Summary

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

Published: January 28, 2018
doi:

Summary

欧洲鳗鲡成熟和精子冷冻保存的协议在过去的几年里得到了改善。这篇文章描述了最佳的协议可利用人绒毛膜促性腺激素 (hCG) 诱导成熟和甲醇为剂。

Abstract

在过去的几年中, 几个研究小组一直致力于开发和改进欧洲鳗鲡处理和成熟的新协议。到目前为止, 每周注射人类绒毛膜促性腺激素 (hCG) 已证明成熟男性经过短短5-6 周的治疗, 生产高品质的精子在几个星期。此外, 使用不同的扩增剂、冷冻、冷却和解冻时间的精子冷冻保存协议已经被描述为欧洲鳗鲡。在这里, 我们表明, Tanaka´s 扩展解决方案可以直接用于施肥或冷冻保存, 使不必要的使用不同类型的解决方案和稀释。此外, 甲醇作为剂的使用使得该协议易于使用, 因为甲醇具有低毒性和不激活精子。精子不需要在加入剂后立即保存, 并且在解冻后可以长时间使用。此外, 在解冻后精子活力仍很高, 但比新鲜精子低。这项工作的目的是为欧洲鳗鱼的处理, 成熟和精子冷冻保存提供最佳的协议。

Introduction

在过去25年中, 欧洲鳗鲡 (安圭拉) 到达欧洲海岸的数量稳步下降, 有 90% 1,2,3。有几个因素解释这一急剧下降, 包括污染, 感染, 过度捕捞和栖息地破坏。所有这一切都对这一物种产生了深远的影响, 导致将欧洲鳗鲡列入国际自然保护联盟 (IUCN) 名单, 作为严重濒危的4。因此, 在圈养的繁殖技术和规程的发展是必要的。

欧洲鳗鲡在圈养中的成熟是通过荷尔蒙治疗来实现的5,6,7但是在两性的配子生产上很难同步8。虽然新的雄激素植入物的发展已经显示出加速卵子在鳗鱼9,10, 雌性的最终成熟时间仍然是高度可变的, 难以控制11。因此, 短期储存精子12,13,14和冷冻保存技术是复制管理所必需的, 使配子同步不必要的8

欧洲鳗鲡精子的超低温保存自 2003年15,16开始开发。一些研究人员设计成功的协议使用二甲基亚砜 (亚砜) 或甲醇作为冷冻16,17,18,19,20。尽管两种协议都已成功使用, 但用亚砜保存的解冻精子所获得的细胞活力低于甲醇20,21。此外, 鳗鱼精子被激活与亚甲基亚砜接触, 需要更繁琐的精子操作19, 因此, 甲醇是一个更适合剂欧洲鳗鲡精子比亚砜。

在这里, 欧洲鳗鲡最佳处理和荷尔蒙治疗的协议将在下面描述。此外, 最好的欧洲鳗鱼精子冷冻保存协议, 使用甲醇作为剂和一个协议的精子质量的评估也将描述。

Protocol

本议定书所述的与欧洲鳗鲡有关的所有程序均由大学 Politècnica de València 的动物实验伦理学委员会批准, 遵循西班牙的法律和法规, 控制实验并活动物的程序。 1. 鱼类保养 把欧洲鳗鲡带到一个研究设施, 并把它们放在一个200升水族馆与循环系统。使用恒温器和冷却机保持恒定的20° c 温度。 保持在黑暗的条件下的鱼, 以避免压力22 , 并在实验中?…

Representative Results

本研究选取了18只有70% 或更高精子活力的鳗鱼精子。结果表明, 与新鲜精子 (表 1和图 2) 相比, 解冻后所有质量参数都有所降低。运动结果 (平均± S.E.M., n=18) 显示了更高的总运动和在新鲜的精子的进步运动比总运动和进步运动发现在后解冻精子样品。 在对快速精子细胞的分析中也发现了同样?…

Discussion

该协议描述了欧洲鳗鲡成熟、处理和精子冷冻保存的完整过程。这里描述的畜牧业条件是最佳的快速成熟和生产高质量精子在这个种类6,7,25。这一冷冻保存协议的成功及其在解冻后的潜在用途很大程度上取决于新鲜精子的质量26。因此, 选择高质量的精子是非常重要的。请注意, 主观的精子质量评估取决于对样…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这份出版物是由欧洲联盟的2020玛丽·斯卡洛多斯卡·居里-居里赠款协议 642893 (打动), 成本办公室 (成本行动法 1205, AQUAGAMETE) 和卓越研究中心资助的研究和创新方案-1476-4/2016/FEKUT。

Materials

AQUACEN BENZOCAÍNA +D2A2:D24 AQUACEN 3394ESP Benzocaine
NaCl Avantor  3624-19
Syringe BD Plastipak 305501 Syringe 1 mL
FC500 Beckman Coulter Flow cytometer
Air pump 100 EHEIM 4011708370032 Modified for suction
Eppendorf 1.5 Eppendorf 175508 Plastic tubes 1.5 mL
Falcon tubes Falcon 175747 Plastic tubes 15 mL
Flexible bucket Fiel 1040101 40 L, Black color.
Ethanol Guinama SL Mg96270
Cyopreservation straws  IMV Technologies 14550 500 µL straws
Live/Dead Sperm Viability Kit Invitrogen L7011 Cytometer Kit
Modelling clay JOVI Art 30 4 different colors
Precision scale PCB KERN & Sohn GmbH PCB35002 Digital scale
Saline solution Vitulia 0.9% Laboratorios Ern, S.A. C.N. 999790.8 Saline solution
Forceps Levantina de Lab. SL 3710025 30 cm metal forceps
Scissors Levantina de Lab. SL 3700014 Scissors 14cm
Ovitrelle (r-hCG) Merck SL EMEA/H/C/000320 Human chorionic gonadotropin
Nikon Eclipse 80i Nikon Microscope
CaCl2 Panreac Quimica SAU 2112211210
Na2SO4 Panreac Quimica SAU 3257091611
NaHCO3 Panreac Quimica SAU 1416381210
782M Camera Proiser 782M Camera for CASA
ISAS v1 Proiser ISAS v1 CASA software
SpermTrack-10 Proiser SpermTrack-10 Countig chamber for CASA
Beaker Pyrex 3L PYREX 2110668 Glass beaker 3L
Flask Pyrex 250 GL-45 PYREX 21801365 Glass flask 250 mL
KCl Scharlab SL PO02000500
Methanol Scharlab SL ME03061000
MgCl2 Scharlab SL MA00370500
BSA Sigma Aldrich 05482 Bovine serum albumina
Styrofoam box 34x34x30 cm and 5cm thick

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Cite This Article
Herranz-Jusdado, J. G., Kása, E., Kollár, T., Gallego, V., Peñaranda, D. S., Rozenfeld, C., Pérez, L., Horváth, Á., Asturiano, J. F. Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation. J. Vis. Exp. (131), e56835, doi:10.3791/56835 (2018).

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