Complement component 3 (C3) is the key molecule of the three pathways of complement activation (alternative, classical, and lectin pathways), which are involved in phagocytosis, inflammation, and immunoregulation processes to destroy infectious microorganisms. In this study, three novel single-nucleotide polymorphisms (SNPs) (g.-1293C>G located in the 5'-flanking region, g.56T>C in exon I, and g.7017C>T in exon XII) of the C3 gene were detected using created restriction site polymerase chain reaction, restriction fragment length polymorphism, and DNA sequencing in 952 cattle from three Chinese breeds. The genotypes and haplotypes were analyzed to investigate the polymorphisms and their possible implications, with particular investigative focus on their associations with serum C3 level, complement hemolytic activity (CH50 and ACH50), and milk production traits. The g.56T>C SNP in exon I affected the serum ACH50 (P<0.01) and the milk somatic cell score (SCS) (P<0.05), and the g.7017C>T SNP in exon XII significantly affected the serum ACH50 values (P<0.01). Moreover, statistical analyses revealed that individuals with genotypic combination CCC/GCC showed significantly lower SCS and the lowest C3 concentration in serum compared with cows with CCC/GTT (P = 0.0007) and CTT/CTT (P = 0.0021); the individuals with CCC/CCT had significantly higher ACH50 values than those with CCC/CTC (P = 0.0008) and CTC/GTC (P = 0.001); cows with CCT/CTT had higher values of CH50 and 305-day milk yield (P>0.05). The C3 expression levels were significantly increased in lung and mammary tissues (P<0.05), while significantly decreased in heart, spleen, liver, and kidney tissues in mastitis cows compared with those in healthy animals (P<0.01), respectively. Bacterial counts of serum antibacterial activities were also completed to verify the effect of SNPs on resistance to mastitis pathogens. Genetically resistant cows (CCC/GCC) had serum with noticeably higher antibacterial activity against S. aureus and E. coli in vitro than the genetically susceptible CCC/GTT cows (P<0.05). Results from this study imply that the C3 gene plays a role in resistance to bacterial infection and that it can be used as a molecular marker for complement activity and traits related to milk production.

驴产业是我国特色畜牧业的重要组成部分,其中山东德州驴是我国较为著名的大型驴品种,是制作上品阿胶的道地药材.德州驴在山东养殖区域主要分布于聊城、德州等地区,现已被列入国家级畜禽遗传资源保护名录.

文章对妊娠母驴在饲料营养和分娩方面作以阐述,提出在饲养和生产管理以及常见产科病处理等方面的技术指导,其对于提升母驴繁育水平、增加种群的数量和新生驴驹的成活率意义重大.

Transition nuclear proteins (TNPs), major proteins found in the chromatin of condensing spermatids, have been implicated in spermatogenesis and male fertility. In this study, DNA samples were collected from 404 Chinese Holstein bulls and sequenced to identify genetic variants in the 3'-untranslated region (UTR) of TNP1 and to investigate genetic variations in the TNP1 gene and their common haplotypes. This study was also conducted to determine whether these variations affect bovine semen quality traits and expression levels by PCR-restriction fragment length polymorphism, bioinformatics analyses, quantitative real-time PCR (qPCR), and fluorescence assay. Results showed that one new single-nucleotide polymorphism (SNP; g. 528 G>A, ss1388116558) and one reported SNP (g. 442 A>G, rs110469441) were found in the 3'-UTR of the TNP1 gene. Bioinformatics analysis results revealed that both loci were located in bta-miR-532-binding and bta-miR-204-binding regions, respectively. Association studies revealed that bulls with H1H1 (AGAG) and H1H3 (AGGG) haplotype combinations exhibited a lower deformity rate than those with other haplotype combinations (P < 0.05). The qPCR results showed that the relative mRNA expression of TNP1 in bulls with H1H1 haplotype combination was significantly higher than that in bulls with H4H4 haplotype combination (P < 0.05). MicroRNA qPCR results suggested that bta-miR-532 expression was downregulated by 5-fold in adult bull testicular tissues compared with that in fetal bull testicular tissues; by contrast, bta-miR-204 expression was downregulated by 1.6-fold. Luciferase assay results also indicated that TNP1 expression was directly targeted by bta-miR-532 and bta-miR-204 in murine Leydig tumor cell lines. These results provide the first indication of g. 442 A>G-mediated and g. 528 G>A-mediated translational suppression in which SNPs altered the binding of bta-miR-204 and bta-miR-532 to the 3'-UTR of TNP1; the mediated translational suppression could be involved in the regulation of TNP1 expression and may influence the morphological characteristics of Chinese Holstein bull sperm. We propose that SNPs on the TNP1 3'-UTR may help select semen quality trait in Chinese Holstein bulls in the dairy industry.

To improve the quality of bovine frozen semen,the effects of different concentrations of cerium chloride on the sperm motility,survival time,abnormal rate,acrosome integrity rate,and membrane integrity rate of bovine semen after freezing were compared using the method of adding the rare earth cerium chloride to the freezing diluents. The results showed that the addition of cerium chloride with a concentration of 0. 30μmol /L,could significantly improve( P 0. 05) the acrosome integrity rate of the frozen sperm. The additions of cerium chloride with the concentration of 0. 10 ~ 3. 00 μmol / L,could significantly or highly significanty improve( P 0. 05 or P 0. 01) the fluidity of bovine sperm cell membranes. It is suggested that the mechanisms of rare earth cerium chloride to improve the acrosome integrity rate of bovine frozen sperm may be related with the increase of the sperm membrane fluidity by cerium chloride.

Peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. In this research, polymerase chain reaction (PCR) technique was used to amplify 766 and 589 bp fragments of intron 3 and 7 of PPARα gene in Chinese Holstein (n = 771). Sequencing results showed that three novel single nucleotide polymorphisms (SNPs) were identified at position 44087 (G/A), 65550 (G/A), and 65676(G/A) in the PPARα gene. PCR–restriction fragment length polymorphism technology was used to genotype the three SNPs. Association analysis showed that cows with H1H8 (P < 0.05), H2H8 (P < 0.01), H5H7 (P < 0.05), H5H8 (P < 0.05), and H8H8 (P < 0.05) haplotype combinations had lower potassium content in erythrocytes than those with H2H6 haplotype combination. Cows with H1H8, and H8H8 haplotype combinations had lower decrease rate of milk yield than those with H2H6 and H6H8 haplotype combinations (P < 0.05). Cows with H2H8 and H8H8 haplotype combinations had lower rectal temperature than those with H5H8 and H7H7 haplotype combinations (P < 0.05). In conclusion, H8H8 haplotype combination may be advantageous for heat resistance traits in Chinese Holstein cattle.

Alpha-2-macroglobulin (A2M) binds proteases, thereby acting as defense barriers against pathogens in the plasma and tissues of vertebrates and invertebrates. Quantitative real-time polymerase chain reaction (PCR) and the isobaric tags for relative and absolute quantitation method were used to determine the expression levels of A2M mRNA and proteins in mastitis-infected mammary tissues. A2M mRNA and protein expression were significantly higher in mastitis-infected mammary tissues than those in healthy tissues. We also identified 23 novel A2M splice variants in the bovine mammary tissues using reverse transcription PCR combined with clone sequencing. These splice variants predominantly affected the bait region, the inhibitory region, and the thioester region of the protein, which have the functional key roles in inhibiting the proteases of pathogens. Genomic sequencing analysis revealed a nonsynonymous c.3535A>T single-nucleotide polymorphism (SNP) in exon 29, which is located within a putative exonic splice enhancer and may be the reason why the A2M gene produces the aberrant splice variant A2M-AS4. Our findings suggest that the A2M gene can play its role by alternative splicing mechanism and it may be of significance against mastitis. This study provides clues to better understand the function of the bovine A2M gene and the effects of the exonic SNP on the production of aberrant splice variants.

[Objective] The aim was to study the genetic polymorphisms of HSF1 and HSBP1 gene in Holstein ox.[Method] DNA direct sequencing was used for SNPs loci scanning,and CRS-PCR and PCR-RFLP method were used for genotyping of four SNPs in 162 Holstein ox,so as to analyze the polymorphism of HSF1 and HSBP1 gene.[Result] The scanning results showed that a novel SNP was discovered at 1451(G/T)in HSF1 gene and three novel SNPs of 324(G/C),589(C/T),651(C/G) were discovered in the second intron of HSBP1 gene.The polymorphism of HSF1 and HSBP1 gene analysis results showed that,for HSF1,the frequency of the AA genotype was higher and A allele was the dominant allele.While for HSBF1,AB,AA and BB appeared more frequently,dominant allele at 589(C/T) was A,and that at 324(G/C) and 651(C/G) was B.Chi-square test indicated that 1451(G/T) polymorphic site in the HSF1 gene and 589(C/T) polymorphic site in HSBP1 gene were met Hardy-Weinberg equilibrium in Holstein(P>0.05),while 324(G/C) and 651(C/G) polymorphic sites in the HSBP1 gene were not meet Hardy-Weinberg equilibrium(P<0.05).[Conclusion] The study can provide trial basis for deeply researching the function of HSF1 and HSBP1 gene.

Three novel SNPs were found by DNA sequencing, PCR-RFLP and CRS-PCR methods were used for genotyping in 979 Chinese Holstein cattle. One SNP, G1178C, was identified in exon 2 of POU1F1 gene. Two novel SNPs, A906G and A1134G, were identified in 5'-flanking regulatory region (5'-UTR) of PRL gene. The association between polymorphisms of the two genes and milk performance traits were analyzed with PROC GLM of SAS. The results showed that GC genotype at 1178 locus of POU1F1 gene was advantageous for milk yield, milk protein yield, and milk fat yield. AG genotype at 906 locus was advantageous for milk yield. There was no significant difference between 1134 locus and milk performance traits of 5'-UTR of PRL gene. Analysis of genotype combination effect on milk production traits showed that the effect of combined genotype was not simple sum of single genotypes and the effects of gene pyramiding seemed to be more important in molecular breeding.

Object: To develop a typing method for Staphylococcal hemolysin genes, and to research on the distribution of hemolysin phenotype and hemolysin genes in Staphylococcus aureus isolates obtained from milk of cow suffered mastitis and to analysis there relevance. Methods: The hemolysin phenotype was oberserved with the method of 5% blood plate. Hemolysin genes were assessed via polymerase chain reaction. Results: The typing method for Staphylococccal hemolysin phenotype and genes with PCR was developed. The isolates with alpha- hemolysis are 56 in 129 isolates, account for 43.4%; 43 show beta-hemolysis, account for 34.11%; Another 29 isolates showed no hemolysis phenotype, and it account for 22.48%. The isolates with hla gene account for 34.88%, hlb gene account for 42.60%. Conclusion: In the hemolytic phenotype, alpha- hemolysis are more than beta-hemolysis, however the hlb gene detection rate is higher than the hla gene. The distribution of hemolysin phenotype and hemolysin genes is not a corresponding relationship of Staphylococcus aureus isolates from raw milk of cow. The study can provide a basis for prevention and treatment of mastitis in dairy cows caused by Staphylococcus aureus.

Toll-like receptor 2 (TLR2), a key component of the innate immune system, plays an important role in the initiation of the inflammatory response to foreign pathogens. This study was carried out to investigate the TLR2 gene expression difference in mammary gland tissues of the cows infected with mastitis and healthy cows and to search for variants of the bovine TLR2 gene for further clarifying the relationship between TLR2 gene and mastitis in cattle. The quantitative real-time polymerase chain reaction (qPCR) revealed that clinical mastitis caused by Staphylococcus aureus could significantly increased the LTR2 mRNA abundance (p<0.05). Seven novel single nucleotide polymorphisms (SNPs) in the 5' upstream of the TLR2 gene in six cattle breeds (Hereford, Simmental, Limousin, Chinese indigenous Lind Yellow cattle, Bohai Black cattle and Chinese Holstein cattle) were identified by the direct sequencing method. Four SNPs are located at the putative promoter region and their variants can alter the transcriptional factor binding sites. Genetic diversity showed that the SNP (4bp deletion) in the promoter was unique and two distinct haplotypes were found in Hereford cattle. Six SNPs in the TLR2 gene exon2 and 3'UTR were genotyped by PCR-RFLP and CRS-PCR methods in 398 Chinese Holstein cattle. Case-control study and associated analysis revealed that the cow with the genotype TT (3.15 +/- 0.52) in c.+189T>G has significantly lower (p<0.05) somatic cell score (SCS) than that with genotype TG (4.84 +/- 0.21) or GG (5.31 +/- 0.27) in Chinese Holstein cattle. In conclusion, haplotype analysis and linkage disequilibrium findings showed that the hapoltype TG of two loci (c.+189T>G and c.+631A>G) in the coding region may be used as a tolerance haplotype for the bovine mastitis.

Protein degradation in bovine milk affects the quality of dairy products. Alpha 1-antitrypsin (AAT) can protect vulnerable elastic tissues from degradation by neutrophil elastase. The aim of this study was to assess the association of polymorphisms in bovine AAT gene with milk yield and milk composition in Chinese Holstein. Traits analyzed were fat percentage, protein percentage, 305-day milk yield and somatic cell score (SCS). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), created restriction site-polymerase chain reaction (CRS-PCR) and allele specific-polymerase chain reaction (AS-PCR) methods were used to genotype five loci in coding regions of the sequence, including position 5504, 5609, 5624, 5747 and 8178 in Chinese Holstein. The five mutations were all silent mutation that caused no alteration in the amino acid sequence. In order to determine the relationship between the polymorphisms of the AAT gene and milk production traits and SCS, the General Linear Model (GLM) procedure from the Statistical Analysis Software was used. SNP5504 affected milk fat percentage, SNP8178 affected milk protein percentage and SNP5609 and SNP5624 affected 305-day milk yield. These results suggest that AAT is a candidate gene that influences milk production traits and it could be implemented in breeding programmes to improve the production performance of Chinese Holstein cattle. Keywords : Dairy cattle, α1-protease inhibitor, SNPs, milk traits, somatic cell score

In order to construct the prokaryotic expression vector pET32a-VP4,total RNA were extracted form MA104 cell infected by bovine rotavirus G6 strain CHLY.The open reading frame of VP4(2331bp) was amplified by RT-PCR and cloned into pEASY-T3 vector by using T-A cloning technique to get cloning vector pEASY-T3-VP4.After,the VP4 gene was subcloned into the expression vector pET32a and transformed into E.coli BL21(DE3).SDS-PAGE showed that the bacteria induced by IPTG successfully expressed,and approximately 108KD exogenous protein was observed on the SDS-PAGE.The with of VP4 was successfully constructed which is expected to lay a foundation for further studies of the subunit vaccine and DNA vaccine to prevent bovine rotavirus.

无脊椎动物体内不存在特异性的免疫球蛋白,因而非特异性免疫因子如凝集素、溶菌酶、溶血素、补体等就起着非常重要的作用.凝集素在无脊椎动物血液中的重要作用是起选择凝集作用,是免疫防御的重要体液因子之一.除血清中有凝集素存在外,在水生动物的皮肤粘液、体液、血细胞和受精卵、未受精卵及胚胎中皆发现存在凝集素.作者从研究简史、检测方法、物理化学性质、生物学性质和功能等几方面对水生无脊椎动物的凝集素研究作一阐述.

本文应用聚丙烯酰胺凝胶电泳结合生化染色方法分析了雌雄文昌鱼中苹果酸酶、苹果酸脱氢酶、酸性磷酸酶和酯酶四种同工酶的酶谱.首次发现苹果酸酶、苹果酸脱氢酶和酸性磷酸酶表型在文昌鱼雌性和雄性个体之间存在差异,而在同一性别不同个体之间无差异.酯酶表型较复杂,不但在不同性别个体之间而且在同一性别不同个体之间都出现一定差异.

概述了染色体的发现和基因在染色体上定位的荧光原位杂交技术,放射杂交体法,重叠群拼接和染色体步移及基因定位克隆的常用方法以及基因定位的应用.

进入20世纪80年代,随着动物养殖业的迅猛发展,兽药行业也进入了一个快速发展时期.企业数量、品种大幅度增长.目前,全国有兽药企业2600家,年产值达200亿元,生产兽药的品种、规格多达3000余个.国家针对兽药行业研发水平低、发展后劲不足,低水平重复建设现象比较严重、产品质量低劣,价格恶性竞争等问题,提出实施兽药GMP达标论证要求,目的是限制低水平重复建设、重复生产,提高兽药行业总体水平,提高兽药产品质量,制止恶性竞争.这对中小兽药企业来说,既是机遇,又是挑战.笔者认为中小兽药企业是指:目前的年销售额不足1000万元,或不能在通过GMP验收后短时间内达到1000万元的企业.这类企业约占全国兽药企业总数的85%左右.能否在短时间内实现营销机制创新,使企业的营销工作有个质的飞跃、质的提高,是摆在中小兽药企业销售部门面前的亟需解决的一个问题.

介绍兽药产品研发两个开发方向:应用开发和创新开发.结合我国中小型兽药企业,尤其是山东省中小兽药企业产品研发的现状,提出"以应用开发为基础,模仿创新为重点,自主创新为方向"的开发模式,并对这一思路进行具体阐述.