Function of berberine on porcine in vitro fertilization embryo development and differential expression analysis of microRNAs

The effect of berberine (Ber) on in vitro fertilization (IVF) embryo development in pigs and the associated differential expression of microRNAs (miRNAs) in the embryo were investigated. NCSU‐23 embryonic culture medium was used for a control group, while NCSU‐23 embryonic culture medium added with Ber was used for a Ber group. The embryo development rates in these groups were determined, and the zygotes, 4‐ and 8‐cell embryos, and blastocysts were collected for cDNA microarray analysis. The development rates of 2‐, 4‐, 8‐cell embryos and blastocysts were significantly higher in the Ber group than those in the control group (p < 0.01). The differentially expressed miRNAs in the 8‐cell versus the 4‐cell stage in control group as well as in the 8‐cell Ber group versus the 8‐cell control group overlapped, and it was found that nine miRNAs were commonly upregulated and two of them were downregulated, while there was no overlap among the other groups. The target genes of Ber‐regulated miRNAs at the 8‐cell stage were mainly associated with the molecular pathway of nucleic acid and protein synthesis. These findings suggest that Ber may regulate the expression of miRNAs at the 8‐cell stage, which is beneficial to provide material reserves for the maternal to zygote transition of porcine embryos, thereby increasing the porcine IVF embryo development rate.     

蜱MicroRNA研究进展

蜱是一类专门寄生于脊椎动物的体表寄生虫,是继蚊子之后的第二大传播媒介。它们传播多种疫病,不仅给畜牧业造成极大的经济损失,而且还引起人类发生疾病甚至死亡。MicroRNA(简称miRNA)是18nt~25nt的非编码小RNA,具有组织特异性和阶段特异性的特点。miRNA作用于靶基因的3′端非翻译区以抑制其表达或者降解mRNA,进而在后转录水平发挥着重要的生物学调控作用,如生长发育和先天性免疫等。有研究显示,miRNA可能调控幼蜱的生长发育,并对蜱虫吸血和血液消化有一定的作用。论文综述了miRNA在蜱体内的研究概况及其生物学功能的研究进展,以期为蜱的研究提供参考。     

山羊皮肤新microRNA的挖掘

已有证据表明,microRNA在动物皮毛发育和形态发生过程中发挥重要的作用.在山羊皮肤microRNA的研究中,数据库不完善仍是限制其功能研究的主要原因.本研究以山羊基因组为参考,对高通量测序中未注释的占总reads 5.40%的数据,使用Mireap软件预测的方法进行数据挖掘,并对新的microRNA的长度、表达量、基因组定位、靶基因预测及KEGG pathway进行分析.研究预测到28个新的microRNA,其长度分布与已知山羊、牛等动物的分布一致.对表达量统计表明,表达量在100以上的有4个,最高的是Novel-15,达到555;表达量在10~100的有19个;低于10的有5个.基因组定位研究表明,新microRNA在山羊染色体上分布均匀,在5、6、7、19和X染色体上分别有两个microRNA被定位.预测和分析得到6 519个靶基因.KEGG分析结果表明,有3 552个靶基因定位到51条通路中,占前3位的通路是Metabolic pathways、Pathways in cancer、MAPK signaling pathway.本研究发掘了更多山羊的microRNA,为今后山羊皮肤发育调控的研究奠定了基础.     

microRNAs作为结核分枝杆菌感染标志物的研究进展

微小RNA(microRN.As)是一类长度约为22个核苷酸的非编码单链RNA分子,它参与基因转录后的表达调控,在免疫系统的调控和运行中具有重要作用。结核分枝杆菌是典型的胞内寄生菌,其感染能够引起人畜共患结核病。从microRNAs与结核免疫机制的相关性以及在结核分枝杆菌感染过程中巨噬细胞、外周血单核细胞及血清中microRNAs表达谱变化等方面,进行综述microRNAs作为结核分枝杆菌感染标志物的研究现状,以期为动物结核病检测提供理论参考。     

microRNAs在动物皮肤及毛囊发育中的调控作用研究进展

基因表达调控是动物被毛生长和发育的决定性因素。microRNA作为一种新发现的基因调控元件,在多种哺乳动物皮肤及毛囊中均有表达,并在转录后水平调节皮肤和毛发的生长和发育过程。从microRNA水平上解析绵羊、山羊及羊驼等被毛生长特点及发生机理,为提高毛用型经济动物的毛发品质和产量提供新的思路,同时也为更深入地研究皮肤组织中microRNA的功能提供更多的理论依据。作者主要针对目前已报道的在绵羊、山羊及羊驼等哺乳动物皮肤及毛囊发育中microRNA的调控作用进行综述。     

Improvement,identification, and target prediction for miRNAs in the porcine genome by using massive,public high-throughput sequencing data

Despite the broad variety of available microRNA (miRNA) research tools and methods, their application to the identification, annotation, and target prediction of miRNAs in nonmodel organisms is still limited. In this study, we collected nearly all public sRNA-seq data to improve the annotation for known miRNAs and identify novel miRNAs that have not been annotated in pigs (Sus scrofa). We newly annotated 210 mature sequences in known miRNAs and found that 43 of the known miRNA precursors were problematic due to redundant/missing annotations or incorrect sequences. We also predicted 811 novel miRNAs with high confidence, which was twice the current number of known miRNAs for pigs in miRBase. In addition, we proposed a correlation-based strategy to predict target genes for miRNAs by using a large amount of sRNA-seq and RNA-seq data. We found that the correlation-based strategy provided additional evidence of expression compared with traditional target prediction methods. The correlation-based strategy also identified the regulatory pairs that were controlled by nonbinding sites with a particular pattern, which provided abundant complementarity for studying the mechanism of miRNAs that regulate gene expression. In summary, our study improved the annotation of known miRNAs, identified a large number of novel miRNAs, and predicted target genes for all pig miRNAs by using massive public data. This large data-based strategy is also applicable for other nonmodel organisms with incomplete annotation information.     

Oviduct epithelial cell co-culture of early porcine embryos.

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144-168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4- and 8-16-cell embryos formed blastocysts in culture at a rate of 80-90%.     

猪轮状病毒的分离与鉴定

将疑似感染猪轮状病毒的内蒙古某猪场的腹泻仔猪粪便样品在MA104细胞上分离培养,得到一株能产生明显细胞病变的毒株(命名为PRV L1株).经纯净性检测,该毒株无菌生长、无支原体污染及外源病毒污染.特异性检测结果显示该PRV L1株能被猪轮状病毒单特异性血清中和,并可被猪轮状病毒单克隆抗体识别,其VP7基因序列与G5型猪轮状病毒VP7基因序列同源性为99％.动物回归试验结果表明,该毒株口服攻击3日龄仔猪,可引起典型的猪轮状病毒病发病症状,并能在发病仔猪小肠内容物中检测到猪轮状病毒.     

Serological identification of a new porcine mycoplasma species, M. flocculare

In a preceding paper (Friis 1972), the so-called SAR (M. suipneumoniae antibody resistant) mycoplasma strains were described. As the reported findings indicated that these isolates belonged to a separate species, a representative cloned strain, Ms42, was selected for serological comparison with the known acid-producing mycoplasma species.     

Composition and physiological functions of the porcine colostrum

The first secretion, 24-h post parturition of the mammary glands of sows, known as colostrum, is high in protein and low in lactose and fat. As a consequence of an insufficient ingestion of colostrum, more than 50% of piglets fail to reach weaning and die. The composition and some functions of colostrum have been previously reported. For example, colostrum carbohydrates consist of mainly lactose. Lipids in the colostrum are mostly triacylglycerols, but <1% is fatty acids, which may act as homeostasis regulators. Similarly, proteins are found mostly as casein and whey, the latter being ≥80% immunoglobulins. Colostrum-derived immunoglobulins and bioactive proteins such as azurocidin help the immune system of the piglet fend off infections. In addition, leukocytes and exosomes are other minor but nonetheless equally crucial bioactive components in the porcine colostrum. Modern pig farming has achieved increases in pig productivity and litter size, but this has been accomplished in detriment of the health and the survival rate of piglets. Therefore, porcine colostrum is now even more important in pig farming. In the present review, we discuss the current knowledge on the composition and physiological functions of the porcine colostrum and briefly propose future research directions.     

DNA分子标记与动物遗传育种

DNA分子标记技术的出现,对动物遗传育种产生了深远的影响,利用DNA标记可以研究动物整个基因组水平的遗传变异,进行动物遗传资源研究、标记辅助选择、标记辅助导入、杂种优势预测、选配、品种与品系确认、构建高分辨率遗传连锁图谱、QTL搜寻定位等。目前在动物遗传中广泛使用的标记主要有mtDNA、RFLP、RAPD、AFLP、SSR、SNP和EST标记。本文综述了这些标记技术的原理、特点,并根据其特点,分析其在动物遗传育种中的应用,并通过维普数据库搜索比较分析研究者对各种技术的使用情况。     

Generation of anti‐porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis

T cell‐mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti‐porcine CD69 mAb‐producing mouse hybridomas, 01‐14‐22‐51 (IgG2b–κ) and 01‐22‐44‐102 (IgG2a–κ), both showing fine reactivity with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin‐stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti‐interferon‐γ mAb and anti‐tumor necrosis factor‐α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.     

猪、鸡、鸭Myostatin成熟区段DNA的克隆及其同源性分析

克隆了金华猪、仙居鸡和绍鸭Myostatin成熟区段的DNA，并测定了各自的序列，通过与GenBank中登录的相应物种的序列比较，发现金华猪存在1个碱基的变异(T^241→A^261)，仙居鸡存在2个碱基的变异(T^218→C^218，C^219→T^219)，鸭的序列在GenBank中还未见报道。用软件对三者及其它物种的序列做了同源性分析，结果表明，Myostatin在不同物种间具有高度的保守性，DNA同源性均在81％以上(除鱼外)，提示其具有重要的生理功能，并用软件分析了该基因在不同物种中的变异，构建了系统进化树，并对各物种之间的亲缘进化关系进行了分析。     

The risk of using monoclonal or polyclonal commercial antibodies: controversial results on porcine sperm CD44 receptor identification

Presence of the hyaluronan (hyaluronic acid, HA) receptor CD44 on spermatozoa has been difficult to pursue, mostly obeying to the use of different commercial mono‐ and/or polyclonal antibodies, often lacking proper controls. Here, we describe how the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor differs in ejaculated pig spermatozoa depending on the type of antibody and protocol used. While we were able to detect binding to spermatozoa and mark its presence in the sperm membrane, the use of blocking peptides clearly indicated that only the monoclonal antibody could confirm the specific presence and location of the CD44 receptor, whereas the polyclonal antibody was detecting multiple presumed CD44 isoforms or degraded proteins thus proving unspecific. These results call for strict protocols when attempting immunological determination of sperm membrane receptors.     

A new rolling culture‐based in vitro fertilization system capable of reducing polyspermy in porcine oocytes

The high incidence of polyspermy is one of the major obstacles during in vitro fertilization (IVF) in pigs. To overcome this, we developed a novel IVF method, which involves constant rotation. Oocytes matured in vitro were mixed with spermatozoa (0.2 × 10⁵ sperm/mL) in an IVF medium (200 μL) using a 200 μL PCR tube. This tube was then rotated at 1 rpm for 6 h at 38.5°C in a rotation mixer (experimental group). A second PCR tube was simultaneously cultured without rotation (control group). The rate of polyspermy was evaluated 12 h after insemination and was significantly (P < 0.05; 21.0% vs. 48.3%) lower in the experimental group than in the control group. Sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, monospermic fertilization rate of the oocytes was significantly (P < 0.05; 44.8% vs. 21.2%) higher in the experimental group than in the control group. Furthermore, the rate of blastocyst formation (30.1% vs. 20.8%) increased in the experimental group, as compared to the control group. This present system will contribute to increase the efficacy of blastocyst production through reduction of polyspermic penetration.     

猪胸膜肺炎放线杆菌的分离和鉴定

某猪场哺乳仔猪及断奶猪临床表现喘气、咳嗽、皮肤发绀以及胸膜肺炎的病理特征，从病猪体内分离出3株菌，根据细菌培养特性、CAMP试验及生化反应等鉴定为猪胸膜肺炎放线杆菌（App)。该菌对小白鼠有一定毒力，回归猪可致死猪只，对常用治疗药有一定的抵抗力，用分离菌制备自家苗可预防本病发生。