Analysis and Validation of the Differentially Expressed ENO1 in the Maternal Placenta of Cow with Retained Foetal Membrane

The paper aimed to analyze and validate the function of differentially expressed ENO1 in the cow with retained foetal membrane.In our research,we chose three healthy Holstein dairy cows and three Holstein dairy cows with retained foetal membrane of similar age,foetal times,weight and milk yield and divided them into two groups.The total protein of maternal placenta was extracted in the control and retained foetal membrane of cow.The differential expression of proteins were found out by the 2-DIGE,and the differential expression of ENO1 was validated by the Western blotting and Real-time quantitative PCR.The results showed that ENO1 was significant expression and large multiple in the two groups,and it was significant increased expression in the retained foetal membrane of cow after Western blotting and Real-time quantitative PCR (P=0.015<0.05;P=0.001<0.01).The ENO1 participated in the energy homeostasis,immune and fibrinolysis process which related to the retained foetal membrane of cow.It suggested that ENO1 was likely connected with the retained foetal membrane.     

奶牛胎衣不下的病因分析

奶牛产后,胎衣在12 h内不能自然完全脱落下来称胎衣不下（Retention Fetal Membrane,简称RFM）。有资料显示,奶牛胎衣不下的发病率通常在10%～25%,有的奶牛合作社为30％～40％,夏季可高达60％[1]。高清友等[2]研究报道,在717头分娩奶牛中有148头发生产后胎衣不下,发病率为21％。靳国旺等[3]于2007-2011年间,对安阳地区奶牛场的奶牛胎衣不下病例进行了统计分析,结果发现在856头分娩奶牛中,有188头在产后发生了胎衣不下,发病率22%。该病作为奶牛产后常发、多发病之一,在导致奶牛产奶量下降的同时,可继发子宫内膜炎,造成繁殖障碍,导致奶牛因不孕而被提前淘汰,使奶牛养殖业遭受经济损失。     

奶牛胎衣不下的病因分析

奶牛胎衣不下是影响奶牛养殖业的重要因素之一。从营养、饲养管理、应激、激素、胎次、异常分娩、疾病等方面综述了奶牛胎衣不下的发病原因,为防治奶牛胎衣不下疾病提供了科学的理论依据。     

奶牛胎儿胎盘解剖学与胎衣不下的关系

本文对12头奶牛的分娩过程、胎儿胎盘的形态和产后状况进行观察,对胎儿站立时间、胎儿重量、胎衣排出时间、胎衣重量、胎衣面积、空角孕角面积、子叶数及子叶的分布方式等指标进行了定量对比分析。结果表明:胎衣正常排出组与胎衣不下疾病组在胎衣重量、空角孕角子叶数量和空角子叶面积差异显著(P<0.05);胎衣排出时间、孕角子叶面积差异极显著(P<0.01)。     

胎衣不下奶牛母体胎盘miRNA-181a异常表达分析及验证

为了对胎衣不下奶牛母体胎盘组织中差异miRNA-181a进行分析及验证,探讨miRNA-181a在奶牛胎衣不下发生中的作用,试验选取年龄、胎次、泌乳量、体重都相近并无其他疾病影响的胎衣正常排出奶牛和胎衣不下奶牛各3头作为研究对象,运用HiSeq高通量测序技术,对正常和胎衣不下奶牛母体胎盘组织间miRNA-181a的差异表达情况进行分析,并进行qRT-PCR验证。结果表明:发生胎衣不下奶牛母体胎盘中miRNA-181a的相对表达量明显低于胎衣正常排出的奶牛,经SPSS19.0分析可得P=0.041 7(P0.05),差异显著。说明发生胎衣不下的奶牛母体胎盘中miRNA-181a异常表达,提示miRNA-181a与胎衣不下的发生密切相关。     

奶牛胎衣不下的病因分析

作者结合国内外奶牛胎衣不下的研究概况,综述了奶牛产后胎衣不下的病因,病因主要包括营养、饲养管理、应激、激素、胎次、异常分娩、疾病等。     

奶牛胎衣不下的病因及防治

本文对奶牛胎衣不下的病因进行了细致、确切的分析,主要有胎盘分离障碍、子宫无力排出胎衣胎衣排除障碍等三类病因,并提出了相应预防措施及治疗方案。     

奶牛胎衣不下的病因及防治

胎衣不下是奶牛产后的一种常见病,一般母牛产后经过12小时胎衣尚未全部排出即称为胎衣不下.胎衣不下不但可引起奶牛产奶量下降,还可引起子宫内膜炎、子宫复旧延迟和子宫脱出,从而导致不孕,致使许多奶牛被迫提前淘汰.现将奶牛胎衣不下的发病原因和防治措施,简单介绍如下,仅供广大养奶牛户参考.     

奶牛胎衣不下的病因分析及防治措施

胎衣不下又称胎衣滞留或胎盘停滞，一般母牛于产后4～8h胎衣自行排出，若产后经过12h胎衣尚未全部排出，称为胎衣不下。胎衣不下是奶牛产后常见病，易继发其他产后疾病，已成为影响奶牛繁殖的主要疾病之一。特别是由胎衣不下引起的子宫内膜炎的发病率显著增高，致使母牛不孕、发情延迟、增加配种次数，影响母牛繁殖效率和产奶量，给奶牛饲养业造成巨大的经济损失。     

奶牛胎衣不下的治疗及预防

奶牛胎衣不下是养牛业的一个多发病,不仅影响奶牛的繁殖率,更降低了经济效益,成为制约养牛业发展的一大障碍。笔者对内蒙古地区某奶牛场的3例病牛,进行了奶牛胎衣不下的兽医常规方法检验,并对该病的症状、治疗方法进行了摘录,以供同行参考及应用。同时提出了该病的预防措施。     

不同发育阶段延边黄牛阉牛背最长肌差异表达基因的筛选

应用引物退火控制技术(ACP)筛选8月龄和30月龄延边黄牛阉牛背最长肌差异表达基因,寻找与延边黄牛生长发育相关的候选基因。相同饲养条件下,分别检测3头8月龄和30月龄延边黄牛阉牛背最长肌组织的m RNA表达水平变化。利用20对随机引物差异显示扩增,共获得9条ESTs。对Telethonin、MYH7、MSTN、Camk2d和SPP1基因进行荧光定量PCR检测。结果表明:MYH7、MSTN和SPP1基因在8月龄阉牛组背最长肌中的表达量显著高于30月龄(P0.05);Telethonin和Camk2d在30月龄阉牛背最长肌中的表达量显著高于8月龄(P0.05)。结果提示,这些基因可能是影响延边黄牛生长及肉质性状的重要调控基因。     

Activity of selected glycosidases and availability of their substrates in bovine placenta during pregnancy and parturition with and without retained foetal membranes

The activity of glycosidases is crucial for the function and biological activity of proteins conjugated with sugar moieties, which play an important role in adhesion of cells during attachment and detachment of the foetal membranes. The aim of study was to describe the ability of bovine placental tissues to break down O-glycosidic bonds in different glycoproteins by the determination of activity of β-galactosidase, α-l-fucosidase, β-N-acetyl-hexosaminidase and sialidase in early–mid-pregnancy as well as at parturition with released and retained foetal membranes. Moreover, the availability of substrates for these glycosidases in placental homogenates was evaluated. Placental samples were collected from pregnant (2–4 months) cows in slaughterhouse (n = 8) as well as during Caesarean section and divided into released foetal membranes (n = 8) and retained foetal membranes (n = 8). Tissue homogenates were subjected to spectrofluorimetric and spectrophotometric determinations of enzyme activities as well as electrophoretic separations. Enzyme activities expressed changes within examined time with significant (p < .05) differences between pregnancy and physiological parturition in β-N-acetyl-hexosaminidase and α-l-fucosidase in foetal part of placenta while in maternal part only in the latter one. Decreasing tendency in enzyme activity was noticed in foetal part of retained samples in comparison with released ones with significant (p < .05) differences in α-l-fucosidase activity. The analysis of staining of sugar moieties attached to selected proteins depicted availability of sugar molecules in examined tissues, but their patterns differed between samples. In conclusion, sugar moieties in conjugated proteins express changes in the course of pregnancy which is reflected by the alterations in activities of placental glycosidases.     

奶牛产后胎衣不下原因调查分析及防治

在奶牛养殖领域有不少牛因为长时间胎衣不下滞留在子宫中造成终身不孕,使奶牛利用年限极大缩短,养殖场淘汰率显著升高。为进一步探明青海省门源县奶牛产后胎衣不下的主要原因,于2020年对门源县养殖的奶牛胎衣不下进行深入调查,并明确了造成胎衣不下的主要原因,提出相应的防治措施。     

家蚕具有CHCH结构域的蛋白家族基因BmCH的原核表达和亚细胞定位

含有CHCH保守结构域的蛋白在不同物种中行使不同的功能。从家蚕蛹cDNA文库筛选获得一条cDNA序列,经生物信息学分析发现其编码蛋白含有一个CHCH保守结构域,将该基因命名为BmCH(GenBank登录号:DQ443425.1)。该序列开放阅读框大小为435 bp,编码144个氨基酸残基。将成功构建的重组质粒pET-28a(+)-BmCH转化到E.coli Rosetta(DE3)菌株,经IPTG诱导表达BmCH融合蛋白,用亲和层析进行纯化后,将融合蛋白免疫新西兰大白兔制备多克隆抗体。通过Westernblotting检测BmCH在家蚕4个发育时期和5龄幼虫组织中的表达情况与用荧光定量PCR检测BmCH在家蚕不同发育时期及5龄幼虫组织中的转录结果基本一致:该蛋白在卵、幼虫、蛹、蛾4个发育时期均有表达,其中在5龄幼虫期的表达量最高;该蛋白在5龄幼虫的表皮、精巢、中肠、丝腺和卵巢中有明显表达,在血淋巴、马氏管、脂肪体中的表达量较少,但差异不大,而在气管和头部没有表达。利用抗体的免疫荧光标记对BmCH在家蚕BmN细胞中的定位进行研究,观察到该蛋白主要分布在细胞质中。初步推测BmCH可能是家蚕生长发育的必需蛋白,参与多种细胞生命活动的过程。     

Effect of Cryopreservation on the Expression of Tetraspanin CD9 in Sheep Oocytes

The experiment was aimed to explore the expression of CD9 in the follicles at different developmental stages, and investigate the effect of in vitro maturation and cryopreservation on CD9. The expression of CD9 protein in the follicle was detected by immunohistochemistry technique, the expression of CD9 mRNA was detected by Real-time quantitative PCR, CD9 protein was detected by Western blotting. The results showed that the CD9 fluorescence signal was detected by immunohistochemistry technique, and the fluorescence signal gradually increased with the maturation of the follicle, and fluorescence signal was the strongest in mature follicle;Real-time quantitative PCR showed that the expression of CD9 mRNA in fresh MⅡ oocytes was significantly increase (P<0.05), and was the least in frozen GV oocytes;The result of Western blotting indicated that CD9 protein was the most in fresh MⅡ oocytes and the expression of frozen GV oocytes was the least, consistent with the results of Real-time quantitative PCR. Cryopreservation of oocytes was more extensive than embryo cryopreservation. Tetraspanins CD9 played a very important role in sperm egg fusion. The above experimental results showed that the cryopreservation made damage to oocytes, decrease content of tetraspanins CD9 protein, and CD9 injury was one of the important reasons for the decline of fertilization rate.     

ENO1过表达对籽鹅卵泡颗粒细胞凋亡基因Bcl-2表达的影响

本试验将原代培养的籽鹅卵泡颗粒细胞转染ENO1腺病毒过表达载体,应用实时荧光定量PCR方法检测ENO1过表达对颗粒细胞Bcl-2 mRNA表达量的影响。结果发现ENO1过表达使卵泡颗粒细胞Bcl-2 mRNA表达量极显著增加(P<0.01)。提示ENO1可能通过使Bcl-2基因表达量增加,从而抑制籽鹅卵泡颗粒细胞凋亡,促进卵泡的发育。     

绵羊卵母细胞超低温冷冻对四跨膜蛋白CD9表达的影响

试验旨在探讨不同发育时期卵泡上四跨膜蛋白CD9的表达,以及体外成熟和超低温冷冻对绵羊卵母细胞CD9的影响。采用免疫组织化学技术检测CD9蛋白在卵泡上的表达部位,实时荧光定量PCR技术检测CD9 mRNA的表达量,Western blotting技术检测CD9蛋白的含量。免疫组织化学结果发现,在绵羊原始卵泡上就开始检测到CD9荧光信号,且随着卵泡的成熟荧光信号逐渐增强,成熟卵泡时荧光信号达到最强;实时荧光定量PCR检测新鲜MⅡ期卵母细胞中CD9 mRNA的表达量显著增高（P<0.05）,冷冻GV期卵母细胞CD9 mRNA的表达量最少;Western blotting检测得出新鲜MⅡ期卵母细胞中CD9蛋白表达最多,冷冻GV期卵母细胞上表达最少,与实时荧光定量PCR结果一致。卵母细胞的冷冻保存要比胚胎保存应用前景广泛,而四跨膜蛋白CD9在精卵融合中更有着重要的作用,以上结果均显示,冷冻保存降低卵母细胞四跨膜蛋白CD9含量,而CD9的损伤是受精率下降的重要原因之一。     

秦川牛ACTC1基因的表达特性分析

试验旨在揭示ACTC1基因在秦川牛中的时空表达规律,为进一步研究ACTC1基因在肉牛肌肉发育和脂肪沉积等方面的功能奠定基础。利用实时荧光定量PCR技术检测了ACTC1基因在24月龄成年秦川牛13种组织和4日龄新生秦川牛12种组织中的表达规律,同时研究分析了该基因在秦川牛成肌细胞和前体脂肪细胞不同分化阶段（0、2、4、6、8 d）的表达特性。结果显示,ACTC1基因在秦川牛心脏中的表达量最高,骨骼肌中次之;除瘤胃和肾脏外,24月龄成年牛各组织中的表达量显著或极显著高于4日龄新生牛（P<0.05;P<0.01）;ACTC1基因在成肌细胞中的表达随着分化程度的增加呈现先升高后降低的趋势,在成肌细胞分化第2、4、6和8天ACTC1基因的表达量与第0天相比差异极显著（P<0.01）,分别是第0天的2.6、4.7、5.6和4.2倍,这与成肌细胞的分化速率一致;在脂肪细胞中ACTC1基因的表达趋势为先降后升,第2、4天的表达量与第0天相比差异极显著（P<0.01）,从分化第2天开始表达量随脂肪细胞分化程度增加而增加。ACTC1基因在不同年龄牛肌肉组织（心肌和骨骼肌）中的表达量最高,且其表达特性与肌细胞分化整体趋势一致;另外,ACTC1基因在脂肪细胞中的表达也有一定的规律。综上所述,推测ACTC1基因可能会影响牛肌肉组织的生长发育和脂肪沉积。     

家蚕耐氟近等基因系SSH文库的构建及部分差异表达基因的分析

为了获取与家蚕耐氟性状相关的基因信息,利用家蚕耐氟品种T6和氟化物敏感品种733新建立耐氟近等基因系,以回交11代的家蚕耐氟近等基因系群体中的耐氟个体和敏感个体的中肠为材料,构建家蚕耐氟近等基因系抑制消减杂交(SSH)cDNA文库。通过对抑制消减杂交cDNA文库中随机挑选的153个阳性克隆测序和聚类拼接,得到19个重叠群(con-tig)、47个已知功能基因(un igene)和28个未知功能基因。获得的表达序列标签(EST)经BLASTx比对和同源性分析,初步发现这些基因参与家蚕体内物质转运、能量代谢、基因转录、蛋白质合成与降解、蛋白质加工、信号转导、机体免疫防御、细胞结构形成等诸多生物学过程。采用实时定量RT-PCR方法,对部分基因在家蚕耐氟近等基因系群体中的耐氟个体和敏感个体的不同组织的表达进行定量分析,发现磷酸根离子转运蛋白基因、三磷酸腺苷(ATP)合成酶基因、液胞型H+-ATP酶基因、脂肪酶基因以及主要过敏原蛋白基因在耐氟个体的中肠、马氏管、脂肪体组织中的表达量有较明显上调。