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研究生:楊翊
研究生(外文):Yi Yang
論文名稱:豬生發泡卵母細胞之玻璃化冷凍保存
論文名稱(外文):Cryopreservation of Porcine Oocytes at Germinal Vesicle Stage by Vitrification
指導教授:陳銘正陳銘正引用關係
指導教授(外文):Ming-Cheng chen
口試委員:吳信志宋麗英林育安
口試委員(外文):Shinn-Chig WuLi-Ying SungYu-An Lin
口試日期:2014-06-30
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術與動物科學系動物科學碩士班
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:2014
畢業學年度:103
語文別:中文
論文頁數:110
中文關鍵詞:卵母細胞玻璃化冷凍
外文關鍵詞:porcineoocytevitrification
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豬卵母細胞冷凍保存可做為種原保存之方法,搭配體外胚生產系統與胚移置等生殖技術,可以應用於種原交流與傳播,加速育種改良之速率。本試驗之目的乃應用乙二醇 (Ethylene glycol, EG) 搭配雙甲基亞砜 (Dimethyl sulfoxide, DMSO) 作為冷凍保護劑,以固體表面玻璃化冷凍法 (solid surface vitrification, SSV) 冷凍保存豬生發泡卵母細胞。試驗首先評估冷凍保護劑組合對豬生發泡卵母細胞滲透性緊迫與毒性傷害之影響,隨後進行SSV法冷凍保存豬卵母細胞,並評估冷凍保護劑組成對卵母細胞之存活率、成熟率與細胞內活性氧之含量,最後評估冷凍保存對豬卵母細胞於孤雌致活後胚發育能力之影響。
試驗一,首先將豬生發泡卵母細胞逐步平衡於30% EG、30% DMSO、15% Mix (15% EG+15% DMSO) 與20% Mix (20% EG+20% DMSO)共4組之冷凍保護劑組合,雖然比對照組有較低之卵母細胞與卵丘細胞存活率,但皆未達顯著水準。試驗二,進一步將豬生發泡卵丘卵母細胞複合體 (cumulus-oocytes complexes, COCs) 平衡於4種冷凍保護劑30秒、2分鐘與5分鐘,評估體外成熟培養48 h後卵母細胞之存活率與成熟率,試驗結果顯示,豬生發泡卵母細胞隨著冷凍保護劑處理時間之增加,其存活率與成熟率均顯著下降,且僅以30% EG、15% Mix與20% Mix處理30 秒者,卵母細胞之存活率與成熟率與對照組無顯著差異。試驗三,比較SSV法與麥管 (straw) 冷凍經30% EG、15% Mix或20% Mix 處理之豬生發泡卵丘卵母細胞,評估體外成熟48h 後卵母細胞之存活率與成熟率,試驗結果顯示,以SSV法冷凍保存豬生發泡卵母細胞之存活率與成熟率均顯著高於麥管冷凍組,且以30% EG搭配SSV法冷凍保存之卵母細胞存活率與成熟率分別為73.7%與42.5%,均顯著高於其他組別,但仍顯著低於對照組之100%與86.5% (P < 0.05)。試驗四,將豬生發泡階段之卵丘卵母細胞經30% EG、15% Mix與20% Mix 冷凍保護劑處理,再搭配SSV法冷凍保存,評估卵母細胞於體外成熟48h後ROS之含量及電激活後胚發育情形,試驗結果顯示,30% EG之ROS含量顯著低於15% Mix與20% Mix (P < 0.05) ,但與對照組並無顯著差異,所有冷凍豬生發泡卵母細胞經孤雌致活後,卵裂率與發育至8細胞以上之能力均顯著低於新鮮卵母細胞之對照組 (P < 0.05)。
綜上所述,豬生發泡卵母細胞以30% EG搭配SSV法冷凍保存能獲得較高之存活率、成熟率與較低之氧化傷害,然而胚發育之能力仍然低於未冷凍者。

The cryopreservation of porcine oocytes accompanied by in vitro embryo production and embryo transfer can be used for preserving genetic resources and genetic information from various species and accelerating the breeding rate. The objective of this study was to study the effects of cryoprotectan components containing ethylene glycol (EG) and dimethyl sulfoxide (DMSO) on the cryopreservation of porcine oocytes at the germinal vesicle stage through solid surface vitrification (SSV). The first experiment was to evaluate the effect of cryoprotectant components on the osmotic stress and toxic injury of porcine oocytes at the germinal vesicle stage. Subsequently, the porcine oocytes were cryopreserved by SSV to evaluate the effect of the cryoprotectant components on the survival rate and maturation rate of cryopreserved oocytes and reactive oxygen species (ROS) in the oocytes. Finally, we analysed the effects of oocytes cryopreservation on the development of porcine oocytes after parthenogenetic activation.
In Experiment 1, the porcine oocytes were gradually treated with 4 cryoprotectant components that contained 30% EG, 30% DMSO, 15% Mix (15% EG + 15% DMSO), or 20% Mix (20% EG + 20% DMSO). The results indicated that the survival rate of oocytes and cumulus cells treated with cryoprotectant components was lower than control, but it was not significantly different. In Experiment 2, the porcine oocytes were treated with 4 cryoprotectant components for 30 s, 2 min, and 5 min. The survival and maturation rates of oocytes were evaluated after they were cultured in vitro for 48 h. The results indicated that both the survival and maturation rates were significantly reduced when the oocytes were treated with cryoprotectants for longer than 30 s. However the survival and maturation rates of oocytes treated with 30% EG, 15% Mix and 20% Mix for 30 s did not significantly differ from those of the control. In Experiment 3, the porcine oocytes at the germinal vesicle stage were cryopreserved by using straw and SSV after cryoprotectant treatment (30% EG, 15% Mix, and 20% Mix) and were cultured in vitro for 48 h. The survival and maturation rates of oocytes cryopreserved by SSV were significantly higher than those cryopreserved in straw. However, the survival and maturation rates of oocytes vitrified by SSV with 30% EG cryoprotectant were 73.7% and 42.5% respectively, which were significantly lower than that of the control (100% and 86.5%) and significantly higher than that produced using other treatments (P < 0.05). In Experiment 4, the porcine oocytes at the germinal vesicle stage were cryopreserved by SSV after treatment with 30% EG, 15% Mix, or 20% Mix cryoprotectants. The level of ROS in the oocytes cultured in vitro for 48 h was evaluated. The results indicated that the ROS level in the oocytes cryopreserved with 30% EG cryoprotectant was significantly lower than that caused by using 15% Mix and 20% Mix treatment (P < 0.05), but was no difference from those of the control. The rates of cleavage embryo development over 8 cells stage after the maturation and activation of cryopreserved porcine oocytes at the germinal vesicle stage were significantly lower than those of the control (P < 0.05).
In conclusion, porcine oocytes at the germinal vesicle stage vitrified with cryoprotectants containing 30% EG cryoprotectant by SSV had comparatively higher survival rates, maturation rates, and a lower ROS level; however, embryo development was still lower than that of fresh oocytes.

中文摘要 I
Abstract III
致謝 VI
表次 X
圖次 XII
壹、前言 1
貳、文獻檢討 4
一、種原保存 4
(一) 胚 4
(二) 精子 5
(三) 卵母細胞 6
二、卵母細胞之冷凍技術 9
(一) 慢速冷凍法 (slow freeze) 9
(二) 玻璃化冷凍法 (vitrification) 10
三、冷凍傷害 10
(一) 核成熟階段 10
(二) 細胞骨架 11
(三) 細胞內脂質小滴 12
(四) 氧化傷害 13
四、冷凍器材之改良 14
(一) 開放式手拉麥管 (open pulled straw, OPS) 14
(二) 冷凍環 17
(三) 電子顯微鏡載物格 19
(四) Cryotop 21
(五) 固體表面玻璃化冷凍 23
五、冷凍保護劑 25
(一) 非滲透性冷凍保護劑 25
(二) 滲透性冷凍保護劑 26
六、冷凍卵母細胞之現況 27
(一) 人 27
(二) 牛 27
(三) 綿羊 29
(四) 豬 30
參、材料與方法 31
一、 卵母細胞來源 31
二、 卵母細胞體外成熟 (in vitro maturation, IVM) 31
三、 電激活 (electrical activation) 36
四、體外胚培養 (in vitro culture, IVC) 38
五、卵母細胞冷凍與解凍 38
(一) 固體表面玻璃化冷凍法 38
(二) 0.25 ml 麥管 39
六、評估指標 40
(一) 存活率 40
(二) 核成熟 44
(三) 活性氧 46
(四) 發育評估 48
七、試驗設計 48
(一) 逐步提升冷凍保護劑濃度對豬生發泡卵丘卵母細胞存活之影響 48
(二) 冷凍保護劑組合對豬生發泡卵丘卵母細胞存活之影響 49
(三) 冷凍保護劑組合對豬生發泡卵母細胞體外成熟後存活及成熟之影響 50
(四) 冷凍保護劑組合與冷凍器材對冷凍保存豬生發泡卵母細胞解凍後2h存活率與體外成熟之影響 50
(五) 冷凍保護劑組合對固體表面玻璃化冷凍保存豬生發泡卵母細胞氧化傷害之影響 51
(六) 冷凍保護劑組合對固體表面玻璃化冷凍保存豬生發泡卵母細胞經孤雌致活後胚發育能力之影響 51
肆、結果 53
一、逐步提升冷凍保護劑濃度對豬生發泡豬卵丘卵母細胞存活之影響 53
二、冷凍保護劑組合對豬生發泡卵丘卵母細胞存活之影響 60
三、冷凍保護劑組合對豬生發泡卵母細胞體外成熟後存活及成熟之影響 62
四、冷凍保護劑組合與冷凍器材對冷凍保存豬生發泡卵母細胞解凍後2h存活率與體外成熟之影響 65
五、冷凍保護劑組合對固體表面玻璃化冷凍保存豬生發泡卵母細胞氧化傷害之影響 68
六、冷凍保護劑組合對固體表面玻璃化冷凍保存豬生發泡卵母細胞經孤雌致活後胚發育能力之影響 71
伍、討論 73
一、逐步提升冷凍保護劑濃度對豬生發泡豬卵丘卵母細胞存活之影響 73
二、冷凍保護劑組合對生發泡豬卵丘卵母細胞存活之影響 74
三、冷凍保護劑組合對生發泡豬卵母細胞體外成熟後存活及成熟之影響 74
四、冷凍保護劑組合與冷凍器材對冷凍保存生發泡豬卵母細胞解凍後2h存活率與體外成熟之影響 76
五、冷凍保護劑組合對固體表面玻璃化冷凍保存生發泡豬卵母細胞氧化傷害之影響 77
六、冷凍保護劑組合對固體表面玻璃化冷凍保存生發泡豬卵母細胞經孤雌致活後胚發育能力之影響 78
陸、結論 79
柒、參考文獻 80

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