Sperm cryopreservation of green swordtail Xiphophorus helleri, a fish with internal fertilization
Sperm cryopreservation of green swordtail Xiphophorus helleri, a fish with internal fertilization
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DOI：
10.1016/j.crybiol.2004.02.004
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发表时间：
2004-06-01
期刊：
影响因子：
2.7
通讯作者：
Tiersch, TR
中科院分区：
文献类型：
--
作者：
Huang, CJ;Dong, QX;Tiersch, TR
尽管许多鱼类是有价值的生物医学研究模型，但内部受精鱼类精子冷冻保存基本上是未知的。为探索剑尾鱼属活鱼精子冷冻保存的方法，本研究采用X。Helleri的研究，以评估冷冻保护剂、渗透压、冷却速率、平衡时间和精子与扩展剂比率的影响。不同冷冻保护剂对精子活力和解冻后存活时间的影响有显著性差异，其中14%甘油组解冻后10 min精子活力最高。随后使用14%甘油作为冷冻保护剂，在300 mOsmol/kg的Hanks平衡盐溶液（HBSS）中观察到解冻后的最高运动性。以20 ℃/min从5 ℃冷却至-80 ℃的样品产生了最高的解冻后运动性，尽管在解冻后的前4小时内，在20-35 ℃/min的冷却速率范围内没有发现显著差异。平衡时间的评估显示，20 min和2 h之间没有显著差异，但在解冻后10分钟时发现平衡20分钟时的运动性最高。1：20、1：60和1：120的精子稀释液对解冻后精子活力和存活时间无显著影响，但解冻后立即用HBSS（320 mOsmol/kg）稀释精子溶液可减轻精子活力的下降，并显著延长存活时间。基于这些发现，当精子以300 mOsmol/kg的浓度悬浮于HBSS中，用14%甘油作为冷冻保护剂，以精子与HBSS-甘油1：20的比例稀释，平衡10 min，以20 ℃/min的速度从5 ℃冷却至-80 ℃，然后投入液氮中，并在40 ℃水浴中解冻7 s时，解冻后10 min的平均精子活力最高（77%）。如果解冻后立即稀释，按上述方案冷冻的精子在4 ℃下储存时，解冻后仍能保持连续活力8天以上。(C)2004年爱思唯尔公司All rights reserved.
Sperm cryopreservation for fishes with internal fertilization is essentially unexplored although many species of these fishes are valuable biomedical research models. To explore methods for sperm cryopreservation within the live-bearing genus Xiphophorus, this study used X. helleri to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio. Sperm motility and survival duration after thawing showed significant differences among different cryoprotectants with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as the cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) at 300 mOsmol/kg. Samples cooled from 5 to -80 degreesC at 20 degreesC/min yielded the highest post-thaw motility although no significant difference was found in the first 4 h after thawing for cooling rates across the range of 20-35 degreesC/min. Evaluation of equilibration time revealed no significant difference between 20 min and 2 h, but the highest motility at 10 min after thawing was found with a 20-min equilibration. Dilution ratios of sperm-to-extender at 1:20, 1:60, and 1:120 showed no significant differences in motility and survival duration after thawing, but the dilution of sperm solutions with HBSS (320 mOsmol/kg) immediately after thawing reduced the decline of sperm motility, and significantly prolonged the survival duration. Based on these findings, the highest average sperm motility (77%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsmol/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 20 degreesC/min from 5 to -80 degreesC before being plunged in liquid nitrogen, and thawed in a 40 degreesC water bath for 7 s. If diluted immediately after thawing, sperm frozen by the protocol above retained continuous motility after thawing for more than 8 days when stored at 4 degreesC. (C) 2004 Elsevier Inc. All rights reserved.