大鳞副泥鳅早期发育及耐氨机制研究

发布时间：2018-06-20 17:35

本文选题：大鳞副泥鳅 + 异速生长　；参考：《华中农业大学》2016年博士论文

【摘要】：大鳞副泥鳅是东亚地区近几年养殖需求非常高的水产养殖品种之一,其营养价值和药用价值都非常可观,市场潜力巨大。本文在借鉴前人的研究基础上,对大鳞副泥鳅(Paramisgurnus dabryanus)早期发育阶段的生长模式、核酸含量变化、消化酶活性变化及骨骼畸形及其耐氨机制进行了相关研究,主要研究结果如下:(1)大鳞副泥鳅早期发育阶段的生长模式以初孵至60日龄(DAH)大鳞副泥鳅仔鱼作为实验材料,评价了大鳞副泥鳅早期阶段体重体长关系以及身体各部分异速生长模式。结果发现,大鳞副泥鳅仔鱼阶段最为适合的体重体长关系为指数模型,BW=0.025×TL2.649(R2=0.996)。大鳞副泥鳅仔鱼头长、头高、躯干长、尾长及眼径在早期阶段均表现为正异速生长,而体高、尾柄高、尾鳍长、胸鳍长及须长在早期阶段则表现为负异速生长。随后,大鳞副泥鳅仔鱼身体各部分又表现出相似的异速生长模式,均向着等速生长逐渐变化。结果说明大鳞副泥鳅早期发育阶段身体各部分异速生长的发育顺序遵循对提高存活率的重要性。(2)大鳞副泥鳅早期发育阶段核酸及蛋白的变化测定了大鳞副泥鳅从孵化至60 DAH期间核酸及蛋白质含量以评价其早期阶段的生长。以紫外分分光光度法测定大鳞副泥鳅仔稚鱼的核酸含量(n=3,养殖温度24.4±0.4℃,溶解氧7.1±0.5 mg L-1,p H 7.9±0.4)。结果发现核糖核酸(RNA)含量在2-5 DAH期间显著下降,在5-10 DAH之间快速上升,随后持续降低,直至试验结束。脱氧核糖核酸(DNA)含量也在2-5 DAH期间上升,在5-9 DAH期间下降,随后表现为缓慢上升,直至26 DAH,随后降低至一个相对较为稳定的水平直至试验结束。RNA-DNA比和protein-DNA比均与生长速率表现出明显的相关性,大鳞副泥鳅早期发育阶段RNA-DNA比与生长速率之间表现为明显的正相关关系。结果表明大鳞副泥鳅早期发育阶段细胞水平上的生长模式为从孵化到卵黄囊耗尽主要表现为细胞数量的增生,初次摄食之后则是细胞体积、质量的增大。此外,大鳞副泥鳅早期发育阶段的关键期应在17 DAH之前。(3)大鳞副泥鳅早期发育阶段消化酶活力的变化研究了从孵化至40 DAH期间大鳞副泥鳅仔稚鱼不同胰酶(胰蛋白酶,糜蛋白酶,淀粉酶及脂肪酶)、胃酶(胃蛋白酶)及肠酶(碱性磷酸酶及亮氨酸氨基肽酶)的比活力和总活力以评价其早期阶段的消化生理。大鳞副泥鳅仔鱼养殖温度为24.4±0.4℃,在初次摄食(4 DAH)至15 DAH期间投喂轮虫,10-35 DAH期间投喂小型枝角类,30-40 DAH期间投喂人工配合饲料。胰蛋白酶、糜蛋白酶、淀粉酶和脂肪酶活性在仔鱼孵化当天即可检出,说明这些消化酶是受基因调控的。多数胰酶活性均在20 DAH之前增加,而在随后开始降低。大鳞副泥鳅仔鱼胃蛋白酶在30 DAH时开始检出,表明了其消化系统中功能性胃腺的出现。碱性磷酸酶也在仔鱼孵化时即可检出,随后快速上升,并在20 DAH时达到第二个峰值。而亮氨酸氨基肽酶活性在整个试验过程中均表现为逐渐上升的趋势。因此,大鳞副泥鳅仔鱼在外源性营养开始之前就有一个功能性的消化系统,而且其消化能力随着个体发育而增加。肠酶活力在10-20 DAH之间的显著增加意味着大鳞副泥鳅成鱼消化模式的开端。30DAH之后胃蛋白酶活力的显著上升,标志着大鳞副泥鳅仔鱼由碱性消化方式向酸性消化方式的转变,而这一时期是大鳞副泥鳅仔鱼转食人工配合饲料的适当时机。(4)大鳞副泥鳅早期阶段骨骼畸形的研究采用硬骨-软骨双染色法研究了从孵化至60 DAH期间大鳞副泥鳅仔稚鱼骨骼畸形的发生。结果发现大鳞副泥鳅早期阶段骨骼畸形发生的部位主要在脊椎骨的4个分区,以及背鳍、臀鳍和尾鳍,共观察到14种骨骼畸形类型。大鳞副泥鳅仔稚鱼不同时期骨骼畸形分布不同,本试验中未发现脊椎骨畸形,神经棘畸形在阶段A(TL12 mm)出现频率最低,在阶段D(TL50 mm)出现率最高;脉棘畸形在阶段B(TL 12-30 mm)出现频率最低,在阶段C(TL 30-50 mm)出现频率最高;鳍畸形在阶段C(TL 30-50 mm)出现频率最低,在阶段B(TL 12-30 mm)出现频率最高。大鳞副泥鳅常见的畸形类型包括鳍条骨畸形、神经棘及脉棘畸形等。仔稚鱼骨骼畸形的高发可能与其机体的剧烈生理变化以及内脏器官的增殖、分化相关。鳍条畸形、神经棘分叉和脉棘融合出现的频率最高,说明了这些组织最易受到环境变化的影响。直至试验结束仍未观察到脊柱骨畸形,证明大鳞副泥鳅脊柱畸形的发生时期比较晚(60DAH之后)。(5)空气和氨氮暴露下大鳞副泥鳅体内氨的累积将大鳞副泥鳅暴露于30 mmol L-1 NH4Cl溶液和空气中,以研究其在氨氮和空气暴露条件下体组织中氨、尿素含量以及谷丙转氨酶和谷草转氨酶活性的变化。研究发现30 mmol L-1 NH4Cl溶液暴露下,随着暴露时间的增加,大鳞副泥鳅血浆和脑组织中氨含量显著增加;肝脏组织和肌肉组织中氨含量随着暴露时间的延长在前24 h内表现为少量的增加,而在暴露48 h后则显著上升。空气暴露下,随着空气暴露时间的延长,大鳞副泥鳅血浆、脑组织、肝脏组织和肌肉组织中氨含量在前24 h内表现为少量的增加,而在暴露48 h后则显著上升,血浆、脑、肝脏和肌肉中氨含量分别上升为对照组的2.2倍、3.3倍、2.5倍和2.9倍。大鳞副泥鳅无论暴露于30 mmol L-1 NH4Cl溶液或者空气中,不同暴露时间对其血浆、肝脏组织和肌肉组织中尿素含量的影响均非常的小,其体内尿素含量在氨氮和空气暴露下一直处于较为稳定的水平,不受机体氨累积的影响。大鳞副泥鳅暴露于30 mmol L-1的NH4Cl溶液中,暴露时间对其血浆中谷丙转氨酶活性的影响是很小的。而暴露于空气中时,暴露时间显著影响其血浆中谷丙转氨酶活性(P0.05),空气暴露48 h后,其血浆中谷丙转氨酶活性显著升高。然而,无论是暴露于30 mmol L-1的NH4Cl溶液中还是暴露于空气中,大鳞副泥鳅血浆谷草转氨酶活性及肝脏组织中谷丙转氨酶、谷草转氨酶均不受暴露时间的影响。结果表明说明大鳞副泥鳅组织和细胞具有很高的氨耐受性,也可能具有以NH3形式挥发部分体内氨以达到应对氨氮毒性的机制。大鳞副泥鳅无论暴露于30 mmol L-1 NH4Cl溶液或者空气中,不同暴露时间对其血浆、肝脏组织和肌肉组织中尿素含量的影响均非常的小,说明大鳞副泥鳅并不是以合成尿素作为其主要的氨解毒策略。通过大鳞副泥鳅血浆中谷丙转氨酶活性的显著升高,可推测其在空气暴露下可能会通过部分氨基酸代谢生成丙氨酸以应对体内高浓度的氨累积。(6)谷氨酰胺在大鳞副泥鳅应对氨氮和空气暴露中的作用将大鳞副泥鳅暴露于30 mmol L-1 NH4Cl溶液和空气中,以研究其在氨氮和空气暴露条件下体组织中谷氨酰胺含量以及谷氨酰胺合成酶和谷氨酸脱氢酶活力的变化。研究发现大鳞副泥鳅暴露于30 mmol L-1 NH4Cl溶液和空气,随着暴露时间的延长,大鳞副泥鳅肝脏和肌肉组织中的谷氨酰胺含量有明显累积的趋势,脑、肝脏和肠道组织中谷氨酰胺合成酶活性均显著上升,说明了大鳞副泥鳅可通过体组织中累积谷氨酰胺来应对体内氨浓度的上升,其可刺激体内谷氨酰胺的合成,将氨转化为无毒性的谷氨酰胺。30 mmol L-1 NH4Cl溶液和空气暴露显著影响大鳞副泥鳅脑和肠道组织中谷氨酸脱氢酶活性,但对肝脏组织中谷氨酸脱氢酶活性并没有显著性影响。肠道中谷氨酸脱氢酶活性显著上升,可能其在鱼类应对氨氮毒性中起到了比肠道谷氨酰胺合成酶更加重要的作用。而大鳞副泥鳅肝脏组织中谷氨酸脱氢酶活性并不受氨氮和空气暴露的影响,这可能是由于肝脏组织中转氨酶催化生成了足量的谷氨酸。(7)氨氮和空气暴露下大鳞副泥鳅体表碱化及氨气挥发为确定大鳞副泥鳅是否具有以NH3形式排泄体内氨的能力,设计了氨氮暴露和空气暴露两组试验。结果发现,大鳞副泥鳅在NH4Cl溶液中暴露24 h后,试验组产生氨的量显著高于对照组。空气暴露导致大鳞副泥鳅NH3挥发量的显著提高。试验结果说明大鳞副泥鳅在氨氮和空气暴露下能够以NH3形式排泄体内过量的氨。大鳞副泥鳅NH3挥发量随着暴露时间和温度的增加而升高。空气和氨氮暴露致使大鳞副泥鳅后肠壁明显碱化,说明后肠是其挥发NH3的位点之一。皮肤在空气暴露下也呈现明显的碱化,说明其也可能是一个NH3挥发位点。简而言之,本研究结果表明了大鳞副泥鳅在氨氮和空气暴露下可以气态形式挥发30-40%左右的氨,且高温促进了NH3的挥发。后肠和皮肤的碱化意示着其两个NH3挥发位点。
[Abstract]:Mariculture loach is one of the very high aquaculture varieties in East Asia in recent years. Its nutritional value and medicinal value are very considerable and the market potential is great. On the basis of previous studies, the growth pattern, the change of nucleic acid content and the digestive enzyme in the early development stage of the Paramisgurnus dabryanus were used for reference. The main research results were as follows: (1) the growth pattern of the early development stage of the loach of the large scale of the loach was 60 days old (DAH) as the experimental material, and the weight body length relationship of the early stage of the loach and the different speed growth modes of the body parts were evaluated. The results showed that the most suitable body weight body length relationship was the index model, BW=0.025 x TL2.649 (R2=0.996). The head length, head height, trunk length, tail length and eye diameter of the larva were positive different at the early stage, while the body height, the tail stalk was high, the tail fin long, the pectoral fin length and the length of the whiskers were shown in the early stage. Then, the body parts of the larvae of the loach of the large scales showed a similar pattern of different speed growth and gradually changed toward the constant speed. The results showed that the development sequence of the different speed growth of the body parts in the early development stage of the large scale loach follows the importance of increasing the survival rate. (2) the nucleic acid at the early stage of the development stage of the loach. The content of nucleic acid and protein was measured from hatching to 60 DAH to evaluate its early stage growth. The nucleic acid content of juvenile loach (n=3, culture temperature 24.4 + 0.4 C, dissolved oxygen 7.1 + 0.5 mg L-1, P H 7.9 + 0.4) was determined by UV spectrophotometry. The results showed that ribonucleic acid (RNA) contained RNA. The amount dropped significantly during the 2-5 DAH period, rising rapidly between 5-10 DAH and then decreasing until the end of the test. The content of DNA increased in the period of 2-5 DAH, decreased during the 5-9 DAH period, followed by a slow rise to 26 DAH, and then decreased to a relatively stable level until the test ended the.RNA-DNA ratio and the end of the test. The protein-DNA ratio has a significant correlation with the growth rate. The RNA-DNA ratio and the growth rate in the early development stage of the large scale loach are positively correlated. The results show that the growth pattern of the cell level in the early development stage of the loach of the large scale loach is the proliferation of the cell number from hatching to the oval sac. After the first feeding, the volume and mass of the cells were increased. In addition, the key stage of the early development stage of the loach should be before 17 DAH. (3) the changes of digestive enzyme activity in the early development stage of the loach of the large scale pheid loach studied the difference of trypsin, chymotrypsin, amylase and fat from the hatching to 40 DAH during the incubation period. Enzyme), gastric enzyme (pepsin) and intestinal enzyme (alkaline phosphatase and leucine aminopeptidase) activity and total vitality to evaluate the digestive physiology at the early stage. The culture temperature of the larvae of paramidoside loach was 24.4 + 0.4 C, rotifer was fed during the first feeding (4 DAH) to 15 DAH, and the small Cladocera was fed during the 10-35 DAH period, and the 30-40 DAH was fed to the feeding person. The activity of trypsin, chymotrypsin, amylase and lipase could be detected on the day of hatching, indicating that the digestive enzymes were regulated by genes. Most of the enzyme activities were increased before 20 DAH, and then began to decrease. The pepsin began to be detected at 30 DAH, indicating the digestive system in the digestive system. The appearance of functional gastric glands. Alkaline phosphatase can also be detected when larvae hatch, and then rapidly rise and reach second peaks at 20 DAH. The activity of leucine aminopeptidase is gradually rising in the whole process. The digestibility of the digestive system increased with the development of the individual. The significant increase of the activity of the intestinal enzyme between 10-20 DAH means that the pepsin activity in the digestible pattern of the fish of the loach of the large scale loach was significantly increased after.30DAH, indicating the transformation of the larva from the alkaline digestion to the acid digestion. The time is the appropriate time to feed the artificial mixed feed for the larva of the loach of the large scale. (4) the bone deformity of the early stage of the loach of the large scale loach was studied by the hard bone cartilage double staining method. The bone deformity of the juvenile loach of the loach of the large scale was studied during the incubation period to 60 DAH. 14 kinds of skeletal deformities were observed mainly in 4 sections of the spine, as well as the dorsal fin, the hip fin and the caudal fin. The bone malformation of the juvenile loach larvae was different in different periods. There was no spinal deformity in this experiment. The frequency of A (TL12 mm) was the lowest in the stage of the neurospinous deformity, and the highest incidence of D (TL50 mm) in the stage. The frequency of stage B (TL 12-30 mm) is the lowest, and the frequency of C (TL 30-50 mm) is the highest in stage; fin malformation is the lowest in phase C (TL 30-50 mm), and the frequency of B (TL 12-30 mm) is the highest in phase B. The severe physiological changes of the body, the proliferation of viscera and differentiation, and the highest frequency of fin deformity, nerve spinous branching and spinal cord fusion showed that these tissues were most vulnerable to environmental changes. Until the end of the experiment, the spinal deformity was not observed, which proved that the period of the deformity of the Spina loach was late (6 After 0DAH) (5) (5) the accumulation of ammonia in the body of panaceus loach under air and ammonia nitrogen exposure was exposed to 30 mmol L-1 NH4Cl solution and air to study the changes in the activity of ammonia, urea, alanine aminotransferase and cereal transaminase in the body tissues under ammonia nitrogen and air exposure. The study found 30 mmol L-1 NH4Cl solution. Exposure, with the increase of exposure time, the ammonia content in the plasma and brain tissue of the loach of the large scale increased significantly, and the content of ammonia in the liver tissue and muscle tissue increased with the exposure time in the first 24 h, and increased significantly after exposure to 48 h. The ammonia content in the loach plasma, brain tissue, liver tissue and muscle tissue showed a small increase in the first 24 h, and increased significantly after exposure to 48 h. The ammonia content in the plasma, brain, liver and muscles increased 2.2 times, 3.3 times, 2.5 times and 2.9 times respectively in the control group, no matter exposed to 30 mmol L-1 NH4Cl solution or air, The effect of exposure time on the urea content in the plasma, liver and muscle tissues was very small. The urea content in the body was at a stable level under ammonia nitrogen and air exposure. It was not affected by the accumulation of ammonia in the body. The exposure time was in the NH4Cl solution of 30 mmol L-1. The effect of aminase activity was very small. While exposure to air, exposure time significantly affected the activity of alanine aminotransferase (P0.05) in plasma. After exposure to air for 48 h, the activity of alanine transaminase in the plasma was significantly increased. However, in the NH4Cl solution exposed to 30 mmol L-1 or in the air, the plasma of the loach The activity of aminotransferase and the glutamic pyruvine aminotransferase in liver tissues were not affected by the exposure time. The results showed that the tissues and cells of the loach of the large scale loach had a high ammonia tolerance and could volatilize some ammonia in the form of NH3 to meet the toxicity of ammonia nitrogen. No matter exposed to 30 mmol L-1 NH4C The effect of different exposure time on the urea content in the plasma, liver and muscle tissues in L solution or air is very small. It shows that the large scale loach is not a major ammonia detoxification strategy. Alanine may be generated through partial amino acid metabolism in order to cope with high concentration of ammonia in the body. (6) glutamine is exposed to 30 mmol L-1 NH4Cl solutions and air in the presence of ammonia and air in the loach, to study the glutamine content in the body tissues under ammonia nitrogen and air exposure. The changes in the activity of glutamine synthetase and glutamate dehydrogenase were found. It was found that the amount of glutamine in the liver and muscle tissues of the loach of the large scale loach was obviously accumulated with the exposure time prolonged, and the glutamine synthetase activity in the brain, liver and intestinal tissues of the loach was exposed to 30 mmol L-1 NH4Cl solution and air. The sex of the loach was significantly increased, indicating that the accumulation of glutamine in body tissues could be used to cope with the increase of ammonia concentration in the body. It could stimulate the synthesis of glutamine in the body. The transformation of ammonia to non-toxic glutamine.30 mmol L-1 NH4Cl solution and air exposure significantly affected the dehydrogenation of glutamate in the brain and intestinal tissues of the loach. Enzyme activity has no significant effect on glutamate dehydrogenase activity in the liver tissue. The activity of glutamate dehydrogenase in the intestine increases significantly. It may play a more important role in the fish's response to ammonia nitrogen toxicity than the intestinal glutamine synthetase. And the effect of air exposure, this may be due to the formation of a full amount of glutamic acid catalyzed by transaminase in the liver tissue. (7) ammonia nitrogen and air exposure to the body surface alkali and ammonia volatilization to determine whether large scale loach has the ability to excretion in the form of NH3 in the form of ammonia in the form of two groups of ammonia exposure and air exposure. It was found that after exposure to 24 h in NH4Cl solution, the amount of ammonia produced in the test group was significantly higher than that in the control group. The air exposure resulted in a significant increase in the volatilization of NH3 in the loach of the loach. The results showed that the large scale of the loach could excretion excess ammonia in the form of NH3 under ammonia and air exposure. The volatilization of NH3 in the NH3 of the loach The exposure to the exposure time and temperature increased. Air and ammonia nitrogen exposure resulted in the obvious alkalinity of the intestinal wall of the loach, indicating that the hindgut was one of its volatile NH3 sites. The skin also showed obvious alkalinity under air exposure, indicating that it was also a NH3 volatilization site. And air exposure can be volatile gaseous form about 30-40% ammonia, and the high temperature promoted the volatilization of NH3. Italy intestinal and skin alkaline showing the two volatilization of NH3 sites.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4

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