ACE inhibition in goats under fixed‐time artificial insemination protocol increases the pregnancy rate and twin births

To assess the effect of the angiotensin‐converting enzyme (ACE) inhibition on the efficiency of the fixed‐time artificial insemination (TAI), 69 goats were divided randomly into two groups: enalapril (n = 35) and control (n = 34). In the experiment, all animals underwent the protocol of fixed‐time artificial insemination for 12 days. Enalapril group received enalapril maleate dissolved in saline (Enalapril, Lab Teuto Ltda) subcutaneously at the following doses: 0.2 mg/kg/day in D0‐D2; 0.3 mg/kg/day in D3‐D6 and 0.4 mg/kg/day in D7‐D11. The control group received the corresponding volume of 0.9% saline solution. We performed a single insemination 36 hr after sponge removal using frozen semen from two adult male goats with recognized fertility. The ultrasound pregnancy diagnosis was 30 days after the artificial insemination (AI). There was significant increase in pregnancy rates and twinning as well as a decrease in foetal loss in animals receiving enalapril (p < .01). The use of ACE inhibitors during the TAI protocol was shown to be a promising alternative to increase the efficiency of such reproductive biotechnology.     

Bird flu (H7N9) outbreak and its implications on the supply chain of poultry meat in China

During the past two decades, food safety issues in China not only posed serious threats to Chinese consumers but also damaged the image of Chinese products internationally. In China, food safety is not only about scientific discoveries, advanced laboratories, and sanitation equipment; it is more about the role of different players in the food supply chain. The poultry meat supply chain is instrumental in the spread of the avian influenza A virus (H7N9), raising questions about how policymakers respond to such threats and whether industries need to be restructured to manage and control this epidemic so that it does not recur. As a short-term measure, to prevent the spread of this disease, government authorities enforced the closure of live bird markets (LBM) in disease-affected areas of China. However, in the long term, the poultry meat supply chain needs to be restructured. The aim of the current study was to analyze distribution channels for chicken meat in China and then describe arrangements in poultry meat sectors that incorporate small- and medium-scale producers into the supply chain while responding to shifts in LBMs. We also assessed the role of LBMs in spreading H7N9 and how these interventions affect the poultry meat supply chain in the Chinese market.     

Cloning of Buffalo BMP1 Gene and its Expression Pattern Investigation in Different Tissues

Buffalo BMP1 gene was cloned in the present study, the BMP1 sequence was systemically analysed by bioinformatics techniques, and the expression level of BMP1 gene in different tissues were also assayed with Real-time fluorescence quantitative PCR (QRT-PCR).The results showed that with RT-PCR a 3 195 bp buffalo BMP1 gene was cloned and sequenced, including the whole ORF of 2 967 bp (coding 988 amino acid).The sequence multialigned results showed that buffalo BMP1 gene shared 99%, 96%, 96%, 96% and 95% of similar amino acid sequence with Bos taurus, Sus scrofa, Equus, Homa sapiens and Mus musclus, respectively.It was predicted that buffalo BMP1 protein contained a signal peptide domain, a preregion, a metalloproteinases domain, five complement-Uegf-BMP-1 domain (CUB domain) and two epitheloid growth factor-like domain (EGF-like domain).In addition, we also analyzed the expression level in different tissues through QRT-PCR, the results showed that BMP1 gene mRNA existed in all nine tissues with the most abundant expression in heart, followed by testis, ovary, genital ridge, while with lower amount of bone and other tissues, the minimal expression in liver was observed.     

Bioinformatics Analysis of Bone Morphogenetic Protein 4 (BMP4) of Sheep

The assay was aimed to explore the biological characteristics of bone morphogenetic protein 4 (BMP4) of sheep,NCBI,DNAMAN DNAStar,TMHMM Server v.2.0,PsortⅡ,SignalP various bioinformatical softwares were used to speculate the physical and chemical properties,hydrophobic property,phosphorylation site,conservative structure domain,protein secondary structure of BMP4 protein.Also,the three-dimensional structure was forecasted with the SWISS-MODEL Workspace software.The results indicated that the BMP4 of sheep had high homologies with the BMP4 of various species.The encoded protein was a hydrophilic protein which was unstable.There was no transmembrane regions and it was likely to be located in the nucleus.What was more,there was signal peptide and eighteen phosphorylation sites.Through the forecast of functional domains,the protein had two functional domains,including the transforming growth factor-beta (TGF-beta) superfamily and TGF-beta propeptide superfamily.The result was consistent with the function of BMP4 gene family,it also demonstrated that BMP4 was a growth factor and it had the function of signal transduction.The amino acid homology between the predicted 3D structure of protein and template 3bmp.1.A was 88.29%.The bioinformatics analysis of BMP4 gene could provide reference for the further study in practice.     

三种融合标签对大熊猫轮状病毒结构蛋白VP6-VP7可溶性表达的影响

大熊猫轮状病毒(giant panda rotavirus,GPRV)是引起幼龄大熊猫腹泻的主要病原,对圈养大熊猫产生了较大的危害。轮状病毒结构蛋白VP6是一种载体蛋白,可介导黏膜免疫反应。VP7是轮状病毒结构蛋白中主要的中和抗原。因此,VP6-VP7的融合表达作为候选抗原对该病的防治具有重要的意义。传统大肠埃希菌原核表达存在表达量低、可溶性差以及纯度低等弊端。本研究使用醛缩酶(EDA)、谷胱甘肽S-转移酶(GST)、麦芽糖结合蛋白(MBP)3种融合标签,以实现获得表达量高和纯度高的GPRV-VP6-VP7重组表达蛋白。将扩增的VP6、VP7基因片段利用同源重组酶构建到含3种融合标签的表达载体pET21b上,将重组质粒转化至大肠埃希菌Rosetta(DE3)感受态细胞中进行低温诱导表达。用Ni-柱亲和层析法纯化目的蛋白,SDS-PAGE和Image J分析蛋白表达量和可溶性,Western blot分析得到表达的重组表达蛋白正确且具有蛋白活性。实验结果证明,EDA标签能显著促进VP6-VP7蛋白的原核可溶性表达,提高VP6-VP7蛋白表达量。     

甲状腺素对猪小肠上皮细胞miR-let-7a基因表达及细胞增殖的影响

miR-let-7a在动物细胞的分化、增殖与凋亡等方面发挥越来越重要的作用。甲状腺激素(TH)作用非常广泛,机体的每个细胞几乎都是TH作用的靶细胞,其可以促进组织分化、生长和成熟。本实验用甲状腺素(T4)浓度分别为(0、0.02、0.03、0.05、0.075、0.1、0.2μmol/L)在体外培养猪的小肠上皮细胞。结果表明:T4处理组的细胞体积形态相对于空白对照组没有明显变化;当T4添加浓度为0.03μmol/L时,细胞的增殖率显著低于其他组(P0.05);当T4浓度为0~0.03μmol/L时,let-7a的表达随着添加剂量的增加而升高,浓度从0.03~0.2μmol/L变化时,let-7a的表达呈现降低趋势,浓度为0.03μmol/L时表达量极显著高于其他组(P0.01)。let-7a的表达量与细胞增殖呈负相关。     

绵羊BMP15基因B2突变可视化检测技术的建立

骨形态发生蛋白15（bone morphogenetic protein 15,BMP15）基因突变对绵羊排卵率、产仔数以及繁殖力有很大影响,本研究旨在建立快速可视化检测BMP15基因B2突变的技术。将改良的扩增阻滞突变系统（amplification refractory mutation system,ARMS）与核酸染料SYBR Green Ⅰ结合,针对BMP15基因B2突变设计ARMS特异性引物,使引物下游3'端的最后一个碱基为A,并在下游引物3'端的第3位碱基处设计额外的错配,以提高引物的特异性;经ARMS PCR扩增后,向产物中加入SYBR Green Ⅰ,通过肉眼观察PCR管内的颜色变化来检测BMP15基因的B2突变。经测序发现所采集的样本均为野生型,因此,利用重叠延伸PCR构建BMP15基因B2突变型模板,测序结果显示BMP15基因B2突变型模板构建成功。ARMS PCR结果显示,ARMS特异性引物能够只扩增突变型模板,而不会扩增野生型模板,且扩增效果良好。ARMS PCR扩增后加入SYBR Green Ⅰ发现已知野生型模板与阴性对照一致,呈SYBR Green Ⅰ原始颜色橙黄色,而已知突变型模板呈亮绿色,且色差明显,肉眼可辨。用建立的可视化检测BMP15基因B2突变的技术检测50个小尾寒羊基因组DNA （随机向其中20个样本中加入构建的BMP15基因B2突变型模板）,将可视化检测的突变型样本编号与混合样本时记录的编号对比,结果表明该技术能够准确检测绵羊BMP15基因的B2突变,且准确率高达100%。将构建的BMP15基因B2突变型的模板进行梯度稀释,对本试验可视化检测体系的灵敏度进行检测,发现ARMS可视化检测技术的灵敏度达到0.006 ng/μL。因此,本研究建立的技术能够可视化地检测绵羊BMP15基因B2突变,且准确率高,有望为高繁殖力绵羊的选育提供技术支持。     

噬菌体Bp7尾丝蛋白gp37的原核表达与鉴定

为了研究噬菌体尾丝蛋白gp37在识别宿主菌大肠埃希菌K12过程中的作用,PCR扩增获得gp37基因C端1 161bp序列,以EGFP为荧光标签,构建重组表达质粒pET-28a-EGFP-gp37,在大肠埃希菌BL21(DE3)中IPTG诱导表达重组蛋白EGFP-gp37,亲和层析纯化蛋白。SDS-PAGE及Western blot检测结果表明,重组蛋白EGFP-gp37在大肠埃希菌BL21(DE3)为可溶性表达,表达量占菌体总蛋白量的23%。EGFP-gp37融合蛋白与大肠埃希菌K12相互作用,激光共聚焦显微镜检测结果显示,尾丝蛋白gp37能够吸附到宿主菌K12表面,表明尾丝蛋白gp37参与宿主菌受体的识别和吸附过程。