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05 February 2024, Volume 51 Issue 2
Biotechnology
Expression and Bioinformatics Analysis of Cat CDH1 Gene in c-MYC Overexpressed Fibroblasts
AN Jie, YANG Jie, DOU Minmin, SUN Nannan, ZHAO Dipeng, DU Rong, QIN Jian
2024, 51(2):  443-452.  doi:10.16431/j.cnki.1671-7236.2024.02.001
Abstract ( 60 )   PDF (5408KB) ( 30 )  
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【Objective】 This study was aimed to investigate the effect of myelocytomatosis viral oncogene homolog (c-MYC) on cat fibroblasts and its relationship with cadherin 1 (CDH1) gene expression and molecular characteristics, so as to provide a basis for application in the prevention and treatment of tumor diseases and the repair of tissue damage in cat.【Method】 Cat fetal fibroblasts were isolated and cultured by tissue adherence, and the PB-TRE-c-MYC plasmid was transfected into the cells by electroporator, then the cell morphology was observed.The expression of CDH1 gene was detected by Real-time quantitative PCR, and the physicochemical properties and structural characteristics of CDH1 protein were analyzed by bioinformatics software.【Result】 c-MYC overexpression led to the changes in cell morphology, with a transition from the long shuttle shape of mesenchymal cells to the pebble shape of epithelial cells, and the expression of epithelial cell marker gene CDH1 was extremely significantly upregulated (P<0.01).Bioinformatics analysis results showed that CDH1 protein in cat had 881 amino acids, of which the most abundant was leucine, localized in cell membrane, with 4 CA domains, which mediated cell-cell contact.CDH1 protein belonged to hydrophilic acidic proteins, mainly composed of random coil, and might have signaling peptide sites transported by Sec translocons and cleaved by signal peptidase Ⅰ (Sec/SPⅠ).【Conclusion】 The expression of CDH1 gene in cat could be activated by exogenous reprogramming factor c-MYC, and its encoding protein cadherin could promote the mesenchymal-to-epithelial transition of cat fetal fibroblasts to inhibit cell carcinogenesis.
Effect of Long Non-coding RNA MSTRG.14200 on Porcine Skeletal Muscle Satellite Cell Differentiation and Muscle Fiber Transformation
YANG Yuming, ZHAO Xinming, TAN Baohua, XIAO Liyao, LU Geyan, ZHAI Lijun, HUANG Yiyang, HONG Linjun, GU Ting
2024, 51(2):  453-461.  doi:10.16431/j.cnki.1671-7236.2024.02.002
Abstract ( 33 )   PDF (4878KB) ( 18 )  
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【Objective】 This experiment was conducted to investigate the effects of long non-coding RNA (lncRNA) MSTRG.14200 on myogenic differentiation of porcine skeletal muscle satellite cells and the transformation of different types of muscle fibers.【Method】 Three 6-month-old Duroc pigs were slaughtered, and the heart, liver, spleen, lung, kidney, stomach, longissimus dorsi muscle, leg muscle, soleus and extensor digitorum longus were collected.Real-time quantitative PCR was used to detect the expression of MSTRG.14200 in different tissues.The MSTRG.14200 overexpression vector pcDNA3.1-14200 was constructed and transfected into PSCs cells, and the cells were collected after 3 days of induced differentiation.The mRNA and protein expressions of myosin heavy chain (MyHC), myogenin (MyoG), mydifferentiation factor (MyoD), MyHCⅠ, MyHCⅡa, MyHCⅡb and MyHCⅡx were detected by Real-time quantitative PCR and Western blotting, respectively.The percentage of MyHC, MyHCⅠ and MyHCⅡb positive cells was detected by immunofluorescence assay.【Result】 MSTRG.14200 was expressed in different tissues of Duroc pigs, and the expression was the highest in longissimus dorsi muscle, which was extremely significantly higher than other tissues (P<0.01), and there were significant differences in the expression of extensor digitorum longus and soleus (P<0.05).After overexpression of MSTRG.14200, mRNA levels of MyoD and MyHC genes related to cell differentiation were extremely significantly increased (P<0.01), and protein levels of MyoD, MyoG and MyHC were significantly or extremely significantly increased (P<0.05 or P<0.01), the percentage of MyHC positive cells was extremely significantly increased (P<0.01).The mRNA and protein expressions of MyHCⅠ gene related to muscle fiber transformation and the proportion of positive cells were significantly or extremely significantly decreased (P<0.05 or P<0.01), the mRNA and protein expression of MyHCb gene and the percentage of positive cells were significantly increased (P<0.05).【Conclusion】 MSTRG.14200 could promote myogenic differentiation of porcine skeletal muscle and promote the transformation of slow muscle fibers into fast muscle fibers.The results provided a theoretical basis for exploring the molecular mechanism of transformation of porcine skeletal muscle fiber type.
Effect of Leukemia Inhibitory Factor on Developmental Capacity of Bovine Oocyte
MO Xianhong, TAO Qinghua, FANG Yi, LIANG Jing, ZHANG Jiayin, LIANG Xiaohang, XU Zhenjun
2024, 51(2):  462-469.  doi:10.16431/j.cnki.1671-7236.2024.02.003
Abstract ( 28 )   PDF (1235KB) ( 12 )  
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【Objective】 This experiment was to explore the effect of leukemia inhibitory factor (LIF) on the development ability of bovine oocytes and early embryo and the expression of multipotent genes.【Method】 The cumulus-oocyte-complexes (COCs) of bovine were divided into 4 groups.In the control group, no LIF solution was added during in vitro maturation (IVM) and embryo in vitro culture (IVC).In treatment group 1 (T1 group), 25 ng/mL LIF was added to the IVM process and no LIF was added to the IVC process.In treatment group 2 (T2 group), the IVM process did not add LIF, and the IVC process added 25 ng/mL LIF.In treatment group 3 (T3 group), 25 ng/mL LIF was added to the whole process of IVM and IVC.The concentration of intracellular free calcium ([Ca2+]i) in oocytes was detected by Fluo-3 fluorescence indicator.Oocytes with the first polar body were selected for parthenogenetic activation, and the cleavage rate and blastocyst rate of each group were calculated.Real-time quantitative PCR was used to detect the relative expression of multipotent genes.【Result】 Compared with the control group, intracellular [Ca2+]i concentration of bovine oocytes in T1 group was extremely significantly decreased after 8 h of cultivation (P<0.01).After 24 h of cultivation, the intracellular [Ca2+]i concentration of bovine oocytes in T1 group was extremely significantly increased (P<0.01).The cleavage rate and blastocyst rate in T1 and T3 groups were significantly higher than those in control group, and the blastocyst rate in T3 group was significantly higher than that in T1 and T2 groups (P<0.05).The results of Real-time quantitative PCR showed that the expression levels of POU5F1 and NANOG genes in T3 group were significantly higher than those in control group, and the expression level of CDX2 gene was significantly lower than that in control group (P<0.05).【Conclusion】 The addition of LIF during IVM could improve the developmental ability of oocytes, and the addition of LIF during the whole process of IVM+IVC had a positive effect on early embryonic development and the expression of multipotent genes.
Study on the Co-regulation of m6A and miRNA on Skeletal Muscle Development of Peking Ducks in Embryonic Stage
LI Ying, GUO Xu, JIANG Qicheng, GU Lihong
2024, 51(2):  470-481.  doi:10.16431/j.cnki.1671-7236.2024.02.004
Abstract ( 17 )   PDF (6639KB) ( 4 )  
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【Objective】 The aim of this experiment was to study the regulation of miRNA and N6-methyladenosine (m6A) in the development of breast muscle of Peking ducks at embryonic stage, in order to lay a foundation for understanding the development of breast muscle of Peking ducks.【Method】 The breast muscles of duck embryo at E13 and E27 stages were collected.Methylated RNA immunoprecipitation with next generation sequencing (MeRIP-Seq) and microRNA sequcencing (miRNA-Seq) were used to identify potential differential genes regulated by m6A in the differentiation process of breast muscle cells in Beijing duck embryos, and their functions were analyzed in combination with miRNA and its target genes.【Result】 The miRNA-Seq results showed that there were 181 significantly different miRNAs, and a total of 11 176 targets were predicted through target gene prediction, including m6A related genes and muscle development related genes.14 344 and 14 016 m6A peaks were detected by MeRIP-Seq in E13 and E27 groups, respectively, mapping to 8 248 known genes and 7 763 known genes.A total of 3 240 m6A were mapped to 2 767 differentially methylated genes (DMGs) during E13 and E27 stages.GO and KEGG analysis showed that DMGs were mainly enriched in pathways related to muscle development and fat deposition, as well as many genes related to muscle development, such as MTPN, MYF6 and MYF5 genes.556 differentially expressed genes (DEGs) were detected by miRNA-Seq.GO and KEGG pathway analysis results indicated that these DEGs were significantly enriched in the Wnt signaling pathway, metabolic pathway, and fatty acid, suggesting that DEGs might be involved in adipocyte differentiation and fat metabolism processes.The association analysis between miRNA-Seq and m6A-Seq showed that a total of 1 517 differential expression and methylation genes (DEMGs) were detected.GO and KEGG analysis of these DEMGs showed that DEMGs were mainly enriched in muscle development related pathways.【Conclusion】m6A methylation modification and miRNA had important effects on skeletal muscle development and fat deposition in Peking ducks.
Research Progress of RPA Detection Technology in the Detection of Avian Pathogens
BAO Taotao, YANG Xianfu, LIAO Fei, WU Tongkui, MO Xinghu, WANG Zhongrong
2024, 51(2):  482-490.  doi:10.16431/j.cnki.1671-7236.2024.02.005
Abstract ( 22 )   PDF (1016KB) ( 11 )  
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Recombinant polymerase amplification (RPA) is a new type of constant temperature nucleic acid amplification technology that has attracted more attention from researchers in recent years.Due to its convenient operation, fast amplification speed, high specificity, and high sensitivity, without the need for precision instruments, and being able to complete nucleic acid detection in a constant temperature environment (25-45 ℃), it is highly likely to replace traditional PCR detection technology.Since the development of RPA for over a decade, it has been widely applied in the fields of bacteria, viruses, fungi, parasites, and drug resistance.This method has been recognized by researchers as the most promising rapid molecular diagnostic tool currently, it has a wide range of application prospects.The author summarized the RPA detection, technological advantages, common detection methods, and research progress in the detection of avian pathogens, aiming to provide theoretical reference for the research of new detection technologies for clinical avian pathogens.
Physiological and Biochemical
Effects of Tumor Necrosis Factor-α on Mouse Small Intestinal Organoid Growth,Barrier Function and Intestinal Functional Cells
HE Wensheng, XIE Wenshuai, LI Shunkang, KUANG Yanling, LIU Yulan, WANG Dan
2024, 51(2):  491-499.  doi:10.16431/j.cnki.1671-7236.2024.02.006
Abstract ( 24 )   PDF (4321KB) ( 10 )  
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【Objective】 The effects of tumor necrosis factor-α (TNF-α) on small intestinal organoid growth, tight junction proteins, and various functional cell marker genes were studied to establish disease damage models for small intestinal organoids.【Method】 The crypt cells were isolated from mouse small intestine with gentle cell dissociation reagent (GCDR) digestion solution and cultured in intestinal organoid medium.Small intestinal organoids were stimulated with 0 (control group), 50, 250 and 500 ng/mL TNF-α for 48 h.The growth of organoids was observed under an optical microscope, and cell proliferation was tracked by Edu staining.The expression levels of cell proliferation, barrier function and intestinal functional cell marker genes mRNA were detected by Real-time fluorescent quantitative PCR.【Result】 ①Compared with control group, 50 and 250 ng/mL TNF-α significantly reduced the germination rate of small intestinal organoids (P<0.05), and TNF-α stimulation had no effect on organoid formation rate (P>0.05).250 and 500 ng/mL TNF-α resulted in a significantly higher rate of small intestianl organoid necrosis (P<0.05).②Compared with control group, 250 ng/mL TNF-α significantly reduced the mRNA expression of the small intestinal organoid tight junction protein Occludin (P<0.05).500 ng/mL TNF-α increased mRNA expression of Claudin-1 (P<0.05), a tight junction protein in small intestines.③Compared with control group, different concentrations of TNF-α led to a significant increase in the expression of mRNA of TNF-α (P<0.05), but had no significant effect on the expression of IL-6 (P>0.05).250 and 500 ng/mL TNF-α resulted in a significant increase in mRNA expression of IL-1β (P<0.05).④Compared with control group, 250 ng/mL TNF-α led to a significant decrease in mRNA expression of the marker genes Ki67 and Pcna of proliferating cells (P<0.05).⑤Compared with control group, 50 ng/mL TNF-α stimulation significantly reduced the mRNA expression of Lgr5 gene (P<0.05), 250 and 500 ng/mL TNF-α stimulation significantly reduced mRNA expression in Muc2, Chga and Lyz genes.250 ng/mL TNF-α stimulation significantly reduced the mRNA expression of Alpi gene (P<0.05).【Conclusion】 250 ng/mL TNF-α stimulation could inhibit the growth of small intestinal organoids, inhibit the proliferation of intestinal stem cells and the differentiation of various functional cells, which could provide a reference for future clinical applications.
Regulation of Epithelial Mesenchymal Transition in Mammary Epithelial Cells of Dairy Cow by RUNX1
WANG Kuan, YANG Bowen, WANG Juyu, DENG Jianming, XU Hui, YANG Yang, CHEN Hongjian, DAI Feiyan, GU Xiaolong, QU Weijie, ZHANG Limei
2024, 51(2):  500-512.  doi:10.16431/j.cnki.1671-7236.2024.02.007
Abstract ( 18 )   PDF (4322KB) ( 5 )  
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【Objective】 The aim of this study was to investigate the effect and mechanism of Runt-associated transcription factor 1 (RUNX1) on epithelial mesenchymal transition (EMT) in bovine mammary epithelial cells (MAC-T) induced by Staphylococcus aureus (S. aureus).【Method】 CCK-8 method was used to detect the effect of different multiplicity of infection of heat-inactivated S. aureus on the viability of MAC-T cells.The morphological changes of MAC-T cells treated by S. aureus were observed by inverted microscope.The expression changes of EMT marker molecules (E-cadherin, N-cadherin, α-SMA and Vimentin), TGF/Smad pathway signal molecules (TGF-β1, Smad2, Smad3 and Smad4)and RUNX1 in MAC-T cells treated by S. aureus were detected by Real-time quantitative PCR and Western blotting.The expression levels of EMT markers, TGF/Smad pathway signaling molecules and RUNX1 were compared before and after treatment with RUNX1 and TGF-β-specific inhibitors.【Result】 After 72 h of treatment in different concentration groups, there was extremely significantly decreased in cell viability compared with the 24 and 48 h (P<0.01), and the observation by microscope revealed that the cells appeared to float and die at this time.Therefore, the time settings for subsequent observation of cell morphology changes were 12, 24, 36 and 48 h.When MAC-T cells were treated with heat inactivated S. aureus (MOI=100) for 48 h, the morphology of MAC-T cells changed from the typical 'pebble’ shape of epithelial cells to the 'long spindle’ shape of interstitial cells.The results of Real-time quantitative PCR and Western blotting detection showed that, compared with normal cells, the mRNA expression of EMT marker E-cadherin was extremely significantly down-regulated (P<0.01), and the mRNA expression levels of Vimentin, α-SMA, N-cadherin, TGF-β1, Smad2, Smad3, and Smad4 and RUNX1 were extremely significantly or significantly up-regulated (P<0.01), and the protein expressions of Vimentin, α-SMA, P-Smad2 and RUNX1 obviously up-regulated.After treatment with the LY2109761 inhibitor, compared to the infection group, the mRNA expression of Smad2, Smad3, Smad4, N-cadherin, Vimentin, α-SMA, and RUNX1 was extremely significantly down-regulated in MAC-T cells in the two inhibitor groups (P<0.01), E-cadherin mRNA expression was extremely significantly up-regulated (P<0.01).After treatment with the inhibitor Ro5-3335, there was no significant interstitial cell-like change in the cell morphology of MAC-T cells.Compared to the infection group, the mRNA expression of E-cadherin was extremely significantly up-regulated in the cells of the two inhibitor Ro5-3335 groups (P<0.01), and N-cadherin, Vimentin, α-SMA (except α-SMA in the 10 μmol/L Ro5-3335 inhibitor group), TGF-β1, Smad2, Smad3 and Smad4 mRNA expression levels were all extremely significantly down-regulated (P<0.01).【Conclusion】 Heat-inactivated S. aureus treatment of MAC-T cells for 48 h could activate the TGF/Smad pathway to induce EMT changes in MAC-T cells.RUNX1 could regulate MAC-T cells EMT process induced by S. aureus by mediating TGF/Smad pathway.
Research Status on the Mechanism of SIRT2 Regulating Autophagy
QIN Tianmiao, FANG Xiaohuan, LI Junjie
2024, 51(2):  513-520.  doi:10.16431/j.cnki.1671-7236.2024.02.008
Abstract ( 14 )   PDF (887KB) ( 9 )  
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Sirtuin 2(SIRT2) is a nicotinamide adenine dinucleotide (NAD+) -dependent deacetylase capable of regulating protein acetylation modification, and is a member of the silence-message regulator family (Sirtuins), which has a variety of biological functions.It plays a regulatory role in neurodegeneration, cell differentiation, glucose and lipid metabolism, tumorigenesis, autophagy and other processes.Cellular autophagy is a protective mechanism that maintains body homeostasis by removing abnormal proteins or organelles, and has the role of promoting cell renewal and maintaining cell quality.Celluar autophagy can be divided into three main forms, including macroautophagy, microautophagy and chaperon-mediated autophagy, all of which use lysosomes to degrade or recycle damaged organelles, misfolded and aggregated proteins, and other macromolecules, among which macroautophagy is the most studied, which is divided into macroautophagy (commonly known as autophagy) and selective macroautophagy.Mitochondrial autophagy is a major pathway for selective removal or degradation of damaged and redundant mitochondria in eukaryotic cells, and is also a selective macroautophagy mainly regulated by SIRT2 in cells.The authors mainly review the regulatory role and mechanism of SIRT2 on autophagy and mitochondrial autophagy, in order to provide reference and direction for further research on more regulatory functions of SIRT2 in mammalian physiology and its therapeutic effects on various diseases.
Nutrition and Feed
Effects of Ferulic Acid on Growth Performance and Intestinal Anti-oxidant Indicators of Jilin White Geese Under Lipopolysaccharide Stress
LIU Yingkun, GUO Wei, FU Zengyu, SUN Meng, LI Haojia, ZHOU Haizhu
2024, 51(2):  521-529.  doi:10.16431/j.cnki.1671-7236.2024.02.009
Abstract ( 25 )   PDF (1044KB) ( 17 )  
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【Objective】 The aim of this study was to investigate the effects of ferulic acid (FA) on the growth performance, organ indexes, and intestinal anti-oxidant indicators of Jilin White geese induced by lipopolysaccharide (LPS), and provided a reference for alleviating oxidative stress in goose breeding.【Method】 A total of 120 healthy male Jilin White geese aged 7 days were randomly divided into 6 groups (5 replicates per group, 4 geese per replicate):Group K (blank control group), group L (LPS control group), and groups A, B, C, and D (supplemented with 60, 120, 180, and 240 mg/kg FA, respectively).On days 14, 17, and 20 of the formal experiment, the geese in groups L, A, B, C, and D were injected with 0.5 mg/kg LPS intraperitoneally, while the geese in group K was injected with an equal volume of saline.On day 21, 10 geese were randomly selected from each group for slaughter, and the hearts, livers, spleens, kidneys, bursa of Fabricius, and thymuses were removed and weighed to calculate the organ indexes.The duodenum, jejunum, and ileum were separated for determination of intestinal antioxidant indicators.【Result】 ① On the 1st to 14th day, compared with the group L, the final body weight (FB), average daily feed intake (ADFI), and average daily gain (ADG) in group C were significantly increased, while the feed-to-gain ratio (F/G) was significantly decreased (P<0.05).Except for ADFI, which was significantly higher than that of group K, other indicators in Group C were not significantly different from Group K (P>0.05).On the 15th to 21st day, compared with group L, FB, ADG, and ADFI in groups A, B, C, and D were significantly increased, while F/G was significantly decreased (P<0.05), and some indicators showed no significant difference compared to group K (P>0.05).On the 1st to 21st day, compared with the group L, the ADG and ADFI in groups B and C were significantly increased, while F/G was significantly decreased (P<0.05), and there was no significant difference compared to group K (P>0.05).②The thymus index in group D was significantly higher than that in groups L and K in organ indexes (P<0.05).③ In the duodenum, the content of malondialdehyde (MDA) in groups A, C, and D were significantly lower than that in group L (P<0.05), and there was no significant difference compared to group K (P>0.05).④ In the jejunum, MDA content and glutathione peroxidase (GSH-Px) activity in group C were significantly higher than those in group L (P<0.05), and there was no significant difference compared to group K (P>0.05);The activity of catalase (CAT) in group C was significantly higher than that in groups L and K (P<0.05). ⑤In the ileum, MDA content in groups B and C was significantly lower than that in group L, and GSH-Px activity in group C was significantly higher than that in group L (P<0.05), and there was no significant difference compared to group K (P>0.05).【Conclusion】 The addition of 180 mg/kg ferulic acid in the diet could promote the growth of geese and alleviate oxidative stress damage in various intestinal segments.
Effects of Whole-plant Corn Silage on the Gut Microbiota of Tibetan Pigs
WU Danning, ZHANG Yanan, HUANG Tao, ZHOU Xiaolong, WANG Han, LI Xiangchen, LI Hongjun, WEN Jun, ZHANG Qi, ZHAO Ayong, YANG Songbai
2024, 51(2):  530-539.  doi:10.16431/j.cnki.1671-7236.2024.02.010
Abstract ( 15 )   PDF (5268KB) ( 9 )  
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【Objective】 This study aimed to explore the effects of adding whole-plant corn silage to the feed on the gut microbial community of Tibetan pigs.【Method】 A total of 40 Tibetan pigs with similar age and weight ((24.26±2.78) kg), half male and half female, were randomly divided into control and experimental groups, with 20 pigs in each group.The pigs in control group were fed basal diet, while the pigs in experimental group were fed the basal diet with 30% whole-plant corn silage replacing.Feeding time was 7 months.After the feeding period, 40 fecal samples were collected, 20 from each group.The 16S rRNA gene sequencing technology was used to analyze the effects of whole-plant corn silage on the composition and structure of the gut microbiota in Tibetan pigs.【Result】 The results showed that a total of 19 phyla, 33 classes, 67 orders, 123 families, 277 genera, and 524 species were identified in both groups.The dominant phyla were Firmicutes and Bacteroidota.The Shannon index of gut microbiota in the experimental group was significantly lower than that of the control group (P<0.05).A Wilcoxon rank-sum test was used to compare the abundance of microbial taxa between two groups.The experimental group showed a significant increase in the abundance of Spirochaetota, Fibrobacterota, Treponema, Lactobacillus, and Streptococcus, while Actinobacteriota, Proteobacteria, and Christensenellaceae_R-7_group were significantly decreased (P<0.05).Results of the LEfSe analysis showed that the abundance of Streptococcus alactolyticus and Lactobacillus amylovorus were significantly increased in the experimental group compare to control group (P<0.05).【Conclusion】 Feeding whole-plant corn silaga altered the structure of gut microbiota, increased the relative abundance of beneficial bacteria in Tibetan pigs, and might have a positive impact on gut health.
Effects of Feed Mulberry on Production Performance,Egg Quality and Intestinal Tissue Morphology of Laying Hens
HUANG Jing, ZHAO Na, GUO Wanzheng, JIN Feng, CHEN Fang, ZHU Wei, FAN Qiwen, DU Encun, TAO Wenjing, HUANG Shaowen, WEI Jintao
2024, 51(2):  540-548.  doi:10.16431/j.cnki.1671-7236.2024.02.011
Abstract ( 21 )   PDF (985KB) ( 13 )  
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【Objective】 This experiment was conducted to investigate the appropriate amount of feed mulberry and its application effect in laying hens.【Method】 Two hundred and seventy Jinghong No.1 laying hens at 36 weeks old were assigned to three dietary treatments randomly.Each treatment consisted of six replicates with 15 hens per replicate.The hens in control group were fed with the basal diet, and the hens in two test groups were fed basal diet added with 1%, 3% feed mulberry meal, respecyively.The pre-trial period lasted for 2 weeks, and the experimental period lasted for 12 weeks.During the trial period, daily feed feeding, eggs production, unqualified eggs, eggs weight, and number of hens deaths and culling were recorded every day in each replicate, and the residual feed was recorded once a week.At the end of week 4, 8 and 12, 5 eggs were randomly selected from each replicate for egg quality determination.At the end of week 12, 1 laying hens were randomly selected from each replicate for slaughter, and duodenum, jejunum and ileum samples were separated to determine the intestinal tissue morphology.【Result】 Compared with control group, ①During 1 to 4 weeks, the laying rate of 1%, 3% mulberry meal groups, and the average egg weight of 1% mulberry meal group were decreased significantly (P<0.05).During 9 to 12 weeks, the laying rate of 1%, 3% mulberry meal groups were decreased significantly (P<0.05).During the whole experiment period, the laying rate and the average egg weight of 1%, 3% mulberry meal groups, and the death and culling rate of 1% mulberry meal group were decreased significantly (P<0.05).②At the end of week 4, the eggshell strength was increased significantly (P<0.05), the L* value of egg yolk was decreased significantly (P<0.05), and the a* value of egg yolk was increased significantly (P<0.05) in 1% mulberry meal group.At the end of week 8, the total cholesterol content in egg yolk of 1% mulberry meal group was decreased significantly (P<0.05).At the end of week 12, the shape index of egg was decreased significantly (P<0.05), and the eggshell strength was increased significantly (P<0.05) in 1% mulberry meal group.The total cholesterol content in egg yolk of 1%, 3% mulberry meal groups were decreased significantly (P<0.05).③ At the end of week 12, the muscular thickness of ileum of 3% mulberry meal group was increased significantly (P<0.05), the villus height and the V/C of ileum of 1% mulberry meal group was increased significantly (P<0.05).【Conclusion】 Feed mulberry could reduce death and culling rate and improve egg quality of laying hens, but it had some adverse effects on the production performance and intestinal tissue morphology of laying hens.Diet used with 1% feed mulberry meal was the most appropriate for laying hens under this experimental conditions.
Analysis of Microbial Colony Diversity in Cecum of Wannan Yellow Rabbits for Different Residual Feed Intake Based on the 16S rDNA Sequencing
WANG Yuanlang, QI Pingping, DING Haisheng, ZHAO Huiling, HUANG Dongwei
2024, 51(2):  549-557.  doi:10.16431/j.cnki.1671-7236.2024.02.012
Abstract ( 20 )   PDF (2695KB) ( 10 )  
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【Objective】 The aim of this study was to explore the relationship between the level of residual feed intake in Wannan Yellow rabbits and the diversity of cecal microbiota.【Method】 A total of 110 rabbits with similar body weight and similar days of age were selected for the feeding experiment lasting 60 days.The residual feed intake (RFI) was measured after the experiment.Ten rabbits with high (n=5) and low (n=5) RFI were selected and divided into the H (high RFI) and L (low RFI) groups.16S rDNA amplification sequencing technique was used to compare and analyze the cecal contents of H and L groups.【Result】 There was no significant difference in the abundance between these two groups (P>0.05).However, the bacterial diversity was different.The relative abundance of Fusicatenibacter, Blautia, Lachnospira, Oxalobacter and Intestinibacter in L groups was significantly higher than that in the H groups (P<0.05), while the abundance of paludicola, Mailhella, Helicobacter, Pseudomonas was significantly higher in L groups (P<0.05).【Conclusion】 There were some differences in the diversity and structure of cecal microflora in different RFI of Wannan Yellow rabbits, including Lachnospira, Fusicatenibacter, Colidextribacter, which were related to lipid metabolism and blood glucose regulation.Adjusting the structure and abundance of intestinal microflora may be an effective way to improve the feed efficiency of rabbits.
Effect of Feeding Zinc Lysine on Plasma Biochemical Indices and Sperm Quality of Hu Sheep Rams in Autumn in Northeastern China
WANG Hechuan, LI Tianfeng, ZHANG Han, SONG Yulun, FENG Heze, WEI Yao, CHENG Ye, LI Yongxin, DONG Yingying, YIN Guoan, GUO Qing, CUI Weiguo, LI Jingchun
2024, 51(2):  558-567.  doi:10.16431/j.cnki.1671-7236.2024.02.013
Abstract ( 17 )   PDF (1108KB) ( 4 )  
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【Objective】 This experiment was aimed to investigate the effects of feeding zinc lysine on plasma biochemical indices and sperm quality of Hu sheep in autumn in Northeastern China.【Method】 Thirty-two 1-2 years old Hu sheep rams with well body condition were randomly divided into four groups fed with 0, 25, 50 and 100 mg/d zinc lysine, respectively.The experiment was conducted in Yichun, Heilongjiang from August to October 2022, with an average temperature of 10.3 ℃.The transition period was 7 days, and the experimental period was 56 days.On the 28th, 42nd and 56th days, the jugular vein blood of Hu sheep was collected to determine plasma testosterone, melatonin, interleukin-2, interleukin-4, interleukin-6, interleukin-10, adrenocorticotropic hormone, and tumor necrosis factor-α contents, as well as total antioxidant capacity, catalase, superoxide dismutase, glutathione peroxidase activities and malondialdehyde content.Hu sheep semen was collected to determine sperm viability, plasma membrane integrity, and abnormality rate.【Result】 On the 28th day, the melatonin content of the 50 mg/d group was significantly higher than that of the 0 and 100 mg/d groups (P<0.05), the total antioxidant capacity was significantly higher than that of the 0 mg/d group (P<0.05), the interleukin-4 content was significantly lower than that of the 25 mg/d group (P<0.05), and the interleukin-10 content was significantly lower than that of the 0 mg/d group (P<0.05).On the 42nd day, the superoxide dismutase activity of the 50 mg/d group was significantly higher than that of the 0 mg/d group (P<0.05), the interleukin-4 content was significantly lower than that of the other groups (P<0.05), and the adrenocorticotropic hormone content was significantly lower than that of the 0 and 100 mg/d groups (P<0.05).On the 56th day, the sperm plasma membrane integrity rate of the 50 mg/d group was significantly higher than that of the 0 mg/d group (P<0.05), the superoxide dismutase activity was significantly higher than that of the 0 mg/d group (P<0.05), and the interleukin-4 content was significantly lower than that of the 0 and 100 mg/d groups (P<0.05).【Conclusion】 Supplementing with zinc lysine could ameliorate the plasma reproductive hormones content, antioxidant capacity, immune indexes content and sperm quality of Hu sheep rams in autumn in Northeastern China.The additive amount was recommended 50 mg/d.
Effects of Conditioning Training at Different Stages on Fecal Metabolomics in Yili Horses
HAN Zixiang, YAO Xinkui, ZANG Changjiang, MENG Jun, ZENG Yaqi, WANG Chuankun, YANG Liping, XUE Yuheng, LI Pengcheng, WANG Jianwen
2024, 51(2):  568-580.  doi:10.16431/j.cnki.1671-7236.2024.02.014
Abstract ( 16 )   PDF (7023KB) ( 3 )  
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【Objective】 This study was aimed to explore the change characteristics of fecal metabolites in Yili horses at different stages of conditioning training, and initially screen the differential metabolites affected by conditioning training in Yili horses.【Method】 Ten galloping Yili horses with consistent management and untrained training were selected from Zhaosu horse farm in Yili, Xinjiang, and divided into two groups, one group for special training, and the other group for untrained.Feces samples were collected on the 30th and 60th day of training, non-targeted metabolome detection was performed by LC-MS method, and the detection results were analyzed.【Result】 Compared with training (QY) and untraining (QB) groups at the 30th day, there were significant differences in 27 positive ion mode metabolites and 20 negative ion mode metabolites, and metabolites such as 2-arachidyl glycerol, propionyl L-carnitine and creatine were significantly upregulated.The metabolites were mainly concentrated in β-alanine metabolism, arginine and proline metabolism, pentose phosphate pathway, arachidonic acid metabolism, 5-hydroxytryptaminergic synapse and other metabolic pathways.There were 57 positive ion mode metabolites and 33 negative ion mode metabolites between training (HY) and untraining (HB) groups at the 60th day.The metabolites of palmitoleic acid, palmitic acid, methyltestosterone and melatonin were significantly upregulated, while the metabolites of cortisol and folate were significantly downregulated.The metabolites were mainly concentrated in steroid hormone biosynthesis, Cushing syndrome, cortisol synthesis and secretion, neuroactive ligand-receptor interaction, fatty acid biosynthesis, unsaturated fatty acid biosynthesis and other metabolic pathways.【Conclusion】 There were differences in fecal metabolites of Yili horses at different stages of conditioning training, which provided theoretical basis for improving the overall health level of horses and promoting exercise performance.
Research Advances on the Effects of Heat Stress on Production Performance, Serum Biochemistry and Gut Microbes in Chickens
SUN Pengfei, WANG Xiaohan, HU Yun, WU Huiguang, ZHAO Jingwen, LUO Xugang
2024, 51(2):  581-590.  doi:10.16431/j.cnki.1671-7236.2024.02.015
Abstract ( 21 )   PDF (2682KB) ( 23 )  
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Heat stress is one of the major environmental stress challenges facing chicken production worldwide, and is the sum of a number of non-specific stress responses that animals exhibit when exposed to the external environment.Heat stress can lead to reduced feed intake, reduced growth performance, altered blood biochemical parameters and disturbances in the gut microflora, which can seriously affect chicken productivity.Heat stress can also reduce the defense mechanisms and immune function of chickens, increase the expression of inflammatory factors, cause an increase in body antioxidant indicators, and significantly affect the composition and diversity of gut microbiota, with Escherichia coli and Bacillus methanogens as important predictors that play an important role in foresight.Physiological adjustments in chickens under heat stress conditions include increased heart rate, rapid respiration and increased oxidative metabolism in the body.In the actual production process, the harm caused by heat stress can be effectively alleviated by strengthening breeding management, improving the breeding environment, and adding anti-stress substances, such as trace element zinc and various vitamins.The authors synthesize the effects of heat stress on the production performance, serum biochemistry, antioxidants and the structure of gut microbiota in chicken, so as to provide a theoretical basis for preventing heat stress during chicken breeding.
Research Progress in Odor Reduction Technology for Large-scale Pig Farms
SUN Yaowei, LIU Jinsong, LIU Yulan, DAI Bing, NIU Yu, ZHANG Ruiqiang
2024, 51(2):  591-600.  doi:10.16431/j.cnki.1671-7236.2024.02.016
Abstract ( 13 )   PDF (1195KB) ( 13 )  
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With the rapid development of large-scale pig farming, a large amount of odor is generated, which affects the living environment of surrounding residents and poses a threat to human and animal health.At present, the odor control technology of large-scale pig farms mainly focuses on feeding management technology, nutrition technology, and centralized collection and treatment technology.This article comprehensively references a large number of literature and combines practical production to analyze the sources, components, hazards, and influencing factors of odor.It introduces the dietary additives used to reduce the generation of odor in pig houses and the method of adjusting the proportion of dietary nutrients to promote the reduction of intestinal odor in live pigs.It proposes feeding management methods and intelligent processing systems for reducing odor in pig houses, and outlines centralized control and treatment technologies for reducing gas, solid, and liquid pollutants at the end of pig houses, and prospects were made for the application of comprehensive deodorization technologies for source reduction, process control, and end treatment, as well as intelligent control technologies for odor reduction, with the aim of providing reference for large-scale pig farm odor reduction.
Genetics and Breeding
Genetic Diversity and Origin Evolution Analysis of Qingyang Donkey Based on mtDNA D-loop Region Sequence
SHI Haina, WANG Yongjie, LIANG Wanpeng, GENG Zhiguang, LI Shien, XU Zhenfei, LIU Gang, LIU Zhe
2024, 51(2):  601-613.  doi:10.16431/j.cnki.1671-7236.2024.02.017
Abstract ( 12 )   PDF (4911KB) ( 5 )  
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【Objective】 This study was aimed to explore the genetic diversity and maternal origin of Qingyang donkey breeding population, understand its genetic information, and provide theoretical basis for protecting germplasm resources, breeding, and genetic improvement in Qingyang donkey.【Method】 133 Qingyang donkeys were randomly selected, the mitochondrial DNA (mtDNA) D-loop region sequences were analyzed by PCR amplification, sequencing and alignment, and the genetic diversity and maternal origin of Qingyang donkey were explored.【Result】 520 bp D-loop base sequence were obtained, the AT content (57.3%) was higher than GC content (42.8%), indicating there was bias in base composition.There were 38 SNPs of D-loop region, including 8 conversions.The nucleotide diversity (Pi), haplotype diversity (Hd) and average nucleotide difference (K) were 0.01591, 0.895 and 8.274, respectively, which was lower than the average of European and Chinese domestic donkey studies, indicating that the nucleotide variation of Qingyang donkey breed was relatively poor.The genetic distances of 35 haplotypes of D-loop region in Qingyang donkey ranged from 0.002 to 0.042.Phylogenetic results showed that there were two mitochondrial lineages, indicating that there were two mitochondrial maternal origins, and the genetic distance between Qingyang donkey and Croatian domestic donkey was relatively close.【Conclusion】 The experiment preliminarily revealed the nucleotide variation of Qingyang donkey was poor from the molecular level, the degree of hybridization was high, and the mtDNA genetic polymorphism was gradually lost, so the protection of genetic resources of Qingyang donkey breed should be strengthened.
Research Progress on the Primary Sex Determination Mechanisms in Mammals
JIANG Bowen, LIU Guoshi, ZHANG Lu
2024, 51(2):  614-623.  doi:10.16431/j.cnki.1671-7236.2024.02.018
Abstract ( 18 )   PDF (2191KB) ( 5 )  
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Sex determination in mammals is an important event that occurs during early embryonic development, and is crucial for the formation of an animal's reproductive capability.In recent years, with the development of high-throughput sequencing techniques, the transcriptome, proteome and spatial transcriptome analyze methods at the single-cell level and the cell lineage tracking and precise gene manipulation methods have made a great progress.All these techniques enabled the investigation of sex determination and gonadal development of mammals have made remarkable progress.Meanwhile, along with the continuous increase in the demand for sex-specific animals in livestock and poultry breeding, a series of breakthroughs have been made in the development of new sex sorting technologies based on sex determination mechanisms.With the elevated requirements of animal welfare, the application of postnatal sex sorting methods is limited, the sex sorting methods at sex determination stage shows great potential for application in the field of livestock and poultry production.The authors summarize the regulatory network of sex determination in mammals by analyzing the classical experiments related to sex determination and combining with the case reports of genetic defects clinical cases, and the entire regulatory network of mammalian primary gender determination is connected by studying the gender reversal phenomenon and gender specific differentiation process in gene expression manipulation experiments related to gender determination.In addition, this article traces the progress and development trend of sex sorting technologies utilizing gene editing techniques based on sex determination mechanisms.Thus, the review of mammalian sex determination mechanism in recent years will provide theoretical references for the innovative of livestock sex sorting technologies.
Study on the Genetic Structure and Litter Size Candidate Genes of Pishan Red Sheep Population Based on Whole Genome Resequencing
SHI Lan, MA Meilan, MUHETAPA Maimaitijiang, YANG Huiguo, YIMING Sulaiman
2024, 51(2):  624-638.  doi:10.16431/j.cnki.1671-7236.2024.02.019
Abstract ( 23 )   PDF (7094KB) ( 19 )  
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【Objective】 Pishan Red sheep is a newly bred prolific breed of sheep distributed in Hotan area of Xinjiang.Based on the whole genome resequencing technology, genetic structure of the population and the selection signal region of litter size traits of Pishan Red sheep were understood at the genome level, and the candidate genes were verified.【Method】 Thirty ewes prolific of Pishan Red sheep with 2-3 lambs born were selected as high fertility group (HF) and 30 prolific Pishan Red sheep ewes with single lambs born were selected as low fertility group (LF).The whole genome of these two populations was re-sequenced.Comprehensive analysis methods such as principal component analysis (PCA), phylogenetic tree, population genetic structure and genome-wide scanning (Fst & θπ) were used to determine candidate regions and further screen candidate genes for lambing traits in Pishan Red sheep.The candidate genes were genotyped by flight mass spectrometry.【Result】 The attenuation curves of linkage disequilibrium (LD) analysis of the high and low fertility groups were similar.The phylogenetic tree showed that the degree of differentiation between the two groups were not obvious.The PCA results showed that the two groups were obviously clustered into a cluster, individuals were out of the group, and their location and relationship were consistent with the evolutionary tree structure and population structure results.The window with both the Top 1% Z(Fst) value and the θπ value were set as the candidate region, and a total of 229 strong selection signals were annotated.The annotated genes in HF and LF groups were 86 and 143, respectively.Among them, 42 candidate genes that might be related to reproductive traits were screened, such as MARF1, CHGA, BMPR1B, IMMP2L, CDK14, ZDHHC3, CCDC71, DSCAML1, LIMK2, P2RY14, etc.Through GO function and KEGG pathway analysis, it was found that the GO significant enrichment terms were in the regulation of sodium ion transmembrane transporter activity, regulation of apoptosis process, sodium ion channel regulator activity, G protein-coupled receptor binding, G protein-coupled purine nucleotide receptor activity and other pathways.The KEGG significant enrichment pathways were mainly related to signal transmission, signal pathway, material metabolism and so on.The results of genotyping showed that 11 SNPs of MARF1 gene existed in the population of Pishan Red sheep and g.14023542 T>A, g.14036507 A>G and g.14046123 C>T loci were significantly associated with the average litter size of the population, and the first two loci showed strong linkage relationship (r2>0.3), and the TT genotype of g.14023542 T>A locus had the highest average litter size (1.901±0.675).【Conclusion】 The genetic background of the two groups of high and low fertility groups was similar, and there was a certain degree of differentiation, but the differentiation was not obvious.MARF1, CHGA, BMPR1B, IMMP2L, CDK14, ZDHHC3, CCDC71, DSCAML1, LIMK2, P2RY14 and other genes might be candidate genes affecting the lambing traits in Pishan Red sheep.The three SNPs of MARF1 gene could be used as potential molecular breeding markers for lambing traits of Pishan Red sheep.
Advances in Boar Semen Cryopreservation Technologies
WANG Jialun, CHEN Hui, SUN Haowei, GUO Yaoyao, LIANG Guodong, MA Yunhui, GAO Lei
2024, 51(2):  639-648.  doi:10.16431/j.cnki.1671-7236.2024.02.020
Abstract ( 18 )   PDF (1274KB) ( 12 )  
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Boar semen cryopreservation, as a vital technology for swine industry, is conducive to improve the utilization rate of excellent breeding boars and realize the long-term preservation of superior germplasm resources, as well as the elevation of efficiency in breeding farms.At present, various progress has been made in boar semen cryopreservation, the conception rate and the number born alive by domestic application of pig frozen semen have basically reached the level of foreign frozen semen breeding.However, due to the certain degree of damage of pig spermatozoa in frozen-thawed process which caused sperm fertility declined and the fertilization window period shortened, hence there is still a big gap in application of frozen semen.The article reviews the development process of cryopreservation technology for pig semen at home and abroad, introduces the basic characteristics of pig semen and sperm, briefly describes the mechanism of sperm damage caused by the cooling process, and analyzes and introduces the mechanism and methods of reducing freezing damage from the perspective of the composition of cryodiluents, such as adding cryoprotectants to reduce the formation of ice crystals inside and outside cells, maintaining osmotic pressure inside and outside cells, and stabilizing cell membrane structure.Adding antibacterial substances to inhibit the rapid reproduction of bacteria after thawing.Adding antioxidants to reduce reactive oxygen species levels, alleviate oxidative stress, protect sperm structure and fertilization ability, etc.Finally, the future development direction of boar semen cryopreservation technology is expected to provide reference for related research and practical applications.
Research Progress on Bone Morphogenetic Protein Receptor Type-1B Gene in Sheep
YANG Chulei, LI Xingyao, ZHANG Yiyuan, TANG Hong, GUO Yanhua, WANG Limin, ZHOU Ping
2024, 51(2):  649-658.  doi:10.16431/j.cnki.1671-7236.2024.02.021
Abstract ( 17 )   PDF (2253KB) ( 5 )  
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Sheep reproductive performance holds significant importance in the sheep industry.In addition to various technical methods such as periodic estrus, artificial insemination, and superovulation, among the 20 mutations of 16 genes known to enhance sheep reproductive performance, the bone morphogenetic protein receptor type-1B (BMPR-1B) gene exhibits the most profound impact.BMPR-1B gene stands as the pioneering major lambing gene discovered worldwide.The A746G mutation within the coding region of BMPR-1B gene leads to arginine substitution for glutamine at position 249 in the protein sequence (Q249R), ultimately resulting in increased ovulations and lambs.This paper presents an introduction to the discovery and structure of BMPR-1B gene along with its mutant FecB (A746G).It briefly elucidates on the molecular mechanism of this gene, regulation of BMP/Smad signaling pathway, relationship between sheep reproduction, and provides a concise analysis on how FecB mutation affects ovarian function, follicles, other tissues, hormone regulation and related gene expression in sheep.Further comprehension of BMPR-1B gene will offer valuable insights for researchers exploring regulatory mechanisms inducing increased lambing numbers in sheep and other animals through manipulation of related ligands, regulatory factors as well as upstream and downstream signal proteins.Additionally it can accelerate efficient breeding and reproduction processes among mammals by expanding population size through establishment of multiple litter strains thereby increasing farmers’ economic income.
Gene Modification Technology and Its Application in Animal Breeding and Biomedicine
YAN Yanxia, LI Zicong, DONG Yazheng, LI Zheng, HUANG Sixiu
2024, 51(2):  659-667.  doi:10.16431/j.cnki.1671-7236.2024.02.022
Abstract ( 16 )   PDF (1119KB) ( 8 )  
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Gene modification is a technology that can be used to achieve site-specific integration of exogenose and knockout of endogenous genes. The early form of gene modification was mainly transgene.With the deepening of scientific research, new gene modification methods were gradually developed, such as knockout, knock in, site-specific mutation, etc.According to the purpose of research or application, gene modification technology can be divided into transgenic and gene knockout.In recent years, with the rapid development of modern molecular technology, gene modification technology has been continuously improved and innovated, and its related methods and technologies have been gradually applied in the fields of improving livestock traits, studying gene function, making animal bioreactors and constructing animal models of human diseases, making the research of gene function and transgenic breeding of livestock and poultry more efficient.Remarkable achievements have been made in the fields of animal genetic breeding and biomedicine, which have made up for the defects of random integration and genetic instability of traditional transgenic technology, and have broad prospects for development.In this paper, the development status and trend of gene modification technology were described from the aspects of animal transgenic and gene knockout technologies, and the application status of gene modification technology in the field of animal breeding and biomedicine was briefly summarized.
Preventive Veterinary Medicine
Construction of Codon-optimized RHDV2 Dual VP60 Recombinant Baculovirus and Immunogenicity Analysis of the Expressed Protein
YU Jifeng, XIE Jing, HUANG Yong, XIAO Lu, LIN Yi, CAO Ye, YE Yonggang, WEI Yong, WU Xuejing, LI Jiangling, KANG Runmin
2024, 51(2):  668-677.  doi:10.16431/j.cnki.1671-7236.2024.02.023
Abstract ( 19 )   PDF (4007KB) ( 11 )  
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【Objective】 This study was aimed to optimize the preparation strategy for virus-like particle (VLP) vaccine of Rabbit hemorrhagic disease virus 2 (RHDV2), investigate the immunogenicity of the optimized VLP vaccine on domestic rabbits, and provide new insights for the development of a cost-effective, high-yield RHDV2 novel vaccine.【Method】 RHDV2 VP60 full gene was synthesized based on the codon preference of insect cells, and the dual VP60 gene was inserted into the eukaryotic vector pFastBacTM Dual.The recombinant plamid was transformed into Escherichia coli DH10Bac competent cell carrying Bacmid plasmid, constructing the recombinant Baculovirus Bacmid-VP60-VP60 with dual VP60 genes.The Sf9 insect cells were transfected and the expression of the recombinant Baculovirus Bacmid-VP60-VP60 was verified through Western blotting, indirect immunofluorescence assay (IFA) and transmission electron microscopy (TEM).The recombinant protein antigen prepared by the optimized strategy was combined with aluminum hydroxide adjuvant at a 9∶1 ratio to prepare the VLP inactivated vaccine.The protective effect of the optimized VLP vaccine was evaluated through safety testing, minimum immunization dose and duration of immunity.【Result】 The recombinant Baculovirus Bacmid-VP60-VP60 was successfully constructed.The results of Western blotting showed that the recombinant Baculovirus transfected into Sf9 cells expressed RHDV2 VP60 protein about 60 ku in size.IFA identification results showed that Sf9 cells infected with the recombinant Baculovirus produced a large amount of yellow-green fluorescence, indicating a massive expression of VP60 protein in Sf9 cells.TEM observation revealed that the VP60 protein folded into VLP approximately 40 nm in size, presenting a spherical and smooth surface structure.The VLP vaccine prepared using the optimized strategy exhibited good safety and immunogenicity in rabbits, with a minimum immunization dose of 0.5 mL and an immunity duration of over 210 days.【Conclusion】 A recombinant Baculovirus containing codon-optimized dual VP60 gene was constructed and successfully expressed RHDV2 VP60 protein in insect cells.The protein-prepared VLP vaccine exhibited robust immunogenicity in rabbits.
Preparation of Polyclonal Antibody and Development of an Indirect ELISA Method for Senecavirus A VP2 Protein
REN Jianle, LIN Yiting, JI Kang, TAN Shanshan, CHEN Xinxin, JIN Yi, WANG Ying, NIU Sheng, LIANG Libin, LI Junping, ZHAO Yujun, TIAN Wenxia
2024, 51(2):  678-688.  doi:10.16431/j.cnki.1671-7236.2024.02.024
Abstract ( 16 )   PDF (3653KB) ( 9 )  
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【Objective】 This study was aimed to prepare polyclonal antibodies (pAbs) against the VP2 protein of Senecavirus A (SVA), and establish an indirect ELISA method to specifically detect SVA antibodiy, so as to provide a research basis for the pathogenesis and diagnosis of SVA.【Method】 VP2 gene was cloned into the prokaryotic expression vector pET-28a by homologous recombination, the recombinant expression plasmid pET-28a-VP2 were designated and transformed into Escherichia coli BL21 (DE3) competent cell for IPTG induced expression.The New Zealand White rabbits were subcutaneously immunized with the purified recombinant protein to prepare its pAbs.The specificity, reactivity and neutralizing activity of pAbs were analyzed via indirect immunofluorescence assay (IFA), Western blotting and neutralization test.An indirect ELISA based on VP2 coating antigen was established by matrix optimization, determination of cut-off value, specificity identification, sensitivity and repeatability analysis.A total of 50 serum samples were collected, which were tested by indirect ELISA and IFA to analyze the coincidence rate.【Result】 The recombinant VP2 protein was expressed in the form of inclusion body with a size of 40 ku.The pAbs could specifically react with recombinant VP2 protein and SVA, and had no cross-reactivity with other viruses.Moreover, the pAbs had a higher neutralizing activity.After optimizing the conditions of indirect ELISA, the optimal VP2 coating concentration was measured at 4 μg/mL, and optimal serum sample dilution was 1∶250.Furthermore, the best blocking solution was selected as 5% skimmed milk +5% BSA.The optimal reaction time for serum, secondary antibodies and TMB solution were 90, 90 and 10 min, respectively.Finally, the value of cut-off was calculated to 0.182.No cross-reactivity was observed with other virus antibody-positive pig sera, and indirect ELISA showed a 94.0% coincidence rate compared with IFA.【Conclusion】 VP2 protein expressed in the prokaryotic expression system had good immunogenicity, and the prepared pAbs could react specifically with SVA and recombinant VP2 protein.The established indirect ELISA had high specificity and consistency with IFA, which could be suitable for clinical SVA antibody detection and vaccine efficacy evaluation.
Prokaryotic Expression of EF-Tu Protein of Mycoplasma ovipneumoniae and Preparation of Polyclonal Antibody
WANG Yongfei, DENG Bowen, LIU Xiaoyan, HAERLEHA·Amantai, GUO Jiadong, ZHOU Zhengguo, CAI Jiang, LI Youwen
2024, 51(2):  689-699.  doi:10.16431/j.cnki.1671-7236.2024.02.025
Abstract ( 15 )   PDF (4681KB) ( 6 )  
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【Objective】 The purpose of this study was to clone EF-Tu gene in Mycoplasma ovipneumoniae (Mo), obtain EF-Tu protein by prokaryotic expression, prepare rabbit-derived polyclonal antibody against EF-Tu protein, laying the foundation for the study of the structure and function of Mo EF-Tu protein.【Method】 TGA codon in the middle of EF-Tu gene in pET-28a-EF-Tu plasmid was mutated to TGG by overlapping extension PCR, and the similarity alignment and genetic evolution analysis were conducted between the sequencing results and other Mycoplasma reference strains, and the putative protein sequences were analyzed for bioinformatics using online software.The mutated recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells for prokaryotic expression, identified by SDS-PAGE and Western blotting, and purified using Ni-chelating affinity chromatography.The polyclonal antibodies were prepared by immunizing rabbits with the purified EF-Tu fusion protein.The polyclonal antibody potency and immunological reactivity were detected by indirect ELISA and Western blotting.【Result】 The TGA site in EF-Tu gene was successfully mutated, and a prokaryotic expression vector expressing the His tag pET-28a-EF-Tu' was constructed.Bioinformatics analysis results showed that the cloned EF-Tu gene had the highest similarity and the closest genetic relationship to Mycoplasma ovipneumoniae strain MoGH3-3, and encoded 387 amino acids, without N-glycosylation site and transmembrane region.There were 10 serine, 20 threonine and 4 tyrosine phosphorylation sites of EF-Tu protein, and the secondary structure consists of random coil (35.14%), alpha helix (26.87%), extended chain (26.87%) and beta turn (11.11%).SDS-PAGE and Western blotting results showed that the size of the target protein was about 43 ku, and the protein purification concentration was 0.615 g/L.ELISA and Western blotting results showed that the prepared polyclonal antibody had a potency of up to 1∶128 000, and could specifically recognize the EF-Tu fusion protein, which had good immunological reactivity.【Conclusion】 The TGA codon of EF-Tu gene was successfully mutated in this study, the EF-Tu fusion protein was obtained by prokaryotic expression and purification, and its polyclonal antibody was prepared with a potency of 1∶128 000.This result provided an experimental basis for the subsequent study of the structure and biological function of Mo EF-Tu protein and its vaccine development.
Progress on in vitro Culture of Bovine Coccidia
HU Junhao, LIU Jibing, ZHOU Jiali, MA Yue, ZHAO Hongxi
2024, 51(2):  700-708.  doi:10.16431/j.cnki.1671-7236.2024.02.026
Abstract ( 13 )   PDF (1011KB) ( 9 )  
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Bovine coccidiosis is a highly pathogenic parasitic protozoan disease caused by one or more species of Eimeria, with a wide range of epidemiological scope, which can lead to malabsorption of nutrients, developmental delays, watery or bloody diarrhoea and other symptoms, and even cause death, which seriously harms the development of the cattle industry.Currently, bovine coccidiosis is mainly prevented and controlled through drug prevention, but with the emergence of drug-resistant strains of worms and the concern about veterinary drug residues, the research and development of new anticoccidial drugs is urgent.As an alternative to experimental animal research, in vitro culture can artificially simulate the in vivo environment and conditions of the natural host of bovine coccidia at relatively low cost, which is of great significance for the study of the invasion mechanism of coccidia, the interaction between coccidia and the host, as well as the evaluation of the efficacy of anti-coccidial drugs.In the established in vitro culture system, bovine coccidia can complete part or all of the endogenous development process from ascospores to oocysts, which opens up new ways to study the life cycle stages of the worm and new control strategies.By reviewing the relevant data on in vitro culture of bovine coccidia at home and abroad, the author elaborated on the isolation and purification of coccidian ascospores, primary cell and cell line culture, in vitro culture conditions and influencing factors, and the application aspects of in vitro culture, with a view to providing a reference for the research and development of a safe and effective vaccine and preventive drug for bovine coccidia.
Effects of Different Environmental and Nutritional Conditions on Growth Characteristics,Spore Production and Chitinase Expression of Nematode-trapping Fungi Arthrobotrys conoides
SUN Dianming, ZHANG Huimei, LI Ningxing, SUN Yansen, LI Ruobing, QIAO Jun, CAI Xuepeng, MENG Qingling
2024, 51(2):  709-718.  doi:10.16431/j.cnki.1671-7236.2024.02.027
Abstract ( 13 )   PDF (3482KB) ( 6 )  
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【Objective】 This experiment was conducted to understand the effects of temperature, pH and different nutritional conditions on the growth and spore production of the nematode-trapping fungi Arthrobotrys conoides (A. conoides), analyse the expression level of chitinase-encoding genes under carbon and nitrogen starvation, and optimise the culture conditions to provide a basis for the expansion culture of the fungi.【Method】 The hyphae of A. conoides AC-shz1 were inoculated at different temperatures (15, 20, 25, 30 and 35 ℃), pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0), carbon resource (fructose, galactose, glucose, sucrose, maltose, lactose, starch, cellulose and chitin), nitrogen resource (glycine, phenylalanine, peptone, yeast powder, ammonium sulphate and urea) and carbon-nitrogen ratio (1∶1, 5∶1, 10∶1, 20∶1 and 40∶1) of YPSSA medium, and the growth rate and morphology were observed and recorded, and its sporulation capacity was measured.The expression levels of four chitinase-encoding genes was also analyzed by Real-time quantitative PCR under carbon and nitrogen starvation.【Result】 The results showed that A.conoides could grow at a temperature of 15-30 ℃, among which hyphae grew the fastest and had the strongest sporulation capacity at 25 ℃.When the pH was 6.5-7.5, the hyphae growth was the better and the sporulation capacity was the stronger at pH 7.0.It was found that the most suitable sources for hyphae growth and spore production were glucose medium and sucrose medium, among which glucose medium spore production was the highest.The most suitable nitrogen sources were yeast powder and peptone, among which yeast powder medium had the highest spore yield, and the optimal carbon-nitrogen ratio with glucose and yeast powder as carbon and nitrogen sources was 5∶1.Compared with control group, under carbon starvation, the transcript levels of the chitinase of the nematode-trapping fungi A. conoides-14 (AC-14, P<0.01) and chitinase of the nematode-trapping fungi A. conoides-190 (AC-190, P>0.05) genes were up-regulated, the chitinase of the nematode-trapping fungi A. conoides-379 (AC-379, P>0.05) and chitinase of the nematode-trapping fungi A. conoides-483 (AC-483, P<0.01) genes were down-regulated.Under nitrogen starvation, the transcript levels of all four chitinase-encoding genes were extremely significantly down-regulated (P<0.01).【Conclusion】 The optimum temperature for hyphae growth and spore production was 25 ℃, the optimum pH was 7.0, and the optimum carbon-nitrogen sources were glucose and yeast powder, respectively, the optimum carbon-nitrogen ratio of 5∶1, which laid the foundation for the further development of highly effective nematode-trapping fungal biocontrol agents.
Prokaryotic Expression and Immunogenicity of lpxM Protein of Avibacterium paragallinarum
LIANG Zhixuan, MEI Chen, ZHANG Xue, XU Haojun, ZHI Yan, LI Huanrong, WANG Hongjun
2024, 51(2):  719-727.  doi:10.16431/j.cnki.1671-7236.2024.02.028
Abstract ( 13 )   PDF (1720KB) ( 9 )  
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【Objective】 This experiment was airxed to investigate the immunogenicity of Avibacterium paragallinarum outer membrane protein lpxM, and provide a basis for the prevention of infectious rhinitis in chickens.【Method】 The pET-28a-lpxM prokaryotic expression vector was constructed.The positive plasmid identified by PCR amplification and sequencing was transformed into Escherichia coli BL21 (DE3) competent cells.Induced expression was performed with IPTG, and the induction time and concentration were optimized.The recombinant protein lpxM was purified using urea buffer and identified by SDS-PAGE.The specificity of the recombinant protein was detected by Western blotting.After the recombinant protein was combined with MONTANIDEISA 71 VG adjuvant to form the vaccine v-lpxM, and its immunization effect was observed by animal experiments.【Result】 Recombinant plasmid pET-28a-lpxM transformed Escherichia coli BL21 (DE3) competent cells and was successfully expressed after induction by IPTG, with specific bands of target protein at 37 ku.Protein expression was highest at 37 ℃, 700 mmol/L IPTG and 4 h after induction.Recombinant protein lpxM was expressed in both supernatant and precipitation, and the expression level was relatively high in precipitation.Western blotting results showed that the positive chicken serum of Modesto strain of serotype C Avibacterium paragallinarum could react with the purified recombinant protein. The results showed that the protection rate of v-lpxM vaccine against serotype A, B and C Avibacterium paragallinarum was 0, 20% and 60%, respectively.【Conclusion】 The prokaryotic expression vector pET-28a-lpxM was successfully constructed, achieving the efficient expression of lpxM protein.The prepared lpxM protein showed good immunogenicity.The results laid a foundation for exploring the immunological properties of Avibacterium paragallinarum outer membrane protein lpxM, as well as for the prevention of chicken infectious rhinitis and the development of vaccines.
Prediction and Analysis of Potential Distribution Area and Risk Factors of Bathochytrium dendrobatidis
BIE Jia, SHA Longqian, YAN Jianyu
2024, 51(2):  728-735.  doi:10.16431/j.cnki.1671-7236.2024.02.029
Abstract ( 15 )   PDF (2611KB) ( 4 )  
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【Objective】 Chytridiomycosis is a disease caused by the infection of the aquatic fungus Batrachyttrium dentrobatidis (B.dendrobatidis), which has been one of the main reasons for the significant reduction or even extinction of amphibian populations for over half a century.At present, there is no effective treatment method for chytridiomycosis yet.The investigation and research on the distribution and spread of B.dendrobatidis worldwide, as well as the analysis of the distribution risks and influencing factors of B.dendrobatidis at the global and regional scales, can provide basic research data for the formulation of preventive measures.【Method】 The distribution data of B.dendrobatidis in the world were used and 20 climate environment variables were considered.After the B.dendrobatidis distribution data and variables were screened, a MaxEnt model was established to compare the relationship between six variables and B.dendrobatidis distribution risk, and predicted the distribution risk of B.dendrobatidis in the world and mainland China.【Result】 The results showed that the top four variables with the highest contribution to the probability of B.dendrobatidis distribution were in order of annual average temperature, annual precipitation, temperature seasonality and precipitation seasonality.The overall relationship between annual average temperature, temperature seasonality and the distribution probability of the B.dendrobatidis showed a positive correlation first and then a negative correlation.The relationship between annual precipitation, precipitation seasonality and the distribution probability of the B.dendrobatidis were positively and negatively correlated, respectively.The high-risk areas of global distribution of B.dendrobatidis were mainly distributed in Southern mainland China, Southeastern Australia, Central Papua New Guinea, Southern Sweden, Germany, Poland, Romania, Southern United Kingdom, Ireland, France, Eastern Madagascar, Sudan, Southern United States, Eastern Peru, The Federal Democratic Republic of Ethiopia and Japan etc.And the high-risk areas of B.dendrobatidis in mainland China were mainly concentrated in Northern Guangdong, Central and Northern Guangxi, Yunnan, Guizhou, Hunan, Jiangxi, Fujian, Zhejiang, Southern Jiangsu, Southern Anhui, Southern Hubei, Chongqing, Eastern and Southern Sichuan, Southern Shaanxi, etc.【Conclusion】 The distribution of B.dendrobatidis was closely related to temperature and precipitation.The high-risk areas of B.dendrobatidis in the world were mainly in Europe, Southern Africa and Southern North America, and the high-risk areas in mainland China were mainly in Central China, Eastern China, Southern China and the Southeast of Southwest region of China.
Advances in Research on Hepatitis E Virus
LIU Dingyu, HE Zhenwen, LIU Baoling, WANG Gang, WANG Xiaohu, TAN Chen, CAI Rujian
2024, 51(2):  736-747.  doi:10.16431/j.cnki.1671-7236.2024.02.030
Abstract ( 11 )   PDF (1537KB) ( 8 )  
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Hepatitis E virus (HEV) is a single-stranded, non-enveloped RNA virus, which can be divided into 8 genotypes.As a zoonotic pathogen, HEV can cause acute viral hepatitis in humans and infection in animals.Its main transmission route is fecal-oral transmission, which can also be transmitted through blood transfusion and mother-to-child transmission.HEV is mainly prevalent in developing countries, while sporadic cases are also found in developed countries such as Europe and the United States.At present, the common methods for detecting HEV are fluorescent quantitative Real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA).Some scholars also use new detection methods such as reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR system to detect HEV.In terms of drug treatment, compounds such as ribavirin, interferon or polyene phosphatidylcholine (PPC) can be used to treat infections caused by HEV, and single or compound Chinese medicines can be used to improve clinical symptoms and be used as synergistic treatment.Vaccination is considered to be an effective means to prevent and control HEV infection, but there is still no effective HEV in vitro cell culture system, and traditional inactivated and attenuated vaccines cannot be produced in batches.The current research on HEV vaccines mainly includes the development of recombinant protein vaccines, DNA vaccines, combined vaccines and oral vaccines.Vaccination is an important measure to prevent hepatitis E, and comprehensive preventive measures based on cutting off the transmission route can also control the prevalence of the disease.This paper mainly summarized the research progress of HEV detection methods, drug treatment and vaccine immunity, and looked forward to the prevention and control of HEV, in order to provide new ideas for the prevention and control of HEV.
Research Progress on Immune Escape Mechanism of Staphylococcus aureus in Dairy Cow Mastitis
HE Xingli, WANG Zhaoyuan, ZHOU Peiyao, MOU Quanzhou, SHEN Binglei
2024, 51(2):  748-758.  doi:10.16431/j.cnki.1671-7236.2024.02.031
Abstract ( 16 )   PDF (1982KB) ( 10 )  
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Dairy cow mastitis is one of the most common diseases in dairy farming.Mastitis will seriously affect the milk yield and milk quality of dairy cows and increase the elimination rate of dairy cows.Feeding conditions, environmental factors and pathogens can induce mastitis in dairy cows, with Staphylococcus aureus (S. aureus) being the most difficult to cure.S. aureus is a highly pathogenic, drug-resistant, and immune-avoiding bacterium mediated by its own secreted enzymes and virulence factors.The long-term survival of S. aureus in the host can lead to the occurrence of long-term chronic mastitis in dairy cows.In this paper, we firstly revealed a series of immune defenses produced by the mammary gland of dairy cows when S. aureus invaded, and then we elaborated on S. aureus relying on the virulence factors, biofilms, surface proteins expressed by S. aureus, as well as small colony variants and persisters produced in the course of evolution to enhance its pathogenicity and drug resistance, revealing that S. aureus was highly pathogenic and drug-resistant, and why it was difficult to cure mastitis in dairy cows caused by S. aureus.Finally, we further elucidated how S. aureus survived under the host’s strong immune defense, and how S. aureus colonized in the mammary gland and evaded phagocytosis through capsular polysaccharides and fibronectin, and secreted neutrophil serine protease inhibitor protein, staphylococcal protein A, extracellular vesicles and other virulence factors to evade the body’s immune phagocytosis.The immunophagocytosis of S. aureus was described, revealing that S. aureus had a high degree of environmental adaptability and numerous means of immune evasion.This paper summarized the mechanism of S. aureus evading mammary gland natural immunity and related research from the above three aspects, which would provide a basis for maintaining the health of dairy cows, preventing the occurrence of mastitis disease in dairy cows, and resisting the invasion of pathogens into the organism.
Study on the Anti-AIV-H9N2 Activity of the Ethyl Acetate Extract from Alternanthera philoxeroides in vitro and in vivo
LIAO Siyu, ZHENG Xin, DAI Qi, XU Shiyi, ZHANG Tianxu, ZHANG Xiuqiao, GUI Chun
2024, 51(2):  759-769.  doi:10.16431/j.cnki.1671-7236.2024.02.032
Abstract ( 19 )   PDF (3974KB) ( 6 )  
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【Objective】 The objective of this study was to investigate the activity and mechanism of ethyl acetate extract from Alternanthera philoxeroides (AETAC) against H9N2 subtype Avian influenza (AIV-H9N2) virus in vitro and in vivo.【Method】 The inhibitory effect of AETAC at different concentrations on chicken embryo fibroblasts (DF-1) at different time was detected by CCK-8 method.The toxicity of AETAC to DF-1 cells was detected by preventing infection, influence replication, preventing adsorption and directly kill, and the best anti-AIV-H9N2 mode was selected in vitro.The maximum safe concentration of AETAC was determined by inoculation of different concentrations in allantoic cavity.AETAC was administered to AIV-H9N2 infected chicken embryo by three different administration methods, and the best administration method was selected in vivo.AETAC and AIV-H9N2 were simultaneously inoculated through allantoic cavity, the survival number of chicken embryos in each group was counted, the hemagglutination titer was determined, and the development of embryo and pathological changes of the bursa of Fabricius of chicken embryos were observed.【Result】 Within a certain range, as the concentration of AETAC increases or the duration of action prolongs, the inhibitory rate of AETAC on DF-1 cells increased.Compared with blank control group, the cell survival rate of virus control and drug groups was significantly decreased under four anti-AIV-H9N2 modes (P<0.05).Compared with virus control group, the cell survival rate of the four anti-AIV-H9N2 modes was significantly increased when AETAC concentration was 5-30 μg/mL (P<0.05).The survival rates of DF-1 cells were 8.15%-64.96% (prevent infection), 8.56%-83.83% (influence replication), 1.82%-5.90% (prevent adsorption) and 6.90%-40.12% (directly kill), respectively.The maximum safe concentration of AETAC for chicken embryos was 16.41 mg/mL, and the best administration method for infected chicken embryos was mixed AETAC and virus inoculation.After the optimal in vivo administration, the survival rate of embryos in medium and low AETAC concentration groups was 40%-60% and those in high concentration group were all alive. In high concentration group, the beak development of embryos was obvious, the lymphoid follicles of the bursa of Fabricius of chicken embryos were intact, the size and number of follicles were normal, the amount of congestion was less, and the histopathological status was improved.【Conclusion】 AETAC had significant inhibitory effect on AIV-H9N2 both in vivo and in vitro, and was mainly related to affecting the mode of viral replication.
Research Progress on Klebsiella pneumoniae from Giant Pandas
ZHAN Tongtong, WANG Pingfeng, ZHENG Xiaomei, WEN Qiqi, YE Gang, DENG Linhua, YOU Yuyan, HE Ming, WANG Qian, LI Caiwu, LING Shanshan
2024, 51(2):  770-778.  doi:10.16431/j.cnki.1671-7236.2024.02.033
Abstract ( 14 )   PDF (1124KB) ( 8 )  
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In recent years, the increasing drug resistance of Klebsiella pneumoniae in captive giant pandas, even the emergence of multi-drug resistance, poses a serious threat to the health of giant pandas.Infections with this bacterium may lead to various diseases such as hemorrhagic colitis and urinary tract infections.With the rise in the number of captive giant pandas and the widespread use of antimicrobial drugs, the problem of drug resistance in Klebsiella pneumoniae has become increasingly prominent.Its pathogenic mechanism involves factors such as capsules, lipopolysaccharides, pili, and iron carrier systems, all of which play a role in the colonization and invasion process of the bacterium.From traditional cultivation to modern molecular detection, techniques for pathogenic identification and diagnosis of Klebsiella pneumoniae in giant pandas have become more convenient, rapid, and reliable.However, there is an urgent need to actively engage in antimicrobial drug development, seek new treatment options, and explore alternative treatment methods such as traditional Chinese medicine and plant extracts to inhibit bacterial growth and slow down the development of drug resistance.This article reviewed the research findings on the biological characteristics of Klebsiella pneumoniae in giant pandas, its current drug resistance status, and diagnostic and therapeutic methods, both domestically and internationally.A review and analysis of existing literature provide new insights for further controlling and delaying the emergence of drug-resistant strains and the spread of drug resistance and offers a valuable reference for the prevention and control of Klebsiella pneumoniae in giant pandas.
Screening of the Interacting Proteins with BTV NS4 by Co-immunoprecipitation Combining with Mass Spectrometry
MA Xianping, CHEN Yujuan, LUO Shimei, ZHUO Xiaojing, YANG Yibin, LIU Yiyi, CAI Xuyan, TANG Yiyun, CHEN Changchang, WEI Xiaorong, YI Huashan
2024, 51(2):  779-789.  doi:10.16431/j.cnki.1671-7236.2024.02.034
Abstract ( 15 )   PDF (3851KB) ( 5 )  
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【Objective】 The purpose of this study was to screen the endogenous interaction protein of NS4 protein of Bluetongue virus (BTV) against interferon (IFN) signaling pathway, in order to further investigate the mechanism of NS4 inhibition of IFN signaling.【Method】 On the basis of previous studies on the gene expression of IFN signaling pathway induced by Sendai virus (SeV), NS4 interacting proteins were extracted from HEK-293T cells transfected with NS4 fusion eGFP label expression vector pcDNA3.1-NS4-eGFP and empty vector pcDNA3.1-eGFP by co-immunoprecipitation method.The immunoprecipitates were analyzed by SDS-PAGE, and the immunocoprecipitated eluents were identified by mass spectrometry and bioinformatics.Real-time quantitative PCR was used to detect the expression characteristics of candidate protein-coding genes.【Result】 A total of 189 differentially expressed proteins were obtained by co-immunoprecipitation, mass spectrometry analysis and database comparison.The results of bioinformatics analysis showed that the candidate proteins were mainly involved in protein transcription, translation, viral infection and immune regulation.The results of subcellular localization analysis showed that the differentially expressed proteins were mainly located in the nucleus, cytoplasm and cytoplasm-nucleus, and four candidate proteins interacting with BTV NS4 protein were screened out, which were polypyrimidine bunch-binding protein 1 (PTBP1), cell cycle dependent protein kinase 9 (CDK9), N-terminal myristylase 1 (NMT1) and Y-box binding protein 1 (YBX1).Real-time quantitative PCR results showed that compared with control group, the mRNA expressions of PTBP1, NMT1, YBX1 and CDK9 genes in experimental groups were significantly or extremely significantly up-regulated after 72 h of SeV stimulation (P<0.05 or P<0.01).【Conclusion】 The antagonistic IFN signaling pathway of BTV NS4 protein was related to the up-regulation of PTBP1, NMT1, YBX1 and CDK9 proteins, which provided a target reference for further research on the molecular mechanism of BTV NS4 protein antagonizing host innate immune response.
Research Progress on Regulation of Parasitic Infections by Regulatory B Cells
FENG Anni, LI Qilong, ZHANG Yiwei, SANG Xiaoyu, CHEN Ran, FENG Ying, JIANG Ning
2024, 51(2):  790-798.  doi:10.16431/j.cnki.1671-7236.2024.02.035
Abstract ( 10 )   PDF (1535KB) ( 9 )  
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Parasitic diseases are infectious diseases that are widespread throughout the world and pose a serious threat to human and animal health.Parasites activate the body’s immune system when they invade the host.The host fights the parasitic infection through innate and acquired immune responses and protects itself from an excessive immune response through a variety of regulatory mechanisms.Regulatory B cells (Bregs) are a subset of B cells with immunosuppressive functions during parasitic infections.They have been more extensively studied in autoimmune diseases, cancer and allergic reactions.Currently, an increasing number of studies have shown that parasitic infections can induce the production of Bregs, and that Bregs can inhibit the inflammatory response by secreting the anti-inflammatory cytokine interleukin-10 (IL-10), thus reducing the damage of parasitic infections to the host.However, the signaling pathways and activation mechanisms of Breg proliferation are still unclear and require further investigation.In this review, the author briefly described the different types of Bregs and their phenotypes, and summarized the immunomodulatory roles of Bregs during Plasmodium, Schistosoma, Leishmania, Toxoplasma and Trypanosoma infections, with the aim of providing new insights into parasite treatment and prevention strategies.
Basic Veterinary Medicine
Study on Therapeutic Effect of Aqueous Extract of Vitex negundo L.var.cannabifolia (Sieb.et Zucc.) Hand.-Mazz.on Castor Oil Induced Diarrhea in Mice
TANG Xinggang, LUO Shengjun, YUAN Minggui, TIAN Ya, SHU Bingyao, YANG Xi, LI Yuelong, XIANG Rong
2024, 51(2):  799-808.  doi:10.16431/j.cnki.1671-7236.2024.02.036
Abstract ( 12 )   PDF (3168KB) ( 4 )  
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【Objective】 This study was aimed to explore the therapeutic effect of aqueous extract of Vitex negundo L.var.cannabifolia (Sieb.et Zucc.) Hand.-Mazz.on diarrhea induced by castor oil in mice, evaluate its anti diarrhea effect, and provide theoretical basis for the development of new natural drugs or plant extract feed additives.【Method】 48 SPF KM mice weighing 18-22 g were selected.The mice were randomly divided into six groups which including control, model, loperamide, Vitex negundo L.var.cannabifolia(Sieb.et Zucc.)Hand.- Mazz.aqueous extract high, medium and low dose groups, 8 in each group, half male and half female.Mice were gavaged with 16, 8 and 4 g/kg BW in high, medium and low dose groups, respectively, equal doses of saline were gavaged in control and model groups, and 5 mg/kg BW loperamide were practiced in loperamide group.The mice were gavaged for 5 d.0.5 h after administration on the 5th day, mice in model, loperamide, and the high, medium and low dose groups were gavaged with 0.5 mL castor oil for modeling, and mice in control group were gavaged with the same amount of saline.The mice were caged one by one after modeling and were observed the diarrhea for 4 h.After 4 h, blood samples were collected and mice were executed by cervical dislocation, two copies of liver and small intestine were randomly taken from 4 mice in each group, and one copy was for tissue section, the other copy was for RNA extraction to detect mRNA expression of jejunal channel protein and acute phase protein of liver.【Result】 The diarrhea score and diarrhea index of mice in model group were extremely significantly higher than those in control group (P<0.01), indicating that the castor oil diarrhea model was manufactured successfully.The diarrhea score and diarrhea index of mice in loperamide and high dose groups were extremely significantly or significantly lower than those in model group (P<0.01 or P<0.05).The concentration of interleukin-1β (IL-1β), IL-6 and IL-10 in serum of mice in high dose group were significantly or extremely significantly lower than those in model group (P<0.05 or P<0.01).The histopathological results of liver showed that the hepatic cords were clear and the hepatocytes were closely arranged in high, medium and low dose groups, and only a few hepatocytes were mildly swollen and infiltrated with a few inflammatory cells.The results of the intestine histopathology showed that the mucosal layer, muscle layer and plasma layer of jejunum were clearly visible in high, medium and low dose groups, and a large number of vacuoles were seen in villous epithelium of mucosal layer, but no obvious necrosis or inflammatory reactions were observed.Compared with model group, the expression of NHE8, NHE3, AQP3 and AQP4 of jejunum in mice were not significantly different in high dose group (P>0.05), while the expression of NHE2 was significantly lower (P<0.05), and the expression of the acute phase proteins TRF and CRP were significantly lower (P<0.05).【Conclusion】 The therapeutic effect of aqueous extract of Vitex negundo L.var.cannabifolia (Sieb.et Zucc.) Hand.-Mazz.on castor oil-induced diarrhea model in mice was good, and could protect liver and small intestine mucosa.The mechanism might be related to the reduction of IL-6 and IL-10 levels in serum and decreasing NHE2 expression and the inhibition of TRF and CRP expression.
Mechanism of Modified Yujin San in Treatment of Transmissible Gastroenteritis of Swine Based on Network Pharmacology and Molecular Docking
GUO Ziyan, SONG Yanping, NIAN Xia, SU Min
2024, 51(2):  809-819.  doi:10.16431/j.cnki.1671-7236.2024.02.037
Abstract ( 15 )   PDF (9549KB) ( 12 )  
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【Objective】 Based on network pharmacology and molecular docking technology, the target and the mechanism of modified Yujin San in the treatment of transmissible gastroenteritis(TGE) in pigs were studied.【Method】 The active ingredients and targets of modified Yujin San were searched by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and converted by UniProt platform.TGE related targets were searched in CTD database.The intersection targets of modified Yujin San and TGE were imported into Cytoscape 3.9.1 software to construct the modified Yujin San-component target-disease network diagram.Protein-protein interaction (PPI) network analysis was carried out in STRING database and Cytoscape 3.9.1 software.GO function and KEGG signaling pathway were analyzed in the DAVID database.Molecular docking was performed with AutoDock software.【Result】 A total of 126 active ingredients of modified Yujin San were collected, including quercetin and formononetin, and 74 related targets of modified Yujin San were obtained.The results of the network diagram of modified Yujin San-ingredient-target-disease showed that the core components of modified Yujin San for TGE were quercetin, kaempferol, etc.PPI results showed that the core targets of modified Yujin San were tumor suppressor gene P53 (TP53), tumor gene FOS, tumor necrosis factor (TNF), matrix metalloproteinase 9 (MMP9) and Caspase 3 (CASP3), etc.GO function and KEGG signaling pathway analysis results showed that the mechanism of modified Yujin San in the treatment of TGE involved immune response, anti-inflammatory and other processes, and regulated MAPK, IL17, TNF, TH-17 cell differentiation, anti-inflammatory bowel disease, NF-κB signaling pathways and so on.The molecular docking between the core components and the key receptor aminopeptidase N (APN) indicated that modified Yujin San had good binding ability with APN.【Conclusion】 Modified Yujin San mainly used core ingredients quercetin, 4'-methoxyglabridin, frmononetin and other core components to act on TP53, FOS, TNF, MMP9, CASP3 and other targets, and regulated signaling pathways such as MAPK, IL7, TNF, TH-17 cell differentiation, inflammatory bowel disease, and NF-κB to treat TGE.
In vitro and in vivo Antibacterial Effect of Isopropoxy Benzene Guanidine Combined with Colistin Against Pasteurella multocida
XUE Yaqian, QIAO Weimei, HONG Xiaoxin, JIN Yuhang, LU Yixing, PENG Xianfeng, ZENG Zhenling
2024, 51(2):  820-828.  doi:10.16431/j.cnki.1671-7236.2024.02.038
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【Objective】 This study was aimed to investigate the antibacterial effect of isopropoxy benzene guanidine (IBG) with colistin against Pasteurella multocida (P.multocida) in vivo and in vitro, in order to provide data support for the development of effective new colistin synergists and clinical prevention and treatment of P.multocida.【Method】 The minimum inhibitory concentration of IBG and colistin was determined by broth microdilution.The in vitro antibacterial activity against P.multocida was evaluated by checkerboard assays and time-kill curves, and the in vivo antibacterial effect was further evaluated by establishing a mouse lung infection model.【Result】 The drug sensitivity test results showed that IBG had no antibacterial effect on P.multocida (MIC>256 μg/mL), the MIC range of colistin on the tested P.multocida strains was 1-8 μg/mL.When IBG was combined with colistin, it could enhance the antibacterial activity of colistin (FIC was 0.094-0.313), demonstrating an excellent synergistic antibacterial effect.The time-kill curves further indicated that the combination group could significantly reduce the number of bacteria.The bactericidal effect could be achieved when colistin with sub inhibitory concentration (0.5 μg/mL) was combined with IBG.In the mouse lung infection model, the combination group could significantly or extremely significantly reduce the bacterial burden of P.multocida in lungs compared to the colistin or IBG alone group (P<0.05 or P<0.01).The pathological tissue sections of mouse lungs showed that the alveolar structure was normal and the alveolar cavities were clean in the combined group, which was better than IBG and colistin alone.【Conclusion】 The combination of IBG and colistin could enhance the antibacterial effect of colistin against P.multocida in vivo and in vitro, and was expected to be further developed as a new antibacterial synergist.
Probiotic Characteristics and Antibacterial Activity of Lactobacillus casei Strain YG-01
LIU Yimei, ZHOU Jiaying, HAN Yanyan, LI Yuan, SONG Houhui, XU Yigang, WANG Mei
2024, 51(2):  829-836.  doi:10.16431/j.cnki.1671-7236.2024.02.039
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【Objective】 The present study was aimed to evaluate the probiotic properties and inhibitory activity against pathogenic bacteria of Lactobacillus casei (L. casei) YG-01 isolated from cheese, and provide a theoretical reference for the application of this strain in food animal production practices.【Method】 This study evaluated the drug sensitivity, intestinal colonization ability, digestive tract environmental tolerance, modulation of intestinal microbiota, inhibition of pathogenic bacteria activity, and growth promotion effect of the L. casei YG-01 strain.【Result】 The drug sensitivity test showed that L. casei YG-01 was highly sensitive to common antibiotics such as minocycline, erythromycin, clarithromycin, clindamycin, and penicillin.Using carboxyfluorescein diacetate, succinimidyl ester (cFDA-SE) labeled L. casei YG-01 for oral administration in rabbits, the detection rates of L. casei YG-01 in rabbit jejunum, ileum and colon on the first day were 70.9%, 59.3% and 66.0%, respectively, and the high colonization levels (>35%) were maintained on the 11th day after oral administration.The viable numbers of L. casei YG-01 were higher than 1×105 CFU/mL in simulated gastric juice at pH 2.5, 3.5 and 4.5, simulated intestinal juice, 2 g/L cholate concentration and 9% NaCl solution. In vitro antibacterial tests showed that L. casei YG-01 significantly inhibited the growth of pathogenic bacteria in the intestines, such as Escherichia coli O157, Yersinia enterocolitica, Salmonella, Shigella, and Vibrio parahaemolyticus, while having no inhibitory effect on the probiotic bacterium Lactobacillus rhamnosus.Oral administration of L. casei YG-01 significantly increased average daily feed intake and average daily gain, but inhibited the proliferation of bacteria from the phyla Firmicutes and Proteobacteria.【Conclusion】 L. casei YG-01 exhibited excellent probiotic properties, as it could significantly inhibit the proliferation of pathogenic bacteria in the intestines and improve food animal growth performance.These findings laid a foundation for the development of microbiota-based preparations using the L. casei YG-01 strain.
Drug Resistance and Prevalence of Escherichia coli Isolated from Livestock and Poultry in Areas of Guangdong
LI Yafei, LIANG Xiaoyun, WU Liqin, XIAO Tianan, WANG Fuhua, ZENG Zhenling
2024, 51(2):  837-849.  doi:10.16431/j.cnki.1671-7236.2024.02.040
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【Objective】 The aim of this experiment was to investigate the prevalence and drug resistance of Escherichia coli (E. coli) from livestock and poultry in some areas of Guangdong, and provide reference for drug resistance prevention and control of E. coli derived from animals.【Method】 Samples of feed, drinking water and anal (cloaca) swabs of healthy animals collected from 10 farms in some areas of Guangdong in 2019 were subjected to E. coli isolation and identification, drug sensitivity test, detection of drug resistance genes and insertion sequence common region (ISCR), as well as ethnic phylogenetic analysis.【Result】 A total of 416 E. coli strains were isolated, including 78 strains from breeding pigs, 77 strains from broilers, 122 strains from laying hens and 139 strains from broilers.These strains showed the characteristics of multiple drug resistance, and the drug resistance rate of E. coli from broilers was the highest among different animal sources.79.6% (331/416) strains were resistant to 3 or more drugs, up to 13 drugs, and a total of 156 drug resistance profiles were formed.The main resistance profiles was sulfamethoxazole-trimethoprim/florfenicol/doxycycline/amoxicillin/ampicillin, accounting for 11.1%. floR gene showed the highest detection rate of 50.0%, followed by sul2 and sul3 genes, which were 44.2% and 42.5%, respectively.The detection rates of tet(X3), tet(X4) and mcr-1 genes associated with tigecycline and colistin resistance phenotypes were all lower than 10%.Insert sequence common region (ISCR) elements for ISCR1, ISCR2 and ISCR3 were present in 14.4%, 10.8% and 10.6% isolates, respectively.The coincidence rate of phenicols, sulfonamides and β-lactam resistance genes with drug-resistant strains was more than 60%, respectively.The coexistence of drug resistance genes cmlA, tetA, qnrS, blaCTX-M-9G with ISCR1, tet(X4), sul2 with ISCR2, and tet(X4), sul3, qnrS, blaSHV with ISCR3, were statistically significant (P<0.05), respectively.E. coli from different animals and ISCR-related mobile element-positive bacteria were both mainly distributed in phylogenetic group A.【Conclusion】 E. coli isolates from animals in some areas of Guangdong presented multiple drug resistance, and carried multiple drug resistance genes, as well as mobile elements ISCR.These results provided reference for understanding the drug resistance characteristics and prevalence of resistant genes in animal-derived bacteria in Guangdong province.
Study on the Mechanism of Total Glucosides of Paeony in the Treatment of Pulmonary Interstitial Fibrosis in Rheumatoid Arthritis Based on Network Pharmacology
XING Qinghua, LI Songwei, GONG Xiaohong, HU Wenying, LU Chaoqun
2024, 51(2):  850-863.  doi:10.16431/j.cnki.1671-7236.2024.02.041
Abstract ( 16 )   PDF (11911KB) ( 11 )  
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【Objective】 The aim of this experiment was to explore the targets and mechanisms between the main drug components of total glucosides of paeony (TGP) and pulmonary interstitial fibrosis in rheumatoid arthritis (RA) through network pharmacology.【Method】 By searching multiple databases, according to two ADEM parameters of oral bioavailability (OB)>30% and drug likeness (DL)>0.18, the main active ingredients and gene targets of total glucosides of paeony were screened out.The gene targets of RA and pulmonary interstitial fibrosis were screened by searching DrugBank, GeneCards, OMIM, TTD, DisGenet and other databases.The gene names of target proteins were converted by UniProt database, and the targets of total glucosides of paeony in the treatment of RA and pulmonary fibrosis were collected.The intersection of ingredients and disease targets were obtained, Venn diagram was made.The target intersection was imported into the STRING database to draw the PPI network diagram, and the intersection was copied and pasted into the DAVID database for GO function and KEGG signaling pathway analysis, and the mechanism of its treatment of RA with pulmonary interstitial fibrosis was analyzed in depth. Wistar rats were divided into blank group, model group, total glycosides of peony group, and tofacitinib group. The lung index was measured to reflect the edema and inflammatory response of lung tissue, HE staining was used to observe the inflammatory response of lung tissue and ankle joint synovium in each group, and Masson staining was used to observe the collagen deposition in lung tissue.【Result】 A total of 85 components of total glucosides of paeony were screened by searching the TCMSP database.According to OB>30% and DL>0.18, 9 main components were screened.A total of 1 625 RA gene targets and 1 930 pulmonary interstitial fibrosis gene targets were screened from DrugBank, DisGenet, OMIM, TTD and GeneCards databases.A total of 65 terms of molecular functions, 37 terms of cell components, 350 terms of biological processes and 141 enrichment pathways were obtained by GO function and KEGG signaling pathway enrichment analysis.Through the Chinese medicine-target-pathway network diagram, TNF, IL6, AKT1, VEGFA, MMP9, JUN, PPARG, CASP3, PTGS2, and ICAM1 in the top 10 of Degree were used as the key targets for the treatment of RA pulmonary interstitial fibrosis.The results of animal experiments showed that compared with model group, the arthritis score and lung index of the treatment group decreased. HE staining revealed a decrease in the infiltration of inflammatory cells in the joint and lung tissues of the total glycosides of peony group. Masson staining found that total glycosides of peony ccould reduce fibrous lesions in the form of strips. It indicated that total glucosides of paeony could not only treat RA, but also delay collagen deposition and inflammation of pulmonary interstitial fibrosis.【Conclusion】 Total glucosides of paeony acted on multiple targets and pathways in inflammatory response, immunoregulation, cell differentiation and proliferation through various components, and delayed the development of pulmonary interstitial fibrosis in RA through the synergistic interaction between targets and pathways.
Alleviating Effects of Inonotus obliquus Extract on Liver Injury Induced by AFB1 in Mice
ZHAO Chengming, XIE Weitian, LIN Hongying, HU Yingxin, MA Wenao, LI Ying, CHEN Zhibao
2024, 51(2):  864-874.  doi:10.16431/j.cnki.1671-7236.2024.02.042
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【Objective】 The objective of this study was to investigate the protective effect and mechanism of Inonotus obliquus extract (IOE) on liver injury in aflatoxin B1 (AFB1) exposed mice.【Method】 Thirty 6-week-old SPF male C57BL/6 mice with similar body weight were randomly divided into 5 groups.The control group was continuously gavage with distilled water for 10 days.AFB1 group received continuous intragastric administration of distilled water from day 1 to day 7, and 2 mg/kg AFB1 from day 8 to 10.Groups with different concentrations of IOE were given continuous intragastric administration of 25, 50 and 100 mg/kg IOE on days 1 to 7, and 2 mg/kg AFB1 on days 8 to 10, respectively.The intragastric dose was 0.2 mL, once a day.After the experiment, the mice were weighed, blood and liver tissues were collected, and liver index was calculated.The pathological changes of liver were observed by hematoxylin-eosin (HE) staining.The apoptosis of liver cells was analyzed by flow cytometry.The contentss of inflammatory factors interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) in serum and cells were detected by ELISA.The mRNA expression of TNF-α, IL-6, IL-1β, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) genes in liver and AML12 cells were detected by Real-time quantitative PCR.The expression of NLRP3, Bax and Bcl-2 proteins in liver and AML12 cells were detected by Western blotting.【Result】 Compared with control group, hepatocyte arrangement was disordered, cell swelling and apoptosis rate were extremely significantly increased in AFB1 group (P<0.01).The contents of TNF-α, IL-6 and IL-1β in serum were extremely significantly increased (P<0.01).The expressions of TNF-α, IL-6, IL-1β, NLRP3 and Bax genes in liver were extremely significantly increased, and the expression of Bcl-2 gene was extremely significantly decreased (P<0.01).The protein levels of NLRP3 and Bax were extremely significantly increased, and the level of Bcl-2 protein was extremely significantly decreased (P<0.01).Compared with AFB1 group, inflammatory cell infiltration was decreased, cells were arranged neatly, and cell apoptosis rate was extremely significantly decreased in IOE groups (P<0.01).Serum TNF-α, IL-6 and IL-1β contents were extremely significantly decreased (P<0.01).The mRNA expressions of TNF-α, IL-6, IL-1β, NLRP3 and Bax genes in liver were extremely significantly decreased, and the expression of Bcl-2 gene was extremely significantly increased (P<0.01).NLRP3 and Bax proteins levels were extremely significantly decreased, while Bcl-2 protein levels was extremely significantly increased (P<0.01).The results of cytotoxicity test showed that the optimal condition for AFB1-induced AML12 cell damage was 10 μg/mL AFB1 for 24 h, and the concentration gradients of IOE for prevention and treatment of AFB1-induced AML12 cell damage were 1.5, 3 and 6 μg/mL, respectively.The results of in vitro test showed that the secretion of inflammatory factors, apoptosis-related genes and protein expression of AML12 cells were consistent with the results of in vivo test.【Conclusion】 IOE could inhibit the secretion of inflammatory factors and reduce apoptosis by regulating the expression of NLRP3, Bax and Bcl-2, thus alleviating the liver injury induced by AFB1 in mice.
Research Progress on the Protective Effect of Baicalin on Intestinal of Animals
HE Zhengke, SU Lijuan, TAN Xiaoyan, LI Zhangxun, CAO Liting, MA Yue, DU Hongxu
2024, 51(2):  875-882.  doi:10.16431/j.cnki.1671-7236.2024.02.043
Abstract ( 21 )   PDF (2025KB) ( 18 )  
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Baicalin is a kind of flavonoid active substance extracted from the root of Scutellaria baicalensis.Under alkaline environment, the chemical properties of baicalin are unstable, and the glycoside bond in its molecular structure is easily hydrolyzed and the o-diphenol hydroxyl group is easily oxidized.The presence of sugar groups in the molecular structure of baicalin makes it almost insoluble in water, which limits its clinical application.Baicalin is not easily absorbed in the intestine, but under the action of β-glucuronidase produced by intestinal microbes, its glycosyl is removed and converted into baicalin, which is more easily absorbed by the intestine.The metabolites of baicalein were excreted in bile or urine after glucosylation, glucosylation, methylation and hydrolysis in animals.Pharmacological studies have found that it has anti-inflammatory, antibacterial, antiviral, anti-tumor and anti-intestinal damage pharmacological activities.Based on the bibliometrics analysis of the relevant literature in recent years, it is found that the research attention of baicalin in animal intestinal protection is increasing.In order to more comprehensively review the current research status of baicalin in animal intestinal protection, the author reviewed the effects of baicalin on animal intestinal pathogenic microorganisms and maintenance of intestinal barrier function homeostasis, aiming to provide a reference for further research of baicalin and its clinical application in veterinary medicine.
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Effect of Different Storage Conditions and Treatments on the Content of Sodium Thiocyanate in Raw Milk
YE Qiaoyan, HAO Xinyu, LIU Huimin, YUAN Ning, GUO Hongxia, ZHENG Nan
2024, 51(2):  883-892.  doi:10.16431/j.cnki.1671-7236.2024.02.044
Abstract ( 20 )   PDF (3415KB) ( 7 )  
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【Objective】 The aim of this study was to explore the effects of different storage conditions and treatment methods on the content of sodium thiocyanate in raw milk samples.【Method】 Raw milk samples were treated differently, including storage conditions (frozen at -20 ℃ for 1, 7 and 30 days, chilled at 0-6 ℃ for 4, 8, 24 and 48 h), thawing temperature (thawed at 25, 40 and 60 ℃ after being frozen for 24 h), thawing frequency (frozen at 24 h, after thawed in water bath at 40 ℃ for 1, 3 and 5 times respectively) and preservatives (sodium thiocyanate, potassium dichromate, sodium azide, propanediol bromide and formaldehyde).Then, sodium thiocyanate was detected in the sample, and the results were analyzed using One-Way ANOVA, principal component analysis, and recovery analysis to explore the impact of different treatments on the content of sodium thiocyanate in raw milk.【Result】 After 1, 7, and 30 days of frozen storage, there was no statistically significant difference in the content of sodium thiocyanate in milk (P>0.05), with a recovery rate of 97.4% to 103.1%.After being chilled for 4, 8, 24, and 48 h, the content of sodium thiocyanate in milk showed a trend of first decreasing and then increasing, with a recovery rate of 94.5% to 102.1%.After 24 hours of freezing, there was no statistically significant difference in the content of sodium thiocyanate in milk at three different thawing temperatures (P>0.05), with a recovery rate of 97.8% to 99.0%.The results of thawing 1, 3 and 5 times after freezing showed that the content of sodium thiocyanate in milk decreased with the increase of thawing times, and the assay recoveries were 95.3% to 101.8%, which were in the acceptable range.There were significant differences in the content of sodium thiocyanate in milk with the addition of different preservatives (P<0.05), in which the addition of sodium thiocyanate as a preservative led to high results, the addition of bromonitropropanediol led to low assay results, and the addition of formaldehyde led to non-detection of sodium thiocyanate.【Conclusion】 When raw milk samples were frozen at -20 ℃ for 30 days, at 0 to 6 ℃ for 48 h, thawing temperature below 60 ℃ and thawing frequency less than 5 times, there was no effect on the content of sodium thiocyanate in raw milk samples.Sodium azide or potassium dichromate did not affect the content of sodium thiocyanate in raw milk samples.