本研究用濾網收集海鱺魚鰓上的卵圓鞭毛蟲（Amyloodinium ocellatum），進行4℃低溫冷藏實驗、抗凍劑毒性測試、冷凍保存測試及利用魚類細胞株體外培養卵圓鞭毛蟲測試等，來探討卵圓鞭毛蟲可行的冷凍保存和體外培養方法。以營養體（trophont）活存率、孢囊體（tomont）分裂情形和浮游孢子（dinospore）的數量，來評估可行的冷凍保存和體外培養方法。結果顯示，長時間4℃低溫保存，會影響trophont分裂，產生不規則延緩分裂、不規則的分裂及停止分裂等現象，而4℃冷藏24小時組，1×103個trophont可產生1.08×104 cell/ml個dinospore明顯較其他冷藏組別高（p＜0.0001），因此trophont可利用4℃低溫短期保存24小時。經過抗凍劑毒性測試，可用的抗凍劑種類和濃度有DMSO 3~10﹪、Glycerol 3~10﹪、Methanol 3~10﹪、Ethanol 3~5﹪、PrOH 3~5﹪、DMAc 3~5﹪、Sucrose 3~15﹪、Trehalose 3~15﹪、Dextran 3~5﹪及Ficoll 3~10﹪。在冷凍保存方面，使用10﹪DMSO和Glycerol兩種抗凍劑及直接液態氮低溫冷凍、不同-20℃冷凍時間處理、-1℃ min-1冷凍盒冷凍和不同抗凍劑平衡時間處理等四種冷凍處理，皆無法成功有效的冷凍保存卵圓鞭毛蟲trophont蟲體。在本研究中使用單、雙環U形管收集dinospore，可以收集到無細菌污染的dinospore。將收集的dinospore，分別加入含不同培養液之鰻魚表皮細胞株及海鱺鰭細胞株培養皿中，進行體外培養卵圓鞭毛蟲測試，各組皆沒有發現trophont或tomont，表示含不同培養液之各組細胞株無法成功體外培養卵圓鞭毛蟲。

The Amyloodinium ocellatum was collected from cobia ( Rachycentron canadum ) gill and four tests including 4 ℃ storage, toxicity of cryoprotectant, cryopreservation and in vitro cultivation on fish cell line were conducted to establish the methods of preservation of Amyloodinium ocellatum. Survival of trophont, morphology and division of tomont and number of dinospore released were evaluated the effects of this study. The results showed that division irregulated, delayed and stopped of the tomont were found after stored at 4 ℃ over 48 hours. It was produced 1.08 x 10 4 cell/ml dinospores from 1 x 10 3 trophont at 4 ℃, 24 hours storage group and significant higher ( p＜0.0001 ) than other storage groups. For the toxicity of cryoprotectant, the concentration of DMSO 3~10﹪, Glycerol 3~10﹪, Methanol 3~10﹪, Ethanol 3~5﹪, PrOH 3~5﹪, DMAc 3~5﹪, Sucrose 3~15﹪, Trehalose 3~15﹪, Dextran 3~5﹪and Ficoll 3~10﹪were safety to use on A. ocellatum trophont preservation. It was unsuccessful to cryopreserve the trophont of A. ocellatum when stored at direct liquid N2 freezing, different -20 ℃ freezing time, -1 ℃ min-1 freezing container and different cryoprotectant equilibration time contain 10﹪Glycerol and DMSO, respectively. Using the U-shaped tube of sigle and double loop could gain pure and bacteria-free dinospores. The results of in vitro cultivation of A. ocellatum showed that eel epidermis and cobia fin cell line with different culture mediums were unable to grow the trophont and tomont of A. ocellatum.

目錄
章次 頁次
謝辭……………………………………………………………………I
中文摘要………………………………………………………………II
英文摘要………………………………………………………………III
目錄……………………………………………………………………V
圖目錄…………………………………………………………………VI
表目錄…………………………………………………………………VII
文獻整理………………………………………………………………1
前言……………………………………………………………………7
材料與方法……………………………………………………………9
結果……………………………………………………………………16
討論……………………………………………………………………44
參考文獻………………………………………………………………48
附錄……………………………………………………………………52
個人履歷………………………………………………………………55

圖目錄
圖次 頁次
一、卵圓鞭毛蟲 Amyloodinium ocellatum 生活史………………… 2
二、卵圓鞭毛蟲 Amyloodinium ocellatum 分裂形態………………17
三、4 ℃ 冷藏後卵圓鞭毛蟲 trophont活存率………………………22
四、4 ℃ 冷藏後卵圓鞭毛蟲 trophont、tomont 死亡情形…………24
五、DMSO - Glycerol抗凍劑處理卵圓鞭毛蟲 trophont 死亡率……27
六、DMSO - Glycerol抗凍劑處理卵圓鞭毛蟲dinospore孵出數量…28
七、解凍後卵圓鞭毛蟲 Amyloodinium ocellatum 的情形………… 32
八、單環和環U形管收集卵圓鞭毛蟲dinospore 結果………………34

表目錄
表次 頁次
一、 4 ℃ 冷藏後卵圓鞭毛蟲產生dinospore的數量…………………23
二、十種抗凍劑毒性測試種類間的比較……………………………30
三、十種抗凍劑毒性測試濃度間的比較……………………………31
四、鰻魚表皮細胞株培養液的pH值、鹽度、滲透壓和Na、K、Ca
離子量 ………………………………………………………………37
五、在鰻魚表皮細胞株和各組培養液中Amyloodinium ocellatum
dinospore 活動情形…………………………………………………38
六、在各組培養液中鰻魚表皮細胞株生長情形……………………39
七、海鱺鰭細胞株培養液的pH值、鹽度、滲透壓和Na、K、Ca離
子含量 ………………………………………………………………41
八、在海鱺鰭細胞株和各組培養液中Amyloodinium ocellatum
dinospore 活動情形…………………………………………………42
九、在各組培養液中海鱺鰭細胞株生長情形………………………43

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