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研究生:劉映均
研究生(外文):Liu, Ying-Jyug
論文名稱:海藻糖對冷凍乾燥公豬精液品質之影響
論文名稱(外文):Effect of trehalose on the quality of freeze-dried boar semen
指導教授:陳銘正陳銘正引用關係
指導教授(外文):Chen, Ming-Cheng
口試委員:陳銘正劉秀洲林育安
口試委員(外文):Chen, Ming-ChengLiu, Hsiu-ChouLin, Yu-An
口試日期:2011-07-14
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:動物科技學系碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:148
中文關鍵詞:海藻糖冷凍乾燥公豬精液
外文關鍵詞:TrehaloseFreeze-driedBoar semen
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冷凍乾燥保存精液有著保存時效長、運輸方便與成本較低之優點,但因冷凍乾燥後精子已喪失活動力,無法以體外受精 (in vitro fertilization, IVF) 或人工授精 (artificial insemination, AI) 達成受精之目的,惟仍可應用胞質內精子顯微注射 (intracytoplasmic sperm injection, ICSI) 結合胚移置以產生後代,但如何降低冷凍乾燥過程對精子之傷害,仍是目前主要之課題。本試驗之目的乃建立最佳化公豬精液冷凍乾燥平台,並比較海藻糖濃度 (0 M、0.1 M 與 0.2 M)、保存溫度 (25℃、4℃ 與 -80℃) 與保存時間 (1個月與 6 個月) 對凍乾精液品質及 ICSI 後豬卵母細胞雄原核形成 (male pronuclear formation, MPN) 及胚發育之影響。考量凍乾精液之含水量及精液品質,公豬稀釋精液經 -54℃ 與 5 mTorr 條件下冷凍乾燥 16 小時,含水量降低至 3.55%,精子品質不再有變化。與新鮮精液相比,冷凍乾燥後,無論是否添加海藻糖,稀釋精液精子之存活率 (viability)、頭巾完整率 (acrosomal integrity) 與粒線體功能性 (mitochondrial function) 皆顯著下降,而 DNA 片段化指數 (DNA fragmentation index, DFI) 則顯著上升;惟添加 0.1 與 0.2 M 海藻糖之凍乾精液比未添加者有較高之頭巾完整率與粒線體功能性,且 DFI 也顯著低於未添加者;凍乾精液保存在 -80℃,無論在精子存活率、頭巾完整率、粒線體功能性與 DNA 完整性均比保存在 4℃ 或 25℃ 者為佳;凍乾精液之保存時間不影響精子存活率與粒線體功能性,惟保存一個月之凍乾精液有較高頭巾完整率與較低之 DFI。冷凍乾燥精液進行 ICSI 後,卵母細胞之雄原核形成率、卵裂率 (cleavage rate) 與隨後胚發育率各處理組間皆無顯著差異。綜合上述,添加海藻糖可以提升冷凍乾燥精液精子之頭巾完整率、粒線體功能性與 DNA 完整性,有效降低冷凍乾燥對精子之傷害,但對 ICSI 後胚發育則無明顯改善效果。
The freeze-dried semen was lower cost and more convenience, not only for long-term preservation but also for long-distance transportation. Being freeze-dried, the spermatozoa lost their motility and could not be used for in vitro fertilization (IVF) or artificial insemination (AI) for the purpose of fertilization. Only intracytoplasmic sperm injection (ICSI) with embryo transfer could be possible for producing offspring. How to reduce the damage to sperm during the freeze-drying process is the major issue in this research. The objective of the present study was to establish the optimal condition for freeze-drying boar semen and evaluated the effect of the concentration of trehalose (0 M, 0.1 M and 0.2 M), storage temperature (25℃, 4℃ and -80℃) and storage time (1 and 6 months) on the quality of freeze-dried semen, male pronuclear formation (MPN) and embryo development after ICSI. Considering the water content and quality change of boar semen after freeze-dried, we decided the freeze-dried condition with 5 mTorr and -54℃ for freeze-drying 16 hours. The water content of freeze-dried semen was reduced to 3.55%, the quality of freeze-dried semen was not affects for 1 week preservation at 18℃. Compared with fresh semen, the viability, acrosomal integrity and mitochondrial function of freeze-dried semen were significantly decreased and DNA fragmentation index (DFI) was increased significantly. However, the 0.1 M or 0.2 M of trehalose freeze-dried semen possessed higher acrosomal integrity and mitochondrial functional and indicated a significantly lower DNA fragmentation index than that without trehalose. Being stored at -80℃ the viability, acrosomal integrity, mitochondrial functional and DNA integrity of freeze-dried semen were better than that stored at 25℃ or 4℃. Despite that the time in storage did not affect its viability and mitochondrial functional, freeze-dried semen in storage for one month indicated higher acrosomal integrity and lower DFI than that in six months. After the injection of freeze-dried sperm into the oocytes, the male pronuclear formation rate, embryos cleavage and embryo development rate between the treatment groups had no significant difference. Therefore, adding the trehalose can enhance the acrosomal integrity, mitochondrial functional and DNA integrity and effectively reduce the damage to the sperm during the freeze-drying process, but it provides no significant improvement to MPN and embryos development after ICSI.
中文摘要………………………………………………………………………...I
英文摘要…………………………………………………………………….....III
表目錄………………………………………………………………………….XI
圖目錄…………………………………………………………………...…...XIII
附次…………………………………………………………………………...XV
壹、 前言……………………………………………………………………….1
貳、 文獻回顧………………………………………………………………….3
一、 前言……………………………………………………………………….3
二、 公豬精液保存…………………………………………………………….4
(一) 液態稀釋保存……………………………………………………………..4
1. 稀釋精液備製………………………………………………………………..5
2. 影響稀釋精液保存之因素…………………………………………………..5
3. 液態稀釋保存所面臨之問題………………………………………………..9
(二) 固態冷凍保存……………………………………………………………..9
1. 公豬冷凍精液之製作與保存……………………………………..…………9
2. 影響精液冷凍保存之因素……………………………..…………………..10
3. 冷凍保存所面臨之問題…………………………………………...……….12
(三) 冷凍乾燥保存……………………………………..……….……...……..13
1. 冷凍乾燥原理與流程……………………………………….………….…..13
2. 影響冷凍乾燥精液保存之因子……………………………….…………..14
3. 冷凍乾燥保存面臨之課題…………………………………………………18
三、 精液品質檢測………………………………………………………...…18
(一) 例行性之精液檢測………………………….....………………………...18
1. 物理性狀…………...……………………………………………………….19
2. 精子活力………………………………………...………………………….19
3. 精子存活率………………………………...……………………………….19
4. 精子形態…………………...……………………………………………….20
5. 精子濃度…………………...……………………………………………….20
(二) 非例行性之精液檢測…...………………………………………....…….21
1. 低滲透壓腫脹測試……………………………………………….…..…….21
2. 流式細胞儀……………………………………………….…..…………….22
四、 保存精液之授精………………………….…..…………………………..25
(一) 人工授精……………………….…..…………………………….………25
(二) 體外受精……………………….…..…………………………….………26
(三) 精子顯微注射……………………….…..…………………………….…27
1. 精子顯微注射之過程與處理……….…..………………………..…….…..27
2. 豬精子顯微注射之應用與發展..…………………………….…….28
3. 豬精子顯微注射所面臨之問題與改善策略……………………….…….29
五、 總結……………………….……..........................................................30
參、 材料與方法…………………………………………………….…..31
一、 精液來源………………………………………………………….….31
(一) 精液品質評估…………………………………………………….……...31
1. 活力………………………………………………………………..………31
2. 濃度………………………………………………….…………………....31
3. 形態………………………………………………….…………………….31
4. 流式細胞儀評估精液品質………………………….…………………..32
(二) 精子之冷凍乾燥……….…………………………………………..….…34
(三) 精子之體外獲能……….………………………………….……..…......34
二、 精子顯微注射……….……….…………………………….…….…..37
(一) 卵母細胞之來源與體外成熟……….…………………………………37
(二) 顯微注射針之製作與架針步驟.……………………………………….38
(三) 精子顯微注射之步驟.…………………………………………………38
三、電激活之處理……………………………………………..……………….41
四、體外培養…………………………………………………..……………….41
五、卵母細胞與胚形態之觀察………………………………..……………….42
六、 評估指標……………………………..…………………………………42
(一) 成熟評估……………………………..…………………..………………42
(二) 受精評估……………………………..…...…..………………………….42
(三) 發育評估…………………………....…...……………………………….43
七、 統計分析……………………..…...……………………………………..43
八、 試驗設計……………………………………………….…………………45
(一) 建立 Acridine orange 染色檢測精子 DNA 之完整性…….…..……...45
(二) 公豬精液冷凍乾燥條件之試驗……………………….……………...…45
(三) 海藻糖對冷凍乾燥後公豬精液精子品質之影響….………………...…45
(四) 公豬精液冷凍乾燥後保存狀態對精子品質之影響….……………...…45
肆、 結果….………………………………………………………………......47
一、 建立Acridine orange 染色檢測精子 DNA 之完整性………………..47
二、 公豬精液冷凍乾燥條件之設定……………………………………...….51
三、 海藻糖對冷凍乾燥公豬精液精子品質之影響…………………...……62
四、 公豬精液冷凍乾燥後保存狀態對精子品質之影響……………….…..72
伍、討論………………………………………………………………..….…..90
一、 建立Acridine orange 染色檢測精子 DNA 完整性……………………90
二、 公豬精液冷凍乾燥條件之建立…………………..………………….…..90
三、 海藻糖對冷凍乾燥公豬精液品質之影響……..………………….……91
四、 海藻糖對凍乾精子 ICSI 之影響……..………………….………….…93
五、 保存狀態對公豬凍乾精液品質之影響..………………….………….…94
陸、 結論.………………….………….………………………………………95
柒、 參考文獻…………….………….……………………..…………………96
捌、 附錄.……………………………………………………………………122

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