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研究生:翁珮鈴
研究生(外文):Weng Pay Ling
論文名稱:影響豬卵母細胞體外受精及後續發育之研究
論文名稱(外文):The Study of Fertilization and Subsequent Development In Vitro of Pig Oocytes.
指導教授:謝來安
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
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:2002
畢業學年度:90
語文別:中文
論文頁數:106
中文關鍵詞:冷凍-解凍精子豬卵母細胞體外受精
外文關鍵詞:frozen-thawed spermatozoaporcine oocytein vitro fertilization
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本研究的目的旨在探討體外受精時不同精子濃度、精卵共培養的時間、受精時卵母細胞不同處理方式及不同培養液等條件,對精子穿入成熟卵母細胞以及後續發育之影響,以建立應用源自冷凍-解凍精子進行豬卵母細胞之體外生產系統。同時為了能得到穩定及可靠之卵母細胞來源,本研究也對豬隻屠宰時,有無燙毛處理,對其卵丘-卵母細胞複合體體外成熟與體外受精作一探討。
試驗結果顯示:屠宰時有無燙毛處理豬隻之卵母細胞,培養於NCSU-23體外成熟培養液中44∼48小時,成熟率分別為86.94%與85.77%,二者無顯著差異。進一步以新鮮精液進行體外受精,其精子穿入率、多精入卵率與雄原核形成率彼此間亦無顯著差異。以精子濃度1 × 106 cells / ml精卵分別共培養4、5、6小時,結果顯示,5小時組別可獲得較佳結果,精子穿入源自燙毛或無燙毛豬隻卵母細胞之比例分別為78.31%與80.70%,多精入卵率為51.39%與58.04%。顯示豬隻屠宰時,有無燙毛處理,所取得之卵母細胞於體外成熟及受精時,不會影響成熟率及精子穿入率。
應用冷凍-解凍精子,以1 × 106與1 × 107 cells / ml精子濃度進行體外受精,結果顯示精子穿入率分別為83.18%與88.23%,彼此間無顯著差異;多精入卵率分別為59.54%與90.98%,有顯著差異。精子濃度以1 × 106 cells / ml精卵分別共培養4、5及6小時,共培養5及6小時組之精子穿入率為84.54%及84.24%,顯著高於4小時組(27.27%),共培養6小時組,多精入卵率為62.57%,顯著高於4及5小時組(12.50%與57.72%)。豬卵母細胞於受精時去除卵丘細胞(精子濃度為1 × 106 cells / ml精卵共培養5小時),其精子穿入率(87.09%)與多精入卵率(55.51%)均顯著的高於受精時不去除卵丘細胞(精子濃度為1 × 107 cells / ml精卵共培養8小時)(25.34%與6.12%)。以NCSU-23與mTBM不同受精培養液進行豬卵母細胞體外受精,結果顯示精子穿入率分別為82.63%與14.21%,多精入卵率為54.53%與14.00%兩處理組間均達顯著差異(p<0.05)。如以上述最佳條件進行體外受精,將受精卵置於NCSU-23與CZB胚培養液,經144小時後其卵裂率分別為43.35%與49.57%,彼此間無顯著差異,發育至桑椹胚分別為5.80%與2.16%,但均無發育至囊胚階段。
結果顯示,從有無燙毛處理豬隻所取得之卵丘-卵母細胞複合體,進行體外成熟與體外受精試驗,顯示並不會影響其成熟率及精子穿入率。經冷凍-解凍精子進行體外受精,可以使精子穿入卵母細胞,但後續發育能力仍有待改善。
The objects of this study were to evaluate factors, including sperm concentration for fertilization in vitro, time for sperm-oocytes coincubation, without cumulus or with cumulus and medium for in vitro fertilization, may be important for sperm penetration and reduce the incidence of polyspermy. In order to get stable and reliable source of oocytes, this study was examined the maturation and fertilization rate of oocytes collected from slaughtered porcine with or without scalded-hair on maturation and fertilization were examined.
The maturation rates of porcine oocytes collected from slaughtered porcine with or without scalded-hair and cultured in NCSU-23 medium for 44〜48 hr were 86.94% and 85.77%, respectively, There was no significant difference between the two group. For In vitro fertilization of maturation oocytes with fresh boar spermatozoa, sperm penetration rate, polyspermy rate and male pronuclei formation rate were no significant difference between with or without sacaled-hair groups. The results of sperm penetration of oocytes collected from slaughtered porcine with or without scalded-hair and inseminated with 1 ×106 cells/ml for 5 hr were 78.31% and 80.70%, which polysperm rate were 51.39% and 58.04%, respectively. The oocytes from slaughtered porcine with or without scalded-hair were not different on maturated rate and sperm penetration rate.
The sperm penetration rate on oocytes inseminated with frozen-thawed spermatozoa by 1 ×106 cell/ml and 1 × 107 cell/ml concentration were 83.18% and 88.23%, respectively. There was not difference between two concentrations group. The polyspermy rate(90.98%)of 1 × 107 cell/ml group was higher than 1 ×106 cells/ml(59.54%) group. IVF medium containing spermatozoa(1 ×106 cells/ml)and then incubated for 4、5 and 6 hr, respectively. Penetration rate was low at 4 hr group, but it is increased to 84.54% and 84.24% at 5 and 6 hr group. Compared to the value at 6 hr group, the polyspermy(62.57%)had increase significant than 4 and 5 hr group(12.50% and 57.72%).In vitro fertilization, oocyts without cumulus cell(sperm concentration were 1 × 106 ml/cells and coincubation time of 5 hr), The sperm penetration were 87.09% and polyspermy were 55.51%;this value had then increased significantly by cumulus cell were not removed(sperm concentration were 1 × 107 ml/cells and coincubation time of 8 hr)(25.34% and 6.12%). In vitro fertilization medium for NCSU-23 and mTBM, sperm penetration rate were 82.63% and 14.21%, respectively and polyspermy rate were 54.53% and 14.00%, respectively. The sperm penetrated and polysperm have significant differences among these two medium(p<0.05).Sanctus aforesaid optimal condition prosecuted in vitro fertilization After fertilization, oocytes transferred into NCSU-23 and CZB embryo medium. At 144 hr, cleavage rate were 43.35% and 49.57%, respectively. There was no significant difference among the cleavage rate. The embryos development rate form morula stage was 5.80% and 2.16%. The difference between two media was not significant however NCSU-23 shown higher tendency. Embryos did not develope to blastocsyt stage.
The result showed that the maturation and fertilization rates of porcine oocytes from slaughtered porcine with or without scalded-hair were no difference. Via in vitro fertilization with frozen-thawed ejaculated spermatozoa were able to penetrate oocytes, however but ootide supervene development capacility was open to objection.
中文摘要……………………………………………….………Ⅰ
英文摘要…………………………………..…………….……..Ⅳ
誌謝…………………………………………………………….Ⅷ
目錄……………………………………….……………………Ⅸ
圖次索引………………………………………………………ⅩⅣ
表次索引………………………………………………………ⅩⅥ
附表索引………………….…………………………………ⅩⅧ
壹、緒言………………………………………………………..1
貳、文獻探討…………………………….…………………….3
一、 濾泡及卵母細胞之發育……………………..……..3
(一)、濾泡之生長發育與調控…………………………3
1、濾泡生長發育………………………………..3
2、濾泡生長調控…………………………………..5
(二)、卵母細胞成熟作用………………………………..6
(三)、卵母細胞成熟作用之調控因子………………….7
1、環化腺苷單寧酸……………………………………7
2、成熟抑制因子………………………………………8
3、成熟促進因子……………………………………….8
4、細胞靜止因子……………………………………11
5、鈣離子…………………………………………….12
二、 影響卵母細胞體外成熟之因子………………...…12
(一)、濾泡大小………………………………………….12
(二)、卵丘細胞………………………………………….13
(三)、內泌素…………………………………………….14
(四)、豬濾泡液………………………………………….15
(五)、血清……………………………………………….16
(六)、其他……………………………………………….17
三、 卵母細胞之受精作用…………………………...…18
(一)、精子的獲能作用…………………………………18
(二)、頭巾反應…………………………………………20
(三)、精子穿透…………………………………………20
(四)、精卵融合………………………………………….21
(五)、原核的形成及遷徙……………………………….22
(六)、防止多精入卵之可能機制………………………22
四、 影響體外受精因子之探討………………………...24
五、 胚之早期發育……………………………………...27
(一)、卵裂………………………………………………27
(二)、早期胚的營養……………………………………28
參、材料與方法……………………………………………….32
一、 卵母細胞之來源及前處理………………..….32
二、 卵母細胞之體外成熟……………………………...35
(一)、培養液……………………………………………35
(二)、體外成熟培養之流程……………………………35
三、 卵母細胞之體外受精……………………...………36
(一)、精液的採集………………………………………36
(二)、精子冷凍及解凍方式……………………………39
(三)、體外受精前精子之製備………………………….40
(四)、卵母細胞體外受精………………………………41
四、 受精卵之體外培養…………………………...……44
五、 卵或胚固定、染色及發育評估……………………45
(一)、卵母細胞成熟之評估……………………………45
(二)、受精卵發育之評估………………………………47
六、 試驗處理……………………………………………53
試驗一、屠宰時豬隻有無燙毛對其卵母細胞體外成熟之影響……………………………...….53
試驗二、屠宰時有無燙毛豬隻之卵母細胞以不同精子濃度對其體外受精之影響……………53
試驗三、屠宰時有無燙毛豬隻之卵母細胞受精時,精卵共培養時間對其體外受精之影響………………………………………..53
試驗四、不同冷凍-解凍精子濃度對豬卵母細胞體外受精之影響…………………………..54
試驗五、精卵共培養時間對冷凍-解凍精子體外受精之影響……………………………….54
試驗六、豬卵母細胞受精時,有無去除卵丘細胞對冷凍-解凍精子體外受精之影響…………………………………………54
試驗七、不同受精培養液對豬冷凍-解凍精子體外受精之影響…………………………..…55
試驗八、不同胚培養液對受精卵發育之影響……………………………………..55
七、 統計分析……………………………………………56
肆、結果與討論……………………………………………….57
試驗一、屠宰時豬隻有無燙毛對其卵母細胞體外成熟之影響……………………………………………57
試驗二、屠宰時有無燙毛豬隻之卵母細胞以不同精子濃度對其體外受精之影響………………………59
試驗三、屠宰時有無燙毛豬隻之卵母細胞受精時,精卵共培養時間對其體外受精之影響…………62
試驗四、不同冷凍-解凍精子濃度對豬卵母細胞體外受精之影響………………………………….......65
試驗五、精卵共培養時間對冷凍-解凍精子體外受精之影響…………………………………………..68
試驗六、豬卵母細胞受精時,有無去除卵丘細胞對冷凍-解凍精子體外受精之影響………………..69
試驗七、不同受精培養液對豬冷凍-解凍精子體外受精之影響…………………………………………72
試驗八、不同胚培養液對受精卵發育之影響………………………...………………...75
伍、結論………………………………………………………..79
陸、參考文獻………………………………………………….80
作者簡介………………………………………………...……106
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