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20 June 2020, Volume 47 Issue 6
Biotechnology
Tissue Expression,Cloning and Bioinformatics Analysis of ERBB4 Gene in Muscovy Duck (Cairna moschata)
ZHOU Xuan, YANG Shenglin, YANG Shihao, LUO Linli, ZHU Yongcai, ZHANG Yun
2020, 47(6):  1637-1647.  doi:10.16431/j.cnki.1671-7236.2020.06.001
Abstract ( 311 )   PDF (3249KB) ( 96 )  
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This study was aimed to detect the relative expression level of receptor tyrosine protein kinase 4 (ERBB4) gene in brain under different physiological conditions of muscovy ducks,analyze its bioinformatics function,and explore whether ERBB4 gene affected the aggressive behavior of ducks.The relative expression of ERBB4 gene in striatum and hypothalamus of muscovy ducks was detected by Real-time quantitative PCR.The CDS core region of ERBB4 gene was cloned and sequenced by RT-PCR,analyze the homology and genetic evolution of ERBB4 gene,and predict the physicochemical properties,subcellular localization,signal peptide,secondary structure and tertiary structure by bioinformatics analysis tools.The results showed that ERBB4 gene was expressed in both brain tissues of muscovy ducks,the expression in brain of muscovy ducks with aggressive behavior was higher than that of normal group,and the expression in striatum was slightly higher than that of hypothalamus.The homology of nucleotide and amino acid sequences showed that ERBB4 gene had certain genetic diversity among different species and was closely related to Anas platyrhynchos.ERBB4 protein was hydrophilic and stable.There was no signal peptide at the N-terminal of the amino acid sequence,5 furan-like cysteine (FU) rich domains and 1 tyrosine kinase (TyrKc) protein domain.The subcellular localization was in cytoplasm,and the spatial structure was mainly right-handed alpha helix and random coil.In addition,ERBB4 gene might play an important role in the nervous system of muscovy ducks,this results provided a reference for further exploring the mechanism of ERBB4 gene in aggressive behavior of muscovy ducks.
Bioinformatics Analysis and Prokaryotic Expression of Porcine Aminopeptidase N Gene in Longlin Black Pigs
YUAN Tingting, QIN Yibin, WANG Ruomu, LIU Fang, CHEN Ying, WEI Zuzhang, HE Ying, HUANG Weijian, ZHAO Wu, OUYANG Kang
2020, 47(6):  1648-1658.  doi:10.16431/j.cnki.1671-7236.2020.06.002
Abstract ( 270 )   PDF (2657KB) ( 53 )  
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The aim of this study was to amplify the porcine aminopeptidase N (pAPN) gene and analyze with bioinformatics,and express it in Escherichia coli.pAPN gene was amplified from three-way hybrid commercial pigs and Guangxi local Longlin Black pigs,and its bioinformatics and phylogenetic analysis were carried out by ExPASy and Mega 6.0 software.Then pAPN gene was cloned into prokaryotic expression vector pET-32a(+) to construct the recombinant plasmid pET-32a-pAPN,and expressed in Escherichia coli Rosetta(DE3).The expression was induced by IPTG,the product was analyzed by SDS-PAGE and Western blotting.The results showed that pAPN gene of Longlin Black pigs and three-way hybrid commercial pigs was 2 949 bp in length,the open reading frame was 2 892 bp,encoding 963 amino acids,and the homology was 100%.Compared with standard pAPN gene (accession No.:NM_214277) sequence in GenBank database,there were 5 amino acid mutations in Longlin Black pigs,of which Phe82Asn,Leu107Phe,Leu108Ile and Ser330Pro were located in the catalytic activity region of pAPN enzyme and Val621Ile mutation was located in APN virus combined area.PAPN was an unstable hydrophilic protein,the transmembrane region was between 17th and 33th amino acid.There were 12 N-glycosylation sites,33 B cell antigen epitope prediction sites,and the tertiary structure was homologous dimer.Val621Ile mutation might affect the ability of APN binding virus.The recombinant plasmid pET-32a-pAPN was successfully constructed and expressed in the form of inclusion body by SDS-PAGE.The purified protein had obvious band at 123 ku and had good antigenicity,which laid a foundation for further obtaining polyclonal antibody and studying the relationship between the gene and TGEV,PEDV and PDCoV.
Cloning,Bioinformatics Analysis and Tissue Expression Detection of IGF-1 Gene CDS Regions in Kunming Dogs
WEI Yunfang, LI Feixiang, MA Weiguo, XU Tao, LI Judong, CHEN Fangliang, WAN Jiusheng, CHEN Chao
2020, 47(6):  1659-1667.  doi:10.16431/j.cnki.1671-7236.2020.06.003
Abstract ( 212 )   PDF (1898KB) ( 50 )  
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This study was aimed to systematically analyze the gene structure and function of insulin-like growth factor-1 (IGF-1) in Kunming dogs,in order to better understand the gene tissue expression pattern,and provide basic data for the study of body size and growth characteristics of working dogs.Kunming dogs was taken as the research object to amplify the CDS region of IGF-1 gene by molecular cloning technique.Sequence homology alignment and phylogenetic tree of IGF-1 gene were constructed by bioinformatics method.The physical and chemical property,subcellular structure,hydrophilicity and hydrophobicity and phosphorylation site of the corresponding amino acid sequence were analyzed by online predicition software.The secondary and tertiary structures of IGF-1 protein were predicted by SOPMA and SWISS-MODEL software.Meanwhile,the expression of IGF-1 gene in different tissues of Kunming dogs was detected by Real-time quantitative PCR.The results showed that the CDS region of IGF-1 gene in Kunming dogs was 462 bp which encoding 153 amino acids.IGF-1 gene CDS in Kunming dogs had higher homology with Canis lupus familiaris (100%),but lower homology with Mus musculus and Gallus gallus (82.6% and 81.2%).The theoretical molecular weight and isoelectric point of IGF-1 protein was 16.97 ku and 9.36,respectively.It was a hydrophilic and secretory protein with no transmembrane structure and its signal peptide region was 1-48 amino acids.The results of subcellular localization showed that IGF-1 protein was mainly located in the nucleus (56.5%) and mitochondria (17.4%).There were 15 phosphorylation sites.The secondary structure of the protein was mainly composed of alpha helix and random coil.Tissue expression showed that IGF-1 gene was expressed in different tissues of Kunming dogs,especially in spleen,liver and kidney.The results laid a theoretical reference for a better study of IGF-1 gene functions,and provided certain basic data for further exploration of growth and development characters of IGF-1 gene in Kunming dogs.
Prokaryotic Expression and Antigenicity Analysis of S2 Protein of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A Strain
LIU Lei, QIN Yibin, LU Bingxia, JIANG Dongfu, CHEN Zhongwei, HE Ying, ZHAO Shuo, ZHOU Yingning, LI Bin, DUAN Qunpeng, LIANG Jiaxing, ZHAO Wu
2020, 47(6):  1668-1676.  doi:10.16431/j.cnki.1671-7236.2020.06.004
Abstract ( 245 )   PDF (2904KB) ( 59 )  
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The purpose of this study was to understand the antigenicity of porcine epidemic diarrhea virus (PEDV) S2 protein and lay a foundation for the further study of diagnostic kit and subunit vaccine.In this study,the partial fragment of S2 gene (S2A) of PEDV CH/GX/2015/750A strain was amplified by RT-PCR and inserted into prokaryotic expression vector pET-32a(+) to construct prokaryotic expression plasmid pET32a-S2A.The prokaryotic expression plasmid pET32a-S2A was transformed into E.coli BL21(DE3).The recombinant protein S2A was induced by IPTG and purified by affinity chromatography on Ni column.The activity of S2A protein was detected by Western blotting.The purified recombinant protein S2A was used to immunize Kunming mice to prepare polyclonal antibody.The titer of the polyclonal antibody was detected by indirect ELISA,and the specificity of the polyclonal antibody was verified by indirect immunofluorescence.The results showed that the optimal induction condition of recombinant S2A protein was that when the final concentration of IPTG was 0.2 mmol/L,the induction temperature was 37 ℃,and the induction time was 3 h.The recombinant protein mainly existed in the form of inclusion body.Western blotting results showed that the purified and renatured recombinant protein S2A could specifically bind to PEDV positive serum and had good react ogenicity.The titer of the polyclonal antibody was 1:32 000.IFA results showed that the polyclonal antibody could specifically recognize and bind PEDV virus.The results of this study showed that PEDV S2A protein had good antigenicity and could be used as a candidate antigen of diagnostic kit or subunit vaccine.
Identification of Subviral Particles Efficiently Self-assembled by Infectious Bursal Disease Virus rVP2 Protein
WANG Yanwei, ZHANG Suling, WU Peng, GENG Xiaolin, HUANG Yuxin, LI Penghao, WANG Mengyue, LI Xiangdong, PANG Wenqiang, TIAN Kegong
2020, 47(6):  1677-1684.  doi:10.16431/j.cnki.1671-7236.2020.06.005
Abstract ( 211 )   PDF (2373KB) ( 45 )  
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In order to obtain highly purified subviral particles (SVPs) of VP2 protein of infectious bursal disease virus (IBDV),and evaluate the assembly efficiency and homogeneity of rVP2 SVPs,IBDV rVP2 was expressed in Escherichia coli (E.coli).The protein expression and immunoreactivity were identified by SDS-PAGE and Western blotting.rVP2 was purified by anion exchange chromatography and tangential flow ultrafiltration concentration system.The assembly morphology of single SVP and the overall assembly of rVP2 SVPs were analyzed by a variety of detection technologies,such as transmission electron microscopy (TEM),high performance liquid size exclusion chromatography (HPSEC),sucrose density gradient centrifugation,particle size and Zeta potential,and systematic methods for measuring the assembly efficiency of IBDV rVP2 SVPs were preliminarily established.The results showed most of the rVP2 was expressed as soluble protein in E.coli,and had obvious immunoreactivity with IBDV positive serum.After purification,the purity of rVP2 was increased 66.9 times and the recovery was 93.3%.The observation by TEM showed rVP2 self-assembled into SVPs with a diameter of about 25 nm,and the size of SVPs in the field of vision was uniform and evenly dispersed.The molecular weight of rVP2 SVPs detected by HPSEC was about 2 482 ku,which was consistent with the molecular weight of T=1 SVPs assembled by 20 VP2 trimers.The results of sucrose density gradient centrifugation showed rVP2 protein was efficiently self-assembled into SVPs with a uniform volume distribution.The results of particle size distribution and Zeta potential showed that the system of rVP2 SVPs was stable with a good particle dispersion,and was beneficial to the long-term preservation of rVP2 SVPs.The results of the assembly of rVP2 SVPs analyzed by various detection technologies showed that rVP2 SVPs were self-assembled efficiently,and provided a strong support for the quality control of IBDV SVPs vaccine.
Construction and Screening of Phage Library Against VHH Antibody of Canine Distemper Virus
WANG Zhaoyang, JIANG Yajun, LIU Xueting, LIN Weidong, XIN Ting, HOU Shaohua, GUO Xiaoyu, ZHU Hongfei, JIA Hong
2020, 47(6):  1685-1693.  doi:10.16431/j.cnki.1671-7236.2020.06.006
Abstract ( 387 )   PDF (2438KB) ( 79 )  
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In order to construct a specific nanobody library of canine distemper virus (CDV) and obtain VHH antibody against CDV,alpaca was immunized with CDV.After four immunizations,peripheral blood lymphocytes were collected,total RNA was extracted and transcribed into cDNA,and the nanoantibody sequence was amplified by nested PCR.The target fragment was connected to the pComb3x phage display vector,and transferred to the TG1 host bacteria.40 clones were selected for liquid PCR verification.13 positive monoclonals were selected randomly for sequencing.The auxiliary phage was added to save the phage display antibody library.After three rounds of panning,the phages with high binding capacity to CDV were enriched.Pichia pastoris was used to express two phages with high binding capacity.After purification by Ni column,the binding capacity of phages was identified by ELISA.The results showed that the titer of alpaca serum reached 1:25 000 after four immunizations,and the capacity of phage display library reached 3.41×109 PFU.After three rounds of panning,the specific antibody library was diluted 100 times,and the ELISA was still positive,indicating that the phage with specific binding to CDV significantly enriched.The results of ELISA showed that the reactivity of the two purified antibodies to CDV was significantly higher than that of the control group.These results suggested that two specific CDV binding VHH antibodies were successfully screened in this study,which laid a foundation for the application of VHH antibody in the diagnosis and treatment of canine distemper.
Reverse Genetics and Its Research Progress of Porcine Epidemic Diarrhea Virus
LU Ying, SU Xueli, DU Chen, WANG Ruomu, ZHONG Lian, CHEN Ying, WEI Zuzhang, HUANG Weijian, OUYANG Kang
2020, 47(6):  1694-1701.  doi:10.16431/j.cnki.1671-7236.2020.06.007
Abstract ( 314 )   PDF (778KB) ( 128 )  
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Porcine epidemic diarrhea virus (PEDV) is the pathogen of porcine epidemic diarrhea (PED),a highly-contact intestinal infectious disease of pigs.PEDV is a single-stranded positive-stranded RNA virus with a cystic envelope,its genome is approximately 28 kb.Since 2010,the highly pathogenic PEDV G2 genotype strains have been continuously mutated,which has caused huge economic losses to the pig industry nationwide and even globally.Reverse genetic system is the full-length infectious clone of RNA virus construction.In recent years,PEDV is mainly based on the three methods of targeted RNA recombination,BAC system and in vitro connection to establish infectious cDNA cloning.The principle and method of reverse genetics were briefly summarized in this paper.Targeted RNA recombination utilizes the high homologous recombination of coronavirus RNA to achieve viral salvation,BAC system utilizes pBeloBAC11 vector to overcome the difficulty of cDNA instability in high-copy plasmid caused by the toxic sequence contained in PEDV genome.In vitro linkage technique mainly uses restriction endonuclease restriction sites existing in PEDV genome itself or modified restriction sites to connect virus fragments to full-length cDNA clones in vitro.In addition,this paper also summarized the recent progress in PEDV-related research based on reverse genetics technology.PEDV reverse genetics is an effective tool for studying the structure and function of PEDV virus genome and designing live attenuated vaccine,using reverse genetic technology to explore virulence related genes such as S gene,to explore the effect of mutations or deletions on the pathogenesis of the virus,to reveal the molecular mechanism of PEDV virulence attenuation,and to design live attenuated vaccine with good immunogenicity and avoid the return of virus and recombination of the virus strain.In conclusion,PEDV reverse genetics is an important method to study the genomic structure and function of PEDV,viral host interaction and pathogenic mechanism,as well as a reasonable and effective way to design a live attenuated PEDV vaccine.
Physiology and Biochemistry
Effect of Riemerella anatipestifer on the Morphological Structure and TLR4 Signal Pathway Related Gene Expression of Cecum in Ducks
TAO Zhiyun, ZHU Chunhong, SHI Zuhao, XU Wenjuan, ZHANG Shuangjie, SONG Weitao, LIU Hongxiang, LI Huifang
2020, 47(6):  1702-1708.  doi:10.16431/j.cnki.1671-7236.2020.06.008
Abstract ( 192 )   PDF (3224KB) ( 35 )  
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In order to investigate the possible pathogenic mechanism of Riemerella anatipestifer,the morphological structure was observed using microscope,the thickness of intestinal mucosa,the height of intestinal villi and the depth of crypt were measured and counted,and the expression of TLR4 signal pathway related genes in the cecum of ducks after infected with Riemerella anatipestifer was detected by Real-time quantitative PCR.Pathological observation results showed that the intestinal structure was damaged after Riemerella anatipestifer infection,which was manifested as intestinal villus shedding,congestion,bleeding and lymphocyte infiltration and lymphocyte proliferation.The statistical results showed that the thickness of intestinal mucosa in infection group (383.58 μm) was significantly lower than that in control group (643.39 μm) at 2 d (P<0.05).The height of intestinal villi in infection group (173.04 and 168.68 μm) were significantly lower than that in control group (355.79 and 276.54 μm) at 2 and 5 day (P<0.05),respectively.The ratio of height of intestinal villi to crypt depth in infection group (3.42) was significantly lower than that of control group (5.34) at 9 d (P<0.05).Real-time quantitative PCR results showed that the expression of TLR4 signal pathway related genes of TLR4,MD2,MyD88,TRAF6,NFB,IL-4 and IL-8 mRNA was up-regulated at 2 and 5 d after infection.These results suggested that the infection of Riemerella anatipestifer could lead to the physical damage and inflammatory lesions of the cecum,and TLR4 signal pathway was involved in the inflammatory response process.
Research Progress on Structure,Function and Its Key Immune Genes of Spleen in Pigs
SONG Yukun, HE Jun
2020, 47(6):  1709-1718.  doi:10.16431/j.cnki.1671-7236.2020.06.009
Abstract ( 233 )   PDF (1049KB) ( 116 )  
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The spleen,as the largest secondary lymphoid organ in pigs,contains a variety of immune-active cells and immune factors.It is an important organ of response and regulation for innate and adaptive immunity,and has extensive immune regulatory functions.At the same time,it can remove old aged and damaged red blood cells,filter out pathogens,play an important role in hematopoiesis and blood cells storage.These functions are closely related to the structure of the spleen.Among them,the red pulp is mainly used as a blood filter to perform the functions of hematopoiesis,blood storage and foreign objects removal.The white pulp is the main area ofimmunization,containing varieties of immune cells,which can perform the functions of specific immunity and response regulatory.The marginal zone is a key bridge which connects the red and white pulps,allowing all immune cells and antigens to enter different parts of spleen.Different key genes are fundamental for development and immunity to the realization of spleen function.The expression of developmental related genes guarantees the structural integrity and provides chance for the spleen to perform various functions.The expression of key immune genes ensures the immune response,in which the expression of different pattern recognition receptor genes ensures the phagocytosis and recognition response of innate immunity,and a variety of cytokines and immunoactive substances secreted by various immune cells are acquired.The basis of the immune response is the physiological process of antigens clearance and monitoring.With the development of spleen research,a new understanding of its various immune cells and immune functions has been gained.This article firstly explained the biological functions of different components of spleen,then discussed their functions of innate and specific immune,and the role and connection of the vital immune cells and immune factors.In the end,the development of spleen tissues and immune related genes which in embryonic and postnatal period were introduced,and the important pattern recognition receptors and their gene family in innate immunity of spleen in pig were summarized,which can provide reference for the study of innate immune function of spleen in pig.
Monitoring of Blood Biochemical Indicators in Ketosis Cattle
GUO Shihui, YU Yongtao, ZHAO Qingmei, XU Lihua, WAN Jiahong, HE Shenghu, YANG Qi, LI Ke
2020, 47(6):  1719-1728.  doi:10.16431/j.cnki.1671-7236.2020.06.010
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In order to compare the difference of blood biochemical indexes between ketosis dairy cows and healthy dairy cows in a period of postpartum,and provide theoretical basis for prevention and control of ketosis dairy cows and veterinary clinical diagnosis and treatment.In this study,30 cows in an intensive dairy farm in Wuzhong city were selected for blood biochemical monitoring for 3 weeks.Dairy cows were divided into ketosis group and control group according to the concentration of β-hydroxybutyric acid (BHBA) which be used to compare significance.The results showed that there was no significant difference between the ketosis group and the control group on the day of calving (0 d);The concentration of BHBA in the ketosis group was extremely significantly higher than that in the control group on the 7th postnatal day (P<0.01);The concentrations of AST and T-bil were significantly higher on the 7th and 14th postnatal day (P<0.05);The concentrations of blood glucose (Glu) decreased significantly at 7th postpartum (P<0.05),and it decreased extremely significantly on the 14th postpartum (P<0.01);The concentrations of calcium (Ca) and phosphorus (P) did not show significant differences,but both were lower than the normal reference range which resulted in hypocalcemia and hypophosphatemia.It was found that the ketosis of dairy cows in this field was type Ⅱ ketosis after analysis and the high parity of ketosis cattle was 3-6 parities and the postpartum hypocalcemia,hypophosphatemia,and hypoglycemia were serious.At the same time,cow ketosis could cause hepatic dysfunction and affect cow performance.
Animal Nutrition and Feed Science
Effects of Dietary Crude Protein Level on Laying Raws,Reproductive Hormone Concentrations and Related Gene mRNA Expression in Reproductive Axis of Yili Geese
ZHAO Xiaoyu, SHAERSHANBIEKE Abudilida, MA Chen, WU Yingping, WANG Junhua, LI Haiying
2020, 47(6):  1729-1738.  doi:10.16431/j.cnki.1671-7236.2020.06.011
Abstract ( 200 )   PDF (1488KB) ( 206 )  
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This experiment was conducted to study the effects of dietary crude protein level on laying raws,reproductive hormone concentrations and related gene mRNA expression in reproductive axis of Yili geese.200 Yili geese aged 3 years old with similar body weight and good health were selected,and were randomly divided into 4 groups with 10 replications.The crude protein level of control group and male geese (goose field self-administered) was 11.20%.The experimental groups fed the test diets with crude protein levels of 13.86%,15.20% and 16.48%,respectively.The nutritional levels of other diets remained the same.The pre-trial period was 1 week,the formal period was 9 weeks.The results showed that:①The egg production rate of Yili geese showed fluctuating changes during the trial period.The egg production rate of Yili geese in control group gradually increased in the first to second weeks,and slightly increased in the fourth to fifth weeks,the other stages showed a downward trend.The egg production rate of Yili geese in each experimental group significantly increased during the first to second and the fourth to fifth weeks,and reached the peak of egg production at the fifth week,and then gradually declined.The broodiness rate of 15.20% crude protein group in Yili geese was significantly lower than that of 11.20% and 13.86% crude protein groups.②Compared with control group,the serum GnRH content of 13.86% crude protein group were extremely significantly increased (P<0.01),and the serum E2 and LH contents significantly increased (P<0.05).The serum GnRH,E2 and LH content of 15.20% and 16.48% crude protein groups were extremely significantly increased (P<0.01),and the serum FSH content was significantly increased (P<0.05).The serum PRL concentration of each test group was significantly lower than that of control group (P<0.05).③Compared with control group,the GnRH,LHR mRNA relative expression of hypothalamus in 15.20% crude protein group were significantly increased (P<0.05),and the PRL and PRLR mRNA relative expression was significantly decreased (P<0.05).The LH mRNA relative expression in pituitary was significantly increased (P<0.05).The LH and ESR2 mRNA relative expression in ovary were significantly or extremely significantly increased (P<0.05 or P<0.01).In summary,based on the comprehensive evaluation of the effects of dietary crude protein level on the laying rate,broodiness rate,serum reproductive hormone concentration and the related gene mRNA expression in reproductive axis of Yili geese.It suggested the suitable crude protein level of the diet in Yili geese was 15.20% during laying period.
Advance on Application and Influence Factors of Medium Chain Fatty Acids in Animal Production
FENG Xin, ZHANG Luomeng, LUAN Jiaming, ZHOU Jinying, ZHANG Min, GENG Chunyin
2020, 47(6):  1739-1749.  doi:10.16431/j.cnki.1671-7236.2020.06.012
Abstract ( 339 )   PDF (1221KB) ( 88 )  
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Medium-chain fatty acid (MCFA) is a kind of energy materials with special physiological functions.Comparing with long-chain fatty acid (LCFA),MCFA has the advantage of shorter carbon chains and faster approach to the liver in a direct way through portal vein,directly entering hepatocyte mitochondria for β-oxidation without transport of carnitine acyl transferase.So MCFA can be absorbed by the animals in a direct and faster way while supplying energy.Besides,MCFA can not only provide energy for the animal,but also possess special physiological functions beyond its energy value,including inhibiting the growth of bacteria,improving the microecology and structure of the animal intestines,participating in the immune regulation and hormone regulation,etc.The nutritional characteristics and physiological functions of MCFA provide the basis for its application in animal production.In terms of animal production and application,MCFA can improve the growth performance of animals,reduce the rumen methane production of ruminants,prevent animal diseases,delay the corruption of livestock and poultry products,and effectively control the infestation of mosquitoes and flies on livestock.Furthermore,the joint use of MCFA and some feed additives not only has synergistic effect,but also can overcome the shortcomings in actual production and application.In recent years,with the increasing calling for safe alternatives to antibiotics,MCFA is getting more and more attention.However,there are differences in the effects of MCFA in research and production application,which may be related to the type,level and way of MCFA addition.In this paper,the nutritional characteristics,physiological functions,the effects of MCFA on animal production and its influence factors were reviewed to provide theoretical basis for further development and rational utilization of MCFA.
Effects of Dietary Copper on Growth Performance,Nutrient Digestibility of Growing Raccoon Dog
ZHANG Xinyu, ZHANG Haihua, LIU Hanlu, ZHONG Wei, SI Huazhe, HAN Feifei, CAI Xiheng, LI Guangyu
2020, 47(6):  1750-1758.  doi:10.16431/j.cnki.1671-7236.2020.06.013
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This experiment was investigated to study the effects of dietary copper on the growth performance,nutrient digestibility,nitrogen,copper metabolism and serum biochemical indexes of male raccoon dog.105 healthy male raccoon dogs with (60±5) days old whose body weight was (2.83±0.37) kg were randomly divided into 7 groups with 15 replicates per group and 1 raccoon dog per replicate.The raccoon dogs were fed 7 diets with different copper supplemental levels,0 (control group),20 (group Ⅰ),30 (group Ⅱ),40 (group Ⅲ),50 (group Ⅳ),60 (group Ⅴ) and 200 mg/kg (group Ⅵ).The pre-test period lasted for 7 d and the trial period lasted for 55 d.During the experiment,8 raccoon dogs in each group were randomly selected for digestion and metabolism test by total fecal and urine collection.The results showed as follows:The final weight in the group Ⅲ and Ⅳ were significantly higher than control group (P<0.05).There was no significant difference in body weight between the other groups and control group.The average daily gain (ADG) in the groupsⅠ,Ⅱ,Ⅲ,Ⅳ,Ⅴand Ⅵ were significantly higher than control group (P<0.05).The fat digestibility in the group Ⅴ was significantly higher than that in the group Ⅰ and control group (P<0.05).The copper digestibilities in control group and group Ⅵ were significantly higher than that in the groups Ⅰ,Ⅱ,Ⅲ,Ⅳ and Ⅴ (P<0.05).The nitrogen deposition in the group Ⅳ was significantly higher than that in the group Ⅰ and control group (P<0.05).There were significant difference in copper intakes between the two groups (P<0.05).The fecal copper contents increased with the increase of copper intake (P<0.05).The urinary copper in the groups Ⅴ and Ⅵ was significantly higher than that in the group Ⅰ,Ⅱ,Ⅲ,Ⅳ and control group (P<0.05).There was significant difference in copper deposition between each copper addition group and control group (P<0.05).The serum low density lipoprotein (LDL) in the group Ⅵ was significantly higher than that in the groups Ⅰ and control group (P<0.05).The serum albumin (ALB) in the groups Ⅱ,Ⅲ,Ⅳ,Ⅴ and Ⅵ were significantly higher than control group (P<0.05).The serum urea in the groups Ⅳ was significantly lower than control group (P<0.05).It was concluded that the dietary copper supplemental level arrives at 30 to 50 mg/kg,the growth performance was better.In order to reduce copper emissions,protect the environment and reduce the cost of feeding,it was recommended to add 30 mg/kg in practice.
Effects of Feeding Pregnant Ewes with Bamboo Vinegar Powder on Growth Performance,Blood Physiological and Biochemical Indexes, Antioxidant and Immune Ability of Lambs
LIU Mingmei, DAI Lejun, HE Congcong, ZHENG Qiangqiang, CHANG Yan, WANG Weiwei
2020, 47(6):  1759-1764.  doi:10.16431/j.cnki.1671-7236.2020.06.014
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The study was aimed to investigate the effects of feeding pregnant ewes with bamboo vinegar power on growth performance,blood physiological and biochemical indexes,antioxidant and immune ability of lambs.A total of 50 healthy,primiparous pregnant ewes at about 120 d pregnant age were chosen and randomly divided into 2 groups.The control group was fed basic diet and the experimental group was fed basic diet with 2% of bamboo vinegar power.Feeding period was 3 weeks (1 week for preliminary feeding).After delivery,the growth performance,blood physiological and biochemical indexes,antioxidant and immune ability of twenty three-week-old lambs with similar delivery date (1-4 d) were detected.The results showed as follows:The difference of BW and ADG between the two groups were not significant (P>0.05),but the ADG of experimental group was increased by 4.74% compared to control group.Compared with the control group,blood leukocyte,erythrocyte,platelet and hemoglobin contents of experimental group had no significant change (P>0.05),but GOT activity decreased significantly (P<0.05).TP,ALB,GLB and A/G increased in a certain extent,while the levels of TC,MDA,SOD activity and T-AOC had an decreased trend (P>0.05).Compared with the control group,there was an increased trend in IgA,IgM and IgG of experimental group,but the differences were all not significant (P>0.05).In conclusion,supplementing bamboo vinegar power in diets for pregnant ewes could improve ADG,increase serum TP,ALB,GLB,IgA,IgM and IgG contents in a certain degree.
Optimization of Steam Explosion Pretreated Wheat Straw Using Response Surface Methodology
DU Chunmei, XIONG Benhai
2020, 47(6):  1765-1772.  doi:10.16431/j.cnki.1671-7236.2020.06.015
Abstract ( 236 )   PDF (2500KB) ( 49 )  
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The purpose of this study was to optimize the parameters of steam explosion pretreatment of wheat straw by Box-Behnken response surface method,in order to solve the problem of its low utilization rate.The steam pressure (1.5-2.5 MPa),moisture content (10%-50%) and treatment time (90-210 s) were choosed as the independent variables,the rate of gas production in the in vitro rumen fermentation was the response value,and then the regression model were established and fitted.The results demonstrated that the influence of the treatment time on the rate of gas production in the in vitro rumen fermentation was extremely significant (P<0.01).The effect of steam pressure and moisture content on the rate of gas production in the in vitro rumen fermentation was not significant (P>0.05).According to the F value,the effect of three factors on the rate of gas production in the in vitro rumen fermentation was as treatment time > steam pressure > moisture content.The optimal conditions for steam explosion pretreatment by response surface methodology were steam pressure 1.77 MPa,moisture content 49.91% and treatment time 209.98 s.Under this condition,the rate of gas production in the in vitro rumen fermentation could reach 0.0794 mL/h.The average rate of gas in vitro was 0.0786 mL/h and the relative error was only 1.02%.The wheat straw was treated under the optimal steam explosion condition and subjected to in vitro rumen fermentation.Compared with untreated wheat straw,steam explosion extremely significantly decreased the content of NDF,ADF and hemicellulose in wheat straw (P<0.01),and extremely significantly increased the yield of VFA and NH3-N in vitro rumen incubation (P<0.01).In conclusion,the operational parameter of steam explosion was optimized using the response surface method,which was reasonable and reliable.The steam explosion condition optimized in the present study could improve the nutritional value of wheat straw as forage.
Application Status and Prospect of Several Unconventional Plant Materials in Pig Industry
ZHANG Fusheng, WEN Ge, ZHANG Yichan, WEN Lixin
2020, 47(6):  1773-1780.  doi:10.16431/j.cnki.1671-7236.2020.06.016
Abstract ( 229 )   PDF (821KB) ( 79 )  
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China is a big country of agriculture,among which pig breeding is the pillar industry.However,the shortage of feed materials and long-term dependence on imports are the basic status of China's livestock industry.Therefore,to exploit unconventional feed is of great significance for pig breeding.Unconventional feed derived from plants,including Broussonetia papyriferal, Folium mori,Cichorium intybus L.and Medicago sativa have the advantages of high nutritional value and yield,besides,their feature of convenient processing and storage,make them potential to replace conventional feed.Both fresh leaves and dried leaves of Broussonetia papyriferal can be used for pig breeding,and the best additive amount of dried leaves is about 10%.Cichorium intybus L.contains polysaccharides,organic acids,flavonoids and other biological active substances,in addition to its good palatability,it is also a potential feed source in pig breeding.An appropriate amount of Folium mori addition can not only improve the production performance of sows,but also improve the meat quality of fattening pigs.The optimal addition amount of Folium mori powder is 6%-10%,which can be further improved after fermentation.As the "king of herbage",Medicagosativa has the advantages of high protein content and balanced amino acid content,its biological function can be achieved at various growth stages of pigs,including weaned piglets,growing pigs,fattening pigs and pregnant sows.These kinds of unconventional feed have the potential to replace partly of the diet in pig breeding.This paper introduced the application of four kinds of plants derived unconventional feeds from the aspects of nutritional value,feeding method and application status.
Genetics and Breeding
Cloning,Sequence Analysis and Tissue Expression of INSIG1 Gene in Yuxi Fat-tailed Sheep
QUAN Kai, SONG Feihu, LI Jun
2020, 47(6):  1781-1790.  doi:10.16431/j.cnki.1671-7236.2020.06.017
Abstract ( 247 )   PDF (2341KB) ( 39 )  
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Insulin induced gene 1 (INSIG1) was an important regulatory gene of lipid synthesis and lipolysis.This study was aimed to clone INSIG1 gene,analyze the sequences by bioinformatics software,and measure the expression of INSIG1 gene in different tissues of Yuxi Fat-tailed sheep.The tissue RNA was extracted by Trizol method,and the INSIG1 gene sequence was obtained by RT-PCR and cloning.The expression of INSIG1 mRNA was detected by Real-time PCR.The results showed that the complete CDS of sheep INSIG1 gene was successfully cloned which was 831 bp and encoded 276 amino acids.The homology analysis showed that the INSIG1 coding region in Yuxi Fat-tailed sheep was 99.64% similar to that of sheep (XM_015095466.2),and the similarity of coding amino acid sequence was 99.28%.The physical and chemical properties of INSIG1 protein showed that the molecular weight was 29.58 ku and the theoretical isoelectric point (pI) was 9.07,which belonged to stable alkaline hydrophobic protein.There was 5 transmembrane structures and no signal peptide in INSIG1 protein, mainly located in cytoplasm.The tertiary structure of INSIG1 protein mainly consisted of 6 alpha helices and random coils.Real-time PCR results showed that the expression of INSIG1 gene in liver was obviously higher than other tissues,which followed by lung,intestine,and adipose of tail,and the lowest was in muscle.This study improved the database of Yuxi Fat-tailed sheep,and provided basic data for further study on the function of INSIG1 gene in fat deposition and regulation of mutton quality in sheep.
Analysis on Disease Resistance of Immunity+ Bulls' Daughters
AN Tao, ZHANG Hailiang, WANG Yachun
2020, 47(6):  1791-1799.  doi:10.16431/j.cnki.1671-7236.2020.06.018
Abstract ( 303 )   PDF (831KB) ( 73 )  
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The objective of current study was to explore daughters' disease resistance of Immunity+ bulls selected for disease resistance.A total of 5 112 individual disease records were collected from 3 dairy farms that using both frozen semen from Immunity+ bulls and ordinary bulls.A Logistic regression was used to analyze the impacts of birth season of calves,age of cows and sire type (Immunity+ bull or ordinary bull) on the risk of some common diseases in dairy cattle.The results showed that birth season and age had significant impacts on the risk of various diseases in calves and cows (P<0.05),respectively.The sire type had a significant impact on the risk of mastitis in some populations (P<0.05).Compared with the daughters of ordinary bulls,the daughters of Immunity+ bulls had a lower risk of mastitis.In conclusion,the breeding technology of selecting for disease resistance in Immunity+ bulls was effective,and their progeny had better disease resistant ability,especially on resistance to mastitis,which provided a feasible technical route to improve the disease resistance and profitability of dairy population by selective breeding.
Association Analysis on HLCS Gene Polymorphic Site with Growth and Development Traits in Duroc Pigs
PU Lei, DAI Yuxing, ZHANG Yuebo, ZHANG Longchao, GUO Liang, WANG Lixian
2020, 47(6):  1800-1808.  doi:10.16431/j.cnki.1671-7236.2020.06.019
Abstract ( 238 )   PDF (876KB) ( 36 )  
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The objective of this study was to clarify the correlation between SNP of holocarboxylase synthetase (HLCS) gene and growth and development traits in Duroc pigs.SNP of HLCS gene was obtained using the sequencing results of Illumina SNP60 chip,the polymorphism information of SNP,the correlation between SNP of HLCS gene and growth and development traits,and the haplotype of SNP were analyzed in this study.The results indicated that 4 SNPs (Ssc13:200455646 T>C,Ssc13:200458655 G>C,Ssc13:200504530 G>T and Ssc13:200551864 T>G) of HLCS gene were detected in Duroc pigs,which were in Hardy-Weinberg equilibrium (P>0.05),showing moderate polymorphism.The correlation analysis results showed that the 100 kg back fat (100 kg BF) and remaining food intake (RFI) of TT genotype in Ssc13:200455646 T>C were significantly higher than that of TC and CC genotypes (P<0.05).The 100 kg BF and RFI of GG genotype in Ssc13:200458655 G>C were significantly higher than that of CG and CC genotypes (P<0.05).The average daily feed intake (ADFI) and average daily gain (ADG) of GT genotype in Ssc13:200504530 G>T were significantly higher than that of TT genotype (P<0.05),the 100 kg age of GT genotype in Ssc13:200504530 G>T were significantly lower than that of TT genotype (P<0.05).The ADFI and ADG of TG genotype in Ssc13:200551864 T>G were significantly higher than that of GG genotype (P<0.05),the 100 kg age of TG genotype in Ssc13:200551864 T>G were significantly lower than that of GG genotype (P<0.05).There was no significant difference of other traits in different genotypes of SNPs (P>0.05).It showed high linkage of Ssc13:200455646 T>C and Ssc13:200458655 G>C,Ssc13:200504530 G>T and Ssc13:200551864 T>G,respectively.The results indicated that there were significant correlation between 4 SNPs of HLCS gene and RFI,ADFI,ADG,100 kg age and 100 kg BF in Duroc pigs,it could lay a foundation for the genetic improvement of growth and development in pigs.
Research Progress on Genetic Mechanisms of Coat Color Trait and Cultivation of Improved Varieties in Mink(Neovison vison)
SONG Xingchao, ZHANG Ru, XU Chao, WANG Shuming, ZHANG Zhiming, YANG Fuhe, PENG Yinghua
2020, 47(6):  1809-1818.  doi:10.16431/j.cnki.1671-7236.2020.06.020
Abstract ( 285 )   PDF (3383KB) ( 43 )  
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Coat color is an important intuitive and easily recognized economic trait,and also attracts much attention in the new breed development of mink.There are some differences in the quality and price of fur with different coat colors.Thus,it is inevitable to investigate the genetic mechanisms of mink coat color for breeders.In this review,we classify mink coat colors from the perspective of genetics,and the relationship between the coat color traits and DNA sequence polymorphisms of major candidate genes MLPH,LYST,TYRP1,MITF,TYR,ASIP,EDNRB and Pax3 in mink were summarized.At the same time,the important achievements of improved breed development of mink in China were overviewed,which would provide references for further systematic study on mink coat color patterns,designing breeding goals and program,and cultivating new coat color varieties.
Effects of Estrus Induction on Lambing and Serum Index of Cashmere Goats in Non-breeding Season
WANG Tao, LI Yurong, HE Yunmei, XU Fuxun, SUN Xuefeng, A Lamusi, SUN Chunhe, ZHAO Shengguo, LIU Bin
2020, 47(6):  1819-1827.  doi:10.16431/j.cnki.1671-7236.2020.06.021
Abstract ( 234 )   PDF (918KB) ( 27 )  
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The aim of the paper was to study Chinese herbal medicine concentration aphrodisiac and PMSG exogenous induction to the non-breeding season goat lambing and serum index of Inner Mongolia cashmere goats.272 Inner Mongolia cashmere goats of similar weight were selected and divided into Chinese herbal medicine test group Ⅰ (112 goats),hormone test group Ⅱ (100 goats) and control group (60 goats).The group Ⅰ was fed Chinese herbal medicine (50 g/goat),starting 7 days before estrus,once every two days;The group Ⅱ sponge plug+PMSG 250 IU per goat,normal feeding of control group.Blood samples were collected from jugular vein of 5 sheep in each group before early feeding on the second day after the end of the experiment,and the changes of physiological,biochemical and reproductive hormones in serum were measured and analyzed.The results showed that the oestrus rate,lambing rate and double lambing rate of the group Ⅰ and the group Ⅱ were significantly higher than that of control group (P<0.01),the lambing rate and double lambing rate of the group Ⅱ were significantly higher than that of the group Ⅰ (P<0.01),and the oestrus rate was significantly higher than that of the group Ⅰ (P<0.05).The contents of TP,ALB,UREA and GSH-Px in the group Ⅱ were significantly higher than those in the group Ⅰ and control group (P<0.05).The content of MDA and SOD activity in the group Ⅰ were significantly higher than those in the group Ⅱ and control group (P<0.05).The contents of FSH,E2 and LH in the group Ⅱ were significantly higher than those in the control group (P<0.05),and the differences of P4 and T among the three groups were not significant (P>0.05).The reproductive hormones in the group Ⅰ were higher than those in the control group,but the differences were not significant (P>0.05).Correlation analysis of hormones and antioxidant enzymes showed that the group Ⅰ P4 was significantly negatively correlated with GSH-Px (P<0.05),with a correlation coefficient of -0.594,while the group Ⅱ LH was significantly positively correlated with GSH-Px (P<0.01),with a correlation coefficient of 0.757.In conclusion,Chinese herbal medicine and hormone treatment could significantly promote the oestrus concentration and increase lambing rate of Inner Mongolia cashmere goats in non-breeding season.Hormone treatment could improve the digestion and utilization of protein and antioxidant function of Inner Mongolia cashmere goats in non-breeding season,and Chinese herbal medicine treatment could increase the activity of serum SOD.It was found that there was significant antagonistic effect between GSH-Px and P4,and significant promoting effect between GSH-Px and LH.
Screening of Candidate Genes Related to Intramuscular Fat Deposition in Chickens
LI Jinghui, XING Siyuan, WANG Xicai, LI Qinghe, ZHAO Guiping, ZHANG Yonghong, WEN Jie, LIU Ranran
2020, 47(6):  1828-1836.  doi:10.16431/j.cnki.1671-7236.2020.06.022
Abstract ( 290 )   PDF (1452KB) ( 134 )  
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In this study,14 candidate genes related to the differential deposition of chicken intramuscular fat (IMF) screened by the research team in the transcriptome study of Jingxing-Huang chicken were performed for verification.To detect the association between the expression level of candidate genes and breast muscle IMF deposition in two breeds.The breast muscle of 98-day-old slow-growing local Jingxing-Huang chickens and 42-day-old fast-growing white feather Cobb broilers were used to distinguish the high and low phenotypic groups based on the content of triglycerides in the breast muscle.The results showed that there were significant differences in expression of 10 genes of ABCB8,ADIPOQ,BEND6,CD74,FKTN,HAT1,HS3ST5,MED4,TNFSF8 and TNIP1 between Jingxing-Huang chicken breast muscle TG high and low groups (P<0.05).There were significant differences in 7 genes of ADIPOQ,BEND6,FKTN,HAT1,HS3ST5,MED4 and TNIP1 between Cobb broiler breast muscle TG high and low groups (P<0.05).6 genes including ADIPOQ,FKTN,HAT1,HS3ST5,MED4 and TNIP1 were significantly expressed in both varieties (P<0.05).This study provided new candidate genes related to chicken IMF deposition,laying a good foundation for the study of IMF molecular regulation mechanism and related molecular marker screening research.
Effects of Different Diluents on Sperm Motility and Insemination Results of Chicken Semen in Low-temperature Storage
ZHOU Lele, LI Chengde, HUANG Jianhao, CHEN Yun, ZENG Fanwen, HUANG Xiaogang, ZHANG Faliang, ZHENG Xican, YAN Changyan, WEI Hengxi, ZHANG Shouquan
2020, 47(6):  1837-1843.  doi:10.16431/j.cnki.1671-7236.2020.06.023
Abstract ( 252 )   PDF (727KB) ( 57 )  
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For the improvement of the skills of semen preservation and the usage of semen of chicken farms in Southern China,the effects of different storage time (0,4,8,24 h) and different diluents (the original、formula Ⅰ and formula Ⅱ) on sperm motility and the results of artificial insemination in low-temperature (4 ℃) storage were studied in this trial.192 Huang hens of 33 weeks old were selected and divided into 12 groups as (3 sorts of processed semen×4 storage time) with 16 hens for each,42 Huang cocks of 33 weeks old were selected as one group.After collecting semen,two kinds of diluents was used to dilute semen and kept them in 4 ℃ for 0,4,8,24 hours,then the sperm motility and inseminated were observed,eggs were collected from all groups for measurement of the fertilization rate,birth rate and healthy chicks rate of each group.The experiment was taken with the original semen kept in 4 ℃ as control.The results showed that sperm motility of the two dilute semen was extremely significantly higher than that of the semen group (P<0.01) and the sperm motility of formula Ⅱ group was higher than that of formula Ⅰ group (P>0.05) after storage of 4,8 and 24 h.Fertilization rates of the two dilute semen were significantly higher than that of the semen group (P<0.05) after storage of 4 h;Fertilization rates of the two dilute semen were extremely significantly higher than that of the semen group (P<0.01) after storage of 8 h.The two semen diluents were more effective for semen preservation,and the two dilute semen could significantly improve the fertilization rate.The result provided a powerful support for the improvement of semen preservation skills in chicken farms in Southern China.
Research Progress of IGF2 Gene Expression Regulation and Its Genetic Variation in Animal Growth and Development
ZHAO Haidong, WU Mingli, CHEN Pingbo, WEI Yanpei, WANG Shuhui, SUN Xiuzhu
2020, 47(6):  1844-1852.  doi:10.16431/j.cnki.1671-7236.2020.06.024
Abstract ( 410 )   PDF (1343KB) ( 174 )  
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Insulin-like growth factor 2(IGF2),one of the insulin-like hormone family,widely involved in many physiological and metabolic processes of the body,which has an important role in cancer,neuroregulation,metabolic disease,osteoporosis,muscle development and lipidosis.IGF2 functions mainly through competitive binding to the IGF1R,IGF2R or IGF2BPs.Base on the IGF2 plays an important role in musle develop and lipidosis,it makes sense to mine molecular markers of IGF2.Furthermore,the internal regulation mechanism of IGF2 in livestock production was further analyzed.It was found that there was a significant correlation between the genetic variation of IGF2 gene and animal development.Genetic variation functions by affecting IGF2 gene imprinting,methylation,transcription factors binding or post-transcriptional regulation due to miRNA targeting binding.Therefore,this paper focused on the research progress of IGF2 gene expression regulation,gene imprinting,and epigenetic regulation about miRNA,in order to provide relevant reference for the study of IGF2 gene in the regulation of animal growth and development,and provide effective clues for molecular breeding in animal husbandry.
Preventive Veterinary Medicine
Rescue of Recombinant Feline Panleukopenia Virus Carrying GFP Gene
YIN Juanbin, ZHOU Yahua, YIN Xiangping, ZHANG Zhidong
2020, 47(6):  1853-1860.  doi:10.16431/j.cnki.1671-7236.2020.06.025
Abstract ( 258 )   PDF (9325KB) ( 36 )  
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The purpose of this study was to construct a recombinant feline panleukopenia virus (FPV) carrying green fluorescent protein (GFP) in order to detect neutralizing antibody more quickly and effectively.GFP specific primers were designed by referring to the GFP gene sequence in GenBank,and the GFP gene carrying AgeⅠ and NheⅠ digestion sites was amplified by PCR,and plasmid pFPV preserved in laboratory was subjected to site-directed mutagenesis to obtain the Age Ⅰ and Nhe Ⅰ restriction sites.pFPV and GFP genes were double-digested with Age Ⅰ and Nhe Ⅰ,and ligated at 4 ℃ overnight and transformed to obtain the recombinant plasmid pFPV-GFP.The recombinant plasmid pFPV-GFP was successfully transfected into feline kidney cells (F81) by Lipofection 3000.Using the unique luminescence mechanism of GFP,the labeled recombinant virus rFPV-GFP could be observed in vitro under fluorescence microscope,and Western blotting was used to identify the expression of GFP in the recombinant virus rFPV-GFP.The results showed that green fluorescent protein could be observed when the recombinant plasmid pFPV-GFP was transfected into F81 cells for 24 hours,and with the extension of transfection time,the amount of green fluorescent protein was increased,indicating that the recombinant plasmid pFPV-GFP was successfully transfected into F81 cells and GFP was stably expressed in the recombinant virus rFPV-GFP.In this study,GFP was successfully inserted into pFPV and rescued recombinant virus,indicating that foreign genes could be inserted into pFPV,laying a foundation for the insertion of other foreign genes into pFPV.At the same time,GFP insertion provided a more favorable research tool for the research of FPV,which laid the foundation for the basic research of FPV and the rapid detection of neutralization antibodies.
Isolation,Identification and Biological Characteristics of Streptococcus suis Serotype 14
WANG Zhifang, XU Yindi, ZHANG Qingxian, ZHU Wenhao, BAI Hongjie, JIAO Wenqiang, LI Haili, XU Feng, WANG Keling, ZHANG Bin, LOU Zhiguo
2020, 47(6):  1861-1869.  doi:10.16431/j.cnki.1671-7236.2020.06.026
Abstract ( 288 )   PDF (2073KB) ( 89 )  
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In order to detect and determine the pathogen of the diseased piglets in a large-scale pig farm in Henan province in May 2019,a strain of bacteria was isolated from the joint fluid of diseased piglets.The isolated strain was identified as Streptococcus suis by purification culture,Gram staining,morphological observation and PCR amplification of gdh gene of Streptococcus suis.PCR amplification and software were used to identify the Streptococcus suis.The results showed that the strain was Streptococcus suis serotype 14 and 100% homologous with js14 (GenBank No.:CP002465.1).The results of virulence gene detection showed that epf,mrp,sly,fbps and orf2 virulence genes were carried by the strain at the same time.The results of mouse pathogenicity test also showed that the strain was a highly pathogenic Streptococcus suis.The results of drug sensitivity test showed that the strain was sensitive to β-lactams and quinolones,and highly resistant to aminoglycosides,tetracyclines,macrolides and sulfonamides,showing multiple drug resistance.The resistance genes of blaTEM,aadA1,strA,strB,aacC2,aphA1,tet(B),gyrA,parC and sul2 were detected.This study laid a foundation for further research on the epidemic characteristics and pathogenic mechanism of Streptococcus suis serotype 14,provided theoretical basis for clinical prevention and control of Streptococcus suis serotype 14,and had important public health significance.
Epidemiological Investigation of CSFV in Shandong Province from 2017 to 2019
WANG Jiayun, WANG Xin, WANG Bingyu, ZENG Hao, SHEN Wenhui, YU Jiang, CHEN Zhi, ZHANG Yuyu, SUN Wenbo, GUO Lihui, REN Sufang, LI Yubao, WU Jiaqiang
2020, 47(6):  1870-1881.  doi:10.16431/j.cnki.1671-7236.2020.06.027
Abstract ( 239 )   PDF (4191KB) ( 116 )  
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To investigate the molecular epidemiology and genetic diversity of CSFV in Shandong province,1 687 tissue samples collected from January 2017 to December 2019 from suspected pigs in 10 regions (including Jinan,Taian,Liaocheng,Qingdao cities and so on) of Shandong province were detected for CSFV,the CSFV-positive samples were amplified and sequenced for E2 and CSFV gene,and the bioinformatics software DNAStar and Mega 7.0 were used to analyze the genetic evolution of CSFV E2 protein and the complete gene compared with reference strain.In addition,4 866 serum samples collected from the same regions as tissue samples were tested for E2-ELISA antibodies.The results showed that 188 out of 1 687 cases were positive for CSFV,with a total positive rate of 11.1%,and the annually positive rates from 2017 to 2019 were 8.5%,13.1% and 15.1%,respectively,it could be clearly seen that the antigen-positive rate had been increasing year by year,which might indicate that there was a certain risk and pressure in the immunization of swine fever rabbit attenuated vaccine in large-scale swine farms.While 4 146 of 4 866 blood samples (85.2%) were positive for E2 antibody,and the annually positive rates of E2 antibodies from 2017 to 2019 were 84.3%,83.0% and 92.3%,respectively.The positive rate of antibody detection had increased year by year,indicating that the overall level of swine fever vaccine protection in large-scale farms in Shandong province had increased year by year.In addition,5 strains of CSFV genome and 5 strains of virus E2 gene sequences were obtained from the positive disease material.The sequence homology between the 5 clinical isolates and the 2.1d subtype CSFV genome were 97.5% to 99.5%,indicating that the 5 isolates were closely related to the CSFV of the 2.1d subgenotype,mutations in the amino acid sites of the isolated strains were at positions 102 (L→M) and 159 (K→R).This study analyzed the prevalence and variation of CSFV in pigs in parts of Shandong from January 2017 to December 2019,and the results could provide a reference for the prevention and control in Shandong.
Detection of Resistance and Resistance Genes of Dairy Cow Pastureland Environment in Gansu Province
ZHANG Xuejing, HE Zhuolin, HOU Xiao, PU Wanxia
2020, 47(6):  1882-1891.  doi:10.16431/j.cnki.1671-7236.2020.06.028
Abstract ( 205 )   PDF (1041KB) ( 63 )  
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In order to investigate the distribution of major resistant bacteria and the resistant genes around the environment of dairy cow pastureland in Gansu province,the resistant bacteria were isolated and purified by resistance plate screening method,and then identified by 16S rRNA in the study.The results showed that a total of 665 strains of different species of resistant bacteria were isolated from the two fields (farm A and farm B).The major strains were Escherichia coli,Enterococcus faecalis and Proteus genera by PCR identification of 16S rRNA.The number of resistant Escherichia coli was the largest (398 strains).The number of resistant bacteria in farm A increased by 28.50% over the past two years,while the number of resistant bacteria in farm B remained the same.The results of 10 kinds of resistant bacteria screening showed that the isolation rates of the first three categories were glycopeptide (11.13%,74/665),tetracycline and sulfonamide (10.98%,73/665),and beta-lactam and aminoglycoside (10.23%,68/665).PCR results showed that the resistant genes in dairy cow pastureland during two years period of time were almost similar.The prevalence rates of quinolone resistant genes qnrB and qnrS were 10.81% and 83.78%,respectively.The prevalence rates of sulfanilamide resistant genes Sul1,Sul2 and Sul3 were 100%,100% and 56.76%,respectively.The prevalence rates of glycopeptide resistance genes VanA,VanB and VanC were 78.38%,100% and 100%,respectively.The prevalence rates of tetracycline resistant genes tetA,tetB, tetC,tetM,tetO and tetL were 100%,100%,100%,100%,81.08% and 75.68%,respectively.The prevalence rates of KPC,NDM and aac(6')-Ⅰb -cr resistance genes were 32.43%,29.73% and 24.32%,respectively.However,qnrC,tetK,tetW,VIM and OXA resistance genes were not detected.The homology between resistance gene and the gene sequence of reference strain in GenBank was 98% to 99%.The resistant bacteria carrying corresponding resistance gene was very common in Gansu province.Especially,carbapenems and aminoglycoside resistance genes were detected in high number.Therefore,regular surveillance of drug-resistant bacteria was highly essential in order to minimize the risk caused by resistant bacteria to human.In addition,antibiotic drugs should be used wisely and in appropriate dose during clinical treatment.
Molecular Characteristics Analysis of penton Gene of Ankara Virus Guizhou Strains
HE Ling, ZHANG Yundan, LI Qiaobin, YANG Yuan, ZHOU Bijun, WEN Ming, CHENG Zhentao, YUE Jun
2020, 47(6):  1892-1901.  doi:10.16431/j.cnki.1671-7236.2020.06.029
Abstract ( 248 )   PDF (3032KB) ( 133 )  
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To understand the molecular characteristics of penton gene of Ankara virus Guizhou strains,specific primer was designed and synthesized based on the sequence of FAdV gene,and the penton gene CDS regions were amplified.The nucleotide sequence,physical and chemical properties of protein,signal peptide,secondary structure,tertiary structure and B cell epitope of penton gene were analyzed by bioinformatics software.The results showed that three FAdV (GZ-BJ,GZ-QL and GZ-LPS) penton gene fragments were 1 578 bp.Phylogenetic tree analysis showed that GZ-BJ,GZ-QL and GZ-LPS all belonged to FAdV-4 type.The nucleotide homology of three FAdV-4 strains ranged from 99.9% to 100%,and the amino acid sequence homology ranged from 99.8% to 100%.There was a little difference with other domestic isolates and foreign isolates.The physical and chemical properties of the protein showed that the protein was unstable and had no transmembrane structure.The secondary structure was mainly composed of random coil (54.29%).The domain prediction results showed that the penton protein contained only a low complexity region.B cell epitope prediction showed that there were three dominant epitopes in the amino acid encoded by penton gene:5-171,207-236 and 420-462.The results indicated that the Ankara virus penton gene was relatively conserved and there were multiple dominant epitopes.It could be used as a target protein for Ankara virus prevention and control research,laying a foundation for the virus prevention and control and pathogenic mechanism research.
Molecular Epidemiological Investigation and Genetic Evolution Analysis of Theileria luwenshuni in Sheep and Goats in Different Regions of China
CHEN Qian, WANG Kunlun, YAN Yaqun, ZHOU Yongchun, ZHAO Cong, JIAN Fuchun, WANG Rongjun, ZHANG Longxian, NING Changshen
2020, 47(6):  1902-1909.  doi:10.16431/j.cnki.1671-7236.2020.06.030
Abstract ( 248 )   PDF (1050KB) ( 117 )  
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To understand the prevalence of T.luwenshuni in goats and sheep in different regions of China,in this study,281 sheep blood samples collected from 7 regions of Henan,Gansu,Shaanxi,Shanxi,Guizhou,Yunnan and Xinjiang were tested by PCR based on the 18S rRNA gene locus of T.luwenshuni,and positive samples were sequenced for sequence analysis.The results showed that the total infection rate of T.luwenshuni was 35.23% (99/281).The infection rate of T.luwenshuni differed significantly among sampling sites (P<0.01),among them,the infection rate of T.luwenshuni in Henan province was the highest (98%,49/50),and the lowest in Xinjiang (0).The infection rates of T.luwenshuni in sheep and goats were 51.67% (31/60) and 30.77% (68/221),respectively,and the differences were extremely significant (P<0.01).The infection rate of T.luwenshuni in grazing sheep (41.55%,86/207) was significantly higher than that in stabling sheep (17.57%,13/74) (P<0.01).The infection rates of T.luwenshuni were 56.00% (28/50),42.75% (59/138),9.43% (5/53) and 17.50% (7/40) in spring,summer,autumn and winter,respectively,and the differences were extremely significant (P<0.01).The infection rates of T.luwenshuni in sheep aged ≥ 12 months and <12 months were 33.50% (67/200) and 39.51% (32/81),respectively,with no significant difference (P>0.05).In addition,the genetic and evolutionary analysis indicated that the isolates of T.luwenshuni obtained from this study were more than 99.60% homologous to the isolates of T.luwenshuni isolated from China and they were located on the same cluster.This survey provided an important reference for further understanding of the prevalence and distribution of T.luwenshuni in sheep and goats in different regions of China.
Study on Serotyping and Detection of Virulence-associated Genes of Pasteurella multocida Isolated from Bovine
WU Qin, ZHANG Xingxing, GU Xiaoxiao, TAOQIAO Xiaoci, HUANG Xin, HAN Mengli, WU Tongzhong, ZHOU Xia, ZHONG Fagang
2020, 47(6):  1910-1920.  doi:10.16431/j.cnki.1671-7236.2020.06.031
Abstract ( 276 )   PDF (2368KB) ( 128 )  
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In order to determine the distribution of serotypes,ST types and virulence related genes of Pasteurella multocida (Pm) isolated from suspected cases in Northern Xinjiang,17 strains of Pm isolated from cattle were studied in this study.The methods of capsule multiplex PCR,LPS multiplex PCR,multilocus sequence typing (MLST) and PCR were used to detect the capsule type,lipopolysaccharide type,MLST type and the distribution of 7 classes of 25 virulence related genes in 17 strains of Pm isolates.The results showed that 13 strains of Pm were A:L3,ST was ST1,4 strains of Pm were B:L2,ST was ST44.The detection rate of 17 Pm virulence related genes (exbB,exbD,fimA,fur,hgbA,hsf2,nanB,oma87,ompA,ompH,plpB,psl,ptfA,sodA,sodC,tonB and tbpA) was 100%,and the detection rate of toxA gene was 0.The results showed that the main serotype of Pm,which isolated from parts of northern Xinjiang large-scale cattle farms, was A:L3:ST1.
Basic Veterinary Medicine
Isolation,Identification and Biological Characteristics of Probiotics from Swine Origin in Guangxi
ZHANG Zai, ZHENG Ruicheng, FANG Qingli, HUANG Weijian
2020, 47(6):  1921-1933.  doi:10.16431/j.cnki.1671-7236.2020.06.032
Abstract ( 272 )   PDF (2597KB) ( 175 )  
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In order to investigate the prevalence of probiotics in pig feces and microecological fermented feeds in Guangxi area,and to rationally develop and utilize porcine probiotics,in this experiment,15 pig feces samples and 5 probiotic fermentation feeds were collected and the probiotic bacteria were isolated.The morphological characteristics,physiological and biochemical characteristics and 16S rRNA molecular sequence of the probiotics were studied.The growth curve,bile salt resistance test,temperature sensitivity test,gastrointestinal tolerance test,in vitro and in vivo bacteriostasis test,drug resistance test,mouse safety test were carried out to study the biological characteristics of the isolated strains.In this experiment,6 species and 29 probiotics strains were isolated and identified,including 1 Enterococcus faecium (C2),2 Enterococcus lactate (C1 and C3),3 Lactobacillus plantarum (R20,R30 and R37),4 Lactobacillus casei (R41-R44),7 Bacillus subtilis (K1-K7) and 12 Bacillus cereus (Y1-Y12).Six probiotics with good growth performance,bile salt resistance,high temperature resistance,gastrointestinal tolerance and strong antibacterial ability in vitro were screened out,including Enterococcus lactis C1,Enterococcus faecium C2,Lactobacillus plantarum R20,Lactobacillus casei R41,Bacillus subtilis K1 and Bacillus cereus Y3.The safety test of the selected strains in mice showed that six probiotics were safe and harmless to mice at the concentration of 109 CFU/mL.At the same time,C2,R20,R41 and Y3 probiotics could extremely significantly increase the weight of mice (P<0.01),K1 could markedly increase the weight of mice (P<0.05).The results of bacteriostatic experiment in vivo showed that C2,R20 and K1 could reduce the mortality of mice infected by Salmonella G21,and had a certain degree of antibacterial effect in vivo.All test results showed that R20 and K1 could be selected as pig probiotic strains.
Study on Pharmacokinetics of Cyclodextrin-encapsulated Flufenicol in Pigs
ZHANG Le, ZHANG Guangfeng, ZHU Chongmiao, WU Zhi, ZHU Shanyuan, YU Shenglan, FENG Qi, XING Xiaoling, ZHAO Qiannan
2020, 47(6):  1934-1941.  doi:10.16431/j.cnki.1671-7236.2020.06.033
Abstract ( 251 )   PDF (1004KB) ( 110 )  
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In order to evaluate the bioequivalence of flufenicol (test reagent F) produced by the new coating process with the similar foreign products (R1) and domestic products (R2),and to explore its pharmacokinetic characteristics in pigs.The design of random triplet and triplet self-crossing test was adopted.Six healthy castrated male pigs (15 kg±2 kg) were respectively to take three formulations by oral administration (20 mg/kg).Flufenicol plasma concentration was analyzed by high performance liquid chromatography.Pharmacokinetic characteristics were analyzed by Kinetica 5.0,and bioequivalence was evaluated by SAS statistical software.The results showed that the drug time curve of the tested preparation in pigs conformd to the first-order absorption one-chamber open model.The Cmax of F,R1 and R2 were 16.0845,18.3287 and 21.1678 μg/mL respectively,Tmax were 5.0,1.9 and 1.5 h respectively;AUC0-∞ were 144.7327,118.2670 and 123.3715 μg/mL·h,respectively.The relative bioavailability of the two reference preparations was 122.51% (R1) and 117.52% (R2),respectively.The results showed that cyclodextrin coated with flurbenicol had better sustained-release effect,better biosafety,longer duration of efficacy,and higher bioavailability.
Drug Resistance and Detection of HPI Related Genes in E.coli from Calves with Diarrhea
GAO Haihui, LI Yuqiong, GAO Xiaofei, WANG Peizhu, WU Xueqing, KANG Xiaodong, LIANG Xiaojun
2020, 47(6):  1942-1951.  doi:10.16431/j.cnki.1671-7236.2020.06.034
Abstract ( 256 )   PDF (3973KB) ( 126 )  
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The purpose of this study was to determine the drug resistance of Escherichia coli (E.coli) from calf diarrhea,the carrying of high pathogenicity island (HPI) marker gene and related genes,and the relationship between E.coli isolates and HPI carrying.158 pathological diarrhea samples of calves were collected and screened by McConkey medium and Eosin-Methylene blue medium.VITEK 2 compact biochemical identification and PCR identification were carried out for those with the morphology of E.coli.The results showed that 75 strains of E.coli were isolated.The drug resistance rate of the isolates to amoxicillin,piperacillin,ampicillin,cefazolin,florfenicol,enrofloxacin and tetracycline was >90%.The drug resistance rate to cefalexin,cefradine,ceftriaxone,ciprofloxacin,norfloxacin and ofloxacin was ≥ 60.00%.The drug resistance rate to amikacin was 9.33%.75 strains of E.coli were resistant to more than 3 kinds of drugs,85.33% of them were multi resistant (≥ 10),the resistant spectrum was concentrated in 14 to 17 kinds of drugs,and 5 strains were resistant to all 19 kinds of drugs.The positive rate of HPI marker gene irp2 was 100%,and that of other related genes fyuA,irp3,irp5,irp8 and ytbA was over 66.00%.In conclusion,the drug resistance of E.coli from diarrhea in Ningxia was widespread,and the multiple drug resistance was serious.
Isolation,Identification and Biological Characteristics of a Pathogon of Salmonella Agona Originated from Pigeon
LI Tingcui, LI Ke, YAN Hongya, ZHAO Rong, TANG Yaqi, CHANG Zhishun, ZOU Fengcai, XIN Aiguo
2020, 47(6):  1952-1960.  doi:10.16431/j.cnki.1671-7236.2020.06.035
Abstract ( 286 )   PDF (2971KB) ( 83 )  
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In order to understand the cause of the disease in a pigeon farm in Yunnan,a treatment plan was developed.The liver tissues of sick pigeons were collected aseptically and streaked on MacConkey medium for bacterial isolation and culture,biochemical identification,serotype identification,16S rDNA homology analysis,animal regression experiments,drug sensitivity tests and virulence analysis.The results showed that round,smooth,colorless and translucent colonies were observed on the MacConkey medium,which were initially identified as Gram-negative bacteria.The strain was identified as Salmonella Agona by Salmonella serum agglutination test and 16S rDNA homology analysis.Its antigenic structural formula was O antigen 1(+),4(+),12(+),H antigen fg(+),s(+),which was named SSYN036 strain.The growth curve of bacteria showed that the strain grow faster.Animal experiments showed the strain was highly pathogenic to the test mice,the inoculated with 5×108,5×107,5×106 and 5×105 CFU/mL of mice,within 7 days,which mortality were 100%,100%,60% and 30%,respectively.The pathological sections showed more obvious focal necrotic inflammation and inflammatory cell infiltration.The drug sensitivity test showed that the strain was resistant to penicillin,oxacillin,streptomycin,doxycycline and tetracycline,moderately sensitive to rifampicin,and sensitive to amoxicillin,ampicillin,gentamicin,tobramycin,ceftazidime,etc.11 antibacterial drugs.Virulence gene test showed that the strain contains spvB,spiA,pagC,msgA,invA,sipB,prgH,spaN,tolC,iroN,sitC,lpfC,sifA,sopB and pefA genes.This study provided new data information of the pathogenicity of Salmonella and the biological characteristics of strains,and its public health significance was worthy of attention.
Products Quality and Safety
Determination of Robenidine and Its Metabolites in Animal Derived Food by UPLC-MS/MS
ZHENG Xingguo, GUO Lingan, ZHAO Shan, LI Xi, LEI Xinyu, ZHONG Lingli
2020, 47(6):  1961-1970.  doi:10.16431/j.cnki.1671-7236.2020.06.036
Abstract ( 202 )   PDF (1446KB) ( 114 )  
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In order to detect robenidine and its metabolites residues in animal derived food rapidly and effectively,an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of robenidine and its metabolites (4-chlorobenzoic acid,4-chlorobenzoylamino-acetic acid) residues in five kinds of animal derived food,including eggs,chicken,beef,fish and pork.Samples were extracted with 2% (V/V) formic acid-acetonitrile solution,removed water with anhydrous sodium sulfate,fixed volume with methanol,then excepted fat with n-hexane.The purified samples were analyzed by UPLC-MS/MS after freezed and high speed centrifuged.UPLC was achieved using a Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm,1.7 μm);the mobile phase consisted of methanol and 0.1% formic acid-water solution.The qualitative analysis was performed with MS/MS under multiple reaction monitoring (MRM) mode with positive and negative electrospray ionization;the matrix matched external standard method was used for quantitation.The results showed that there were good linearities of the three compounds in their respective concentration range (R2>0.999).The limits of detection (LODs) and the limits of quantity (LOQs) for robenidine were 0.5 and 1.0 μg/kg,for 4-chlorobenzoic acid were 2.5 and 5.0 μg/kg,for 4-chlorobenzoylamino-acetic acid were 1.0 and 2.5 μg/kg,respectively.While blank samples spiked four levels (robenidine:1.0,25,50,100 μg/kg;4-chlorobenzoic acid:5.0,25,50,100 μg/kg;4-chlorobenzoylamino-acetic acid:2.5,25,50,100 μg/kg),the recoveries and RSDs (n=6) of the three compounds were 76.0%-95.9% and 2.6%-10.6%,respectively.The matrix effects (|ME|) of the developed method were 0.2%-26.2%.There was a strong matrix effect (|ME|>20%) of robenidine in eggs and 4-Chlorobenzoylamino-acetic acid in chicken,beef and fish;blank pork could be used as representative matrix for quantitative analysis of three compounds.Therefore,the method was simple in pretreatment,high in sensitivity and good in reproducibility,and could be used for the determination of robenidine and its metabolites in five kinds of animal derived food.