1 Zebrafish International Resource Center, University of Oregon , Eugene, Oregon.
2 Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center , Baton Rouge, Louisiana.
Zebrafish. 2018 Jun;15(3):279-290. doi: 10.1089/zeb.2017.1521. Epub 2018 Jan 25.
Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.
精子冷冻保存是一种高效的保存遗传资源的方法。它延长了雄性动物的繁殖期，并显著降低了通常与维持活体动物群体相关的成本。然而，以前的斑马鱼（Danio rerio）冷冻保存方法产生了不同的结果和低的解冻后受精率。为了提高冷冻保存后的解冻后受精率，我们开发了一种新的稀释液和冷冻保护剂（CPM），引入了质量评估（QA），确定了最佳冷却速率，并改进了解冻后的体外受精过程。我们发现高渗稀释液 E400 可以保持精子在冰上至少 6 小时的活力。我们通过使用 NanoDrop 分光光度计测量精子细胞密度和计算机辅助精子分析（CASA）测量精子活力来实施 QA。我们开发了一种包含棉子糖、脱脂乳、甲醇和 bicine 缓冲液的 CPM，RMMB。解冻后活力表明，在两种类型的低温小瓶中的最佳冷却速率在 10 到 15°C/分钟之间。该方法的测试解冻产生了 20%±13%的平均活力和 68%±16%的平均解冻后受精率。
