臺灣博碩士論文加值系統

本研究旨在應用牛胚體外生產系統，包括牛卵之體外成熟、受精及受精卵之體外培養等技術，冀能於體外產生數目眾多處於特定發育階段之受精卵或胚源，供往後進行基因轉殖、冷凍保存及胚移置之使用。
試驗結果證明，源自屠宰場屠宰之荷蘭乳牛，取得卵巢濾泡內具完整卵丘-卵母細胞複合體之初級卵母細胞，合計6,479個，經應用5% FCS+ Medium-199之成熟液，於38.5oC、2% CO2及98%空氣與100%相對濕度等條件下進行體外培養24h後，平均可獲得69.6%之卵母細胞發育達第二次減數分裂中期(2nd meiotic metaphase, MetII)階段。進一步將源自體外成熟之牛卵丘-卵母細胞複合體與業經體外獲能之公牛冷凍暨解凍精子（1×107個精子/ml）於體外受精用培養液中進行共培養4、8、12、16、20及24h後，經固定染色證明，其受精率與原核形成百分率分別為55.5、88.9、82.8、90.0、80.0、85.4% 與0、69.1、85.3、86.6、100、85.2%。另一方面，於體外受精培養終了時，將卵母細胞取出經清洗移去粒性細胞後，再置回原含有卵丘細胞飼養層之成熟培養液中進行共培養5~7日，證明發育達桑椹胚期~囊胚期者顯著高於未經共培養者（22.1 vs 9.4%）；此外，利用此等體外生產發育至桑椹胚期~囊胚期之牛胚，進行冷凍暨解凍後之存活率約為45.7%。至於牛胚外源基因之顯微注射係於受精後16~20h予以進行，並經與卵丘細胞飼養層共培養5~7日後，發育至桑椹胚期~囊胚期之百分率可達13.6%；進一步將其移置於發情同期化之受胚牛後，更可順利懷孕並獲得正常活仔牛之出生。綜合上述結果，顯示本培養系統確能提供牛胚生產，以進行基因轉殖、冷凍保存及胚移置試驗之使用。

The aim of this study was to apply in vitro produced (IVP) embryos from oocytes matured, fertilized and cultured system in vitro which were capable of large-scale materials for transgenic, embryos frozen and embryos transfer in cattle.
Oocyte-cumulus complexes in 6,479, aspirated from antral follicles of Holstein ovaries, were cultured with Medium-199, supplemented with 5% FCS, in a CO2 incubator ( 2% CO2 in air at 38.5℃, 95% humidity) for 24h. These results showed that the precentage of these oocytes developed to second meiotic metaphase was 69.6%. Moreover, frozen-thawed bull sperm after capacitation in vitro were co-cultured with the in vitro matured oocytes for 4, 8, 12, 16, 20 and 24h . Stain results observed there were 55.5, 88.9, 82.8, 90.0, 80.0, and 85.4% and 0, 69.1, 85.3, 86.6, 100 and 85.2% of eggs appeared to be fertilized and showing clear configuration of pronuclei within ooplasm, respectively. On the other hand, at the end of culture, oocytes were removed from cumulus cells and transfered back into the maturation drop and co-cultured 5-7 days with the cumulus feeder layers. The proportion of oocytes cleaved to morula-blastocyst stage was significantly higher in co-culture than culture-free cumulus feeder layers (22.1 vs. 9.4% ). Besides, we employed in vitro- produced morula and blastocyst for embryos frozen showed that the proportions frozen-thawed survived embryos after the in vitro culture was 45.7%. Pronuclear DNA inject was performed at 16-20 hr postinsemination. The rates of in vitro developed to morula-blastocyst stage with co-culture cumulus feeder layers was 13.6%. Furthermore, these transgenic embryos were transferred to recipient cow on day 6 to 8 post-estrus and resulted in a live calf was born. It is concluded that these IVM, IVF and IVC system could fit properly to materials for transgenic, embryos frozen and embryos transfer in cattle.

目 錄
摘要……………………………………………………….1
緒言……………………………………………………….3
文獻檢討
壹、卵母細胞之成熟作用………………………………4
一、卵巢濾泡之發育……………………………………4
二、卵巢細胞之成熟過程………………………………5
三、卵母細胞成熟作用之調節…………………………8
貳、受精作用……………………………………………18
一、受精過程……………………………………………18
二、影響受精過程之因素………………………………24
參、受精卵於體外發育之潛能…………………………31
一、胚之體外培養………………………………………31
二、體內環境與體外培養之差異………………………36
肆、應用體外生產系統產製基因轉殖牛之方法………39
一、產製基因轉殖家畜之經濟價值與面臨之挑戰……39
二、胚源之取得…………………………………………42
三、源核注射之時機與部位……………………………44
四、轉殖基因的效率……………………………………45
五、胚之體外培養與胚移置……………………………46
伍、牛胚之冷凍保存……………………………………47
材料與方法
一、 卵巢及卵母細胞之來源與處理………………………………49
二、 卵母細胞之體外成熟培養……………………………………50
三、 卵母細胞之體外受精…………………………………………52
四、 受精卵之體外培養……………………………………………54
五、 卵母細胞、受精卵、及胚等之固定、染色及其發育評估…57
六、 外源基因之顯微注射…………………………………………60
七、 牛胚之冷凍保存………………………………………………63
八、 胚移置之進行…………………………………………………65
九、 出生仔牛之DNA分析…………………………………………67
十、 統計分析………………………………………………………67
結果與討論
一、 卵母細胞之體外成熟率………………………………………68
二、 體外成熟牛卵之受精率與原核發育率………………………74
三、 牛胚體外培養之發育潛能……………………………………82
四、 離心與顯微操作處理對體外發育潛能之影響………………89
五、 牛胚冷凍暨解凍後之存活率…………………………………95
六、 胚移置後之體內發育潛能……………………………………97
七、 出生子代之外源基因檢測……………………………………103
結論………………………………………………………………….105
參考文獻…………………………………………………………….106
英文摘要…………………………………………………………….122
小傳………………………………………………………………….124
表 次
表1. 早期胚之雄原核與雌原核之發育過程…………………………22
表2. 原核顯微注射法產製基因轉殖家畜之效率……………………41
表3. 原核顯微注射法產製基因轉殖牛之效率………………………43
表4. Medium-199培養液之組成分……………………………………51
表5. 精子洗滌液及獲能液之成分……………………………………53
表6. 修正之磷酸緩衝液成分…………………………………………59
表7. 卵丘-卵母細胞複合體完整性對卵母細胞體外成熟
之影響…………………………………………………………………70
表8. 體外受精後不同時段對源自體外成熟牛卵原核發育
之影響…………………………………………………………………76
表9. 源自體外成熟及體外受精之牛胚於不同體外培養
系統中發育潛能之比較………………………………………………86
表10. 源自不同卵巢來源之牛卵母細胞經體外成熟及體外
受精後與卵丘細胞進行共培養五至七日之發育潛能………………90
表11. 離心與基因顯微注射處理對源自體外成熟及體外
受精之牛受精卵與卵丘細胞進行共培養五至七日
之發育潛能……………………………………………………………92
表12. 源自體外成熟、體外受精與體外培養後所產生之
牛胚經冷凍暨解凍後之存活率………………………………………99
表13. 源自體外成熟、體外受精與體外共培養五至七日
所產生之牛胚經胚移置後於體內之發育潛能………………………101
圖 次
圖1. 哺乳動物卵母細胞減數分裂過程………………………………7
圖2. 牛卵受精時，精卵間之交互作用………………………………21
圖3. 體外成熟及體外受精期間GSH含量變化及作用之假說………27
圖4. 不同培養方式對胚源性因子濃度之影響………………………34
圖5. 各不同階段之牛胚正常發育之形態……………………………56
圖6. 塑膠麥管構造……………………………………………………66
圖7. 牛卵丘卵母細胞複合體於體外成熟培養前後之外觀…………71
圖8. 牛卵母細胞減數分裂之各階段發育……………………………73
圖9. 牛卵於體外成熟及體外受精後不同時段原核發育曲
線圖……………………………………………………………………79
圖10. 牛卵於體外成熟及體外受精後各不同時段原核發育
之情形…………………………………………………………………80
圖11. 牛卵之多精入卵經固定並用aceto- lacmoid染色後之
位向差顯微照相圖……………………………………………………81
圖12. 飼養層卵丘細胞………………………………………………85
圖13. 牛胚經體外培養後不同時段之外觀…………………………87
圖14. 外源基因經顯微注射技術注入牛受精卵之原核內…………91
圖15. 源自體外生產牛胚經冷凍暨解凍後之發育潛能……………98
圖16. 源自體外成熟、體外受精、基因顯微注射並於體外
培養七日後之牛胚經移置於同期發情母牛之子宮後
所產生之荷蘭仔牛…………………………………………………102
圖17. 應用PCR方法檢測8888仔牛之耳組織及臍帶DNA
中之αLA-hFIX基因片段……………………………………………104

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