本文選題：大鱗副泥鰍 + 異速生長　； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：大鱗副泥鰍是東亞地區(qū)近幾年養(yǎng)殖需求非常高的水產(chǎn)養(yǎng)殖品種之一,其營養(yǎng)價值和藥用價值都非常可觀,市場潛力巨大。本文在借鑒前人的研究基礎(chǔ)上,對大鱗副泥鰍(Paramisgurnus dabryanus)早期發(fā)育階段的生長模式、核酸含量變化、消化酶活性變化及骨骼畸形及其耐氨機制進行了相關(guān)研究,主要研究結(jié)果如下:(1)大鱗副泥鰍早期發(fā)育階段的生長模式以初孵至60日齡(DAH)大鱗副泥鰍仔魚作為實驗材料,評價了大鱗副泥鰍早期階段體重體長關(guān)系以及身體各部分異速生長模式。結(jié)果發(fā)現(xiàn),大鱗副泥鰍仔魚階段最為適合的體重體長關(guān)系為指數(shù)模型,BW=0.025×TL2.649(R2=0.996)。大鱗副泥鰍仔魚頭長、頭高、軀干長、尾長及眼徑在早期階段均表現(xiàn)為正異速生長,而體高、尾柄高、尾鰭長、胸鰭長及須長在早期階段則表現(xiàn)為負(fù)異速生長。隨后,大鱗副泥鰍仔魚身體各部分又表現(xiàn)出相似的異速生長模式,均向著等速生長逐漸變化。結(jié)果說明大鱗副泥鰍早期發(fā)育階段身體各部分異速生長的發(fā)育順序遵循對提高存活率的重要性。(2)大鱗副泥鰍早期發(fā)育階段核酸及蛋白的變化測定了大鱗副泥鰍從孵化至60 DAH期間核酸及蛋白質(zhì)含量以評價其早期階段的生長。以紫外分分光光度法測定大鱗副泥鰍仔稚魚的核酸含量(n=3,養(yǎng)殖溫度24.4±0.4℃,溶解氧7.1±0.5 mg L-1,p H 7.9±0.4)。結(jié)果發(fā)現(xiàn)核糖核酸(RNA)含量在2-5 DAH期間顯著下降,在5-10 DAH之間快速上升,隨后持續(xù)降低,直至試驗結(jié)束。脫氧核糖核酸(DNA)含量也在2-5 DAH期間上升,在5-9 DAH期間下降,隨后表現(xiàn)為緩慢上升,直至26 DAH,隨后降低至一個相對較為穩(wěn)定的水平直至試驗結(jié)束。RNA-DNA比和protein-DNA比均與生長速率表現(xiàn)出明顯的相關(guān)性,大鱗副泥鰍早期發(fā)育階段RNA-DNA比與生長速率之間表現(xiàn)為明顯的正相關(guān)關(guān)系。結(jié)果表明大鱗副泥鰍早期發(fā)育階段細(xì)胞水平上的生長模式為從孵化到卵黃囊耗盡主要表現(xiàn)為細(xì)胞數(shù)量的增生,初次攝食之后則是細(xì)胞體積、質(zhì)量的增大。此外,大鱗副泥鰍早期發(fā)育階段的關(guān)鍵期應(yīng)在17 DAH之前。(3)大鱗副泥鰍早期發(fā)育階段消化酶活力的變化研究了從孵化至40 DAH期間大鱗副泥鰍仔稚魚不同胰酶(胰蛋白酶,糜蛋白酶,淀粉酶及脂肪酶)、胃酶(胃蛋白酶)及腸酶(堿性磷酸酶及亮氨酸氨基肽酶)的比活力和總活力以評價其早期階段的消化生理。大鱗副泥鰍仔魚養(yǎng)殖溫度為24.4±0.4℃,在初次攝食(4 DAH)至15 DAH期間投喂輪蟲,10-35 DAH期間投喂小型枝角類,30-40 DAH期間投喂人工配合飼料。胰蛋白酶、糜蛋白酶、淀粉酶和脂肪酶活性在仔魚孵化當(dāng)天即可檢出,說明這些消化酶是受基因調(diào)控的。多數(shù)胰酶活性均在20 DAH之前增加,而在隨后開始降低。大鱗副泥鰍仔魚胃蛋白酶在30 DAH時開始檢出,表明了其消化系統(tǒng)中功能性胃腺的出現(xiàn)。堿性磷酸酶也在仔魚孵化時即可檢出,隨后快速上升,并在20 DAH時達(dá)到第二個峰值。而亮氨酸氨基肽酶活性在整個試驗過程中均表現(xiàn)為逐漸上升的趨勢。因此,大鱗副泥鰍仔魚在外源性營養(yǎng)開始之前就有一個功能性的消化系統(tǒng),而且其消化能力隨著個體發(fā)育而增加。腸酶活力在10-20 DAH之間的顯著增加意味著大鱗副泥鰍成魚消化模式的開端。30DAH之后胃蛋白酶活力的顯著上升,標(biāo)志著大鱗副泥鰍仔魚由堿性消化方式向酸性消化方式的轉(zhuǎn)變,而這一時期是大鱗副泥鰍仔魚轉(zhuǎn)食人工配合飼料的適當(dāng)時機。(4)大鱗副泥鰍早期階段骨骼畸形的研究采用硬骨-軟骨雙染色法研究了從孵化至60 DAH期間大鱗副泥鰍仔稚魚骨骼畸形的發(fā)生。結(jié)果發(fā)現(xiàn)大鱗副泥鰍早期階段骨骼畸形發(fā)生的部位主要在脊椎骨的4個分區(qū),以及背鰭、臀鰭和尾鰭,共觀察到14種骨骼畸形類型。大鱗副泥鰍仔稚魚不同時期骨骼畸形分布不同,本試驗中未發(fā)現(xiàn)脊椎骨畸形,神經(jīng)棘畸形在階段A(TL12 mm)出現(xiàn)頻率最低,在階段D(TL50 mm)出現(xiàn)率最高;脈棘畸形在階段B(TL 12-30 mm)出現(xiàn)頻率最低,在階段C(TL 30-50 mm)出現(xiàn)頻率最高;鰭畸形在階段C(TL 30-50 mm)出現(xiàn)頻率最低,在階段B(TL 12-30 mm)出現(xiàn)頻率最高。大鱗副泥鰍常見的畸形類型包括鰭條骨畸形、神經(jīng)棘及脈棘畸形等。仔稚魚骨骼畸形的高發(fā)可能與其機體的劇烈生理變化以及內(nèi)臟器官的增殖、分化相關(guān)。鰭條畸形、神經(jīng)棘分叉和脈棘融合出現(xiàn)的頻率最高,說明了這些組織最易受到環(huán)境變化的影響。直至試驗結(jié)束仍未觀察到脊柱骨畸形,證明大鱗副泥鰍脊柱畸形的發(fā)生時期比較晚(60DAH之后)。(5)空氣和氨氮暴露下大鱗副泥鰍體內(nèi)氨的累積將大鱗副泥鰍暴露于30 mmol L-1 NH4Cl溶液和空氣中,以研究其在氨氮和空氣暴露條件下體組織中氨、尿素含量以及谷丙轉(zhuǎn)氨酶和谷草轉(zhuǎn)氨酶活性的變化。研究發(fā)現(xiàn)30 mmol L-1 NH4Cl溶液暴露下,隨著暴露時間的增加,大鱗副泥鰍血漿和腦組織中氨含量顯著增加;肝臟組織和肌肉組織中氨含量隨著暴露時間的延長在前24 h內(nèi)表現(xiàn)為少量的增加,而在暴露48 h后則顯著上升�？諝獗┞断�,隨著空氣暴露時間的延長,大鱗副泥鰍血漿、腦組織、肝臟組織和肌肉組織中氨含量在前24 h內(nèi)表現(xiàn)為少量的增加,而在暴露48 h后則顯著上升,血漿、腦、肝臟和肌肉中氨含量分別上升為對照組的2.2倍、3.3倍、2.5倍和2.9倍。大鱗副泥鰍無論暴露于30 mmol L-1 NH4Cl溶液或者空氣中,不同暴露時間對其血漿、肝臟組織和肌肉組織中尿素含量的影響均非常的小,其體內(nèi)尿素含量在氨氮和空氣暴露下一直處于較為穩(wěn)定的水平,不受機體氨累積的影響。大鱗副泥鰍暴露于30 mmol L-1的NH4Cl溶液中,暴露時間對其血漿中谷丙轉(zhuǎn)氨酶活性的影響是很小的。而暴露于空氣中時,暴露時間顯著影響其血漿中谷丙轉(zhuǎn)氨酶活性(P0.05),空氣暴露48 h后,其血漿中谷丙轉(zhuǎn)氨酶活性顯著升高。然而,無論是暴露于30 mmol L-1的NH4Cl溶液中還是暴露于空氣中,大鱗副泥鰍血漿谷草轉(zhuǎn)氨酶活性及肝臟組織中谷丙轉(zhuǎn)氨酶、谷草轉(zhuǎn)氨酶均不受暴露時間的影響。結(jié)果表明說明大鱗副泥鰍組織和細(xì)胞具有很高的氨耐受性,也可能具有以NH3形式揮發(fā)部分體內(nèi)氨以達(dá)到應(yīng)對氨氮毒性的機制。大鱗副泥鰍無論暴露于30 mmol L-1 NH4Cl溶液或者空氣中,不同暴露時間對其血漿、肝臟組織和肌肉組織中尿素含量的影響均非常的小,說明大鱗副泥鰍并不是以合成尿素作為其主要的氨解毒策略。通過大鱗副泥鰍血漿中谷丙轉(zhuǎn)氨酶活性的顯著升高,可推測其在空氣暴露下可能會通過部分氨基酸代謝生成丙氨酸以應(yīng)對體內(nèi)高濃度的氨累積。(6)谷氨酰胺在大鱗副泥鰍應(yīng)對氨氮和空氣暴露中的作用將大鱗副泥鰍暴露于30 mmol L-1 NH4Cl溶液和空氣中,以研究其在氨氮和空氣暴露條件下體組織中谷氨酰胺含量以及谷氨酰胺合成酶和谷氨酸脫氫酶活力的變化。研究發(fā)現(xiàn)大鱗副泥鰍暴露于30 mmol L-1 NH4Cl溶液和空氣,隨著暴露時間的延長,大鱗副泥鰍肝臟和肌肉組織中的谷氨酰胺含量有明顯累積的趨勢,腦、肝臟和腸道組織中谷氨酰胺合成酶活性均顯著上升,說明了大鱗副泥鰍可通過體組織中累積谷氨酰胺來應(yīng)對體內(nèi)氨濃度的上升,其可刺激體內(nèi)谷氨酰胺的合成,將氨轉(zhuǎn)化為無毒性的谷氨酰胺。30 mmol L-1 NH4Cl溶液和空氣暴露顯著影響大鱗副泥鰍腦和腸道組織中谷氨酸脫氫酶活性,但對肝臟組織中谷氨酸脫氫酶活性并沒有顯著性影響。腸道中谷氨酸脫氫酶活性顯著上升,可能其在魚類應(yīng)對氨氮毒性中起到了比腸道谷氨酰胺合成酶更加重要的作用。而大鱗副泥鰍肝臟組織中谷氨酸脫氫酶活性并不受氨氮和空氣暴露的影響,這可能是由于肝臟組織中轉(zhuǎn)氨酶催化生成了足量的谷氨酸。(7)氨氮和空氣暴露下大鱗副泥鰍體表堿化及氨氣揮發(fā)為確定大鱗副泥鰍是否具有以NH3形式排泄體內(nèi)氨的能力,設(shè)計了氨氮暴露和空氣暴露兩組試驗。結(jié)果發(fā)現(xiàn),大鱗副泥鰍在NH4Cl溶液中暴露24 h后,試驗組產(chǎn)生氨的量顯著高于對照組�？諝獗┞秾�(dǎo)致大鱗副泥鰍NH3揮發(fā)量的顯著提高。試驗結(jié)果說明大鱗副泥鰍在氨氮和空氣暴露下能夠以NH3形式排泄體內(nèi)過量的氨。大鱗副泥鰍NH3揮發(fā)量隨著暴露時間和溫度的增加而升高�？諝夂桶钡┞吨率勾篦[副泥鰍后腸壁明顯堿化,說明后腸是其揮發(fā)NH3的位點之一。皮膚在空氣暴露下也呈現(xiàn)明顯的堿化,說明其也可能是一個NH3揮發(fā)位點。簡而言之,本研究結(jié)果表明了大鱗副泥鰍在氨氮和空氣暴露下可以氣態(tài)形式揮發(fā)30-40%左右的氨,且高溫促進了NH3的揮發(fā)。后腸和皮膚的堿化意示著其兩個NH3揮發(fā)位點。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S917.4

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