臺灣博碩士論文加值系統

本試驗之主要目的，在比較不同精液保存液及貯藏方法對於山羊精子之影響。試驗選用本校乳羊場經過訓練之成熟賴滿嬌(LaMancha)公羊2頭，使用人工陰道採集精液，並予以混合。在繁殖季節外共進行12次之精液採集，採集之精液區分為去除精漿組及含精漿組，並分別依精子濃度加入不同量之蛋黃及脫脂乳保存液進行稀釋。將稀釋過之精液於4℃平衡2小時，並加入第2段保存液(含甘油)以進行冷藏(4℃)及冷凍(-196℃)保存。冷藏保存之精液樣品，連續7天分別評估精子活力評分(sperm motility scale, SMS)、精子存活率(viability, VIA)及頭帽完整率(acrosome integrity, AIR)。
試驗結果顯示：新鮮精液以蛋黃保存液稀釋，並於4℃冷藏保存1週後，不論是SMS、VIA (%)或AIR (%)均以去除精漿組顯著較含精漿組為佳(2.72 ± 0.14 vs. 1.37 ± 0.18；72.33 ± 0.03 vs. 29.01 ± 0.04；92.97 ± 0.03 vs. 42.86± 0.04)；而以脫脂乳保存液稀釋，並保存1週後，前述各項精子性狀在去除精漿組與含精漿組均無顯著差異。綜合在4℃冷藏保存之結果，去除精漿組以蛋黃保存液對頭帽之保護效果顯著較佳(92.97 ± 0.01 vs. 76.8 9 ± 0.01) (P＜0.05)；而含精漿組則以脫脂乳保存液之結果較佳。在冷凍保存方面，兩種保存液在SMS、VIA (%)及AIR (%)均無顯著差異；而含精漿組之SMS及VIA (%)顯著較去除精漿組為高(2.60 ± 0.07 vs. 1.98 ± 0.07；32.74 ± 0.02 vs. 19.44 ± 0.02 ) (P＜0.05)。在降溫程序方面，加入第2段含甘油之保存液後平衡90分鐘再予冷凍保存顯著較未平衡者之SMS、VIA (%)及AIR (%)為佳(P＜0.05)。綜合-196℃冷凍保存之結果，將含有精漿之精液以蛋黃保存液稀釋，並在加入第二段保存液後平衡90分鐘之處理組，其精子性狀均較其他處理組為佳。在繁殖季節外8隻母羊經發情同期化處理，並以由蛋黃或脫脂乳保存液稀釋之冷凍精液人工授精。在人工授精後19天，以糞孕酮濃度作早期懷孕診斷，結果共有4隻母羊懷孕，其中3隻為以脫脂乳保存液稀釋之精液，另1隻則為以蛋黃保存液稀釋之精液。

The purpose of this research is to compare the effect of different storage method and extenders on goats spermatozoa. Two trained mature male LaMancha goats were chosen to collect the semen by artificial vagina (AV), and the semen were pooled, 12 ejaculations were collected in non-breeding seasons. Each pooled semen were separated into two treatments, with or without seminal plasma semen. In addition, different volumes of egg yolk or skimmed milk extenders were added to the pooled semen depend on the concentrations of sperm. Second part of extenders (including glycerol) were added to the semen after 120 minutes of balanced. After adding second part of extenders, semen samples were kept at -196℃ and 4℃ for preservation. The sperm motility scale (SMS), viability (VIA), and acrosome integrity (AIR) of semen samples kept at 4℃ were evaluated respectively for a week.
The result of this research: The SMS, VIA (%) and AIR (%) of goats semen without seminal plasma diluted with egg yolk extender (EYE) for storage 1 week at 4℃ were significantly higher than those of goats semen with seminal plasma(2.72 ± 0.14 vs. 1.37 ± 0.18；72.33 ± 0.03 vs. 29.01 ± 0.04；92.97 ± 0.03 vs. 42.86 ± 0.04). And the SMS, VIA (%) and AIR (%) of goats semen with or without seminal plasma diluted with skimmed milk extender (SKM) for storage 1 week at 4℃ were no significantly difference. The acrosome integrity of goats semen without seminal plasma diluted with EYE and preserved under 4℃ were higher than SME(92.97 ± 0.01% vs. 76.89 ± 0.01%) (P＜0.05). The goat semen with seminal plasma diluted with SME and storage under 4℃, had a better effect on all parameters(P＜0.05). In cryopreservation, there was no significant difference between extenders on all parameters. The SMS and VIA (%) of semens without seminal plasma were higher than those of semen with seminal plasma (2.60 ± 0.07 vs. 1.98 ± 0.07；32.74 ± 0.02 vs. 19.44 ± 0.02 ). The SMS, VIA (%) and AIR (%) of goats semen equilibrated over 90 minutes after added second part of extender were higher than those without equilibration .In summary, goat semen diluted with EYE and equilibrated over 90 minutes after added second part of extender could get good cryopreservation results in this research. Eight does synchronized were chosen for artificial insemination. Fecal progesterone were detected for pregnancy diagnosis after 19 days of artificial insemination. The result showed that 4 does were pregnant, which 3 of SME, and 1 of EYE.

壹、 前言 1
貳、 文獻探討 2
一、 公山羊之生殖生理 2
(一) 生殖系統 3
(二) 公山羊之生精作用或精子生成作用 5
(三) 影響精液品質之因子 7
1. 環境溫度 7
2. 營養 8
3. 季節 8
二、 精液之採集方法 9
(一) 附睪採精法 10
(二) 按摩採精法 10
(三) 電激採精法 10
(四) 人工陰道法 11
三、 精液之一般性狀分析 12
(一) 精液量 12
(二) 精液外觀 12
(三) 精子之活力 12
(四) 精子之濃度 13
(五) 精子之存活力 14
(六) 精子頭帽之完整率 14
四、 精液之保存 15
(一) 影響精子存活之因子 15
1. 溫度 15
2. 滲透壓 16
3. 稀釋倍率 17
4. pH值 17
5. 光線 17
6. 精漿 17
(二) 保存液成分 18
1. 冷凍保護劑 19
2. 緩衝溶液 20
3. 醣類 20
4. 抗生素 21
5. 其他 21
(三) 冷藏保存 21
(四) 冷凍保存 22
參、 材料與方法 24
一、 試驗動物 24
二、 飼養與管理 24
三、 試驗設計 24
(一) 實驗室檢測 24
(二) 田間試驗 25
四、 樣品之採集及處理 25
(一) 精液採集 25
(二) 精液品質之評估 27
1. 精液量 27
2. 精子濃度 27
3. 精子活力評分 28
4. 精子存活率 30
5. 精子頭帽完整率 31
五、 冷藏及冷凍精液之製作 33
(一) 冷藏及冷凍保存液之製作 33
(二) 冷藏及冷凍精液之製作流程 35
(三) 冷藏及冷凍精液之回溫及解凍 36
六、 公羊冷凍精液之現場製作與母羊之發情同期化及人工授精 36
(一) 公羊冷凍精液之現場製作流程 36
(二) 母羊之發情同期化及人工授精 37
七、 糞孕酮濃度之測定及早期懷孕診斷 38
八、 試驗結果之統計分析 39
肆、 結果與討論 40
一、 試驗期間新鮮精液之一般性狀分析 40
(一) 精液量 40
(二) 精子活力評分 40
(三) 精子存活率 41
(四) 精子頭帽完整率 41
二、 冷藏精液之性狀分析 42
(一) 精子直線運動活力評分 42
(二) 精子存活率 44
(三) 精子頭帽完整率 46
三、 冷凍精液之性狀分析 48
(一) 精子直線運動活力評分 48
(二) 精子存活率 50
(三) 精子頭帽完整率 52
四、 田間試驗之早期懷孕診斷 54
伍、 結論 56
陸、 參考文獻 57
柒、 附表 66

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