Kong Dewei, Jiang Song, Shi Jianzhi, Yang Qibin, Huang Jianhua, Li Yundong, Ding Yangyang, Wang Jieyi, Qi Xinyu, Liu Tianmi, Zhou Falin
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China.
State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Qingdao 266071, China.
Biology (Basel). 2025 Apr 11;14(4):408. doi: 10.3390/biology14040408.
(black tiger shrimp) is one of the important shrimp species in aquaculture. Cryopreserving its sperm not only provides technical support for breeding but also effectively prevents the decline of genetic resources, promoting the sustainable development of its aquaculture industry. This study screened different types of diluents, cryoprotectants, and concentrations and explored equilibration time, cooling protocols, and thawing conditions, ultimately determining the optimal cryopreservation protocol for sperm. The results showed that the optimal cryopreservation protocol involved using natural seawater as the diluent with 10% dimethyl sulfoxide (DMSO) as the cryoprotectant, in which the sperm suspension and cryoprotectant were mixed at a 1:1 (/) ratio and equilibrated at 4 °C for 30 min. Subsequently, cooling was performed using a programmable controlled-rate freezer: the temperature was reduced to -20 °C at -5 °C/min and held for 5 min; then cooled to -80 °C at -10 °C/min and held for 5 min; finally, the temperature was reduced to -180 °C at -20 °C/min. After cooling, the sperm samples were transferred to liquid nitrogen for long-term storage. The results demonstrated that thawing in a 37 °C water bath achieved the highest sperm motility compared to conditions at 27 °C, 32 °C, 42 °C, and 60 °C. After 15 days of liquid nitrogen storage, the sperm survival rate was 53.33 ± 9.18%. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed that the sperm structure was intact before freezing, with a rounded head, a distinct acrosomal spike anterior to the head, a concentrated nucleus in the head, dense chromatin, and a smooth cell membrane surface. However, after freezing and thawing, the acrosomal spikes of some sperm were fractured, and the membrane structure was damaged. Enzyme activity analysis showed that during liquid nitrogen storage from 0 to 15 days, the enzyme activity of alkaline phosphatase (AKP) and acid phosphatase (ACP) in sperm gradually increased with significant differences observed compared to day 0 ( < 0.05). The activity of malondialdehyde (MDA) showed a gradual increase at 0, 5, and 10 days, but then decreased at day 15. The enzyme activity of catalase (CAT) showed no significant changes from 0 to 10 days ( > 0.05) but significantly increased on day 15 ( < 0.05). The activity of total superoxide dismutase (T-SOD) showed no significant changes from 0 to 5 days ( > 0.05) but significantly increased from days 10 to 15 ( < 0.05). These findings provide valuable insights into the cryopreservation of sperm and will guide the optimization of cryoprotectant combinations and freezing protocols aimed at improving sperm survival rates.
（黑虎虾）是水产养殖中的重要虾类品种之一。冷冻保存其精子不仅为育种提供技术支持，还能有效防止遗传资源衰退，推动其水产养殖业的可持续发展。本研究筛选了不同类型的稀释剂、冷冻保护剂及其浓度，并探索了平衡时间、降温程序和解冻条件，最终确定了精子的最佳冷冻保存方案。结果表明，最佳冷冻保存方案是使用天然海水作为稀释剂，10%二甲基亚砜（DMSO）作为冷冻保护剂，精子悬浮液与冷冻保护剂按1:1（/）比例混合，在4℃平衡30分钟。随后，使用程序控制速率冷冻机进行降温：以-5℃/分钟的速度将温度降至-20℃并保持5分钟；然后以-10℃/分钟的速度冷却至-80℃并保持5分钟；最后，以-20℃/分钟的速度将温度降至-180℃。降温后，将精子样本转移至液氮中进行长期保存。结果表明，与27℃、32℃、42℃和60℃的条件相比，在37℃水浴中解冻可使精子活力达到最高。液氮保存15天后，精子存活率为53.33±9.18%。扫描电子显微镜（SEM）和透射电子显微镜（TEM）观察显示，冷冻前精子结构完整，头部圆润，头部前方有明显的顶体棘，头部细胞核浓缩，染色质致密，细胞膜表面光滑。然而，冷冻和解冻后，部分精子的顶体棘断裂，膜结构受损。酶活性分析表明，在液氮保存0至15天期间，精子中碱性磷酸酶（AKP）和酸性磷酸酶（ACP）的酶活性逐渐升高，与第0天相比差异显著（<0.05）。丙二醛（MDA）的活性在第0、5和10天呈逐渐上升趋势，但在第15天下降。过氧化氢酶（CAT）的酶活性在0至第10天无显著变化（>0.05），但在第15天显著升高（<0.05）。总超氧化物歧化酶（T-SOD）的活性在0至5天无显著变化（>0.05），但在第10至15天显著升高（<0.05）。这些发现为精子冷冻保存提供了有价值的见解，并将指导旨在提高精子存活率的冷冻保护剂组合和冷冻程序的优化。
