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論文寫作技巧
模板資源
精華論文
Abstract Ill
目錄 1
1文獻綜述 2
1.1淀粉的營養研究進展 2
1.1.1淀粉的來源 2
1.1.2淀粉的理化特性 2
1.1.3淀粉在羊體內的消化代謝特點 4
1.1.4影響淀粉消化利用的因素 6
1.2反芻動物小腸淀粉消化 6
1.2.1淀粉在反芻動物小腸內的消化機理 6
1.2.2羔羊小腸消化酶的分布特點 7
1.2.3小腸消化淀粉的限制因素 7
1.3反芻動物小腸葡萄糖吸收 8
1.3.1反芻動物體內葡萄糖來源 8
1.3.2小腸上皮細胞內葡萄糖的轉運機制 8
1.3.3反芻動物葡萄糖營養調控 9
1.4不同淀粉源調控動物腸道健康的研究進展 10
1.4.1不同淀粉源對動物腸道形態的影響 10
1.4.2不同淀粉源對動物腸道消化酶活性的影響 10
1.4.3不同淀粉源對動物腸道發育相關基因及葡萄糖轉運載體蛋白基因表達量的影響11
1.5本研究的目的與意義 11
1.6技術路線 13
2不同淀粉源對羔羊小腸形態結構發育的影響 15
2.1材料與方法 15
2.1.1試驗設計 15
2.1.2試驗精料 15
2.1.3試驗動物的飼養管理 16
2丄4樣品的采集及預處理 16
2.1.5測定指標及方法 16
2.1.6數據處理 17
2.2結果與分析 17
2.2.1不同淀粉源對羔羊各腸段相對質量的影響 17
2.2.2不同淀粉源對羔羊小腸形態結構的影響 23
2.3 i寸i侖 28
2.3.1不同淀粉源對羔羊各腸段相對質量的影響 28
2.3.2不同淀粉源對羔羊小腸形態結構的影響 28
2.4 29
3不同淀粉源對羔羊小腸揮發性脂肪酸的影響 31
3.1材料與方法 31
3.1.1試驗設計 31
3.1.2試驗精料 31
3.1.3試驗動物的飼養管理 31
3.1.4樣品采集及預處理 31
3.1.5小腸食糜中揮發性脂肪酸含量的測定 31
3.1.6數據處理 31
3.2結果與分析 31
3.2.1不同淀粉源對羔羊十二指腸食糜中揮發性脂肪酸的影響 31
3.2.2不同淀粉源對羔羊空腸食糜中揮發性脂肪酸的影響 34
3.2.3不同淀粉源對羔羊回腸食糜中揮發性脂肪酸的影響 36
5.3 38
5.4 /J、參吉 39
4不同淀粉源對羔羊小腸食糜中消化酶活性的影響 41
4.1材料與方法 41
4.1.1試驗設計 41
4.1.2試驗精料 41
4.1.3試驗動物的飼養管理 41
4.1.4小腸食糜樣品的采集及處理 41
4.1.5小腸食糜消化酶活性的測定 41
4.1.6數據處理 41
4.2結果與分析 41
4.2.1不同淀粉源對羔羊小腸食糜中淀粉酶活性的影響 41
4.2.2不同淀粉源對羔羊小腸食糜中麥芽糖酶活性的影響 44
4.2.3不同淀粉源對羔羊小腸食糜中乳糖酶活性的影響 46
4.2.4不同淀粉源對羔羊小腸食糜中胰蛋白酶活性的影響 48
4.2.5不同淀粉源對羔羊小腸食糜中糜蛋白酶活性的影響 50
5.4 i寸i侖 52
5.4 /J、參吉 53
5不同淀粉源對羔羊小腸發育相關基因及葡萄糖轉運載體蛋白基因mRNA表達量的影響55
5.1材料與方法 55
5丄1試驗設計 55
5丄2試驗精料 55
5.1.3試驗動物的飼養管理 55
5丄4樣品的采集及預處理 55
5.1.5測定指標及方法 55
5.1.6數據處理 57
5.2結果與分析 57
5.2.1羔羊小腸組織中RNA純度及完整性分析 57
5.2.2基因表達定量的溶解曲線 57
5.2.3不同淀粉源對羔羊小腸發育相關基因表達量的影響 59
5.2.4不同淀粉源對羔羊小腸葡萄糖轉運載體蛋白基因的影響 64
(1) 67
5.3.1不同淀粉源對羔羊小腸發育相關基因表達量的影響 67
5.3.2不同淀粉源對羔羊小腸葡萄糖轉運載體蛋白基因的影響 67
(2) ^ 68
6總體討論與結論 69
6.1總體討論 69
6.2總體結論 70
6.3研究的創新點 70
6.4有待進一步石開宄的問題 70
Contents
Abstract in Chinese I
Abstract in English Ill
Introduction 1
Literature review 2
Research progress of starch nutrition 2
The source of starch 2
Starch physical and chemical properties 2
The characteristics of digestion and metabolism of starch in sheep 4
Factors affecting the digestion and utilization of starch 6
Intestinal starch digestion in ruminants 6
The digestion mechanism of starch in the small intestine of ruminant 6
Distribution of digestive enzymes in the small intestine of lamb 7
The limiting factors of digestive starch in the small intestine 7
Absorption of glucose in the small intestine of ruminants 8
Glucose sources in ruminants 8
The mechanism of glucose transport in small intestinal epithelial cells 8
Regulation of glucose nutrition in ruminants 9
Research progress on regulation of animal intestinal health by different starch sources.... 10
The effect of different starch sources on the intestinal morphology of animals 10
Effects of different starch sources on digestive enzyme activities in animal intestine.. 10
Effects of different starch sources on expression of genes related to intestinal
development and glucose transporter protein in animals 11
The purpose and significance of this study 11
Technical route 13
Effects of different starch sources on the development small intestine morphology and
structure in lambs 15
Materials and methods 15
Eperimental design 15
Eperimental concentrate 15
Feeding and management of experimental animals 16
Sample collection and pretreatment 16
Measurement index and method 16
Data processing 17
Results and analysis 17
Effects of different starch sources on relative quality of intestinal segments in lambs. 17
Effects of different starch sources on the morphology and structure of small intestine
in lambs 23
Discussion 28
Effects of different starch sources on relative quality of intestinal segments in lambs. 28
Effects of different starch sources on the morphology and structure of small intestine
in lambs 28
Summary 29
Effects of different starch sources on volatile fatty acids in small intestine of lambs 31
Materials and methods 31
Eperimental design 31
Eperimental concentrate 31
Feeding and management of experimental animals 31
Sample collection and pretreatment 31
Determination of volatile fatty acid content in intestinal chyme 31
Data processing 31
Results and analysis 31
Effect of different starch sources of volatile fatty acids in the duodenum lamb 31
Effect of different starch sources of volatile fatty acids in the jejunal digesta of lamb.34
Effect of different starch sources of volatile fatty acids in lamb ileal digesta 36
Discussion 38
Summary 39
Effect of different starch sources on digestive enzyme activities of intestinal chyme in lamb* 41
Materials and methods 41
Eperimental design 41
Eperimental concentrate 41
Feeding and management of experimental animals 41
Acquisition and processing of intestinal chyme samples 41
Determination of enzyme activity in intestinal Digesta 41
Data processing 41
Results and analysis 41
Effect of different starch sources on amylase activity of intestinal chyme in lamb 41
Effect of different starch sources on maltase activity of intestinal chyme in lamb 44
Effect of different starch sources on the lactase activity of intestinal chyme in lamb.. 46
Effect of different starch sources on trypsin activity of intestinal chyme in lamb 48
Effect of different starch sources on chymotrypsin activity of intestinal chyme in
lamb 50
Discussion 52
Summary 53
Effects of different starch sources on mRNA expression of small intestine development related genes and glucose transporter protein genes in lambs 55
Materials and methods 55
Eperimental design 55
Eperimental concentrate 55
Feeding and management of experimental animals 55
Sample collection and pretreatment 55
Measurement index and method 55
Data processing 57
Results and analysis 57
Analysis of purity and integrity of RNA in small intestinal tissue of lambs 57
Quantitative dissolution curve of gene expression 57
Effects of different starch sources on the expression of small intestine development
related genes in lambs 59
Effects of different starch sources on gene expression of glucose transporter in small intestine of lambs
Discussion
Effects of different starch sources on the expression of small intestine development
related genes in lambs
Effects of different starch sources on gene expression of glucose transporter in small
intestine of lambs
Summary
General discussion and conclusion
General discussion
General conclusion
Innovation of research
Questions to be further studied
Reference
Acknowledgement
Appendix
本試驗系統地研究了日糧中不同淀粉源對不同日齡羔羊小腸發育、發酵、消化酶活性 和小腸發育相關基因及葡萄糖轉運載體蛋白基因mRNA表達量的影響。試驗采用完全隨 機試驗設計，分別以淀粉源和羔羊日齡作為試驗因素。試驗選擇48只初生公羔（東北細 毛羊x小尾寒羊），隨機分為4組，每組3個重復，每重復4只。試驗中分別選擇木薯淀 粉、玉米淀粉、小麥淀粉和豌豆淀粉作為羔羊精料中唯一的淀粉來源。試驗全期共77天， 在第21、35、56和77天時，每重復屠宰1只羔羊，檢測其各腸段相對質量、小腸形態結 構、小腸食糜揮發性脂肪酸、小腸消化酶活性、小腸發育相關基因及葡萄糖轉運載體蛋白 基因mRNA的表達量。試驗結果如下：
不同淀粉源對羔羊各腸段相對質量和小腸形態結構的影響
試驗精料中不同淀粉源未影響羔羊各腸段相對質量（％活體質量、％腸道質量） (P>0.05)。與木薯、玉米和小麥淀粉組相比，豌豆淀粉組羔羊十二指腸、空腸絨毛高度 最高（P<0.05)，豌豆淀粉組羔羊空腸、回腸絨毛寬度最寬（P<0.05)，豌豆淀粉組羔羊小 腸各段的隱窩深度和絨毛表面積均最大（P<〇.〇5)。生長時期對羔羊直腸的相對質量（％ 活體質量)、小腸、空腸、大腸和直腸的相對質量（％腸道質量)、十二指腸的絨毛寬度和 隱窩深度無顯著影響（P>0.05)，而對小腸、十二指腸、空腸、回腸、大腸、盲腸和結腸 的相對質量（％活體質量)、十二指腸、回腸、盲腸和結腸的相對質量（％腸道質量)、空 腸和回腸的絨毛高度、絨毛寬度、隱窩深度、絨毛表面積、絨毛高度/隱窩深度的值和十 二指腸的絨毛高度、絨毛表面積、絨毛高度/隱窩深度的值有顯著影響（P<〇.〇5)。
不同淀粉源對羔羊小腸食糜揮發性脂肪酸的影響
試驗精料中不同淀粉源對羔羊十二指腸食糜乙酸和異丁酸濃度、空腸和回腸食糜乙酸 /丙酸均無顯著影響（P>0.05)。與其他3個淀粉組相比，豌豆淀粉組35日齡羔羊十二指 腸食糜丙酸濃度和乙酸/丙酸最大（尸<0.05)，豌豆淀粉組77日齡羔羊回腸食糜乙酸濃度 和空腸食糜異丁酸濃度最大（尸<0.05)，豌豆淀粉組羔羊十二指腸、空腸和回腸中丁酸濃 度均最大（P<〇.〇5)。生長時期對羔羊空腸食糜乙酸濃度無顯著影響（P>0.05)，而對十二 指腸和回腸食糜中乙酸、丙酸、丁酸、異丁酸、乙酸/丙酸的值和空腸食糜中丙酸、丁酸、 異丁酸、乙酸/丙酸的值均有顯著影響（P<0.05)。
不同淀粉源對羔羊小腸消化酶活性的影響
試驗精料中不同淀粉源對羔羊回腸中麥芽糖酶活性、十二指腸和回腸中胰蛋白酶活性 和十二指腸中糜蛋白酶活性均無顯著影響（P>〇.〇5)。與木薯、玉米和小麥淀粉組相比， 豌豆淀粉組羔羊十二指腸中淀粉酶和乳糖酶活性最大（P<〇.〇5)，豌豆淀粉組羔羊空腸中 淀粉酶、麥芽糖酶、乳糖酶、胰蛋白酶和糜蛋白酶活性均最大（P<〇.〇5)，豌豆淀粉組羔 羊回腸中淀粉酶和糜蛋白酶活性最大（P<〇.〇5)。生長時期對羔羊小腸各段的淀粉酶、麥 芽糖酶、乳糖酶、胰蛋白酶和糜蛋白的活性均有顯著影響（P<〇.〇5)，小腸中除了乳糖酶 活性是隨著生長時期的增加而減小，其他都隨著生長時期的增加而增大（P<〇.〇5)。
不同淀粉源對羔羊小腸發育相關基因及葡萄糖轉運載體蛋白基因mRNA表達量 的調節
試驗精料中不同淀粉源對羔羊空腸和mRNA的表達量均無顯著影響 (P>0.05)。與其他3個淀粉組相比，豌豆淀粉顯著提高了羔羊十二指腸、回腸中 和/GF-LR mRNA的表達量及小腸各段中五GF、和GZLT-2 mRNA的表達量 (戶<0.05)。生長時期對羔羊回腸GZt/T-2 mRNA的表達量無顯著影響（P>0.05)，而對羔 羊十二指腸和空腸八以?、五和GZLT-2 mRNA的表達量和回腸八 /GF-LR、五仰、5dT-/mRNA的表達量均有顯著影響（尸<0.05)。
綜上所述，飼喂不同淀粉源的精料未影響羔羊各腸段相對質量，但與木薯、玉米和小 麥淀粉相比，豌豆淀粉日糧改善了羔羊小腸組織的形態結構，促進了丁酸的產生、消化酶 的分泌和小腸發育相關基因及葡萄糖轉運載體蛋白基因的上調。因此，飼喂豌豆淀粉日糧 能夠促進羔羊早期小腸的發育及功能的完善。
關鍵詞：淀粉源；羔羊；小腸發育；消化酶活性；基因表達
Abstract
The effects of different starch sources on small intestine development, fermentation, digestive enzyme activity, small intestine development related genes and glucose transporter protein gene mRNA of lambs of different ages were determined systematically in this study. A completely randomized trial was designed, starch source and lamb day-old were used as the experimental factors. Forty-eight male lambs (Northeast fine wool sheepx Small-tailed Han sheep) were randomly divided into 4 groups with 3 replicates in each group and 4 lambs per replica. The tapioca starch, corn starch, wheat starch and pea starch were selected as the sole source of starch in lamb concentrate. The whole experiment lasted for 77 days, at the 21st, 35th, 56th and 77th days, one lamb was slaughtered for each repetition, the relative mass of each intestinal segment, the morphology of the small intestine, the volatile fatty acids in the intestinal chime, the digestive enzyme activity of the small intestine, the expression of small intestine development related genes and glucose transporter gene mRNA were measured. The results are as follows:
Effect of different starch sources on the relative mass of the intestinal segments and the morphology and structure of small intestine in lambs.
The relative mass of each intestinal segment of lamb was not significantly affected by different starch sources in this test (% live weight, % intestinal weight) (P>0.05). Compared with tapioca, corn and wheat starch group, the height of duodenum and jejunum villi of lambs in pea starch group was highest (P<0.05), while the width of jejunum and ileum villus of lambs in pea starch group was widest (P<0.05), and the depth of recess and the surface area of small intestine of lambs in the pea starch group were largest (P<0.05). In addition to the relative mass of the rectum (% live weight), the relative mass of the small intestine, the jejunum, the large intestine and rectum(% intestinal weight), the villi width and the crypt depth of duodenum of the lamb had no significant influence (P<0.05), the growing period had significant influence on the other indexes (尸<0.05).
Effects of different starch sources on volatile fatty acids in small intestinal chyme of Lambs
The concentration of acetic acid and isobutyric acid in duodenum, chyme acetic acid/propionic acid in jejunum and ileum were not significantly affected by different starch sources in the concentrate (P>0.05). Compared with the other three starch groups, the concentration of propionic acid and acetic acid/propionic acid in duodenum of 35 days old lambs in pea starch group was highest (P<0.05), and the concentration of acetic acid in ileum and jejunum chyme isobutyric acid of 77 days old lambs in pea starch group were highest (P<0.05), the concentration of butyric acid in jejunum and ileum was highest (P<0.05). The growth period had no significant effect on the concentration of chyloacetic acid in the jejunum of lambs, but had a significant effect on the other indexes (P<0.05).
Effect of different starch sources on intestinal digestive enzyme activity of lambs
There were no significant effects of different starch sources on the activity of maltase in
ileum of lambs, trypsin activity in duodenum and ileum and chymotrypsin activity in duodenum (P>0.05). Compared with tapioca, corn and wheat starch groups, the amylase and lactase activities in duodenum of lambs in pea starch group were the highest (P<0.05), the activity of amylase, maltase, lactase, trypsin and chymotrypsin in jejunum of lambs in pea starch group was the highest (P<0.05), and the activity of amylase and chymotrypsin in the ileum of lambs in pea starch group was the highest (P<0.05). The activities of digestive enzymes in the small intestine of lambs were significantly affected by the growth period (P<0.05), except that the activity of lactase in the small intestine decreased with the increase of the growth period, and the other activities increased with the increase of the growth period (P<0.05).
Regulation of mRNA expression of small intestine development related genes and glucose transport carrier protein genes in lambs by different starch sources
There was no significant effect on the expression of IGF-1 and IGF-1R mRNA in jejunum of lamb with different starch sources (P>0.05). Compared with the other three starch groups, the expression of IGF-1, IGF-1R mRNA in duodenum and ileum and the expressions of EGF, SGLT-1 and GLUT-2 mRNA in small intestine of lambs were significantly increased by pea starch (P<0.05). In addition to having no significant effect on the expression of GLUT-2 mRNA in the ileum of lambs, the expression of IGF-1, IGF-1R, EGF, SGLT-1 and GLUT-2 mRNA in the small intestine of lambs was significantly affected by growth period (P<0.05).
To sum up, the concentrate fed with different starch sources did not affect the relative quality of the intestinal segments of lambs, but compared with the other three starch diets, the diets of pea starch improved the morphology of intestinal tissues in lambs, promoted the production of butyric acid, the secretion of digestive enzymes and the up-regulation of genes associated with small intestine development and glucose transport carrier protein. It can be speculated that feeding pea starch diet can promote the development and function of small intestine in early stage of lamb.
Key words: starch source; lamb; small intestine development; digestive enzyme activity; gene expression
不同淀粉源對羔羊小腸發育、消化酶活性及相關基因表達量的影響
符號說明 | ||
符號 | 英文全稱 | 中文全稱 |
ATP | Adenosine triphosphate | 三磷酸腺苷 |
BSEG | Exogenous glucose | 外源葡萄糖 |
CS | Corn starch | 玉米淀粉 |
EGF | Epidermal growth factor | 表皮生長因子 |
GLUT-2 | Facilitated glucose transporter | 促葡萄糖轉運載體 |
H-E | Hematoxylin/eosin | 蘇木精-伊紅 |
IGF-1 | Insulin-like growth factor-1 | 類胰島素生長因子-1 |
IGF-1R | Insulin-like growth factor-1R | 類胰島素生長因子-1受體 |
mRNA | Mmessenger RNA | 信使核糖核酸 |
POEG | Endogenous glucose | 內源葡萄糖 |
PS | Pea starch | 豌豆淀粉 |
RES | Rumen bypass starch | 過瘤胃淀粉 |
RT-PCR | Real-time polymerase chain reaction | 實時熒光定量PCR |
SGLT-1 | Apical sodium-dependent glucose co-transporter-1 | Na+依賴型葡萄糖轉運載體 |
TS | Tapioca starch | 木薯淀粉 |
VFA | Volatile fatty acids | 揮發性脂肪酸 |
WS | Wheat starch | 小麥淀粉 |
不同淀粉源對羔羊小腸發育、消化酶活性及相關基因表達量的影響
腸道是動物消化和吸收營養物質的主要場所，腸道的健康水平決定動物整體健康和生 產性能的發揮。腸道健康包括宏觀和微觀結構的完整性、腸道發育、腸道微生物菌群結構 和腸道免疫系統的狀態等m。對于新生羔羊而言，腸管占整個消化道的比例與單胃動物相 近，能達到70%左右，但隨著日齡的增長和日糧的改變，小腸所占比例逐漸降低。羔羊 早期腸道發育程度會直接影響其成年后消化和吸收能力的發揮。因此，在畜牧經濟迅速發 展的今天，為了確保羔羊生產性能和效率的發揮，營養學家們將通過營養調控羔羊的腸道 健康列入了重點研究內容之一。
生命早期，腸道組織分化、細胞增殖、酶的分泌以及機體其它重要物質的合成都需要 供應大量的營養素M。淀粉作為羔羊所需能量的重要來源，對其腸道的健康起著至關重要 的作用。玉米、大米、小麥等在世界范圍內被廣泛地用作動物飼料中的淀粉源，尤其是在 幼齡反芻動物的初始飼料中。近些年來，研究者對這些淀粉源進行了大量的研究，發現不 同淀粉源的淀粉含量以及理化特性不同，進而對動物生長發育和健康的影響也不同。目前， 關于羔羊日糧中淀粉的研究主要集中在淀粉源、淀粉水平和抗性淀粉對羔羊生產性能、瘤 胃發酵和瘤胃微生物影響的研究。然而，關于日糧中不同來源的淀粉是否會引起不同日齡 羔羊小腸形態結構、小腸發酵、小腸中消化酶活性和小腸發育相關基因及葡萄糖轉運載體 蛋白基因改變的報道較少，有待于進一步探討和研究。
基于以上原因，本文系統地研究了日糧中不同淀粉源對不同日齡羔羊小腸形態結構、 小腸發酵、小腸中消化酶活性和小腸發育相關基因（/GF-/、/(^-以?、及葡萄糖轉 運載體蛋白基因（SGZr-/、GZLT-2) mRNA的影響，分析不同淀粉源影響羔羊小腸發育 的作用機制。為探索通過營養素調控羔羊腸道健康和生產性能提供理論依據和基本參數。
