Yang Huiping, Hu E, Buchanan John T, Tiersch Terrence R
School of Forest Resources and Conservation, IFAS, University of Florida, 7922 NW 71 Street, Gainesville, FL 32653.
Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources Louisiana State University Agricultural Center, 2288 Gourrier Avenue, Baton Rouge, LA 70820.
J World Aquac Soc. 2018 Feb;49(1):96-112. doi: 10.1111/jwas.12431. Epub 2017 May 18.
Sperm cryopreservation is an essential tool for long-term storage of genetic resources for aquaculture fishes. The goal of this study was to develop an efficient and streamlined protocol for high-throughput processing for sperm cryopreservation in Atlantic salmon, . The objectives were to evaluate: 1) osmolality of blood serum for determining extender osmolality; 2) effects of extenders for fresh sperm dilution and refrigerated storage; 3) effects of methanol and dimethyl sulfoxide (DMSO) on fresh sperm motility, and 4) motility and fertility after thawing. In this study, sperm samples were collected at a hatchery site in Canada, and shipped to a freezing site located 2200 miles (3550 km) away in the United States. Evaluation of three extenders indicated that Mounib solution was suitable for diluting dry sperm for sample processing. Ten percent of methanol or DMSO was less toxic to sperm cells than was 15% within 30 min. Further testing with methanol at 5, 10, and 15%, and sperm solution:extender dilutions (v:v) of 1:1, 1:3, 1:19 (at concentrations of ~5×10; 3×10, and 1×10 cells/mL) indicated that methanol at 5% and 10% showed less toxicity to fresh sperm within 1 hr at sperm: extender dilutions of 1:1 and 1:3. Post-thaw motility of sperm cryopreserved with 10% methanol was significantly higher than that with 10% DMSO, and fertility reflected those results (0-1% in DMSO vs. 38-55% in methanol). Further evaluation of sperm cryopreservation with 10 and 15% methanol at sperm dilution ratios of 1:1, 1:3, 1:19 indicated post-thaw motility in 10% methanol was significantly higher than that in 15% methanol, and post-thaw fertility in 10% methanol at 1:1 and 1:3 dilution ratios had fertilization rates similar to that of fresh sperm controls. Sperm samples from 12 males cryopreserved with 10% methanol showed male-to-male variation in post-thaw motility (0-36%). Overall, a simplified standard protocol was established for cryopreservation of shipped sperm of Atlantic salmon using extender without egg yolk and yielded satisfactory post-thaw motility and fertilization rates. This procedure can be readily adopted by aquaculture facilities to take advantage of high-throughput cryopreservation capabilities at remote service centers. Most importantly, this approach lays the groundwork for an alternative commercial model for commercial-scale production, quality control and development of industrial standards. Control of male variability and sperm quality remain important considerations for future work.
精子冷冻保存是水产养殖鱼类遗传资源长期储存的一项重要手段。本研究的目的是开发一种高效且简化的方案，用于大西洋鲑鱼精子冷冻保存的高通量处理。目标是评估：1）血清渗透压以确定稀释液渗透压；2）稀释液对新鲜精子稀释和冷藏保存的影响；3）甲醇和二甲基亚砜（DMSO）对新鲜精子活力的影响，以及4）解冻后的活力和受精率。在本研究中，精子样本在加拿大的一个孵化场采集，并运往2200英里（3550公里）外位于美国的冷冻地点。对三种稀释液的评估表明，穆尼布溶液适用于稀释干精子以进行样本处理。在30分钟内，10%的甲醇或DMSO对精子细胞的毒性低于15%的甲醇或DMSO。进一步用5%、10%和15%的甲醇以及精子溶液与稀释液的体积比为1:1、1:3、1:19（细胞浓度分别约为5×10、3×10和1×10个/毫升）进行测试，结果表明，在精子与稀释液体积比为1:1和1:3时，5%和10%的甲醇在1小时内对新鲜精子的毒性较小。用10%甲醇冷冻保存的精子解冻后的活力显著高于用10%DMSO冷冻保存的精子，受精率也反映了这一结果（DMSO组为0 - 1%，甲醇组为38 - 55%）。进一步评估在精子稀释比为1:1、1:3、1:19时用10%和15%甲醇进行精子冷冻保存，结果表明，解冻后10%甲醇组的精子活力显著高于15%甲醇组，在1:1和1:3稀释比下，10%甲醇组解冻后的受精率与新鲜精子对照组相似。用10%甲醇冷冻保存的来自12条雄鱼的精子样本解冻后的活力存在雄鱼个体间差异（0 - 36%）。总体而言，建立了一种简化的标准方案，用于使用不含蛋黄的稀释液对运输来的大西洋鲑鱼精子进行冷冻保存，解冻后的活力和受精率令人满意。水产养殖设施可轻易采用此程序，以利用远程服务中心的高通量冷冻保存能力。最重要的是，这种方法为商业规模生产、质量控制和行业标准制定的替代商业模式奠定了基础。控制雄鱼个体差异和精子质量仍是未来工作的重要考虑因素。
