臺灣博碩士論文加值系統

熱緊迫使畜禽繁殖力下降，造成畜牧產業巨額經濟損失。成熟母禽卵巢內處於未進入排序階段的小黃濾泡(small yellow follicles, SYFs；6-8 mm)對濾泡發育與排序以及產蛋表現有關鍵性影響。為瞭解急性熱緊迫影響母雞產蛋的分子機制，本研究探討急性熱緊迫對台灣母土雞之生理指標及SYFs mRNA與蛋白質表現之效應，以期作為改善台灣土雞耐熱力之基礎。本研究使用30週齡國立中興大學育成之L2品系及B品系台灣土雞母雞進行功能性基因體調控之分析，並記錄急性熱緊迫前後之產蛋性狀變化。所有母雞於溫溼度環控氣候室(25°C)適應至少兩週，將母雞分為急性熱緊迫處理組及對照組(25°C)，L2品系及B品系的急性熱緊迫處理組分別接受36°C 4小時及38°C 2小時熱處理，隨後於25°C恢復0、2及6小時，並於恢復期後採SYFs供後續分析。所有雞隻在熱處理或恢復期皆測量呼吸速率及體溫。母雞SYFs於熱緊迫處理後之mRNA及蛋白質表現差異分析以商業生產含44K基因之微陣列晶片及二維差異膠體電泳(two-dimensional difference gel electrophoresis, 2D-DIGE)進行。所有母雞之呼吸速率及體溫皆在熱處理開始短時間內顯著提高；不論是L2品系或是B品系母雞的產蛋率皆在急性熱緊迫後顯著下降而至一周後漸恢復。急性熱緊迫處理組L2品系及B品系母雞之SYFs分別有351及406個mRNA表現量顯著差異於對照組；L2品系及B品系母雞之SYFs在熱緊迫處理後分別有142及119個蛋白質表現量顯著差異。經胜肽質量指紋分析差異表現蛋白質可分別鑑定出93及76個蛋白質。基因功能分析結果顯示，上述差異表現的基因及蛋白質主要參與代謝過程、細胞過程(細胞骨架、脂質運輸)、生物調控(細胞凋亡調控、ATP合成調控)發育過程、緊迫反應及免疫反應等過程；急性熱緊迫顯著改變SYFs之熱緊迫蛋白質家族(HSP25、HSP47、HSP60、HSP70、HSC70、HSP90、HSPA8及HSPA9)、抗氧化物(PRDX1、3、4及6)及蛋黃合成相關蛋白質(VTG、VTG2、Apo A1及Apo B)之表現量。由本研究結果可知，急性熱緊迫可能改變台灣土雞SYFs內與細胞凋亡、過氧化物質傷害及蛋黃沉積速率相關mRNA及蛋白質表現而保護其免於細胞凋亡及過氧化物質傷害，然而，不論是L2品系或是B品系，其濾泡皆有可能因急性熱緊迫而發生閉鎖且產蛋率下降。

Heat stress causes the decline of fertility and results in enormous economic losses in farm animals. The small yellow follicles (SYF, 6-8 mm) are classified in a crucial pre-hierarchical stage related to the development of follicles, selection into the hierarchy, and laying performance. To understand the mechanisms of acute heat stress response on reproductive efficacy of hen, this study aimed to evaluate the effect of acute heat stress on SYFs of Taiwan country chickens (TCCs) through proteomic and genomic approaches. 30-wk-old hens of L2 strain and B strain TCCs selected in National Chung Hsing University were used to study the genomic regulation and the laying performances thereafter. After two weeks of adaption period (25°C), the L2 strain and B strain TCCs were subjected to acute heat stress of 36°C for 4 h and 38°C for 2 h, respectively. hens were then allowed recovery for 0, 2, and 6 h at 25°C. The respiratory rate and body temperature were recorded during the treatment and recovery periods. The control group was kept at 25°C. The mRNA and protein expressions in SYFs were evaluated with a chicken 44K oligo microarray and by two-dimensional difference gel electrophoresis (2D-DIGE). Results showed that the respiratory rate and body temperature increased significantly immediately after the treatment started. Laying rate was decreased significantly after acute heat stress in all hens. Totally 351 and 406 genes differed in transcript levels by heat stress groups in both strains of TCCs. 2D-DIGE analysis revealed that 142 and 119 protein spots were differentially affected by acute heat stress in SYFs of L2 strain and B strain TCCs. Peptide mass fingerprinting identified the differentially expressed protein spots belonging to 93 and 76 unique proteins. Gene ontology analysis indicated that these differentially expressed mRNAs and proteins were associated with biological processes of metabolic process, cellular process (cytoskeleton, lipids transports), biological regulation (regulation of apoptosis, regulation of ATP synthesis), developmental process, response to stimuli, and immune system process. Specifically, the expression of mRNA and protein of HSP family (HSP25, HSP47, HSP60, HSP70, HSC70, HSP90, HSPA8, and HSPA9), antioxidative defense (PRDX1, 3, 4, and 6), and egg yolk related proteins (VTG, VTG2, Apo A1, and Apo B) in SYFs of TCCs were significantly changed after acute heat stress, suggesting a protective mechanism of SYFs against damages caused by acute heat stress. In conclusion, acute heat stress resulted in differentially expressions of mRNA and proteins related to the responses to stimuli, metabolic processes and the cargo lipid for yolk deposition of SYFs to protect them from oxidative insults and death in TCCs. However, acute heat stress decrease laying rate in both strains of TCCs and might be attributed to follicular atresia.

摘要……………………………………………………………………………………i
Abstract……………………………………………………………………………….ii
List of Tables vii
List of Figures viii
Chapter 1. General introduction 1
1.1. Introduction of ovary in poultry 2
1.2. The effect of heat stress on female reproduction and folliculogenesis 3
1.3. The genomic regulation of female reproductive tissue in response to heat stress 5
1.4. Introduction of layer-type L2 strain and broiler-type B strain Taiwan country chickens 6
Chapter 2. Materials and Methods 8
2.1. Experimental animals 9
2.2. Conditions of acute heat stress, measurements of physiological parameters, and sample collection 9
2.3. Laying traits and egg characteristics 9
2.4. Hematoxylin-eosin staining 10
2.5. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay 10
2.6. Gene expression by microarray analysis 10
2.7. Validation of gene expressions by using quantitative real-time polymerase chain reaction 11
2.8. Protein expression analysis by two-dimensional difference gel electrophoresis (2D-DIGE) 15
2.9. Protein identification by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/TOF MS 16
2.10. Western blot analysis of proteins 17
2.11. Bioinformatic analysis 18
2.12. Immunohistochemistry of proteins in SYFs 18
2.13. Statistical analysis 19
Chapter 3. Differential genomic regulation in small yellow follicles of broiler-type B strain Taiwan country chickens in response to acute heat stress 20
3.1. Abstract 21
3.2. Introduction 22
3.3. Results 22
3.3.1. Effect of acute heat stress on physiological parameters, egg laying traits and egg characteristics in broiler-type B strain TCCs 22
3.3.2. Morphology and apoptosis in chicken SYFs of broiler-type B strain TCCs after acute heat stress 23
3.3.3. Effects of heat stress on gene expressions in the SYFs of broiler-type B strain TCCs after acute heat stress 25
3.3.4. Functional categories of the differentially expressed genes in the SYFs of broiler-type B strain TCCs after acute heat stress 26
3.3.5. Validation of representative differentially expressed genes in the SYFs of broiler-type B strain TCCs after acute heat stress 28
3.3.6. Comparison of protein profiles and identification of differentially expressed proteins in the SYFs of broiler-type TCCs after acute heat stress 30
3.3.7. Bioinformatic analysis of the differentially expressed proteins in broiler-type B strain TCCs 42
3.3.8. Validation of protein expression in SYFs of heat-stressed broiler-type B strain TCCs 46
3.4. Discussion 48
3.5. Conclusion 55
Chapter 4. Differential genomic regulation in small yellow follicles of layer-type L2 strain Taiwan country chickens in response to acute heat stress 56
4.1. Abstract 57
4.2. Introduction 57
4.3. Results 58
4.3.1. Effect of acute heat stress on physiological parameters, laying traits and egg characteristics layer-type L2 strain TCC hens 58
4.3.2. Gene expression and annotation of differentially expressed genes in the SYFs of layer-type L2 strain TCCs after acute heat stress 58
4.3.3. Validation of representative differentially expressed genes in the SYFs of layer-type L2 strain TCCs after acute heat stress 60
4.3.4. Comparison of protein profiles and identification of differentially expressed proteins in SYFs after acute heat stress 61
4.3.5. Validation of protein expression in SYFs of layer-type L2 strain TCCs 73
4.4. Discussion 73
4.5. Conclusion 80
Chapter 5. General discussion 81
5.1. The differentially responses to acute heat stress in mRNA and protein regulation 82
5.2. The differentially responses to acute heat stress in small yellow follicles of layer-type and broiler-type TCCs 82
Chapter 6. General conclusion 85
References… 87
Appendices 100

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