【作者】 赵建华；

【导师】 熊邦喜； 杨德国；

【作者基本信息】 华中农业大学 ， 水产养殖， 2014， 博士

【摘要】 长江是我国第一大河,共有鱼类约350余种,是我国鱼类资源的宝库。长江上游以山区为主,坡陡水急,流态复杂多样,特有鱼类数量达124种。近年来,由于各类水电工程设施的兴建,长江流域生态环境急剧恶化,鱼类洄游通道受阻、造成生境破碎、产卵场和栖息地被破坏或丧失,加之缺乏严格和科学的管理,多种鱼类资源接近衰竭。长江鱼类特别是珍稀特有鱼类资源已经成为目前急需解决的问题。圆口铜鱼隶属于鲤形目、鲤科、鮈亚科,属河道洄游鱼类,产漂流性卵,是长江上游特有鱼类和重要的经济鱼类。然而,近年来,由于过度捕捞,特别是受水电工程设施建设等人类活动的影响,圆口铜鱼资源量急剧下降,渔获物中低龄鱼和幼鱼个体比重逐年增加。更为严峻的是,长江干流和金沙江中下游水电工程的建设,已经阻挡了圆口铜鱼的洄游通道,并将淹没其大部分产卵场。如果不采取有效措施,这种长江上游特有的重要经济鱼类将可能面临灭绝境地。增殖放流被认为是保护珍稀濒危鱼类的有效措施之一,驯养足够的亲鱼是增殖放流的前提。然而,由于圆口铜鱼具有激烈的应激反应特征,且在人工养殖时极易感染小瓜虫病,死亡率较高,导致人工驯养圆口铜鱼难度较大。目前,除了零星的试验性养殖外,尚未见室内驯养成功的报道。本研究针对圆口铜鱼移养驯化过程中的关键操作程序,通过对圆口铜鱼正常、不同程度操作胁迫、麻醉与低温运输状态下其血液生理生化、关键组织形态结构以及应激蛋白HSP70/HSC70变化特征的观测,探究圆口铜鱼应激反应的基本规律,并寻找缓解圆口铜鱼应激反应的措施,具体内容及主要研究结果如下：对船体网箱养殖近一年的圆口铜鱼血细胞形态及生理生化参数进行了测定,建立了网箱养殖圆口铜鱼生理生化指标的参考区间。同其他鱼类相比,圆口铜鱼血液皮质醇基础水平较高(185.70ng/mL),这可能是和其具有发达的肝胰联合体、摄食能力强、代谢水平高、喜急流环境等生物学习性有关。加之圆口铜鱼驯化程度低,对人工养殖环境适应周期长等因素导致其基础皮质醇处于较高水平。和野生圆口铜鱼相比,网箱养殖圆口铜鱼血糖、甘油三酯、总蛋白和球蛋白水平均明显偏高,这可能是由于人工养殖环境下圆口铜鱼能获得更加充足的高能饵料,以及网箱高密度养殖条件下鱼类的活动范围被限制、活动量减少等原因所致。对网箱养殖圆口铜鱼施以低强度(30s)和高强度(60s)的急性离水胁迫,并分别于刺激后的0、1、4、12、24、72、120、168h采取血液、肝、鳃、肾组织,测定血液生理生化参数的变化特征及关键组织器官的形态结构变化。结果显示,不同强度操作胁迫刺激后的前24h,圆口铜鱼均表现出以血液皮质醇、溶菌酶活性、葡萄糖和谷丙转氨酶急剧升高为典型特征的急性应激反应。组织形态学的观察显示,不同强度的离水胁迫均可诱发圆口铜鱼鳃、肾形态结构的变化或损伤。离水胁迫后的24h发现两处理组均自然感染了小瓜虫,且随时间的延长有加剧的趋势,至实验结束时(168h),虽然圆口铜鱼血液皮质醇、谷丙转氨酶、谷草转氨酶水平已回复到正常水平,但其血液葡萄糖、乳酸和溶菌酶活性均维持在较高水平,说明圆口铜鱼依旧处于疾病感染和适应新环境的慢性应激阶段。通过行为观察和实践运输成活率等预实验研究,得出适于圆口铜鱼长途运输的MS-222的麻醉浓度为20-40mg/L。为考查圆口铜鱼对长途运输胁迫的应激反应特征和麻醉、低温以及二者联合使用对圆口铜鱼长途运输应激反应的缓解效果,设置了常温不麻(Hna)、常温麻醉(Ha)、低温不麻(Lna)和低温麻醉(La)四个处理组,于运输后的0h采取水样,并分别于运输后的0、4、24、72和168h采取血液。检测结果显示,经14h的长途运输,四个处理组总氨氮(TAN)、非离子氨氮(NH3-N)、亚硝酸氮(NO2-N)和总氮(TN)均明显升高(P<0.05)；且除NO2-N外,这些指标在高温处理组的浓度均明显高于低温处理组。运输后及恢复过程中,血液皮质醇、葡萄糖、乳酸、转氨酶等生化指标多数呈现出明显的先升高后降低的典型急性应激反应特征。麻醉处理在抑制运输过程中圆口铜鱼的活动、能量消耗方面作用明显,低温处理则在抑制长途运输过程中圆口铜鱼皮质醇的升高、提高溶菌酶活力、降低鱼类活动方面发挥重要作用。因此,建议在高温条件下对圆口铜鱼进行长途运输时,低温麻醉处理是首选,其次是单独使用低温或MS-222处理,而且运输前的禁食时间控制在48-72h为宜。此外,运输后的前两周圆口铜鱼尚处在急性或慢性应激状态,此期间要特别注重鱼体健康护理。利用圆口铜鱼心脏组织克隆出HSC70和HSP70基因全长的cDNA序列,其cDNA序列全长分别为2330bp、2290bp,编码氨基酸数目为649个、643个。通过比对分析发现,圆口铜鱼HSC70和HSP70两种基因与其他动物的同源性较高。组织差异性表达结果显示,HSP/HSC70基因mRNA在圆口铜鱼不同组织中的表达有所差异,HSP70和HSC70在不同组织中的表达量从高到低依次分别为肌肉、眼、肾、鳃、脑、脾、心脏和鳍,以及肝、眼、鳃、鳍、心脏、脾、脑、肌肉和肾脏。急性操作胁迫过程中圆口铜鱼HSC70除在肝中的表达外,HSP70/HSC70两种基因mRNA在其他不同组织中的变化并不明显,与血液生理生化指标的变化一致性程度较低。因此,在圆口铜鱼的急性操作胁迫和长途运输胁迫的应激评估中,不易将HSP70和HSC70作为应激的评价指标,此方面的研究有待于深入。更多还原

【Abstract】 As the longest river of china, Yangtze River is a treasure trove of fish resources fostering more than350fish species. The upper reaches of the Yangtze River is mainly located in the mountainous areas with steep slopes and swift streams.Due to the complicated flow pattern there are approximately124kinds of endemic fish. In recent years, as a result of the damming, thefish migration routes had been blocked, causing habitat fragmentation, spawning grounds and habitat destruction or loss. To be worse, a variety of fish stocks are close to be exhausted due to the lack of strict and scientific management. Consequently, species conservation of the endemic fish in Yangtze River are badly in need. Largemouth bronze gudgeon, Coreius guichenoti belongs to Cyprinidae in the Cypriniformes, and is one of the rare endemic fishes in the upper Yangtze River in China. The fish is a potamodromous migration species living in schools and producing pelagic eggs, and can usually be found in rivers with torrential flow. Due to its tender meat, delicious taste and high nutrition, C. guichenoti used to be an important economic fish in the upper Yangtze River. However, in recent years, the catch of this species has declined dramatically and the fish tends to become stunted owing to overcatching and construction of hydroelectrical projects. The construction of hydropower stations in the Yangtze River and in the middle and lower reaches of the Jinsha River has blocked most of the passage of C. guichenoti and submerged most of its spawning fields. This endemic and important economic fish is facing the threat of extinction if measures are not taken in the succeeding period.Fishery stock enhancement and releasing has been proved to be an effective measure to protect the rare and endangered fish species. Culturing adequate broodstocks is the precondition to start the artificial propagation, but intense stress response and high susceptibility to ichthyophthiriasis are critical challenges to the collection, domestication and culture of C. guichenoti during the aquaculture practice. To date, the culture of C. guichenoti can only be performed in cages of vessels near the shore in the upper reaches of the Yangtze River from Luzhou to Hejiang. To the best of our knowledge, no report has been published about the successful indoor culture of C. guichenoti, except for experimental culture. For the key parts of the transfering and domestication in C. guichenoti, we tested the changes of blood physiology and biochemistry, the morphology and strcture of the key tissues, as well as the stress protein HSP70/HSC70. We tried to explore the basic laws of stress response of Coreius, and find measures to mitigate the corresponding stress response. And the specific content and key findings are as follows:We tested the morphology of blood cell and physiological and biochemical parameters of C. guichenoti cultured for almost one year in the cage; and established the corresponding reference intervals. Compared with other fish, the blood cortisol levels of C.guichenoti remained at a relatively higher level (185.70ng/mL), which may be related to the preference and physiological traits of C.guichenoti, such as preference for torrent, high swimming speed, and long-distance migration, well-developed liver and pancreas combination, and high efficiency in ingestion, digestion, and transformation. In addition, C.guichenoti the low degree of domesticatiton and the long period adapt to farmed environment may cause the high cortisol level. Compared with the wild C.guichenoti, the levels of glucose, triglycerides, total protein and globulin are singnificantly higher. This can be attributed to the large amount of high-energy food (relatively high protein, lipid and carbohydrates) that the fish get and the reduction of activities in the culturing environment versus the wild.We expoursed the Cage-cultured C.guichenoti to a low intensity(out of water for30s) and high intensity(out of water for50s) acute handling stress; and the tissues including blood, liver, gill and kidney were sampled at0,1,4,12,24,72,120and168h after the handling stress. We tested the changes of the blood physiology and biochemistry as well as the morphology and strcture of the key tissues. The results suggest that the treated fish all displayed typical acute stress response including the abrupt increase in plasma cortisol, lysozyme, glucose and ALT. the abservation of tissues morphology suggest that the low and high intensity acute handling stress both caused the changes or damage of the gill and kidney. The treated fish in both groups were spontaneously infected with Ichthyophthirius multifiliis. By the end of the experiment168h), the of plasma glucose, lysozyme and lactate remainded at relitively high levels, although the levels of plasma cortisol, ALT and AST had retained to the resting levels. This suggested that the fish also remainded in the state of diseases and adapting the new environment of chronic stress.We get the appropriate anaesthesia concentration of MS-222(20-40mg/L) used in the long-distance transport of C.guichenoti by the preliminary experiment including behavior observation and the survival rate of the pratical transport. In order to investigate the basic characteristics of the physiological stress response of fish subjected to transport stress and evaluate the effects of MS-222anesthesia, temperature reduction and the combination of both practices as sedative during transportation, we set up the four groups including Ana (ambient temperature with no anesthetic) group, Aa (ambient temperature with MS-222anesthetic) group, Lna (low temperature with no anesthetic) group, and La (low temperature with MS-222anesthetic) group. And we sampled the blood at0,4,24,72and168h. The results suggested the concentrations of TAN, NH3-N, and TN among the four treatment groups were significantly higher than before transport. Most of the examined plasma biochemical parameters, such as plasma COR, GLU, LA, ALT, and AST, displayed an increase, followed by a gradual decrease, similar to the stress responses of other bony fish. MS-222anesthesia, temperature reduction or the combination of the both had the effect of decreasing the metabolism and activity of C. guichenoti to different degrees. The results suggest that for a successful long-term transportation of C. guichenoti employing the combination of temperature reduction and MS-222is most preferable, followed by the use of temperature reduction alone, or MS-222at a relatively high temperature, and the fasting period before transport will be ensured for48-72h. In additon, the fish also remained in the acute or chronic stress state in the first two week after the transport, so it is critical to nurse the fish.Full length cDNAs of HSC70and HSP70were cloned from the liver SMART cDNA library. The full length cDNA sequences of HSC70and HSP70are2,330bp and2,290bp respectively and encoding649amino acids and643amino acids respctively. Multiple amino acid sequence alignment suggests that the two gens HSC70and HSP70have high homology to other animals. Tissue distribution analysis revealed that the transcription levels of HSP70and HSC70were different in various tissues, with HSP70transcribed in a reduced trend in muscle, eye, kidney, gill, brain, spleen, heart and fin, together with HSC70transcribed most in liver followed by eye, gill, fin, heart, spleen, brain, muscle and kidney. Under acute handling stress, the transcription levels of HSP70and HSC70in different tissues changed not variously except in livers, and showed little consistency with the changes of the parameters of blood physiology and biochemistry. Therefore, it is not applicable to include the transcription levels of HSP70and HSC70as one of the evaluation parameters during the acute handling stress and long-term transportation stress in C. guichenoti, and further research is needed in this area.更多还原