本文選題：仔豬 + 低蛋白日糧��； 參考：《華中農業(yè)大學》2017年博士論文

【摘要】：充足的底物濃度和平衡的氨基酸模式是豬骨骼肌高效蛋白質合成的必要條件。其中,采食量是保障充足的底物濃度的關鍵。除作為合成蛋白質底物外,支鏈氨基酸(branched chain amino acids,BCAAs)(包括亮氨酸、異亮氨酸、纈氨酸)可能還發(fā)揮生物學調控作用。比如,纈氨酸能夠調控豬采食量。此外,幾乎所有谷氨酸和天冬氨酸以及大部分谷氨酰胺在腸道發(fā)生了分解代謝,導致不可逆的損失。骨骼肌蛋白質合成所需的這三個非必需氨基酸必須要由BCAAs代謝生成。而且BCAAs僅有部分被腸道和肝臟等器官截留。因此,通過日糧中添加BCAAs可能增加骨骼肌內自身代謝生成谷氨酸、谷氨酰胺和天冬氨酸,優(yōu)化胞內氨基酸模式,促進蛋白質合成。目前已有的研究表明,日糧添加BCAAs可以提高豬生長性能,但其對采食量及骨骼肌蛋白質沉積的調控機制尚待進一步闡明。本研究圍繞BCAAs提高仔豬采食及骨骼肌生長的關鍵問題,通過兩輪仔豬飼養(yǎng)試驗證實了低蛋白日糧添加BCAAs通過提高采食量和直接促進肌肉生長,改善仔豬生長性能,并進一步闡明了下丘腦感應BCAAs調節(jié)仔豬采食量的機制。應用血插管結合代謝組學技術,研究了日糧BCAAs對攝食狀態(tài)下骨骼肌的氨基酸凈利用及代謝的影響,并探究了BCAAs代謝與肌肉氨基酸凈利用的關系;應用血插管結合穩(wěn)定同位素示蹤技術,進一步闡明了BCAAs通過自身代謝調控骨骼肌蛋白質合成的機制。最后研究了日糧BCAAs對禁食狀態(tài)下骨骼肌內調節(jié)蛋白質合成與降解的信號通路活性的影響。主要研究內容及結果如下:第一部分:通過兩輪仔豬飼養(yǎng)試驗研究了低蛋白日糧添加BCAAs對仔豬生長性能、采食量及骨骼肌生長的影響。在試驗一中,選取28頭斷奶仔豬分為4個組,即正對照組、負對照組、試驗1組以及試驗2組,日糧粗蛋白水平依次為19.5%、16.7%、16.7%和17.2%。其中,試驗1組在負對照組基礎上添加纈氨酸、異亮氨酸和亮氨酸保證日糧中三種BCAAs水平與正對照組一致,并增加一倍的添加劑量形成試驗2組的日糧。自由采食。試驗二為配對試驗,選取21頭斷奶仔豬分為3個組,即負對照組、試驗1組以及配對組。其中,負對照組和試驗1組同試驗一。配對組試豬飼喂試驗1組的日糧,但采食量與負對照組保持一致。28 d的飼養(yǎng)試驗結束后,每組隨機選擇6頭屠宰,測定胴體性狀,采集下丘腦及背最長肌樣。同時,剝離背最長肌等主要的23塊肌肉,分別稱量肌肉塊重量。主要結果如下:1.與負對照組相比,試驗1組和試驗2組顯著提高了平均日增重、末重、采食量、前軀、中軀和后軀肌肉總重、背最長肌等大多數肌肉塊重量以及氮沉積(P0.05),并能恢復到正對照組的水平。當采食量保持一致時,添加BCAAs的配對組仍能顯著提高仔豬平均日增重和肉料比(P0.05),并也顯著提高了岡上肌、背闊肌、背最長肌等大塊肌肉重量(P0.05)。2.與負對照組相比,日糧添加BCAAs以劑量依賴的方式上調下丘腦Agrp和NPY mRNA水平,下調MC4R mRNA水平,并在試驗2組達到差異顯著水平(P0.05)。此外,試驗1組顯著下調了CART mRNA水平(P0.05)�；貧w分析顯示,采食量隨NPY mRNA水平增加呈二次曲線增加(P=0.02),而隨MC4R mRNA水平增加呈線性降低(P=0.02)。表明日糧添加BCAAs對采食量的提高與其上調下丘腦NPY基因表達以及下調MC4R基因表達有關。3.與負對照組相比,試驗1組和試驗2組顯著降低了下丘腦eIF2α磷酸化水平,以及顯著提高了S6K1磷酸化水平(P0.05),并能恢復到正對照組的水平。此外,試驗1組的mTOR磷酸化水平顯著高于負對照組(P0.05)。表明日糧添加BCAAs對采食量的提高與其降低GAAC通路活性以及提高mTORC1通路活性有關。4.與負對照組相比,試驗1組和配對組均顯著提高了背最長肌S6K1和mTOR磷酸化水平(P0.05),但試驗1組和配對組之間差異不顯著。說明BCAAs能夠激活骨骼肌mTORC1通路活性,且不依賴于采食量的變化。第二部分:利用血插管結合代謝組學技術研究了日糧添加BCAAs對仔豬肌肉氨基酸凈利用和股靜脈代謝產物譜的影響。試驗動物同第一部分的配對試驗。28 d的飼養(yǎng)試驗結束后,對負對照組及試驗1組的試豬分別在股動脈、股靜脈、頸動脈和頸靜脈安裝插管。在術后的第5 d,每組各選擇6頭健康且插管通暢的試豬進行正式試驗。通過股動脈持續(xù)9 h灌注對氨基馬尿酸(pAH)以測定肌肉血流速度。每隔1 h飼喂以保持試豬始終處于攝食狀態(tài),連續(xù)飼喂8次。攝食前及攝食后每隔1 h分別采集頸動脈和股靜脈血樣以供分析pAH、游離氨基酸及支鏈α酮酸,連續(xù)采集8次。待血樣采集完成后屠宰,采集股二頭肌樣用于游離氨基酸及3-甲基組氨酸分析,股靜脈血樣同時進行代謝組學分析。主要結果如下:1.肌肉血流速度在攝食后顯著提高了(P0.05),但不受日糧處理的影響。日糧添加BCAAs顯著增加了動脈和肌肉游離的異亮氨酸、亮氨酸以及纈氨酸濃度(P0.05),但顯著降低了動脈游離的苯丙氨酸和絲氨酸濃度以及肌肉游離的絲氨酸濃度(P0.05)。同時,添加BCAAs顯著降低了肌肉游離的3-甲基組氨酸濃度(P0.05),表明BCAAs抑制骨骼肌蛋白質動員。2.日糧添加BCAAs顯著提高了總BCAAs、總必需氨基酸、總非必需氨基酸及總氨基酸的肌肉凈利用量(P0.05),從而維持高效的蛋白質沉積。此外,添加BCAAs顯著增加了異亮氨酸、亮氨酸、纈氨酸及其代謝產物丙氨酸、谷氨酸和谷氨酰胺的肌肉凈利用量(P0.05),同時還顯著增加了組氨酸、蛋氨酸以及甘氨酸、脯氨酸和絲氨酸等功能性非必需氨基酸的肌肉凈利用量(P0.05)。3.日糧添加BCAAs對股靜脈代謝物有顯著影響,這些差異化合物與BCAAs代謝和蛋白質合成相關。例如,添加BCAAs顯著提高了三種支鏈α酮酸、谷氨酰胺以及天冬酰胺等代謝產物含量(P0.05);同時也顯著提高了二十碳五烯酸等脂質代謝相關產物含量(P0.05)。進一步對產物定量的結果顯示,添加BCAAs顯著提高了KIC和KMV(分別為亮氨酸和異亮氨酸的轉氨產物)的肌肉凈生成量(P0.05),提示添加BCAAs增加了骨骼肌內自身的代謝。4.肌肉氨基酸凈利用量分別與動脈的亮氨酸、異亮氨酸及纈氨酸濃度呈顯著正相關(P0.05),而與肌肉的BCAAs濃度之間無相關性。有趣的是,肌肉氨基酸凈利用量分別與KIC和KMV凈生成量呈顯著正相關(P0.05)。這些結果暗示添加BCAAs可能通過增加動脈濃度及胞內自身代謝來提高肌肉氨基酸凈利用量。第三部分:利用血插管結合穩(wěn)定同位素示蹤技術研究了日糧BCAAs通過調控自身代謝影響仔豬骨骼肌蛋白質合成與降解的機制。攝食狀態(tài)下機制研究的試驗動物同第二部分試驗。在試豬第一次采食完,通過頸靜脈灌注NaH~(13)CO_3,持續(xù)2 h。隨后灌注[1-~(13)C]亮氨酸,持續(xù)6 h。pAH灌注以及血樣采集同第二部分試驗。血樣用于分析亮氨酸、KIC和CO_2濃度及其同位素豐度。禁食狀態(tài)下機制研究的試驗動物同第一部分試驗的試驗一。采集負對照組和試驗1組的背最長肌樣。主要結果如下:1.日糧添加BCAAs顯著提高亮氨酸的動脈攝入量和凈利用量(P0.05),從而顯著提高蛋白質合成量以及沉積量(P0.05)。添加BCAAs也顯著增加凈轉氨量(P0.05),但不影響氧化脫羧量。有趣的是,蛋白質降解在添加BCAAs后亦顯著增加了(P0.05)。進一步的代謝命運分析顯示,肌肉攝取亮氨酸約有30%發(fā)生代謝,并主要經轉氨作用生成KIC。2.肌肉的[1-~(13)C]亮氨酸凈攝取以及[1-~(13)C]KIC凈生成在日糧添加BCAAs后也顯著提高了(P0.05)。相關分析顯示,骨骼肌蛋白質合成分別與蛋白質降解及[1-~(13)C]KIC凈生成之間呈顯著正相關(P0.05),暗示日糧BCAAs可能通過增加蛋白質降解以及自身的轉氨代謝,提高攝食狀態(tài)下蛋白質合成。3.日糧添加BCAAs顯著提高了背最長肌的Akt、mTOR及其下游靶蛋白S6K1的磷酸化水平(P0.05),提示飼喂BCAAs仔豬在禁食狀態(tài)下骨骼肌具有更高的蛋白質合成能力。此外,添加BCAAs顯著增加了FoxO1磷酸化水平(P0.05),相應地下調了Atrogin-1、MuRF1以及LC3-II蛋白的表達(P0.05),提示飼喂BCAAs仔豬在禁食狀態(tài)下骨骼肌呈現更低水平的蛋白質降解。本研究的主要結論為:(1)低蛋白日糧添加BCAAs可以提高采食量以及直接促進肌肉生長,從而改善仔豬生長性能,并能恢復到飼喂高蛋白日糧的水平;(2)日糧BCAAs可以通過影響下丘腦的食欲調節(jié)基因表達及通路活性,提高采食量;(3)日糧BCAAs通過促進自身在肌細胞內的代謝以及蛋白質降解,進而增加氨基酸凈利用量,提高攝食狀態(tài)下骨骼肌蛋白質合成速率;同時,日糧BCAAs通過下調泛素-蛋白酶體以及自噬-溶酶體途徑的關鍵蛋白表達,抑制禁食狀態(tài)下骨骼肌蛋白質降解,并最終提高蛋白質沉積,從而促進骨骼肌生長。
[Abstract]:Sufficient substrate concentration and balanced amino acid patterns are essential for the synthesis of high efficient protein in porcine skeletal muscles. In addition, feed intake is the key to ensuring sufficient substrate concentration. The branched chain amino acids (BCAAs) (including leucine, isoleucine, valine) may also be produced as a synthetic protein substrate. In addition, almost all glutamic acid and aspartic acid, as well as most glutamine, are metabolized in the intestines, causing irreversible loss. The three non essential amino acids needed for the protein synthesis of skeletal muscle must be metabolized by BCAAs. And only part of the BCAAs Therefore, the addition of BCAAs to the diet may increase the metabolism of the skeletal muscle to produce glutamic acid, glutamine and aspartic acid, optimize the intracellular amino acid pattern and promote protein synthesis. Current studies have shown that dietary supplementation of BCAAs can improve the growth performance of pigs, but it is for food intake and bone. The regulation mechanism of muscle protein deposition has yet to be further elucidated. This study focuses on the key problems of improving the growth of piglets and the growth of skeletal muscle around BCAAs. Through two rounds of piglet feeding tests, it is proved that the addition of BCAAs to the low protein diet improves the growth performance of the piglets by increasing the feed intake and directly promoting the growth of the muscle, and further clarifies the hypothalamus sense. The effect of dietary BCAAs on the net use and metabolism of amino acids in skeletal muscles under feeding condition was studied by using blood intubation and metabonomics, and the relationship between the metabolism of BCAAs and the net use of amino acids in the muscle was investigated by using the blood cannula and metabonomics, and the BCAAs was further elucidated by the use of blood intubation and stable isotope tracer technology. The effect of dietary BCAAs on the signaling pathway activity of protein synthesis and degradation in skeletal muscles under fasting state was studied by self metabolism. The main contents and results were as follows: the first part was to study the growth of low protein diet supplemented with BCAAs for piglet birth through two rounds of piglet feeding tests. In the experiment one, 28 weanling piglets were divided into 4 groups, namely, the normal control group, the negative control group, the test 1 and the 2 groups, and the dietary crude protein levels were 19.5%, 16.7%, 16.7% and 17.2%. respectively. The experiment 1 added valine, isoleucine and leucine on the basis of the negative control group. The three BCAAs levels in the diet were the same as those in the normal control group, and increased the dosage form of the 2 groups to form the diet. The test two was paired test, and 21 weanling piglets were divided into 3 groups, that is, negative control group, test 1 group and paired group. Among them, the negative control group and the test 1 groups were the same test. 1 groups of paired test pig feeding test. Diet, but after the feeding test and the negative control group kept the same.28 D feeding test, each group randomly selected 6 butchers, measured the carcass traits, collected the hypothalamus and the longest muscle in the back. At the same time, the main 23 muscles, such as the longest muscle of the peeling back, were weighed respectively. The main results were as follows: 1. compared with the negative control group, 1 groups and tests were tested. The 2 groups significantly improved the average daily gain, the end weight, the feed intake, the body weight of the anterior body, the middle body and the posterior body, the most weight of the muscle mass as well as the nitrogen deposition (P0.05), and it could be restored to the level of the control group. When the feed intake remained consistent, the average daily gain and the meat ratio (P0.05) of the piglets could still be significantly increased by the addition of BCAAs. Compared with the negative control group, the weight (P0.05).2. of the supraspinatus, latissimus dorsi and the longest muscle of the dorsal muscle (P0.05) was significantly higher than that in the negative control group. The level of Agrp and NPY mRNA in the hypothalamus was up-regulated in a dose-dependent manner, and the level of MC4R mRNA was down regulated in a dose dependent manner, and the difference was significant (P0.05) in the 2 groups. Moreover, the 1 groups were significantly reduced to CART mRNA water. Regression analysis (P0.05). The regression analysis showed that the feed intake was increased two times with the increase of NPY mRNA level (P=0.02), but linearly decreased with the increase of MC4R mRNA level (P=0.02). Compared with the increase of BCAAs for the feed intake and the up-regulation of the NPY gene expression in the hypothalamus and the.3. and negative control of MC4R gene expression, 1 groups and tests were tested. The 2 groups significantly reduced the level of eIF2 alpha phosphorylation in the hypothalamus, and significantly increased the level of S6K1 phosphorylation (P0.05), and was able to restore to the level of the control group. In addition, the level of mTOR phosphorylation in the 1 groups was significantly higher than that of the negative control group (P0.05). The increase of the amount of food added to the diet by adding BCAAs to the diet and the reduction of the activity of GAAC pathway and the increase of mTORC1 in the diet. Compared with the negative control group, the pathway activity related.4. significantly increased the S6K1 and mTOR phosphorylation level (P0.05) of the longest muscle in the 1 groups and the paired groups, but there was no significant difference between the 1 groups and the paired groups. It showed that BCAAs could activate the mTORC1 pathway activity of skeletal muscle and did not depend on the change of feed intake. The second part was the use of blood intubation in combination. The effects of dietary supplementation of BCAAs on the net use of amino acids and the profile of femoral vein metabolites in the muscle of piglets were studied. After the test animals and the first part of the paired test of.28 D, the test pigs in the negative control group and the 1 groups were installed in the femoral artery, femoral vein, carotid artery and jugular vein respectively. Fifth after the operation. D, a formal test was conducted in each group of 6 healthy and unobstructed pigs. The muscle blood flow velocity was measured by continuous perfusion of 9 h in the femoral artery with aminaminic acid (pAH). Every 1 h was fed to keep the test pigs in the feeding state and feeding continuously for 8 times. The carotid and femoral vein blood samples were collected at 1 h every septum before feeding and after feeding. PAH, free amino acid and branched chain alpha ketoacid were collected for 8 consecutive times. After the collection of blood samples, the two head samples were collected for free amino acids and 3- methyl histidine analysis, and the femoral vein blood samples were analyzed simultaneously. The main results were as follows: 1. muscle blood flow velocity increased significantly after feeding (P0.05), but not in the diet. Dietary supplementation of BCAAs significantly increased the dissociative isoleucine, leucine and valine concentration (P0.05) in the arteries and muscles, but significantly reduced the concentration of phenylalanine and serine and the free serine concentration (P0.05) of the free artery (P0.05). At the same time, the addition of BCAAs significantly reduced the concentration of 3- methylhistidine in the muscle. P0.05) showed that BCAAs inhibited skeletal muscle protein mobilization by adding BCAAs to the total BCAAs, total essential amino acids, total non essential amino acids and total amino acids (P0.05), thus maintaining efficient protein deposition. In addition, the addition of BCAAs significantly increased isoleucine, leucine, valine and its metabolite C The muscle net benefit of ammonia, glutamic acid, and glutamine (P0.05), and also significantly increased the muscle net benefit of functional non essential amino acids such as methionine, glycine, proline and serine (P0.05).3. diet added BCAAs to the femoral vein metabolites significantly, these differential compounds and BCAAs metabolism and protein For example, adding BCAAs significantly increased the content of three kinds of branched chain alpha ketoacid, glutamine and asparagine (P0.05), and increased the content of lipid metabolism related products such as twenty carbon five enoic acid (P0.05). Further quantitative results showed that adding BCAAs significantly increased KIC and KMV (respectively Muscle net production (P0.05) of leucine and isoleucine (leucine and isoleucine), suggesting that adding BCAAs increases the metabolism of.4. muscle amino acids in the skeletal muscle itself and has a significant positive correlation with the arterial leucine, isoleucine and valine concentration (P0.05), but no correlation with the BCAAs concentration in the muscles. Interestingly, the muscle is the muscle. The net yield of meat amino acids was positively correlated with net production of KIC and KMV respectively (P0.05). These results suggest that adding BCAAs may increase the net benefit of muscle amino acids by increasing arterial concentration and intracellular metabolism. The third part: using blood intubation and stable isotope tracing technique to study the regulation of BCAAs by regulating self generation. The mechanism of protein synthesis and degradation in the skeletal muscle of piglets. Experimental animals studied in the feeding state were tested in second parts. After the first feeding of the pig, NaH~ (13) CO_3 was perfused through the jugular vein, [1-~ (13) C] leucine was perfused after 2 h., and the continuous 6 h.pAH perfusion and blood samples were collected with second parts of the experiment. Blood samples were used in the test. Analysis of leucine, KIC and CO_2 concentrations and their isotopic abundances. Experimental animals studied in the fasting state of the first part of the first part of the test. A negative control group and 1 groups of the longest dorsal muscle samples were collected. The main results were as follows: the addition of BCAAs to the 1. diet increased significantly the arterial intake and net benefit of leucine (P0.05). Protein synthesis and deposition (P0.05). Adding BCAAs also significantly increased the net ammonia conversion (P0.05), but did not affect the oxidation decarboxylation. Interestingly, the protein degradation was also significantly increased after the addition of BCAAs (P0.05). Further metabolic fate analysis showed that muscle uptake of leucine was about 30% metabolism, and mainly through the action of ammonia to produce KIC The net uptake of [1-~ (13) C] leucine and net production of [1-~ (13) C]KIC in.2. muscles also increased significantly (P0.05) after dietary supplementation with BCAAs. The correlation analysis showed that the protein synthesis in skeletal muscle was positively correlated with protein degradation and [1-~ (13) C]KIC net formation respectively (P0.05), suggesting that dietary BCAAs may be degraded by protein degradation and The addition of BCAAs to the dietary protein synthesis.3. diet enhanced the Akt, mTOR and its downstream target protein S6K1 phosphorylation level (P0.05), suggesting that the BCAAs piglets had a higher protein binding ability in the fasting state of the skeletal muscle. In addition, the addition of BCAAs significantly increased FoxO1 phosphoric acid. The expression of Atrogin-1, MuRF1 and LC3-II protein (P0.05) was adjusted accordingly, suggesting that the skeletal muscles of BCAAs piglets were reduced to a lower level of protein degradation in the fasting state. The main conclusions of this study were: (1) the addition of BCAAs to low protein diet could improve the feed intake and direct the growth of muscle, thus improving the piglets. Growth performance, and can be restored to the level of feeding high protein diet; (2) diet BCAAs can increase feed intake by affecting the appetite regulating gene expression and pathway activity in the hypothalamus, and increase feed intake. (3) dietary metabolism and protein degradation in the muscle cells, and then increase the net benefit of amino acids, and increase the skeleton of the feeding state. The rate of muscle protein synthesis; at the same time, dietary BCAAs can inhibit the protein degradation of skeletal muscle in the fasting state by down regulation of the key protein expression of ubiquitin proteasome and autophagy lysosome pathway, and ultimately improve protein deposition, thus promoting skeletal muscle growth.

【學位授予單位】：華中農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S828.5

【參考文獻】

相關期刊論文 前4條

1 Yinghui Li;Fengna Li;Li Wu;Hongkui Wei;Yingying Liu;Tiejun Li;Bie Tan;Xiangfeng Kong;Kang Yao;Shuai Chen;Fei Wu;Yehui Duan;Yulong Yin;;Effects of dietary protein restriction on muscle fiber characteristics and m TORC1 pathway in the skeletal muscle of growing-finishing pigs[J];Journal of Animal Science and Biotechnology;2017年01期

2 Jaap van Milgen;Jean-Yves Dourmad;;Concept and application of ideal protein for pigs[J];Journal of Animal Science and Biotechnology;2015年03期

3 Agus Suryawan;Teresa A Davis;;Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs[J];Journal of Animal Science and Biotechnology;2014年03期

4 鄭溜豐;彭健;;中樞神經系統(tǒng)整合外周信號調節(jié)采食量的分子機制[J];動物營養(yǎng)學報;2013年10期

相關博士學位論文 前1條

1 羅杰;長鏈n-3 PUFA對仔豬免疫成熟和生長的影響及調控作用研究[D];華中農業(yè)大學;2010年

，

本文編號：1831237