过瘤胃胆碱对肉羊脂肪代谢和肌内脂肪形成的调控作用及分子机制

发布时间：2018-06-20 21:53

本文选题：过瘤胃胆碱 + 肉羊　；参考：《扬州大学》2017年博士论文

【摘要】：肌内脂肪含量是评定羊肉品质的关键指标,探索肌内脂肪营养调控技术和沉积机制成为目前生产和科研关注的重点。肌内脂肪沉积,是肉羊生长、体脂分配、血液脂质代谢、脂肪酸组成、脂肪代谢关键基因及转录调控因子等综合作用的结果。胆碱在调控单胃动物脂肪分配方面发挥重要作用,但是其对肉羊生长及各部位脂肪组织中脂肪酸代谢变化的调控作用及机制缺乏研究。本研究采用过瘤胃胆碱(Rumen Protected choline,RPC)作为营养调控剂,借助动物饲养试验、代谢组学、分子生物学技术以及生物信息学分析技术,研究胆碱对肉羊脂肪代谢和肌内脂肪形成的调控作用及分子机制。全文分为4个部分进行。试验一、RPC对肉羊生长性能及体脂肪分配的影响试验选取3-5月龄和10-12月龄杜泊羊X湖羊杂交F1代公羔开展两期试验,每期试验选用24只,随机分成4组(6头/组,组间体重差异不显著(P0.05)),分别为对照组、0.25%RPC、0.50%RPC和0.75%RPC添加组,每期试验60d,试验结果如下:3-5月龄肉羊阶段,添加RPC有提高肉羊采食量、日增重、料肉比的作用(P0.05)。RPC对3-5月龄肉羊血液甘油三酯、总胆固醇、HDL和LDL均无显著影响(P0.05)。RPC有降低3-5月龄肉羊血液FFA含量的作用(P0.01),对血清皮质醇、瘦素、脂联素和胰岛素无显著影响(P0.05)。RPC对屠宰率、眼肌面积、肋肉厚度、皮下脂肪厚度无显著影响(P0.05),试验组腹脂率、肾脂率和盆脂率较对照组有所降低,但并不显著(P0.05)。添加RPC有提高肾、脾脏、胃、小肠重量的作用,但差异并不显著(P0.05);对背最长肌脂肪含量无显著影响(P0.05);对背最长肌、前腿肌pH具有显著的升高作用(P0.05)。RPC降低3-5月龄肉羊前腿肌、后腿肌失水率(P0.05),对背最长肌失水率调控效果不显著(P0.05);RPC有提高背最长肌、前腿肌、后腿肌嫩度的潜在作用,但差异并不显著(P0.05)。10-12月龄肉羊阶段,添加RPC对肉羊采食量、日增重、饲料利用率无显著影响(P0.05)。RPC对10-12月龄肉羊血液甘油三酯、胆固醇、HDL、LDL含量无显著影响(P0.05)。RPC对10-12月龄肉羊血液FAA有先升高后降低作用,但差异并不显著(P0.05),RPC显著降低胰岛素水平(P0.05);对抵抗素、脂联素有趋势性降低效果,但差异不显著(P0.05)。RPC对10-12月龄肉羊的屠宰率无显著影响(P0.05),降低腹脂率、肾脂率和盆脂率,对眼肌面积和皮下脂肪厚度有提高作用,增加肝、肾、胃和大肠重量,但差异均不显著(P0.05);显著增加脾脏和小肠重量(P0.05)。RPC对10-12月龄肉羊背最长肌脂肪含量略有降低,但差异并不显著(P=0.73),对调节背最长肌、前腿肌和后腿肌pH值无显著影响(P0.05),降低背最长肌、前腿肌和后腿肌失水率、剪切力,但差异并不显著(P0.05);对肉色调控无显著效果(P0.05)。RPC能够提高10-12月龄肉羊皮下脂肪和背最长肌中PUFA含量,但差异并不显著(P0.05),提高n-3、n-6脂肪酸含量,降低背最长肌中SFA含量,但均无显著影响(P0.05),RPC对背最长肌中C18:0、C18:1trans、C18:1cis、C18:2cis含量略有提升,但差异并不显著(P0.05)。添加RPC能够降低10-12月龄肉羊皮下脂肪、腹脂、肾周脂肪和盆腔脂肪组织的细胞直径(P0.01),对各部位脂肪细胞降脂调控效果依次是肾脂腹脂盆腔脂肪皮下脂肪,细胞直径分别较对照组降低33.52%、18.23%、17.47%、5.10%。试验二、RPC对肉羊尿液代谢组的影响试验选用体重相近的杜泊羊X湖羊杂交F1代公羔36头,随机分成3组,每组12头,组间体重差异不显著(P0.05)。在基础日粮中分别添加0%、0.25%和0.75%RPC水平,饲养管理相同,预饲期10天,正试期5天,在第3-5天收集试验羊尿样,测定不同RPC添加水平对肉羊尿样代谢组的影响,分别采用PCA、PLS-DA和OPLS-DA分析。结果表明:0.25%和0.75%RPC添加水平显著改变肉羊的尿样代谢组,差异代谢物主要有丙酮酸、氧化三甲胺、对甲酚、苯乙酰甘氨酸和马尿酸。RPC添加组较对照组尿液中丙酮酸含量减少,尿样中氧化三甲胺、对甲酚、苯乙酰甘氨酸和马尿酸含量有所提高。这些差异代谢物参与的生物反应过程主要有能量代谢、脂肪、蛋白质和AA代谢以及肠道微生物代谢。RPC能够改变肉羊机体脂肪和能量代谢,主要相关代谢物是丙酮酸和PAG。试验三、RPC对肉羊脂肪代谢关键基因表达的影响3-5月龄肉羊采集对照组、0.25%RPC和0.75%RPC试验组样品,10-12月龄肉羊采集对照组和0.25%RPC试验组样品,测定各组织样品中脂肪代谢关键基因mRNA相对表达量。结果发现:对于3-5月龄肉羊,RPC促进背最长肌中LPL、CD36、ACC、FASN、AGPAT1和AC ADVL mRNA 表达(P0.05),对 DGAT1、DGAT2、ATGL、HSL、MGLL 和 CPT-1βmRNA 表达无显著影响(P0.05)。RPC通过促进脂肪酸摄入、合成、酯化和氧化关键基因表达,,促进肉羊背最长肌脂肪酸沉积。RPC抑制3-5月龄肉羊盆腔脂肪中LPL、FASN和AGPAT1mRNA表达(P0.05),促进 CD36、GPAT1 和 CPT-1βmRNA 表达(P0.05),对 DGAT1、DGAT2、ATGL、HSL、MGLL和ACADVLmRNA表达无显著影响(P0.05)。RPC主要通过降低3-5月龄肉羊盆脂脂肪酸摄入、合成和酯化关键基因mRNA表达,加强脂肪酸氧化关键基因表达,整体降低肉羊盆脂沉积。RPC降低3-5月龄肉羊肾周脂肪中的LPL、FASN、GPAT1、AGPAT1mRNA表达(P0.01),提高脂肪酸氧化关键基因CPT-1β和ACADVLmRNA表达(P0.01),对CD36、ACC、DGAT1、DGAT2、ATGL、HSL 和 MGLLmRNA 表达无显著影响(P0.05)。RPC主要通过降低脂肪酸摄入、合成和酯化关键基因表达,加强脂肪酸氧化关键基因表达,降低肾周脂肪沉积。RPC降低3-5月龄肉羊皮下脂肪中CD36、ACC、GPAT1和ACADVL mRNA表达(P0.05),提高 LPL、FASN、AGPAT1、DGAT1、ATGL 和 CPT-1βmRNA 表达(P0.05),RPC通过降低摄入、合成、酯化和氧化关键基因表达,促进脂肪酸水解关键基因表达,整体降低皮下脂肪沉积。RPC 降低 3-5 月龄肉羊腹脂 CD36、ACC、FASN、GPAT1、DGAT2 和 ACADVLmRNA表达(P0.05),促进 LPL、AGPAT1、DGAT1 和 CPT-1β mRNA 表达(P0.01),RPC通过降低脂肪酸摄入、合成、酯化和氧化关键基因表达,整体降低腹脂沉积。对于10-12月龄肉羊,RPC降低0.25%RPC试验组肉羊背最长肌LPL和CD36 mRNA表达(P0.01),促进 HSL、MGLL 和 CPT-1β的 mRNA 表达(P0.01),对 ACC、FASN、GPAT1、AGPAT1、DGAT1、DGAT2、ATGL 和 ACADVLmRNA 表达均无显著影响(P0.05)。RPC通过降低脂肪酸摄入关键基因表达,加强脂肪酸水解和氧化关键基因表达,整体降低10-12月龄肉羊背最长肌脂肪沉积。RPC抑制10-12月龄肉羊盆腔脂肪中LPL、ACC和FASNmRNA表达(P0.01),促进 CD36、GPAT1、AGPAT1、ATGL 和 ACADVL mRNA 表达(P0.05),对 ATGL、DGAT1、DGAT2、HSL和CPT-1β mRNA表达无显著影响(P0.05),RPC通过降低中脂肪酸摄入、合成关键基因表达,促进酯化、水解和氧化关键基因的表达,整体降低10-12月龄肉羊盆腔脂肪沉积。RPC 降低 10-12 月龄肉羊肾周脂肪中 LPL、ACC、FASN、GPAT1、AGPAT1、DGAT2、ATGL 和 ACADVLmRNA 表达(P0.05),促进 MGLLmRNA 表达(P0.05),RPC 通过降低脂肪酸摄入、合成、酯化和水解关键基因表达,整体降低肾周脂肪沉积。RPC降低10-12月龄肉羊皮下脂肪组织CD36、GPAT1和ACADVL mRNA表达水平(P0.05),促进FASN和ATGLmRNA的表达(P0.05)。RPC通过降低脂肪酸摄入、合成、酯化和氧化关键基因表达,加强脂肪酸水解关键基因表达,整体降低10-12月龄肉羊皮下脂肪沉积。RPC 降低 10-12 月龄肉羊腹脂 CD36、ACC、FASN、GPAT1、DGAT2、ATGL 和 ACADVL mRNA表达(P0.05),增加 LPL、DGAT1 和 MGLLmRNA 表达水平(P0.05)。RPC通过降低脂肪酸摄入、合成、酯化、水解和氧化关键基因表达,整体降低10-12月龄肉羊腹脂沉积。试验四、RPC对肉羊肌内脂肪沉积调控的分子机制添加RPC对3-5月龄肉羊背最长肌中LXRα、LXRβ mRNA表达无显著影响(P0.05),对PPARα转录有上调作用,但差异并不显著(P0.05),说明RPC对3-5月龄肉羊肌内脂肪沉积的调控机制主要通过维持LXRα和促进PPARα mRNA表达,进而上调脂肪代谢关键基因,促进肉羊背最长肌脂肪沉积的效果。添加RPC对10-12月龄肉羊背最长肌中LXRα和PPARα mRNA表达均产生抑制效果,整体降低脂肪沉积。
[Abstract]:Intramuscular fat content is a key index to assess the quality of mutton, explore the intramuscular fat deposition mechanism and nutrition control technology become the focus of production and scientific research. Intramuscular fat deposition, is the growth of sheep, body fat distribution, blood lipid metabolism, fatty acid composition, the comprehensive effect of fat metabolism key genes and transcription factors. The knot Fruit. Choline plays an important role in the regulation of monogastric animal fat distribution, but the change of fat tissue and the growth of sheep in different parts of acid metabolism regulation and mechanisms of the lack of research. This research adopts the Rumen Protected Choline (Rumen Protected, choline, RPC) as the nutrition regulation agent, by means of animal experiment, metabonomics learning points Molecular biology technology and bioinformatics analysis technology, the research on the regulation of cholinergic function and molecular mechanism of the formation of fat and intramuscular fat metabolism in sheep. The text is divided into 4 parts. The first experiment, experiment on the effect of RPC on growth performance and body fat distribution on sheep were selected from 3-5 month old and 10-12 month old Dorper sheep sheep hybrid X F1 generation male lamb open Show two experiments, each experiment 24 rats were randomly divided into 4 groups (6 / group, difference in weight between the groups was not significant (P0.05), respectively) as the control group, 0.25%RPC, 0.50%RPC and 0.75%RPC are added in each experiment group, 60d test results are as follows: 3-5 month old sheep stage, adding RPC to improve sheep production food intake, daily gain, feed gain ratio (P0.05) of.RPC for 3-5 months The age of sheep blood triglyceride, total cholesterol, there were no significant effects of HDL and LDL (P0.05).RPC can decrease the sheep blood content of FFA at the age of 3-5 months (P0.01), the effect of leptin on serum cortisol, and had no significant effect on adiponectin and insulin (P0.05).RPC on the slaughter rate, eye muscle area, rib thickness, no significant effect the subcutaneous fat thickness (P0.05), test group The rate of abdominal fat and kidney fat and fat basin rate than the control group decreased, but not significant (P0.05). Adding RPC improve the kidney, spleen, stomach, small intestine weight, but the difference was not significant (P0.05); no significant effect on longissimus muscle fat content (P0.05) on the back; long muscle, leg muscle pH significantly increased the role of (P0.05).RPC decreased 3-5 month old sheep Leg muscle, leg muscle water loss rate (P0.05), there was no significant difference on the longissimus muscle water loss rate control effect (P0.05); RPC enhanced the longissimus muscle, leg muscle, leg muscle potential tenderness, but the difference was not significant (P0.05).10-12 month old sheep stage, adding RPC on nutrient intake the daily gain, feed utilization rate has no significant effect on.RPC (P0.05) 10-12 months The age of sheep blood triglyceride, cholesterol, HDL, had no significant effect on the content of LDL (P0.05).RPC has the effect of lowering the increased after the first of 10-12 months old sheep blood FAA, but the difference was not significant (P0.05), RPC (P0.05) significantly reduced insulin levels of resistin, adiponectin; a trend of decreasing effect, but the difference was not significant. (P0.05).RPC on the 10-12 month old sheep The slaughter rate had no significant effect (P0.05), decrease the rate of abdominal fat and kidney fat and fat percentage of the basin, eye muscle area and subcutaneous fat thickness have improved, increase the liver, kidney, stomach and intestine weight, but the difference was not significant (P0.05); spleen and intestinal weight increased slightly (P0.05).RPC to reduce the 10-12 month old sheep longissimus muscle fat content, but the difference Is not significant (P=0.73), on the regulation of longissimus muscle, leg muscle and leg muscle pH value had no significant effect (P0.05), reduce the longissimus muscle, leg muscle and leg muscle water loss rate, shear stress, but the difference was not significant (P0.05); no significant effect on color control (P0.05) to.RPC to improve the PUFA content of meat sheep fat and longissimus muscle in 10-12 months, but the poor Difference is not significant (P0.05), n-3, n-6 fatty acid content, reduce the content of SFA in longissimus dorsi muscle, but had no significant effect (P0.05), RPC of longissimus dorsi muscle C18:0, C18:1trans, C18:1cis, C18:2cis content increased slightly, but the difference was not significant (P0.05). The addition of RPC can be reduced by 10-12 month old meat sheep fat, abdominal fat, perirenal fat and pelvic fat Fatty tissue cell diameter (P0.01), each part of the lipid regulation effect of fat cells are kidney fat and abdominal fat pelvic fat subcutaneous fat cell diameter were lower than the control group 33.52%, 18.23%, 17.47%, two 5.10%. test, the effect of RPC on sheep urine metabolic tests with similar weight Dorper sheep sheep hybrid X F1 generation of male lambs and 36 heads, with The machine is divided into 3 groups, 12 pigs in each group, differences in weight between the groups was not significant (P0.05). The basal diet was supplemented with 0%, 0.25% and 0.75%RPC, the same feeding management, pre feeding period of 10 days, during the period of 5 days, 3-5 days in the collection of the sheep urine, affected by different RPC levels the sheep urine group, respectively PCA, PLS-DA and OPLS-DA analysis. The results showed that: 0.25% and 0.75%RPC levels changed significantly in sheep urine group, there are differences in the metabolite pyruvic acid, trimethylamine, oxidation of p-cresol, phenylacetylglycine and hippuric acid added in.RPC group decreased compared with the control group of pyruvic acid content in urine, urine in three oxidation of methylamine, p-cresol, phenylacetylglycine and hippuric acid content The biological process improved. These different metabolites mainly involved in energy metabolism, fat, protein and AA metabolism and intestinal microbial metabolism of.RPC can change the sheep body fat and energy metabolism, the main metabolites of pyruvate and PAG. test is three, effect of RPC on the expression of key genes in lipid metabolism in sheep meat 3-5 months The sheep acquisition control group, 0.25%RPC group and 0.75%RPC test samples, 10-12 month old sheep acquisition control group and test group 0.25%RPC samples, determination of key genes in lipid metabolism of mRNA the relative expression of tissue samples. The results showed that: for the 3-5 month old sheep, promoting RPC in longissimus dorsi muscle LPL, CD36, ACC, FASN, expression AGPAT1 and AC ADVL mRNA (P0.05), D GAT1, DGAT2, ATGL, HSL, MGLL and CPT-1 beta mRNA expression had no significant effect (P0.05).RPC by promoting fatty acid intake, synthesis, expression, esterification and oxidation of key genes, promote the sheep longissimus muscle fatty acid deposition.RPC inhibited the sheep pelvic fat LPL 3-5 months of age, the expression of FASN and AGPAT1mRNA (P0.05) CD36, promote the expression of GPAT1 and CPT-1, beta mRNA (P 0.05), DGAT1, DGAT2, ATGL, HSL, MGLL and ACADVLmRNA expression had no significant effect (P0.05.RPC) mainly by reducing the 3-5 month old sheep pot of fatty acid intake, expression of mRNA key gene synthesis and esterification of fatty acid oxidation, strengthen the expression of key genes, reduce the whole sheep fat deposition in lower.RPC basin sheep perirenal fat in the 3-5 month old LPL, FASN, GPAT 1, the expression of AGPAT1mRNA (P0.01), increased expression of fatty acid oxidation of key gene CPT-1 beta and ACADVLmRNA (P0.01), CD36, ACC, DGAT1, DGAT2, ATGL, HSL and MGLLmRNA expression had no significant effect (P0.05.RPC) mainly by reducing the intake of fatty acid synthesis and esterification, the expression of key genes, enhance the expression of fatty acid oxidation key genes, reduce the perirenal fat To reduce the fat deposition of.RPC CD36, 3-5 month old meat sheep ACC, expression of GPAT1 and ACADVL mRNA (P0.05), FASN, AGPAT1, LPL, DGAT1, expression of ATGL and CPT-1 beta mRNA (P0.05) RPC, by reducing the intake, synthesis, esterification and oxidation of key genes expression, promote the expression of key genes in fatty acid hydrolysis overall, reduced subcutaneous fat deposition of.RPC reduced 3- 5 month old sheep abdominal fat CD36, ACC, FASN, GPAT1, expression of DGAT2 and ACADVLmRNA (P0.05), LPL AGPAT1, promote the expression of DGAT1, CPT-1 and beta mRNA (P0.01) RPC, by reducing the intake of fatty acid synthesis, esterification and oxidation, the expression of key genes, the overall lower abdominal fat deposition. For the 10-12 month old sheep, RPC to reduce the 0.25%RPC test group of sheep longissimus muscle LPL The expression of CD36 and mRNA (P0.01), promote the expression of MGLL and HSL, CPT-1 beta mRNA (P0.01), ACC, FASN, GPAT1, AGPAT1, DGAT1, DGAT2, ATGL and ACADVLmRNA expression had no significant effect (P0.05) by reducing.RPC fatty acid intake key gene expression, enhance the expression of fatty acid hydrolysis and oxidation key gene, reduce the whole sheep back 10-12 months most Long muscle fat deposition in LPL sheep.RPC inhibited the pelvic fat at the age of 10-12 months, the expression of ACC and FASNmRNA (P0.01), GPAT1, AGPAT1, promoting CD36, expression of ATGL and ACADVL mRNA (P0.05), ATGL, DGAT1, DGAT2, HSL and CPT-1 beta mRNA expression had no significant effect (P0.05, RPC) by reducing fat acid intake, expression, synthesis of key genes promoting esterification, hydrolysis The expression of key genes and oxidation, the overall decrease of 10-12 month old sheep pelvic fat deposition decreased.RPC sheep kidney fat LPL, 10-12 month old ACC, FASN, GPAT1, AGPAT1, DGAT2, expression of ATGL and ACADVLmRNA (P0.05), promote the expression of MGLLmRNA (P0.05), RPC by lowering lipid fatty acid intake, synthesis, expression the esterification and hydrolysis of key genes, overall Reduce the perirenal fat deposition of.RPC reduced CD36 in adipose tissue of meat sheep under 10-12 months of age, GPAT1 and ACADVL expression level of mRNA (P0.05), promote the expression of FASN and ATGLmRNA (P0.05).RPC by reducing the intake of fatty acid synthesis, esterification and oxidation, the expression of key genes, strengthen key gene expression of fatty acid hydrolysis, reduce the whole sheep skin at the age of 10-12 months .RPC CD36 reduced fat deposition, abdominal fat sheep of the 10-12 month old ACC, FASN, GPAT1, DGAT2, ATGL and ACADVL expression of mRNA (P0.05), DGAT1 and LPL increased, the expression level of MGLLmRNA (P0.05).RPC by reducing the intake of fatty acid, synthesis, esterification, hydrolysis and oxidation of the expression of key genes, reduce the whole 10-12 month old sheep abdominal fat deposition. In Experiment four, RPC of meat The molecular mechanism of sheep intramuscular fat deposition in regulation of addition of RPC on the 3-5 month old sheep longissimus muscle in LXR alpha, LXR beta mRNA expression had no significant effect (P0.05), with up regulation of PPAR transcription, but the difference was not significant (P0.05, RPC) on fat deposition in 3-5 month old sheep muscle tone the main control mechanism by maintaining the LXR and promote the expression of alpha PPAR alpha mRNA, And the up regulation of fat metabolism key genes, promote the sheep longissimus muscle fat deposition. The effect of adding RPC expression had inhibitory effect on the 10-12 month old sheep longissimus muscle in LXR and alpha PPAR alpha mRNA, reduce the overall fat deposition.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S826.5

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