This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me2SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me2SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me2SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN2) vapor for 10 min at 5 cm above the LN2 surface and then submerging them in LN2 for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me2SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me2SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me2SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.

中文翻译：

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养殖太平洋鲍鱼精子的冷冻保存，Haliotis discus hannai

本研究旨在改进养殖太平洋鲍鱼（Haliotis discus hannai）的精子冷冻保存方案。选择二甲基亚砜 (Me2SO)、甘油、乙二醇 (EG)、丙二醇 (PG) 和甲醇作为冷冻保护剂 (CPA)。本实验选择了四种不同的平衡时间（5、10、30 和 60 分钟）和两种类型的平衡温度（4 °C 和 20 °C）。每种 CPA 的大多数平衡温度在不同的平衡时间之间显示出显着差异。5种CPA解冻后精子活力在两种平衡温度下均无显着差异。根据这些结果，选择 8% Me2SO、8% EG、6% PG、2% 甘油和 2% 甲醇来确定 H. discus hannai 精子冷冻保存的最佳条件。解冻后精子活力最高（8% Me2SO: 50.6%, 8% EG: 45.6%, 2% 甘油: 44.5%, 6% PG: 28.7%, 2% 甲醇: 25.4%) 在将精子暴露在液氮 (LN2) 蒸气中 10 分钟后，在液氮 (LN2) 表面上方 5 厘米处，然后将其浸没在 LN2 中至少 2 小时，然后在 60 °C 下用海水解冻，并在 20 °C 下用海水恢复。在这项研究中，选择 8% Me2SO 和 2% 甘油来检查解冻后精子质量，以使用荧光技术估计质膜完整性 (PMI)、线粒体电位分析 (MP) 和顶体完整性 (AI) 的百分比。在用 8% Me2SO 冷冻保存的精子和用 2% 甘油冷冻保存的精子之间没有发现 PMI、MP 和 AI 的显着差异。目前的研究表明，8% Me2SO 最适合于 5 分钟平衡时间的 H. discus hannai 精子冷冻保存，5 厘米的机架高度和 60 °C 的解冻温度。本研究为汉奈铁饼精子提供了比以往研究更有效的冷冻保存方法。