臺灣博碩士論文加值系統

乳牛場之搾乳作業為飼養管理之重要一環，而提升乳量乳質與降低生產成本則為乳牛產業最終目標。本研究旨在探討台灣荷蘭乳牛生長素基因（bovine growth hormone, bGH）、粒線體DNA（mitochondrial DNA, mtDNA）增殖環區（D-loop）多態性與下乳速率（milking speed, MS）對乳牛產乳性狀之影響。試驗運用PCR-RFLP技術與配合MspI限制酵素之切位辨識，進行279頭荷蘭泌乳牛生長素基因第三內含子第1557個核苷酸變異點（bGH-intron 3-T1557C）基因型鑑定與乳質乳量分析。生長素基因CC、CT與TT基因型頻率分別為65.9、33.0與1.1%。CC基因型泌乳牛較CT型者有顯著較低乳蛋白質率、乳糖率與總固形物率（P < 0.01）。擴增與定序荷蘭乳牛mtDNA 增殖環區（全長910 bp）經資料檢索合併不同品種資料後，分析發現與其他品種比較，計有45個單核苷多態性（SNP）突變點，包括38個置換（tarnsition）、5個顛換（transversion）、4個刪除（deletion）與1插入（insertion）；其中第169個核苷酸位置有較高之突變率（mtDNA-D loop-A169G）。應用CviAⅡ 限制酵素辨識切位與RFLP技術鑑別209頭荷蘭乳牛前述突變點發現，計有A與G兩種粒線體基因型，頻率分別為36.8與63.2%。不同基因型泌乳牛305-2X-ME校正乳量差異不顯著，然G型者較A型者有顯著較高之乳蛋白質率、乳糖率與總固形物率。應用bGH-intron 3-C1557T與mtDNA-D loop-A169G之組合基因型比較發現，CT-G組合型者有較高之乳蛋白質率、乳糖率與總固形物率；然不同基因型組合之305-2X-ME校正乳量與平均單日乳量差異不顯著。乳牛產乳、體型（乳房結構）與輔助性狀（下乳速率）相關係數估值顯示，體長和臀寬，與305-2X-ME校正乳量具顯著正相關，但與下乳速率呈顯著負相關。同時，305-2X-ME校正乳量與乳房深度呈顯著負相關。臀寬較寬牛隻，有較高之乳蛋白質率；前乳頭較長、乳房深度較深與具前傾式乳房之牛隻，有顯著較高乳脂率及乳總固形物率。單日乳量較高之牛隻，其下乳速率顯著較快；但其乳蛋白質率、乳糖率與總固形物率則顯著較低。此外，體細胞數（未罹患臨床性乳房炎牛隻）與本研究所評估之各性狀（產乳、體型與輔助性狀）間相關係數估值皆不顯著。綜合言之，應用乳牛bGH-intron 3-T1557C與mtDNA-D loop-A169G點突變之基因多態性，配合體型與輔助性狀資訊於乳牛群生產性狀選拔，以提升乳牛場生產效率與競爭力，應是可行的。

The purpose of this study was to study the effects of bovine growth hormone gene (bGH), mitochondrial DNA (mtDNA) D-loop polymorphisms, and milking speed (MS) on milking performances of Holstein cattle in Taiwan. Milking procedure is one of the important sections in dairy cattle management. Furthermore, quantity and quality improvement, as well as cost reduction are the final goals of dairy industry. A total of 279 Holstein cows were genotyped for bGH-intron 3-T1557C point mutation using PCR-RFLP via MspI enzyme, and furthered evaluated for milking performances. The genotypic frequencies of CC, CT and TT were 65.9, 33.0 and 1.1%, respectively. Cows with CC genotype produced significantly lower milk protein (%), milk lactose (%) and total solid (%) (P < 0.01) than those of cows with CT gneotype. There were 45 single nucleotide polymorphisms (SNP) observed in bovine mtDNA D-loop (910 bp in total length), including 38 transition, five transversion, and four deletions as well as one insertion. Also, the 169th nucleotide site (mtDNA-D loop-A169G) showed the highest mutation rate in D-loop region. The PCR-RFLP technique with CviAΠ enzyme was applied for mtDNA-D loop-A169G genotyping. The frequencies of A and G genotypes were 36.8 and 63.2%, respectively for 209 Holstein cattle. No significant difference was observed in 305-2X-ME between mtDNA-A and -G cows (P= 0.504). However, cows with mtDNA-G genotype produced significantly higher milk protein (%), milk lactose (%) and total solid (%) than those of mtDNA-A ones (P < 0.05). Milking performances comparisons among combination of bGH-intron 3-C1557T and mtDNA-D loop-A169G genotypes indicated that cows with CT-G genotype showed higher milk protein (%), milk lactose (%), and total solid (%) than others, but no difference was found in 305-2X-ME and average daily milk yield. Correlations among milking performances, body conformation (especially for udder morphology) and auxiliary traits (milking speed) were estimated phenotypically. Both body length and rump width showed positively correlated with 305-2X-ME, and negatively correlated with milking speed (MS). Moreover, negative estimate was obtained for correlation between 305-2X-ME and udder depth. Cows with wider rumps had higher milk peotein (%). Also, cattle with longer fore teats, deeper udder depth and forward leaning udder produced higher milk fat (%) and total solid (%). High MS cows had higher average daily yield with lower milk fat (%), milk protein (%), and total solid (%) when compared with others. Within this limited study, all traits studied did not significantly correlate with somatic cell counts (cows without clinical mastitis only). In conclusion, genotypes of bGH-T1557C and mtDNA-D loop-A169G, conformation and auxiliary traits could be employed to promote production efficiency and thus enhance the international competability for dairy industry.

摘 要 I
Abstract III
謝 誌 V
圖表目錄 IX
壹、前言 1
貳、文獻回顧 2
一、基因多態性對乳牛產乳性狀之影響 2
（一）乳牛經濟性狀之遺傳變異率估值 2
（二）乳牛產乳性狀之候選基因 5
1. 生長素基因介紹 8
2. 粒線體基因介紹 14
二、輔助性狀對產乳性狀之影響 18
（一）下乳速率之特性 18
1. 下乳速率之遺傳參數 18
2. 搾乳作業方式 18
3. 乳房內壓 19
（二）體型性狀 19
1. 體型結構 20
2. 乳房結構 20
參、生長素基因型對產乳性狀之影響 25
一、材料與方法 25
（一）試驗動物來源 25
（二）生長素基因型鑑定 25
1. 血液與乳汁樣本採集 25
2. 基因組DNA萃取 25
3. 基因組DNA濃度定量 26
4. 引子設計 26
5. PCR反應液配置 27
6. PCR條件 27
7. 限制酶切割反應與電泳分析 27
8. 膠體電泳分析 27
（三）PCR產物之核苷酸定序 28
1. PCR產物純化 28
2. 黏接作用（ligation） 28
3. 外源性重組DNA之轉形作用（transformation） 30
4. 質體DNA萃取 30
5. EcoRI 限制酶截切確認外源性重組DNA 30
6. PCR產物之核苷酸定序分析與序列比對 31
（四）產乳性能資料 31
（五）統計分析 32
二、結果與討論 34
（一）bGH-intron 3點突變定序結果 34
（二）應用PCR-RFLP模式分析 34
（三）生長素基因型對乳牛產乳性狀之影響 35
肆、乳牛粒線體增殖環區基因型對產乳性狀之影響 41
一、材料與方法 41
（一）試驗動物來源 41
（二）試驗步驟 41
1. 血液與乳汁樣本採集 41
2. 引子設計 41
3. PCR反應液之配置 42
4. PCR條件 42
5. 限制酶切割與電泳分析 43
（三）PCR產物之核苷酸定序與序列分析 43
（四）統計分析 44
二、結果與討論 45
（一）粒線體增殖環區全長序列比對 45
（二）粒線體增殖環區A169G點突變定序結果 45
（三）mtDNA 增殖環區A169G基因型對產乳性狀影響 46

（四）荷蘭乳牛GH-intron 3-C1557T與mtDNA-D loop-A169G基因組合型效應分析 47
伍、功能體型性狀與下乳速率對乳牛產乳性狀之影響 55
一、材料與方法 55
（一）搾乳機械 55
（二）電子流量紀錄 55
（三）體型與乳房結構性狀量測 55
（四）泌乳性能資料 56
（五）統計分析 56
二、結果與討論 61
（一）下乳速率評估 61
（二）下乳速率對產乳性狀之影響 68
（三）體型性狀評估 72
陸、結論 75
參考文獻 76
附 錄 86
作者簡介 91

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