Effect of Different Selenium Sources on Antioxidant Capacity and Immunity in Lipopolysaccharide Challenged Broilers

摘要

氧自由基由好氧菌产生并被之降解。大量氧自由基的产生会导致氧化应激。硒是非常重要的谷胱甘肽过氧化物酶(GPX)的调节物。硒可以提高营养物质的生物利用率，从而提高机体的抗氧化力和免疫力。GPX4和碘甲状腺原氨酸脱碘酶(D1)都是重要的含硒蛋白。它们可以被用作肉鸡的硒源生物指示剂。脂多糖(LPS)是一种可以促进机体产生一氧化氮的内毒素。它会降低机体的抗氧化能力从而增加脂质过氧化。LPS还会降低机体重要的T3、T4转化物—D1的活性。此外，LPS增加细胞内促炎细胞因子的产生，从而影响机体的免疫机能。
　　 本文研究主要包括以下方面:(1)不同硒源对4℃储藏12天肉鸡生长性能的影响及其对肉鸡胸肌营养组成、硒含量、抗氧化力和肉质感观的影响;(2)不同浓度NaCl溶液对4℃储藏12天的富含硒的肉鸡胸肌的抗氧化酶的活力、总抗氧化力和脂质过氧化的影响;(3)不同硒源对肉鸡肝脏和血清中的硒含量、抗氧化力、硫代巴比妥酸活性物质(TBARS)含量、肉鸡甲状腺含量和肉鸡肝脏中GPX4 mRNA和D1mRNA含量的影响;(4)不同硒源对LPS刺激后肉鸡对肝脏和血清中的硒含量、抗氧化力、硫代巴比妥酸活性物质(TBARS)含量、肉鸡甲状腺含量和肉鸡肝脏中GPX4 mRNA和D1mRNA含量的影响;(5)日粮中添加硒对LPS诱导的肉鸡体内促炎细胞因子的产生的抑制作用。
　　 1、日粮中不同硒源对肉鸡胸肌中硒含量和胸肌抗氧化力的影响
　　 试验研究肉鸡日粮中添加单一的亚硒酸钠(SS)或酵母硒(SY)以及添加两者的混合物对肉鸡胸肌中硒含量和抗氧化力的影响。试验选用600只体重相近的1日龄肉鸡，随机分为五组，每组6个重复，每个重复20只鸡。五组分别为对照组（饲喂基础日粮）、SS组（基础日粮中添加0.3 mg/kg亚硒酸钠）、SYⅠ组（基础日粮中添加0.2mg/kg酵母硒）、SYⅡ组（基础日粮中添加0.3 mg/kg酵母硒）、MS组(基础日粮中添加亚硒酸钠和酵母硒各0.15 mg/kg)。试验分为前期(1-21 d)和后期(22-42 d)两阶段。结果表明:日粮添加有机硒显著降低了肉鸡前期和全期平均日采食量(ADFI)(P＜0.05)，对平均日增重(ADG)和料重比(FCR)无显著影响（P＞0.05）;试验结果表明SS组与其他组相比，肉鸡胸肌中具有较高的谷胱甘肽过氧化物酶(GSH-Px)活性(P＜0.05);SY和MS组肉鸡42日龄时，胸肌中硒含量、过氧化氢酶(catalase，CAT)活性、总超氧化物歧化酶(T-SOD)活力、总抗氧化力(TAC)显著高于对照组(P＜0.05)，丙二醛(MDA)含量显著低于对照组(P＜0.05)。4℃冷藏12天后，GSH-Px活性显著下降(P＜0.05)，CAT、T-SOD和TAC保持稳定。SY组肉鸡胸肌中脂质过氧化程度明显低于SS组。酵母硒组和混合硒组与亚硒酸钠组相比有效提高肌肉的抗氧化力。
　　 2、不同浓度NaCl溶液对富含硒的肉鸡胸肌抗氧化力的影响
　　 试验研究不同浓度NaCl溶液对4℃储藏12天的富含硒的肉鸡胸肌的抗氧化酶的活力、总抗氧化力和脂质过氧化的影响。试验动物选择和分组同试验1。SY-Ⅱ组和MS组肉鸡胸肌pH24h显著升高(P＜0.05)，MS组肉鸡胸肌的滴水损失显著降低。SY组和MS组42日龄肉鸡胸肌的烹饪损失有所降低。SY-Ⅱ组和MS组肉鸡胸肌的亮度值下降，而MS组上升。NaCl显著提高MDA含量。4℃储藏12天后，胸肌的硫代巴比妥酸活性物质(TBARS)含量显著升高(P＜0.05)，GSH-Px含量显著下降(P＜0.05)，CAT、T-SOD和TAC含量差异不显著。胸肌中GSH-Px的活性与NaCl溶液浓度相关。结果表明，LO和GSH-Px呈负相关;采用NaCl处理后，4℃储藏12天肉鸡的胸肌中脂质过氧化增加。
　　 3、不同硒源对肉鸡肝脏和血清中的硒含量、抗氧化力、TBARS含量、肉鸡甲状腺含量和肉鸡肝脏中GPX4 mRNA和D1mRNA含量的影响
　　 试验研究不同硒源对肉鸡肝脏和血清中的硒含量、抗氧化力、TBARS含量、肉鸡甲状腺含量和肉鸡肝脏中GPX4mRNA和D1mRNA含量的影响。试验设计同试验1。试验发现，添加硒后显著增加了21日龄和42日龄肉鸡血清和肝脏中硒、AEA、TAC的含量(P＜0.05)，显著降低了MDA含量(P＜0.05)。酵母硒显著提高了42日龄肉鸡血清中T3的含量(P＜0.05)。较高水平的硒的添加显著提高肉鸡肝脏中D1mRNA的含量(P＜0.05)。MS组GPX4 mRNA含量显著高于其他各组(P＜0.05)。42日龄肉鸡所需使其肝脏中D1mRNA最大表达的硒含量低于使肉鸡肝脏中GPx4mRNA最大表达的硒含量。
　　 4、不同硒源对LPS刺激后肉鸡对肝脏和血清中的硒含量、抗氧化力、TBARS含量、肉鸡甲状腺含量和肉鸡肝脏中GPX4 mRNA和D1 mRNA含量的影响
　　 给肉鸡注射LPS后，其体内免疫反应被激活导致生长性能下降、抗氧化机能下降。酵母硒有更高的生物学效价，因此其可以肉鸡的抗氧化力。因此本试验目的为研究酵母硒对LPS刺激肉鸡的保护作用。试验选用288只1日龄肉鸡，分为3组，每组6个重复，每个重复16只鸡，三组分别为对照组（饲喂基础日粮）、SS组（基础日粮中添加0.3 mg/kg亚硒酸钠）和SY组（基础日粮中添加0.3 mg/kg酵母硒）。在肉鸡16日龄、18日龄和20龄时，每组肉鸡中的一半注射0.9％NaCl，另一半注射LPS(250mg/kg body weight)。试验结果用来验证有机硒对21日龄肉鸡LPS刺激的缓解作用。结果表明，LPS组肉鸡血清丙氨酸氨基转移酶和天冬氨酸氨基转移酶活性显著提高(P＜0.05)。试验研究还发现LPS组肉鸡血清和肝脏中MDA含量显著下降(P＜0.05)，GSH-Px、CAT、T-SOD和T-AOC含量显著下降(P＜0.05)。注射LPS后，肉鸡血清T3、T4和TSH含量显著下降(P＜0.05)。但是，添加硒后可以降低LPS注射组肉鸡的MDA含量，提高GSH-Px、CAT、T-SOD和T-AOC含量，从而缓解LPS的氧化应激。注射LPS后对肉鸡的GPX4mRNA和D1mRNA含量没有显著影响。在肉鸡日粮中添加有机硒和酵母硒可能对于LPS刺激的21日龄肉的氧化应激具有一定的缓解作用。
　　 5、日粮中添加硒对LPS诱导的肉鸡体内促炎细胞因子的产生的抑制作用
　　 LPS会在体内产生氧自由基和一氧化氮。这些分子可以引起动物机体的炎症反应。硒作为一种生物必需微量元素，具有调节细胞生长和成活信号通路上信号转导蛋白的作用。为了研究不同硒源添加对LPS刺激后肉鸡免疫力的影响，试验选用288只1日龄肉鸡，随机分为三组，饲喂大豆玉米日粮，试验期为21天。三组分别为对照组（饲喂基础日粮加011mg/kg硒），SS组（饲喂基础日粮加0.3mg/kg亚硒酸钠），SY组（饲喂基础日粮加0.3mg/kg酵母硒）。在肉鸡16日龄、18日龄和20龄时，每组肉鸡中的一半注射0.9％NaCl，另一半注射LPS(250mg/kg body weight)。结果表明，SY组与其他组相比，肉鸡的肝脏的病原学变化有所减轻(P＜0.05)，且血清PGE2分泌量减低。SS组和SY组肉鸡血清和肝脏的IFN-γ、IL-1β、IL-2和IL-8含量显著提高(P＜0.05)。此外，SS组和SY组肉鸡的一氧化氮合酶含量和磷酸激酶含量显著下降(P＜0.05)。总之，有机硒和无机硒都可以缓解LPS诱导的肉鸡的炎症反应，且有机硒的效果更好。
　　 结论
　　 试验结果表明在肉鸡日粮中添加有机硒可以提高肉鸡器官重硒的含量和其抗氧化力，并且提高LPS致激后肉鸡的免疫力。降低NaCl的浓度可能对于提高4℃冷藏12天肉鸡胸肌的抗氧化力具有较好的作用。

目录

声明

Contents

ABSTRACT

摘要

List of Abbreviations

Introduction

Research Goals

References

Chapter 1 Literature Review

1．1．Meat quality

1．1．1．Color

1．1．2．Breast meat of broilers

1．1．3．Water holding capacity

1．1．4．Sodium chloride

1．1．5．Concerns surrounding sodium chloride use in the meat industry

1．1．6．Thiobarbituric acid reactive substances(TBARS) and sensory characteristics

1．1．7．Sodium chloride as a pro-oxidant

1．1．8．Lipid oxidation and storage

1．2．Selenium

1．2．1．Chemistry of selenium

1．2．2．Bioaccumulation of Se

1．2．3．Selenium distribution in tissues

1．2．4．Inorganic and organic sources of selenium

1．2．5．Selenium bioavailability，absorption and metabolism

1．3．Selenoproteins

1．3．1．Glutathione

1．3．2．Glutathione peroxidases

1．3．3．Glutathione peroxidase 4

1．3．4．Other selenoproteins

1．4．Selenium deficiency in poultry

1．5．Application of selenium in poultry

1．6．Selenium in ruminants

1．7．Reactive oxygen species

1．8．Copper，zinc superoxide dismutases

1．9．Alanine aminotransferase

1．10．Selenium and growth performance

1．11．Iodothyronine deiodinases

1．12．Immunology

1．12．1．Selenium and immune system

Referenees

Chapter 2 Effects of Dietary Sodium Selenite and Selenium Yeast on Antioxidant Enzyme Activities and Oxidative Stability of Broiler Breast Meat

2．1．Introduction

2．2．Material and methods

2．2．1．Materials

2．2．2．Husbandry

2．2．3．Sample collection and preparation

2．2．4．Storage procedure of broilers breast meat

2．2．5．Determination of selenium content of feed and broilers breast meat

2．2．6．Measurements of antioxidant enzyme activities，total antioxidant capacity and thiobarbituric acid reactive substances of raw broiler breast meat

2．2．7．Determination of glutathione peroxidase(GSH-Px)activity in broiler breast meat

2．2．8．Determination of catalase(CAT)activity in broiler breast meat

2．2．9．Determination of total superoxide dismutase(T-SOD)activity in broiler breast meat

2．2．10．Determination of total antioxidant capacity(TAC)in broiler breast meat

2．2．11．Determiantion of maleicdialdehyde(MDA) in broiler breast meat

2．2．12．Sensory evaluation of broiler breast meat

2．2．13．Statistical analysis

2．3．Results

2．3．1．Growth performance of broilers

2．3．2．Influence of Se supplementation on nutritive values of broiler breast meat

2．3．3．Selenium content of broiler breast meat

2．3．4．Influence of selenium supplementation on GSH-Px activity in broiler breast meat

2．3．5．Influence of Selenium supplementation on CAT and T-SOD activities in broiler breast meat

2．3．6．Influence of Se supplementation on TAC in broiler breast meat

2．3．7．Influence of Se supplementation on lipid oxidation in broiler breast meat

2．3．8．Influence of Se supplementation on oxidative stability in refrigerated broiler breast meat

2．3．9 Effect of different Se sources on the sensory characteristics of broiler breast meat

2．4．Discussion

2．4．1．Growth performance of broilers

2．4．2．Influence of Se on nutritive values of broiler breast meat

2．4．3．Selenium content in broiler breast meat

2．4．4．Influence of Selenium supplementation on GSH-Px activity in broiler breast meat

2．4．5．Influence of Se supplementation on CAT and T-SOD activities in broiler breast meat

2．4．6．Influence of Se supplementation on TAC in broiler breast meat

2．4．7．Influence of Se supplementation on lipid peroxidation in broiler breast meat

2．4．8．Influence of Se supplementation on oxidative stability in refrigerated broiler breast meat

2．4．9．Effect of dietary Se sources on the sensory characteristics of broiler breast meat

References

Chapter 3 Effects of sodium chloride on Antioxidant Enzyme Activities and Oxidative Stability of Selenium Enriched Broiler Breast Meat

3．1．Introduction

3．2．Materials and methods

3．2．1．Broilers，diets and feeding protocol

3．2．2．Collection of broiler breast meat samples

3．2．3．pH and color measurement of broiler breast meat

3．2．4．Drip loss of broiler breast meat

3．2．5．Cooking loss and shear force of broiler breast meat

3．2．6．Preparation of broiler breast meat samples

3．2．7．Determination of antioxidant enzyme activities，total antioxidant capacity and thiobarbituric acid reactive substances of selenium enriched breast meat of broilers

3．2．8．Statistical Analysis

3．3．Resuits

3．3．1．Meat quality of broiler breast meat

3．3．2．Effect of difierent concentrations of sodium chloride on antioxidant enzyme actvities，antioxidant capacity and TBARS of Se enriched broiler breast meat

3．4．Discussion

3．4．1．Meat quality of broiler breast meat

3．4．2．Effect of different concentrations of sodium chloride on antioxidant enzyme activities in broiler breast meat

3．4．3．Effect of different concentrations of sodium chloride on antioxidant enzymes stability in refrigerated broiler breast meat

3．4．4．Effect of sodium chloride and ionic strength on antioxidant enzymes in broiler breast meat

Reference

Chapter 4 Effect of Different Selenium Sources on Antioxidant Enzyme Activities，Total Antioxidant Capacity，MDA Content，Thyroid Hormones，Expressions of Hepatic mRNA GPX4 and D1 in Broilers

4．1．Introduction

4．2．Materials and methods

4．2．1．Broilers，diets and design

4．2．2．Determination of selenium content of serum and liver in broilers

4．2．3．Determination of serum and liver antioxidant enzyme activities，total antioxidant capacity and malondialdehyde contents in broilers

4．2．4．Determination of thyroid hormones after supplementation of different Se sources in broilers

4．2．5．Real time polymerase chain reaction

4．2．6．RNA extraction and real-time PCR

4．2．7．Statistical analysis

4．3．Results

4．3．1．Selenium content of serum and liver in broilers

4．3．2．Effect of selenium supplementation OR GSH-Px activity of serum and liver in broilers

4．3．3．Effect of Se supplementation on CAT and T-SOD activities in serum and liver of broilers

4．3．4．Effect of Se supplementation on TAC in serum and liver of broilers

4．3．5．Effect of Se supplementation on lipid peroxidation in serum and liver of broilers

4．3．6．Effect of selenium supplementation on serum T3，T4，and TSH in broilers

4．3．7．Effect of selenium supplementation on hepatic mRNA GPX4 expression in broilers

4．4．Discussion

4．4．1．Selenium content in serum and liver of broilers

4．4．2．Effect of selenium supplementation on serum and liver GSH-Px activity in broilers

4．4．3．Effect of Se supplementation on serum and liver CAT and T-SOD activities in broilers

4．4．4．Efiect of Se supplementation on serum and liver TAC in broilers

4．4．5．Effect of Se supplementation on serum and liver lipid oxidation in broilers

4．4．6．Effect of Se supplementation on serum T3，T4，and TSH in broilers

4．4．7．Effect of Se supplementation on broiler hepatic GPx4 mRNA expression

4．4．8．Effect of selenium supplementation on broilers hepatic type Ⅰ deiodinase mRNA expression

References

Chapter 5 Effect of Selenium Supplementation on Expression of Hepatic GPX4 and D1 mRNA in LPS Challenged Broilers

5．1．Introduction

5．2．Materials and methods

5．2．1．Animals and experimental design

5．2．2．Experimental protocols

5．2．3．Collection of samples

5．2．4．Growth performance of LPS challenged broilers

5．2．5．Determination of Se content of serum and liver of LPS challenged broilers

5．2．6．Determination of serum and liver antioxidant enzyme activities，total antioxidant capacity and malondialdehyde contents of LPS challenged broilers

5．2．7．Determination of thyroid hormones

5．2．8．Determination of serum ALT and AST enzymes activities

5．2．9．Hepatic GPX4 and DI mRNA expression of LPS challenged broilers

5．2．10．Statistical analysis

5．3．Results

5．3．1．Growth performance of LPS challenged broilers

5．3．2．Serum and liver Se content of LPS challenged broilers

5．3．3．Effect of different Se sources on serum and liver antioxidant enzyme activities，total antioxidant capacity and MDA content of LPS challenged broilers

5．3．4．Effect of dietary Se sources on serum ALT and AST enzymes activities of LPS challenged broilers

5．3．5．Effect dietary Se sources on serum T3，T4 and TSH hormones of LPS challenged broilers

5．3．6．Hepatic GPX4 mRNA expression of LPS challenged broilers

5．3．7．Hepatic type I deiodinase mRNA expression of LPS challenged broilers

5．4．Discussion

5．4．1．Growth performance of LPS challenged broilers

5．4．2．Se content of serum and liver of LPS challenged broilers

5．4．3．Effect on antioxidant enzyme activities，total antioxidant capacity and MDA content of serum and liver of LPS challenged broilers

5．4．4．Effect on serum ALT and AST enzymes activities of LPS challenged broilers

5．4．5．Effect on serum T3，T4 and TSH hormones of LPS challenged broilers

5．4．6．Hepatic GPX4 mRNA expression of LPS challenged broilers

5．4．7．Hepatic type I deiodinase mRNA expression of LPS challenged broilers

References

Chapter 6 Dietary Selenium Yeast Attenuates Lipopolysaccharide Induced Inflammatory Cytokines Secretion in Broilers

6．1．Introduction

6．2．Materials and methods

6．2．1．Animals and management

6．2．2 Sample collection and procedures

6．2．3．Histological examination for liver tissues of LPS challenged broilers

6．2．4．Preparation of hepatic tissue homogenization

6．2．5．Determination of reactive oxygen species in hepatocytes

6．2．6．Cytokines and PGE2 assay

6．2．7．Hepatic protein kinase C activity

6．2．8．Determination of serum and hepatic nitric oxide

6．2．9．Statistical analysis

6．3．Results

6．3．1．Histology examination of liver

6．3．2．Effect of different Se Sources on plasma and liver cytokines in LPS challeneged broilers

6．3．3．Effect of dietary SS and SY on protein kinase C activity and reactive oxygen species content of LPS challenged broilers

6．4．Discussion

References

Summary and Conclusions

Future Recommendations

Aknowledgement

List of Published or Submitted Papers

Oral presentations