臺灣博碩士論文加值系統

精液品質影響公鵝受精率與繁殖力，精子活力為精液性狀的主要特性之一，其對受精作用具有重要影響，精漿可提供精子安全環境以利於進入雌性生殖道中，也提供精子所需部分營養，並具有緩衝溶液特性，可吸收精子代謝之產物及影響精子結構與功能，而荷爾蒙機制也會調控繁殖能力。本研究之目的為探討公鵝血清中性腺荷爾蒙是否與精子活力有相關性，以及不同精子活力之公鵝精子與精漿蛋白質差異表現。試驗共使用39隻(平均月齡=24±2)白羅曼公鵝，採集其血液與精液樣品，血液用於分析不同活力群之睪固酮和雌二醇濃度，精液進行精液品質評估，取精子活力前後15%公鵝精液之精子與精漿，以isobaric tags for relative and absolute quantification (iTRAQ)進行蛋白質分析。血清中睪固酮、雌二醇濃度以及睪固酮與雌二醇比值於兩族群之間皆無顯著差異。精子蛋白質分析結果顯示，在3,090個定量蛋白質中有93個蛋白質在組間表現量具顯著差異，且其表現量差異達1.5倍以上，此等差異表現蛋白質中，56個在高活力組表現量較高，37個表現量較低；基因功能分析結果顯示此等差異表現蛋白質主要參與細胞過程(27%)、生物過程調控(24%)、細胞成分組織或生物合成(11%)和代謝過程(11%)等生物過程，主要位於細胞質(44%)和胞外囊泡(10%)，並與蛋白質結合(33%)、水解酶活性(16%)和離子結合(12%)等分子功能有關。精子蛋白質功能分析結果顯示，與能量代謝有關之L-lactate dehydrogenase、NADH dehydrogenase和adenylate kinase 8及與精子結構有關之outer dense fiber 2、tektin 2和sperm-associated antigen 6在高活力精液之精子表現量較高。精漿蛋白質分析結果顯示，在675個定量蛋白質中有27個蛋白質表現量具顯著的組間差異，其中14個於高活力精液之精漿中表現量較高，13個表現量較低。此等差異蛋白質主要參與生物過程調控(30%)和刺激反應(26%)等生物過程，主要位於細胞質(34%)且與蛋白質結合(63%)和水解酶活性(19%)有關，功能分析結果顯示，與氧化傷害有關之glutathione S-transferase Mu 1及與抗蛋白酶有關之alpha-1-antiproteinase 2在高活力精液精漿之表現量較高。綜合本研究結果可知，公鵝血清中性腺荷爾蒙與精子活力可能無一定關聯性，而高精子活力精液精子與精漿蛋白質中，與能量代謝、精子結構、抗氧化和抗蛋白酶相關之蛋白質表現量較高，因而有利於精子活力。

Semen quality affects fertility and fecundity of ganders. Motility is one of the major characteristics of spermatozoa and is important for normal fertility. Seminal plasma provides a safe environment, nourishing and buffering properties and thus influence the structure and function of spermatozoa. Endocrine mechanisms also control the development of reproductive capacity. The purposes of this study was to investigate whether sex hormone in the serum associate with sperm motility and to explore the differentially expressed proteins in spermatozoa and seminal plasma of ganders with different sperm motility. Blood and semen samples were collected from 39 White Roman geese (age = 24 ± 2 months). Blood samples were used to determine the levels of testosterone and estrodial. Protein samples of spermatozoa and seminal plasma from semen with top 15% and bottom 15% sperm motility were subjected to isobaric tags for relative and absolute quantification (iTRAQ) analysis. The concentrations of testosterone and estradiol in serum were not differed between the two groups. Results showed that 93 out of 3,090 identified proteins had differential expression between the two groups with a minimum of 1.5-fold changes. Among these proteins, 56 proteins were upregulated and 37 proteins were downregulated in the sperm with higher motility. Gene ontology analysis revealed that most of the differentially expressed proteins were involved in biological process of cellular process (27%), regulation of biological process (24%), cellular component organization or biogenesis (11%) and metabolic process (11%). The majority of the differentially expressed proteins were located in cytoplasm (44%) and extracellular exosome (10%), and participated in molecular function of protein binding (33%), hydrolase activity (16%) and ion binding (12%). Proteins associated with energy metabolism, including L-lactate dehydrogenase, NADH dehydrogenase, adenylate kinase 8, and with spermatozoa structure, including outer dense fiber 2, tektin 2, sperm-associated antigen 6, were upregulated in sperm with higher motility. Results in the analysis of seminal plasma proteins showed that 27 out of 675 identified proteins had differential expression between the two groups with a minimum of 1.5-fold changes. Among these proteins, 14 proteins were upregulated and 13 proteins were downregulated in the seminal plasma of semen with higher sperm motility. Gene ontology analysis revealed that most of the differentially expressed proteins were involved in biological process of regulation of biological process (30%) and response to stimuli (26%). The majority of the differentially expressed proteins were located in cytoplasm (34%), and participated in molecular function of protein binding (63%) and hydrolase activity (19%). Glutathione S-transferase Mu 1 and alpha-1-antiproteinase 2 of seminal plasma were upregulated in semen with higher sperm motility. In conclusions, the results of this study suggested that sex hormone in the serum may not associate with sperm motility and expressions of proteins associated with energy metabolism, sperm structure, antioxidation and antiproteinase were higher in sperm and seminal plasma with high motility and thus regulate sperm motility of ganders.

中文摘要 i
Abstract ii
目錄 iii
圖次 v
表次 vi
壹、前言 1
貳、文獻探討 2
一、公鵝之繁殖生理 2
(一) 繁殖特性 2
(二) 生殖系統 2
(三) 精子生成作用 3
(四) 精子結構 5
二、影響精液品質之因素 7
(一) 季節 7
(二) 荷爾蒙 7
(三) 年齡 8
(四) 營養 8
(五) 飼養系統 9
三、精液性狀對繁殖力之影響 10
四、精子與精漿之關係 10
五、精液蛋白質體之相關研究 11
(一) 蛋白質體之定義與應用 11
(二) 精液品質相關之精子蛋白質 13
(三) 精液品質相關之精漿蛋白質 14
六、研究目的 14
參、材料與方法 15
一、試驗動物與飼養管理 15
二、精液採集與精液品質評估 15
三、血清中性腺荷爾蒙濃度之分析 16
四、精子與精漿蛋白質體之分析 17
(一) 蛋白質樣品製備 17
(二) 蛋白質的還原(reduction)、烷基化(alkyation)和消化(digestion) 18
(三) 以iTRAQTM試劑標定胜肽 18
(四) 固相萃取(sold phase extraction, SPE)純化 19
(五) 胜肽樣品分離(fractionation) 19
(六) 奈米高解析液相層析-電噴灑-四極棒飛行時間式串聯質譜儀(nanoUHPLC-ESI-Q-TOF-MS)分析 19
五、蛋白質身分鑑定及定量 20
六、差異表現蛋白質之生物資訊分析 20
七、統計分析 21
肆、結果 22
一、不同精子活力之公鵝精液性狀 22
二、不同精子活力公鵝血清中性腺荷爾蒙濃度之比較 23
三、不同精子活力公鵝精子差異表現蛋白質及功能分類 24
四、不同精子活力公鵝精漿差異表現蛋白質及功能分類 34
伍、討論 38
一、公鵝血清中性腺荷爾蒙對精液品質之影響 38
二、不同精子活力公鵝之精子差異表現蛋白質之功能探討 38
(一) 能量代謝相關之精子蛋白質對精子活力之調控 39
(二) 精子結構相關之精子蛋白質對精子活力之調控 40
(三) 其他精子蛋白質對精子活力之影響 42
三、不同精子活力之公鵝精漿差異表現蛋白質之功能探討 43
(一) 抗氧化相關之精漿蛋白質對精子活力之影響 43
(二) 抗蛋白酶和抗菌相關之精漿蛋白質對精子活力之影響 43
(三) 免疫反應相關之精漿蛋白質對精子活力之影響 44
陸、結論 46
柒、參考文獻 47

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