이 게시는 유럽 연합의 수평선 2020 연구에 의해 투자 되었다 및 혁신 프로그램 마리 Sklodowska 퀴리 부여 계약 없음 642893 (감동), 비용 사무실 (비용 액션 FA 1205, AQUAGAMETE), 그리고 우수 연구 센터- 1476-4/2016/FEKUT입니다.

이 프로토콜에서 설명 하는 유럽 장 어와 함께 작업 하기 위한 모든 절차는 위원회의 윤리의 동물 실험 Universitat Politècnica de València, 스페인 법률 및 규정 제어 실험에 의해 승인 및 살아있는 동물에 절차입니다.
1. 물고기 유지 보수
<ol><li>연구 시설에 유럽 뱀장어를 가져오고 재순환 시스템 200 L 수족관에 넣어. 20 ° C에서 일정 한 온도 유지 하기 위해 온도 쿨러를 사용 합니다.</li>
	<li>실험 기간 동안 물고기 음식와 스트레스22 를 피하기 위해 어두운 조건에서 유지.</li>
	<li>첫 3 일 동안 신선한 물에서 물고기를 유지. 다음 물 1/3을 변경 하 고 해 수 37.0 ±의 염 분을 도달할 때까지 매일 리필 0.3 g/l.</li>
</ol>2. 호르몬 치료
참고: 호르몬 치료는 실험의 전체 기간 (9 주)을 통해 인간의 융 생식 샘 자극 호르몬 (hCG)의 주간 주사의 구성 되어 있습니다.
<ol><li>1 IU / µ L의 농도에서 미리 hCG 호르몬 호르몬 생리 식 염 수에 희석 하 여 준비 (0.9 %NaCl). 참고: 호르몬 보존 될 수 있습니다-18 ° c.에 1 주일 이상에 대 한이 농도에 희석</li>
	<li>Anesthetize 물고기, 시스템 물 5 l 40 L 유연한 물통을 준비 합니다.<ol>
<li>250 mL 플라스 크에 300mg benzocaine 70% 에탄올 100ml에 희석.</li>
		<li>양동이에 희석된 benzocaine를 부 어 (최종 농도 0.36 m m)와 적절히 섞어. 이것은 60 mg/l.의 최종 benzocaine 농도 대 한</li>
		<li>Benzocaine와 물으로, 물고기를 개별적으로 전송 하 고는 benzocaine 적용 됩니다 물고기 제대로 마 취 될 때까지 몇 초를 기다립니다. 참고: 물고기 취 것인지 물고기 부정사 자리에 놓고 그것은 여전히 그 자리에에서 유지 하는 확인 하십시오.</li>
	</ol>
</li>
	<li>호르몬 관리, 무게 물고기 고 물고기, 1.5 mL 플라스틱 튜브에서의 1.5 IU/g의 복용량에 hCG 호르몬을 준비.<ol>
<li>플라스틱 튜브에서 hCG 호르몬을 1 mL 주사기를 채우십시오.</li>
		<li>복 지역에서 신중 하 게 호르몬을 주입, 부정사 위치와 도움 또는 주사기 마 취 물고기를 놓습니다.</li>
	</ol>
</li>
	<li>물고기는 수족관에 반환 하 고 완전히 복구 될 때까지 그것을 모니터링 합니다. 참고: 호르몬 관리는 실험 내내 매주 실시 하는. 일반적으로, 유럽 뱀장어는 호르몬 치료의 6-7 주 후 spermating를 시작합니다.</li>
</ol>3. 정자 샘플링
<ol><li>최고의 품질 샘플을 얻기 위해 호르몬 관리6,23후 정자 24 h를 추출 합니다.</li>
	<li>2.2 단계에 설명 된 대로 benzocaine와 물고기를 anesthetize.</li>
	<li>부정사 위치에 마 취 물고기를 배치, 증류수의 물 총으로 성 기 영역을 청소 하 고 종이 수족관에서 해 수와 접촉 하 여 배설물 오염 음부 나 실수로 정자 활성화를 방지 하려면 신중 하 게 건조.</li>
	<li>물고기의 측면 양쪽에 손가락을 배치, 신중 하 게 누르면 옆으로 가슴 지 느 러 미에서 성 기 영역을 밖으로 정자를 마사지.<ol>
<li>마사지를 반복 하 여 더 더 정자 생식 기 오프닝에서 나온다.</li>
	</ol>
</li>
	<li>15 mL 플라스틱 튜브 진공 펌프를 사용 하 여 정자를 수집 합니다.</li>
	<li>4 º C에서 수정된 Tanaka´s 익스텐더 솔루션24 (137 m m NaCl, 76.2 m m NaHCO3, 증류수에)에서 추출한 정자 1시 10분 희석. 참고: extender 솔루션에 정자 4 º C에서 유지 되어야 합니다.</li>
</ol>4. 정자 품질 평가
<ol><li>인공 해 수13 준비 (mm에서: NaCl 354.7, MgCl2 52.4, CaCl2 9.9, 나2등4 28.2, KCl 9.4, 증류수에) 2% 소 혈 청 알 부 민 (BSA) (w:v), pH 8.2, osmolality 1100 mOsm/kg를 조정 하 고 유지 그것은 박테리아의 성장을 피하기 위해 4 º C에서.</li>
	<li>컴퓨터 기반 정자 분석 (카사)에 대 한 소프트웨어를 열고 물고기 정자 모듈을 선택 합니다.<ol>
<li>소프트웨어의 시스템 설치 프로그램을 열려면 속성 클릭 하십시오. 거기, 초당 60 이미지에서 캡처 옵션을 설정 합니다. 선택 부정적인 단계 광학, Makler 챔버와 10 배 규모.</li>
		<li>다음 분석 값에 종 및 입자 영역 2 µ m2 보다 크고 20 µ m2보다 작은 물고기 를 선택 합니다.</li>
		<li>속도 매개 변수를 설정 느린 때 셀 10 그리고 45 µ m/s 사이 이동, 45, 100 µ m/s 사이 이동할 때 보통 으로 설정 빠른 100 µ m/s 보다 빠르게 이동 하는 경우. 참고: 10 µ m/s 보다 느린 속도와 정 충 immotile 간주 됐다.</li>
		<li>직진도 (STR)의 80%로 진보적인 가치를 선택 하 고 속성을 저장 합니다.</li>
		<li>캡처 필드 (카메라에서 라이브 이미지 창이 열립니다)를 클릭 하십시오. 참고: 컴퓨터 기반 정자 분석 시스템 현미경, 카메라 및 이미지 분석 소프트웨어에 의해 형성 된다.</li>
	</ol>
</li>
	<li>세 실에 인공 해 수의 4 µ L를 넣어 하 고 정자 솔루션 (정자 익스텐더 솔루션에 희석)의 0.5 µ L를 추가 합니다.<ol>
<li>이미지에 부정적인 단계에 10 배 확대 현미경 초점. 15 활성화 후 s 클릭 캡처-비디오에서 컴퓨터 기반 정자 분석 소프트웨어에서.</li>
		<li>Cryopreservation 70% 보다 높은 운동 성을 가진 triplicate 선택 샘플에서 모든 샘플을 분석 합니다. 참고: 그것은 매우이 cryopreservation 프로토콜의 성공에 대 한 높은-품질 정자만을 선택 하는 것이 중요입니다.</li>
	</ol>
</li>
</ol>5. 정자 동결 방법
<ol><li>34 cm x 34 cm x 30 cm 및 5 cm 두꺼운 스티로폼 상자에서에서 사전에 액체 질소 (약 2.5 L)를 준비 합니다. 참고: 항상 액체 질소의 4-5 cm 높이의 레벨을 유지 합니다.<ol>
<li>전 정자를 동결 부동 구조 구축. 폴리스 티 렌 (20 cm x 5 cm)의 2 개를 사용 하 여, 2 플라스틱 튜브와 함께 그들을 바인딩할 하 고 액체 질소에 구조를 놓습니다. 이 구조는 표면 (그림 1)에 3 cm의 대략적인 높이에 액체 질소를 통해 플 로트 해야 합니다.</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56835/56835fig1.jpg"> 그림 1 . 미리 이상의 액체 질소 냉동에 사용 되는 부동 구조 그리기 도식. 구조는 낮은 밀도 20 cm x 4 cm x 5의 스티로폼의 두 부분으로 구성 됩니다 cm 14 c m의 플라스틱 튜브와 연결. 빨 대는 액체 질소에 3 cm에 플라스틱 튜브를 통해 배치 됩니다. <a href="//cloudflare.jove.com/files/ftp_upload/56835/56835fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<ol start="2"><li>정자, 익스텐더 솔루션 및 메탄올 1의 비율로 혼합 하 여 cryopreservation 솔루션 준비: 8:1 1.5 mL 플라스틱에서 튜브, 부드럽게 저 어.</li>
	<li>피 펫의 도움으로 500 µ L 빨 대에 cryopreservation 솔루션의 480 µ L을 추가 하 고 필요 하다 면, 클레이 모델링 한 끝을 폐쇄 하 여 표시 합니다.</li>
	<li>3 분 액체 질소에 3 cm의 고도에 떠 있는 장치에 빨 대를 넣어. 그런 다음 액체 질소에 빨 대를 놓습니다.</li>
	<li>10-15 분 후 긴 집게를 사용 하 여 액체 질소 저장 탱크에 빨 대를 전송 하 고 항상 액체 질소에 빠져들 유지 합니다. 여기, 정자는 무기한으로 저장할 수 있습니다.</li>
</ol>6. 해 동 방법
<ol><li>5.1, 단계에서 설명한 대로 액체 질소로 스티로폼 상자를 준비 하 고 준비 물 목욕 40 º C에서 수돗물 3l 비 커를 사용 하 여.</li>
	<li>긴 집게를 사용 하 여 액체 질소 저장 탱크 스티로폼 상자에 냉동된 정자를 빨 대 전송.<ol>
<li>13 40 º C에서 물 목욕으로 각 빨 대를 넣어 s.</li>
		<li>가 위로 빨 대의 닫힌된 끝을 절단 하 여 1.5 mL 플라스틱 관으로 정자를 붓는 다.</li>
	</ol>
</li>
	<li>4.1 단계에 설명 된 대로 컴퓨터 기반 정자 분석 시스템을 사용 하 여 정자의 운동 성 분석.</li>
	<li>교류 cytometer와 정 충의 생존 능력을 분석 하는 정자의 100 µ L를 유지.</li>
</ol>7. Cytometry
<ol><li>Propidium 요오드 화물 (PI), 죽은 세포의 핵을 얼룩 빨간 형광 화합물 이며 녹색 살아있는 세포의 핵 얼룩 막 permeant 핵 형광 성 화합물을 포함 하는 형광 키트를 사용 합니다.<ol>
<li>100 µ M의 작업 솔루션 Tanaka´s 매체에 재고 솔루션 (1mm) 1시 10분에서 그것을 diluting 하 여 녹색 형광 얼룩 솔루션을 준비 합니다.</li>
		<li>PI 솔루션을 희석 하지 마십시오. 2.4 m m 재고 솔루션이입니다.</li>
	</ol>
</li>
	<li>각 샘플에 대 한 신선한 또는 해 동 정자의 50 µ L 고 작업 솔루션 (최종 농도 1 µ M)를 착 색 하는 녹색 형광의 0.5 µ L을 추가 하 고 PI 솔루션 (최종 농도 100 µ M)의 2 µ L.</li>
	<li>5 분 동안 어둠 속에서 염료 (PI와 녹색 형광 성 얼룩)를 포함 하는 샘플을 품 어.<ol>
<li>Tanaka´s 익스텐더 솔루션의 500 µ L의 샘플을 희석 하 고 교류 cytometer 분석.</li>
	</ol>
</li>
	<li>교류 cytometer 설정 하 고 적어도 2 음모를 포함 하는 새로운 프로토콜을 만들: SS 대 FS 로그 및 FL1 vs FL3 로그. 참고: 두, 녹색 형광 얼룩이 지 고 propidium 요오드 화물 보이는 파장 빛으로 흥분 수 있습니다. DNA에 바인딩된 경우,이 염료의 최대 형광 방출은 516 및 617 nm, 각각.<ol>
<li>다른 레이저의 전압 조정: SS = 199; FS = 199; FL1 377; = FL3 = 372</li>
		<li>최대 이벤트 수집 설정 협정 설정 = 5000 또는 15 s (샘플의 최종 농도 약 1 백만 셀/mL의 해야 함). 낮 은 흐름을 사용 하 여 샘플을 읽기.</li>
		<li>읽기 모드 단일 튜브 고정 위치 모드를선택 합니다.</li>
		<li>회전 목마의 오른쪽 숫자에 샘플을 넣어</li>
		<li>(F 9를 누르면) 샘플을 읽고 고 추가 분석에 대 한 (f 7을 누르면) excel 파일에 수집 된 데이터를 저장 합니다.</li>
	</ol>
</li>
</ol>

<ol>
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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. 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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. 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저자 들은 아무 경쟁 금융 관심사 선언 합니다.

이 프로토콜 유럽 장 어 성숙, 처리 및 정자 cryopreservation에 대 한 완전 한 프로세스를 설명합니다. 여기에 설명 된 농업 조건 빠른 성숙 및이 종 6,,725에 높은-품질의 높은 볼륨의 생산을 위한 최적의 있습니다. 이 cryopreservation 프로토콜 및 해빙 후 수정에 대 한 그것의 잠재적인 사용의 성공 신선한 정자 26의 품...

지난 25 년 동안 유럽 뱀장어 (앵귈라 앵귈라) 유럽 해 안에 도착의 수는 꾸준히 90 1,2,3감소. 오염, 감염, overfishing, 서식 지 파괴 등이 급격 한 하락을 설명 하는 몇 가지 요인이 있다. 이 모든 중요 한 멸종 위기 4자연 보존 (IUCN) 목록은 국제 연합에 유럽 장 어의 포함에 지도 하는이 종에 깊은 영향을 했다. 따라서, 기술 및 포로에 복제에 대 한 프로토콜의 개발은 필요 합니다.
포로에서 유럽 장 어의 성숙 호르몬 치료 5,,67 에 의해 이루어집니다 하지만 남녀 모두에서 gametes의 생산 8동기화 할 어렵습니다. 새로운 안 드로 겐 임 플 란 트의 개발 oogenesis 뱀장어 9,에 있는 가속을 보이고 있다 하더라도10, 여성에서 마지막 성숙의 타이밍은 여전히 매우 변수 하 고 11를 제어 하기 어려운. 따라서, 기술의 정자 12,,1314 및 cryopreservation 단기 저장 복제 관리, 생식 체 동기화 불필요 한 8를 만들기 위해 필요 하다.
유럽 장 어 정자의 cryopreservation 2003 15,16이후 개발 되었습니다. 몇몇 연구원은 성공적인 프로토콜 cryoprotectants 16,17,18,,1920디 메 틸 sulfoxide (DMSO) 또는 메탄올을 사용 하 여 설계 되었습니다. 두 프로토콜 성공적으로 사용 되었습니다, 하지만 해 동된 정자 DMSO와 cryopreserved의 얻은 세포 생존 능력에 메탄올 20,21보다 낮은 것입니다. 또한, 장 정자 DMSO와 접촉을 활성화 하 고 더 지루한 정자 조작 19필요 하, 그러므로 메탄올 DMSO 보다 유럽 장 어 정자에 대 한 더 적합 한 cryoprotectant 이다.
여기, 아래 최적의 처리 및 유럽 장 어의 호르몬 치료에 대 한 프로토콜을 설명 합니다. 이것 이외에,이 종에서 정자 품질 평가 대 한 프로토콜은 cryoprotectant로 메탄올을 사용 하 여 최고의 유럽 장 어 정자 cryopreservation 프로토콜도 설명 됩니다.

<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

지난 년 동안 여러 연구 그룹 개발 및 유럽 장 어 처리 및 성숙에 대 한 새로운 프로토콜의 개선에 노력 하고있다. 로 서 아직, 인간 융 생식 샘 자극 호르몬 (hCG)의 주간 주사 치료, 몇 주 동안 높은-품질의 높은 볼륨 생산의 단지 5-6 주 후 남성 maturate를 입증 해야 합니다. 또한, 정자 cryopreservation 프로토콜 다른 extender를 사용 하 여, cryoprotectants 및 냉각 시간을 녹고 있다 이전 설명 유럽 장 어에 대 한. 여기, 우리가 보여줍니다 수정 또는 cryopreservation, Tanaka´s 익스텐더 솔루션 직접 사용 될 수 있다 솔루션 및 희석의 다른 종류의 사용을 불필요 한 만들기. 또한, 메탄올의 사용은 cryoprotectant로 만드는이 프로토콜 사용 하기 쉬운 메탄올은 낮은 독성과 정자를 활성화 하지 않습니다. 정자는 cryoprotectant 추가 직후 cryopreserved 필요 하지 않습니다 그리고 그것은 오래 되 고 해 동 후 사용할 수 있습니다. 또한, 정자 운동 성은 비록 그것이 신선한 정자 보다 낮은 녹고 후 여전히 높다입니다. 이 작품의 목표 처리, 성숙 및 정자 cryopreservation 유럽 장 어에 대 한 가장 사용할 수 있는 프로토콜을 표시 하는 것입니다.

70% 이상, 정자 운동 성 가진 18 뱀장어에서 정자는이 연구에 대 한 선정 됐다. 결과 신선한 정자 (표 1 및 그림 2)에서 그에 비해 녹고 후 모든 품질 매개 변수에서 감소를 보였다. 운동 성 결과 (± S.E.M., 의미 n = 18) 총 운동 성 및 진보적인 운동 후 해 동 정자 샘플에서 발견 보다 신선한 정자에 더 높은 총 운동 성 및 진보적인 운동 성을 보였...

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

유럽 장 어 성숙 및 정자 cryopreservation 프로토콜 지난 수 년에 걸쳐 개선 되었습니다. 이 문서는 성숙과 메탄올 cryoprotectant로 유도 대 한 인간의 융 생식 샘 자극 호르몬 (hCG)을 사용 하 여 사용할 수 있는 최상의 프로토콜을 설명 합니다.

처리와 치료의 남성 유럽 뱀장어 (앵귈라 앵귈라) 호르몬 성숙 및 정자 Cryopreservation

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

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 조회수.  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

비디오: 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.

조작 및<em&gt; 체외</em의&gt; 성숙<em&gt; 아프리카 발톱 개구리 laevis의</em세포질 내 정자 주입에 이어&gt; 난 모세포, 배아 발달을 연구하는

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

연구

발생학

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.

분리 및 신생아 쥐 심근 세포의 냉동 보존

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.

수정 MicroSecure의 유리화 : 인간 배반포를위한 안전 간단하고 매우 효과적인 냉동 보존 절차

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

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

사용 모성 유전자 제품의 기능 조작<em&gt; 체외</em제브라 피쉬&gt; 난자의 성숙

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

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.

전기 자극을 사용하여 Biowires 인간 줄기 세포 유래 심근 세포의 성숙

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.

기계 조직 해리 및 유동 세포 계측법에 의한 포유 동물 수컷 세포의 다중 종 정제에 대한 표준화 된 접근법

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.

유도 및 기본 인간 세포에서 세포 노화의 유효성 검사

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.

정자 세포 막의 평가 대 한 Fluorimetric 기법

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.

정자 지도 및 운동 성 아닌 간 남녀 양성 생식 기관에서 측정

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

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

분화를 촉진하기 위해 DMSO를 사용하여 인간 다능성 줄기 세포의 일시적인 치료

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...