Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.
Cryobiology. 2011 Feb;62(1):62-7. doi: 10.1016/j.cryobiol.2010.12.004. Epub 2010 Dec 17.
The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever's solution prepared at 296mOsmol kg(-1). Sperm were activated with distilled water (24mOsmol kg(-1)) to characterize motility. Maximum motility (90%) was observed within 15s after activation, and sperm remained motile for 35s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ≥287mOsmol kg(-1). To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5°C to -80°C at 10°C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7-15days at -196°C. The highest motility in thawed sperm 61±8% (mean±SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.
本研究专注于开发一种 critically endangered olive barb Puntiussarana (Hamilton, 1822) 的精子冷冻保存方案，该鱼类采集自孟加拉国的两个种群，并在孟加拉农业大学的渔业现场实验室中进行饲养。在 296mOsmol kg(-1) 的 Alsever 溶液中收集精子。用蒸馏水（24mOsmol kg(-1)）激活精子以评估其运动性。激活后 15s 内观察到最大运动性（90%），精子可保持 35s 的运动性。在不同渗透压下评估精子激活情况，当延伸剂的渗透压≥287mOsmol kg(-1) 时，精子完全失去运动性。为了评估冷冻保护剂毒性，将精子分别用 5%、10%和 15%的二甲基亚砜（DMSO）和甲醇平衡。当精子用 15%DMSO 平衡 10min 时，精子运动性明显下降，表明精子对 DMSO 有急性毒性，因此在进一步试验中排除了此浓度。用 5%和 10%DMSO 以及 5%、10%和 15%甲醇对精子进行冷冻保存。采用一步冷冻法（从 5°C 到-80°C，每分钟 10°C）在计算机控制的冷冻器（FREEZE CONTROL® CL-3300；澳大利亚）中进行，将含有精子的 0.25-ml 细管在-196°C 的液氮中储存 7-15 天。解冻后精子的最高运动性为 61±8%（平均值±标准差），使用的是 10%DMSO。冷冻精子的受精率和孵化率分别为 70%和 37%，新鲜精子的受精率和孵化率分别为 72%和 62%。本报告中介绍的方案可用于橄榄巴的孵化场规模生产。冷冻精子的使用可以促进孵化场的运作，并为长期保存遗传资源提供便利，这有助于恢复像橄榄巴这样的极度濒危鱼类。
