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20 November 2019, Volume 46 Issue 11
Biotechnology
Prokaryotic Expression and Identification of Porcine Derived Glucagon Like Peptide-2 Fusion Protein with Site Directed Mutagenesis
HAN Fei, ZHAO Ruixiao, WANG Gang, JIANG Mingfeng
2019, 46(11):  3137-3143.  doi:10.16431/j.cnki.1671-7236.2019.11.001
Abstract ( 245 )   PDF (2382KB) ( 161 )  
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In order to improve the production efficiency of GLP-2 to meet the application needs of animal husbandry,the porcine derived[Gly2]GLP-2(p[Gly2]GLP-2) was induced by site directed mutagenesis using genetic engineering technology.After replacing alanine at the second amino end of GLP-2 with glycine residue,the codon sequence of p[Gly2]GLP-2 was optimized according to the codon preference of Escherichia coli,and enterokinase recognition sites were added to the N end of the target peptide sequence to synthesize the gene sequence.Prokaryotic recombinant expression vector p[Gly2]GLP-2-pET-40b(+) was constructed by connecting Kpn Ⅰ and Xho Ⅰ digestion sites to pET-40b(+).The recombinant plasmid was transformed in E.coli BL21(DE3) competent cell to induced the expression of recombinant protein.The optimum expression parameters were that 0.2 mmol/L IPTG was used to induce the expression of p[Gly2]GLP-2-pET-40b(+) recombinant plasmid for 5 h when the bacterial solution D600 nm value was 0.6,and the recombinant strain with high expression of p[Gly2]GLP-2 was finally obtained.High purity p[Gly2]GLP-2 fusion protein was obtained by purification with nickel ion affinity chromatography column and gradient elution,which laid a foundation for further functional research and application in animal husbandry.
Cloning and Bioinformatics Analysis of Occludin Gene in Luchuan Pig
QU Qiuhong, XIE Hongyue, XIA Qin, PAN Peng, JIANG Qinyang, HUANG Yanna
2019, 46(11):  3144-3153.  doi:10.16431/j.cnki.1671-7236.2019.11.002
Abstract ( 260 )   PDF (2071KB) ( 143 )  
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The aim of this study was to obtain the coding sequence (CDS) of Occludin gene in Luchuan pig and carry out bioinformatics analysis of the sequence.A pair of specific primers were designed according to porcine Occludin gene sequence (accession No.:NM_001163647.2) published on GenBank,the jejunum tissue samples of Luchuan pig were collected,and RNA was extracted and then was reverse transcribed to synthesize cDNA.The target fragment was obtained by RT-PCR amplification,PCR products then were inserted into the pMD18-T vector,and then screened positive clones was sequenced.The obtained sequence was analyzed by bioinformatics software after sequencing.The results showed that the size of Occludin gene CDS in Luchuan pig was 1 569 bp,encoding 522 amino acids.The results of sequence alignment showed that the homology was 99.7% with reference sequence,and there were 3 different sites,the C→T was a missense mutation in the 16th position,which caused the leucine turns to phenylalanine at the 6th position,and the A→G at 1 059 bp position and the C→T at 1 218 bp position were synonymous mutations.This missense mutation site might be the reason of the intestinal barrier function in Luchuan pig was different from other pig breeds.The homology of Occludin gene in Luchuan pig were 83.8%,89.5%,88.1%,84.1%,88.3% and 86.6% with Mus musculus,Bos taurus,Homo sapiens,Mesocricetus auratus,Macaca nemestrina and Canis lupus,respectively.Phylogenetic tree analysis showed that the genetic distance between Luchuan pig and Bos taurus was the closest,and the genetic distance was the furthest with Canis lupus.The molecular weight of Occludin protein was 59.13 ku,the amino acid composition of the serine had a high content (9.2%).At the N-terminus of the peptide chain (Met),Occludin protein had an extinction coefficient of 96 415 in an aqueous solution and an instability index of 62.85,which was an unstable protein.Hydrophobic analysis results indicated that Occludin protein was a hydrophilic protein.There were five transmembrane helix regions of Occludin protein and two superfamily domains (MARVEL superfamily domain and Occludin-ELL superfamily domain) in Luchuan pig,and it had no signal peptide.The secondary structure prediction results showed that the alpha helix,extended chain and random coil of Occludin protein in Luchuan pig accounted for 41.00%,5.36% and 53.64%,respectively,and the tertiary structure was consistent with the secondary structure.This study successfully cloned and analyzed the sequence of Occludin gene CDS in Luchuan pig,which provided a reference for further study of the effect of Occludin gene on the intestinal barrier function of local pig breeds.
Cloning of Acot2 Gene and Its Expression Difference of Different Tissues in Red Steppe
LIU Lixiang, GAO Yi, LYU Yang, XUE Jiajia, HU Zhongchang, ZHANG Guoliang
2019, 46(11):  3154-3162.  doi:10.16431/j.cnki.1671-7236.2019.11.003
Abstract ( 164 )   PDF (2033KB) ( 83 )  
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This experiment was aimed to study the gene function of Acot2 in Red steppe using bioinformatics,and investigate the mRNA expression of Acot2 gene in different tissues of Red steppe.Primers were designed according to the sequence of cattle Acot2 gene (accession No.:NM_001101938.1) in GenBank.The complete CDS region of Acot2 gene was cloned by RT-PCR and sequencing.Sequence homology alignment and phylogenetic tree of Acot2 gene constructed by software.The corresponding amino acid sequence was obtained.The physical and chemical property,subcellular structure,hydrophilicity and hydrophobicity and phosphorylation site were analyzed by online prediction software.Protein secondary and tertiary structures were predicted by DNAStar software and SWISS-MODEL.Real-time PCR was used to detect the mRNA expression of Acot2 gene in different tissues of Red steppe.The results showed that the length of Acot2 gene CDS was 1 395 bp,encoding 464 amino acids.Acot2 gene CDS in Red steppe had higher homology with Bubalus bubalis(98.3%),but lower homology with Macaca mulatta and Pan troglodytes (80.5% and 80.4%).The molecular formula of Acot2 protein was C2317H3606N640O628S14,the molecular weight was 50.924 ku,the theoretical isoelectric point was 8.84.Most of the amino acid residues were hydrophilic residues,and the total average hydrophilicity was -0.094.The results of subcellular localization showed that Acot2 protein was located in the endoplasmic (30.4%),mitochondria (26.1%),golgi (17.4%),cytoplasmic (17.4%),vacuolar (4.3%) and nuclear (4.3%).There were 20 phosphorylation sites.The main forms of the secondary structure were alpha helix (21.8%),beta turn (33.4%),beta fold (18.4%) and random coil (26.4%),which was the same as the prediction of the tertiary structure.Real-time PCR result showed that the expression of Acot2 gene in Red steppe was the highest in stomach,it was rarely expressed in lung.The Acot2 gene was lowly conserved in the process of biological evolution.Acot2 protein was structurally stable,and it belonged to water soluble protein.The protein played a role in mitochondria and endoplasmic.The expression of Acot2 gene in different tissues of Red steppe was significant difference.This study provided the basis for further investigation of the effects of Acot2 gene on lipid metabolism in livestock and the screening of quality candidate genes in Red steppe.
Cloning and Bioinformatics Analysis of IL-6 Gene in Bovine
LYU Yanan, LIU Jingyu, YAO Jingjie, AN Zhixing, BAI Yueyu, ZHANG Xiaojian
2019, 46(11):  3163-3171.  doi:10.16431/j.cnki.1671-7236.2019.11.004
Abstract ( 218 )   PDF (1679KB) ( 116 )  
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The aim of the experiment was to clone the bovine interleukin-6 (IL-6) gene and analyze its coding protein bioinformatically.RNA was extracted from the tail heel blood of Holstein cows and retrieved into the cDNA.Specific primers were designed according to the known bovine IL-6 gene sequence in NCBI database,the full-length sequence of CDS region was cloned by RT-PCR.The biological characteristics of bovine IL-6 gene sequence,homology and physicochemical properties of coding products were analyzed by bioinformatics software.The results showed that the bovine IL-6 gene CDS region was successfully cloned,the total length was 627 bp,and the molecular weight was 23.759 ku,encoding a total of 208 amino acids.The theoretical isoelectric point was 7.58,the fat soluble coefficient was 93.37,which belonged to the hydrophilic protein.The homologies of Bos taurus IL-6 gene with Sus scrofa,Ovis aries,Rattus norvegicus,Homo sapiens,Felis catus,Canis lupus familiaris,Gallus gallus,Mus musculus,Equus caballus and Oryctolagus cuniculus were 84.4%,96.2%,66.7%,77.5%,75.9%,79.3%,48.5%,67.0%,79.5% and 67.5%,respectively.Phylogenetic tree analysis revealed that cattle and sheep had the closest genetic relationship,followed by pigs,and chickens was farthest.In the secondary structure prediction,the random coil and alpha-helix of the protein were 34.6% and 60.1% respectively.Subcellular localization was located in cytoplasm;The protein contained signal peptide and transmembrane helix structure.This study laid a theoretical foundation for further analysis of bovine IL-6 gene function.
RNA Sequencing of Citrobacter fredii Under Fluphenicol Stress
WEI Panqiu, HUANG Jie, HAN Shuyu, LUO Fuguang, LI Ya, YI Yi
2019, 46(11):  3172-3180.  doi:10.16431/j.cnki.1671-7236.2019.11.005
Abstract ( 231 )   PDF (2350KB) ( 195 )  
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In order to explore the resistance mechanism of Citrobacter freundii,florfenicol was used as an inducer,RNA was extracted from normal cultured strains and strains under flufenicol stress at 400 μg/mL.Gene expression levels and differentially expressed genes between strains were analyzed by RNA sequencing (RNA-Seq).The results showed that the average output of each sample was 1.31 Gb.The mean alignment rate with reference genome was 67.01%.Through the detection of differentially expressed genes among samples,a total of 2 019 significantly differentially expressed genes were screened,among which 963 genes were up-regulated and 1 056 genes were down-regulated,and 2 genes of emrA and emrB were found.KEGG Pathway analysis showed that the most differentially expressed genes were enriched in metabolism,accounting for 72% of the total.Carbohydrate metabolism,energy metabolism,amino acid metabolism,membrane transport and other pathways were significantly enriched.GO functional analysis showed that the distribution of significantly different genes in biological processes was the largest,accounting for 46% of all genes.Significant differentially expressed genes mainly involved metabolic process,catalytic activity and binding.It was suggested that the resistance of Citrobacter freundii might be related to active efflux mediated by extracellular pump,target change,enzyme hydrolysis,membrane permeability change and biofilm formation.
Effect of Pathogenic E.coli HPI on the Expression of Burnout-related Molecules in Caspase-1 Cells
CHA Jinlong, LIU Chaoying, GAO Hong, WANG Shiyu, SHAN Chunlan, JING Linxi, WANG Hao, YANG Wei
2019, 46(11):  3181-3189.  doi:10.16431/j.cnki.1671-7236.2019.11.006
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In order to study the molecular mechanism of cell death in porcine macrophages induced by pathogenic E.coli HPI from Saba pig in Yunnan,the pig derived macrophages infected with HPI positive strain of Saba pig in Yunnan were taken as the breakthrough point. E.coli was isolated from feces of yellow-white dysentery in a large scale farm in Chuxiong,Yunnan.The HPI irp2 gene was detected by PCR technology.Macrophages were infected with HPI positive strains (HPI+) and negative strains (HPI-),respectively,and LPS+ATP group and blank control group were set up,cells and their supernatants were collected at 0.5 and 6 h.The expression levels of Caspase-1,IL-1β and IL-18 in different groups were detected by Real-time PCR,and the contents of pro-IL-1β,pro-IL-18,IL-1β and IL-18 in cell supernatant were detected by ELISA.The results showed that pathogenic E.coli was successfully isolated and HPI irp2 gene positive strains were successfully obtained by PCR.Real-time PCR results showed that the expression levels of Caspase-1,IL-1β and IL-18 of HPI+ and HPI- groups were all higher than those of blank control group.It showed an upward trend,and HPI+ group was higher than HPI- group.ELISA results showed that compared with blank control group,the expression of pro-IL-1β,pro-IL-18,IL-1β and IL-18 in HPI+ and HPI- groups were generally up-regulated,and the HPI+ group was higher than that in HPI- group.The results showed that the pathogenic E.coli HPI of Saba pig in Yunnan could promote the release of inflammatory cytokines IL-1β and IL-18 from pig macrophages by up regulating the expression of Caspase-1,IL-1β and IL-18 mRNA in porcine macrophages,and eventually induce inflammation.
Physiology and Biochemistry
Effects of Baicalin on Tight Junction Structure and MLCK-MLC in Piglets Induced by Lipopolysaccharide
ZHAO Wenhua, ZHUANG Feng, XIONG Chunhong, LIU Yu, FU Shulin, QIU Yinsheng
2019, 46(11):  3190-3197.  doi:10.16431/j.cnki.1671-7236.2019.11.007
Abstract ( 258 )   PDF (4361KB) ( 125 )  
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In this study,lipopolysaccharide (LPS) induced immune injury model of piglets was established to study the effect of baicalin on the expression levels of LPS-induced vascular tight coupling protein in piglets and the changes of MLCK-MLC signaling pathway,and to explore its protective mechanism.Forty-two healthy piglets were selected and divided into six groups:Control group (group A),LPS model group (group B),LPS+ethyl pyruvate treatment group (group C),LPS+flunixin meglumine treatment group (group D),LPS+baicalin treatment group (25,50,100 mg/kg,groups E,F,G).After feeding for 7 d,drug treatment was conducted 0.5 h before challenge.Mice in treatment group and model group got LPS intraperitoneally injected,and the same amount of normal saline was injected into mice in the control group.LPS was induced for 6 h and then administered again.After 1 d of the experiment,two aorta blood vessel samples were collected.The structural changes of tight junction proteins (ZO-1,Claudin-1 and Occludin) in the blood vessels were observed by immunohistochemistry or immunofluorescence.Western blotting was used to determine the expression of MLCK-MLC signaling pathway related proteins (MLCK,MLC2 and p-MLC2).The results showed that after LPS induced,the expression levels of ZO-1,Claudin-1 and Occludin were extremely significantly decreased (P<0.01),and structure of tight vascular connections was damaged,while baicalin treatment could significantly increase their expression.In the LPS model group,the expression levels of MLCK and p-MLC2 were extremely significantly increased (P<0.01),and baicalin could significantly down-regulate expression levels of MLCK and p-MLC2(P<0.01).The expression of MLCK in all groups showed no significant difference (P>0.05).In conclusion,LPS induction could cause vascular injury in piglets,inhibit MLCK-MLC signaling pathway,and damage the structure and function of tight junctions.Baicalin had the protective effect on the damage of vascular tight junction structure in piglets induced by LPS.The results could provide a scientific basis for baicalin in the treatment of inflammatory vascular injury in pigs.
Histological Characteristics Analysis of Aging Hadley Pomeranian Testis
LI Chengye, GUO Yansheng, YUAN Ligang, WANG Qianmei, YANG Jie, REN Jianming, GAO Rui
2019, 46(11):  3198-3207.  doi:10.16431/j.cnki.1671-7236.2019.11.008
Abstract ( 237 )   PDF (6783KB) ( 85 )  
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This study was conducted to characterize the histologic structure of testicular tissue and the distribution of related protein between aging and young adult Hadley Pomeranian,and analyze the variation at different ages and influence on reproductive function.The testicular histochemical characteristics of young adult and agin Hadley Pomeranian were compared by special staining,immunohistochemistry and immunofluorescence.The quantitative analysis was carried out by IPP image analysis software.The results showed that compared with the young adult Pomeranian testis,the number and thickness of spermatogenic epithelial of the aging Pomeranian were decreased,and the number of Leydig cells and Sertoli cells were significantly increased (P<0.05).The basal layer of the seminiferous tubules and vascular wall were increased significantly.The results of immunohistochemistry showed that compared with young adult Pomeranian,the content of Col Ⅳ and HSPG in the aging Pomeranian testicular were extremely significantly decreased (P<0.01),the LN significantly decreased (P<0.05).Immunofluorescence results showed that Col Ⅳ,HSPG and LN were strongly expressed in Leydig cells and Sertoli cells.Therefore,the increased collagen and reticular fibers and the number of Leydig cells and Sertoli cells might be closely related to the spermatogenic function maintenance of the aging Pomeranian,and especially related to the Col Ⅳ and HSPG expression.
Animal Nutrition and Feed Science
Effects of Saccharomyces cerevisiae and Bacillus licheniformis on Rumen Fermentation in Sheep in vitro
ZHENG Weicai, HAO Xiaoyan, ZHANG Chunxiang, XIANG Binwei, ZHANG Wenjia, WEN Haoyu, ZHANG Jianxin
2019, 46(11):  3208-3215.  doi:10.16431/j.cnki.1671-7236.2019.11.009
Abstract ( 236 )   PDF (809KB) ( 95 )  
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The aim of this experiment was to study the effect of Saccharomyces cerevisiae and Bacillus licheniformis on rumen fermentation of sheep by in vitro culture method.Six fermentation substrates were prepared by adding 6×1011 CFU/kg Saccharomyces cerevisiae and 5 levels of Bacillus licheniformis (0 (C1 group)、1×1011(C2 group)、2×1011(C3 group)、3×1011(C4 group) and 4×1011 CFU/kg (C5 group))were added to the diet with a concentrate to forage ratios of 50:50,and the control group was set with no Saccharomyces cerevisiae and Bacillus licheniformis (C group).The gas production parameters,CH4 concentration,ruminal fermentation parameters and nutrient degradation rates were determined after fermented for 48 h.The result showed as follows:①The combined effects of Saccharomyces cerevisiae and Bacillus licheniformis had significant effects on the cumulative gas production at 24 and 48 h,the gas production of fast fermentation,the gas production of slow fermentation,the potential gas production and the gas production rate (P<0.05),and they reached the maximum at the addition of 2×1011CFU/kg Bacillus licheniformis.②The interaction of Saccharomyces cerevisiae and Bacillus licheniformis had no significant effect on pH value (P>0.05),but they could significantly affect ammoniacal nitrogen (NH3-N) and microbial proteins (MCP) yields (P<0.05).The concentration of NH3-N reached the minimum and MCP reached the maximum when the dosage of Bacillus licheniformis was 2×1011 CFU/kg.③The interaction effect of Saccharomyces cerevisiae and Bacillus lichenformis on the dry matter degradation rate (DMD),organic matter degradation rate (OMD) and metabolic energy (ME) were significant (P<0.05),but had no significant effects on neutral detergent fiber degradation rate (NDFD) and acid detergent fiber degradation rate (ADFD) (P>0.05).The DMD,OMD,NDFD and ME reached the maximum while ADFD reached the minimum at the addition of 2×1011CFU/kg Bacillus lichenformis.In conclusion,the Saccharomyces cerevisiae and Bacillus lichenformis supplement had significant interaction effects at 6×1011 and 2×1011 CFU/kg on rumen fermentation of sheep in vitro.
Biological Function of Conjugated Linoleic Acid and Its Application in Animal Production
YANG Mei, MA Jie, YANG Tai, YAN Zhaoming, CHEN Qinghua
2019, 46(11):  3216-3224.  doi:10.16431/j.cnki.1671-7236.2019.11.010
Abstract ( 247 )   PDF (925KB) ( 214 )  
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As a natural new functional fatty acid,conjugated linoleic acid (CLA) has anti-hypertensive,anti-cancer,anti-diabetic and other biological properties,and its main active isomers play different roles.In recent years,with the decrease of CLA artificial synthesis cost and the deep researches in the field of animal nutrition,it was found that CLA can inhibit the expression and production of pro-inflammatory factors,promote the production of anti-inflammatory factors,and achieve anti-inflammatory effects,it can also regulate lymphocyte proliferation and differentiation,macrophage activity,promote antibody synthesis,regulate humoral immunity and cellular immunity,thereby enhancing animal immunity.CLA can also regulate the expression of a variety of nuclear transcription factors,affect fatty acid uptake and oxidation,as well as lipid anabolism,thereby reducing body fat deposition,increasing intramuscular fat content,changing fatty acid composition,and improving meat quality.Therefore,CLA has potential application value and broad market prospects in animal husbandry production.The article mainly introduced the roles of CLA in anti-inflammatory,immune function,lipid metabolism and anti-oxidation,and reviewed the biological functions of CLA and its application researches in animal production.In addition,CLA was also discussed as a feed additive,including its synthetic purity,optimal addition amount in different types of animals,different ages,feeding methods,in order to provide a theoretical reference for its rational application in animal production.
Research Progress on Energy and Protein Requirements of Fur Animals
HAN Feifei, ZHONG Wei, WANG Jing, LI Guangyu
2019, 46(11):  3225-3233.  doi:10.16431/j.cnki.1671-7236.2019.11.011
Abstract ( 210 )   PDF (901KB) ( 97 )  
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The levels of energy and protein in diet can directly affect the health and production performance of fur animals,also determine the feed cost and cause environmental pollution effect.The ratio of nutrient to energy in diet and its sources can affect the utilization of dietary energy,thus affect the effective energy value of diet.Therefore,exploring the optimum requirements and sources of energy and protein of fur animals possesses great significance in practical production.In this paper,the authors introduced the energy system of fur animals and summarized recent research progress on nutrient to energy ratio and nutrient sources,metabolism rules and requirements of energy and protein of fur animals,and forecasted the further researches direction,in order to offer worthy insights into precise feeding and diet preparation of fur animals.
The Biological Function of Resveratrol and Its Application in Animal Production
MA Yujing, HE Rongxiang, YANG Ling, HE Jianhua
2019, 46(11):  3234-3243.  doi:10.16431/j.cnki.1671-7236.2019.11.012
Abstract ( 376 )   PDF (1121KB) ( 176 )  
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Resveratrol is a natural non-flavonoid polyphenolic compound containing a quinone structure.It has various biological functions,including inhibiting and eliminating excess free radicals in body,activating the body's antioxidant enzyme system and exerting antioxidant function.By regulating a series of inflammatory or anti-inflammatory pathways,it can reduce the expression of inflammatory factors in the body and the inflammatory response.Resveratrol also has important biological functions such as regulating glycolipid metabolism,protecting cardiovascular,anti-cancer,anti-virus,etc.It has the advantages of safety,high efficiency,no residue,no drug resistance,and has broad application prospects in animal production.Animal experiments show that resveratrol can increase the average daily gain,average feed intake,and feed to gain ratio of broilers.It also can improve the quality of fattening pork,increase the longissimus dorsi muscle,pH24 h,redness,crude protein and myoglobin The compound can also reduce muscle brightness after 24 h,shear force,drip loss and backfat thickness,increase antioxidant enzyme activity in animal body,reduce inflammatory factor production,enhance immunity and improve intestinal flora composition and mucosal morphology,inhibits the formation of cancer cells.The authors will review the biological characteristics,physiological functions of resveratrol and its application status in order to provide reference for its application in livestock and poultry production.
Effects of Dietary Phosphorus Level on Nutrient Digestibility in Non-Pregnant Yunnan Semi-fine Wool Ewes
LI Weijuan, WU Jing, LI Yinjiang, OUYANG Yina, WANG Siyu, XUE Bai, HONG Qionghua
2019, 46(11):  3244-3251.  doi:10.16431/j.cnki.1671-7236.2019.11.013
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25 non-pregnant Yunnan semi-fine wool sheep were allocated to 5 groups corresponding to 5 dietary treatments with P levels at 0.19%,0.32%,0.45%,0.56% and 0.64%,for investigating the effect of dietary P level on nutrient digestibility.19 d digestion trial were conducted,with 14 d preliminary period and 5 d formal period.During the official period,200 g of concentrate and 200 g of crude material were collected in each group every day.Each feces sample was weighed,20% was taken and divided into two parts,one part was added 10% HCL to fix nitrogen.The percentage of 10% HCL in the mixture was 5% (W/W).Each day,the feed and feces were dried at 55℃,followed by rehydration for 24 hours.Then the dry samples were smashed and filtrated through 40 meshes,and the corresponding indicators were determined.The results showed that dietary P levels significantly affected nutrient digestibility,with the exclusion of Ca digestibility.Digestibility of DM and CP decreased with the elevated dietary P levels,and that in 0.19% and 0.32% P level groups was higher than that in 0.64% P group (P<0.01).Digestibility of gross energy,crude fat,ash,NDF and ADF increased with the increasing of P levels,then decreased till dietary P reached 0.45%,after that it increased again with the inflexion of 0.45% P level.Digestibility of P decreased with the elevated dietary P levels,and that in 0.19% P group was higher than that in 0.45%,0.56% and 0.64% P level (P<0.01).There was no differences on P digestibility among other groups (P>0.05).Regression analysis revealed that the linear relationship between dietary P levels and digestibility of DM,CP and P were significant (R2 were 0.7823,0.8386 and 0.9976,respectively).In conclusion,dietary P levels did not affect Ca digestibility.Digestibility of DM,CP and P decreased with the elevated dietary P levels.The highest digestibility of nutrients always happened in 0.32% group.
Effects of Long-term Addition of Ampelopsis grossedentata Extract on Serum Free Amino Acids,Carcass Traits and Meat Quality in Finishing Pigs
XIONG Yunxia, WANG Li, YI Hongbo, WU Qiwen, WEN Xiaolu, HU Youjun, JIANG Zongyong
2019, 46(11):  3252-3261.  doi:10.16431/j.cnki.1671-7236.2019.11.014
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In order to study the effects of Ampelopsis grossedentata extract on the serum free amino acid,carcass traits and meat quality of finishing pigs,90 three line hybrid (Duroc×Landrace×Yorkshire) castrated boars with initial weight of 30 kg were randomly divided into 3 treatments with 6 columns per group and 5 pigs per column,CON group (fed with basal diet),group A(fed with basal diet adding 0.03% plant essential oil complex) and group B (fed with basal diet adding 0.03% Ampelopsis grossedentata extract),respectively.All the pigs were weighed when they were about 130 kg,and one pig with the close body weight to the replicate average was selected to collect blood samples and slaughtered.Group A was used as positive control.The results showed that:① Compared with CON group,the contents of essential amino acids Lys,Met,Ile were significantly increased (P<0.05) and His content was significantly decreased (P<0.05),the content of serum non-essential amino acids Glu,Ala,Pro decreased significantly (P<0.05) in group B.Compared with group A,the contents of essential amino acids Met and Thr were significantly increased (P<0.05),and the content of His was significantly decreased (P<0.05) in group B.There were no significant differences in non-essential amino acids between group A and B (P>0.05).② Compared with the CON group,the abdominal fat weights of both group A and B was significantly increased (P<0.05),but there was no significant difference between group A and B (P>0.05).There was no significant changes in pre-slaughter weight,hot carcass weight,dressing out,backfat thickness and loin-eye area among the three groups (P>0.05).③There was no significant difference in meat color,pH value,drip loss,marble score,intramuscular fat and shear force of longest muscle among the three groups (P>0.05).Compared with the CON group,the score of chewiness in group B was significantly improved (P<0.05);The score of turbidness of soup in group A and B were significantly lower (P<0.05),while the score of appearance,smell,abnormal flavor,juiciness,freshness of soup showed no significant difference among the three groups (P>0.05);And muscle fiber diameter and density were not significant among the three groups (P>0.05).In summary,long-term addition of 0.03% Ampelopsis grossedentata extract to the diet could significantly change the serum amino acid composition of the finishing pigs and improve the tenderness of the pork to some extent.However,the improved effect on carcass traits and muscle quality of Ampelopsis grossedentata extract or plant essential oil complex was not significant,and abdominal fat deposition was easy to occur in both cases.
Advances in the Application of Algae in Pig and Chicken Production
XUAN Xiongzhi, LI Wenjia, LI Shaoyu, WEI Fengxian, XU Bin, LIU Jingen
2019, 46(11):  3262-3269.  doi:10.16431/j.cnki.1671-7236.2019.11.015
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The kinds and nutritional components of the algae which available in livestock and poultry production were summarized in this paper.The effects of algae-containing feed on the growth,immunity,meat quality,egg-laying performance and egg quality of livestock and poultry which represented by pig and chicken were emphatically introduced.The problems existing in the application of algae in livestock and poultry and some suggestions were pointed out to provide reference for the wide application of algae resources in pig and chicken production.Algae are widely distributed,easy to grow and high-yielding,especially the marine algae,which contain bioactive substances and nutrients that terrestrial plants do not possess.Microalgae which is rich in unsaturated fatty acids and pigments adding to the feed could improve the meat and egg quality of breeding animals.Macroalgae which is rich in polysaccharides and other bioactive substances adding to feed could improve the immunity of livestock and poultry.Adding a small amount of algae in feed could improve the feed stability,growth performance and feed utilization of livestock and poultry.Because of its anti-nutritional factors,excessive addition of algae to the feed will have a negative impact on livestock and poultry,but some anti-nutritional factors could be eliminated by proper pretreatment and the negative effects can be alleviated.
Research Advances of Net Energy Application for Broiler Production
ZOU Yi, ZHANG Xiaofeng, LIU Songbo, PENG Yunzhi, GUO Yuming, TAN Huize
2019, 46(11):  3270-3276.  doi:10.16431/j.cnki.1671-7236.2019.11.016
Abstract ( 258 )   PDF (736KB) ( 165 )  
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Energy is the basis of all life activities for broilers,and it is also the most expensive part of broiler diets.Grains,protein meals and edible oils consumed by broilers yield energy when oxidised during metabolism.Accuracy energy requirement continues to gain economic importance and research importance as a component of broiler feed.Almost all poultry feed is currently formulated on the basis of metabolisable energy (ME).Formulation of broiler feed on a net energy (NE) basis may be more accurate than the ME system as it takes into account the energy lost as heat increment (HI).Therefore,combined with the related research progress of net energy of broilers at home and abroad,this paper focuses on the methods of measuring net energy of broilers,the prediction equation of net energy of raw materials and the factors affecting the application of net energy system of broilers,in order to provide reference for the application of net energy system in broiler production.
Genetics and Breeding
Comparative Transcriptome Analysis of Ovary Tissues in Fuqing and Daiyun Goats During Summer Estrus
WU Xianfeng, LI Wenyang, LIU Yuan, ZHANG Ying, HUANG Qinlou
2019, 46(11):  3277-3288.  doi:10.16431/j.cnki.1671-7236.2019.11.017
Abstract ( 196 )   PDF (2842KB) ( 178 )  
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In order to reveal the molecular mechanism of superior fecundity of Daiyun goat in summer season and find out the useful genetic variation in ovary.Transcriptome sequencing of ovary tissue in Fuqing and Daiyun goats were performed by Illumina HiSeq.Differentially expressed genes (DEGs) were screened using gene ontology (GO),cluster of orthologous groups of proteins (COG) and kyoto encyclopedia of genes and genomes (KEGG) databases,which were verified by Real-time quantitative PCR.The results showed that there were 357 DEGs in Fuqing and Daiyun goats,221 DEGs were up-regulated and 136 DEGs were down-regulated.Cluster analysis results showed that 287,78 and 219 DEGs annotated into the GO,COG and KEGG databases,respectively.KEGG analysis results showed that 219 DEGs were mainly enriched in protein digestion and absorption,phagosome,tuberculosis,ECM-receptor interaction, Staphylococcus aureus infection and allograft rejection.Real-time quantitative PCR results showed that the expression patterns of the selected gene (transcript) were consistent with that of transcriptome sequencing,indicating that the sequencing results were reliable.In this study,a large amount of ovarian transcriptome information in goat was obtained by high-throughput sequencing,which was helpful to further study the ovary in goats at the molecular level.
Association Analysis of UCP1 Gene Variation with Growth and Carcass Traits in Ovine
AN Qingming, ZHOU Huitong, WANG Xing, WU Zhenyang, LUO Yuzhu, JON G. Hickford
2019, 46(11):  3289-3298.  doi:10.16431/j.cnki.1671-7236.2019.11.018
Abstract ( 175 )   PDF (1829KB) ( 102 )  
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This study was aimed to analyze the effect of UCP1 gene variation on growth and carcass traits in ovine,so as to screen out the nucleotide variation that could improve growth and carcass traits in ovine and provide materials for molecular genetic markers of important economic traits related to ovine.The mutations in intron 5 and exon 6 regions of ovine UCP1 gene were detected by PCR-SSCP in 9 ovine populations,and the relationships between mutations in growth in male and female lambs and carcass traits in male lambs were investigated using General Linear Models (GLMs) by Minitab 16.0 software.The results showed that 8 SNPs were detected in intron 5 and exon 6 regions of ovine UCP1 gene,the nucleotide substitution c.910 G/A in exon 6 resulted in amino acid change (p.Ala304Thr).The results of correlation analysis of growth traits showed that the allele in intron-5 region of ovine UCP1 gene had different effects on growth traits of male and female lambs.The presence of allele A1 was associated with decreased birth weight in female lambs (P<0.05),and the presence of allele C1 was associated with increased tailing weight in male lambs (P<0.05),no associations were detected between the other allele and growth traits in male and female lambs.The results of correlation analysis of carcass traits showed that the presence of allele A1 was associated with decreased leg yield,loin yield and increased proportion of should yield (P<0.05),and the presence of allele C1 was associated with decreased proportion of should yield (P<0.05),no associations were detected between the other allele and carcass traits in male lambs,and the genotype analysis results was consistent with allele analysis.The results indicated that the effect of ovine UCP1 gene on growth traits was gender-specific,and allele A1 had lower carcass production traits in male lambs,which provided a theoretical basis for improving carcass production in male lambs.
Effects of FSH on Genes Expression Related to Steroidogenesis in Bovine Follicles Cultured in vitro
WANG Yangyang, XU Huitao, ZHAO Shanjiang, PANG Yunwei, HAO Haisheng, ZOU Huiying, ZHU Huabin
2019, 46(11):  3299-3306.  doi:10.16431/j.cnki.1671-7236.2019.11.019
Abstract ( 166 )   PDF (920KB) ( 112 )  
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The purpose of this study was to investigate the effects of FSH treatment on steroidogenesis genes expression in granulosa cells and theca cells of cultured bovine antral follicles in vitro.Cavernous follicles 9-11 mm in diameter on the surface of bovine ovaries were collected and cultured in vitro with DMEM/F12 containing different concentrations of FSH for 24 hours.The total RNA of granulosa cells and theca cells were extracted,and reverse transcribed into cDNA,the expression of steroidogenesis genes (CYP11A1,3β-HSD,CYP17A1,CYP19A1 and 17β-HSD) and gonadotropin receptor genes (FSHR and LHR) in follicular granulosa cells and theca cells were detected by Real-time quantitative PCR.The results in granulosa cells showed that FSH up-regulated the expression of CYP11A1,3β-HSD and CYP19A1 genes,25 ng/mL FSH treatment extremely significantly increased the expression of CYP11A1 gene (P<0.01),10 ng/mL FSH treatment significantly increased the expression of 3β-HSD gene (P<0.05),50 ng/mL FSH treatment significantly increased the expression of CYP19A1 gene (P<0.05).The results in theca cells showed that 50 ng/mL FSH treatment significantly or extremely significantly increased the expression of CYP11A1,3β-HSD and CYP17A1 genes (P<0.05;P<0.01),but 10 and 25 ng/mL FSH treatment significantly or extremely significantly decreased the expression of CYP11A1,3β-HSD and CYP17A1 genes (P<0.05;P<0.01).The results showed that FSH treatment had no significant effect on the expression of FSHR and LHR genes in granulosa cells (P>0.05),only 25 and 50 ng/mL FSH treatments significantly or extremely significantly increased the expression of LHR gene in theca cells (P<0.05;P<0.01).The expression of CYP11A1,3β-HSD and CYP17A1 genes in theca cells among the different treatment groups showed the same trend as that of LHR gene.The results indicated that FSH treatment could increase the expression of CYP11A1,3β-HSD and CYP17A1 genes in granulosa cells of bovine antral follicular,the expression of CYP11A1,3β-HSD and CYP17A1 genes in theca cells were more sensitive to the stimulation of LH.FSH might regulate the expression of steroidogenesis genes in theca cells by affecting the expression of LHR gene.
Research Progress on Genetic Breeding and Reproduction of Domestic and International Rabbit in 2018
ZHANG Zhaoxia, YANG Lin, DING Haisheng, HUANG Dongwei, WANG Yong, CHENG Guanglong, ZHAO Huiling
2019, 46(11):  3307-3315.  doi:10.16431/j.cnki.1671-7236.2019.11.020
Abstract ( 212 )   PDF (969KB) ( 128 )  
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The review summarized research progress on genetics and breeding in domestic and international rabbit in 2018,which mainly included traditional breeding,molecular breeding,and reproductive techniques.Selection effect and trait evaluation,interaction between heredity and environment and measurement of germplasm performance were mainly studied in traditional breeding,while related genes on growth,meat quality,fur and reproduction were mainly researched in molecular breeding.In addition,the studies of rabbit reproduction techniques referred to semen freezing and effects of additives and environment to rabbit reproduction.More researches on traditional breeding and molecular breeding of rabbits were performed at home,domestic traditional breeding mainly included measurement and comparison of genetic performance about native breed,domestic molecular breeding mainly included the researches of meat quality,fur and reproduction trait,and obtained new molecular markers and genes related to meat quality,fur and reproduction traits.However,relatively few researches were found in propagation technology of rabbits.While the research of traditional breeding abroad mainly focuses on selection and trait evaluation,interaction between heredity and environment;Molecular breeding mainly focuses on filtering molecular markers regulating growth,meat quality and reproduction,but relatively insufficient studies focusing on fur were found at abroad.Molecular breeding abroad mainly used traditional research methods to filter molecular markers and research the function of genes,while high-throughput sequencing technology was mostly used to screen genes and regulatory networks important for the studied traits in China.The author summarized the differences in domestic and international rabbit breeding in 2018 and provided references for rabbit breeding in later research.
Correlation Analysis Between GLP2R Gene Polymorphism and Meat Quality Traits in Sheep
LIANG Yifan, CAO Yang, JIN Huazi, WEI Tian, XIAO Cheng, MA Huihai, JIN Haiguo
2019, 46(11):  3316-3322.  doi:10.16431/j.cnki.1671-7236.2019.11.021
Abstract ( 167 )   PDF (1072KB) ( 138 )  
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To explore the correlation between glucagon-like peptide-2 receptor (GLP2R) gene polymorphism and meat quality traits in sheep,the hybrid offspring of 68 South African meat Merino sheep×Northeast Fine wool sheep and 102 Dorper sheep×Small-tail Han sheep as the research object in this study,the Sanger sequencing method was used to find the mutation sites of the sequences of GLP2R gene exon 11,according to the sequencing results,the genotype frequency and gene frequency of the mutation site and the chi-square suitability test,homozygosity (Ho),heterozygosity (He) and polymorphism information content (PIC) were analyzed.The results showed that there were C/T mutations of GLR2R gene exon 11 in sheep,there were three genotypes (CC,TC and TT) and two alleles (C and T).TC genotype was the dominant genotype in South Africa meat Merino sheep×Northeast Fine wool sheep,CC genotype was the dominant genotype in Dorper sheep×Small-tail Han sheep,and C allele was dominant in both two populations.The results of chi-square suitability test showed that C/T site in two populations were in Hardy-Weinberg equilibrium (P>0.05).The intra-population variation was small and moderate polymorphism (0.25 < PIC < 0.5),which had certain genetic significance.The results of different genotypes of meat quality traits showed that the shearing force of TC genotype in South African meat Merino sheep×Northeast Fine wool sheep was significantly higher than that of CC and TT genotypes,and the water loss rate of TT genotype was significantly higher than that of CC and TC genotypes (P<0.05);The shearing force of CC genotype in Dorper sheep×Small-tail Han sheep was significantly higher than that of TC and TT genotypes,and the water loss rate of TT genotype was significantly higher than that of CC and TC genotypes (P<0.05).In this study,a C/T mutation was found in GLP2R gene exon,which might be a potential site affecting meat quality traits,whether it could be used as a genetic marker needed further exploration.
Effects of Somatic Cell Score on Milk Yield and Milk Composition in Holstein Milk in Xinjiang
ZHAO Fanfan, GE Janju, LI Hongyan, ZHANG Menghua, WEI Chen, WANG Dan, DU Jianwen, XU Lei, JIANG Hui, SANG Zhagen, HUANG Xixia, TAN Shixin
2019, 46(11):  3323-3331.  doi:10.16431/j.cnki.1671-7236.2019.11.022
Abstract ( 165 )   PDF (992KB) ( 125 )  
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In order to explore the relationship between daily milk yield (DMY)and milk composition of somatic cell score (SCS) in milk,and to understand the health status of dairy cows' breasts,in this study,33 290 DHI records and production performance data of 2 338 Holstein cattle in Changji area,Xinjiang were used to analyze the effect of SCS on daily milk yield and milk composition after converting SCC recorded by DHI into SCS.The least square variance analysis and multiple comparison of milk yield and milk composition were carried out by using SAS 9.2 software,and the correlation between SCS and DMY and milk composition was analyzed.The fixed effects mainly included SCS,field,year,season and parity effect.The results showed that the factors of SCS,field,year,seasonal and parity had significant effects on DMY and milk composition (P<0.01).With the increase of SCS,the DMY and milk lactose percentage (MLP) decreased.When SCS was 0 and SCC was 9×103 to 17×103,the DMY and MLP were extremely significantly higher than those of other SCS levels (P<0.01).The milk fat percentage (MFP) and milk protein percentage (MPP) showed an upward trend with the increased of SCS,and when SCS was 9 and SCC was 4 529×103 to 4 904×103,the MFP and MPP were highest.The results of correlation analysis showed that SCS,field,year,season and parity all had extremely significant effects on daily milk yield and milk composition (P<0.01);There was a extremely significant negative correlation between SCS and DMY,MLP (P<0.01),while SCS was extremely significantly positively correlated with MFP and MPP (P<0.01).Therefore,by analyzing the relationship between DMY,milk composition and SCS,it could provide scientific basis for daily management of large-scale dairy cows and prevent and reduce the risk of mastitis.
Effects of Variety,the Seasons of Entering into Swinery and Breeding, First Mating Time on Reproduction Performance of Gilts
XU Zhen, LYU Minyong, WEI Hengxi, ZHANG Huiwen, CHEN Guangxin, LI Zan, LI Li, MENG Li, ZHANG Shouquan
2019, 46(11):  3332-3340.  doi:10.16431/j.cnki.1671-7236.2019.11.023
Abstract ( 227 )   PDF (783KB) ( 139 )  
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To explore the effects of variety,the seasons of entering into swinery and breeding,first mating time on reproduction performance of gilts,the breeding status of 1 656 gilts were observed and recorded in this study,and they were grouped according to different varieties,the seasons of entering into swinery,the seasons of breeding and the first mating time,and statistical analysis was performed among the different groups.The results showed that:①The breeding utilization rate of Landrace gilts (89.73%) was extremely significantly higher than those of Yorkshire gilts (75.00%) and Landrace-Yorkshire hybrid gilts (80.57%)(P<0.01);The total number of born (TNB) of Landrace-Yorkshire hybrid gilts in the first parity (11.36) was significantly higher than that of Yorkshire gilts (10.35) (P<0.01),and significantly higher than that of Landrace gilts(10.74) (P<0.05).②The breeding utilization rate (100.00%) of Landrace-Yorkshire hybrid gilts of entering into swinery in the spring and winter was extremely significantly higher than those of entering into swinery in the summer (71.89%) and autumn (51.47%) (P<0.01),and the breeding utilization rate of Landrace-Yorkshire hybrid gilts in the summer was also significant higher than that in the autumn (P<0.01);The delivery rate of conception of breeding Landrace-Yorkshire hybrid gilts in the autumn (86.21%) was significantly lower than that in the spring(94.07%)(P<0.05),and extremely significantly lower than that in the winter (97.42%) (P<0.01).③The number of born alive (NBA) of Landrace-Yorkshire hybrid gilts in the group of 211~230 d first mating time (9.30) was significantly lower than those of the groups of 231~250 d (10.15) and 251~230 d (10.36) (P<0.05),and extremely significantly lower than that of the group of 271~290 d(10.48) (P<0.01);The number of qualified piglets of Landrace-Yorkshire hybrid gilts in the group of 211~230 d first mating time (8.96) was significantly lower than that of the group of 231~250 d (9.82) (P<0.05),and extremely significantly lower than those of the groups of 251~270 d (10.04) and 271~290 d (10.21) (P<0.01).In summary,the breeding utilization rate of Landrace gilts was better,and the litter performance of Landrace-Yorkshire hybrid gilts was better among the different varieties of gilts.The breeding utilization rates of Landrace-Yorkshire hybrid gilts of entering into swinery in the spring and winter seasons were better,and the delivery rate of conception was poor when the season of breeding was in the autumn,and the litter performance of Landrace-Yorkshire hybrid gilts was poor in the group of 211~230 d first mating time,and it had an increasing trend when they were bred in 211~290 d first mating time.
Preventive Veterinary Medicine
Construction and Biological Characteristics Analysis of asd Gene Mutant Balanced Lethal Host-vector System of Salmonella Typhimurium LH430 Strain
ZHANG Mingliang, SUN Changjiang, GU Jingmin, CUI Ziyin, HAN Wenyu
2019, 46(11):  3341-3349.  doi:10.16431/j.cnki.1671-7236.2019.11.024
Abstract ( 212 )   PDF (2916KB) ( 129 )  
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This study was aimed to construct a balanced lethal system based on attenuated Salmonella Typhimurium.Cat gene was recombined into the position of asd gene in Salmonella Typhimurium LH430 strain,then the thermo sensitive plasmid pCP20 was transferred into the strain to eliminate the Cat gene fragment,and the mutant strain Δasd LH430 was identified by PCR.Then the balanced lethal system of Salmonella Typhimurium Δasd LH430(pYA3493) was successfully constructed with the plasmid pYA3493 transferred into Δasd LH430.The mutant strain lost the ability to survive in the medium without DAP,had the same ability of using carbon sources was consistent with parent strains,and inherited the biochemical characteristics from parent strains based on nine biochemical experiments,Δasd LH430(pYA-gfp) carrying green fluorescent gene was successfully constructed,and the green fluorescent protein could be also inherited the deletion fragment of 423 bp.Then the Δasd LH430(pYA-gfp) carrying green fluorescent gene was successfully constructed,and the green fluorescent protein could be effectively expressed by Δasd LH430(pYA-gfp).In conclusion,the balanced lethal system Δasd LH430(pYA3493) was successfully constructed in this study,and could effectively express the carrying genes,the system could be used as a living vaccine vector to carry exogenous genes.
Construction and Identification of Swine O-type Foot-and-mouth Disease Virus-like Particles
LIU Hanping
2019, 46(11):  3350-3357.  doi:10.16431/j.cnki.1671-7236.2019.11.025
Abstract ( 312 )   PDF (4076KB) ( 257 )  
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To develop the virus-like particles (VLPs) genetic engineering subunit vaccine for swine O-type foot-and-mouth disease virus (FMDV),according to the FMDV complete gene sequence available in GenBank (accession No.:JN998085),four pairs of specific primers were designed to amplify VP1,VP2,VP3 and VP4 genes of FMDV O/MYA98/XJ/2010 strain by PCR. VP3, VP1 and VP4,VP2 gene were cloned into p10 and pH promoters of pFastBac Dual vector,respectively.The recombinant plasmid pFBD-VP3-VP1 and pFBD-VP4-VP2 were obtained and identified.Two recombinant bacmid of rBacmid-VP3-VP1 and rBacmid-VP4-VP2 were generated after the E.coli DH10Bac competent cell was transformed by the two recombinant plasmid,respectively.Two recombinant baculovirus of rvAc-VP3-VP1 and rvAc-VP4-VP2 were successfully rescued by transfected two recombinant bacmids into Sf9 insect cells.Two recombinant baculoviruses were coinfected with suspension cultured Sf9 insect cells,and VP1, VP2, VP3 and VP4 genes were expressed via Bac-to-Bac expression system in Sf9 insect cells.The target proteins were identified and analyzed with specific positive antibody through IFA,SDS-PAGE and Western blotting,and the target proteins were checked as VLPs via by electron microscopy (EM).The results showed that two recombinant baculoviruses expressing four structural proteins of FMDV VP1,VP2,VP3 and VP4 were successfully constructed.The specific antibodies detection results showed that four proteins VP1,VP2,VP3 and VP4 were successfully expressed with good specificity.Four proteins could self-assemble in Sf9 insect cells and form VLPs with a diameter of 25-30 nm similar to that of natural viruses.In this study,FMDV VLPs were successfully prepared in Sf9 insect cells by coinfection expression,which laid a foundation for further developing efficient and safe FMDV gene engineering subunit vaccine.
Comparative Analysis of the Antimicrobial Activity of Lactobacillus Supernatants from Swine in Biofilm and Plankton
JIN Shengnan, SHU Huiping, ZHANG Dongxing, ZHAO Zelin, KANG Yuanhuan, ZHANG Lei, SHAN Xiaofeng, QIAN Aidong
2019, 46(11):  3358-3369.  doi:10.16431/j.cnki.1671-7236.2019.11.026
Abstract ( 180 )   PDF (5212KB) ( 111 )  
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The aim of this study was to compare and analyze the bacteriostatic activity of the supernatants of Lactobacillus johnsonii L-76 and Lactobacillus amylovorus L-102 from swine in biofilm and plankton to pathogenic bacteria.The biofilm formation ability of L-76 and L-102 were measured by crystal violet staining assay.Then the supernatants of Lactobacillus in biofilm and plankton were treated with different influencing factors to study the inhibitory effect of Lactobacillus supernatants on pathogenic bacteria.Scanning electron microscopy was used to observe the effect of supernatant on the morphology of indicator bacteria.The results showed that L-76 and L-102 Lactobacillus had strong ability of biofilm formation and that the growth of pathogenic bacteria was inhibited after co-culture with the supernatant of Lactobacillus in biofilm and plankton.After the action of protease,compared with plankton,the inhibitory zone diameters of biofilm L-76 and L-102 supernatant on the indicator bacteria were significantly reduced.After the action of catalase and different temperatures,there was no significant difference in the antimicrobial activity between L76 and L-102 Lactobacillus supernatants in both conditions (P>0.05).However,they had the best inhibitory effect when pH was 3.0.We observed that the supernatant of biofilm Lactobacillus had a slightly greater effect on the morphology of Staphylococcus aureus by scanning electron microscopy.In conclusion,the supernatant of Lactobacillus contained antimicrobial substances in both conditions,and the activity of bacteriostatic substances in the supernatant of biofilm Lactobacillus was slightly higher or the content was slightly higher.
Isolation,Identification and Characteristics Analysis of a Proteus mirabilis from the Aborted Foxes
ZHANG Ping, CHENG Yuening, ZHANG Haiwei, QIAO Lianjiang, ZHOU Yucheng, XU Li, HOU Qingfeng, YANG Yanling
2019, 46(11):  3370-3377.  doi:10.16431/j.cnki.1671-7236.2019.11.027
Abstract ( 153 )   PDF (4601KB) ( 89 )  
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In order to clarify the main causes of fox abortion,identify the main pathogens and propose effective prevention and control measures,the spleen and ovaries of several aborted foxes in Hebei province were collected and analyzed by bacterial isolation,morphological observation,drug sensitivity test,16S rRNA gene sequencing,sequence analysis and homology alignment to isolate and identify the pathogens causing fox abortion and analyze its drug resistance to screen effective therapeutic drugs.At the same time,the pathogenicity of pathogens was tested.The results showed that a single Gram-negative bacterium was isolated from the spleen and ovaries of the aborted fox lesion,named as FOX-abortion-Hebei.The results of drug susceptibility test showed that the isolated strain was sensitive to 11 drugs such as cefoperazone,but was intermediate to 5 antibiotics such as tobramycin.The results of clinical animal tests showed that the strain had strong pathogenicity and could cause animal death in both mice and foxes.16S rRNA sequencing and homology analysis showed that the isolated Gram-negative bacteria were Proteus mirabilis (PM),which had a 93.6% to 99.9% homology with published sequences.The phylogenetic relationship between the bacteria and the published strain from Egypt (AUMC B-199) was the closest.The above research results showed that the main pathogen causing fox abortion was Proteus mirabilis,this bacterium was also isolated for the first time in fox abortion,which could cause serious reproductive disorders,it was necessary to do a good job of prevention and control in fox breeding.
Research Progress on Infection Epidemic Patterns Among Major Dairy Cow Mastitis Pathogenic Bacteria
ZHAO Gaoqian, WANG Suying, LI Guangshan, ZHAO Qingbin
2019, 46(11):  3378-3386.  doi:10.16431/j.cnki.1671-7236.2019.11.028
Abstract ( 192 )   PDF (1038KB) ( 157 )  
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Dairy cow mastitis is one of the major serious diseases in dairy farming industry world wide since its significant impact on economics,dairy farming and human health.More and more study focus on its pathogenesis,diagnosis,prevention,and so on.Our review based upon the recent progress on mastitis pathogen,one of the major causes of dairy cow mastitis and try to know the relationship among infection type,dairy cow and pathogen and pathogen themselves.The development of mastitis in dairy cows is due to many factors,such as invasion of pathogenic bacteria,the capability of cow defence system,the response of breast to pathogen,the synergism or repressive interaction among pathogenic bacteria.Staphylococcus aureus is well-known main infectious pathogen of dairy cow mastitis.It is very hard to cure in dairy cow body and has widespread drug resistance due to its unique biological characteristics.Streptococcus agalactiae belongs to an infectious or environmental pathogen is not definitive so far.It does cause high infection rate in our dairy cow farming.This kind of disease has relative higher cure rate comparing to other pathogenic bacteria since it is sensitive to many antibiotics.Escherichia coli is known as an environmental pathogen,it was detected in many cow mastitis isolates.However,the effect of antibiotics treatment on it requires further investigation since its drug resistance is common exist and becoming more severe.Mycoplasma bovis is one of the world wide infectious pathogens.Its infection rate has risen during past ten years.There is a overall poor therapeutic effect on it and it is difficult in vaccine development.Furthermore its drug resistance is still increasing.This review focused on the recent progresses of epidemiology,disease and prevention of the major cow mastitis pathogens,in order to help researchers in the field of dairy farm management and scientific study.
Construction of Mycoplasma ovipneumoniae pcDNA3.1-TBP30-Hsp70 Fusion Plasmid and Its Effect on Cellular Immune Response in Mice
ZHOU Yi, WANG Bolin, YANG Mei, HE Ling, QIN Lan, YUE Jun, ZHANG Shuangxiang, ZHOU Bijun, WANG Kaigong, CHENG Zhentao
2019, 46(11):  3387-3395.  doi:10.16431/j.cnki.1671-7236.2019.11.029
Abstract ( 168 )   PDF (1445KB) ( 96 )  
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To investigate the effect of the fusion expression plasmid of Mycoplasma ovipneumoniae (Mo) pcDNA3.1-TBP30-Hsp70 on the cellular immune response in mice,a fusion expression plasmid of Mycoplasma ovipneumoniae pcDNA3.1-TBP30-Hsp70 was constructed.Using the constructed plasmids pMD19T-P30 and pMD19-Hsp70 as templates,primers were designed based on the principle of gene site-directed mutagenesis (SDM).The target gene fragments were amplified by SOE-PCR and directionally cloned into the expression vector pcDNA3.1(+) to construct pcDNA3.1(+)-TBP30 and the fusion plasmid pcDNA3.1(+)-TBP30-Hsp70.Mice were immunized with pcDNA3.1-TBP30,pcDNA3.1-TBP30-Hsp70,pcDNA3.1(+) and Elution Buffer.Serum levels of cytokines interleukin-2 (IL-2),IL-4 and interferon-γ (INF-γ) were detected by ELISA kit.The results showed that after digestion of pcDNA3.1-TBP30-Hsp70,the target gene fragments with the size of 1 413 bp and the vector bands with the size of 5 400 bp were visible.Compared with the blank control and the pcDNA3.1(+) groups,the immune recombinant plasmid groups could increase the secretion levels of cytokines INF-γ,IL-2 and IL-4 in the serum of mice.Compared with the blank control and the pcDNA3.1(+) groups,the difference was significant or extremely significant (P<0.05;P<0.01),but there was no significant difference between the blank control and blank plasmid groups (P>0.05).The final secretion of IL-2,INF-γ and IL-4 in serum of mice immunized with pcDNA3.1-TBP30 and pcDNA3.1-TBP30-Hsp70 increased,which indicated that the recombinant plasmids could stimulate the changes of IL-2,INF-γ and IL-4 in serum of mice,and showed the regularity of increasing and decreasing in time.The results showed that after immunizing mice with recombinant plasmid pcDNA3.1(+)-TBP30-Hsp70,the secretion levels of IL-2 and INF-γ increased,which enhanced the cellular immune function of the organism,and then regulated the cellular immunity of the organism to affect the secretion of T cells and macrophages,thereby improving the cellular immunity of the organism.Th2 differentiated into Th1,maintains the dominant state of Th1 and enhanced the cellular immune function of the body.The results provided a reference for the development of genetic engineering vaccine against Mycoplasma ovipneumoniae.
Study on Preparation and Immunogenicity of Subunit Proteins of Pasteurella multocida Toxin from Pigs
HE Hai, HAO Chengwu, LING Chen, ZHANG Fei, HOU Feng, HE Sun
2019, 46(11):  3396-3403.  doi:10.16431/j.cnki.1671-7236.2019.11.030
Abstract ( 191 )   PDF (1893KB) ( 109 )  
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In order to obtain the high immunogenicity antigen of the main pathogen of porcine atrophic rhinitis,Pasteurella multocida toxin,the subunit protein antigen of the Pasteurella multocida toxin was constructed,expressed and verified.The PMT gene of porcine Pasteurella multocida toxin was linked and transformed to pMD19-T vector,and identified by sequence analysis.BamHⅠ,HindⅢ and BlpⅠ restriction endonucleases were used to digest three gene fragments.Fragment 1 (Tox1),fragment 2 (Tox2) and fragment 3 (Tox3) were subcloned into prokaryotic expression vectors pET32-b,pET32-a and pET32-b,respectively,and three recombinant expression vectors were constructed.The recombinant expression vector was transformed into E.coli BL21 (DE3) competent cells.After induction by IPTG,SDS-PAGE and Western blotting were performed respectively.The immunogenicity of the recombinant expression vector was preliminarily studied in mice and guinea pigs.The results showed that the length of three gene fragments were 776,409 and 410 bp,which were highly homologous with the related sequences in GenBank.Three protein fragments were expressed normally,with the expression levels reaching 379.95,447.62 and 459.82 μg/mL,respectively.The SDS-PAGE validation bands were 75,77 and 53 ku,respectively.Because of the different positions of the three fragments,only Tox3 had Western blotting detection bands,which was consistent with the theoretical prediction.After immunizing mice and guinea pigs with three expressed proteins,the positive rate of serum was 100% on the 14th day after immunization,and the protected rate was 93% to mice on the 14th day after immunization.In this study,three subunit active fragments of PMT were successfully constructed,and they had good immunogenicity.
Research Progress on Immune Response and Genetic Engineering Vaccines Against African Swine Fever Virus
MA Xingshu, SONG Jinxiang
2019, 46(11):  3404-3413.  doi:10.16431/j.cnki.1671-7236.2019.11.031
Abstract ( 370 )   PDF (3401KB) ( 301 )  
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African swine fever (ASF) is an acute,hemorrhagic and highly contagious tiboviral disease in domestic pigs,caused by African swine fever virus (ASFV).The disease shows reticuloendothelial hemorrhage and high mortality,is one of arguably the most significant emerging disease threat for the swine industry worldwide.ASFV is a large DNA virus,with the complexity of the structure and the large number of proteins encoded by its genome.This review focuses on recent progress of the ASFV characteristics,mechanisms of immune response including immune escape,humoral immunity,and cellular immunity,genetic engineering vaccines including live attenuated vaccines,subunit vaccines,live virus vector vaccines and DNA vaccines.Finally,the vaccine development and prevention and control-eradiction strategies of ASF in China are prospected.
Basic Veterinary Medicine
Extraction of Effective Fraction of Huanglian Jiedu Decoction and Its Scavenging Effect on Candida albicans in vivo
YAN Lijun, LYU Xinyu, ZHANG Duo, CHEN Changbin, ZHANG Shuang, GAO Yuan, LUO Jianghan, WANG Fuling, CUI Wenyu, ZHANG Chengxian
2019, 46(11):  3414-3421.  doi:10.16431/j.cnki.1671-7236.2019.11.032
Abstract ( 266 )   PDF (849KB) ( 164 )  
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The aim of the experiment was to study the extraction of effective fractions of Huanglian Jiedu decoction and its scavenging effect on Candida albicans in vivo.The fractions of Huanglian Jiedu decoction were extracted by water and ethanol reflux,and then the ethanol extract was extracted with three different polar organic solvents (n-butanol,ethyl acetate,petroleum ether).The extraction process was optimized with the minimum inhibitory concentration (MIC) as the index.The changes of fungal load in kidney and liver,and reactive oxygen species (ROS) levels in a mouse model with systemic Candida albicans infection (SC) before or after taking medicine were observed.The levels of cytokines IL-6,IL-1β,pro-IL-1β and TNF-α in serum were measured by ELISA.The results showed that the optimal extraction process of Huanglian Jiedu decoction was the following:The material-liquid ratio was 1:30,the ethanol concentration was 60%,the temperature was 80℃,the extraction time and frequencey were 1 h and 2 times.Under this condition,the MIC90 of EAHD was 0.3125 mg/mL.Compared with the model group,Huanglian Jiedu decoction ethyl acetate extract (EAHD) high dose group significantly reduced the kidney fungal load of mice (P<0.05),and the complete cure rate was 40% which was obvious higher than that of fluconazole group.The fungal load of liver was significantly decreased in EAHD low,medium and high dose groups (50,100 and 200 mg/(kg·d))(P<0.05),and the effect of EAHD medium dose group and fluconazole group on reducing the hepatic fungal load was similar.The ROS content in kidney and liver of EAHD high dose group were significantly or extremely significantly reduced,respectively (P<0.05;P<0.01),and the levels of IL-6,IL-1β and TNF-α in the serum were significantly or extremely significantly increased,respectively (P<0.05;P<0.01).And the pro-IL-1β content in the serum was reduced.In conclusion,the EAHD obtained by the optimal extraction process could reduce ROS content and regulate inflammatory factors in kidney and liver of model mice,thereby alleviate inflammatory reaction to protect tissues and resist Candida albicans.
Determination of Related Substances in Oxyclozanide and Albendazole Compound Suspension by HPLC
LI Xiaoting, BAI Yubin, LI Bing, DONG Zhen, SUN Jichao, CHENG Fusheng, ZHANG Jiyu
2019, 46(11):  3422-3431.  doi:10.16431/j.cnki.1671-7236.2019.11.033
Abstract ( 278 )   PDF (1083KB) ( 171 )  
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The purpose of experiment was established a high performance liquid chromatography (HPLC) method to determine the related substances of oxyclozanide and albendazole compound suspension.A HPLC was used to determine the related substances of oxyclozanide and albendazole in the compound suspension by self-contrast method.The chromatographic separeation was performed using a Thermo Hypersil BDS C18 column (4.6 mm×150 mm,5 μm) at 30℃.The mobile phase consisted of water containing 0.1% phosphoric acid and 10 mmol/L ammonium dihydrogen phosphate (A) and methanol (B) at a constant ratio of 31:69 (V/V) and a flow rate of 1 mL/min.The detection wavelength was 295 nm,and the injection volume was 5 μL.Under the above chromatographic conditions,the peaks of oxyclozanide,albendazole and impurity peaks could be completely separated.The good linear relationships for peak area for 3,5,6-trichlorosalicylic acid,2-amino-4,6-dichlorophenol hydrochloride,oxyclozanide and albendazole were obtained in the range of 5 to 100 μg/mL (R2 ≥ 0.999).LOQ were 5 μg/mL,750 ng/mL,500 ng/mL and 225 ng/mL,respectively.LOD were 1 μg/mL,500 ng/mL,250 ng/mL and 100 ng/mL,respectively.The average recovery of 3,5,6-trichlorosalicylic acid and 2-amino-4,6-dichlorophenol hydrochloride were 101.15% (RSD was 0.71%) and 100.35% (RSD was 0.71%),respectively.The validation results showed that the HPLC method was simple,rapid and accurate,and could be used to the related substances of oxyclozanide and albendazole compound suspension.
Resistant Phenotype and Molecular Characterization of blaCTX-M Gene Carrying Klebsiella pneumoniae of Pet Dogs in Beijing
ZOU Zhiyu, LEI Lei, HE Junjia, CHEN Qiyan, SHAO Bing, WANG Yang
2019, 46(11):  3432-3439.  doi:10.16431/j.cnki.1671-7236.2019.11.034
Abstract ( 243 )   PDF (2122KB) ( 103 )  
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In order to investigate the presence,resistant phenotype and molecular characterization of blaCTX-M gene positive Klebsiella pneumoniae in pet dogs,we isolated ceftaxime-resistant bacteria from anal and nasal swabs of 355 pet dogs and species were further identified by 16S rDNA and MALDI-TOF-MS.We detected blaCTX-M gene by PCR.All blaCTX-M gene carrying Klebsiella pneumoniae were subjected to antimicrobial susceptibility testing to observe resistance phenotype,PFGE typing and the whole sequencing to know molecular characterization.The results of PCR showed that 7 blaCTX-M gene containing Klebsiella pneumoniae were isolated,of which three were from anal swabs and four were from nasal swabs.All strains were resistant to ceftiofur,cefotaxime and amoxicillin-clavulanate,but were susceptible to colistin,meropenem and tigecycline.The results of PFGE patterns indicated that the similarity of three isolates were 100% and the similarity of the other four were <30%.7 isolates belonged to 5 ST types,and 5 were blaCTX-M-14 gene positive and 2 were blaCTX-M-3 gene positive.However,some β-lactamase-resistant genes,quinolones-resistant genes and aminoglycosides-resistant genes were co-exist with blaCTX-M gene in these strains.In summary,we found that the presence of blaCTX-M gene carrying Klebsiella pneumoniae had a low detection rate,but they were multi-drug resistant,the phenotype of blaCTX-M and blaSHV genes in this study were different from the prevalence phenotype at home and abroad.Until now,the usage and control of antibiotics in companion animals still had limitation,we needed pay more attention to antibiotic resistance in companion animals to provide scientific evidence to control in the future.
Effect of Essential Oil from Artemisia vulgaris on CYP450 Isoforms Activity and Expression
JIANG Zhihui, ZHANG Jingmiao, QU Yicheng, ZHANG Shouquan, CHANG Xuemei, GUO Xiao
2019, 46(11):  3440-3448.  doi:10.16431/j.cnki.1671-7236.2019.11.035
Abstract ( 238 )   PDF (1465KB) ( 135 )  
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Effect of essential oil from Artemisia vulgaris on CYP450 isoforms activity and expression in mouse was detected in this study.Essential oil was extracted from Artemisia vulgaris by steam extraction and was detected by GC-MS.In order to study the effect of essential oil on CYP450 activity,SD rats were randomly divided into control group (CON) and essential oil group (EO).The rats were given 0.1% Tween-80 100 μL/g and 0.1% Tween-80 diluted essential oil (5%) 100 μg/g respectively by gavage.After gavage,20 μg/g probes were given.Cocktail probes were used to measure the concentrations of three probe at 5,10,20 and 30 min,and 1,2,3,6,12,24,36 and 48 h after administration.To study the essential oil on the expression of CYP450,the Kunming mice were randomly divided into two groups.The control group was administrated with 1% Tween-80 (100 μL/g) and the essential oil group with 5% essential oil (100 μg/g).The administration was given twice daily for 3 days.The mRNA and protein expression were tested by Real-time PCR and Western blotting.The results show that:①The major components of essential oil were Eucalyptol;3-Cyclohexen-1-ol,4-methyl-1-(1-methylethyl)-3;(+)-2-Carene,4-.alpha.-isopropenyl-2;m-Mentha-4,8-diene,(1S,3S)-(+)-;Benzoic acid,2,4,6-trimethyl-,2,4,6-trimethylphenyl ester.②Pharmacokinetics of Phenacetin(CYP1A2 substrate),dextromethorphan(CYP2D6 substrate) and chlorzoxazone(CYP2E1 substrate) were analyzed.The results suggested that the essential oil could inhibit the activities of CYP2D6 and CYP2E1,while,had no effect on the activity of CYP1A2.③Real-time PCR and Western blotting results showed that essential oil significantly decreased the CYP2E1 and CYP2D6 expression.In conlusion,essential oil from Artemisia vulgaris could inhibit the activities and expressions of CYP2E1 and CYP2D6.It suggested that the essential oil from Artemisia vulgaris could not be taken together with the drugs in which metabolic pathway CYP2D6 and CYP2E1 were the main factors.The results of this study could provide data basis for the rational use of essential oil from Artemisia vulgaris.
Study on the Anti-porcine Reproductive and Respiratory Syndrome Virus Effect of Gentian and Its Extract
LIU Yun, ZHU Shanyuan, GONG Zhunan, QIN Feng, GU Lingling, WU Xiaojie
2019, 46(11):  3449-3456.  doi:10.16431/j.cnki.1671-7236.2019.11.036
Abstract ( 234 )   PDF (2706KB) ( 125 )  
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In order to study the water extract,total flavonoids and total glycosides of gentian in vitro against porcine reproductive and respiratory syndrome virus (PRRSV),the effect of Marc-145 cell model was established in this experiment.Based on the study of its maximum safe concentration of Marc-145 cells,combined with the observation of cytopathic morphology and MTT method,the drug group,ribavirin positive control group,virus control group and cell control group were set up to determine the three action modes of blocking,inhibition and direct inactivation to the virus,respectively.The results showed that the maximum safe concentrations of the water extract of gentian,total flavonoids of gentian,total glycosides of gentian and ribavirin were 6.250,0.020,0.630 and 0.016 mg/mL,respectively. In vitro anti-PRRSV effect of the test,the highest inhibition rates of the water extract of gentian on virus blocking,inhibition and direct inactivation effect were 43.1%,57.4% and 56.4%,respectively,which were all lower than that of ribavirin in the positive control group,and the cells had obvious pathological changes in each mode of action.The highest inhibition rates of the total flavonoids of gentian on virus blocking,inhibition and direct inactivation effect were 66.1%,41.1% and 42.7%,respectively.The inhibition rate of its inhibitory effect and direct inactivation effect were lower than that of ribavirin in the positive control group.The highest inhibition rates of the total glycosides of gentian on virus blocking,inhibition and direct inactivation effect were 33.4%,78.2% and 81.9%,respectively.The inhibition and direct inactivation were much stronger than that of ribavirin in the positive control group,and the cells basically kept the monolayer intact under the two action modes.Combined with the three action modes,among the three gentian extracts,the total glycosides of gentian had the best anti-PRRSV effect,followed by the water extract and the total flavonoids of gentian.