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Table of Content

05 September 2024, Volume 51 Issue 9
Biotechnology
Analysis of Differentially Expressed Genes and Regulatory Pathways in Intramuscular Fat Deposition of Ningxiang Pigs at Different Developmental Stages
XIA Minglong, XIAO Yintao, ZHENG Saizhen, TAN Bie, YIN Yulong, CHEN Jiashun, YIN Jie
2024, 51(9):  3703-3714.  doi:10.16431/j.cnki.1671-7236.2024.09.001
Abstract ( 136 )   PDF (16463KB) ( 65 )  
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【Objective】 This study was conducted to screen out differentially expressed genes (DEGs) and their regulatory signaling pathways related to lipid metabolism by analyzing mRNA expression profiles in longissimus dorsi muscle tissues of Ningxiang pigs at different ages,and determine the expression characteristics of some key genes during the development of intramuscular fat. 【Method】 Ningxiang pigs of 30 (D30, n=3) and 250 (D250, n=3) days old were selected, the longissimus dorsi muscle tissues were collected to extract total RNA for RNA-Seq, and sequencing data was compared and processed.The DEGs related to lipid metabolism were screened for GO function, KEGG pathway and protein interactions analysis.Six DEGs were randomly selected for Real-time quantitative PCR validation. 【Result】 A total of 30 DEGs related to lipid metabolism were screened by RNA-Seq, among which 9 genes were up-regulated and 21 genes were down-regulated.The DEGs were mainly enriched in biological process such as fatty acid oxidation and long-chain fatty acid metabolic process;Cellular components such as mitochondrion and organelle membrane;Molecular functions such as acylglycerol O-acyltransferase activity and oxidoreductase activity.The DEGs were involved signaling pathway such as fatty acid degradation, glycerolipid metabolism and PPAR.Key protein genes such as ACSL1, ACAA2, HADHB, LIPG, GPCPD1 and ALDH2 were screened out by protein interaction analysis, which were involved in the regulation of lipid metabolism through their mRNA expression.Real-time quantitative PCR validation results of six genes such as ACSL1 and CPT1A were consistent with RNA-Seq results. 【Conclusion】 This study screened some key genes such as ACSL1, ACAA2, HADHB, LIPG, GPCPD1 and ALDH2 participating in the regulation of intramuscular fat in Ningxiang pigs, and played a regulatory role through signaling pathways such as fatty acid degradation and glycerolipid metabolism.The resuts provided a theoretical reference for further research on the molecular regulation of genes related to intramuscular fat deposition in pigs.
Cloning,Molecular Characteristics and Tissue Differential Expression Analysis of KLF15 Gene in Buffalo
ZHOU Fangting
2024, 51(9):  3715-3725.  doi:10.16431/j.cnki.1671-7236.2024.09.002
Abstract ( 79 )   PDF (9137KB) ( 36 )  
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【Objective】 The aim of this study was to obtain Krüppel-like factor 15 (KLF15) gene in buffalo, analyze its molecular characteristics and tissue differential expression, and initially elucidate the structure and function of this gene. 【Method】 The CDS region of KLF15 gene in buffalo was cloned, and comparative analysis were performed on gene structure, amino acid sequence identity, physicochemical properties, motifs, conserved domains, biological functions, and so on.The expression of KLF15 gene in various tissues of buffalo were detected by Real-time quantitative PCR. 【Result】 The length of KLF15 gene CDS in buffalo was 1 212 bp, encoding 403 amino acids.Bioinformatics analysis showed that buffalo KLF15 had a high sequence identity with other bovine species,it was a hydrophilic protein in nucleus with no signal peptide or transmembrane helix, which contained conserved domains of the KLF15_N, COG5048 and zf-C2H2 zinc finger structures.Buffalo KLF15 was mainly regulate DNA transcription, bind DNA, bind zinc finger structure.KLF15 gene was most highly expressed in fat of lactating buffalo while in muscle of non-lactating buffalo.In both lactating and non-lactating buffaloes, KLF15 gene was moderately expressed in mammary gland, heart, liver and spleen, while low or almost no expression was observed in lung, kidney, rumen and small intestine. 【Conclusion】 Buffalo KLF15 was a protein localised in nucleus that binding to zinc finger structures to regulate DNA transcription, which played a role in tissues with vigorous lipid synthesis.
Discovering Key Genes in Hair Follicle Development of Tan Sheep at Different Days of Age Based on Transcriptome Data
MA Sijia, ZHAO Zhengwei, WANG Jin, MA Lina
2024, 51(9):  3726-3738.  doi:10.16431/j.cnki.1671-7236.2024.09.003
Abstract ( 63 )   PDF (14574KB) ( 23 )  
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【Objective】 By comparing the morphological changes of hair follicles of skin tissues in Tan sheep at different days of age, this study was aimed to explore key signaling pathways and candidate genes influencing hair follicle development, which was of great significance to reveal the molecular regulation mechanism of hair follicle development in Tan sheep. 【Method】 The skin tissue samples from Tan sheep at 0, 35 and 55 days of age (n=3) were collected for histological analysis using HE staining method, respectively, RNA-Seq was conducted through Illumina NovaSeq 6000 platform, and differentially expressed genes (DEGs) was analyzed through DESeq2 software.KEGG pathway enrichment analysis was also performed on DEGs to further screen genes related to hair follicle development, and 10 DEGs were randomly selected for Real-time quantitative PCR validation of RNA-Seq results. 【Result】 HE staining results showed that with the days of age in Tan sheep increasing, the number of primary hair follicles was significantly or extremely significantly decreased (P<0.05 or P<0.01), while the number of secondary hair follicles was extremely significantly increased(P<0.01), and follicle cluster structures were formed by secondary hair follicles which were gradually distributed around primary hair follicles.RNA-Seq results indicated that a total of 681 DEGs were screened in B35 vs B0 comparison group, including 368 up-regulated and 313 down-regulated, a total of 852 DEGs were screened in B55 vs B0 comparison group, including 469 up-regulated and 383 down-regulated, and a total of 194 DEGs were screened in B55 vs B35 comparison group, including 99 up-regulated and 95 down-regulated.1 274 DEGs were yielded from the intersection of 3 comparison groups of DEGs, which could divide into 4 clusters (K1-K4).After conducting KEGG enrichment analysis on each cluster, it was found that these genes had a significant enrichment in signaling pathways associated with hair follicle morphogenesis such as PI3K-Akt and MAPK.Among these genes, PDGFRA, PDGFRB, PDGFB, FGFR4 and NR4A1 played important roles in hair follicle development and molecular regulation mechanism.The results of Real-time quantitative PCR showed that the expression of 10 DEGs were consistent with RNA-Seq results, which indicated the reliability of the sequencing results. 【Conclusion】 This study identified that PDGFRA, PDGFRB, PDGFB, FGFR4 and NR4A1 were important candidate genes in the process of hair follicle development through RNA-Seq analysis, which provided a theoretical basis for further elucidation of the molecular mechanisms underlying the fur characters of Tan sheep.
Physiological and Biochemical
Study on Apoptosis of Mouse Leydig Cells Induced by BPA Based on Mitochondrial Damage
YANG Wenzhe, LIU Kexiang, WANG Shirui, ZHAO Tong, PAN Feilong, ZHAO Shuchen, ZHAO Lijia
2024, 51(9):  3739-3751.  doi:10.16431/j.cnki.1671-7236.2024.09.004
Abstract ( 53 )   PDF (11089KB) ( 15 )  
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【Objective】 The aim of this study was to investigate the mechanism of action of apoptosis induced by bisphenol A (BPA) exposure in mouse Leydig cells (TM3) from the perspective of mitochondrial damage. 【Method】 The TM3 cells were treated with different concentrations of BPA (0, 10, 30, 60, 90, 120 and 150 μmol/L) for 24 h, and the lowest BPA concentration causing apoptosis was screened by CCK-8 method.TM3 cells were randomly divided into control group (0 μmol/L BPA) and BPA group (60 μmol/L BPA) with three replicates in each group for 24 h.The state of cells was observed by inverted light microscope and apoptosis was observed by TUNEL staining.The TM3 cells were randomly divided into control group (0 μmol/L BPA), BPA group (60 μmol/L BPA), and BPA and N-acetyl-L-cysteine (NAC) co-treatment group (60 μmol/L BPA+5 mmol/L NAC), with three replicates in each group for 24 h.The content of malondialdehyde (MDA) and the activity of total superoxide dismutase (T-SOD) in the supernatant of cell culture were detected by ELISA method, the level of intracellular reactive oxygen species (ROS) was detected by kit, and the mitochondrial membrane potential was observed by JC-1 staining.The mRNA expression levels of glutathione peroxidase 1 (Gpx1), glutamate-cysteine ligase modifier subunit (Gclm), catalase (Cat), glutamate-cysteine ligase catalytic subunit (Gclc), mitochondrial fusion protein 2 (Mfn2), NAD-dependent protein deacetylase Sirtuin3 (Sirt3), caspase 3 (Casp3), cytochrome C (Cycs), B-cell lymphoma-2(BCL-2) associated X protein (Bax) in cells were detected by Real-time quantitative PCR.The relative expression of BAX and apoptosis regulatory factor BCL-2 proteins were detected by Western blotting, and the cell apoptosis rate was detected by flow cytometry. 【Result】 After treated with 60 μmol/L BPA for 24 h, the relative survival rate of TM3 cells began to decrease extremely significantly (P<0.01), the number of apoptosis was extremely significantly increased (P<0.01), and the number of cells were obviously decreased.Compared with control group, after 24 h of treatment with 60 μmol/L BPA, MDA content in TM3 cells was significantly increased (P<0.01), T-SOD activity was decreased significantly (P<0.01), the mRNA expression of antioxidation related genes (Gpx1, Cat, Gclm and Gclc genes) were significantly or extremely significantly reduced (P<0.05 or P<0.01), ROS production was extremely significantly increased (P<0.01).The red/green fluorescence intensity ratio was extremely significantly decreased (P<0.01).The mRNA expression of mitochondrial function related genes (Mfn2 and Sirt3 genes) were extremely significantly decreased (P<0.01), while the mRNA expression of mitochondrial apoptosis pathway related genes (Casp3, Cycs and Bax genes) were extremely significantly increased (P<0.01), the expression of BAX protein was extremely significantly increased (P<0.01), the relative expression of BCL-2 protein was extremely significantly decreased and the apoptosis rate was extremely significantly increased (P<0.01).Compared with the BPA-treated group, the MDA content in TM3 cells of the BPA+NAC treatment group was significantly reduced (P<0.05), T-SOD activity was significantly increased (P<0.05), the mRNA expression levels of antioxidant-related genes (Gpx1, Cat, Gclm and Gclc genes) were significantly increased (P<0.05), the ROS production was extremely significantly decreased (P<0.01).The red/green fluorescence intensity ratio was increased significantly (P<0.05).The mRNA expression levels of mitochondrial function-related genes (Mfn2 and Sirt3 genes) were significantly or extremely significantly increased (P<0.05 or P<0.01), and the mRNA expression of mitochondrial apoptosis pathway related genes (Casp3, Cycs, Bax genes) were extremely significantly decreased (P<0.01).The expression of BAX protein was extremely significantly decreased (P<0.01), while the relative expression of BCL-2 extremely significantly increased (P<0.01), and apoptosis rate was extremely significantly decreased (P<0.01). 【Conclusion】 BPA exposure induced oxidative stress, resulting in mitochondrial dysfunction and subsequently triggering apoptosis in TM3 cells.
Establishment and Biological Characteristics of Fibroblasts from Different Origins in Asian Elephant
LI Jinda, PENG Ziwei, Muhammad Ameen Jamal, JIAO Deling, WEI Hongjiang
2024, 51(9):  3752-3761.  doi:10.16431/j.cnki.1671-7236.2024.09.005
Abstract ( 47 )   PDF (15617KB) ( 22 )  
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【Objective】 The aim of this study was to establish Asian elephant fibroblast cell lines of different age and tissue source, and conduct cryopreservation and cell biological characteristics, so as to provide theoretical basis for the preservation and development of endangered animal genetic resources. 【Method】 Samples from 6-year-old Asian elephant ear edge tissue (AE00), newborn Asian elephant umbilical cord tissue (AE01), and 4-year-old Asian elephant ear edge tissue (AE02) 16 hours after death were collected, and fibroblast cell lines were established for passage culture and cryopreservation.The biological characteristics of fibroblasts from different age and tissue sources were analyzed by cell morphology, reproductive ability, aging, and apoptosis levels.Green fluorescent protein (GFP) transfection screening and karyotype detection were performed to analyze the genetic stability of Asian elephant fibroblasts. 【Result】 Three Asian elephant fibroblast cell lines were successfully established and cryopreserved.After recovery, the cells maintained a certain level of vitality.After continuous passage and culture, the cell morphology was spindle shaped.AE02 fibroblasts had the best cryopreservation activity and cell proliferation ability, with an apoptosis rate of AE01 (16.7%)>AE00 (6.4%)>AE02 (3.8%), and an aging rate of AE00 (35.9%)>AE01 (17.2%)>AE02 (7.8%).After 19 passages, the number of chromosomes in the AE02 fibroblasts remained stable.GFP transfection screening of AE00 resulted in stable expression of GFP in Asian elephant fibroblast cell lines. 【Conclusion】 In this study, Asian elephant fibroblast cell lines with different ages and tissue sources were successfully established.The cell morphology was spindle shaped, and the proliferation ability, cell aging, and apoptosis levels of fibroblast cell lines with different ages and tissue sources were different.After continuous passage, the number of cell chromosomes remained stable, enabling stable transfection and long-term screening of exogenous genes.It could be an important method for the long-term preservation and revival of endangered animal genetic resources.
Effect of Taraxasterol on Apoptosis and Autophagy of Chicken Primary Hepatocytes Induced by AFB1
YU Minghong, WANG Meng, GE Bingjie, YAN Kexin, WANG Wei, LIU Xinman, LIU Xiaotong, QIU Qian, SANG Rui, ZHANG Xuemei
2024, 51(9):  3762-3770.  doi:10.16431/j.cnki.1671-7236.2024.09.006
Abstract ( 41 )   PDF (3946KB) ( 7 )  
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【Objective】 This experiment was aimed to explore the effect and mechanism of taraxasterol on apoptosis and autophagy of chicken primary hepatocytes induced by aflatoxin B1 (AFB1). 【Method】 The toxicity of taraxasterol to chicken primary hepatocytes was determined by MTT assay and the safe concentration of taraxosterol was determined.Chicken primary hepatocytes were divided into normal, model (AFB1,0.05 μg/mL), silybinin-positive groups (Sil,2 μg/mL) and low (5 μg/mL), medium (10 μg/mL) and high (20 μg/mL) dose of taraxasterol groups.Cells of each test group were collected after treatment with the corresponding concentration of drug.The apoptosis and autophagy were measured by flow cytometry and CYTO-ID.The mRNA expression of cytochrome enzyme, apoptosis and autophagy related genes in chicken primary hepatocytes were determined by Real-time quantitative PCR. 【Result】 Taraxosterol had no obvious toxicity to chicken primary hepatocytes when the concentration of taraxosterol was 5-25 μg/mL.Therefore, the concentrations of taraxosterol in low, medium and high dose groups were set as 5, 10 and 20 μg/mL, respectively.Flow cytometry and CYTO-ID detection results showed that compared with normal group, the number of green fluorescent labeled autophagy follicles in the chicken primary hepatocytes of AFB1 group was significantly increased.Compared with AFB1 group, the number of green fluorescentially labeled autophagy follicles in chicken primary hepatocytes in silybin and taraxosterol different dose groups was significantly reduced.Real-time quantitative PCR results showed that compared with normal group, the mRNA expressions of cytochrome enzyme P450 1A5 (CYP1A5), CYP3A37A, cysteine proteolytic enzyme 3 (Caspase-3), Caspase-9, Beclin-1, autophagy protein 5 (ATG-5), microtubule-associated protein light chain 3 Ⅰ (LC3-Ⅰ) and LC3-Ⅱ genes of chicken primary hepatocytes in AFB1 group were extremely significantly increased (P<0.01).Compared with AFB1 group, mRNA expression levels of CYP1A5, CYP3A37, Caspase-3, Caspase-9, Beclin-1, ATG-5, LC3-Ⅰ and LC3-Ⅱ genes of chicken primary hepatocytes in silybin and taraxosterol different dose groups were extremely significantly decreased (P<0.01). 【Conclusion】 Taraxosterol could protect chicken primary hepatocytes induced by AFB1 by influencing the expression of cytochrome enzyme, apoptosis and autophagy related genes.
Effects of Glutamate Dehydrogenase gdhA Gene Deficiency on Antioxidant Stress in Listeria monocytogenes
TIAN Lanxin, YANG Yuting, QIN Yi, ZHANG Zheng, TIAN Guangming, FANG Chun
2024, 51(9):  3771-3779.  doi:10.16431/j.cnki.1671-7236.2024.09.007
Abstract ( 55 )   PDF (2454KB) ( 11 )  
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【Objective】 The aim of this study was to elucidate the effect of glutamate dehydrogenase gdhA gene on the antioxidant stress ability of Listeria monocytogenes, and explore the regulatory effect of glnR gene on gdhA gene. 【Method】 The gdhA gene deletion strain of Listeria monocytogenes was constructed by homologous recombination technique.The growth ability of parental strain 10403S, deletion strain 10403S-ΔgdhA and complementation strain 10403S-CΔgdhA were measured by growth curves.The antioxidant stress survival test was used to detect the effect of gdhA gene deletion on the antioxidant stress ability of Listeria monocytogenes.The rabbit polyclonal antibody was prepared by inducing the expression and purification of GdhA protein, and the specificity of the antibody was detected by Western blotting.GFP reporter plasmid carrying gdhA gene promoter region was constructed to determine the effect of glnR gene deletion on gdhA gene transcription level under oxidative stress.Western blotting was used to detect the effect of glnR gene deletion on the expression level of GdhA protein under oxidative stress, and to detect the effect of glnR gene deletion on the survival ability of strains. 【Result】 The results of growth curve showed that the deletion of gdhA gene did not affect the growth ability of Listeria monocytogenes under normal conditions.The survival ability of deletion strain 10403S-ΔgdhA was extremely significantly higher than that of the parental strain 10403S under the condition of oxidative stress with 20 mmol/L H2O2 (P<0.01).Western blotting detection indicated that the rabbit polyclonal antibody to GdhA was successfully produced.GFP fluorescence analysis showed that the transcription level of the gdhA gene was extremely significantly increased after the deletion of glnR gene (P<0.01).Under oxidative stress, the expression level of GdhA protein was extremely significantly increased after glnR gene deletion (P<0.01).glnR gene deletion significantly decreased the viability of strains (P<0.05). 【Conclusion】 The deletion of gdhA gene, which encoded for glutamate dehydrogenase, did not affect the normal growth of Listeria monocytogenes parental strain 10403S, but significantly increased the survival ability under the oxidative stress.The transcription level of gdhA gene and the expression level of GdhA protein were negatively regulated by glnR gene.
Nutrition and Feed
Effect of Allium mongolicum Regel Powder on Nutrient Apparent Digestibility and Plasma Metabolomics in Angus Calves
HE Jianjian, GAO Huixia, SUN Chenxu, YAO Haibo, XIE Yaodi, YU Aihuan, HU Jinsheng, WANG He, DUAN Yueyan, TANG Defu, LIU Wangjing
2024, 51(9):  3780-3793.  doi:10.16431/j.cnki.1671-7236.2024.09.008
Abstract ( 74 )   PDF (11690KB) ( 63 )  
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【Objective】 The purpose of this experiment was to explore the effects of different levels of Allium mongolicum Regel powder on nutrient apparent digestibility and the characteristics of plasma metabolomics in Angus calves,in order to provide some theoretical basis for the application of Allium mongolicum Regel as a plant additive in Angus calves production. 【Method】 Twenty-four Angus calves were randomly divided into 4 groups, with 6 calves in each group.The calves in control group (CON) were fed with TMR basal diet, and the calves in experimental groups were fed with Allium mongolicum Regel powder at the dosage of 10 (LAMR), 15 (MAMR) and 20 (HAMR) g/d, respectively.The adaptation period was 15 days and the experimental period lasted for 120 days, the experimental period was divided into 2 parts of 60 days each.Feed and fecal samples were collected for 7 days from 114th-120th days of the experimental period to determine the apparent digestibility of nutrients, and blood samples were collected before the morning feeding on the 113th day of the trial period for the determination of plasma non-targeted metabonomics. 【Result】 Compared with CON group, the apparent digestibility of crude protein, calcium, acidic detergent fiber and neutral detergent fiber of Angus calves in LAMR and MAMR groups were significantly increased (P<0.05), and the effect of LAMR group was the best.The results of metabonomics showed that 62 differential metabolites were screened out, of which 11 were up-regulated and 30 down-regulated in positive ion mode, and 1 was up-regulated and 20 down-regulated in negative ion mode.These differential metabolites were mainly lipids and lipid-like molecules, organic acids and derivatives and organo-heterocyclic compounds, they mainly regulated the metabolic pathway of alpha-linolenic acid by up-regulating metabolites such as PC 19∶0_20∶3, PC 18∶0_18∶1 and 25-hydroxycholecalciferol, and promoted the utilization of nutrients in Angus calves. 【Conclusion】 Adding 10 g/d Allium mongolicum Regel powder to the diet had the best effect on the apparent digestibility of nutrients in Angus calves.The active components in Allium mongolicum Regel powder might regulate the pathway of alpha-linolenic acid metabolic by up-regulating the metabolites such as glycerophospholipid compounds (PC 19∶0_20∶3, PC 18∶0_18∶1) and 25-hydroxycholecalciferol, thus affecting the utilization of nutrients in Angus calves.
Comparative Analysis of Yield and Nutritional Value of Six Perennial Grasses in Alpine Area of Qinghai
LIU Jinping, ZHANG Nannan, ZHAO Xinsheng, HUANG Yayu, YANG Yingkui, ZHOU Xueli, ZHONG Rongzhen, ZHAO Guojun, BAI Binqiang, HAO Lizhuang
2024, 51(9):  3794-3806.  doi:10.16431/j.cnki.1671-7236.2024.09.009
Abstract ( 64 )   PDF (1420KB) ( 13 )  
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【Objective】 The aim of this experiment was to evaluate the nutritional value of artificially cultivated grass perennial forages in alpine region of Qinghai, in order to provide reference for the development of high-altitude animal husbandry and the demand for livestock feed. 【Method】 The experiment focused on common cold regions such as Agropyron cristatum, Lolium perenne L., Triticale, Elymus tangutorum, Poa crymophila and Poa pratensis L.as research objects, and three adult male yaks were selected as rumen fluid donor animals, while the nutritional value of the pasture grasses was evaluated by in vitro gas production method combined with the pasture grass production. 【Result】 ① The fresh grass yield and dry grass yield of Triticale were significantly higher than those of other grasses (P<0.05).The dry to fresh ratio of Triticale and Elymus tangutorum was low, which was significantly lower than that of other grasses (P<0.05).② The dry matter (DM) and crude protein (CP) contents of Triticale were significantly higher than those of Poa crymophila and Poa pratensis L.(P<0.05).The content of crude ash (Ash), neutral detergent fiber (NDF) and acid detergent fiber (ADF) was the highest in Poa pratensis L..The ether extract (EE) content of Agropyron cristatum was significantly higher than that of other grasses (P<0.05).The calcium (Ca) content of Triticale was significantly higher than that of other grasses (P<0.05), while the phosphorus (P) content was the same as that of Agropyron cristatum.③ The in vitro gas production (GP) of Triticale at 24, 48 and 72 h was significantly higher than that of other grasses (P<0.05), and the gas production among six cultivated forages showed an increasing trend over time.④ The pH range of in vitro fermentation broth of six cultivated forages was 6.76 to 6.98.There were significant differences in the total volatile fatty acid, acetic acid and propionic acid content between Triticale and Elymus tangutorum compared with other grasses (P<0.05).Triticale had the highest content of butyric acid and valeric acid.The ratio to acetate to propionate of Poa pratensis L.were significantly higher than that of other grasses (P<0.05).The concentration range of ammonia nitrogen (NH3-N) of six cultivated forages was 84.70-112.60 mg/L.The in vitro dry matter digestibility (IVDMD) of Poa crymophila was significantly higher than that of other grasses (P<0.05).⑤ The yield of nutrients per unit area of Triticale was the highest, which was significantly higher than that of other grasses (P<0.05). 【Conclusion】 In summary, Triticale had the best production performance and high nutritional value, and could be used as a suitable pasture for cultivation in alpine region of Qinghai.
Effects of Fermented Black Solider Fly (Hermetia illucens L.) on Growth Performance, Body Composition,Antioxidant and Immune Ability of Hybrid Snakehead
ZHU Xifeng, HUANG Wenqing, LI Guoli, WANG Menghua, PENG Kai, WANG Guoxia, HUANG Yanhua
2024, 51(9):  3807-3816.  doi:10.16431/j.cnki.1671-7236.2024.09.010
Abstract ( 63 )   PDF (1180KB) ( 16 )  
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【Objective】 This experiment was conducted to investigate the effects of adding fermented Black Solider fly (Hermetia illucens L.) in feed on growth performance, body composition, serum biochemical indexes, and antioxidant and immune ability of hybrid snakehead (Channa maculate ♀×Channa argus ♂). 【Method】 A total of 1 920 hybrid snakehead with an initial weight of (5.88±0.01) g were randomly divided into 6 groups with 4 replicates per group and 80 fish per replicate.Hybrid snakehead were fed 6 experimental diets supplemented with 0 (G0 group, as control group), 2% (G2 group), 4% (G4 group), 6% (G6 group), 8% (G8 group) and 10% (G10 group) of fermented Black Solider fly wet matter for 56 days, respectively.At the end of experiment, the weight, quantity, and food intake of hybrid snakehead in each net cage were counted to calculate their growth performance.Six hybrid snakehead were randomly selected from each pet for body composition analysis.Blood was collected from the caudal vein of sampled hybrid snakehead to separate serum for determination of serum biochemical and serum antioxidant and immune indicators.Liver were collected from sampled hybrid snakehead for determination of antioxidant and immune indicators. 【Result】 Compared with G0 group, the final body weight, weight gain rate, specific growth rate in G2 to G8 groups were significantly increased (P<0.05) and feed intake was significantly increased in G4 group (P<0.05).The feed coefficient rate was significantly decreased (P<0.05) and survival rate was significantly increased in G6 and G8 groups (P<0.05)).There were no significant differences in condition factor, viscerosomatic index and intestosomatic index among groups (P>0.05), and patosomatic index was significantly decreased in G2, G4, G6 and G10 groups compared with G0 group (P<0.05).The contents of moisture, crude protein, crude lipid, ash, calcium and total phosphorus were not significantly different in whole body of hybrid snakehead among groups (P>0.05).There was no significant difference in serum total protein, glucose, cholesterol, urea nitrogen contents and alanine transaminase and aspartate transaminase activities among groups (P>0.05), and the serum triglyceride content was significantly decreased in G2 to G10 groups compared with G0 group (P<0.05).Compared with G0 group, the serum total antioxidant capacity in G2 group was significantly increased (P<0.05) and the activities of peroxidase and superoxide dismutase in liver were significantly increased in G2 to G8 groups (P<0.05), and the serum lysozyme activity was significantly increased in G2 to G10 groups (P<0.05). 【Conclusion】 Fermented Black Solider fly could significantly improve growth performance, decrease patosomatic index and the serum triglyceride content, and increase antioxidant and immune ability of hybrid snakehead.The appropriate addition levels of fermented Black Solider fly in feed for hybrid snakehead were 2%-8%.
Effects of Different Feeding Methods, Castration on Fattening,Slaughtering Performance and Meat Quality of Shorthorn
DAI Sifan, ZHANG Ruiyun, WANG Zibei, ZHI Li, ZHAO Minlin, ZHU Shengquan, YAO Xinrong, LIU Yiduan, WANG Lixing, QU Kaixing, WU Dongwang, MAO Huaming, LI Qing
2024, 51(9):  3817-3827.  doi:10.16431/j.cnki.1671-7236.2024.09.011
Abstract ( 63 )   PDF (1188KB) ( 15 )  
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【Objective】 The aim of this study was to investigate the effects of different feeding methods, castration on growth and fattening, slaughtering performance and meat quality of Shorthorn were studied. 【Method】 18 Shorthorn with similar body weight at about 17 months of age were selected, including 12 bulls (divided into 2 groups:Grazing bull group and fattening bull group, with 6 bulls in each group) and 6 steers (fattening steer group).The bulls in grazing bull group were raised by grazing, and in fattening bull group and fattening steer group were fattened in a fence style and freely fed with total mixed ration (TMR).All bulls in three groups were fed and fattened until 24 months of age and slaughtered.The fattening performance, slaughter performance and meat quality of Shorthorn were measured. 【Result】 Under different feeding methods, the final weight, average daily gain (ADG), body slanting length, chest girth and abdominal circumference, carcass weight, slaughter rate, meat percentage, meat-bone ratio, carcass meat production rate, backfat thickness, loin muscle area, marling, total meat weight, as well as the weight of heart, liver, spleen, lungs, kidneys, and the crude fat and protein content of the longissimus dorsi muscle and biceps femoris muscle in fattening bull group were extremely significantly or significantly higher than those in grazing bull group (P<0.01 or P<0.05).The shear force and water content of the longissimus dorsi muscle and biceps femoris were significantly or extremely significantly lower than those in the grazing bull group (P<0.05 or P<0.01). Under the same feeding method, the ADG, loin muscle area, weight of choicest and premium cuts, heart and liver weight, shear force of the longissimus dorsi muscle, water content of the biceps femoris and longissimus dorsi muscle in fattening bull group were significantly or extremely significantly higher than those in the fattening steer group (P<0.05 or P<0.01), while marling,crude fat content of biceps femoris and longest back muscle, and crude protein content of the biceps femoris were significantly or extremely significantly lower than those in fattening steer group (P<0.05 or P<0.01). 【Conclusion】 Fattening could improve growth and slaughter performance, beef tenderness and nutritional levels, while castration could reduce the fattening and slaughter performance of Shorthorn.However, it could improve the marbling grade, tenderness, and beef quality of Shorthorn.Therefore, castration should be decided based on market demand.
Protective Effect of Selenomethionine on Spleen Injury Induced by Aflatoxin B1 in Rabbits
LIU Shiyang, ZHANG Ziqiang, WANG Shishi, NIU Bingyu, KONG Dejing, ZHANG Zhikai, LIU Yumei
2024, 51(9):  3828-3836.  doi:10.16431/j.cnki.1671-7236.2024.09.012
Abstract ( 61 )   PDF (15948KB) ( 20 )  
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【Objective】 To investigate the protective effect of selenomethionine (SeMet) on spleen injury induced by aflatoxin B1 (AFB1) in rabbits. 【Method】 50 rabbits aged 35 days were randomly divided into 5 groups, 10 in each group, namely control group (basal diet), model group (0.3 mg/kg BW AFB1+basal diet), Se-1 group (0.2 mg/kg SeMet+0.3 mg/kg BW AFB1+basal diet), Se-2 group (0.4 mg/kg SeMet+0.3 mg/kg BW AFB1+basal diet) and Se-3 group (0.6 mg/kg SeMet+0.3 mg/kg BW AFB1+basal diet).The experimental period was 21 days.After the experiment, the spleen was taken out to observe the morphological changes of spleen tissue by HE staining, PCNA protein expression by immunohistochemical staining, apoptosis rate by TUNEL staining, and the oxidative stress level and inflammatory factors content in spleen tissue were detected. 【Result】 Compared with control group, the red pulp and white pulp of the spleen of rabbits in model group were arranged disorder, the number of lymphocytes decreased obviously, PCNA protein expression decreased and a large number of apoptotic cells appeared.Compared with model group, SeMet supplementation in diet could significantly improve the pathological damage of spleen induced by AFB1 poisoning in rabbits.At the same time, SeMet could also increase the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in spleen tissue (P<0.01), and significantly reduce the contents of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β (P<0.01). 【Conclusion】 SeMet supplementation could effectively improve AFB1-induced spleen injury in rabbits by increasing the activity of antioxidant enzymes and decreasing the expression of inflammatory factors.Among them, 0.2 mg/kg SeMet had the best response.
The Effect of Compound Bacteria and Cellulase on Nutritional Value of Hedysarum scoparium Microbial Fermentation
YANG Shuangming, TIAN Mei, SHEN Yan, ZHAO Xiaoqing, WANG Linian, WANG Wenqing, ZHAO Xiaoying, XU Xiaofeng, ZHANG Lili
2024, 51(9):  3837-3846.  doi:10.16431/j.cnki.1671-7236.2024.09.013
Abstract ( 54 )   PDF (1173KB) ( 13 )  
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【Objective】 The aim of this study was to investigate the effects of adding Lactobacillus plantarum, Bacillus licheniformis, and cellulase on the fermentation quality and microbial community structure of Hedysarum scoparium microbial fermentation. 【Method】 The experiment was conducted using Hedysarum scoparium as raw materials.Four groups were set up, named control group (no microbial fermentation agents added), group J (Bacillus licheniformisLactobacillus plantarum), group M (cellulase), and group JM (Bacillus licheniformisLactobacillus plantarum+cellulase), respectively.The substrate and the additives were mixed well according to the experimental design and sealed in transparent polyethylene bag for fermentation at room temperature in a dark place.Samples were taken on the 7th, 14th, 30th, and 60th days of fermentation.The nutritional value,fermentation quality, and phenolic content of microbial fermented feed from Hedysarum scoparium was measured. 【Result】 The results showed that the synergistic fermentation of Lactobacillus plantarum, Bacillus licheniformis, and cellulase could avoid dry matter loss of Hedysarum scriums and preserve nutritional components.On the 60th day of fermentation, the neutral and acidic washing fibers of the three treatment groups were significantly lower than control group (P<0.05), while the neutral washing fibers of the group M were significantly lower than those of the groups J and JM (P<0.05).On the 30th and 60th day of fermentation, the content of lactic acid and acetic acid in the groups J and JM were significantly higher than that in control group (P<0.05). And the pH of each treatment group was below 4.2, indicating excellent quality of microbial fermentation.As the fermentation time increased, the tannin content in each treatment group showed a decreasing trend.On the 60th day of fermentation, the tannin, plant anthocyanins, and total plant phenols content in group JM were significantly lower than those in control group (P<0.05). 【Conclusion】 All microbial fermentation agents could improve the nutritional composition and fermentation quality of Hedysarum scopariums to a certain extent.The addition of Lactobacillus plantarum, Bacillus licheniformis, and cellulase for collaborative fermentation for 60 days had the best effect on Hedysarum scopariums microbial fermentation.
Effects of Morinda officinalis Polysaccharide on Growth Performance,Serum Immune and Antioxidant Indexes and Immune Organs Development of Wenchang Chickens
WU Wei, SUN Ruiping, LIU Jie, LIU Quanwei, LIU Yuhang, ZHANG Yan, OUYANG Kun, ZHAO Guiping, ZHANG Xuli, WANG Xiuping, WEI Limin, ZHANG Haojie
2024, 51(9):  3847-3856.  doi:10.16431/j.cnki.1671-7236.2024.09.014
Abstract ( 59 )   PDF (1171KB) ( 26 )  
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【Objective】 This experiment was conducted to investigate the effects of Morinda officinalis polysaccharides (MOP) on growth performance, serum immune and antioxidant function and immune organs development of Wenchang chickens, in order to provide reference for the application of MOP in healthy breeding of broilers. 【Method】 Six hundred 6-day-old healthy female Wenchang chickens were selected and randomly divided into 5 groups, with 8 replicates in each group and 15 chickens in each replicate.The chickens in control group was fed with basal diet, and the chickens in the experiment groups were fed with basal diet supplemented with 250, 500, 1 000 and 2 000 mg/kg MOP, respectively.The experiment lasted for 40 days.Body weights were taken on the 1st and 40th day of the trial to calculate average daily gain (ADG).During the trial period, the leftover feed was recorded in repetition, and average daily feed intake (ADFI) and feed-to-gain ratio (F/G) were calculated.On the 40th day of the trial, one chicken from each replicate was selected for blood sampling and tissue collection.Serum biochemical indicator, antioxidant indicator, immune indicator and immune organ index of Wenchang chickens were determined. 【Result】 Compared with control group, ① Adding different levels of MOP had no significant effects on the final body weight, ADG and ADFI of Wenchang chickens (P>0.05),but F/G of 250 mg/kg MOP group was significantly increased (P<0.05), and F/G of other experiment groups did not have significant changes (P>0.05).②The serum IgG content in 250 mg/kg MOP group and the serum IgM content in 500 mg/kg MOP group were significantly increased (P<0.05), serum MDA contents in 500 and 1 000 mg/kg MOP groups were significantly reduced (P<0.05), and serum GSH-Px activity in 2 000 mg/kg MOP group was significantly increased (P<0.05).③Dietary addition of 500, 1 000 and 2 000 mg/kg MOP could significantly increase the bursa of Fabricius index of Wenchang chickens (P<0.05). 【Conclusion】 The addition of 250 and 500 mg/kg MOP in this experiment could increase the serum immunoglobulin content of Wenchang chickens, and the addition of 500, 1 000 and 2 000 mg/kg MOP could improve the serum antioxidant indicator of Wenchang chickens and promote the growth and development of the bursa of Fabricius.In this experiment, the appropriate amount of MOP addition was 500 mg/kg.
Role of ROS Accumulation in Apoptosis of Jejunal Epithelial Cells Induced by Aflatoxin B1 in Piglets
HANG Man, WANG Yanhui, LI Hanxiao, LIU Enci, LI Qinghao, YU Rui, LUO Qin, JIN Xin
2024, 51(9):  3857-3866.  doi:10.16431/j.cnki.1671-7236.2024.09.015
Abstract ( 55 )   PDF (4311KB) ( 14 )  
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【Objective】 To investigate the effect of aflatoxin B1 (AFB1) on the apoptosis of porcine jejunum epithelial cells (IPEC-J2) by inducing intracellular reactive oxygen species (ROS) production, providing a theoretical basis for the pathogenic mechanism of AFB1. 【Methods】 Cells were stimulated with AFB1 at concentrations of 0, 20, 40, 60, 80 and 100 μg/mL for 12 h.CCK-8 assay kit was used to detect cell viability and determine the toxic effect of AFB1 on IPEC-J2 cells, and then immunofluorescence assay were used to detect intracellular ROS level after the cells were stimulated with 0, 10, 20 and 30 μg/mL AFB1.At the same time, Real-time quantitative PCR and Western blotting were used to detect the expression of key factors related to apoptotic pathways in cells.ROS inhibitor N-acetyl-L-cysteine (NAC) was used to stimulate cells, and then the effect of AFB1 on the expression of apoptotic factors in cells was detected. 【Result】 The results showed that AFB1 at concentrations of 0-20 μg/mL had no significant effect on cell viability (P>0.05), and as the concentration of AFB1 increased, the viability of IPEC-J2 cells showed a significant or extremely significant decrease trend compared with blank control group (P<0.05 or P<0.01).ROS immunofluorescence analysis showed that ROS level was highly dependent on AFB1 concentration, and as the concentration of AFB1 increased, the intracellular ROS level was significantly or extremely significantly increased compared with blank control group (P<0.05 or P<0.01).Real-time quantitative PCR and Western blotting results showed that when the concentration of AFB1 was 20 μg/mL, the expression of Caspase-3 mRNA and protein were significantly higher than that of blank control group (P<0.05).When the concentration of AFB1 was 30 μg/mL, the expression of Bax and Bax/Bcl-2 mRNA and protein were significantly higher than that of blank control group (P<0.05), while the protein expressions of Bax and Bcl-2 were significantly lower than those of blank control group(P<0.05).Pretreatment of cells with ROS inhibitor NAC followed by AFB1 addition resulted in a decrease in apoptotic factors to varying degrees compared with the AFB1-only treatment group (P<0.05). 【Conclusion】 AFB1 could promote the upregulation of apoptotic factors Bax, Bcl-2, and Caspase-3 in IPEC-J2 cells, and this process might be related to the accumulation of intracellular reactive oxygen species.
Research Progress on the Nutritional Value of Cottonseed Protein and Its Application in Animal Feed
LI Min, LYU Dan, CHEN Bing, CAO Junming, ZHAO Hongxia
2024, 51(9):  3867-3877.  doi:10.16431/j.cnki.1671-7236.2024.09.016
Abstract ( 100 )   PDF (1222KB) ( 38 )  
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Due to the rapid development of livestock, poultry, and aquaculture industries, high-quality feed protein is facing challenges such as increasing demand, rising raw material prices, and inconsistent quality.Finding high-quality alternative proteins has become the key to solving these problems.Cottonseed protein, as a high-quality protein, is widely used in animal feed.It has a protein content of over 40%, is abundant in resources, and has a low price.However, the presence of anti-nutritional factors in cottonseed protein limits its application in production.With the continuous development of cottonseed protein production technology, the removal of anti-nutritional factors from cottonseed protein and further optimization of its nutritional composition through reasonable and effective scientific and technological methods have continuously improved its nutritional value and palatability.Rational utilization of cottonseed protein can not only ensure animal growth performance but also significantly reduce animal feed costs.However, the appropriate addition amount and form of cottonseed protein in different aquaculture animals and growth stages, as well as its impact on animal absorption, metabolism, physiological functions, and mechanisms, have not been clearly studied.The author elucidates the nutritional value, production technology, research status of alternative feed protein sources, and factors influencing the utilization value of cottonseed protein.This provides a reference for the rational use of cottonseed protein and further improvement of its utilization value.
Genetics and Breeding
Comparison of Growth and Slaughtering Performances and Meat Quality Differences of Xinjiang Brown Cattle of Different Generations
MA Zhen, YE Zhibing, CUI Fanrong, YAN Xiangmin, WANG Xiao, CHEN Wenzhong, SU Nan, LI Haojie
2024, 51(9):  3878-3891.  doi:10.16431/j.cnki.1671-7236.2024.09.017
Abstract ( 67 )   PDF (6202KB) ( 34 )  
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【Objective】 This study was aimed to provide data reference for the quality breeding of Xinjiang Brown cattle by comparing the differences in growth performance and slaughter performance of three generations, and analyzing the quality differences of the striploin, eye round and chuck tender. 【Method】 A total of 313 Xinjiang Brown cattle bulls that reached 24-month-old (24m) and 30-month-old (30m) of age after 200 days of fattening were measured, and they were grouped into 6 groups according to generation class,marking as G1-24m (n=101),G2-24m (n=75),G3-24m (n=20),G1-30m (n=53),G2-30m (n=44) and G3-30m (n=20).Eight bulls of each group were randomly selected for slaughter performance measurement, and the physicochemical properties, nutrient components and texture parameters of striploin, eye round and chuck tender in Xinjiang Brown cattle were analyzed. 【Result】 The chest width, hip width, pin bone width, carcass weight, lean meat weight and bone weight of bulls in G3-30m group were significantly higher than those in G1-30m and G2-30m groups (P<0.05).Compared with the Evaluation Standard for New Types of Xinjiang Brown cattle (DB65/T 3791―2015), the relative growth rate of G1-24m, G2-24m and G3-24m was positive except for the body height growth of G1-24m by ―0.53%, the waist angle width by ―0.64%, and the waist angle width of G2-24m by ―1.18%.In terms of meat quality, the tenderness of striploin was better than that of eye round and chuck tender, but the water holding capacity of striploin was poorer.There was no significant difference in the palatability among three parts (P>0.05). 【Conclusion】 Through selective breeding, the growth and meat production performance of Xinjiang Brown cattle were significantly improved, which was reflected in the enlargement of physique, increased chest circumference and compact body size.
Transcriptomic Analysis of Blood in Early Pregnancy at Different Time Points in Sika Deer
SUN Huimin, FAN Bingfeng, ZHANG Xulin, SHAO Jing, LIU Lixiang, XU Baozeng
2024, 51(9):  3892-3908.  doi:10.16431/j.cnki.1671-7236.2024.09.018
Abstract ( 50 )   PDF (35608KB) ( 9 )  
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【Objective】 The objective of this study was to analyze the blood transcriptome data at different stages of early pregnancy in sika deer and identify key genes involved in early pregnancy. 【Method】 Blood samples from female sika deer were collected at 0, 7, 15, and 20 days after artificial insemination.Transcriptome sequencing was performed using the Illumina HiSeqTM3000C sequencing system to select differentially expressed genes.Principal component analysis and inter-group expression correlation analysis were conducted on blood samples at different time points to identify key stages of early pregnancy in sika deer.DESeq was used to screen differentially expressed genes(DEGs) at each time period, followed by GO, KEGG, and protein-protein interaction network analysis. 【Result】 The transcriptome sequencing results revealed a transcriptional peak in sika deer blood on day 15 of pregnancy.Comparing the groups D7 vs D0, D15 vs D7, and D20 vs D15, 181, 996, and 265 DEGs were identified, respectively.Among them, a total of 7 key genes, including ERAS, MET and KDR, were screened in comparison group D7 vs D0.Comprehensive analysis of GO and KEGG enrichment results indicated that these genes were mainly enriched in biological processes such as cellular response to stimuli and signal transduction, as well as pathways including the PI3K-Akt signaling pathway, and MAPK signaling pathway.A total of 10 key genes, including HRAS, HIF1A, MAPK1, and JAK1, were screened in comparison group D15 vs D7.The genes were mainly enriched in immune system processes, immune response, chemokine signaling pathway, and C-type lectin receptor signaling pathway.A total of 4 key genes, including MAPK1 and MMP2, were identified in comparison group D20 vs D15.The genes were mainly enriched in the estrogen signaling pathway and relaxin signaling pathway. 【Conclusion】 The dynamic expression of blood transcriptome at key time points during early pregnancy in Sika deer was analyzed, and key genes such as KDR, HRAS, MAPK1, and JAK1 were screened, providing reference for the exploration of diagnostic markers for early pregnancy in sika deer.
Research Progress on Boar Semen Preservation
ZHAO Yijia, LI Dong, HU Jianhong, YANG Gongshe, YU Taiyong
2024, 51(9):  3909-3920.  doi:10.16431/j.cnki.1671-7236.2024.09.019
Abstract ( 76 )   PDF (3939KB) ( 47 )  
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In response to the demands of social and economic growth, the pig industry is steadily transitioning towards large-scale, intensive and highly efficient breeding methods.Artificial insemination (AI) is currently used in pig reproduction as one of the most effective biological technology, in which the preservation of high-quality boar semen is a key step in artificial insemination.The quality of pig semen after preservation directly affects the fertility rate of sows and the survival rate of piglets, and its quality will be influenced by various factors such as temperature, pH, microorganisms and semen extenders during semen preservation.Nowadays, three methods of pig semen preservation are mainly adopted in production practice:Room temperature preservation, low temperature preservation and cryopreservation.Room temperature preservation utilizes the acidic metabolites produced by the semen at 15-25 ℃ to achieve short-term preservation by acid inhibition.Low temperature preservation involves slowly cooling the semen to 0-5 ℃ under the protection of cryoprotectants to preserve the sperm by keeping it in a semi-dormant state.Cryopreservation utilizes cryogenic sources such as liquid nitrogen to rapidly freeze the semen to ―196 to ―79 ℃, thereby inhibiting physiological metabolic activities of sperm to achieve long-term preservation of pig semen.The authors aim to comprehensively review three distinct methods of pig semen preservation technology, provide a concise overview of the primary factors that impact the efficacy of this preservation process, and strive to present improvement strategies tailored to each preservation method, so as to contribute valuable references and insights to the research and implementation of artificial insemination technology, as well as the preservation of porcine germplasm resources within China’s pig industry.Ultimately, its objective is to facilitate the sustainable and healthy growth of China’s pig industry.
Transcriptome Construction of F3 Follicle and Analysis of Genes Related to Follicle Atresia in Pigeons
WANG Ying, CHEN Jing, MIAO Dongzhi, ZHANG Chi, CHEN Junhong, YANG Haiming, WANG Zhiyue
2024, 51(9):  3921-3929.  doi:10.16431/j.cnki.1671-7236.2024.09.020
Abstract ( 41 )   PDF (14887KB) ( 4 )  
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【Objective】 This study was aimed to screen key genes involved in follicular atresia by high-throughput sequencing of granulosa cell of F3 follicle in pigeons. 【Method】 Three White King pigeons at laiying interval 5 (LI5) and 7 (LI7) days were selected to construct the transcriptome library of granulosa cell of F3 follicle, respectively, and RNA-Seq was used to screen and annotate the differentially expressed genes (DEGs), the expression of 5 randomly selected DEGs were verified by Real-time quantitative PCR. 【Result】 The clean reads of 41.96-43.62 Mb were obtained from 6 cDNA libraries of granulosa cells of F3 follicle in pigeons at LI5 and LI7 stages, the value of Q30 was greater than 92%, and the comparison rate was ranged from 82.21% to 85.94%.A total of 6 587 DEGs were obtained from granulosa cell of F3 follicle in pigeons at LI5 and LI7 stages, of which 2 318 DEGs were up-regulated and 4 269 were down-regulated.GO function enrichment analysis results of DEGs was performed, mainly in important biological pathways such as cell process, metabolic process, binding and catalytic activity.KEGG pathway enrichment analysis results showed that DEGs were mainly involved in focal adhesion, protein processing in endoplasmic reticulum, ovarian steroidogenesis and steroid hormone biosynthesis.Combining protein-protein interaction analysis and key DEGs in critical pathways, it found that StAR, HSD3B1, MARCH6, BCL2, BOK and CDCA7 genes played an important role in follicular atresia in pigeons.Real-time quantitative PCR results showed that the expression trends of 5 genes in granulosa cells of F3 follicle in pigeons at LI5 and LI7 stages were consistent with the results of RNA-Seq. 【Conclusion】 Six key genes (StAR, HSD3B1, MARCH6, BCL2, BOK and CDCA7) were obtained, which played an important role in follicular atresia in pigeons.The results provided a basis for further understanding of the molecular mechanism of follicular atresia and a theoretical basis for inhibiting follicular atresia and improving egg production in pigeons.
Polymorphism of TBX15 Gene and Its Association Analysis with Body Size Traits in Leizhou Goats
ZHOU Guangxian, TAN Jiayong, YANG Jian, LIU Yanfen, GAN Shangquan, ZHAO Zhihui, KANG Danju
2024, 51(9):  3930-3938.  doi:10.16431/j.cnki.1671-7236.2024.09.021
Abstract ( 51 )   PDF (5240KB) ( 12 )  
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【Objective】 The aim of this study was to screen the potential single nucleotide polymorphism (SNP) of T-box transcription factor 15 (TBX15) gene and analyze its correlation with body size traits in Leizhou goats, so as to provide theoretical support for molecular breeding of Leizhou goats. 【Method】 DNA were extracted from blood samples of 78 Leizhou goats.TBX15 gene SNP was detected by PCR amplification and direct sequencing, and its genetic diversity was analyzed.The correlation between the polymorphism of TBX15 gene and body size traits such as body height, body length and chest circumference in Leizhou goats were analyzed using SPSS 23.0 software.The linkage disequilibrium and haplotype of TBX15 gene SNP were analyzed by HaploView software. 【Result】 Three SNPs were found on the intron of TBX15 gene, namely g.96346139 A>G, g.96345894 T>C and g.96330058 G>A.There were three genotypes in all the three SNPs, and the dominant genotypes were GG, CC and AA, respectively.The chi-square test indicated that three SNPs were all in Hardy-Weinberg equilibrium, the polymorphic information content (PIC) were 0.309, 0.285 and 0.294, respectively, indicating moderate polymorphism (0.25<PIC<0.50).The association analysis results showed that the body height, cross height and body length of GG genotype at g.96346139 A>G of TBX15 gene in Leizhou goats were significantly higher than those of AA genotype (P<0.05).The body height, cross height, body length and chest depth of CC genotype at g.96345894 T>C of TBX15 gene were significantly higher than those of TT genotype (P<0.05).The chest depth of GG genotype at g.96330058 G>A of TBX15 gene was significantly lower than that of the other two genotypes (P<0.05).In the analysis of haplotypes and linkage disequilibrium, it was found that there was a strong linkage relationship among three SNPs (R2>0.33), with five main haplotypes, among which the dominant haplotype was ACG, with a probability of 0.716. 【Conclusion】 Three SNPs of TBX15 gene in Leizhou goats had significant effects on body height, body length and other body size indicators, and the three SNPs were in linkage disequilibrium with a strong linkage relationship.Haplotype ACG could serve as candidate markers in Leizhou goat breeding.
Study on the Differences in Meat Quality and Sensory Evaluation of Leg Muscle Between Pekin Ducks and Liancheng White Ducks
LI Shunan, LIU Dapeng, LI Zhinan, LI Yating, ZHANG Beibei, HOU Shuisheng, ZHOU Zhengkui
2024, 51(9):  3939-3947.  doi:10.16431/j.cnki.1671-7236.2024.09.022
Abstract ( 43 )   PDF (3678KB) ( 14 )  
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【Objective】 The aim was to explore the differences in leg muscle quality and sensory fingerprint between 42-day-old Pekin ducks and Liancheng White ducks in order to provide basic data for further analysis of the flavor characteristics of leg muscles between Pekin ducks and Liancheng White ducks. 【Method】 Eight 42-day-old Pekin ducks and Liancheng White ducks were selected to collect thigh muscles for testing their meat quality, including pH, meat color, shear force, and water loss rate.At the same time, electronic tongue and electronic nose were used for biomimetic sensory evaluation, and human sensory evaluation was conducted on them. 【Result】 In terms of meat quality indicators, L* and a* values of thigh muscles of 42-day-old Liancheng White ducks were extremely significantly higher than those of Pekin ducks (P<0.01), and the water loss rate and shear force of thigh muscles of Liancheng White ducks were extremely significantly lower than those of Pekin ducks (P<0.01). The electronic tongue results showed that the response values of thigh muscles of Pekin ducks in bitterness, aftertaste-B, umami, and sweetness were extremely significantly or significantly higher than those of Liancheng White ducks (P<0.01 or P<0.05), but there was no significant difference in richness between the two (P>0.05).The electronic nose results indicated that the thigh muscles of Liancheng White ducks contained higher levels of methane, with notable differences between the two breeds in inorganic sulfides and ethanol.Pekin duck thigh muscles contained higher levels of aromatic compounds, ammonia, hydrogen, alkanes, methane, and organic sulfides.In human sensory evaluation, the scores of saltiness, sweetness,fishyl of thigh muscles of Liancheng White ducks were extremely significantly higher than those of Pekin ducks (P<0.01), while the score of aroma of thigh muscles of Pekin ducks were extremely significantly higher than those of Liancheng White ducks(P<0.01). 【Conclusion】 The overall flavor profile of 42-day-old Pekin ducks were richer compared to Liancheng White ducks, while Liancheng White ducks were juicier in texture.The results provided basic data for further analyzing the flavor characteristics of leg muscles of two duck breeds.
Paternal Genetic Molecular Assessment of Four Yak Populations in Golog Tibetan Autonomous Prefecture, Qinghai
CAO Ping, LI Ruizhe, CHEN Shengmei, XUE Bin, HAN Shenglan, MEI Sa, DA Sang, CAI Jia, SUN Yonggang, MA Zhijie
2024, 51(9):  3948-3957.  doi:10.16431/j.cnki.1671-7236.2024.09.023
Abstract ( 81 )   PDF (4063KB) ( 23 )  
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【Objective】 The objective was to explore the paternal origin, population genetic structure, genetic diversity and differentiation of yaks in Golog Tibetan Autonomous Prefecture, Qinghai. 【Method】 The paternal genetic analysis of 144 yaks from 4 populations of Maduo, Jiuzhi, Gande and Banma was carried out through genomic DNA extraction, PCR amplification and sequencing.It mainly included 5 Y chromosome single nucleotide polymorphism (Y-SNPs) markers (SRY4, USP9Y, UTY19, AMELY3 and OFD1Y10) and 1 Y chromosome microsatellite (Y-STR) marker (INRA189).Haplotype diversity (Hd) and fixed differentiation index (Fst) were analyzed to evaluate the paternal genetic diversity and population genetic differentiation degree of yak populations.The UPGMA tree map and Network mediator map were constructed by Mega 5.05 and Network 10.1, respectively, to explore the paternal genetic relationship and population structure of yak populations. 【Result】 A total of 7 Y chromosome haplotypes were identified from 4 yak populations in Golog Tibetan Autonomous Prefecture, Qinghai.6, 4, 4 and 4 haplotypes of Maduo, Jiuzhi, Gande and Banma yaks were identified respectively.2 specific haplotypes (H2Y1 and H3Y1) and 1 specific haplotype (H7Y1) were detected in Maduo and Banma yaks, respectively.Genetic diversity analysis showed that the paternal genetic diversity of the 4 yak populations was relatively rich (Hd=0.506), among which the haplotype diversity of Madao yak was the highest (Hd=0.632), and that of Gande yak was the lowest (Hd=0.324).The results of genetic differentiation showed that the genetic differentiation between Banma and Gande, and Madao yak populations was moderate (0.05<Fst<0.15), and the differentiation degree among other yak populations was weak (0<Fst<0.05).Cluster analysis showed that the 4 yak populations were obviously grouped into 2 groups, among which Jiuzhi and Maduo yak populations were first grouped together, and then grouped together with Gande yak populations, while Banma yak population was grouped separately.Phylogenetic analysis showed that the Banma yak was composed of 1 paternal lineage (Y1), while the other 3 yak populations were composed of 2 paternal lineages (Y1 and Y2). 【Conclusion】 This study preliminarily revealed the paternal genetic diversity, degree of differentiation, and genetic structure of 4 yak populations in Golog Tibetan Autonomous Prefecture, Qinghai, using Y chromosome molecular markers, which indicated that the 4 yak populations possessed relatively rich paternal genetic diversity and had 2 paternal origins.The results provided a theoretical basis for the preservation, breeding, development and utilization of local yak genetic resources.
Preventive Veterinary Medicine
Isolation,Identification and Genome Sequence Analysis of Two Recombinant Strains of Avian Infectious Bronchitis Virus
LI Feng, XIE Bin, ZHANG Shuai
2024, 51(9):  3958-3969.  doi:10.16431/j.cnki.1671-7236.2024.09.024
Abstract ( 57 )   PDF (4596KB) ( 15 )  
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【Objective】 The experiment was aimed to understand the genetic variation of avian Infectious bronchitis virus (IBV). 【Method】 Samples suspected to be infected with IBV was collected from partial farms in Shandong, inoculated with SPF chicken embryos for virus isolation and identification.Whole genome sequencing was performed using second-generation sequencing technology, and genetic evolution and key amino acid sites were analyzed based on the whole genome sequence. 【Result】 Autopsy of infected chickens showed hemorrhages in the tracheal rings and yellow or black-yellow embolism in the lumen.Two strains of IBV were isolated from the samples, and named SDJN and SDLY, respectively.Similarity analysis showed that isolates SDJN and SDLY had high similarity with GⅠ-19 genotype reference strains, with nucleotide similarity of 96.0%-97.3% and amino acid similarity of 94.9%-100%.Amino acid sequence analysis showed that the two IBV isolates had multiple amino acid variation sites compared with other strains.Phylogenetic tree results showed that isolates SDJN and SDLY were located in the same branch as the reference strains of the IBV GⅠ-19 genotype.Recombinant analysis showed that isolates SDJN and SDLY were recombinant strains of multiple genotypic strains, both with the ck/CH/LDL/091022 strain as the main parental strain. 【Conclusion】 In this study, two IBV isolates were isolated from Shandong province.The two isolates were closely related to GⅠ-19 genotype, and were a new recombinant IBV strain.The results laid a foundation for further study on the prevalence and evolution of IBV and the prevention and control of avian infectious bronchitis.
Exploration of Respiratory Tract Response Patterns in Chickens Infected with H9N2 Subtype Avian Influenza Virus
CHI Zhouying, LI Tianxu, WU Yaxin, QU Xiaoyun, LI Sijie, SUN Minhua, ZHANG Jianfeng, LIAO Ming, DU Shouwen
2024, 51(9):  3970-3979.  doi:10.16431/j.cnki.1671-7236.2024.09.025
Abstract ( 54 )   PDF (12173KB) ( 14 )  
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【Objective】 This study was conducted to establish a model of upper respiratory tract inflammation in chickens infected with H9N2 subtype Avian influenza virus (AIV) and explore the inflammatory response caused by its infection. 【Method】 H9N2 subtype AIV epidemic strain GD10142 was infected with SPF chickens by nose and eye drops at a dose of 100 μL/bird (108 EID50/100 μL).The clinical characteristics of chickens were observed after infection, and throat swabs and serum samples were collected at 0, 1, 3, 5, 7 and 9 days after infection.The pathological changes of respiratory tract and lung were observed 5 and 9 days after infection, and tracheal lavage fluid, trachea and lung tissues were collected.The viral load in throat swab, trachea lavage fluid, trachea and lung tissue was detected by Real-time quantitative PCR.The changes of serum total IgG and H9N2 subtype AIV-specific IgG were detected by ELISA, and the neutralization activity of antiserum was analyzed by serum neutralization test.The levels of inflammation-related cytokines interleukin-6 (IL-6), IL-10, IL-1β, tumor necrosis factor α (TNF-α) and nuclear transcription factor κB (NF-κB) in serum and trachea lavage fluid were detected by ELISA. 【Result】 After infection with H9N2 subtype AIV, chickens did not show typical clinical features, but viral nucleic acid could be detected in throat swabs 1-5 days after infection, and the viral load was highest 3 days after infection, and no viral nucleic acid could be detected 5 days after infection.After 5 days of infection, viral nucleic acid was detected in trachea lavage fluid, trachea and lung, and the first two viral nucleic acid loads were higher than those in lung.The total serum IgG and H9N2 subtype AIV-specific IgG increased 3 days after infection, maintained at high levels 5-9 days after infection, and exhibited strong virus neutralizing activity.IL-10, IL-1β, TNF-α and NF-κB in serum also showed an upward trend starting from 3 days after infection, however, the contens of IL-6, IL-10, IL-1β, TNF-α and NF-κB in tracheal lavage fluid were very low, and there was no significant change before and after infection. 【Conclusion】 In this study, the respiratory tract model of chickens infected with H9N2 subtype AIV was successfully established, and the upper respiratory tract was determined to be the main site of infection and proliferation of H9N2 subtype AIV.The infection of H9N2 subtype AIV induced the production of pro-inflammatory and anti-inflammatory cytokines, which might be an important factor in the maintenance of immune homeostasis in infected animals.This study laid a foundation for further exploring the pathogenic mechanism and host defense mechanism of H9N2 subtype AIV.
Prokaryotic Expression,Purification and Polyclonal Antibodies Preparation of African Swine Fever Virus B602L Protein
GUO Chenyun, SONG Jinxing, WANG Mengxiang, SUN Junru, YAN Ruoqian, XIE Caihua, WU Yanan, ZHANG Gaiping
2024, 51(9):  3980-3989.  doi:10.16431/j.cnki.1671-7236.2024.09.026
Abstract ( 61 )   PDF (6255KB) ( 14 )  
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【Objective】 African swine fever virus (ASFV) is a pathogen that is very harmful to the swine industry, and the B602L protein, as an important non-structural protein, is one of the chaperone molecules for the folding of the coat protein p72 into the correct tertiary structure, which plays a key role in the assembly of the virus into an icosahedral structure.Specific antibodies are of great importance for the study of the mechanism of the anti-African swine fever immune response and serological detection methods.This study was aimed to obtain ASFV B602L protein and prepare polyclonal antibodies against ASFV B602L protein, and provide materials for the functional research of ASFV B602L protein and the development of related diagnostic reagents. 【Method】 The present study was conducted on the B602L gene of ASFV China/2018/AnhuiXCGQ strain.After the prediction of the physicochemical properties and the analysis of the antigenicity of B602L protein, the B602L gene was cloned into the prokaryotic expression vector pET-28a(+) by homologous recombination and expressed in Escherichia coli BL21(DE3) competent cells.The protein was purified by nickel column affinity chromatography, the purification effect and specificity were verified by SDS-PAGE and Western blotting.And the purified B602L protein was immunized against BALB/c mice to prepare polyclonal antibodies.The titer of polyclonal antibodies was determined by indirect ELISA method, and the specificity of polyclonal antibodies was detected by Western blotting and Dot blotting. 【Result】 Bioinformatics analysis showed that B602L protein was an unstable hydrophilic protein without transmembrane region and signal peptide.The secondary structure mainly consisted of alpha helix (54.91%), extended chain (9.81%) and random coil (32.08%).ASFV B602L recombinant plasmid was successfully constructed.SDS-PAGE and Western blotting results showed that the recombinant strain pET-28a-B602L-BL21 was highly expressed under the condition of 16 ℃ and 0.5 mmol/L, and the obtained recombinant protein was mainly expressed in the supernatant of strain BL21.It was about 70 ku in size and binded specifically to ASFV-positive serum.Indirect ELISA showed that the prepared murine polyclonal antibody against B602L had a titer of 1∶409 600.Western blotting and Dot blotting results showed that the murine polyclonal antibody against B602L could specifically recognize B602L protein. 【Conclusion】 The prokaryoticly expressed B602L recombinant protein had good immunogenicity, which could specifically bind to ASFV-positive serum, and the prepared polyclonal antibody against mouse anti-ASFV B602L protein had good biological activity.The results could provide experimental materials and theoretical support for further research on the biological function of ASFV B602L protein and the establishment of diagnostic methods for African swine fever.
Biological Characteristics and Whole Genome Analysis of Klebsiella pneumoniae Phage kp6
RONG Ruifan, GUO Haiyue, HU Pengcheng, ZUO Junhao, ZHANG Dongchao, GUO Zhiliang, LI Ying, ZHANG Yuxin, WANG Xinru, ZHAO Ruili, LIU Dingkuo
2024, 51(9):  3990-4001.  doi:10.16431/j.cnki.1671-7236.2024.09.027
Abstract ( 57 )   PDF (11971KB) ( 8 )  
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【Objective】 The purpose of this study was to explore the biological characteristics and whole genome characteristics of Klebsiella pneumoniae phage isolated from sewage samples, and provide a basis for the prevention and control of clinical Klebsiella pneumoniae infection and the development of new phage related alternative antibody products. 【Method】 A Klebsiella pneumoniae TJAU-K11 was used as the host bacteria, the phage of Klebsiella pneumoniae was isolated and purified from sewage samples by double plate method.The morphology of the isolated phage was observed by transmission electron microscopy, and the host spectrum, optimal multiplicity of infection (MOI) and one-step growth curve were determined,and the stability of phage was evaluated.The effect of phage on the biofilm formation and cleavage of Klebsiella pneumoniae TJAU-K11 was investigated, and the whole genome was sequenced to understand the biological characteristics and whole genome characteristics of phage. 【Result】 3 Klebsiella pneumoniae phage were successfully isolated from sewage samples and named kp4, kp5 and kp6, respectively.Transmission electron microscopy showed that phage kp6 was a short-tailed phage with regular icosahedral morphology.The one-step growth curve showed that the incubation period of phage kp6 was 20 min and the outbreak period was 30 min.The phage titer remained stable at -20-50 ℃, pH 4.0-10.0 and UV irradiation for 12 min.The results of single bacteriophage inhibition test in vitro showed that the D600 nm value of phage kp6 groups with different MOI was always less than 0.4 within 7 h.The results of in vitro bacteriostatic test of phage cocktail showed that the D600 nm value of phage kp6 within 12 h was less than 0.1 when MOI was 1 and 100.The amount of host bacteria biofilm formation in phage kp6 groups with different MOI was extremely significantly lower than that in positive control group (P<0.01), and both single phage kp6 and bacteriophage cocktail could lysate host bacteria biofilm.The whole genome sequencing showed that the phage kp6 genome was double-linked looped DNA, with a total length of 40 971 bp and GC content of 53.13%.Among 49 coding sequences, 23 encoded proteins with known functions, including gp12, a tail fibrillar protein for short tail iophage adsorption and injection, and lytic functional protein endolysin. 【Conclusion】 In this study, Klebsiella pneumoniae lytic phage kp6 was isolated, and there was an endolysin in its genome.The phage cocktail prepared by phage kp6 could completely inhibit the growth of host bacteria within 12 h.The results laid a foundation for the subsequent development of lytic enzymes and phage preparations.
Research Progress on Biofilm Formation and Its Drug Resistance Mechanism of Streptococcus suis
CHEN Xiangyu, HUANG Yuan, LIU Baoling, LUO Qin, LU Yongkun, HE Zhenwen, LIU Dingyu, QIAO Changhong, WANG Xiaohu, WANG Gang, BAI Aiquan, CAI Rujian
2024, 51(9):  4002-4013.  doi:10.16431/j.cnki.1671-7236.2024.09.028
Abstract ( 62 )   PDF (1243KB) ( 35 )  
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Streptococcus suis is a zoonotic pathogen that poses a significant threat to both pigs and humans. Streptococcus suis has the ability to form biofilms in its natural state, which is one of the main factors contributing to its drug resistance.It is currently known that Streptococcus suis forms biofilm in the three stages of adhesion, aggregation and dispersion of infection, and its virulence factors (capsular poly saccharide, muramidase-released protein and suilysin) play a major role, and the whole formation process is regulated by the signal transduction system of bacterial cells.The quorum-sensing (QS) system is an important intracellular signaling transduction system in bacteria, which significantly regulates the formation of bacterial biofilms.It enhances bacterial drug resistance by regulating gene transfer, replication, biofilm formation, and plays an important role in increasing bacterial density.The formation of biofilms enhances the drug resistance of Streptococcus suis, and its drug resistance mechanism mainly involves the regulation of QS system, the formation of extracellular polymeric substances, extracellular DNA action, the role of efflux pumps, the structure of the biofilm and the horizontal gene transfer.Understanding the formation and regulation of Streptococcus suis biofilms and the mechanisms of drug resistance caused by biofilms is of great significance for the prevention and treatment of Streptococcus suis.Chemical disinfectants, high temperatures, ultraviolet light, and ozone can destroy the biofilms of Streptococcus suis and kill the bacteria.The use of antibiotics or antimicrobials and other chemical synthetic drugs can inhibit the formation of biofilm or destroy biofilm, so as to achieve the role of killing bacteria and achieve the purpose of treating Streptococcus suis infection.In addition, some traditional Chinese medicines have an inhibitory and destructive effect on the biofilms of Streptococcus suis, and their active ingredients have anti-drug-resistant properties, showing promising prospects for development.The author summarizes the research progress on the formation and regulation of Streptococcus suis biofilms and the mechanisms of drug resistance, and summarizes the current prevention and treatment strategies, in order to provide new ideas for the prevention and control of Streptococcus suis.
Prokaryotic Expression and Monoclonal Antibody Preparation of Bovine Promyelocytic Leukemia Protein
CHENG Jing, CHEN Peilin, GUO Yu, CUI Jinqiang, JIANG Bo, ZHOU Linyi, LIU Wenxiao, LI Huanrong, LI Yongqing
2024, 51(9):  4014-4024.  doi:10.16431/j.cnki.1671-7236.2024.09.029
Abstract ( 45 )   PDF (7616KB) ( 13 )  
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【Objective】 Bovine promyelocytic leukemia protein (PML),a constituent of nuclear substructure called PML nuclear bodies (PML NBs), was crucial for antiviral intrinsic immunity.In this study, monoclonal antibody against bovine PML was generated to facilitate further investigation into the structure and function of PML NBs. 【Method】 Recombinant plasmid pET-32a-bPML expressing bPML gene truncator was constructed.The recombinant plasmid was transformed into Escherichia coli Transetta (DE3) competent cells, and induced by IPTG, and the expression products were analyzed by SDS-PAGE and Western blotting.The recombinant protein purified by nickel column affinity chromatography was utilized for immunization of mice, and monoclonal antibody was prepared.ELISA was applied to determine the classes/subclasses and subtypes of the monoclonal antibody, and the epitope recognized by the monoclonal antibody was further identified.The specificity of monoclonal antibody was detected by detecting cells of different species origin.The monoclonal antibody against bovine PML was analyzed by Western blotting and indirect immunofluorescence assay (IFA) to detect of exogenously transfected bovine PML cells.IFA established by this monoclonal antibody was used to detect the endogenous PML localization in different bovine-derived cells. 【Result】 Bovine PML was solubly expressed in Escherichia coli with a molecular weight size of 50 ku, a monoclonal antibody targeting bovine PML, bPML-2G5, was prepared from this purified protein as an immunogen.bPML-2G5 belonged to the IgG1 subclass, its light chain was Kappa type.The epitope recognized by bPML-2G5 was located in the structural domain 78EQPRPSTSRA88 of the bovine PML.Western blotting and IFA analysis demostrated that bPML-2G5 not only specifically recognized the exogenously transfected bovine PML, but also specifically recognized PML localized within PML NBs in bovine kidney cells, bovine nasal turbinate cells, and bovine embryonic tracheal cells. 【Conclusion】 In this study, a hybridoma cell line secreting bovine PML monoclonal antibody was successfully prepared, and its secreted monoclonal antibody could be used to detect exogenously and endogenously expressed bovine PML proteins.
Investigation and Identification of Species of the Culex pipiens Complex in Kunming
LI Susheng, YANG Zhenxing, HE Yuwen, WANG Jinglin
2024, 51(9):  4025-4035.  doi:10.16431/j.cnki.1671-7236.2024.09.030
Abstract ( 46 )   PDF (10249KB) ( 3 )  
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【Objective】 The experiment was aimed to investigate and identify the species of Culex pipiens complex in Kunming, Yunnan, and provide data for the prevention and control of mosquito borne infectious diseases. 【Method】 Morphological and molecular biological identification were conducted to the male Culex pipiens complex collected from Qinglong mountain next to the Jindian and Huangtupo,Kunming,in June 2022 and May 2023.The morphological characteristics of mosquitoes were observed under stereomicroscope,and the male mosquito phallosomes was cut off for observation, and the mosquito species were identified according to the morphological characteristics.The mitochondrial cytochrome C oxidase subunit Ⅰ(COⅠ) gene and acetylcholinesterase gene 2 (AChE2) of mosquitoes were amplified and their sequences were analyzed. 【Result】 Morphology showed that the middle end of the phallosomes of mosquitoes collected from Qinglong mountain next to the Jingdian was pointed,and the extension part of the inner leaf was broad leaf.The middle end of the phallosomes of mosquitoes collected from Huangtupo of Kunming is flush,and the inner leaf of the stem was blade like.The results of gene sequence similarity analysis showed that there was no significant difference in the COⅠ gene of the collected mosquitoes.The AChE2 gene distinguished mosquitoes into two types.Mosquitoes collected from Qinglong mountain next to the Jingdian had high similarity with Culex pipiens quinquefasciatus,with the highest similarity ranging from 98.1% to 100%.Mosquitoes collected from the Huangtuppo had high similarity with Culex pipiens pipiens,with the highest similarity of 99.7%.The results of COⅠ gene phylogenetic analysis showed that the six mosquitoes were in the same evolutionary branch as Culex quinquefasciatus, Culex pipiens, Culex pipiens molestus, and Culex pipiens pallens, without significant genetic evolutionary differences.The phylogenetic analysis of AChE2 gene showed that mosquitoes collected from Qinglong Mountain next to the Jingdian were clustered with Culex quinquefasciatus, while mosquitoes collected from Huangtupo were clustered with Culex pipiens, located in different evolutionary branches. 【Conclusion】 Two kinds of the Culex pipiens complex were collected.The mosquitoes collected from Qinglong mountain next to the Jingdian were Culex pipiens quinquefasciatus,while the mosquitoes collected from the Huangtupo were Culex pipiens pipiens.Research had shown the discovery of Culex pipiens pipiens in Southern regions such as Kunming in China, and the monitoring of West Nile virus and related viruses should be strengthened.
Prokaryotic Expression of Anaplasma phagocytophilum MSP4 Protein and Preparation of Its Polyclonal Antibodies
CUI Yanyan, WANG Guan, HAO Junfang, LI Zhiqiang, YU Fuchang, SHI Ke, XI Li, QI Meng
2024, 51(9):  4036-4042.  doi:10.16431/j.cnki.1671-7236.2024.09.031
Abstract ( 47 )   PDF (3444KB) ( 13 )  
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【Objective】 The purpose of this study was to express the protein MSP4 of Anaplasma phagocytophilum (AP), then prepare the polyclonal antibody against MSP4 protein, so as to provide experimental materials and theoretical basis for the establishment of plasma free detection method for phagocytic cells. 【Method】 According the gene sequence published in GenBank, MSP4 gene was optimized and synthesized.Then the MSP4 gene was inserted into pET-30a vector to construct recombinant plasmid pET-30a-MSP4 and then was transformed into Escherichia coli BL21(DE3) competent cells.The factors affecting the expression effect (temperature and IPTG) were optimized to select the best induced expression conditions.The expression of protein was analyzed by SDS-PAGE.The recombinant protein was purified by Ni-IDA affinity chromatography, and was identified by SDS-PAGE and Western blotting.The polyclonal antibody against MSP4 was prepared by immunizing New Zealand White rabbits with the purified recombinant protein MSP4, and then the titer and specificity of the polyclonal antibody were determined by indirect ELISA and Western blotting. 【Result】 The results showed that the pET-30a-MSP4 recombinant plasmid was successfully constructed.The protein expression level was high under the induction conditions of 37 ℃ and 0.8 mmol/L IPTG, and it was mainly expressed in the form of inclusion bodies with molecular weight of about 28 ku.Western blotting showed that it had good immunogenicity.The titer of rabbit-derived polyclonal antibodies were 1∶64 000 (rabbit 1) and 1∶128 000 (rabbit 2), respectively and the prepared polyclonal antibodies had high specificity. 【Conclusion】 In this study, MSP4 recombinant protein was obtained, and polyclonal antibody was successfully prepared, which could provide reference for the establishment of a method for detecting antigen and antibody of AP and the evaluation of vaccine efficacy, and also laid the foundation for the study of MSP4 protein function.
Research Progress on Brucellosis Vaccine for Sheep and Goat
QIU Dongxu, LYU Lang, PI Xiangcheng, FENG Yu, JIANG Hui, CHEN Xiang, DING Jiabo
2024, 51(9):  4043-4051.  doi:10.16431/j.cnki.1671-7236.2024.09.032
Abstract ( 84 )   PDF (1162KB) ( 51 )  
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Brucellosis is an important zoonotic disease caused by Brucella, which is widely prevalent worldwide, causing serious economic losses and threatening public health safety. China is one of the countries with severe outbreaks of brucellosis.Implementing vaccination in high prevalence areas is one of the effective strategies to prevent the spread of brucellosis.Currently, the main prevalent strain in China is Brucella melitensis, and the infected sheep/goat are the main sources of infection for Brucella.At present, vaccines available in China for the prevention of sheep/goat brucellosis include conventional attenuated vaccines such as Brucella melitensis strain Rev.1, Brucella melitensis strain M5/M5-90 and Brucella suis strain S2, as well as newly approved Brucella melitensis strains M5-90Δ26 and BA0711.Although there are many types of vaccines that can be used to prevent sheep/goat brucellosis, existing vaccines have two obvious shortcomings.Firstly, their safety is insufficient, and immunization of pregnant animals can cause miscarriage, and the vaccine strain and the wild strain are both smooth, which interferes with clinical diagnosis after immunization.Therefore, it is still urgent to develop a highly safe and non-invasive brucellosis vaccine.This paper provides a review of the application of sheep/goat brucellosis vaccines and the research status of new vaccines.
Isolation, Identification and Mouse Pathogenicity Analysis of a Strain of Bovine Viral Diarrhea Virus from Imported Fetal Bovine Serum
LIU Zhao, DONG Qianqian, WU Aodi, WANG Kaiyue, LIANG Chengzhe, Adnan ALI, SHENG Jinliang
2024, 51(9):  4052-4059.  doi:10.16431/j.cnki.1671-7236.2024.09.033
Abstract ( 48 )   PDF (2981KB) ( 12 )  
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【Objective】 The experiment aims to isolate, identify and analyze the genetic evolution of Bovine viral diarrhea virus (BVDV) in imported fetal bovine serum. 【Method】 The presence of BVDV nucleic acid contamination in the imported fetal bovine serum purchased by the laboratory was detected by RT-PCR, the contaminated serum was inoculated with MDBK cells, and the cells were subcultured to the 7th passage, and the cultures of each generation of cells were amplified by BVDV 5'-UTR, and the PCR amplification product was ligated with the pMD19-T vector and sequenced for genetic evolution analysis.Indirect immunofluorescence assay (IFA) was used to detect the antigen and virus titer of the isolates.BALB/c mice were inoculated with the isolates, and orbital blood was collected for blood routine detection and RT-PCR detection. 【Result】 The results showed that the virus strain failed to cause cytopathy during MDBK cell culture, and the RT-PCR amplification of cell culture was positive.Analysis based on the similarity of the 5'-UTR sequence indicated that the isolated strain had 99.6% similarity in its 5'-UTR sequence to the BVDV UDEC-COY7-17 (OL860952.1) strain, both belonged to the BVDV-1e subtype.IFA result detected specific green fluorescent signals in the cytoplasm of MDBK cells infected with the isolated strain, while no signals were observed in the control group.The 50% tissue culture infectious dose (TCID50) was determined to be 10-4.7/0.1 mL.Blood routine tests conducted on BALB/c mice on the 7th day after immunization revealed extremely significantly lower counts of white blood cells (WBC) and lymphocytes (LYM) compared with control group (P<0.01).Total RNA extracted from the blood was subjected to 5'-UTR RT-PCR testing, which yielded positive results with an amplified product size of 298 bp, consistent with expectations. 【Conclusion】 A NCP BVDV-1e subtype strain was isolated in this study.It was proved that there was BVDV antigen contamination in imported fetal bovine serum, and also provided experimental materials for further study of the related molecular mechanisms of BVDV.
Construction of Machine Learning Models to Predict the Cross-host Infection Risk of Diarrheagenic Escherichia coli Based on CRISPR Sequence
FENG Xinyuan, ZHAO Jiaxue, LONG Jinzhao, HU Jingyan, XI Yanyan, CHEN Shuaiyin, YANG Haiyan, DUAN Guangcai
2024, 51(9):  4060-4065.  doi:10.16431/j.cnki.1671-7236.2024.09.034
Abstract ( 52 )   PDF (1452KB) ( 8 )  
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【Objective】 This study was aimed to predict the cross-host infection risk of diarrheagenic Escherichia coli and identify the zoonotic isolates based on CRISPR sequences by machine learning. 【Method】 The genome sequence information of 806 strains of diarrheic Escherichia coli isolated in China was obtained from Enterobase database.The spacer sequence construction features of CRISPR sites were extracted.Subsequently, the machine learning models were established and their performances were evaluated using 10-fold cross-validations.Moreover, the zoonotic risk for each isolates was obtained by the best-fitted model and the zoonotic potential risks with different animal sources were compared. 【Result】 A total of 1 093 spacer sequence clusters were obtained from 806 isolates, containing 196 unique spacer sequence clusters of human, 291 unique spacer sequence clusters of animal, and 606 spacer sequence clusters shared between human and animal.Linear discriminant analysis showed that there were significant differences in the distribution of interval sequence clusters between human and animal strains.Subsequently, random forest, logistic regression, support vector machine and gradient boosting decision tree models were established and successfully predicted the source for their accuracy were all >0.82 and their area under receiver operating characteristic curve (AUC) value were all close to 0.9.Finally, the random forest model performed best after optimization, its accuracy was 0.844 and its AUC value was 0.915.According to infected risk of each isolates generated by the best model, the swine isolates displayed the highest risk to infect human, the ovine isolates performed a low risk to infect human, and only a few poultry isolates might exhibit the potential to infect human. 【Conclusion】 The machine learning model based on spacers sequences could identify isolates with the zoonotic potential, which provided new insights in control and prevention of infectious disease.
Basic Veterinary Medicine
Comparative Analysis of miRNA Expression Profiles in Bovine Mammary Epithelial Cells Treated with Lipopolysaccharide and Calcium Ions
XU Haotian, YU Yuetong, LI Jing, MA Zhiyuan, YANG Bin, WANG Zekun, TUO Haixin, QI Meng
2024, 51(9):  4066-4079.  doi:10.16431/j.cnki.1671-7236.2024.09.035
Abstract ( 42 )   PDF (11291KB) ( 3 )  
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【Objective】 This study was aimed to investigate the regulation of calcium ions on inflammatory responses in bovine mammary epithelial cells, and compare and analyze miRNA expression profiles of bovine mammary epithelial cells co-treated with lipopolysaccharide and calcium ions. 【Method】 A model of inflammatory response was created by treating bovine mammary epithelial cells with lipopolysaccharide.The transcript levels of inflammatory factors were measured in bovine mammary epithelial cells co-treated with different concentrations (0.5-6.6 mmol/L) of calcium ions and lipopolysaccharide (50 μg/mL).RNA was extracted from bovine mammary epithelial cell samples with normal, lipopolysaccharide treated and co-treated with lipopolysaccharide and calcium ions, with 3 samples in each group.Quality assessment was performed on 9 RNA samples, followed by miRNA sequencing and bioinformatics analysis.Finally, Real-time quantitative PCR validation was performed on common differentially expressed miRNA among 3 groups. 【Result】 In a model of inflammation in bovine mammary epithelial cells, high concentrations of calcium ions were found to inhibit the transcription of inflammatory factors.Nine RNA samples were obtained, with clean reads accounting for between 93.86% and 98.18% of raw reads.The sequence distribution lengths were all concentrated between 21 and 24 nt.The samples from all three groups exhibited high intra-group similarity but also high inter-group variability.A total of 47 miRNA were identified as differentially expressed across 3 groups of samples.The samples among 3 groups showed differential expression of bta-miR-11980, bta-miR-1246 and bta-miR-677.The results of GO function and KEGG pathway enrichment analysis showed that the target genes of differentially expressed miRNA were significantly enriched in GO entries related to regulation of cell communication, signaling regulation, endomembrane system, phylogeny, cellular developmental process and cell differentiation, etc.The genes targeted by the differentially expressed miRNA were significantly enriched in various pathways, such as the HIF-1 signaling pathway, ErbB signaling pathway, TNF signaling pathway, synaptic vesicle cycle and inositol phosphate metabolism pathway.Real-time quantitative PCR results confirmed that the expression of common differentially expressed miRNA among 3 sample groups (bta-miR-677 and bta-miR-1246) were consistent with RNA-Seq results. 【Conclusion】 Calcium ions indirectly affected the inflammatory response in bovine mammary epithelial cells by regulating the expression of differentially expressed miRNA, such as bta-miR-11980, bta-miR-1246 and bta-miR-677.
Study on Allicin Alleviating the Liver Injury of Quail Caused by Fumonisin B1
LI Xinran, ZHU Lingxin, SONG Huanni, ZHU Xueyan, LI Ruobin, LIU Yang, CAO Changyu
2024, 51(9):  4080-4091.  doi:10.16431/j.cnki.1671-7236.2024.09.036
Abstract ( 60 )   PDF (10076KB) ( 25 )  
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【Objective】 The objective of this experiment was to explore the mechanism of allicin in alleviating liver injury caused by fumonisin B1 (FB1) in quail during egg-laying period, so as to provide experimental basis for the prevention of FB1-induced liver injury in quail, and reduce the economic loss caused by FB1 poisoning to the breeding industry. 【Method】 The Yellow-feathered female quails were used as test subjects, and 120 quails were randomly divided into 4 groups:Control group, allicin group (500 mg/kg allicin), fumonisin group (30 mg/kg FB1), and remission group (500 mg/kg allicin+30 mg/kg FB1), with 30 quails in each group.The histopathological and ultrastructural changes of liver in quails were observed, the indexes of liver function, antioxidant function and the expression of related inflammatory factors in quail were detected. 【Result】 The results of liver pathology and ultrastructure showed that allicin could effectively alleviate the pathological damage caused by FB1, such as the disappearance of mitochondrial cristae and the increase of collagen fibers.Compared with control group, the concentration of aspartate aminotransferase (AST) in serum of quails in fumonisin group was significantly increased (P<0.05), the activitiy of catalase (CAT) and total antioxidant capacity (T-AOC) were significantly decreased (P<0.05), and the malondialdehyde (MDA) content was significantly increased (P<0.05).The expressions of interleukin-8 (IL-8), IL-6, IL-18, IL-12β, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), related apoptosis factor ligand (FASL) and Toll-like receptor 4 (TLR4) genes were also significantly increased (P<0.05).Compared with fumonisin group, the AST content in serum of quails in remission group was significantly decreased (P<0.05), the activitiy of CAT and T-AOC were significantly increased (P<0.05), and MDA content was decreased significantly (P<0.05).The expressions of IL-8, IL-6, IL-18, IL-12β, TNF-α, COX-2, iNOS, FASL and TLR4 genes were also significantly decreased (P<0.05). 【Conclusion】 Allicin could effectively alleviate FB1-induced liver injury in quails, and its mechanism was closely related to the anti-inflammatory and antioxidant effects of allicin.
Characteristics of Microbiota and Metabolites in Feces of Calves with Diarrhea
ZHAO Qingmei, CUI Shengwei, GUO Shihui, YU Yongtao, LIANG Taiyu, LI Huanyu
2024, 51(9):  4092-4105.  doi:10.16431/j.cnki.1671-7236.2024.09.037
Abstract ( 66 )   PDF (20380KB) ( 36 )  
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【Objective】 The objective of this study was to reveal the characteristics of fecal microbiota and metabolites of calves with diarrhea. 【Method】 According to the fecal score and clinical symptoms, the suckling calves within 30 days of age were divided into healthy group (CK) and diarrhea group (D).The fecal microbiota of calves in CK and D groups were analyzed using 16S rRNA amplification sequencing technology.The non-targeted metabolome analysis was performed to screen differential metabolites in the feces of CK and D groups through LC-MS, and the differential metabolites were conducted for KEGG pathway enrichment analysis.The Spearman correlation analysis was performed on fecal microbiota and differential metabolites in feces. 【Result】 Compared with CK group, the OTUs and Chao1 indexes of fecal microbiota in D group were extremely significantly increased (P<0.01), and the Shannon and Simpson indexes were extremely significantly decreased (P<0.01).The relative abundances of Fusobacteriota,Proteobacteria, Acidobacteriota, Chloroflexi and 9 bacteria genera that including Escherichia-Shigella, Fusobacterium and Clostridium_sensu_stricto_1, etc.were extremely significantly or significantly increased in D group (P<0.01 or P<0.05), while the relative abundances of Actinobacteriota, Firmicutes, Euryarchaeota and 11 bacteria genera that including Faecalibacterium, Subdoligranulum, Olsenella and Bifidobacterium, etc.were extremely significantly or significantly decreased in D group (P<0.01 or P<0.05).Fusobacteriota, Proteobacteria, Fusobacterium, Escherich-Shigella, Fusobacterium mortiferium and Escherichia coli were biomarkers in the feces of calves with diarrhea.A total of 54 different metabolites were screened from the feces of CK and D groups.PC(16∶1(9Z)/16∶1(9Z)) was enriched into the glycerophospholipid metabolism pathway, which was significantly positively correlated with Fusobacterium (P<0.01) and significantly or extremely significantly positively correlated with Collinsella, Megasphaera and Olsenella(P<0.05 or P<0.01).Dethiobiotin was enriched into the biotin metabolic pathway.It was significantly negatively correlated with Fusobacterium (P<0.01), while significantly positively correlated with Subdoligranulum, Megasphaera and Rolstonia (P<0.05).Dihydrobiopterin and dihydroneopterin triphosphate enriched into the folate biosynthesis pathway were extremely significantly or significantly negatively correlated with Fusobacterium and Peptestreptococcus (P<0.01 or P<0.05), which extremely significantly or significantly positively correlated with Subdoligranulum, Megasphaera, Olsenella, Bifidobacterium, Prevotella, Collinsella, Faecalibacterium, and Parabacteroides (P<0.01 or P<0.05). 【Conclusion】 The richness, diversity, species composition of fecal microbiota and the composition of fecal metabolites were significantly changed in calves with diarrhea.Theglycerophospholipid metabolism, biotin metabolism and folate biosynthesis were affected in the gut of calves with diarrhea.The dysbiosis of intestinal microbiota of calves was closely related to the significant changes of fecal metabolites.
Study on the Mechanism of Yinchenhao Decoction in Treating Diabetes Mellitus Based on Network Pharmacology and Experimental Verification
SONG Yu, SUN Yangang, ZHANG Yiyuan, ZHOU Siqi, ZHANG Lu, LI Wei, ZHANG Minghao, ZHANG Zijuan, MIAO Jinxin, CHEN Fang, CAO Shan, ZHANG Xiaoli, XIE Sha
2024, 51(9):  4106-4119.  doi:10.16431/j.cnki.1671-7236.2024.09.038
Abstract ( 65 )   PDF (15555KB) ( 48 )  
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【Objective】 This study was aimed to explore the action mechanism of Yinchenhao decoction (YCHD) in the treatment of diabetes mellitus (DM) by network pharmacology, molecular docking and experimental validation. 【Method】 TCMSP, ETCM, UniProt, SwissTargetPrediction databases and literature reviews were utilized to find the active ingredients and their related targets.OMIM, PharmGkb, TTD and DrugBank databases were utilized to obtain the disease targets of DM.The protein-protein interaction (PPI) network diagrams of YCHD and DM were constructed, and the key targets were analyzed by GO function and KEGG signal pathway enrichment.Molecular docking was performed using AutoDockTool 1.5.7 software to predict the binding energy of active ingredients and core targets.The cells were divided into control group (Control), model group (Model), and YCHD-containing serum low (YCHD-L), medium (YCHD-M) and high (YCHD-H) dose groups.The levels of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (Cox2), Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB) p65, interleukin-1β (IL1β), IL6 mRNA and iNOS, COX2, Myd88, NF-κB P65 protein were examined by in vitro cell assay to verify the key pathways. 【Result】 A total of 147 YCHD active ingredients were obtained, 486 DM disease targets were identified, and 57 genes related to the action of YCHD on DM were identified, including insulin (INS) and IL1β, tumor necrosis factor (TNF), nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NFKB1), signal transducer and transcriptional activator 1 (STAT1), albumin (ALB), TLR4, IL1A, IL10, and IL8 were key target genes.According to the counts value, the top ten entries in GO included 1 biological process, 7 cellular components, and 2 molecular functions, which were the positive regulation of RNA polymerase Ⅱ promoter transcription, endoplasmic reticulum membrane, plasma membrane, extracellular region, nucleoplasm, cytosol, cytoplasm, integral component of membrane, identical protein binding, and protein binding.KEGG pathway analysis showed that it was closely related with NOD-like receptors and NF-κB, Toll-like receptors and other inflammatory signaling pathways.The molecular docking results showed that the active ingredients of YCHD had good binding ability with the main targets.The experimental results showed that compared with control group, the mRNA levels of TLR4, iNOS, COX2, NF-κB p65, IL1β, IL6 and the protein expression levels of INOS, COX2, MyD88, NF-κB p65 of cells were significantly increased (P<0.05) in model group.Compared with model group, the mRNA levels of iNOS, COX2, IL1β, IL6 and the protein expression levels of iNOS and COX2 of cells were significantly reduced (P<0.05) in YCHD-L, YCHD-M and YCHD-H groups.The mRNA levels of TLR4 and NF-κB p65 of cells in YCHD-M and YCHD-H groups were significantly decreased (P<0.05), and the protein levels of Myd88 and NF-κB p65 of cells in YCHD-H group were significantly decreased (P<0.05). 【Conclusion】 YCHD might regulate TLR4, NF-κB and other inflammatory signaling pathways through multiple components and multiple targets to treat the DM.The YCHD-H group showed the most significant inhibition of inflammatory signaling pathway related indicators.
Preparation Process and Quality Control of Compound Ku Xuan Shen Oral Liquid
LI Jiada, XIANG Yifei, NIU Mengchu, LIU Chengzhi, ZHONG Yawen, WU Chunxuan, ZHANG Siyuan, NIE Jing, HOU Yunfeng, HE Jiakang
2024, 51(9):  4120-4131.  doi:10.16431/j.cnki.1671-7236.2024.09.039
Abstract ( 45 )   PDF (3981KB) ( 23 )  
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【Objective】 The experiment was aimed to enrich the dosage form of compound Ku Xuan Shen granules for the prevention and control of bacterial diarrhea in livestock and poultry, develop compound Ku Xxuan Shen oral liquid, and control its quality, lay a pharmacological foundation for later clinical trials. 【Method】 Based on the basis of the previous experiments, the applicability of the joint use of 0.25% benzoic acid and 0.075% sorbic acid as preservatives was evaluated, and the suitable antioxidants among 0.025%-0.100% sodium bisulfite, sodium metabisulfite and ascorbic acid were screened.TLC identification and analysis method for the four medicinal materials of Picria felterrae Lour., Cortex ilicis Rotundae, root of Rhodomyrtus tomentosa and Artemisia anomala S.Moore in the prescription and a HPLC method for the content determination and analysis of the standard component of picroside IA in the royal medicine Picria felterrae Lour. were established.The quality control of oral liquid was carried out according to the relevant provisions of the 2020 edition of the Veterinary Pharmacopoeia of the People’s Republic of China. 【Result】 Adding 0.25% benzoic acid and 0.075% sorbic acid as preservatives, the viable counts of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus niger and Candida albicans in the compound Ku Xuan Shen oral liquid met the criteria for bacteriostatic efficacy on the 14th and 28th day, which indicated that the combination of the preservative had a good anticorrosive effect.Compared with the negative samples, the degradation of picroside IA decreased from 7% to 1.38% at 10 d with the addition of 0.075% sodium metabisulphite as antioxidant, which significantly enhanced the stability of the compound Ku Xuan Shen oral liquid.TLC results showed that the fluorescent spots of Picria felterrae Lour., Cortex ilicis Rotundae, root of Rhodomyrtus tomentosa and Artemisia anomala S.Moore in the oral liquid of compound Kuan Xuan Shen and the control or control herbs were in the same colour in the corresponding positions, which indicated that the specificity of the established TLC method was good.The HPLC results showed good linearity in the range of 14.063 to 450 μg/mL (R2=0.9996) for picroside IA, the active ingredient of Picria felterrae Lour., and the relative standard deviation (RSD) of the results of the precision test, reproducibility test, stability test were 1.02%, 0.44% and 0.60%, respectively.The average recovery was 98.50% with the RSD of 0.56%, which indicated that the established HPLC method of compound Ku Xuan Shen oral liquid met the methodological requirements in terms of specificity, linearity, stability and accuracy.The average values of the content of picroside IA in the three batches of compound Ku Xuan Shen oral liquid were 214.97, 213.85 and 214.82 μg/mL, respectively, and the RSD was 0.38%.Other quality control indicators such as yellow-brown appearance and properties, relative density was 1.039 to 1.055 g/mL, with RSD of 0.88%, pH was 5.50 to 5.66, with RSD of 0.72%, microbiological limits, etc.were in accordance with the relevant provisions of the 2020 edition of the Veterinary Pharmacopoeia of the People’s Republic of China for oral liquids. 【Conclusion】 The compound Ku Xuan Shen oral liquid prepared in this study had a simple process, accurate and reliable quality control method, and could be used for subsequent pilot production and quality control.
Expression and Antibacterial Activity Verification of Endolysin in Prophages of Klebsiella pneumoniae from Cattle
LIU Chuyang, HU Ruirui, LI Fulin, CUI Chaowen, HU Shengwei
2024, 51(9):  4132-4142.  doi:10.16431/j.cnki.1671-7236.2024.09.040
Abstract ( 66 )   PDF (13127KB) ( 18 )  
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【Objective】 The purpose of this experiment was to predict and analyze the prophage carried by Klebsiella pneumoniae and its carrying endolysin gene, explore the antibacterial activity of endolysin in vitro, and provide a new treatment for Klebsiae pneumoniae infection. 【Method】 Using 134 strains of bovine-origin Klebsiella pneumoniae as the research subjects, the prophages in bovine-origin Klebsiella pneumoniae were predicted through the PhiSpy software.Functional annotation and identification of endolysin were performed on the predicted prophages.The recombinant protein was induced and purified by constructing endolysin prokaryotic expression vector, and its lytic activity against Klebsiella pneumoniae NMG10, GS4 and ATCC 13883 was verified in vitro. 【Result】 The prophage sequence carriage rate in the genomes of 134 strains of bovine-origin Klebsiella pneumoniae was 98.5%, with a total of 414 predicted prophage sequences.Among them, 112 belonged to Klebsiella pneumoniae phages, while the other were either phages or phage components of other bacteria.The proportion of all prophages carried by each bovine-origin Klebsiella pneumoniae genome was approximately 1.4%.A total of 44 prophage genes encoding endolysin were found in the genome of 112 prophages of Klebsiella pneumoniae, which was named LyKb.Endolysins LyKb exhibited both lytic and antibacterial activities against Klebsiella pneumoniae strains NMG10, GS4 and ATCC 13883.Among these, LyKb showed the strongest lytic and antibacterial activities against Klebsiella pneumoniae NMG10, followed by Klebsiella pneumoniae GS4 and ATCC 13883. 【Conclusion】 134 strains of bovine-origin Klebsiella pneumoniae commonly carried prophages, and some of the prophage genomes contained genes encoding endolysins.The endolysins of Klebsiella pneumoniae prophages had antibacterial activity against various strains of Klebsiella pneumoniae.
Protective Effect of Cangpu Wumei Powder on Diarrhea Mice Induced by Lipopolysaccharide
FANG Renlai, CHEN Jingyi, LI Hanglin, LIU Qing, CHEN Qi, HU Tingjun
2024, 51(9):  4143-4152.  doi:10.16431/j.cnki.1671-7236.2024.09.041
Abstract ( 49 )   PDF (6628KB) ( 17 )  
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【Objective】 The aim of this study was to evaluate the protective effect of Cangpu Wumei powder on lipopolysaccharide (LPS)-induced diarrhea in mice, and provide theoretical basis for the protection of Cangpu Wumei powder against enteritis diarrhea. 【Method】 60 ICR mice were randomly divided into 6 groups with 10 mice each group and half male and half female.Mice in blank control group and LPS model group were given 20 mL/kg of purified water by gavage.Berberine hydrochloride group was given 5 mg/mL berberine hydrochloride solution as 20 mL/kg.The Cangpu Wumei powder low, medium and high dose groups were given the suspension of 0.8, 1.6 and 3.2 g/kg of Cangpu Wumei powder (the final concentrations were 40, 80 and 160 mg/mL, respectively) according to 20 mL/kg.After 7 consecutive days of administration, except for blank control group, mice in the other groups were intraperitoneally injected with 0.5 mg/mL LPS solution at the dose of 20 mL/kg at 24 h after the last administration, which induced diarrhea in mice.The defecation of mice within 2 h was observed and recorded, and the starting time of diarrhea, diarrhea rate and diarrhea index were calculated.The initial body weight, pre-modeling body weight and final body weight of mice were counted.After 2 h post LPS treatment, the blood of the mice was collected and routine blood test was performed.The ileum, cecum and colon tissues were collected and pathological sections were observed. 【Result】 The starting time of diarrhea of male and female mice in LPS model group was 8 and 6 min, respectively.The starting time of diarrhea of male mice in the Cangpu Wumei powder low, medium and high dose groups was 23, 29 and 41 min respectively, and the starting time of diarrhea of female mice was 22, 27 and 29 min, respectively.Compared with LPS model group, the diarrhea rate and diarrhea index of male and female mice in Cangpu Wumei powder medium and high dose groups were significantly decreased (P<0.05).There was no significant difference in white blood cells and lymphocytes counts between male and female mice in the low, medium, and high dose groups of Cangpu Wumei powder compared to the berberine hydrochloride group (P>0.05).There were no significant differences in diarrhea index and weight gain rate of male and female mice in Cangpu Wumei powder medium and high dose groups compared with blank control group (P>0.05).The histopathological observation showed that the inflammatory cell infiltration of ileum, cecum and colon was more serious and the villi was shorter in the LPS model group.A small number of inflammatory cells infiltrated the male and female mice in Cangpu Wumei powder low dose group.The intestinal villi and goblet cells of male and female mice in berberine hydrochloride group and Cangpu Wumei powder medium and high dose groups were not significantly changed. 【Conclusion】 Cangpu Wumei powder treatment could prolong the starting time of diarrhea, reduce the rate of loose stool and diarrhea index, and relieve LPS-induced enteritis diarrhea.The recommended dose of Cangpu Wumei powder was 1.6 g/kg under the experimental conditions.
Establishment and Index Evaluation of Inflammatory Bowel Disease Model in C57BL/6 Mice
HE Jiayu, YANG Mingqi, WU Jiao, YANG Yu, YAO Huan, YI Jine, KONG Li
2024, 51(9):  4153-4163.  doi:10.16431/j.cnki.1671-7236.2024.09.042
Abstract ( 66 )   PDF (9437KB) ( 24 )  
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【Objective】 The study was aimed to construct a mouse inflammatory bowel disease (IBD) model by stimulating with dextran sulfate sodium (DSS) or lipopolysaccharide (LPS), providing an efficient and economical model reference for IBD research, and providing reliable methodological support for the screening and efficacy evaluation of IBD anti-inflammatory drugs. 【Method】 C57BL/6 male mice were chosen and exposed to different concentrations of DSS (3%, 4% and 5%) for 7 d or LPS (5, 10 and 20 mg/kg) for 4 h in the current study.The model was evaluated by measuring colon length, observing intestinal morphology and structure with HE staining, and detecting mRNA expression of intestinal inflammatory factors by Real-time quantitative PCR. 【Result】 DSS could decrease the colon length of mice, led to pathological bleeding, atrophy of glands and disordered structure in colon.Moreover, DSS significantly induced inflammatory cell infiltration and the mRNA expression of the inflammatory cytokines interleukin-1β (IL-1β), IL-6, IL-10 and tumor necrosis factor-α (TNF-α) in the colon of mice, which implied that 4% DSS could establish the IBD efficiently.Meanwhile, LPS could cause mesenteric hemorrhage, intestinal swelling and congestion of mice.Moreover, LPS could decrease the villus height and increased the crypt depth in the intestine.In addition, LPS could also upregulate mRNA levels of intestinal inflammatory cytokines IL-10 and IL-1β, downregulate IL-6 mRNA level and elevate TNF-α mRNA level in the duodenum and ileum, and reduce TNF-α mRNA level in the jejunum.It was found that the most significant effect of intestinal damage was 5 mg/kg LPS treatment. 【Conclusion】 4% DSS or 5 mg/kg LPS treatment were the optimal concentrations to establish IBD model in C57BL/6 mice.Intestinal pathomorphological changes, intestinal inflammatory cell infiltration and inflammatory factors disorder such as the mRNA expression of IL-1β, IL-6, IL-10 and TNF-α were important indicators to evaluate the models of IBD.
Safety Test of Banqing Granules on Target Animal Pigs
DING Mengjie, WANG Pingping, HU Longfei, ZHANG Qiuna, CHEN Jing, LIU Jia, LIU Ruonan, HAO Zhihui
2024, 51(9):  4164-4173.  doi:10.16431/j.cnki.1671-7236.2024.09.043
Abstract ( 44 )   PDF (15888KB) ( 43 )  
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【Objective】 This experiment was aimed to evaluate the safety of Banqing granules on pigs, and provide reference for clinical rational drug use. 【Method】 32 healthy pigs of about 110 days of age were selected and randomly divided into recommended dose (100 mg/kg BW), triple dose (300 mg/kg BW), fivefold dose (500 mg/kg BW) groups of Banqing granules, and control group, with 8 pigs in each group.Pigs in experimental groups were administered in drinking water for 14 days and continued observation for 10 days after withdrawal.Pigs in control group were raised normally without medication.Clinical manifestations of experimental pigs were observed and recorded one by one in the morning and afternoon every day.The pigs were weighed before administration (experiment day 0, D0), after administration (experiment day 15, D15) and after observation (experiment day 25, D25), and blood was collected after fasting weighing to detect blood routine and blood biochemical indexes.Histopathological examinations were performed on animals in fivefold dose group and control group after administration and after observation, respectively. 【Result】 During the experiment, the mental state, appetite and bowel movements of pigs in each dose group were normal, and the growth performance indexes were not significantly different (P>0.05).Blood routine index results showed that the number of red blood cells and hemoglobin content of pigs increased with the increase of dose, but both fluctuated within the normal range, and the other individual indicators had significant differences but no dose correlation and the index values were within the normal range.Blood biochemical index results showed that serum total protein of pigs increased with the increase of administration concentration, blood glucose content of pigs decreased with the increase of administration concentration but all fluctuated within the normal range, and other individual indexes showed significant differences but no dose correlation and the index values were all within the reference range of historical data.The histopathological results showed that the heart, liver, spleen, lung and kidney of pigs in fivefold dose group had clear structure, distinct layers and normal development, and no pathological changes related to the action of Chinese medicine were found, such as cell degeneration, necrosis and inflammatory cell infiltration, and no significant differences were found between the tissues and control group. 【Conclusion】 Fivefold dose of Banqing granules (500 mg/kg BW) was safe for pigs.
Effect of Bamboo Leaf Flavonoid on Immune Efficacy of Chick Embryo Inoculation with Newcastle Disease Vaccine
JIANG Xinrui, FENG Helong, YANG Hongchun, JIANG Liren, WANG Hongcai, ZENG Zhe, ZUO Bo, ZHANG Tengfei, LUO Qingping, SHANG Yu, WEN Guoyuan
2024, 51(9):  4174-4181.  doi:10.16431/j.cnki.1671-7236.2024.09.044
Abstract ( 45 )   PDF (1656KB) ( 26 )  
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【Objective】 The aim of this experiment was to explore the effect of bamboo leaf flavonoid (BLF) as a vaccine adjuvant on the immune effect of Newcastle disease (ND) vaccine in chicken embryos. 【Method】 The experiment selected 108 18-day embryonic SPF hatching eggs and randomly divided into 3 groups:Control group (no injection of any substance), TS group (injection of 0.1 mL 103.0 EID50/0.1 mL of Newcastle disease virus (NDV) TS09-C vaccine strain) and BLF+TS group (injection of 0.1 mL of a mixture of 2% BLF and 103.0 EID50/0.1 mL NDV TS09-C vaccine strain), with 36 eggs in each group.During the experiment, the hatching rate of eggs and the survival rate of chicks were calculated.When the chicks were 7,14,21 and 28 days old, 3 chicks from each group were randomly selected for slaughter, then spleen, bursa of Fabricius, and thymus tissue were collected, and immune organ indexes were counted.The subwing vein blood of chicks was collected and serum antibody and cytokine levels were measured.The challenge protection test was carried out with NDV strain F48E9 at 28 days of age.After challenge, the status of the chicks was observed every day, and the weight change was recorded.On the 3rd day after the challenge, 3 chicks in each group were randomly selected for slaughter.Duodenum, lung and trachea tissues were collected and viral load in different tissues of chicks were detected. 【Result】 After chicken embryo inoculation, the hatchability rate of chicks in BLF+TS group was 100%, and the survival rate was 97.22%.Compared with TS group, the spleen, bursa of Fabricius and thymus indices as well as the serum levels of interleukin 6 (IL-6) and interferon-γ (IFN-γ) contents in BLF+TS group were significantly increased (P<0.05).The results of the challenge test showed that the survival rate of chicks in BLF+TS group after challenge was 100%.The body weight increase rate of BLF+TS group was significantly lower than that of healthy control group at 2-4 days after challenge (P<0.05).The body weight growth rate of chicks in BLF+TS group was significantly higher than that of TS group at 3-7 days after challenge (P< 0.05).The viral load in the respiratory tract and digestive tract tissues of chicks in BLF+TS group after challenge was significantly lower than that in TS group (P<0.05). 【Conclusion】 BLF could be safely used as an immune adjuvant for chicken embryo vaccination, and could help chicks establish early immune protection and further improve the immune effect of ND vaccine.The results provided reference for the development of efficient chicken embryo immune vaccine adjuvants.
Environmental Safety
Study on Bacterial Flora Changes in Producing Bedding Process Using Cow Manure by Anaerobic Fermentation Technology in Dairy Farms
WANG Wenkai, SUI Jie, XU Yijing, TAO Qiyuan, RU Caixia, WEI Zehui, XIN Yaping, BASANG Zhuzha, JIA Cunling
2024, 51(9):  4182-4189.  doi:10.16431/j.cnki.1671-7236.2024.09.045
Abstract ( 48 )   PDF (5529KB) ( 10 )  
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【Objective】 The changes of bacterial flora were investigated for producing bedding process using cow manure by anaerobic fermentation technology in dairy farms, in order to provide some theoretical references for standardized production,the reasonable and safe use of cow manure as bedding. 【Method】 Samples were collected from four large-scale dairy farms, including cow manure before anaerobic fermentation, cow manure after solid-liquid separation during anaerobic fermentation, and cow manure bedding on the bed.Bacterial DNA was extracted for high-throughput 16S rRNA sequencing, and the microbial diversity, composition, and significant differences in characteristic microbial communities at different stages were analyzed. 【Result】 Compared with cow manure before fermentation, the diversity and richness of microbial communities in cow manure after anaerobic fermentation solid-liquid separation were reduced, but were similar to those in cow manure substrate.The phyla and genus with the highest abundance in cow manure before fermentation were Firmicutes (55.15%) and UCG-005 (16.68%), respectively.The phyla and genus with the highest abundance in cow manure after anaerobic fermentation solid-liquid separation were Firmicutes (48.12%) and Bacillus (12.16%), respectively.The phyla and genus with the highest abundance in cow manure bedding were Actinobacteriota (37.02%) and Glutamicibacter (11.65%), respectively.LEfSe analysis showed that there were a total of 14 bacterial genera with significant differences between the three groups of cow manure before fermentation, cow manure after anaerobic fermentation solid-liquid separation, and bedding. 【Conclusion】 Through the study of the microbial community composition during anaerobic fermentation of cow manure production in dairy farms, it was found that the dominant bacterial phyla and genus had undergone significant changes.After fermentation treatment, the diversity and richness of the microbial community had decreased.This production process could meet the safety requirements of dairy farms for cow manure production.