【作者】 李艳；

【导师】 计成； 马秋刚；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 本研究首先通过体内与体外试验研究硫辛酸(ALA)对肉鸡骨骼肌发育相关基因表达、肌纤维类型转化和抗氧化能力的影响。另外,利用ALA具有抗氧化和抗炎性的特性,研究ALA缓解肉鸡黄曲霉毒素中毒的效果及机制。试验一研究日粮中添加ALA对肉鸡生产性能、骨骼肌发育相关基因表达、肌纤维类型转化和肌肉抗氧化能力影响。选取80只健康公雏,随机分成2个处理组,在基础日粮上添加ALA(300mg/kg),以饲喂基础日粮为对照组,试验期21天。结果表明：ALA显著降低肉仔鸡平均日增重(ADWG)(P<0.05),对平均日耗量(ADFI)有降低趋势(P=0.0532);日粮中添加ALA显著降低肉仔鸡胸肌绝对重量(P<0.05),对胸腿肌相对重量和腿肌绝对重量没有影响(P>0.05); ALA显著降低肉仔鸡血液中甘油三酯、葡萄糖和甲状腺激素3水平(P<0.05); ALA显著降低胸肌中丙二醛(MDA)水平(P<0.05),显著增加胸腿肌中谷胱甘肽(GSH)、谷胱甘肽还原酶(GR)、Na+-K+-ATP和Ca2+-Mg2+-ATP酶(P<0.05)水平,对腿肌中总超氧化物歧化酶(T-SOD)活性有增加趋势(P=0.0622); ALA显著增加肉仔鸡胸腿肌慢肌(SM)和腿肌快红肌(FRM)肌球蛋白重链基因(MyHC) mRNA相对表达量(P<0.05),并显著降低胸腿肌快白肌(FWM) MyHC基因相对表达量(P<0.05);另外,显著降低胸肌生肌决定因子(MyoD)、生肌因子(Myf5)和肌肉生长抑制素基因表达量(P<0.05),显著降低胸肌MyoD、Myf5、胰岛素样生长因子Ⅰ和1胰岛素样生长因子Ⅰ受体基因表达量(P<0.05)。本试验结果提示：ALA能降低肉鸡体重、胸肌绝对含量和抑制骨骼肌发育,但明显提高肉仔鸡肌肉抗氧化性能,并调控肌纤维类型转化,从而改善肉品质。试验二主要研究ALA对鸡胚骨骼成肌细胞增殖、Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性、骨骼肌增殖与分化相关调节因子基因表达,及MyHC基因相对表达量影响。通过噻唑蓝(MTT)比色法测定发现,与对照组相比,ALA显著降低成肌细胞吸光值,且随着时间推移和浓度增加差异更显著(P<0.05)。通过细胞形态学角度看基本与MTT结果一致,另外还发现1000μmol/LALA组在处理72h后有大量细胞脱落现象发生。细胞周期测定结果可知,ALA浓度达到100μmol/L以上时就已经开始显著增加处于G1/GO期细胞数,且显著降低S期细胞数,使得细胞主要停留G1/G0期。200μmol/L ALA显著降低成肌细胞融合率。相关基因表达方面：500μmol/L ALA对增殖期成肌细胞Pax7基因表达有降低趋势(P=0.0622)；200μmol/L ALA显著增加处理后期MyoD、肌细胞生长素(MyoG)和Myf5基因表达量(P<0.05),而对MyoG基因表达在处理24h是显著降低(P<0.05);随着ALA添加量增加,FWM MyHC基因mRNA相对表达量呈线性降低趋势,FRM和SM MyHC mRNA相对表达量呈线性增加趋势。另外,ALA显著抑制成肌细胞Na+-K+-ATP和Ca2+-Mg2+-ATP两种酶活性(P<0.05)。本试验结果提示：高浓度ALA抑制骨骼成肌细胞增殖,对分化影响也许是先降低后升高,并调控肌纤维类型转化。试验三主要研究ALA对缓解采食被黄曲霉毒素污染花生粕肉鸡机体损伤的机制。选取160只健康公雏,随机分配成4个处理组：对照组(Control)为基础日粮组；硫辛酸组(ALA)：基础日粮中添加300mg/kg ALA;黄曲霉毒素霉变组(AFB1)：用霉变花生粕替代基础日粮中正常花生粕(AFB1检测浓度是(74μg/kg));霉变+硫辛酸组(AFB1+ALA),在霉变日粮中添加300mg/kg ALA,处理时间为3周。结果显示：AFB1没有影响到肉鸡生产性能(P>0.05),而添加ALA后显著降低肉鸡ADWG;AFB1显著降低血浆中白蛋白(ALB)和总蛋白(TP)含量(P<0.05),显著增加血浆中碱性磷酸酶(AKP)活性(P<0.05),添加ALA后增加了血浆中ALB和TP含量,且AKP活性被显著降低(P<0.05); AFB1显著降低肝脏中代谢酶谷草转氨酶(GOT)和谷丙转氨酶(GPT)活性(P<0.05),并对肝脏组织形态学造成一定损伤,添加ALA后显著改善AFB1对肝脏形态学损伤,显著增加GOT活性,对GPT有增加趋势；AFB1显著增加肉鸡肝脏中丙二醛(MDA)和一氧化氮(NO)水平(P<0.05),显著降低肝脏中谷胱甘肽过氧化物酶(GSH-Px)活性(P<0.05),消耗了肝脏中GSH水平(P<0.05),添加ALA后显著抑制这些指标变化,而单独添加ALA后显著降低MDA含量和提高T-SOD、GR、GSH-Px活性与GSH水平(P<0.05)；AFB1显著下调肉鸡肝脏GSH-Px基因表达量,添加ALA后其表达量得到恢复；AFB1显著上调肝脏Ⅰ相代谢酶细胞色素氧化酶(CYP1A1和CYP2H1)基因表达量(P<0.05),下调Ⅱ相酶谷胱甘肽巯基转移酶α(GSTα)基因表达量(P<0.05),对环氧化物水解酶基因表达量无影响,添加ALA后显著下调CYP1A1和CYP2H1基因表达量,对GSTα基因表达量有上调趋势；AFB1显著上调肝脏促炎性因子白介素-6(IL6)表达水平(P<0.05),添加ALA后其表达量得到抑制；AFB1显著上调肉鸡肝脏核因子-κB(NF-κB)和诱导性一氧化氮合酶(iNOS)蛋白表达量(P<0.05),对NF-κB基因表达量无影响,添加ALA后显著抑制NF-κB和iNOS蛋白表达量；AFB1显著上调肉鸡脾脏促炎性因子肿瘤坏死因子α、γ-干扰素和IL6(P<0.05)表达水平,对NF-κB和血红素氧合酶基因表达无影响,添加ALA后促炎性因子基因表达量被抑制。本试验结果提示：ALA虽然降低采食AFB1污染日粮肉鸡生产性能,但能提高肉鸡健康水平,主要体现在缓解AFB1对肝脏功能的损伤,提高肝脏抗氧化能力,改善肝脏生物转化,抑制肝脏和脾脏的炎性反应,以及降低核转录因子NF-κB蛋白表达量。更多还原

【Abstract】 This study was designed to evaluate the effect of alpha lipoic acid (ALA) on skeletal muscle development, transformation of myofiber type and antioxidant ability in vovo and in vitro in broiler chickens. In addition, the protective mechanisms of lipoic acid on aflatoxicosis were investigated in broiler chickens, based on the characteristics of its antioxidant and anti-inflammatory.In Exp.l, the experiment was conducted to study the effects of dietary ALA on the growth performance, the expression of skeletal muscle development-related genes, transformation of myofibre type and antioxidant ability of skeletal muscle in broiler chickens. A total of80male broilers were randomly divided into2groups and assigned different diets:basal diet (Control),300mg/kg ALA supplementation in basal diet, for3weeks. Results showed that LA significantly decreased the average daily weight gain (ADWG), have a decrease tend on the average daily feed intake (ADFI). ALA had no marked change on the relative percentage of breast and thigh muscle, but significantly decreased the breast muscle weight. Triglyceride, glucose, and triiodothyronine were significantly decreased among chickens in the treatment group. In ALA treated broiler chickens, a decrease in the breast muscle malondialdehyde (MDA) level, an increase in the activities of glutathionereductase (GR), Na+-K+-ATPase and Ca2+-Mg2+-ATPase (P<0.05) and a deletion of the glutathione (GSH) in in breast and thigh muscle, while had an increase in total superoxide disumutase (T-SOD) activity in thigh muscle. LA increased the expression of breast muscle slow myofiber (SM)(P<0.05), thigh muscle SM (P <0.05), thigh muscle fast red myofiber (FRM) MyHC mRNA (P<0.05), and decreased the expression of breast muscle fast white myofiber (FWM)(P<0.05), thigh muscle FWM MyHC mRNA (P<0.05) in broilers. ALA administration down-regulated the mRNA expression of myogenic determining factor (MyoD), myogenic factor5(Myf5) and myostatin in breast muscle, and down-regulated the mRNA expression of MyoD, Myf5, insulin-like growth factor I and insulin-like growth factor I receptor in thigh muscle. Results indicated that ALA decreased the growth performance and breast muscle weight, inhibited the expression of skeletal muscle development-related genes, but enhance the antioxidant ability of chicken muscle and regulated the transformation of myofibre type, and then might improve meat quality.In Exp.2, this study was designed to evaluate the effect of lipoic acid on skeletal myoblasts proliferation, the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase, the expression of skeletal muscle cell transcription factors (involved in proliferation and differentiation) and MyHC genes. The results by MTT method showed that, the value of myoblast absorbance in the ALA-treated group was significantly decreased than the control group, especially high doses (P<0.05). Similar with the results by the cell morphology, beside that, we also found that a large number of cells were shed after72h in1000μmol/L ALA treated group. Results of cell cycle showed that ALA (exceed to100umol/L) significantly increased the number of cells in G1/G0phase, and significantly reduced the number of cells in the S phase. The fusion index was inhibited in the200umol/L ALA group as compared to the control group.500μmol/L ALA down-regulated the expression of the myoblast Pax7gene in the proliferation period.200μmol/L ALA up-regulated the expression of MyoD, myogenin (MyoG) and Myf5in the later stages of ALA treatment, however, the expression of MyoG was down-regulated in24h. With the increasing ALA level, significantly increased the expression of skeletal cell myofibre SM, FRM MyHC mRNA, and decreased the relative expression of FWM MyHC mRNA. In addition, ALA inhibited the activities of myoblast Na+-K+-ATP and Ca2+-Mg2+-ATPase. Results suggest that a high dose of ALA can inhibit the myoblast proliferation, maybe have a tendency of decreasing firstly and then increasing with treatment time, and alter the transformation of myofiber type.In Exp.3, LA, an antioxidant and anti-inflammatory compound, is evaluated in this study for its ability to protect the body from Aflatoxin B1(AFB1) caused injury in broiler chickens. A total of160male broilers were randomly divided into4groups and assigned different diets:basal diet,300mg/kg ALA supplementation in basal diet, diet containing74μg/kg AFB1, and300mg/kg ALA supplementation in diet containing74μg/kg AFB1, for3weeks. The results revealed that AFB1(74ppb) had no change on growth performance, however, the addition of300mg/kg ALA to the AFB1diet significantly decreased the ADWG and had a decrease trend on ADFI in broilers. Addition of300mg/kg ALA protected against the liver function damage of broilers induced by chronic low dose of AFB1as estimated by significantly (P<0.05) change in levels of plasma total protein, albumin, alkaline phosphatase and the activities of liver glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase, and histological morphology. After fed diet containing AFB1, MDA (P<0.05) and nitric oxide (NO) levels (P<0.05) increased; the activity of glutathione peroxidase (GSH-Px)(P<0.05) decreased and GSH (P<0.05) content depleted in the chicken liver, while the expression of GSH-Px (P <0.05) mRNA was also down-regulated, but these negative effects were inhibited by the addition of300mg/kg ALA. Meantime, ALA alone can decrease the levels of MDA and NO, increase the activities of T-SOD, GR and GSH-Px and GSH level. AFB1significantly up-regulated the expression of the hepatic CYP1A1and CYP2H1, down-regulated the expression of the hepatic glutathione S-transferase (GSTa), and had no any change on the expression of epoxide hydrolase (P>0.05). However, the supplementation of ALA in AFB1-diet down-regulated the expression of the CYP1A1and CYP2H1, and tended to up-regulate the expression of GSTa gene. The pro-inflammatory factor IL-6(P<0.05) mRNA level increased in the AFB1group as compare to the control group. Additionally, the protein expression of the nuclear factor kappa B (NF-κB) p65and inducible nitric oxide synthase increased significantly (P<0.05) in the AFB1group compared to the corresponding control group. In addition, the increased expressions of IL-6, TNFa and IFNy were observed in birds exposed to the AFB1-contaminated diet. However, these negative effects were inhibited by treatment of ALA. The results suggest that ALA inhibits the performance in broiler chickens exposed to AFB1, but improves birds health, alleviates AFB1induced liver damage, enhance liver antioxidant ability, modulates the liver biotransformation, inhibits the inflammatory response in liver and spleen, and reduce the protein expression of NF-κB.更多还原