跳到主要內容區塊

研究報告

更新日期: 2015-02-09
點閱數: 2,238

建立石斑魚及觀賞魚關鍵生物技術

年度 2013
計劃名稱 建立石斑魚及觀賞魚關鍵生物技術
中文摘要 「建立石斑魚及觀賞魚關鍵生物技術」之中文摘要: 一、「利用分子技術進行石斑魚育種選拔研究」       本計畫為科技保密計畫,故不引入相關資訊,請另參照細部計畫期末報告紙本。 二、「觀賞魚分子育種技術研發」       本計畫為科技保密計畫,故不引入相關資訊,請另參照細部計畫期末報告紙本。 三、「適應環境之蝦類種蝦及淡水長臂大蝦的選殖與保種研究」        採用低溫(臨界温度)適應的連續選育的途徑,藉以遺傳改良某些亞熱帶魚蝦優良品種抗寒能力差的性狀。在對蝦耐低溫系統研究中,本計畫分子選殖出腦乙醯膽鹼酯酶(Acetycholinesterase,AchE)系基因;另外同工酶開關合成isoenzyme switch system以及能量代謝相關基因正在分析中。   為瞭解泰國淡水長臂大蝦抗病選育群體的抗病力和遺傳信息!通過人工注射溶藻弧菌感染16 個泰國淡水長臂大蝦(M. rosenbergii)選育群體,並利用微衛星分子標記對抗病品種選育進行遺傳結構分析。16 個選育群體抗溶藻弧菌感染能力存在明顯差別!初步鑒定出抗病力強的選育3個群體(SCR-11-6,SCR11-11 和SCR11-16);抗病力較強的選育7個群體SCR11-10, SCR11-5, SCR11-7, SCR11-12, SCR11-15,SCR11-1 和SCR11-14);抗病力一般的選育4 個群體(SCR11-9, SCR11-13, SCR11-2和SCR-11-4);抗病力差的選育2個群體(SCR11-3, SCR11-8)。其中中鑒定出抗病力強的3個群體其在感染溶藻弧菌感染後的成活率達80%。其中SUGbp8-103b, MRMB-11, MRMC2; 具有203,263, 185, 96bpDNA片段;與泰國淡水長臂大蝦(M. Rosenberii )抗弧菌感染有關。經進一步分析MRMBbp185 對偶基因座(allele locus )與M. rosenbergii抗弧菌感染有關。   本研究目第二目的為篩選和白蝦生長性狀相關之第Ⅰ型和第Ⅱ型分子標記。對兩個經成長試驗的白蝦群體(命名為G–1和G–2),以TUMXLv6.63、TUMXLv6.124、TUMXLv7.121、TUMXLv9.145、TUMXLv10.411等五個微衛星標記(Microsatellite DNA markers),利用分組分析法和群體分析法探討標記基因型和體重的關係。結果顯示,兩個白蝦群體的平均對偶基因數皆為3.8、平均異型合子觀測值(Ho)為0.457和0.382、平均異型合子期望值(He)為0.507和0.478、平均多型性訊息含量(PIC)為0.488和0.462。遺傳多樣性(Genetic diversity)和標記多型性(Marker polymorphism)皆為中等水平。在G–1群體中以TUMXLv6.63的AC基因型之平均體重顯著較高,而G–2群體則為TUMXLv7.121的AB基因型平均體重顯著較高,推測這兩個微衛星標記可能與白蝦生長性狀有某種關聯性。   海洋大學利用過去三年所完成的EST(Expressed Sequence Tag) 序列定序資訊,已完成;1)利用草蝦與白蝦DNA晶片與特定草蝦候選標記基因I型,2)篩選出4個南美白蝦先天型免疫(innate immunity)相關候選標記基因I型(candidate marker genes)。建立以對蝦免疫晶片比較高病力之先天免疫基因表現模式(pattern)技術。其主要目標是研究在分子層次上對蝦類免疫系統之抗病基因與抗病基因之調控系統,並將此對蝦生物資訊科技與DNA晶片應用在高抗病力(specific pathogen resistant; SPR)種蝦之分子育種與篩選免疫刺激飼料添加物。 2)  設計高病力種蝦保種的SPF對蝦繁養殖設施 SPF水產動物舍設計之需求 1. 動線規劃 國際畜衛組織雖無明列相關人與魚類共通之傳染病,但參考現有陸生動物疫苗設施使用均為符合生物安全之防疫要求,魚用動物舍區亦規劃為密閉設計。 動物舍前後區分有淨走道及污(回收)走道,所有單一動物舍由淨走道端之入口進入後,只可由污走道端之出口離開,且污走道所有出口只出不進以防止工作人員由污染區誤入試驗區,造成試驗區之交互污染。污走道後側出口通往焚化爐,用以處理廢棄物及試驗斃死或淘汰試驗魚,污走道前側則為清洗區,工作人員在此區清洗消毒器械及更換乾淨工作服後,由準備區可再進入淨走道至不同動物舍工作,或離開動物舍。動物舍前後區分有淨走道及污(回收)走道,所有單一動物舍由淨走道端之入口進入後,只可由污走道端之出口離開,且污走道所有出口只出不進以防止工作人員由污染區誤入試驗區,造成試驗區之交互污染。污走道後側出口通往焚化爐,用以處理廢棄物及試驗斃死或淘汰試驗魚,污走道前側則為清洗區,工作人員在此區清洗消毒器械及更換乾淨工作服後,由準備區可再進入淨走道至不同動物舍工作,或離開動物舍。   2. 空調設備 魚用動物舍區皆為氣密設計,空調設備之容量需能使各區間造成壓差,如同動線規劃一般,使空氣之流動由淨走道進入每單一動物舍,然後進入污走道。同時依實驗需求可以空調系統溫度調節,輔助動物舍內養殖施水溫維持恆定 (20~25℃,養殖設施另有加溫、冷卻系統)。 3. 水產特殊病原菌檢疫舍 為建立無特定病原(SPF)之親魚,因此所有進入動物舍之試驗魚,需於檢疫舍內進行檢疫測試及觀察至少14~21日,確認實驗魚健康無虞,始可進入檢定舍進行疫苗免疫試驗。依動物統進統出原則,檢疫舍規劃為一間,每單一循環水槽為1.2 噸共5組。  4. SPF繁殖與孵化室 設立大型循環設施二座,每座循環設備均有10 組0.5噸水槽。每組水槽容量至少可放養200尾20~60g種蝦,於10 組水槽滿載下,可維持正常運轉 60 天以上。 5. SPF舍-獨立水體 以日本 NVAL 標準,每間共設有5組獨立循環之0.5 噸水槽,每組水槽容量至少可飼養200尾20~60g種蝦60 天以上。另預留配置管線之檢定舍一間,以為特殊需求之試驗彈性運用之設施。   6. 其他設施 工作準備室、飼料儲藏室、儲物室、電氣室、清洗室各一間。    (四)水處理系統 1. 海水進水處理系統 參照中研院臨海研究站之設計,於室外設置20噸儲存槽,室內設置3組10 噸海水緩衝槽。海水經砂濾機過濾後,以臭氧氣水混合機處理,導入室內3組緩衝槽內稍作停留,以使水溫保持恆定並以ORP監測 TRO 含量至容許範圍後,再經過線流式紫外燈照射,供應各檢疫室及檢定室用水。採用低溫(臨界温度)適應的連續選育的途徑,藉以遺傳改良某些亞熱帶魚蝦優良品種抗寒能力差的性狀。在對蝦耐低溫系統研究中,本計畫分子選殖出腦乙醯膽鹼酯酶(Acetycholinesterase,AchE)系基因;另外同工酶開關合成isoenzyme switch system以及能量代謝相關基因正在分析中。 2. 試驗廢排水臭氧滅菌處理系統 參照中研院臨海研究站之設計,於室外設置20噸廢排水儲存槽,所有魚用動物舍設施內之排水經袋濾機過濾後,以高量臭氧進行氣水混合的殺菌處理,暫存於排水集水槽中並以ORP監測TRO 含量至容許範圍後,再予以排放至污水處理場。 3.淡水進水處理系統 設置曝氣設備,淡水經設備曝氣處理,進入室內淡水緩衝槽以使水溫恆定後,再經活性碳濾過及線流式紫外燈照射,供應各檢疫室及檢定室用水。 4. 污水處理系統 污水處理為環保署法規要求,所有魚用動物舍之廢排水經臭氧滅菌處理過程後,需進入污水處理場處理後,再予以排放。 5.水電、照明及空調配管 1.) 電氣及照明系統 需具一般供電及以柴油發電機緊急全區供電之電力系統,並具有獨立電錶。 2. )供排水系統 單一動物舍皆需有冷、熱水清洗裝置及排水陰井系統。 3. )空調配管 單一動物舍需具有獨立空調開關,送風需有小型冷風機及配有高效率過濾網之獨立風車濾網箱組。  6.土木隔間 1.) 庫板氣密隔間 使用鍍鋅防鏽耐燃二級鋼板,施工需符合氣密要求。 2. )地板施作 環氧樹脂耐磨地坪最小厚度5mm,全區施作。 7.焚化爐 實驗過程中死亡或實驗後淘汰之實驗動物,予以焚燒處理。設施建置之執行與成果。 8.成立專家諮詢小組 邀請國內各單位水產養殖、遺傳學與魚病領域之專家成立專家諮詢小組,參考中央研究院臨海研究站之養殖設備、入水與排放處理經驗,進行水產魚用SPF檢定設施之規劃、設計與改建進行水產SPF設施之工程規劃、設施設計與圖說審查,以利設施之完善與符合SPF飼養之需求。 四、「海龍孵化繁殖技術之建立」        不論是淡水、半淡鹹水或海洋棲性的海龍科(Family Syngnathidae)物種,皆因為過度利用、環境汙染與棲地喪失,而使野生動物資源受到影響並成為瀕危物種。而人工繁殖培育可能成危在傳統藥材、觀賞水族與工藝品需求上的解決之道。悉數種類不僅因為具有特殊型態與獨特的生理行為,且所有種類皆具有相對較高的附加價值。本研究探討影響淡水、半淡鹹水與海洋棲性,分別以Microphis屬、Hippichthys屬、Doryrhamphus屬及Doryichthys屬為代表之海龍物種,其種別、棲性、水質條件與各項參數、性比、不同投餵餌料種類與組成等因素對於人工繁殖培育海龍之影響。在為期6個月的試驗中,發現光照週期18:6(光照:黑暗)對個體活存、成長與交配具良好效果,而將諸如微藻、輪蟲、豐年蝦及其無節幼蟲、絲蚯蚓、微蟲、米蝦與卵胎生魚類混合投餵並經滋養強化處理,則對海龍稚幼苗階段具成長助益。淡水(genusMicrophis)、半淡鹹水(genusHippichthys and Doryichthys)與海洋棲性物種(genus Doryrhamphuss),仔稚魚孵化後培養至90天之活存率分別為60-85%, 25-85%及25-40%,試驗證實適當的光罩、性比、充滿變化之多樣性餌料投餵、投餵頻率可促進不同階段海龍之活存、成長、攝食與配對行為。 五、「小丑魚育種及分子標記技術之開發」       小丑魚為重要的海水觀賞魚種,因為具有鮮豔的體色及形態,因此小丑魚受歡迎的程度日趨增加。然而目前小丑魚的主要來源以野生捕捉為主,對野生族群造成威脅,為了減少自然資源的耗竭,減少人為捕撈及開發小丑魚的人工繁養殖技術已經成為最重要的課題。目前小丑魚的繁養殖關鍵技術在於生產過程(產卵、胚胎發育、孵化及訓餌),有部份小丑魚的繁養殖技術已漸趨成熟,但仍有待加強。為了區分小丑魚,需要建立一種鑑定方法,本計劃擬利用粒線體DNA分析,建立DNA密碼資料庫,提供區別小丑魚的鑑定方法。我們已完成收集五種台灣不同品系之小丑魚,也利用PCR及DNA定序分析小丑魚粒線體16S rDNA,並以MEGA軟體Test Maximum Likihood Tree分析。我們也已建立了公子小丑微衛星DNA資料庫。 六、「開發觀賞魚蝦繁養殖關鍵技術」       本計畫為科技保密計畫,故不引入相關資訊,請另參照細部計畫期末報告紙本。 七、「市售功能性觀賞魚飼料評析」        本研究分成二部份,第一部份為全球觀賞魚水族飼料產學概況調查與台灣觀賞水族飼料產業優勢分析及未來產業發展;第二部份為分析市售10家觀賞魚血鸚鵡增艷飼料色素之效能與兩家配合廠商建立觀賞魚飼料最低添加量及增艷最低標準。 本次調查主要針對台灣觀賞水族飼料產業,旨調查台灣地區觀賞水族飼料近年內外銷產業概況,並於製造技術、品質、品牌行銷等方面進行國內外品牌之比較分析。本調查對廠商進行問卷及訪談以瞭解現今市場概況,並於2013年觀賞魚博覽會進行消費者對觀賞魚飼料認知之問卷調查。 廠商為常見市售觀賞魚飼料品牌廠商、生產者及代工廠訪談共十一家。問卷調查結果顯示101年度觀賞水族飼料內銷值為1億6500萬元,外銷值為2億6500萬元,內外銷總值約4億3500萬元。外銷地區以去除中國、日本之亞洲其他國家(如東南亞)為最多,為23.81%。 在消費者方面,共取得492份問卷,有效問卷為470份,結果顯示34%消費者購買飼料之最大考量為廠牌,單次購買預算59%的消費者落於100~500元之間。觀賞飼料最廣為人知的品牌為56%之消費者知道此品牌;使用者最多的品牌有30%之消費者正在使用。國外品牌中市場上知名度及使用度最高為同一品牌,40%之消費者知道該品牌,使用此品牌者佔國外品牌使用者的58%。 競爭力分析上顯示,臺灣觀賞魚飼料產業之最大優勢為製成技術及飼料配方,國內廠商雖具有生產高品質飼料之技術,但品牌行銷上仍不如歐美日等國,未來須強化行銷策略,我國觀賞飼料才能擴大世界市場。 本實驗利用色卡比色、數位影像擷取和高效液相層析之方法,來比較不同商業觀賞魚飼料投餵血鸚鵡魚後的增豔效果。另外製作觀賞魚問卷來分析消費者對國內外商業飼料之喜好程度。實驗使用十一家商業觀賞魚飼料,含蝦紅素濃度5.65至1164.32 mg/kg。血鸚鵡魚初重平均為4.09克,實驗為期14天。實驗結果中成長部分,十一組各自與控制組無顯著差異。第一週時, D組在魚鰭有最高含量26.96±1.75 mg/kg的蝦紅素。C組在魚皮有最高含量22.28±0.11 mg/kg。I組在肌肉有最高含量18.55±0.14 mg/kg。第二週時, B組在魚鰭有最高含量26.96±1.75 mg/kg的蝦紅素。E組在魚皮和肌肉有最高含量,分別為20.74±0.37和7.6±0.17 mg/kg。數位影像擷取分析部分, H組在L*值有最大值55±2。J組在a*值有最大值22±8。D組在b*值有最大值30±2。 E組在C*值有最大值35±10。H*值在控制組有最大值1.36±0.06。色卡比色的部分,D組有最高的等級43±5, K組有最低的等級16±0。問卷部分則是國內廠商的認知度和喜好度較國外廠商高。 八、「東星斑養殖關鍵技術研發」       本計畫為科技保密計畫,故不引入相關資訊,請另參照細部計畫期末報告紙本。
英文摘要 Abstract of "To establish the key technology for grouper and ornamental fish culture": 1. "Utilization of molecular technology to screen high quality grouper for breeding" :      本計畫為科技保密計畫,故不引入相關資訊,請另參照細部計畫期末報告紙本。 2. "Development of molecular breeding on ornamental fish" :      本計畫為科技保密計畫,故不引入相關資訊,請另參照細部計畫期末報告紙本。 3. "Selection breeding program of environmental adaptation in penaeid shrimp and freshwater shrimp" :       White shrimp (Litopenaeus vannamei) is the important aquaculture species in the world. Using the molecular markers in genetic selection and process can improve the efficiency and accuracy of breeding. Acetylcholinesterase (EC 3.1.1.7; AChE), a key enzyme in the nervous system, regulates nerve impulse transmission by catalyzing hydrolysis of the neurotransmitter, acetylcholine, It has been shown that low temperature stress caused decrease in the level of AchE in warm water fish and shrimp. In present study, suing AChE gene activity in shrimp as a potential biomarker against cold stress is under investigation. In present study, five microsatellite DNA markers (TUMXLv6.63, TUMXLv6.124, TUMXLv7.121, TUMXLv9.145, TUMXLv10.411) were selected to analyze two different white shrimp groups (G–1 and G–2) and compared the correlation between markers and body weight. The mean number of alleles, mean Ho, mean He, and mean PIC were 3.8, 0.457 and 0.382, 0.507 and 0.478, 0.488 and 0.462, in G–1 and G–2, respectively, which indicated this groups was medium genetic diversity. The mean body weight of G–1 with the genotype AC at locus TUMXLv6.63 was significantly higher than other two genotypes, and G–2 with the genotype AB at locus TUMXLv7.121 was significantly higher than other five genotypes. These data indicated that these two markers and growth traits may have some relationship. A cDNA microarray encompassing 6000 putative unigenes expressed in gills, circulating haemocytess, and hepatopancreas of L. vannamei was applied in nutrigenomic study and the result revealed that the expression of innate immune genes ( such as, crustin, lysozyme, mo-penaeidin, transglutaminase and kazal-type proteinase inhibitor ) were increased after WSSV infection. In present study, we have demonstrated that shrimp immune DNA microarray might provide a useful tool to identify disease resistant gene markers for marker-assisted selection program for paneid shrimps.     4. "Establishment of the hatching and propagation techniques of Syngnathidae" :      Almost sea dragon and/or pipe fish (Syngnathidae)(including freshwater, brackish water and marine species) are imminent danger species for many issues in wild animal resource conservation including overexploitation, aquatic environment polluted and habitat loss. Artificial propagation has been proposed as one solution to address unsustainable trade for traditional medicine, ornamental fishes and curios. Almost species have not only specific morphological model but also special physiological behavior. And all species have higher price and additive value in ornamental fish market. This investigation examined the effects of species, habitat, water quality and parameters, sex ratio, various feed organisms culture, maintain and feeding, and propagation, survival ration (PHD 90) and growth of different species sea dragon (including genus Microphis, Hippichthys, Doryrhamphus and Doryichthys). During the 6-month study, almost species of sea dragon cultured at a photoperiod L:D=16:8 had higher survival ratio, mating frequencies and breed. Juvenile growth rate increased with mixture and enhance feed organisms, including microalgae, rotifer, Artemia and nauplii, tubifex, microworm, Caridina spp. and livebearer fish (platy, molly and guppy). Survival ratio of freshwater(genusMicrophis), brackish (genusHippichthys and Doryichthys)and marine(genus Doryrhamphuss) species were 60-85%, 25-85% and 25-40% when juvenile after PHD 90, respectively. Results demonstrated that appropriate photoperiods, sex ratio, various feed organisms and feeding frequency can improve the survival, growth, feeding and mating of the juvenile, sub-adult and adult sea dragon. 5. "Clownfish breeding and developing molecular marker technique" :       The clownfish is one of the most important marine ornamental fish and is becoming increasingly popular in the aquarium fish. The main source of the clownfish is capture from the natural sources. Therefore the resource of clownfish is gradually decreased. In order to reduce the exhaustion of the natural sources, reducing capture and developing the technologies for the farming of clownfish have already become a most important subject. Currently, the development of an ornamental aquaculture protocol faces several critical bottlenecks related to production processes (i.e. spawning induction, embryo development, hatching and transition from endogenous to exogenous feeding). In order to distinguish the clownfish species, we will analyze mitochondrial DNA and construct a DNA barcode database to provide a new species identification tool for the clownfish. We have finished collect five kinds of Taiwan clownfish. We also established the clownfish (Amphiprion ocellaris) microsatellite DNA library. 6. "Develop the key techniques of ornamental fish and shrimp" :      本計畫為科技保密計畫,故不引入相關資訊,請另參照細部計畫期末報告紙本。 7. "commercial funtional ornamental fish feed analysis" :       There are two parts in this project. The first part focused on the competition of worldwide ornamental fish feed among Mainland China and southeast Asia countries and analysis of the dominance and future strategies of Taiwan ornamental fish feed. The second part focused on the estimation of the efficiency and the enhanced pigments of eleven ornamental fish feeds and the certification standards for enhanced pigment of ornamental fish feed and formula standards for special functional fish feed. Taiwan has advantages in ornamental fish culturing environment, advanced culturing system, breeding technology and biotechnology. Ornamental fish markets have been shown increasing demand, and following up the demand of ornamental fish feed. Hence, government should encourage ornamental fish industry to develop and promote products in the worldwide. We focused on four topics concerning the ornamental fish:1) the competition of worldwide ornamental fish feed among Mainland China, and southeast Asia countries; 2) to analyze the dominance and future strategies of Taiwan ornamental fish feed; 3) to estimate the efficiency and to analyze the enhanced pigments of ornamental fish feed; 4) to set the certification standards for enhanced pigment of ornamental fish feed and formula standards for special functional fish feed. Also, we visited two local feed manufacture companies and discuss how much pigment should be included in the diets. Through the development of certification standard of ornamental fish feed, we can promote this industry into the top one level of the worldwide. The survey on ornamental fish feed industry in Taiwan is to understand the value of domestic sale and export in recent year and compare the domestic and international brand in Taiwan on manufacturing technology, quality, marketing and etc. The survey questionnaire and interviews with producers and consumers in 2013 ornamental fish Expo to understand the current market situation and preference of ornamental fish feed. Totally, 11 feed producers and brand owners were interviewed face to face or on phone.  Survey results show that in 2012 year the domestic value and export value of ornamental fish feed is 165 million and 265 million NT, respectively and the total value is 430 million NT. The Asia area exclude China and Japan has the highest percentage of 23.8% of the total value. On the results of consumer survey, 470 among the total of 492 questionnaires is valid. It showed that 34% consumers concerned the brand of the feed and 56% of consumer is willing to pay 100-500 NT in single purchase. The most well-known domestic brand for consumers in Taiwan is 56% and the most users’ brand is 30% of those responders. Among those international brands in Taiwan, the most well-known brand for consumers is 40% and the most users’ brand is 58% of those responders. On the competitive analysis, the biggest advantage of Taiwan's ornamental fish feed industry is processing technology and formulation. Although domestic feed company have the ability to produce high-quality ornamental fish feed, the ability of marketing brand in the world is inferior to Europe, America and Japan. In the future, Taiwan has to strengthen the marketing strategy, which is the way to expand world market for Taiwanese ornamental fish feed.4.09 g fish were fed with eleven commercial enhanced pigment feeds coming from domestic and foreign countries for two weeks. A control diet without pigment included was made by us. The efficiency of pigmentation was estimated by the color chip, digital image acquisition and processing (DIAP), and HPLC analysis. We also made questionnaires to investigate the behavior of the ornamental fish consumers. There were no significant different weigh gain of fish fed control and commercial diets. The astaxanthin concentration of commercial diets ranged from 5.7 to 1164.2 mg/kg. After one week feeding, the astaxanthin concentrations of fish fin of D grouper, the fish skin of C group and the fish muscle of I group showed the highest levels, 26.96 + 1.75, 22.28 + 0.14 and 18.55 + 0.14 mg/kg, respectively. However, after two weeks feeding, the astaxanthin concentrations of fish fin of B group, and the fish skin and fish muscle of E group showed the highest levels, 26.97 + 1.75, 20.74 + 0.37, and 7.6 + 0.17 mg/kg, respectively. Through DIAP observation, the highest level of L*, a*, b*, C* and H* values were found in H group (55), J group (22), D group (30), E group (35) and control group (1.4), respectively. Through color chip observation, the highest level was found in D group (43) and the lowest level was found in K group (16). The questionnaire was distributed to the ornamental fish trade show during October. From this survey, we know the following facts: 1) Taiwanese consumers like to raise small size ornamental fish; 2) they like the colorful ornamental fish; 3) they can afford 100g diet with below 250 NT dollars; 4) they are used to buy the domestic diets rather than alien diets. 8. "Development of key technology of leopard coral grouper" :      本計畫為科技保密計畫,故不引入相關資訊,請另參照細部計畫期末報告紙本。
計畫編號 102農科-11.3.3-漁-F3(Z)
網站導覽錨點