长期超低温冷冻保存对鞍带石斑鱼精子超微结构及酶活性的影响

Effects of long-term cryopreservation on ultrastructure and enzyme activity of Epinephelus lanceolatus sperm

为探究长期超低温冷冻保存中鞍带石斑鱼精子质膜、活力、超微结构及酶活性的变化,阐明影响鞍带石斑鱼精子冷冻保存质量的相关机制。实验采集2022年鞍带石斑鱼鲜精及储存时间分别为23、49和61个月的冷冻保存精液,用伊红-苯胺黑染色方法检测精子质膜完整性;用计算机辅助精子分析仪(CASA)检测精子运动参数;测量精浆和精子中琥珀酸脱氢酶(SDH)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)、总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-Px)和肌酸激酶(CK)共6种酶活性的变化及三磷酸腺苷(ATP)浓度变化;用扫描电镜和透射电镜观察鲜精和冻精超微结构。结果显示,伊红-苯胺黑染色后,鲜精质膜完整性最高,为83.43%±2.73%,经过超低温冷冻后,精子质膜完整性显著降低,且随着冷冻保存时间的延长而逐渐降低。CASA结果显示,鲜精活力最高,为90.47%±3.34%,经过超低温冷冻后精子活力显著降低,但长期保存23~61个月的精子活力无显著差异,精子活力保持在(63.95%±3.66%)~(68.58%±2.73%),具有稳定的活力,且鲜精与冻精之间精子平均直线运动速度(VSL)、平均曲线运动速度(VCL)和平均路径速度(VAP)均没有显著差异。精子超微结构显示,鲜精形态结构正常、线粒体排列结构规则、形态大小正常。经过超低温冷冻保存后,精子形态结构损伤明显,表现为精子头部质膜破损、细胞质外漏、细胞核膜破损、尾部鞭毛断裂或脱落等。鞍带石斑鱼精浆和精子超低温冷冻前后6种酶活性的变化及ATP含量结果显示,经过超低温冷冻后,精子内SOD、GSH-Px和CAT及ATP含量均显著降低。精浆中酶活性升高,除GR和CAT外,其余酶活性均差异显著。研究表明,长期超低温冷冻对鞍带石斑鱼精浆和精子酶活性、精子活力及精子超微结构均具有较显著的影响。本研究结果为鱼类精子冷冻损伤机理研究积累了丰富的数据,为鱼类精子长期冷冻保存提供了技术参考和评价指标。

This paper aims to explore the changes in plasma membrane,vitality,ultrastructure and enzyme activity of giant grouper sperm in cryopreservation,and provides a theoretical basis for elucidating the relevant mechan-isms affecting the cryopreservation quality of sperm.The semen of giant grouper was collected and stored for 0,23,49 and 61 months.Plasma membrane integrity was assessed in fresh sperm and cryopreserved sperm by Y-aniline black staining.The motion parameters of giant grouper sperm before and after cryopreservation were ana-lyzed using computer assisted sperm analysis system.Changes in the activity of six enzymes(SDH,CAT,GR,T-SOD,GSH-Px and CK)ATP content were measured for seminal plasma and sperm.The ultrastructure of fresh and cryopreserved sperm was observed by scanning electron microscope and transmission electron microscope.Fresh sperm had the highest plasma membrane integrity of 83.43%±2.73%,and sperm plasma membrane integrity was significantly reduced after cryopreservation(P<0.05).CASA results showed that the highest mobility was 90.47%±3.34%.Sperm motility was significantly reduced after cryopreservation(P<0.05),but its value stayed within the range of 63.95%±3.66%-68.58%±2.73%after 23-61 months of storage.The VSL VCL and VAP value of cryopreserved sperm had no significant difference compared to fresh sperm(P>0.05).The ultrastructure of fresh sperm showed that the sperm morphology and structure were normal,the mitochondria were arranged regularly,with normal morphology and size.After cryopreservation,the sperm morphology and structural damage was obvious.The damage is manifested as sperm head plasma membrane damage,cytoplasmic leak-age,cell nuclear membrane damage,flagella fracture or shedding of the tail.The results of changes in the activities of six enzymes and ATP contents of seminal plasma and spermatozoa before and after cryopreserva-tion freezing of giant grouper sperm showed that after cryopreservation,three enzymes,SOD,GSH-Px and CAT,and ATP contents in spermatozoa were significantly reduced.The enzyme activities in seminal plasma were increased,and all the enzyme activities were significantly different except for GR and CAT.Cryopreser-vation had a great effect on the enzyme’s activities,ultrastructure and spermatozoa energy of fish.The results have accumulated rich data for the mechanism of frozen fish sperm damage,and provided a technical refer-ence and evaluation index for the long-term cryopreservation of fish sperm.