本文選題：奶牛　切入點(diǎn)：laminin　出處：《河南農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：本試驗(yàn)主要探討了laminin在奶牛體外受精及胚胎發(fā)育中的影響,同時對提高牛體外胚胎數(shù)量與質(zhì)量的方法進(jìn)行了研究,旨在建立一套比較完善的IVP技術(shù)體系。主要研究內(nèi)容為:卵母細(xì)胞的體外成熟、體外受精及整合素β1在精卵融合中的作用、評價(jià)胚胎質(zhì)量技術(shù)的優(yōu)化和早期胚胎的體外培養(yǎng)。研究結(jié)果如下:1.卵丘細(xì)胞對牛卵母細(xì)胞體外成熟的影響試驗(yàn)結(jié)果顯示:添加離散卵丘細(xì)胞能夠顯著促進(jìn)DOs的體外成熟(57.98±14.27 vs35.53±14.00,P0.05),對COCs具有促進(jìn)趨勢但差異不顯著(76.66±5.77 vs 66.76±9.46,P0.05);卵母細(xì)胞胞外卵丘細(xì)胞對與之共培養(yǎng)的DOs的體外成熟促進(jìn)作用效果不明顯(35.83±18.32 vs 35.53±14.00,P0.05)。分析結(jié)果顯示:卵母細(xì)胞胞外卵丘細(xì)胞主要通過間隙連接促進(jìn)卵母細(xì)胞的體外成熟,添加的離散卵丘細(xì)胞主要以旁分泌途徑促進(jìn)卵母細(xì)胞的體外成熟;3層及其以上胞外卵丘細(xì)胞能夠提高其包裹的卵母細(xì)胞平均體外成熟能力的85.34%,添加的離散卵丘細(xì)胞能夠提高COC平均體外成熟能力的16.18%,能夠提高DO平均體外成熟能力的60.61%。2.不同受精液及有無卵丘細(xì)胞對牛體外成熟卵母細(xì)胞體外發(fā)育的影響試驗(yàn)研究了體外成熟卵母細(xì)胞在IVF-100、BO液和TALP液三種體外受精液中的受精效果,同時比較了裸卵(NOs)與卵丘卵母細(xì)胞復(fù)合體(COCs)對受精效果的影響。結(jié)果顯示體外成熟卵母細(xì)胞在受精液IVF-100中的卵裂率顯著高于BO液的(0.7055±0.0745 VS0.5794±0.0956,P0.05),而TALP液中的卵裂率與IVF-100和BO液的差異都不顯著。NOs的卵裂率雖然低于COCs的,但差異不顯著;NOs能夠發(fā)育到囊胚階段,但囊胚率極顯著低于COCs的。說明BO液和TALP液作為體外受精液還有很大的改進(jìn)空間,NOs的體外發(fā)育能力顯著低于COCs的。3.laminin在牛早期胚胎中的分布及添加層粘連蛋白對體外胚胎發(fā)育的影響將經(jīng)IVM-IVF的牛卵子移入含不同濃度laminin(0、5、10 and 20μg/ml)的體外培養(yǎng)基中進(jìn)行培養(yǎng),并對不同階段胚胎的發(fā)育情況進(jìn)行檢測。結(jié)果顯示10μg/ml組卵裂率及囊胚率顯著高于對照組(0.8031±0.0667 vs 0.7039±0.0688,P0.05;0.4835±0.0167 vs0.3765±0.0257,P0.05)。雖然受精48h后各試驗(yàn)組4-8cell數(shù)與對照組之間差異不顯著,但5、10μg/ml組的囊胚細(xì)胞團(tuán)總數(shù)顯著高于對照組(97.00±8.24、98.71±13.59 vs87.86±6.22,P0.05)。在滋養(yǎng)層細(xì)胞數(shù)上試驗(yàn)組與對照組之間無差異,各組內(nèi)細(xì)胞團(tuán)數(shù)與囊胚細(xì)胞團(tuán)總數(shù)的比值均接近0.33。在對0-8天牛體外胚胎層粘連蛋白的分布檢測試驗(yàn)顯示,最早可于8細(xì)胞胚胎檢測到laminin的表達(dá)分布,在16細(xì)胞到囊胚階段層laminin的表達(dá)呈遞增趨勢,并于桑囊胚階段大量表達(dá)。在囊胚及擴(kuò)張囊胚階段同時還能檢測到滋養(yǎng)層細(xì)胞laminin部分的表達(dá)分布情況。結(jié)果表明:laminin隨胚齡的增加需求量不斷加大,添加一定量的的外源性laminin能夠在不同程度上提高體外胚胎的卵裂率、囊胚率以及囊胚細(xì)胞數(shù);運(yùn)用免疫熒光技術(shù)最早可于8細(xì)胞階段檢測到奶牛胚胎laminin的表達(dá)分布,而且在桑囊胚階段能夠檢測到laminin的大量表達(dá)。4.牛體外胚胎質(zhì)量評定技術(shù)的優(yōu)化為建立快速、可靠的評估牛囊胚質(zhì)量的雙重染色方法,本試驗(yàn)對4種雙重染色方案進(jìn)行了改進(jìn)。結(jié)果顯示改進(jìn)的方案一、方案三及方案四都能達(dá)到牛囊胚雙重染色的效果。改進(jìn)的方案三、方案四囊胚細(xì)胞總數(shù)與Hoechst33342染色結(jié)果無差異(84.33±9.98 VS83.63±9.74,P0.05;93.00±10.50 VS83.63±9.74,P0.05),而且囊胚內(nèi)細(xì)胞團(tuán)(ICM)數(shù)與囊胚細(xì)胞總數(shù)的比值分別為0.3382與0.3356,均接近0.33。改進(jìn)的方案四染色最為穩(wěn)定,效果較好,整個染色時間不到3分鐘,極大的提高了染色效率。試驗(yàn)結(jié)果表明改進(jìn)的方案三、方案四都可以作為一種快速簡潔有效的牛囊胚質(zhì)量鑒別方法。5.整合素β1在精卵融合中的作用此試驗(yàn)通過不同條件下laminin與anti-β1對整合素β1的阻斷來探索整合素β1在精卵融合中的作用,同時對laminin特意消失于受精階段的原因進(jìn)行分析。結(jié)果顯示當(dāng)分別用laminin與anti-β1對卵子上的整合素β1進(jìn)行阻斷時,laminin能極顯著的影響受精(10μg/ml:67.38±5.41,20μg/ml:71.51±4.96 VS 86.04±3.73,P0.01)及卵裂(10μg/ml:41.43±8.33VS 59.30±7.85,P0.01),而anti-β1對受精及卵裂的影響不大。當(dāng)分別將laminin與anti-β1添加到受精液中,對laminin肝素受體進(jìn)行封閉時,結(jié)果顯示隨laminin濃度的升高,對精卵融合的影響顯著增加(10μg/ml:80.36±1.92 VS 86.04±3.73,P0.05;20μg/ml:64.95±7.61 VS86.04±3.73,P0.01),但對卵裂的影響不大;而anti-β1能極顯著的影響受精(50μg/ml:29.49±11.39,100μg/ml:34.98±7.54 VS 86.04±3.73,P0.01)及卵裂(50μg/ml:5.44±7.37,100μg/ml:3.65±6.84 VS 59.30±7.85,P0.01)。當(dāng)分別用laminin與anti-β1對精子表面上的整合素β1進(jìn)行阻斷時,結(jié)果顯示兩者都顯著的影響受精及卵裂(P0.05),而且laminin濃度越大,顯著性越強(qiáng)。同時,當(dāng)分別用laminin與anti-β1對精子表面和卵子表面整合素β1進(jìn)行阻斷再進(jìn)行體外受精時,結(jié)果顯示兩者都極顯著的影響精卵的融合(P0.01),卵裂率上除50μg/ml anti-β1組外都極顯著的低于對照組(P0.01)。結(jié)果表明,整合素β1參于了奶牛體外精卵融合的過程;阻斷精子表面上的整合素β1能極顯著的阻礙精卵的融合,而阻斷卵子表面上的整合素β1對受精影響不大;laminin在受精過程中的消失可能是由于對整合素β1及肝素的阻斷而阻礙了精卵的正常融合。
[Abstract]:This experiment mainly discusses the influence of laminin in dairy cows in vitro fertilization and embryo development, and the methods to improve the quantity and quality of bovine embryo in vitro were studied to establish a perfect IVP system. The main contents are as follows: in vitro maturation of oocytes, in vitro fertilization and integrin beta 1 in sperm egg fusion and optimization of cultivation and early embryo quality evaluation technology of embryo in vitro. The results are as follows: 1. effects of cumulus cells on in vitro maturation of bovine oocytes showed that the addition of discrete cumulus cells could significantly promote DOs maturation in vitro (57.98 + 14.27 vs35.53 + 14, P0.05), but the difference is to promote the trend there was no significant difference on the COCs (76.66 + 5.77 vs 66.76 + 9.46, P0.05); in vitro oocyte cumulus cells on extracellular co cultured with DOs maturation promoting effect is not obvious (35.83 + 18.32 vs 35. 53 + 14, P0.05). The analysis results show that the oocyte cumulus cell extracellular mainly through gap junctions promote oocyte maturation in vitro, add the discrete cumulus cells mainly by paracrine pathway promote oocyte maturation in vitro; the 3 layer and above extracellular cumulus cells can improve the inclusion of oocytes the average maturation capacity of 85.34%, add the discrete cumulus cells can improve the average COC in vitro maturation capacity of 16.18%, affected by semen and without cumulus cells of bovine oocytes matured in vitro experimental study on the in vitro development of oocytes matured in vitro in IVF-100 can enhance the DO average in vitro maturation ability of 60.61%.2., BO and TALP solution three kinds of liquid in vitro fertilization effect in semen, and made a comparison of naked oocytes (NOs) and cumulus oocyte complexes (COCs) effect on fertilization effect. The results showed that the in vitro maturation of oocytes Cells in the cleavage of semen IVF-100 was significantly higher than those in the BO solution (0.7055 + 0.0745 VS0.5794 + 0.0956, P0.05), while the difference of TALP in liquid IVF-100 and liquid BO and cleavage rate were not significant.NOs although the cleavage rate of less than COCs, but the difference was not significant; NOs can develop to the blastocyst stage, but the blastocyst rate was significantly lower than that of COCs. BO and TALP solution as in vitro semen, there is still much room for improvement, and add the laminin effects on embryonic development in vitro by IVM-IVF in bovine oocytes with different concentration of laminin, the distribution of NOs in vitro development ability was significantly lower than that of COCs.3.laminin in bovine embryos in (0,5,10 and 20 g/ml) in the medium were cultured in vitro, and the development of different embryonic stages were detected. The results showed that 10 g/ml group, the cleavage rate and blastocyst rate was significantly higher than the control group (0.8031 + 0.0667 vs 0.7039 + 0.068 8, P0.05; 0.4835 + 0.0167 vs0.3765 + 0.0257, P0.05). Although 48h after fertilization in each experimental group and control group the difference between the 4-8cell number is not significant, but the total number of blastocysts group 5,10 g/ml group was significantly higher than the control group (97 + 8.24,98.71 + 13.59 vs87.86 + 6.22, P0.05). The number of cells in trophoblastic layer between the experimental group and the control group had no difference. The ratio of the total number of groups and the number of blastocyst inner cell mass cells were close to 0.33. in the display of the detection of 0-8 beetles in vitro embryo distribution of laminin expression distribution test, the earliest in 8 cell embryos detected by laminin, in the 16 cell to blastocyst stage presentation layer laminin a trend of increasing, and a large number of expressions. At the same time on the blastocyst stage but also in the expansion of the blastocyst stage to blastocyst and expression of trophoblast cell laminin detection part of the distribution. The results show that the laminin increase with the increasing demand of embryo age, add A certain amount of exogenous laminin can improve the in vitro embryo cleavage rate in different degrees, and the rate of blastocyst cell number of blastocyst; distribution of expression by immunofluorescence technique was first detected in 8 cell stage laminin of bovine embryos, but also in mulberry to blastocyst stage expression quality assessment technique of.4. optimization detection of bovine embryo in vitro to laminin in order to establish a rapid, reliable method of double staining evaluation of bovine blastocyst quality, this experiment was improved. 4 kinds of scheme of double staining results show that the improved scheme, scheme three and scheme four can reach the bovine blastocyst double staining results. The improved scheme three, and the total number of Hoechst33342 staining showed no difference scheme four blastocysts (84.33 + 9.98 VS83.63 + 9.74, P0.05; 93 + 10.50 VS83.63 + 9.74, P0.05), and the inner cell mass (ICM) and the ratio of number of total cells were blastocysts 0.3382 and 0.3356 are close to the 0.33. improvement plan four was the most stable, the effect is good, the dyeing time less than 3 minutes, which greatly improves the efficiency of dyeing. The experimental results show that the improved scheme three, scheme four can be used as a rapid method for identification of bovine blastocyst quality effective.5. integrin beta 1 the role of the sperm egg fusion test by laminin and anti- under different conditions of beta 1 integrin beta 1 integrin beta 1 to explore the blocking effect on sperm egg fusion, to analyze the causes of laminin specially disappeared from the stage. The results showed fertilization when using laminin and anti- beta 1 on the egg integrin beta 1 blocking, laminin can significantly affect the fertilization (10 g/ml:67.38 + 5.41,20 g/ml:71.51 + 4.96 VS 86.04 + 3.73, P0.01) and cleavage (10 g/ml:41.43 + 59.30 8.33VS + 7.85, P0.01), and anti- beta 1 on fertilization and egg Little effect on crack. When laminin and anti- were added to the beta 1 in semen of laminin, heparin receptor blocking, results show that with the increase of laminin concentration, the effect of sperm egg fusion increased significantly (10 g/ml:80.36 + 1.92 VS 86.04 + 3.73, P0.05; 20 g/ml:64.95 + 7.61 VS86.04 + 3.73 P0.01),, but the effect on the cleavage of the little; anti- beta 1 can affect the fertilization significantly (50 g/ml:29.49 + 11.39100 g/ml:34.98 + 7.54 VS 86.04 + 3.73, P0.01) and cleavage (50 g/ml:5.44 + 7.37100 g/ml:3.65 + 6.84 VS 59.30 + 7.85 P0.01, respectively). When using laminin and anti- beta 1 on sperm on the surface of the integrin beta 1 block, the result shows that the influence of both fertilization and cleavage significantly (P0.05), and higher laminin concentration, significantly stronger. At the same time, when using laminin and anti- beta 1 on the surface of the egg and sperm surface integrin beta 1 blocked again In vitro fertilization, sperm egg fusion results show that both are affected significantly the cleavage rate (P0.01), except 50 g/ml anti- beta 1 group were significantly lower than the control group (P0.01). The results showed that integrin beta 1 participate in the process of cattle sperm egg fusion in vitro; integrin blocking fusion sperm on the surface of beta 1 can significantly hinder the sperm egg, and the egg on the surface of the block is not 1 integrin on fertilization effect; Laminin in the process of fertilization was probably due to the blocking of the disappearance of integrin beta 1 and heparin and hinder the normal sperm egg fusion.

【學(xué)位授予單位】：河南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823

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