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23 October 2024, Volume 55 Issue 10
Review
Effects and Regulation Methods of Progesterone on Reproduction in Dairy Cows During Estrus and Early Pregnancy
Guohan SUN, Hao ZHENG, Zhuo YANG, Wenjuan SHEN, Jinzhong TAO
2024, 55(10):  4241-4249.  doi:10.11843/j.issn.0366-6964.2024.10.001
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Insufficient progesterone levels during estrus and early pregnancy in dairy cows is one of significant factors contributing to low reproductive efficiency. Elevated progesterone levels have been shown to enhance oocyte quality, regulate hormone secretion, control the uterine environment, and promote embryonic development, thereby improving reproductive efficiency in dairy cows. Consequently, the relationship between progesterone levels during estrus and early pregnancy and reproductive efficiency in dairy cows has attracted significant attention. This paper summarizes the effects of progesterone on various stages of estrus and early pregnancy in dairy cows and explores methods to enhance progesterone levels during these critical periods. The objective is to provide production guidance for dairy farming industry and to improve the quality and efficiency of dairy cow breeding.

Progress in Research and Application of Fermented Chinese Herbal Feed Additives in Livestock and Poultry Production
Zeqing LIU, Yiwen GENG, Longlong ZHU, Chao MA, Guoshun CHEN, Jing WANG
2024, 55(10):  4250-4262.  doi:10.11843/j.issn.0366-6964.2024.10.002
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With the comprehensive prohibition of antibiotic feed additives in livestock production, Chinese herbal feed additives have gradually become the potential alternative antibiotics. After microbial fermentation, Chinese herbal medicine not only can increase the content of active substances and enhance the efficacy, but also produce new active ingredients and reduce the toxic side effects of Chinese herbal medicine. Supplementation of fermented Chinese herbal medicine can improve the production performance, alleviate the health statues, and enhance the products quality of livestock animals. This review summarized the characteristics of the fermented Chinese herbal medicines, discussed the influence of fermented Chinese herbal feed additive on animal production, and explored the problems existing in its application. Hoping to provide reference for the research and application of fermented Chinese herbal feed additives in livestock and poultry production.

Research Progress of Gut Microbiota Regulating Fat Deposition and Metabolic Related Diseases
Lanmeng XU, Yuzhi HUANG, Yuzhu HAN, Changying LI, Jie ZHANG
2024, 55(10):  4263-4277.  doi:10.11843/j.issn.0366-6964.2024.10.003
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Adipose tissue is an important component of the body, which not only stores energy, protects tissues, and regulates body temperature, but also participates in metabolic regulation by secreting cytokines, playing an important role in the pathogenesis of obesity and related complications. Numerous studies have confirmed that, there exists a close relationship between gut microbiota and host fat metabolism and related diseases. This article reviews the main factors that gut microbiota affects fat deposition, including adipocytes, fatty acid composition, and fat related blood indicators. Exploring how gut microbiota participates in regulating fat absorption, generation, and decomposition processes through a series of pathways, and elaborating in detail the association between gut microbiota and diseases caused by lipid metabolism disorders. This article aims to improve and deepen the understanding of the regulation of fat deposition and related metabolic diseases by gut microbiota, providing a theoretical basis and reference for further research and clinical practice.

Research Progress on in vitro Culture and Induced Differentiation of Non-human Primate Pluripotent Stem Cells
Kecheng SHEN, Jiaqiao ZHU, Zongping LIU
2024, 55(10):  4278-4289.  doi:10.11843/j.issn.0366-6964.2024.10.004
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Stem cells have great value in disease occurrence, cell therapy, drug screening, and clinical applications. Since Shinya Yamanaka, a Japanese scientist, discovered induced pluripotent stem cells (iPSCs), the relevant research has become a research hotspot and up to the present. The study of mouse stem cells has opened up a path for the related research in other animals, but there are significant limitations in drug screening and disease treatment. Non-human primates have higher genetic homology with humans and are highly similar to humans in physiology, pathology, reproduction, pharmacology, nerves, and other aspects. They are ideal animal models for studying human diseases and developing drugs for human. Research on non-human primates is also a necessary step for a new drugs before entering clinical trials. The study of non-human primate stem cells is becoming important in the mechanism of diseases and drug development, while avoiding ethical issues. This article reviews the related research on non-human primate pluripotent stem cells including optimization of in vitro culture systems and methods of inducing differentiation, aiming to provide reference for further in-depth research on non-human primate stem cells.

Progress on Single-cell Transcriptomics Technology and Its Applications in Research on Parasites
Tingting ZHOU, Li LI, Yantao WU, Wenying LU, Baoquan FU, Hong YIN, Wanzhong JIA, Hongbin YAN
2024, 55(10):  4290-4301.  doi:10.11843/j.issn.0366-6964.2024.10.005
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The single cell transcriptome represents the total expression of all mRNA in a single cell at a certain time, and its expression level reflects the overall characteristics of the cell. The entire RNA of a single cell is reversely transcribed, amplified and sequenced by single cell RNA sequencing (scRNA-seq). The extensive information contained in the sequencing results has the potential to reshape the understanding of developmental biology, gene regulation and cellular heterogeneity in health and disease. With the continuous development of science and technology, sequencing technologies are also constantly improving. The omni-directional, high-throughput and high-resolution characteristics of scRNA-seq can build a more detailed, comprehensive and accurate single cell map. This paper summarizes the commonly used scRNA-seq methods, the main processes of data analysis, and the application of single cell transcriptome technology in the field of parasitology and parasitic diseases, in order to provide reference materials for related research in this field.

Animal Genetics and Breeding
Changes in Gut Microbiota after ZBED6 Knockout were Analyzed Based on 16S rRNA Sequencing
Cheng XU, Wenjie TIAN, Yuehui MA, Shengnan WANG, Lin JIANG, Dandan WANG
2024, 55(10):  4302-4310.  doi:10.11843/j.issn.0366-6964.2024.10.006
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The aim of this study was to investigate the association between changes in gut microbiota and muscle phenotype in ZBED6-knockout Bama pigs. In this study, 5-month-old wild-type and ZBED6-knockout Guangxi Bama mini-pigs were divided into 3 groups based on sex and genotypes: wild-type boars compared with wild-type sows (male WT: female WT), wild-type sows compared with knock-out sows (female WT: female Ko), and wild-type boars compared with knock-out boars (male WT: male Ko). The microbial composition of each intestinal segment was analyzed by high-throughput sequencing of 16S rRNA gene. The results showed that: 1) The community diversity of large intestine in Bama pigs was significantly higher than that of small intestine; 2) Compared with female wild-type pigs, male wild-type pigs have higher lean meat percentage. Turicibacter in ileum, Streptococcus in cecum and Peptostreptococcaceae in rectum were found to be concentrated in male wild-type pigs, while Lactobacillus in ileum was found to be concentrated in female wild-type pigs; 3) After knockout of ZBED6, muscle growth increased in pigs. The content of Lactobacillus in ileum of sows was significantly decreased and SMB53 in cecum was significantly increased. After knockout of ZBDE6, Prevotella and Ruminococcus increased significantly in boar rectum, and SMB53 increased significantly in cecum. The results suggest that the differences in abundance of Turicibacter, Streptococcus, Peptostreptococcaceae and Lactobacillus in the gut may affect energy metabolism and promote muscle growth in male wild-type pigs; After ZBED6 knockout, an increase abundance in SMB53 in the cecum of pigs may be a factor contributing to muscle gain.

Detection and Comparative Analysis of Genomic Genetic Variations in Trace Cells Using Different Methods
Qingzhen SHI, Hongyang XU, Yan ZHANG, Yi ZHANG, Yachun WANG, Jianyong HAN, Li JIANG
2024, 55(10):  4311-4324.  doi:10.11843/j.issn.0366-6964.2024.10.007
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The aim of this study was to detect and compare the genetic variation of the whole genome of trace cells by different methods. In this study, 3, 5, 7 and 10 cell numbers from pig ear fibroblasts were used as experimental materials, and trace cell genomes were extracted by different methods for whole genome sequencing (WGS) to compare the performance of genetic variation detection among different cell numbers and extraction methods. Subsequently, comparative analysis of whole genome sequencing and SNP chip technology was conducted using the genomes obtained from 5 or 7 bovine trophectoderm cells, with 3 replicates per group. The results showed that the multiple displacement amplification (MDA) method based on whole genomic amplification (WGA) technology had better DNA product concentration, sequencing data quality and SNP detection performance than other methods for different cell numbers. The DNA concentrations of 7 and 10 cell numbers amplified by REPLI-g® Single Cell Kit were significantly higher than those of 3 and 5 cell numbers, but there was no significant difference in the performance of quality assessment, and the number of SNPs detected was similar in different cell numbers. The call rates of Illumina Bovine GGP chip with 5 and 7 cell numbers were 74.09% and 81.52%, respectively. The results showed that more genetic variation information could be obtained by whole genome sequencing compared to SNP chips, but the proportion of SNPs and the number of loci with the same genotype detected by both methods was low. In conclusion, this study systematically compared the performance of genome extraction methods and whole genome genetic variation detection techniques for different cell numbers. The results showed that NGS amplification of 7 cells using the REPLI-g® Single Cell Kit could obtain relatively accurate and stable results. This study established a reliable genome extraction and genetic variation detection scheme for trace cell samples of livestock embryos, which is of important significance and value for accurate embryo genomic selection and embryo quality assessment in the future.

Accuracy Analysis of Genotype Imputation from 10K to 50K SNP Loci in Layers
Junfeng WU, Yiyuan YAN, Ning YANG, Congjiao SUN, Guangqi LI, Bin WANG, Guiqin WU, Ling LIAN
2024, 55(10):  4325-4333.  doi:10.11843/j.issn.0366-6964.2024.10.008
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The aim of this study was to analyze the accuracy of using low-density chip genotyping data to obtain high-density genotype data through genotype imputation. In this study, the genotypic consistency between the imputed genotypes based on 10K and the 50K real genotypes was analyzed. The 4 435 healthy brown laying hens from pure-line were selected, and 50K SNP array of laying hens was used for genotype determination. Some individuals were randomly selected as test and reference populations, respectively. The 10K genotype data was evenly selected from 50K typing data as known genotypes, and Beagle 4.0 software was used to impute genotypes of the rest 40K to obtain 50K data. The consistency of the imputed genotype and the real genotype was compared. The accuracy of genotype filling was evaluated by genotype filling consistency. We also analyzed the influence of following 3 aspects on the accuracy of genotype filling: 1) usage of pedigree or not (100 individuals in test population vs. 1 000 individuals in reference population); 2) kinship between reference and test population (100 individuals in test population vs. 1 000 individuals in reference population); 3) reference population size (100 individuals in test population vs. 500, 1 000, 2 000, 3 000 individuals in reference population). The results showed that the consistency of genotypic imputation was not affected by the use of genealogical information or not (0.973 vs. 0.973). The consistency of genotypic imputation was changed as the change of inter-population kinship. Using the individuals from 18th generation (1 000 individuals) as reference population to impute the genotypes of 19th generation population (100 individuals), the consistency of genotypic imputation was to 0.972. When using individuals from the 16th, 17th and 18th generations as reference population to impute the genotypes of 19th generation population, and the consistency of genotypic imputation was decreased to 0.968. The imputation consistency of genotype was increased with the increase of reference population size. The consistency of genotype imputation was 0.959, 0.973, 0.980, and 0.984 when the reference population size was 500, 1 000, 2 000, and 3 000, respectively. Collectively, this study shows that the imputation of layer SNP array from 10K to 50K is feasible, and it can be applied in genome selection breeding to reduce genotyping costs.

Evaluation of the Population Structure of the Ziwuling Black Goat Based on Super-GBS
Xiangzhen GOU, Junxiang YANG, Zihui ZHAO, Lingxia FENG, Wanhui CHEN, Yujie LI, Zhongyu ZHANG, Keyan MA, Dongping JIANG, Rong CHANG, Yazhou WEN, Ke WANG, Youji MA
2024, 55(10):  4334-4345.  doi:10.11843/j.issn.0366-6964.2024.10.009
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This study aimed to assess the genetic diversity and population structure of the Ziwuling black goat, laying the groundwork for the preservation and utilization of this breed. The whole-genome SNP analysis of a total of 99 adult Ziwuling black goats (10 males and 89 females) was performed by super-genotyping by sequencing(super-GBS). Genetic diversity parameters including observed heterozygosity (Ho), expected heterozygosity (He), polymorphic information content (PIC), nucleotide diversity (Pi), effective allele number (Ne), and minor allele frequency (MAF) were calculated using Plink software. Principal component analysis and construction of genetic relationship G matrix were conducted using GCTA software. IBS genetic distance matrix was built using Plink software, and a heatmap was plotted using R language. A phylogenetic tree was constructed using PHYLP, while runs of homozygosity (ROH) were detected using the detect RUNS tool. A total of 996 042 SNPs were detected in the 99 individuals of Ziwuling black goats. The PIC, Pi, Ne, and MAF values of Ziwuling black goats were 0.161, 0.193, 1.295, 0.130, respectively, indicating a relatively low genetic diversity with Ho(0.167) lower than He(0.192). Both the G matrix and IBS genetic distance results showed that most individuals in the Ziwuling black goat population had distant genetic relationships. The principal component analysis revealed no obvious differentiation within the Ziwuling black goat population, while the phylogenetic tree indicated that the rams could be roughly divided into 6 lineages, with few rams in each lineage. The inbreeding coefficient FROH of the Ziwuling black goat population was 0.049 6, suggesting a relatively low level of inbreeding within the population. In conclusion, the genetic diversity of the Ziwuling black goat population is relatively low, with most individuals having distant kinship relations and a low level of inbreeding within the group. It is recommended to pay attention to the breeding of offspring in order to avoid loss of genetic diversity in the future.

Genome-Wide Association Study of Wool Economic Traits in Aohan Fine Wool Sheep
Xiaokun LIN, Mengmeng DU, Lisheng ZHOU, Zhengang HUANG, Di WANG, Donghui ZHOU, Xinxin CAO, Jianning HE, Jinshan ZHAO, Hegang LI
2024, 55(10):  4346-4359.  doi:10.11843/j.issn.0366-6964.2024.10.010
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The study aimed to identify new molecular markers and candidate genes for wool traits in Aohan fine wool sheep using genome-wide association study. The ear tissue and wool of 1-2 years old healthy Aohan fine wool sheep were collected as experimental materials, 248 ewes and 81 rams, totaling 329. The wool traits were detected (including fiber diameter, natural length, elongation length, and elongation ratio), and the phenotypic data were subjected to descriptive statistics and correlation analysis. All individuals were genotyped using the 40K liquid phase SNP chip for sheep. The chip data were quality controlled using PLINK 1.07 software, and the quality-controlled data were analyzed for population structure using GCTA software and PopLDdecay software. The GMEMA mixed linear model was used to perform a genome-wide association study (GWAS) on 4 wool traits, and online software was used to conduct GO and KEGG enrichment analyses on candidate genes. After quality control, 329 individuals with 30 079 SNP loci were used for subsequent analysis. Through GWAS analysis, 4 SNP loci were identified as significantly associated with wool economic traits across the genome, located on chromosomes 1, 6, and 8. Additionally, 9 SNP loci were identified as significantly associated with wool traits at the chromosome level, located on chromosomes 3, 5, 8, 11, 18, 21, 22, and 25, and 39 candidate genes were identified as potentially affecting wool traits. The results of this study provide important references for further exploring the genetic mechanisms and molecular breeding markers of wool traits in Baohan fine wool sheep.

Effects of CHRNG Gene on Proliferation and Differentiation of Bovine Myoblasts and Its Mechanism
Xiaoyu MAO, Jiawei DU, Jiayu TANG, Jinhai PAN, Lei JIANG, Xiaolei SUN, Linsen ZAN, Hongbao WANG
2024, 55(10):  4360-4376.  doi:10.11843/j.issn.0366-6964.2024.10.011
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This study aimed to investigate the effect of CHRNG gene on the proliferation and differentiation of bovine myoblasts and its potential molecular pathways. In this study, adenovirus was used to overexpress and interfere with CHRNG gene in Qinchuan cattle myoblasts. This study isolated myoblasts from the longissimus dorsi and hind leg muscles of healthy 3-day-old Qinchuan cattle. Adenovirus overexpression and interference with CHRNG gene were used in Qinchuan cattle myoblasts, which were divided into interference group (sh-CHRNG), interference control group (sh-NC), overexpression group (OE-CHRNG), and overexpression control group (OE-NC), with 3 replicates in each group. CCK-8, EdU, qRT-PCR, Western blot, immunofluorescence staining and other methods were used to detect the effects of interference and overexpression of CHRNG gene on the proliferation and differentiation of bovine myoblasts; Further screening of differential genes and enrichment of signaling pathways were performed through RNA-Seq. The results showed that overexpression and interference with CHRNG significantly downregulated the expression of cell cycle factors PCNA, CCNB1, and CCND2 (P < 0.01), significantly upregulated the expression of CDKN1A (P < 0.01), reduced the number of proliferating cells (CCK-8, P < 0.01), and reduced the proportion of positive cells in the S replication phase (EdU). Overexpression and interference of CHRNG on bovine myoblasts and induction of differentiation were observed by morphological observation and immunofluorescence staining on D2, D4, and D6. The results showed that interference and overexpression of CHRNG inhibited the differentiation of bovine myoblasts and myotube formation. The qRT-PCR and Western blot results showed that overexpression and interference with CHRNG downregulated the mRNA and protein levels of MYOD1, MYOG and MYH3 genes (P < 0.01). Through RNA-Seq sequencing analysis, it was found that the differentially expressed genes screened by overexpressing CHRNG were mainly enriched in endoplasmic reticulum protein processing, IL-17, thyroid hormone synthesis, PPAR, PI3K-Akt and other signaling pathways. Among the top 20 enriched pathways analyzed by KEGG, 10 pathways were related to cell differentiation; The differentially expressed genes screened by CHRNG were mainly enriched in signaling pathways such as axon guidance, MAPK, and PI3K-Akt. Among the top 20 enriched pathways analyzed by KEGG, 8 pathways were related to cell differentiation. The results suggested that overexpression and interference with CHRNG gene can inhibit the proliferation and differentiation of bovine myoblasts, and the pathways enriched by differential genes caused by overexpression and interference with CHRNG are mostly related to cell differentiation.

Study on the Genetic Composition of Four Local Tibetan Cattle Breeds Based on SNP Chip Analysis
Longgang MA, Nan LIU, MA Qunzong NI, Xin WANG, Jian LU, Huaming MAO, Gong CHEN, ZENG Ouzhu DAN, BA Ciren LA, Nan ZHANG, Fuqing YU, Yachun WANG
2024, 55(10):  4377-4390.  doi:10.11843/j.issn.0366-6964.2024.10.012
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The study aimed to explore the genetic structure and bloodline composition of 4 local cattle breedings in Tibet. In this study, 22 plateau Holstein cattle, 20 plateau Jersey cattle, 45 American Brahmin cattle, 46 Yunnan Zebu and 11 Brahmen cattle as reference breeds, the samples to be tested were 177 cattle of 4 local breeds in Tibet. SNPs data were obtained by Illumina GGP100K chip hybridization, and the genetic structure and bloodline composition of SNPs were analyzed by MEGA X and Admixture. The results showed that Tibetan cattle had a closer genetic relationship with plateau Holstein cattle and plateau Jersey cattle than other local cattle breeds in Tibet. Some Zhangmu cattle and Apeijia cattle divided Tibetan cattle into two large subpopulations; There was a close genetic relationship between Shigatse hump cattle and Yunnan Zebu. In the Shigatse hump cattle population, the proportion of plateau cattle bloodline was more than 90%, in the Apaijia cattle population and Tibet cattle population, the proportion of plateau cattle bloodline was more than 50%, only in the Zhangmu cattle population, the proportion of plateau cattle bloodline was 33.15%. Secondly, bos indicus bloodline accounted for 18.27% in Zhangmu cattle population, while bos indicus bloodline was less (5%) in Apaijiza cattle, Tibet cattle and Shigatse hump cattle population. There were 35.94% dairy cattle bloodline in Apaijia population and 48.58% dairy cattle bloodline in Zhangmu population. This study provided important guidance for the future cross-breeding and cross-fixing of the native cattle species in Tibet, and also provided theoretical reference for the conservation and utilization of the resources of the two endangered breeds of Apaijiza cattle and Zhangmu cattle.

Study on the Function of PPP5C Gene in Regulating the Proliferation and Differentiation of Bovine Adipocytes
Lan FENG, Xue FENG, Yulin MA, Lingkai ZHANG, Yanfen MA, Dawei WEI, Fen LI, Lupei ZHANG, Runjun YANG, Yun MA, Bei CAI
2024, 55(10):  4391-4402.  doi:10.11843/j.issn.0366-6964.2024.10.013
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The aim of this study was to investigate the role of PPP5C gene in the proliferation and differentiation of bovine adipocytes. Loss-of-function and gain-of-function experiments of PPP5C gene were performed in precursor adipocytes of Guyuan cattle. mRNA expression levels of marker genes and cell proliferation viability were detected using qRT-PCR, EdU, CCK-8 and flow cytometry. The results of TG assay, Oil Red O, BODIPY and Nile red staining provided phenotypic data support of lipid droplets accumulation during adipocytes differentiation. The results showed that, compared with the control group, interfering with the PPP5C gene significantly up-regulated the mRNA expression of proliferation marker genes (CDK1, CDK2, and PCNA) (P < 0.01), and cell viability was significantly increased (P < 0.01), which facilitated the transition of bovine precursor adipocytes from the S-phase to the G2-phase (P < 0.01). The results also suggested that PPP5C might also play a negative regulatory role in bovine precursor adipose differentiation. This was mainly manifested in the significant up-regulation of mRNA expression of lipogenic marker genes (PPARγ, C/EBPα, and FABP4) (P < 0.01) and the significant increase of TG content in adipocytes (P < 0.01) after interfering with the PPP5C gene. Meanwhile, the results of cellular phenotypic data of Oil Red O, BODIPY and Nilered staining suggested that interfering with PPP5C promoted bovine precursor adipocyte differentiation and increased lipid droplet accumulation. The results of functional acquisition experiments with the PPP5C gene were just the opposite. In summary, interfering with the PPP5C gene promotes bovine precursor adipocyte proliferation and differentiation, and overexpression of the PPP5C gene inhibits bovine precursor adipocyte proliferation and differentiation, suggesting that the PPP5C gene may play a role as a negative regulator in bovine precursor adipocyte proliferation and differentiation. This study provides basic data for further investigation of the molecular mechanism of PPP5C gene in regulating bovine fat deposition.

Analysis of the Effects of Age and Variety on the Flavor of Duck Breast Muscle Based on Lipidomics and Flavoromics
Yating WANG, Yating ZENG, Xingzhe ZHANG, Qiong WU, Xu WU
2024, 55(10):  4403-4416.  doi:10.11843/j.issn.0366-6964.2024.10.014
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This study aimed to investigate the effects of age and breed on the lipid composition and flavor compounds of duck breast muscles. Using liquid chromatography-mass spectrometry technology and headspace solid phase microextraction combined with comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry to determine lipid composition and detect volatile flavor compounds in the breast muscle of 120 days old Liancheng White duck (LC120), 500 days old Liancheng White duck (LC500), and 500 days old Longyan Shanma duck (SM500).Univariate statistical analysis and partial least squares discriminant analysis (PLS-DA) were used to screen for differential lipids and volatile flavor compounds, then relative odor activity values (ROAV) were calculated to determine the main substances affecting duck flavor. The results showed that a total of 1 615 lipids were identified in LC120, LC500, and SM500. In the comparison between LC120 and LC500 were identified 26 differential lipids, with the main types of different lipids were triglycerides (TG). The 38 differential lipids were screened in the comparison between SM500 and LC500, mainly reflected in phosphatidylcholine (PC). Further analysis revealed that unsaturated fatty acids on the structural sites of TG and PC could affect the formation of volatile compounds in meat. In LC120 and LC500, the sn-2 positions of the TG side chain contained a few polyunsaturated fatty acids, but it was not found in SM500. The results of flavor substance detection showed that one differential volatile compound, 3-acetyl-2, 4-dimethylfuran, was selected in the comparison between LC120 and LC500. The 29 differential volatile compounds were selected in the comparison between SM500 and LC500, mainly reflected in hydrocarbons, aldehydes, ketones and others. In addition, there were 16 key aroma compounds in 3 groups of duck meat by calculating ROAV, among which 2-methylbutanal and 2, 3-butanedione were the main characteristic flavor compounds. In summary, this study identified 64 differential lipids that distinguish the breast muscles of 3 groups of ducks, and identified 16 volatile organic compounds as key aroma compounds in the breast muscles of the 3 groups. Indicated that the flavor of Liancheng White duck is superior than Longyan Shanma duck, and the influence of variety on meat flavor is greater than that of age. TG and PC play a crucial role in the formation of volatile compounds in duck meat. The results provide theoretical basis and data support for the development of duck germplasm resources and the evaluation of flavor and quality of local duck varieties in China.

Mitochondrial Genome Assembly and Phylogenetic Analysis of the Jiuyishan Rabbit
Congcong LI, Zike HUANG, Nianni HUANG, Shiyu MA, Han LIU, Zhibiao XIAO, Guo SONG, Liang JIANG, Weibo PENG, Lianxi YANG, Yuntao GUO, Shengqiang HUANG
2024, 55(10):  4417-4427.  doi:10.11843/j.issn.0366-6964.2024.10.015
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The aim of the study was to assemble the mitochondrial genome of the Jiuyishan rabbit and to investigate its phylogenetic evolution and taxonomic position. High-throughput sequencing technology was employed to perform whole-genome sequencing on ear tissues from 20 Jiuyishan rabbits at 150 days of age. Bioinformatics software was employed for the assembly of the mitochondrial genome, followed by conducting sequence analysis, gene prediction and annotation of the obtained mitochondrial genome. Subsequently, multiple sequence alignment and phylogenetic analysis were conducted based on the published mitochondrial genome sequences of rabbits, hares, economically important animals and model animals. Polymorphic sites within the study population were identified. The complete mitochondrial genome sequence of the Jiuyishan rabbit was successfully obtained, the length was 17 306 bp. The nucleotide composition and gene arrangement of the mitochondrial genome were found to be largely consistent with those reported for 5 previously published domestic rabbit breeds. Phylogenetic analysis based on the D-loop region indicated a closer evolutionary relationship between the Jiuyishan rabbit and the Fujian Yellow rabbit, while Jiuyishan rabbit was more distantly related to the European rabbit, Yimeng Wool rabbit, Chuanbai Rex rabbit and New Zealand White rabbit. Interspecific phylogenetic analysis involving 14 wild rabbit species and 11 economic or model animal species demonstrated that the Jiuyishan rabbit shared a closer phylogenetic relationship with the European wild rabbit, Hainan rabbit, and the White-booted rabbit but was further with others. A total of 12 mutation sites were identified in the conserved regions of the mitochondrial genome of the study population, with 3 mutations having allele frequencies greater than 0.8, indicating polymorphism. This study has successfully assembled the first complete mitochondrial genome sequence of the Jiuyishan rabbit, clarifying its phylogenetic relationships and taxonomic status in comparison to other domestic rabbit breeds, wild rabbits and common economic or model animals. This work provides foundational data for the utilization of genetic resources and the breeding of the Jiuyishan rabbit.

Comparative Analysis of the Contents of Amino Acids, Fatty Acids and Volatile Flavor Compounds in the Muscles of Hu Sheep and Their Different Hybrid Combinations
Liwa ZHANG, Yaojing YUE, Xuejiao AN, Jianye LI, Bohui YANG, Zhenfei XU, Jinxia ZHANG, Zhiguang GENG, Yanli GUO, Rui ZHANG
2024, 55(10):  4428-4442.  doi:10.11843/j.issn.0366-6964.2024.10.016
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The aim of this study was to analyze the differences in muscle cholesterol, amino acids, fatty acids and volatile flavor compounds between Hu sheep and their hybrid combinations. Sixteen male lambs each of Hu×Hu (HH), Poll Dorset×Hu (TH) and Southdown×Hu (NH) F1. generations were selected at about 3 months of age, and were reared in a single pen under the same nutritional level and feeding management conditions, and the experiment lasted for a total of 95 days (with a pre-test period of 15 days). At the end of the feeding trial, seven test lambs from each group that were close to the average weight of the group were selected for slaughter, and the longissimus dorsi was collected for the determination of cholesterol, amino acids, fatty acids, and volatile flavor substances content. The results were showed as follows: Muscle cholesterol content in NH group was significantly higher than that in TH group (P < 0.05); A total of 17 amino acids were identified in the muscle of the three groups. Among essential amino acids, the contents of threonine, isoleucine, phenylalanine and lysine in TH and NH groups were extremely significantly higher than those in HH group (P < 0.01); Among non-essential amino acids, the contents of serine, histidine and proline in TH and NH groups were extremely significantly higher than those in HH group (P < 0.01), while the contents of aspartic acid and arginine were significantly higher than those in HH group (P < 0.05). The contents of sweet amino acid and umami amino acid in NH group were significantly higher than those in HH group (P < 0.05). Thirty fatty acids were identified in the muscles of all three groups. Among the saturated fatty acids, the contents of stearic acid and behenic acid in group HH were significantly higher than those in group NH (P < 0.05), and among the monounsaturated fatty acids, the cil-10-pentadecanoic acid was significantly higher in the NH group than in the HH group (P < 0.05). Among polyunsaturated fatty acids, the arachidonic acid content was significantly higher (P < 0.05) in the HH group than in the TH group, while C20:5n3 content in HH group was significantly higher than that in NH group; A total of 33 volatile flavor substances were identified in muscle of the three groups. The content of 2-heptanol in NH group was significantly higher than that in TH and HH groups, the content of 3-hydroxy-2-butanone in HH group was significantly higher, and the content of thiazole in TH group was significantly higher. The content of 2-heptanone was significantly lower than that in HH group (P < 0.05). In summary, the progeny muscles of local Hu sheep by crossing breed with Poll Dorset and Southdown have more desirable cholesterol, amino acid and fatty acid contents as well as rich volatile flavor substances, which provide a reference for local high-quality lamb meat production.

Animal Biotechnology and Reproduction
Study on the Separation Effect of Pig X, Y Sperm of Different Acid-Base Diluents Combined with Creatine Monohydrate
Bingyan HU, Qihao LIU, Chaoyue CAO, Mengxuan LI, Weijun PANG
2024, 55(10):  4443-4454.  doi:10.11843/j.issn.0366-6964.2024.10.017
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The study aimed to explore the separation effect of porcine X, Y sperm by adjusting the acidity and alkalinity of porcine semen diluent combined with creatine monohydrate (CMH). In this study, eight healthy Duroc boars aged 2-4 years were used to collect semen. By adjusting the pH of semen diluent Beltsville (BTS), the optimal pH of the X, Y sperm sorting system was determined by sperm computer-aided analysis system (CASA), and the optimal concentration of creatine monohydrate was determined by CASA. The effect of X, Y sperm sorting was evaluated by immunofluorescence staining and flow cytometry, and sperm quality indexes such as acrosome, plasma membrane integrity, and reactive oxygen species content (ROS) were detected by fluorescence labeling and MitoSOX Red staining of sperm ROS. The results showed that when the pH of the diluent was 6.4 and 7.1, there was no significant effect on sperm viability after incubation for 60 min (P>0.05), and the proportion of sperm in the upper layer moving forward was significantly higher than that in the lower layer (P < 0.01). Adding 750 μmol·L-1 creatine monohydrate to the diluent and incubating for 60min significantly improved sperm motility, linear movement rate, curve movement rate, and wobble (P < 0.05). Using acid diluent combined with creatine monohydrate to separate pig X, Y sperm, the proportion of X sperm in the upper semen after separation was 63.32%. Alkaline diluent combined with creatine monohydrate was used to separate pig X, Y sperm. After separation, the proportion of Y sperm in the upper semen reached 67.19%. Further study showed that there was no significant difference in acrosome integrity, plasma membrane integrity, and ROS level between the two groups (P>0.05), while ATP level, mitochondrial membrane potential, and exercise performance were significantly higher than those of the control group (P < 0.05) after separation. To sum up, this study established a simple and effective separation system of X and Y sperm in pigs by adjusting the acidity and alkalinity of the diluent in parallel with creatine monohydrate. This method did not affect the quality of sperm and improved the motility of sperm after separation, which further provided a theoretical basis for the research and development of new technologies for controlling semen in pigs.

Mining of Key Candidate Genes Involved in Bone Metabolism Differences at Pre- and Post-laying Stage Based on Transcriptome Data in Laying Hens
Yinliang ZHANG, Ran ZHANG, Wenjun WANG, Dehe WANG, Lanhui LI, Rongyan ZHOU
2024, 55(10):  4455-4465.  doi:10.11843/j.issn.0366-6964.2024.10.018
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The study aimed to explore the regulatory pathways and important candidate genes that affect the differences in bone metabolism before and after egg laying in laying hens, providing a theoretical basis for further research on the regulatory mechanisms for maintaining bone health in laying hens. A total of 15 at 15-week-old and 12 at 22-week-old Hyline Grey laying hens with similar body weights and reared under identical conditions were selected. Blood and tibia samples were collected to measure plasma levels of calcium, phosphorus, estrogen, and other biochemical markers related to bone metabolism. Based on estrogen levels, tibia tissues from 6 hens within each age group were selected to construct 12 transcriptome libraries. Differentially expressed genes (DEGs) were then screened from these libraries, and GO functional annotation and KEGG pathway enrichment analyses were performed with DEGs. Additionally, 6 randomly selected DEGs were validated for their relative expression levels using qRT-PCR. When comparing 15-week-old hens to 22-week-old hens, significant changes (P < 0.05) were observed in plasma bone metabolism markers and estrogen levels in the laying hens. The clean reads of 40 082 240-186 154 554 were obtained from the 12 constructed tibial cDNA libraries, with a minimum Q30 value of 92.18% and a contrast rate between 76.52% and 90.55%. A total of 1 832 DEGs were screened from the tibia of laying hens at 15 and 22 weeks of age. Among them, 914 genes were downregulated and 918 genes were upregulated. GO functional annotation revealed that differentially expressed genes were significantly enriched in protein processing, response to endoplasmic reticulum stress, and collagen fibril organization (P < 0.05). KEGG enrichment analysis revealed significant enrichment pathways related to bone metabolism, including steroid biosynthesis, thyroid hormone synthesis, and TGF- β signal pathway, MAPK signaling pathway, PI3K-Akt signaling pathway. Screening out 18 genes were shared by these pathways as key candidate genes for regulating bone metabolism. This study revealed that there are differences in gene expression in the tibia tissue of laying hens, and multiple DEGs and pathways affecting bone metabolism at pre- and post-laying stages were screened in laying hens. These results provide theoretical basis for further research on the mechanism of bone metabolism changes in laying hens before and after egg laying.

Effect of Sericin on Cryopreservation of Semen of Chengde Hornless Black Goat
Wentao ZHANG, Yang YU, Yatian QI, Wei ZHAO, Hongjun ZHANG, Chenyu TAO, Wei XIA, Junjie LI
2024, 55(10):  4466-4474.  doi:10.11843/j.issn.0366-6964.2024.10.019
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The aim of this study was to explore the effect of sericin on cryopreservation of semen of Chengde hornless black goat. Semen samples of 3 healthy Chengde hornless black goats were collected by the sham vaginal method and randomly divided into 6 groups: control group and experimental groups with different sericin concentrations (0.2%, 0.4%, 0.6%, 0.8%, 1%) added to the OptiXcell 2 dilution. Each group was set up with 5 replicates. Sperm motility, acrosome integrity rate, plasma membrane integrity rate, total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) activity, malondialdehyde (MDA) content, mitochondrial membrane potential (MMP) and ATP content were detected after freezing and thawing. The results showed that the addition of different concentrations of sericin to the semen frozen dilution of Chengde hornless black goat could improve the quality of sperm after freezing and thawing. Compared with other concentrations, the addition of 0.6% sericin significantly increased sperm motility, acrosome integrity rate, plasma membrane integrity rate, mitochondrial membrane potential and ATP content after sperm freezing and thawing, and increased the activities of antioxidant enzymes T-AOC, GSH-Px, SOD and CAT to (13.61±0.39) mmol·g-1, (26.52±1.30) U·mg-1, (165.45±11.10) U·mg-1 and (13.76 ±0.30) U·mg-1, respectively. Meanwhile, the MDA value was reduced to (3.15±0.20) μmol·mg-1. In summary, sericin at different concentrations can improve the quality of frozen semen of Chengde hornless black goat, and the most suitable concentration is 0.6%.

Highly Efficient BLG Knockout in Bovine Mammary Epithelial Cells by Using CRISPR/Cas9
Xiuhu DING, Zhiping LIN, Fang ZHAO, Kunlin CHEN, Jifeng ZHONG, Yan ZHANG, Yundong GAO, Huixia LI, Huili WANG, Jianli ZHANG, Qiang DING
2024, 55(10):  4475-4488.  doi:10.11843/j.issn.0366-6964.2024.10.020
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This study aimed to establish CRISPR/Cas9 mediated BLG-knockout (BLG-KO) system in bovine mammary epithelial cells (bMECs), and to explore the function effect of BLG-KO on bMECs. Three sgRNAs were designed from the first exon of the bovine BLG gene sequence and screened by using in vitro cleavage test. The Cas9 expression vector and sgRNAs vectors were cotransfected into bMECs by electroporation. Monoclonal cell lines were screened with 1.8 μg·mL-1 puromycin and 6 μg·mL-1 blasticidin. BLG-KO gene editing type were verified by using PCR amplification and sequencing analysis. The potential off-target sites were selected according to the sgRNA sequence and detected by PCR sequencing. The BLG expressing level of knockout cell was detected by Western blot (WB). Cell viability was tested by cell counting kit-8 (CCK-8) assay, further the growth curve was drawn to analyze the proliferation effect of bMECs cells after BLG-KO. Two sgRNAs were screened and used for further gene edit. 10 monoclonal cell lines were obtained after screening and 9 BLG-edit lines verified by PCR sequencing, 4 clones with large fragment deleted at BLG sequence. Sequence analysis showed that the editing sites contained multiple gene-editing types, including insertion, deletion and base substitution. WB result showed that BLG-KO cell lines with low BLG protein expressing level compare to none edit cell. Furthermore, the BLG-KO promote cell growth and proliferation according to the results of cell growth curve and CCK-8 assay. In this study, we constructed an efficient BLG-KO method in bMECs by using CRISPR/Cas9 system, and the results demonstrated that BLG-KO significantly affected the cell proliferation in bMECs, the results provided a cellular model for exploring the functional mechanism for BLG-KO cattle.

Application Study of Chinese Cow's SNPs Chip-Ⅰ in Chinese Holstein and Pakistani Indigenous Dairy Cattle Populations
Wanyi LAI, Xinyue TAO, Gengxin YANG, Wenli YU, Shujing LI, Tahir USMAN, Ying YU
2024, 55(10):  4489-4499.  doi:10.11843/j.issn.0366-6964.2024.10.021
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The purpose of this study was to identify significant SNP loci or combinations relevant to cow udder health, so as to improve udder health in dairy cattle and promote the healthy development of the dairy industry in "the Belt and Road" countries. In this study, genomic DNA was collected from the tail vein blood of 1 097 Chinese Holstein and 161 Pakistani Holstein, and the tail root hair follicles of 116 Achai cattle and 104 Red Sindhi. Chinese Cow's SNPs Chip-I (CCSC-I) was used to obtain the genotype. In conjunction with udder health indicators from corresponding DHI reports—somatic cell count (SCC) and somatic cell score (SCS), significant SNPs or combinations were identified through single SNP and pairwise SNP association analysis. The unit point association analysis showed that SNP3, 6, 8, 11, 19, and 20 were significant loci in the Chinese Holstein(P < 0.05), with SNP3 and 11 identified as significant loci in the Pakistani dairy cattle population(P < 0.05). In pairwise SNP association analysis, stable combinations of SNP11-SNP15 were retained for the Chinese Holstein using large samples (farms in Beijing, Zhejiang, and Hebei provinces) and samples from Hebei province specifically, with the AA genotype predominating for SNP19 and SNP20. Significant combinations of SNP11-SNP2, 4, 8, 9, 12, 16, 19 were identified for Pakistani Holstein(P < 0.01), and significant combinations like SNP3-SNP11, 15 were identified for the Pakistani indigenous Achai(P < 0.01). The results of this study show that Chinese Cow's SNPs Chip-I exhibits wide applicability. SNP11, 15, 19, 20 and other loci have important effects on cow udder health. In Holstein populations, CD4, TRAPPC9, and PTK2 showed potential resistance to mastitis. In the Pakistani indigenous dairy cattle populations, CD4, DGAT1, and TRAPPC9 showed significant resistance characteristics. These findings provide a strong scientific basis for future accurate breeding.

Animal Nutrition and Feeds
Study on Growth Model and Nutrient Deposition Pattern of Rapidly-growing Yellow-feathered Chickens
Dan GOU, Yunjie ZHAO, Qi CHANG, Xinxiong ZHUO, Jian XIAO, Haihan ZHANG, Xi HE, Zehe SONG
2024, 55(10):  4500-4516.  doi:10.11843/j.issn.0366-6964.2024.10.022
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The purpose of this study was to investigate the growth pattern and dynamic nutritional deposition pattern of rapidly-growing yellow-feathered chickens (Xiangjia Yellow Chicken No.2) at 0-56 days of age. Aim to establish growth curves of body weight(BW) and average daily gain(ADG), deposition models of energy(TE), body protein(BP), body fat(BF), total amio acids(TAA). In order to provide scientific proof for accurate feeding of rapidly-growing yellow-feathered chickens. In this study, 672 healthy 0-day-old rapidly-growing yellow-feathered chickens (No.2 Xiangjia, half male half female) were selected from the same batch of chicks. The growth performance indexes and body composition of male or female chickens were measured at the 0th, 7th, 14th, 21st, 28th, 35th, 42nd, 49th, and 56th days of age. The results showed that BW, empty weight (EW), feather weight (FW), ADG, and the deposition of TE, BP, BF and TAA of chickens were significantly increased with the age (P < 0.001). The growth curves of BW, EW, FW and ADG, and the model for the weights and deposition of TE, BP, BF, and TAA were used to fit by three non-linear regression model, Gompertz, Logistic and Bertalanffy.Based on the coefficients of determination, residual sum of squares, mean squared error and the red pool information criterion of the regression model, the Gompertz model was more accurate than Logistic or Bertalanffy model. Using the Gompertz function equation, growth curves for BW, ADG, and the model for deposition of TE, BP, BF and TAA were developed for the Xiangjia No.2 yellow-feathered chickens. In conclusion, Gompertz model can accurately predict BW, ADG and the nutrient weights and deposition of the Xiangjia No.2 yellow feathered chicken, and also provide a basis for the study of its nutrient dynamic requirements and precise feeding.

Preventive Veterinary Medicine
Application of the Cre-LoxP System to Construct the Deleted TK Gene of the Lumpy Skin Disease Virus
Yuzhe ZHANG, Shanhui REN, Wei YAO, Zhenli GONG, Hongqiang ZHANG, Minyi LIU, Ting YOU, Xiangwei WANG, Jiyun LI, Xiangping YIN, Yuefeng SUN, Haotai CHEN, Xuerui WAN
2024, 55(10):  4517-4529.  doi:10.11843/j.issn.0366-6964.2024.10.023
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A TK-deficient Lumpy skin disease virus (LSDV) strain was constructed by homologous recombination technology and the Cre-LoxP system platform to provide an alternative strain for developing a safe and efficient LSDV genetic engineering vaccine. Using the TK gene as the target gene, using the Cre-LoxP system platform and red fluorescent protein (RFP) as screening marker, the left and right homologous arms and RFP gene expression cassettes were amplified by overlapping PCR and fused, and then cloned into pUC19T vector to construct gene deletion transfer vector pUC19T-LSDVΔTK-RFP. The transfer vector recombinant plasmid was transfected into Vero cells and infected with LSDV/CHA/FJ/2021 strain to construct LSDV-ΔTK-RFP recombinant virus. LSDV-TK-RFP was screened and purified by the plaque method and limited dilution method. Then, using the Cre-LoxP system, RFP tags were cut off from the viral genome in MDBK-Cre cells, and LSDV-TK strains were screened and purified by the plaque method and limited dilution method. PCR and sequencing identified the recombinant strain, and its one-step growth curve was determined. The recombinant strain (LSDV-ΔTK) with deletion of the TK gene was successfully constructed, and the replication ability of the recombinant strain was slightly lower than that of the wild-type strain. The LSDV strain with deletion of the TK gene constructed by the Cre-LoxP system has good genetic stability and replication growth characteristics, so it can be used as an alternative strain for the development of the LSDV genetic engineering vaccine and expands the application range of the Cre-LoxP system.

Construction and Growth Characteristics of ORF123 Deleted Lumpy Skin Disease Virus Strain
Fangping WANG, Shanhui REN, Xiaohong GAO, Xue YANG, Jiyun LI, Xiangwei WANG, Xiangping YIN, Yuefeng SUN, Haotai CHEN, Xuerui WAN
2024, 55(10):  4530-4541.  doi:10.11843/j.issn.0366-6964.2024.10.024
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The aim of this study was to analyze the growth characteristics of the Lumpy skin disease virus (LSDV) strain with the ORF123 gene deleted, which laid a foundation for the functional research of LSDV ORF123. ORF123 was used as the target gene and enhanced green fluorescent protein (EGFP)was used as the screening marker, the homologous left and right arm sequences and the EGFP gene expression frame were amplified and fused by overlapping PCR and cloned into pUC-19T vector to construct pH5-LSDVΔORF123-EGFP. The plasmid pH5-LSDVΔORF123-EGFP was then transfected into Vero cells and after that the cells were infected with the LSDV/CHA/FJ/2021 strain. The recombinant virus strain LSDVΔORF123-EGFP was purified through multiple plaque screening using EGFP as a screening markers, and its viral genetic stability and growth characteristics were characterized. The results revealed that the recombinant virus was able to stably express green fluorescent protein across at least 8 cell passages and the one-step growth curve following MDBK cell inoculation indicated that the titer of the recombinant virus was slightly lower than that of the parental strain. In conclusion, the recombinant virus strain LSDVΔORF123-EGFP was successfully obtained, which laid a foundation for the study of the biological function of LSDV ORF123 protein and the development of a live attenuated LSDV vaccine.

Suppressive Effect of Histone Deacetylase Inhibitor MGCD0103 on Peste Des Petits Rumants Virus Replication in vitro
Ruixue DENG, Chunrong PAN, Xueliang ZHU, Linjie HU, Yuefeng SUN, Qiaoying ZENG, Xuelian MENG
2024, 55(10):  4542-4552.  doi:10.11843/j.issn.0366-6964.2024.10.025
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In this study, the effect of MGCD0103 (Mocetinostat), a selective HDACi, on the replication of peste des petits ruminants virus (PPRV) in goat endometrial epithelial cells (EEC cells) was investigated, aiming to clarify the role and activity of MGCD0103 in PPRV multiplication. The transcription levels of HDACs and PPRV N gene in different treated cells were determined by RT-qPCR, and the action stage of MGCD0103 in the viral proliferation cycle of PPRV was determined by the time of addition assay. The effect of MGCD0103 on the expression level of PPRV N protein and viral titer were further analyzed by Western blot and TCID50, respectively. The results showed that the mRNA expression levels of HDAC1 (P < 0.001) and HDAC2 (P < 0.002) were increased significantly in PPRV-infected EEC cells. Although MGCD0103 has an excellent inhibitory effect on PPRV multiplication, its inhibitory effect in the virus entry is limited. MGCD0103 significantly reduced the transcription and expression levels of PPRV N gene, as well as the viral titer (P < 0.002). The half-maximal effective concentration (EC50) and selectivity index (SI) of MGCD0103 against PPRV were 0.43 μmol·L-1 and 4.35, respectively. These results indicated that MGCD0103 significantly inhibited PPRV replication, which is of great significance for the development of anti-PPRV drugs and provides a new idea for constructing sensitive cell lines for PPRV proliferation.

Pathogenicity of a Newly Akabane Virus on Goats from Bamboo Rat
Shuying QIN, Jinfeng LIU, Ling MA, Lishi XU, Lei YANG, Fenglian CHEN, Shanshan WEI, Chenyang LU, Jun LIN, Jue WEI, Shaomin QIN, Jianmin WU
2024, 55(10):  4553-4561.  doi:10.11843/j.issn.0366-6964.2024.10.026
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In order to determine the pathogenicity of Akabane virus (AKAV) from bamboo rat in goats, GXLCH16-70 strain was used to infect goats via intracranial(IC), intravenous (IV)and subcutaneous (SC) inoculation. The clinical manifestations, viremia, pathological changes, histopathology, and pathogenic distribution in tissue or organ of goats were observed. The 4th to 9th day post infection (dpi), goats in the IC group showed obvious neurological symptoms such as intermittent spasm, convulsion, while goats in the IV and SC groups showed symptoms as such diarrhea and increased secretions in the eyes at the 3rd to 8th dpi. All goats infected by GXLCH16-70 developed viremia at the 2nd to 10th dpi, and produced AKAV neutralizing antibodies at the 8th dpi. The antibodies continued to exist up to the 21st dpi. Autopsy in the intracranial group showed non-suppurative encephalomyelitis of the brain, pulmonary congestion and hemorrhage, marginal hemorrhagic spots of the spleen and other pathological changes. Histopathological examinations on the IC group showed the multiple layers of inflammatory cells infiltrating to the meninges and cortical blood vessels, forming a "vascular sheath" like structure. Pathogen detection by fluorescence quantitative RT-PCR (qRT-PCR) showed that AKAV-S RNA fragments were detected in almost all regions of the brain in the IC group, and in some brain tissue and spinal cord in the IV group and SC group. The results showed that AKAV GXLCH16-70 strain from bamboo rat causes systemic lesions of multiple organs and tissues of goats and induce encephalomyelitis.

Isolation, Purification and Immunogenicity Evaluation of Senecavirus A Intact Particle and Empty Capsid
Mei LI, Suyu MU, Hu DONG, Shuo LI, Songjia PAN, Huichen GUO, Shiqi SUN
2024, 55(10):  4562-4570.  doi:10.11843/j.issn.0366-6964.2024.10.027
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Senecavirus A (SVA) has the ability to form intact virions and empty capsids during replication; however, the conditions for isolating them and their differences in antibody response are still unknown. In this study, we utilized guanidine hydrochloride to generate SVA empty capsids and determined the optimal conditions for separating SVA intact virions and empty capsids. This was achieved through ultracentrifugation using different density gradients, media, rotation rates, and durations. Subsequently, SVA intact virions and empty capsids were intramuscularly immunized into BALB/c mice at a dosage of 12.5 μg per mouse. Specific and neutralizing antibodies were then monitored 1-7 weeks post-immunization. The findings indicated that the addition of 100 mmol·L-1 guanidine HCl for 2 hours in infected cells at MOI=1 of SVA resulted in the complete formation of empty capsids. The most effective method for distinguishing SVA intact virions from empty capsids was centrifugation at 36 000 r·min-1 for 2.5 hours, using a cesium chloride gradient ranging from 10% to 50%. Furthermore, the specific and neutralizing antibody levels for SVA intact virions were comparable to those of empty capsids. This study provides a new reference for the isolation and purification of the complete and empty capsid of Seneca virus A, as well as for the development of recombinant SVA virus-like particle vaccines.

Expression and Identification of Porcine Encephalomyocarditis Virus-like Particles Based on Baculovirus-insect Cell Expression System
Yunhang WANG, Wei JING, Suyu MU, Shiqi SUN, Huichen GUO, Manyuan BAI
2024, 55(10):  4571-4578.  doi:10.11843/j.issn.0366-6964.2024.10.028
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Porcine encephalomyocarditis is a viral zoonotic disease caused by encephalomyocarditis virus and characterized by porcine encephalitis and myocarditis, which can infect a variety of mammals. Currently, there is no effective vaccine in China, so developing a safe and effective vaccine is an effective measure to prevent and control this disease. In this study, virus-like particals of porcine encephalomyocarditis virus were assembled by using the baculovirus-insect cell expression system with P1 and 3CD co-expression strategy, and the immune response effect of the virus in mice was preliminatively evaluated. The results showed that the specific antibody titer of mice in the immune group reached more than 1∶192, the neutralizing antibody titer reached more than 1∶64, and the combination with ISA206 adjuvant can induce higher antibody levels. The levels of IL-4, IFN-γ, IL-5 and TNF-α cytokines all reached 20 pg·mL-1, and were significantly higher than those in PBS group. Therefore, the EMCV virus-like particle vaccine obtained in this study can induce the body to produce good cellular and humoral immune responses, and can also induce the production of inflammatory cytokines, which helps to stimulate the body's immune response, enhance the protective effect of the vaccine, and provide theoretical basis and technical reserve for the prevention and control of EMCV.

Development of Monoclonal Antibodies against Classical Swine Fever Virus E2 Protein by Single B Cell Sorting Technology and Its Application in ELISA
Zhongyuan MA, Junzuo ZHENG, Zhibo LIANG, Li PAN, Qiaoying ZENG
2024, 55(10):  4579-4589.  doi:10.11843/j.issn.0366-6964.2024.10.029
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The purpose of this study was to develop a classical swine fever virus (CSFV) E2 monoclonal antibody-based competition ELISA (Enzyme Linked Immunosorbent Assay) for evaluating the efficiency of the live attenuated C-strain and E2 subunit vaccines. Firstly, the E2 gene of CSFV was constructed into pFast BAC vector, and the E2 protein was efficiently expressed in SF9 cells; Secondly, 6-8 weeks of BABL/c mice were immunized at intervals with purified E2 protein, and the single B cells were then sorted out at the gate of IgM-/E2+ by flow cytometry. Then, the heavy and light chains of E2 antigen-specific IgG antibodies were amplified by semi-nested PCR, after sequencing, the heavy and light chains of the antibodies were constructed into pCDNA3.1vector, and then were co-transfected into HEK293 cells to prepare the monoclonal antibodies against CSFV E2. The results showed that the two monoclonal antibodies derived from single B cells, namely mAb3A9 (IgG1, kappa) and mAb4F7 (IgG2a, lambda), could efficiently reacted with the linear epitopes25GLTTTWKEYSHDLQL39 and259GNTTVKVHASDERGP273 of CSFV E2 protein, respectively. In addition, the competitive ELISAs developed using the monoclonal antibodies and E2 protein mentioned above exhibited an excellent diagnostic sensitivity (97.49%, 95.97%) and specificity (96.08%, 94.38%) in the process of detecting serum samples, which provides a favorable technical support for the gradual elimination of CSF in China.

Correlation Analysis of Neutralizing Antibody and E2 Antibody against Classical Swine Fever Virus
Yingju XIA, Yan LI, Jun LIU, Yuan XU, Fangtao LI, Xingqi ZOU, Qi LI, Jiaxin LI, Junjie ZHAO, Qianyi ZHANG, Yebing LIU, Lu XU
2024, 55(10):  4590-4596.  doi:10.11843/j.issn.0366-6964.2024.10.030
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In order to explore the correlation between neutralizing antibody and E2 antibody against classical swine fever virus (CSFV), 289 swine sera were detected using both neutralization assay and ELISA among which neutralizing antibody titers of 15 sera were further tested by neutralization assay using different CSFV strains. The results showed that E2 antibody level against CSFV was positive correlated with neutralizing antibody titer. When the individual percentage inhibition of E2 antibody was over 60%, the neutralizing antibody titer of more than 70% vaccinated pigs in the herd meet the protection standard. These results could help the local veterinaries to evaluate the efficacy of CSFV vaccine better in the field and reduce the risk of CSF outbreak.

Evaluation of the Immune Efficacy of Attenuated Strain of Subtype B Avian Metapneumovirus Disease on Commercial Broilers
Zhuangzhuang XU, Suyan WANG, Yuntong CHEN, Tao ZHANG, Mengmeng YU, Lingzhai MENG, Wenrui FAN, Xiaole QI, Yulong GAO
2024, 55(10):  4597-4604.  doi:10.11843/j.issn.0366-6964.2024.10.031
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In order to study the immune effect of attenuated strain of subtype B avian metapneumovirus (LN16-A strain) on commercial broilers, 3-week-old commercial broilers were selected from large-scale farms and immunized with attenuated strain of subtype B avian metapneumovirus (LN16-A strain) by dropping nose (200 μL, virus content≥103 TCID50/piece), and the blank control group was given the same dose of PBS. Twenty-one days after immunization, blood serum was collected under the wings and corresponding antibodies were detected using ELISA and neutralization tests. At 6-week-old, the B subtype avian metapneumonia virus (LN16-V strain) was administered via nasal drip (200 μL, 5000 TCID50 per chicken) to chickens for virus challenge. After the challenge, the virus copy number of nasal swabs from each group of chickens was continuously observed for 7 days. RT-qPCR was used to detect the virus copy number. On the 9th day after the challenge, 2 chickens from each group were randomly selected for dissection. Nasal turbinate, tracheal, and lung tissues were collected, and pathological sections were made using HE staining to observe the histopathological changes of the tissues. The results of antibody showed that 21 days after immunization, the positive rate of ELISA antibody and neutralizing antibody in the immunization group was 100%, with an average ELISA antibody titer of 1 860 and an average neutralizing antibody titer of 7.44 log2. The results of clinical symptoms showed that the commercial broilers in the control group had obvious clinical symptoms, such as cloudy, viscous, and filamentous nasal juice, and the incidence rate was 100%. While the broilers in the immunization group had no clinical symptoms. In addition, the results of RT-qPCR showed that the virus copy number of nasal swab in the immunization group decreased by 78% to 96%, compared to the control group. The results of pathological sections showed that the turbinate bone, trachea and lung of broilers in the control group had different degrees of pathological damage, while no obvious pathological changes were observed in the organs of broilers in the immunization group. The above results indicate that the attenuated strain of subtype B avian metapneumovirus (LN16-A strain) could induce the production of specific antibodies in commercial broilers, which could significantly reduce the virus shedding level of broilers after challenge, and had a good protective effect on commercial broilers. This study may provide important theoretical guidance and technical support for the prevention and control of avian metapneumovirus disease in large-scale broiler farms.

Effect of Plant Essential Oil on Prevention and Treatment of Chicks Artificially Infected with Infectious Bronchitis Virus
Jiajie LIN, Zixue LIN, Lijia WANG, Guowei WANG, Yanping HUANG, Yu ZHANG, Taoni ZHANG, Yinghao JIN, Meilan MO
2024, 55(10):  4605-4619.  doi:10.11843/j.issn.0366-6964.2024.10.032
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The aim was to investigate the efficacy of plant essential oil (Commercial name: Shu-ning 500) on chicks artificially infected with avian infectious bronchitis virus (IBV). A total of 260 1-day-old non-immunized healthy chicks were divided into 13 groups, including blank group, model control groups (A, B, C), preventive groups (A, B, C), treatment groups (A, B, C), and positive drug groups (A, B, C), with 20 chicks in each group. Except the blank group, all the chicks in group A were mono-infected with IBV strain GX-QZ20181028 (LX4 type) at 8-day-old, and all the chicks in group B were mono-infected with IBV strain GX-NN20200723 (Taiwan type) at 8-day-old. All group C chicks were co-infected with IBV strains GX-QZ20181028 (LX4) and GX-NN20200723 (Taiwan type) at 8-day-old. The preventive groups were given the drug(essential oil, 1 mL·L-1 water)in drinking water from 3-day-old (5 days before infection). Both the treatment groups and the positive drug group (Shuanghuanglian Oral Liquid, 0.7 mL·L-1 water) were administered when more than half of the chicks showed clinical symptoms, and the drug was administered continuously for 5 days. Clinical symptoms and efficacy were evaluated at 8, 9, 13, 14, 15, 16, 17, 18, 19, and 20 days old, while pathological histopathological changes, immune organ indexes, and viral loads in trachea and kidneys were observed at 19 and 22 days old. IBV-specific antibodies, as well as cytokines IFN-γ, IL-4, and IL-6 in sera were detected at 8, 9, 15, 17, 19, and 22 days old. The results showed that the protection rates of prevention group A, B and C were 75.00%, 85.00% and 70.00%, respectively. The effective rates of treatment group A, B and C were 92.31%, 78.57% and 77.78%, respectively. In the model control groups, cilia of a large number of epithelial cells in the tracheal mucosa decreased or disappeared, and degeneration and necrosis occurred, and lymphocytes were infiltrated in the renal mesenchyme, and no obvious lesions were observed in other groups. The viral loads of trachea and kidney in the prevention, treatment and positive drug groups were significantly lower than those in the model control groups (P < 0.05). The thymus and bursa indices in the model control groups were significantly lower than those in the remaining groups (P < 0.05). At 17 days old (the 5th day after administration), the antibody levels of prevention, treatment and positive drug groups increased, and at 22 days old (the 5th day after withdrawal), the antibody levels were significantly higher than those in the model control groups (P < 0.05). The levels of IL-4 and IFN-γ in the model control groups were significantly lower than those in other groups from pre-administration to the 5th day after withdrawal (P < 0.05) at 13 to 22 day-old (5 days before treatment to 5 days after withdrawal), and the contents of IL-6 were significantly higher than those in other groups (P < 0.05). In conclusion, under the condition of the current study, the plant essential oil has good preventive and therapeutic effects on chicks both mono-infected and co-infected with IBV, and the preventive and curative effects are mainly exerted through inhibiting viral replication, promoting immune organ development and antibody production, and regulating cytokines. The present study provides a new idea for the prevention and treatment of avian infectious bronchitis (IB), and also provides a reference for the effective treatment and prevention of coronavirus diseases in other animals.

Experimental Study on the Production of Cysts in Mice Infected with Toxoplasma gondii Tachyzoites of PRU Strain
Ruifang LI, Manyu ZHANG, Qing SUN, Jingying DU, Wei JIANG, Zengqiang LI, Luming XIA, Quan WANG
2024, 55(10):  4620-4629.  doi:10.11843/j.issn.0366-6964.2024.10.033
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This experiment was designed to obtain Toxoplasma cysts, which are often used as infectious materials in experimental studies related to chronic infection of Toxoplasma gondii, and to search for the most suitable mouse model for cysts passage. In this study, mice with KM, ICR, BALB/c and C57BL/6 strains were intraperitoneally inoculated with different numbers of tachyzoites of PRU strains. After 40 days of infection, antibodies in the serum of surviving mice were detected, and cysts were examined and isolated from the tissues of seropositive mice. Different strains of mice were infected by cyst intragastric administration to explore the cysts passage dose and the best passage mouse model. We successfully isolated cysts from the brains of C57BL/6 mice infected with low doses of Toxoplasma tachyzoites. When passing cysts, ICR mice had low mortality and significantly more cysts in the brain than other strains (P < 0.05). C57BL/6 mice can survive and form cysts in the brain when 10-50 tachyzoites are intraperitoneally inoculated; A larger number of toxoplasma cysts could be obtained by intragastric infecting ICR mice with 20-50 cysts.

Establishment of a Duplex Real-time PCR Method for Tetracycline Resistance Gene Detection in Clostridium perfringens
Lanxin OU, Bijin YE, Mingfei SUN, Nanshan QI, Juan LI, Minna LÜ, Xuhui LIN, Haiming CAI, Junjing HU, Yongle SONG, Xiangjie CHEN, Yibin ZHU, Lijun YIN, Jianfei ZHANG, Shenquan LIAO, Haoji ZHANG
2024, 55(10):  4630-4637.  doi:10.11843/j.issn.0366-6964.2024.10.034
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The purpose of this study was to establish a rapid and sensitive duplex real-time PCR method for tetA(P) and tetB(P) tetracycline resistance genes detection in Clostridium perfringens, aiming at providing a new strategy for the expedited identification of tetracycline antibiotic resistance within C. perfringens. Primers and probes were designed to target the conserved sequences of tetA(P) and tetB(P), construct recombinant plasmid, and optimize the annealing temperature and system. At the same time, validation of the method was substantiated through disc diffusion test for the determination of strain of tetracycline drugs sensitivity. The results showed that, the optimized reaction program was 95 ℃ 30 s; 95 ℃ 5 s; 56 ℃ 30 s, 39 cycles. And this method exclusively amplified the recombinant plasmid with no amplification curve observed for control strains. Intra-assay and inter-assay coefficients of variation were observed to be less than 1.3% and 1.8%, respectively. The plasmid standard had a minimum detection threshold of 102 copies·μL-1. Using this method, resistance gene detection was performed on 33 clinical isolates of C. perfringens, complemented by antimicrobial susceptibility testing. The detection method established by the current study yielded a detection rate of 75.8% (25/33) for tetracycline resistance genes, aligning significantly with the antimicrobial susceptibility testing results. In conclusion, this study successfully established a specific, sensitive, and highly reproducible duplex real-time PCR method for the detection of tetA(P) and tetB(P) tetracycline resistance genes in C. perfringens.

Basic Veterinary Medicine
Establishment of Cattle Precision-Cut Lung Tissue Slices (PCLS) Models Infected with Mycoplasma bovis in vitro
Hui ZHANG, Doukun LU, Yiqiu ZHANG, Gang ZHAO, Yingyu CHEN, Xi CHEN, Changmin HU, Aizhen GUO
2024, 55(10):  4638-4645.  doi:10.11843/j.issn.0366-6964.2024.10.035
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Mycoplasma bovis (M. bovis) is an important pathogen of bovine respiratory disease complex (BRDC), mainly causing pneumonia, mastitis, arthritis, keratoconjunctivitis, otitis, and genital disorders, leading to high economic losses in dairy and beef cattle production. Lack of small animals' models have greatly hindered the progress of effective vaccines and drugs for prevention and control in Mycoplasma bovis. In this study, we have established an in vitro infection model of M. bovis in Precision-cut lung tissue slices (PCLS). We collected bovine lungs from apparently healthy two-months cattle after slaughter, filled the lung airway with low melting point agarose, transferred it to the vibratome tissue slicer and then made the 250 μm-thickness precision cut lung slices. After M. bovis wildtype strain HB0801(P1) and its attenuated strain P150 at a MOI of 108 CFU infected, quantitative real-time PCR, lactate dehydrogenase cytotoxicity assay, 3D fluorescence microscope panoramic scanning, immunohistochemistry analysis were performed. The results showed that cultured PCLS remained 60% cell viability for at least 72 h and maintained normal structural integrity including edge sharpness and alveolar structure well in uninfected PCLS. After infected with wildtype and attenuated M. bovis strains respectively, we found that both M. bovis could survive and proliferate in PCLS, tropism to alveolar and bronchial space, and showed cytotoxic reactions until 18 h post-infection. We also observed that after 36 h incubation with M. bovis wildtype strain P1 and its attenuated strain P150, cultured PCLS produced pro-inflammatory factors IL-1β and IL-8 in PCLS, and M. bovis wildtype strain HB0801 could induce significantly higher level than P150 (P < 0.05). In conclusion, this study provides a model and method to investigate M. bovis infection in vitro which can mimic M. bovis infection in vivo. It is expected to provide reference for subsequent research on host-pathogen interaction in M. bovis.

Construction of a Passaged Porcine Alveolar Macrophage Cell Line Stably Expressing the Porcine BRD4-BD1/2 Protein and Its Effects on ASFV Proliferation
Mengli WU, Hualin SUN, Jifei YANG, Yaru ZHAO, Guiquan GUAN, Hong YIN, Qingli NIU
2024, 55(10):  4646-4659.  doi:10.11843/j.issn.0366-6964.2024.10.036
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Previous studies have identified that the host epigenetic regulator, bromodomain-containing protein 4 (BRD4) facilitates the replication of the African swine fever virus (ASFV). To further investigate the impact of BRD4 on ASFV replication, the BRD4-BD1/2 domain, which significantly enhances ASFV replication, was identified. Using a lentiviral expression system, a stably expressing 3D4/21 cell line of the BRD4-BD1/2 domain was successfully constructed to analyze replication differences between the 3D4/21-BRD4-BD1/2 cell line and the wild-type (WT) cell line. Initially, a recombinant plasmid, pLVX-IRES-puro-3x Flag-BRD4-BD1/2, targeting the porcine gene BRD4-BD1/2 and containing a 3x Flag tag, was constructed. This plasmid, along with plasmids pMD2.G and pSPAX2, was transfected into HEK-293T cells for lentiviral packaging to obtain infectious lentiviruses. Following lentiviral infection of 3D4/21 cells and selection with puromycin, a cell line stably expressing the BRD4-BD1/2 domain was established. The transcription levels of ASFV genes CP204L and B602L, and the expression levels of their corresponding proteins in ASFV-infected 3D4/21-BRD4-/BD1/2 cells, were detected using RT-qPCR and Western blot techniques, respectively. ASFV replication capacity was assessed using the HAD50 assay. The results showed that the stably expressing BRD4-BD1/2 domain in the 3D4/21 cell line significantly enhanced ASFV replication compared to the 3D4/21-WT cell line. This study provides biological material for in-depth research on the function of the BRD4 protein in ASFV replication and lays a theoretical foundation for the development of ASFV vaccine candidates.

Study on the Antibacterial Effect of T6SS Effector Protein Tae4 on S. aureus and L. monocytogenes
Leyang ZHAN, Jingxuan GOU, Manqi ZHANG, Weixuan FU, Shouxin LAN, Jian TU, Zhenyu WANG, Ying SHAO, Xiangjun SONG
2024, 55(10):  4660-4669.  doi:10.11843/j.issn.0366-6964.2024.10.037
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The aim of this study was to investigate the antibacterial effect of Tae4 amidase, a virulent effector protein of Type Ⅵ secretion systems, on Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes). The recombinant plasmid pET-28a-Tae4 was transformed into E. coli expression strain BL21 to induce expression, and the induction time, concentration and temperature of IPTG were optimized. Finally, the purified Tae4 protein was tested by Agar pore diffusion test, minimal inhibitory concentration test and antibacterial test combining with antibiotic on S. aureus and L. monocytogenes, respectively. SDS-PAGE results showed that the optimal expression conditions were as follows: the final concentration of IPTG was 0.5 mmol·L-1 at 16 ℃ for 16 h, the size of the recombinant protein was about 21 ku, and existed in the form of soluble supernatant. The results of Agar pore diffusion test showed that when the concentration of Tae4 protein was 500 μg·mL-1, S. aureus strains showed bacteriostatic zones above 13mm, and the effect was related to the serotype of strains, and L. monocytogenes strains showed bacteriostatic zones above 18 mm. Minimum inhibitory concentration test showed that MIC of Tae4 against S.aureus was about 250 μg·mL-1, MIC of Tae4 against L. monocytogenes was about 125 μg·mL-1, which were consistent with spot plate method and microbroth dilution method. The combination test showed that the bacteriostatic effect of Tae4 protein combined with penicillin was better than that of penicillin alone. Furthermore, the antibacterial effect of L. monocytogenes was better than that of S. aureus. In summary, Tae4 protein has bacteriostatic effect on S.aureus and L. monocytogenes, and the bacteriostatic effect combined with penicillin has been enhanced, which provides a theoretical basis for subsequent antibiotic substitution.

The Mechanism of Chelerythrine against Methicillin-resistant Staphylococcus aureus
Qingxin YUAN, Kuo LIU, Xuhua BAO, Dongyang GAO, He LI, Jun SONG, Zhixin ZHOU
2024, 55(10):  4670-4678.  doi:10.11843/j.issn.0366-6964.2024.10.038
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The objective of this study was to investigate the antibacterial effect of chelerythrine (CHE) against methicillin-resistant Staphylococcus aureus (MRSA) and approach its mechanism. The antibacterial activity of CHE against MRSA was determined by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). After treatment with CHE, the cell membrane and permeability of MRSA were assessed, and the morphology and ultrastructure of MRSA cells were observed by electron microscopy to preliminarily analyze the antibacterial mechanism of CHE. Additionally, the effect of CHE on reactive oxygen species (ROS) levels and ROS-related factors in MRSA strains was detected, combined with fluorescence quantification and transcriptomics to further explore the antibacterial mechanism of CHE. The results showed that the MIC of CHE against MRSA was 6.25 μg·mL-1, MBC is 12.5 μg·mL-1, and CHE can destroy cell membrane integrity and permeability to achieve antibacterial effect. CHE may block the electronic transmission of respiratory chain and promote ROS production by inhibiting the expression of gene SdhABC and other genes to exert activity against MRSA. In conclusion, this study explored the antibacterial mechanism of CHE against MRSA, which provided a new reference for clinical prevention of MRSA infection.

Effects of Methanomassiliicoccus DZ1 on Serum Trimethylamine-N-oxide and Inflammatory Factors, Liver Antioxidant Capacity and Cecum Microbiota in Mice
Xiaoxiu ZHAN, Pengyu LIU, Xiao'e XIANG, Shengyong MAO, Wei JIN
2024, 55(10):  4679-4689.  doi:10.11843/j.issn.0366-6964.2024.10.039
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Methanomassiliicoccales is an indigenous group of methanogens in the mammalian gut that can use trimethylamine (TMA) to produce methane, but the mechanisms of its action on the host remain unclear. The aim of this study was to investigate the effects of Methanomethylophilus sp. DZ1 on serum trimethylamine-N-oxide (TMAO) and inflammatory factors, liver antioxidant capacity, and cecum microbiota in mice. Fourteen five-week-old male B7/6J mice (body weight 18.4±1.1g) were randomly divided into a control group (n=7) and a treatment group (n=7), single cage feeding. Each mouse in the treatment group was given 200 μL DZ1 (1.09×109 cells·mL-1) daily, and each mouse in the control group was given 200 μL sterile PBS solution daily. The experiment lasted for 4 weeks. The results showed that the body weight and feed intake of mice in the treatment group had no significant changes (P>0.05). Serum TMAO concentration and inflammatory factor levels of mice in the treatment group were significantly decreased (P < 0.05). The activities of superoxidase dismutase (SOD) in the liver of the treatment group were significantly increased (P=0.035), and the total antioxidant capacity (T-AOC) of the liver was significantly increased (P=0.039). The results of 16S rRNA gene sequencing of cecal bacteria showed no significant difference in bacterial community structure between the treatment group and control group (ANOSIM, P=0.161). The relative abundance of Verrucomicrobia in the treatment group significantly decreased (P=0.064). The relative abundances of Lawsonibacter, Ruminococcus, and Akkermansia were significantly decreased (P < 0.05). In summary, the strain DZ1 had no significant effect on the bacterial community structure of the cecum in mice, but decreased the levels of serum TMAO and inflammatory factors, and increased the total antioxidant capacity of the liver.

Effects of Heat Stress on Duodenal Mucosal Structure, HIF-1 and Its Related Protein Expression in Congjiang Xiang Pigs
Yongqing LIU, Gang ZHANG, Yanling XIONG, Zhongxin SUN, Fan GAO, Ting LIU, Hui LI
2024, 55(10):  4690-4699.  doi:10.11843/j.issn.0366-6964.2024.10.040
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The aim of this study was to investigate the effects of heat stress (HS) on the duodenal mucosal barrier structure and the character of hypoxia inducible factor 1 (HIF-1) expression and regulation in Congjiang Xiang pigs. Twenty-four fattening pigs were divided into 6 groups randomly, the control group (Con) kept under normal conditions, and the other 5 groups were treated with heat stress for 6, 12, 24, 48, 72 h), respectively. Rectal temperature and respiratory rate were measured during treatment, and the duodenum of each group was collected at the end of the experiment. The change of duodenal tissue structure and goblet cell number in each group were observed with hematoxylin-eosin staining (HE) and alcian blue-periodate Schiff reaction (AB-PAS). The apoptosis cell of duodenal was detected by TUNEL staining. Real-time fluorescence quantification (qRT-PCR), Western blot and immunohistochemistry were used to detect the expression changes of tight junction related proteins, HIF-1 and its upstream regulatory factors respectively. The results showed that, compared with the control group, HS could induce rectal temperature and respiratory rate increase, and the villus height kept decreasing with extension of HS time, while the crypt depth increased synchronously, the ratio of villus height/gland depth and the expression of Occludin and ZO-1 decreased significantly. On the contrary, the number of goblet cells kept increasing. TUNEL showed the apoptosis rate of group HS 72 h was significantly higher than that of control group (P < 0.001).The positive expression of HIF-1α was mainly observed in small intestinal epithelial cells, and which expression enhanced at HS 72 h, the trend was as same as which HIF-1α mRNA and protein expression. The character of HSP90 expression were basically consistent with HIF-1α while the profile of PHD-2 was obviously the opposite. These results indicated that HS could induce apoptosis of small intestinal epithelial cells and damage duodenal mucosal barrier. The increase of HIF-1 expression is closely related to the apoptosis of small intestine cells, and may be regulated by both HSP90 and PHD-2.

Clinical Veterinary Medicine
Effects of Abortion on the Diversity of Vaginal and Intestinal Flora in Mares and the Isolation and Identification of Vaginal Bacteria
Han FU, Chong LU, Ronghao MIAO, Yabin LU, Jianlong LI, Jianhua LIU, Mingyang GENG, Qingyong GUO, Zhanhai MAI, Ling KUANG
2024, 55(10):  4700-4719.  doi:10.11843/j.issn.0366-6964.2024.10.041
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The aim of this experiment was to investigate the differences in the vaginal and intestinal flora structure of mares affected by abortion, and to isolate and identify vaginal pathogens and explore the biological characteristics of vaginal pathogens responsible for equine abortion. Vaginal secretion and faecal samples were collected from 10 mares in the aborted mare group and 6 mares in the healthy group. High-throughput sequencing of vaginal and faecal samples was performed in the V3-V4 region of the 16S rRNA to compare the differences in the vaginal and intestinal flora between the two groups, and to isolate and identify the vaginal bacteria in the aborted mares. Alpha diversity showed a trend of increased vaginal and intestinal flora richness and diversity in the aborted group compared with the healthy group. The similarity, dispersion, abundance and evolutionary relationship of the vaginal and intestinal flora of the two groups were found to be different by both Binary jaccard and unweighted unifrac distance matrix analyses. At the phylum level, compared with the healthy group, the vaginal flora of the abortion group showed a decrease in the abundance of Anaplasma and Clostridium phyla and an increase in the abundance of Helicobacter phyla and Thick-walled phyla, while the intestinal flora of the abortion group showed a decrease in the abundance of Thick-walled phyla and Anaplasma phyla, and an increase in the abundance of Aspergillus phyla and Actinobacterium phyla. LEfSe analysed Lactobacillaceae, Lactobacillus, Enterobacterales, Enterobacteriaceae, and Fusarium as the dominant vaginal flora species in the abortion group; and Actinomyces, Actinobacteria, Actinomycetia, Corynebacteriaceae, Corynebacterium, Peptoniphilus, and Peptinophilae were the dominant intestinal species in the abortion group, and the altered abundance of these bacteria may be correlated with the development of the disease, as well as with the prognosis. Functional prediction and correlation analyses revealed a positive correlation between normal vaginal bacteria and health-related microorganisms. Normal vaginal microbes were negatively correlated with abortion-associated pathogens. Metabolic pathways in the gut of aborted mares were highly correlated with vaginal disease pathways, and immune disease pathways in the gut were positively correlated with changes in the vaginal immune system, suggesting that metabolic dysfunction in the gut may trigger the development of certain vaginal diseases. Four main pathogenic bacteria were isolated and identified from the vagina of aborted mares: Salmonella, Streptococcus equi subspecies zoonoticus, Klebsiella and Escherichia coli. Vaginal and intestinal flora of mares are highly involved in the process of abortive disease and immunity, and the metabolic pathways of the flora may play a role in bridging the intestinal and vaginal flora and host immunity at the time of gestation, with the phyla Thick-walled and Actinobacteria in the vaginal flora and the phyla Arcanobacterium hippocoleae and Streptococcus infantarius were negatively correlated with the abortion-associated Aspergillus phylum. Salmonella may be the main causative agent of equine abortion and that Aspergillus phylum, Peptostreptococcus spp. and Campylobacter spp. may be the vaginal flora biomarkers for the occurrence of abortion.

The Therapeutic Effect of the Fecal Microbiota Transplantation on Calf Non-specific Pathogenic Diarrhea and Bacterial Diarrhea in Association with Their Gut Microbiota Changes
Zuobin YANG, Jincheng SHI, Ziwei MA, Rulong CHEN, Zhan SHU, Xin LI, Jinquan WANG, Qi ZHONG, Xuelian MA, Gang YAO
2024, 55(10):  4720-4734.  doi:10.11843/j.issn.0366-6964.2024.10.042
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The aim of this study was to compare the therapeutic efficacy and the changes of gut microbiota by faecal microbiota transplantation (FMT) treatment between non-specific pathogenic diarrhea and the bacterial diarrhea in calves. In the study, 8 healthy newborn calves were selected as the health (Health, H) group, 24 newborn calves with clinical symptoms of diarrhea were selected, in which 16 diarrheic calves without diarrheic related pathogens were assigned to the non-specific pathogenic diarrhea (Diarrhea, D) group, and the other 8 diarrheic calves with STEC infection were assigned to the STEC diarrhea (STEC-Diarrhea, SD) group after diarrhea-associated pathogens detection. The average age of all calves was 14.8±6.1 days. After FMT group D was named FMT-D and group SD was named FMT-SD. Donor calves were screened by clinical symptoms of diarrhea with pathogenic test, and then the fecal microbiota solution was prepared for oral treatment (250 mL each calf, contains 40 g feces of single donor). Bristol Stool Scale (BSS) was used to assess the effectiveness of FMT. The healing day and the daily gain (DG) were recorded, and calves' blood physiological parameters and inflammatory cytokines were determined. Rectal feces were collected for 16S rRNA gene sequencing to analyze the changes of gut microbiota. The results showed that after FMT, BSS of calves in group D and SD was extremely decreased (P < 0.0001) from type 6-7 to type 4-5, and after decline was not significantly different from that in group H (P>0.05). There was no significant difference in the healing day between group FMT-D and group FMT-SD (P>0.05). At day 150 after FMT, DG of FMT-D group was not significantly different from that of group H (P>0.05), whereas DG of group FMT-SD was still significantly lower than that of group H (P < 0.05). IL-1β and IL-6 as well as IL-10 were extremely higher in group D and group SD than those in group H (P < 0.01), and after FMT they were decreased to the level of H group (P>0.05); Secretory immunoglobulin A was extremely lower in group D and group SD than that in group H (P < 0.001), and IL-22 was significantly lower in group D than that in group H (P < 0.05), which were both increased to the level of group H after FMT (P>0.05). The richness and diversity of gut microbiota in group D and group SD were significantly lower than that in group H (P < 0.01), while increased after FMT. There were extremely significantly different gut microbiota structure of group D and group SD from that of group H (P < 0.001). The relative abundance (RA) of Fusobacteriota was extremely higher in both group D and group SD than that in group H (P < 0.001), the RA of genera Escherichia_Shigella, Tyzzerella, Faecalibacterium, Fusobacterium, [Ruminococcus]_gnavus_group, Butyricicoccus, Collinsella and Prevotella was significantly higher in group D and group SD than those in group H (P < 0.05), After FMT they were decreased to the level of group H. Moreover, The RA of genera Muribaculaceae, Rikenellaceae_RC9_gut_group, [Eubacterium]_coprostanoligenes_group, Intestinimonas, Clostridia_UCG_014, Subdoligranulum and Breznakia was extremely reduced in group D and group SD in comparison with group H (P < 0.01), then increased in both group FMT-D and group FMT-SD after FMT, which was reached to the level of H group (P>0.05). FMT treatment has a significant therapeutic effect on both aforementioned kinds of diarrhea in calves via reducing the relative abundance of some pathogenic genera, while increasing that of some potentially probiotic ones in the gut microbiota of diarrheic calves, thus resulting in the composition and structure of the gut microbiota tended to be healthier, and host immune function is enhanced, FMT treatment may have long-term beneficial impact on calves' gain and grows. However, the gut microbiota restoration by FMT treatment between these two kinds of diarrhea calves still remains some difference.

The Preventive Effect of Schisandrin A on Chicken Colibacillosis and Its Impact on Immune Function
Jialu BAO, Yan ZHANG, Xiaodan WANG
2024, 55(10):  4735-4746.  doi:10.11843/j.issn.0366-6964.2024.10.043
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To investigate the preventive effect of schisandrin A against chicken colibacillosis, 180 one-day-old chicks were divided into the following groups: control group, Escherichia coli infection group, 50 mg·kg-1 schisandrin A group, 100 mg·kg-1 schisandrin A group, 200 mg·kg-1 schisandrin A group, and antibiotic group (florfenicol). After 5 days of administration, except for the control group, all other groups were intraperitoneally inoculated with E. coli suspension. The chicks' body weight, immune organ index, spleen histopathological and ultrastructural changes, blood cell count, serum inflammatory factors, antibody levels, and immunoglobulin changes were evaluated. The results demonstrated that pre-feeding schisandrin A improved the body weight of infected chicks, significantly reduced the spleen index, alleviated spleen tissue damage and cellular nuclear and mitochondrial injuries caused by E. coli infection. Furthermore, 200 mg·kg-1 schisandrin A significantly reduced the white blood cell count in infected chickens, while both 100 mg·kg-1 and 200 mg·kg-1 schisandrin A groups significantly decreased the levels of TNF-α, IL-1β, IL-6, and IL-8 in the serum of infected chickens, and significantly increased the antibody levels of IgM, IgG, and NDV. In summary, schisandrin A exerted a disease-resistant effect against avian colibacillosis by modulating the immune response.

High Levels of Boron Exposure Aggravated Hepatocytes Damage in Broilers via Pyroptosis
Ting HE, Siying LIU, Jinwen QUAN, Weiqian SU, Jiangli HUANG, Yumeng LI, Zhonghua LIU, Wenlan YU
2024, 55(10):  4747-4759.  doi:10.11843/j.issn.0366-6964.2024.10.044
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We aim to explore the mechanism of boron-induced hepatocytes damage in broilers. Thirty AA broiler chickens were randomly divided into control group (CG), low boron group (LBG) and high boron group (HBG). They were fed with different additional doses of boron in the basal diet: the basal diet (boron 0 mg·kg-1), low boron diet (boron 120 mg·kg-1) and high boron diet (boron 240 mg·kg-1), respectively. Liver tissues were collected 7 and 14 days after treatment. The content of boron in liver tissue was determined by ICP-MS, the ASL and ALT levels were tested by automatic blood biochemistry analyzer, the pathological changes in liver tissue were observed by H & E staining, ultrastructural changes in liver tissue were observed by transmission electron microscopy, and the localized expressions of NLRP3 and IL-1β in liver tissue were detected by immunohistochemistry and immunofluorescence. The mRNA and protein expression levels of NLRP3, Casapse-1, IL-1β, IL-18, and GSDMD were detected by RT-qPCR and Western blot. The results showed that the amount of boron stored in the liver tissue as along dietary boron levels increased, with the duration and dosage of exposure. In addition, the structural integrity of the liver tissue of 14-day-low boron group (14 d-LBG) and 14-day-high boron group (14 d-HBG) was significantly compromised, with an increase in pyroptosis levels with an increase in boron dosage. Compared with CG at the same time, a significant upregulation of mRNA expression levels of NLRP3, Caspase-1 and IL-18 was observed in the 14 d-LBG(P < 0.05). Furthermore, a significant upregulation of both mRNA and protein expression levels of NLRP3, Caspase-1, IL-1β, IL-18 and GSDMD was observed in the 14 d-HBG (P < 0.05). High levels of boron exposure could induce hepatocytes damage in broilers through the pyroptosis pathway.

Effect of Rumen Acidosis on Gastrointestinal Function, Morphology, and Microflora in Goats
Daoliang ZHANG, Hongyan DING, Liuxing WANG, Wenjun TAI, Hao KONG, Chang ZHAO, Shibin FENG, Xichun WANG, Yanfeng XUE, Jinjie WU, Yu LI
2024, 55(10):  4760-4772.  doi:10.11843/j.issn.0366-6964.2024.10.045
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This study utilized a model of rumen acidosis induced by the administration of corn flour to investigate its impact on the gastrointestinal tract flora structure of goats. In this experiment, eight healthy Boer goats were selected as test subjects, 4 in the control group and 4 in the acidosis group after modeling. Gastrointestinal(GI) tissues were collected for HE staining, RT-qPCR, and Western blot assay to investigate the effects of rumen acidosis on the morphology and functional proteins of goat GI tissues. Illumina MiSeq high-throughput sequencing was used to determine the 16S rRNA of the bacteria in rumen fluid and cecum content, aimed to sutdy the effect of acidosis on the bacterial communities in the gastrointestinal tracts. The results showed that, compared with the control group, the tissue morphology and structural integrity of the rumen and small intestine were damaged in the acidosis group. The relative expression levels of MCT1 mRNA and protein in rumen and duodenum were extremely significantly increased (P < 0.01), while the relative expression levels of SLC5A8 mRNA and protein were extremely significantly decreased (P < 0.01). The results of high-throughput sequencing in rumen contents showed that, the Shannon index of Alpha diversity analysis in the acidosis group was significantly lower than that in the control group (P < 0.01), and the relative richness of Bacteroidetes and Synergistetes was significantly decreased (P < 0.01). The relative abundance of Fibrobacteres and Tenericutes was significantly increased (P < 0.05), while the relative abundance of Ruminococcus and Prevotella was significantly decreased (P < 0.05). After high-throughput sequencing of cecum contents, compared with the control group, the relative richness of Ruminococcus and Oscillospira in acidosis group was significantly decreased (P < 0.05), while the relative richness of Clostridium was significantly increased (P < 0.05). In conclusion, rumen acidosis can destroy the integrity of digestive tract morphology, affect the expression of related proteins, and disturb digestive tract flora structure.

Research Notes
Preparation and Application of N Protein Polyclonal Antibody of Feline Infectious Peritonitis Virus SH2021 Strain
Fukang LIU, Ligang YUAN, Da ZHANG, Aoxing TANG, Guangqing LIU, Jie ZHU
2024, 55(10):  4773-4778.  doi:10.11843/j.issn.0366-6964.2024.10.046
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This study aimed to express the nucleocapsid protein (N) of feline infectious peritonitis virus (FIPV) strain SH2021 in vitro and prepare rabbit polyclonal antibodies. Primers were designed based on the N gene sequence of FIPV strain SH2021 to construct the recombinant expression plasmid pCold-I-N. Subsequently, the recombinant plasmid was transformed into expression-competent cells BL21 (DE3), and expression was induced under conditions of 1.0 mmol·L-1 IPTG and 16 ℃. Finally, purification was performed using a His column, and the purified recombinant protein was used to immunize New Zealand white rabbits to prepare polyclonal antibodies. The experimental results showed that the purified recombinant N protein had a size of approximately 45 ku, and the titer of the prepared rabbit polyclonal antibodies against the N protein reached 1∶512 000. The antibodies exhibited good reactivity and specificity towards the N protein. This study successfully prepared rabbit polyclonal antibodies against the FIPV N protein, which showed good reactivity and specificity, providing important tools for FIPV antigen-antibody detection and research on the biological functions of the N protein.

The Establishment of a SYBR Green Fluorescence Quantitative PCR Detection Method by Targeting the G-protein Coupled Chemokine Receptor Gene of Goat Poxvirus
Hongqiang ZHANG, Shanhui REN, Wei YAO, Zhenli GONG, Xue YANG, Chunling MA, Ting YOU, Yuzhe ZHANG, Minyi LIU, Wenjie QIAN, Liuyang LI, Zhipeng YU, Yuefeng SUN, Haotai CHEN, Jiangfeng FAN
2024, 55(10):  4779-4784.  doi:10.11843/j.issn.0366-6964.2024.10.047
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Based on the genomic sequence of the goat pox virus (GTPV), we have established a fluorescence quantitative PCR method targeting the G-protein-coupled chemokine receptor (GPCR) gene. Six pairs of specific qPCR primers were designed based on the whole genomic sequence of GTPV. The specificity and sensitivity of these six qPCR primers were screened and detected. A pair of qPCR primers with high specificity and sensitivity was finally screened and obtained. According to the GPCR gene sequence of the GTPV AV41 vaccine strain, we designed an ordinary PCR primer pair to amplify the GPCR gene to construct the eukaryotic expression vector, which was used to establish the standard curve. Results showed that A pair of specific qPCR primers targeting the GPCR gene was obtained. The eukaryotic expression plasmid named pCAGGS-GPCR was constructed. The standard curve of y=-3.5289x+49.07 was established, whose linear correlation coefficient is R2=0.997 2 and amplification efficiency is 92.7%. The results of the replication experiment suggested that the lowest detective limitation of this primer was 2.0 copies·μL-1, and the coefficient variation of the reproducibility results within and between batches was 2%, indicating the advantages of reasonable specificity, repeatability, and sensitivity. We have successfully established a specific fluorescence quantitative PCR method targeting the GPCR gene of GTPV, which provides adequate detecting support for the veterinary clinical diagnosis and the prevention and control of goat poxvirus.