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05 July 2024, Volume 51 Issue 7
Biotechnology
Exploring Candidate Genes Related to Kidney-adjacent Lipid Deposition in Yak Calves Based on Transcriptome Sequencing
ZHAO Yutian, CHEN Fuju, YI Pingchang, MA Min, WANG Shouning, QUAN Qishiliu
2024, 51(7):  2727-2738.  doi:10.16431/j.cnki.1671-7236.2024.07.001
Abstract ( 49 )   PDF (7709KB) ( 21 )  
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【Objective】 This study was aimed to compare the changes in transcriptome expression profiles of kidney-adjacent adipose tissue in yak calves at different ages,and explore the signaling pathways and key genes that regulated kidney-adjacent lipid deposition in yak calves.【Method】 Three healthy male yak calves at 1,7 and 30 day-old were bled to death,and the kidney-adjacent adipose tissue were collected for transcriptome sequencing using Illumina HiSeq platform,after which the transcriptome data were subjected to quality control,comparison,differentially expressed gene (DEGs) screening,and the DEGs were performed to GO function and KEGG pathway enrichment analysis.In addition,seven genes were randomly selected for Real-time quantitative PCR verification.【Result】 Transcriptome results showed that a total of 1 627 significantly DEGs were identified in the comparison group of 1- and 7-day-old,of which 939 were up-regulated and 688 were down-regulated.Also,a total of 3 979 significantly DEGs were identified in the comparison group of 7- and 30-day-old,of which 1 925 were up-regulated and 2 054 were down-regulated.GO function and KEGG pathway enrichment analysis showed that the DEGs were enriched in acyl-CoA dehydrogenase activity to participate in fatty acid biosynthesis,insulin resistance,PPAR signaling pathway, sphingolipid signaling pathway, etc. in the comparison group of 1- and 7-day-old.In addition,the DEGs were enriched in several biological processes,including fatty acid metabolism,lipid metabolism,phosphohydrolase activity and lipase activity to participate in butyrate metabolism,fatty acid metabolism,glycerophospholipid metabolism,phospholipase D signaling pathway,AMPK signaling pathway,etc. in the comparison group of 7- and 30-day-old.The enriched pathways showed that SCD,ADIPOQ,PLIN1,ACADM and LPL were identified as candidate genes associated with kidney-adjacent lipid deposition in yak calves.Real-time quantitative PCR results showed that the expression of seven genes were consistent with RNA-Seq results.【Conclusion】 Five genes related to adipocyte differentiation and fat deposition were screened in this study,including SCD,ADIPOQ,PLIN1,ACADM and LPL.The results provided a theoretical reference for further research on kidney-adjacent lipid deposition in yak calves at different growth stages.
Screening of Differential lncRNAs and Construction of ceRNA Network in Ovary Tissue of Guangxi Ma Chicken
ZHANG Ke, ZHANG Min, LI Tianbao, ZHANG Jie, LU Shengsheng, JIANG Mingsheng
2024, 51(7):  2739-2750.  doi:10.16431/j.cnki.1671-7236.2024.07.002
Abstract ( 35 )   PDF (14890KB) ( 10 )  
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【Objective】 This study was aimed to screen differentially expressed long non-coding RNA (lncRNA),messenger RNA (mRNA) and microRNA (miRNA) of ovary in Guangxi Ma chicken,and further select lncRNA related to egg production traits,providing theoretical support for the study of lncRNA regulation of egg production traits in chickens.【Method】 Based on the egg production of Guangxi Ma chickens from start of production to 41 weeks of age,three high-yielding and three low yielding Guangxi Ma chickens were selected,and their ovarian tissues were used as materials.RNA sequencing technology was used to sequence them.Differentially expressed lncRNA,mRNA,and miRNA were screened from the sequencing data using edgER and DESeq2 software.the target genes of miRNA and lncRNA were predicted by miRanda software and cis-acting of lncRNA,respectively.GO function and KEGG pathway enrichment analysis were performed on the target genes of differentially expressed lncRNA,and a ceRNA network diagram was constructed using Cytoscape(v 3.8.2) software The transcriptome sequencing results were verified by Real-time quantitative PCR.【Result】 RNA sequencing technology was used to obtain 438 differentially expressed lncRNAs,18 differentially expressed miRNAs,and 301 differentially expressed mRNAs in the ovaries of Guangxi Ma chicken.GO function and KEGG pathway enrichment analysis of the target genes of the differentially expressed lncRNAs showed significant enrichment in bioprocesses related to hormone secretion regulation,mesoderm formation,mesoderm development,and mesoderm morphogenesis related to egg production,as well as signaling pathways such as MARK,biosynthesis of glycosaminoglycans,oocyte meiosis,progesterone-mediated oocyte maturation,and gonadotropin-releasing hormone.Through GO function and KEGG pathway enrichment analysis,six lncRNAs related to egg production traits were obtained,including TCONS_00091814,TCONS_00051866,XR_006939520.1,XR_005839807.1,TCONS_00088588 and TCONS_00037324.A ceRNA network map containing 9 miRNAs with 60 lncRNAs and 3 mRNAs was successfully constructed based on the sequencing results,and all the genes in the network were differentially expressed in the low and high yield groups.Real-time quantitative PCR results of the selected differentially expressed genes were consistent with the gene expression levels from transcriptome sequencing,indicating that the transcriptome sequencing results were reliable.【Conclusion】 A series of differentially expressed lncRNAs were screened from ovary in Guangxi Ma chicken using RNA sequencing technology,and six lncRNAs possibly associated with egg production traits were identified,providing theoretical support for further enhancing egg production performance in chickens.
Screening of Key Homologous Genes for Bovine Fertility Based on High Folic Acid Mouse Testicular Transcriptome
JI Hongjin, YE Qiannan, LI Wenlong, LI Zhipeng, FENG Xia, MI Siyuan, SHI Yuanjun, ZHANG Jinning, GUO Jian, YU Ying
2024, 51(7):  2751-2765.  doi:10.16431/j.cnki.1671-7236.2024.07.003
Abstract ( 30 )   PDF (9087KB) ( 5 )  
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【Objective】 Based on transcriptome data of folic acid mice testis,the homologous genes of folic acid regulating bovine and mouse fertility were screened,which provided a theoretical foundation for improving male animal reproductive capacity through folic acid.【Method】 Twenty four healthy and similarly growing 11 weeks old C57BL/6 male mice were randomly divided into three groups (n=8):CON,A,and B groups,fed with 2,7,and 15 mg/kg folic acid,respectively,for 57 consecutive days.After feeding,the weight of the left and right testes and seminal vesicles,sperm density,and the proportion of slow forward sperm were measured in three groups of mice.The transcriptome of mouse testes was sequenced using Illumina NovaseqTM 6000 high-throughput sequencing.The sequencing results were analyzed using differentially expressed genes (DEGs),GO functional annotation,and protein-protein interaction network (PPI) analysis to screen for key genes regulating semen quality and reproductive capacity by folic acid,and combined with homologous genes and quantitative trait genes related to reproductive capacity in Holstein bulls.Quantitative trait loci (QTLs) data were used to further screen for homologous genes that regulated male reproductive capacity in cattle and mice through folate regulation.【Result】 Compared with CON group,the weight of the left and right seminal vesicles of mice in B group was extremely significantly increased (P<0.01),and the unfavorable trait of the proportion of sperm slow foward in the semen of mice in both A and B groups were extremely significantly reduced (P<0.01).Through RNA-Seq analysis,it was found that 423 DEGs were screened in A vs CON group,and 661 DEGs were screened in B vs CON group.A total of 85 upregulated DEGs and 40 downregulated DEGs were found in both A vs CON and B vs CON groups.Through GO functional annotation analysis,it was found that the DEGs in A vs CON group were significantly enriched in GO entries related to folate metabolism and semen quality regulation, while those in B vs CON group were significantly enriched in GO entries related to folate metabolism and reproductive organ development. The upregulated DEGs shared by both A vs CON and B vs CON groups were significantly enriched in folate metabolism and semen quality regulation,while the downregulated DEGs shared were significantly enriched in one-carbon metabolism and single sperm fertilization functions.Through PPI network analysis of DEGs,it was found that the selected hub genes might affect male animal fertility by affecting semen quality.At the same time,the hub genes screened in this study were all homologous genes of Holstein bulls,and homologous genes and their neighboring QTLs that affected the reproductive capacity of Holstein bulls at different concentrations of folic acid were identified,including inseminations per conception QTL and sperm motility QTL.Further comparing the hub gene with differentially expressed genes in the transcriptome of previous Holstein bull sperm,the homologous gene Serpinb1b (bovine homologous gene SERPINB1) was screened out,which regulated fertility in Holstein bulls and mice through folate regulation.【Conclusion】 Feeding 7 and 15 mg/kg folic acid could reduce the proportion of slow forward sperm in semen.The Serpinb1b gene (bovine homologous gene: SERPINB1) screened by RNA-Seq was one of the key genes for folic acid regulation of male animal fertility,which could provide reference information for the breeding and breeding of large dairy animal such as Holstein cows in the actual production of animal husbandry.
Physiological and Biochemical
Effects of Chlorogenic Acid on Cholesterol and Bile Acid Metabolism in Calf Hepatocytes Treated with High FFA
TIAN Yan, LIU Jiachen, WANG Shuang, FAN Wenwen, CHANG Yaqi, YANG Tianjiao, ZHANG Zexin, ZHANG Bingbing, XU Chuang, YANG Wei
2024, 51(7):  2766-2775.  doi:10.16431/j.cnki.1671-7236.2024.07.004
Abstract ( 21 )   PDF (2344KB) ( 17 )  
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【Objective】 The objective of this study was to investigate the effects of chlorogenic acid (CGA) on cholesterol and bile acid metabolism in calf hepatocytes treated with high free fatty acid (FFA).【Method】 The primary calve hepatocytes were separated by two-step collagenase perfusion method,and the cells were identified by immunofluorescence and divided into 4 groups.Cells in control group were cultured with RPMI-1640 medium containing 2% BSA.The cells in FFA group were cultured after adding 1.2 mmol/L FFA in RPMI-1640 medium containing 2% BSA,and CGA group were cultured after adding 20 μg/mL CGA in RPMI-1640 medium containing 2% BSA.Cells in CGA+FFA group were cultured after adding 1.2 mmol/L FFA and 20 μg/mL CGA in RPMI-1640 medium containing 2% BSA.Cells were collected after 12 h,and the contents of triglyceride (TAG) and total cholesterol (TC) were detected by the kit.Real-time quantitative PCR and Western blotting were used to detect the mRNA and proteins expression levels of cholesterol synthesis-related factors,including sterol regulatory element binding transcription factor 2 (SREBF2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR),and cholesterol effection-related factors,including acetyl-CoA acetyltransferase 2(ACAT2),ATP-binding box subfamily A member 1(ABCA1),ATP-binding box subfamily G member 5(ABCG5),and bile acid metabolism related factors,including cholesterol 7α-hydroxylase (CYP7A1),cholesterol 12α-hydroxylase (CYP8B1),cholesterol of 27 α-hydroxylase (CYP27A1),farnesol X receptor (FXR) and fibroblast growth factor receptor 4(FGFR4).【Result】 After different treatments of calf hepatocytes,compared with control group,the TC content of calf hepatocytes in FFA group was extremely significantly reduced (P<0.01),and the TAG content was extremely significantly increased (P<0.01),and the mRNA expression levels of HMGCR,ABCA1,ABCG5,ABCG8,APOA1,ACAT1,NPC1L1,FXR and FGFR4 genes and the protein expression levels of SREBF2,ACAT2,ABCA1 and ABCG5 were extremely significant or significantly reduced (P<0.01 or P<0.05),the mRNA expression of CYP8B1 gene and the protein expression of CYP7A1 and CYP8B1 were extremely significant or significantly increased (P<0.01 or P<0.05).The TC content of calf hepatocytes in CGA group was extremely significantly increased (P<0.01),the protein expression levels of SREBF2,ABCA1,CYP27A1,FXR and FGFR4 were extremely significantly increased (P<0.01),the protein expression of CYP7A1 was extremely significantly reduced (P<0.01).Compared with FFA group,the TC content of calf hepatocytes in CGA+FFA group was significantly increased (P<0.05),and the TAG content was significantly reduced (P<0.05),the mRNA expression levels of HMGCR,ACAT2,ABCA1,ABCG5 and APOA1 genes and the protein expression levels of SREBF2,ACAT2,ABCA1,ABCG5,FXR and FGFR4 were extremely significant or significantly increased (P<0.01 or P<0.05),the protein expression levels of CYP7A1 and CYP8B1 were extremely significantly reduced (P<0.01).【Conclusion】 CGA could participate in the regulation of intracellular cholesterol homeostasis in calf hepatocytes while activating FXR and FGFR4,which in turn alleviates high FFA-treated bile acid accumulation.
Study on the Optimization of Medium Composition to Increase the Viable Counts of Streptococcus suis Type 2 NT15B Strain
MA Linli, XU Guoqiang, ZHANG Qiuping, CHE Qiaolin, SHI Jinsong, XU Zhenghong
2024, 51(7):  2776-2786.  doi:10.16431/j.cnki.1671-7236.2024.07.005
Abstract ( 27 )   PDF (1426KB) ( 6 )  
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【Objective】 The experiment took Streptococcus suis type 2 NT15B strain as the research object.By optimizing the fermentation medium components,the viable counts of the strain was increased and the production cost was reduced,so as to provide a reference for high-density culture of Streptococcus suis type 2 NT15B strain and the research related to the large-scale production and promotion of the vaccine against porcine streptococcal disease.【Method】 The maximum viable counts of Streptococcus suis type 2 NT15B strain was used as the screening index,and the optimal components and concentrations of carbon sources,nitrogen sources (organic and inorganic nitrogen sources),inorganic salt metal ions,and phosphoric acid buffers in the fermentation medium of the strain were screened by single-factor test.According to the results of single factor screening,3 levels of each of 4 factors,carbon source concentration,total organic nitrogen source concentration,organic nitrogen source proportion and phosphate buffer concentration,were selected to design orthogonal tests,and further multi-factor screening and verification were carried out.【Result】 The optimal fermentation medium of Streptococcus suis type 2 NT15B strain was 6.0 g/L sucrose,1.0 g/L urea,10 g/L yeast extract YE7 mixed with 20 g/L yeast peptone Nucel 887 as nitrogen sources,1.0 g/L NaCl,9.58 g/L K2HPO4·3H2O,1.09 g/L KH2PO4,1 mmol /L MgSO4·7H2O and 0.3 g/L L-ascorbic acid.Using this optimal combination,the viable counts of Streptococcus suis type 2 NT15B strain at the shaking flask level was 80.83×108 CFU/mL,which was 256.08% higher than before optimization.【Conclusion】 The optimized medium effectively increased the viable counts of Streptococcus suis type 2 NT15B strain,reduced production costs and provided a reference for optimization in fermenters.
Nutritionand Feed
Effects of Dietary Crude Fiber Levels on Growth Performance, Carcass Traits and Meat Quality in Pigs
YUN Hongmei, ZHAO Xinyu, ZHAO Xiaolei, ZHAO Yan, YANG Yang, NIU Jin, GUO Xiaohong, LI Bugao, CAO Guoqing
2024, 51(7):  2787-2798.  doi:10.16431/j.cnki.1671-7236.2024.07.006
Abstract ( 32 )   PDF (3328KB) ( 20 )  
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【Objective】 The purpose of this study was to investigate the effects of dietary crude fiber levels on growth performance,carcass traits and meat quality in Mashen (MS) pigs and Duroc×Landrace×Yorksire (DLY) pigs,so as to provide the theoretical basis for the construction of the precise nutrition requirements and production of high-quality meat in pigs.【Method】 Eighty healthy MS pigs and eighty DLY pigs with an initial body weight of (49.67±5.85) kg were selected and randomly divided into four treatment groups with five replicates per treatment and four pigs per replicate within breed.Four diets with crude fiber level of 2%,5%,8% and 11% were fed to pigs in the corresponding treatment group,which named 2% CF,5% CF,8% CF and 11% CF groups,respectively.The pre-test period was 7 days and the formal test period was 30 days.At the end of the experiment,three MS pigs and three DLY pigs with similar body weight of the treatment average weight were selected from each group and slaughtered,carcass traits and meat quality were measured.【Result】 The dietary crude fiber levels had significant effects on growth performance of pigs.The average daily gain of MS pigs in 11% CF group was significantly lower than those in other three groups (P<0.05),and the feed-to-gain of DLY pigs in 11% CF group was significantly higher than those in other three groups (P<0.05).The dietary crude fiber levels had significant effects on carcass traits,such as the backfat thickness,eye muscle area and hind leg ratio.The backfat thickness of MS pigs in 2% CF group was significantly higher than those in other three groups (P<0.05),and the backfat thickness of DLY pigs in 11% CF group was significantly lower than those in other three groups (P<0.05).The eye muscle area was gradually increased with the increase of the dietary crude fiber levels both in MS and DLY pigs,which in 11% CF group was extremely significantly higher than those in other three groups (P<0.01),and which in 8% CF group was significantly higher than those in 2% and 5% CF groups (P<0.05).The hind leg ratio of MS pigs in 5% and 8% CF groups was significantly higher than those in other two groups (P<0.05).The meat quality was also affected by the dietary crude fiber levels.The intramuscular fat (IMF) content of MS pigs in 2% and 5% CF groups was extremely significantly higher than those in 8% and 11% CF groups (P<0.01). The cysteine content in 5% CF group of MS pigs were significantly higher than those in other three groups (P<0.05),and the methionine content in 2% CF group were significantly lower than those in other three groups (P<0.05).The contents of palmitic acid,stearic acid,oleic acid and linoleic acid of MS pigs in 8% CF group were the highest,and the change trend in DLY pigs was in opposition.【Conclusion】 When feeding MS pigs with a diet with 8% CF,the feed conversion rate and eye muscle area increased,the backfat thickness decreased,and the meat color improved.When feeding DLY pig with a diet containing 5% CF,the daily weight gain,feed conversion rate and eye muscle area increased,and the body structure and muscle tenderness were better.
Effects of Alfalfa Saponin Extract on Immunity,Antioxidant Capacity and Rumen Microflora of Weaned Hu Sheep
ZHANG Yan, MA Sen, LI Shouren, HUANGFU Weikang, LA Shaokai, MA Jixiang, WANG Zhichang, SHI Yinghua
2024, 51(7):  2799-2809.  doi:10.16431/j.cnki.1671-7236.2024.07.007
Abstract ( 31 )   PDF (7809KB) ( 10 )  
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【Objective】 As a natural bioactive substance,alfalfa saponins have many effects,such as improving immunity,promoting growth,and regulating the gastrointestinal function of livestock and poultry.The experiment was aimed to investigate the effects of alfalfa saponin extract on the growth performance,immune function,antioxidant capacity and rumen microflora of weaned Hu sheep.【Method】 Thirty-two 45-day-old weaned Hu sheep with similar body weight (15.67 kg±1.18 kg) were randomly divided into 2 groups with 4 replicates per group and 4 sheep per replicate.Hu sheep in control group (CON) were fed a conventional diet,and Hu sheep in saponins group (AS) were fed a basal diet supplemented with 2 g/kg (dry matter basis of the diet) of alfalfa saponin extract.The pre-trial period was 7 days,and the trial period was 60 days.At the end of the experiment,the rumen fluid of weaned Hu sheep was collected to determine the microbial composition,and the jugular vein blood was collected to determine the serum immune,and antioxidant performance-related indicators.【Result】 ①There was no significant difference in average daily gain (ADG) and feed/gain (F/G) between CON and AS groups of Hu sheep (P>0.05).②Compared with CON group,the levels of immunoglobulin (IgA and IgM),anti-inflammatory factor interleukin-10 (IL-10),CD4+T cells,and CD4/CD8 in serum of Hu sheep in AS group were extremely significantly or significantly increased (P<0.01 or P<0.05),while the levels of pro-inflammatory factors tumor necrosis factor-α (TNF-α) and IL-6 were extremely significantly decreased (P<0.01).③Compared with CON group,the total antioxidant capacity (T-AOC) in serum of Hu sheep in AS group was significantly increased (P<0.05),while the content of malondialdehyde (MDA) was extremely significantly decreased (P<0.01).④There were no significant differences in rumen fluid pH,ammonia nitrogen (NH3-N),volatile fatty acids,and total volatile fatty acids (TVFA) concentrations between CON and AS groups (P>0.05).⑤ There was no significant difference in Shannon index,Simpson index,and principal coordinate analysis (PCoA) between CON and AS groups.At the genus level,the relative abundance of sulfate-reducing bacteria Desulfovibrio,beneficial bacteria Roseburia, Eubacterium_ventriosum_group,etc. in rumen fluid of Hu sheep in AS group was significantly higher than that in CON group (P<0.05).【Conclusion】 Adding alfalfa saponin extract to the diet could affect the composition of rumen microorganisms in weaned Hu sheep,improve the body’s immunity and antioxidant capacity,and was beneficial to the health of the body to a certain extent.
Effects of Active Dry Yeast on Growth Performance,Slaughter Performance and Serum Biochemical Indexes of Jinlan Cashmere Goats
LIU Yin, WANG Zhiwu, ZHAO Peng, ZHANG Li, GUO Hongyu, WANG Haojun
2024, 51(7):  2810-2822.  doi:10.16431/j.cnki.1671-7236.2024.07.008
Abstract ( 21 )   PDF (1210KB) ( 8 )  
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【Objective】 This study was aimed to explore the effects of active dry yeast on growth performance,slaughtering performance and serum biochemical,immune and antioxidant indexes of Jinlan cashmere goats,and select the appropriate amount of active dry yeast.【Method】 A total of 100 3-month-old Jinlan cashmere male lambs were randomly divided into 5 groups,with 20 lambs in each group and 4 lambs in each replicate.Goats in experimental groups ADY1,ADY2,ADY3 and ADY4 were fed with 1,2,3 and 4 g/d active dry yeast on the basis of the basal diet,respectively,while goats in control group (ADY0) was not added active dry yeast.The growth performance and serum biochemical,immune and antioxidant indexes were measured every 30 days,and the slaughter performance was measured on days 90 after the initial experiment for 15 days and the formal experiment for 90 days.【Result】 Compared with control group,① The final body weight of goats in ADY3 and ADY4 groups was significantly increased (P<0.05),and the feed to gain was significantly decreased (P<0.05).On days 90,the chest circumference and body length of goats in ADY3 group were significantly increased (P<0.05),and the chest circumference of goats in ADY4 group was significantly increased (P<0.05).② The live weight before slaughter,carcass weight and eye muscle area of goats in ADY3 and ADY4 groups were significantly increased (P<0.05),while the carcass fat content and backfat thickness were significantly decreased (P<0.05).The net meat weight and net meat percentage of goats in ADY2,ADY3 and ADY4 groups were significantly increased (P<0.05),and the heart weight of goats in ADY3 and ADY4 groups was significantly increased (P<0.05).③ On days 30,60 and 90,the serum triglyceride (TG) content of goats in 4 experimental groups was significantly increased (P<0.05),and the total cholesterol (TC) content of goats in ADY3 group was significantly decreased (P<0.05).On days 90,the contents of total protein (TP) and globulin (GLB) of goats in ADY2 and ADY3 groups were significantly increased (P<0.05).④ On days 60 and 90,the contents of immunoglobulin A (IgA) and tumor necrosis factor-α (TNF-α) in ADY3 group were significantly increased (P<0.05).⑤ On days 60,the total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity of goats in ADY3 group were significantly increased (P<0.05).On days 60 and 90,the malondialdehyde (MDA) content of goats in ADY3 group was significantly decreased (P<0.05).⑥ On days 90,the serum insulin growth factor-1 (IGF-1) content of goats in ADY2 and ADY3 groups was significantly increased (P<0.05).【Conclusion】 Adding an appropriate amount of active dry yeast to the feed could improve the growth performance and slaughtering performance of Jinlan cashmere goats,enhance immunity and antioxidant capacity.Considering the comprehensive indicators,the appropriate adding amount was 2-3 g/d.
Expression and Bioactivity of Porcine Epidermal Growth Factor in Lactococcus lactis
ZHOU Xinxin, GUO Yingying, XIAO Yuncai, WANG Jinshuo, ZHOU Zutao, LIU Huazhen, SHI Deshi
2024, 51(7):  2823-2836.  doi:10.16431/j.cnki.1671-7236.2024.07.009
Abstract ( 25 )   PDF (12398KB) ( 9 )  
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【Objective】 The objective of this experiment was to construct the prokaryotic expression system of Lactococcus lactis expressing porcine epidermal growth factor (pEGF) and to explore its effect on diarrhea of weaned piglets. 【Method】 Using pUC57-SP45-LEISSTCDA-pEGF sequence as the template,the fusion product of the missing LEISSTCDA fragment was obtained by PCR amplification,which was connected with the expression vector pNZ8149 and transferred to Lactococcus lactis NZ3900 by electric transmission,and the recombinant pEGF-NZ was obtained by using LacF as the screening marker.Recombinant strain PEGF-NZ was induced by nisin to express recombinant protein pEGF,and its expression was detected by Tricine-SDS-PAGE.The pEGF expression was increased by improving the laboratory and industrial fermentation conditions.The bioactivity of pEGF expressed in porcine ileal epithelial cells (IPEC-J2) was verified in vitro by proliferation assay.The effects of recombinant protein pEGF on diarrhea in mice and weaned piglets were verified by animal experiments.【Result】 The recombinant strain PEGF-NZ was successfully constructed and secreted to express pEGF protein. In vitro experiment results showed that pEGF protein could promote cell proliferation.pEGF-N recombinant bacteria and its expression of pEGF could delay the occurrence time of diarrhea in mice,reduce the diarrhea rate,and protect the integrity of intestinal villi in mice.Compared with control group,after treatment with recombinant pEGF-NZ,the number of lactic acid bacteria of mice was significantly increased (P<0.05),the number of Escherichia coli and Enterococcus were significantly decreased (P<0.05),and the contents of IL-1α and IL-1β were significantly decreased (P<0.05).The content of IL-13 were significantly increased (P<0.05).After supplemented with pEGF-NZ,the diarrhea of weaned piglets was significantly relieved,and the average daily gain tended to increase,but the difference was not significant (P>0.05).【Conclusion】 Recombinant PEGF-NZ and its expression pEGF could repair intestinal barrier,inhibit the adhesion of pathogenic bacteria,regulate intestinal flora,and exert anti-inflammatory effects mainly by down-regulating the expression of some pro-inflammatory factors and up-regulating anti-inflammatory factors,so as to protect intestinal health of weaned piglets and effectively relieve diarrhea.
Effect of Manganese Level in Breeding Pigeon Diets on Egg Quality of Breeding Pigeons and Slaughtering Performance,Meat Quality and Antioxidant Properties of Squabs
LI Haoxuan, WANG Zheng, QIN Shizhen, ZHANG Bo, LI Jing, SUO Yanrui, ZHAO Dongdong, WANG Yangyang, LIU Wenli, SHEN Li, SUN Hong, SHI Zhaoguo, SHAO Yuxin
2024, 51(7):  2837-2848.  doi:10.16431/j.cnki.1671-7236.2024.07.010
Abstract ( 28 )   PDF (1386KB) ( 4 )  
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【Objective】 In this experiment, the effects of different manganese levels in pigeon diets on egg quality and squab weight, slaughter performance, meat quality and serum antioxidant indexes were investigated through the addition of exogenous manganese preparation (MnSO4·H2O) to provide experimental references for pigeon farming in China. 【Method】 One hundred and twenty pairs of healthy, adult Silver King pigeons of similar body weight and laying performance were selected and randomly divided into five groups, with eight replicates per group, each replicate containing three pairs of pigeons, and each pair of pigeons rearing two squabs. These five groups used a basal diet without additional manganese (control group) and experimental diets with 35, 70, 105 and 140 mg/kg manganese added to the basal diet, respectively. The ration was fed to the pigeons in the form of pellets. The test period consisted of an incubation period of 18 days and a lactation period of 28 days, for a total of 46 days. During the experiment, the feed intake of adult pigeons and the weight of squabs were recorded, and the average daily feed intake of adult pigeons and the average daily gain of squabs were calculated. The pigeon eggs, serum from breeding pigeons and 28-day-old squabs, breast muscle, heart and liver samples from squabs were collected for the measurement of the quality of pigeon eggs, serum antioxidant index, the meat quality and manganese content in the breast muscle of squabs, and the slaughtering performance of 28-day-old squabs was calculated. 【Result】 ① The eggshell weight of pigeon eggs in 70 and 140 mg/kg manganese groups was significantly higher than that in 35 and 105 mg/kg manganese groups (P<0.05). The yolk color value of pigeon eggs in 70 and 105 mg/kg manganese groups was significantly higher than that in control and 35 mg/kg manganese groups (P<0.05). The HU value of pigeon eggs in 35 mg/kg manganese group was significantly lower than that in other groups (P<0.05). The eggshell reflectance in 70, 105 and 140 mg/kg manganese groups were significantly lower than that in control group (P<0.05). ② The average daily gain of squabs aged 1 to 28 days in 35 and 140 mg/kg manganese groups was significantly lower than that in control group (P<0.05). ③ The slaughter rate of 28-day-old squabs in 35 and 140 mg/kg manganese groups was significantly higher than that in control group (P<0.05). The evisceration rate of 28-day-old squabs in 35, 105 and 140 mg/kg manganese groups was significantly higher than that in control group (P<0.05). The semi-evisceration rate of 28-day-old squabs in 35 mg/kg manganese group was significantly higher than that in other groups (P<0.05). ④ There were no significant differences in breast muscle meat quality and manganese content in breast muscle, myocardium and liver of 28-day-old squabs among all groups (P>0.05). ⑤ The serum catalase activity of 28-day-old squabs in 35 and 140 mg/kg manganese groups were significantly lower than that in other groups (P<0.05). 【Conclusion】 The addition of 35 mg/kg manganese in the basal diet of breeding pigeons could improve the slaughter rate, full evisceration rate and half evisceration rate of 28-day-old squabs. The addition of an additional 70 mg/kg manganese could improve the egg shell quality and deepen the yolk color.
Effects of Different Compound Additives on Quality and Microbial Diversity of Mixed Silage of Forage Mulberry and King Grass
CHEN Rongqiang, LEI Xiaowen, WU Lijuan, OU Xiang, LIAN Hai, ZHANG Qiang, QIU Jingyun, CAO Xianhong, YAN Lei, CAI Xiaofen
2024, 51(7):  2849-2859.  doi:10.16431/j.cnki.1671-7236.2024.07.011
Abstract ( 29 )   PDF (1166KB) ( 6 )  
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【Objective】 This experiment was conducted to study the effects of different combinations of additives on the quality and microbial diversity of mixed silage of forage mulberry (Morus alba L.) and king grass (Pennisetum purpureum×Pennisetum americanum cv.Reyan No.4).【Method】 A randomized block design was used to set up four treatment groups:control group (CK),compound bacteria preparation group (J,Lactobacillus plantarumBacillus subtilis),compound enzyme preparation group (M,cellulase+xylanase) and compound bacteria enzyme preparation group (JM,Lactobacillus plantarumBacillus subtilis+cellulase+xylanase),with 6 replicates in each group.After 60 days of silage,the nutrient content,fermentation quality and microbial diversity of silage were analyzed,and the fermentation effect was evaluated by V-score scoring system and fuzzy mathematics membership function method.【Result】 Compared with CK group,the silage quality of the other groups of mixed silage was improved to varying degrees,the contents of crude protein and soluble carbohydrate in J,M and JM groups were significantly increased (P<0.05),and the contents of neutral detergent fiber,lignin,ammonia nitrogen and pH were significantly decreased (P<0.05),the content of acid detergent fiber in group M was significantly decreased (P<0.05),and the content of lactic acid and acetic acid in group JM was significantly increased (P<0.05).The propionic acid and butyric acid was not detected in each group.The V-score of each group was above 85 points,and the JM group had the highest score of 88.93 points.The order of the mean value of membership function in each group was JM>J>M>CK.Compared with the CK group,the Shannon index,Simpson index,Chao1 index and Ace index of silage microorganisms in other groups decreased significantly (P<0.05).At the phylum level,the dominant microbial phylum in each group of mixed silage was Firmicutes and Proteobacteria.Compared with CK group,the relative abundance of Firmicutes in J and JM groups was significantly increased,and the relative abundance of Proteobacteria was significantly decreased (P<0.05).At the genus level,compared with CK group,the relative abundance of Lactiplantibacillus in J,M and JM groups was significantly increased,and the relative abundance of unclassified_Enterobacteriaceae was significantly decreased (P<0.05).Additionally,compared with CK group,the relative abundance of Bacillus in J and JM groups was significantly increased (P<0.05),and the relative abundance of Enterobacter in JM group was significantly decreased (P<0.05).【Conclusion】 In conclusion,the addition of compound bacteria preparation,compound bacteria enzyme preparation or compound bacteria enzyme preparation could improve the nutrient content and fermentation quality of mixed silage of forage mulberry and king grass to varying degrees,and could increase the abundance of beneficial bacteria and reduce the abundance of harmful bacteria.Comprehensive consideration,adding compound bacteria enzyme preparation was more suitable for mixed silage of forage mulberry and king grass.
Effect of Mannitol on Rumen and Fecal Microbiome Structure,Serum Antioxidant and Immune Indices of Grazing Sheep
XIAO Min, Alder, GONG Jian
2024, 51(7):  2860-2870.  doi:10.16431/j.cnki.1671-7236.2024.07.012
Abstract ( 35 )   PDF (8712KB) ( 15 )  
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【Objective】 This experiment was aimed to study the effects of mannitol on the gastrointestinal microflora and serum antioxidant and immune indexes of grazing sheep.【Method】 Ten healthy 7-8 months old sheep with similar body weight (27.10 kg±3.55 kg) were selected and randomly divided into two groups by single factor complete randomized trial design,with five sheep in each group.Sheep in control group was supplemented with 100 g/d of corn powder without mannitol,and in experimantal group was supplemented with 100 g/d of corn powder containing 1 g mannitol.A 35 day feeding experiment was conducted under natural grazing conditions,with 5 d for adaptation period and 30 d for experimental period.Weights were weighed before grazing on the start and end days of the positive trial period and daily average weight gain (ADG) was calculated.On the last experimental day,rumen fluid,feces samples,and jugular vein blood were collected,respectively.The 16S rRNA sequencing method was used to analyze the changes in rumen and fecal microflora of sheep.Serum antioxidant and immune indexes were determined using relevant kits.【Result】 ①There was no significant difference in ADG,pH and NH3-N content of rumen fluid between the control and experimental groups (P>0.05),while the concentration of acetic acid,propionic acid and total SCFAs in rumen fluid in experimental group were significantly higher than those in control group (P<0.05).②There were no significant difference in the number of OTUs and the Alpha diversity index of rumen and faeces microorganisms in the control and experimental groups (P>0.05).However,PCoA based on abund_jaccard proximity distance for both rumen and facecal microorganisms were significantly separated in experimental group from control group (P<0.05).③At the phylum level,there was no significant difference between control and experimental groups in terms of rumen microbes (P>0.05),the abundance of Firmicutes was significantly lower,and the abundance of Bacteroidetes was significantly higher in the faeces of experimental group than that of control group (P<0.05).At the genus level,the relative abundance of Ruminococcaceae_NK4A214 in the rumen of experimental group was significantly higher than that of control group (P<0.05),the relative abundance of Unclassified_f_Achnospiraceae in the faeces of experimental group was significantly lower than that of the control group (P<0.05).④There was no significant difference in the serum immune index between control and experimental groups (P>0.05).The serum contents of glutathione (GSH) and malondialdehyde (MDA) were significantly lower in the experimental group than in control group (P<0.05).【Conclusion】 Supplementation with mannitol to grazing sheep diets could increase the rumen fluid concentration of acetic acid,propionic acid and total SCFAs,as well as the relative abundance of the Ruminococcaceae_NK4A214 in the rumen;Reduce the relative abundance of Firmicutes phyla and Unclassified_f_achnospiraceae,and increase the relative abundance of Bacteroidetes phyla in faeces;Improve the body’s antioxidant capacity.
Effect of Dietary Addition of Soy Isoflavones on Production and Reproductive Performance of Angus Beef Cattle
DING Hui, LIU Xin, WANG Ruiling, MAO Yongxia, WANG Jiandong, GUO Yansheng
2024, 51(7):  2871-2879.  doi:10.16431/j.cnki.1671-7236.2024.07.013
Abstract ( 29 )   PDF (1127KB) ( 10 )  
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【Objective】 The aim of this experiment was to study the effects of adding different levels of soy isoflavones to feed rations on the growth performance,serum biochemical indexes,antioxidant capacity,immune performance and reproductive hormones of Angus beef cattle,and explore the appropriate amount of soy isoflavones to be used in the practice of beef cattle production.【Method】 Forty healthy Angus heifers with similar body weight and 2-3 lactations were randomly divided into 4 groups of 10 heifers each.The cattle in control group was fed a basal diet,while cattle in groups Ⅰ,Ⅱ and Ⅲ were given 10,20 and 40 mg/kg of soy isoflavones in diet,respectively.The cattle were weighed at the beginning and at the end of the experiment,and the feed intake was recorded daily to calculate the average daily feed intake (ADFI),average daily gain (ADG) and feed-to-gain ratio (F/G).At 40th days,blood samples were collected for the detection of serum biochemistry,antioxidant,immune indexes and reproductive hormone level.【Result】 Compared with control group,①The addition of soy isoflavones in each experiment group had no significant effect on ADFI,ADG and F/G in Angus beef cattle (P>0.05).② The total protein content of bovine serum in groups Ⅰ and Ⅱ was significantly increased (P<0.05),and the glutamine aminotransferase activity was significantly decreased (P<0.05).The contents of total protein and globulin of bovine serum in group Ⅲ were extremely significantly increased (P<0.01),and the activities of glutamic oxaloacetic transaminase and glutamic alanine aminotransferase were extremely significantly decreased (P<0.01).The levels of immunoglobulin (IgA,IgM and IgG) and interferon-γ of bovine serum in group Ⅲ were extremely significantly or significantly increased (P<0.01 or P<0.05).③The total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) activity of bovine serum in group Ⅱ were extremely significantly increased (P<0.01).The T-AOC,GSH-Px and superoxide dismutase levels of bovine serum in group Ⅲ were extremely significantly increased (P<0.01),and the malondialdehyde content was extremely significantly decreased (P<0.01).④ The estradiol level of bovine serum in group Ⅱ was significantly increased (P<0.05).The levels of progesterone,estradiol,follicle stimulating hormone and luteinizing hormone of bovine serum in group Ⅲ were extremely significantly increased (P<0.01).【Conclusion】 Addition of soy isoflavones to feed was found to be effective in improving the immune function and reproductive performance of Angus beef cattle,and the best results were obtained at a level of 40 mg/kg.
Research Progress on Antioxidant Mechanism of Flavonoids Regulating the Keap1-Nrf2/ARE Signaling Pathway and Its Application in Livestock and Poultry
E Xiaodi, ZHAO Xiaonan, ZHAO Jinbiao, HU Youjun
2024, 51(7):  2880-2889.  doi:10.16431/j.cnki.1671-7236.2024.07.014
Abstract ( 32 )   PDF (4295KB) ( 14 )  
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Flavonoids are polyphenolic compounds that are widely found in natural plants and have strong antioxidant capabilities.In recent years,more and more studies have proved that flavonoids are beneficial to livestock production as feed additives.Kelch-like ECH-associating protein 1-nuclear factor erythroid 2 related factor 2/antioxidant response element (Keap1-Nrf2/ARE) is a key signaling pathway that enhances the body’s antioxidant capacity and is involved in resisting damage from external oxidative stress.Flavonoids mainly regulate the Keap1-Nrf2/ARE pathway,activate the expression of upstream and downstream key factors,activate the activity of antioxidant enzymes,improve the body’s antioxidant capacity,and thus achieve the improvement of animal growth performance,reproductive performance,and immune system.In this article the molecular structure of Keap1-Nrf2/ARE and its antioxidant mechanism,as well as the regulation of Keap1-Nrf2/ARE signaling pathway by flavonoids and their application in animal production were reviewed.The aim of this paper is to provide reference for the further development and application of flavonoids as feed additives.
Effects of Substituting Soybean Meal with Fermented Cottonseed Meal Equivalently on Growth Performance,Meat Quality and Antioxidation Activity of Yellow-Feathered Broilers
LIN Xiajing, GOU Zhongyong, WANG Yibing, FAN Qiuli, YE Jinling, ZHANG Sai, JIANG Shouqun, RUAN Dong
2024, 51(7):  2890-2898.  doi:10.16431/j.cnki.1671-7236.2024.07.015
Abstract ( 32 )   PDF (5435KB) ( 7 )  
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【Objective】 This experiment was conducted to study the effects of dietary fermented cottonseed meal level on growth performance, meat quality and antioxidation activity of Yellow-feathered broilers aged 1 to 63 days.【Method】 A total of 960 1-day-old Lingnan Yellow-feathered broilers were randomly divided into 4 treatment groups with 6 replicates per treatment and 40 chickens per replicate.The supplemental levels of fermented cottonseed meal in starter (1-21 d) diets was 0,5%,7.5% and 10%,in grower diets (22-42 d) was 0,6%,9% and 12%,and in finisher diets (43-63 d) was 0,10.5%,14.0% and 17.5%,respectively.The experiment lasted for 63 days.During the experiment,ADFI was recorded every day, and body weigh was recored at 1,21,42 and 63 days of age, and growth performance was calculated in units of replicate.Two chickens were selected from each replicate for blood collection and sample collection,and immune organ index,plasma and liver antioxidant indexes and meat quality were determined.【Result】 Compared with the control group,the F/G in high-supplemental group was significantly decreased from 1 to 21 days of age (P<0.05). At 43 to 63 days of age,the ADFI of low-supplemental group was significantly decreased(P<0.05),but the F/G had no significant difference (P>0.05).The ADFI and ADG were significantly decreased in low-supplemental groups during the whole experiment period (1 to 63 days of age) (P<0.05),but the F/G had no significant difference (P>0.05).There were no significant difference on thymus index,spleen index,bursa index,shear force of chest muscle,water drop loss,pH value (45 min and 96 h) and meat color (45 min and 96 h) of 63-day-old Yellow feathered broilers among 4 groups (P>0.05),and no obvious changes were found in liver section analysis.Plasma MDA content in medium-supplemental group was significantly higher than that in other experimental groups (P<0.05).The liver T-AOC in low and medium supplemental groups were significantly higher than that in control group (P<0.05).【Conclusion】 In conclusion,dietary supplementation of 10%,12% and 17.5% fermented cottonseed meal (free gossymol content was 12.21 mg/kg) to replace the same amount of soybean meal in Yellow-feathered broilers had no negative effects on the growth performance and meat quality,and the lowest F/G could be obtained by supplemented with 7.5%,9.0% and 14.0% fermented cottonseed meal in starter,grower and finisher stage,respectively.
Effects of Different Levels of Dietary Ellagic Acid Supplementation on Growth Performance,Blood Biochemical Indices and Antioxidant Capacity of Kazakh Sheep
REN Feier, YANG Fan, LI Minghao, LIANG Yingtao, REN Jie, ABDURAHMU Ehmeti, CHEN Kaixu
2024, 51(7):  2899-2907.  doi:10.16431/j.cnki.1671-7236.2024.07.016
Abstract ( 26 )   PDF (1127KB) ( 10 )  
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【Objective】 The purpose of the experiment was to explore the effects of different levels of ellagic acid on growth performance,blood biochemical indexes and antioxidant capacity of Kazakh sheep,and to provide reference basis for the application of ellagic acid in ruminants.【Method】 Twenty 3-month-old Kazakh rams with a body weight of (25.58±2.85) kg were randomly divided into 4 groups with 5 sheep in each group.The ellagic acid supplementation levels in control,15 EA,30 EA and 45 EA groups were 0,15,30 and 45 mg/kg BW, respectively.The 60-day feeding experiment was carried out after 5 days of pre-feeding.The fasting weight was weighed every 30 days.Blood samples were collected before feeding on the morning of the 60th days.Growth hormone,insulin-like growth factor-Ⅰ,triiodothyronine,thyroid hormone,glucose,total protein,albumin,globulin,urea nitrogen,triglyceride,total cholesterol,total antioxidant capacity,superoxide dismutase,catalase,glutathione peroxidase and malondialdehyde were measured.【Result】 ① There was no significant difference in initial body weight,final body weight,feed intake,daily gain and F/G of experimental sheep among four groups during the whole trial period (P>0.05).② There was no significant difference in plasma growth hormone,insulin-like growth factor-Ⅰ,triiodothyronine and thyroid hormone levels of experimental sheep among four groups (P>0.05).③ Plasma TP contents of experimental sheep in 30 EA and 45 EA groups were increased significantly by 11.28% and 10.46% compared to control group (P<0.05),and it was increased linearly with the increase of ellagic acid supplementation (P<0.05).Compared with control group,the plasma ALB content of experimental sheep in three test groups were increased significantly by 17.47%,25.38% and 24.85%,respectively (P<0.05),and it was increased linearly with the increase of ellagic acid supplementation (P<0.05).There were no significant differences in the levels of glucose,globulin,urea nitrogen,and total cholesterol in the plasma of experimental sheep among four groups (P>0.05).The plasma triglyceride content of experimental sheep in 30 EA group was significantly higher than that in control group (P<0.05).④Compared with control group,plasma total antioxidant capacity and glutathione peroxidase activities of experimental sheep in three test groups were increased linearly with the increase of ellagic acid supplementation (P<0.05),and plasma total antioxidant capacity in 30 EA and 45 EA groups increased significantly by 10.24% and 11.02% (P<0.05).Plasma glutathione peroxidase activities in three test groups increased significantly by 29.49%,55.78% and 52.36%,respectively (P<0.05).【Conclusion】 Adding different levels of ellagic acid to the diet did not have a significant effect on the feed intake,daily weight gain,and plasma levels of growth hormone and insulin-like growth factor in Kazakh sheep.However,it could affect fat and protein metabolism to a certain extent and significantly enhance the body’s antioxidant capacity.The appropriate addition level of ellagic acid was 30 mg/kg BW.
Genetics and Breeding
Genetic Parameters for Conformation Traits of Dairy Cattle in Ningxia
HUANG Yuechuan, ZHANG Hailiang, XU Wei, HAN Liyun, ZHOU Jiamin, MA Liqin, WEN Wan, WANG Yachun
2024, 51(7):  2908-2922.  doi:10.16431/j.cnki.1671-7236.2024.07.017
Abstract ( 24 )   PDF (1862KB) ( 3 )  
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【Objective】 Conformation traits have been the focus in dairy cattle breeding,and the selection of conformation traits is crucial for achieving production performance in dairy cattle.This study was conducted to reveal the phenotypic and genetic characteristics of conformation traits,and provide data basis for dairy cattle population in Ningxia.【Method】 Conformation linear classification records of 32 532 Holstein cows from 32 farms in Ningxia were collected.The mixed procedure of SAS 9.2 software was used to analyze the influencing factors of conformation traits of dairy cattle in Ningxia.The DMUAI module of DMU software was used to estimate the genetic parameters for conformation traits,including the heritability and the genetic correlations among 20 conformation traits,5 composite traits and final score.【Result】 The deviations between the average score and optimal score for conformation traits in dairy cattle of Ningxia ranged from 0.03 (udder depth) to 3.73 (central ligament),and the average score of 5 composite traits and final score ranged from 80.77 (rump) to 87.02 (body capacity).The age of first calving,lactation month and parity had significant impacts on 5 composite traits of dairy cattle in Ningxia.The results of unisex animal model estimation showed that the conformation traits of dairy cattle of Ningxia had low to high heritabilities,ranging from 0.04 (chest width) to 0.45 (stature).The results of doublesex animal model estimation showed that the genetic correlations among traits related with body capacity ranged from 0.04 (chest width and loin strength) to 0.72 (stature and body capacity score).The genetic correlations among traits related with rump ranged from 0.32 (rump width and loin strength) to 0.78 (rump angle and rump score).The genetic correlations among traits related with feet and leg ranged from -0.36 (heel depth and rear legs rear view) to 0.78 (heel depth and feet and leg score).The genetic correlations among traits related with lactation system ranged from -0.13 (central ligament and rear udder height) to 0.73 (udder depth and fore udder attachment),and the genetic correlations among traits related with dairy character ranged from 0.54 (bone consistency and angularity) to 0.96 (angularity and dairy character score).【Conclusion】 The conformation of dairy cattle in Ningxia performed well,with heritabilities ranging from 0.04 to 0.45 and genetic correlations ranging from -0.36 to 0.96.The genetic correlations among traits in a same body system were found to be medium to high.The results could provide genetic parameters for genetic selection of dairy cattle in Ningxia.
Genotyping of ALDH7A1 and EDNRB2 Genes and Their Association with Skin Blackness in Black-bone Chickens
TU Yunjie, LUAN Deqin, ZHANG Ming, JU Xiaojun, LIU Yifan, SHAN Yanju, JI Gaige, ZOU Jianmin, SHU Jingting, ZHAO Weidong, ZHENG Guoqing
2024, 51(7):  2923-2932.  doi:10.16431/j.cnki.1671-7236.2024.07.018
Abstract ( 32 )   PDF (3418KB) ( 7 )  
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【Objective】 In order to further verify the role of ALDH7A1 and EDNRB2 in melanin deposition in Black-bone chickens,single nucleotide polymorphisms (SNPs) of ALDH7A1 and EDNRB2 genes were genotyped,and the association between polymorphic loci and skin blackness was analyzed,to find out SNP markers that had significant effects on skin blackness.It would provide reference for marker assisted selection breeding of slaughtered Black-bone chickens with higher blackness.【Method】 Genotyping of SNPs of ALDH7A1 and EDNRB2 genes in 192 individuals of Silky fowls using time-of-flight mass spectrometry analysis technology.the degree of linkage disequilibrium (LD) of these SNPs were analyzed by HaploView 4.1 software,and the association between different genotypes and haplotypes with skin brightness (L*) values was analyzed.【Result】 The mass spectrometry detection rate of two SNPs in ALDH7A1(rs317018616 C>A(SNP1) and rs15992676 T>C(SNP2)) and two SNPs in EDNRB2 genes(rs739725493 G>A(SNP3) and rs316614064 C>T(SNP4)) were 100%.There were only two homozygous genotypes at the two SNPs of ALDH7A1 gene,while there were three genotypes at both SNPs of EDNRB2 gene.Chi-square test showed that the two SNPs of ALDH7A1 gene deviated from Hardy-Weinberg equilibrium (P<0.05),while the two SNPs of EDNRB2 gene were in Hardy-Weinberg equilibrium (P>0.05).The genetic diversity of SNP1 was low (PIC<0.25),and the other three SNPs were moderately polymorphic (0.25<PIC<0.5).Association analysis showed that there was no significant difference in L* values of the back skin and breast skin among different genotypes of the four SNPs (P>0.05).The thigh skin L* value of TT genotype at SNP2 was significantly lower than that of CC genotype (P<0.05).The thigh skin L* values of CC and CT genotypes at SNP4 were significantly lower than that of TT genotype (P<0.05).There was no significant difference in thigh skin L* values between different genotypes at SNP1 and SNP3 (P>0.05).The analysis of linkage disequilibrium indicated that SNP1 and SNP2 of ALDH7A1 gene were in a strong linkage imbalance state,three haplotypes were generated after SNP1-SNP2 linkage.The thigh skin L* values of AATT and CCTT haplotypes of Black-bone chickens were significantly lower than that of CCCC haplotype (P<0.05),but there was no significant difference in L* values of the back skin and breast skin among the three haplotypes (P>0.05).【Conclusion】 The ALDH7A1 and EDNRB2 gene were closely related to the thigh skin blackness.The AATT and CCTT haplotypes of ALDH7A1 gene and TT genotype of EDNRB2 gene could be used as a candidate molecular marker for studying the thigh skin blackness in Silky fowls.It would provide a theoretical reference for using molecular marker assisted breeding to accelerate the cultivation of new Silky fowls with higher blackness.
Genetic Diversity and Population Structure Analysis of Jiaxian Red Cattle Based on Whole Genome Sequencing
ZHANG Yan, WEI Zhitong, HU Yehao, ZHANG Huaju, ZHANG Man, FU Tong, LIU Xian, LI Zhigang, QI Xinglei, WANG Eryao, ZHANG Zijing, LIANG Dong, GAO Tengyun
2024, 51(7):  2933-2942.  doi:10.16431/j.cnki.1671-7236.2024.07.019
Abstract ( 28 )   PDF (7943KB) ( 8 )  
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【Objective】 This study was aimed to understand the genetic diversity and population structure of Jiaxian Red cattle conservation population,make a better protection and utilization of the genetic resources of Jiaxian Red cattle.【Method】 Based on whole genome sequencing technology,the single nucleotide polymorphism (SNP) of 30 Jiaxian Red cattle were detected,and a comprehensive assessment of the conservation effectiveness in Jiaxian Red cattle conservation population was conducted by analyzing population genetic diversity,population structure,distribution characteristics of runs of homozygosity (ROH),and kinship relationships.【Result】 A total of 41 215 797 high-quality SNPs were detected among 30 individuals of Jiaxian Red cattle.The genetic diversity within Jiaxian Red cattle population was extensive,with a minimum allele frequency of 0.13±0.13,polymorphic marker ratio of 0.58±0.33,expected heterozygosity of 0.192±0.152,observed heterozygosity of 0.191±0.158,and nucleotide diversity of 0.003±0.001.The effective population size of Jiaxian Red cattle population showed a decreasing trend over the generations,with an effective population size of 2 716 individuals before 1 000 generations ago, and 143 individuals before 20 generations ago.The genetic distance among individuals of Jiaxian Red cattle ranges from 0.80 to 0.89,with an average genetic distance of 0.83±0.02.Cluster analysis revealed that 30 individuals of Jiaxian Red cattle were divided into a total of 7 families,with 15 bulls being divided into 5 families.A total of 5 947 ROH were detected among 30 individuals of Jiaxian Red cattle,with a cumulative length of 2.8 Gb.The segments ranging from 0 to 0.5 Mb represented the highest proportion at 73.08%,while segments of 2 to 4 Mb represented the lowest proportion at 0.40%.The average inbreeding coefficient based on ROH was 0.19±0.07,and the average inbreeding coefficient of 15 bulls was 0.15±0.05.【Conclusion】 The conservation population of Jiaxian Red cattle exhibited rich genetic diversity and no obvious stratification,maintaining higher levels of inbreeding overall.For the emerging risk of inbreeding,it was necessary to strengthen breeding selection and matching efforts to ensure the sustainable development of genetic resource in Jiaxian Red cattle.
Effect of Cepharanthine on Anti-apoptotic Ability of Bovine Oocytes and Early Embryos in vitro
ZHENG Xiangmin, WANG Yuqi, XU Bingjie, SUN Zhaoyang, GAO Qingshan, FANG Nanzhu, JIN Qingguo
2024, 51(7):  2943-2952.  doi:10.16431/j.cnki.1671-7236.2024.07.020
Abstract ( 22 )   PDF (5424KB) ( 3 )  
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【Objective】 This study was aimed to explore the effect of cepharanthine (Ceph) on the antiapoptotic capacity of bovine oocytes and early embryos,so as to provide reference for improving the efficiency of bovine embryo production in vitro and new data for the pharmacological action of Ceph in anticancer process.【Method】 Bovine oocytes were cultured in in vitro mature medium containing different concentrations (0,10,100,500 and 1 000 μmol/L) of Ceph to observe the oocyte maturation rate and early embryo development after fertilization,the optimal concentration of Ceph was determined.The cathepsin B (CB) activity,the cell autophagy-related protein (LC3) level,the mRNA expression of apoptosis-related genes Survivin and Caspase-3,and the apoptosis rate of blastocysts in bovine oocyte were detected.【Result】 The maturation rate of bovine oocytes and the division rate of early embryo treated with 500 μmol/L Ceph were significantly higher than that of other groups (P<0.05).The addition of Ceph significantly reduced the CB activity and LC3 level in bovine oocytes (P<0.05),significantly increased the mRNA expression of Survivin gene (P<0.05),and significantly reduced the mRNA expression of Caspase-3 gene and the apoptosis rate of blastocysts (P<0.05).【Conclusion】 The addition of Ceph during in vitro embryo production could regulate the expression of apoptosis-related genes Survivin and Caspase-3 to affect the levels of CB and LC3,and enhance the anti-apoptotic capacity of bovine oocytes and in vitro embryo production efficiency.
Isolation and Culture of Equine Umbilical Vein Endothelial Cells and Their Functional Validation
ZHAO Bilig, WEN Xin, YI Minna, Tseweendolmaa, SHEN Yingchao, SI Qin, Zhaolunqiqig, REN Hong, MANG Lai, Gerelchimeg
2024, 51(7):  2953-2962.  doi:10.16431/j.cnki.1671-7236.2024.07.021
Abstract ( 26 )   PDF (18823KB) ( 2 )  
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【Objective】 The aim of this study was to establish a simple and efficient method for isolation and culture of equine umbilical vein endothelial cells (eUVECs) in vitro and to provide a reliable cell model for the study of angiogenic functions in horses.【Method】 Veins were isolated from equine umbilical cords under sterile conditions and rinsed thoroughly with PBS solution.The umbilical veins were digested using 0.2% type Ⅰ collagenase,and after digestion for 25-30 min,the isolated eUVECs were collected for culture.The growth of eUVECs was observed every 12 h using an inverted microscope.The D450 nmvalue of first-generation (P1) and P3 generation eUVECs was determined over a 7 d period using the CCK-8 method,and the growth curves were plotted.The expression of endothelial cell markers CD31 and CD34 was identified by immunofluorescence staining. eUVECs were induced using MSC adipogenic and osteogenic differentiation induction reagents to verify whether they had differentiation properties.After the endothelial cells were identified,eUVECs were stimulated by adding a gradient of increasing concentrations of tumour necrosis factor-α (TNF-α) (5,10,15,20 and 25 ng/mL) and cell activity was detected and morphological changes were observed using the CCK-8 assay.eUVECs were cultured using the Matrigel method,and ImageJ software was used to analyse the data and count the four indexes of neovascularisation:Junctions number,branching lenght,tube formation rate and meshes area.The optimal TNF-α stimulation concentration for in vitro angiogenic capacity was searched.【Result】 The eUVECs isolated using 0.2% type Ⅰ collagenase digestion reached 90% confluence within 4-5 days.These cells were observed under an inverted microscope to be well-grown and arranged in a paving stone-like pattern.Positive expression of CD31 and CD34 in eUVECs was confirmed by immunofluorescence staining,and eUVECs showed weak adipogenic and osteogenic differentiation. Under the culture conditions of the Matrigel method,the indicators of angiogenic capacity,such as the junctions number,branching lenght,tube formation rate and meshes area,were highest in the 20 ng/mL TNF-α-treated group,and were extremely significantly higher than those in the control group (P<0.01).However,under conventional culture conditions,with the increase of TNF-α concentration up to 15 ng/mL,the proliferation of eUVECs was significantly inhibited or even started to lead to apoptosis.【Conclusion】 Type Ⅰ collagenase umbilical cord-filled digestion was able to successfully isolate and culture primary eUVECs.20 ng/mL TNF-α significantly enhanced the angiogenic ability of eUVECs in vitro,and eUVECs could be used as a cellular model to study angiogenesis in vitro.
Research Progress on the Role of lncRNA in Mammalian Male Reproduction
WANG Meijie, LIU Xingwang, BAI Man
2024, 51(7):  2963-2972.  doi:10.16431/j.cnki.1671-7236.2024.07.022
Abstract ( 42 )   PDF (4133KB) ( 15 )  
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The development of mammalian testes and spermatogenesis form the foundation for male animals to acquire normal reproductive capacity and maintain overall health.The seminiferous tubules within testicular tissue serve as crucial sites for sperm production.Spermatogenesis encompasses the entire process,starting from the proliferation and differentiation of spermatogonia until the formation of mature spermatozoa.With the advancement of genomics and molecular biology technologies,a plethora of unidentified long non-coding RNA (lncRNA) have been unearthed.lncRNA,characterized by their transcript length exceeding 200 nt,lack of protein coding potential,absence of open reading frames (ORF),and involvement in gene expression regulation,have emerged as a prominent research focus in recent years.Notably,lncRNA play an indispensable role in mammalian testicular spermatogenesis process with some functional mechanisms being preliminarily elucidated.However,the majority of these mechanisms remain enigmatic and necessitate further investigation.The article comprehensively reviews the research progress on the role of lncRNA in mammalian male reproduction,encompassing the classification and origin of lncRNAs,their mechanisms of action,involvement in testicular development,regulation of proliferation and differentiation of spermatogonia stem cells (SSCs),impact on spermatocyte meiosis,as well as their contribution to sperm cell formation and maturation.This review aims to provide a solid theoretical foundation and serve as a valuable reference for gaining a deeper understanding and conducting further investigations into the functions and underlying mechanisms of lncRNA in mammalian testicular development and spermatogenesis.
Cloning,Sequence Analysis and Expression Characteristics of Carboxylesterase 2 Gene in Dermanyssus gallinae
ZHANG Xuedi, XU Kai, YIN Shuo, LIU Jing, WANG Zhonghao, QIN Jianhua, WU Pengyuan, WANG Chuanwen
2024, 51(7):  2973-2983.  doi:10.16431/j.cnki.1671-7236.2024.07.023
Abstract ( 23 )   PDF (17600KB) ( 1 )  
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【Objective】 The aim of this study was to clone the CDS region sequence of the carboxylesterase 2 (CarE2) gene in Dermanyssus gallinae and conduct bioinformatic analysis,as well as detect its expression characteristics in Dermanyssus gallinae,so as to explore the role of CarE2 gene in the formation and development of metabolic resistance in Dermanyssus gallinae.【Method】 Fifty adult female Dermanyssus gallinae were selected,and the CDS region of CarE2 gene was amplified by PCR and sequenced.Multiple sequence alignment and phylogenetic tree construction were carried out for its coding amino acid sequence.The physical and chemical properties and structural functions of CarE2 protein were predicted by bioinformatics online software.Real-time quantitative PCR was used to detect the expression patterns of CarE2 gene in different developmental stages and different resistant strains of Dermanyssus gallinae,and to analyze the induction effect of cypermethrin on CarE2 gene in sensitive and resistant strains of Dermanyssus gallinae.【Result】 The CDS region of CarE2 gene in Dermanyssus gallinae was 1 665 bp,encoding 554 amino acids. Multiple sequence alignment results showed that the amino acid sequence of CarE2 gene had highly conserved catalytic triad (Ser-Glu-His) and pentapeptide motifs (Gly-X-Ser-X-Gly).Phylogenetic tree analysis showed that CarE2 in Dermanyssus gallinae was closely related to Drosophila melanogaster, and was divided into epidermal esterase branch.Bioinformatics analysis results showed that CarE2 protein was a stable hydrophilic protein with signal peptide cleavage site,glycosylation site and multiple phosphorylation modification sites,but it did not have a transmembrane structure.The secondary structure of CarE2 protein was dominated by random coil,and the predicted tertiary structure was consistent with the secondary structure.The results of Real-time quantitative PCR showed that the expression of CarE2 gene was the highest in the nymph stage,which was significantly higher than that in other stages (P<0.05),followed by in adult stage,and was relatively low in egg and larva stages. The relative expression of CarE2 gene in beta-cypermethrin resistant strain of Dermanyssus gallinae was 2.78 times that of sensitive strain.After treatment with beta-cypermethrin,the expression of CarE2 gene in sensitive strain was not significantly different compared with control group (P>0.05),but the expression of CarE2 gene in beta-cypermethrin cypermethrin resistant strain was significantly higher than that of control group (P<0.05).【Conclusion】 In this study,the CDS region of CarE2 gene in Dermanyssus gallinae was cloned successfully,and it was expressed in all developmental stages of Dermanyssus gallinae,and overexpressed in beta-cypermethrin resistant strain.The expression of CarE2 gene was significantly up-regulated after treatment with cypermethrin,suggesting that CarE2 gene was involved in the metabolic resistance of beta-cypermethrin to carboxylesterase detoxification in Dermanyssus gallinae.
Preventive Veterinary Medicine
Study on the Function of PCYOX1L Gene Regulating LPS-induced Bovine Endometrial Epithelial Cells
JI Guoshang, SHENG Hui, ZHANG Junxing, FENG Xue, HU Chunli, WANG Yachun, MA Yanfen, LI Li, YANG Wenfei, MA Yun
2024, 51(7):  2984-2997.  doi:10.16431/j.cnki.1671-7236.2024.07.024
Abstract ( 25 )   PDF (29451KB) ( 11 )  
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【Objective】 This study was aimed to investigate the regulatory effects of prenylcysteine oxidase 1 like protein (PCYOX1L) on lipopolysaccharide (LPS)-induced inflammation,proliferation,and apoptosis in bovine endometrial epithelial cells (BEECs),so as to clarify the regulatory mechanism of PCYOX1L gene on the occurrence of endometritis in dairy cows.【Method】 An in vitro inflammation model was constructed by stimulating BEECs with LPS,and small interfering RNA (si-PCYOX1L) and overexpression vector (pcDNA3.1-PCYOX1L) of PCYOX1L gene were designed and synthesized,Lipofectamine 3000 transfection reagent was used to transfect the BEECs with pcDNA3.1-PCYOX1L.The effects of interference and overexpression of PCYOX1L gene on the mRNA expression of cellular inflammation,proliferation and apoptosis marker genes were detected by Real-time quantitative PCR.Reactive oxygen species (ROS) level in cell was detected by kit,the protein expression of interleukin-1 (IL-1) was detected by ELISA method,and the cell viability,proliferation and cycle were detected by EdU,CCK-8 and flow cytometry.Cellular mitochondrial damage was detected by a mitochondrial membrane potential kit,and the apoptosis was detected by flow cytometry.【Result】 pcDNA3.1-PCYOX1L overexpression vector was successfully constructed in this experiment,and the interference effect of si-PCYOX1L-385 was screened out to be the best.Compared with control group,after interfering with PCYOX1L gene,the mRNA expression of inflammatory marker genes (IL-1β,IL-6 and IL-8) was extremely significantly down-regulated (P<0.01),the IL-1 protein content was significantly increased (P<0.05),and the intracellular ROS level was extremely significantly decreased (P<0.01). The mRNA expressions of proliferative marker genes (CDK2,CDK4,PCNA and CCND2) were extremely significantly or significantly up-regulated (P<0.01 or P<0.05),and the cell viability was increased,promoting the transition of cells from S phase to G2 phase. The expression of pro-apoptotic gene Bax was significantly decreased (P<0.05),and the expression of anti-apoptotic gene BCL2 was increased,reducing the apoptosis rate of BEECs(P<0.01).However,the results were exactly the opposite after overexpression of PCYOX1L gene.【Conclusion】 PCYOX1L gene promoted inflammation,inhibited cell proliferation,and promoted apoptosis in BEECs.This results provided basic data for further investigation of the molecular mechanism of PCYOX1L gene regulating the occurrence of endometritis in dairy cows.
Bioinformatics Analysis and Prokaryotic Expression of SipD Protein of Salmonella Enteritidis in Chickens
YUAN Liang, HAN Xiaoxi, GONG Yingchao, FAN Xianan, HAO Beili, HAO Shuangshuang, LI Changwen, CHANG Yicong, LI Rui, LIU Fangping
2024, 51(7):  2998-3007.  doi:10.16431/j.cnki.1671-7236.2024.07.025
Abstract ( 31 )   PDF (9955KB) ( 6 )  
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【Objective】 This study was aimed to predict the potential biological functions of Salmonella Enteritidis (SE) SipD protein,and express and purify SipD protein,so as to provide a theoretical basis for exploring SipD protein as a candidate antigen for anti-Salmonella nanobodies.【Method】 DNAStar software was used to perform similarity alignment on the amino acid sequences of SipD protein among different serotypes of Salmonella strains published in GenBank.Subsequently,the bioinformatics analysis of SipD protein was performed using online software.Primers were designed based on SipD gene sequence in GenBank,using the standard strain of Salmonella Enteritidis (ATCC 13076) as a template.PCR was applied to amplify SipD gene and construct a pET-32a(+)-SipD recombinant plasmid,which was then transformed into Escherichia coli BL21 (DE3) competent cells.IPTG was applied to induce the expression of recombinant SipD protein and Ni2+ affinity chromatography was used to purify.SDS-PAGE and Western blotting was applied to detect the expression of SipD protein.【Result】 SipD protein was highly conserved among different serotypes of Salmonella strains. SipD protein,with the molecular formula C1619H2573N441O540S8,was an extracellular protein without transmembrane domain and signal peptide.The amino acids 1 to 343 of SipD protein had conserved structural domains from the superfamily of invasive plasmid antigen IpaD.The secondary structure prediction of SipD protein showed that alpha helix,extended chain,beta turn and random coil accounted for 59.18%,6.41%,3.21% and 31.20%,respectively.There were 12 antigenic epitopes that bound SipD protein to B cells.The size of SipD gene was 1 029 bp,which was expressed as a soluble protein in Escherichia coli BL21 (DE3) competent cells.After purification,dialysis and concentration,the protein concentration of SipD protein was 8.6 mg/mL.SDS-PAGE and Western blotting analysis revealed that SipD protein with high purity could be used to immunize alpacas for the preparation of nanobody.【Conclusion】 SipD protein was an extramembrane protein with many antigen binding sites.The recombinant protein of SipD with high purity was obtained by expression and purification,which provided materials for further preparation of nanobody targeting SipD protein of Salmonella Enteritidis in chickens.
Prokaryotic Expression of Chicken Interferon-γ and Preparation and Identification of Its Monoclonal Antibodies
CUI Jinqiang, CHENG Jing, JIANG Bo, ZHOU Linyi, LIU Wenxiao, LI Yongqing, SONG Qinye
2024, 51(7):  3008-3019.  doi:10.16431/j.cnki.1671-7236.2024.07.026
Abstract ( 26 )   PDF (12816KB) ( 6 )  
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【Objective】 The purpose of this study was to express chicken interferon-gamma (ChIFN-γ) by prokaryotic expression system and prepare its monoclonal antibody to provide crucial reagents for the detection of natural ChIFN-γ.【Method】 Recombinant plasmid pET21a-ChIFN-γ was constructed by cloning codon optimized ChIFN-γ gene into pET-21a(+) vector,and then transformed into Escherichia coli BL21(DE3) competent cells.The recombinant protein was induced by IPTG.BALB/c mice were immunized with this purified protein.The splenocytes of immune-qualified mouse was hybridized with myeloma cells (SP2/0) by cell fusion technique.Positive hybridoma secreting specific antibodies against ChIFN-γ were identified through screening using indirect ELISA,and prepare ascites,purified ascites with Protein A/G to obtain specific antibodies.The classes and subclasses of monoclonal antibodies were identified.Western blotting and indirect immunofluorescence assay (IFA) were used to detect the reactivity,affinity,and specific cross reactivity of monoclonal antibodies.Monoclonal antibodies were labeled with horseradish peroxidase (HRP),and antibody pairing were conducted using pairwise methods.【Result】 Soluble recombinant ChIFN-γ protein were successfully prepared.Western blotting showed that ChIFN-γ binded to the corresponding antibody,presenting specific bands(16 ku).Seven hybridoma cell lines specific to ChIFN-γ were obtained which were 3E8,5F8,2G12,5A10,3D8,3B5 and 3G2,respectively.Western blotting and IFA results showed that all 7 monoclonal antibodies had good reactivity, and could specifically recognize eukaryotic expressed ChIFN-γ.The results of class and subclass identification of monoclonal antibodies showed that the heavy chains of 3E8,5F8,3D8,3B5 and 2G12 were all IgG1. The heavy chains of 5A10 and 3G2 were IgG2a and IgM,respectively.The light chains of the 7 monoclonal antibodies were all Kappa type.The 7 monoclonal antibodies obtained had dissociation constants (Kd) between 1.04 and 58.33 nmol/L,indicating high affinity.Five pairs of paired antibodies were also obtained.【Conclusion】 This study successfully prepared recombinant ChIFN-γ protein and its monoclonal antibodies,laying the foundation for further research on ChIFN-γ and its related studies in avian immunology,virology and vaccine efficacy evaluation.
Isolation,Identification and Antimicrobial Properties of Enterococcus faecium FDT2
ZHANG Xiaoyong, LAN Wei, WANG Ya, LAO Jianlong, LI Zhi, JIA Dan, KONG Xiangying, HU Kaiyin, WEN Yongsen, LYU Shuting, CHEN Man, YIN Hong, LI Youquan
2024, 51(7):  3020-3029.  doi:10.16431/j.cnki.1671-7236.2024.07.027
Abstract ( 28 )   PDF (6155KB) ( 10 )  
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【Objective】 The purpose of this experiment was to isolate and identify Enterococcus faeciums isolates from yak faeces and conduct in vitro antibacterial studies,in order to screen probiotics with excellent antibacterial properties.【Method】 In this experiment,Enterococcus spp.were isolated from faeces of healthy yaks in Qinghai province utilizing MRS medium,the strains were identified by combining morphology and molecular biology and conduct antibacterial research.The supernatants of the strains were isolated by fermentation filtration,and the corresponding antibacterial effect was observed using punching method.The antibacterial substances were analyzed by changing pH,adding catalase,pepsin and trypsin,and the effects of temperature and pH on the fermentation supernatants were explored.【Result】 The results of morphology and phylogenetic tree analysis showed that the two new isolates (2 and D2) belonged to Enterococcus faecium. The antibacterial experiment results showed that Enterococcus faecium FDT2 had inhibitory effect on all five pathogenic bacteria,and the inhibitory effects on enterotoxigenic Escherichia coli K88 and Escherichia coli ATCC 43888 were more stronger,while D2 had no inhibitory effect on all five pathogenic bacteria.The acid excretion test results proved that the acidic substance in the fermentation supernatant of Enterococcus faecium FDT2 did not have antibacterial effects.The experiment of adding catalase proved that hydrogen peroxide existed in the bacteriostatic substance and played partly bacteriostatic effect.After addition of pepsin and trypsin,it was found that the diameter of inhibition zone was extremely significantly lower than that of control group (P<0.01),indicating that antibacterial substances contained bacteriocin and played a major role in antibacterial effects.With the gradual increase of temperature,the antibacterial activity of Enterococcus faecium FDT2 decreased,and it remained antibacterial activity at 100 ℃,indicating its strong thermal stability.With the increase of pH,the bacteriostatic activity of Enterococcus faecium FDT2 decreased gradually,which also indicated that bacteriocin had strong activity in acidic environment.【Conclusion】 Two strains of bacteria were isolated in the experiment,among which Enterococcus faecium FDT2 had good bacteriostatic properties and contained bacteriocin and other antibacterial substances,and it was necessary to further study on the bacteriocin of Enterococcus faecium FDT2.
Whole Genome Sequencing and Virulence and Drug Resistance Analysis of a Strain of Proteus mirabilis Derived from Peacock
JIAO Fengchao, LEI Zhen, LI Yingxiao, WU Xian, QU Zhehui
2024, 51(7):  3030-3040.  doi:10.16431/j.cnki.1671-7236.2024.07.028
Abstract ( 22 )   PDF (11423KB) ( 4 )  
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【Objective】 The aim of this study was to investigate the virulence,drug resistance and genomic characteristics of Proteus mirabilis PM19 isolated from blue peacock.【Method】 Animal experiments,drug sensitivity tests and PCR methods were used to detect the virulence,drug resistance and virulence genes of the isolated bacteria.The Illumina NovaSeq6000 platform was used to sequence the genome frame diagram,and the functional genes,pathogenic host interaction protein,carbohydrate active enzyme,drug resistance gene and virulence factor prediction were carried out by NR,KEGG,Swiss-Prot,COG,PHI-base,CAZy,CARD and VFDB databases.【Result】 The isolate PM19 was a multidrug-resistant bacterium and was resistant to 14 antimicrobial agents including ampicillin,amoxicillin/clavulanate potassium,ceftriaxone,cefotaxime,amikacin,gentamicin,kanamycin,ciprofloxacin,tetracycline,doxycycline,sulfadiazine sodium,tilmicosin,florfenicol and chloramphenicol.The isolated bacteria had strong virulence,and the median lethal dose to mice was 6.19×106 CFU.Eight virulence genes including FliL,zapA,rsmA,hmpA,mrpA,atfA,pmfA,ureC were detected in the strain.The whole genome sequencing analysis showed that the genome size of the strain was about 3.98 Mb,the GC content was 38.94%,and 3 715 coding genes were predicted.158 protein genes related to pathogen-host interaction were annotated by PHI-base database.101 carbohydrate active enzyme genes were annotated by CAZy database.92 drug resistance genes and 8 virulence-related genes were annotated by CARD database and VFDB database,respectively.【Conclusion】 The Proteus mirabilis strain PM19 was virulent and multidrug-resistant,carrying a large number of pathogenic host interaction protein genes,carbohydrate active enzyme genes,drug resistance genes and virulence genes.
Research Progress on the Pathogenic Mechanism and Prevention Strategy of African Swine Fever Virus
WANG Meile, YU Yangfan, XU Peng, CHEN Qing, AKILA·ANWAR, LI Shuxuan, YANG Songhua, YIN Sugai, FENG Shuying
2024, 51(7):  3041-3055.  doi:10.16431/j.cnki.1671-7236.2024.07.029
Abstract ( 32 )   PDF (4442KB) ( 25 )  
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African swine fever (ASF) is an acute,hemorrhagic and highly contagious disease,which has a huge negative impact on the world pig industry and food security.There are still no specific drugs and vaccines for the prevention and treatment.African swine fever virus (ASFV) is a large,complex double-stranded DNA virus,but the function of 80% of the encoded proteins is unknown.Therefore,its pathogenic mechanism and drug research status are of great significance for prevention and control. The author summarized the pathogen characteristics,transmission routes and clinical manifestations of ASFV.The latest progress of viral pathogenesis was reviewed from the aspects of virus invasion process,immune cell function changes and related pathway changes.The latest detection technology and vaccine progress of ASFV were introduced.This paper systematically summarized the latest research status of Chinese and Western medicine treatment,the future prospects of traditional Chinese medicine in the prevention and treatment of ASFV were prospected,and the key research directions and related problem solving strategies in the future were put forward,in order to provide reference and practical guidance for the treatment of ASF.
Truncated Expression of Cap Protein of Porcine Circovirus Type 3 and Establishment of Indirect ELISA Method for Its Antibody
OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHAI Jiajia, ZHANG Jie
2024, 51(7):  3056-3066.  doi:10.16431/j.cnki.1671-7236.2024.07.030
Abstract ( 25 )   PDF (2782KB) ( 11 )  
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【Objective】 This study was aimed to express truncatly Cap protein (1-197 amino acids) of Porcine circovirus type 3 (PCV3) Sichuan strain in prokaryotic expression system,and develop an indirect ELISA (i-ELISA) method for rapid detection of PCV3 antibody,and provide material for serological investigation of PCV3.【Method】 The genome of PCV3 Sichuan strain was used as template.The Cap protein coding gene of PCV3 was obtained by PCR amplification and inserted into the prokaryotic expression vector pET-30a(+) to construct the recombinant expression plasmid pET30a-PCV3-Cap.The recombined plasmid pET30a-PCV3-Cap was expressed in E.coli BL21 (DE3) competent cells and was induced by IPTG.The recombinant Cap protein with good antigenicity was purified by Ni-NTA,and analyzed by SDS-PAGE and Western blotting.The i-ELISA was developed using the recombinant Cap protein as coating antigen.The optimal concentration of antigen,the optimal dilution ratio of serum to be tested,the dilution ratio of enzyme-labeled antibody and the threshold of negative and positive were determined,and the specificity,sensitivity,repeatability,correlation and coincidence rate of the i-ELISA were analyzed.The established i-ELISA method was used to detect 351 pig serum samples collected from pig farms in Northeast Sichuan from 2018 to 2022 to analyze the prevalence of PCV3 in the region.【Result】 The recombinant expression plasmid pET30a-PCV3-Cap was successfully obtained,and the recombinant Cap protein (1-197 amino acids) was successfully expressed in E.coli BL21(DE3) competent cells could react specifically with positive pig serum of PCV3 with a relative molecular weight of 26 ku.The coating concentration of Cap protein was 1 ng/μL,and the detection positive threshold was D450 nm>0.684.The dilution concentration of serum and HRP-rabbit anti-pig IgG were 1∶100 and 1∶60 000,respectively.The i-ELISA was not cross reaction with positive sera of CSFV,PRRSV,PCV2,PRV,JEV and PPV1.The sensitivity of i-ELISA was 1∶1 600.The method was of high duplicability with less than 10% variation of intra-and inter-assay coefficients.The result of i-ELISA was positive correlation with that of virus neutralization test,and the coincidence rate with Western blotting method was 95.8%.The positive rate of PCV3 antibody was 7.12% in 351 pig serum samples collected in Northeast Sichuan from 2018 to 2022.【Conclusion】 This experiment successfully truncated expression Cap protein (1-197 amino acids) and established i-ELISA of PCV3 with better specificity,sensitivity,repeatability and coincidence rate,which provided material for serological investigation and test kit development of PCV3.
Effect of Different Factors on the Infection of Gastrointestinal Nematodes in Teha Crossbred Sheep
WANG Yuqi, HE Sangang, LIANG Long, HAILATI Kasimu, JIAO Wenjing, YUE Chenbing, HAN Bing, SONG Xuebin, YAN Xiaofei
2024, 51(7):  3067-3076.  doi:10.16431/j.cnki.1671-7236.2024.07.031
Abstract ( 32 )   PDF (3768KB) ( 6 )  
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【Objective】 This experiment was aimed to study the effects of season,gender,group and flock on the infection intensity of gastrointestinal nematodes (GIN),weight,hematocrit and specific antibody immunoglobulin G (IgG) content in Texel and Kazakh (hereinafter referred to as Teha) crossbred sheep,in order to provide a reference basis for research on the prevention and treatment of GIN in sheep and anti-GIN breeding.【Method】 Feces were collected rectally,blood was collected from the jugular vein,and weights were weighed in Teha crossbred sheep.The eggs of GIN were qualitatively detected by saturated brine flotation method.The modified McMaster method was used for egg counting of GIN,Infection rate and infection intensity were calculated.Hematocrit was measured using a veterinary auto hematology analyzer.Nematode L3 larval somatic cell antigens were prepared,and specific antibody IgG content in the serum of the sheep was determined using indirect ELISA method.【Result】 The infection rate of GIN in Teha crossbred sheep was high at 94.44%,which was moderate. And total of nine GIN were identified which were Trichostrongylus spp.,Haemonchus contortus,Ostertagia spp.,Bumastomum trigonocephalum,Chabertia spp.,Marshallagia spp.,Nematodirus spp.,Oesophagostomum spp.and Trichuris globuulosa.Season had a significant effect on the infection intensity of GIN and weight of sheep (P<0.05),the infection intensity of GIN was significantly higher in spring than that in summer (P<0.05),and the weight of sheep was significantly higher in summer than that in spring (P<0.05).Gender had a significantly effect on the infection intensity,weight and hematocrit (P<0.05),with the infection intensity of GIN significantly higher in rams than in ewes (P<0.05),the weight was significantly higher in rams than in ewes (P<0.05),and hematocrit was significantly higher in ewes than in rams (P<0.05).Group had a significant effect on weight (P<0.05),the weight of F2×Ha sheep was significantly higher than that of F2 and F2×F1 sheep (P<0.05),the weight of F1×F1 sheep was significantly higher than that of F2×F1 sheep (P<0.05),and the weight of F2×F2 sheep was significantly lower than that of other groups (P<0.05).Flock had a significant effect on the infection intensity in sheep (P<0.05),with the infection intensity of GIN in flock 1 being significantly higher than that in flocks 2 and 3 (P<0.05),and the infection intensity of GIN in flock 3 was significantly higher than that in flock 2 (P<0.05).All factors had no effect on specific antibody IgG content (P>0.05).【Conclusion】 Season,gender,group and flock had influence on the infection of GIN in sheep,and different control measures could be selected for sheep through the effect of factors on the infection and the differences between different levels of each factor,to provide a theoretical basis for anti-GIN breeding strategies.
Effect of gltC Gene Deletion on Acid Stress Resistance of Listeria monocytogenes 10403S
YANG Yuting, HAN Xu, XIAO Jinhua, FANG Xiaowei, LIANG Xiongyan, LIU Jing, FANG Chun
2024, 51(7):  3077-3085.  doi:10.16431/j.cnki.1671-7236.2024.07.032
Abstract ( 30 )   PDF (2188KB) ( 2 )  
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【Objective】 The experiment was aimed to construct a strain of Listeria monocytogenes with gltC gene deletion and elucidate the effect of gltC gene on the acid stress resistance of Listeria monocytogenes.【Method】 The effect of glutamic acid on the survival ability of the 10403S was determined by acid stress test with or without the addition of exogenous glutamic acid under lethal acidic conditions.The homologous recombination method was used to construct a strain with a deletion of gltC gene.The effect of gltC gene deletion on the growth capacity of the strain was determined by growth curves.The effect of gltC gene deletion on the acid stress capacity of Listeria monocytogenes was determined by the acid stress survival assay.The effect of the gltC gene deletion on the expression level of GadD2 protein was determined by Western blotting.By constructing the gfp reporter plasmid carrying the gltBD promoter region,the effect of gltC gene deletion on the transcription level of the gltBD promoter region was measured under acidic and neutral conditions.【Result】 Acid stress results showed that adding exogenous glutamic acid could extremely significantly improve the survival ability of strain 10403S under acidic conditions at pH 2.5 (P<0.01).PCR results showed that the deletion strain 10403SΔgltC was successfully constructed.The growth curve results showed that gltC gene deletion did not affect the growth ability of Listeria monocytogenes.The results of acid stress showed that the growth ability of the deletion strain 10403SΔgltC was weaker than that of the parental strain 10403S in a lethal acidic environment at pH 2.5 (P<0.01).The results of the Western blotting showed that the deletion of gltC gene did not affect the expression level of GadD2 protein (P>0.05).PCR results showed that gfp with gltBD promoter region reported plasmid was successfully constructed.GFP fluorescence analysis showed that the deletion of gltC gene extremely significantly decreased the transcription level of the gltBD promoter region under both acidic and neutral conditions (P<0.01).【Conclusion】 Deletion of gltC gene did not affect the normal growth of the Listeria monocytogenes 10403S,but the deletion strain 10403SΔgltC reduced resistance to acid stress and decreased the level of transcription in the gltBD promoter region,without affecting the level of GadD2 protein expression.
Advances in Cytotoxic T Lymphocyte Epitopes of Porcine Viruses
XIAN Yuhan, FENG Hongsheng, GAO Yongyu, LI Haiyang, YANG Siyu, SANG Chenjun, CAO Yudie, TANG Yue, LI Zibin, GAO Fengshan
2024, 51(7):  3086-3099.  doi:10.16431/j.cnki.1671-7236.2024.07.033
Abstract ( 27 )   PDF (6258KB) ( 3 )  
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The major histocompatibility complex (MHC) class Ⅰ molecules,also known as swine leukocyte antigen (SLA) class Ⅰ molecules,play an important role in antigen presentation,organ transplantation,immune response and regulation.Cytotoxic T lymphocyte (CTL) epitopes are linear peptides that bind and present SLA-Ⅰ molecules after antigen processing by antigen-presenting cells.With high specificity,CTL epitopes play a key role in antigen recognition and presentation and cellular immunity against viral infection.CTL epitopes can stimulate CTLs to play the role of cell killing,which is mainly manifested as killing virus.The author reviewed the current status of CTL epitopes of several important porcine viruses,such as Foot-and-mouth disease virus,African swine fever virus and Porcine reproductive and respiratory syndrome virus,and analyzed the binding motif characteristics of SLA-Ⅰ and CTL epitopes by bioinformatics,in order to provide a reference for studying CTL epitopes derived from porcine viruses and designing the epitope vaccine in future.
Isolation,Identification and Genetic Evolution Analysis of a Strain of Bovine Coronavirus
LIU Wenkai, WANG Ping, JIN Yinghong, XUE Jing, LI Yue, LIANG Qianqian, LI Xiaozhuo, HAN Xiangshu, ZHENG Qiming, JIANG Song, XIA Jun
2024, 51(7):  3100-3108.  doi:10.16431/j.cnki.1671-7236.2024.07.034
Abstract ( 28 )   PDF (7003KB) ( 7 )  
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【Objective】 The experiment was aimed to obtain the Bovine coronavirus (BCoV) strain in Xinjiang,explore the best conditions for in vitro isolation of BCoV,understand the genetic evolution of BCoV Xinjiang strain,and provide candidate strain research basis for BCoV vaccine.【Method】 The small intestine and the contents (n=185) of diarrhea calves from 7 cattle farms in Xinjiang were detected for BCoV by RT-PCR.The positive samples were inoculated into HCT-8 cells after treatment.The isolated virus was further identified by indirect immunofluorescence assay (IFA),Western blotting and transmission electron microscopy.Median tissue culture infective dose (TCID50) was determined by Reed-Muench method.The isolated virus was subjected to whole genome sequencing,and the sequencing results were subjected to genetic evolution analysis.【Result】 The detection rate of BCoV in diarrhea samples was 64.9%.After inoculation with HCT-8 cells,a strain of BCoV was successfully isolated and named as XJ-SHZ-37 (GenBank accession No.:OR750853). The green specific fluorescence of the cells incubated with the virus could be observed by IFA,however,the control group did not.Western blotting test results showed that the isolated strain had a specific reaction with BCoV N protein polyclonal antibodies.The crown-like virus particles could be observed by transmission electron microscopy.The TCID50 of the isolates measured by Reed-Muench method was 10-7.9/0.1 mL.The results of genome sequencing and genetic evolution showed that XJ-SHZ-37 had the highest similarity with ZJNB2106,a BCoV isolate from the genus Coronaviruses of the Coronaviruses family,isolated from cattle.【Conclusion】 There was a high detection rate of BCoV in 7 cattle farms in Xinjiang.A strain of virus that could be stably inherited and specifically bind to the polyclonal antibody of BCoV N protein was successfully isolated.The isolate had a high virus titer,and the genetic evolution relationship showed that it had the closest evolutionary relationship with the Chinese bovine strain ZJNB2106.
Bactericidal Effect and Safety Evaluation of 222 nm Far-ultraviolet
XIANG Yifei, MA Qingyang, ZHONG Yawen, ZHU Shuxin, HE Ruini, WEI Hua, ZHANG Haishan, LIU Chengzhi, NING Junkai, HE Jiakang
2024, 51(7):  3109-3117.  doi:10.16431/j.cnki.1671-7236.2024.07.035
Abstract ( 23 )   PDF (9826KB) ( 5 )  
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【Objective】 The spread of pathogenic bacteria in the environment was an urgent problem to be solved in the prevention and control of current diseases. Traditional ultraviolet (254 nm) was the main mode of environmental disinfection at present, which was limited due to its irreversible damage to the immune system, skin and eyes, so exploring an uninterrupted, safe and effective means of disinfection was a promising study. 【Method】 A poor site was selected to investigate the effect of uninterrupted irradiation with 222 nm far-ultraviolet light for 8 h on the elimination of pathogenic bacteria in the environment, in which the negative control group was not treated, the positive control group was placed openly, and the far-ultraviolet group was irradiated with the far-ultraviolet light from a distance of 30 cm at the same time as it was placed openly. The common clinical pathogenic bacteria (Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli) were coated with continuous irradiation for 30 min, and negative and positive control groups were set up, so as to compare the difference between the far-ultraviolet rays and the traditional ultraviolet rays. The surface bactericidal effect of the far-ultraviolet rays on the common pathogenic bacteria was examined by comparing the irradiation distances (20 and 30 cm) and the action times (15, 30, 45, 60, 75 and 90 s). Common pathogenic bacteria were inoculated into liquid culture medium to investigate the bactericidal effect of ultraviolet radiation on bacteria in the liquid. After uninterrupted irradiation of mice with 222 nm far-ultraviolet radiation for 8 h, the clinical status, blood routine, and skin histological changes were observed to preliminarily determine the safety of far-ultraviolet radiation on the body.【Result】 Compared with the negative control group, a variety of bacteria were observed on the culture medium of the positive control group, while no bacteria were colonized on the culture medium of the far-ultraviolet group. By continuously irradiating with 222 nm far-ultraviolet light for 30 minutes, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli could be killed. The bactericidal rate could reach 99.9% after 15 s of irradiation. The bactericidal effect of 222 nm far-ultraviolet light irradiated at a distance of 20 cm was generally better than that irradiated at a distance of 30 cm. In the liquid bactericidal evaluation, the sterilizing effect of far-ultraviolet light was not good. The results of the safety test in mice showed that the uninterrupted irradiation of 222 nm far-ultraviolet light for 8 h had no significant effect on the clinical symptoms, internal organs, blood and skin tissues of the mice, while the traditional ultraviolet light might cause damage to them. 【Conclusion】 222 nm far-ultraviolet light had a strong bactericidal effect on the surface colonization of common pathogenic bacteria, and the bactericidal rate could be as high as 99.9% in 15 s. The irradiation distance was directly proportional to the bactericidal effect, but the bactericidal effect on the liquid still needed to be improved and upgraded. Moreover, the continuous irradiation for 8 h would not have a significant effect on the clinical status, blood routine and skin histology of mice, and it had a good safety, providing a new solution for the prevention and control of the disease in the environment.
Research Progress on S and HE Proteins of Bovine Coronaviruses
JIN Shuangyuan, DU Jiawei, WANG Xueyan, GUO Xuelian, XU Lihua
2024, 51(7):  3118-3127.  doi:10.16431/j.cnki.1671-7236.2024.07.036
Abstract ( 26 )   PDF (4290KB) ( 6 )  
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Bovine coronavirus (BCoV) is a pathogenic agent that causes diarrhoea in newborn calves,respiratory infections in cattle and winter dysentery in adult cattle,and is mainly transmitted by faecal-oral infection and respiratory aerosols.Studies have shown that BCoV can persist in subclinically infected adult cattle,which may explain the widespread presence of BCoV in herds and its ability to circulate over time.The prevention and control of Bovine coronavirus disease is extremely critical,as the occurrence of the disease can cause great economic losses to intensive cattle farms.The BCoV S protein contains major antigenic sites that bind to salivary acid (SA) on the host surface,and the S protein is involved in adsorption and membrane fusion between the virus and the host cells,which promotes the production of potent neutralising antibodies by the host.The BCoV HE protein has acetyl esterase activity and haemagglutination activity,and can synergise with the S protein to attach to the cell surface and initiate infection.Both S and HE proteins play important roles in the tissue or host tropism and virulence of the virus,and these roles also provide references for the further study of the pathogenesis of BCoV,and the subsequent development of vaccines and drugs.The author focuses on the molecular structure and function of the BCoV S and HE proteins,and the role of BCoV in receptor recognition based on the S and HE proteins (including the use of N-acetyl-9-O-acetylneuraminic acid as a sugar-binding receptor,and the use of human leukocyte antigen class Ⅰmolecules as a protein-binding receptor).Receptor recognition is an important determinant of Coronavirus infection and pathogenesis,and it is also one of the most important targets for host immune surveillance and human intervention strategies.Therefore,a full understanding of the structure-function and receptor recognition roles of the S and HE proteins is important for understanding the viral pathogenesis as well as for the development of antiviral drugs and vaccines.
Basic Veterinary Medicine
Isolation,Identification and Pathogen Characteristics Evaluation of a Strain of Pasteurella multocida D:L3:L5 from Cattle
ZHAO Yiduo, ZHANG Kang, WANG Lei, LU Xiaorong, ZHANG Jingyan, CHENG Jia, GUO Zhiting, XU Guowei, HUANG Liping, NIU Yuhui, MA Ting, FENG Xiaowei, EGIDE, LIU Mingchao, LI Jianxi
2024, 51(7):  3128-3141.  doi:10.16431/j.cnki.1671-7236.2024.07.037
Abstract ( 28 )   PDF (5431KB) ( 5 )  
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【Objective】 The experiment was aimed to explore the distribution of capsule serotypes,drug resistance and virulence genes of Pasteurella multocida from a large-scale dairy farm in Xining city,Qinghai province,in order to provide reference for the treatment and prevention of respiratory diseases caused by Pasteurella multocida in cattle.【Method】 Bacteria were isolated from the lung sample of a dead cattle.And then,through the methods of colony observation,dyeing microscopy,biochemical identification,specific primer amplification,16S rRNA clone sequencing analysis,phylogenetic tree construction,capsular serum and lipopolysaccharide type,virulence gene detection,pathogenicity test and drug sensitivity test,the isolated Pasteurella multocida was identified and its pathogen characteristics were evaluated.【Result】 The colonies isolated from the lung samples were dewdrop-like with a slight depression in the gray middle,and no hemolysis ring appeared.Gram staining showed coccidia pink,Wright’s staining showed blue coccidia with dense staining at both ends.Biochemical identification showed that the results of D-glucose,D-mannitol,D-mannose,tyrosine arylaminase,phosphatase,COURMARATE and ELLMAN reaction were positive.16S rRNA sequencing results showed that the strain was more than 95% similar to a variety of Pasteurella multocida,and the isolated strain was identified as Pasteurella multocida and named XN222.The results of capsule serum typing and lipopolysaccharide type showed that XN222 was classified as type D,and its lipopolysaccharide types were L3 and L5.The results of virulence gene amplification showed that XN222 carried 19 virulence genes,including ptfA,fimA,hsf-2, sodA, tbpA, sodC,and so on.The results of pathogenicity test in mice showed that injection of 0.2 mL 1.5×108 CFU/mL bacterial solution resulted in 100% (10/10) death of mice,indicating that the isolated strain XN222 had strong pathogenicity. The results of drug sensitivity test showed that XN222 was sensitive to 15 kinds of antimicrobial agents,such as ceftiofur,levofloxacin,enrofloxacin,marbofloxacin,tetracycline and penicillin,and was moderately sensitive to tylosin.【Conclusion】 In this study,a highly pathogenic Pasteurella multocida type D strain was isolated.The discovery of this strain enriched the domestic bovine subtype of Pasteurella multocida,and provided biological materials for the prevention and control of bovine respiratory diseases caused by Pasteurella multocida,etiological investigation and development of Pasteurella multocida multivalent vaccine.
Optimization of Streptomyces sp. Fermentation Medium for Production of Alkylated Anthraquinone by Response Surface Methodology
LIU Fang, QUAN Tingting, ZHANG Fei, SHI Liqiao, LIU Manli, WU Zhaoyuan
2024, 51(7):  3142-3154.  doi:10.16431/j.cnki.1671-7236.2024.07.038
Abstract ( 25 )   PDF (7869KB) ( 6 )  
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【Objective】 This study was aimed to optimize the fermentation conditions and medium composition of Streptomyces sp. WS-13394 by response surface methodology,so as to improve the production level of alkylated anthraquinones produced by the strain,and provide technical support for the application of this compound in animal husbandry.【Method】 The optimal shake-flask fermentation conditions and the proper combination of carbon and nitrogen sources and mineral salts for the yield of alkylated anthraquinone produced by Streptomyces sp.WS-13394 were achieved through single-factor experiments.The significant factors affecting the yield of alkylated anthraquinone were fixed through Plackett-Burman design.The optimized medium for the yield of alkylated anthraquinone was achieved through the steepest ascending experiment,response surface design by fitting the significant factors with the yield of alkylated anthraquinone.【Result】 The composition of the optimized medium for the yield of alkylated anthraquinone produced by Streptomyces sp. WS-13394 was as followings:Soluble starch 28.12 g/L,fish meal 10.00 g/L,yeast extract 6.34 g/L,CaCO3 0.71 g/L,MgSO4 0.50 g/L,and K2HPO4 0.50 g/L. The theoretical yield of alkylated anthraquinone based on the nonlinear regression formula was 408.35 mg/L.The optimal combination was applied to shake bottle fermentation test,and the yield of alkylated anthraquinone was 411.73 mg/L,which was 3.59 times higher than that before optimization (89.70 mg/L).【Conclusion】 Response surface optimization was used to obtain the shaking flask fermentation medium formulation that significantly improved the yield of alkylated anthraquinone produced by Streptomyces sp.WS-13394.The results provided technical support for the studies of the antibacterial activity mechanism of this compound and the evaluation of its effect in animal models.
Screening and Action Pathway of Compounds Antagonistic Senacavirus A in vitro from Natural Product Library
WANG Xitong, SHA Zhou, CHI Tianying, CUI Jin, ZHENG Hui, CHEN Feng, CAO Zhenshan, ZHANG Hui, GE Shengqiang, WEI Rong, NI Bo, HUANG Baoxu
2024, 51(7):  3155-3165.  doi:10.16431/j.cnki.1671-7236.2024.07.039
Abstract ( 24 )   PDF (4805KB) ( 5 )  
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【Objective】 The experiment was aimed to search for natural products with inhibitory activity against Senecavirus A (SVA) and further investigate its antagonistic pathway.【Method】 The concentration of the natural products were uniformly diluted to 10 μmol/L.Combined with the recombinant Senecavirus A with luciferase (rSVA-NLuc),compounds with luciferase inhibitory activity were screened from the natural products library,and their activity was further verified using Real-time quantitative RT-PCR.The maximum non-toxic concentration was determined by cytotoxicity testing using lactate dehydrogenase (LDH) method.Virus titer determination,Real-time quantitative RT-PCR,Western blotting,IFA and other techniques were used to study the effect and pathway of antagonism against SVA.【Result】 Five compounds,monensin sodium salt,progesterone,hypophyllanthin,4-hydroxyderricin and 2-methoxyestrone,which could antagonize SVA in vitro,were screened from 136 natural products.Progesterone,a natural product that could antagonize SVA in vitro,was screened by Real-time quantitative RT-PCR and cytotoxicity detection.Compared with blank control group,the mRNA expression level of SVA was extremely significantly decreased in vitro (P<0.01),and the maximum non-toxic concentration reached 50 μmol/L.Further study showed that progesterone could continuously inhibit the proliferation of SVA at 8,12,24 and 36 h after infection,and the virus titer was significantly decreased at 24 and 36 h after infection compared with control group (P<0.05).Compared with control group,different concentrations of progesterone (10,20 and 50 μmol/L) extremely significantly decreased the adsorption level of SVA at the adsorption stage (P<0.01).In the replication stage,the level of SVA VP3 and virus titer were extremely significantly decreased (P<0.01).The proportion of released extracellular virus extremely significantly decreased (P<0.01).But there was no significant effect on other approaches.These results indicated that progesterone could inhibit the adsorption,replication and release of SVA in vitro,but it had no obvious effect on its induction and assembly pathway.【Conclusion】 In this study,progesterone,a natural product with low cytotoxicity and excellent antagonistic effect against SVA,was screened from the small molecule library of natural products.The product could inhibit the adsorption,replication and release of SVA,thus preventing the infection of SVA.This study provided a scientific reference for promoting the development of anti-SVA drugs and studying the infection bias of SVA.
Physicochemical Properties Research of Lactiplantibacillus plantarum MU-6 Bacteriocin and Its Coding Gene Mining
SU Hui, WANG Yunfei, ZHAO Xinling, LIU Mimi, ZOU Mingxiao, DONG Wenlong, LI Guojiang, YIN Baishuang
2024, 51(7):  3166-3175.  doi:10.16431/j.cnki.1671-7236.2024.07.040
Abstract ( 26 )   PDF (8800KB) ( 3 )  
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【Objective】 The purpose of this experiment was to study the stability of bacteriocin produced by a Lactobacillus strain and to mine its coding gene cluster.【Method】 Traditional kimchi from Bayannur city,Inner Mongolia Autonomous Region was placed in MRS broth and cultured at 37 ℃ for 12 h to isolate bacteria.Salmonella Typhimurium was used as indicator bacteria for bacteriostatic tests.The isolated strains were screened and analyzed by 16S rRNA sequencing and BLAST comparison.The stability of bacteriocin produced by isolates was evaluated according to protease,temperature,pH and UV.Whole genome sequencing technology was used to obtain the genomic characteristics of isolates and explore the potential bacteriocin coding gene clusters.【Result】 A Lactobacillus strain producing bactericin against Salmonella Typhimurium was screened from kimchi.Similarity comparison results showed that the isolated strain was 98% similar to Lactiplantibacillus plantarum,which was named MU-6.After the fermentation supernatant concentrate of MU-6 was treated with acid discharge the MRS inhibition zone diameter of the test group was significantly smaller than that of the blank control group. In the hydrogen peroxide exclusion experiment,the inhibition zone diameter did not change. The antibacterial activity decreased after protease treatment.The bacteriocin produced by MU-6 still had high bacteriostatic activity under 100 ℃,pH 3.0-5.0 and UV irradiation.Whole genome sequencing showed that the genome length of MU-6 was 2 782 959 bp,3 129 genes were predicted,and the GC content was 44.43%.The results of gene cluster mining showed that strain MU-6 had 7 core peptides,including Plantaricin J,Plantaricin A,Plantaricin E and Plantaricin F,and so on,most of which belonged to Class Ⅱb bacteriocins.【Conclusion】 In this study,a kind of Lactiplantibacillus plantarum bacteriocin with stable physical and chemical properties and good antibacterial activity was screened,which had good antibacterial effect and had broad application prospects as a natural antiagent.
Effect of Dexmedetomidine on Intestinal Barrier Function and Intestinal Microbiota in Laparoscopic Pneumoperitoneum Rats
NING Xiaqing, WANG Huiying, ZHANG Nuannuan, WANG Jiaqi, LI Xueqin, JIANG Congcong, ZHAI Fan, ZHANG Shixia
2024, 51(7):  3176-3184.  doi:10.16431/j.cnki.1671-7236.2024.07.041
Abstract ( 28 )   PDF (4751KB) ( 4 )  
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【Objective】 The purpose of this study was to investigate the effects of dexmedetomidine (DEX) on intestinal barrier function and intestinal microbiota in rats undergoing laparoscopic pneumoperitoneum.【Method】 Thirty SD rats were randomly divided into three groups:Sham group (n=6),CO2 pneumoperitoneum group (CO2) (n=12) and DEX group (n=12).The rats in CO2 and DEX groups were intraperitoneally injected with 1 mL/kg normal saline and 50 μg/kg DEX 30 min before pneumoperitoneum,respectively,and then maintained at 15 mmHg pneumoperitoneum pressure for 90 min to establish an pneumoperitoneum model.Pneumoperitoneum was impassable in Sham group,and other operations were the same as those in CO2 group.The ileum tissues were collected 12 and 24 h after pneumoperitoneum,and the histopathologic changes were observed.The contents of malondialdehyde (MDA),glutathione (GSH),interleukin-6 (IL-6),IL-1β,tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) activity of ileum in rats were determined by the kit.The mRNA relative expression of occlusive zonal-1 (ZO-1),occlusive protein-1 (Claudin-1) and occlusive protein (Occludin) was detected by Real-time quantitative PCR.The contents of ileal tissue were collected and the changes of ileal microflora were detected by high-throughput paired-end sequencing. 【Result】 Compared with Sham group,the structure of ileum was disordered,intestinal villi deformed and inflammatory cell infiltrated in CO2 group.MDA,IL-6,IL-1β and TNF-α contents, and MPO activity were extremely significantly increased (P<0.01),GSH content and mRNA expression of ZO-1,Occludin,Claudin-1 genes were extremely significantly decreased (P<0.01).The diversity of intestinal flora decreased.At the phylum level,the relative abundance of Firmicutes showed a decreasing trend (P>0.05),while the relative abundance of Actinobacteria and Proteobacteria showed an increasing trend (P>0.05).At the genus level,the relative abundance of Lactobacillus and Allobaculum showed a decreasing trend (P>0.05),while the relative abundance of Bifidobacterium showed an increasing trend (P>0.05).Compared with CO2 group,the ileal tissue structure disorder and intestinal villi deformation of rats in DEX group were relieved,and inflammatory cell infiltration was reduced.GSH content and mRNA expression of ZO-1,Claudin-1 and Occludin genes were extremely significantly or significantly increased (P<0.01 or P<0.05),MDA,IL-6,IL-1β and TNF-α contents, and MPO activity were significantly or extremely significantly decreased (P<0.05 or P<0.01).The diversity of intestinal flora increased.At the phylum level,the relative abundance of Firmicutes showed an increasing trend (P>0.05),while the relative abundance of Actinobacteria and Proteobacteria showed a decreasing trend (P>0.05).At the genus level,the abundance of Lactobacillus showed an increasing trend (P>0.05),while the abundance of Bifidobacterium and Allobaculum showed a decreasing trend (P>0.05).【Conclusion】 DEX could increase the abundance and diversity of intestinal microbiota by improving oxidative stress,the release of inflammatory factors,and the structure of intestinal flora,so as to alleviate intestinal damage caused by laparoscopic pneumoperitoneum.
Isolation,Identification and Drug Resistance Analysis of Riemerella anatipestifer from Some Areas in Guangdong
CHEN Yixing, ZHANG Meilin, LI Shikai, YANG Zhican, LI Wanjia, CHEN Jidang, ZHU Wanjun, ZHANG Jipei
2024, 51(7):  3185-3194.  doi:10.16431/j.cnki.1671-7236.2024.07.042
Abstract ( 27 )   PDF (1879KB) ( 20 )  
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【Objective】 The experiment was aimed to understand the prevalence of Riemerella anatipestifer (RA),drug resistance and drug resistance gene carrier in some areas of Guangdong province. 【Method】 In this study,101 liver,heart,brain and other tissues suspected to be infected with RA were collected from Guangdong during 2020-2022.Bacteria were isolated from the collected tissues,and the isolates were identified by Gram staining,biochemical experiments and PCR.The serotype of isolates identified as RA was identified,and K-B disk diffusion method and PCR were used to conduct drug sensitivity tests and corresponding drug resistance gene detection for isolates.Statistical software SPSS 22.0 was used to analyze the drug-resistant phenotypes and drug-resistant genotypes of these strains.【Result】 The small round,slightly protruding,shiny and creamy colonies of the isolated bacteria were found in the plate culture containing 5% newborn bovine serum TSA.Gram-stained microscopic examination showed that they were negative short bacilli.The results of biochemical identification showed that the isolates did not ferment carbohydrates,and were positive for oxidase,negative for nitrate reduction,Simon’s citrate,peptone water,MR,VP,H2S,and some strains could liquify gelatin and produce urease.The ompA gene was identified by PCR,and 51 strains of RA were isolated in this experiment.Serotype identification of the isolates showed that 5 serotypes were detected,which were serotypes 1,2,4,11 and 18,respectively.Analysis of serotypes of isolates from different host sources showed that serotype 18 was detected only in geese.The results of drug sensitivity test showed that RA was sensitive to flufenicol,ofloxacin,doxycycline,daguamycin,ceftiofurme and cephradine among the 25 drugs selected.The resistance of RA to β-lactam and aminoglycoside was the most serious,among which the resistance rate of β-lactam and aminoglycoside were above 50.0% and 80.0%,respectively.The results of multi-drug resistance showed that the strains with three or more drug resistance were up to 96.08%.Analysis of drug resistance of different host sources showed that the resistance rate of RA isolates from geese to penicillin,amoxicillin,ampicillin and cefazoline was higher than that from ducks.There was no significant difference between duck and goose sources of other drugs.Fifteen drug resistance genes were tested,among which the detection rates of blaCTX-M,aph(3')Ⅱa,qnrB,qnrS,floR,tetM and ermF genes were relatively high,with detection rates of 70.59%,68.63%,76.47%,96.08%,88.24%,82.35% and 96.08%,respectively.The correlation analysis of drug resistance phenotype and drug resistance gene showed that the drug resistance gene aph(3')Ⅱa was significantly associated with the development of neomycin and streptomycin resistant strains (P<0.05),and the drug resistance gene ermF was significantly associated with the emergence of azithromycin-resistant strains (P<0.05). The drug resistance gene tetX was significantly associated with the generation of doxycycline resistant strains (P<0.05). 【Conclusion】 In this study,51 isolates of RA were successfully isolated,and the isolates were resistant to multiple drugs,and the phenomenon of multiple drug resistance was serious.The results of this experiment could provide reference for guiding the formulation of clinical treatment of RA infection in Guangdong province.
Clinical Veterinary Medicine
Imaging Findings and Lamellar Clinicopathological Features of Laminitis in Horse in Parts of Yili Prefecture of Xinjiang
PAN Pingping, HAO Jiangfan, MA Yuhui, MIAO Ronghao, XU Wenkui, SAILEJIBULI Xiarefu, LI Caishan, LI Jianlong, GUO Qingyong
2024, 51(7):  3195-3205.  doi:10.16431/j.cnki.1671-7236.2024.07.043
Abstract ( 25 )   PDF (11548KB) ( 4 )  
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【Objective】 This study was aimed to investigate the lameness of horses in some areas of Yili,Xinjiang,and explore the imaging findings and histopathological features of laminitis in natural clinical cases,so as to provide reference for further elucidating the pathogenic mechanism of laminitis.【Method】 Through clinical examination and lameness grade evaluation,the lameness of 655 horses in part of Yili,Xinjiang was investigated,and imaging diagnosis was made for horses with limb and hoof disease.At the same time,the lamellar tissues of 3 horses with laminitis were collected to make pathological sections,and the histopathological features were observed by hematoxylin-eosin (HE) and periodic acid Schiff (PAS) staining.【Result】 A total of 103 horses in this area showed clinical lameness,and the proportions of lameness≥grade 3 in small,medium,and large scale farms were 10.9%,14.4% and 17.2%,respectively.Imaging differential diagnosis showed that lameness horses had different degrees of arthritis,laminitis and flexor tendinitis.X-ray imaging showed that the third hoof bone remodeled,the dorsal hoof distance widened,the dorsal hoof bone was not parallel to the dorsal hoof wall,the ratio between the measured dorsal hoof distance and the lateral volar length of the road bone was greater than 30%,and the crown extension distance increased.HE staining result showed that the epidermal lamellar and dermal lamellar were loosely connected,the tip of the epidermal lamellar was sharp,the primary epidermal layer was edema to varying degrees,the secondary epidermal lobules were scattered,and there were a large number of hemosiderin-containing and fibroblasts scattered in some areas of the dermal lamellar,and occasionally a small number of eosinophils.PAS staining result showed that the basal membrane and epidermal basal cells were separated to varying degrees,the boundary between epidermis and dermis was blurred,there was no clear boundary of the basal membrane,the number of secondary basal cells increased and their distribution was chaotic,but the hoof layer of the secondary epidermis was shortened,the tip became blunt and the nucleus was elongated.【Conclusion】 The lameness of horses in some areas of Yili,Xinjiang showed an increasing trend with the increase of the scale of horse farms.Common limb and hoof diseases were the main causes of lameness.The imaging findings of some clinical cases of natural laminitis were consistent with the criteria of chronic laminitis,and the histopathology showed that it had developed to a chronic irreversible stage.Imaging and histopathological features could correlate apparent imaging changes and microscopic epidermal changes with the severity of clinical lameness,which could provide theoretical basis for the diagnosis of chronic laminitis.
Observation on the Anesthetic Effect of Two Administration Regimens of Zoletil Combined with Dexmedetomidine on Bama Miniature Pig
LI Junfeng, CHEN Dongni, DOU Xinyi, HOU Xiaoyu, PIAO Xue, ZHAO Yuan, YANG Tianyuan, YANG Haotian, FAN Honggang
2024, 51(7):  3206-3214.  doi:10.16431/j.cnki.1671-7236.2024.07.044
Abstract ( 21 )   PDF (1408KB) ( 10 )  
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【Objective】 This experiment was aimed to explore the effect of different zoletil combined with dexmedetomidine on the anesthetic effect of Bama miniature pigs.【Method】 Eight Bama miniature pigs were randomly divided into intravenous injection (IV) group and intramuscular injection (IM) group,and the anesthesia interval was 14 days.Group IV received zoletil 3.3 mg/kg+dexmedetomidine 8 μg/kg intravenously at the ear edge.In group IM,zoletil 4.6 mg/kg+dexmedetomidine 11.2 μg/kg was injected into the muscles of the neck behind the ear.The period of anesthesia,anesthetic effect,biological reflex and physiological indexes of Bama miniature pigs were monitored during anesthesia.【Result】 The results showed that the induction time,maintenance time and recovery time of anesthesia in group IV were 3.75,85.75 and 30.25 min,respectively.The induction time,maintenance time and recovery time of anesthesia in group IM were 7.13,107.00 and 41.00 min,respectively.Groups IV and IM could maintain surgical anesthesia for 50 and 65 min,respectively.The anesthesia effect score in group IM was significantly higher than that in group IV during 80-110 min (P<0.05).During anesthesia,body temperature decreased.Heart rate and blood oxygen saturation decreased first and then increased.Respiratory rate,systolic pressure,stretch pressure and mean arterial pressure increased first and then decreased.Respiratory rate in group IM was significantly higher than that in group IV during 50-80 min (P<0.05).The systolic blood pressure in group IM was lower than that in group IV during 10-15 min (P<0.05).The total inhibition time of biological reflex in groups IV and IM were 45 and 50 min,respectively.【Conclusion】 Zoletil combined with dexmedetomidine had a good anesthesia effect on Bama miniature pigs,with rapid induction,long maintenance time,stable recovery,and minimal impact on physiological indicators and biological reflexes.Among them,group IM anesthesia plan was more suitable for clinical surgery and scientific research teaching than group IV.
Research Progress on Mitral Degenerative Diseases of Dogs
CHEN Shumin, ZENG Liwei, CHEN Yu, LIU Mengmeng
2024, 51(7):  3215-3223.  doi:10.16431/j.cnki.1671-7236.2024.07.045
Abstract ( 25 )   PDF (1148KB) ( 11 )  
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Myxomatous mitral valve disease (MMVD),also known as degenerative mitral valve disease,is the result of valve degeneration.The disease is one of the most common heart diseases in dogs and causes serious consequences such as mitral regurgitation (MR) and left side congestive heart failure (CHF).The incidence of MMVD increases with age and has a high genetic characteristic in certain breeds of dogs.The pathological changes of MMVD are mainly mitral valve thickening,prolapse and elongation or rupture of the tendinous cord.The pathogenesis of the disease is still not completely clear,and the existing studies have obtained certain results from the aspects of tissue cell level,genetics and proteomics,and signaling pathways.Studies at the tissue and cell level have shown that MMVD is associated with the activation of valve interstitial cell (VIC) and the deposition of collagen and proteoglycan in the extracellular matrix (ECM) in dogs and other species.Genetic and proteomic studies have shown that miRNAs may be involved in the genetic regulation of MMVD,and blood circulating miRNAs are expected to be biomarkers of CHF in dogs with MMVD.From the molecular signaling pathway level,MMVD is closely related to many signaling pathways.Among them,the well-studied 5-hydroxytryptamine (5-HT) and transforming growth factor-β (TGF-β) pathways play important roles in the activation of VICs.At the same time,the mitral valve is also affected by the mechanical stress of pulling and blood flushing during the contraction and relaxation of the heart,and the two pathways interact with each other to promote the occurrence and development of MMVD.In this review,the epidemiological characteristics,pathological changes,clinical symptoms,diagnostic stages,therapeutic methods and disease-related mechanisms of MMVD in dogs were reviewed.