Seroprevalence,associated risk factors analysis and first molecular characterization of chlamydia abortus among Egyptian sheep

Chlamydia abortus is one of the most common abortive agents worldwide in sheep. Few studies have been reported C. abortus infection among sheep in Egypt but the available data is scarce. The objective of the present study was to determine the seroprevalence of C. abortus among sheep, the associated risk factors and its molecular characterization. The present study was conducted on 675 sheep in six Governorates at Northern Egypt. Data analysis confirmed the presence of antibodies against C. abortus in 93 out of 675 sheep. The logistic regression model was fitted to identify the associated risk factors with C. abortus infection. The results revealed that C. abortus increased significantly in ewes (OR = 4.04, 95 %CI: 1.44−11.28) during autumn season (OR = 3.6, 95 %CI: 1.64–8.28), in ewes with a history of abortion (OR = 1.4, 95 %CI: 0.87–2.50) and in farm where no lambing pen (OR = 2.2, 95 %CI: 1.30–3.94) or abscence of post abortion measures (OR = 1.96, 95%CI: 1.23-3.12). In addition, age, flock size and exchange of breeding ram had no significant effect on prevalence of chlamydiosis. Also, PCR assay was confirmed presence of C. abortus as accusative pathogen in aborted ewe and the genetic characterization of Egyptian C. abortus strain revealed 100 % identity with another strain from Iraq. A control program should be applied to reduce economic losses and risk of human infection.     

绵羊Musclin基因真核表达载体的构建及其对肌细胞糖代谢和胰岛素作用的影响

为了研究绵羊Musclin对糖代谢和胰岛素作用的影响,本试验运用生物信息学软件对其结构特征进行预测,并构建绵羊Musclin基因真核表达载体。分4组对绵羊胎儿成肌细胞进行处理：空载体组（pcDNA3.1）、Musclin过表达组（pcDNA3.1-Musclin）、空载体+胰岛素组（pcDNA3.1+Insulin）、Musclin过表达+胰岛素组（pcDNA3.1-Musclin+Insulin）。然后分别在分化48和72 h时收集细胞,检测Musclin对正常或添加胰岛素培养的分化状态绵羊肌细胞糖代谢的影响,并利用荧光定量PCR检测相关基因表达的变化。酶切鉴定和测序结果表明,成功构建了表达载体pcDNA3.1-Musclin。生物信息学分析表明,绵羊Musclin蛋白含有一个信号肽,并在28氨基酸位点处存在一个酶切位点。亚细胞定位在胞外,属于分泌蛋白。其二级和三级结构以α螺旋和无规则卷曲为主。细胞试验表明,绵羊Musclin基因过表达明显减少了正常或胰岛素处理的绵羊肌细胞糖原含量并增加了培养液中葡萄糖含量。诱导分化72 h时,过表达Musclin抑制了Musclin过表达组和Musclin过表达+胰岛素组绵羊肌细胞中IRS1、AKT1、AKT2、GLUT1、GLUT4基因mRNA表达,并促进了GSK3β mRNA表达;而诱导分化48 h时,只有GLUT4基因的表达受到抑制。综上所述,绵羊Musclin基因过表达可以影响肌细胞糖代谢并减弱胰岛素的作用,二者相互协调以维持糖代谢稳态。其作用机制涉及到上述相关基因的表达变化,且与绵羊成肌细胞的分化状态有关。     

NPFFR1基因对鹅卵泡颗粒细胞激素分泌和细胞凋亡的影响研究

神经相关肽受体（RFamide-related peptide receptor,NPFFR1）是促性腺激素抑制激素的主要亲和受体,它在调控动物繁殖方面起着重要作用。为了解NPFFR1对鹅卵巢卵泡发育的作用,本研究以42周龄健康产蛋四川白鹅为试验材料（n=9）,利用RT-qPCR法检测NPFFR1基因在等级前和等级卵泡颗粒细胞中的mRNA表达规律;在颗粒细胞中过表达NPFFR1基因,酶联免疫吸附法检测颗粒细胞上清液（n=9）中雌二醇（estradiol,E2）、孕酮（progesterone,P4）和抗缪勒管激素（anti-Mullerian hormone,AMH）的浓度变化,剩余贴壁细胞作一步法TUNEL检测细胞凋亡情况;转录组测序方法筛选大黄卵泡（8~10 mm）颗粒细胞过表达NPFFR1前后表达差异显著基因,并对差异表达基因进行功能聚类分析。结果显示,除F1等级外,其余等级卵泡颗粒细胞NPFFR1表达量均极显著高于等级前卵泡（P<0.01）;过表达NPFFR1后,等级颗粒细胞上清液中的E2和等级前颗粒细胞上清液AMH的含量显著（P<0.05）降低,但孕酮P4含量变化不显著（P>0.05）;转录组测序共筛选到267个差异表达基因（119个下调,148个上调）,这些基因主要富集在生物节律过程、繁殖进程等生物学过程中;同时,与对照组相比,差异基因AMH显著下调表达（P<0.05）,Clock（clock circadian regulator）、FOS（proto-oncogene,AP-1 trans-cription factor subunit）、Per（period circadian regulator）和ANTXR2（cell adhesion molecule 2）分别极显著（P<0.01）或显著（P<0.05）上调表达。上述试验结果提示,NPFFR1可从激素、细胞凋亡和生物节律等多个环节影响卵泡颗粒细胞,参与调控卵泡的时序等级发育。     

猪急性腹泻综合征冠状病毒SYBR Green荧光定量PCR检测方法的建立及应用

为建立快速、灵敏且特异的检测猪急性腹泻综合征冠状病毒（swine acute diarrhea syndrome coronavirus,SADS-CoV）检测方法,本试验扩增SADS-CoV N基因保守区域将其克隆至pMD18-T载体。所构建的重组质粒pMD18-T-SADS-qN作为阳性质粒标准品,以其为模板建立一种SYBR Green荧光定量PCR检测方法。结果显示,所建立方法在3.31×10¹~3.31×10⁷拷贝·μL^-1模板量时,呈良好的线性关系,相关系数（R²）为0.997,斜率为-3.318。该方法特异性检测SADS-CoV;而猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）、猪德尔塔冠状病毒（PDCoV）和猪繁殖与呼吸综合征病毒（PRRSV）检测结果均为阴性。所构建的标准品检测灵敏度下限可以达到3.31×10¹拷贝·μL^-1,组内和组间变异系数均小于1%,表明其具有良好的灵敏性和重复性。用该方法检测SADS-CoV感染IPI-2I和IPEC-J2细胞后不同时间点和不同接毒剂量的复制情况,结果显示,SADS-CoV感染细胞后2 h病毒含量较低,在12~36 h病毒含量迅速增长,36 h后增长速度减缓且病毒含量维持在较高水平。分别用0、0.1、1 MOI SADS-CoV感染细胞结果显示病毒的mRAN转录水平呈现剂量依赖性增加,当MOI为1时,IPI-2I和IPEC-J2细胞病毒含量分别为10^6.7、10^5.3拷贝·mL^-1。进一步利用所建立的方法对经口服攻毒SADS-CoV仔猪的临床样本进行检测,结果发现病毒在空肠回肠含量较高,表明病毒主要定殖于空肠和回肠。综上表明,本研究建立SYBR Green荧光定量PCR检测方法能灵敏特异地检测SADS-CoV,为SADS-CoV的诊断和病毒相关基础研究提供可靠的检测手段。     

牦牛StAR基因克隆及其生物信息学与组织表达特性的研究

旨在克隆获得牦牛StAR基因编码序列（CDS）并进行生物信息学分析,探究其mRNA组织表达特性。本研究以屠宰场采集的成年母牦牛心、肝、脾、肺、肾、卵巢、输卵管、子宫组织（n=5）,不同年龄（胎牛、1岁、2岁）牦牛的卵巢（n=3）,不同发情周期（卵泡期、黄体期）的牦牛卵巢（n=3）,黄体期黄牛的卵巢（n=3）及实验室冻存的牦牛颗粒细胞为研究材料。以牦牛黄体期卵巢cDNA为模板,用逆转录PCR克隆StAR基因,并使用MEGA7.0和ExPASy-ProtParam等软件分析其生物信息学特性;采用实时荧光定量PCR技术分析牦牛StAR基因组织表达特性。结果发现,StAR基因CDS区长858 bp,编码285个氨基酸,StAR蛋白总体带正电荷,属于碱性亲水稳定蛋白,无跨膜结构及信号肽,主要存在于细胞质和线粒体; StAR基因具有较高的保守性,符合物种进化规律。牦牛StAR基因在卵巢表达水平最高（P<0.01）,且2岁时卵巢表达水平极显著高于胎牛和1岁龄（P<0.01）,黄体期卵巢表达水平极显著高于卵泡期（P<0.01）;黄体期黄牛卵巢中StAR基因的表达量极显著高于牦牛（P<0.01）;在颗粒细胞的体外培养过程中StAR基因表达量逐渐上升,在培养24 h时达到高峰（P<0.01）,随后显著降低。综上所述,StAR基因序列较为保守,在牦牛卵巢组织中表达最高,且表达水平随年龄与卵巢周期而变化,提示StAR基因可能参与牦牛卵巢及黄体功能相关的繁殖调控。     

猪C1QTNF3基因可变剪接体的鉴定及介导miR-101调控成脂分化的研究

旨在研究猪C1QTNF3基因可变剪接体的特性及miR-101通过C1QTNF3基因促进猪脂肪SV细胞成脂分化的机制。本研究采集3头30日龄健康马身猪仔公猪心、肝、脾、肺、肾、胃、股二头肌、腰大肌、皮下脂肪和背部脂肪组织样品,首先利用RT-PCR技术和生物信息学方法对C1QTNF3基因的不同转录本进行扩增和生物学特性分析,采用qRT-PCR技术检测C1QTNF3基因不同转录本在猪组织中的表达变化。随后,利用生物信息学预测发现,C1QTNF3上游的调控因子是miR-101,采用双荧光素酶报告试验验证miR-101对C1QTNF3的调控作用。最后,利用油红O染色和qRT-PCR技术检测过表达miR-101对猪脂肪SV细胞成脂分化的影响。基因克隆得到猪C1QTNF3存在两个转录本C1QTNF3和C1QTNF3-1,其中C1QTNF3-1为新鉴定的转录本;测序分析显示,与C1QTNF3相比,C1QTNF3-1的第1外显子区域缺失了219个碱基,少编码73个氨基酸。进化树分析显示,猪C1QTNF3和C1QTNF3-1蛋白序列与人和马来亚穿山甲等物种的亲缘关系较近。C1QTNF3和C1QTNF3-1在猪各组织中均有表达,且在各组织中C1QTNF3-1的表达量均极显著高于C1QTNF3（P<0.01）。过表达miR-101极显著下调C1QTNF3 3'UTR区域的荧光素酶活性（P<0.01）和C1QTNF3 mRNA的表达（P<0.01）,同时增加了脂肪细胞成脂分化关键基因PPARγ、C/EBPβ、SREBP-1c和FABP4的mRNA表达量（P<0.01）。本试验成功克隆了猪C1QTNF3基因的两个可变剪接体,其中C1QTNF3-1在猪不同组织中均高表达,推测该转录本为基因发挥功能的主要亚型。并深入研究了C1QTNF3基因的上游调控因子,揭示了miR-101靶向C1QTNF3促进成脂分化的机制,丰富了C1QTNF3的生物学作用和调控网络。     

戴瑞奶绵羊产奶性状的全基因组关联分析

旨在通过对奶绵羊产奶性状的全基因组关联分析（GWAS）,寻找和定位与奶绵羊产奶性状相关的遗传标记和功能基因。本研究以135只戴瑞奶绵羊为试验材料,基于全基因组重测序技术,通过SAMTOOLS进行单核苷酸多态性（SNP）检测,使用PLINK v1.90进行质控,利用GEMMA v 0.98.1的混合线性模型对质控结果进行奶绵羊产奶性状相关的全基因组关联分析。结果表明,有1个SNP与泌乳后90天日均产奶量在全基因组范围内显著相关,8个SNPs达到潜在显著关联,相关的候选基因包括TRNAQ-CUG-2、LOC114117240、ACADL、MYL1、CHD6、SLCO3A1;有2个SNPs与150天日均产奶量达潜在显著关联,相关的候选基因包括PRMT6、RNF180;有2个SNPs与泌乳周期达潜在显著关联,相关的候选基因包括PRMT6、TRNAW-CCA-68、TRNAS-GGA-61。进一步基因功能分析推测,ACADL、SLCO3A1可能是影响奶绵羊产奶性状的候选基因。本研究为奶绵羊产奶性状的分子机制解析提供了一定的基础,为我国奶绵羊新品种培育提供了一定的理论参考。     

犏牛和牦牛ESCO2基因的序列特征及其在睾丸中的表达对比分析

旨在克隆犏牛和牦牛姐妹染色单体内聚建立蛋白2（establishment of sister chromatid cohesion N-acetyltransferase 2,ESCO2）基因,并分析其在不同发育阶段睾丸中的表达与定位,为进一步解析ESCO2在减数分裂过程中的作用机制提供理论依据。本研究以健康雄性犏牛及牦牛为试验动物,根据年龄分为胎牛组（5~6月龄）、幼年组（1~2岁）和成年组（3~4岁）,每组各3头。通过RT-PCR技术克隆犏牛和牦牛ESCO2基因并进行生物信息学分析,采用实时荧光定量PCR （quantitative real-time PCR,qRT-PCR）检测ESCO2基因在犏牛不同组织中的表达谱,比较分析ESCO2在犏牛和牦牛不同时期睾丸中的表达规律,利用免疫组织化学（immunohistochemistry,IHC）技术检测ESCO2蛋白的细胞定位和表达差异。结果显示,犏牛ESCO2基因（GenBank登录号：MW198470） CDS区为1 833 bp,编码610个氨基酸,与牦牛相比,犏牛ESCO2序列第301~319位多19个氨基酸,另有3个氨基酸突变;犏牛ESCO2蛋白序列与黄牛的同源性高于其他哺乳动物;ESCO2可能与SMC3、SMC1A、PDS5A、PDS5B、STAG2等蛋白相互作用,互作蛋白功能与姐妹染色单体凝聚、减数分裂细胞周期、DNA修复、细胞分裂和染色体重构等生物学过程相关。ESCO2在犏牛各组织中均有表达,但在睾丸中的相对表达水平显著高于其它组织（P<0.05）;在犏牛睾丸中的表达随年龄增长呈上升趋势,幼年和成年时期犏牛睾丸中ESCO2的表达显著低于同时期牦牛（P<0.05）;IHC染色结果发现,雄性犏牛减数分裂阻滞于初级精母细胞,ESCO2蛋白在犏牛初级精母细胞中无表达并与牦牛存在差异。本研究结果表明,犏牛与牦牛的ESCO2基因、蛋白序列差异较大,且在睾丸发育过程中的表达模式差异显著,这可能是引起雄性犏牛减数分裂阻滞及不育的原因之一,其具体作用机制有待进一步研究。     

Carrier rate of the c.235G>C mutation in the bovine isoleucyl-tRNA synthetase (IARS) gene of Japanese Black cows at Kagoshima prefecture, Japan,and analysis of the metabolic profiling and reproductive performance of heterozygous cows

Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.     

Developmental competence of heat stressed oocytes from Holstein and Limousine cows matured in vitro

The negative effects of heat stress on dairy cattle's fertility have been extensively studied, but the relevant knowledge for beef cattle is rather limited. The aims of this study were to investigate the effects of HS during in vitro maturation on the developmental potential of oocytes derived from Limousine and Holstein cows and to estimate the effect of the differential gene expression of important genes in oocytes, cumulus cells and blastocysts in the growth competence between the breeds. In seven replicates, cumulus oocyte complexes from Holstein and Limousine cows were matured for 24 hr at 39°C (controls C; Hol_39, Lim_39) or at 41°C from hour 2 to hour 8 of IVM (treated T; Hol_41, Lim_41), fertilized, and presumptive zygotes were cultured for 9 days at 39°C. Cleavage and embryo formation rates were evaluated 48 hr post-insemination and on days 7, 8 and 9, respectively. From all groups, subsets of cumulus cells, oocytes and blastocysts were analysed for the relative expression of genes related to metabolism, stress, apoptosis and placentation. No difference was detected in cleavage rate or in blastocyst formation rate among the control groups. In both breeds, heat stress reduced blastocyst yield, but at all days the suppression was higher in Limousines. In Holsteins, altered gene expression was detected in cumulus cells (G6PD, GLUT1) and blastocysts (PLAC8), while in Limousines, differences were found in oocytes (G6PD, HSP90AA1), in cumulus cells (CPT1B, HSP90AA1, SOD2) and blastocysts (DNMT, HSP90AA1, SOD2). It appears that Holstein COCs are more tolerant than Limousine COCs, possibly due to compulsory, production driven selection.