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>

<ol>
	<li>ICES_Working_Group_on_Eels. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Report of the 2011 Session of the Joint EIFAAC/ICES Working Group on Eels. , 246 (2011).</li><li>Moriarty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+Catches+of+Elver+of+1928-1988.">European Catches of Elver of 1928-1988.</a> Int. Rev. Hydrobiol. 75 (6), 701-706 (1990).</li><li>Dekker, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fractal+geometry+of+the+European+eel+stock.">The fractal geometry of the European eel stock.</a> ICES J. Mar. Sci. 57 (1), 109-121 (2000).</li><li>Jacoby, D., Gollock, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anguilla anguilla. The IUCN red list of threatened species. Version 2014.2. , (2014).</li><li>Asturiano, J. F., 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=Effects+of+hCG+as+spermiation+inducer+on+European+eel+semen+quality.">Effects of hCG as spermiation inducer on European eel semen quality.</a> Theriogenology. 66 (4), 1012-1020 (2006).</li><li>P&#233;rez, L., 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=Induction+of+maturation+and+spermiation+in+the+male+European+eel:+assessment+of+sperm+quality+throughout+treatment.">Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment.</a> J. Fish Biol. 57 (6), 1488-1504 (2000).</li><li>Gallego, V., 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=Study+of+the+effects+of+thermal+regime+and+alternative+hormonal+treatments+on+the+reproductive+performance+of+European+eel+males+(Anguilla+anguilla)+during+induced+sexual+maturation.">Study of the effects of thermal regime and alternative hormonal treatments on the reproductive performance of European eel males (Anguilla anguilla) during induced sexual maturation.</a> Aquaculture. 354, 7-16 (2012).</li><li>Asturiano, J. F., S&#248;rensen, S. R., P&#233;rez, L., Lauesen, P., Tomkiewicz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+Production+of+Larvae+Using+Cryopreserved+Sperm:+Effects+of+Preservation+Temperature+and+Cryopreservation+on+European+Eel+Sperm+Fertilization+Capacity.">First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity.</a> Reprod. Domestic Anim. 51 (4), 485-491 (2016).</li><li>Di Biase, A., 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=Co-treatment+with+androgens+during+artificial+induction+of+maturation+in+female+eel,+Anguilla+anguilla:+Effects+on+egg+production+and+early+development.">Co-treatment with androgens during artificial induction of maturation in female eel, Anguilla anguilla: Effects on egg production and early development.</a> Aquaculture. 479, 508-515 (2017).</li><li>Lokman, P., Wylie, M. J., Downes, M., Di Biase, A., Damsteegt, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+induction+of+maturation+in+female+silver+eels,+Anguilla+australis:+The+benefits+of+androgen+pre-treatment.">Artificial induction of maturation in female silver eels, Anguilla australis: The benefits of androgen pre-treatment.</a> Aquaculture. 437, 111-119 (2015).</li><li>Mylonas, C. C., Duncan, N. J., Asturiano, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+manipulations+for+the+enhancement+of+sperm+production+in+cultured+fish+and+evaluation+of+sperm+quality.">Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality.</a> Aquaculture. , (2016).</li><li>Ohta, H., Izawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diluent+for+cool+storage+of+the+Japanese+eel+(Anguilla+japonica)+spermatozoa.">Diluent for cool storage of the Japanese eel (Anguilla japonica) spermatozoa.</a> Aquaculture. 142 (1-2), 107-118 (1996).</li><li>Pe&#241;aranda, D. S., 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=European+Eel+Sperm+Diluent+for+Short-term+Storage.">European Eel Sperm Diluent for Short-term Storage.</a> Reprod. Domestic Anim. 45 (3), 407-415 (2010).</li><li>Ohta, H., Kagawa, H., Tanaka, H., Unuma, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+by+the+environmental+concentration+of+ions+of+the+potential+for+motility+in+Japanese+eel+spermatozoa.">Control by the environmental concentration of ions of the potential for motility in Japanese eel spermatozoa.</a> Aquaculture. 198 (3), 339-351 (2001).</li><li>Asturiano, J., 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=Media+and+methods+for+the+cryopreservation+of+European+eel+(Anguilla+anguilla)+sperm.">Media and methods for the cryopreservation of European eel (Anguilla anguilla) sperm.</a> Fish Physiol. Biochem. 28 (1), 501-502 (2003).</li><li>Asturiano, J., 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=Physio-chemical+characteristics+of+seminal+plasma+and+development+of+media+and+methods+for+the+cryopreservation+of+European+eel+sperm.">Physio-chemical characteristics of seminal plasma and development of media and methods for the cryopreservation of European eel sperm.</a> Fish Physiol. Biochem. 30 (3), 283-293 (2004).</li><li>Szab&#243;, G., 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+of+European+eel+(Anguilla+anguilla)+sperm+using+different+extenders+and+cryoprotectants.">Cryopreservation of European eel (Anguilla anguilla) sperm using different extenders and cryoprotectants.</a> Acta. Biol. Hung. 56 (1-2), 173-175 (2005).</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=Cryopreservation+of+sperm+of+farmed+European+eel+Anguilla+anguilla.">Cryopreservation of sperm of farmed European eel Anguilla anguilla.</a> J World Aquac Soc. 35 (2), 225-231 (2004).</li><li>Pe&#241;aranda, D. S., P&#233;rez, L., Gallego, V., Jover, M., Asturiano, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+European+eel+sperm+cryopreservation+method+by+preventing+spermatozoa+movement+activation+caused+by+cryoprotectants.">Improvement of European eel sperm cryopreservation method by preventing spermatozoa movement activation caused by cryoprotectants.</a> Cryobiology. 59 (2), 119-126 (2009).</li><li>Marco-Jim&#233;nez, F., 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+of+European+eel+(Anguilla+anguilla)+spermatozoa:+effect+of+dilution+ratio,+foetal+bovine+serum+supplementation,+and+cryoprotectants.">Cryopreservation of European eel (Anguilla anguilla) spermatozoa: effect of dilution ratio, foetal bovine serum supplementation, and cryoprotectants.</a> Cryobiology. 53 (1), 51-57 (2006).</li><li>Asturiano, J. F., 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=Effect+of+sperm+cryopreservation+on+the+European+eel+sperm+viability+and+spermatozoa+morphology.">Effect of sperm cryopreservation on the European eel sperm viability and spermatozoa morphology.</a> Reprod. Domestic Anim. 42 (2), 162-166 (2007).</li><li>Mordenti, M., Di Biase, A., Sirri, R., Modugno, S., Tasselli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Sexual+Maturation+in+Wild+Female+European+Eels+(Anguilla+anguilla)+in+Darkness+and+Light.">Induction of Sexual Maturation in Wild Female European Eels (Anguilla anguilla) in Darkness and Light.</a> Isr. J. Aquac. 64, (2012).</li><li>Ohta, H., 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=Artificial+induction+of+maturation+and+fertilization+in+the+Japanese+eel,+Anguilla+japonica.">Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica.</a> Fish Physiol. Biochem. 17 (1), 163-169 (1997).</li><li>Tanaka, S., 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=Long-term+cryopreservation+of+sperm+of+Japanese+eel.">Long-term cryopreservation of sperm of Japanese eel.</a> J. Fish Biol. 60 (1), 139-146 (2002).</li><li>Gallego, V., 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=Subpopulation+pattern+of+eel+spermatozoa+is+affected+by+post-activation+time,+hormonal+treatment+and+the+thermal+regimen.">Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen.</a> Reprod. Fertil. Dev. 27 (3), 529-543 (2015).</li><li>Asturiano, J. F., Cabrita, E., Horv&#225;th, &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress,+challenges+and+perspectives+on+fish+gamete+cryopreservation:+A+mini-review.">Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review.</a> Gen. Comp. Endocrinol. , (2016).</li><li>Kime, D. 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 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

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 ...

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 Görüntüleme.  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.

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

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.

İzolasyon ve Neonatal Rat kardiyomiyositlerde Kryoprezervasyon

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.

Modifiye MicroSecure Vitrifikasyon: İnsan Blastosistlerinin A, Güvenli Basit ve Etkili Kryoprezervasyon Prosedürü

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.

Elektriksel Uyarı Biowires İnsan Kök Hücre kaynaklı kardiyomiyositlerde Olgunlaşma

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.

Memeli Erkek Germ Hücrelerinin Çok Denekli Saflaştırılmasında Standart Bir Yaklaşım, Mekanik Doku Ayrılma ve Akış Sitometrisi ile

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.

İndüksiyon ve birincil insan hücrelerinde hücresel yaşlanma doğrulama

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.

Fluorimetric teknikleri Sperm membran değerlendirilmesi için

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.

Ölçme sperm rehberlik ve motilite içinde Caenorhabditis elegans hermafrodit üreme yolu

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.

Tek Bir Oocyte Reporter Tahlil Kullanarak In vitro Olgunlaşma Sırasında Maternal mRNA Çeviri Programını Tanımlama

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

Canlı Fare Spermini İncelemek için Görüntülemeyi Uyarıcı Dağıtım Sistemi ile Birleştirme

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.

Yazar Spotlight: Fare Sperminde Kalsiyum Sinyalini ve Akrozom Ekzositozunu İncelemek İçin Canlı Hücre Görüntüleme Tekniği 

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.

Canlı fare sperminde akrozomal kalsiyum dinamiği ve ekzositozun gerçek zamanlı görüntülenmesi

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