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研究生:陳奕竹
研究生(外文):Chen, Yichu
論文名稱:開發新型稀釋液改善公豬精液之品質
論文名稱(外文):Develop New Extenders To Improve The Boar Semen Quality
指導教授:陳銘正陳銘正引用關係
指導教授(外文):Chen, Mingcheng
口試委員:林佳靜劉秀洲林育安
口試委員(外文):Lin,ChaichingLiu, HsiuchouLin, Yuan
口試日期:2012-07-18
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:動物科技學系碩士班
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:2012
畢業學年度:100
語文別:中文
論文頁數:111
中文關鍵詞:公豬精液流式細胞儀明膠
外文關鍵詞:Boar semenFlow cytometryGelatin
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家畜精液之保存主要有液態與冷凍兩種方式,目前豬人工授精產業使用之精液仍以液態保存為主,藉由添加不同類型之稀釋液以延長保存時間。稀釋精液在液態保存期間會因為少數精子死亡,造成精子之凝集現象,使得更多精子死亡。在兔子與綿羊之稀釋精液中添加明膠 (gelatin) 可以使精子均勻分佈,避免精子凝集而沉澱,維持精子之活動力及頭巾膜完整性,並改善生育力,但尚未應用於豬人工授精產業;三仙膠 (Xanthan gum) 是一種廣泛應用於化妝品之增稠劑,也可以使精子均勻懸浮於稀釋精液中,但未有對公豬精液品質影響之研究。本試驗擬開發含有明膠或三仙膠之稀釋液,應用於公豬精液冷藏保存,首先將稀釋液分別保存在 16℃ 3 日、4℃ 14 日或 -20℃ 90 日,檢測稀釋液之 pH 值、滲透壓與總生菌數之變化,評估保存期間稀釋液品質之變化;之後進行精液之稀釋保存,應用流式細胞儀與螢光顯微鏡檢測精液之品質,且以體外受精技術驗證生育力。試驗結果顯示,於 4℃ 保存 14 日與 16℃ 保存 3 日之稀釋液,其滲透壓可以穩定維持,但 pH 值皆顯著較新鮮配製者高 0.2;然而於 -20℃ 保存 90 日之稀釋液相較於 16℃ 保存 3 日者,除了含有 1.5% 明膠之稀釋液外,其他稀釋液之 pH 值顯著較高 (P < 0.05),但仍在精子可耐受之範圍內。以含有 1.5% 明膠或 0.3% 三仙膠之稀釋液稀釋公豬精液,保存 3 日後,仍可以使精子懸浮於上層,然而未添加明膠或三仙膠之稀釋精液,在保存 6 小時後,精子皆沉澱於下層。添加明膠或三仙膠之稀釋精液,保存至第 3 日時,其精子活力與頭巾完整性均顯著較第 0 日者為低 (P < 0.05),但仍符合人工授精之合格標準;在保存第 0 日時,添加明膠之稀釋精液之精子膜完整性 (93.8 ± 1.5%) 與粒線體活性 (89.9 ± 0.4%) 均顯著高於其他稀釋精液 (P < 0.05),但保存至第 3 日時,其精子存活率 (79.3 ± 1.7%) 則顯著低於其他稀釋精液 (P < 0.05)。進一步以體外受精技術驗證精子之生育力,比較有進行搖晃或不搖晃,與添加明膠或三仙膠之稀釋精液,保存 3 日後進行體外受精,其精子穿透率分別為:86.3 ± 20.3%、70.4 ± 7.0%、88.6 ± 7.1% 與 76.6 ± 1.3% 並無顯著差異。綜合上述,添加明膠或三仙膠之稀釋精液,可以維持良好精液品質與良好之生育力。
Livestock semen is preserved in liquid or frozen states. The principal preservation mode of semen used for pig artificial insemination (AI) is liquid storage, which prolongs the storage time of boar semen to different extenders, depending on the extender used. During extended semen storage, the death of some spermatozoa causes agglutination of the cells, causing more of the live sperm to die. The addition of gelatin to semen extenders used for storage of rabbit and sheep semen has resulted in a more uniform distribution of spermatozoa and prevents sedimentation. This has not only maintained sperm motility and membrane integrity but also improved fertility. However, the gelatin-supplemented extenders used for boar are not applicable to pig AI. Xanthan gum (X-gum) is a widely used cosmetic thickener, and it can be used to uniformly suspend semen; however, the effect of X-gum on boar semen quality is unknown. The objective of this study was to develop gelatin- or X-gum-supplemented extenders applicable to refrigerated preservation of boar semen. Initially, the extenders were stored at 16°C for 3 days, 4°C for 14 days, or -20°C for 90 days, and we examined changes in pH value, osmotic pressure, and total bacteria count to evaluate the effect of storage on the quality of the extender. Fresh boar semen was then diluted in the extenders and stored. Semen quality was examined by flow cytometry and fluorescence microscopy, and fertility was verified by in vitro fertilization (IVF). The results indicated that the osmotic pressure of extenders could be maintained during storage at 16°C for 3 days and 4°C for 14 days, but that the pH value was significantly 0.2 higher than that of fresh extenders. Nevertheless, when comparing extenders stored at -20°C for 90 days and at 16°C for 3 days, the pH values and osmotic pressures were significantly higher expected mBTS (+G) (P < 0.05), but were still tolerated by the sperm. Boar semen was extended in 1.5% gelatin- or 0.3% X-gum-supplemented extenders for 3 days. In these extenders, the spermatozoa remained suspended in the upper layer; however, in semen extenders lacking gelatin or X-gum, the semen sedimented after 6 hours. When the semen was extended in gelatin- or X-gum-supplemented extenders, the sperm motility and membrane integrity were significantly lower on the 3rd day than on the 1st day (P < 0.05); however, these factors still met the eligibility criteria for pig AI. The sperm membrane integrity (93.8 ± 1.5%) and mitochondrial function (89.9 ± 0.4%) on the 1st day (P < 0.05) were significantly higher for semen extended in gelatin-supplemented extenders than those of semen extended in other types of extenders. However, the sperm viability (79.3 ± 1.7%) was significantly lower in gelatin-supplemented extenders than in other types on the 3rd day (P < 0.05). Using IVF of pig oocytes, we then compared the fertility of the extended semen between that stored with shaking or non-shaking, and that stored in gelatin- or X-gum-supplemented extenders for 3 days. The sperm penetration rates were 86.3 ± 20.3%, 70.4 ± 7.0%, 88.6 ± 7.1%, and 76.6 ± 1.3%, respectively; there were no significant differences in fertility between semen stored under these conditions. In conclusion, boar semen extended with gelatin- or X-gum-supplemented extenders can effectively maintain semen quality and fertility.
目錄
中文摘要 I
英文摘要 III
誌謝 VI
表次 XI
圖次 XII
附次 XV
壹、前言 1
貳、文獻檢討 3
一、豬人工授精 3
(一) 國外現況 4
(二) 國內現況 4
(三) 授精方式 5
二、公豬精液保存 7
(一) 液態保存 7
(二) 冷凍保存 8
三、稀釋液 9
(一) 稀釋液成分 10
(二) 種類 12
四、精液品質檢測 14
(一) 顯微鏡檢測 14
1. 物理性狀 14
2. 精子濃度 14
3. 精子活力 14
4. 精子形態 15
(二) 流式細胞儀檢測 15
1. 精子存活率 17
2. 精子頭巾完整性 17
3. 精子粒線體功能性 18
4. 精子獲能狀態及膜流動性 18
5. 精子染色質結構分析 19
五、公豬生育力之評估 20
(一) 人工授精 20
(二) 體外受精 20
参、材料與方法 22
一、精液之收集與稀釋 22
二、稀釋液之製備 22
三、稀釋液 pH 值、滲透壓與總生菌數檢測 22
四、精液品質檢測 23
(一) 精子濃度 23
(二) 顯微鏡檢查 23
1. 活力 23
2. 形態 23
(三) 流式細胞儀評估精液品質 26
1. 存活率 26
2. 頭巾膜完整率 26
3. 粒線體功能性 27
五、精子之體外受精 34
(一) 卵母細胞之體外成熟 34
(二) 卵母細胞與精子之體外受精 35
(三) 卵母細胞形態之評估 36
(四) 體外受精之評估指標 36
六、試驗設計 36
(一) 以流式細胞儀檢測公豬精液性狀之有效性 37
(二) 稀釋液保存期間 pH 值、滲透壓與總生菌數之變化 37
(三) 在精液保存期間不同稀釋液對精液品質之影響 38
(四) 應用體外受精技術評估稀釋液對保存精液生育力之影響 38
七、統計分析 39
肆、 結果 40
一、以流式細胞儀檢測公豬精液性狀之有效性 40
二、稀釋液保存期間 pH 值、滲透壓與總生菌數之變化 48
三、在精液保存期間不同稀釋液對精液品質之影響 60
四、應用體外受精技術評估稀釋液對保存精液生育力之影響 68
伍、討論 72
一、以流式細胞儀檢測公豬精液性狀之有效性 72
二、稀釋液保存期間 pH 值、滲透壓與總生菌數之變化 72
三、在精液保存期間不同稀釋液對精液品質之影響 75
四、應用體外受精技術評估稀釋液對保存精液生育力之影響 77
陸、結論 78
柒、參考文獻 79
捌、附錄 86
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