本文選題：過瘤胃膽堿 + 肉羊　； 參考：《揚州大學》2017年博士論文

【摘要】：肌內(nèi)脂肪含量是評定羊肉品質(zhì)的關(guān)鍵指標,探索肌內(nèi)脂肪營養(yǎng)調(diào)控技術(shù)和沉積機制成為目前生產(chǎn)和科研關(guān)注的重點。肌內(nèi)脂肪沉積,是肉羊生長、體脂分配、血液脂質(zhì)代謝、脂肪酸組成、脂肪代謝關(guān)鍵基因及轉(zhuǎn)錄調(diào)控因子等綜合作用的結(jié)果。膽堿在調(diào)控單胃動物脂肪分配方面發(fā)揮重要作用,但是其對肉羊生長及各部位脂肪組織中脂肪酸代謝變化的調(diào)控作用及機制缺乏研究。本研究采用過瘤胃膽堿(Rumen Protected choline,RPC)作為營養(yǎng)調(diào)控劑,借助動物飼養(yǎng)試驗、代謝組學、分子生物學技術(shù)以及生物信息學分析技術(shù),研究膽堿對肉羊脂肪代謝和肌內(nèi)脂肪形成的調(diào)控作用及分子機制。全文分為4個部分進行。試驗一、RPC對肉羊生長性能及體脂肪分配的影響試驗選取3-5月齡和10-12月齡杜泊羊X湖羊雜交F1代公羔開展兩期試驗,每期試驗選用24只,隨機分成4組(6頭/組,組間體重差異不顯著(P0.05)),分別為對照組、0.25%RPC、0.50%RPC和0.75%RPC添加組,每期試驗60d,試驗結(jié)果如下:3-5月齡肉羊階段,添加RPC有提高肉羊采食量、日增重、料肉比的作用(P0.05)。RPC對3-5月齡肉羊血液甘油三酯、總膽固醇、HDL和LDL均無顯著影響(P0.05)。RPC有降低3-5月齡肉羊血液FFA含量的作用(P0.01),對血清皮質(zhì)醇、瘦素、脂聯(lián)素和胰島素無顯著影響(P0.05)。RPC對屠宰率、眼肌面積、肋肉厚度、皮下脂肪厚度無顯著影響(P0.05),試驗組腹脂率、腎脂率和盆脂率較對照組有所降低,但并不顯著(P0.05)。添加RPC有提高腎、脾臟、胃、小腸重量的作用,但差異并不顯著(P0.05);對背最長肌脂肪含量無顯著影響(P0.05);對背最長肌、前腿肌pH具有顯著的升高作用(P0.05)。RPC降低3-5月齡肉羊前腿肌、后腿肌失水率(P0.05),對背最長肌失水率調(diào)控效果不顯著(P0.05);RPC有提高背最長肌、前腿肌、后腿肌嫩度的潛在作用,但差異并不顯著(P0.05)。10-12月齡肉羊階段,添加RPC對肉羊采食量、日增重、飼料利用率無顯著影響(P0.05)。RPC對10-12月齡肉羊血液甘油三酯、膽固醇、HDL、LDL含量無顯著影響(P0.05)。RPC對10-12月齡肉羊血液FAA有先升高后降低作用,但差異并不顯著(P0.05),RPC顯著降低胰島素水平(P0.05);對抵抗素、脂聯(lián)素有趨勢性降低效果,但差異不顯著(P0.05)。RPC對10-12月齡肉羊的屠宰率無顯著影響(P0.05),降低腹脂率、腎脂率和盆脂率,對眼肌面積和皮下脂肪厚度有提高作用,增加肝、腎、胃和大腸重量,但差異均不顯著(P0.05);顯著增加脾臟和小腸重量(P0.05)。RPC對10-12月齡肉羊背最長肌脂肪含量略有降低,但差異并不顯著(P=0.73),對調(diào)節(jié)背最長肌、前腿肌和后腿肌pH值無顯著影響(P0.05),降低背最長肌、前腿肌和后腿肌失水率、剪切力,但差異并不顯著(P0.05);對肉色調(diào)控無顯著效果(P0.05)。RPC能夠提高10-12月齡肉羊皮下脂肪和背最長肌中PUFA含量,但差異并不顯著(P0.05),提高n-3、n-6脂肪酸含量,降低背最長肌中SFA含量,但均無顯著影響(P0.05),RPC對背最長肌中C18:0、C18:1trans、C18:1cis、C18:2cis含量略有提升,但差異并不顯著(P0.05)。添加RPC能夠降低10-12月齡肉羊皮下脂肪、腹脂、腎周脂肪和盆腔脂肪組織的細胞直徑(P0.01),對各部位脂肪細胞降脂調(diào)控效果依次是腎脂腹脂盆腔脂肪皮下脂肪,細胞直徑分別較對照組降低33.52%、18.23%、17.47%、5.10%。試驗二、RPC對肉羊尿液代謝組的影響試驗選用體重相近的杜泊羊X湖羊雜交F1代公羔36頭,隨機分成3組,每組12頭,組間體重差異不顯著(P0.05)。在基礎(chǔ)日糧中分別添加0%、0.25%和0.75%RPC水平,飼養(yǎng)管理相同,預飼期10天,正試期5天,在第3-5天收集試驗羊尿樣,測定不同RPC添加水平對肉羊尿樣代謝組的影響,分別采用PCA、PLS-DA和OPLS-DA分析。結(jié)果表明:0.25%和0.75%RPC添加水平顯著改變?nèi)庋虻哪驑哟x組,差異代謝物主要有丙酮酸、氧化三甲胺、對甲酚、苯乙酰甘氨酸和馬尿酸。RPC添加組較對照組尿液中丙酮酸含量減少,尿樣中氧化三甲胺、對甲酚、苯乙酰甘氨酸和馬尿酸含量有所提高。這些差異代謝物參與的生物反應過程主要有能量代謝、脂肪、蛋白質(zhì)和AA代謝以及腸道微生物代謝。RPC能夠改變?nèi)庋驒C體脂肪和能量代謝,主要相關(guān)代謝物是丙酮酸和PAG。試驗三、RPC對肉羊脂肪代謝關(guān)鍵基因表達的影響3-5月齡肉羊采集對照組、0.25%RPC和0.75%RPC試驗組樣品,10-12月齡肉羊采集對照組和0.25%RPC試驗組樣品,測定各組織樣品中脂肪代謝關(guān)鍵基因mRNA相對表達量。結(jié)果發(fā)現(xiàn):對于3-5月齡肉羊,RPC促進背最長肌中LPL、CD36、ACC、FASN、AGPAT1和AC ADVL mRNA 表達(P0.05),對 DGAT1、DGAT2、ATGL、HSL、MGLL 和 CPT-1βmRNA 表達無顯著影響(P0.05)。RPC通過促進脂肪酸攝入、合成、酯化和氧化關(guān)鍵基因表達,,促進肉羊背最長肌脂肪酸沉積。RPC抑制3-5月齡肉羊盆腔脂肪中LPL、FASN和AGPAT1mRNA表達(P0.05),促進 CD36、GPAT1 和 CPT-1βmRNA 表達(P0.05),對 DGAT1、DGAT2、ATGL、HSL、MGLL和ACADVLmRNA表達無顯著影響(P0.05)。RPC主要通過降低3-5月齡肉羊盆脂脂肪酸攝入、合成和酯化關(guān)鍵基因mRNA表達,加強脂肪酸氧化關(guān)鍵基因表達,整體降低肉羊盆脂沉積。RPC降低3-5月齡肉羊腎周脂肪中的LPL、FASN、GPAT1、AGPAT1mRNA表達(P0.01),提高脂肪酸氧化關(guān)鍵基因CPT-1β和ACADVLmRNA表達(P0.01),對CD36、ACC、DGAT1、DGAT2、ATGL、HSL 和 MGLLmRNA 表達無顯著影響(P0.05)。RPC主要通過降低脂肪酸攝入、合成和酯化關(guān)鍵基因表達,加強脂肪酸氧化關(guān)鍵基因表達,降低腎周脂肪沉積。RPC降低3-5月齡肉羊皮下脂肪中CD36、ACC、GPAT1和ACADVL mRNA表達(P0.05),提高 LPL、FASN、AGPAT1、DGAT1、ATGL 和 CPT-1βmRNA 表達(P0.05),RPC通過降低攝入、合成、酯化和氧化關(guān)鍵基因表達,促進脂肪酸水解關(guān)鍵基因表達,整體降低皮下脂肪沉積。RPC 降低 3-5 月齡肉羊腹脂 CD36、ACC、FASN、GPAT1、DGAT2 和 ACADVLmRNA表達(P0.05),促進 LPL、AGPAT1、DGAT1 和 CPT-1β mRNA 表達(P0.01),RPC通過降低脂肪酸攝入、合成、酯化和氧化關(guān)鍵基因表達,整體降低腹脂沉積。對于10-12月齡肉羊,RPC降低0.25%RPC試驗組肉羊背最長肌LPL和CD36 mRNA表達(P0.01),促進 HSL、MGLL 和 CPT-1β的 mRNA 表達(P0.01),對 ACC、FASN、GPAT1、AGPAT1、DGAT1、DGAT2、ATGL 和 ACADVLmRNA 表達均無顯著影響(P0.05)。RPC通過降低脂肪酸攝入關(guān)鍵基因表達,加強脂肪酸水解和氧化關(guān)鍵基因表達,整體降低10-12月齡肉羊背最長肌脂肪沉積。RPC抑制10-12月齡肉羊盆腔脂肪中LPL、ACC和FASNmRNA表達(P0.01),促進 CD36、GPAT1、AGPAT1、ATGL 和 ACADVL mRNA 表達(P0.05),對 ATGL、DGAT1、DGAT2、HSL和CPT-1β mRNA表達無顯著影響(P0.05),RPC通過降低中脂肪酸攝入、合成關(guān)鍵基因表達,促進酯化、水解和氧化關(guān)鍵基因的表達,整體降低10-12月齡肉羊盆腔脂肪沉積。RPC 降低 10-12 月齡肉羊腎周脂肪中 LPL、ACC、FASN、GPAT1、AGPAT1、DGAT2、ATGL 和 ACADVLmRNA 表達(P0.05),促進 MGLLmRNA 表達(P0.05),RPC 通過降低脂肪酸攝入、合成、酯化和水解關(guān)鍵基因表達,整體降低腎周脂肪沉積。RPC降低10-12月齡肉羊皮下脂肪組織CD36、GPAT1和ACADVL mRNA表達水平(P0.05),促進FASN和ATGLmRNA的表達(P0.05)。RPC通過降低脂肪酸攝入、合成、酯化和氧化關(guān)鍵基因表達,加強脂肪酸水解關(guān)鍵基因表達,整體降低10-12月齡肉羊皮下脂肪沉積。RPC 降低 10-12 月齡肉羊腹脂 CD36、ACC、FASN、GPAT1、DGAT2、ATGL 和 ACADVL mRNA表達(P0.05),增加 LPL、DGAT1 和 MGLLmRNA 表達水平(P0.05)。RPC通過降低脂肪酸攝入、合成、酯化、水解和氧化關(guān)鍵基因表達,整體降低10-12月齡肉羊腹脂沉積。試驗四、RPC對肉羊肌內(nèi)脂肪沉積調(diào)控的分子機制添加RPC對3-5月齡肉羊背最長肌中LXRα、LXRβ mRNA表達無顯著影響(P0.05),對PPARα轉(zhuǎn)錄有上調(diào)作用,但差異并不顯著(P0.05),說明RPC對3-5月齡肉羊肌內(nèi)脂肪沉積的調(diào)控機制主要通過維持LXRα和促進PPARα mRNA表達,進而上調(diào)脂肪代謝關(guān)鍵基因,促進肉羊背最長肌脂肪沉積的效果。添加RPC對10-12月齡肉羊背最長肌中LXRα和PPARα mRNA表達均產(chǎn)生抑制效果,整體降低脂肪沉積。
[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.
【學位授予單位】：揚州大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S826.5

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