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05 December 2022, Volume 49 Issue 12
Biotechnology
Cloning, Bioinformatics and Expression Analysis of DJ-1 Gene in Large White×Landrace Pigs
CHUI Linya, XIA Boce, HUANG Jiaojiao
2022, 49(12):  4513-4523.  doi:10.16431/j.cnki.1671-7236.2022.12.001
Abstract ( 316 )   PDF (3999KB) ( 120 )  
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【Objective】 The purpose of this study was to clone the CDS region of DJ-1 gene in Large White×Landrace pigs and analyzed by bioinformatics, explore the expression pattern of DJ-1 gene in porcine adipose tissue, and lay a foundation for further studies on the role of DJ-1 gene in porcine adipose deposition.【Method】 The CDS region of DJ-1 gene was amplified by PCR using the cDNA of inguinal subcutaneous adipose tissue in Large White×Landrace pigs as a template, and the bioinformatics analysis of DJ-1 protein was performed using online tools.Real-time quantitative PCR was used to measure the expression of DJ-1 gene in adipose tissue of Large White×Landrace pigs, Large White pigs (lean-type) and Laiwu pigs (obese-type), and the expression differences were compared.【Result】 The length of DJ-1 gene CDS in Large White×Landrace pigs was 570 bp, which coded 189 amino acids.According to the phylogenetic tree, Large White×Landrace pigs was most closely related to Bos taurus and most distantly related to Danio rerio.The molecular weight of DJ-1 protein was 19.94 ku, it was stable, acidic and hydrophilic protein, which had one conserved domain called GAT_1 superfamily Thij.The DJ-1 protein had 21 phosphorylation sites, 4 N-glycosylation modification sites and 1 O-glycosylation modification site, but no signal peptide and transmembrane domain.The secondary structure of DJ-1 protein was composed of alpha helix, extended chain, beta turn and random coil, the proportions were 42.86%, 17.99%, 7.94% and 31.22%, respectively, and the tertiary structure prediction results were consistent with them.Protein interaction analysis revealed that DJ-1 protein interacted with SOD2, NDUFV2, SNCA, BCL2L1 and MAP3K.Real-time quantitative PCR results showed that DJ-1 gene was generally expressed in backfat, inguinal fat and perirenal fat of Large White×Landrace pigs, there were no significant difference among them (P>0.05).The expression of DJ-1 gene in Large White pigs was significantly or extremely significantly lower than that of Laiwu pigs (P<0.05 or P<0.01).【Conclusion】 This study successfully cloned the CDS region of DJ-1 gene in Large White×Landrace pigs, and DJ-1 was a stable, acidic and hydrophilic protein.The expression of DJ-1 gene in Laiwu pigs was significantly or extremely significantly higher than that of Large White pigs. The results provided a theoretical reference for further study the function of porcine DJ-1 gene.
Construction and Identification of Recombinant Replication-deficient HAdV-5 Expressing VP2 Protein of Infectious Bursal Disease Virus
XU Ting, XIONG Ting, LI Linyu, LIU Yufu, WEN Lianghai, XIE Wenting, YANG Zekun, CHEN Ruiai
2022, 49(12):  4524-4534.  doi:10.16431/j.cnki.1671-7236.2022.12.002
Abstract ( 203 )   PDF (6356KB) ( 33 )  
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【Objective】 The purpose of this study was to construct recombinant replication-deficient Human adenovirus serotype-5 (HAdV-5), that could express the VP2 protein of Infectious bursal disease virus (IBDV) by using the AdEasy system, in order to provide a new idea for the development of IBDV live vector vaccine.【Method】 By homologous recombination, VP2 gene was cloned into the shuttle plasmid pAdTrack-GOI, and finally the recombinant shuttle plasmid named pAdTrack-VP2 was constructed.After linearization of pAdTrack-VP2 by PmeⅠ enzyme, it was transformed into E.coli BJ5183-AD-1 competent cells by heat shock.The recombinant Adenovirus plasmid named pAd-VP2 was constructed by homologous recombination in bacteria.After that, pAd-VP2 was digested with PacⅠ enzyme, and the purified product was transfected into HEK293A cells to package the recombinant replication-deficient HAdV-5 (rAd5-VP2) expressing VP2 protein.The identified recombinant viruses were infected with non-replicating cells such as Vero cells, CEF cells and DF1 cells, and the expression of the target protein was analyzed.【Result】 rAd5-VP2 was expanded by HEK293A cells and the titer of the 10th generation Adenovirus was 7.9×109 IFU/mL.RT-PCR showed that VP2 gene could translate stably in recombinant Adenovirus.Both Western blotting and indirect immunofluorescence assays detected the expression of VP2 protein with a molecular weight of 41 ku.The expression of VP2 protein was also detected in Vero cells, CEF cells and DF1 cells infected with recombinant Adenovirus, indicating that HAdV-5 has the potential to be developed as a live vector vaccine for IBDV.【Conclusion】 Recombinant replication-deficient HAdV-5 was constructed, which could infect some mammalian cells and poultry cells, and the expression of the target protein could be detected.
Effects of circRNA-Zfp609 on Proliferation and Differentiation of C2C12 Myoblasts
LIU Zhuang, HE Ruyue, CAO Yang, LI Cunyuan, ZHANG Yunfeng, HU Shengwei
2022, 49(12):  4535-4544.  doi:10.16431/j.cnki.1671-7236.2022.12.003
Abstract ( 189 )   PDF (2847KB) ( 42 )  
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【Objective】 The purpose of the experiment was to explore the potential molecular mechanism of circRNA-Zfp609 regulating the proliferation and differentiation of C2C12 myoblasts.【Method】 The expression of circRNA-Zfp609 in mouse skeletal muscle tissues and C2C12 myoblasts were analyzed by RT-PCR and sequencing.The relative expression of circRNA-Zfp609 in heart, liver, spleen, lung, kidney, stomach, small intestine and skeletal muscle tissues of mice, in C2C12 myoblasts after 12, 24, 36 and 48 h proliferation and 0, 1, 3 and 5 d differentiation was detected by Real-time quantitative PCR.The relative expression of myogenin (MyoG) and myosin heavy chain (MyHC) in C2C12 myoblasts after 0, 1, 3 and 5 d differentiation was detected by Real-time quantitative PCR.The cells were interfered with the interfering expression vector (siRNA) of circRNA-Zfp609 and the effect of siRNA on the proliferation rate of C2C12 myoblasts was determined by CCK-8, and the effect of siRNA interference on the relative expression of circRNA-Zfp609, MyoG and MyHC were detected by Real-time quantitative PCR.The sites of muscle differentiation-related miRNAs on circRNA-Zfp609 were predicted by TargetScan 7.0 and miRDB softwares.The overexpression vectors of the screened miRNA were transfected into HEK293T cells, and the interaction between circRNA-Zfp609 and corresponding miRNA was verified by dual-luciferase reporter assay.According to the interaction of miRNAs with circRNA-Zfp609, the wild-type and mutant vectors of circRNA-Zfp609 were constructed and transfected into HEK293T cells, and the targeted relationship of circRNA-Zfp609 and miRNA was verified by dual-luciferase reporter assay.【Result】 The PCR and sequencing results showed that circRNA-Zfp609 was expressed in mouse skeletal muscle.circRNA-Zfp609 had the highest expression level in mouse skeletal muscle, and the expression in other tissues from high to low were kidney, lung, heart, liver, stomach, spleen and small intestine.Compared with 12 h, the relative expression of circRNA-Zfp609 was significantly increased at 36 and 48 h of C2C12 myoblast proliferation (P<0.05).Compared with differentiation day 0, the relative expression of circRNA-Zfp609 was significantly increased on days 1, 3 and 5 (P<0.05), and the relative expression of MyoG and MyHC were extremely significantly increased (P<0.01).Compared with the NC group, the proliferation rate and the relative expression of circRNA-Zfp609 of C2C12 myoblasts in the siRNA group were both extremely significantly decreased (P<0.01), and the relative expression of MyoG and MyHC were significantly decreased (P<0.05).There were 4 miRNAs (miR-150-5p, miR-327, miR-344g-3p and miR-615-5p) on circRNA-Zfp609 were related to muscle differentiation.circRNA-Zfp609 had the strongest adsorption capacity with miR-615-5p, and had a targeted binding effect.circRNA-Zfp609 could act as a molecular sponge to interact with miR-615-5p that regulated muscle differentiation.【Conclusion】 circRNA-Zfp609 was widely expressed in mouse tissues, with the highest expression level in skeletal muscle.circRNA-Zfp609 was differentially expressed in C2C12 myoblasts at different stages of proliferation and differentiation, circRNA-Zfp609 had 4 miRNAs related to muscle differentiation, and miR-615-5p had a targeting relationship with circRNA-Zfp609.The results could provide references for the research related to the growth and development of domestic animal skeletal muscle.
Analysis of Enterotoxigenic Escherichia coli Induced Transcriptomic Changes of Porcine Intestinal Epithelial Cell Based on the Inflammatory Model
ZHU Yue, LIU Yingying, LI Yinghui, LI Pei, ZHU Kaiqing, JIA Tong, LI Yan
2022, 49(12):  4545-4553.  doi:10.16431/j.cnki.1671-7236.2022.12.004
Abstract ( 193 )   PDF (1515KB) ( 117 )  
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【Objective】 This study was aimed to analyze the key host-regulated genes and related molecular pathways in the inflammatory response induced by enterotoxigenic Escherichia coli (ETEC) in porcine small intestinal epithelial cells (IPEC-J2), in order to provide theoretical basis for the investigation of inflammatory mechanism induced by ETEC infection.【Method】 The IPEC-J2 cells were infected with ETEC bacterial solution with a multiplicity of infection (MOI) of 100, and the medium supernatant was collected at 18 h post infection.The secretion of proinflammatory factor interleukin 1β (IL-1β) was detected by ELISA method.High throughput transcriptome sequencing was performed by Illumina PE150 sequencing platform.Differentially expressed analysis was performed on the sequencing results by edgeR v 3.12.1 software.GO function and KEGG pathway enrichment analysis were performed on the differentially expressed mRNA obtained by GOseq and KOBAS 2.0 softwares.Five differentially expressed mRNA were randomly selected and verified by Real-time quantitative PCR.【Result】 A model of ETEC infection-induced inflammation in IPEC-J2 cells was successfully established.Compared with control group, a total of 529 differentially expressed mRNA were identified in inflammatory model group, of which 236 were up-regulated and 293 were down-regulated, including immune-related genes such as HSP90AA1, CGNL1, CADPS2 and EHBP1.GO function analysis showed that differentially expressed mRNA after ETEC infection were significantly enriched in cellular component and biological process, such as the intracellular part, cytoplasm, nuclear part, intracellular membrance-bound organelle, membrance-bound orgabelle and cellular process.KEGG pathway analysis showed that most of differentially expressed genes after ETEC infection were annotated as disease-related pathways as well as immune-related signaling pathways such as Toll-like receptor signaling pathway, mTOR signaling pathway, cell cycle and adhesion junctions.Real-time quantitative PCR was used to validate the expression of five randomly selected differentially expressed mRNA, the results were consistent with the trend of differentially expressed mRNA in the sequencing result, which indicated that the sequencing results were reliable.【Conclusion】 HSP90AA1, CGNL1, CADPS2 and EHBP1 genes might play a key role in ETEC adhesion to IPEC-J2 cells and induce inflammatory responses, and immunoregulated through Toll-like receptors, mTOR and other signaling pathways.The results provided a reference for further understanding the molecular mechanisms and regulatory networks involved in the inflammatory response induced by ETEC infection.
Cloning, Bioinformatics Analysis and Tissue Expression Study of FGF2 Gene in Guanzhong Dairy Goats
WU Shuanghu, WANG Pengfei, LI Hong, CHAO Juanjuan, WANG Zhanhang, ZHU Junru, AN Xiaopeng
2022, 49(12):  4554-4563.  doi:10.16431/j.cnki.1671-7236.2022.12.005
Abstract ( 162 )   PDF (3571KB) ( 34 )  
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【Objective】 The purpose of this study was to perform cloning and bioinformatic analysis of fibroblast growth factor 2 (FGF2) gene in Guanzhong dairy goats, and detect the expression differences of FGF2 gene in various tissues of goats, so as to lay the foundation for the subsequent exploration of the function of FGF2 gene.【Method】 Using mammary gland cDNA of Guanzhong dairy goat as template, the complete CDS region of FGF2 gene was amplified by RT-PCR and cloned, the similarity alignment, phylogenetic tree and bioinformatics analysis were carried out.Meanwhile, Real-time quantitative PCR was used to detect the expression of FGF2 gene in heart, liver, spleen, kidney, longissimus dorsi muscle, lung, mammary gland and ovary of Guanzhong dairy goats.【Result】 The coding region of FGF2 gene in Guanzhong dairy goats was 468 bp, encoding 155 amino acids, the molecular mass was 17.26 ku and the theoretical isoelectric point was 9.58, the highest content was glycine, accounting for 10.3%.The similarity alignment results showed that the similarity of FGF2 amino acid sequence of Guanzhong dairy goat with Homo sapiens, Bos taurus, Equus caballus, Oryctolagus cuniculus, Ovis aries, Orcinus and Sus scrofa were 98.7%, 99.4%, 100.0%, 98.7%, 99.4%, 99.4% and 62.6%, respectively.The phylogenetic tree showed that FGF2 gene was highly conserved among different species and had the closest homologous relationship with Equus caballus.The instability index of FGF2 protein of Guanzhong dairy goat was 38.39, which was an stable hydrophilic protein without signal peptide and transmembrane domain.The secondary structure of FGF2 protein contained alpha helix, beta turn, extended chain and random coil, accounting for 10.32%, 14.19%, 30.97% and 44.52%, respectively.Protein interaction prediction analysis found that FGF2 protein interacted with FGFR, KDR, FGF1, FGFR4, MET and FGFR1 proteins related to mammary gland growth and development.Real-time quantitative PCR detected that FGF2 gene of Guanzhong dairy goat was expressed in heart, liver, spleen, kidney, longissimus dorsi muscle, lung, mammary gland and ovary.The expression in mammary gland was highest, followed by ovary, and the lowest expression in longissimus dorsi muscle.【Conclusion】 The CDS region of FGF2 gene sequence in Guanzhong dairy goat was 468 bp, and encoded 155 amino acids, which was a hydrophilic protein.The secondary structure of FGF2 protein was mainly extended chain and random coil.FGF2 gene in Guanzhong dairy goat was expressed in different tissues during the peak lactation period.The results of this study provided a reference for further exploring the role of FGF2 gene in Guanzhong dairy goat mammary gland development and its specific functions.
Construction and Main Biological Characteristics Determination of RIA_0940 Gene Deleted Strain of Riemerella anatipestifer
LI Linlin, DONG Jiawen, ZHANG Junqin, HUANG Yunzhen, SUN Minhua
2022, 49(12):  4564-4572.  doi:10.16431/j.cnki.1671-7236.2022.12.006
Abstract ( 158 )   PDF (1900KB) ( 25 )  
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【Objective】 In this study, Riemerella anatipestifer RIA_0940 gene deletion strain was constructed, and its main biological characteristics were studied to explore potential functions of RIA_0940 gene.【Method】 Taking the isolated Riemerella anatipestifer RA-GD strain as the parent strain, the left and right homologous sequences and erythromycin resistance gene fragments were amplified, and the left homologous arm-ermR resistance gene box--right homologous arm fusion fragment was constructed.The RIA_0940 gene of RA-GD was deleted by natural transformation, and the gene deleted strains RA-GDΔRIA_ 0940 were screened and identified by PCR.Growth curves, LD50 to ducklings, blood and tissues bacterial load and the ability of cell adhesion and invasion were tested and analyzed of wild-type strain RA-GD and deletion mutant RA-GDΔRIA_0940, respectively.【Result】 The results showed that the RA-GD RIA_0940 gene deletion strain was successfully constructed.The results of biological characteristics showed that the growth ability of strain RA-GDΔRIA_0940 in vitro was significantly or extremely significantly inhibited compared with its wild-type(P<0.05;P<0.01), the LD50 of RA-GDΔRIA_0940 was 114 times that of wild-type strain.Compared with wild-type strain, the blood and tissues bacterial load of RA-GDΔRIA_0940 infected ducks were significantly or extremely significantly decreased (P<0.05;P<0.01);The adherence and invasion capacities of RA-GDΔRIA_0940 to Vero cells were extremely significantly decreased(P<0.01).【Conclusion】 In summary, RA-GDΔRIA_0940 deletion mutant was constructed successfully.The growth trend of the deletion strain was slow under the condition of in vitro culture.The pathogenicity to ducklings, the amount of bacteria in infected duck blood fluid and tissues, and the ability of adhesion and invasion to Vero cells were significantly or extremely significantly lower than those of the wild-type strain.The results laid a foundation for the further study of the molecular pathogenesis of Riemerella anatipestifer and the development of genetic engineering vaccine.
Screening of Sex-specific Genes in vivo-Produced Bovine Blastocysts Based on Single-cell RNA Sequencing Technology
CUI Baoshan, HUANG Fei, WANG Jie, LI Nan, GAO Qinghua
2022, 49(12):  4573-4581.  doi:10.16431/j.cnki.1671-7236.2022.12.007
Abstract ( 607 )   PDF (2532KB) ( 46 )  
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【Objective】 The purpose of this study was to explore the specific differences in mRNA levels between female and male blastocysts in cattle and to explore candidate genes related to the development of female and male blastocysts.【Method】 The primers were designed concerning the GenBank sequence of the bovine enamel gene (AMEL) (AMELX gene, accession No.:NM_001014984.1;AMELY gene, accession No.:NM_174240.2), and the intra-embryonic cellular DNA was used as the template to identify the sex of individual bovine blastocysts by nested PCR amplification.Single female and male blastocysts of definite sex were selected as test materials, and a single embryo sequencing library was constructed using Smart-Seq2 amplification technology.Illumina HiSeq Xten high-throughput sequencing technology was applied to single-cell transcriptome sequencing (scRNA-Seq) of single embryos, and differential gene expression analysis, GO functional analysis and KEGG pathway analysis were performed.【Result】 Determination of embryo sex by nested PCR amplification of the AMEL gene present in intra-embryonic cellular. Gene expression analysis screened 6 160 differentially expressed genes (DEGs) between the two groups, including 675 female-specific genes and 305 male-specific genes.GO function annotation revealed that female-specific genes were significantly annotated in nucleotide binding, signal transduction regulation, multicellular biogenesis, cytoskeleton and other entries, and male-specific genes were significantly annotated in oxidative phosphorylation, mitochondria, mitochondrial inner membrane, ribosome and other items.KEGG pathway enrichment analysis identified seven pathways associated with differences in the development of female and male blastocyst, including metabolic pathway, glycolysis, pentose phosphate pathway, cellular senescence, oxidative phosphorylation, signaling pathway regulating stem cell pluripotency and Wnt signaling pathway.Five genes with direct or indirect roles in the development of bovine embryos were screened by GO and KEGG:FBP1, GADD45G, FHL2, FOSB and WNT2B.【Conclusion】 Extensive transcriptional differences existed between female and male blastocysts in cattle, and sex-specific genes FBP1, GADD45G, FHL2, FOSB and WNT2B might be key genes that directly or indirectly influence embryo development.
Nutrition and Feed
Developmental Changes of Muscle Fiber Histology and Myogenic Regulatory Genes with Different Slaughter Weights in Yuxi Black Pigs
SHI Zhuoyan, LIU Xiaoping, JI Xiang, CHU Xiaoran, HOU Junjie, SONG Zhen, LI Xiaoxia, YANG Youbing, ZHANG Guluan, WANG Jianping, WEN Fengyun
2022, 49(12):  4582-4592.  doi:10.16431/j.cnki.1671-7236.2022.12.008
Abstract ( 232 )   PDF (2177KB) ( 41 )  
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【Objective】 The aim of this study was to reveal the developmental changes of muscle fiber histology, muscle fiber type and myoblast-related regulatory genes of longissimus dorsi muscle in Yuxi Black pigs and their relationship.【Method】 The average weight with 60, 75, 90, 105 and 120 kg Yuxi Black pigs were selected, each weight slaughtered 3 pigs, a total of 15 pigs, and the male and female randomly selected.HE staining and immunofluorescence staining were used to determine the composition of fast and slow muscle types and fibrous histological characteristics, and the mRNA expressions of myogenic differentiation (MyoD), paired-box 7 (Pax7), myostatin (MSTN), myogenin (MyoG), myocyte enhancer factor 2C (MEF2C) and myosin heavy chain (MyHC) subtypes were measured by Real-time quantitative PCR.【Result】 With the increase of weight in Yuxi Black pigs, the overall muscle fiber density showed a downward trend, which was extremely significantly higher than that of other groups at 75 kg (P<0.01), and the area and diameter of muscle fibers showed an overall upward trend.At 60 and 105 kg, the percentage of slow muscles area were significantly or extremely significantly lower than other weight stages (P<0.05 or P<0.01), and the total percentage of the area occupied by the fast and slow muscles were extremely significantly lower than that of other weight stages at 90 kg (P<0.01).Real-time quantitative PCR results found that the mRNA expression of MyHCⅠ gene at 75 and 105 kg was extremely significantly higher than other body weight (P<0.01), the mRNA expression of MyHCA gene as a whole showed a downward trend, the mRNA expression of MyHCX gene at 60 kg reached the highest, and the mRNA expression of MyHCⅡB gene at 75 kg was extremely significantly lower than other body weight (P<0.01).The mRNA expression of MyoD, Pax7, MSTN, MyoG and MEF2C genes reached their maximum values at 60, 75, 90, 120 and 120 kg, respectively.The correlation analysis results showed that in addition to MSTN gene, MyoG, MyoD, Pax7 and MEF2C genes had significant or extremely significant correlations with muscle fiber morphology and MyHC gene subtypes (P<0.05 or P<0.01).Through the comparison of 120 kg of Yuxi Black pigs and Duroc pigs, it was found that the diameter, area, density of muscle fiber, and the mRNA expression of MyHC gene and related muscle-forming regulatory genes between the two were extremely significant differences (P<0.01).【Conclusion】 During the growth stage of 60 to 120 kg in Yuxi Black pigs, the area and diameter of muscle fiber increased and the density of muscle fiber decreased, and the change of muscle fiber type composition was mainly the transformation of types Ⅰ, ⅡA and ⅡX muscle fiber to type ⅡB muscle fiber.Compared with Duroc pigs, the area and diameter of muscle fiber in Yuxi Black pigs were larger, the density of muscle fiber was lower, and the mRNA expression of MyHCⅠ and MyHCA genes were higher.
Effects of Bacillus licheniformis on Growth Performance, Serum Biochemical Indexes and Intestinal Morphology of Broilers
WANG Jiaxin, HAN Yunsheng, CAI Hongying, WEN Zhiguo, XU Xin, ZHAO Zitao, MENG Kun, YANG Peilong
2022, 49(12):  4593-4603.  doi:10.16431/j.cnki.1671-7236.2022.12.009
Abstract ( 339 )   PDF (1065KB) ( 130 )  
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【Objective】 This experiment was conducted to investigate the effects of different levels of Bacillus licheniformis BCG on growth performance and health of broilers, explore the probiotic effect of BCG on broilers and provide theoretical basis for the application in production.【Method】 A single factor experimental design was adopted, 180 1-day-old Arbor Acres broilers with similar body weight (42.62 g±0.82 g) and good health conditions were selected and randomly divided into 3 treatments, and each treatment included 6 replicates with 10 chickens per replicate.The broilers in control group (CT group) were fed with the basal diet, and the broilers in Bacillus licheniformis treatment groups (BCG1 and BCG2 groups) were supplemented with 1.0×108 and 1.0×109 CFU/kg of Bacillus licheniformis BCG in the basal diet, respectively.The trial period was divided into two feeding stages, 1 to 21 days and 22 to 42 days, with a duration of 42 days.On days 21 and 42, the broilers in each group were weighed on an empty stomach in the unit of replication and feed intake at each stage was recorded to calculate average daily gain (ADG) and average daily feed intake (ADFI).Two chickens per replication were selected to collect blood and to measure blood routine indexes, serum biochemistry and serum immunity related indexes.On day 42, small intestine was separated after slaughtering, and the intestinal morphological structure was observed to measure villus height (VH), crypt depth (CD) and to calculate the ratio of villus height to crypt depth (VH/CD).【Result】 ① Compared with CT group, the body weight of broilers in BCG2 group was significantly increased on day 42 (P<0.05).During days 22 to 42, the ADG and ADFI in BCG2 group were significantly increased (P<0.05), and the former was significantly higher then that in BCG1 group (P<0.05).During days 1 to 42, both ADG and ADFI in BCG1 and BCG2 groups were significantly increased (P<0.05), and there was no significant difference between the BCG1 and BCG2 groups (P>0.05).② On day 21, compared with CT group, the contents of the serum immunoglobulin A (IgA), IgM and IgG in BCG2 group were significantly increased (P<0.05), and the endotoxin activity was significantly decreased (P<0.05).The content of serum IgA and IgM in the BCG 2 group were significantly higher than those in BCG1 group (P<0.05).On day 42, the addition of Bacillus licheniformis BCG had no significant effect on blood routine indexes and IgA, IgM and IgG contents (P>0.05).③ On day 42, the VH of jejunum in BCG2 group was significantly higher than that in CT group (P<0.05), and VH/CD of jejunum in BCG2 group was significantly higher than that in CT and BCG1 groups (P<0.05).【Conclusion】 Bacillus licheniformis BCG supplementation could improve the growth performance of broilers, enhance humoral immunity and intestinal barrier function in the early stage of broilers, and improve the morphological structure of the small intestinal tract.The prebiotic effect was better with the addition amount of 1.0×109 CFU/kg.
Effects of Taurine Supplementation on Glycolipid Metabolism in C57BL/6J Mice with High-fat Diet
ZHANG Yuanyuan, TIAN Zhichen, SUN Yujia, ZHANG Huimin, YANG Zhangping, WANG Cong, CAO Ke, MAO Yongjiang, LI Mingxun
2022, 49(12):  4604-4613.  doi:10.16431/j.cnki.1671-7236.2022.12.010
Abstract ( 234 )   PDF (5487KB) ( 63 )  
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【Objective】 The experiment was conducted to study the effect of taurine on glucose and lipid metabolism in obese mice induced by high-fat diet.【Method】 20 SPF C57BL/6J male mice aged 5 weeks were randomly divided into 4 groups (blank, model, 3% taurine and 5% taurine groups) with 5 mice in each group.The experiment lasted for 15 weeks.The mice in blank group were fed a control diet, the mice in model group were fed a high-fat diet, and the mice in other two groups were supplemented with 3% and 5% taurine respectively on the basis of the model group diet.Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed 2 weeks before the end of the trial.At the end of the experiment, the eyeballs were anesthetized with ether and blood was collected.The mice were sacrificed by cervical dislocation and the liver, kidney, spleen and epididymal adipose tissue were collected and weighed to calculate the organ/fat coefficient.The serum biochemical indexes and the contents of leptin (LEP) and adiponectin (ADPN), triglyceride (TG) and total cholesterol (TC) in liver were determined.Oil red O staining and hematoxylin-eosin (HE) staining were used to observe the distribution of lipid droplets and the morphological changes of adipocytes in liver.【Result】 ①Compared with blank group, the body weight of mice in model group was extremely significantly increased (P<0.01), and the indexes of blood glucose (GLU) and blood lipids were abnormal.The glucose tolerance and insulin tolerance increased extremely significantly under the curve (P<0.01), which was consistent with the characteristics of the obesity model. ②Compared with model group, the total weight gain, liver coefficient and fat coefficient of taurine supplementation were extremely significantly decreased (P<0.01), the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and content of low density lipoprotein (LDL) in serum were extremely significantly increased (P<0.01), while the content of GLU was significantly decreased (P<0.05). The contents of TG and TC in liver were extremely significantly decreased (P<0.01), the content of ADPN was extremely significantly increased (P<0.01), glucose tolerance and insulin tolerance were positively and extremely significantly decreased under the curve (P<0.01). The number of lipid droplets in liver decreased, and the cross section of adipocytes decreased extremely significantly (P<0.01), the number of adipocytes increased extremely significantly (P<0.01), and the distribution of adipocytes was uniform.【Conclusion】 Taurine might have a protective effect on liver by regulating glucose and lipid metabolism and improving histopathological morphology of liver and fat in obese mice induced by high-fat diet.
Effects of Fermented Andrographis Powder on Production Performance, Serum Biochemical Indexes, Immune Related Indexes, Antioxidant Indexes and Fatty Acid Contents of Muscle in Muscovy Ducks
ZHANG Qiang, LIU Zhenni, LIAO Qiang, LEI Xiaowen, KONG Zhiwei, XIE Hualiang, TAN Donghai, LI Jianjun, ZHONG Yunping
2022, 49(12):  4614-4624.  doi:10.16431/j.cnki.1671-7236.2022.12.011
Abstract ( 236 )   PDF (993KB) ( 41 )  
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【Objective】 The purpose of this experiment was to study the effects of fermented Andrographis powder on production performance, serum biochemical indexes, immune related indexes, antioxidant indexes and fatty acid content of muscle in Muscovy ducks.【Method】 Andrographis powder was fermented by compound bacteria (the mass ratio of yeast and lactic acid bacteria was 1:1), and its microecological function and medicinal value were analyzed according to the changes of components after fermentation.450 15-day-old Black feather muscovy ducks weighing about 250 g were randomly divided into 5 groups, 6 repetitions in each group, 15 ducks each repetition.The ducks in control group Ⅰ were fed with basic diet, while in control group Ⅱ were added with 3% Andrographis powder in basic diet, in fermentation group Ⅰ, fermentation group Ⅱ and fermentation group Ⅲ were added with 1%, 3% and 5% fermented Andrographis powder in basic diet respectively.The pre-feeding period was 7 d and the feeding period was 48 d.The muscovy ducks were weighed at the age of 16, 21, 42 and 70 days respectively.At the age of 70 days, 6 muscovy ducks (1 muscovy duck for each repetition) were randomly selected from each group for slaughter determination.The samples of pectoral muscle in left and blood were collected to determine serum biochemical indexes, immune related indexes, antioxidant indexes and muscle fatty acid content.【Result】 ① After Andrographis powder fermented by compound bacteria, the number of viable bacteria was more than 2.0×108 CFU/g and the crude fiber content was decreased, but the difference was not significant (P>0.05).② The feed weight ratio of 16-70 days old in fermentation group Ⅱ was significantly or extremely significantly lower than that of control group Ⅰ and fermentation group Ⅲ (P<0.05;P<0.01).③ The slaughter rates of fermentation group Ⅰ and Ⅱ were significantly higher than those of control group Ⅰ and Ⅱ (P<0.05).④ The serum IL-2 level of muscovy ducks in fermentation group Ⅱ, Ⅲ and control group Ⅱ were extremely significantly higher than those in control group Ⅰand fermentation group Ⅰ (P<0.01).⑤ The serum level of malondialdehyde in fermentation group Ⅱ was extremely significantly or significantly lower than that of control group Ⅰ and control group Ⅱ (P<0.01;P<0.05).⑥ The contents of unsaturated fatty acids, polyunsaturated fatty acid and essential fatty acids in muscle of fermentation group Ⅱ were significantly or extremely significantly higher than those of control group Ⅰ (P<0.05;P<0.01).【Conclusion】 Compared with the basic diet group, 3% fermented Andrographis powder could significantly improve the production performance, serum IL-2 level, unsaturated fatty acid and essential fatty acid content in muscle of muscovy ducks, reduce lipid peroxidation and improve antioxidant capacity of muscovy ducks.In addition, the effects of 3% fermented Andrographis powder on slaughter rate and antioxidant capacity of muscovy ducks were significantly better than that of unfermented Andrographis powder in the same proportion.
Biological Function of β-glucan and Its Application in Aquaculture
HOU Dongqiang, ZHAO Hongxia, PENG Kai, CAO Junming
2022, 49(12):  4625-4634.  doi:10.16431/j.cnki.1671-7236.2022.12.012
Abstract ( 515 )   PDF (1066KB) ( 81 )  
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β-glucan comes from a wide range of sources, it mainly exists in plant and microbial cell walls and is composed of glucan monomers.β-glucan has a variety of structures, which has a variety of biological functions due to the variety and number of side chain residues.β-glucan can regulate immunity by reducing the expression of inflammation related genes and increasing the expression of immune factors, it plays a role in regulating lipid metabolism by reducing the production and absorption of lipids. By improving the scavenging activity of antioxidant enzymes, free radicals and superoxide anions, activating Nrf2 signaling pathway and up-regulating the expression of antioxidant genes, β-glucan can enhance the antioxidant capacity of the body and play the role of antioxidant.β-glucan can enhance the hypoxia tolerance, improve the ability to resist ammonia nitrogen stress, energy metabolism and the expression of antioxidant enzyme genes, and play an anti-stress effect.Since the prohibition of antibiotics, the research and application of β-glucan in aquatic animals have been gradually deepened, and β-glucan plays an important role in preventing aquatic animal diseases and reducing the use of antibiotics.In aquaculture, β-glucan can improve the growth performance of aquatic animals by increasing digestive enzyme activity, improving intestinal structure, optimizing intestinal flora and enhancing non-specific immunity.The combination of β-glucan has a better immune effect than the addition of a single type of immune stimulant.In this paper, the structural characteristics, biological activities, mechanism of action and application effects of β-glucan in aquatic animals were summarized, and the future development direction of β-glucan was prospected, which provides materials for realizing the ecological and healthy aquaculture of aquatic animals.
Effect of Taurine Additive Package on the Immune Function of Immunosuppressed Mice
GUO Zimeng, WANG Fuzheng, WANG Chengzhi, DU Peng, ZHANG Mingyue, LI Zeyao, SU Xiaochen, WU Gaofeng, YANG Jiancheng
2022, 49(12):  4635-4645.  doi:10.16431/j.cnki.1671-7236.2022.12.013
Abstract ( 162 )   PDF (2122KB) ( 35 )  
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【Objective】 This experiment was conducted to study the effect of adding taurine additive package in basic diet on the immune function of immunosuppressed mice.【Method】 80 mice were randomly and equally divided into four groups, mice in C and M groups were given basic diet, mice in H and MH groups were given diet containing 8.8% taurine additive package.The experiment was lasted for 51 days, the immunosuppressed mice model was established by intraperitoneal injection of 70 μg/g BW cyclophosphamide (CTX) for four consecutive days from the 31st day (in M and MH groups), while the other mice were intraperitoneally injected with the equal volume of normal saline.The feed intake and body weight were recorded to calculate the average daily gain (ADG) and average daily feed intake (ADFI).Effects of this immunopotentiator on immune organ index, cellular immunity, humoral immunity, non-specific immune function, levels of cytokines and immunoglobulins of mice were detected.【Result】 Compared with C group, mice in M group showed significant reduction in ADFI, ear swelling, serum hemolysin level, and immunoglobulin G(IgG) (P<0.05), while ADG, thymus index, splenic lymphocyte proliferation capacity, antibody-producing cell amount, abdominal macrophage phagocytosis, interleukin-6(IL-6) and immunoglobulin A(IgA) levels were extremely significantly reduced (P<0.01), and the spleen index was extremely significantly increased (P<0.01).There were no significant differences in carbon clearance index and immunoglobulin M(IgM) level between C and M groups (P>0.05).Compared with M group, mice in MH group had significantly increased ADFI, ADG, thymus index, spleen lymphocyte proliferation, ear swelling and IgM levels (P<0.05), extremely significantly increased serum hemolysin level, antibody-producing cell amount, abdominal macrophage phagocytosis, IL-6 and IgA levels (P<0.01), and significantly decreased spleen index (P<0.05).There was no significant difference in carbon clearance index and IgG levels between M and MH group (P>0.05).【Conclusion】 The taurine additive package administered in the present study could improve the innate and adaptive immunity, improve the levels of cytokines and immunoglobulins of mice with low immunity caused by CTX, and play a role in enhancing the immunity of mice.
Effects of Rumen-protected Choline and Organo-chromium on Immunity and Antioxidant Properties of Perinatal Dairy Cows
LI Xiangbao, XU Xiaohui, AN Xiuxiu, WEI Xueliang, XU Yuwei, WANG Yuanyuan, CHEN Shi
2022, 49(12):  4646-4653.  doi:10.16431/j.cnki.1671-7236.2022.12.014
Abstract ( 158 )   PDF (791KB) ( 24 )  
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【Objective】 The aim of this study was to investigate the effects of rumen-protected choline and organic-chromium on immunity and antioxidant properties of perinatal dairy cows, and provide scientific guidance and theoretical basis for the application of rumen-protected choline and organo-chromium in perinatal dairy cow diets.【Method】 Forty healthy Chinese Holstein cows (average parity was 1 to 2) were randomly divided into 4 groups with 10 cows per group.Cows in control group were fed the basal diet, cows in the choline group were fed the basal diet +30 g/d rumen-protect choline, cows in the organic chromium group were fed the basal diet +8 g/d chromium methionine, and cows in the mixed group were fed basal diet + 30 g/d rumen-protect choline +8 g/d chromium methionine.The experiment lasted from day 20 before delivery to day 20 after delivery.The average daily milk production was recorded within 4 weeks postpartum.The blood samples were collected at 20 and 10 days before parturition, delivery day, 10 and 20 days after parturition, respectively, before morning meal.The contents of serum antioxidant(GSH-Px, CAT and SOD) and immunity properties indexs (IL-1, IL-6 and TNF-α) were dectected.【Result】 Compared with the control group, the average daily milk yield of choline, organic-chromium and mixed groups were significantly increased within 4 weeks postpartm (P<0.05).There was no significant difference in the serum GSH-Px and CAT contents of the dairy cows among groups prepartum(P>0.05), but the serum SOD contents in three experimental groups were significantly decreased (P<0.05).On the day of delivery, day 10 after delivery, the contents of serum GSH-Px, CAT and SOD in choline and organic-chromium groups were significantly higher than those in control group (P<0.05).On the day 20 after delivery, serum GSH-Px and CAT contents in choline group were significantly lower than those in control, organic chromium and mixed groups (P<0.05), while serum SOD contents were significantly decreased (P<0.05).In the whole trail period, the content of serum IL-1 was significant lower than that in control, choline and organic-chromium groups, but the contents of serum IL-6 and TNF-α were significantly increased (P<0.05).【Conclusion】 Supplement of 30 g/d remen-protect choline or 8 g/d organic chromium could significantly decrease the oxidative stress level, increase milk yield and immunity, and improve the heath status of dairy cows. There was no signicant difference between the effect of combined supplement and single supplement.
Isolation and Identification of a Bacillus subtilis S1 Involved in Aflatoxin B1 Detoxification
SUN Xiangli, LIU Yuxuan, SHI Ziyao, WEN Changyin, TIAN Yadong, KANG Xiangtao, WANG Yanbin
2022, 49(12):  4654-4664.  doi:10.16431/j.cnki.1671-7236.2022.12.015
Abstract ( 152 )   PDF (2270KB) ( 39 )  
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【Objective】 This study was aimed to screen out strains that could efficiently degrade alflatoxin, and to detect the degrading activity and residual toxicity, in order to provide solutions for the prevention and control of alflatoxin B1(AFB1) pollution.【Method】 Samples were collected from feces of herbivorous animal such as yak and sheep, as well as moldy feed from livestock farms.The strain with the highest efficiency in degrading AFB1 was screened using coumarin, a structural analog of AFB1, as the sole carbon source, and the strains were identified by morphology, physiology and biochemistry and 16S rDNA sequencing.AFB1 was mixed with an appropriate amount of fermentation broth, the final concentration of AFB1 was 1 μg/mL, and AFB1 was cultured at 37 ℃ at 150 r/min, and the degradation rate of AFB1 at different time was measured.The supernatant, bacterial suspension and bacterial cells were prepared by differential centrifugation, and the bacterial cells were ultrasonically lysed to obtain an intracellular solution, and the degradation rates of different components to AFB1 were determined.The supernatant was subjected to proteinase K, proteinase K+SDS and heat treatment to detect the degradation rate of AFB1.Precipitation of the supernatant with different concentrations of saturated ammonium sulfate, and the supernatant concentrate solutinon was prepared using 7 ku dialysis bags, that were for the measurement of degradation efficiency of AFB1.So as to determine the nature and distribution of the active ingredients of the strain.Chicken hepatocytes were divided into AFB1, AFB1-strain, strain and blank control groups.The relative expression of the inflammatory factor interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and Bcl-2-associated X protein (Bax) genes were evaluated.【Result】 After initial screening and rescreening, one strain was isolated from the sheep feces sample numbered S1, which had the highest activity of degrading AFB1 with a degradation rate of 60.36%.The strain S1 was cultured on the LB solid medium for 12 h, and pale yellow opaque colonies could be observed, which had smooth surface and bumps, the microscopic examination was Gram-positive and rod-shaped.The 16S rDNA gene was amplified by PCR and further sequenced, and the sequence identity with LC55966.1 was 100%.Based on nucleotide sequence analysis and physiological and biochemical results, the strain was comprehensively identified as Bacillus subtilis.The bacterial fermentation broth was reacted with AFB1 (1 μg/mL) for 4, 12, 24, 48 and 72 h, and the degradation rates reached 10.98%, 25.36%, 46.24%, 52.65% and 80.84%, respectively.The degradation rate of AFB1 in the supernatant was significantly higher than that in the bacterial suspension and intracellular solution (P<0.05).The degradation activity of the supernatant was decreased after heat treatment, while the degradation activity was basically lost after treatment with proteinase K and SDS.There was no significant difference in the degradation rate of supernatants treated with different concentrations of saturated ammonium sulfate (P>0.05).However, the degradation rate of AFB1 in the supernatant after dialysis concentration was significantly higher than that in the supernatant precipitated by ammonium sulfate (P<0.05).In chicken embryonic liver primary cells, the expressions of IL-6, TNF-α and Bax induced by degradation product of AFB1 with Bacillus subtilis S1 were significantly lower than that in AFB1 group (P<0.05).【Conclusion】 The strain of Bacillus subtilis S1 was screened out, which could degrade AFB1 efficiently.Its degradation activity was from a protein mainly distributed in the culture medium supernatant, and its residual toxicity of AFB1 was significantly reduced after degradation.
Genetics and Breeding
Research Progress on Application of CRISPR/Cas9 Technology in Pigs and Chickens
LI Xiaojiao, HE Yanhua, ZHU Xinyu, ZOU Xian, LUO Chenglong
2022, 49(12):  4665-4673.  doi:10.16431/j.cnki.1671-7236.2022.12.016
Abstract ( 298 )   PDF (1338KB) ( 122 )  
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Gene editing relies on nucleases to generate DNA double-strand breaks at specific sites in the genome, thereby triggering the cell’s own DNA damage repair mechanism to achieve site-specific modification of target gene sequences.Clustered regularly interspersed short palindromic repeats (CRISPR) and its related protein 9 (Cas9) system play an important role in agriculture and gene therapy research as a third-generation gene editing technology.Compared with the gene editing method of transcription activator-like effector ribozyme, zinc finger ribozyme, it can target any site of gene to cause DNA double-strand break, achieve precise knockout of target gene or insert foreign fragment, and can quickly efficiently modifying the genome, including gene knockout, knockin, repression, activation, etc., is one of the most widely used gene editing tools.The emergence of CRISPR/Cas9 technology has revolutionized the research status of life science, medicine and genetics.In recent years, the use of CRISPR/Cas9 technology to modify animal genomes has opened a new era of molecular breeding of livestock and poultry, which not only greatly promoted the development of modern livestock and poultry breeding technology, but also made special contributions to human medical research, especially in pigs and chickens.Taking pigs and chickens as objects, the author reviewed the recent research progress on the CRISPR/Cas9 system in allogeneic organ transplant donors, biological models of human diseases, preparation of disease-resistant breeding materials, and genome-wide high-throughput screening, and how to use CRISPR technology to quickly and simply produce gene editing pigs and chickens, so as to provide a reference basis for promoting CRISPR technology to prepare other gene editing animals.
Construction of Ovarian Transcriptional Profiles and Analysis of Follicle Development-Related Genes in Yili Geese at Various Stages Before and After Egg Laying
ZHAO Xiaoyu, WU Yingping, LI Haiying, YAO Yingying, LI Jiahui, YAO Yang, LU Qingqing
2022, 49(12):  4674-4687.  doi:10.16431/j.cnki.1671-7236.2022.12.017
Abstract ( 202 )   PDF (6336KB) ( 42 )  
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【Objective】 The experiment was aimed to construct a transcriptome library of Yili goose ovaries before and after egg laying, combined with bioinformatics analysis, reveal differentially expressed genes in ovarian tissues of Yili geese at different egg laying stages, identify key genes affecting goose ovarian development, and provide theoretical references for reproductive regulation of Yili geese.【Method】 Four Yili geese at the prelaying period(KL), laying period(CL), and ceased period(XL) were selected, and the ovarian tissues were obtained after slaughter for the construction of ovaries transcriptome libraries.Candidate genes associated with follicle development were screened by novel gene mining, differential gene expression, gene annotation, and protein interaction network analysis.Eight differentially expressed genes were randomly selected and Real-time quantitative PCR was used to verify their expression.【Result】 Histological results of the ovaries of Yili geese showed that a large number of primary follicles on the surface of the ovaries at the prelaying period, while the ovaries at the laying period showed a hierarchy of follicles, and follicles at the ceased period could be observed to appear sunken and atretic inwards.By RNA sequencing (RNA-Seq), 57 811 186 to 85 328 377 clean reads were obtained from the 12 constructed Yili goose ovary cDNA libraries, all with Q30 values were greater than 93.38%, and the sequencing reads produced by each sample were compared to the goose reference genome at a rate of 82.79% to 89.24%.A total of 1 112 novel genes were annotated in ovaries, and the total number of single nucleotide polymorphism loci (SNP) was ranged from 1 642 273 to 2 425 069.SNP and InDel were mainly annotated in the intron region.The alternative splicing in each period were mainly concentrated in TSS and TTS.There were 337, 1 136 and 525 differentially expressed genes in KL vs CL, XL vs CL and XL vs KL groups, respectively, and the total differentially expressed genes were adrenoceptor alpha 2A (ADRA2A), cuticular protein (CP), glycoprotein non-metastatic melanoma protein B (GPNMB) and alpha-1-antitrypsin-like (LOC106033756).GO function enrichment analysis revealed that the differential mRNAs were mainly enriched in positive regulation of peptidyl-tyrosine phosphorylation, cell adhesion and external side of plasma membrane.KEGG pathway enrichment analysis revealed that differentially expressed genes were significantly enriched mainly in neuroactive ligand-receptor interaction, ECM-receptor interaction and steroid biosynthesis.Combined with protein interaction network analysis, potential regulatory factors Bruton’s tyrosine kinase (BTK), platelet derived growth factor receptor alpha (PDGFRA), integrin subunit beta 3 (ITGB3) associated with goose ovarian development were screened.The results of Real-time quantitative PCR showed that RNA-Seq results were accurate and reliable.【Conclusion】 This study revealed the gene expression differences in ovarian tissues of Yili geese before and after egg laying, and screened out important pathways (neuroactive ligand-receptor interaction, ECM-receptor interaction, steroid biosynthesis) and key candidate genes (BTK, PDGFRA and ITGB3), which could provide theoretical support for understanding the molecular mechanism of ovarian tissues in regulating egg laying performance of Yili geese.
Polymorphism of SLC8A1 Gene and Its Effect on Eggshell Quality in Changshun Blue-eggshell Chickens
WANG Yingtong, YANG Suan, TAN Yuancheng, WANG Chunyuan, ZHANG Yiyu
2022, 49(12):  4688-4696.  doi:10.16431/j.cnki.1671-7236.2022.12.018
Abstract ( 156 )   PDF (2492KB) ( 29 )  
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【Objective】 This study was aimed to explore the effect of solute carrier family 8 member A1 (SLC8A1) gene polymorphism on the eggshell quality in Changshun Blue-eggshell chickens, so as to provide reference for improving eggshell quality in the future.【Method】 The sequence of SLC8A1 gene in chicken was searched by GenBank database (accession No.:NC_052534.1), Primer Premier 3.0 software was used to design primers for PCR amplification, and the SNP of SLC8A1 gene was screened by direct sequencing.The differences of eggshell indexes of different genotypes of SNP were detected by SPSS 22.0 software.【Result】 Three new SNPs were identified in exon 11 of SLC8A1 gene:g.16085681 T>C, g.16085766 C>T and g.16085781 T>C.A total of 3 haplotypes and 6 diplotypes were detected, the haplotypes were H1(TCT), H2(CTC) and H3(TTC), and the diplotypes were H1H1(TTCCTT), H1H2(TCTCTC), H1H3(TTTCTC), H2H2(CCTTCC), H2H3(CTTTCC) and H3H3(TTTTCC).χ2 test showed that three SNPs were in Hardy-Weinberg equilibrium (P>0.05), all showed moderate polymorphism.The results of correlation analysis showed that the eggshell strength of TC genotype individual of g.16085681 T>C was significantly higher than that of TT genotype (P<0.05).g.16085681 T>C and haplotype H2 (C16085681T16085766C16085781) had significant effects on the secondary structure of SLC8A1 gene mRNA.The egg shape index of CC genotype individual of g.16085766 C>T was significantly higher than that of TT genotype (P<0.05).The egg shape index of H1H1 and H1H3 individuals were significantly higher than that of H2H3 (P<0.05).The eggshell strength and eggshell thickness of H2H3 individual were significantly higher than those of H1H3 (P<0.05).The egg weight of H1H2 and H2H2 individuals were significantly higher than that of H3H3 (P<0.05).【Conclusion】 g.16085681 T>C could be used a marker site affecting eggshell strength, g.16085766 C>T mutation could be used a marker site affecting egg shape index, and diplotype H2H3 might be a favorable diploid of eggshell strength and eggshell thickness.
Research Progress on Application of Lipidomic Technology in Quality Assessment and in vitro Storage of Livestock Semen
CHEN Jiakang, LYU Chunrong, MIAO Yongwang, QUAN Guobo
2022, 49(12):  4697-4706.  doi:10.16431/j.cnki.1671-7236.2022.12.019
Abstract ( 145 )   PDF (1431KB) ( 53 )  
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Semen quality evaluation is one of important means to assess the fecundity of male animals.With the fast development of omics technologies, researchers can obtain high-throughput information of genes, transcripts, proteins, metabolites, etc.The information is of great value for research on semen quality evaluation and sperm-related assisted reproductive technologies.In recent years, researchers have attempted to use lipidomic technologies to investigate the factors affecting sperm cell fertility in mammals.Lipidomics mainly uses high-throughput technologies to explore the composition and distribution of lipids in cells, tissues, or organisms, and accurately quantify the content of lipids, so as to elucidate the relationship between lipid diversity and cell metabolism regulation and corresponding biological functions.As the downstream products of proteins and nucleic acids, lipids can more directly reflect the real-time physiological state of tissues and cells.The plasma membrane of sperm is rich in various lipids, which have important impacts on the structure and function of sperm.In addition, some potential lipid markers can be obtained by lipidomic studies for the evaluation of semen quality in livestock.At present, lipidomics has been used to study the features of fatty acids in sperm.However, despite the rapid development of lipidomics studies in recent years, this method still has some limitations.There is no unified standard for the technical process of lipid extraction from various samples, which leads to different interpretations of the same result.Therefore, there is still a large space for improvement of lipidomic technologies.This review introduces the lipidomic characteristics of livestock semen, summarizes the development status of lipidomics in the field of semen quality evaluation and in vitro storage, and forecasts its future developmental trend.
Effects of Vitamin A on the Maturation and Subsequent Development of Yak Oocytes in vitro
JI Wenhui, WANG Yuling, HE Honghong, FU Wei, LAN Daoliang
2022, 49(12):  4707-4714.  doi:10.16431/j.cnki.1671-7236.2022.12.020
Abstract ( 164 )   PDF (1192KB) ( 37 )  
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【Objective】 The aim of this study was to explore the effects of vitamin A on in vitro maturation and subsequent embryo development of yak oocytes.【Method】 Yak oocytes were taken as the research object, and vitamin A of 0 (control group), 2, 5, 10 and 20 μmol/L was added to the in vitro maturation culture medium, and the first polar body excretion rate was counted after in vitro culture for 24 h.Parthenogenetic activation was carried out on matured oocytes in each group.After activation, the cleavage rate and blastocyst rate were counted at the 2nd and 8th days, respectively.The relative expression of node genes, including RARα, RARβ, RARγ, RXRα, RXRβ, RXRγ and STRA8 genes in typical signaling pathways and MEK and ERK1 genes in atypical signaling pathways were detected by Real-time quantitative PCR in MⅡ oocyte stage, and the optimal treatment concentration of vitamin A was selected.The optimal concentration of vitamin A was added to the in vitro maturation medium, and oocytes in MⅠ and MⅡ stage were collected at 6 and 24 hours of maturation.Some MⅡ oocytes were parthenogeneously activated, and blastocysts were collected at 8 days after activation.The relative expression of RARα, RXRα and STRA8 genes in GV, MⅠ and MⅡ oocytes and parthenogeneously activated blastocysts were detected by real-time fluorescence quantitative PCR.【Result】 Compared with control group, the first polar body excretion rate and cleavage rate of 2, 5 and 10 μmol/L vitamin A groups were significantly increased (P<0.05), and reached the highest in 2 μmol/L vitamin A group.The blastocyst rate of 2 μmol/L vitamin A group was increased significantly (P<0.05), while the first polar body excretion rate, cleavage rate and blastocyst rate of 20 μmol/L vitamin A group were decreased significantly (P<0.05).The results of Real-time quantitative PCR showed that compared with control group, the relative expressions of RARα, RXRα and STRA8 genes in 2, 5, 10 and 20 μmol/L vitamin A groups were significantly increased (P<0.05), among which the 2 μmol/L vitamin A group was the highest, so 2 μmol/L vitamin A had the best effect on the in vitro maturation of yak oocytes.Compared with GV stage oocytes, the relative expression of STRA8, RXRα and RARα genes in MⅡ stage oocytes were extremely significantly increased (P<0.01), but there was no significant difference between MⅠ stage oocytes and blastocyst (P>0.05).【Conclusion】 The addition of 2 μmol/L vitamin A could promote the maturation of yak oocytes in vitro and significantly increase the cleavage rate of the parthenogeneously activated blastocysts, and vitamin A regulated the maturation of yak oocytes mainly through typical signaling pathways.
Research Progress on Fish Antifreeze Proteins in Cryopreservation of Animals Semen, Cells and Embryos
XUE Li'e, CAO Jiacheng, CHEN Dejun, RAO Yongyong, LIN Ruiyi, XIAO Tianfang, FANG Shaoming
2022, 49(12):  4715-4724.  doi:10.16431/j.cnki.1671-7236.2022.12.021
Abstract ( 213 )   PDF (828KB) ( 39 )  
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Antifreeze proteins (AFPs) are proteins that inhibit the growth of ice crystals and improve the body’s antifreeze function.AFPs are widely distributed and have been identified in animals, plants, bacteria, and fungi.Until now, accumulating researches have been performed on fish AFPs which are expected to be applied in the production process of animal husbandry.Previous studies indicated that fish AFPs possess thermal hysteresis activity and ice crystal recrystallization inhibition activity.Fish AFPs are consisted of AFPⅠ, AFPⅡ, AFPⅢ, AFPⅣ and antifreeze glycoprotein (AFGP).Although these AFPs exert the same antifreeze effect, differences in compositions and structures were presented and homologous sequences can not be found in the corresponding genes.The antifreeze mechanism of fish AFPs mainly includes adsorption-inhibition theory, rigid body energy theory, inclusion complex anchoring theory, affinity interaction coupled agglomeration theory and so on.Fish AFPs can protect sperm by maintaining cell membrane integrity, reducing reactive oxygen species production and DNA damage in cryopreserve of human, fish, livestock and mouse semen.In cryopreservation of oocytes, spermatogonia, red blood cells and animal embryos, fish AFPs can improve the preservation effect by preventing ice crystallization and antioxidant function.In this paper, the authors summarized the species, structural characteristics and mechanism of fish AFPs, and summarized the application progress of fish AFPs in cryopreservation of semen, oocytes, spermatogonial cells, red blood cells and embryos, in order to provide scientific basis for the application of fish AFPs as candidate antifreeze agents in germplasm conservation and animal husbandry production.
Research Progress on Effects of Granulosa Cell Apoptosis on Follicle Atresia in Poultry
ZHANG Ying, LI Xianqiang, LI Yana, WANG Jiaxiang, WU Yan, PI Jinsong, CHEN Lin
2022, 49(12):  4725-4733.  doi:10.16431/j.cnki.1671-7236.2022.12.022
Abstract ( 164 )   PDF (907KB) ( 69 )  
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Ovary is an important reproductive organ of poultry, which can produce a large number of follicles, and follicles may be atresia in each stage of growth and development due to the regulation of different factors, resulting in the decline of reproductive performance.Granulosa cells have been proved to play an important role in regulating follicular growth and development, and their apoptosis can induce follicular atresia.There are many regulatory factors that induce apoptosis in granulosa cells, including hormones, cytokines, oxidative stress, mitochondria and other external factors.Granulosa cell apoptosis is mainly caused by the mitochondrial pathway, which involves the Caspase family, the release of cytochrome C (Cyt-C) when mitochondria cleaved, then the formation of apoptotic body activated Caspase-3 and Caspase-8, finally, Caspase-9 was activated to induce apoptosis of granulosa cells.When granulosa cells apoptosis occurs, follicles in poultry lose biological function and imbalances in regulation between cells within the follicle promotes the apoptosis of oocytes and membrane cells in follicles, and finally leads to follicle atresia.The growth factors, gonadal steroids and cytokines secreted by granulosa cells in the survival state can help oocytes reduce oxidative damage and prevent mitochondrial DNA damage caused by excessive intracellular reactive oxygen species (ROS), thus avoiding apoptosis of granulosa cells caused by mitochondrial dysfunction.In order to provide theoretical basis for reducing follicular atresia and improving reproductive performance of poultry, this paper discussed the apoptosis of granulosa cells and its influencing factors, the relationship between apoptosis of granulosa cells and follicular atresia, and the effect of apoptosis of granulosa cells on follicular atresia.
Preventive Veterinary Medicine
Prokaryotic Expression and Bioinformatics Analysis of Salmonella Typhimurium Virulence Gene SptP
ZHOU Nanlong, DING Yonghui, LI Tiansen
2022, 49(12):  4734-4744.  doi:10.16431/j.cnki.1671-7236.2022.12.023
Abstract ( 173 )   PDF (3415KB) ( 42 )  
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【Objective】 The purpose of the experiment was to obtain the highly expressed SptP protein of Salmonella Typhimurium and carry out bioinformatics analysis, so as to provide a theoretical basis for its function research and screening of interacting proteins.【Method】 The SptP gene was amplified by PCR technology, and the sequence was ligated to pET-32a(+) vector to construct the prokaryotic expression vector pET32a-SptP of Salmonella Typhimurium SptP gene.The recombinant plasmid was introduced into Escherichia coli BL21(DE3) competent cells by heat shock method, the recombinant protein was induced to express by IPTG, purified, and verified by SDS-PAGE and Western blotting.Then the SptP protein was analyzed by bioinformatics using online softwares.【Result】 The SptP gene was successfully amplified by PCR with a size of 1 632 bp.The recombinant SptP protein was successfully induced, expressed and purified in E.coli BL21(DE3) competent cells to obtain a protein with a molecular mass of 79.7 ku.The molecular formula of the SptP protein was C2625H4257N745O812S25, no transmembrane structure, no signal peptide, and a molecular mass of 60 047.68 u, the theoretical isoelectric point was 8.75, and there were 57 possible phosphorylation sites.The SptP protein was mainly located in the nucleus, cytoplasm, Golgi apparatus, cytoskeleton, secretion system vesicles, and plasma membrane, accounting for 43.5%, 34.8%, 8.7%, 4.3%, 4.3% and 4.3%, respectively.SptP protein was mainly composed of alpha helix, extended strand, beta turn and random coil, accounting for 43.65%, 14.92%, 4.42% and 37.02%, respectively.【Conclusion】 In this study, a recombinant plasmid pET32a-SptP expressing SptP protein was constructed, and a SptP recombinant protein with a molecular mass of 79.7 ku was obtained.The basic physical and chemical properties and biological functions of SptP protein were clarified, which provided theoretical and experimental basis for the interaction mechanism between SptP protein and host cells and the preparation of new Salmonella Typhimurium vaccine.
Bioinformatics Analysis of African Swine Fever Virus D250R Protein and Preparation of Polyclonal Antibodies
XU Chunmei, SHAO Mingzhu, WANG Xinyue, LIN Jiadi, GUO Jianxiong, ZHANG Xiangyin, TONG Dewen, LIANG Ruiying, ZHAO Xiaomin
2022, 49(12):  4745-4755.  doi:10.16431/j.cnki.1671-7236.2022.12.024
Abstract ( 184 )   PDF (4144KB) ( 30 )  
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【Objective】 The aim of this study was to analyze the potential biological function of Africa swine fever virus (ASFV) D250R protein and prepare polyclonal antibodies against it, and provide materials for the development of ASFV D250R protein function and related diagnostic reagents.【Method】 Bioinformatics softwares were applied to analyze the physicochemical properties, signal peptide, transmembrane structure, phosphorylation site, biological function and protein structure of D250R protein.The recombinant D250R protein was expressed by E.coli expression system, purified using nickel affinity chromatography column and molecular sieve, and the reactogenicity of the purified protein was detected by Western blotting.BALB/c mice were immunized with purified D250R protein to prepare polyclonal antibody.The titer of the polyclonal antibody was determined by indirect ELISA, and the specificity of the polyclonal antibody was detected by indirect immunofluorescence assay (IFA).【Result】 The bioinformatics analysis showed that D250R protein consisted of 250 amino acids with a theoretical molecular mass of 29 822.54 u, the theoretical isoelectric point was 8.96, it was a hydrophilic protein with no signal peptide region and no transmembrane region.The modification site prediction results showed that D250R protein might had 15 phosphorylation modification sites, including 6 serine (Ser), 6 tyrosine (Tyr) and 3 threonine (Thr), and had 2 N-glycosylation modification sites at positions 61 and 197, respectively.Biological function prediction showed that D250R protein contained a Nudix sequence with a dehydrolytic enzyme activity. The structure prediction results showed that the secondary structure of D250R contained 49.20% alpha helices, 7.60% beta turns, 15.20% extended strands and, 28.00% random coils, and more alpha helices existed in the three-dimensional spatial structure, which was basically consistent with the secondary structure prediction results. ASFV D250R gene was cloned into pET-32a(+) to obtain the pET-32a-D250R recombinant plasmid, which was transformed into E.coli BL21(DE3) receptor cells, and D250R protein was expressed in two forms of supernatant soluble and inclusion body under the induction conditions of 16 ℃ and 1 mmol/L IPTG.The protein supernatant was purified by nickel affinity chromatography column and molecular sieve chromatography to obtain a high purity protein.Western blotting results showed that the recombinant protein had good reactivity.The results of indirect ELISA showed that the antibody titer of D250R protein reached 1:256 000, and the polyclonal antibody was successfully prepared.The IFA test showed that the polyclonal antibody had good specificity.【Conclusion】 In this study, the physical and chemical properties and protein structure of D250R protein were analyzed at the molecular level, and the high-efficiency expression of ASFV D250R protein was realized in the E.coli expression system.The polyclonal antibody prepared by immunizing mice with the purified D250R protein had high specificity, which laid a foundation for further exploring the biological function of ASFV D250R protein and the development of ASFV related diagnostic reagents.
Advances on Trained Immunity of Poultry
MA Xingshu
2022, 49(12):  4756-4775.  doi:10.16431/j.cnki.1671-7236.2022.12.025
Abstract ( 192 )   PDF (5259KB) ( 54 )  
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Microbial resistance is a major problem threatening human care, animal health and food safety.To reduce drug resistance and residues in animal-derived foods, there is an urgent need to explore alternative strategies for prophylactic and therapeutical interventions.One of them is the activation of the innate immune system to enhance a strong and durable non-specific immune responsiveness to subsequent triggers.This process has been termed trained immunity, a de facto innate immune memory.More and more studies have shown that innate immune cells and even tissue-resident stem cells have an immune memory function to protect against reinfection for certain infections and vaccinations, that is, the innate immune system also exhibits adaptive immunity characteristics.In the veterinary field, the concept of improving disease resistance in poultry by enhancing the innate immune system is not new, however, there are few available, purposeful applied research on trained immunity.Therefore, enhancing animal immunity by trained immunity pathways is a new field worthy of attention, which will open new avenues for designing novel broad-spectrum vaccines and finding new drug targets.In this review, we provided an updated advances in the field of trained immunity and elaborated modulation and future research directions for trained immunity in poultry.
Construction and Immunogenicity of Recombinant Rabies Virus with Canine Distemper Virus H Gene
ZHAO Jing, CHU Ying, LUO Jun, GUO Xiaofeng
2022, 49(12):  4776-4785.  doi:10.16431/j.cnki.1671-7236.2022.12.026
Abstract ( 159 )   PDF (3646KB) ( 31 )  
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【Objective】 The aim of this experiment was to construct recombinant Rabies virus (RABV) expressing the hemagglutinin H gene of Canine distemper virus (CDV), and study its biological characteristics and immunogenicity.【Method】 H gene of CDV was inserted between G and L genes of RABV HEP-dG genome and the full-length cDNA plasmid pHEP-dG(H) was constructed.The plasmid pHEP-dG(H) and help plasmid were co-transfected into BHK cell and the recombinant RABV carrying double G and H genes was rescued.The recombinant virus was inoculated into BHK cells to analyze the diffusion ability of the virus.Mice were inoculated with the recombinant virus, and the pathogenicity and immunogenicity of the virus were analyzed.【Result】 RT-PCR and direct immunofluorescence showed that H gene could still be detected in the third generation virus, indicating that the CDV H gene had been successfully inserted into the RABV genome and stably inherited and correctly expressed in HEP-dG(H).The growth curves of HEP-dG(H) virus and HEP-dG strain maintained similar and the titers of two viruses both reached the peak at 96 h.But the titer of recombinant virus HEP-dG(H) was slightly lower than that of HEP-dG at each time point.Spread and infection experiments showed when the multiplicity of infection (MOI) was 0.005, proliferation ability of HEP-dG(H) was lower than HEP-dG strain.HEP-dG(H) and HEP-dG were not lethal to 6 weeks adult mice, however, HEP-dG(H) had a weaker effect on the body weight of adult mice than HEP-dG.HEP-dG (H) and HEP-dG were both able to induce mice to produce antibody, On the 7th day post infection, antibody reached the level of protection (0.5 EU/mL), and the recombinant virus HEP-dG(H) could also induce neutralizing antibody against CDV.Virus challenge test indicated that antibody induced by HEP-dG(H) and HEP-dG could resist the attack of RABV CVS-24 after immunization for three weeks.【Conclusion】 In this study, the recombinant RABV HEP-dG(H) was successfully constructed, which had good immunogenicity and safety, and could be used as a new dual genetic engineering candidate vaccine of RABV-CDV.
Isolation and Identification of Pasteurella multocida from Swine and Genetic Evolution Analysis of oppA Gene
LI Hongjie, DONG Zifan, CHE Yongliang, ZHOU Lunjiang, WANG Longbai, BAI Dingping
2022, 49(12):  4786-4794.  doi:10.16431/j.cnki.1671-7236.2022.12.027
Abstract ( 146 )   PDF (2253KB) ( 36 )  
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【Objective】 The purpose of the experiment was to understand and master the epidemic situation of Pasteurella multocida (Pm) and the genetic evolution of oppA gene in pig farms in Fujian province.【Method】 In this study, bacterial isolation and culture, biochemical test, 16S rRNA PCR amplification and sequencing, PCR capsule typing, oppA gene cloning and similarity analysis, animal regression test and other methods were used to identify and analyze the isolated strains.【Result】 A total of 10 strains were isolated in this study.The isolated bacteria formed pale grayish white, moist and smooth, butter dew beads on the blood plate.The results of biochemical tests showed that the isolated strains could ferment sucrose, fructose, maltose and mannitol, and could not decompose glucose, citrate, lactose and hydrogen sulfide, which were basically consistent with the biochemical characteristics of Pasteurella multocida.The 16S rRNA sequence of the isolated strains was more than 99.9% similarity to that of Pasteurella multocida in GenBank, and 10 strains were Pasteurella multocida.PCR capsular typing showed that 6 isolates were capsular serotype A and 4 isolates were capsular serotype D.The genetic evolution tree based on oppA gene showed that all 10 isolates were located in the same branch.Animal regression test demonstrated that the mortality of infected mice was higher within 24 h (21/30), and the isolated bacteria strains had strong pathogenicity.【Conclusion】 The results indicated that the capsular serotypes of Pasteurella multocida strains in Fujian pig farms were mainly types A and D, and most of the strains were from common ancestors, which enriched the epidemiology of Pasteurella multocida and laid the foundation for the prevention and control of Pasteurella multocida infection.
Basic Veterinary Medicine
Optimization of Fermentation Process of Oregano Powder by Response Surface Method and Study on Its Antibacterial Ability
LI Yang, ZHU Siming, ZHANG Jianling, ZHENG Yue, CHEN Yingqiang, LUO Dizhou, YU Ting, ZHANG Yanbin, WANG Zhilin
2022, 49(12):  4795-4806.  doi:10.16431/j.cnki.1671-7236.2022.12.028
Abstract ( 207 )   PDF (2799KB) ( 161 )  
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【Objective】 The aim of this study was to ferment oregano powder using compound bacteria, in order to enhance its nutritional quality.【Method】 Antimicrobial capacity was used as an evaluation indicator, oregano powder, corn powder and soybean meal powder were used as substrates to optimize the solid-state fermentation process of oregano herb by compound probiotics using the Plackett-Burman experiment, the steepest climb experiment and the central composite experiment in turn.The antibacterial ability, pH, lactic acid content, antioxidant ability, protein content and volatile oil content of fermented oregano were measured.【Result】 The fermentation time, moisture content of substrate and inoculum of lactic acid bacteria were the main effect factors affecting fermentation.The optimal fermentation process was as follows.The composition ratio of the fermentation substrate was oregano meal:corn meal:soybean meal=4:1:5, the inoculum amount of Bacillus subtilis, Bacillus licheniformis and yeast inoculum were all 5×106 CFU/g, lactic acid bacteria inoculum amount was 1.87×107 CFU/g, fermentation temperature was 25 ℃, fermentation time was 130 h, and water content was 46.7%.Under these conditions, the antibacterial ability of fermented oregano against Escherichia coli reached 99.98%.Compared with unfermented oregano, pH of the fermented combination was significantly decreased (P<0.05), the antioxidant capacity was significantly increased (P<0.05), the water-soluble proteins and acid-soluble proteins were significantly increased (P<0.05), and the content of thymol and carvacrol were both significantly increased by more than 40% (P<0.05).Compared with unfermented oregano, the antibacterial ability of fermented oregano against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Clostridium perfringens and Vibrio parahaemolyticus was significantly increased(P<0.05).【Conclusion】 After optimizing the fermentation process of oregano powder by response surface method, the contents of active ingredients thymol and carvacrol were significantly increased by more than 40%, and the antibacterial ability and nutritional quality were significantly improved.
Study on the Mechanism of Lianshenzhilikeli in the Treatment of Swine Diarrhea Based on Network Pharmacology and Molecular Docking
ZHANG Lu, SUN Yangang, XIE Sha, ZHANG Xiaoli, WANG Shaopei, LI Xiaofei, YUAN Yueyue, SONG Yu, LI Wei, CHEN Siqing
2022, 49(12):  4807-4819.  doi:10.16431/j.cnki.1671-7236.2022.12.029
Abstract ( 255 )   PDF (8567KB) ( 77 )  
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【Objective】 This study was conducted to reveal the mechanism of Lianshenzhilikeli in the treatment of swine diarrhea by network pharmacology and molecular docking.【Method】 Traditional Chinese Medicine Systems Pharmacology Database and Analysis platform (TCMSP) and Encyclopedia of Traditional Chinese Medicine (ETCM) platform were used to search for the active ingredients and targets of Coptidis Rhizoma, Pulsatilliae Radix, Sophorae flavescentis Radix, Chebulae fructus and licorice in Lianshenzhilikeli, and the network diagram of active ingredients and their targets was constructed by the Cytoscape 3.7.2 software.Swine diarrhea disease targets were obtained from NCBI, OMIM, GeneCards and MalaCards platform.The intersections of disease and ingredient targets were uploaded to the STRING 11.5 database, and the protein-protein interaction (PPI) network diagram was built by the Cytoscape 3.7.2 software.The GO function and KEGG signal pathway enrichment of the core targets in the PPI network were analyzed via microscopic letter platform.The key active ingredients and the core targets were verified by molecular docking technology.【Result】 96 active ingredients of Lianshenzhilikeli, 208 drug targets and 1 785 disease targets were obtained after screening.PPI analysis showed that the key targets of Lianshenzhilikeli directly targeting to swine diarrhea disease were Jun proto-oncogene (JUN), mitogen-activated protein kinase 14 (MAPK14), MYC proto-oncogene (MYC), NFKB inhibitor alpha (NFKBIA), NF-κB p65 (RELA), caspase-3 (CASP3), tumor necrosis factor (TNF), MAPK1, interleukin 6 (IL6), estrogen receptor 1 (ESR1) and so on.Six items of biological process, two items of cellular component and two items of molecular function were obtained from GO function screening.Biological processes included positive regulation of transcription from RNA polymerase Ⅱ promoter, positive regulation of interleukin-8 production, positive regulation of sequence-specific DNA binding transcription factor activity, cellular response to lipopolysaccharide, negative regulation of apoptotic process and regulation of transcription from RNA polymerase Ⅱ promoter.Cellular components included nucleus and cytoplasm.Molecular functions included RNA polymerase Ⅱ core promoter proximal region sequence-specific DNA binding and DNA binding.A total of 53 signal pathways were obtained from KEGG pathway enrichment analysis, and two of them closely were relating to the disease.The results of molecular docking showed that the key active ingredients of Lianshenzhilikeli had excellent binding activity to the swine diarrhea disease targets.【Conclusion】 Lianshenzhilikeli might regulate Salmonella infection disease pathway and IL17 signaling pathway by targeting to JUN, RELA, MYC, NFKBIA, MAPK14, CASP3, TNF, MAPK1 and IL6 through the active ingredients, such as formononetin, 3', 7-dihydroxy-4', 6-dimethoxyisoflavone, 3, 3'-dimethylquercetin, pinocembrin, (R)-(6-methoxy-4-quinolyl)-[(2R, 4R, 5S)-5-vinylquinuclidin-2-yl]methanol, inermine, 1-methoxyphaseollidin, 2, 4, 4'-trihydroxychalcone, so as to achieve the purpose of treating the disease.
Detection of Virulence and Resistance Genes and Drug Sensitivity Analysis of Escherichia coli Derived from Diarrheic Calves in Beijing-Tianjin-Hebei Partial Regions
JIA Tong, ZHANG Di, ZHANG Zhaotian, CHEN Panliang, ZHANG Jie, LI Yinghui, ZHU Yue, QIN Jianhua, LI Yan
2022, 49(12):  4820-4831.  doi:10.16431/j.cnki.1671-7236.2022.12.030
Abstract ( 174 )   PDF (2106KB) ( 30 )  
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【Objective】 The purpose of this study was to explore the prevalence of virulence genes and drug resistance genes of Escherichia coli (E.coli) isolated from diarrheic calves in Beijing-Tianjin-Hebei regions, providing basis for the screening of sensitive drugs.【Method】 In this study, 146 fecal samples of diarrheic calves were collected from different cattle farms in Beijing-Tianjin-Hebei regions from December 2020 to July 2021. E.coli was isolated, purified and identified by Gram staining microscopy and 16S rRNA sequencing.The virulence genes (F17, K99, F41, STa, stx1, irp2 and fyuA genes) and drug resistance genes (aac(6')-Ⅰb, blaCTX-M, blaTEM, OqxB, tetA and sul1 genes) were identified by PCR.The drug sensitivity test was performed using K-B paper method.【Result】 The growth morphology of the isolates on the medium and the gram staining results was consistent with the physiological and biochemical characteristics of E.coli.The 16S rRNA sequencing results of the isolates showed a single peak, and more than 96% of the sequences were identical to E.coli genome using NCBI BLAST, indicating the clinical isolates were E.coli.A total of 142 strains of E.coli were isolated, of which 88 strains carried virulence genes, accounting for 61.97% (88/142).The positive rates of virulence genes F17, K99, F41, STa, stx1, irp2 and fyuA were 24.65%, 0.70%, 0, 2.11%, 1.41%, 45.07% and 21.83%, respectively.Therefore, F17, irp2 and fyuA were the dominant virulence factors, while E.coli carrying multiple virulence factors was less frequently detected.Six resistance genes aac(6')-Ⅰb, blaCTX-M, blaTEM, OqxB, tetA and sul1 were detected.blaTEM gene had the highest detection rate of 45.77%.In addition, aac(6')-Ⅰb and OqxB genes had the lowest detection rate of 9.15%, and the isolated strains mainly carried 1 to 3 resistance genes.The results of drug susceptibility test on 142 isolates showed the highest susceptibility rate of norfloxacin, followed by ciprofloxacin.The susceptibility rate to penicillin was 0, and the drug resistance phenomenon was severe, with 86.62% of isolates resistant to more than 2 antimicrobial agents.【Conclusion】 The virulence genes and drug resistance genes of E.coli isolated from diarrheic calves in Beijing-Tianjin-Hebei regions were widespread distributed and highly drug resistance.The phenomenon of multiple drug resistance was severe.This study provided a theoretical basis for the prevention and treatment of E.coli induced calf diarrhea in Beijing-Tianjin-Hebei regions.
Study on in vitro Antibacterial and Antiviral Activity of Compound Shuanghuanglian Preparation
GUO Bin, SHI Han, FENG Haibo, LIU Qun
2022, 49(12):  4832-4842.  doi:10.16431/j.cnki.1671-7236.2022.12.031
Abstract ( 447 )   PDF (2096KB) ( 63 )  
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【Objective】 The aim of this study was to investigate the in vitro antibacterial and antiviral activity of the compound Shuanghuanglian preparation, and provide a scientific basis for the in-depth development of compound Shuanghuanglian and the rational selection of drugs for poultry and livestock.【Method】 The granules of honeysuckle, scutellaria and forsythia was made into infusion, respectively, and then the drug infusion and Andrographis paniculata were used to make the oral solution of Andrographis paniculata, double forsythia (honeysuckle:scutellaria:forsythia was 1:1:2) and compound double forsythia (honeysuckle:scutellaria:forsythia: Andrographis paniculata was 1:1:2:2) according to the ratio, and the raw drug concentration was 100%, pH was adjusted as 7.0.12 strains of Escherichia coli, Salmonella and Staphylococcus aureus were used for bacterial susceptibility testing, and the sensitive strains were screened by Oxford cup combined with test tube twofold dilution method.The in vitro antibacterial activity was investigated based on the minimum inhibitory concentration (MIC) of the drugs.The drugs were diluted by the twofold dilution method, rhesus monkey embryonic kidney cells (MA-104) and chicken embryonic fibroblasts (DF-1) were cultured, cell viability was detected by CCK-8 method, and the safe concentrations of the three drugs were determined in combination with cytopathic lesions (CPE).The maximum safe concentration of the drug was diluted in three gradients by twofold dilution, Newcastle disease virus (NDV) and Rotavirus were used to test MA-104 and DF-1 in three ways: Drug+virus interactions, drug addition followed by attack, and attack followed by drug addition.The cell viability was detected by CCK-8 method, and the in vitro antiviral activity of the drugs was explored in combination with CPE.【Result】 The antibacterial effect of compound Shuanghuanglian was better than that of Andrographis paniculata and Shuanghuanglian, and its MICs against Escherichia coli, Salmonella and Staphylococcus aureus were 0.20-1.56, 0.20-1.56 and 0.78-1.56 mg/mL, respectively.CPE and cell viability results showed that the safe concentration ranges of Shuanghuanglian, Andrographis paniculata and compound Shuanghuanglian against DF-1 cells were 0-6.25, 0-0.78 and 0-0.78 mg/mL, respectively, and the safe concentration ranges for MA-104 cells were 0-3.13, 0-0.78 and 0-0.78 mg/mL, respectively.The TCID50 of Newcastle disease virus strain 17E on DF-1 cells was 10-6.19/100 μL, and the TCID50 of yak Rotavirus G6P[1] type SDA2 strain on MA-104 cells was 10-6.24/100 μL.The effective inhibition rates of compound Shuanghuanglian against Newcastle disease virus by directly inactivating virus (drug+virus interactions), blocking virus adsorption (drug addition followed by attack) and interfering virus replication (attack followed by drug addition) pathways were 28.82%, 55.92% and 42.45%, respectively, and the corresponding effective inhibition rates of Rotavirus were 48.84%, 26.52% and 15.40%, respectively.【Conclusion】 The antibacterial and antiviral effects of the compound Shuanghuanglian were stronger than those of Shuanghuanglian and Andrographis paniculata, and its antibacterial effect on Escherichia coli and Salmonella was better than that on Staphylococcus aureus.The results could provide scientific basis for the clinical application and further development of compound Shuanghuanglian in veterinary medicine.
Simultaneous Determination of 4 Isoquinoline Alkaloids in Broiler Tissues by Ultra-performance Liquid Chromatography-tandem Mass Spectrometry
LIU Mengting, HU Nanxi, ZOU Xianglin, YANG Zihui, ZENG Jianguo
2022, 49(12):  4843-4853.  doi:10.16431/j.cnki.1671-7236.2022.12.032
Abstract ( 151 )   PDF (2361KB) ( 78 )  
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【Objective】 The purpose of the experiment was to establish an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of protopine, allocryptopine, chelerythrine and sanguinarine in broiler tissues, so as to better monitor the distribution and residue of active ingredients of Boluohui powder and Bopuzongjian powder in broiler tissues.【Method】 The pretreatment method of broiler tissues was optimized, with 0.1% formic acid water and acetonitrile as the mobile phase, gradient elution on Agilent Zorbax SB-C18, the flow rate was 0.3 mL/min, the column temperature was 35 ℃, and the injection volume was 1 μL, multiple reaction monitoring mode for data acquisition in positive ion mode, the content was calculated by the external standard method, corrected by the internal standard, and the linearity, limit of detection and limit of quantification, recovery, intra-and inter-run precision, accuracy and stability of the method were investigated.【Result】 The results showed that the linear range of chelerythrine, protopine and allocryptopine were 0.5-200 μg/L, sanguinarine was 2-200 μg/L, the linear relationship was good, and the correlation coefficients were greater than 0.999.The detection and quantification limit of chelerythrine, protopine and allocryptopine were 0.2 and 0.5 μg/L respectively, and sanguinarine were 1 and 2 μg/L respectively.The recovery rate of the four alkaloids were 86.61%-111.17% at high, middle and low concentrations, the precision and accuracy were ≤14.93% within and between batches, and the matrix effect was 81.73%-120.78%, RSD of stability were all less than 13.55%.【Conclusion】 The established ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous detection of four isoquinoline alkaloids in the heart, liver, kidney, chicken breast and chicken thigh of broilers was consistent with various methodological investigations, and it was specificity, sensitivity, convenience and rapidness.It was suitable for accurate and rapid quantification of trace alkaloids in broiler tissues.
Preparation and Preliminary Evaluation of Minocycline Hydrochloride Complex Temperature-sensitive Gel
WANG Tianyang, MA Bei, DING Ze, DONG Tianzhen, LIU Junfeng
2022, 49(12):  4854-4865.  doi:10.16431/j.cnki.1671-7236.2022.12.033
Abstract ( 189 )   PDF (4129KB) ( 34 )  
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【Objective】 The aim of this experiment was to prepare a complexed temperature-sensitive gel capable of local delivery of minocycline hydrochloride.【Method】 Minocycline hydrochloride complexes formed with calcium ions were loaded into a temperature-sensitive gel formed by poloxamer 407 (P407) and poloxamer 188 (P188). Its temperature sensitivity, characterized structure, drug content, stability, in vitro release effect and antibacterial properties were then investigated.【Result】 The temperature-sensitive gel of minocycline hydrochloride complex was prepared [JP2]with the formula of minocycline 0.25 g, P407 8 g, P188 1.5 g, CaCl2 0.01 g, [JP]acetic acid adjusted to pH 4.0±0.2 and deionized water in 50 mL of gel, and the appearance of minocycline hydrochloride complex temperature-sensitive gel showed a light yellow clarified solution with uniform transparency.The temperature-sensitive performance was good (gelling occurred at 31 ℃), the grid of pores formed by the gel was dense and uniformly distributed, the average particle size was 11.5 nm and the zeta potential was -3.8 mA, and the in vitro release time was up to 48 h, which was significantly longer compared with that of the raw materials.In the antibacterial performance test, the concentration of minocycline hydrochloride complex was the same as that of the raw materials.In the antibacterial performance test, the inhibition ability of minocycline hydrochloride complex warming gel did not decrease, and the antibacterial time in vitro was longer compared with that of the raw materials, the inhibition effect was good.【Conclusion】 In this study, a temperature-sensitive gel of minocycline hydrochloride complex was successfully prepared.The simulated in vitro release test and in vitro inhibition curve showed that the gel could significantly prolong the drug action time and improve the drug utilization, providing a new way of drug delivery for the clinical use of minocycline hydrochloride.
Optimization of Foaming Performance of Compound Quaternary Ammonium Salt Foam Disinfectant and Evaluation of Its Killing Effect on Viruses
LIU Huan, ZHANG Mengmeng, CUI Yifang, LIU Jing, ZHANG Jingju, CHEN Dengjin, LI Xiubo, LI Cun, XU Fei
2022, 49(12):  4866-4877.  doi:10.16431/j.cnki.1671-7236.2022.12.034
Abstract ( 289 )   PDF (3138KB) ( 68 )  
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【Objective】 The purpose of the test was to study the foaming performance of compound quaternary ammonium salt foam disinfectant, and evaluate the killing effect of the disinfectant on Newcastle disease virus (NDV) and Classical swine fever virus (CSFV), as well as the effect on the disinfection effect at different action concentrations and different action time.【Method】 The specific contents of benzalkonium bromide, decylmethyl ammonium bromide and decyl glucoside in the compound quaternary ammonium salt foam disinfectant were optimized by the response surface method.Suspension killing test and cell infection method were used to determine the inactivation rate of the disinfectant against NDV LaSota strain at dilution of 1:100, 1:200, 1:400, 1:500, 1:600, 1:800 and 1:1000 for 5, 10 and 15 min respectively.Suspension quantitative method was used to determine the inactivation rate of the disinfectant against CSFV Thiveral strain when the dilution was 1:200, 1:300, 1:400, 1:500 and 1:600 for 3, 5, 7, 10 and 15 min respectively.【Result】 When the content ratio of benzalkonium bromide, decylmethyl ammonium bromide and decyl glucoside in the compound quaternary ammonium salt foam disinfectant was 1.4%, 3.8% and 0.75%, the foam forming time reached 83 min.When the compound quaternary ammonium salt foam disinfectant was diluted up to 1:400 (about 137.5 mg/L in terms of total content), the minimum action time was 5 min, and the inactivation rate of NDV LaSota strain was 100%, and the inactivation rate of CSFV Thiveral strain reached 100% at 3 min, when the action time was extended to 15 minutes, and the inactivation rate of both viruses was stable at 100%.When the dilution multiple of compound quaternary ammonium salt foam disinfectant was less than 1:400, the killing effect on NDV LaSota strain was not good.When the dilution multiple was 1:500 (about 110 mg/L in terms of total content), 1:600 (about 91.67 mg/L in terms of total content), the action time was 10 min, and the inactivation rate of CSFV Theiveral strain reached 100%, and when the action time was extended to 15 min, the inactivation rate was stable at 100%.【Conclusion】 When the content of benzalkonium bromide, decylmethyl ammonium bromide and decyl glucoside in the compound quaternary ammonium salt foam disinfectant was 1.4%, 3.8% and 0.75%, the foaming effect was the best.The disinfectant had a good killing effect on NDV and CSFV, which provided reference and theoretical basis for the establishment of its disinfection effect on NDV and CSFV and its standardized use in farms, and also provided more possibilities for the evaluation of the environmental disinfection effect of livestock and poultry breeding sites.
Effects of EGF and FGF-2 on Proliferation and Differentiation of Porcine Subcutaneous Adipose Neural Crest Stem Cells
DONG Bowen, CUI Xiaozhen, BAI Rui, ZHENG Mingxue, ZHANG Li, LYU Xiaoling, REN Yuhong
2022, 49(12):  4878-4886.  doi:10.16431/j.cnki.1671-7236.2022.12.035
Abstract ( 156 )   PDF (4023KB) ( 16 )  
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【Objective】 The aim of this study was to investigate the effects of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) on proliferation and differentiation of porcine subcutaneous adipose neural crest stem cells (NCSCs), so as to optimize the culture conditions of porcine subcutaneous adipose NCSCs.【Method】 The primary porcine subcutaneous adipose NCSCs were isolated and cultured in vitro, and the NCSCs marker p75 NTR was identified by immunofluorescence staining.The porcine subcutaneous adipose NCSCs were treated with different concentrations of EGF and FGF-2 (0 and 0, 10 and 10, 10 and 20, 20 and 10, 20 and 20, 30 and 30 ng/mL).The cell proliferation rate was tested by CCK-8 kit to determine the optimal concentration of EGF and FGF-2 for cell growth.The experiment was divided into control and optimal concentration groups.The cell growth curve of two groups was drawn, and the lipid droplet production of cells in two groups was compared by oil red O staining after the adipogenic induction.【Result】 The results of immunofluorescence staining showed that the primary porcine subcutaneous adipose NCSCs were positive by p75 NTR.The results of CCK-8 cell proliferation showed that EGF and FGF-2 had the best proliferative effect on porcine subcutaneous adipose NCSCs when the concentration of EGF and FGF-2 was 20 ng/mL, respectively.The results of growth curve showed that the cells in two groups were in logarithmic growth phase from 5th to 9th days, and the cell proliferation slowed down from 10th to 15th days, and gradually reached stagnation stage.The results of oil red O staining showed that the amount of lipid droplets in the cytoplasm of the optimal concentration group was much more than that of control group.【Conclusion】 Adding 20 ng/mL EGF and 20 ng/mL FGF-2 in culture medium could promote the proliferation and differentiation of porcine subcutaneous adipose NCSCs.
Isolation, Identification, Drug Resistance and Pathogenicity Analysis of Salmonella from Duck
ZHANG Haolei, SALANG Wenzhu, HUANG Zhihong, WEN Yongping, ZHANG Huanrong
2022, 49(12):  4887-4897.  doi:10.16431/j.cnki.1671-7236.2022.12.036
Abstract ( 159 )   PDF (3511KB) ( 35 )  
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【Objective】 In order to prevent and control duck Salmonella infection, this test was conducted to study the infection of duck Salmonella in Deyang, Sichuan.【Method】 222 samples of different types were collected from 3 duck breeding farms and 1 duck incubator in the region, and Salmonella was isolated and identified according to GB 4789.4-2016. Further, the sensitivity of the isolated bacteria to 14 antibiotics was detected by K-B method, and the pathogenicity of the identified rare Salmonella serotype was studied.【Result】 The suspected Salmonella isolates were black, gray or brownish, metallic colonies on BS agar and colorless, transparent, or black or almost all black colonies on XLD agar.The suspected Salmonella isolates were inoculated with trisaccharide iron agar slant for biochemical identification.The suspected Salmonella isolates could make the trisaccharide iron slant turn red and the bottom turn yellow with black, which conformed to the biochemical characteristics of Salmonella.The specific gene invA of Salmonella was amplified by PCR.After agarose gel electrophoresis, the target band was observed above 500 bp.It was confirmed that 17 Salmonella strains were isolated, with a total isolation rate of 7.7% (17/222).The 17 strains of Salmonella included three serotypes, namely Salmonella Typhimurium, Salmonella Hato and Salmonella Bonn, with the proportions of 47.1% (8/17), 29.4% (5/17) and 23.5% (4/17) respectively.The predominant serotype is Salmonella Typhimurium.The isolates were the most resistant to β-lactams and aminoglycosides, and the resistant rates to ampicillin and streptomycin were 82.4% (14/17) and 88.2% (15/17), respectively.The isolates were the most sensitive to quinolones, and 100% (17/17) to nalidixic acid.The isolated bacteria had serious multi-drug resistance, six strains were resistant to more than 10 kinds of antibiotics (including 10 kinds), accounting for 35.2%.And the 10 kinds of antibiotics were from at least 6 different kinds of antibiotics.Two strains were resistant to 8 kinds of antibiotics, accounting for 11.8%.Six strains were resistant to 5-7 kinds of antibiotics, accounting for 35.3%.A total of 3 strains were resistant to 2-3 antibiotics, accounting for 17.6%.The LD50 of rare serotype strains H4 and B2 were determined to be 3.98×106 and 1.58×106 CFU respectively by Karber’s modified method.The results of pathogenicity test showed that both Salmonella Hato and Salmonella Bonn could cause acute death, organ congestion, hemorrhage, cell degeneration and other pathological changes in mice.【Conclusion】 In this study, 17 strains of Salmonella from ducks were successfully isolated, it was the first time that Salmonella Hatto was isolated from ducks at home and abroad.The isolates had serious drug resistance and multiple drug resistance, the two rare serotypes of Salmonella had strong pathogenicity to mice, and the pathogenic effects were similar.This study could provide a reference for the prevention and treatment of salmonellosis in duck farms.
Investigation of the Effective Components and Active Mechanism of Qiweng Huangbo Powder in the Treatment of Piglet Diarrhea Based on Network Pharmacology
CHEN Wenguang, ZHANG Yang, XIAO Xiaoyue, LIU Yanyan, CHEN Xueying, WANG Xiumin, QIN Junjie, LI Yanhua
2022, 49(12):  4898-4908.  doi:10.16431/j.cnki.1671-7236.2022.12.037
Abstract ( 250 )   PDF (7872KB) ( 43 )  
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【Objective】 The purpose of this research was to predict the effective components and explore the mechanism of Qiweng Huangbo powder in treating diarrhea of piglets based on network pharmacology.【Method】 Potential active ingredients and target proteins of Qiweng Huangbo powder were screened through TCMSP database website, and Cytoscape 3.9.1 software was used to construct a drug components-targets network.GeneCards and OMIM databases were used to retrieve and remove repeat targets, and the common targets of disease and drugs were obtained.The common targets were imported into STRING database and the protein-protein interaction (PPI) network was constructed.GO function and KEGG pathway enrichment analysis were performed for the common targets.【Result】 A total of 37 active components and 74 targets of Qiweng Huangbo powder were obtained, among which 68 common targets intersected with the disease.147 biological process, 15 cell component and 32 molecular function were obtained by GO functional annotation.98 pathways were obtained by KEGG pathway enrichment analysis.Quercetin, kaempferol, stigmasterol, tetrahyroberberine, berberine and so on were active components.Tumour necrosis factor (TNF), tumor protein p53(TP53), vascular endothelial growth factor A(VEGFA), interleukin 6 (IL6), IL10, chemokine 2(CCL2), epidermal growth factor (EGF), NF-kappa-B inhibitor alpha(NFKBIA) and so on were shown as key targets of the drug.Cancer, phosphatidylinositol-3/kinase protein kinase B(PI3K-Akt), mitogen-activated protein kinase (MAPK), IL17, inflammatory bowel disease and other signal pathways were the key pathways, and which also involved in immune response, transcription, apoptosis, cytokine activity, extracellular aspects.【Conclusion】 This study predicted the effective components and action mechanism of Qiweng Huangbo powder in the treatment of piglet diarrhea, which reflected the characteristics of multiple components, multiple targets and multiple pathways of Traditional Chinese medicine, and provided direction and reference for subsequent research.
Identification and Biological Characteristics Evaluation of an Achromobacter Strain Isolated from Rumen of Yak
LI Dapeng, CAO Chunhua, FU Pengcheng, ZHANG Jingyan, ZHANG Kang, WANG Lei, ZHANG Kai, LIU Qin, BAO Yongzhan, LI Jianxi
2022, 49(12):  4909-4919.  doi:10.16431/j.cnki.1671-7236.2022.12.038
Abstract ( 234 )   PDF (2668KB) ( 35 )  
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【Objective】 In order to develop a new type of animal-derived probiotics, a yak rumen strain was identified and its biological characteristics were analyzed.【Method】 The tested strains were identified by microscope and colony observation, and its biochemical characteristics was identified by VITEK2 microbiological automatic identification instrument.The 16S rDNA was amplified by PCR, and similarity comparison and genetic evolution tree construction were carried out.The growth performance, acid producing ability, self-coagulation, hydrophobicity, hemolysis and drug resistance, acid and bile salt resistance and gastrointestinal environmental adaptability were studied.【Result】 After morphological, molecular identification and 16S rDNA amplification, the test strain was identified as Achromobacter and named as Achromobacter YKS2.The strain entered logarithmic phase at 2 h, and continued to grow to 20 h, and began to plateau and slowly decline.The pH did not decrease after 48 h fermentation, indicating that the strain did not have acid production ability.The self-cohesion was 28.7%, the surface hydrophobicity was 77.1% in chloroform, and the other solvents were more than 60.0%, with an average of more than 60%, indicating that the bacteria had certain adhesion ability.There was no hemolytic ring after 48 h incubation on blood agar plate.The sensitivity to 14 antibiotics was tested by K-B method, and it was found that it was sensitive to 12 common antibiotics.The strain could not survive in the medium with pH 2.0, and the survival rate was 61.4% in the medium with pH 4.0.It had high tolerance to bile salt medium containing 0.1% and 0.3%.The survival rate of bacteria cultured in artificial gastric juice for 3 h was 78.18%.After treatment in artificial intestinal juice for 4 and 8 h, the number of colonies increased significantly compared with the control group (P<0.05), indicating that YKS2 strain could tolerate artificial intestinal juice to a certain extent.【Conclusion】 YKS2 strain derived from yak rumen was a subspecies of Achromobacillus of Enterobacteriaceae with no hemolysis, acid production and strong acid resistance.It was a non-drug resistant strain with good proliferation performance, and had the properties of bile salt resistance, gastric fluid resistance, self-condensation and surface hydrophobicity.It could be used as a candidate probiotic strain for further evaluation and study.
Establishment of 3D4/2 Cell Inflammatory Model Induced by PCV2 in vitro
ZHAO Yi, ZHANG Wen, CHEN Jiaji, SONG Manling, CHEN Qi, XIE Xiaodong, HU Tingjun
2022, 49(12):  4920-4927.  doi:10.16431/j.cnki.1671-7236.2022.12.039
Abstract ( 213 )   PDF (2859KB) ( 79 )  
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【Objective】 The experiment was to establish a model of the inflammatory response of 3D4/2 cells infected with Porcine circovirus type 2 (PCV2) in vitro by exploring the relationship between the concentration, time and the inflammatory level of PCV2 in vitro infection, and lay a foundation for the study of the later drug regulation of PCV2-induced 3D4/2 cells inflammatory response.【Method】 3D4/2 cells were divided into control group and different dilutions PCV2 infected groups (100, 10-1, 10-2 and 10-3 dilutions), with 3 replicates per group.The control group was treated with DMEM, and PCV2 infected groups were treated with PCV2 in different concentrations, then replaced with DMEM maintenance solution containing 5% FBS for culture after 2 h.After 4, 8, 12 and 24 h of culture, cells and cell supernatant were collected, respectively.NO level was detected by Griess method, ROS level was detected by DCFH-DA fluorescent probe method, GSH level was detected by enzyme-conjugated method, XOD and MPO enzyme activities were detected by spectrophotometry.IL-1β, IL-6, TNF-α, IL-10, IFN-γ, IL-8, MCP-1, COX-1 and COX-2 inflammatory factor levels were determined by ELISA method.【Result】 100 to 10-3 concentrations of PCV2 could successfully infect 3D4/2 cells after 4, 8, 12 and 24 h.Compared with control group, after 3D4/2 cells were infected with PCV2 at 100 concentrations for 4, 8, 12 and 24 h, the ROS level was extremely significantly increased (P<0.01), and after 3D4/2 cells were infected with PCV2 at 10-1 to 10-3 concentrations for 8, 12 and 24 h, the ROS level was significantly or extremely significantly increased (P<0.05;P<0.01).After 3D4/2 cells were infected with 100 to 10-3 concentrations of PCV2 for 8, 12 and 24 h, the intracellular NO concentration and MPO activity were significantly increased (P<0.05).The levels of IL-1β, IL-6, TNF-α, IL-10, IFN-γ, IL-8, MCP-1 and the activity of COX-1 in cell supernatant or cells were significantly or extremely significantly increased (P<0.05;P<0.01).Among which, after 100 concentration of PCV2 infected 3D4/2 cells, the increase of each inflammatory factor was the most significant, and with the prolongation of time, the NO concentration was gradually increased, and the activity of XOD was gradually decreased.【Conclusion】 PCV2 could induce inflammatory responses in 3D4/2 cells, and infection of 3D4/2 cells with 100 dilution PCV2 in vitro for 4-12 h was the best condition for establishing an inflammatory model.
Study on Repair Effect and Mechanism of Nigella sativa Seed Extract on Alcoholic Liver Injury in Mice
ZHAO Weijie, SUN Yaogui, SUN Panpan, SUN Na, YANG Huizhen, LI Hongquan
2022, 49(12):  4928-4935.  doi:10.16431/j.cnki.1671-7236.2022.12.040
Abstract ( 197 )   PDF (2626KB) ( 75 )  
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【Objective】 This study was aimed to verify the repairing effect of Nigella sativa seed extract regarding to alcoholic liver injury in mice, and explore its mechanism, so as to provide theoretical basis for its clinical development and research.【Method】 Fifty-six 3-week-old Kunming mice were randomly divided into 7 groups:Blank control group, model group, high, medium and low dose Nigella sativa seed extract groups, Nigella sativa seed powder group and silybin positive control group, with 8 mice in each group.Except for blank control group, the mice in other groups were given 10 mL/kg 60% alcohol by gavage, once a day for 15 consecutive days.On the 16th day, the mice in high, medium and low dose Nigella sativa seed extract groups were given 8, 4 and 2 mL/kg Nigella sativa seed extract, respectively, and the mice were sacrificed for 10 consecutive days.The liver index of mice in each group was calculated, the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum were detected.Paraffin sections were prepared to observe the pathological changes of liver, the protein expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cysteinyl aspartate specific proteinase 1 (Caspase-1) and interleukin-1β (IL-1β) were detected by Western blotting.【Result】 Compared with blank control group, the liver index of mice in model group was significantly increased (P<0.05), and the liver cells showed obvious granular degeneration, vesicular degeneration, nucleus lysis, unclear differentiation boundaries and disappearance of hepatic cords.The activities of ALT and AST in serum were significantly increased (P<0.05), the activity of SOD, CAT and GSH-Px were significantly decreased (P<0.05), indicating the liver damage model successfully established, and the expression of NLRP3, Caspase-1 and IL-1β proteins in liver were significantly increased (P<0.05).Compared with model group, the activity of ALT and AST in serum showed a downward trend in three dose Nigella sativa seed extract groups, and the activity of SOD, CAT and GSH-Px showed an upward trend, among which the above indicators in high-dose group were significantly different (P<0.05), the liver tissue structure was significantly improved, and the expression of NLRP3, Caspase-1 and IL-1β proteins were decreased significantly (P<0.05).【Conclusion】 High doses of Nigella sativa seed extract (8 mL/kg) could significantly increase the antioxidant levels in liver injury mice, and reduce the synthesis of IL-1β by inhibiting the expression of NLRP3 inflammatory body, thereby improving liver function and repairing damaged liver tissue structure.
Effect of Supplementary Ellagic Acid on Parasitic Infection in Lactating Foals
GUO Cuijie, ZANG Changjiang, HUANG Xinxin, LI Jiahao, HE Linjiao, YANG Fan, REN Fei'er, CHEN Kaixu
2022, 49(12):  4936-4944.  doi:10.16431/j.cnki.1671-7236.2022.12.041
Abstract ( 205 )   PDF (840KB) ( 26 )  
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【Objective】 The aim of this study was to explore the effect of supplementary feeding ellagic acid on intestinal parasite infection in foals with pure blood during lactation, reveal the role of tannic acid in the prevention and treatment of digestive tract parasites in equines, and provide references for the screening of new anthelmintic drugs.【Method】 15 lactating pure blood foals with average body weight (143.33 ±16.10) kg, date of birth (±5 d) and similar parasite infection rate were randomly divided into control group and test groups Ⅰ and Ⅱ, with 5 foals in each group.Under the same feeding conditions, foals in the control group did not receive any treatment, foals in group Ⅰ were supplemented with 15 mg/kg BW of ellagic acid daily, and those in group Ⅱ were supplemented with 30 mg/kg BW of ellagic acid for a 60-day feeding experiment, and foal fecal samples were collected at 0, 15, 30, 45 and 60 days of the experiment to detect egg species, count the number of eggs and evaluate the deworming effect.【Result】 There were 10 species of parasites with high infection rates in mammalian thoroughbred foals, among which the most infected parasites were parasites, equine roundworm and slender-necked trilodonde.With the prolongation of ellagic acid feeding time and the increase of dose, the infection rate of parasites showed a decreasing trend, and the excretion of Trichodon tenuis eggs, Strongylus equi eggs, Parascaris equi and Strongylus westermani eggs were decreased significantly(P<0.05).The EPG values of test groups Ⅰ and Ⅱ were 66.59% and 97.06% lower than those of the control group on the 60th day after feeding with ellagic acid.The egg reduction rates of the 30th and 60th days of test group Ⅰ were 30.10% and 42.97%, respectively.The egg reduction rates in test group Ⅱ were 37.51% and 49.86%, respectively.【Conclusion】 Under the experimental conditions, the supplementary feeding of ellagic acid to suckling foals could significantly reduce the parasite infection and the output of Trichodon tenuis, Strongylus equi, Parascaris equi and Strongylus westermani in feces, and the supplementary feeding dose of 30 mg/kg BW was more effective.
Environmental Safety
Aerobic Composting Treatment of Pig Manure with Corn Straw and Its Safety Evaluation
LIU Xin, CHEN Qun, QIU Yulang, HOU Guoxi, LI Lin, YAN Xiaogang, LIU Dongmei, LYU Zhichao, GAO Xingai, LI Zhonghe, ZHANG Yunying, YAN Qiuliang
2022, 49(12):  4945-4952.  doi:10.16431/j.cnki.1671-7236.2022.12.042
Abstract ( 227 )   PDF (1647KB) ( 42 )  
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【Objective】 To explore the influence of aerobic composting with different proportions of pig manure and corn straw on the composting quality, and to evaluate the safety of composting products to provide reference for the resource utilization of pig manure and straw.【Method】 Using pig manure and corn straw as materials, three groups were set in the experiment, which were composted according to the volume ratio of pig manure and corn straw of 4:6 (group A), 6:4 (group B) and 8:2 (group C).The composting period lasts for 55 days.The temperature of the fermentation reactor and the ambient temperature were recorded daily, the reactor samples were collected on days 0, 5, 15, 25, 35, 45 and 55, and the water content, pH and seed germination index of the samples were measured, as well as the total nutrient content(total phosphorus, total potassium, total nitrogen), organic matter content, heavy metal content (total arsenic, total mercury, total lead, total cadmium, total chromium), fecal coliform group number and Ascaris egg mortality on days 0 and 55.【Result】 In groups A, B and C, the composting temperature increased first and then decreased with the increase of composting time, and the difference between groups was not significant (P>0.05);The water content decreased continuously, and the difference between groups was not significant (P>0.05);pH fluctuated between 7.5 and 9.1, and the change trend was basically the same, with no significant difference between groups (P>0.05).At the end of composting, the total nutrient contents of the samples in groups A, B and C were 6.94%, 7.36% and 6.95%, respectively, and the organic matter contents were 48%, 45% and 39%, respectively, which met the standard requirements of NY/T 525-2021.The seed germination indexes of the three groups were more than 80% at the end of composting, indicating that the decompositions were complete, in which the groups A and B exceeded 100% after 35 days.At the end of composting, the content of heavy metals, the number of fecal coliforms and the mortality of Ascaris eggs in groups A, B and C were all lower than the limits of the organic fertilizer standard.The number of fecal coliforms in groups A and B were <3/g, and the mortality of Ascaris eggs was 100%.【Conclusion】 The ratio of pig manure to corn straw by volume was 4:6, which was more conducive to composting maturity, could shorten the composting cycle, and met the standard requirements of harmless manure and organic fertilizer.