【摘要】：當前,卵巢靜止已成為集約化牛場高產(chǎn)奶牛產(chǎn)后常見的卵泡生長異常的疾病。其特征為卵巢或相關的卵巢結構出現(xiàn)無周期性特征的靜止狀態(tài)。奶牛產(chǎn)后卵巢上卵泡生長受到諸多因素的影響。盡管能量負平衡被公認為奶牛產(chǎn)后卵巢靜止的主要病因,但是奶牛產(chǎn)后發(fā)生卵巢靜止時,機體整體蛋白表達的情況,以及與該病的關系還未見報道。為此,本試驗應用蛋白組學中熒光差異雙向凝膠電泳(Fluorescence Two-dimensional Differential Gel Electrophoresis,2D-DIGE)與基質輔助激光解析離子化飛行時間質譜(MatrixAssisted Laser Desorption/Lonization Time of Flight Mass Spectrometry,MALDI-TOF-MS)聯(lián)用技術,結合生物信息學分析,獲得了能量負平衡所致的卵巢靜止奶牛血清差異表達蛋白,探究了這些差異表達蛋白與卵巢靜止之間的關系,為今后深入探索奶牛產(chǎn)后卵巢靜止發(fā)生機制提供科學依據(jù)。本試驗在黑龍江某集約化奶牛場選取了年齡、胎次相近的經(jīng)產(chǎn)奶牛,在產(chǎn)后14~21d根據(jù)血清中β-羥丁酸(β-hydroxybutyrate,BHBA)、游離脂肪酸(Non esterified fatty acid,NEFA)和葡萄糖(Glucose,Glc)含量將其分為能量正平衡和能量負平衡組。兩組奶牛跟蹤至產(chǎn)后60~90d,根據(jù)有無發(fā)情表現(xiàn),結合B超與直腸檢查卵巢卵泡狀態(tài),最終確定了30頭能量負平衡的卵巢靜止奶牛為實驗組(T)和30頭能量正平衡的發(fā)情奶牛為健康對照組(C)。隨機選取兩組(T或C)奶牛各15頭,應用2D-DIGE/MS技術檢測兩組奶牛血清樣品獲得兩組奶牛的差異表達蛋白。通過生物信息學分析,確定差異蛋白與卵巢靜止的關系。應用Western-blot技術對血清中AMBP蛋白復合體(alpha-1-microglobulin/bikunin precursor,α1微球蛋白/bikunin蛋白前體)進行驗證。兩組(T或C)中剩余15頭奶牛,檢測血清中生殖激素和AMBP蛋白復合體含量,并進行彼此的相關性分析,結合回歸分析和受試者工作特征曲線(receiver operating characteristic curve,ROC),確定AMBP蛋白復合體對卵巢靜止的預警作用。結果顯示:(1)通過2D-DIGE/MALDI-TOF-MS檢測和差異分析,發(fā)現(xiàn)卵巢靜止組和發(fā)情組奶牛之間有12種差異表達蛋白質,其中AMBP蛋白復合體(Protein AMBP)、α-S1-酪蛋白(Alpha-S1-casein)、血紅蛋白(Hemoglobin subunit beta)和結合珠蛋白(Haptoglobin)等4種蛋白表達上調,另外載脂蛋白A1(Apolipoprotein A-I)、載脂蛋白A4(Apolipoprotein A-IV)、α-1-酸性糖蛋白(Alpha-1-acid glycoprotein)、α-2-HS-糖蛋白(Alpha-2-HS-glycoprotein)、亮氨酸富集α-2-糖蛋白1(Leucine-rich alpha-2-glycoprotein 1)、補體3d(Complement component 3d(Fragment))、補體C3(Complement C3)和絲氨酸蛋白酶抑制劑E2C(Endopin 2C)等8種蛋白表達下調。通過生物信息學分析發(fā)現(xiàn)這些差異表達蛋白的基因功能主要與類固醇和脂質代謝,酶活性和抗氧化作用有關;參與的主要通路是脂肪和維生素消化與吸收,PPAR信號通路和補體與凝血級聯(lián)。AMBP蛋白的Western-blotting結果與2D-DIGE的一致,都上調。(2)能量負平衡的卵巢靜止組奶牛產(chǎn)后60~90d血清NEFA含量均顯著高于健康發(fā)情組(p0.05),血清雌激素(Estrogen,E2)含量顯著低于發(fā)情奶牛(p0.05),血清AMBP蛋白復合體濃度極顯著高于健康發(fā)情組奶牛(p0.01);血清中E2濃度與試驗動物分組情況呈極顯著正相關(p0.01),AMBP蛋白復合體與試驗動物分組呈極顯著負相關(p0.01);血清NEFA與E2含量呈顯著負相關(p0.01);通過回歸分析和ROC分析確定了當奶牛血清中AMBP4268.59 ng/L時,奶�；悸殉察o止的風險增大。結論:本研究應用蛋白質組學技術獲得了能量負平衡所致的卵巢靜止奶牛血清差異表達蛋白,探討了差異表達蛋白質與奶牛產(chǎn)后卵巢靜止之間的關系,確定了AMBP蛋白復合體在奶牛產(chǎn)后卵巢靜止發(fā)生的風險預警作用,為今后深入揭示能量負平衡所致的卵巢靜止發(fā)生機理提供了新的方向。
[Abstract]:At present, ovarian motility has become a common postpartum follicular growth disorder in high-yielding dairy cows in intensive farms. It is characterized by a stationary state of ovaries or related ovarian structures with no periodic characteristics. However, the expression of whole body protein and its relationship with the disease have not been reported in postpartum dairy cows with ovarian motility. In this study, fluorescence Two-dimensional Differential Gel Electrophoresis (2D-DIGE) and matrix-assisted laser resolution were applied to isolate the disease. Matrix Assisted Laser Desorption/Lonization Time of Flight Mass Spectrometry (MALDI-TOF-MS) combined with bioinformatics analysis was used to obtain differentially expressed proteins in ovarian resting cow serum induced by negative energy balance. In this study, we selected the age and parity of the same type of dairy cows from an intensive dairy farm in Heilongjiang Province. According to the serum levels of beta-hydroxybutyrate (BHBA), non-esterified fatty acid (NEFA) and glucose (Glucose, Glc) from 14 to 21 days postpartum. Two groups of dairy cows were followed up to 60-90 days postpartum. According to oestrus and ovarian follicular status, 30 calorie-balanced ovarian stationary cows were selected as experimental group (T) and 30 calorie-balanced calorie cows as healthy control group (C). Fifteen dairy cows in each group (T or C) were used to detect the differentially expressed proteins in the serum of the two groups. The relationship between the differentially expressed proteins and ovarian motility was determined by bioinformatics analysis. The serum AMBP protein complex (alpha-1-microglobulin/bikunin precursor, alpha-1 microspheres) was detected by Western blot. Serum levels of reproductive hormones and AMP protein complexes were measured in 15 dairy cows in two groups (T or C), and their correlation was analyzed. Combined with regression analysis and receiver operating characteristic curve (ROC), the resting ovarian function of AMBP protein complexes was determined. The results showed that: (1) Through two-dimensional DIGE/MALDI-TOF-MS detection and differential analysis, 12 differentially expressed proteins were found between ovarian resting group and estrus group, including AMBP protein complex (Protein AMBP), alpha-S1-casein (Alpha-S1-casein), hemoglobin subunit beta (Hemoglobin subunit beta) and Haptoglobin (Haptoglobin). Apolipoprotein A-I, Apolipoprotein A-IV, Alpha-1-acid glycoprotein, Alpha-2-HS-glycoprotein, Leucine-rich alpha-2-glycoprotein 1, Complement 3D Bioinformatics analysis showed that the gene functions of these differentially expressed proteins were mainly related to steroid and lipid metabolism, enzyme activities and antioxidant activities, and the main pathways involved were fat and vitamin digestion and absorption, and PPA. Western-blotting results of AMBP protein were consistent with those of 2D-DIGE. (2) Negative energy balance ovarian motility group had significantly higher serum NEFA content than healthy estrus group (p0.05), lower serum estrogen (Estrogen, E2) content than estrus cows (p0.05), and serum AMBP protein complex. Body concentration was significantly higher than that of healthy estrous cows (p0.01); E2 concentration in serum was significantly positively correlated with the grouping of experimental animals (p0.01); AMBP protein complex was significantly negatively correlated with the grouping of experimental animals (p0.01); serum NEFA and E2 content was significantly negatively correlated (p0.01); regression analysis and ROC analysis showed that the serum of dairy cows was positively correlated with the grouping of experimental animals (p0.01). CONCLUSION: In this study, the serum differentially expressed proteins in ovarian quiescent cows induced by negative energy balance were obtained by proteomics, and the relationship between differentially expressed proteins and postpartum ovarian quiescent in dairy cows was discussed. The risk warning function of resting ovarian motility provides a new direction for further revealing the mechanism of resting ovary caused by negative energy balance.
【學位授予單位】：黑龍江八一農(nóng)墾大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.23

【相似文獻】

相關期刊論文 前10條

1 程云;中藥促孕灌注液對奶牛卵巢靜止的療效驗證[J];畜牧與獸醫(yī);1989年03期

2 王愛春;;低功率激光療法對乳牛卵巢靜止的效果[J];青海畜牧獸醫(yī)雜志;1989年01期

3 梁光華;鄧敏;徐雷;劉賢俠;高樹;羅燕;賽務加甫;谷新利;;促孕散治療卵巢靜止不孕奶牛血清白介素與激素的相關性分析[J];中獸醫(yī)學雜志;2013年05期

4 鐘清蓮，林峰，林嵩海;母牛卵巢靜止的治療[J];甘肅畜牧獸醫(yī);1994年04期

5 鄧敏;梁光華;徐雷;劉賢俠;高樹;羅燕;賽務加甫;谷新利;;促孕散治療卵巢靜止不孕奶牛血清微量元素與激素的相關性分析[J];黑龍江畜牧獸醫(yī);2014年03期

6 韓學義;徐大文;;淺議奶牛產(chǎn)后卵巢靜止及萎縮的治療[J];科學種養(yǎng);2011年10期

7 鄧敏;梁光華;谷新利;劉賢俠;高樹;徐雷;;促孕散對卵巢靜止不孕奶牛血清中激素和微量元素含量的影響[J];石河子大學學報(自然科學版);2012年03期

8 康曉冬;吳韶儒;范春梅;;奶牛產(chǎn)后卵巢靜止的診斷與治療[J];黃牛雜志;2005年06期

9 侯樂樂;代永聯(lián);郝小靜;張廷榮;;奶牛卵巢靜止及其中西療法的療效比較[J];北方牧業(yè);2014年11期

10 祁生旺;喬振國;娜仁;康美榮;劉新陸;段愛蓮;;奶牛高燒性疾病后繼發(fā)卵巢靜止的治療試驗[J];畜牧與飼料科學(畜牧版);2006年01期

相關會議論文 前2條

1 高樹;楊恒;胡路鋒;劉賢俠;谷新利;王建梅;陳愛江;汪嘉來;李明;楊文生;;口服促孕散治療卵巢靜止奶牛B超影像學的研究[A];第四屆中國奶業(yè)大會論文集[C];2013年

2 張?zhí)於?李俊松;陳亮;;GnRH對治療奶牛卵巢靜止的效果分析[A];中國奶業(yè)協(xié)會第24次繁殖學術年會暨國家奶牛/肉牛產(chǎn)業(yè)技術體系第一屆全國牛病防治學術研討會論文集[C];2009年

相關重要報紙文章 前1條

1 上海光明荷斯坦牧業(yè)有限公司 張金華;生殖激素在實際生產(chǎn)中的應用[N];中國畜牧報;2004年

相關博士學位論文 前1條

1 胡元亮;中藥治療母畜某些不孕癥及其機理研究[D];南京農(nóng)業(yè)大學;1999年

相關碩士學位論文 前3條

1 許楚楚;基于氣相色譜/質譜聯(lián)用技術的產(chǎn)后卵巢靜止奶牛血漿代謝組學輪廓分析[D];黑龍江八一農(nóng)墾大學;2016年

2 肖鑫煥;能量負平衡奶牛產(chǎn)后卵巢靜止血清蛋白質組學分析及其預警評估[D];黑龍江八一農(nóng)墾大學;2017年

3 富宏騫;中藥治療奶牛卵巢靜止的研究[D];東北農(nóng)業(yè)大學;2008年

，

本文編號：2225709