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研究生:潘秀敏
研究生(外文):Hsiu-Min Pan
論文名稱:卵母細胞品質和培養液組成分對孤雌激活豬胚及體細胞複製豬胚發育能力之影響
論文名稱(外文):Effects of Oocyte Quality and Medium Components on the Development of Parthenogenetic and Cloned Porcine Embryos
指導教授:沈朋志沈朋志引用關係
指導教授(外文):Perng-Chih Shen
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:畜產系所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:95
中文關鍵詞:Trichostatin ABrilliant cresyl blue培養液成分孤雌激活豬胚複製豬胚
外文關鍵詞:Trichostatin ABrilliant cresyl blueMedium componentsParthenogenetic porcine embryoCloned porcine embryo
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本研究旨在探討卵母細胞生長期別及培養液中組成分對孤雌激活豬卵母細胞及複製豬胚激活後發育能力之影響。於試驗一係評估利用亮鉀酚藍(brilliant cresyl blue, BCB)染色所篩選已達完全生長期卵母細胞之發育能力,及探討胚培養液成分對孤雌激活豬卵母細胞發育能力之影響。結果顯示,孤雌激活豬卵母細胞經PZM-3培養液培養後之分裂率(97.0 vs. 82.6%)與囊胚率(61.8 vs. 32.8%)皆顯著高於培養於NCSU-23培養液組者(P < 0.05)。然而,利用含13 μM BCB培養液染色呈藍色之未成熟猪卵母細胞,經體外成熟培養後之成熟率(77.2 vs. 78.0%)及激活後之囊胚率(26.8 vs. 25.6%),均與未染色之對照組卵母細胞者相近(P > 0.05),但若BCB之濃度增加為26 μM時,則其成熟率反之顯著下降(63.2%)(P < 0.05)。此外,在評估移除部份細胞質對豬卵母細胞於孤雌激活後之體外發育效率時則發現,未去卵質之激活豬卵母細胞的體外囊胚率顯著高於去卵質組之激活豬卵母細胞者(44.5 vs. 19.9%;P < 0.05);惟於未去卵質(36.7 ~ 56.3%)與去卵質(12.9 ~26.7%)組間之激活豬卵母細胞,不論培養於PZM-3液或另添加PVA或sorbitol或PVA + sorbitol者之體外囊胚率於組內之各處理組別間均無顯著差異(P >0.05)。
於試驗二乃評估供核細胞及複製胚經去乙醯化抑制劑(trichostatin A, TSA)培養後,其複製豬胚之後續發育能力。結果顯示,經含40 nM TSA培養之供核細胞所產製複製豬胚之卵裂率(66.8 vs. 32.5%)及發育至四細胞期之百分比(53.4 vs. 14.0%),皆顯著高於未經TSA處理之供核細胞所產製之對照組複製胚者(P < 0.05);惟若培養供核細胞之TSA濃度增加至80 nM時,則各胚期之發育率均與對照組者相近(P > 0.05)。進一步探討TSA應用於複製豬胚發育中之合適添加時機的結果顯示,無論單獨於供核細胞培養過程或/和胚培養液中給予TSA之處理,各處理組複製豬胚於體外發育至各胚期之效率均無顯著差異(P > 0.05)。綜合上述結果說明,相較於NCSU-23培養液而言,利用PZM-3培養液進行孤雌激活豬卵母細胞之體外培養,可得更佳之體外囊胚率;而應用BCB染色以獲得達完全生長期卵母細胞,及在體外培養液中添加PVA或/和Sorbitol之策略,似無助於孤雌激活豬卵母細胞之體外發育能力;惟供核細胞經40 nM TSA培養後,確實能提升由之所生產複製豬胚之早期發育能力。
The objective of this study was to investigate the effect of oocyte quality and medium components on the development of parthenogenticed and cloned porcine embryos. Experiment 1 was carried out to evaluate the brilliant cresyl blue (BCB) test as a means of selecting porcine oocytes that have finished their growth phase for in vitro embryonic development, and the effect of culture medium with different supplements on the in vitro development of embryos. Results showed that the percentages of porcine parthenotes cultured in PZM-3 had significantly higher percentage of the cleavage (97.0 vs. 82.6%) and blastocyst stage (61.8 vs. 32.8%) in comparison with those cultured in NCSU-23 (P < 0.05). Compared to the control, no significant differences in the maturation rate (77.2 vs. 78.0%) and blastocyst formation (26.8 vs. 25.6%) were found in porcine oocytes selected by 13 μM BCB (P > 0.05). However, when BCB concentration was increased to 26 μM, the maturation rate (63.2%) was significantly (P < 0.05) decreased in comparison with the other groups. In
addition, results from evaluating the influence of partially removing cytoplasm in porcine oocytes on in vitro development after parthenogenetic activation revealed that intact porcine parthenotes had significantly higher blastocyst rate than those with cytoplasm partially removed (44.5 vs. 19.9%; P < 0.05). PZM-3 supplementation with polyvinyl alcohol (PVA) and/or sorbitol had no significant effects on the blastocyst rate of intact (36.7~56.3%) and cytoplasm-removed (12.9~26.7%; P > 0.05) porcine oocytes. In experiment 2, the optimal exposure time of Trichostatin A (TSA) for cloned embryo development in vitro was determined. Result showed that TSA-treated donor cells had improved early development depending on the concentration of TSA. When 40 nM TSA was applied, clones showed a better development those without TSA treatment (cleavage rate: 66.8 vs. 32.5%; rate of 4-cell stage: 53.4 vs. 14.0%; P < 0.05). However, when the concentration of TSA was increased to 80 nM, the rate of development to all stages were no significantly different from the control (P > 0.05). Furthermore, treatment of TSA at different time points did not show any significant effects on the in vitro development of donor cells and/or cloned embryos (P > 0.05). In conclusion, addition of PZM-3 provided a more suitable environment for supporting the development of parthenogeticed and cloned porcine embryos in comparison with using NCSU-23. Selection of oocytes by BCB test and supplementation of PVA and/or sorbitol in culture medium failed to improve the in vitro development of parthenogeticed and cloned porcine embryos. Finally, donor cells cultured with 40nM TSA significantly improved the early development of cloned porcine embryos in vitro.
中文摘要 I
Abstract III
謝 誌 V
目 錄 VI
圖表目錄 VIII
壹、前言 1
貳、文獻探討 3
一、影響豬胚體外培養效率之因素 3
(一)豬胚體外培養液 3
(二)外源氨基酸與能量受質之影響 5
(三)胎牛血清與牛血清白蛋白之影響 6
(四)培養環境中養分壓之影響 7
(五)培養液滲透壓之影響 8
二、影響複製豬產製效率之主要因素 9
(一)供核細胞於複製豬產製效率之影響 9
(二)卵母細胞成熟度之影響 12
(三)受核與供核之細胞週期及兩細胞間之交互作用 14
(四)複製胚之激活處理 17
三、TSA處理對複製胚發育能力之影響 18
(一)染色質結構與基因轉錄之關係 19
(二)複製胚之乙醯化調控 21
四、體細胞複製胚之再程序化現象 22
五、體細胞複製豬之應用 23
參、試驗研究 25
研究一:卵母細胞品質及培養液組成分對豬胚孤雌激活後發育能力之影響 25
(一)前言 25
(二)材料與方法 26
(三)結果 37
(四)討論 44

研究二:TSA之處理濃度及時機對體細胞複製豬胚體外發育能力之影響 49
(一)前言 49
(二)材料與方法 50
(三)結果 62
(四)討論 67
參考文獻 70
作者簡介 95
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