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研究生:蔡馥嚀
研究生(外文):Fu-Ning Tsai
論文名稱:利用冷凍乾燥法保存淡水魚類精子相關因素之研究
論文名稱(外文):Study on Lyophilization-related Factors in Freshwater Fish Sperm
指導教授:陳鴻鳴陳鴻鳴引用關係趙乃賢
指導教授(外文):Hong-Ming ChenNai-Hsien Chao
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:2004
畢業學年度:92
語文別:中文
論文頁數:120
中文關鍵詞:冷凍乾燥魚類精子流式細胞儀掃瞄式電子顯微鏡
外文關鍵詞:lyophilizationfishspermFlow cytometerSEM
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摘要
應用冷凍乾燥保存動物精子之技術,目前仍屬於新的研究領域,現階段科學家只以少數物種,包括小鼠、牛、及兔子為對象來探討並已發表研究結果;至於在魚類方面目前正處於初期試驗階段。本研究之目的在應證首創將哺乳類精液冷凍乾燥粉末之同類技術應用於台灣淡水魚類配子保存之可行性,以期提供保有種原多樣性之另一途徑。本實驗針對冷凍乾燥技術應用於魚類精子保存的研究,已初步完成淡水魚類精液冷凍乾燥之基本流程及冷凍乾燥精子復水流程,並探討可茲改善的諸多方向。
精液與稀釋緩衝液EGTA buffer容積比,對冷凍乾燥後精細胞之復水性及細胞膜活性影響實驗之結果顯示,以精液比例愈高之組別進行冷凍乾燥後,其復水後細胞群大小和DNA量集中現象愈明顯,即復水率越高,且不同濃度之EGTA buffer間具有相同之趨勢。而細胞膜活性維持結果,則大致和復水率結果相反。鯉魚精液實驗結果,以採EGTA buffer:純精液容積比為2:1,且EGTA buffer濃度為50/50/10之冷凍效果最佳,其具完整細胞膜之精細胞比例高達33.94 %。
本文著重鯉魚、鯝魚、台灣石鲋、台灣石賓、泥鰍等五種魚類精子,比較添加EGTA buffer下精子之敏感性,及最適冷凍乾燥濃度效果之差異。結果顯示:泥鰍精子對於EGTA buffer之耐受性較低,存活率以濃度為20/50/10組為最低,為10.05 %,控制組為78.67 %。而台灣石賓精子之耐受性較高,濃度為20/50/10組之存活率最低,也仍有80.32 %,控制組為93.10 %。結果顯示精細胞對於EGTA buffer之敏感性,會因濃度及實驗魚種而有差異。整體言之,EGTA buffer對於精細胞具有某種程度之毒性,會影響精細胞存活,應減少暴露於EGTA buffer的時間。
最適冷凍乾燥濃度效果方面,上述五種魚類精子於冷凍乾燥後之復水率,皆以不加任何媒介物之效果最好。而以細胞膜完整性之維持為指標,各魚種之最適冷凍乾燥之媒介物及濃度分別為,EGTA buffer (50/50/10)、(50/80/10)、(PBS)、(PBS)及(50/20/10)。可見EGTA buffer對冷凍乾燥之細胞膜完整性維持效果,依魚種不同而有差異。
SEM電子顯微鏡分析鯉魚冷凍乾燥精子粉末結果:復水前,精子外觀完整程度,依序為PBS、EGTA buffer (不同五組濃度,無顯著差異)、pure sperm。而依據電顯圖檢視各組之精細胞密度結果,也和流式細胞儀分析之復水率結果相符。經由EGTA buffer處理之組別,其復水後,組織凝集現象明顯,精子濃度明顯下降。
目前完成之冷凍乾燥之基要流程,可望提供未來以凍乾法長期保存之後續研究,以提供魚類配子保存法之另一種亟待深度開發途徑。
Abstract
Lyophilization, also known as freeze-drying, has been generally applied in food science, microbiology, chemistry and pharmaceuticals. It is advantageous in terms of stability of important components, economics in storage, and convenience. This study aims to study the related factors with purpose to apply in future lyophilization on male gametes of fishes for the feasibility of long term preservation. Treatment of collected milt with simple EGTA buffer (composed of Tris-HCl containing EGTA and NaCl of various concentrations) but without cryoprotectant allowed the subsequent lyophilization of sperm. Experiments were conducted to compare different factors on lyophilization of sperm of several local freshwater fishes. These factors include: combination concentrations of EGTA, NaCl and Tris-HCl; ratios of milt to buffer; durations of lyophilization; and protocols and media of rehydration. Results of these preliminary experiments are reported and discussed. Efforts were made to decrease the percentage of dead sperm cells after buffer treatment but before lyophilization and to increase the percentage of lyophilized sperm cells with membrane integrity. The follow-up study will focus on microinjection of immotile sperm heads into eggs using intracytoplasmic sperm injection technique (ICSI) as platform. It is hoped that the study of the optimal protocol with favorable factors of lyophilization of fish sperm will lead to an ideal and novel method of preserving gametes in addition to traditional cryopreservation in selected and endangered domestic finfish species.
目錄
中文摘要…………………………………………………………………I
Abstract………………………………………………………………Ⅲ
謝辭………………………………………………………………………Ⅳ
目錄………………………………………………………………………Ⅵ
表目錄…………………………………………………………………Ⅸ
圖目錄………………………………………………………………Ⅹ
壹、前言…………………………………………………………………1
一、文獻回顧………………………………………………………2
二、研究動機及目的………………………………………… 10
貳、材料與方法……………………………………………………13
一、材料………………………………………………………13
二、方法…………………………………………………………17
(一)、建立冷凍乾燥之最適流程…………………………………17
1. 實驗魚種精液特性之掌握………………………………17
2. 冷凍乾燥螫合劑 EGTA buffer之配製..................20
3. 流式細胞儀應用-檢測精子存活比率及計數……………21
4. 記錄精子數位影像檔…………………………………21
5. 冷凍乾燥之最適凍結點測試……………………………22
6. 冷凍乾燥系統及冷凍乾燥所需時間……………………22
7. 封管及保存………………………………………………23
8. 乾燥精子之復水程序及復水用液………………………23
9. 乾燥精子之復水率、細胞膜及細胞核完整性分析……24
(二)、流式細胞儀於魚類精子冷凍乾燥保存法之相關應用………24
1. 精子計數…………………………………………………25
2. 新鮮精液品質檢測………………………………………26
3. 進行EGTA buffer敏感性實驗之存活率檢測…………29
4. 冷凍乾燥後細胞活性及細胞核完整性檢測……………30
(三)、EGTA buffer與精液容積比對復水性及細胞膜活性之影響.30
1. 定量精液與EGTA buffer之比例…………………………30
2. 實驗設計…………………………………………………31
3. 統計分析…………………………………………………32
(四)、EGTA buffer對精子之敏感性測試及最適冷凍乾燥濃度…32
1. 敏感性測試………………………………………………32
2. 最適冷凍乾燥濃度………………………………………33
(五)、利用掃瞄式電子顯微鏡觀察冷凍乾燥精子粉末之外部特徵.34
1. 掃描式電子顯微鏡( SEM )之樣品製作…………………34
2. 冷凍乾燥精子粉末(復水後)之樣品製作………………35
3. 冷凍乾燥精子粉末(復水前)之樣品製作………………36
4. SEM上機操作……………………………………………37
參、結果…………………………………………………………………38
(一)、冷凍乾燥最適流程………………………………………………38
(二)、流式細胞儀分析模式之探討…………………………………43
1. 樣品染色時間………………………………………………43
2. 細胞濃度……………………………………………………44
(三)、精液與EGTA buffer容積比對復水性及細胞膜活性之影響…44
1. 復水性……………………………………………………45
2. 完整細胞膜比例……………………………………………45
(四)、EGTA buffer對五種魚類精子之敏感性及最適冷凍乾燥濃度46
1. 敏感性測試…………………………………………………46
2. 復水率………………………………………………………48
3. 細胞膜完整性及細胞核收集………………………………49
(五)、以掃瞄式電子顯微鏡觀察精子粉末復水前後之外觀……50
1. 復水前………………………………………………………50
2. 復水後………………………………………………………51
肆、討論……………………………………………………………53
伍、參考文獻………………………………………………………59
附表…………………………………………………………………66
附圖…………………………………………………………………71
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