韓玉山Han, Yu-San臺灣大學:漁業科學研究所簡卉君Chien, Hui-ChunHui-ChunChien2010-05-102018-07-062010-05-102018-07-062008U0001-2207200818205800http://ntur.lib.ntu.edu.tw//handle/246246/181669日本鰻 (Anguilla japonica) 為一降海洄游魚類，野生日本鰻在海中產卵並且孵化後，鰻線經洋流漂送到河口，進入河川成長 4-10 年，直至變態成銀鰻後再洄游至產卵場產卵而後死亡，完成整個生活史。不同於一般野生鰻洄游、成熟、成長及生殖的整個生活史，養殖鰻是由河口捕撈鰻苗後直接移至養殖場，之後整個生活史都在養殖池中完成，在充分供應餌料下進行高速成長，僅需兩年就可達到銀鰻階段。本研究旨在探討野生和養殖日本鰻在生活棲地與成長速率不同下，是否也在形態與腦下垂體激素的表現上反應出差異? 論文中所使用之樣本於 1998 年 5 月到 2008 年 5 月採自台灣西南部高屏溪下游的野生鰻，以及採集自雲林、嘉義、台南、屏東等地，養殖 1-2 年的黃鰻。根據鰻魚的成長體型，探討在同一體型下，養殖黃鰻、野生黃鰻及野生銀鰻，在形態以及促濾泡激素 (FSH)、促黃體激素 (LH) 、前腦啡黑色素腎上腺皮質素 (POMC) mRNA 表現的差異。結果顯示，在同體長的鰻魚中，肥滿度 (CF) 與生殖腺指數 (GSI) 養殖雌鰻較野生雌鰻者為佳，雄鰻則無顯著差異。在眼徑指數 (OI) 和胸鰭指數 (FI) 方面，不論雌雄皆是野生鰻較養殖鰻者為佳。而在腦下垂體的內分泌激素表現上，GPα 、 LHβ 、以及 POMC mRNA的表現，野生雄鰻表現較養殖雄鰻佳，但在 FSHβ 上則無顯著差異。在雌鰻的結果中，GPα、FSHβ、LHβ 和 POMC mRNA 的表現，在養殖及野生鰻中皆無顯著差異。綜合形態和內分泌表現兩點，只有在雄鰻有較一致的結果，在雌鰻則各有高下。活在穩定環境中的養殖鰻，少了鹽度、滲透壓改變等的環境刺激以及食物的競爭，再加上養殖鰻由於有大量且充足的食物供給，使得在同年齡的養殖鰻生長速度較野生鰻快，以致造成同體型的養殖鰻與野生鰻在形態與內分泌上表現有所差異。The Japanese eel (Anguilla japonica) is a catadromous fish with a complex life cycle, spending their lives both in sea and freshwater environments. The wild eels hatch in the sea, and the leptocephali are transported from the spawning ground to the continental shelf. They metamorphose into glass eels in the coastal waters then become yellow eels in rivers where they may live for 4–10 years until silvering. After silvering, eels migrate back to the spawning area to spawn and die. In contrast with the wild eels that follow this normal migratory pattern to grow, mature and reproduce, the cultured eels that are collected from the wild spend their whole life cycle in aquaculture farms. In captivity, they grow fast when fed with commercial diet and may reach silver eel stage in two years. This study aims to investigate the differences between wild and cultured eels on morphological indices and pituitary hormone gene expression profiles due to different growth rates and habitats. The morphological indices and pituitary hormone gene expression profiles were compared between wild and cultured eels of the same size range and same developmental stage.ild eels were collected from Kaoping River estuary in southwestern Taiwan from May 1998 through May 2008 and the cultured eels, cultivated about 1-2 years, were collected from Yun Lin , Chiayi , Tainan and Pingdong. Morphological indices like CF, GSI, OI and FI, together with mRNA expression profiles of GPα, FSHβ, LHβ and POMC were examined. The results showed that cultured female eels, with the same size, have higher mean CF and GSI than wild ones. No significant differences were found between males of wild and cultured eels. Irrespective of the sex, mean OI and FI are significantly higher in the wild eels than cultured ones. In pituitary hormones, the expression of GPα, LHβ and POMC mRNA in males were higher in the wild than in the cultured eels. However, no difference on mean FSHβ mRNA expression was found between wild and cultured males. In female eels, there were no significant differences between wild and cultured eels in mRNA expression profiles of GPα, FSHβ, LHβ and POMC. Taken together, the results revealed only that male but not female eels were found to have the consistence aptitude in morphology and endocrine. The cultured eels spend their life in a more stable environment where they do not experience drastic environmental changes like salinity variation, osmotic stress and competition in food. In addition, in the same age eels, cultured eels grow faster than wild eels due to the ample amount of food supplied to them. These conditions may have caused the endocrine and morphological differences between wild and cultured eels.目 錄文摘要 ……………………………………………………………………………………………………… ii文摘要 …………………………………………………………………………………………………………… iv、 前言 ……………………………………………………………………………………………………… 1 日本鰻生活史 ………………………………………………………………………………………… 1 日本鰻銀化的形態變化 ………………………………………………………………………… 1 養殖日本鰻歷史及特性 ………………………………………………………………………… 2 鰻魚的人工繁殖 ……………………………………………………………………………………… 3 硬骨魚生殖內分泌主軸 ………………………………………………………………………… 4 51 促性腺激素 (Gonadotropins, GtHs) …………………………………………………… 4 52 魚類 HPG axis 與青春期的關係 ……………………………………………………… 5 53 促性腺激素的功能 ……………………………………………………………………………… 6 POMC 與銀化的關係 ……………………………………………………………………………… 6 61 銀化與 POMC …………………………………………………………………………………… 6 62 其他 POMC 相關研究 ……………………………………………………………………… 7 研究動機目的 ………………………………………………………………………………………… 7、 實驗材料………………………………………………………………………………………………… 9 生物材料…………………………………………………………………………………………………… 91 鰻魚樣本取得……………………………………………………………………………………… 92 菌株……………………………………………………………………………………………………… 93 質體……………………………………………………………………………………………………… 9 儀器及器材……………………………………………………………………………………………… 9 反應試劑………………………………………………………………………………………………… 10 31 細菌培養基………………………………………………………………………………………… 10 32 酵素…………………………………………………………………………………………………… 10 33 生物反應試劑組 (Kits) 及化學試劑……………………………………………… 11、 實驗方法 ……………………………………………………………………………………………… 12 犧牲採樣………………………………………………………………………………………………… 12 形態與生理指數之計算………………………………………………………………………… 12 腦下垂體 total RNA 萃取……………………………………………………………………… 12 cDNA 製備 ………………………………………………………………………………………… 13 日本鰻完整 POMC 選殖 ……………………………………………………………………… 13 51 POMC 蛋白質轉譯區 (CDS) 選殖………………………………………………… 13 52 3’RACE …………………………………………………………………………………………… 14 53 5’RACE …………………………………………………………………………………………… 15定量分析 腦下垂體 GPα、LHβ、FSHβ、POMC mRNA 表現量……………… 16 61 Real-Time PCR 引子合成 ……………………………………………………………… 16 62 標準品備製 ……………………………………………………………………………………… 16 621 基因放大 ……………………………………………………………………………………… 16 622 膠質萃取 (Gel Elution) ………………………………………………………………… 17 623 銜接反應及轉形作用 (Ligation Reaction And Transformation) ……… 17 624 小量質體抽取 (Plasmid Extraction) …………………………………………… 18 625 連續稀釋 ……………………………………………………………………………………… 19 63 Real-Time PCR ………………………………………………………………………………… 19 64 資料分析 …………………………………………………………………………………………… 20 統計分析…………………………………………………………………………………………………… 21、 結果 ………………………………………………………………………………………………………… 22 體長對應養殖黃鰻、野生黃鰻以及野生銀鰻之形態指數差異………………… 221 肥滿度 (CF) …………………………………………………………………………………… 222 生殖腺指數 (GSI) …………………………………………………………………………… 223 眼徑指數 (OI) ………………………………………………………………………………… 224 胸鰭指數 (FI) ………………………………………………………………………………… 235 肝指數 (HSI) …………………………………………………………………………………… 23 成熟度對應養殖野生鰻眼徑指數及胸鰭指數 ……………………………………… 23 21 GSI vs OI ………………………………………………………………………………………… 23 22 GSI vs FI ………………………………………………………………………………………… 23 日本鰻腦下垂體 POMC 基因選殖 ……………………………………………………… 23 日本鰻腦下垂體 GPα、FSHβ、LHβ 及 POMC 之 Real-Time PCR 引子設計專一性測試 ………………………………………………………………………………………… 24 日本鰻腦下垂體 GPα、FSHβ、LHβ 及 POMC 在不同體長之基因表現 51 GPα…………………………………………………………………………………………………… 25 52 FSHβ …………………………………………………………………………………………… 25 53 LHβ ……………………………………………………………………………………………… 25 54 POMC ……………………………………………………………………………………………… 26、 討論……………………………………………………………………………………………………… 27 養殖與野生鰻之體色差異……………………………………………………………………… 27 養殖與野生鰻銀化年齡之差異 …………………………………………………………… 27 養殖及野生鰻形態指數之差異 …………………………………………………………… 27 31 體脂與生殖腺發育的關係 ………………………………………………………………… 27 32 眼徑指數和胸鰭指數 ……………………………………………………………………… 28 33 肝指數 ……………………………………………………………………………………………… 28 日本鰻腦下垂體 POMC 之選殖……………………………………………………………… 29 養殖及野生鰻腦下垂體 GtHs 表現的異同……………………………………………… 29 養殖及野生鰻腦下垂體 POMC 表現的異同 ………………………………………… 30 形態與生理差異綜合討論 ……………………………………………………………………… 31、結論…………………………………………………………………………………………………………………… 33、參考文獻…………………………………………………………………………………………………………… 34、表 ……………………………………………………………………………………………………………………… 43、圖 ……………………………………………………………………………………………………………………… 44application/pdf1791079 bytesapplication/pdfen-US日本鰻養殖鰻形態指數促性腺激素POMCJapanese eelsCultured eelsMorphologic indicesGonadotropinshttp://ntur.lib.ntu.edu.tw/bitstream/246246/181669/1/ntu-97-R94b45023-1.pdf