Abstract This study was performed as part of the effort to develop artificial seed production techniques for stone flounder Kareius bicoloratus, which has a great potential for farming in northeast Asian countries. To develop a sperm cryopreservation protocol that can be applied for artificial fertilization, we carried out experiments on the key factors for successful fish sperm cryopreservation, including cryoprotective agents (CPAs), diluents, equilibration times, and dilution ratios. The CPAs tested in this study were dimethyl sulfoxide (DMSO), glycerol, methanol, and ethylene glycol, and the diluents were glucose and sucrose. The equilibration times tested in the experiments ranged from 0 to 210 s and the dilution ratios ranged from 1:1 to 1:1000. According to the results of our experiments, the optimal CPA for stone flounder sperm cryopreservation was DMSO in concentrations ranging from 7.5% to 10% when 300 mM glucose was used as diluent and DMSO in concentrations ranging from 15% to 17.5% when 300 mM sucrose was used as diluent. Post-thaw sperm motility was high, with equilibration times up to 150 s, and dilution ratios up to 1:10 (semen:CPA + diluent) showed no significant differences in the effect on post-thaw sperm motility. The cryopreserved sperm showed more significant DNA damage than the fresh sperm with the severity of DNA damage being in inverse proportions to post-thaw sperm motility and survival rate. It is expected that these results will be useful for applications of artificial fertilization at stone flounder hatchery farms.

中文翻译：

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冷冻保护剂及处理方法对双色鲽鱼精子冷冻保存的影响

摘要 本研究是开发在东北亚国家具有巨大养殖潜力的石鲽（Kareius bicoloratus）人工种子生产技术的一部分。为了开发可用于人工受精的精子冷冻保存方案，我们对成功进行鱼类精子冷冻保存的关键因素进行了实验，包括冷冻保护剂 (CPA)、稀释剂、平衡时间和稀释比。本研究中测试的 CPA 为二甲亚砜 (DMSO)、甘油、甲醇和乙二醇，稀释剂为葡萄糖和蔗糖。实验中测试的平衡时间范围为 0 到 210 秒，稀释比范围为 1:1 到 1:1000。根据我们的实验结果，石鲽精子冷冻保存的最佳 CPA 是浓度范围为 7.5% 至 10% 的 DMSO，当使用 300 mM 葡萄糖作为稀释剂时，DMSO 的浓度范围为 15% 至 17.5%，当使用 300 mM 蔗糖作为稀释剂时。解冻后精子活力高，平衡时间长达 150 秒，稀释比高达 1:10（精液：CPA + 稀释剂）对解冻后精子活力的影响没有显着差异。冷冻保存的精子比新鲜精子表现出更显着的DNA损伤，DNA损伤的严重程度与解冻后精子的活力和存活率成反比。预计这些结果将有助于石鲽孵化场人工施肥的应用。当 300 mM 葡萄糖用作稀释剂时为 5% 至 10%，而当 300 mM 蔗糖用作稀释剂时，DMSO 的浓度范围为 15% 至 17.5%。解冻后精子活力高，平衡时间长达 150 秒，稀释比高达 1:10（精液：CPA + 稀释剂）对解冻后精子活力的影响没有显着差异。冷冻保存的精子比新鲜精子表现出更显着的DNA损伤，DNA损伤的严重程度与解冻后精子的活力和存活率成反比。预计这些结果将有助于石鲽孵化场人工施肥的应用。当 300 mM 葡萄糖用作稀释剂时为 5% 至 10%，而当 300 mM 蔗糖用作稀释剂时，DMSO 的浓度范围为 15% 至 17.5%。解冻后精子活力高，平衡时间长达 150 秒，稀释比高达 1:10（精液：CPA + 稀释剂）对解冻后精子活力的影响没有显着差异。冷冻保存的精子比新鲜精子表现出更显着的DNA损伤，DNA损伤的严重程度与解冻后精子的活力和存活率成反比。预计这些结果将有助于石鲽孵化场人工施肥的应用。10（精液：CPA + 稀释剂）对解冻后精子活力的影响没有显着差异。冷冻保存的精子比新鲜精子表现出更显着的DNA损伤，DNA损伤的严重程度与解冻后精子的活力和存活率成反比。预计这些结果将有助于石鲽孵化场人工施肥的应用。10（精液：CPA + 稀释剂）对解冻后精子活力的影响没有显着差异。冷冻保存的精子比新鲜精子表现出更显着的DNA损伤，DNA损伤的严重程度与解冻后精子的活力和存活率成反比。预计这些结果将有助于石鲽孵化场人工施肥的应用。