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論文名稱: | 升溫與氯化銨緊迫對吳郭魚血液之特性及血漿中脂氧合酶活性之影響
| Elevated habitat temperature and ammonium chloride on blood characteristics and plasma lipoxygenase activity
研究生: | 周秉則 Ping-Tse Chou
指導教授: | 孫寶年 Pan, Bonnie Sun
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
畢業學年度: | 92
語文別: | 中文
關鍵字: | 脂氧合酶 lipoxygenase
| 高效能液相層析儀 high-performance liquid chromatography
| 飽和脂肪酸 saturated fatty acid
| 升溫緊迫 heat stress
全文點閱次數: | 16

全文影像連結:

論文全文(pdf)

論文頁數: | 95
摘要: | 摘  要
| 吳郭魚生長的溫度範圍為14℃~ 35℃，本研究探討在由25℃馴養後，升溫 10℃與含氯化銨的環境，吳郭魚行為、血液生化性質與脂氧合酶活性之相互關係，期找出會反映魚體受緊迫的指標。
| 吳郭魚在快速(2℃/h)或緩慢升溫(0.5℃/h)及氯化銨(1.5-3.0 mg /L)緊迫環境中，出現泳動及呼吸急促、排便量增多，紅血球數、血紅素、平均血紅素濃度均隨著緊迫時間的延長而增加，與控制組間有顯著差異；但白血球、血比容積與控制組無顯著差異；血糖濃度在升溫緊迫初期呈現增加的趨勢，隨緊迫時間延長血糖濃度逐漸下降，而在氯化銨緊迫中則無顯著差異。升溫或氯化銨緊迫環境中，紅血球變小，紅血球膜之不飽和與飽和脂肪酸比值即不飽和指數 (UFA/SFA)由正常時的1.80經快速升溫24小時下降至0.72及緩慢升溫經4日後降至0.73，以氯化銨緊迫試驗經24小時由1.72下降至0.12最為明顯，以HPLC測得血漿之脂氧合酶 (lipoxygenase; LOX)，在快速升溫中以5-及15-LOX活性顯著增加，而緩慢升溫中15-LOX活性較5- 及12-LOX明顯增加，但在氯化銨緊迫中僅偵測出15-LOX有明顯增加。
| 在升溫與氯化銨兩種不同性質之緊迫實驗中，紅血球數、紅血球膜之UFA/SFA、血紅素、血糖與LOX活性反映出魚體受緊迫的程度，而血糖濃度與LOX活性在升溫過程中即被誘發而增加，其斜率為8.3較血糖的斜率3.3增加顯著，以LOX較為靈敏，比在行為出現異常早14小時，比血糖增加及行為改變更為顯著。
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| Abstract
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| Tilapia survive at a temperature range of 14~35℃. In this study, tilapia were preadapted at 25℃ and then exposed to 37℃ at two temperature increase rates (2℃/h and 0.5℃/h) in addition to NH4Cl as a stressor. the behavior, blood characteristics and lipoxygenase activities of tilapia were determined to identify a sensitive biomarker reflecting the fish was stressed before damage occurred.
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| Tilapia appeared swimming fastly breathed rapidy and increase in feces. There a increased with extension of exposure time at 37℃ either by rapid (2℃/h) or slow (0.5℃/h) increase and in the presence of NH4Cl (1.5 or 3.0 mg / L) showed significant difference in erythrocyte count, hemoglobin and mean corpuscular hemoglobin (Hb) concentration from those of control (p<0.05), but showed no significant difference (p>0.05) in leucocyte count and hematocrit. The plasma glucose concentration increased from the initial stage of heat stress, decreased gradually after 4 days, NH4Cl at 1.5 and 3.0 mg/L did not cause the tilapia plasma to elevate. Tilapia cultured in water of temperature elevated to 37℃ or containing NH4Cl resulted in the erythrocytes to decrease in size. The unsaturation index of unsaturated / saturated fatty acid ratio of erythrocyte membrane dropped with exposure to heat stress and NH4Cl, the UFA/SFA dropped 93％ from 1.80 to 0.12 in the presence of NH4Cl and 60％ from 1.80 to 0.72 at temperature increased rapidly and 57％ from 1.70 to 0.73 at slow increase of temperature. Lipoxygenase (LOX) activities measured with high performance liquid chromatography (HPLC), showed that the 5-, 15-LOX activities increased with water temperature quickly elevated from 25℃ to 37℃ at 2℃/h, and only 15-LOX activity increased significantly as the water temperature increased at 0.5℃/h and in tilapia exposed to NH4Cl as stressor.
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| Erythrocyte counts, Hb, plasma glucose and LOX activity of tilapia reflected the exposure of fish to environmental stressors, the plasma glucose concentration and LOX activities were induced as the water temperature increased, but LOX activities increased an UFA/SFA decreased within 14 hours being more sensitive than plasma glucose or behavioral changes.
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論文目次: | 目錄
| 目錄    Ⅱ
| 圖目錄    Ⅲ
| 縮寫表    VI
| 中文摘要    Ⅶ
| 英文摘要    Ⅷ
|
| 壹、前言    1
| 貳、文獻回顧    3
| 一、環境緊迫對魚類之影響    3
| (一) 魚類的緊迫反應    3
| (二) 溫度的影響    6
| (三) 氨氮化合物之影響    7
| 二、細胞受緊迫壓力時的反應    8
| 三、脂氧合酶的性質    9
| 四、生物指標的建立    11
|
| 參、材料與方法    12
| 一、吳郭魚    12
| 二、採樣    12
| 三、蓄養條件    13
| (一) 快速升溫實驗    13
| (二) 緩慢升溫實驗    13
| (三) 氨化銨與升溫實驗    13
| 四、魚體測定    14
| 五、採血    14
| 六、血液分析    14
| 血液基本參數值    15
| （a）紅血球個數    15
| （b）白血球個數    15
| （c）血色素    15
| （d）血比容積    15
| （e）滲透壓    16
| （f）血糖測定    16
| （g）紅血球抹片染色    16
| 七、紅血球膜脂肪酸分析    17
| 1. 紅血球細胞膜分離    17
| 2. 微量甲酯化    18
| 八、血漿中脂氧合酶之測定    18
| 1. 粗酵素製備    18
| 2. 脂氧合酶活性測定    19
| a. 冷光測定法    19
| b. 吸光法    19
| c. 高效液相層析法    20
| 九、蛋白質定量    21
| 十、統計分析    22
| 肆、結果與討論    23
| 一、快速升溫緊迫對吳郭魚之影響    23
| 二、緩慢升溫與快速升溫之差異    40
| 三、氨氮濃度與升溫緊迫之影響    58
| 伍、結論    75
| 陸、參考文獻----------------------------------------------------------------------80
| 圖次
| 圖一、吳郭魚於25℃馴養後快速升溫至37℃恆溫蓄養血液性質之變化。    25
| 圖二、吳郭魚於25℃馴養後快速升溫至37℃恆溫蓄養平均血紅素
| 濃度之變化。    26
| 圖三、吳郭魚於25℃馴養後快速升溫至37℃恆溫蓄養血糖之變化。    36
| 圖四、吳郭魚於25℃馴養後快速升溫至37℃恆溫蓄養滲透壓的變化。    37
| 圖五、吳郭魚於25℃馴養後快速升溫至37℃後恆溫蓄養，以冷光儀檢測血漿脂氧合酶活性的的變化。    39
| 圖六、吳郭魚於25℃馴養後快速升溫至37℃後恆溫蓄養，以吸光度法檢測脂氧合酶活性的變化。    40
| 圖七、吳郭魚在25℃恆溫馴養後曝露在37 ℃以逆相HPLC偵測HETEs做
| 為脂氧合酶異構酶活性的變化。    41
| 圖八、吳郭魚於25℃馴養後快速升溫至37℃恆溫蓄養血球外觀之變化。    46
| 圖九、吳郭魚於25℃馴養後緩慢升溫至37℃血液性質之變化。    47
| 圖十、於25℃馴養後緩慢升溫至37℃恆溫蓄養平均血紅素濃度之變化。    48
| 圖十一、吳郭魚於25℃馴養後緩慢升溫至37℃後恆溫蓄養血糖之變化。    50
| 圖十二、吳郭魚於25℃馴養後緩慢升溫至37℃滲透壓的變化。    51
| 圖十三、吳郭魚於25℃馴養後緩慢升溫至37℃以冷光檢測血漿中
| 脂氧合酶之變化。    52
| 圖十四、吳郭魚在25℃馴養後緩慢升溫至37 ℃以吸光度脂氧合酶
| 活性的變化。    52
| 圖十五、吳郭魚在25℃馴養後緩慢升溫至37 ℃後血漿中脂氧合酶
| 異構酶活性的變化。    54
| 圖十六、吳郭魚於25℃馴養後緩慢升溫至37℃後血球外觀之變化。    55
| 圖十七、吳郭魚於25℃馴養後快速升溫至35℃，添加氯化銨後
| 血液性質的變化    56
| 圖十八、吳郭魚於25℃馴養後快速升溫至35℃，添加氯化銨
| 後平均血紅素濃度之變化    57
| 圖十九、吳郭魚於25℃馴養後快速升溫至35℃，添加氯化銨
| 後血醣濃度之變化。    58
| 圖二十、吳郭魚於25℃馴養後快速升溫至35℃，添加氯化銨後血
| 漿滲透壓之變化。    60
| 圖二十一、吳郭魚於25℃馴養後快速升溫至35℃，添加氯化銨後
| 血漿脂氧合酶之變化。    61
| 圖二十二、吳郭魚於25℃馴養後，曝露於16 ± 1℃72小時血漿中脂氧合
| 酶活性之變化。    62
| 圖二十三、吳郭魚於25℃馴養後快速升溫至35℃，添加氯化銨
| 後血球型態之變化。    63
|
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2009 年 11 月 22 日 23:44