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05 November 2024, Volume 51 Issue 11
Biotechnology
Cloning,Expression and Immune Function Analysis of MAVS Gene in Carp
ZUO Mingzhong, ZHANG Meina, LIU Yuqing, CHEN Mengjuan, LIU Bianzhi, LI Ming, YU Guangqing
2024, 51(11):  4653-4664.  doi:10.16431/j.cnki.1671-7236.2024.11.001
Abstract ( 109 )   PDF (9360KB) ( 106 )  
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【Objective】 This study was aimed to obtain the CDS region of mitochondrial antiviral signaling protein (MAVS) gene in carp,perform bioinformatics analysis on it,and explore the function of MAVS gene in immune response. 【Method】 Referring to the predicted sequence of MAVS gene in Cyprinus carpio published in NCBI (GenBank accession No.:XM_019072216.2),primers were designed for its cloning and sequencing,the property and function of MAVS protein in carp were analyzed using bioinformatics softwares.Carp was subjected to Aeromonas hydrophila challenge experiment,and Real-time quantitative PCR was used to detect the expression of MAVS gene in various tissues of carp before and after challenge.Experimental subcellular localization of MAVS and mitochondria was performed by immunofluorescence assay.The expression of MAVS-regulated downstream genes was detected using dual-luciferase reporter gene system. 【Result】 The CDS region of MAVS gene in carp was cloned to a length of 1 749 bp,encoding a total of 582 amino acids.The similarities of the nucleotide sequences of MAVS gene in carp with those of Carassius auratus,Onychostoma macrolepis,Puntigrus tetrazona,Labeo rohita,Ctenopharyngodon idella,Megalobrama amblycephala,Rhinichthys klamathensis goyatoka,Sinocyclocheilus anshuiensis and Pimephales promelas were 92.3%,89.5%,85.1%,79.1%,73.6%,73.1%,72.0%,70.1% and 69.8%,respectively. The results of phylogenetic tree showed that carp clustered into a single unit with Carassius auratus,Labeo rohita,Puntigrus tetrazona,etc.MAVS protein of carp was an acidic,unstable protein with strong hydrophilicity,containing 133 phosphorylation sites and 1 transmembrane helix,which belonged to transmembrane proteins.MAVS protein did not contain a signal peptide,and did not belong to secretory proteins.Protein conservation analysis results showed that the N-terminal CARD structural domain,the middle proline-rich structural domain (PRR) and the C-terminal transmembrane structural domain (TM) of MAVS were highly conserved among different species.The secondary structure and tertiary structure of MAVS protein in carp showed that the protein was mainly composed of random coil (58.93%) and alpha helix (23.37%),with low contents of extended chain (13.92%) and beta turn (3.78%),suggesting that it was a hybrid protein.Protein-protein interaction results showed that there were interactions between MAVS protein and immune proteins such as NLRP3,TRAF6,ATG5,DDX58 and TBK1.Real-time quantitative PCR results showed that compared with control group, the expression of MAVS gene in heart,brain,spleen,liver,intestines,muscle and gill of carp were significantly or extremely significantly increased after challenge (P<0.05 or P<0.01),the expression in kidney was extremely significantly decreased (P<0.01),and there was no significant difference in eye tissues (P>0.05).Cellular immunofluorescence results showed that the expression location of MAVS protein was consistent with mitochondria.The results of dual luciferase gene assay reported that MAVS gene could extremely significantly activate IFN and ISRE promoters (P<0.01). 【Conclusion】 MAVS gene was highly conserved across species and during genetic evolution,and was widely expressed in different tissues of carp.The results provided a theoretical basis for further study on the function and molecular mechanism of MAVS gene in innate immune response of carp.
Joint Analysis of Transcriptome and Translatome During the Development of Bovine Skeletal Muscle Satellite Cells
MA Sufang, YANG Wenqing, ZHANG Bingbing, GUO Ruonan, XU Yingxin, ZHANG Linlin, GUO Yiwen, HU Debao, GUO Hong, DING Xiangbin, LI Xin
2024, 51(11):  4665-4677.  doi:10.16431/j.cnki.1671-7236.2024.11.002
Abstract ( 86 )   PDF (13539KB) ( 128 )  
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【Objective】 The aim of this study was to analyze and compare the growth and development process of bovine skeletal muscle at two different levels of transcription and translation using transcriptome sequencing (RNA-Seq) and ribosome-nascent-chain-complex sequencing (RNC-Seq), analyze the developmental characteristics and patterns,and screen out key genes that might be involved in the growth and development process of bovine muscle in the transcriptome and translatome,in order to provide a target for the subsequent screening and utilization of functional genes. 【Method】 Samples of the proliferative period (GM) and the second day of differentiation (DM2) of skeletal muscle satellite cells from 4-month-old bovine embryos were collected for RNA-Seq and RNC-Seq analysis.The differentially expressed genes were analyzed using bioinformatics,and the differentially expressed genes related to skeletal muscle development were analyzed from the perspective of translational regulation. 【Result】 The differential gene screening of BSMSCs in DM2 phase compared to GM phase showed that a total of 2 808 differential genes (1 749 up-regulated genes and 1 059 down-regulated genes) were obtained from transcriptome screening,and 3 740 differential genes (1 769 up-regulated genes and 1 971 down-regulated genes) were obtained from translatome screening.There were 725 differentially expressed genes shared between transcriptome and translatome,including 618 genes with similar expression trends and 107 genes with opposite expression trends.The functional enrichment analysis results showed that 103 genes related to skeletal muscle development were obtained.Further in-depth analysis showed that the transcriptional and translational regulation were relatively independent and the translational regulation had a certain effect of retrogression on the transcriptional regulation,22 target genes closely related to the growth and development of bovine muscle (CDK1,SERPINE1,THBS1,IGFBP3,CNB1,GADD45A,CYCS,CDK2,CCND1,CCNE2,ZMAT3,ACTB,ACTA1,TNNT1,MYH7,ACTG1,TUBA1C,TNNC1,KIF20A,TUBB4B,MYL3 and TUBB3) and 13 key genes with translation retrogression (GREM1,EGR1,SFRP4,NKD2,DKK3,IGFBP3,CTSK,ITGAV,CTSV,CDKN1A,SESN3,GADD45A and ZMAT3) were obtained. 【Conclusion】 In this study,22 key genes that might be closely related to the growth and development of bovine muscles and 13 key genes with translation callbacks were screened,providing important targets for further regulating the process of bovine muscle growth and development through molecular means for new breed cultivation.
Physiological and Biochemical
PS-MPs and DEHP Combined Induce Autophagy in Mouse Colonic Epithelial Cells via NO/iNOS/NF-κB Pathway
AI Jingzhu, XU Shiwen
2024, 51(11):  4678-4689.  doi:10.16431/j.cnki.1671-7236.2024.11.003
Abstract ( 102 )   PDF (9251KB) ( 58 )  
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【Objective】 This study was aimed to investigate the mechanism of combined exposure of polystyrene microplastics (PS-MPs) and di(2-ethylhexyl) phthalate (DEHP) in inducing autophagy in mouse colonic epithelial cells (MCEC) via NO/iNOS/NF-κB pathway. 【Method】 According to the half inhibitory concentrations (IC50) of PS-MPs and DEHP on MCEC measured by CCK-8 method,MCEC were divided into control,PS-MPs,DEHP,combination,and inhibition groups.They were treated with culture medium containing 200 μg/L PS-MPs,100 μmol/L DEHP,200 μg/L PS-MPs+100 μmol/L DEHP,and 200 μg/L PS-MPs+100 μmol/L DEHP+5 nmol/L bortezomib (PS-341) for 24 h,respectively.Then,the NO content and iNOS activity were detected by biochemical kit,autophagosomes were detected by MDC method,and the expression of mRNA and protein of autophagy and NO/iNOS/NF-κB pathway-related genes were detected by immunofluorescence staining,Real-time quantitative PCR and Western blotting,respectively. 【Result】 Both PS-MPs and DEHP could inhibite the viability of MCEC with IC50 of 2 315 μg/L and 1 477 μmol/L,respectively.Compared with control group,the NO content,iNOS activity and the expression of mRNA and protein of genes related to NO/iNOS/NF-κB pathway in PS-MPs,DEHP and combination groups were significantly increased (P<0.05),the fluorescence intensity of MDC method and immunofluorescence staining were significantly enhanced (P<0.05),and the expression of mRNA and protein of the autophagy-related genes (Beclin1,ATG5 and LC3-B) were significantly upregulated (P<0.05),PS-341 could significantly alleviate the changes of the above detection indexes caused by PS-MPs and DEHP. 【Conclusion】 The combined exposure to PS-MPs and DEHP could activate iNOS,lead to a sharp increase in NO content,and upregulate of NO/iNOS/NF-κB pathway,which induced autophagy in MCEC.
Research Progress on the Regulation of Autophagy by Acetylation Modification
QIN Kening, CHEN Dandan, XU Peng, WANG Xiaomin
2024, 51(11):  4690-4701.  doi:10.16431/j.cnki.1671-7236.2024.11.004
Abstract ( 98 )   PDF (1227KB) ( 176 )  
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Autophagy is a process in which cells degrade their own contents through the function of lysosomes.Mutations in autophagy-related processes can lead to serious human diseases,such as cancer,cardiovascular diseases,neurodegenerative diseases,metabolic diseases,lung diseases and kidney diseases.Autophagy can combine some biochemical reactions of the body to form a dynamic circulatory system,which can eliminate harmful substances and provide new energy for cell renewal and maintaining balance in the body.Therefore,autophagy plays an important role in the physiological and pathological processes of the body.In recent years,some medical and animal physiological and pathological studies have pointed out that acetylation modification plays a key role in regulating cell autophagy.As a kind of modification methods in protein post-translational modification,acetylation is a reversible post-translational modification mediated by acetyltransferase and deacetylase.Among them,acetyltransferase transfers the acetyl group of acetyl coenzyme A to the amino acid residue of the substrate protein,and the removal of acetyl group is completed by deacetylase.Acetylation modification often occurs on the histone lysine ε-amino group and the N-terminal α-amino group of non-histone proteins.The authors describe the effect of acetylation modification on cell autophagy from the level of histone and non-histone acetylation modification,in order to provide reference and direction for the further study of the role of acetylation modification in cell autophagy and its therapeutic effect on various diseases.
Research Progress on Regulation of Thermogenic Adipose Tissue by m6A Methylation Modification
FAN Yanyan, DU Jiawei, SUN Tianhao, FU Shaoyin, HE Xiaolong, ZHANG Lin, DA Lai, Terigele, WANG Liwei, HE Jiangfeng, LIU Yongbin, WANG Biao
2024, 51(11):  4702-4710.  doi:10.16431/j.cnki.1671-7236.2024.11.005
Abstract ( 80 )   PDF (1159KB) ( 55 )  
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Excessive deposition of adipose tissue can lead to obesity and related metabolic diseases,and also directly affect the productivity and product quality of livestock and poultry.Therefore,how to regulate adipose tissue deposition is of great significance for safeguarding human health,increasing animal production performance and improving the quality of animal products.The thermogenic function of adipose tissue has become an area of great concern,which can regulate the body’s thermogenesis through the consumption of glucose,fatty acids,and other energy substances,so as to achieve the purpose of maintaining the energy balance of the body.N6-methyladenosine (m6A) modification,as a major epigenetic mark on RNA molecules,is activated in brown adipose tissue,adipose tissue browning and thermogenesis.The author outlines the thermogenesis of adipose tissue and mRNA m6A-related modification proteins,and focuses on the regulation of energy metabolism in thermogenic adipose tissue by mRNA m6A methylation modification,which is crucial for energy homeostasis,thermogenic adipose activation and metabolic diseases,so as to lay a foundation for the study of the specific thermogenic mechanism of thermogenic fat,and provide new ideas for the precise targeting and regulation of thermogenic mechanism of animal fat,and thus the regulation of animal production performance and product quality.
Study on Regulation of Receptivity Gene Expression in Bovine Endometrial Epithelial Cells by Progesterone
TANG Ying, LI Qi, WANG Hongzhan, LI Bo, CHEN Yanru, ZHENG Peng
2024, 51(11):  4711-4721.  doi:10.16431/j.cnki.1671-7236.2024.11.006
Abstract ( 82 )   PDF (5497KB) ( 40 )  
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【Objective】 This study was aimed to explore the effect of progesterone on the expression of receptivity marker genes in bovine endometrial epithelial cells,and provide theoretical basis for studying the relationship between progesterone and endometrial receptivity and its regulatory mechanism. 【Method】 Bovine endometrial epithelial cells were treated with 0, 10 and 100 μg/L progesterone,and the cell morphology was observed by microscope.The cell proliferation level was detected by CCK8 assay,and the mRNA expression of phosphatidylinositol 3 kinase (PI3K),protein kinase B (Akt),vascular endothelial growth factor (VEGF) and homeobox structure gene 10 (HOXa10) were detected by Real-time quantitative PCR,and the optimal concentration of progesterone was screened.Bovine endometrial epithelial cells were treated with progesterone, transcription activator 3 (STAT3) inhibitor (1.5 μmol/L),and progesterone +STAT3 inhibitor,respectively.The mRNA and protein expression of VEGF and HOXa10 were detected by Real-time quantitative PCR and Western blotting,respectively.With Lipofectamine 3000,VEGF targeting miRNA (miR-497 mimic and miR-497 inhibitor) and HOXa10 targeting miRNA (miR-27a-3p mimic and miR-27a-3p inhibitor) and negative control (mimic-NC and inhibitor-NC) were transfected into endometrial epithelial cells,respectively.Real-time quantitative PCR was used to detect the expression of miR-497,miR-27a-3p,VEGF and HOXa10. 【Result】 Compared with 0 μg/L group,there were no significant differences in cell proliferation level,mRNA expression of PI3K and Akt genes in 10 and 100 μg/L progesterone treatment groups (P>0.05),while VEGF and HOXa10 genes mRNA expression were significantly increased (P<0.05).Subsequently,10 μg/L progesterone was selected for the test.Compared with 10 μg/L progesterone treatment group,the mRNA and protein expression of VEGF and HOXa10 in STAT3 inhibitor treatment group and progesterone+STAT3 inhibitor combined treatment group were significantly decreased (P<0.05),but there was no significant difference compared with control group (P>0.05).Compared with mimic-NC group,mRNA and protein expression of VEGF and HOXa10 in cells transfected with miR-497 mimic and miR-27a-3p mimic were significantly decreased (P<0.05).Compared with inhibitor-NC group,mRNA and protein expression of VEGF and HOXa10 in cells transfected with miR-497 inhibitor and miR-27a-3p inhibitor were significantly increased (P<0.05). 【Conclusion】 Progesterone regulated the expression of VEGF and HOXa10 genes in bovine endometrial epithelial cells by activating STAT3 signaling pathway,miR-497 negatively regulated the expression of VEGF gene,and miR-27a-3p negatively regulated the expression of HOXa10 gene.
Research Progress on the Effect of Endoplasmic Reticulum Stress on Testosterone Synthesis in the Leydig Cells
LI Jingxuan, LONG Cheng, QI Xiaolong
2024, 51(11):  4722-4731.  doi:10.16431/j.cnki.1671-7236.2024.11.007
Abstract ( 97 )   PDF (3490KB) ( 182 )  
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The endoplasmic reticulum is a multifunctional organelle that participates in material transport and plays an important role in biological activities such as protein modification and processing,synthesis of lipids and steroid hormones,cellular stress,and maintenance of calcium homeostasis.When there is an excessive accumulation of misfolded or unfolded proteins on the endoplasmic reticulum,it can cause an endoplasmic reticulum stress response that cannot be regulated,leading to the initiation of cell apoptosis.Leydig cells are the primary site of testosterone synthesis,converting cholesterol into testosterone through a series of enzymes,which is crucial for maintaining animal reproductive performance.Recent studies have shown that during the process of testosterone synthesis in male animal Leydig cells,endoplasmic reticulum stress,and the unfolded protein response are often involved,leading to cell apoptosis through different signaling pathways and affecting testosterone synthesis.However,the specific mechanisms by endoplasmic reticulum stress influences testosterone synthesis remain unclear.The authors reviewed the effects and possible molecular mechanisms of endoplasmic reticulum stress mediated apoptosis pathways,oxidative damage,and calcium signaling pathways on testosterone synthesis in male animal Leydig cells,in order to provide new insights into improving testosterone synthesis by regulating endoplasmic reticulum stress.
Nutrition and Feed
Effect of Dietary Zinc on Growth Performance,Apparent Digestibility and Serum Biochemical Indices of High-temperature Stressed Xueshan Broilers
SUN Pengfei, WANG Xiaohan, HU Yun, WU Huiguang, ZHAO Jingwen, LUO Xugang
2024, 51(11):  4732-4742.  doi:10.16431/j.cnki.1671-7236.2024.11.008
Abstract ( 90 )   PDF (1192KB) ( 58 )  
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【Objective】 The aim of this study was to assess the effect of diverse forms and doses of zinc on the growth performance,apparent digestibility and serum biochemical indices of Xueshan broilers subjected to high-temperature stress conditions,so as to provide a reference for zinc nutrition of Xueshan broilers in high-temperature environments. 【Method】 The experiment employed a 2 (zinc sources)×2 (zinc supplementation levels) completely randomized factorial design.The 2 zinc sources were inorganic zinc sulphate and moderate chelated zinc proteinate,and 2 zinc supplementation levels were 30 and 60 mg/kg,resulting in a total of four experimental groups.In addition,a control group was established to meet the zinc requirement of broilers.A total of 320 61-day-old Xueshan broilers were randomly divided into five groups,with eight replicates per treatment and eight birds per replicate.The pre-trial period was 7 d,followed by a formal trial period of 28 d,both conducted under high-temperature stress conditions.Samples of feed,faeces and blood were collected on days 14 (75 days of age) and 28 (89 days of age) of experiment for determining the growth performance,apparent digestibility of nutrients and serum biochemical indices of broilers. 【Result】 ① The zinc source,zinc supplementation level and their interaction had no significant effect on the growth performance of broilers under high-temperature stress (P>0.05).② Compared with control group,dietary zinc supplementation significantly increased the apparent digestibility of crude protein and crude fat in broilers (P<0.05).The zinc source had a significant effect on the digestibility of dry matter.In the main effect of zinc source,the addition of moderate chelated zinc proteinate significantly decreased the apparent digestibility of dry matter in high-temperature stressed broilers compared with the addition of inorganic zinc sulfate (P<0.05).③ Compared with control group,dietary zinc supplementation significantly increased the serum levels of total protein,albumin and globulin in broilers on days 14 of the experiment (P<0.05),and significantly increased the serum levels of total protein and albumin in broilers on days 28 of the experiment (P<0.05).The zinc source had a significant effect on serum total protein and albumin levels in broilers on days 14 of the experiment (P<0.05).The zinc level had a significant effect on serum total protein,albumin and globulin levels on days 14,as well as total protein and albumin levels on days 28 of the experiment (P<0.05).The interaction between zinc source and zinc level had a significant effect on serum total protein levels on days 14 and serum albumin levels on days 28 in broilers (P<0.05).Among these,the addition of 60 mg/kg moderate chelated zinc proteinate in the diet showed the best effect.④ In the terms of zinc source,zinc level and their interaction,dietary zinc supplementation had no significant effect on the activities of serum alanine aminotransferase and aspartate aminotransferase in high-temperature stressed broilers (P>0.05).⑤ Compared with control group,dietary zinc supplementation significantly increased the serum levels of total cholesterol and high-density lipoprotein cholesterol in broilers on days 14 of the experiment,and significantly decreased the serum levels of total cholesterol and low-density lipoprotein cholesterol in broilers on days 28 of the experiment (P<0.05).The zinc level had a significant effect on the serum levels of total cholesterol,high-density lipoprotein cholesterol and low-density lipoprotein cholesterol in broilers (P<0.05). 【Conclusion】 Despite the absence of a notable effect on the growth performance in broilers under high-temperature stress conditions,dietary zinc supplementation demonstrated a substantial influence on the apparent digestibility of crude protein and crude fat in broilers.Additionally,it exhibited a favorable effect on serum protein and lipid metabolism indices in high-temperature stressed broilers,thereby contributing to the maintenance of physiological stability.
Effect of Compound Preparation of Chinese Herbal Aqueous Extract on Ruminal Fermentation and Bacterial Composition in Tan Lambs
ZHOU Ying, WANG Honghao, LIU Weiping, XU Yuzhang
2024, 51(11):  4743-4754.  doi:10.16431/j.cnki.1671-7236.2024.11.009
Abstract ( 88 )   PDF (5292KB) ( 118 )  
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【Objective】 The experiment was conducted to investigate the effect of Chinese herbal compound additive of Astragalus membranaceus,Licorice and Sophora alopecuroides on the ruminal fermentation parameters and bacterial composition in Tan lambs. 【Method】 A single factor design was used in the experiment,and forty 3-month-old healthy Tan lambs with similar weight were randomly divided into four groups: Control group (k0),experimental group 1 (s1),experimental group 2 (s2) and experimental group 3 (s3), with ten sheep in each group. The Tan lambs were fed with diets containing 0,0.50%,1.00% and 1.50% compound preparation of Chinese herbal aqueous extract,respectively. The pre trial period for the experiment was 10 days,and the main trial period was 60 days.The rumen fluid was collected orally to measure pH,NH3-N and volatile fatty acids and other related rumen fermentation parameters.Subsequently,high-throughput sequencing technology was used to determine the diversity of rumen bacteria,then the correlation between the main differential bacterial genera in the rumen and ruminal fermentation parameters was analyzed by sequencing platform. 【Result】 There were no significant changes in levels of total volatile fatty acids (TVFA),propionate,isobutyrate,isovalerate,valerate,NH3-N and pH in the rumen fluid among the groups (P>0.05).Compared with k0 group,there were no significant changes in the proportion of acetate and butyrate in s1 and s2 groups (P>0.05),in s3 group,the proportion of acetate in rumen fluid was significantly increased (P<0.05),while the proportion of butyrate was significantly reduced (P<0.05).In s1,s2 and s3 groups,the proportions of propionate in rumen fluid were significantly reduced (P<0.05),and the ratios of acetate to propionate were significantly increased (P<0.05).From the analysis of rumen bacterial diversity,at the phylum level,compared with k0 group,the relative abundance of Firmicutes in s2 and s3 groups were significantly reduced (P<0.05),the relative abundance of Spirochaetota in s2 group was significantly reduced (P<0.05),and the relative abundance of Patescibacteria in s1,s2 and s3 groups were significantly reduced (P<0.05).At the genus level,compared with k0 group,the relative abundance of Selenomonas in s1 group was significantly increased (P<0.05),while the relative abundance of Treponema in s1 and s2 groups were significantly reduced (P<0.05).Through correlation analysis,it was found that Fibrobacter was significantly positively correlated with the proportion of acetate and the ratio of acetate to propionate,negatively correlated with the proportion of propionate.Rikenellaceae RC9_gut_group was significantly positively correlated with valerate.Treponema was significantly negatively correlated with pH value (P<0.05). 【Conclusion】 0.50% compound preparation of Chinese herbal aqueous extract was the optimal concentration for adding to the dietary of Tan lamb,which could adjust the proportion of volatile fatty acids in the rumen,and improve the diversity of rumen bacterial communities.To some extent,the relative abundance of rumen fiber degrading bacteria(such as Fibrobacter) was increased,while the relative abundance of lactic acid inhibiting bacteria (such as Selenomonas) was significantly increased,and the relative abundance of semi fiber degrading bacteria (such as Treponema) was significantly reduced.0.50% compound preparation of Chinese herbal aqueous extract was more suitable for the growth needs of Tan lambs.
Study on the Structure and Function of the Archaea Community and Its Correlation with Diarrhea of Weaned Piglets
PAN Chenglin, SONG Tongxing, ZHANG Xiaojun, LOU Fangfang, HU Xujin, TU Pingguang, XIAO Yingping, LYU Wentao
2024, 51(11):  4755-4764.  doi:10.16431/j.cnki.1671-7236.2024.11.010
Abstract ( 82 )   PDF (10419KB) ( 46 )  
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【Objective】 The aim of this experiment was to study the structure and function of Archaea community in intestine of weaned piglets,and analyze the correlation between Archaea and diarrhoea in weaned piglets,so as to provide theoretical basis for the prevention and treatment of post-weaning piglet diarrhea. 【Method】 Thirty-two healthy weaned piglets (H group) and thirty-two diarrheal weaned piglets (D group) at 24 days of age were selected from the same batch.Fecal samples were collected and the structure of Archaea community of the fecal samples from both groups was analyzed using 16S rRNA gene sequencing.PICRUSt2 software was used to predict the function of Archaea community in intestine,and the correlation between differential Archaea and diarrhea in weaned piglets was analyzed. 【Result】 There was significant difference of diarrhea scores between healthy and diarrheal weaned piglets (P<0.05).Euryarchaeota,Crenarchaeota and Thaumarchaeota were the dominant Archaea phyla in healthy and diarrheal weaned piglets.The dominant genera in healthy weaned piglets were Methanosarcina,Euryarchaeota_norank and Methanobrevibacter,while the dominant genera in diarrheal weaned piglets were Euryarchaeota_norank,Methanosarcina and Methanobrevibacter.The diversity and richness of Archaea community in diarrheal weaned piglets was higher than those in healthy weaned piglets.There were significant difference in the structure and function of the Archaeal community between healthy and diarrheal weaned piglets (P<0.05).The difference in the structure of Archaea community between healthy and diarrheal weaned piglets were mainly reflected in the genus Methanosarcina,Euryarchaeota_norank,Sulfophobococcus,Nitrosarchaeum,Pyrodictium,etc.The difference in the function of Archaea community primarily existed in lipopolysaccharide biosynthesis,biosynthesis of amino acids,antigen processing and presentation,etc.The results of correlation analysis indicated that the relative abundance of Methanosarcina,Sulfophobococcus,Euryarchaeota_norank,Methanocaldococcus,Pyrococcus,Methanocalculus were significantly correlated with diarrhea in weaned piglets (P<0.05). 【Conclusion】 There was significant difference in the structure and function of Archaea community between healthy and diarrheal weaned piglets.The relative abundance of Euryarchaeota_norank was higher in diarrheal weaned piglets,while the relative abundance of Methanosarcina was lower.There was a correlation between Euryarchaeota_norank,Methanosarcina,Sulfophobococcus and diarrhea in weaned piglets.
Effect of Caragana korshinskii Silage on Rumen Fermentation Performance of Dairy Cows
SHEN Yao, XUE Fuguang, KANG Yajie, XIONG Benhai, YANG Liang
2024, 51(11):  4765-4773.  doi:10.16431/j.cnki.1671-7236.2024.11.011
Abstract ( 75 )   PDF (2571KB) ( 27 )  
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【Objective】 The purpose of this study was to improve the digestibility and utilization of Caragana korshinskii (CK) in the form of silage,and explore the effect of CK silage on the rumen dry matter degradation rate and fermentation performance of dairy cows,so as to provide a potential feed resource for ruminants. 【Method】 The fresh CK was randomly divided into fresh sample and silage groups,with 9 replicates in each group,and each replicate contained 1 000 g matters.The silage quality and nutrient composition of CK were determined after 30 days ensilaging process,following by the weighing of 18 samples at 10 g/part to determine the degradation rate of nylon bag dry matter (DM) in the rumen fermentation of dairy cows for 2,4,8,24,48 and 72 h,respectively.Further,18 samples were weighed at 0.5 g/part to determine the ruminal fermentation parameters of in vitro for 2,4,8,24,48 and 72 h,respectively. 【Result】 The contents of DM,crude protein (CP),neutral detergent fiber (NDF) and acid detergent fiber (ADF) of CK in silage group were significantly lower than those in fresh group (P<0.05).After 30 days of ensilaging,pH of CK reached to 4.51,the ammonia nitrogen content was 5.12 g/kg,the lactic acid content was 14.53 mmol/g,and the acetic acid content was 8.45 g/kg.The nylon bag DM degradation rate of CK in fresh group was significantly lower than that in silage group at 2,4,8,24 and 48 h of rumen fermentation (P<0.05).The results of rumen in vitro fermentation parameters showed that the DM degradation rate,gas production,and the contents of acetic acid,propionic acid and total volatile acid of CK in silage group were significantly higher than those in fresh group (P<0.05).The contents of ammonia nitrogen and methane in silage group were significantly higher than those in fresh group at 24,48 and 72 h of in vitro fermentation (P<0.05). 【Conclusion】 In this study,ensilaging process of CK could effectively reduce the crude fiber content of CK,and improve the rumen DM degradation rate,in vitro gas production,and the contents of volatile fatty acid and ammonia nitrogen of CK.
Effects of Alkaloids from Sophora alopecuroides on Growth Performance and Serum Biochemical Indexes of Dumeng Lambs Fed with High Concentrate Diet
ZHAO Jianxin, LI Shufang, LU Henan, LI Shaojun, GAO Aiwu, WANG Hairong
2024, 51(11):  4774-4782.  doi:10.16431/j.cnki.1671-7236.2024.11.012
Abstract ( 84 )   PDF (1138KB) ( 44 )  
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【Objective】 This experiment was conducted to investigate the effects of Sophora alopecuroides alkaloids on growth performance and blood indexes of Dumeng lambs fed with high-concentrate diet. 【Method】 Thirty-two Dumeng lambs with body weight of (25.76±2.35) kg were randomly divided into 4 groups with 8 lambs in each group.The basal diet of each experimental group was the same,which was a high-concentrate diet with a concentrate to forage ratio of 7∶3,and the lambs in control group were fed with the basal diet.The lambs in groupⅠ were fed with the diet added 0.2% Sophora alopecuroides,in group Ⅱ were fed with the diet supplemented with 0.121 g/kg Sophora alopecuroides alkaloids (low-dose alkali group),and in group Ⅲ were fed with the diet suplemented with 0.169 g/kg Sophora alopecuroides alkaloids (high-dose alkali group).The pre-feeding period was 15 days,and the feeding period was 60 days.The lambs were fed in a single cage and fed ad libitum.During the experiment,the daily feed intake of each sheep was recorded.The lambs were weighed with an empty stomach on days 0,30 and 60 of the experiment to calculate growth performance indicators.After weighing on the 60th day,blood was collected from the jugular vein of the lambs for measurement of serum biochemical index. 【Result】 The average daily gain of lambs in groups Ⅰ and Ⅱ were significantly higher than that in control group and group Ⅲ from 0 to 30 d (P<0.05).From 30 to 60 d,the average daily gain and daily feed intake of lambs in group Ⅱ were significantly higher than those in group Ⅲ (P<0.05).From 0 to 60 d,the average daily gain of lambs in group Ⅱ was significantly higher than that in group Ⅲ (P<0.05).On the 60 th day of the experiment,compared with group Ⅲ,the activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in groups Ⅰ and Ⅱ were significantly decreased (P<0.05),and the contents of total protein (TP) and albumin (ALB) in serum of lambs in group Ⅱ were significantly increased (P<0.05).Compared with control group,the serum glucose (GLU) contents of groups Ⅰ, Ⅱ and Ⅲ were significantly decreased (P<0.05),the content of serum immunoglobulin M (IgM) in group Ⅱ was extremely significantly increased (P<0.01),and the content of serum IgA in group Ⅱ was significantly higher than that in group Ⅲ (P<0.05). 【Conclusion】 The addition of 0.121 g/kg Sophora alopecuroides alkaloids under high-concentrate diet could improve the growth performance of Dumeng lambs,alleviate liver damage,reduce GLU content and regulate humoral immune function.
Comparison of Follicle Development,Antioxidant Capacity and Autophagy Level Between Shan-ma Duck at Peak and Late Stages of Laying
JIANG Liying, MA Manting, LI Kaichao, ZHANG Yanan, LAI Yue, CHEN Wei, WANG Shuang, XIA Weiguang, JIN Chenglong, GUO Liang, ZHENG Chuntian
2024, 51(11):  4783-4791.  doi:10.16431/j.cnki.1671-7236.2024.11.013
Abstract ( 79 )   PDF (1895KB) ( 42 )  
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【Objective】 To investigate the changes of follicle development,gonadotropin levels,antioxidant capacity and follicle autophagy related genes in ducks at different laying stages,and to lay a research foundation for developing new technology to prolong laying period of laying birds. 【Method】 The single factor completely randomized experimental design was adopted,and 84 normal laying ducks (Longyan Shan-ma duck) in the peak laying stage (25 weeks) and the late laying stage (70 weeks) were selected.These ducks were randomly divided into 6 replicates,with 14 ducks in each replicate.The ducks were fed with the same corn-soybean meal basal diet for 8 weeks.At the end of the 8th week,3 healthy and normal laying ducks were randomly selected from each replicate for blood collection and slaughter sampling to detect blood biochemical indicators and ovarian development indicators. 【Result】 Compared with the duck in peak laying stage,①The number of small yellow follicles,the index of small yellow follicles and the average weight of small yellow follicles of laying duck in late laying stage were significantly reduced,and the weight of atretic follicles was significantly increased (P<0.05).②The plasma follicle-stimulating hormone (FSH) level of laying duck in late laying stage was significantly lower (P<0.05),the activities of glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) in small yellow follicles and the malondialdehyde (MDA) level were significantly higher,and the activities of GSH-Px and T-SOD in atretic follicles were significantly lower (P<0.05).③The mRNA expression level of follicle-stimulating hormone receptor (FSHR) in follicles of laying duck in late laying stage was reduced,and forkhead transcription factor 1 (FOXO1) was activated and regulated autophagy-related genes (selective autophagy receptor (SQSTM1) and beclin 1 (BECN1)) (P<0.05). 【Conclusion】 When Shan-ma ducks entered the late laying period,the content and sensitivity of FSH in follicles were decreased due to the increase of oxidative stress,and FOXO1 was activated to regulate the autophagy of follicular granulosa cells and inhibit follicular development.
Effects of Dietary Metabolizable Energy Levels on Intestinal Development and Cecal Microbial of Yanjin Silky Chickens
TANG Anxing, ZHANG Zhengfei, SUN Jindong, FU Lixiang, WANG Xingxian, ZHANG Shiyun, LI Cien, YANG Liangyu, TAO Linli, NIU Guoyi
2024, 51(11):  4792-4801.  doi:10.16431/j.cnki.1671-7236.2024.11.014
Abstract ( 70 )   PDF (3188KB) ( 69 )  
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【Objective】 The aim of this experiment was to study the effects of dietary metabolizable energy level on the intestinal development and cecal microbial diversity of Yanjin Silky chickens aged 1-42 days. 【Method】 A total of 200 healthy 1-day-old Yanjin Silky chickens with similar body weight were randomly divided into 5 groups with 5 replicates in each group and 8 chickens in each replicate.The chickens were fed five experimental diets with metabolizable energy levels of 11.40,12.00,12.60,13.20 and 13.80 MJ/kg,respectively.At 42 days of age,one chicken was taken from each replicate for slaughter,each intestinal segment was separated and its length and mass were determined,and the contents of the cecum were taken to analyze the diversity of cecal microbial flora by the Illumina NovaSeq high-throughput sequencing platform. 【Result】 The mean cecal length of Yanjin Silky chickens in 11.40 and 12.00 MJ/kg groups were significantly higher than that of 13.20 and 13.80 MJ/kg groups (P<0.05).The absolute and relative mass of rectum in 11.40 MJ/kg group was significantly higher than that in 12.60 and 13.80 MJ/kg groups (P<0.05).Dietary metabolizable energy level had no significant effect on cecal microbial Alpha diversity index of Yanjin Silky chickens (P>0.05),but the microbial community structure in 11.40 MJ/kg group was obvious different from that in 12.60,13.20 and 13.80 MJ/kg groups.At the phylum level,Firmicutes,Bacteroidetes and Desulfurobacteria were the dominant phyla in the caecum of the five groups of Yanjin Silky chickens,and the relative abundance of Cyanobacteria of 12.00 MJ/kg group was significantly higher than that in 11.40, 12.60 and 13.80 MJ/kg groups (P<0.05).At the genus level,the dominant genera in the caecum of the five groups were Faecalibacterium,Bacteroides,Alistipes and unclassified_Oscillospiraceae,and the relative abundance of [Ruminococcus]_torques_group in 11.40 and 12.60 MJ/kg groups were significantly higher than that in 13.20 and 13.80 MJ/kg groups (P<0.05). 【Conclusion】 Under these experimental conditions,the dietary metabolizable energy levels of 13.20 and 13.80 MJ/kg could inhibit the development of caecum and rectum and the growth of some pathogenic bacteria to a certain extent.Dietary metabolizable energy level had little effect on cecal microbial Alpha diversity of Yanjin Silky chickens,but it could affect the structure of cecal microbial community and caused changes in the dominant microbial flora and their relative abundance.
Metabolomics Analysis of Fermented Elephant Grass and It’s Effects on the Growth Performance and Serum Biochemical Indicators of Pigs
ZHANG Xingyan, LAN Haien, YI Xianfeng, YANG Kai, ZHU Wen, LAN Xi, ZHOU Junhua, CHEN Zhonghua
2024, 51(11):  4802-4811.  doi:10.16431/j.cnki.1671-7236.2024.11.015
Abstract ( 70 )   PDF (3829KB) ( 97 )  
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【Objective】 To comprehensive analysis the nutritional value of fermented elephant grass used as feed stock for pigs. 【Method】 Gas chromatography-mass spectrometry (GC-MS) was used to comparatively analysis the metabolites of fermented elephant grass.The different levels of fermented elephant grass mixed with cassava and fish sauce were added into the basal diet for pigs.Pigs in control group were fed with basal diet,in groups Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ and Ⅵ were fed with test diets supplemented by 0,2%,3%,4%,6% and 9% fermented elephant grass.The feeding period was 28 days.After the feeding experiment,the growth performance of pigs were measured,and blood was collected for measuring serum biochemical indicators. 【Result】 ①There were 121 different metabolites in fermented elephant grass with 92 differential metabolites up-regulated.Most of them were sugars,amino acids sand fatty acids.Among them,the levels of tricose,L-aspartic acid,glutamate,inositol,methionine and N-acetyl-ornithine were increased more than 1 000 times.There were 11 extremely significantly different metabolic pathways between elephant grass and fermented elephant grass (P<0.01).②There were no significant differences in serum biochemical indicators of pigs between the experimental groups and control group (P>0.05),except for the levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDLC) in group Ⅱ which were significantly higher than control group (P<0.05).The levels/activities of total protein (TP),TC,LDLC,albumin (ALB),alanine transaminase (ALT),triglycerides (TG),aspartate aminotransferase (AST) and high-density lipoprotein cholesterol (HDLC) in group Ⅱ were significantly higher than those in other groups (P<0.05).In the feeding time,the diarrhea rate of pigs in all seven groups were 0. 【Conclusion】 The nutritional value of fermented elephant grass had been significantly enhanced with significant increases in amino acids,carbohydrates and lipids.Fermented elephant grass could partially replace the basic daily feed for pigs and significantly reduce the fat deposition in pig.Adding 2% fermented elephant grass to diet could get the best comprehensive production efficiency.
Advancements in Research on Methane Emission Reduction in Ruminant Animals
LI Gaolong, WU Zhaohai, ZHAO Liansheng, BU Dengpan, WANG Jianping
2024, 51(11):  4812-4823.  doi:10.16431/j.cnki.1671-7236.2024.11.016
Abstract ( 104 )   PDF (1240KB) ( 181 )  
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Methane is an important component of greenhouse gases,and ruminant production is an important source of methane emissions.In recent years,the research of methane emission reduction in ruminants has become a hot topic.How to safely and efficiently reduce ruminant methane emissions is crucial to promoting sustainable and environmentally friendly animal husbandry,and plays an important role in improving feed conversion,reducing environmental pollution and improving ruminant production performance.In order to promote the green development of animal husbandry,the author starts from the elaboration of the methane production mode of ruminants,and adjusts the total food structure (including the proportion of dietary concentrate and crude,feed raw material quality,feed raw material mix,etc.),optimizing feeding management (including improving the feeding environment,changing grazing methods,and processing feed raw materials,etc.),selecting suitable varieties and animals with physiological status (selecting varieties with low methane emission or individuals with physiological status),and utilizing plant secondary metabolites (such as tannins,saponins,and essential oils),microecological preparations (such as yeast,Lactobacillus and Bacillus subtilis),organic acids (such as malic acid,fumaric acid and tannic acid,etc.),inorganic compounds (3-NOP) and algae (algae and microalgae),and the specific effects of various measures in reducing methane emissions were summarized. And seek a more suitable emission reduction plan.Based on the literature review,specific measures and suggestions for methane emission reduction are proposed,hoping to provide a reference for further research in related fields in the future,and also provide reference experience for reducing methane emissions in production.
Effects of Lactobacillus acidophilus Post-Biotic on Production Performance, Blood Indexes and Egg Quality of Laying Hens in the Initial Laying Stage
QIU Kai, GUAN Xiaofeng, SANG Yang, LIU Zhiyun, LIU Guohua
2024, 51(11):  4824-4832.  doi:10.16431/j.cnki.1671-7236.2024.11.017
Abstract ( 79 )   PDF (1128KB) ( 110 )  
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【Objective】 The purpose of this study was to reveal the effect of dietary supplementation with Lactobacillus acidophilus post-biotic (LAPB) on the production performance,blood indexes and egg quality of laying hens in the initial laying stage. 【Method】 A total of 216 23-week-old healthy Hy-line Brown laying hens were randomly divided into 3 treatment groups,with 6 replicates and 12 chickens per replicate.The control group was fed a basal diet,and the experimental groups were fed the basal diet supplemented with 0.1% or 0.2% LAPB,respectively.One week was used for feed adaption.The formal feeding period was 8 weeks.The egg production was recorded every day,and the feed intake was recorded weekly.At weeks 4 and 8,5 eggs were collected per replicate for egg quality determination.At the end of the feeding,one layer per replicate was selected for blood samples collection,and then blood routine and serum biochemical indexes were measured. 【Result】 Compared with control group,the addition of 0.1% or 0.2% LAPB in diets significantly increased the average egg weight and average daily egg mass (P<0.05),reduced the feed-to-egg ratio (P<0.05),improved the egg yolk color,thick/thin albumen ratio and albumen height (P<0.05),had no effect on blood routine indicators (P>0.05),and increased the contents of total protein,total cholesterol,triglycerides,high-density lipoprotein and low-density lipoprotein in serum (P<0.05),among which,the 0.1% addition level increased the serum creatinine content and reduced the activity of aspartate aminotransferase of laying hens in the initial laying stage (P<0.05). 【Conclusion】 Dietary addition of 0.1% or 0.2% LAPB could effectively improve the production performance and egg quality of laying hens in the initial laying stage,and regulate fat metabolism,enhance liver and kidney health,immunity and antioxidant capacity,and the recommended dosage was 0.1%.
Evaluation of Nutrient Utilization of Sugar Residue and Soy Sauce Residue in Meat Ducks
WU Zhanyue, GUO Yanhong, ZHUANG Lei, WANG Ning, LU Zhentao, REN Wenwen, XU Tong, WANG Qimeng, NIU Zhili, CAO Junting, WU Yongbao, WU Sen, WEN Zhiguo
2024, 51(11):  4833-4841.  doi:10.16431/j.cnki.1671-7236.2024.11.018
Abstract ( 77 )   PDF (1123KB) ( 138 )  
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【Objective】 The nutritional value,nutrient digestibility,metabolizable energy,and ileal amino acid digestibility of sugar residue and soy sauce residue were evaluated and determined in meat ducks. 【Method】 Metabolic energy and nutrient digestibility were measured using the empty-force-feeding method.A total of 24 ducks were selected and randomly divided into sugar residue and soy sauce residue groups,with 12 replicates in each group and 1 duck per replicate.The feed weight was 60 g,and the experimental duration for feed withdrawal and collection of manure samples was both 48 hours.Meat duck feces samples were collected to determine the contents of dry matter,crude protein,ether extract,crude fiber,neutral detergent fiber,acid detergent fiber,and total energy.The digestibility of amino acids in the ileum was determined using nitrogen free diet method and terminal ileum method.A total of 60 ducks were randomly divided into the sugar residue,sugar residue N-free diet,soy sauce residue,and soy sauce residue N-free diet groups,with 5 replicates in each group and 3 ducks per replicate.After the adaptation period of 3 days,the experimental feed was fed separately,and after 4 h,the intestinal contents were collected and measured for amino acid content. 【Result】 The dry matter content of sugar residue was 89.70%,the crude protein was 21.98%,and the gross energy was 26.47 MJ/kg.The dry matter content of soy sauce residue was 89.53%,the crude protein was 26.65%,and the gross energy was 24.06 MJ/kg.The metabolic test results showed that the apparent metabolizable energy of sugar residue and soy sauce residue in meat ducks was 18.09 and 8.59 MJ/kg,dry matter digestibility was 57.46% and 44.61%,and ether extract digestibility was 84.88% and 84.43%,respectively.The results of ileal amino acid digestibility showed that the highest apparent ileal digestibility in sugar residue and soy sauce residue was lysine,which were 92.75% and 55.60%,respectively. 【Conclusion】 Sugar residue and soy sauce residue were rich in crude protein and amino acids,and the utilization rate of crude fat and crude protein of meat ducks were obviously high,which could be developed as feed materials for meat ducks.
Research Progress on Feed Utilization of Grape Residueand and Its Application in Sheep Production
CHANG Anqi, MAO Shuaixiang, LIU Guangbin
2024, 51(11):  4842-4850.  doi:10.16431/j.cnki.1671-7236.2024.11.019
Abstract ( 86 )   PDF (1156KB) ( 80 )  
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Grape residue is a by-product in the wine making process,rich in nutrients such as proteins,crude fat,crude fiber,and active substances such as polyphenolic compounds,grape seed oil,oleanolic acid and dietary fiber.It has a large yield and is easy to collect.In recent years,grape residue have been widely used in livestock and poultry feed production as a natural unconventional feed raw materials and additives to alleviate China’s feed shortage problem.It can not only promote the growth and development of livestock and poultry,improve their health and product quality,but also effectively develop and utilize agricultural by-products,addressing environmental pollution and resource waste issues.However,in actual production,the large-scale production and use of grape residues as feed raw materials and additives are restricted by factors such as grape origin,varieties and feed processing technology.Sheep is one of the main economic animals in China,and in recent years,the scale of China’s sheep industry have been steadily increasing.However,with the continuous rise in prices of feed raw materials such as corn and soybean meal,the cost of sheep farming has also been increasing,making the development of unconventional feed for sheep significant.Therefore,the author systematically summarizes the nutrient content of grape residue,its special active substances and biological functions,feed utilization techniques,limitations,and its application in sheep production,providing reference for the rational development and application of grape residues in sheep feed.
Genetics and Breeding
Analysis of the Population Characteristics and Influencing Factors of Superovulation Response Traits in Northern Chinese Holstein Cattle
HUANG Yuechuan, ZHANG Hailiang, LIU Zhanpeng, ZHAO Shanjiang, SUN Wei, LI Xihe, WANG Lin, ZHU Huabin, HUANG Xixia, WANG Yachun
2024, 51(11):  4851-4859.  doi:10.16431/j.cnki.1671-7236.2024.11.020
Abstract ( 63 )   PDF (2088KB) ( 30 )  
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【Objective】 In vivo superovulation is one of the key technologies in modern dairy cattle breeding,which is of great significance for genetic improvement of dairy cattle populations.This study was conducted to reveal the phenotypic characteristics and main influencing factors in Chinese Holstein cattle,so as to provide foundation for the genetic analysis of superovulation response traits in dairy cattle. 【Method】 In this study,1 855 in vivo superovulation records of 1 842 Chinese Holstein cattle from 9 pastures in Ningxia and Inner Mongolia from March 2021 to August 2023 were collected,7 superovulation response traits were statistically analyzed,including total number of oocytes,number and percentage of transferable embryos,number and percentage of unfertilized oocytes,and number and percentage of degenerated embryos.The Mixed model of SAS 9.2 software was used to analyze the influencing factors on each trait,and the Pearson correlation coefficient among 7 superovulation response traits were calculated using a bivariate correlation program of SPSS 21.0 software. 【Result】 The average number of total oocytes and transferable embryos in Chinese Holstein cattle were 9.49 and 6.01,while the average percentage of transferable embryos was 61.95%.The average number and percentage of unfertilized oocytes were 1.50 and 17.16%,while the average number and percentage of degenerated embryos were 2.29 and 23.34%,respectively.There was varying degrees of phenotypic correlation among 7 superovulation response traits,with correlation coefficients ranging from ―0.629 to 0.882.Seasons had significant influence on all traits except for the percentage of transferable embryos (P<0.05). Hormone types and doses had significant impact on 7 superovulation response traits (P<0.05).Semen dosage significantly affected the number of transferable embryos,degenerated embryos,and the number and percentage of unfertilized oocytes (P<0.05).Donor age had a significant impact only on the percentage of transferable embryos and degenerated embryos (P<0.05). 【Conclusion】 The phenotype of superovulation response traits in Chinese Holstein cattle was similar to the average level in global cattle.The traits were influenced by various non-genetic factors.The results laid a model foundation for further genetic analysis of superovulation response traits in dairy cattle.
Research Progress on the Application of Genome-wide Selection in Goat Breeding
ZHAO Yanpin, HAN Yong, SU Chaozhi, LONG Yong, XIAO Wen, YANG Xiaoling
2024, 51(11):  4860-4870.  doi:10.16431/j.cnki.1671-7236.2024.11.021
Abstract ( 114 )   PDF (3771KB) ( 96 )  
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Genome-wide selection (GS) is a breeding method based on genomic data.By analyzing the relationship between genetic markers and traits,it predicts the genetic and phenotypic values of individuals,thereby achieving precise breeding.With the development of high-throughput sequencing technology,GS has become an important tool in modern livestock and poultry breeding,demonstrating significant potential and application prospects in goat breeding.Researchers use GS to accurately assess the reproductive performance of goats,improve reproductive efficiency,and select superior genotypes to enhance traits such as growth,development and meat quality,thus achieving higher economic benefits.The authors primarily introduce the principles and methods of GS,focusing on its application in key traits such as reproductive performance,growth characteristics and meat quality in goats,discusse the advantages and challenges of GS in goat breeding and provide a summary and outlook on its future development and application.This review aims to offer valuable references and theoretical support for the further advancement of goat breeding.
Effects of Ola1 on Early Embryonic Development of Mouse in vitro
LUO Anfeng, ZHANG Yuqing, WANG Xinxin, YANG Caixia, XIE Di
2024, 51(11):  4871-4879.  doi:10.16431/j.cnki.1671-7236.2024.11.022
Abstract ( 61 )   PDF (6210KB) ( 51 )  
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【Objective】 The objective of this experiment was to explore the effects of Obg-like ATPase 1 (Ola1) on early embryonic development in mouse. 【Method】 The mouse zygotes were obtained through in vitro fertilization,followed by the culture of embryos in vitro.Ola1 gene was knocked down using electric siRNA interference technology.The interference efficiency of Ola1 gene was measured,and the cleavage rate and blastocyst rate were calculated.DNA damage and early apoptosis of embryos were detected by immunofluorescence and TUNEL.Real-time quantitative PCR was used to detect the transcriptional levels of genes related to pluripotency and apoptosis in embryos. 【Result】 Compared with control group,the relative expression of Ola1 gene in mouse embryos was extremely significantly decreased after the knockdown of Ola1 gene (P<0.01).Successful knockdown of Ola1 gene in mouse embryos.After knocking down Ola1 gene,there was no significant difference in the early embryo cleavage rate compared with control group (P>0.05),but the blastocyst rate was extremely significantly lower than that of control group (P<0.01),the γ-H2A.X fluorescence intensity and blastocyst apoptosis rate were extremely significantly higher than that of control group (P<0.01).These results indicated that knockdown of Ola1 gene caused early embryo development to be hindered in vitro.Real-time quantitative PCR results showed that,compared with control group,the expression of pluripotency related genes,sex determining region Y box protein 2 (SOX2) and Nanog in early embryos were extremely significantly decreased after Ola1 gene knockdown (P<0.01).The expressions of pro-apoptotic genes,B-lymphocytoma-2-associated X protein (Bax) and Caspase3 were significantly or extremely significantly up-regulated (P<0.05 or P<0.01).The expression of anti-apoptotic gene,B-lymphoblastoma-2 (Bcl-2) was significantly down-regulated (P<0.05). 【Conclusion】 Ola1 might be involved in DNA damage and early embryo apoptosis by regulating the expression of pluripotent genes in early embryos,thus affected the development potential of early embryos.
Research Progress on CRISPR-Cas9 Gene Editing Technology in Cattle and Sheep Production
PAN Dongxia, WANG Hui, XIONG Benhai, TANG Xiangfang
2024, 51(11):  4880-4889.  doi:10.16431/j.cnki.1671-7236.2024.11.023
Abstract ( 151 )   PDF (1207KB) ( 145 )  
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In recent years,gene editing technology has developed rapidly as a genome modification tool.Gene editing tools including zinc-finger nuclease (ZFNs),transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats associated protein (CRISPR-Cas) system make it possible to target genome DNA modification of organisms.The emergence of the CRISPR-Cas9 system,in particular,accelerated the development of gene editing technology as a revolutionary tool in basic and applied science.Compared with ZFNs and TALENs technologies,CRISPR-Cas9 systems are widely used due to their high flexibility,sensitivity,specificity and cost effectiveness.CRISPR-Cas9 gene editing technology has made significant contributions to different aspects of livestock production by precisely cutting DNA using targeting specific sequences and introducing site-specific modifications such as adding,removing or replacing nucleotides by generating double-strand breaks at specific genomic sites,producing a variety of animal models such as improved livestock reproductive production and disease resistance,so as to study the key gene functions of livestock and poultry and accelerate the improvement of traits.In this paper,the mechanism and function of CRISPR-Cas9 system and its application in the production of cattle and sheep are reviewed,in order to provide reference for future research.
Assessment of Conservation Effects of Lingxian White Goose Based on Whole Genome Resequencing
WAN Weican, ZHANG Xu, HUANG Xuan, YAN Haifeng, JIANG Guitao, LI Chuang
2024, 51(11):  4890-4898.  doi:10.16431/j.cnki.1671-7236.2024.11.024
Abstract ( 62 )   PDF (7604KB) ( 72 )  
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【Objective】 Lingxian White goose is a great indigenous goose breed in Hunan province.To further understand the conservation effect of Lingxian White goose,an evaluation of the conservation work was conducted among various families in the conservation farm. 【Method】 One male goose was selected from each of the 42 families in the Lingxian White goose conservation farm,and blood was collected from the wing vein to extract DNA for whole genome resequencing.Based on the resequencing data of 42 Lingxian White goose male geese in the conservation farm,the population genetic structure and genetic diversity were analyzed and evaluated. 【Result】 A total of 14 270 647 SNPs were detected in the sampled population,and 13 696 453 SNPs were selected through data quality control and filtering.The results of principal component analysis (PCA) showed that 42 male geese were significantly clustered into three subgroups.Population admixture study revealed that there was no substantial hybridization taking place among the three subgroups (K=3).The fixation index (Fst) between subgroup 1 and subgroup 3 was 0.0842.A total of 832 runs of homozygosity (ROH) were identified among Lingxian White geese,all of ROH lengths concentrated in the range of 0 to 2 Mb.FROH which resulting inbreeding coefficient based on ROHs was 0.0130.The expected heterozygosity for the group was 0.2918,the observed heterozygosity was 0.2968,inbreeding coefficient (Fis) was 0.036.The polymorphism information content (PIC) was 0.266,and the gene diversity index (Nei) was 0.336. 【Conclusion】 The conserved population of Lingxian White goose was found to have a low degree of inbreeding and a comparatively high genetic diversity.The observed heterozygosity was slightly higher than the expected heterozygosity,but without a significant difference,suggesting that the Lingxian White goose population was in a balanced state.Although some degree of moderate genetic differentiation had emerged among certain subgroups.In subsequent conservation plan,it was necessary to promote interbreeding among these subgroups to reduce the genetic differentiation of Lingxian White goose.
Preventive Veterinary Medicine
Genetic Evolution Analysis of H9N2 Subtype Avian Influenza Viruses in Wild Birds from Yunnan Province
YANG Jia, YANG Qinhong, WANG Wei, ZHANG Yongxian, DAI Hongyang, YIN Hongbin, LI Suhua
2024, 51(11):  4899-4910.  doi:10.16431/j.cnki.1671-7236.2024.11.025
Abstract ( 64 )   PDF (2267KB) ( 43 )  
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【Objective】 This study was aimed to analyze the genetic evolution and molecular characteristics of H9N2 subtype Avian influenza virus (AIV) originating from Napahai,an international important wetland in Shangri-La,Yunnan province,and clarify the epidemic characteristics of H9N2 subtype AIV in wild birds,so as to provide data for the prevention and control of avian influenza epidemics. 【Method】 Fecal samples of wild birds,and the oropharyngeal,cloacal swab and and environmental water samples of surrounding poultry from Napahai international important wetland of Yunnan province during 2022-2023 were collected.Allantoic cavity inoculation in 10-day-old SPF chicken embryo was used for isolating virus,and AIV subtype was identified through hemagglutination (HA) assay and RT-PCR amplification.Firstly,all the eight gene (HA,NA,M,PB2,NS,NP,PA and PB1) segments of H9N2 subtype AIV isolates were simultaneously amplify using MBTuni-12/13 primers,followed by the second-generation sequencing to obtain full genome sequences.Finally,the genetic evolutionary relationship and amino acid mutation analysis were performed. 【Result】 Four H9N2 subtype AIV isolates were identified,which exhibited G57 genotype characteristics that had been prevalent in China since 2013,with HA and NA genes from Y280-like subtype,M and PB2 genes from G1-like subtype,and NS,NP,PA and PB1 genes from F/98-like subtype.Four H9N2 subtype AIV might be the recombinant strains from multiple AIV subtypes.The results of sequence similarity analysis showed that the internal genes of four H9N2 subtype AIV isolates had the higher similarity with H7N9,H5N6 and H5N2 subtype AIV isolates,the genetic diversity characteristic was prominent.The HA cleavage sites of four strains were consistent with characteristic of cleavage of low-pathogenic avian influenza.Multiple amino acid mutations associating with increasing the risk of cross-species transmission and enhancing virulence in mammals were acquired by the isolates,including A198T, Q234L and N166D (HA),I292V and A558V (PB2),I368V and L13P (PB1),N30D and T215A (M1),P42S (NS1), etc.Four wild bird-derived H9N2 subtype AIV strains exhibited characteristics similar to those of current Chinese chicken-derived AIVs,indicated the potential for transmission route from poultry to wild birds. 【Conclusion】 Four H9N2 subtype AIV from wild birds in Napahai international important wetland of Yunnan province belonging to G57 genotype,were likely to be reassortants and may originate from poultry,and had the molecular characteristics related to the adaptation to mammalian hosts.
Research Progress on the Pathogenicity and Immune Evasion Mechanism of Haemophilus parasuis
HAO Yue, DUAN Yu, CHAI Wenqin, FENG Huapeng, SHU Jianhong, HE Yulong
2024, 51(11):  4911-4922.  doi:10.16431/j.cnki.1671-7236.2024.11.026
Abstract ( 95 )   PDF (1230KB) ( 95 )  
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Haemophilus parasuis (Hps) is the pathogen of Glasser’s disease in pigs,which can cause serofibrinous pleurisy,pericarditis,peritonitis,arthritis and other diseases in pigs.There are many Hps serotypes,and there are differences in antigenic heterogeneity and pathogenicity among different strains.The pathogenic process of Hps infection involves a variety of virulence factors,such as biofilm,liposaccharide,capsule,fimbrial and other structural substances, as well as functional substances such as cytolethal distending toxin,ceramidase,α-2,3-sialyltransferase,and other outer membrane proteins,ect.A variety of virulence factors participate in the adhesion,invasion and colonization of Hps to host cells,and then cause inflammatory responses,including crossing the blood-brain barrier to cause encephalitis,and eventually cause damage to host cells and tissues and organs.Hps has a variety of complex immune evasion mechanisms,such as breaking through the epithelial cell barrier,degrading sIgA on the mucosal surface,resisting macrophage phagocytosis and the killing effect of complement,so as to help it resist the killing of the host immune system.The authors reviewed the different types of Hps virulence factors and their roles in the pathogenesis,and summarized the research progress of immune evasion mechanism of Hps,in order to provide new information for effective prevention and control of the disease and the development of novel vaccines.
Prokaryotic Expression,Preparations of Polyclonal Antibodies and Immunogenicity Evaluation of NS1 Protein of Minute Virus of Mice
ZHANG Xuliang, CHEN Li, ZHAO Zhigang, MA Chang, YOU Jinwei, DONG Min, YUN Shifeng
2024, 51(11):  4923-4931.  doi:10.16431/j.cnki.1671-7236.2024.11.027
Abstract ( 72 )   PDF (7327KB) ( 47 )  
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【Objective】 The objective of this experiment was to express nonstructural protein 1 (NS1) of Minute virus of mice (MVM) through prokaryotic expression system and prepare polyclonal antibody,which provided research materials for the study of biological function of MVM NS1 protein and the establishment of related detection methods. 【Method】 MVM NS1 gene was linked to plasmid pET-N-His-C-His through homologous recombination to construct recombinant plasmid,which was transformed into Escherichia coli BL21 (DE3) competent cells for induced expression.Soluble proteins without renaturation were screened and purified by different induction conditions.The purified recombinant protein NS1 was immunized with New Zealand White rabbits and polyclonal antibodies were prepared.The titer of the antibodies was determined by indirect ELISA.Western blotting and indirect immunofluorescence assay (IFA) were used to determine the expression and immunogenicity of the recombinant protein. 【Result】 SDS-PAGE analysis showed that the recombinant protein was 76 ku in size,and the optimal expression was achieved under the conditions of IPTG concentration of 0.6 mmol/L and induction temperature of 28 ℃.Indirect ELISA results showed that the titer of the prepared polyclonal antibody was 1∶32 000.Western blotting and IFA detection results showed that the prepared NS1 polyclonal antibody could bind specifically to NS1 protein and MVM. 【Conclusion】 This study successfully expressed and purified MVM NS1 protein,and successfully prepared polyclonal antibody with good immunogenicity,which laid a foundation for further study of the biological function of NS1 protein and its role in the mechanism of MVM replication infection,and the establishment of clinical diagnostic methods for MVM.
Progress on Delivery Systems of mRNA Vaccine
LI Ziwei, ZHANG Lei, CHEN Feng, ZHENG Hui, CUI Jin, CAO Zhenshan, ZHANG Hui, GE Shengqiang, WEI Rong, LIU Fuxiao, NAN Fulong, SHA Zhou, NI Bo
2024, 51(11):  4932-4942.  doi:10.16431/j.cnki.1671-7236.2024.11.028
Abstract ( 105 )   PDF (1189KB) ( 135 )  
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Compared with traditional vaccines,mRNA vaccines have the advantages of easy compilation,high immunogenicity,strong safety and low production cost.The efficient delivery of mRNA to cells and successful expression is the key link for mRNA vaccines to play a role,and also the main bottleneck and pain point of mRNA vaccine research and development.Therefore,screening for cost-effective nucleic acid delivery systems is crucial for mRNA vaccine development.At present,nucleic acid delivery systems with more attention mainly include physical delivery,lipid nanoparticle delivery,polymer delivery,exosome delivery and cationic emulsion delivery.Physical delivery is basically the physical process of getting naked mRNA directly across the cell membrane into the cell.Lipid nanoparticle,polymers,exosomes and cationic emulsions protect mRNA by using themselves as delivery carriers,thereby improving delivery efficiency and reducing immune response.Based on the latest research progress of mRNA vaccines,the author summarized the application prospects and limitations of different delivery methods,and prospected their research prospects.It is expected to provide a theoretical basis for the development of a new delivery system,promote the clinical application of mRNA therapy,and provide a new mRNA vaccine solution for the prevention and treatment of more diseases.
Molecular Characteristics and Evolutionary Analysis of the Spike Protein Gene of Bovine Torovirus Strain from Yunnan
GENG Juan, QU Weijie, ZHANG Xiaomei, WEI Zhijie, ZHONG Dekai, WANG Xinwen, LI Wengui, ZHANG Shuangling, LI Jincun, ZHANG Zhenxing, ZHANG Yifang, ZHENG Jinling, SONG Jianling
2024, 51(11):  4943-4955.  doi:10.16431/j.cnki.1671-7236.2024.11.029
Abstract ( 64 )   PDF (9012KB) ( 31 )  
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【Objective】 This study was aimed to investigate the prevalence of Bovine torovirus (BToV) and the genetic evolution of the S gene in cattle herds in some areas of Yunnan province. 【Method】 655 faecal samples were collected from cattle of different ages from 15 cattle farms in 13 regions of Yunnan province,and BToV was detected by RT-PCR.6 representative positive samples were subjected to the S gene amplification,cloning,sequencing and genetic evolution analysis. 【Result】 RT-PCR results showed that there were 71 BToV positive samples were detected for the 655 samples with a total detection rate of 10.8%.The detection rate of diarrhea and non-diarrhea samples were 21.2% (59/278) and 3.2% (12/377),respectively.The detection rate of the samples from cattle at 1 week old,1 week to 1 month old,1 to 6 months old,and over 1 year old were 15.0% (3/20),17.7% (20/113),14.0% (45/321)and 1.5% (3/201) respectively.The results of similarity analysis showed that the six S gene sequences with a nucleotide similarity from 96.0% to 99.8%,and nucleotide similarity with the original strain Breda1 was from 94.7% to 96.0%.The similarity with domestic epidemic strains was 95.2% to 99.7%. Genetic evolutionary analysis showed that the six S gene were not in the same branch with the original strain Breda1,and formed a branch with the domestic strains in Sichuan and Henan as well as the Turkish strains,indicating that the BToV strains prevalent in Yunnan province were consistent with those of the currently prevalent strains in China,and that they were closely related to the domestic strains.Amino acid mutation site analysis showed that there were a small number of unique amino acid mutation sites of S gene in Yunnan province.The results of recombination analysis showed that BTOV-China/YN03-2021,BTOV-China/YN01-2021 and BTOV-China/YN06-2022 were predicted to be recombinant sequences,in which BTOV-China/YN06-2022 had the highest recombination score,with recombination region was located in 1 438 to 1 609 bp,and the main parent was strain BTOV-China/YN05-2021.The recombination region of BTOV-China/YN03-2021 was located in the 1 668 to 3 569 bp region,and the main parent was the Turkish strain BToV-HT2-TUR.The recombination region of BTOV-China/YN01-2021 was located in 261 to 2 552 bp,and the main parent was the Turkish strain BToV-HT2-TUR.The results of gene selection pressure analysis showed that the evolutionary mode of S gene was mainly dominated by neutral selection,there were the influence of purifying selection and positive selection pressure,too. 【Conclusion】 This study revealed that BToV had existed in Yunnan province cattle populations and the situation of prevention and control was relatively serious,which provided a reference for the study of epidemiology for BToV and the genetic evolutionary relationship of the spike protein gene in China,as well as a theoretical basis for the development of preventive and control measures for BToV infection in Yunnan province.
Bioinformatics Analysis and Validation of PCNA Protein in Echinococcus granulosus sesensu lato
XU Shaoquan, MAIERHABA·Maimaitiaili, LYU Guodong, ZHOU Run, ZHAO Jinlong, LI Jing, XIAYIDANMU·Tuniyazi, ZHAO Jun
2024, 51(11):  4956-4963.  doi:10.16431/j.cnki.1671-7236.2024.11.030
Abstract ( 66 )   PDF (4397KB) ( 29 )  
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【Objective】 The objective of this study was to analyze the bioinformatic properties of proliferating cell nuclear antigen (PCNA) protein in Echinococcus granulosus sensu lato (Eg) and effect of harmine (HM) and its derivatives (H-2-168 and H-2-104) on the content of protein EgPCNA,in order to treat cystic echinococcosis and establish a basis for the hunt for potential therapeutic targets. 【Method】 The full length sequence of EgPCNA gene was amplified by PCR and cloned.Biological information related to EgPCNA was predicted and analyzed by bioinformatics softwares.Mega 7.0 was used to construct phylogenetic tree of EgPCNA protein,and the effects of HM and its derivatives (H-2-168 and H-2-104) on EgPCNA protein content were analyzed by Western blotting. 【Result】 The total length of EgPCNA gene was 783 bp,encoding 260 amino acids,the molecular weight of protein EgPCNA was 28.36435 ku,the isoelectric point was 4.62,and the fat index was 96.81.With a hydrophilic value of -0.015,it was a hydrophilic protein with no transmembrane domain.The subcellular localization prediction results showed that the protein was distributed in the cytoplasm.It contained PCNA superfamily structure and PCNA conserved functional domain.The secondary structure of this protein was mainly random coil,followed by alpha helix,and had two reliable B-cell epitopes,which was far from the relationship with mammals such as Homo sapiens and Mus musculus.Western blotting analysis showed that compared with DMSO group, the expression of EgPCNA protein in HM group was extremely significantly up-regulated (P<0.01),and the expression of EgPCNA protein in H-2-168 and H-2-104 groups was significantly down-regulated (P<0.05). 【Conclusion】 In this study,the full length of EgPCNA gene was successfully cloned.Bioinformatics analysis predicted that protein EgPCNA had a regulatory role in DNA replication and repair of Echinococcus granulosus,and H-2-168 and H-2-104 could down-regulate protein EgPCNA content.
Differential Expression Analysis of RLCht1 Gene in Rhipicephalus linnaei and Detection of Enzyme Activity
LI Yao, ZHAO Peizhen, XIE Zifang, PENG Weiqi, CHEN Jie, ZHAO Jianguo, GUAN Qingfeng
2024, 51(11):  4964-4973.  doi:10.16431/j.cnki.1671-7236.2024.11.031
Abstract ( 65 )   PDF (1865KB) ( 44 )  
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【Objective】 The molting process of ticks was closely related to chitin synthesis and hydrolysis,and chitin degradation is mainly done by chitinase,analysis of different periods and tissue in the tick (Rhipicephalus linnaei) chitinase RLCht1 sequence expression profile,in vitro heterologous expression protein and verify the activity of chitinase,for the tick and other vector biological control of reference were carried out,so as to provide a new target for developing safe and efficient biological tick. 【Method】 Different tissue samples of different period ticks and female blood-fed ticks were collected for differential expression analysis.Based on the putative chitinase sequence screened from the transcriptome of Rhipicephalus sanguineus,the recombinant prokaryotic plasmid was amplified and constructed in Rhipicephalus linnaei,and the E.coli system was used to express the chitinase protein RLCht1.After the inclusion body treatment,the protein was purified by nickel column and the RLCht1 protein domain was predicted. After purification using a nickel column,RLCht1 protein domain structure was predicted.The catalytic activity size and mode of action of purified chitinase RLCht1 were tested by a catalytic reaction with three different fluorescent substrates. 【Result】 The RLCht1 gene was expressed at different stages of satiety and in different tissues of satiated female adult ticks.The expression of RLCht1 gene in the satiated tick stage was extremely significantly higher than that in the egg stage (P<0.01) and other stages (P<0.05),and had a higher expression in the midgut of satiated female adult ticks.The recombinant plasmid RLCht1-pET-28a was constructed and the RLCht1 protein was successfully expressed.After inclusion body treatment,the purified RLCht1 protein was obtained by elution at a concentration of 150 mmol/L imidazole.The protein prediction results showed that RLCht1 belonged to the GH18 family chitinase.Fluorescent substrate enzyme activity detection showed that RLCht1 inclusion body dissolution solution,refolded unpurified prokaryotic protein,and refolded purified concentrated prokaryotic protein had chitin exonuclease activity of 0.078 and 0.350 U/mL,and β-N-acetylglucosaminidase activity of 0.034 U/mL,respectively. 【Conclusion】 RLCht1 might be related to the activity of ticks,affect life activities related to immunity,digestion and humoral regulation.The study successfully expressed chitinase protein for the first time in the Rhipicephalus linnaei,demonstrating that the purified RLCht1 protein had chitin catalytic function and provided a theoretical basis for the application of chitinase RLCht1 protein in the development of ticks in the future.
Effect of Trichomonas on the Structure of Macaca mulatta Gut Microbiota Based on 16S rDNA Sequencing Technology
LI Heling, CHEN Zhigang, ZONG Faliang, NING Qing, YAN Yulin, WANG Hong
2024, 51(11):  4974-4983.  doi:10.16431/j.cnki.1671-7236.2024.11.032
Abstract ( 70 )   PDF (11892KB) ( 61 )  
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【Objective】 This experiment was aimed to study the changes and diversity of gut microbiota in rhesus monkeys(Macaca mulatta) infected with Trichomonas using 16S rDNA sequencing technology,provide a reference for clinical research on using microbiota as an alternative antibiotics for treating trichomoniasis. 【Method】 Six samples of rhesus monkeys infected with Trichomonas and six samples of Trichomonas-negative fresh feces from rhesus monkeys were collected for predictive analysis of gut microbiota and functional genes through metagenomic sequencing. 【Result】 The diversity of gut microbiota in Trichomonas-infected rhesus monkeys decreased significantly compared to healthy rhesus monkeys,leading to significant changes in microbial community composition.Specifically,there was a significant decrease in Bacteroidota and Fibrobacterota (P<0.05),while Campylobacta and Desulfobacterota showed a significant increase (P<0.05).Additionally,Lactobacillales,Campylobacter and Campylobacteraceae were significantly enriched in the intestinal microbes of Trichomonas-infected rhesus monkeys,whereas Prevotella_9,Bacteroidales and Bacteroidota were significantly enriched in the intestinal microbes of Trichomonas-negative rhesus monkeys.Furthermore,Brachyspira_hampsonii and Prevotellaceae_bacterium_Marseille_P2831 were identified as biomarkers of Trichomonas-infected rhesus monkeys.The functional gene prediction of gut microbiota also revealed a significant decrease in the biosynthesis function of amino acids and antibiotics in the Trichomonas-infected rhesus monkeys (P<0.05). 【Conclusion】 Infection with Trichomonas could lead to a reduction in the diversity of gut microbiota and an alteration in the composition of the microbial community in rhesus monkeys.Additionally,it could also decrease the biosynthetic function of gut microbiota.
Whole Genome Sequencing and Genetic Evolution Analysis of Bovine Coronavirus in Inner Mongolia
QI Lemuge, WANG Xufen, HOU Lin, ZHANG Jialei, MA Yutian, ZHANG Zhidan, ZHOU Weiguang
2024, 51(11):  4984-4995.  doi:10.16431/j.cnki.1671-7236.2024.11.033
Abstract ( 67 )   PDF (7260KB) ( 55 )  
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【Objective】 This study was aimed to understand the whole genome characteristics and genetic evolution of Bovine coronavirus (BCoV) epidemic strains in Inner Mongolia,provide reference for effective prevention and control of BCoV infection. 【Method】 This study performed metavirome sequencing and stepwise RT-PCR amplification on the spleen of calves suspected of being infected with BCoV,obtaining the complete genome sequence of BCoV and conducting bioinformatics and genetic evolution analysis on it.Specific primers were designed to amplify the S gene using Oligo 7.0 software,and genetic evolution,amino acid sequence variation,epitope prediction and tertiary structure prediction were also performed. 【Result】 This study successfully obtained the 30 971 bp BCoV whole genome sequence of the spleen of calves,with NCBI registration number PP352170.1.The nucleotide sequence of the whole genome had the highest similarity with the whole genome sequence of BCoV ABGEB0-62 in Ireland,with a similarity of 99.2%,and was most closely related to the whole genome sequence of the strain.At the same time,it belonged to the same evolutionary branch as BCoV from France (BCoV_2014_13) and Norway (Nes_2012-01-03).The nucleotide length of the amplified S gene was 4 092 bp,which was closest to the S gene sequence of the BCoV ABGEB0-62 in Ireland and belonged to the same evolutionary branch.However,the nucleotide sequence had the highest similarity with S gene of BCoV NMG1 in Inner Mongolia,which was 99.4%.Comparing the amino acid sequences encoded by S gene with those of the Mebus strain,it was found that there were 15 amino acid mutation sites (12 in S1 subunit and 3 in S2 subunit),among which the mutated amino acid residues 154,260,499-520,718,1 192 and 1 344 were located in the antigenic epitope region,leading to changes in the tertiary structure of S protein. 【Conclusion】 This study obtained the complete genome sequence of Inner Mongolia BCoV using metavirus sequencing and genomic 5'end shift RT-PCR method.The variation of the S protein antigen epitope amino acid site and tertiary structural changes indicated a change in S protein antigenicity.The variation of amino acid sites in the polymorphic region of S1 subunit indicated a change in the pathogenicity of S protein.
Diagnosis of Bovine Akabane Disease in an Area of Jilin Province
YANG Baolin, MA Xiushan, LIN Jian, WANG Yuzhen, WANG Jinling, ZHAO Jicheng, LIU Yuehuan, YANG Zhiyuan
2024, 51(11):  4996-5003.  doi:10.16431/j.cnki.1671-7236.2024.11.034
Abstract ( 79 )   PDF (10463KB) ( 62 )  
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【Objective】 In 2022,the suspected case of bovine Akabane disease occurred in a region of Jilin province.To clarify the cause of infected cattle,a diagnosis was conducted on the affected cattle. 【Method】 Epidemiological investigation and clinical symptom analysis were conducted on the cattle farm,followed by comprehensive diagnosis through laboratory histopathological observation and serological testing.Tissue samples from diseased and dead fetal cows and placental tissue grinding solution from diseased cows were inoculated into Vero cells,SPF chicken embryos,and mouse brains for virus isolation. 【Result】 The clinical signs were abortion,stillbirth in cow,and congenital anomaly in fetuses.The epidemiological statistical analysis revealed that the empty pregnant rate of local cows was 29%,and the incidence rate of stillbirth and malformed birth of cows was 24%.The infected cows could be pregnant and deliver calves normally.Deformed calves showed congenital arthrogryposis,visual impairment,delayed neurological reactions due to abnormal brain development.The typical lesions of stillborn calves exhitited hydranencephaly syndrome,with abnormal head,malformation of brain and massive hydrocephalus,consistent with the clinical characteristics of Akabane disease.Pathological examination of the brain tissue of deformed calves showed eosinophilic necrosis of neurons,with microglia engulfing necrotic neurons and lymphocytes and monocytes surrounding blood vessels to form a "sheath",which was a typical non suppurative encephalitis and consistent with the pathological characteristics of bovine brain tissue affected by Akabane disease.No viral nucleic acid was detected in the tissue homogenate supernatant and cell culture supernatant of deceased fetal cattle and cow placenta,which might be related to the short duration of viremia caused by Akabane disease.The ELISA antibody test results for the serum Akabane disease in cows and calves with the disease were both positive. 【Conclusion】 The present results indicated that abortion,stillbirth and calf deformities in the area were associated with Akabane disease.The results of this study providesd experimental data and reference materials for the diagnosis and prevention of Akabane disease.
Research Progress on Porcine Epidemic Diarrhea Vaccine
ZHANG Yang, OU Yunwen, MA Chunxia, REN Shaoke, PAN Qin, DENG Shuming, ZHAI Jiajia, YANG Shanshan
2024, 51(11):  5004-5013.  doi:10.16431/j.cnki.1671-7236.2024.11.035
Abstract ( 116 )   PDF (1741KB) ( 143 )  
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In recent years,with the rapid development of pig industry in China,porcine epidemic diarrhea (PED) has become an important infectious disease in pigs.PED is an acute viral infectious disease caused by Porcine epidemic diarrhea virus (PEDV),characterized by diarrhea and dehydration.PED is mainly transmitted through the respiratory and digestive tracts,and lactating piglets are most susceptible to it,and the mortality rate can be as high as 100%,which has caused great economic losses to the pig industry.PEDV is a single-stranded positive-sense RNA virus,it has 4 major structural proteins,16 non-structural proteins,and 1 auxiliary protein ORF3.Since 2010,the spread of the mutated strain of the virus has been expanding and the epidemiological trends have become increasingly complex,the classical strain (CV777)-related vaccines have been unable to provide effective protection.Therefore,it is urgent to develop a safer and more effective vaccine.Inactivated vaccines are safe,but require multiple vaccinations and are not as protective.Attenuated vaccine have good immunogenicity,but there is a risk of virulence regression.Subunit vaccines do not carry other protein structures of the virus and are safe and inexpensive to use,but they are poorly immunogenic.Live virus vector vaccines induce specific immune responses but have an inadequate safety profile.Live bacterial vector vaccines are able to stimulate mucosal immunity in the body by oral administration and the vectors are adjuvant,but their immunization efficiency is not high.Transgenic plant vaccines are safe and stable,but have low expression and long development cycles.Nucleic acid vaccine has a short development cycle and high flexibility,but the safety is not high,and the foreign gene is easy to recombine with the host gene.With the deepening of related research,various types of vaccine research have achieved important research results.This article provided an overview of PED inactivated vaccines,attenuated vaccine,subunit vaccine,live virus vector vaccine,live bacterial vector vaccine,transgenic plant vaccine,and nucleic acid vaccine,in order to provide reference for the prevention and treatment of PED and the development of new vaccines.
Basic Veterinary Medicine
Study on the Prevention and Treatment of Fermented Compound Traditional Chinese Medicine of Pleuron ostreatus HXS-M1 on Escherichia coli-induced Diarrhea in Mice
BAI Changsheng, WANG Huan, TIAN Qiufeng, LIU Qiujin, YIN Junyi, WANG Yan, JIANG Botao, SHI Tongrui
2024, 51(11):  5014-5022.  doi:10.16431/j.cnki.1671-7236.2024.11.036
Abstract ( 62 )   PDF (1154KB) ( 76 )  
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【Objective】 The objective of this experiment was to study the clinical therapeutic effect of fermented compound traditional Chinese medicine of Pleuron ostreatus HXS-M1 on Escherichia coli-induced diarrhea in mice,and provide a new therapeutic method for clinical Escherichia coli diarrhea. 【Method】 The diarrhea model of mice was established by intraperitoneal injection of Escherichia coli suspension (0.2 mL/mouse).40 mice with diarrhea were randomly divided into model group,antibiotic group (AB),traditional Chinese medicine group (TCM) and fermented traditional Chinese medicine group (FTCM),with 10 mice in each group.Another 10 healthy mice were assigned to the blank group with intraperitoneal injection of normal saline (0.2 mL/mouse).Mice in AB group were given enrofloxacin solution (50 mg/mL),mice in TCM group were given traditional Chinese medicine extract (1.0 g/mL),mice in FTCM group were given fermented traditional Chinese medicine extract (1.0 g/mL),mice in model and blank groups were given normal saline intragastric administration once a day (0.2 mL/mouse).The drug was given by continuous intragastric administration for 6 days.The diarrhea,body weight and feed intake of mice were recorded daily.The heart,liver,kidney,spleen and lung tissues of mice were collected 12 h after the last administration,weighed and organ coefficients were calculated.The intestinal contents of mice were collected for colony counting. 【Result】 The mouse model of Escherichia coli diarrhea was successfully constructed.Compared with blank group,the diarrhea rate of model group was 100%,body weight,average daily gain,average daily feed intake and intestinal lactobacillus number were extremely significantly decreased (P<0.01),liver index,spleen index and intestinal Escherichia coli and Salmonella number were extremely significantly increased (P<0.01).Compared with model group,the diarrhea rate of AB and TCM groups was 30%,and the diarrhea rate of FTCM group was 0.In AB group,the final body weight,average daily gain and average daily feed intake were extremely significantly increased (P<0.01),while liver index,spleen index and the number of Escherichia coli and Salmonella in intestinal were extremely significantly decreased (P<0.01).The final body weight,average daily gain,average daily feed intake and intestinal lactobacillus quantity in TCM and FTCM groups were extremely significantly increased (P<0.01),while the ratio of feed to gain,liver index and spleen index,intestinal Escherichia coli and Salmonella quantity were extremely significantly decreased (P<0.01). 【Conclusion】 The fermented compound traditional Chinese medicine of Pleurotus ostreatus HXS-M1 could reduce the diarrhea rate,increase the average daily feed intake and average daily gain,reduce the liver index and spleen index,and the number of Escherichia coli and Salmonella in intestine,and increase the number of lactic acid bacteria,so as to have a synergistic protective effect on Escherichia coli diarrhea in mice.The results provided reference for the development of prevention and treatment of diarrhea caused by Escherichia coli infection.
Alleviation of Lycopene on Reproductive Toxicity Induced by Bisphenol A in Mice
CHEN Yuming, ZHANG Jingjing, ZHAO Yuanhui, FU Guoshuang, WANG Tong, MA Shuang
2024, 51(11):  5023-5032.  doi:10.16431/j.cnki.1671-7236.2024.11.037
Abstract ( 67 )   PDF (16579KB) ( 86 )  
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【Objective】 The aim of this study was to investigate the alleviation effect of lycopene on reproductive toxicity induced by bisphenol A (BPA) in mice. 【Method】 30 pregnant Kunming mice were randomly divided into 3 groups with 10 mice in each group.Mice in control group were gavaged with normal saline from days 1 to 7 of gestation and received conventional feeds.Mice in BPA treatment group were gavaged with normal saline on days 1 to 7 of gestation,and 500 mg/kg BPA on days 8 to 14 of gestation.Mice in lycopene treatment group were gavaged with 20 mg/kg lycopene on days 1 to 7 of gestation,and 500 mg/kg BPA on days 8 to 14 of gestation,with natural birth allowed.The survival number of F1 generation mice was observed and recorded.F1 generation mice were normally fed to 8 weeks and dissected.Testis and ovary were collected,and the structural changes of testicular and ovarian tissues were observed by HE staining.Testosterone (T),follicle-stimulating hormone (FSH),estradiol (E2) and luteinizing hormone (LH) levels of serum in F1 generation mice were detected by ELISA.The expression of B lymphoblastoma-2 (Bcl-2) and B lymphoblastoma-2-associated X protein (Bax) in testis and ovaries of F1 generation mice were detected by immunohistochemistry and immunofluorescence techniques. 【Result】 After BPA treatment,the survival rate of F1 generation mice was 62.50%,which was significantly lower than that of control group (83.65%) and lycopene treatment group (81.25%).Compared with control group,the levels of reproductive hormones T,FSH and LH in F1 generation mice in BPA treatment group were significantly decreased (P<0.05),and E2 level was significantly increased (P< 0.05).The arrangement of cells in seminiferous tubule of mouse testis was loose and disordered,the number of interstitial cells was reduced,the lumen was enlarged and the tube wall was thin.The granulosa cells in ovarian were overapoptosis and the cell arrangement was loose.Compared with BPA treatment group,T,FSH and LH levels in F1 generation mice in lycopene treatment group were significantly increased (P<0.05),while E2 level was significantly decreased (P< 0.05).The structure of mouse testis was complete,and spermatogenic cells at all levels of spermatogenic tubule were arranged closely and neatly.Follicles at different developmental stages were observed in ovary,the granulosa cells were arranged neatly,and a few granulosa cells were regulated and died.The results of immunohistochemistry and immunofluorescence showed that Bax and Bcl-2 proteins were expressed in testicular stromal cells and ovarian granulosa cells.Compared with control group,the expression of Bax protein in ovary of F1 generation mice in BPA treatment group was significantly increased (P< 0.05),and the expression of Bcl-2 protein in testes and ovaries was significantly decreased (P<0.05).Compared with BPA treatment group,the expression of Bax protein in ovaries of F1 generation mice in lycopene treatment group was significantly decreased (P<0.05),and the expression of Bcl-2 protein in testes and ovaries was significantly increased (P<0.05). 【Conclusion】 Lycopene could alleviate the disturbance of reproductive hormone level caused by BPA,improve the structure of testis and ovary,balance apoptosis and anti-apoptosis levels,and thus antagonize the reproductive damage of mice caused by BPA.The results provided experimental basis for the clinical application of lycopene.
Isolation,Identification and Detection of Drug Resistance and Virulence Genes of Salmonella Pullorum from a Breeding Chicken Farm of Guangdong
ZHOU Di, WEI Ke, DUAN Qianxi, CAO Weisheng
2024, 51(11):  5033-5042.  doi:10.16431/j.cnki.1671-7236.2024.11.038
Abstract ( 82 )   PDF (7266KB) ( 85 )  
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【Objective】 The purpose of this experiment was to understand the infection of Salmonella Pullorum in chickens of a breeding farm,and analyze the drug resistance and virulence genes,in order to provide evidence for the prevention and control of pullorosis. 【Method】 The liver tissue of sick chickens suspected to be suffering from pullorosis was collected and Salmonella was isolated and cultured.The isolates were identified by PCR amplification using specific primers.Molecular typing was obtained by multilocus sequence typing (MLST) technique.The minimum inhibitory concentration of the isolates was determined by agar dilution method.Next-generation sequencing (NGS) technology was utilized to analyze the drug resistance and virulence genes. 【Result】 A total of 5 isolates of Salmonella Pullorum were isolated and named SPNH05-1 to SPNH05-5,respectively.MLST typing results showed that all the 5 isolates were ST92.The results of drug sensitivity test showed that the isolates were multi-drug resistant,and there were 2 drug resistance profiles,ampicillin + nalidixic acid + sulfamisoxazole and ampicillin + nalidixic acid + sulfamisoxazole + polycolistin B.NGS was performed on isolates SPNH05-1 and SPNH05-2,which showed that there were 28 drug resistance genes,including β-lactam,quinolones,aminoglycosides,sulfonamides and polypeptides,and 151 virulence genes,which were mainly related to Salmonella adhesion,type Ⅲ secretion system,stress and toxin. 【Conclusion】 The results of this study showed that multi-drug resistant Salmonella Pullorum infection existed in this breeding farm,and the isolates carried multiple drug resistance and virulence genes.The results provided reference for the purification of pullorosis.
Inhibitory Effect of Quercetin on Biofilm Formation of Salmonella
WANG Qiaoyu, ZHEN Mingzhe, ZHANG Nianjie, YAN Dongxue, WANG Xin
2024, 51(11):  5043-5050.  doi:10.16431/j.cnki.1671-7236.2024.11.039
Abstract ( 74 )   PDF (2501KB) ( 33 )  
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【Objective】 The objective of this experiment was to study the inhibitory effect of quercetin on the biofilm formation of Salmonella and its mechanism. 【Method】 The growth curve of Salmonella biofilms was plotted by crystal violet staining,and the minimum biofilm inhibitory concentration (MBIC) of quercetin on Salmonella was determined.The bacteria were treated with quercetin,and the metabolic activity and morphology of the bacteria in Salmonella biofilms were observed by XTT method and scanning electron microscope.The contents of exopoly saccharides,protein and DNA of Salmonella biofilm were detected by phenol-sulfuric acid method,BCA method and ultraviolet spectrophotometer,respectively.The hydrophobicity and motility of Salmonella were determined by bacteria adhesion to hydrocarbons and soft agar method. 【Result】 The results of growth curve showed that the adhesion,aggregation,maturation and dispersion periods of Salmonella biofilms were 0-48,48-72,72-120 and 120-168 h,respectively.The MBIC of quercetin against Salmonella biofilms was 256 μg/mL.The metabolic activity of bacteria in Salmonella biofilm treated with quercetin was extremely significantly decreased at different times (P<0.01).Scanning electron microscopy observation showed that after quercetin treatment,the structure of Salmonella biofilms was destroyed and the membrane-like substance disappeared.Compared with control group,the exopoly saccharides,protein and DNA contents of Salmonella biofilms in quercetin group were extremely significantly decreased (P<0.01).The hydrophobicity index,swimming motility diameter and swarming motility diameter of Salmonella decreased extremely significantly (P<0.01). 【Conclusion】 Quercetin inhibited biofilm formation by affecting the metabolic activity of bacteria in Salmonella,reducing the production of extracellular polymeric substances,and interfering with surface hydrophobicity and motility of Salmonella.
Study on Extraction Process and Anti-inflammatory Effect of Total Flavonoids from Viola yedoensis
CHEN Zhiliang, LIU Cong, LIAO Ziwen, WU Yujiao, XING Jiankang, LONG Xiaoqing, ZHAO Wenxue, WANG Zhen, ZHAO Xin, SHAO Yongbin, LUO Yan
2024, 51(11):  5051-5063.  doi:10.16431/j.cnki.1671-7236.2024.11.040
Abstract ( 67 )   PDF (11348KB) ( 6 )  
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【Objective】 The aim of this study was to optimize the extraction process of total flavonoids from Viola yedoensis by response surface methodology,identify the composition of compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS),and study its anti-inflammatory activity. 【Method】 The absorbance at 510 nm was determined by rutin standard,and the standard curve of flavonoids was established with absorbance (Y) as ordinate and sample concentration (X) as abscissa.The powder of Viola yedoensi was sieved through 100 mesh,soaked in ethanol for 12 h,and ultrasonically extracted by ultrasonic instrument.The ratio of Viola yedoensis powder to ethanol (solid-liquid ratio),ultrasonic extraction time (ultrasonic time),and ethanol concentration (ethanol concentration) were used as investigation factors.The extraction process of total flavonoids from Viola yedoensis was optimized by Box-Behnken response surface methodology with comprehensive score of total flavonoids extraction.The total flavonoids extracted from Viola yedoensis were purified by D101 macroporous resin,and the composition of the compounds was identified by LC-MS/MS method.Sixty 6-week-old healthy Kunming mice were randomly divided into blank,model,low dose (150 mg/kg),medium dose (300 mg/kg),high dose (600 mg/kg) and levofloxacin (5 mg/kg) groups.Except for blank group,LPS (1 g/L,50 μL/ mouse) was instilled into the nasal cavity to establish an acute lung injury (ALI) model.After successful modeling,the drug intervention was performed for 1 week (1 time/d,continuous 7 days).Blood was collected 1 h after the last administration,followed by anesthesia,execution,and lung collection.The degree of lung injury was observed by HE staining.The levels of interleukin-1 β (IL-1β),IL-6,IL-10 and tumor necrosis factor-α (TNF-α) in serum of mice were detected by ELISA. 【Result】 When the solid-liquid ratio was 1∶20,the extraction time was 22 min,and the ethanol concentration was 73%,the extraction amount of total flavonoids was the highest,which was 25.134 mg/g.It was identified that the extract contained 37 flavonoids (quercetin,luteolin,apigenin,etc.),a small amount of isoflavones,sugars,coumarins,lignans,monoterpenes,and sesquiterpenes.Pathological sections showed that compared with model group,the bleeding points of lung tissue in each administration group were gradually reduced,the area was reduced,and there were different degrees of recovery.The lung tissue of the high dose and the levofloxacin groups showed slight damage in color and morphology.Compared with model group,the damage degree of lung tissue in the low and medium dose groups was reduced,but there was still a certain degree of edema and some bleeding points.The results of ELISA showed that after administration,the levels of TNF-α,IL-β,IL-6 and IL-10 in the low,medium and high dose groups were lower than those in the model group to varying degrees.Except that the levels of TNF-α and IL-β in the serum of mice in the low dose group were not significantly different from those in the model group,the rest of the administration groups were significantly reduced (P<0.05).The levels of inflammatory factors in the serum of mice in the high dose group were slightly different from those in the levofloxacin group,which were slightly higher than those in the blank group. 【Conclusion】 The response surface method was used to effectively improve the extraction amount of total flavonoids from Viola yedoensis,and the active components of total flavonoids from Viola yedoensis were identified by LC-MS/MS technology.At the same time,through the model of acute lung injury in mice,it was proved that the total flavonoids of Viola yedoensis could improve the acute lung injury in mice and reduce the levels of IL-1β,IL-6,IL-10 and TNF-α in the blood.The total flavonoids of Viola yedoensis had good anti-inflammatory effect.
Screening and Application Research of Traditional Chinese Medicine Formulas for Anti-heat Stress in Dairy Cows
WANG Xiaofang, WANG Yawen, LI Jiefeng, FU Le, ZHANG Ning, QIN Jianhua
2024, 51(11):  5064-5073.  doi:10.16431/j.cnki.1671-7236.2024.11.041
Abstract ( 65 )   PDF (4928KB) ( 63 )  
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【Objective】 This study was aimed to screening traditional Chinese medicine formulas and their applicable dosages for anti-heat stress in dairy cows,and providing reference for traditional Chinese medicine in anti-heat stress in dairy cows. 【Method】 With the principle of “supplementing qi and nourishing blood,nourishing yin and generating fluids” and “clearing heat and cooling blood”,中 国 畜 牧 兽 医51卷 11期王晓芳等:抗奶牛热应激中药组方筛选及应用研究 three traditional Chinese medicine formulas were formulated,which were named TCM1,TCM2 and TCM3,respectively.40 dairy cows with similar milk production and milk composition were selected,and randomly divided into 4 groups TM1,TM2,TM3 and control (CON) groups (n=10).Dairy cows in CON group were fed with conventional diet,while in the other three groups were fed with diets supplemented with TCM1,TCM2 and TCM3,respectively.On the 1st,7th,14th,21st,28th and 30th day of the experiment,the milk production of each group of dairy cows was recorded,and the milk fat percentage,milk protein percentage,lactose percentage,and somatic cell number in milk were measured to screen for the best traditional Chinese medicine formula.Based on the selected formula,40 healthy dairy cows with similar milk production and milk indicators were selected and randomly divided into high-dose group (30 g/(head·d)),medium dose group (20 g/(head·d)), low-dose group (15 g/(head·d)) and control group (n=10).Dairy cows in control group were fed with conventional diet,while each traditional Chinese medicine group was fed with a diet with different doses of traditional Chinese medicine,respectively.The milk production of each cow was recorded on the 1st,15th,31st day of the experiment.The milk samples were collected from each group,and the milk fat percentage,milk protein percentage,lactose percentage,and somatic cell number were measured for screening the optimal dosage of medication. 【Result】 Results of formula screening test:①The results of milk fat percentage measurement showed that,On the 21st day,the milk fat percentage in TCM1 group was significantly higher than of CON and TCM2 groups (P<0.05),and there was no significant difference compared to TCM3 group (P>0.05).On the 28th day,the milk fat percentage in TCM1,TCM2 and TCM3 groups was significantly higher than that of CON group (P<0.05),and there was no significant difference among them (P>0.05).On the 30th day,the milk fat percentage of TCM1 and TCM3 groups was significantly higher than that of TCM2 and CON groups.The milk protein percentage in TCM1 was significantly higher than that of CON group (P<0.05) on the 14th and 21th day.And there were no significant differences between the experimental groups at other time points.There was no significant difference in lactose percentage among the groups during the experiment (P>0.05).②The results of milk production measurement showed that,on the 14th,21st,28th and 30th day,milk production in all traditional Chinese medicine groups was significantly higher than that of CON group (P<0.05).On the 28th day,milk production in TCM3 group was significantly higher than that in TCM2 group (P<0.05).The test results of 4% FCM showed that,at the 14th day and subsequent time points,4% FCM of all traditional Chinese medicine groups showed a significantly higher level than that of CON group (P<0.05).On the 14th day,TCM3 group was significantly higher than that of TCM1 group (P<0.05).On the 28th and 30th day,4% FCM of TCM3 group was significantly higher than that of TCM2 group (P<0.05).③The results of somatic cell count (SCC) measurement showed that,on the 14th day,the SCC of TCM3 group was significantly lower than that of CON group (P<0.05).On the 21st,28th and 30th day,the SCC of TCM3 group was significantly lower than that of TCM1 group.On the 21st and 30th day,the SCC of TCM2 group was significantly lower than that of TCM1 group (P<0.05).The dose screening results showed that:①There were no significant difference in milk fat percentage,milk protein percentage,lactose percentage among all the groups (P>0.05).② The results of milk production level measurement showed that,on the 15th day,the actual milk production of medium and high dose groups were significantly higher than that of low dose group and control group (P<0.05),and there was no significant difference in the 4% FCM among all the groups (P>0.05).On the 31st day,the actual milk production and 4% FCM of medium and high dose groups were significantly higher than that of control group (P<0.05).③ The results of SCC measurement showed that,on the 15th day,each SCC of medium and high dose groups was significantly lower than that of low dose group and control group (P<0.05).On the 31st day,each SCC of all traditional Chinese medicine groups was significantly lower than that of control group (P<0.05),and SCC of medium and high dose groups were significantly lower than that of low dose group (P<0.05). 【Conclusion】 The traditional Chinese medicine formula selected with the best anti-heat stress effect on dairy cows was TCM3.The recommended amount of additives was 20 g/(head·d).
Construction of Phage Display Library and Perliminary Screening of Trimethoprim Nanobody
LIU Xiangyu, YIN Yongkang, ZHANG Hong, SONG Qian, WANG Ling, ZHANG Qidi, LIANG Xiao
2024, 51(11):  5074-5085.  doi:10.16431/j.cnki.1671-7236.2024.11.042
Abstract ( 68 )   PDF (12982KB) ( 78 )  
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【Objective】 This study was aimed to construct an trimethoprim (TMP) nanobody phage display library and prepare a high-affinity TMP nanobody,provide novel antibody materials for the establishment of TMP immunoassay methods. 【Method】 Two immunogens were prepared by conjugating Hapten B to bovine albumin (BSA) and keyhole hemocyanin (KLH) by the active ester method.And Hapten A and Hapten B were conjugated to ovalbumin (OVA) to prepare two coating antigens using the same method.The conjugating effect was identified by indirect ELISA with the monoclonal antibody 5C4 against sulfonamide synergists.The immunogens were mixed with Freund’s adjuvants for primary and booster immunizations of alpacas,antiserum titers and TMP affinity were monitored after each immunization.Peripheral blood samples with the highest titer and maximal affinity for TMP was selected as the source of nanobody genes to construct the phage display library.Alpaca peripheral blood lymphocytes were isolated,variable domain of heavy-chain antibody (VHH) gene was amplified by PCR,and then ligated into phagemids.The recombinant phagemids were transformed into E.coli XL1-Blue competent cells to construct the initial phage display library,and then rescued with the helper phage.High-affinity TMP phages were obtained by screening using the decreasing concentration gradient coating of homologous coating antigen,acid elution and the decreasing concentration gradient elution of TMP.The sequence of the high-affinity TMP nanobody was obtained by sequencing.The nanobody fusion protein was expressed in prokaryotes and identified by SDS-PAGE and Western blotting. 【Result】 The results of indirect ELISA showed that the D450 nm values of two immunogens and two coating antigens were higher than 1.25 when they were diluted 18 000 times.Following five immunizations with Hapten B-BSA,antiserum titers reached 1∶88 000 with an half maximal inhibitory concentration (IC50) of 12.21 μg/L for TMP.And antiserum titers reached 1∶11 000 with an IC50 of 30.56 μg/L for TMP following four immunizations with Hapten B-KLH.The peripheral blood of alpaca immuned with Hapten B-BSA for five times was selected as the source of VHH and the phage display library was constructed,which exhibited a capacity of 6.08×107 CFU/mL,a 96% insertion rate of target fragment and considerable diversity.After 4 rounds of screening,three high-affinity TMP phages were obtained and 1 sequence of high-affinity TMP nanobody was obtained by sequencing.The size of fusion protein was approximately 17 ku,with more than 80% protein content determined by SDS-PAGE and Western blotting analysis. 【Conclusion】 The TMP nanobody phage display library was successfully constructed,and a high-affinity TMP nanobody was selected from this library.This study provided theoretical basis and technical support for the preparation of nanobodies against sulfonamide synergists,and offered new insights for the preparation of novel recognition materials for veterinary drugs and other small molecules.
Identification of Exosomes from Supernatant of IPEC-J2 Cells Before and After Infected with Salmonella,and Expression and Transcriptome Analysis of m6A Related Proteins
ZHANG Xinguo
2024, 51(11):  5086-5096.  doi:10.16431/j.cnki.1671-7236.2024.11.043
Abstract ( 58 )   PDF (13048KB) ( 60 )  
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【Objective】 The aim of this study was to investigate the effect and molecular mechanism of exosomes in regulating the porcine small intestinal epithelial cells (IPEC-J2 cells) during Salmonella infection. 【Method】 The distribution of Salmonella antibody in IPEC-J2 cells after Salmonella infection was observed by indirect immunofluorescence assay.The exosomes in the cell supernatant of Salmonella-infected IPEC-J2 cells were observed by supercentrifugation.The morphology of the exosomes was observed by transmission electron microscopy (TEM),the particle size of the exosomes was measured by nanoparticle tracking analysis (NTA),and the molecular markers CD9,CD63,CD81 and TSG101 were detected by Western blotting.Additionally,the expression of N6-methyladenosine (m6A) related proteins was detected by Real-time quantitative PCR and Western blotting.Besides,the differentially expressed genes of exosomes in supernatant were screened by transcriptome sequencing. 【Result】 Indirect immunofluorescence assay results showed that Salmonella adhered to IPEC-J2 cells,which indicated that Salmonella was able to infect IPEC-J2 cells. TEM and NTA analysis showed that the exosomes had bilayer membrane vesicles with a diameter of 30-150 nm,and four exosomal protein markers CD9,CD63,CD81 and TSG101 were expressed in the purified exosomes by Western blotting.The expression of m6A modification-related genes and proteins ALKBH5,YTHDC2 and YTHDF1 was significantly or extremely significantly up-regulated in exosomes after Salmonella infection (P<0.05 or P<0.01).Transcriptome sequencing results showed that there were a total of 1 524 differentially expressed genes (DEGs) in exosomes of IPEC-J2 cells after Salmonella infection,including 842 down-regulated genes and 682 up-regulated genes.Go function enrichment analysis showed that Salmonella infection mainly affected the immune system process,cell junction and enzyme regulator activity and other biological processes.KEGG pathway enrichment analysis revealed that differentially expressed genes were closely related to intestinal immune network for IgA production,T cell receptor signaling pathway,Toll-like receptor signaling pathway. 【Conclusion】 In this study, exosomes were successfully isolated and identified in the supernatant of Salmonella infected IPEC-J2 cells, and it indicated that m6A modification could regulate Salmonella infection through the activation of immune signaling pathways, and this study could provide a reference for the subsequent study of exosome-induced release of exosomes from IPEC-J2.
Effect of Plasma-derived Exosomes on LPS-induced MAC-T Cell Inflammation in Dairy Cows with Mastitis
LI Yang, WANG Zhaoyuan, HE Xingli, LIU Peng, WANG Zi, LI Xiaolin, SONG Yangyang, SHEN Binglei
2024, 51(11):  5097-5107.  doi:10.16431/j.cnki.1671-7236.2024.11.044
Abstract ( 61 )   PDF (9504KB) ( 58 )  
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【Objective】 Exosomes are key factors in cellular communication and establishment of tissue microenvironment,and they have a regulatory role in inflammatory processes.This study was aimed to investigate the role of plasma-derived exosomes from dairy cows in the regulation of lipopolysaccharide (LPS)-induced inflammation in mammary epithelial cells (MAC-T) of dairy cows. 【Method】 In this study,venous blood was collected from healthy and mastitis-affected dairy cows,and plasma was obtained after centrifugation,and plasma-derived exosomes were isolated by ultracentrifugation.The expression of pro-inflammatory factors after incubation of plasma-derived exosomes from dairy cows was detected by Real-time quantitative PCR.Western blotting was used to verify the expression of NF-κB pathway proteins TLR4,MyD88 and TNF-α after incubation of plasma-derived exosomes from dairy cows.The effects of plasma-derived exosomes from dairy cows on the proliferation as well as apoptosis of MAC-T cells were verified by CCK8 and Hoechst staining. 【Result】 Real-time quantitative PCR results showed that plasma-derived exosomes from dairy cows with mastitis significantly down-regulated the expression of pro-inflammatory factors IL-6,IL-23,IL-1α,NFRK,MyD88,TNF-α and IL-1β genes mRNA compared with LPS group (P<0.05). Plasma-derived exosomes from healthy cows significantly inhibited mRNA expression of the IL-1β gene (P<0.05).Western blotting results showed that the plasma-derived exosomes from dairy cows with mastitis significantly decreased the expression of the NF-κB pathway proteins TLR4,MyD88 and TNF-α (P<0.05). NF-κB pathway protein expression in plasma-derived exosomes from healthy cows was not significantly different from that of the LPS group (P>0.05). CCK8 and Hoechst staining results showed that plasma-derived exosomes from dairy cows with mastitis could promote cell proliferation and alleviate apoptosis compared with LPS group. Plasma-derived exosomes from healthy cows restored some cell viability. 【Conclusion】 The results indicated that plasma-derived exosomes from dairy cows with mastitis could alleviate LPS-induced inflammation of MAC-T cells through NF-κB pathway.This study would provide a theoretical basis for the anti-inflammatory role played by plasma-derived exosomes in dairy cow mastitis,and at the same time laid the foundation for the clinical application of plasma-derived exosomes.
Selection of a Monensin-resistant Strain of Eimeria tenella and Differences Analysis Between Resistant and Sensitive Strains by SNP Density Distribution
GU Xiaolong, XUE Yi, FANG Sufang, BAN Chengyi, SHI Yubo, YANG Jianhang, DU Fangchen, QI Zhenzhen, CUI Ping
2024, 51(11):  5108-5116.  doi:10.16431/j.cnki.1671-7236.2024.11.045
Abstract ( 58 )   PDF (8708KB) ( 62 )  
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【Objective】 The purpose of study was to determine the genetic differences between Eimeria tenella monensin-resistant and monensin-sensitive strains,and elucidate the molecular mechanism of monensin resistance. 【Method】 Resistance to monensin was induced in Eimeria tenella by simulating field infection.400 broilers were equally divided into two groups,group Ⅰ (inoculated and untreated group) and group Ⅱ (monensin recommended dose group). Monensin was added to the basal diet at 100 mg/kg.10 birds in group Ⅰ were inoculated with 5 000 oocysts on the first day.Litter containing feces with shed oocysts was randomly sampled from group Ⅰ and dispersed to the litter of group Ⅱ from the 8th day to the 25th day.33 days post inoculation,the caeca of 10 chickens in group Ⅱ were removed to collect the resistant oocysts.In the second experiment,100 broilers formulated the group Ⅲ (monensin higher dose group).10 chickens of group Ⅲ were inoculated with 5 000 resistant oocysts.The chickens of group Ⅲ were fed with gradient monensin (from 133 to 400 mg/kg).27 days post inoculation,the caeca of 10 chickens were removed to collect the resistant oocysts.The resistance was evaluated by ACI,POAA,RLS and POP.The genomes of resistant and sensitive strains were sequenced,then single nucleotide polymorphism (SNP) density was compared with the reference genome. 【Result】 In the first experiment,the resistant oocysts against 100 mg/kg monensin (1×monensin induced strain) were generated 33 days post inoculation.In the second experiment,the resistant oocysts against 400 mg/kg monensin 4×monensin induced strain were harvested 27 days post inoculation.Evaluation result of resistance showed ACI,POAA and ROP of 4×monensin induced strain were 121,25.4%,33% and 64%,respectively.Compared with the reference genome,there were 12 191 SNPs in the genome of the sensitive strain and 74 931 SNPs in the genome of the drug-resistant strain.The SNPs of the drug-resistant strain were enriched and distributed in the region of 3.3-4.2 Mb on chromosome 11,and the SNPs of three genes ETH2_1113700,ETH2_1113500 and ETH2_1113600 in the region were located in the coding region. 【Conclusion】 This study obtained a fully resistant strain of Eimeria tenella to monensin.On chromosome 11,there were significant differences in SNP distribution between resistant and sensitive strains,and three candidate genes for drug resistance were identified.