【作者】 杨帆；

【导师】 邴旭文；

【作者基本信息】 南京农业大学 ， 农业硕士（专业学位）， 2019， 硕士

【摘要】 翘嘴鳜作为鳜鱼中最主要的养殖品种,具有突出的营养和滋补保健价值,在上世纪我国就开展了人工养殖。如今,形成了从种质保护、良种选育、种苗繁育及培育一套完整的养殖技术。在高密度精养过程中常常会因为养殖水体中氨氮浓度升高对鱼类产生胁迫作用,一旦控制不当则会对生产造成不可避免的损失。本研究通过氨氮胁迫下翘嘴鳜幼鱼抗氧化和非特异性免疫指标的变化,初步探讨了氨氮胁迫下的生理响应。1.氨氮对翘嘴鳜幼鱼的急性毒性试验以翘嘴鳜幼鱼为研究对象,翘嘴鳜幼鱼氨氮的急性毒性试验分别设置0、30、60、90 mg/L 4个浓度梯度组。通过试验得到翘嘴鳜幼鱼总氨氮和非离子氨96h LC50分别为48.65mg/L、0.47mg/L,通过计算得到安全浓度(SC):分别为4.865 mg/L、0.047 mg/L。随着氨氮浓度的升高,翘嘴鳜幼鱼的死亡率迅速升高,90 mg/L浓度梯度组在96h时翘嘴鳜幼鱼存活率不足3%,可以得出翘嘴鳜幼鱼的死亡率与氨氮浓度之间存在正相关关系。2.氨氮胁迫对翘嘴鳜幼鱼鳃部离子酶及抗氧化功能的影响以翘嘴鳜幼鱼为研究对象,试验设置总氨氮96h LC50浓度作为实验浓度,随时间采样,于0、6、12、24、48、96h进行,采集翘嘴鳜幼鱼鳃组织进行离子酶及抗氧化指标的测定。结果表明:翘嘴鳜幼鱼鳃组织离子酶活性在96h内呈现出下降-升高-下降的变化趋势,48 h时离子酶活性显著高于0h时酶活性(P<0.05),96 h时离子酶活性与48 h时活性相比显著下降(P<0.05);翘嘴鳜幼鱼鳃组织中过氧化氢酶和还原型谷胱甘肽活性呈现出升高-下降的趋势,其中在24h时两者活性都显著高于0h时活性(P<0.05),并在48-96h内活性显著下降(P<0.05);翘嘴鳜幼鱼鳃组织中超氧化物歧化酶和过氧化物酶活性在48 h内呈现出下降-升高-下降的趋势,在48-96 h内超氧化物歧化酶活性无差异显著(P>0.05),而过氧化物酶活性在该时间内差异显著(P<0.05)。研究表明,在短期急性氨氮毒性试验下,翘嘴鳜幼鱼鳃组织离子酶和抗氧化相关指标整体上呈现出先升高后下降的特性,对其抗氧化功能着明显的影响。在养殖过程应注意水体氨氮的变化,减少氨氮对鱼体生长带来的影响。3.氨氮胁迫对翘嘴鳜幼鱼头肾中免疫相关酶活性和基因表达量的影响以翘嘴鳜幼鱼为研究对象,试验设置总氨氮96h LC50浓度作为实验浓度,随时间采样,于0、6、12、24、48、96h进行,采集翘嘴鳜幼鱼头肾组织进行免疫相关酶活性和基因表达量的测定。结果表明:翘嘴鳜幼鱼头肾中过氧化氢酶、碱性磷酸酶和酸性磷酸酶活性呈现出先升高再下降的趋势,在氨氮胁迫的48h时3种酶活性都显著高于0h时(P<0.05),48-96 h时过氧化氢酶和酸性磷酸酶活性显著下降(P>0.05),96 h时碱性磷酸酶活性相比48 h时略有下降但无显著差异(P>0.05);翘嘴鳜幼鱼头肾中IRF-1基因表达量呈现出先下降再升高的趋势,96 h时IRF-1基因表达量显著高于0h时(P<0.05);翘嘴鳜幼鱼头肾中IL-1β基因表达量整体呈现出低于0h时水平,但24hIL-1β基因表达量显著高于0h时(P<0.05);翘嘴鳜幼鱼头肾中IL-8基因表达量呈现出下降的趋势,96 h时IL-8基因表达量显著低于0h时(P<0.05)。通过试验看出,在短期急性氨氮毒性试验下,翘嘴鳜幼鱼头肾中免疫相关酶活性呈现出先升高再下降的特性,免疫相关基因表达量上在96h时处在低于0h时水平,可知氨氮胁迫下对翘嘴鳜幼鱼免疫功能产生了影响。在养殖过程应注意水体氨氮的变化,氨氮浓度过高影响鱼体正常的免疫功能,易遭受病害威胁,对生产造成巨大损失。更多还原

【Abstract】 As the most important breeding species of Siniperca,mandarin fish has prominent nutritional and health value.In the last century,mandarin fish was cultivated artificially in China.Nowadays,a complete set of breeding techniques has been formed,including germplasm protection,breeding of improved varieties,fry breeding and cultivation.In the process of high density intensive farming,the high concentration of ammonia nitrogen in aquaculture water often exerts stress on the fish.Once improperly controlled,it will cause unavoidable losses to production.In this study,the physiological responses of juvenile mandarin fish to ammonia-nitrogen stress were studied through the changes of antioxidant and non-specific immune indices.1.Acute toxicity of ammonia nitrogen to juvenile mandarin fish was studied with 0,30,60 and 90 mg/L concentration gradient groups.The results showed that the total ammonia nitrogen LC50 for 96h and non-ionic ammonia LC50 for 96h were 48.65 mg/L and 0.47 mg/L respectively.According to the formula of safe concentration(SC),the safe concentration of total ammonia nitrogen and non-ionic ammonia were 4.865 mg/L and 0.047 mg/L,respectively.With the increase of ammonia nitrogen concentration,the mortality of juvenile mandarin fish increased rapidly.The mortality of juvenile mandarin fish in the 90 mg/L experimental group reached 97%at 96 h.That is to say,the survival rate of juvenile mandarin fish was negatively correlated with ammonia nitrogen concentration.2.In this study,the concentration of total ammonia nitrogen 96h LC50 was set as the experimental concentration,and the gill tissues were sampled at 0,6,12,24,48 and 96 hours for the determination of ionase and antioxidant index.The results showed the activity of ionase in gill tissue of juvenile mandarin fish showed a decreasing-increasing-decreasing trend within 96 hours.At 48 hours,the activity of ionase was significantly higher than that at 0 hours(P<0.05),and at 96 hours,the activity of ionase was significantly lower than that at 48 hours(P<0.05).The activities of catalase and reduced glutathione in gill tissues of juvenile mandarin fish showed a trend of increase-decrease.The activities of catalase and reduced glutathione at 24 hours were significantly higher than those at 0 hours(P<0.05),and decreased significantly within 48-96 hours(P<0.05).The activities of superoxide dismutase and peroxidase in gill tissue of juvenile mandarin fish showed a downward-upward-downward trend within 48 hours,but there was no significant difference between 48 and 96 hours(P>0.05),while the activities of peroxidase were significantly different during that time(P<0.05).The results showed that in the short-term acute ammonia-nitrogen toxicity test,the ionase and antioxidant related indexes in gill tissue of juvenile mandarin fish increased first and then decreased,which had a significant impact on its antioxidant function.In the process of aquaculture,attention should be paid to the change of ammonia nitrogen in water to reduce the impact of ammonia nitrogen on fish growth.3.The total ammonia nitrogen concentration of 96h LC50 was set as the experimental concentration,and the time gradient was used to sample the juvenile mandarin fish at 0,6,12,24,48 and 96 hours.The activity of immune-related enzymes and gene expression were measured in the head kidney tissue of juvenile mandarin fish.The results showed that the activities of catalase,alkaline phosphatase and acid phosphatase in the head kidney of juvenile mandarin fish increased first and then decreased.At 48 h of ammonia nitrogen stress,the activities of three enzymes were significantly higher than those at 0 h(P<0.05),catalase and acid phosphatase activities decreased significantly at 48-96 h(P>0.05),while at 96 h,the activities of alkaline phosphatase decreased slightly compared with those at 48 h(P>0.05),but there was no significant difference(P>0.05).The expression of IRF-1 gene in head kidney of juvenile mandarin fish decreased first and then increased.The expression of IRF-1 gene in head kidney of juvenile mandarin fish at 96 h was significantly higher than that at 0 h(P<0.05);the expression of IL-1 beta gene in head kidney of juvenile mandarin fish at 24 h was significantly higher than that at 0 h(P<0.05);the expression of IL-8 gene in head kidney of juvenile mandarin fish was significantly lower than that at 0 h(P<0.05).Trend:The expression of IL-8 gene at 96 h was significantly lower than that at 0 h(P<0.05).The results showed that in the short-term acute ammonia nitrogen toxicity test,the activity of immune-related enzymes in head kidney of juvenile mandarin fish increased first and then decreased.The expression level of immune-related genes was lower than 0 h at 96 h.It was known that ammonia nitrogen stress had an impact on the immune function of juvenile mandarin fish.In the process of aquaculture,attention should be paid to the change of ammonia nitrogen in water.Excessive concentration of ammonia nitrogen affects the normal immune function of fish,which is vulnerable to the threat of diseases and causes huge losses to production.更多还原