臺灣博碩士論文加值系統

本試驗乃評估藉由 X 與 Y 精子存活時間之差異，搭配離心程序，藉此分離 X 與 Y 精子，生產較多特定性別豬之可能性。試驗分二部分，試驗一為評估稀釋精液保存條件對 X 與 Y 精子比率及人工授精後裔性比率之影響，試驗二則建立快速準確之 X 與 Y 精子鑑別方法。試驗一將濃厚精液分別以短效型 (BTS) 與長效型 (Safe-Cell) 稀釋液進行稀釋，並將稀釋精液保存於精液冰箱 (16-18℃) 與室溫 (23-25℃)，保存 1 日、3 日與 5 日，最後經由離心分層，評估精液之保存條件與離心分層對精液品質與 X 精子比率之影響。進一步挑選 BTS 與 Safe-Cell 稀釋液，並製成稀釋精液保存於 16-18℃ 之精液冰箱中，保存 3 日後離心分層，分別取上下層精液對繁殖場之母豬進行人工授精，評估對母豬懷孕率、分娩率、產仔數與後裔性比率之影響。試驗結果顯示，稀釋精液之精子濃度受到離心分層、稀釋液x離心分層與保存溫度 x 離心分層之影響；精子活動力受到稀釋液、保存時間、離心分層與稀釋液 x 保存時間之影響；精子頭巾完整性受到稀釋液、保存溫度、保存時間與離心分層之影響；精子存活率受到保存溫度、保存時間、離心分層、稀釋液 x 保存時間與稀釋液 x 保存溫度 x 保存時間之影響；進一步對 X 精子比率進行分析，各組間均無顯著差異；人工授精之結果顯示，不論是在懷孕率、分娩率、平均產仔數與平均活仔數各組間均無顯著差異；後裔性比率部分，BTS 上下層稀釋精液之間無顯著差異，而 Safe-Cell下層稀釋精液之後裔性比率顯著高於上層者。試驗二以齒釉蛋白基因設計專一性引子對與 X、Y 精子核酸探針，進行即時定量聚合酶鏈鎖反應，以絕對定量建立標準曲線後，分別以不同比例之公豬與母豬基因組 DNA 進行混合，評估 X、Y 精子鑑定技術之準確性。試驗結果顯示，應用齒釉蛋白基因之特異性引子對可以成功增幅 AMEL-X 與 AMEL-Y 之 DNA 片段，且利用 AMEL-X 與 AMEL-Y 螢光核酸探針可以專一性偵測即時定量聚合酶鏈鎖反應之 AMEL-X 與 AMEL-Y PCR 產物。本試驗也成功應用 37.5-600 ng 不同數量之公豬肝臟組織基因組 DNA 與 X 與 Y 染色體數量建立標準回歸曲線，R2 值均為 0.99。進一步檢測不同比例混合之公豬與母豬基因組 DNA，顯示具有高度準確性，且可以成功估算 10% 差距之 X 染色體比率。最後檢測 5 頭不同公豬精液之 X 精子比率，發現公豬精液之 X、Y 精子比率具有個體差異。總結上述，本試驗已成功建立齒釉蛋白基因即時定量聚合酶鏈鎖反應檢測 X、Y 精子比率之技術。不論是長效型 Safe-Cell 或短效型 BTS 稀釋液，皆可保存精液 3 日而不影響精液品質，但保存 5 日後之精子活動力與存活率則有顯著性降低。利用不同稀釋液、保存溫度、保存時間與離心分層未能有效分離 X 與 Y 精子，但以 Safe-Cell 稀釋精液並保存於 18℃ 保存 3 日後離心，取上層與下層精子進行人工授精，分別可以獲得較高比率之雌性與雄性仔豬。

The objective of this study was to use centrifugation and differences between X and Y sperms in terms of lifespan to separate the sperms and produce more sex-specific pigs. For this purpose we performed two experiments. First, we estimated the effects of storage conditions and centrifugation on semen quality and X-bearing sperm ratio of diluted boar semen, and determined the sex ratios of offsprings after artificial insemination in pigs. Second, we established an efficient technique to determine the proportions of X- and Y-bearing spermatozoa. In experiment 1, we used short-term (BTS) and long-term (Safe-Cell) extenders to dilute the boar semen. Semen was stored at 16-18°C or 23-25°C for 1, 3, and 5 days. Subsequently, the diluted semen was centrifuged at 250 ×g for 4 min. The effects of storage conditions and centrifugation on semen quality and X sperm ratios were estimated. BTS or Safe-Cell diluted boar semen was also stored at 16-18°C for 3 days and then centrifuged. The semen from upper and lower layers was used to inseminate sows in a breeding farm. The pregnancy rates, farrowing rates, and litter sizes of the sows and the sex ratios of offspring were estimated. The results showed that centrifugation, extender × centrifugation and extender × storage temperature influenced the concentration of diluted semen; extender, storage time, centrifugation and extender × storage time influenced the sperm motility; extender, storage temperature, storage time, centrifugation influenced the sperm acrosome integrity; storage temperature, storage time, centrifugation, extender × storage time and extender × storage temperature × storage time influenced the sperm viability. The proportions of X sperm were not significantly different in all of these groups. The results of artificial insemination showed that pregnancy rate, farrowing rate, litter size, and live piglets of the sows inseminated by these semens were not significantly different in all of the groups. The sex ratio of offspring using the upper layer of BTS semen was not significantly different from that using the lower layer, while the sex ratio of offspring from Safe-Cell semen from the lower layer was higher than that from the upper layer (P < 0.05). In experiment 2, we used one set of primers and two probes designed based on the specific sequences of AMEL-X and AMEL-Y genes on the X and Y chromosomes, respectively. A standard curve for X- and Y-chromosome copies was established using Q-PCR. This was used to detect the different X ratios from mixtures of different male and female gDNA to estimate the accuracies of X- or Y-bearing spermatozoa determination. These results showed that specific AMEL-X and AMEL-Y DNA fragments were amplified successfully using AMEL gene-specific primers, and that the AMEL-X or AMEL-Y probe could specifically detect AMEL-X or AMEL-Y PCR products by Q-PCR. Furthermore we successfully used 37.5–600 ng of male liver gDNA to establish standard curves for X- and Y-chromosome numbers. The R2 values were 0.99 for both chromosomes. Further, we detected different X ratios in male and female gDNA mixtures and found that Q-PCR could quantify the proportion of X chromosomes with 10% differences. Finally, we estimated the proportions of X sperms in semen of 5 individual boars and found that the proportions of X sperm were differenct among 5 boar semen. In conclusion, we established Q-PCR for X- and Y-bearing spermatozoa as determined by the amelogenin gene. Boar semen diluted in Safe-Cell or BTS extenders and stored for 3 days did not influence semen quality. However, diluted semen stored for 5 days had significantly reduced sperm motility and viability. The external factors combined with centrifugation could not efficiently separate X- and Y-bearing spermatozoa. However, we used the upper and lower layers of Safe-Cell semen that were stored at 18°C for 3 days and subsequently centrifuged to inseminate sows which tended to produce more female and male offspring, respectively.

中文摘要……………………………………………………………………… I
英文摘要……………………………………………………………………… III
致謝…………………………………………………………………………… VI
表目錄………………………………………………………………………… VIII
表目錄………………………………………………………………………… XIII
圖目錄………………………………………………………………………… XIV
附次…………………………………………………………………………… XVI
壹、前言……………………………………………………………………… 1
貳、文獻檢討………………………………………………………………… 3
一、性別與性比率…………………………………………………………… 3
二、影響性比率之因素……………………………………………………… 4
(一) 卵母細胞成熟程度……………………………………………… 4
(二) 體外受精時間…………………………………………………… 5
(三) 人工授精時間…………………………………………………… 6
(四) 體外培養液之能量來源………………………………………… 7
(五) 胚移置…………………………………………………………… 7
(六) 雌性動物之營養狀態…………………………………………… 8
(七) 環境因子………………………………………………………… 8
三、選性繁殖之方法………………………………………………………… 9
(一) 胚性別篩選……………………………………………………… 9
(二) 調整受精條件…………………………………………………… 10
(三) X、Y精子分離…………………………………………………… 10
1. 白蛋白管柱分離法……………………………………………… 10
2. 密度梯度離心法………………………………………………… 11
3. 上游法…………………………………………………………… 12
4. 免疫分離法……………………………………………………… 13
5. 流式細胞儀原理與缺點………………………………………… 14
(四) X、Y 精子鑑別方法..………………………………………… 15
1. 巢式聚合酶鏈鎖反應…………………………………………… 15
2. 螢光原位雜交…………………………………………………… 16
3. 流式細胞儀……………………………………………………… 18
4. 即時定量聚合酶鏈鎖反應……………………………………… 19
(五) 性別特異性基因………………………………………………… 20
1. SRY 基因……………………………………………………… 20
2. ZF 基因………………………………………………………… 21
3. Amelogenin 基因…………………………………………… 22
四、選性繁殖之專利趨勢分析…………………………………………… 22
五、豬選性繁殖之發展現況……………………………………………… 24
(一) 流式細胞儀在養豬產業應用之侷限性………………………… 24
(二) 人工授精與生殖技術之改良與應用…………………………… 24
(三) X 與 Y 精子表面特異性抗原及其應用……………………… 25
(四) 應用 X 與 Y 精子差異性開發新穎之選性繁殖方法……… 27
參、材料方法……………………………………………………………… 28
一、精液來源……………………………………………………………… 28
二、母豬來源……………………………………………………………… 28
三、精液品質檢測………………………………………………………… 28
(一) 精子濃度……………………………………………………… 28
(二) 精子活力……………………………………………………… 29
(三) 精子成熟率、形態正常率與頭巾完整性…………………… 29
(四) 精子存活率…………………………………………………… 29
四、核酸萃取……………………………………………………………… 30
(一) 組織基因組 DNA萃取………………………………………… 30
(二) 精子基因組 DNA萃取………………………………………… 31
(三) 質體DNA萃取………………………………………………… 31
五、即時定量PCR檢測X與Y精子比率…………………………………… 32
(一) SYBR green 系統…………………………………………… 32
(二) Taq-Man 系統………………………………………………… 33
六、試驗設計……………………………………………………………… 35
(一) 評估公豬稀釋精液之保存與離心條件對 X、Y 精子比率及人工授精後代性比率之影響……………………………… 35
1. 公豬稀釋精液之離心時間……………………………………… 35
2. 稀釋液、保存溫度、保存時間與離心分層對精液品質與 X 精子比率之影響…………………………………………… 36
3. 稀釋精液保存 3 日後離心對人工授精母豬分娩性能與後代性比率之影響……………………………………………… 37
(二) 建立齒釉蛋白基因即時定量聚合酶鏈鎖反應檢測 X、Y 精子比率……………………………………………………… 37
七、統計分析………………………………………………………………… 39
肆、結果……………………………………………………………………… 40
一、 評估公豬稀釋精液之保存與離心條件對 X、Y 精子比率及人工授精後代性比率之影響……………………………… 40
(一) 公豬稀釋精液之離心時間……………………………………… 40
(二) 稀釋液、保存溫度、保存時間與離心分層對精液品質與 X 精子比率之影響…………………………………………… 43
(三) 稀釋精液保存 3 日後離心對人工授精母豬分娩性能與後代性比率之影響……………………………………………… 57
二、建立齒釉蛋白基因即時定量聚合酶鏈鎖反應檢測 X、Y 精子比率… 59
伍、討論……………………………………………………………………… 69
陸、結論……………………………………………………………………… 78
柒、附錄……………………………………………………………………… 79
捌、參考文獻………………………………………………………………… 81

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