發(fā)布時間：2018-04-20 06:34

  本文選題：大豆異黃酮 + 下丘腦-垂體-卵巢軸��； 參考：《湖南農業(yè)大學》2015年博士論文

【摘要】：大豆異黃酮(Soybean Isoflavones, SIF)是一種植物雌激素,廣泛存在于豆科植物中。目前的觀點認為,SIF會干擾動物的內分泌平衡,引發(fā)內分泌紊亂,導致繁殖力降低。本研究通過在無豆粕的日糧中添加不同濃度的SIF,探討SIF對巴馬香豬初情啟動及卵泡發(fā)育的影響。試驗采用單因子試驗設計,從11窩胎齡相同、胎次相近的仔豬中,每窩選擇5頭35日齡的雌性巴馬香豬,隨機分配到5個處理組,分別給予0、250、500、1250 mg/kg的大豆異黃酮和0.66 mg/kg的戊酸雌二醇(相當于0.5 mg/kg的雌二醇),每個處理組11個重復,每個重復1頭豬,進行動物飼養(yǎng)試驗。試驗期間,準確記錄各組豬的初情日齡；在4月齡、發(fā)情第2天和第4天分別空腹采血,用于血清激素水平測定;在豬啟動初情后的第4天,每組選擇6頭豬屠宰,采集下丘腦、垂體、卵巢等組織樣品,用于熒光定量PCR、western blot、HE染色和免疫組化的檢測；從剩下的豬中,每組選擇3頭豬于第二次發(fā)情時配種,飼喂至分娩,考察SIF對雌性巴馬香豬繁殖性能的影響。此外,從附近屠宰場收集卵巢組織,分離原代顆粒細胞,探討大豆苷元和染料木黃酮對原代顆粒細胞激素合成、GDF-9/BMP-15信號通路及凋亡的影響。試驗結果如下：(1)巴馬香豬日糧中添加不同濃度的SIF導致豬的初情期延遲,240日齡時豬的發(fā)情率降低,且1250 mg/kg的SIF顯著增加了(P0.05)巴馬香豬的初情日齡。日糧中添加SIF顯著降低了(P0.05)4月齡巴馬香豬血清中的GnRH和LH水平,增加了(P0.05)FSH水平。250mg/kg添加水平的SIF顯著降低了巴馬香豬下丘腦中ER β和卵巢中ER α的mRNA表達水平,500mg/kg的SIF顯著降低了下丘腦中ERβ的表達水平,1250mg/kg的SIF顯著降低了垂體和卵巢中ER α的表達水平,且各添加水平的SIF降低了(P0.05)香豬卵巢ER β的蛋白表達水平。1250 mg/kg的SIF增加了(P0.05)垂體FSHβ的表達水平,250 mg/kg的SIF顯著降低了垂體GNRHR的表達。日糧中添加各水平的SIF極顯著降低了(P0.01)巴馬香豬下丘腦KISS1和卵巢StAR、 3β-HSD的mRNA表達水平和卵巢StAR的蛋白水平。250 mg/kg添加水平的SIF顯著降低了(P0.05)卵巢CYP17A1和CYP19A1的表達水平。此外,日糧中添加250 mg/kg的SIF顯著降低了(P0.05)巴馬香豬卵巢組織FSHR mRNA的表達水平,各水平的SIF顯著降低了(P0.05)卵巢中FSHR蛋白的表達水平。結果提示,SIF導致巴馬香豬初情期的延遲,其機制是通過改變下丘腦、垂體、卵巢中雌激素受體的表達,引起下丘腦中KISS1基因、卵巢中StAR和3 β-HSD基因和蛋白的表達下調,從而引起豬血清中激素水平的改變,導致內分泌紊亂。(2)大豆異黃酮對巴馬香豬的卵巢質量、卵巢指數及初情排卵數均沒有顯著影響(P0.05)。日糧中添加SIF減少了巴馬香豬卵巢中各期卵泡的數量,其中,1250 mg/kgSIF顯著減少了(P0.05)原始卵泡數量,500、1250 mg/kg大豆異黃酮顯著減少了(P0.05)生長期卵泡數量。日糧中添加SIF顯著降低了(P0.05)巴馬香豬卵巢組織GDF-9、BMPR-1B mRNA的表達水平,250 mg/kg的SIF顯著降低了(P0.05)卵巢BMP-15、BMPR2的mRNA的表達水平；各水平的SIF顯著降低了(P0.05)巴馬香豬卵巢中BMP-15蛋白的表達水平。日糧添加SIF顯著降低了(P0.05)巴馬香豬卵巢組織HAS2、PTGS2、Ptx3、Tnfaip6以及SMAD2的mRNA表達水平,250 mg/kg組顯著降低了(P0.05) SMAD3和SMAD4的mRNA表達水平。日糧中添加不同水平的SIF減少了產仔母豬的頭數,增加了返情母豬頭數。結果表明：大豆異黃酮還可通過調節(jié)GDF-9/BMP-15信號通路中關鍵因子的表達,來干擾豬的卵泡發(fā)育,進而影響繁殖性能。(3)日糧中添加1250 mg/kg的SIF和0.66 mg/kg的EV減少了(P0.05)巴馬香豬卵巢中閉鎖卵泡數目。500、1250 mg/kg添加水平的SIF顯著增加了(P0.05)巴馬香豬卵巢組織Bc1-2的mRNA表達水平,250 mg/kg組降低了(P0.05)Bax的mRNA表達水平,且隨著SIF含量的增加,Bcl-2的蛋白表達水平逐漸增加,Bax的蛋白表達水平逐漸降低,提示SIF可通過調節(jié)Bcl-2和Bax的表達水平減少卵泡閉鎖。(4)在體外培養(yǎng)的顆粒細胞的培養(yǎng)基中加入100、500 μM的大豆苷元和1、10、100和500州的染料木黃酮,能顯著抑制(P0.05)培養(yǎng)48 h時顆粒細胞的活力。大豆苷元和染料木黃酮均顯著抑制了(P0.05)顆粒細胞ERβ的mRNA表達水平,染料木黃酮還顯著抑制(P0.05)ER α的mRNA表達水平。0.1和1 μM的染料木黃酮則降低了(P0.05)培養(yǎng)基中孕酮的濃度。各添加水平的大豆苷元顯著降低了(P0.05)顆粒細胞3β-HSD的mRNA表達水平,而染料木黃酮則顯著降低了(P0.05)細胞StAR和3β-HSD的mRNA表達水平。同時,在培養(yǎng)基中加入0.1和10 gM的大豆苷元顯著降低了(P0.05)豬顆粒細胞CYP11A1的表達水平,而1和10μM的大豆苷元則降低了(P0.05) CYP19A1的表達水平。說明大豆苷元和染料木黃酮均能通過下調類固醇激素合成通路中關鍵基因的表達來影響顆粒細胞雌二醇和孕酮的合成與分泌。在體外培養(yǎng)的豬顆粒細胞培養(yǎng)基中加入0.1μM的染料木黃酮能顯著增加(P0.05)BMPR2的表達水平。大豆苷元和染料木黃酮均顯著降低了(P0.05)豬顆粒細胞Ptx3和HAS2基因的表達水平,10 μM的大豆苷元降低了(P0.05) PTGS2的表達水平,1、10μM的大豆苷元顯著降低了(P0.05) Tnfaip6的表達水平,且染料木黃酮均顯著降低了(P0.05)顆粒細胞Tnfaip6的表達水平。說明給體外培養(yǎng)的豬顆粒細胞的培養(yǎng)基中加入不同濃度的大豆苷元和染料木黃酮,雖然不改變GDF-9/BMP-15受體的表達水平,但是能顯著下調其下游調控因子的表達水平。在體外培養(yǎng)的豬顆粒細胞培養(yǎng)基中加入1、10 μM的大豆苷元減少了(P0.05)細胞的早期凋亡率,1 μM的大豆苷元還減少了(P0.05)細胞的晚期凋亡率和總凋亡率,0.1、1μM的大豆昔元減少了(P0.05)死亡細胞的百分率。不同濃度的染料木黃酮減少了(P0.05)豬顆粒細胞的早期凋亡率,但是增加了(P0.05)晚期凋亡率、總凋亡率和死亡細胞的百分率。添加0.1μM的大豆苷元顯著增加了(P0.05)顆粒細胞Bcl-2/Bax的比例。在培養(yǎng)基中加入不同濃度的染料木黃酮,降低了顆粒細胞Bcl-2的表達水平,其中添加1 μM的水平達到顯著性水平(P0.05)；添加10 μM的染料木黃酮則增加了Bax的表達水平(P0.05),說明低濃度的大豆苷元和染料木黃酮能抑制顆粒細胞的早期凋亡,而高濃度則能引起顆粒細胞凋亡甚至死亡。由以上試驗結果得出,大豆異黃酮作用于巴馬香豬,導致香豬初情期延遲,卵泡發(fā)育發(fā)生障礙,其機理主要為：1)大豆異黃酮能改變下丘腦、垂體、卵巢組織雌激素受體的表達水平,影響GnRH、LH、FSH及類固醇激素合成中關鍵因子的表達,從而影響激素的合成和分泌,引起香豬激素調節(jié)改變；2)大豆異黃酮能下調GDF-9/BMP-15信號通路中關鍵因子的表達,減少卵巢中各期卵泡的數量,從而影響卵泡的正常發(fā)育和香豬的繁殖性能；3)顆粒細胞的體外培養(yǎng)試驗也表明,大豆苷元和染料木黃酮均能改變細胞雌激素受體、固醇激素合成關鍵基因、GDF-9下游因子的基因表達,從而影響固醇激素的合成和分泌。由此可知,大豆異黃酮作為飼料添加劑,或者后備母豬日糧配方中使用高水平的豆粕需要慎重考慮。
[Abstract]:Soybean Isoflavones (SIF) is a kind of phytoestrogens, which is widely found in leguminous plants. The present view is that SIF interferes with the endocrine balance of animals, causes endocrine disorders and leads to the decrease of fecundity. In this study, the initial condition of SIF to Bama Xiang pigs was discussed by adding different concentrations of SIF in the diet without soybean meal. The effect of dynamic and follicular development. A single factor test was used to select 5 female Bama pigs of 35 days of age at the same age of 11 nests and similar fetal times. 5 treatment groups were randomly assigned to 02505001250 mg/kg of soybean isoflavones and 0.66 mg/kg of valerate (equal to 0.5 mg/kg estradiol). Each treatment group had 11 repetitions, each repeated 1 pigs, and the animal feeding test was carried out. During the trial, the initial age of the pigs was recorded, and the blood was collected on the empty abdomen in 4 month old, second and fourth days, respectively, for the determination of serum hormone levels, and 6 pigs were slaughtered in each group for fourth days after the start of the initial situation, and the hypothalamus, pituitary and ovary were collected. Tissue samples were used for the detection of fluorescent quantitative PCR, Western blot, HE staining and immunohistochemical staining. From the remaining pigs, 3 pigs were selected for second estrus, feeding to childbirth, and the effects of SIF on the reproductive performance of female Bama Xiang Pigs. The effects of glycosides and genistein on the synthesis of hormone, GDF-9/BMP-15 signaling pathway and apoptosis in primary granulosa cells. The results were as follows: (1) the initial stage delay of pigs was delayed by adding different concentrations of SIF in the diet of Bama Xiang pig, and the oestrus rate of pigs decreased at the age of 240 days, and the 1250 mg/kg SIF significantly increased the initial day of (P0.05) Bama Xiang pig The addition of SIF in the diet significantly reduced the level of GnRH and LH in the serum of (P0.05) 4 month old Bama Xiang Pigs, and the SIF significantly reduced the mRNA expression of ER alpha in the hypothalamus of Bama Xiang Pigs, which was significantly reduced by SIF (P0.05) FSH level.250mg/kg addition level. F significantly reduced the expression level of ER alpha in the pituitary and ovary, and the levels of SIF decreased (P0.05) the protein expression level of ER beta in the ovary of the pigs increased (P0.05) the expression level of the pituitary FSH beta, and the 250 mg/kg SIF significantly reduced the expression of pituitary gland. The mRNA expression level of 3 beta -HSD in the hypothalamus of Bama Xiang pig and the mRNA expression level of 3 beta -HSD and the protein level of the ovarian StAR significantly decreased the level of the expression of CYP17A1 and CYP19A1 in the ovary (P0.05). In addition, the expression level of the ovarian tissue of Bama Xiang pig was significantly reduced by the addition of 250 mg/kg in the diet. The horizontal SIF significantly reduced the expression level of FSHR protein in the ovary of (P0.05). The results suggested that SIF led to the delay in the early stage of Bama Xiang pig. The mechanism was to change the expression of estrogen receptor in the hypothalamus, pituitary, and ovary, causing the KISS1 gene in the hypothalamus, the expression of StAR and 3 beta -HSD in the ovary, and thus causing swine blood. The changes in hormone levels in the Qing Dynasty led to endocrine disorders. (2) soybean isoflavones had no significant effects on the ovarian quality, ovarian index and initial oviposit number of Bama Xiang Pigs (P0.05). The addition of SIF to diet reduced the number of follicles in the ovary of Bama Xiang Pigs, of which 1250 mg/kgSIF significantly reduced the number of original follicles (P0.05), 5001250 M G/kg soybean isoflavones significantly reduced the number of follicles in the growth period of (P0.05). The addition of SIF in the diet significantly reduced the expression level of GDF-9 and BMPR-1B mRNA in the ovarian tissue of Bama Xiang Pigs (P0.05), and the SIF of 250 mg/kg decreased significantly (P0.05) ovary BMP-15, and the expression of BMPR2 was flat. BMP-15 protein expression level. Dietary supplementation of SIF significantly reduced the mRNA expression level of HAS2, PTGS2, Ptx3, Tnfaip6 and SMAD2 in the ovarian tissue of (P0.05) Bama Xiang pig. The 250 mg/kg group significantly reduced the level of SMAD3 and the expression level of SMAD2. The results showed that soybean isoflavones could also interfere with the development of porcine follicle by regulating the expression of key factors in the GDF-9/BMP-15 signal pathway, and then affect the reproductive performance. (3) adding 1250 mg/kg SIF and 0.66 mg/kg EV in the diet reduced the SIF display of the number of.5001250 mg/kg in the ovaries of (P0.05) Bama Xiang pig. The mRNA expression level of Bc1-2 in the ovarian tissue of Bama Xiang pig was increased (P0.05). The 250 mg/kg group reduced the mRNA expression level of (P0.05) Bax, and with the increase of SIF content, the expression level of Bcl-2 protein gradually increased and the protein expression level of Bax decreased gradually, suggesting that SIF could reduce the follicle atresia by regulating Bcl-2 and the expression level. (4) 100500 M daidzein and 1,10100 and genistein in 500 state were added to the culture medium of the cultured granulosa cells, which could significantly inhibit the activity of granulosa cells in (P0.05) culture of 48 h. Daidzein and genistein significantly inhibited the mRNA expression level of ER beta in (P0.05) granulosa cells, and genistein also significantly inhibited (P0.05) E The mRNA expression level of R alpha and genistein of 1 mu M decreased the concentration of progesterone in the (P0.05) medium. The levels of daidzein significantly reduced the mRNA expression level of 3 beta -HSD in (P0.05) granulosa cells, while genistein significantly reduced the mRNA expression level of StAR and 3 beta -HSD in (P0.05) cells. Daidzein into 0.1 and 10 gM significantly reduced the expression level of CYP11A1 in pig granulosa cells, while 1 and 10 M daidzein decreased the expression level of (P0.05) CYP19A1. It indicated that daidzein and genistein could affect the estradiol in granular cells by downregulating the expression of the key genes in the steroid hormone synthesis pathway. The synthesis and secretion of progesterone. The addition of 0.1 M genistein to the culture medium of porcine granulosa cells in vitro could significantly increase the expression level of (P0.05) BMPR2. Both daidzein and genistein significantly reduced the expression level of Ptx3 and HAS2 genes in (P0.05) pig granulosa cells, and 10 UG daidzein decreased the expression of (P0.05) PTGS2. Level, 1,10 mu M daidzein significantly reduced the expression level of (P0.05) Tnfaip6, and genistein significantly reduced the expression level of Tnfaip6 in (P0.05) granulosa cells. It indicated that the different concentrations of daidzein and genistein were added to the culture medium of pig granulosa cells in vitro, although the GDF-9/BMP-15 receptor was not changed. The expression level of the downstream regulatory factors was significantly reduced. The early apoptosis rate of (P0.05) cells was reduced by the addition of 1,10 mu M daidzein in the culture medium of porcine granulosa cells in vitro. The 1 u M daidzein also reduced the late apoptosis rate and total apoptosis rate of (P0.05) cells, and the soy celecoxib of 0.1,1 mu M decreased (P0.0). 5) the percentage of dead cells. The different concentrations of genistein reduced the early apoptosis rate of (P0.05) pig granulosa cells, but increased the rate of late apoptosis, the total apoptosis rate and the percentage of dead cells. The proportion of daidzein adding 0.1 mu M significantly increased the proportion of Bcl-2/Bax in the (P0.05) granulocyte. Different concentrations were added to the medium. Genistein reduced the expression level of Bcl-2 in granular cells, in which the level of adding 1 mu M reached a significant level (P0.05), and the expression level of Bax was increased by the addition of 10 M genistein (P0.05), indicating that the low concentration of daidzein and genistein could inhibit the early apoptosis of the granulosa cells, while the high concentration could cause a grain. The effects of soybean isoflavones on Bama Xiang pig, which resulted in the delayed initial stage and the development of follicle development, were mainly: 1) soybean isoflavones could change the expression level of estrogen receptor in the hypothalamus, pituitary and ovary, and affect the synthesis of GnRH, LH, FSH and steroid hormones. The expression of key factors, which affects the synthesis and secretion of hormone, causes the regulation changes of the corticosteroids; 2) soybean isoflavones can downregulate the expression of key factors in the GDF-9/BMP-15 signaling pathway, reduce the number of follicles in each stage of the ovary, and thus affect the normal development of follicles and the reproductive performance of the fragrant pigs; 3) in vitro culture test of granular cells It is also indicated that daidzein and Genistein can change the cell estrogen receptor, the key gene of steroid hormone synthesis, the gene expression of the downstream factor of GDF-9, thereby affecting the synthesis and secretion of steroid hormones. Thus, soy isoflavones as feed additives, or high level soybean meal in the reserve sow formula, need caution. Serious consideration.

【學位授予單位】：湖南農業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S828

【相似文獻】

相關期刊論文 前10條

1 王占元;袁淑芹;盧景都;祝云江;;巴馬香豬寒地擴繁效益分析[J];養(yǎng)殖技術顧問;2010年02期

2 周澤平;;巴馬香豬[J];湖南農業(yè);2013年08期

3 魯春霞;袁淑芹;王占元;徐占東;;巴馬香豬的引進與飼養(yǎng)[J];養(yǎng)殖技術顧問;2010年02期

4 魯春霞;祝云江;楊曉明;袁淑芹;;北方寒地引進繁育巴馬香豬的工作及其效果[J];養(yǎng)殖技術顧問;2010年03期

5 孫強東;;巴馬香豬閹割技術及其注意事項[J];養(yǎng)殖與飼料;2011年07期

6 王愛德，蘭干球，郭亞芬;巴馬香豬耐熱性的探討[J];家畜生態(tài);1995年04期

7 張自勤;長壽之鄉(xiāng)的巴馬香豬[J];中國牧業(yè)通訊;1998年05期

8 張承露;曾德軍;趙貴軍;;巴馬香豬耐熱性解析[J];湖北畜牧獸醫(yī);2014年02期

9 ;廣西巴馬香豬[J];科技致富向導;2009年03期

10 劉偉;;能拎著走的烤全豬——巴馬香豬[J];農業(yè)知識;2012年07期

相關會議論文 前6條

1 曾本華;劉宇;黃亞琴;岑彥艷;鄭書全;魏泓;;洛伐他汀在巴馬香豬體內的絕對生物利用度研究[A];中國實驗動物學會第七屆學術年會論文集[C];2006年

2 劉宇;曾本華;岑艷燕;黃亞琴;李健;魏泓;;巴馬香豬在洛伐他汀體外代謝的酶動力學上與人和大鼠的比較研究[A];中國實驗動物學會第七屆學術年會論文集[C];2006年

3 孔德營;;通過正交實驗設計優(yōu)化巴馬香豬精液凍存方法[A];中國畜牧獸醫(yī)學會——第三屆中國獸醫(yī)臨床大會論文集[C];2012年

4 張小杰;曾俊棋;王麗娟;朱誠其;岳萬福;;高通量16S測序法比較不同食性豬腸道細菌結構差異[A];浙江省動物學會第十二屆會員代表大會暨學術討論會論文摘要集[C];2013年

5 徐漢進;雍艷紅;巨向紅;趙云濤;安立龍;效梅;許英梅;;SYBR GreenⅠ實時熒光定量PCR檢測巴馬香豬Toll樣受體2 mRNA方法的建立[A];中國畜牧獸醫(yī)學會獸醫(yī)產科學分會第五屆全體會議第十次學術研討會論文集[C];2009年

6 楊家大;商海濤;魏泓;楊婉身;劉昕;;實時熒光定量檢測小型豬肝臟CYP3A29 mRNA表達水平[A];中國實驗動物學會第七屆學術年會論文集[C];2006年

相關重要報紙文章 前8條

1 記者/師曉鵑 曾永聯(lián) 見習記者/韋德斌;巴馬香豬亮相博覽會[N];廣西日報;2004年

2 本報記者 王銘澤　于立兵 通訊員 周兵;女礦老板迷上飼養(yǎng)巴馬香豬[N];中國特產報;2010年

3 記者 周映;巴馬香豬獲得地理標志產品保護[N];廣西日報;2005年

4 張元躍;廣西巴馬香豬有了“護身符”[N];中國食品質量報;2005年

5 楊明遠　記者 簡放鵬;“巴馬香豬”落戶天仙示范片[N];瀘州日報;2010年

6 本報記者 張長虹;沒上餐桌卻登“臺”[N];黑龍江日報;2010年

7 陳振 記者 崔海峰;綠野長壽食品喜獲殊榮[N];中國食品質量報;2004年

8 記者 王敏　吳德群;基因“迷你豬”打入寵物市場[N];深圳特區(qū)報;2011年

相關博士學位論文 前6條

1 范覺鑫;大豆異黃酮對巴馬香豬初情啟動及卵泡發(fā)育的影響及其機制[D];湖南農業(yè)大學;2015年

2 孔德營;N-乙基-N-亞硝基脲對巴馬香豬精液品質及精子發(fā)生影響的研究[D];西南大學;2013年

3 江雯;皮膚靶向表達hCTLA-4Ig轉基因巴馬香豬種群建立及其異種移植研究[D];第三軍醫(yī)大學;2012年

4 商海濤;實驗用小型豬細胞色素P450家族新基因克隆及其CYP3A29基因多態(tài)研究[D];第三軍醫(yī)大學;2007年

5 劉宇;以洛伐他汀為典型藥物研究巴馬香豬和人代謝的異同[D];第三軍醫(yī)大學;2008年

6 陳俊穎;巴馬香豬皮膚比較生物學特性及其無細胞真皮的研究[D];第三軍醫(yī)大學;2006年

相關碩士學位論文 前10條

1 祝明皓;巴馬香豬CIK細胞培養(yǎng)體系建立及體外殺傷活性研究[D];東北農業(yè)大學;2016年

2 褚毅;豬高糖高脂飼喂條件下肝臟轉錄組分析[D];廣西大學;2016年

3 徐海艷;低頻強噪聲對巴馬香豬內耳損傷及機制研究[D];中國人民解放軍醫(yī)學院;2016年

4 李艷君;miR-208b和miR-499在巴馬香豬骨骼肌肌纖維類型轉化中的作用研究[D];廣西大學;2015年

5 黃濤;豬皮膚褶皺和厚度的初步遺傳學分析[D];江西農業(yè)大學;2016年

6 黃亞琴;洛伐他汀在巴馬香豬體內的毒代動力學及毒效學研究[D];第三軍醫(yī)大學;2006年

7 段超偉;實驗用巴馬香豬繁殖性能與仔豬生長發(fā)育性能測定[D];西南大學;2012年

8 陳玉龍;巴馬香豬肝硬化模型的建立及其腸道菌群演替的研究[D];西南大學;2010年

9 敖秋桅;骨骼肌肌纖維類型與巴馬香豬肉質性狀的相關性研究[D];廣西大學;2014年

10 宋玉東;巴馬香豬肝硬化形成過程中細菌移位的初步研究[D];西南大學;2010年

，

本文編號：1776670