暗纹东方鲀（Fugu obscurus）呼吸、消化生理生态及皮肤主要生化组分研究

【作者】 王培军；

【导师】 赵清良；

【作者基本信息】 南京师范大学 ， 动物学， 2003， 硕士

【摘要】 暗纹东方鲀（Fugu obscurus）以其独特的经济和科学价值而日益为人们所关注，本文着重从呼吸及消化生理生态（包括呼吸耗氧和摄食消化状态）、皮肤胶原蛋白和肾脏细胞原代培养等三个方面对其进行了较全面的研究，以期为暗纹东方鲀的基础研究、科学养殖以及产品的深度开发提供理论依据。 在暗纹东方鲀规模化养殖过程中早期鱼苗的养殖密度一般都比较高，容易出现鱼苗在高温天气下因缺氧而大规模死亡的情况，因此无论从实践上还是理论上研究暗纹东方鲀鱼苗的呼吸过程和对溶氧的耐受性规律都是十分必要的。为此我们对其幼鱼一天24h的呼吸耗氧状况进行了较详细的跟踪研究。实验发现无论是规格大一些的还是小一些的幼鱼，它们在24h内的呼吸耗氧曲线基本上是一致的即：白天耗氧明显高于夜间，而且都出现几个类似的耗氧高峰和耗氧低谷。但是不同大小的鱼苗耗氧量(mg·尾^-1·h^-1和耗氧率(mg·g^-1·h^-1)存在明显的差异，规格、体重越大，其耗氧量也越大，相反其耗氧率则越低。我们的研究还发现暗纹东方鲀鱼苗的呼吸窒息点（mg/L）远远低于同种规格的四大家鱼、大口鲶等鱼种，因此暗纹东方鲀鱼苗在养殖过程中更容易受到水体溶氧不足的威胁。我们对暗纹东方鲀鱼苗呼吸窒息点、耗氧量、和耗氧率的研究为在养殖过程中有效地预防鱼苗缺氧事故的发生，合理安排活鱼运输，提供了理论依据，从而可以大大提高养殖的经济效益。 投饵次数、投饵的间隔时间和投饵率长期以来一直为水产养殖界所关注，因为这不但关系到鱼类的生长发育，同时也关系到养殖的最终经济效益。为此我们对暗纹东方鲀三种规格大小鱼苗在20℃、24℃、28℃三种不同水温条件下的饱食量、摄饵率进行了比较研究，并对一天24h的消化速度、食物在消化道内的滞留时间进行了详细的观察测定，绘制了相应的消化曲线图。结果证明温度能够明显的影响鱼苗的消化状况，在一定范围内温度越高相应的摄饵率、饱食量也越高，消化速度也越快，排粪量也越多，食物在消化道内的滞留时间则越短。试验还表明在相同环境条件下鱼体越小摄饵率越高，饱食量则越低，食物在消化道内的滞留时间也越短。总体上暗纹东方鲀鱼苗的消化曲线成倒“S”型，摄饵开始一段时间消化较平缓，接下来是一段较快消化的过程，而温度的升高能明显加速这一过程。一次摄入饵料的整个消化过程（至排空）大约需要持续20h左右。根据这一规律，结合实际情况，我们可以安排合理科学的投饵方式，既能满足鱼的正常所需，减少浪费，又能保持水质清洁和较高的溶氧。 暗纹东方鲀皮肤结构特殊，富含胶原蛋白，而且易于提取。随着近年来人们对胶原蛋白的认识不断深入，进一步研究暗纹东方鲀皮肤胶原蛋白生化组分对深入了解暗纹东方鲀的营养价值、资源的综合利用以及提高其经济效益等有着重要意义。本试验对暗纹东方鲀皮肤的生化组分进行了测定，确定其含有较高水平的胶原蛋白。在此基础上参照文献方法，以酸提取法和胃蛋白酶促溶法对皮肤胶原蛋白进行提取。试验发现暗纹东方鲀皮肤胶原蛋白极易提取，在弱酸性条件下即可提取绝大部分的胶原蛋白，酸溶性胶原蛋白占总胶原蛋白产量的82％左右。对试验提取的ASC和PSC进行SDS-PAGE凝胶电泳，电泳条带显示ASC和PSC无明显差异，进一步氨基酸分析，说明所提成分符合胶原蛋白特征。 水产动物细胞培养在病毒分离和纯化、疾病防治、水体监测等方面具有重要意义。本试验以暗纹东方鲀肾脏组织细胞为材料就其原代培养方法进行了初步研 南京师范大学硕士论文 王培军 摘 要究和探索。试验以TC－199为基础培养基，添加了1790020％左右小牛血清，在25OC环境下分别进行组织块贴壁培养和细胞悬液培养。整个试验操作过程保持严格无菌，并及时更换新鲜培养液。经过一周左右的培养之后，培养瓶局部可形成致密单层，细胞单一长纤维状，呈放射状向四周扩散。经0．25％胰酶消化，传代后细胞仍然能够生长增殖，但活力明显降低，仅有极个别区域可形成群落，但最终细胞崩解成碎片死亡，仅留灰色网状痕迹。试验历经了长达半年的反复探索，虽然仍未达到预期的理想效果，但为以后的继续深入研究积累了经验。更多还原

【Abstract】 Fugu Obscurus has been taken into account more and more because of its particular economic value and science research. In this paper the fundamental physiology, ecology and biochemistry on Fugu obscurus were studied, which included oxygen consumption, food retention time, collagen and cell culture. These experimentations tried to provide fundamental theories for progressively research on them.Based on the facts that fishes tend to suffocate in high water temperature if they were fed too densely, the oxygen consumption of Fugu obscurus fry was studied within 24 hours. The results showed that the curves of oxygen consumption were almost agreeable between A and B size of fry. Oxygen consumption in daytime was much higher than in night. Both in daytime and in night had a couple of higher and lower crest value in oxygen consumption within 24 hours. The oxygen intake and the oxygen density causing suffocation increased following the size of fry amplification, but the rate of oxygen consumption was just contrary to the size of fry. According to these results, we could make a reasonable arrangement to deal with the threat caused by oxygen consumption, and the transport of the living fishes.How to feed fishes is an important factor that effects the growth of them and the development of fish farming. The full ingestion, feeding rate and the food retention time of Fugu obscurus fry were discussed in this paper under the water temperature of 20 C,24 C,28 C conditions. In this range of physiological temperature, the results showed that the water temperature effected the digestion of Fugu obscurus fry evidently, and that the full ingestion, the defecation capacity were much more increasing, and the digestion speed was also more accelerating, but the food retention time became shorting. Under the same water temperature conditions, the full ingestion and the retention time increased with the enlargement of the fries, but the feeding rate declined with it. We could see that the digestive curve of Fugu obscurus fry was always appearing the form of reversed "S". At the beginning and end of the phase, digestion speed was much slower than that in the midst of the phase. And we also could see that the water temperature could evidently affect the whole course that continued about 20 hours. According to these, we could feed Fugu obscurus fry scientifically, not only meet with its need, but also reduce the forage waste and keep a higher DO of water.The skin structure of Fugu obscurus is very special, there is a plenty of collagen that can be extracted easily. The comprehensive utilization of Fugu obscurus’ skin will not only enhance economic profit, but also more comprehend its value. The biochemical components of Fugu obscurus’ skin was studied in this paper, the results showed that a plenty of collagen was found in it. According to the methods from literatures, acid-soluble collagen (ASC) and pepsin-solubilized collagen (PSC) were obtained by the acid and enzyme methods. The yield of ASC was about 82% in all of the collagen that was more easily obtained by the mean of acid-extraction. Based on the SDS polyacrylamide gel electrophoresis (SDS-PAGE), there was no obviousdifference between ASC and PSC. According to the amino acid analysis, we concluded that our collagen extracted from Fugu obscurus’skin (by ASC and PSC methods) had the same properties as the typical collagen.It has been proved that the tissue and cell culture of aquatic animals had important value in the fields of the virus isolation and purity, the diagnosing and treating disease, the contamination examine of water and so on. The methods of primary culture in vitro for cells from the kidney of Fugu obscurus fry were first studied in this paper. The cultures of tissue and cell were experimented in TC-199 medium with about 17%-20% calf bovine serum under the temperature of 25 C. The whole course of these experiments seriously kept free germ and changed fresh medium in time. Under above conditions, the monolayers of primary cultured kidney cells could be更多还原