Title

任食影響白肉種雞生殖功能─免疫細胞角色與卵巢顆粒性細胞凋亡之探討

Translated Titles

Voluntary feeding affects reproductive functions in broiler breeder hens - the role of immune cell functionality and apoptosis of ovarian granulosa cells

DOI

10.6845/NCHU.2011.01234

Authors

謝宜倫

Key Words

肥胖 ; 脂肪中毒 ; 排卵 ; 單核球/巨噬細胞 ; 基質金屬蛋白酶 ; Obesity ; lipotoxictity ; ovulation ; monocyte/macrophage ; matrix metalloproteinase (MMPs)

PublicationName

中興大學動物科學系所學位論文

Volume or Term/Year and Month of Publication

2011年

Academic Degree Category

碩士

Advisor

陳洵一

Content Language

繁體中文

Chinese Abstract

白肉雞經過幾十年來的育種選拔其生長效率與飼料效率大幅提高,然而同時也出現許多不良的缺陷,例如食慾調節與體內能量衡定能力的喪失,而過度攝食也造成了肥胖的傾向與繁殖能力低落等情形。許多研究顯示,當過量的脂肪堆積超出脂肪組織肥大增值後之容納量時,會造成脂肪調節系統失調,使過多的脂肪堆積在非脂肪組織如肝臟、肌肉、胰臟、心臟等,進而導致細胞失去正常功能甚至死亡,此種生理現象稱為脂肪中毒(lipotoxicity)。其中過量神經醯胺(ceramide)的堆積被認為是引發脂肪中毒的主因之一,而脂肪中毒理論也部份解釋現今白肉種雞產蛋低落。肥胖已被認為會擾亂免疫系統功能,而排卵為一種局部的生理發炎反應(localized physiological inflammatory reaction),免疫細胞藉由釋放一些protease;如基質金屬蛋白酶(matrix metalloproteinase, MMPs),協同參與濾泡壁分解破裂,以利排卵完成。本試驗將雞隻以任飼及限飼方式以達成肥胖模式,分析血液單核球/巨嗜細胞ceramide堆積,並以RT-PCR分析相關基因表現,另外進行sphingomyelin及ceramide含量檢測,同時分析單核球IL-1β、MMP的分泌及吞噬作用。結果顯示任飼雞隻其腹脂重及腹脂率較高且產蛋率較低,單核球ceramide含量,限飼組顯著高於任飼組,異嗜球ceramide含量則相反,而單核球吞噬作用則是任飼組高於限飼組。在基因表現部分,任飼雞隻單核球的serine palmitoyl transferase(SPT)表現顯著較限飼組高,但sphingomyelinase(SMase)低於限飼組。異嗜球的SPT基因表現限飼組高於任飼組,但SMase基因表現則沒有差異。而限飼組單核球的IL-1β釋放量與分子量72 kDa MMP活性皆顯著高於任飼組。限飼雞隻單核球/巨噬細胞與排卵相關調控因子IL-1β、MMP的分泌皆下降的結果顯示,肥胖雞隻中免疫細胞已出現功能上的損傷。另外將雞隻卵巢濾泡做組織染色及免疫螢光染色,觀察其結構及免疫細胞滲入(主要為單核球/巨噬細胞),並分析卵巢濾泡顆粒細胞凋亡及濾泡壁Akt活性,結果顯示肥胖影響雞隻卵巢濾泡細胞凋亡並且卵巢濾泡具較肥大鬆散之組織結構,而濾泡壁細胞激素IL-1β的分泌提高可能促使免疫細胞移行進入,因而任飼組卵巢濾泡組織間具明顯的炎症細胞分佈。將顆粒細胞於採樣後做活體與體外培養細胞凋亡分析,以禽類生理劑量的棕櫚酸處理以模擬肥胖情況下過量脂肪酸對於細胞的影響,結果顯示經棕櫚酸處理兩天後,細胞凋亡的情況增加,至第四天則更加顯著,而添加acyl-CoA synthetase抑制劑Triacsin C,以阻斷脂肪酸與CoA形成活化的acyl-CoA,結果顯示脂肪酸引發顆粒細胞凋亡顯著受到抑制。以活體雞隻採樣新鮮顆粒細胞並以Annexin V/PI staining方法分析,亦顯示肥胖雞隻排序濾泡有較多的顆粒細胞凋亡。本試驗證實了脂肪酸會誘導細胞凋亡及壞死的現象,而基因表現部分由於皆沒有差異,因此推論在體外培養飽和脂肪酸引發顆粒細胞凋亡可能不是透過ceramide途徑所影響。綜上所述,此實驗結果顯示過度餵飼造成肥胖及相關脂肪中毒情形,進而影響免疫細胞之功能,其中MMP分泌及IL1-β釋放量的減少導致濾泡壁消解緩慢因而使排卵延遲,而肥胖雞隻卵巢濾泡組織內有較多免疫細胞與組織結構鬆散顯示發炎情形,此極可能係因脂肪中毒引發濾泡顆性細胞的死亡,進而導致濾泡閉鎖與卵巢形態異常,故較多免疫細胞滲入。如此脂肪中毒引發的排卵延遲、濾泡閉鎖與卵巢形態異常部份解釋了因過度餵飼造成白肉種雞低產蛋率的原因。

English Abstract

Genetic selection for rapid growth results in a propensity for hyperphagia and obesity-susceptible characteristics in association with poor reproductive performances in modern broiler chickens. A fundamental mechanism elucidating poor egg production in broiler hens remains elusive. Obesity has been considered to cause disruption of the immune system and ovulation is regarded as a localized physiological inflammatory reaction involving participation of immune cells within the follicle and ovary. Synergy of cytokines and enzymes from the filtrated leukocytes administer to implementation of ovarian functions, in which some proteases such as matrix metalloproteinase (MMPs) can degrade the follicle wall and cause follicle rupture to release the oocyte. In the present study, we achieved an obesity model by ad libitum vs. restricted feeding in broiler hens, in attempt to link delayed ovulation due to impaired immune cell functions and poor egg production in overfed broiler hens. Results showed broiler breeders of ad libitum group exhibited significantly higher body weight, abdominal fat weight, but lower egg production. Monocyte ceramide content of restricted group was lower than that of ad libitum group. However, hens fed ad libitum exhibited higher phagocytosis capacity of monocytes/macrophages. Contrary with heterophils, monocyte/macrophage SPT mRNA expression of hens fed ad libitum was significantly higher but SMase mRNA expression was lower than those of restricted group. Hens of ad libitum group exhibited significantly lower macrophage MMP activity at molecular weight around 72 kDa and IL-1 β release as compared to those of their restricted counterparts. At necropsy, histological staining demonstrated thicker but loose and inflammatory structures with more infiltrated immune cells within the connective tissue layers of the F1 follicle in the hens fed ad libitum. Hens with restricted feeding had higher follicle wall Akt activeity. These observations were consistent with a significant higher fraction of granulose cell apoptosis. In vitro, treatment of palmitic acids to mimic obesity-associated excessive accumulation of cellular fatty acids resulted in a higher fraction of cell apoptosis in cultured granulosa cells. In the presence of Triacsin C, an acyl-CoA synthetase inhibitor to block fatty acid activation, the palmitate-induced granulosa cell apoptosis was significantly rescued. Anaylses of related gene expressions suggested the fatty acid-induced apoptosis in culture granulosa cells may not depend on ceramide signaling. These results linked obesity-associated lipotoxicty to hierarchical follicle granulosa cell apoptosis and impaired immune cell functions, which may subsequently result in follicle atresia, abnormal ovarian morphology, delayed ovulation, and finally poor egg production in broiler hens under voluntary feeding.

Topic Category 農業暨自然資源學院 > 動物科學系所
生物農學 > 動物學
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