Effects of four functional feed additives on growth performance, lipid metabolism, non-specific immunity and health of liver and intestine of Anguilla marmorata

学校编码：10384 密级 学号：22320141151340

硕 士 学 位 论 文

四种功能性饲料添加剂对花鳗鲡生长性能、

脂肪代谢、非特异性免疫和肝肠健康的影响

Effects of four functional feed additives on growth

performance, lipid metabolism, non-specific immunity and

health of liver and intestine of Anguilla marmorata

解文丽

指导教师姓名：艾春香 副教授

专 业 名 称：海 洋 生 物 学

论文提交日期：

2017 年 0 5 月

论文答辩时间：

2017 年 0 5 月

2017年05月

厦门大学博硕士论文摘要库

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厦门大学博硕士论文摘要库

目录

目

录

缩略词表

...

I

摘要

...

III

Abstract

...

V

第一章 绪论

...

1 1.1 花鳗鲡的养殖现状及其存在的问题 ... 1 1.1.1 花鳗鲡的养殖现状 ... 1 1.1.2 花鳗鲡养殖存在的问题 ... 2 1.1.3 花鳗鲡养殖的发展对策 ... 3 1.2 功能性饲料添加剂的研发及其应用 ... 3 1.2.1 功能性饲料添加剂概述 ... 3 1.2.2 四种功能性饲料添加剂的研发及其在水产饲料中的应用 ... 4 1.3 本研究的目的与意义 ... 13 1.3.1 本研究的目的与意义 ... 14 1.3.2 本研究的主要内容和研究方案 ... 14

第二章 四种功能性饲料添加剂对花鳗鲡生长性能、脂肪代谢和非特

异性免疫的影响

...

16 2.1 材料和方法 ... 17 2.1.1 实验材料 ... 17 2.1.2 实验方法 ... 19 2.1.3 数据处理 ... 21 2.2 结果 ... 21 2.2.1 四种功能性饲料添加剂对花鳗鲡生长性能的影响 ... 21 2.2.2 四种功能性饲料添加剂对花鳗鲡肠道消化酶活性的影响 ... 22 2.2.3 四种功能性饲料添加剂对花鳗鲡脂肪代谢的影响 ... 22 2.2.4 四种功能性饲料添加剂对花鳗鲡肝脏和血清转氨酶活性的影响 . 24 2.2.5 四种功能性饲料添加剂对花鳗鲡非特异性免疫的影响 ... 24

厦门大学博硕士论文摘要库

2017 届厦门大学硕士学位论文 2.3 讨论 ... 27 2.3.1 四种功能性饲料添加剂对花鳗鲡生长性能的影响 ... 27 2.3.2 四种功能性饲料添加剂对花鳗鲡肠道消化酶活性的影响 ... 28 2.3.3 四种功能性饲料添加剂对花鳗鲡脂肪代谢的影响 ... 29 2.3.4 四种功能性饲料添加剂对花鳗鲡肝脏和血清转氨酶活性的影响 . 31 2.3.5 四种功能性饲料添加剂对花鳗鲡非特异性免疫的影响 ... 32 2.4 小结 ... 35

第三章 四种功能性饲料添加剂对花鳗鲡肝脏和肠道组织形态、肠道

菌群结构的影响

...

37 3.1 材料与方法 ... 38 3.1.1 实验材料 ... 38 3.1.2 实验方法 ... 39 3.2 结果 ... 42 3.2.1 四种功能性饲料添加剂对花鳗鲡肝脏组织结构的影响 ... 42 3.2.2 四种功能性饲料添加剂对花鳗鲡肠道组织结构的影响 ... 43 3.2.3 四种功能性饲料添加剂对花鳗鲡后肠肠道壁菌群结构的影响 ... 45 3.3 讨论 ... 50 3.3.1 四种功能性饲料添加剂对花鳗鲡肝脏组织结构的影响 ... 50 3.3.2 四种功能性饲料添加剂对花鳗鲡肠道组织结构的影响 ... 50 3.3.3 四种功能性饲料添加剂对花鳗鲡后肠肠道壁菌群结构的影响 ... 51 3.4 小结 ... 54

结语

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55

附录

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57

参考文献

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58

致谢

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82

厦门大学博硕士论文摘要库

Contents

Contents

List of abbreviations

...

I

Abstract (In Chinese)

...

III

Abstract (In English)

...

V

Chapter 1 Introduction

...

1

1.1 Current situation and problems Anguilla marmorata culturing ... 1

1.1.1 Current situation of Anguilla marmorata culturing ... 1

1.1.2 Problems of Anguilla marmorata culturing ... 2

1.1.3 Strategies of Anguilla marmorata culturing ... 3

1.2 Study and application of functional feed additives ... 3

1.2.1 Introduction of functional feed additives ... 3

1.2.2 Study and application of four functional feed additives ... 4

1.3 Objective and significance of the research ... 13

1.3.1 Objective and significance of the research ... 14

1.3.2 Content and protocol of the research ... 14

Chapter 2 Effects of four functional feed additives on growth

performance, lipid metabolism and non-specific immunity of Anguilla

marmorata

...

16

2.1 Material and method ... 17

2.1.1 Experimental material ... 17

2.1.2 Experimental method ... 19

2.1.3 Data processing ... 21

2.2 Results ... 21

2.2.1 Effects of four functional feed additives on growth performance of Anguilla marmorata ... 21

2.2.2 Effects of four functional feed additives on digestive enzyme activities of Anguilla marmorata ... 22

2.2.3 Effects of four functional feed additives on lipid metabolism of Anguilla marmorata ... 22

2.2.4 Effects of four functional feed additives on liver and serum transaminase activities of Anguilla marmorata ... 24

厦门大学博硕士论文摘要库

2017 届厦门大学硕士学位论文

2.2.5 Effects of four functional feed additives on non-specific immunity of

Anguilla marmorata ... 24

2.3 Disscussion ... 27

2.3.1 Effects of four functional feed additives on growth performance of Anguilla marmorata ... 27

2.3.2 Effects of four functional feed additives on digestive enzyme activities of Anguilla marmorata ... 28

2.3.3 Effects of four functional feed additives on lipid metabolism of Anguilla marmorata ... 29

2.3.4 Effects of four functional feed additives on liver and serum transaminase activities of Anguilla marmorata ... 31

2.3.5 Effects of four functional feed additives on non-specific immunity of Anguilla marmorata ... 32

2.4 Summary ... 35

Chapter 3 Effects of four functional feed additives on histomorphology of

liver and intestine and intestinal bacteria of Anguilla marmorata

...

37

3.1 Material and method ... 38

3.1.1 Experimental material ... 38

3.1.2 Experimental method ... 39

3.2 Resultes ... 42

3.2.1 Effects of four functional feed additives on histomorphology of liver of Anguilla marmorata ... 42

3.2.2 Effects of four functional feed additives on histomorphology of intestine of Anguilla marmorata ... 43

3.2.3 Effects of four functional feed additives on intestinal bacteria of Anguilla marmorata ... 45

3.3 Disscussion ... 50

3.3.1 Effects of four functional feed additives on histomorphology of liver of Anguilla marmorata ... 50

3.3.2 Effects of four functional feed additives on histomorphology of intestine of Anguilla marmorata ... 50

3.3.3 Effects of four functional feed additives on intestinal bacteria of Anguilla marmorat ... 51

厦门大学博硕士论文摘要库

Contents 3.4 Summary ... 54

Epilogue

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55

Appendix

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57

References

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58

Acknowledgements

...

82

厦门大学博硕士论文摘要库

缩略词表

I

缩略词表

缩略词 英文全称 中文全称

ACP Acid Phosphatase 酸性磷酸酶

AKP Alkaline Phosphatase 碱性磷酸酶

AMS Amylase 淀粉酶

CAT Catalase 过氧化氢酶

CF Condition Factor 肥满度

DGGE Denaturing Gradient Gel

Electrophoresis 变性梯度凝胶电泳 DHA Docosahexaenoic Acid 二十二碳六烯酸

EPA Eicosapentaenoic Acid 二十碳五烯酸 FCR Feed Conversion Rate 饲料转化率

FOS Fructooligosaccharide 果寡糖 GOT Glutamic-oxaloacetic Transaminase 谷草转氨酶 GPT Glutamic-pyruvic Transaminase 谷丙转氨酶 GPx Glutathione Peroxidase 谷胱甘肽过氧化物酶 HDL-C High Density Lipoprotein

Cholesterol 高密度脂蛋白胆固醇

HL Hepatic Lipase 肝脂酶

HSI Hepatosomatic Index 肝体指数

LDL-C Low Density Lipoprotein

Cholesterol 低密度脂蛋白胆固醇

LPS Lipase 脂肪酶

LPL Lipoprotein Lipase 脂蛋白脂酶

LYZ Lysozyme 溶菌酶

2017 届厦门大学硕士学位论文

II

缩略词表(续)

OTU Operational Taxonomic Units 运算分类单元 PCR Polymerase Chain Reaction 聚合酶链式反应 PUFA Polyunsaturated Fatty Acid 多不饱和脂肪酸 rDNA Ribosomal Deoxyribonucleic Acid 核糖体脱氧核糖核酸

ROS Reactive Oxygen Species 活性氧族 SOD Superoxide Dismutase 超氧化物歧化酶 SGR Specific Growth Rate 特定增长率

SI Spleen Index 脾体指数

SR Survival Rate 存活率

T-AOC Total Antioxidant Capacity 总抗氧化能力

TC Total Cholesterol 总胆固醇

TG Triglyceride 甘油三酯

TL Total Lipase 总脂酶

TRY Trypsin 胰蛋白酶

WGR Weight Gain Rate 增重率

摘要 III

摘 要

本论文以初始体长(41.54±0.83) cm，体质量(167.67±13.33) g 的花鳗鲡为研究 对象，在基础饲料中分别添加200 mg/kg 姜黄素(1 组)、1000 mg/kg 丁酸钠(2 组)、 1000 mg/kg 果寡糖(3 组)和 1000 mg/kg 复合中草药提取物(4 组)配制成 4 种等氮 等能的试验饲料，基础饲料为对照组(5 组)饲料，开展为期 60 d 的饲养试验，探 讨4 种功能性饲料添加剂对花鳗鲡生长性能、脂肪代谢、非特异性免疫、肝肠组 织形态和肠道菌群结构的影响，为其在花鳗鲡配合饲料中的应用积累基础资料。 本研究的主要结果如下： 1. 四种功能性饲料添加剂对花鳗鲡生长性能和消化酶活性的影响 添加姜黄素、丁酸钠、果寡糖和复合中草药提取物等 4 种功能性饲料添加剂 均 能 显 著 提 高 花 鳗 鲡 的 增 重 率(WGR) 、 特 定 增 长 率 (SGR) 以 及 饲 料 转 化 率 (FCR)(P<0.05)，未显著影响花鳗鲡的肥满度(CF)(P>0.05)。4 种功能性饲料添加 剂对花鳗鲡消化酶活性的影响各异，均未显著影响花鳗鲡肠道胰蛋白酶(TRY)和 胃蛋白酶活性(P>0.05)，均提高花鳗鲡肠道淀粉酶(AMS)活性，果寡糖显著提高 了花鳗鲡肠道 AMS 活性(P<0.05)，果寡糖和复合中草药提取物均显著提高了花 鳗鲡肠道脂肪酶(LPS)活性(P<0.05)。 2. 四种功能性饲料添加剂对花鳗鲡脂肪代谢的影响 4 种功能性饲料添加剂对花鳗鲡脂肪代谢的影响各异。姜黄素降低了花鳗鲡 肝 脏 总 胆 固 醇(TC) 、 甘 油 三 酯 (TG) 和 低 密 度 脂 蛋 白 胆 固 醇 (LDL-C) 的 水 平 (P>0.05)。丁酸钠降低了花鳗鲡肝脏 TC 和 TG 的水平(P>0.05)，显著降低其肝脏 LDL-C 水平(P<0.05)和血清谷草转氨酶(GOT)活性(P<0.05)。果寡糖降低了花鳗鲡 肝脏 TC 和 TG 的水平(P>0.05)，显著降低其肝脏 LDL-C 水平(P<0.05)。复合中 草药提取物显著提高花鳗鲡血清高密度脂蛋白胆固醇(HDL-C)水平(P<0.05)，降 低了花鳗鲡肝脏TC、TG 和 LDL-C 的水平(P>0.05)。综合上述，200 mg/kg 姜黄 素对花鳗鲡脂肪代谢影响不显著(P>0.05)，1000 mg/kg 丁酸钠、1000 mg/kg 果寡 糖和 1000 mg/kg 复合中草药提取物显著增强花鳗鲡脂肪代谢能力，同时 1000 mg/kg 丁酸钠有利于花鳗鲡的肝脏健康。 3. 四种功能性饲料添加剂对花鳗鲡非特异性免疫指标的影响

厦门大学博硕士论文摘要库

2017 届厦门大学硕士学位论文

IV

4 种功能性饲料添加剂均显著增强花鳗鲡非特异性免疫力。姜黄素显著提高

花 鳗 鲡 脾 体 指 数(SI) 及 鳃 谷 胱 甘 肽 过 氧 化 物 酶 (GPx) 活 性 和 总 抗 氧 化 能 力

(T-AOC)(P<0.05)，提高其血清过氧化氢酶(CAT)活性和 T-AOC(P>0.05)。丁酸钠

显著提高花鳗鲡鳃T-AOC(P<0.05)，提高其血清 CAT 活性和 T-AOC(P>0.05)。果

寡糖显著提高花鳗鲡SI、鳃超氧化物歧化酶(SOD)、血清 CAT 和酸性磷酸酶(ACP)

活性(P<0.05)。复合中草药提取物显著提高花鳗鲡血清溶菌酶(LYZ)活性和 T-AOC(P<0.05)，提高其血清 ACP 和鳃 SOD 活性以及 T-AOC(P>0.05)。

4. 四种功能性饲料添加剂对花鳗鲡肝脏和肠道组织结构的影响 4 种功能性饲料添加剂对花鳗鲡肝脏组织结构影响不大。各组花鳗鲡肝脏细 胞形态和大小正常，细胞边界清晰，细胞核居中。4 种功能性饲料添加剂均显著 改善了花鳗鲡肠道组织结构(P<0.05)。200 mg/kg 姜黄素显著增加花鳗鲡中肠绒毛 个数和肌层厚度(P<0.05)，1000 mg/kg 果寡糖显著促进花鳗鲡中肠绒毛的发育， 增加中肠直径和肌层厚度(P<0.05)，二者对花鳗鲡肠道组织结构改善效果更明显。 1000 mg/kg 丁酸钠显著增加花鳗鲡中肠肌层厚度(P<0.05)。1000 mg/kg 复合中草 药提取物显著改善花鳗鲡中肠绒毛的发育(P<0.05)。 5. 四种功能性饲料添加剂对花鳗鲡肠道菌群结构的影响 4 种功能性饲料添加剂对花鳗鲡肠道菌群结构影响较大。与对照组相比，4 个功能性饲料添加剂组的花鳗鲡肠道菌群多样性均降低了，对照组的花鳗鲡后肠 菌群香农指数最高为3.08，2 组的香农指数最低为 2.32。花鳗鲡后肠的优势菌群 为变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)，4 种功能性饲料添加剂均增 加了变形菌门的细菌数量，降低了厚壁菌门细菌比例。从菌群的属水平分析，4 种功能性饲料添加剂增加了肠道有益菌乳球菌属(Lactococcus)细菌数量，降低了 条件性致病菌梭菌属(Clostridium)细菌和有益菌乳酸菌属(Lactobacillus)细菌的数 量，调节肠道菌群结构。1 组和 3 组的花鳗鲡肠道菌群聚类位置最近，肠道菌群 结构最相似，这与它们显著改善了肠道组织结构的结论一致。 关键词：花鳗鲡；功能性饲料添加剂；脂肪代谢；非特异性免疫；肝肠组织结构； 肠道菌群

厦门大学博硕士论文摘要库

Abstract

V

Abstract

A 60-day feeding trail was conducted to evaluate effects of four functional feed additives on growth performance, lipid metabolism, non-specific immunity, histomorphology of liver and gut and intestinal bacteria of Anguilla marmorata. Five isoenergetic and isolipid diets were formulated: group 1(basal diet + 200 mg/kg curcumin), group 2(basal diet +1000 mg/kg sodium butyrate), group 3(basal diet +1000 mg/kg fructooligosaccharide), group 4(basal diet +1000 mg/kg compound of Chinese herbal medicine extracts) and group 5(basal diet). Each diet was randomly fed to triplicate groups of fish with initial body length of 41.54 ± 0.83 cm and initial body weight of 167.67 ± 13.33 g. The research achievement could provide the basic information for the four feed additives in A. marmorata formulated feed.The main results are as follows:

1. Effects of four functional feed additives on growth performance and digestive enzyme activities of A.marmorata

WGR, SGR and FCR of A. marmorata were significantly increased by adding four functional feed additives (P<0.05) except for CF (P>0.05) respectively. There were different effects of four feed additives on digestive enzyme activities of A. marmorata. The intestinal protease activity was not significantly influenced by four feed additives (P>0.05), while intestinal AMS activity was improved by adding four feed additives. And 1000 mg/kg fructooligosaccharide significantly enhanced intestinal AMS activity (P<0.05). In addition, intestinal LPS activity of A. marmorata was significantly improved by fructooligosaccharide and compound of Chinese herbal medicine extracts (P<0.05).

2. Effects of four functional feed additives on lipid metabolism of A.marmorata Different feed additives had different effect on lipid metabolism. The level of TC, TG and LDL-C of liver were decreased by adding 200 mg/kg curcumin (P>0.05). Sodium butyrate decreased the level of TC and TG in liver (P>0.05), besides it significantly decreased the content of LDL-C of liver and GOT activity of serum

2017 届厦门大学硕士学位论文

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(P<0.05). Similarly, the level of TC and TG of liver were decreased by adding 1000 mg/kg fructooligosaccharide (P>0.05), besides the content of LDL-C of liver was significantly decreased (P<0.05). Compound of Chinese herbal medicine extracts significantly increased the level of HDL-C in serum (P<0.05). Meanwhile, it decreased the level of TC, TG and LDL-C in liver (P>0.05). Above all, 200 mg/kg curcumin has no significant effect on lipid metabolism of A. marmorata (P>0.05). The ability of lipid metabolism of A.marmorata was significantly enhanced by 1000 mg/kg sodium butyrate, 1000 mg/kg fructooligosaccharide and 1000 mg/kg compound of Chinese herbal medicine extracts (P<0.05), besides sodium butyrate was benificial to keep liver health .

3. Effects of four functional feed additives on non-specific immunity of A. marmorata Four functional feed additives all can significantly improved non-specific immunity of A. marmorata. Curcumin significantly enhanced SI, GPx activity and T-AOC in gill (P<0.05), and enhanced CAT activity and T-AOC in serum (P>0.05). Sodium butyrate significantly improved T-AOC of gill (P<0.05) and improved CAT activity and T-AOC of serum (P>0.05). SI, CAT and ACP activity in serum, SOD activity in gill were significantly increased by adding 1000 mg/kg fructooligosaccharide (P<0.05). Compound of Chinese herbal medicine extracts significantly improved LYZ activity and T-AOC of serum (P<0.05) and improved SOD activity and T-AOC of gill and ACP activity of serum (P>0.05).

4. Effects of four functional feed additives on histomorphology of liver and gut of A.marmorata

Four functional feed additives have no significant effect on histomorphology of liver. Each group had the normal liver cell with clear borders and centered nucleus. Four feed additives all significantly improved histomorphology of gut (P<0.05). Curcumin significantly increased the quantity of villus and muscular thickness of midgut (P<0.05). Fructooligosaccharide significantly increased the quantity of villus, diameter and muscular thickness of midgut (P<0.05). In a word, both of them have more positive effect on histomorphology of gut than the others. Sodium butyrate

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