The circadian system is an internal timer system in most mammals, as the main circadian clock, the hypothalamic suprachiasmatic nucleus (SCN) coordinates the timing of the organizational clock to align internal rhythms with environmental cycles. Cells in the SCN core are capable of receiving light signaling directly from intrinsically photosensitive retinal ganglion cells (ipRGCs), whose endogenous oscillations determine behavioral and physiological rhythms throughout the animal. Estrogen is capable of regulating a variety of biological processes of mammals, including but not limited to the circadian rhythmic behavior of the estrous cycle. Estrogen and SCN synergistically regulate GnRH secretion, LH peak, ovulation, and resulting in the regu-lation of the mammalian reproductive process. This paper reviews the recent research progress in the mutual regulation of estrogen and circadian rhythm in female mammals, including the phenomenon of estrogen secretion and circadian rhythm, the molecular link between estrogen and circadian rhythm, and their regulation mechanism on the reproduction of female mammals. These results can provide a reference for further exploration of the reproductive rhythm and hormone regulation mechanism of female mammals.

This article reviewed the clinical diagnostics, laboratory diagnostics and circulating cell-free fetal DNA(ccffDNA) detection of pregnancy from the following four aspects:principle, method, diagnostic time and accuracy. Through the comparison and comprehensive analysis of the above methods, The advantages, limitations, research trends and the actual application effects of the above diagnostic methods were summarized, in order to provide suitable methods of livestock pregnancy diagnosis applied in different situations. The traditional clinical diagnostics have the double advantages of fast and cheap, but the accuracy is susceptible to the artificial operation. Laboratory diagnostics are earlier and more accurate, but they have disadvantages of lacking unified standard and costing more. The detection of ccffDNA can be used to identify fetal sex in domestic animals, whereas the specific markers of non-sex-dependent fetal DNA are still needed to be explored further.

The microorganisms habituated in rumen are essential for rumen development, nutrient digestion, and host physiological health. Rumen microbiota transplantation (RMT) refers to transplant specific donor rumen microbiota and their metabolites to the recipient for improving host functional status, production efficiency and product quality, and treating some gastrointestinal diseases of ruminants, by remodeling microflora or restoring microbiome. This article reviewed the development history, application, safety and potential mechanism of RMT, and further elaborated the roles of RMT in revealing the characteristics or functions of rumen microbiota, regulating the balance of microflora and host metabolism, improving growth performance, treating disease, which provided references for the research and application of RMT.

The gastrointestinal tract of animals is the part which directly contacts the external environment and is always exposed to various exogenous and endogenous substances. Hence, there are functional epithelial barriers in the gastrointestinal tract to prevent various substances from invading the tissue. Gastrointestinal microbes play an important role in maintaining the homeostasis and strengthening of the gastrointestinal epithelial barrier. The gastrointestinal epithelial barrier interacts with microorganisms to regulate the growth, development, metabolism and immune function of the host gastrointestinal tract, and the establishment of the gastrointestinal epithelial barrier function of the host affects the colonization and distribution of gastrointestinal microorganisms. This review summarizes the structure and function of the gastrointestinal epithelial barrier and the interaction between gastrointestinal microorganisms and gastrointestinal epithelial barrier. Aiming to provide reference for the study of the relationship between gastrointestinal microorganisms and gastrointestinal epithelial barrier.

At present, problems such as excessive feeding density, feed waste, low production efficiency and environmental pollution are common in intensive and large-scale laying hen farms in China. The precision feeding technology of commercial laying hens can systematically monitor the organism condition of laying hens at different stages and develop personalized feeding programs based on the characteristics of nutritional requirements and changes in feed intake of laying hens, which can improve feed conversion and production performance, increase economic efficiency, reduce environmental problems such as nitrogen and phosphorus emissions, and promote the sustainable development of laying hen farming. In this paper, the application points and development trends of precision feeding technology for laying hens were reviewed in terms of nutritional control measures, feeding management and environmental control, and immunization and hygiene in different stages of laying hens, with the aim of improving the level of commercial laying hens and improving breeding efficiency.

Invariant chain (Ii) is an important immune molecule. It is found that Ii is not only a MHCII chaperone, but also has multiple functions. In this paper, the basic structure, main functions, intracellular assembly and transport of Ii and the applications of Ii as a carrier are summarized. Secondly, it reviews the new progress of Ii research in recent years:the functional domain of interaction between Ii and MHC molecular and their intracellular localization characteristic, the function and pathway of regulating immune cells such as T and B, Ii and macrophage migration inhibitory factor to initiate signaling pathways and corresponding domains of inflammatory response; Various enzymes and bioactive factors associated with Ii and their action characteristics, diseases associated with Ii; Vaccine vector based on Ii structure and its mechanism of enhancing immunity; And the research results on livestock, poultry and fish. Finally, the development trend of Ii in the theoretical research and practical application in the field of animal husbandry and veterinary is prospected. This review provides scientific knowledge and the latest progress in understanding the multifunctional role and mechanism of Ii in animal immunity from the macro and micro aspects, and provides useful reference for promoting related research.

Toxoplasma gondii is a worldwide obligate intracellular protozoa that infects almost all warm-blooded animals and causes zoonotic toxoplasmosis. Individuals with normal immune function are often infected with Toxoplasma gondii without obvious clinical symptoms and show recessive infection, but pregnant women and individuals with immune deficiency can lead to serious consequences and need effective treatment. Toxoplasmosis is a common cause of abortion in sheep and goats worldwide. And it has a serious impact on human health and livestock production. Therefore, the control of human and animal toxoplasmosis is very important for public health and the economic development of animal husbandry. Previous drugs used to treat toxoplasmosis have been limited in their effectiveness due to side effects and/or disease staging. How-ever, nanoparticles can effectively overcome the limitations of traditional drugs. This article reviews the new progress of nanotechnology in the treatment of toxoplasmosis, in order to provide new ideas for the research of anti-toxoplasmosis drugs.

Contagious ecthyma, commonly known as orf, is a highly contagious and epitheliotropic zoonotic infectious disease caused by orf virus (ORFV), blocking the development of animal husbandry and threatening human health. Although ORFV infection can induce strong immune and inflammatory response, the host can be repeatedly infected. This is mainly because ORFV has developed and evolved multiple immune evasion mechanisms during the long-term interaction with the host. ORFV achieves immune evasion mainly by inhibiting interferon response, inhibiting NF-κB signaling pathway, inhibiting inflammatory response, regulating apoptosis, cell cycle, autophagy and vascular proliferation. The immune evasion of ORFV is mainly due to the multiple immunomodulatory proteins encoded by the virus. In this article, the latest research progress on host immune response against ORFV infection and the immune evasion mechanisms of ORFV are summarized, which provides ideas for orf vaccine development and comprehensive prevention and control of the disease.

In order to better protect and utilize the Hechuan black pigs, the geneitc diversity, kinship, inbreeding coefficient, family structure and selection signatures of Hechuan black pigs conserved population were explored in this study. The 50K SNP chip was used to analyze the SNPs in 58 healthy adult Hechuan black pigs conserved population. The effective population size, polymorphic information content, polymorphic marker ratios, expected heterozygosity, observed heterozygosity, effective allele numbers and minor allele frequency was calculated to analyze the genetic diversity of the conserved population. The identity by state (IBS) diatance matrix and runs of homozygosity (ROH) for each sample was calculated by Plink software and the genomic relationship G matrix was calculated by Gamatrix (V2) software to analyze the kinship of the conserved population. The population cluster analysis was calculated by Mega X (V10.0) software to analyze the family structure of the conserved population. Tajima's D and iHS methods were used to analyze the selected signal regions on the genome of Hechuan black pig population and explore potential candidate genes. The results showed that the effective population size, average polymorphic information content, polymorphic marker ratios, average effective allele numbers, minor allele frequency of the Hechuan black pigs conserved population were 4.2, 0.156, 0.534, 1.38 and 0.141 respectively, which indicated that the genetic diversity of Hechuan black pigs conserved population was relatively abundant. The average expected heterozygosity and observed heterozygosity were 0.255 and 0.271, which indicated that the conserved population has been differentiated. The average IBS genetic distance of 58 Hechuan black pigs was 0.199 2±0.042 9, and the average IBS genetic distance of 26 breeding boars was 0.176 7 ±0.048 4. The results of IBS genetic distance and G matrix both suggested that some individuals had closely relattionship with each other. A total of 2 246 ROH, gatherring in individuals with the most ROH by 1-10 Mb length (79.12%) and 40-50 (48.28%) numbers, were founded in the Hechuan black pigs conserved population.A total of 2 246 ROH fragments were found in the Hechuan black pig population. The number of ROH fragments with a length of 1-10 Mb accounted for the most(79.12%), and the number of individuals with 40-50 ROH accounted for the most(48.28%). The average inbreeding coefficient of conserved population was 0.175. The Hechuan black pigs conserved population includes 7 families with boars and 1 family without boars, and the number of individuals in each family were significantly different. Using Tajima's D and iHS methods, 192 and 303 significant selection signal regions were detected, which contained 193 and 331 candidate genes, respectively. Five candidate genes were detected simultaneously by Tajima's D and iHS. HVCN1 was associated with sperm motility and sperm cryotolerance. TRAF3IP1 and PER2 were associated with immune function. In general, the genetic diversity of Hechuan black pigs conserved population was relatively abundant. However, there was a greater risk of inbreeding among some individuals, and the number of individuals in each family was significantly different. The number of boars in some families is small, thus, there was a risk of ancestry loss in this families. In addition, during the adaptive evolution of Hechuan black pigs, the related genes of reproduction and immune traits were selected to a certain extent.

This study aimed to perform genome-wide association study for candidate gene identification of growth traits in Duroc pigs. In this study, 361 Duroc boars were selected as the experimental population, the days to 100 kg, average daily gain to 100 kg, average backfat thickness to 100 kg and loin muscle area to 100 kg traits were measured. The genotype information was typed by 50K single nucleotide polymorphism array, and 31 618 SNPs were obtained after quality control. The genetic parameters of growth traits were estimated using genome information by GCTA software, and candidate genes related to growth traits were identified by GWAS using R software rMVP package FarmCPU model. The results showed that the heritabilities of the days to 100 kg, average daily gain to 100 kg, average backfat thickness to 100 kg and loin muscle area to 100 kg were 0.27, 0.29, 0.16 and 0.11, respectively, which were moderate heritability traits. The genetic correlation and phenotypic correlation of days to 100 kg and average daily gain to 100 kg were -0.99, indicating the strong negative correlation. The results of GWAS showed that 3 significant SNPs were detected on the days to 100 kg and average daily gain to 100 kg traits, located on chromosome 10. Multiple comparison of allelic genotypes of significant SNPs was performed by Least significant difference test. The dominant alleles of significant SNPs rs81237156, rs81424502 and rs81313018 were G, T, and A, respectively. Candidate genes HACD1 and BAMB were identified to be associated with pig growth traits in the candidate region near significant SNPs. In this study, the newly discovered candidate genes will promote the understanding of growth traits, and the identification of new variants can provide new insights for potential markers in pig breeding.

The purpose of this experiment was to evaluate the differences in carcass traits, meat quality, and flavor substances between Rongchang (RC) pigs and Duroc×Landrace×Yorkshire (DLY) pigs, in order to provide basic data for further interpretation of the flavor characteristics of Rongchang pigs. DLY pigs ((117.75±7.19)kg) (n=8) and RC pigs ((112.40±8.78)kg) (n=10) in good health, with similar weights at slaughtering time were selected for the study, each group consisting of half female and male. Carcass traits and conventional meat quality were measured after slaughter. Sensory bionic evaluation technology was used to determine the taste profile and odor profile of pork.The volatile flavor substances in pork were determined using HS-SPME-GC-MS technique. The Results showed that:1) Compared with the DLY pigs, the straight length, skew length, and eye muscle area of RC pigs decreased significantly (P<0.05), while the average back fat thickness significantly increased (P<0.01). 2) Compared with the DLY pigs, longissimus dorsi a^*_{45 min,} a^*_{24 h} cohesion of RC pigs significantly increased (P<0.05); whereas L^*_{24 h}, dripping loss, shear force and hardness significantly reduced (P<0.05). 3) Electronic tongue analysis showed that the taste profile of the longissimus dorsi of RC pigs were significantly different from that of DLY pigs, and the taste richness and saltiness response value of RC pigs significantly increased (P<0.05). 4) Electronic nose analysis showed that the odor profile of the longissimus dorsi of RC pigs were significantly different from that of DLY pigs. The response value of W2S sensor, which mainly responded to alcohols, aldehydes and ketones, was significantly increased in RC pigs as well (P<0.01). 5) Based on HS-SPME-GC-MS technology, 76 volatile flavor compounds were identified in the longissimus dorsi of RC pigs and DLY pigs. Among them, 16 keys volatile flavor compounds were significantly increased in RC pigs (VIP>1 and P<0.05). RC pig has bright red color, and high quality of meat tenderness and waterholding power. Sixteen volatile flavor substances, including hexanal, valeraldehyde, 2,3-octanedione, ethyl acetate, allyl butyrate, and 2-ethylfuran, may play an important role in the formation of flavor differences between RC pigs and DLY pigs. Overall, these findings provide basic data for promoting the development and utilization of RC pig germplasm resources.

This study aimed to investigate the effect of miR-145-5p on the proliferation and differtiation of porcine skeletal muscle satellite cells. The tissue expression profile and developmental expression pattern of miR-145-5p were detected by qRT-PCR. The skeletal muscle satellite cells were isolated and cultured from the extensor digitorum longus of one-day old Jinfen White pig. The miR-145-5p simulant (mimics) and its control group (mimics NC), miR-145-5p inhibitor (inhibitor) and its control group (inhibitor NC) were transfected respectively. Each treatment had three replicates. The proliferation-related genes, the number of EdU-positive cells and the proliferative activity of cells were detected by qRT-PCR, EdU and CCK-8 methods. After the differentiation of porcine skeletal muscle satellite cells, qRT-PCR, Western blot and immunofluorescence staining were used to investigate the level of mRNA and protein of differentiation related genes and the formation of myotubes. The effects of miR-145-5p on the downstream IGF1R and AKT pathways were investigated by double luciferase test, qRT-PCR and Western blot. The results showed that the expression of miR-145-5p was the highest in the liver and lung, followed by in heart and subcutaneous fat (P<0.05). The expression of miR-145-5p in the longissimus dorsi muscle continued to increase with the increase of age (P<0.05). During the culture procession of porcine skeletal muscle satellite cells in vitro, the expressions of Ki67 and CDK1 (P<0.01), and PCNA and CDK4 (P<0.05) were down-regulated significantly after transfection of miR-145-5p mimics. The positive cell index was very significantly decreased (P<0.01), and the cell viability was also significantly decreased (P<0.05). Regarding to the differentiation of porcine skeletal muscle satellite cells, the expressions of MyOD, MyOG and Myf5 were down-regulated significantly (P<0.01) by overexpression of miR-145-5p, the level of MyOD protein was inhibited (P<0.05), and the MyHC-positive myotube area was also less than that of the control group. Transfection of miR-145-5p inhibitor resulted in the opposite result. Overexpression of miR-145-5p significantly reduced the relative expression of IGF1R mRNA and protein(P<0.05), and significantly reduced the phosphorylation level of AKT protein. Interference with miR-145-5p increased significantly the relative expression of IGF1R mRNA and protein(P<0.01), and rescued the inhibitory effect of si-IGF1R on p-AKT protein(P<0.01). In conclusion, miR-145-5p negatively regulates the relative expression of IGF1R mRNA and protein, affects AKT pathway, inhibits the proliferation and differentiation of porcine skeletal muscle satellite cells, and then participates in the development of skeletal muscle. The results of this study enrich the molecular network of pig muscle growth and development, and provide molecular targets for molecular breeding of muscle traits.

The purpose of this study was to identify the differentially expressed genes (DEGs) and signaling pathways between Beijing You chicken and Guangming No.2 broiler line B(Guangming broiler) in response to heat stress, and to reveal the molecular regulatory mechanisms underlying heat tolerance of different chicken breeds by analyzing spleen transcriptomic data of two chicken breeds Beijing You chicken and Guangming No. 2 broiler line B (Guangming broiler) under heat stress and normal rearing conditions. The animals were 25-day-old Beijing You chickens and Guangming broilers under the same rearing managements. Heterophil to lymphocyte ratio (H/L), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were measured, and spleen tissues were collected for RNA-Seq. WGCNA analysis was performed, in combination with gene expression matrix based on RNA-Seq data and the phenotypes, to screen the modules and genes with high correlation with the traits. Comparison of the transcriptome sequencing results between the control and heat stress groups showed that 313 DEGs in Beijing You chicken before and after heat stress, of which 169 genes were up-regulated and 144 genes were down-regulated; while 235 DEGs in Guangming broiler, of which 152 genes were up-regulated and 83 genes were down-regulated. Through WGCNA, 2 modules with high correlation with H/L were screened in Beijing You chicken, while 4 modules with strong correlation with H/L and T-AOC were screened in Guangming broiler. Screening of the Hub gene in the module revealed the presence of the TRIM29 gene in both breeds, which plays an important role under heat stress conditions. In this study, the differential patterns of gene expression in different chicken breeds under heat stress were revealed by RNA-Seq. The candidate genes related to heat tolerance were identified, which provided novel ideas and clues for further study of heat tolerance mechanism in chickens.

The purpose of this study was to investigate the quantitative differences in tissue morphology and molecular regulatory pathways alterations between the normal individuals and myopathies including wooden breast (WB) and white striping (WS) occur separately and concomitantly of chicken pectoralis major. The research based on the 42-day-old paternal line of the fast growing white feather broiler terminal. According to the apparent judgment criteria of wooden breast and white striping, 4 groups of pectoral muscle tissue samples were screened. The 4 groups were normal group, WB group, WS group, WB accompanied by WS group, respectively. Statistics and analysis of 61 individual myofiber tissue morphological indicators were conducted. According to the apparent judgment score and histological statistics, tissue samples were selected from the normal group and the wooden breast group for mRNA sequencing. The histological results showed that the area, diameter and endomysial thickness of myofibers in the WB group were significantly higher than those in the other 3 groups (P <0.05), while the density of myofibers in the WB group was significantly lower than that in the WS and WS+WB groups (P <0.05). Transcriptome analysis screened out 1 201 upregulated expression difference genes in the WB group compared with the normal group, and there was no significant downregulated expression difference gene. KEGG results showed that the upregulation genes were mainly enriched in connective tissue proliferation related pathways including gap junctions, focal adhesion, ECM-receptor interaction and collagen fibril organization. Real-time PCR verification of genes related to processes such as connective tissue proliferation were screened from the enrichment pathways. The result showed that the expression trend of differentially expressed gene IGF1R, NOS2, ITGB2, ITGA11, FMOD and others were consistent with the transcriptome results between wooden breast and normal groups. In summary, this study report that the average endomysial thickness of normal individual is more than 75% higher than wooden breast of pectoral muscle, which is likely to be related to excessive proliferation of connective tissue. ITGA11 and FMOD can be used as candidate genes for myopathies pectoral muscle for later in-depth research.

The study aimed to determine the regulatory effect of goat apolipoprotein A-IV (APOA4) on intramuscular adipocyte differentiation. The animals used in this experiment were 1 year old Jianzhou big ear goat rams (n=5) with good physiological state. The APOA4 gene sequence of goat was cloned by RT-PCR, and the cloned sequence was analyzed by bioinformatics analysis, and the sequence characteristics were obtained. Temporal expression profiles of APOA4 in tissues and cells of goat were constructed by qPCR. By constructing the goat APOA4 gene overexpression vector, this experiment was divided into an overexpression group (pEGFP-N1- APOA4) and a negative control group (pEGFP-N1), with 3 replicates in each group and 2 samples in each replicate. Transfected pEGFP-N1-APOA4 overexpression vector and pEGFP-N1 into goat intramuscular precursor adipocytes and induced differentiation. Oil red O staining, Bodipy staining, DAPI staining, absorbance (OD490 nm) and qPCR were used to determine the effects of overexpression of goat APOA4 gene on adipocyte differentiation and adipogenesis markers. The results showed as follows:1) The APOA4 gene sequence of goat was 1 467 bp, in which the CDS region was 1 143 bp, encoding 380 AA. 2) Bioinformatics analysis predicted that APOA4 was negatively charged and acid-unstable hydrophobic protein. 3) APOA4 was widely expressed in goat tissues, and the highest expression level was found in liver, which was significantly higher than that in other tissues (P<0.01). APOA4 expression was the highest in intramuscular adipocytes at 48 h after induction of differentiation. 4) Compared with the control group after APOA4 gene overexpression, the accumulation of lipid droplets in intramuscular adipocytes was decreased, and the expression levels of adipose-differentiation markers C/EBPα and PPARγ were significantly down-regulated (P< 0.01), and the expression levels of SREBP1 and C/EBPβ were significantly down-regulated (P<0.05). Lipid metabolism marker genes AP2 and LPL were significantly down-regulated (P<0.05). Based on morphological staining and molecular biology results, APOA4 gene may inhibit the differentiation of adipocytes and lipid droplet accumulation in goats by inhibiting the expression of differentiation markers C/EBPα, PPARγ, SREBP1, C/EBPβ and lipid metabolism markers AP2 and LPL. It was suggested that overexpression of APOA4 gene inhibited the differentiation of intramuscular adipocytes.

The aim of this study was to analyze the genetic diversity and population structure of Yongdeng Qishan sheep by specific-locus amplified fragment sequencing (SLAF-seq), and to provide support for the declaration of Yongdeng Qishan sheep as a newly discovered resource. Four local sheep populations (10 adult healthy ewes randomly selected from each population) in Gansu Province were taken as the research object. Single nucleotide polymorphisms(SNPs) within the whole genome were detected by SLAF technology. Five genetic diversity indexes, such as observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), Shnnon Wiener index (SHI) and Nei diversity index (Nei), were calculated by Perl software. The linkage disequilibrium of each breed was analyzed by PopLDdecay software. elgensoft software was used for principal component analysis (PCA); mega x software was used to build phylogenetic trees of 4 sheep breeds; The structure software was used to analyze the genetic structure of the population; gcta software was used for kinship analysis. A total of 1 658 596 SNPs were detected in 40 sheep individuals, most of which were located in the intergenic region. The Ho, He, PIC, SHI, and Nei values of Yongdeng Qishan sheep were 0.082, 0.277, 0.221, 0.411, and 0.305, respectively. The higher linkage disequilibrium coefficient and slower decay rate in Yongdeng Qishan sheep indicate the low genetic diversity in the population of Yongdeng Qishan sheep. The genetic differentiation coefficients (F_st) between Yongdeng Qishan sheep and Tan sheep, Lanzhou fat tail sheep and Minxian black fur sheep were 0.090 6, 0.098 0 and 0.104 5, respectively, which indicated a high differentiation degree between Yongdeng Qi-shan sheep and the other 3 breeds. Principal component analysis showed obvious differences in clustering patterns among groups, which could distinguish Yongdeng Qishan sheep from Lanzhou fat tail sheep, Tan sheep and Minxian black fur sheep; The results of phylogenetic tree showed that Lanzhou fat tail sheep was clustered as one big branch alone, Yongdeng Qishan sheep and Tan sheep were clustered as another big branch, and the kinship was relatively close, subsequently Yongdeng Qishan sheep was gradually separated into one small branch alone. The structure analysis showed that K=2 was the optimal number of subpopulations, with 4 populations from two original ancestors. As the K value gradually increased, some QS individuals segregated and differed significantly from the genetic background of other breeds. The kinship heat map showed low kinship between individuals of each group. These results indicated that there was low genetic diversity in Yongdeng Qishan sheep and Lanzhou fat tail sheep, and that there was obvious differentiation between Yongdeng Qishan sheep and Tan sheep, Lanzhou fat tail sheep and Minxian black fur sheep. This provides theoretical data for further mining the characteristics of Yongdeng Qishan sheep germplasm resources.

The aim of this study was to screen for differentially expressed lncRNAs, miRNAs and mRNAs in the longissimus dorsi of Leiqiong and Lufeng cattle, construct a competitive lncRNA-miRNA-mRNA regulatory network, search for lncRNA cis-targeted regulatory genes, then performed functional prediction to identify key candidate genes that could be used to enhance the utility value of local yellow beef breeds in South China. Four 4-month-old female Leiqiong cattle and four Lufeng cattle were randomly selected, and longissimus dorsi tissues were taken for RNA sequencing, and the sequencing results were verified using RT-qPCR. The differentially expressed lncRNAs, miRNAs and mRNAs were screened for the construction of a competitive regulatory network and the search for lncRNA cis-targeted regulatory genes. Candidate genes were used for functional prediction using GO and KEGG enrichment analysis. The results showed that, using Leiqiong cattle as the control group, there were 119 differentially expressed lncRNAs in the two breeds, of which 73 were up-regulated and 46 were down-regulated; a total of 13 differentially expressed miRNAs, of which 7 were up-regulated and 6 were down-regulated; and a total of 599 differentially expressed mRNAs, of which 155 were up-regulated and 444 were down-regulated. The GO enrichment analysis of mRNAs in the competitive regulatory network showed that the terms related to myogenesis were mainly phosphorylation and membrane structure. The pathways related to myogenesis in the KEGG enrichment analysis results were mainly signaling pathways such as Rap1, MAPK, PI3K-Akt, etc. The GO enrichment analysis of cis-targeted regulatory genes showed that the GO terms related to myogenesis were mainly the development of muscle structure and muscle organ development, etc. The pathways related to myogenesis in the KEGG enrichment results were mainly MAPK signaling pathways and local adhesion, etc.In this study, we screened the differentially expressed lncRNAs, miRNAs and mRNAs in the longissimus dorsi of Leiqiong and Lufeng cattle, and then constructed a competitive regulatory network and searched for lncRNA cis-targeted regulatory genes. The results of the experiments provide theoretical support for further rational development and utilization of the local breeds of yellow cattle in South China.

The study aimed to explore the characteristics and influencing factors of blood indicators for dairy cows raised at high altitude in China. A total of 435 healthy lactating Holstein cattle of different purity were used to measure the phenotypic data, including 18 hematological parameters, 3 blood gas indexes, 4 electrolyte indexes, and 2 biochemical parameters. Correlation and variance analyses were further carried out to determine the phenotypic correlations among corresponding phenotypes and significant influencing factors that should be addressed in follow-up studies, in which the farm, month age, lactation stage, body condition score, Holstein breed composition, and measuring time were considered in variance analysis. The results showed that the white blood cell count, red blood cell count (RBC), and platelet count of dairy cows raised on plateau were (7.66 ±2.14)×10⁹ cells·L^-1, (6.94 ±0.74)×10¹² cells·L^-1 and (405.73 ±224.48)×10⁹ cells·L^-1, respectively; and their partial pressure of oxygen (PO₂) and the partial pressure of carbon dioxide (PCO₂) were (11.28 ±1.61) kPa and (3.39 ±0.38)kPa, respectively. Among the 27 blood indicators, the mean corpuscular hemoglobin concentration (MCHC), platelet distribution width, blood pH, sodium ion concentration (Na⁺), and chloride ion concentration had small inter-individual variation with variation coefficients being less than 5%. In the correlation analysis, the PCO₂ and PO₂, as well as PCO₂ and pH had correlation coe-fficients of -0.53 and -0.55, respectively, showing moderate negative correlations. Conversely, hemoglobin concentration (HGB) and hematocrit (HCT), mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), platelet count and platelet volume showed highly positive correlations (correlation coefficient ≥ 0.95). In the variance analysis, months of age had significant effects (P<0.05) on 15 hematological parameters, at the same time, body condition score and lactation stage significantly affected 6 indexes in erythrocyte system except MCHC (P<0.05). HGB, HCT, MCV and MCH were significantly negatively correlated with the proportion of Holstein breed (P<0.05). The value of RBC was highest in mid-lactation, while MCV and MCH were lowest at this stage. Moreover, the measuring time had significant effects on PCO₂, Na⁺, calcium ion concentration (Ca²⁺), glucose concentration (GLU), lactic acid concentration (LAC), and arterial blood HCT (P<0.05). Among them, PCO₂, Ca²⁺, and HCT appeared extreme values from 12:41 to 14:10. The effect of lactation stage was also significant on PCO₂, pH, potassium ion concentration, GLU, LAC, and arterial blood HCT (P<0.05). In the early lactation stage, the values of arterial blood GLU and LAC were the lowest, while PCO₂ was the highest, indicating the weakest alkalinity. Taken together, blood indicators are mainly affected by the physiological state of cows and external factors. It is important to use blood indicators to carry out health detection of dairy cattle in high-altitude area, and should take into account key factors such as lactation stage, months of age, and body condition and pay attention to the choice of measuring time.

Low temperature preservation can effectively prolong the storage time of sperm in vitro and improve the semen utilization ratio. However, long-term exposure to low temperature will cause the continuous accumulation of reactive oxygen species (ROS), induce sperm oxidative stress, reduce the integrity of plasma membrane and result in sperm motility decline. This study intends to investigate the effect of adding different concentrations of niacin (nicotinic acid, NA) to the diluents on sperm preservation under the condition of 4℃ storage of sheep sperm, and to explore its mechanism of action. In this study, semen from healthy adult German Merino rams (n=6) were collected and supplemented with 5、10 and 20 mmol·L^-1 NA used as protective agents to clarify the protective effect of NA on sheep sperm. Sperm motility and motility were detected by sperm analyzer, plasma membrane integrity was analyzed by HOST,ROS level and mitochondrial function in sperm were detected by fluorescent staining, while H₂O₂ content, antioxidant factor activity level and ATP level were detected by kit. The results showed that compared with 10% BSA control group, the survival time of sperm treated with NA could be extended to 120 h,and the sperm motility could still reach 70% at 72 h after 10 mmol·L^-1NA treatment. At 24~72 h after NA treatment, sheep sperm maintained higher motility, movement characteristic and plasma membrane integrity (P<0.05), and decreased ROS levels in sperm cells (P<0.05). At 72 h after NA treatment, the content of H₂O₂in sperm was significantly lower than that in the 10% BSA control group (P<0.05), while the antioxidant factors (CAT,GSH-PX,T-SOD,T-AOC) activity levels, mitochondrial function and ATP levels were significantly better than 10% BSA control group (P<0.05). In conclusion, adding NA to sheep semen low temperature dilution under 4℃ storage conditions can improve sperm motility, movement characteristic, plasma membrane integrity, antioxidant capacity, sperm mitochondrial function and ATP. It can effectively improve the low temperature preservation effect of sheep sperm.

The aim of this study was to explore whether 1,25(OH)₂D₃ regulates beta-defensin gene expression in the caprine epididymal caput epithelial cells by the VDR signaling pathway. Three 6-month-old Taihang black goats were selected, and the epididymal caput tissues were collected. The caprine epididymal caput epithelial cells were isolated by differential adherence time, and the purity of epithelial cells was identified by cellular immunofluorescence; The epididymal caput epithelial cells were treated with 100 nmol·L^-1 1,25(OH)₂D₃; And the pCas9/gRNA1 plasmid vector with the highest knockout efficiency was selected for cell transfection. And negative control group and blank control group were set up, with three replicate in each group. After the cells were treated with 1,25(OH)₂D₃ or VDR gene knockout, the expressions of VDR and 17 β-defensin genes were detected by qRT-PCR, and the expression of VDR protein and 3 β-defensin proteins were detected by Western blot. The results showed that 1,25(OH)₂D₃ could significantly increase mRNA and protein expressions of VDR, gBD124, gBD126 and gBD104a(P<0.01), and markedly increased the expression of gBD104, gBD109tr1, gBD109tr2, gBD113, gBD116, gBD120, gBD121 and gBD123 genes(P<0.01), and significantly increased the expression of gBD106, gBD127, gBD129 and gBD134 genes (P<0.05), but had no significant effect on gBD110 like and gBD128 expression (P<0.05); After the three gene knockout vectors were transfected into cells, the expression of VDR protein in pCas9-VDR-V1 group was significantly decreased (P<0.01). VDR gene knockout significantly increased gBD124 mRNA and protein expression (P<0.01), and significantly reduced the mRNA and protein expression of gBD126 and gBD104a (P<0.05), the mRNA level of gBD109tr1, gBD109tr2, gBD116, gBD123, gBD127, gBD128 and gBD134 genes in VDR gene knockout group were significantly lower than those in the other groups (P<0.01), the mRNA level of gBD104, gBD106, gBD120 and gBD129 genes in VDR gene knockout group were significantly lower than those in the other groups (P<0.05), but had no significant effect on gBD121, gBD110 like and gBD113 (P<0.05). In conclusion, 1,25(OH)₂D₃ could up-regulate the expression of VDR and some beta-defensins, VDR gene knockout increased the expression of some beta-defensins. The results showed that 1,25(OH)₂D₃ could increase the expression of part beta-defensins in caprine epididymal caput epithelial cells by up-regulating the expression of VDR and activating the VD/VDR signaling pathway.

The purpose of this study was to explore the expression and possible relationship of main related factors of OPN5-TSH-DIO2/DIO3 pathway and VIP-PRL pathway in the process of short light inhibiting on reproductive function of male Shanma duck. In this experiment, 80 healthy male Shanma ducks with similar body weight and normal breeding period (220 days old) were selected as subjects. The long light (18L:6D) of the normal breeding period was maintained 15 d before the experiment, from d16, the illumination was shortened by 2 h per day, and the illumination time was 8 h per day (8L:16D) until the end of the experiment (d45). Blood samples and tissue samples of hypothalamus, pituitary and testis were collected on d15, d21, d30 and d45, and testicular development and expression of related genes and proteins in hypothalamus, pituitary and testis were detected by HE staining, ELISA, qRT-PCR and Western blot techniques. The results showed that, compared with long light exposure, short light exposure resulted in testicular atrophy, a significant decrease in the number of spermatogenic cells and sperms(P<0.05), a significant decrease in blood LH, PRL and testosterone levels(P<0.05), a decrease in GnRH expression and an increase in GnIH expression in hypothalamus, but the difference was not significant(P>0.05). GnIHR showed an upward trend and FSH showed a downward trend in pituitary, but the difference was not significant(P>0.05). GnRHR increased significantly in the later stage of the experiment(P<0.05). The gene expression levels of LH in pituitary and FSHR, LHR in testis was decreased, but there was no significant difference(P>0.05). In OPN5-TSH-DIO2/DIO3 pathway, the expression gene levels of OPN5, DIO2, TSH-β and the expression protein levels of OPN5 was decreased significantly(P<0.05), the expression protein levels of DIO2 was decreased, but there was no significant difference(P>0.05). While the expression trend of DIO3 gene was opposite to that of DIO2. Shortening light exposure time led to the increase of gene(P>0.05) and protein(P<0.05) expression level of DIO3. In VIP-PRL pathway, the gene expression of VIP in hypothalamus and PRL in pituitary decreased significantly(P<0.05), while the gene expression of PRLR in pituitary increased significantly(P<0.05), and the gene expression of PRLR in testis decreased significantly(P<0.05). The results showed that both OPN5-TSH-DIO2/DIO3 pathway and VIP-PRL pathway were involved in the process of short light inhibiting on reproductive performance of male Shanma duck, which mediated the inhibitory effect of short light on reproductive axis. Compared with the light conditions needed in normal breeding period, short light could inhibiting the efficiency of two pathways and down-regulate the activity of reproductive axis, but the mutual regulation between the two pathways remains to be further studied.

The objective of this experiment was to explore the effects of dietary different levels of lysine on the hair production performance and hair follicle development of Angora rabbits. One hundred and sixty Angora rabbits with good growth and development status (10 months old) were selected and randomly divided into 4 groups. The control group was fed basic diet, and the experimental groups were fed basic diet with an addition of 0.25%, 0.35% or 0.45% lysine, respectively. The experimental period lasted for 65 days. The results showed that:The addition of 0.25% lysine to diet significantly increased hair yield, hair yield ratio, feed conversion rate, total follicle density and secondary follicle density (P<0.05). The addition of 0.25% lysine in diet significantly increased the gene expression of the Wnt family 10b protein (Wnt10b), β-catenin (β-catenin), and rapamycin target protein (mTOR) in the skin (P<0.05). The addition of 0.35% lysine in diet significantly increased the expression of keratin-associated protein (Kap6.1) gene and also significantly reduced the expression of transforming growth factor (TGF-β1) gene (P<0.05). Dietary addition of lysine can promote the development of skin follicles in rabbits, and Wnt10b/β-catenin, TGF-β and Kap signaling pathways may be involved in the regulatory process.

In order to solve the problem of how to optimize and innovate the existing methods to establish a new method for FMD antibody detection, this study aimed to optimize the method based on liquid phase blocking ELISA to achieve rapid and sensitive detection of FMD virus antibody. In this study, a novel liquid phase blocking ELISA method (SA-LPBE) was established by combining the purification of IgG labeled biotin (dolphin anti-foot-and-mouth disease) from guinea pig serum with HRP labeled streptavidin (HRP-SA). SA-LPBE has a high consistency rate in bovine and sheep serum samples. Combined with the actual situation, the optimized serum antibody titer of cattle and sheep was determined to be ≥ 128, and that of pigs serum sample was determined to be ≥ 64. The results were determined to be positive, which was consistent with the results of commercial detection kits. And after compliance analysis, it has a compliance rate of 92.3% with the original kit, and the intra-batch variation coefficient is less than 5%, and the inter-batch variation coefficient is less than 10%. This method changed the original route of preparing rabbit anti-guinea pig antiserum and labeling HRP, improved the efficiency of enzyma-tic reaction, guaranteed the original sensitivity of the method and improved the specificity of the method; Using the advantages of quick reaction between biotin-labeled antibody and HRP-SA, stable labeling raw material and detection background value, the operation time of liquid phase blocking ELISA kit was shortened from 2 days to 3 hours, which solved the problems of tedious, long time, operation fatigue, instability and error-prone of the original operation. It can be applied to FMDV antibody detection, providing a technical method for FMD epidemic and clinical detection.

Bovine respiratory syncytial virus (BRSV) is an important pathogen of bovine respiratory disease complex (BRDC). This experiment aims to investigate whether BRSV were present in Tibetan plateau yaks and its molecular prevalence in yaks with respiratory diseases. One hundred and twenty-two nasal swabs from Ganzi (Garzê) Tibetan Autonomous Prefecture and Aba (Ngawa) Tibetan and Qiang Autonomous Prefecture were collected from Yak with BRDC in 10 farms across Sichuan provinces from March 2021 to July 2022, among them, 91 samples were from Ganzi Prefecture and 31 samples were from Aba Prefecture. Using reverse transcription insulated isothermal PCR (RT-iiPCR), 64.75% of samples tested positive for BRSV. Among them, the positive rate in Ganzi Prefecture and Aba Prefecture was 73.63% and 38.71% respectively, and the farm positive rate was 100%. Further, 10 complete G genes were identified as subgroup Ⅲ strains, and 9 complete F genes were amplified from positive samples. Compared to known BRSV strains in GenBank, G proteins and F proteins from yaks with BRSV strains previously amplified by our laboratory shared several identical amino acid mutations. Moreover, complete genomes from Yaks were obtained, which was closest to the BRSV strain (GenBank accession number:OP137030-OP137034) uploaded recently by our laboratory. In conclusion, this study confirmed the existence and prevalence of BRSV in yaks for the first time, obtained a genome sequence of BRSV in the Ⅲ subgroup from yaks, enriched the pathogenic spectrum of respiratory diseases in yaks, and provided a reference for the prevention and control of respiratory diseases in yaks.

African swine fever virus (ASFV) pD205R protein is involved in virus gene transcription, which belongs to ASFV RNA polymerase, similar with RPB5 of eukaryotic RNA polymerase II. In this study, in order to further understand the structure, function of pD205R and interaction mechanism between virus and host, the sequence of D205R gene from different ASFV strains deposed in GenBank were analyzed by using DNAStar and Mega7.0 software. Physicochemical Properties and Structure of Protein were predicted by using ExPASy, GOR4 and AlphaFold software, and its prokaryotic expression and preparation of anti-pD205R protein polyclonal antibody were performed. Furthermore, the D205R gene was amplified and cloned into pET-28a vector, named as pET-28a-D205R. The results showed that the D205R gene was highly conserved among different genotypes of ASFV isolates. The physicochemical properties analysis showed that pD205R protein was hydrophilic with poor stability, its molecular formula was predicted as C₁₀₈₈H₁₇₁₂N₂₇₈O₂₉₅S₈. The secondary structure and tertiary structure indicated that α helix was the main structural form, proportion for 35.12%. Furthermore, the pET-28a-D205R recombinant plasmids were constructed successfully using ASFV CN/SC/2019 strain genome as template and transformed into E. coli Rosetta (DE3) competent cells, the recombinant proteins were successfully induced and expressed by IPTG, with the size of 23.3 ku. Then the obtained protein was purified by nickel column affinity chromatography, which could react specifically with ASFV positive serum by Western blot identification. The titer of polyclonal antibody prepared by immunizing New Zealand white rabbits could reach to 1:51 200, and could specially recognize the native pD205R protein expressed in ASFV-infected porcine alveolar macrophages (PAMs) by Western blot and IFA detection. It laid a foundation for further study the function of D205R gene and its role in host-pathogen interaction.

The purpose of this study was to isolate a novel Duck adenovirus 3 (DAdV-3) YN strain and elucidate its genetic characteristics and pathogenicity. LMH cells were selected for virus isolation and purification, and then the virus genes, including hexon, penton base, fiber1, and fiber2 were sequenced and analyzed. Seven-days-old Muscovy ducks were infected by intramuscular injection in the legs, and the mental state of the ducks was observed. Besides, the body weight changes, necropsy characteristics of organs, histopathological changes, viral load of organs, cloaca swab detoxification, and serum biochemistry were recorded and analyzed. Furthermore, the transcription levels of apoptosis-related genes in the bursa and the spleen were measured to explore the effect of YN strain on immune function. The results showed that the weight gain of infected ducks was significantly inhibited (P<0.01, P<0.000 1), and different degrees of lesions appeared in various organs. The coefficients of livers and kidneys of the experimental group were larger than those of the control group, and the difference was extremely significant on the 7th day after the challenge. Alanine aminotransferase, aspartate aminotransferase, and urea nitrogen in the serum of the Muscovy ducks in the challenge group were significantly higher than those in the control group (P<0.001, P<0.0001). At the same time, the mRNA levels of apoptosis-related genes in the bursa and the spleen increased significantly (P<0.01, P<0.001). (Conclusion) The above results suggest that DAdV-3 YN strain can damage livers and kidneys when challenging the 7-days-old Muscovy ducks, simultaneously, it can affect the immune function of Muscovy ducks by causing apoptosis of cells in the bursa and the spleen.

This study aimed at establishing an indirect-ELISA method for the titer detection of Mycoplasma gallisepticum (MG) antibodies in chicken, which could provide a set of effective technology for large-scale clinical monitoring of MG infection combined with HI test established. Prokaryotic-expressed protein VlhA3.03 (rVlhA) was obtained as envelope antigen. The linear equation lg (antibody titer)=1.257×lg (S/P value at 1:500) +3.709 of serum antibody titer and S/P value at 1:500 was established by a single serum dilution test and the correlation coefficient (R²) was 0.917 6. The cutoff value of negative and positive S/P was determined to be 0.32, and the corresponding titer is 1 200. The rVlhA-ELISA showed good specificity and repeatability. The detection limit of MG positive sera is 1:2 000. An HI assay was also established using domestic MG isolate SH/2020-1 as hemagglutination inhibition test antigen. The combined rVlhA-ELISA method and HI test, and IDEXX-MG were compared in serum sample detection, with RT-qPCR method to monitor MG infection. The results showed that the trend of serum antibody monitored by rVlhA-ELISA and IDEXX-MG was consistent with the result of HI assay. The coincidence rate between combined rVlhA-ELISA and HI, and IDEXX-MG was 91.53%. These results indicated that the rVlhA-ELISA and HI test established in this study were beneficial to clinical application, which can be applied to large-scale and rapid clinical screening of MG infection and recheck the results.

To obtain subunit antigens with good immune effect, in this study, six potentially protective subunit proteins of G. parasuis were expressed in prokaryotic cells. After immunizing mice with proteins, challenge experiments were performed to screen antigen candidates with better immune protection effects, thus laying the foundation for developing a subunit vaccine of G. parasuis with cross-protection effects. Sixty-three BALB/c mice were randomly divided into 9 groups, including HPSNAG-1330, CyaY, HPSNAG-0978, HPSNAG-0140, AfuA, Fe³⁺ABC-tbp, inactivated G. parasuis, challenge group and blank group. The mice were immunized the second time with an interval of 14 days. Among them, the 6 subunit vaccine groups were immunized with 50 μg of each protein per mouse, and the inactivated vaccine group was immunized with 4×10⁹ CFU per mouse. After 14 days of the secondary immunization, the mice were challenged with a dose of 1.26×10⁹ CFU. At the same time, the antibody levels, lymphocyte proliferation, IL-2, IL-6, IL-10, IFN-γ and TNF-α were detected. After the challenge, the clinical symptoms and survival rate of the mice were observed and recorded, and the pathological variations of the lung and spleen were also observed. Mice in all immunized groups can produce antibodies against the corresponding proteins. The lymphocyte proliferation experiment showed that recombinant proteins, except for the rAfuA, stimulated lymphocyte proliferation significantly in the inactivated vaccine group (P<0.05). At the same time, there were no significant changes in the lymphocyte proliferation of the mice in the subunit vaccine group and the blank group when stimulated with the corresponding immune proteins. The survival rate (60%) of the CyaY and HPSNAG-1330 groups was higher than that of the inactivated vaccine group (40%), and which of the other groups was lower than that of the inactivated vaccine group. The above results proved that CyaY and HPSNAG-1330 proteins can induce effective immune protection responses in mice. At the same time, the lesions of the spleens and lungs of the mice in CyaY and the HPSNAG-1330 are relatively mild. So, the protective effect of these two proteins is better than that of inactivated vaccine. In this study, two subunit antigens with immune-protective properties were screened by mouse immunization experiments, which provided materials for subsequently combined immunization of multiple antigens using pig model experiments.

The aim of this study was to prepare monoclonal antibodies against Brucella outer membrane protein 16 (OMP16), establish a competitive ELISA method for OMP16 antibody, and provide an immune serological detection method for clinical prevention and control of brucellosis. In this study, Brucella OMP16 with high conservation and good immunogenicity was selected, and recombinant OMP16 carrying GST or His tag was obtained by prokaryotic expression. Hybridoma cell lines stably secreting OMP16 specific monoclonal antibodies were screened by hybridoma technique. The properties of monoclonal antibodies were analyzed by subtype identification, hybridoma cell karyotype analysis and antibody cross-reactivity test. Preparation and purification of mouse ascites and establishment of competitive ELISA for Brucella OMP16 antibody by square titration. The prokaryotic expression system of OMP16 was constructed, and rGST-uOMP16 and rHis-OMP16 were obtained by expression and purification. A genetically stable OMP16-specific hybridoma cell line was screened and named B7. After identification, the antibody subtype was identified as IgM-κ, recognizes the target protein, and has no cross reaction with the tag protein. The Brucella OMP16 antibody competition ELISA method was established. Compared with the results of the standard tube agglutination test, the overall coincidence rate of this method was 90.53%, showing good consistency. It is expected to provide an immune serological detection method with high specificity and good sensitivity for the clinical prevention and control of brucellosis.

In July 2021, a severe flood occurred in some area of Henan Province, pig farms in these areas suffered heavy losses. The purpose of the current research is to explore the bacteria in the flood-affected water samples of pig farms, so as to provide technical support for the prevention and control of possible diseases. Different types of water samples including 19 drinking water, 22 rainwater, and 7 waste sewage samples were collected by field sampling and airmail from 26 farms in the affected areas. Using colony counting and high-throughput sequencing technology, the total number and the abundance of bacteria in the collected samples were analyzed, and disinfection tests were carried out for the collected water samples. The results showed that the total bacterial counts in drinking water, rainwater and sewage were 1.55×10⁵, 1.88×10⁸and 3.70×10⁹ CFU·mL^-1, respectively. The total number of bacteria in the sewage was the highest, followed by the rainwater, and that of the drinking water was the lowest, but still higher than the national standard of drinking water. By mean of high-throughput detection technology, potential pathogenic genus such as Streptococcus, Escherichia, Arcobacter, Actinobacter and Pseudomonas, as well as waterborne pathogenic bacteria such as Arcobacter cryaerophilus, Streptococcus suis, Escherichia coli, Acinetobacter baumannii and Aeromonas caviae, were detected in water samples. Bactericidal tests showed that when the final concentration of chlorine reaches 2 500 mg·L^-1, all bacteria in rainwater and sewage were killed within 30 minutes. It was demonstrated that water samples in pig farms after floods contained a variety of pathogenic bacteria and pig farms can choose chlorine-containing disinfectants to kill the bacteria in polluted water in the farm to ensure the health of humans and pigs.

Potassium diformate (KDF) is one of the representatives of acidifiers, which was approved for use as a feed additive by the European Union in 2001 and by China in 2005. Currently, there are more studies showing that KDF can improve the growth performance of animals. However, there are less researches to provide direct evidence of the prevention effect of KDF to infectious disease. The aim of this study was to evaluate the preventive effect of KDF on pathogenic bacteria in vitro and in vivo through bacterial inhibition assay and mouse infection model, and to detect the effect of KDF on intestinal flora of mice in order to investigate the mechanism of KDF action. First, detection of the minimum inhibitory concentration (MIC) of KDF against common pathogenic bacteria in the farm. Subsequently, C57/BL6 female mice aged 3-4 weeks and weighing 12-15 g were selected and examined for changes in body weight, tissue load, inflammatory cytokine levels and cecum pathology after infection with Salmonella Typhimurium in KDF treatment group (n=6) and control mice (n=6). The effects of feeding KDF on the cecum flora of mice were analyzed using 16S rRNA sequencing technology. The results showed that the MIC of KDF was 3.125 mg·mL^-1 for pathogenic bacteria such as Salmonella and Escherichia coli, and 6.25 mg·mL^-1 for bacteria such as Staphylococcus aureus and Pasteurella. In vivo, KDF pretreatment significantly reduced the bacterial loads in the blood and cecum (P<0.05), significantly reduced the levels of inflammatory cytokines IL-6, IL-12 and TNF-α in the blood (P<0.05), and alleviated the pathological changes in the cecum. Simultaneously, KDF significantly reduced the diversity of the intestinal flora of the cecum (P<0.01), which allowed the differential species presence. In addition, the species richness of the cecum flora of mice in the KDF treatment group decreased. At the phylum level, the relative abundance of the Proteobacteria was significantly higher in the KDF group (P<0.001). At the family level, the relative abundance of the Mogibacteriacceae was markedly lower in the KDF group (P<0.05). At the genus level, the relative abundance of the Akkermansia, Blautia, Lactobacillus and Eubacterium were increased. In summary, KDF showed significant inhibitory effects against common pathogenic bacteria in vitro, and prevents infection with the Salmonella in mice in vivo. The intestinal flora of mice changed significantly after KDF supplementation, and the increase in the abundance of beneficial genera such as Akkermansia, Blautia, Lactobacillus and Eubacterium may have a positive effect on the intestinal health of the animals.

In order to explore the existence of ornithine-dependent anti-acid system in Trichinella spiralis(T. spiralis, Ts)muscle larvae, three specific siRNAs molecules were designed according to the complete genome sequence of Ts-ODC(Trichinella spiralis ornithine decarboxylase) in GenBank. Three siRNAs were transfected into T. spiralis muscle larvae by liposome transfection. The best interference siRNA molecules and the best interference conditions were determined by qPCR, Western blot and indirect immunofluorescence assays. The muscle larvae were cultured at pH 1.5, 2.5, 4.5 and 6.6 for 0.5, 1 and 2 h, respectively. The effects of Ts-ODC gene interference on the acid resistance of muscle larvae were evaluated from three aspects of the ODC transcription level, ornithine decarboxylase activity and larvae survival rate. Mice were infected with muscle larvae of T. spiralis after Ts-ODC gene interference. Adults and muscle larvae were collected at 7 d and 42 d, respectively, and the worm reduction rates were calculated. The first-generation muscle larvae were cultured under acidic conditions at pH 2.5 for 2 h, and the survival rate was calculated to analyze the heritability of Ts-ODC gene interference. The results showed that Ts-ODC siRNA-757 was the optimal interference molecule, and the best interference conditions were transfection for 12 h and 3 d culture at a concentration of 2 μmol·L^-1; the highest transcription level of Ts-ODC gene was reached at pH 2.5 for 2 h, and the survival rate was significantly lower than other culture conditions at pH 1.5 for 2 h (P<0.01). The ornithine decarboxylase activity was the highest at pH 4.5 for 0.5 h. After the mice were orally gavaged with muscle larvae transfected with Ts-ODC siRNA-757, the worm reduction rates were 58.95% and 65.91% at 7 d and 42 d, respectively. The survival rate of the first-generation muscle larvae was 49% when cultured in a pH 2.5 medium for 2 h. The above experiments concluded that T. spiralis muscle larvae has an ornithine-dependent acid-resistant system. In this study, the Ts-ODC gene of T. spiralis muscle larvae was interfered by siRNA technology for the first time, and it was confirmed that interfering with the Ts-ODC gene could reduce the acid resistance of muscle larvae, which provided a new idea for the prevention and treatment of Trichinellosis.

XRCC1 is a molecular scaffold protein that plays a central role in the base excision repair pathway. Here, we examined the subcellular localization of XRCC1 and investigated its effects on meiotic maturation of oocytes, subsequent embryonic development, and global DNA methylation in mice. XRCC1 was found to be localized mainly in the nuclei during oocyte maturation and subsequent embryonic development. Blocking XRCC1 by microinjection with antibodies partly reduced oocyte germinal vesicle breakdown (GVBD) and embryo cleavage, and also disrupts DNA demethylation in the 2-cell embryos. The results indicates that XRCC1 is mainly located in the nuclei at the interphase of cell cycle, and XRCC1 with normal function contributes to the maintenance of the quality of mouse oocytes during meiosis and the normal development of early embryos.

The aim of this study was to investigate the role of perilipin 2(PLIN2) in the regulation of autophagy in BCG infected mouse macrophages RAW264.7. In this study, we used small interfering RNA technology to knock down the expression of PLIN2 in murine macrophages RAW264.7, combined with BCG infection, and detected intracellular PLIN2 protein expression changes and indicators of autophagy, endoplasmic reticulum stress and fatty acid metabolism-related factors by immunoblotting, flow cytometry and immunofluorescence. BCG infection significantly upregulated the expression of PLIN2 (P<0.05) and highly significantly upregulated the expression of autophagy-associated proteins ATG5 (P<0.01), ATG12 (P<0.001) and LC3 (P<0.001) in macrophages RAW264.7 and was accompanied by intracellular neutral lipid accumulation. Knockdown of PLIN2 significantly downregulated autophagy-related proteins ATG5 (P<0.05) and ATG12 (P<0.05) and LC3 (P<0.001) in BCG-infected macrophages RAW264.7, significantly reduced autophagy rate (P<0.001) and significantly decreased intracellular neutral lipid content (P<0.001). At the same time, knockdown of PLIN2 significantly downregulated CHOP (P<0.05) and highly significantly downregulated ATF4 (P<0.001) endoplasmic reticulum stress-related protein expression, and intracellular Ca²⁺ concentration was highly significantly reduced (P<0.001). Finally, we treated cells with the endoplasmic reticulum stress agonist Tunicamycin and found no significant difference in intracellular autophagy-related protein. BCG infection of mouse macrophages RAW264.7 promoted the expression of PLIN2 and increased the accumulation of neutral lipids, which in turn activated the PERK-eIF2α-ATF4-CHOP signaling pathway in the endoplasmic reticulum stress pathway and induced the onset of cellular autophagy.

Clostridium perfringens (C. perfringens) disease in deer mainly manifests as enterotoxemia with high lethality. The aim of this study was to investigate the key genes and pathways of C. perfringens type C infection in the intestine of deer. A C. perfringens-infected deer jejunum ligation loop model was constructed, HE staining was used to detect pathological changes in the jejunum after C. perfringens infection, Illumina NovaSeq was used to sequence the transcriptome of C. perfringens-infected deer intestine, and qPCR was used to further validate some differentially expressed genes (differentially expressed genes (DEGs); finally, the multigene protein interaction network was predicted by STRING website. Morphological analysis revealed significant necrosis and hemorrhage in deer intestinal tissues following C. perfringens infection. Transcriptome analysis indicated the presence of a total of 705 DEGs, of which 276 DEGs up-regulated and 429 DEGs down-regulated genes were present in the infected group intestine. GO enrichment analysis showed that DEGs was mainly enriched in biological regulation, response to stimulus and immune system process; KEGG enrichment analysis showed that the pathways enriched in DEGs were mainly T cell receptor signaling pathways, Toll-like receptor signaling pathway and hematopoietic cell lineage, which caused intestinal damage and bleeding, and promoted the progression of deer enterotoxemia. In addition, the qPCR results confirmed that the analysis was accurate and reliable. The results of protein interaction analysis of DEGs showed that TLR6, CLDN1, CD3E, IL1A and CCL20 had more interactions with other proteins, and the pathways significantly enriched by GO analysis and KEGG analysis were highly overlapping. Among them, TLR6 may play a key role in C. perfringens-induced enterotoxemia in deer through inhibition of tight junction protein expression and immunoinflammation response. C. perfringens type C induced immunoinflammation response by disrupting cell membranes and inhibiting tight junction protein expression, caused intestinal bleeding and intestinal injury. This study provided new ideas on the molecular mechanism of regulation after C. perfringens infection in deer and provided a theoretical basis for the treatment of the disease.

In this study, we aimed to explore the impact of exogenous multidrug resistant (MDR) Salmonella Typhimurium on gut microbiota of healthy mice. Twenty-five mice were randomly divided into quality control group (C), one day gavage group (G1), three days gavage group (G2), five days gavage group (G3) and seven days gavage group (G4), with 5 mice in each group. Resistant Salmonella Typhimurium from swine carrying drug resistance genes was administered to mice in experimental groups except the quality control group by gavage at a concentration of 10⁶ CFU·mL^-1. Fresh fecal samples were collected on day 0 before gavage and day 1-14 after gavage. High-throughput sequencing technology was used to analyze the diversity of gut microbiota in feces of mice after gavage. The results showed as follows:1) Compared with group C, all experimental groups showed mild diarrhea after gavage, MDR Salmonella Typhimurium was isolated from fecal samples with the same type as gavage bacteria, and there was no significant difference in the isolation rate. 2) The Chao1, Goods_coverage and Observed_species indices in Alpha diversity of gut microbiota in experimental group were significantly higher than those in group C; 3) The relative abundance of Epsilonbacteraeota and Helicobacteraceae at phylum and genus level in groups G2, G3 and G4 was significantly lower than that in group C (P<0.05). According to LEfSe analysis, Epsilonbacteraeota phylum and Helicobacter genus were significantly enriched and had the highest abundance in group C. 4) A total of 10 metabolic pathways were screened out from the gut microbiota of mice in each experimental group and group C. Compared with group C, the glycogen degradation Ⅱ metabolic pathway in G1 group was down-regulated, while the thiol biosynthesis and NAD biosynthesis Ⅱ metabolic pathways in G2, G3 and G4 groups were significantly up-regulated (P<0.05) to regulate gut balance. In conclusion, persistent infection with MDR Salmonella Typhimurium can increase the diversity and richness of intestinal flora in mice. Long-term oral administration of MDR Salmonella Typhimurium can promote the regulation of intestinal flora through metabolic pathways. However, due to the complexity of intestinal flora itself, the self-regulation and related mechanisms of intestinal flora need to be further studied.

The aim of this study was to investigate the material basis and potential mechanism of action of the antioxidant function of Taraxacum mongolicum (T. mongolicum) based on network pharmacology and to provide guidance for the development of T. mongolicum as a functional plant extracts feed additive. The active ingredients and potential targets in T. mongolicum were screened by HERB database, TCMSP database and SwissTargetPrediction web tool, and the physicochemical properties of the active ingredients were calculated using SwissADME tool, and the target genes related to antioxidant function were obtained from GeneCards database. Compound-target-function, compound-target-pathway visualization networks and protein-protein interaction (PPI) networks were constructed using Cytoscape and STRING databases. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by DAVID database. In vitro antioxidant activities of different extract fractions of T. mongolicum were determined by ABTS and FRAP assays. The screening yielded 28 active ingredients, 296 compound prediction targets, 1 371 antioxidant targets and 135 intersection targets. Physicochemical calculations showed that most of the active ingredients were water soluble. Protein-protein interaction analysis indicated that 20 key proteins, including JUN, VEGFA, SRC, HSP90AA1 and MMP9, may play key roles in antioxidant function. GO and KEGG enrichment analysis indicated that antioxidant function may be related to vascular endothelial growth factor pathway, TNF signaling pathway, MAPK signaling pathway, IL-17 signaling pathway and estrogen signaling pathway. In vitro antioxidant tests showed higher antioxidant activity of aqueous extracts in T. mongolicum. The results suggest that the water-soluble active ingredients in T. mongolicum are the main material basis for its antioxidant function, providing theoretical support for further development of T. mongolicum as an antioxidant functional feed additive.

The object of this study was to investigate the effect of arginine kinase of Psoroptes ovis (PsoAK) on cutaneous eosinophil recruitment in rabbit. In vitro of HaCaT cells and in vivo models of P. ovis-sensitization rabbits, qRT-PCR analysis were used to evaluate whether the recombinant PsoAK (rPsoAK) has effect on the eosinophils-related genes expression in vitro and in vivo, such as IL-4, IL-5, IL-13, TSLP, IL-25, IL-33, CCL5, CCL11, IL-4R. Additionally, chromotrope-2R staining was employed to evaluate whether rPsoAK can induce eosinophil recruitment to skin in vivo. Compared to the blank and pET32a control groups, 0.1 μg·mL^-1 rPsoAK significantly promoted the relative transcript levels of IL-13, IL-33,CCL5 and CCL11 mRNA in HaCaT cells (P<0.05). In vivo, rPsoAK was also significantly upregulated in the mRNA levels of IL-4R and CCL5 in rabbit skin compared with the blank and pET32a control groups (P<0.01), and there was no significant effect on other factors (P>0.05). Meanwhile, rPsoAK significantly induced the increased number of eosinophil recruitment in rabbit skin as compared with PBS and vector control groups(P<0.001). In conclusion, PsoAK can promote the increase of some eosinophil-related genes expressions and induce cutaneous eosinophil recruitment in rabbit, and serves as a molecule contributing to skin eosinophils accumulation in P. ovis infection and involves in the pathogenesis of P. ovis infestation.

In order to investigate the current prevalence and variation of pseudorabies virus (PRV) in Guangdong Province, blood samples of diseased pigs with suspected PRV infection were collected from a large-scale pig farm in Foshan City, Guangdong Province in 2020. PRV was identified by transmission electron microscopy and indirect immunofluorescence, and which was named as GDFS2020 strain. Molecular characteristics and genetic evolution of gB, gC, gD, gE and TK deduced amino acid sequences were analyzed. The results indicated that GDFS2020 strain was a domestic variant, but there were individual amino acid mutations. The results of the pathogenicity test showed that there were infarcts at the edge of liver, hyperemia, swelling, and congestion in heart, kidney, spleen, and brain tissues. The results of the histopathological section showed obvious pathological changes such as degeneration and atrophy of neurons, nucleus slightly shrunken, and increase of voids in brain. Our results indicated that a mutant PRV strain was successfully isolated in this study and had high pathogenicity in rabbits, providing reference data for the epidemiological research and genetic evolution of PRV in Guangdong Province.

Interferon-induced transmembrane protein 3 (IFITM3) plays an important role in the regulation of viral infection. However, the role of IFITM3 in peste des petits ruminants virus (PPRV) infection remains unclear. This study aimed to investigate the effects of IFITM3 on virus infection in goat endometrial epithelial cells (EECs) infected with PPRV. We performed Western blot assay to detect the kinetics of IFITM3 expression in PPRV-infected EECs. Then, the effect of IFITM3 on PPRV replication was further explored by gene overexpression and knockdown techniques. Our data showed that, compared with mock-infected cells, virus replication increased in PPRV-infected cells in post infection time-dependent manner, which was accompanied with an increased IFITM3 expression followed by decreased expression. The expression of IFITM3 levels increased starting at 2 hours post infection (hpi) (P<0.001), and reached peak at 3-4 hpi (P<0.0001). At 24 hpi, there was no significant difference in IFITM3 expression between PPRV-and mock-infected cells. Moreover, IFITM3 silencing with siRNA significantly inhibit PPRV N expression levels compared to negative control cells at 0-6 hpi, particularly, at 6 hpi. In contrast, over expression of IFITM3 by pcDNA3.1-IFITM3 transfection enhanced PPRV replication levels compared to untransfected cells at 6 hpi (P<0.001). There was no significant difference of virus replication levels in pcDNA3.1-IFITM3 transfected-cells compared to negative control at 12-24 hpi. Together, these results demonstrate that PPRV-induced IFITM3 expression facilitate virus replication during early infection.

In order to diagnose transmissible gastroenteritis virus(TGEV)efficiently and easily, specific primers, probes and recombinant standard plasmids TGEV-N were designed. By screening the primers and optimizing the reaction conditions, a real-time reverse-transcription recombinase-aid amplification (RT-RAA) assay was established. The assay can be completed within 20 min at 42℃ and there was no cross-reactivity with porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, porcine deltacoronavirus and porcine enteric alphacoronavirus. Sensitivity test conducted with serial dilutions of TGEV-N ranging from 10⁰-10⁶ copies·μL^-1 showed the minimum detection was 6.62×10¹copies·μL^-1. 50 porcine samples were tested by this assay and the positive rate of TGEV was 6%, which was consistent with RT-PCR. The TGEV real-time RT-RAA assay was specific, sensitive and efficient and could be used for clinical screening of TGEV.