Flask Pyrex 250 GL-45</td><td>PYREX</td><td>21801365</td><td>Glass flask 250 mL</td></tr><tr><td>KCl</td><td>Scharlab SL</td><td>PO02000500</td><td></td></tr><tr><td>Methanol</td><td>Scharlab SL</td><td>ME03061000</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Scharlab SL</td><td>MA00370500</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>05482</td><td>Bovine serum albumina</td></tr><tr><td>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

Son yıllarda birkaç araştırma grubu geliştirme ve iyileştirme Avrupa yılan balığı kullanım ve olgunlaşma için yeni iletişim kuralları üzerinde çalışıyoruz. Derecede-in henüz, insan Korionik gonadotropin (hCG) haftalık enjeksiyonlari tedavi sırasında birkaç hafta yüksek miktarlar yüksek kaliteli Sperm üreten, sadece 5-6 hafta sonra erkek maturate kanıtladılar. Buna ek olarak, sperm dondurma farklı Extender kullanarak iletişim kuralları, cryoprotectants ve soğutma ve kez çözme daha önce için Avrupa yılan balığı tarif edilmistir. İşte, Tanaka´s extender çözüm doğrudan veya dondurma, gübreleme için kullanılabilir çözümler ve dilutions farklı kullanımı gereksiz yapma göstermektedir. Gibi metanol düşük toksisite ve sperm etkinleştirmez Ayrıca, bir cryoprotectant olarak metanol kullanımı bu iletişim kuralı kullanımını kolaylaştırır. Sperm cryoprotectant eklenmesinden sonra hemen cryopreserved gerekmez ve uzun çözdürülen sonra kullanılabilir. Ayrıca, sperm motilitesi taze sperm daha düşük olmasına rağmen çözdürme sonra hala yüksektir. Bu çalışmanın amacı, Avrupa yılan balığı için en iyi kullanılabilir iletişim kuralı işleme, olgunlaşma ve sperm dondurma göstermektir.

Spermi ile % 70 veya daha yüksek, sperm motilitesi 18 yılan balıkları bu çalışma için seçildi. Çözdürme için taze sperm (Tablo 1 ve Şekil 2) insanlardan karşılaştırıldığında sonra sonuçları tüm kalite parametreleri bir azalma gösterdi. Hareketliliği sonuçları (yani ± S.E.M., n = 18) daha yüksek toplam hareketliliği ve ilerici bir hareketliliği toplam hareketliliği ve ilerici hareket sonrası tezcan sperm örn...

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

İletişim kuralları Avrupa yılan balığı olgunlaşma ve sperm dondurma için son yıl içinde geliştirilmiştir. Bu makalede, en iyi Protokolü olgunlaşma ve cryoprotectant olarak metanol inducing için insan Korionik gonadotropin (hCG) kullanarak kullanılabilir.

Kullanım ve tedavi, erkek Avrupa yılan balığı (Anguilla anguilla) Hormonal olgunlaşma ve Sperm dondurma için

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

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 Görüntüleme.  Universitat Politècnica de València. İletişim kuralları Avrupa yılan balığı olgunlaşma ve sperm dondurma için son yıl içinde geliştirilmiştir. Bu makalede, en iyi Protokolü olgunlaşma ve cryoprotectant olarak metanol inducing için insan Korionik gonadotropin (hCG) kullanarak kullanılabilir.

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.

Manipülasyon ve<em&gt; İn Vitro</emVe&gt; Olgunlaşma<em&gt; Xenopus laevis</emIntrasitoplazmik sperm enjeksiyonu izledi&gt; oositler, Embriyonik Geliştirme Çalışması

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

Gelişim Biyolojisi

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Atıksu Arıtma Süreçlerinin Etkinliğinin Değerlendirilmesi için Kimya ve Ekotoksikolojik Yöntemlerinin bir pil kullanılması östrojenik Potency çıkarmak

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Çevre

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.

Isıl Şok 2 Tedavi ile Zebra balığı Embriyolar ploidisi Manipülasyon

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

Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology has been identified as a way to obtain more detailed information on movement and behavior across a broader spatial scale than is possible with other known technology. The purpose of this method is to provide a detailed step by step instruction on tag implantation for both lampreys and eel. For laboratory studies using actively migrating juvenile Pacific Lamprey (120-160 mm), we determined that the presence of the tag did not alter the swimming ability between tagged and untagged Individuals or have any significant tag loss (&lt;3%). Similar results were determined during laboratory testing of Yellow phase American Eels (113-175 mm). No mortality occurred during a 38-day holding period and there was minimal tag loss (3.8%). The presence of the tag did not have any significant effect on the swimming ability or survival of tagged eels compared to untagged controls and there was minimal tag loss.

This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

Yeni bir mikro akustik etiketi Juvenil Pasifik taşemen ve Amerikan yılan balığı implantasyonu

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology ...

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.

Zebra balığı sperm Kriyoprezervasyon için bir Yüksek Verim Yöntem ve In Vitro Gübreleme

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 ...

Biyoloji

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Yüksek Verimli<em&gt; Danio rerio</em&gt; Enerji Harcaması Deneyi

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

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.

Zebra balığı Spermatogonia bütün testis iğne tarafından dondurma hızlı soğutma dalmış

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 ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

Yazılımı kullanarak ve soğutma aygıtlarının balık Sperm değerlendirmesi

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

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Gamete koleksiyonu ve Astyanax mexicanus In vitro fertilizasyon

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

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).

Teleost Modelinde Sperm Toplama ve Bilgisayar Destekli Sperm Analizi Japon Medakası (Oryzias latipes)

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

Kullanım ve tedavi, erkek Avrupa yılan balığı (Anguilla anguilla) Hormonal olgunlaşma ve Sperm dondurma için

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