瑪拉巴石斑稚魚之必須脂肪酸營養及其對免疫反應之影響

研究生: 吳豐成
指導教授；陳宏遠 博士
本研究以三實驗探討瑪拉巴石斑稚魚對餌料必需脂肪酸營養及其對免疫反應之影響。各實驗中，各試驗飼料的油脂添加量均為10 g/100 g 乾餌重(DW)，並以一添加含鱈魚肝油、亞麻籽油和紅花籽油 (2:1:1, wt/wt/wt)混合天然油脂的飼料為參考飼料。實驗一以七種試驗飼料、一種對照飼料和參考飼料探討瑪拉巴石斑稚魚(平均體重11.8 ± 0.7 g)對餌料DHA和EPA的需求情形以及其對免疫反應之影響。試驗飼料的油脂添加為不等比例的DHA與EPA 1 g/100 g DW及三硬脂酸9 g/100 g DW。對照組不添加EPA或DHA，而含 1 g/100 g DW 混合trilinolenin與trilinolein (3:1, wt/wt)以及三硬脂酸9 g/100 g DW。12週飼育結果顯示餌料DHA/EPA比例顯著影響石斑魚成長與其頭腎白血球的吞噬作用及T細胞的增生活性，但對活存率和相對肝臟重則沒有影響。DHA/EPA比例大於1時對石斑魚具有促進成長的效果，顯示做為必需脂肪酸，DHA促進石斑魚成長的效果是優於EPA，而且DHA是(n-3)高度不飽和脂肪酸(HUFAs)中唯一可促進白血球之吞噬功能和T細胞增生的脂肪酸。實驗二以八種試驗飼料和參考飼料探討瑪拉巴石斑稚魚(11.3 ± 0.6 g)對18:3(n-3) (LNA)和18:2(n-6) (LA)的餌料需求，及餌料LNA與LA含量和其混合的比例對石斑魚非特異性細胞免疫反應的影響。試驗組包括僅添加LA 或 LNA 1 或 2 g/100 g DW等四處理組，和添加2 g/100 g DW 的LNA及LA混合油脂，分別有LNA/LA為3、1.4、0.7和0.4等四處理組；各試驗飼料除原料所殘留的HUFAs外，並不另外添加。12週飼育結果顯示餌料添加LNA和高LNA/LA比例均會顯著促進石斑稚魚的成長及其頭腎白血球吞噬活性和呼吸猝發活性等非特異性細胞免疫反應，但是與攝食參考飼料(含有HUFA)者間沒有顯著差異；餌料中添加LNA/LA (3:1) 2 g/100 g 乾餌重(DW) 可以滿足瑪拉巴石斑稚魚的必需脂肪酸需求。實驗三為一2 × 3複因子試驗，由兩(n-3)HUFAs濃度(1 或2 g/100 g DW)與三花生四烯酸(AA) 濃度(0、1 或2 g/100 g DW)所配合成的六種處理，目的在探討瑪拉巴石斑稚魚(13.2 ± 0.9 g)對(n-3)HUFAs和AA的餌料需求與可能的交互關係，及其對石斑魚免疫反應的影響。十二週飼育試驗結果顯示適量餌料AA和 (n-3)HUFA之添加顯著促進瑪拉巴石斑稚魚的成長，而AA和 (n-3)HUFA兩者之交感並不顯著，試驗魚的活存率也不受餌料(n-3)HUFA或AA濃度等影響；肝臟(n-6)HUFA 濃度顯著與石斑稚魚的吞噬活性、呼吸猝發活性和白血球增生等免疫反應呈正相關。綜合本研究結果，當(n-3)HUFA中，DHA與EPA的比例為 3:1(wt/wt)時，餵飼含有(n-3)HUFA 1 g/100 g DW和AA 1 g/100 g DW飼料之瑪拉巴石斑稚魚可達最佳成長狀態，且其免疫反應可受最大的激化。

Essential fatty acids nutrition and its effects on immune responses of the juvenile grouper, Epinephelus malabaricus

Feng-Cheng Wu
(Advisor: Dr. Houng-Yung Chen)
Institute of Marine Biology, National Sun Yat-sen University
Kaohsiung 804 Taiwan.

A series of three experiments was conducted to study the essential fatty acids nutrition and its effects on immune responses (IR) of the juvenile grouper, Epinephelus malabaricus. All experimental diet contained 10 g/100 g diet supplemental lipids from various sources. A reference diet was used in all experiments and contained natural oil mixture of cod liver oil, linseed oil, and safflower oil at a rate of 2:1:1 (wt/wt/wt). In experiment 1, juvenile grouper (11.8 ± 0.7 g) were fed for 12 wks on one of the seven experimental diets, control diet and the reference diet to investigate dietary requirement for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and effects on IR of grouper. Seven experimental diets contained 1 g/100 g diet of DHA and EPA in various combinations and 9 g/100 g diet of tristearin. The control diet contained 1 g/100 g diet of trilinolenin and trilinolein (3:1, wt/wt). The results indicate that there was a significant difference among dietary treatments in growth, phagocytosis and leucocytes proliferation, when stimulated by Con A and PHA-P but not by LPS. However there was no difference in survival rate and relative liver weight. Enhanced growth was observed when the dietary DHA/EPA was greater than 1, indicating that DHA was superior to EPA in promoting fish growth. DHA is the only member in the family of (n-3) highly unsaturated fatty acid (HUFA) that stimulates phagocytic functions of leucocytes and T cell proliferation of the juvenile grouper. In experiment 2, juvenile grouper (11.3 ± 0.6 g) were fed for 12 wks on one of the eight experimental diets or the reference diet to investigate dietary requirement for linolenic acid (LNA) and linoleic acid (LA), as well as effects on nonspecific IR of grouper. The test diets were supplemented with LNA or LA at a rate of 1 or 2 g LNA or LA/100 g diet or 2 g/100 g diet of LNA and LA in various ratios (3, 1.4, 0.7 and 0.4). Tristearin was used to fill the lipid supplemental level to 10 g/100 g diet. The results show that enhanced growth and optimal non-specific cellular IR were observed when the grouper were fed on the diet having the highest LNA/LA ratio (3:1) or on the diets supplemented with LNA. But the enhancement was not different (P>0.05) from that of the reference diet group. Thus, incorporating 2 g/100 g LNA/LA (3:1, wt/wt) in diet ensures adequacy of the grouper for essential fatty acid. In experiment 3, juvenile grouper (13.2 ± 0.9 g) were fed on one of the six experimental diets in the 2 × 3 factorial design or on the reference diet for 12 wks to investigate dietary requirement for (n-3)HUFAs and arachidonic acid (AA), as well as effects on IR of grouper. Two levels of (n-3)HUFAs (1 or 2 g/100 g ) in combination with 3 levels of AA (0, 1 or 2 g/100 g) were tested. The results show an enhanced growth when optimal concentrations of AA and (n-3)HUFA were incorporated to the diets. Liver (n-6)HUFAs concentration reflects IR of the juvenile grouper. Interaction of AA and (n-3)HUFAs in affecting fish growth and IR was insignificant (P>0.05), and concentrations of dietary (n-3)HUFAs or AA did not significantly affect fish survival rate. The results of the 3 experiments show that the grouper will benefit most in growth and IR when their diets contain 1 g/100 g diet of (n-3)HUFA and 1 g/100 g diet of AA, when (n-3)HUFA is a mixture of DHA and EPA at a ratio of 3:1 (wt/wt).

目錄


謝辭......................................Ⅱ
中文摘要……………………………………………Ⅲ
英文摘要……………………………………………Ⅴ
目錄…………………………………………………Ⅶ
表目錄………………………………………………Ⅸ
圖目錄………………………………………………XI

第一章 研究背景與目的…………………………1
第二章 綜合材料與方法…………………………16
2.1. 試驗動物………………………………………17
2.2. 飼料配方與配置法……………………………17
2.3. 養殖系統和飼育實驗…………………………25
2.4. 採樣、血清收集和頭腎白血球之製備………26
2.5. 一般成分分析和脂肪萃取……………………28
2.5.1. 水分分析……………………………………28
2.5.2. 灰份分析……………………………………28
2.5.3. 粗蛋白質分析………………………………28
2.5.4. 脂肪萃取及中性脂質與極性脂質之分離…29
2.6. 脂肪酸分析……………………………………30
2.6.1. 脂肪酸甲基酯化步驟………………………30
2.6.2. 脂肪酸之定性和定量………………………30
2.7. 免疫分析………………………………………31
2.7.1. 吞噬活性分析……………………………..31
2.7.2. 呼吸猝發活性分析………………………..32
2.7.3. 白血球細胞增生分析………………………33
2.8. 統計分析………………………………………34
第三章 瑪拉巴石斑稚魚之(N-3)高度不飽和脂肪酸需求及其對免疫反應之影響(實驗一)…………………………….....................36
3.1. 摘要……………………………………………37
3.2. 前言……………………………………………38
3.3. 材料與方法……………………………………41
3.4. 結果……………………………………………47
3.5. 討論……………………………………………57
第四章 瑪拉巴石斑稚魚之次亞麻油酸和亞麻油酸需求及其對非特異性免疫反應之影響(實驗二)……………………...................68
4.1. 摘要……………………………………………69
4.2. 前言……………………………………………70
4.3. 材料與方法……………………………………71
4.4. 結果……………………………………………78
4.5. 討論……………………………………………84
第五章 瑪拉巴石斑稚魚對花生四烯酸需求之交互作用及對免疫反應之影響與(N-3)高度不飽和脂肪酸需求之再確認 (實驗三)............91
5.1. 摘要……………………………………………92
5.2. 前言……………………………………………93
5.3. 材料與方法……………………………………95
5.4. 結果………………………………………….101
5.5. 討論………………………………………….110
第六章 結論與建議………………………………118
第七章 參考文獻…………………………………121
附錄 由本研究所正式發表論文之全文

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