跳到主要內容

臺灣博碩士論文加值系統

(44.201.99.222) 您好!臺灣時間:2022/12/09 00:43
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:李尚親
研究生(外文):Shang-Chin Lee
論文名稱:應用臭氧消毒、微粒飼料和病毒分子檢測方法降低點帶石斑(Epinepheluscoioides)魚苗之死亡率
論文名稱(外文):Employments of ozone disinfection, microfeed and viral nucleic acid diagnosis to reduce larval moralities of orange-spotted grouper (epinephelus coioides)
指導教授:陸振岡
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:71
中文關鍵詞:PCR/RT-PCR檢測臭氧海水魚卵消毒甲狀腺激素石斑魚血清動素
外文關鍵詞:PCR/RTPCR diagnosisozonated seawater egg-disinfectionthyroid hormonesgrouperserotonine
相關次數:
  • 被引用被引用:0
  • 點閱點閱:320
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:5
台灣是全球水產養殖產業和技術最佳的國家之ㄧ,特別是在魚苗育成的領域更是東南亞首強。台灣更是幼魚和魚苗出口的重要供應國。神經壞死病毒(Nervous necrosis virus, NNV)屬於野田病毒科(Nodaviridae)內的β-野田病毒屬(Betanodavirus),它通常感染海水魚類如海鱸、鲹、鸚哥魚、河豚和鰈魚,且引起高死亡率的病毒型神經壞死症造成經濟上的嚴重損失。在本研究應用RT-PCR和PCR建立快速、可信度且具可重複性的方法來偵測NNV的受精卵和幼魚感染,可以經由腦和水晶體樣本來篩選出NNV帶原的種魚。ㄧ般養殖業者通常以牡蠣受精卵作為石斑魚苗開口餌料餵食,再配合輪蟲(Brachionus sp.)混合餵食,等更大時再使用水蚤幼生(Harpacticoid and Calanoid)作為石斑魚苗之餌料生物。本研究中NNV-RT-PCR分析結果顯示在上述的四種常用幼期餌料生物均可偵測到NNV病毒核酸,這顯示魚苗業者所使用在石斑魚苗幼期階段的活餌均有可能成為NNV的帶原者。經由餵食15日齡的石斑魚苗含0.01 ppm甲狀腺激素T3/T4以及血清動素(5-HT)的微粒飼料一週,可以改善魚隻的變態發育和平均體長增加11.3%。為了降低病毒垂直感染,本研究以5 mgO3/L或是更低的總臭氧濃度浸泡30秒,對魚卵的活存率和對魚苗發育無不良的影響。本研究應用病原分子檢測、臭氧消毒法和變態促進物質補充微粒飼料可以降低點帶石斑魚魚苗死亡率。
Taiwan is one of the aquaculture countries in world and has the best aquaculture techniques especially for larval culture in Southeast Asian. Taiwan also is an important country for fry and larvae export. Nervous necrosis virus, NNV belongs to Nodaviridae, Betanodavirus. It usually infects marine fishes such as sea bass, jack, parrotfish, pufferfish and, flatfish and causes viral nervous necrosis with a high mortality rate and results in further severe economic loss. In the present study, RT-PCR and PCR methods were employed to establish a quick, reliable and reproducible assay to screen NNV infected fertilized eggs and fries of grouper. It was demonstrated that blood, and retina samples can be used to screen NNV carrier of brood stocks. Grouper larvae are initially fed on oyster eggs, often in combination with rotifers (Brachionus sp.), and subsequently use of copepod nauplii (Harpacticoid and Calanoid) as prey during the early larval rearing of groupers has been shown considerable promise in improving larval growth and survival. In the present study, NNV can be detected in all these four larval live foods, it strongly suggests that current grouper larval live foods might be the NNV carriers. Metamorphosis were improved and mean body length 1.13% than control fish when feed the 15-day-old grouper larvae with microfeed containing 0.01 ppm thyroid hormones (T3/T4) and serotonin (5-HT) for a week. To reduce viral vertical transmission, the fertilized eggs groups which had been treated with 5 mg O3 ‘L or lower dose exposures for 30 s had a lower viral content and no adverse effects on the survival rate and development of the larvae after hatching were observed. Employments of pathogen molecular diagnosis, ozone disinfection, and metamorphosis stimulators supplementary microfeed has been shown these modern techniques can reduce the moralities of orange-spotted grouper larva.
目 錄
謝辭…………………………………………………………………………………….i
中文摘要…………………………………………………………………………...…iii
英文摘要………...……………………………………………………………………iv
目錄…………………………………………………………………..……………….vi
表目錄………………………………………………………………………………viii
圖目錄……………………………………………………………………………...…ix
第一章、前言
(一)、文獻整理
一、石斑魚簡介………………………………………………………………..…1
二、台灣石斑魚養殖所面臨的問題………………………………………..……1
三、石斑魚生產所遭遇的困難…………...…………………………………..….3
四、石斑魚的病毒性疾病………………………………………………..….…...4
五、建立無病原的石斑魚養殖環境…………………………………..…………7
六、分子病毒檢測…………………………………………….…………………8
七、以臭氧消毒受精卵去除感染………………………………….…………… 10
八、微粒飼料…………………………………………………….………………11
九、甲狀腺激素與變態期發育…………………………………….……………11
十、血清動素與水產生物變態期之影響………………………….……………12
十一、豬胎盤………………………………………………….…………………13
(二)、本研究之目標……………………………………………………………… 13
第二章、材料與方法
1.病魚病毒核酸檢測…………………………………….……….……………….…15
2.臭氧消毒之石斑魚受精卵病毒核酸檢測………………………………………...20
3.幼魚期餌料生物之病毒核酸檢測……………………………………..……….…21
4.疫區無病徵魚的NNV病毒核酸檢測…………………………………………….22
5.臭氧濃度試驗…………………………………………………………….………..23
6.石斑魚卵臭氧消毒及微粒飼料投餵之現場養殖……………….…………..……24
第三章、實驗結果
1.分子病毒檢測結果……………………………………………………….………28
2.不同臭氧濃度對石斑魚卵消毒的結果………………………………….….…...30
3.石斑魚卵臭氧消毒及餵食微粒飼料之現場養殖成果……………..….………..31
第四章、討論………………………………………………………..……………..33
參考文獻………………………………………………..…………………………..39
圖表………………………………………………………………………..…….….48
附錄…………………………………………………………………….……………70
表 目 錄

表-1、以不同濃度臭氧消毒石斑魚受精卵之孵化率、畸形率、孵化後ㄧ日(1dph)及孵化後二日(2dph)之死亡率………...…….…….48
表-2、以5 mg O3/L消毒魚卵後放養之魚苗孵化率……………….….49
表-3、海王星超微粒飼料添加物……………………………………....50
圖 目 錄

圖-1、石斑魚苗變態各時期之體型與魚鰭變化圖及常用餌料生物轉換…………………………….………………………………….51
圖-2、微粒飼料餵食示意圖……..……………………………………...52
圖-3、輪蟲外觀圖……………………………………………………….53
圖-4、水蚤外觀圖………………………………….……………………54
圖-5-1、石斑魚虹彩病毒PCR分子檢測…….…………………………55
圖-5-2、石斑魚傳染性胰臟壞死病毒RT-PCR分子檢測…………….55
圖-5-3、石斑魚神經壞死病毒RT-PCR分子檢測…….………………..56
圖-5-4、Multiplex-PCR同時檢測三種石斑魚病毒…………………..56
圖-6、石斑魚苗NNV RT-PCR分析結果………………….……………57
圖-7、石斑魚卵臭氧消毒前後NNV RT-PCR分析結果………….……57
圖-8、臭氧消毒受精卵孵化後之魚苗NNV RT-PCR檢測結果….……58
圖-9、牡蠣受精卵NNV RT-PCR分析結果…………………………….59
圖-10、輪蟲NNV RT-PCR分析結果…..……………………………….59
圖-11、水蚤Harpacticoid copepod NNV RT-PCR分析結果…………..60
圖-12、Calanoid copepod NNV RT-PCR分析結果…………….……. 60
圖-13、魚隻血液NNV RT-PCR分析結果……………………..……..61
圖-14、以不同濃度臭氧消毒30秒之石斑魚受精卵之孵化率……......62
圖-15、以不同濃度臭氧消毒30秒之石斑魚受精卵之畸型率……..…62
圖-16、以不同濃度臭氧消毒30秒之石斑魚受精卵孵化後1日之死亡率圖………….………………………………………………….63
圖-17、以不同濃度臭氧消毒30秒之石斑魚受精卵孵化後2日之死亡率圖…………………………………….……………………….63
圖-18、各臭氧處理組孵化率、畸形率與死亡率對臭氧濃度關係圖………………………………………………………………..64
圖-19、飼養第21日之控制組與OT-2組體長抽樣體長分布圖……………………………………….……………………….65
圖-20、飼養第37日之控制組與OT-2組體長抽樣體長分布圖…..…...66
圖-21、餵食促變態微粒飼料對石斑魚苗變態發育之影響…………...67
圖-22、甲狀腺激素分子結構…..…………….…………………………68
圖-23、血清動素(5-hydroxytryptamine, 5-HT)分子結構…….…….…69
李國誥,劉秉忠 (2001)。石斑魚細菌性疾病。石斑魚繁養殖技術改進與疾病防治研討會,9-12頁。
邱義雄(2000)。臭氧在點帶石斑孵化上之應用。碩士論文。國立台灣海洋大學水產養殖研究所。
陳瑤湖(2001)。石斑魚養殖產業分析。石斑魚繁養殖技術改進與疾病防治研討會,1-8頁。
黃貴民(1999)。實用石斑魚養殖。水產出版社。1-89頁。
葉信利(1995)。台灣農家要覽 漁業篇。190-199頁。
張正芳(1994)。石斑魚之病害與防治。國立中山大學漁業推廣工作專刊 ,12期,1-18頁。
趙嘉本(1995)。金目鱸虹彩病毒感染症。台灣省各縣市家畜、水產疾病檢驗技術研究會 八十五年 第三次。
蘇惠美(2005)。水產種苗的生產。科學發展,385期,32-41頁。
Arimoto M., J. Sato, K. Maruyama , G. Mimura and I. Furusawa (1996). Effect of chemical and physical treatments on the inactivation of striped jack nervous necrosis virus (SJNNV). Aquaculture 143, 15–22.
Ball L. A., D. A. Hendry, J. E. Johnson, R. R. Ruechert and P. D. Scotti (2000). Seventh report of the international committee on taxonomy of virus. Academic Press, New York.
Bercovier H., Y. Fishman, R. Nahary, S. Sinai, A. Zoltkin, M. Eyngor, O. Gilad, A. Eldar and Hedrick R.D.(2005). Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis. BMC Microbiol. 5, 5-13.
Brown C. L., S. I. Doroshov, M. D. Cochran and H. A. Bern (1989). Enhanced survival of striped bass fingerlings after maternal triiodothyronine treatment. Fish Physiol. Biochem. 7, 295-299.
Brown C. L., S. I. Doroshov, J. Nunez, C. Hadley, R. S. Nishioka and H. A. Bern (1988). Maternal triiodothyronine injection cause increases in swimbladder inflation and survival rates in larval striped bass, Morone saxatillis. J. Exp. Zool. 46, 59-63.
Chi S. C., C. F. Lo, G. H. Kou, P. S. Chang, S. E. Peng and S. N. Chen (1997). Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared groupers, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel). J. Fish Dis. 20, 1185-1193.
de Jesus E. G., J. D. Toledo and S. M. Simpas (1998). Thyroid hormones promote early metamorphosis in grouper Epinephelus coioides larvae. Gen. Comp. Endocrinol. 112, 10-16.
Eales J.G. and Brown S.B. (1993). Measurement and regulation of thyroidal statu in teleost fish. Rev. Fish Biol. 3, 299-347.
Gomez D. K., J. Sato, K. Mushiake, T. Isshiki, Y. Okinaka and Nakai T. (2004). PCR-based detection of betanodaviruses from cultured and wild marine fish with no clinical signs. J. Fish Dis. 27, 603-608.
Grotmol S., E. Dahl-Paulsen and G. K.Totland (2003). Hatchability of eggs from Atlantic cod, turbot and Atlantic halibut after disinfection with ozonated seawater. Aquaculture 221, 245-254.
Grotmol S. and G. K. Totland (2000). Surface disinfection of Atlantic halibut Hippoglossus hippoglossus eggs with ozonated sea-water inactivates nodavirus and increases survival of the larvae. Dis Aquat Organ. 39, 89-96.
Hall, L., J. Sato, K. Maruyama, G. Mimura and I. Furuzawa (1981). Comparison of ozone and chlorine toxicity to the developmental stages of Striped Bass Morone saxatilis. Can. J. Fish Aquat. Sci. 38, 752–757.
Hedge A., H. C. Teh, T. J. Lam and Y. M. Sin (2003). Nodavirus infection in freshwater ornamental fish, guppy, Poicelia reticulate-comparative characterization and pathogenicity studies. Arch. Virol. 148, 575-586.
Hirata Y., H. Kurokura and S. Kasahara (1989). Effects of thyroxine and thiourea on the development of larval red sea bream Pagrus major. Nippon Suisan Gakkaishi 55, 1189-1195.
Hseu, J. R., F. I. Lu, H. M. Su, L. S. Wang, C. L. Tsai and P. P. Hwang (2003). Effect of exogenous tryptophan on cannibalism, survival, and growth in juvenile grouper, Epinephelus coioides. Aquaculture. 218, 251-263.
Hsu H. C., C. F. Lo, S. C. Lin, K. F. Liu, P. E. Peng, Y. S. Chang, L. L. Chen, W. J. Liu and G. H. Kou (1999). Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders. Dis. Aquat. Organ. 22, 13-19.
Hsu Y. L., K. H. Wang, Y. H. Yang, M. C. Tung, C. H. Hu, C. F. Lo, C. H. Wang and T. Hsu (2000). Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies. Dis. Aquat. Organ. 40, 93-99.
Inui Y. and S. Miwa (1985). Thyroid hormones induce metamorphosis of flounder larvae. Gen. Comp. Endocrinol. 74, 68-76.
Inui Y., S. Miwa, K. Yamano and T. Hirano (1994). Hormonal control of flounder metamorphosis. In: Davey, K.G., Peter R.G., Tobe S.S. (Eds.), Perspectives in Comparative Endocrinology. National Research Council, Canda, pp. 408-411.
Iuchi I. (1973). The post-hatching transition of erythrocytes from larval to adult type in the rainbow trout, Salmo gairdnerii irideus. J. Exp. Zool. 184, 383-396.
Johansen R., S. Grove, A. K. Svendsen, I. Modahl and B. Dannevig (2004). A sequential study of pathological findings in Atlantic halibut, Hippoglossus hippoglossus, throughout one year after an acute outbreak of viral encephalopathy and retinopathy. J. Fish Dis. 27, 327-341.
Liltved H., H. Hektoen and H. Efraimsen (1995). Inactivation of bacterial and viral fish pathogens by ozonation or UV irradiation in water of different salinity. Aquat. Eng. 14, 107-122.
Mata A. I., A. Gibello, A. Casamayor, M. M. Blanco , L. Domínguez, J. F. and Ferna´ndez-Garayza´bal (2004). Multiplex PCR Assay for Detection of Bacterial Pathogens Associated with Warm-Water Streptococcosis in Fish. App. Env. Microbio. 70, 3183-3187.
Mimura G., T. Nagase, Y. Katayama , T. Nagamitu and K. Namba (1998). Effect of ozone-produced oxidants on eggs of Japanese flounder Paralichthys olivaceus. Fish Pathol. 46, 101–110
Miwa S. and Y. Inui (1991). Thyroid hormones stimulates the shift of erythrocyte population during metamorphosis of the flounder. J. Exp. Zool. 259, 222-228.
Mori K. I., T. Nakai, K. Muroga, M. Arimoto, K. Mushiake and I. Furusawa (1992). Properties of a new virus belonging to Nodaviridae found in larval striped jack Pseudocaranx dentex with nervous necrosis. Virology. 187, 336-371.
Nishizawa T., M. Furuhashi, T. Nagai and K. Muroga (1997). Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. App. Env. Microbiology Apr., 1633-1636.
Pantoja C. R. and D. V. Lightner (2000). A non-destructive method based on the polymerase chain reaction for detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp. Dis. Aquat. Organ. 39, 177-182.
Power D. M., L. Llewellyn, M. Faustino, M. A. Nowell, B. Th-Björnsson, I. E. Einarsdottir, A. V. M. Canario and G. E. Sweeney (2001c). Thyroid hormones in growth and development of fish. Comp. Biochem. Physiol. 130, 447-459.
Power D.M., N. P. Ekias, S. J. Mendes, J. Soares and C.M. Santos (2000) Evolution of the thyroid hormone binding protein, transthyretin. Gen. Comp. Endocrinol. 119, 241-255.
Richardson L.B., D.T. Burton, R.M Block and Stavola A.M. (1983) Lethal and sublethal exposure and recovery effects of ozone-produced oxidants on adult white perch(Morone americana Gmelin). Water Res. 17, 205-213.
Schreiber G. and S. J. Richardson (1997) The evolution of gene expression, structure and function of transthyretin. Comp. Biochem. Physiol. 116B, 137-160.
Seng E. K., Q. Fang, T. J. Lam and Y. M. Sin (2004). Development of a rapid, sensitive and specific diagnostic assay for fish Aquareovirus based on RT-PCR. J. Viro. Methods. 118, 111-122.
Shi Y. B., J. Wong, K. M. Puzianowaska and M. A. Stolow (1986). Tadpole competence and tissue-specific temporal regulation of amphibian metamorphosis : roles of thyroid hormone and its receptors. BioEssays 18, 391-399.
Sugita H., T. Asai, K. Hayashi , T. Mitsuya, K. Amanuma, C. Maruyama, and Y. Deguchi (1992). Application of ozone disinfection to remove Enterococcus seriolicida, Pasteurella piscicida, and Vibrio anguillarum from seawater. App. Env. Microbiol. 58, 4073-4075.
Sukhumsirichart W., C. Wongteerasupaya, V. Boonsaeng, S. Panyim, S. Sriuairatana, B. Withyachumnarnkul and T. W. Flegel (1999). Characterization and PCR detection of hepatopancreatic parvovirus (HPV) from Penaeus monodon in Thailand. Dis. Aquat. Organ. 39, 1-10.
Szisch V., N. Papandroulakis, E. Fanouraki and M. Pavlidis (2005). Ontogeny of the thyroid hormones and cortisol in the gilthead sea bream Sparus aurata. Gen. Comp. Endocrinol. 142, 186-192.
Tan C., Huang B., Chang S.F., Ngoh G.H., Munday B.3, Chen S.C., Kwang J., 2001. Determination of the complete nucleotide sequences of RNA1 and RNA2 from greasy grouper (Epinephelus tauvina) nervous necrosis virus, Singapore strain. J. Gen. Virol. 82, 647-653.
Tnaka M. (1971). Studies on the structure and function of the digestive system in teleost larvae-Ⅲ. Development of digestive system during postlarval stage. Japan. J. ichthyol. 18, 164-174.
Thakur P. C., F. Corsin, J. F. Turnbull, K. M. Shankar, N. V. Hao, P. A. Padiyar, M. Madhusudsan, K. L. Morgan and C. V. Mohan (2002) . Estimation of prevalence of white spot syndrome virus (WSSV) by polymerase chain reaction in Penaeus monodon postlarvae at time of stocking in shrimp farms of Karnataka, India: a population-based study. Dis. Aquat. Organ. 49, 235-243.
Tsai C. T., J. W. Ting, M. H. Wu, M. F. Wu, I. C. Guo and C. Y. Chang (2005). Complete Genome Sequence of the Grouper Iridovirus and Comparison of Genomic Organization with Other Iridoviruses. J. Virol. 79, 2010-2023.
Yada T., I. Misumi, K. Muto, T. Azuma and C. B. Schreck (2004). Effects f prolactin and growth hormone on proliferation and survival of cultured trout leucocytes. Gen. Comp. Endocrinol. 136, 298-306.
Yada T., K. Muto, T. Azuma, S. Hyodo and C. B. Schreck, (2005). Cortisol stimulates growth hormone gene expression in rainbow trout leucocytes in vitro. Gen. Comp. Endocrinol. 142, 248-255.
Yamamoto M. and I., Iuchi (1975). Electron microscopic study of erythrocytes in developing rainbow trout, Salmo gairdnerii iridovirus, with particular reference to changes in the cell line. J. Exp. Zool. 191, 407-426.
Yamano K., H. Takano-Ohmuro, T. Obinata and Y. Inui (1994b). Effect of thyroid hormone on developmental transition of myosin light chains during flounder metamorphosis. Gen. Comp. Endocrinol. 93, 321-326.
Yan D.C., Dong S.L., Huang J., Yu X.M., Feng M.Y., Liu X.Y.(2004) White spot syndrome virus (WSSV) detected by PCR in rotifers and rotifer resting eggs from shrimp pond sediments. Dis. Aquat. Organ. 59, 69-73.
Yoshikoshi K. and K. Inoue (1990).Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel). J. Fish Dis. 13, 69-77.
Yoshimizu M., M. Sami and T. Kimura (1989). Survivability of infectious hematopoietic necrosis virus (IHNV) in fertilized eggs of masu (Oncorhynchus masou) and chum salmon (O. keta). J. Aquat. Anim. Health. 1, 13–20.
Yoshimizu M., S. Hyuga, M.J. Oh, Y. Ezura, S. Ito and G. Minura (1995). Disinfectant effect of oxidant produced by ozonization of seawater on fish pathogenic viruses, bacteria, and ciliata. Dis. Asian Aquac. 1, 203–209.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top