Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes.
Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes.
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DOI：
10.1016/j.cryobiol.2010.07.006
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发表时间：
2010-10
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：
Yang H;Norris M;Winn R;Tiersch TR
青鳉因其体型小、胚胎透明、基因敲除和修饰技术成熟等优点，是公认的生物医学鱼模型。本研究的目的是评估 0.25 毫升法国吸管中精子冷冻保存的两个关键因素：冷冻保护剂和冷却速率。目的是： 1) 评估浓度为 5%、10% 和 15% 的甲醇、2-甲氧基乙醇 (ME)、二甲亚砜 (Me2SO)、N,N-二甲基乙酰胺 (DMA)、N,N,-二甲基甲酰胺 (DMF) 和甘油在 4°C 下孵育 60 分钟的急性毒性； 2) 评估 5 至 25 °C/min 的冷冻冷却速率及其与冷冻保护剂的相互作用，以及 3) 测试用选定冷冻保护剂冷冻保存的解冻精子的生育力和相关冷却速率。冷冻保护剂毒性评估表明，甲醇和ME（5％和10％）30分钟后没有改变精子活力； Me2SO、DMA 和 DMF（10 和 15%）以及甘油（5、10 和 15%）在混合后 1 分钟内显着降低精子的活力。根据这些结果，选择甲醇和 ME 作为冷冻保护剂 (10%)，以不同的冷却速率（从 5 °C/min 到 25 °C/min）进行评估，并与 Me2SO 和 DMF (10%) 进行比较（基于它们在之前出版物中作为冷冻保护剂的用途）。解冻后活力受冷冻保护剂、冷却速率及其相互作用的影响 (P ≤ 0.000)。使用甲醇作为冷冻保护剂，在 10 °C/min 的冷却速率下观察到最高的解冻后活力 (50 ± 10%)。使用 ME 作为冷冻保护剂，在 15 °C/min 的冷却速率下获得了相当的解冻后活力 (37 ± 12%)。使用 DMF，所有冷却速率下的解冻后活力均≤ 10%，明显低于甲醇和 ME。使用 Me2SO 时，在所有冷却速率下解冻后的迁移率均低于 1%，并且明显低于其他三种冷冻保护剂 (P ≤ 0.000)。当将雄性个体的精子分别用 10% 甲醇和 10% ME 以 10 °C/min 的冷却速度和 15 °C/min 的冷却速度冷冻保存时，解冻后的活力没有发现差异。用 10% 甲醇以 10 °C/min 的速度冷冻保存的解冻精子的生育力测试显示，平均孵化率为 70 ± 30%，与新鲜精子 (86 ± 15%) 相当。总的来说，这项研究建立了青鳉高通量精子冷冻保存的基线，为未来的协议标准化和自动化处理设备的使用提供了轮廓。
Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: 1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me2SO), N, N- dimethylacetamide (DMA), N, N,-dimethyl formamide (DMF), and glycerol with concentrations of 5, 10, and 15% for 60 min of incubation at 4 °C; 2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and 3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5 and 10%) did not change the sperm motility after 30 min; Me2SO, DMA, and DMF (10 and 15%) and glycerol (5, 10 and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 °C/min to 25 °C/min) and were compared to Me2SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ≤ 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ≤ 10% which was significantly lower than that of methanol and ME. With Me2SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ≤ 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.
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