Sheep is one of the earlier domesticated livestock, which can provide human with meat, wool, milk and other products, and occupies an important position in Chinese animal husbandry industry. Due to the differences in the natural environment and social needs in various regions, hundreds of sheep breeds have been formed in the long-term selection process, each with its own characteristics in appearance and physiological habits. As a phenotypic characteristic of sheep breeds, the horns bear the dual functions:the developed horns not only help the male sheep to improve its position in the population, have priority to mate, but also can effectively resist attacks from natural enemies and protect its population from predation. However, with the popularization of large-scale breeding, the existence of sheep horns is not conducive to the feeding management in the house, which puts forward new requirements for the breeding of sheep breeds. At present, although sheep horns have received extensive attention in actual production and breed breeding, the research on its morphological diversity and genetic regulation mechanism is still insufficient. This review focused on the number and morphology of horns, development process, genetic regulation mechanism and other aspects of current sheep breeds in China, which can not only provide a reference for identifying the mutation sites and major genes affecting sheep horns diversity in the later work, but also lay the theoretical foundation for the breeding of new sheep breeds.

Bovine mastitis is one of the diseases that seriously affect the body health status and milk quality of dairy cows. The infection of mammary gland caused by the invasion of exogenous pathogenic bacteria has long been considered to be the main factor of bovine mastitis. However, recent studies have shown that the gastrointestinal microorganisms can also influence and regulate the development of mastitis. The main mechanism may involve the entero-mammary pathway, that is, certain bacteria from the gastrointestinal tract may migrate to the mammary gland via an endogenous cellular pathway through a mechanism involving monocytes (mainly phagocytes). In this review, the pathogenic factors of bovine mastitis and its influence, the relationship between gastrointestinal microflora and mastitis as well as the regulation of gastrointestinal microbiome on mastitis (including dietary, short-chain fatty acids (SCFAs), probiotics and commensal bacteria) etc., were reviewed, aiming to provide several new information for the pathogenesis and mitigation measures of bovine mastitis.

In the spring of 2013, human infections with H7N9 subtype avian influenza emerged in China, posing serious threats to both poultry farming and public health. Additionally, highly pathogenic H7N9 variants with insertional mutation at the cleavage site of hemagglutinin (HA) protein yet evolved during the fifth epidemic wave. As the most abundantly expressed glycoprotein on the surface of influenza A virus, HA protein plays crucial roles in the viral lifecycle such as mediating the linkage between virus and host cell surface receptors, promoting the fusion between viral envelope and cell membrane, and stimulating the production of neutralizing antibodies. In this review, we briefly summarized the structure and function of HA protein, and the research progress of functional amino acid variations effecting on H7N9 biological properties, in order to provide important reference for in-depth analysis of the role of HA protein in H7N9 infection and pathogenesis.

Endogenous retroviruses (ERVs) are the remains of ancient retroviruses that were infected and integrated into the host genome millions of years ago and inherited through Mendelian law. ERVs have important biological functions and play important roles influencing host phenotype, productivity, embryonic development and immune regulation. In this article, the genomic structure, activity regulation of ERVs by epigenetic modification and integration locus was reviewed. The relation between host embryonic development, immune regulation and ERVs was also discussed. It was expected to provide reference for further exploring the function of endogenous retrovirus in the genome and its influence and evolution in the host.

This study aimed to predict and analyze the effects of fixed effects such as the year of birth, birth season, birth weight and the age at the beginning of test on the growth traits of Landrace and Large White pigs, and calculate the genetic parameter estimated values (heritability, genetic variance, phenotypic correlation, and genetic correlation), which would provide a basic reference for pig genetic improvement. In this experiment, the GLM model was used to analyze the effects of fixed effects on growth traits in the experimental pig herd (398 Landrace pigs and 1 176 Large White pigs), and a multi-trait animal model was used to perform the genetic parameters estimation to the target traits. The target growth traits included age to 100 kg (AGE), backfat to 100 kg (BF), and average daily gain to 100 kg (ADG). The study showed that, in Large White and Landrace pigs, the year of birth, birth season, birth weight, and the age at the beginning of test all had extremely significant effects on growth traits (P<0.001); The heritability of AGE, ADG and BF were 0.321, 0.327 and 0.324 in Landrace pigs, the heritability of the corresponding traits in Large White pigs were 0.454, 0.469 and 0.408, respectively. The genetic correlation and phenotypic correlation between ADG and AGE in Landrace pig were -0.990, -0.995, respectively; the genetic correlation and phenotypic correlation between ADG and AGE in Large White pigs were -0.993 and -0.998, respectively, showing a strong negative correlation. The growth traits (AGE, ADG, BF) of Landrace and Large White pigs were the traits with medium heritability. The year of birth, birth season, birth weight, and the age at the beginning of the test have a greater impact on the growth traits of pigs. When genetic parameters are estimated and analyzed, the reliability of genetic parameter estimation can be increased by increasing the number of samples and improving the quality of phenotypic data. In this study, the estimation results of the genetic parameters of growth traits are relatively reliable and can provide references for subsequent genetic improvement.

The study aimed to identify and analyze CIITA splicing mutants in SPF Yorkshire and Landrace pigs and study their expression patterns in different pig tissues. In this study, the CIITA gene was amplified using specific primers, cloned and sequenced in 8 SPF Yorkshire and 7 SPF Landrace pigs aged 5 weeks, and the splicing mutant characteristics of the obtained sequences were analyzed. The online software was used for bioinformatics analysis of different splicing mutants. The expression patterns of CIITA splicing mutants in 8 tissues of 2 SPF Landrace pigs were detected by fluorescence quantitative PCR method. The results showed that 3 different CIITA splicing mutants (CIITA-A, CIITA-B and CIITA-C) in SPF Yorkshire and Landrace pigs were identified with CDS lengths of 939, 984 and 1 698 bp, respectively. The deletion of partial nucleotides (40 and 199 bp) in Exon 11 in CIITA-A and CIITA-B mutants made the termination codon appeared in advance, which resulted in the splicing of the domain encoding CIITA protein and the deletion of 4 leucine rich repeat domains, the structure of the encoded protein had changed obviously. The results of phylogenetic tree showed that pigs had close relationship with sheep and cattle, and had higher conservation in different pig breeds for CIITA. Moreover, different splicing mutants of CIITA were expressed in 8 pig tissues, and the expression levels tended to be consistent, with high expression levels in spleen or lung, followed by kidney, spinal cord, thyroid, liver and thymus, and the lowest expression level in heart. A total of 3 CIITA splicing mutants were obtained in SPF Yorkshire and Landrace pigs, two of which deleted 4 leucine rich repeat domains. Different splicing mutants were expressed in different pig tissues with slightly different levels. The results of this study lay a theoretical foundation for the further studies on the immune regulation function of CIITA gene in the host.

The aim of this study was to investigate molecular mechanism of the effects of immunological stress on pectoralis major (PM) meat quality of AA broilers. The effects of immunological stress on the proteomic changes of PM of AA broilers were studied by using the label-free LC-MS proteomics technique. One hundred and twenty healthy one-day-old male AA broilers were randomly divided into control group and immunological stress group challenged with Escherichia coli lipopolysaccharide (LPS), with 6 replicates per treatment and 10 chickens per replicate. At the age of 36, 38 and 40 days, 1 mL of sterile saline for control group or LPS dissolved in saline at an approximate dose of 5.0 mg·kg^-1 of bodyweight for immunological stress group was injected intraperitoneally, respectively. At the age of 42 days, 2 broilers were selected from each replicate randomly, and the tissue samples of PM were collected for measuring the drip loss, cooking loss, inosine monophosphate (IMP) content and pH, and the qualitative and quantitative analysis of proteomics were performed. The results showed that:1) Compared with the control group, immunological stress significantly increased the drip loss and cooking loss of PM of broilers (P<0.05), and significantly reduced the content of IMP (P<0.05); Immunological stress had no significant effect on pH of PM of broilers slaughtered after 24 and 48 h (P>0.05); The cross sectional perimeter and area of PM muscle fiber in immunological stress group were significantly increased (P<0.01). 2) The results of qualitative proteomics analysis showed, in the control group, the specially expressed proteins were enriched in cell respiration, aerobic respiration, hexose metabolism and biosynthesis, monosaccharide biosynthesis, glucose metabolism and gluconeogenesis; While, in the immunological stress group, the specially expressed proteins were enriched in the tight junction metabolic pathway, the biological processes included macromolecular complex decomposition and protein metabolism, decomposition and depolymerization of protein, synthesis and metabolism of nucleic acid and glycosyl compounds, metabolism of nucleobase-containing small molecule. 3) Quantitative proteomics analysis showed that:21 proteins were differentially expressed in the immunological stress group and the control group, 15 of which were up-regulated and 6 were down-regulated in the immunological stress group; The up-regulated proteins in the immunological stress group included the proteins involved in carbohydrate metabolism and energy production (AMPD1, GPD1L2, LDHA, UQCRC2), muscle contraction related proteins (MYH1C, MYH1A, CASQ2, TMEM38A) and proteins involved in immunological response to protect cells from damage (HMGB1, PPP1R1A, LOC101747971, PRDX1,1ABCF2); The down-regulated protein in the immunological stress group were HBBA, CS, ATP5A1WL, HSP90B1, ABCB6 and SYNJ2. The results indicated that immunological stress induced by LPS increased the muscle fiber area, reduced the cell hydraulic holding capacity of muscle cells, and resulted in the poor meat quality by changing the expression of proteins related to the energy metabolism and muscle contraction in PM.

This study aimed to explore the genetic parameters of feed utilization efficiency traits in fast-growing yellow-feathered broilers and evaluate the accuracy of the estimated breeding values obtained by different methods. A total of 1 923 fast-growing yellow-feathered broiler E line (1 199 males and 724 females) were genotyped using a 55K SNP chip. The genetic parameters were estimated based on the additive effects model using 3 methods:conventional best linear unbiased prediction (BLUP), genomic best linear unbiased prediction (GBLUP) and single-step genomic best linear unbiased prediction (SSGBLUP), and the accuracy of the estimated breeding values obtained by the 3 methods were compared by 10-fold cross-validation. The studied traits included 4 growth traits and 4 feed utilization efficiency traits:body weight at 42 days (BW42D), body weight at 56 days (BW56D), average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR), residual feed intake (RFI), residual body weight gain (RG), residual intake and body weight gain (RIG). The results showed that all 4 feed utilization efficiency traits had low heritability (0.08-0.20), and the other growth traits had moderate to low heritability (0.11-0.35). All 4 feed utilization efficiency traits were high genetically correlated with each other, RFI and RIG were moderate genetically correlated with ADFI, RFI was not significantly correlated with ADG, RIG was low genetically correlated with ADG. On the basis of the obtained best genotype and pedigree matrix weight ratios for the SSGBLUP method, the accuracy of the estimated breeding values for the 8 traits were compared, and the accuracy of the SSGBLUP method was 3.85%-14.43% and 5.21%-17.89% higher than the conventional BLUP and GBLUP, respectively. In conclusion, using RIG as a selection indicator can reduce the average daily feed intake while maintaining the average daily gain, and it is more suitable than RFI for the selection and breeding of fast-growing yellow-feathered broilers; SSGBLUP analysis using the best weight ratio has the best predictive performance for the estimated breeding value of the target characteristics, and it is recommended as a genomic selection method for fast-growing yellow-feathered broilers.

Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle. In this study, the MSTN gene exon of Kazakh sheep was edited to create a stop codon in order to knock out the MSTN gene by using cytidine base editor (CBE). Two sgRNAs targeting to loci across exons of MSTN gene of Kazakh sheep were designed in the study. The sgRNAs were ligated to pGL3-U6-sgRNA-PGK-puromycin plasmid and then co-transfected with pCMV-AncBE4 max-P2A-GFP plasmid into Kazakh sheep fetal fibroblasts. The Cruiser^TM assay, Sanger sequencing and TA clone analysis were performed. The results showed that two sgRNAs could create stop codons in the MSTN gene exons of Kazakh sheep. The editing efficiency of the STOPsg-1 target site was 26.7%, and the editing efficiency of the STOPsg-2 target site was 6.7%. In this study, the CBE (AncBE4 max) technique was successfully used to edit the coding region of MSTN gene in fetal fibroblasts of Kazak sheep in the present study, which lay a technical foundation for breeding Kazakh sheep with precise editing of MSTN gene.

This study aimed to explore the molecular mechanism and key genes that affect the development of arrector pili muscle in Tibetan sheep dorsal skin. In this study, the dorsal skin tissues of 30 healthy Tibetan sheep embryos aged 75-110 days(E75-E85) were collected. The morphological changes of the arrector pili muscle on different days in the dorsal skin were observed by paraffin tissue section and hematoxylin-eosin (HE) staining. It was inferred that the key period of the occurrence of arrector pili muscle was E75-E85.RNA was extracted from 6 dorsal skin samples(E75 vs. E85, n=3), respectively, and transcriptome sequencing was carried out on the Illumina Hiseq platform. The sequencing data were qualitatively controlled, filtered and compared, and differentially expressed genes were screened. Functional annotation, enrichment analysis, protein-protein interaction network and RT-qPCR were carried out to explore the potential regulatory genes affecting arrector pili muscle development. A total of 1 159 differentially expressed genes were identified (P<0.05), 900 up-regulated genes and 259 down-regulated genes. Six differentially expressed genes were randomly selected for RT-qPCR verification, and the results were consistent with transcriptome sequencing, which indicated that the sequencing results were reliable. Forty seven non-redundant genes (such as BAMBI, TNNI3 and HOXA9, etc) in 5 biological process items and 5 signaling pathways related to arrector pili muscle development were screened through GO function annotation and KEGG enrichment analysis of differentially expressed genes. This study shows that the E75-E85 is probably the key period for the development of arrector pili muscle in Tibetan sheep skin. During this period, SPP1, BAMBI, TNNI3, HOXA9, and SOX15 may be the key genes regulating morphogenesis of arrector pili muscle in Tibetan sheep skin.

The study aimed to explore the imprinting status of AQP1 gene in various tissues and placenta of bovine and to determine the role of DNA methylation in regulating imprinted expression of AQP1 gene. The direct sequencing of RT-PCR products was used based on SNP, 5 heterozygous cows and 3 heterozygous placentas were identified from the heart tissues of 32 healthy female adult Holstein cows and 15 placentas after natural delivery, respectively. The allele expression and imprinting status of AQP1 gene in 7 tissues(heart, liver, spleen, lung, kidney, muscle and fat) and placentas of heterozygous cattle were analyzed. The DNA methylation status of the CpG island located the promoter and exon 1 of AQP1 gene was analyzed in heart, liver, two placentas and corresponding sperms by bisulfite sequencing. The results showed that AQP1 gene was in biallelic expression in 7 tissues tested of heterozygous cows. In the placenta, AQP1 gene was in maternal monoallelic expression by analyzing the parental genotype of heterozygous placenta. Furthermore, analysis of the DNA methylation status of CpG island in AQP1 gene promoter in tissues, placenta and corresponding sperms of bovine showed that there were no differentially methylated regions (DMR) in the biallele expressed heart and liver tissues, but in placenta with monoallelic expression, there were DMR, while in sperm with paternal allele expression, there was hyper-methylation. These results suggest that AQP1 gene is a paternal imprinting gene, and DNA methylation modification of the AQP1 CpG island is involved in the regulation of imprinting expression in bovine placenta.The expression of AQP1 gene is biallelic in the tested tissues.

The aim of this study was to explore the effect of egg yolk (EY) treated by high pressure homogenization on the freezing effect of boar semen. Semen were collected from 5 healthy, mature Duroc boars. The ordinary egg yolk+Tris-citric acid-glucose (TCG) diluent was the control group, and the high pressure homogenized egg yolk+Tris-citric acid-glucose (TCG) diluent was the treatment group. Frozen semen samples were thawed, and the sperm motility, DNA integrity, changes in mitochondrial membrane potential, number of apoptotic cells, Caspases activity were detected. The expression of apoptosis-related genes mRNA was measured by qRT-PCR, and extracting raw data and performing data were analyzed. The results showed that, after the high pressure homogenization treatment, the vitality, motility and acrosomal integrity of pig sperms after freezing were significantly higher than those of the control group (P<0.05), which were 87.64%, 87.14% and 66.61%, respectively, higher than those of the control group for 12.58%, 5.82% and 12.48%; mitochondrial membrane potential and DNA integrity were significantly higher than those of the control group (P<0.05), which were 0.74 and 61.76%; the level of sperm apoptosis was significantly reduced (P<0.05), which was 37.74%; the Caspase-3, Caspase-8 and Caspase-9 activities of the experimental group were 17.15, 11.19 and 15.18, which were reduced by 6.17, 2.18 and 3.51 compared with the control group (P<0.05); the expression of Bax, TNF-α, and Caspase-9 genes in the control group were significantly higher than those in the treatment group (P<0.05), and the Bcl-2 gene expression was lower than that in the treatment group (P>0.05). In summary, high pressure homogenous egg yolk can improve the quality of pig sperms after freezing, promote the integrity of mitochondrial function, reduce the level of early sperm cell apoptosis and intracellular Caspase activity, and reduce the mRNA expression of apoptosis-related genes.

This study aimed to evaluate the effect of stevia chlorogenic acid on enhancing immunity of layers infected with E.coli O₇₈ via abdominal air sac artificially, and to provide basic parameter support for the development of functional antibiotic substitutes. A total of 360 1-day-old healthy Hailan layers with no significant difference in body weight were randomly divided into 6 groups:control group (C), E.coli O₇₈ treatment group (EC0), 1.0 g·L^-1 eucommdin + E.coli O₇₈ treatment group (ED1), 1.0 g·L^-1 stevia chlorogenic acid + E.coli O₇₈ treatment group (EC1), 2.0 g·L^-1 stevia chlorogenic acid + E.coli O₇₈ treatment group (EC2), 4.0 g·L^-1 stevia chlorogenic acid + E.coli O₇₈ treatment group (EC4), the formal test started after 7 days of pre-feeding. On the 7th day, the layers was infected with E.coli O₇₈through the abdominal air sac. The treatment lasted for 3 days, and medicine was dissolved into drinking water once a day. Subsequently, the serum levels of IL-1β, IL-2, IL-6, IgM, IgA and TNF-α were detected by ELISA; the expression level of IL-1β, IL-2, TNF-α, Claudin-1 and ZO-1 genes in jejunum and ileum were detected by RT-qPCR; the high-throughput sequencing was used to analyze the types of microorganisms in the cecum contents.The results showed that:1) E.coli O₇₈ significantly increased the mortality of layers (P<0.05), while the mortality of layers in EC2 and EC4 groups were significantly reduced (P<0.05). 2) Stevia chlorogenic acid had a tendency to increase serum IgA and IgM levels in E.coli O₇₈-infected layers, and reduce serum inflammatory factor levels to varying degrees. Among them, serum TNF-α levels of layers in EC1, EC2 and EC4 groups were significantly reduced (P<0.05); the gene expression of ileal pro-inflammatory cytokines IL-1β, IL-2 and TNF-α in layers infected with E.coli O₇₈ were significantly reduced in EC2 group (P<0.05). 3) Stevia chlorogenic acid could promote the expression of jejunum tight junction protein gene, and improve the intestinal barrier damage caused by E.coli O₇₈ infection. 4) E.coli O₇₈ infection would lead to the increase of specific OTUs in intestine of layers, and affect the composition of cecum microflora. The relative abundance of Bacteroidetes, Proteobacteria and Fusobacteria were increased and the relative abundance of Firmicutes was decreased by the injection of E.coli O₇₈. However, stevia chlorogenic acid treatment group (EC2) could improve the relative abundance of Firmichoides in the cecum and reduce the relative abundance of Bacteroidetes and Proteobacteria in layers infected with E. coli. Stevia chlorogenic acid can enhance the immune function of layers infected with E.coli O₇₈ in the abdominal air sacs, and resist the invasion of E.coli O₇₈ to layers. The application dose of 2.0 g·L^-1 stevia chlorogenic acid has a better effect. This indicates that chlorogenic acid has the effect of an antibiotic substitute. Its positive effect on the immunity of E.coli-infected layers may be achieved by regulating immune-related genes and maintaining the steady state of the cecal microbial flora, but its mechanism still needs in-depth study.

This study aimed to evaluate the effect of microecological preparation of Lactobacillus paracasei KL1 screened from Tibetan kefir, which could significantly reduce serum cholesterol and tolerate the gastrointestinal environment, on production performance, egg quality and cholesterol levels of laying hens producing low cholesterol eggs. A total of 120 30-week-old healthy Nongda No.3 laying hens with similar laying rate were randomly divided into 4 groups with 5 replicates per group, and 6 hens per replicate. Hens in control group were fed the basal diet, hens in low, medium and high dose groups were fed the basal diet supplemented with different doses of Lactobacillus paracasei KL1 microecological preparation(10⁵, 10⁶,10⁷CFU·(hen·d)^-1), which lasted for 10 weeks. The production performance of laying hens, egg quality and cholesterol content in eggs were measured and calculated. The results showed that:1) the hen-day laying rate of the laying hens in the medium dose group was significantly higher than that of the control group (P<0.05). 2)Compared with the control group, the eggshell strength, eggshell thickness and serum calcium level of hens in the medium and high dose groups significantly increased (P<0.05), and the relative weight of egg yolk extremely significantly increased (P<0.01). 3)The contents of egg yolk cholesterol and serum TC and TG of hens in the low, medium and high dose groups were extremely significantly lower than that of the control group (P<0.01). The serum HDL-C levels of hens in the high-dose group was extremely significantly higher than those in the control group (P<0.01), and the serum LDL-C levels of hens in the high-dose group was significantly lower than that of in the control group (P<0.05). Lactobacillus paracasei KL1 has significant effects on laying rate, egg quality, egg yolk cholesterol levels and serum cholesterol levels. The results indicate that adding Lactobacillus paracasei KL1 probiotics to the diet can significantly improve the production capacity and eggshell quality of laying hens, and reduce the cholesterol content in eggs. In general, 10⁶ CFU· (hen·d)^-1 is the best dosage, which has good application value in the production of low cholesterol eggs.

The objective of this study was to assess the effect of grazing and confinement feeding systems on meat quality of Small-tailed Han sheep. One hundred male weaning lambs of 2-month-old were randomly divided into grazing and confinement groups. The nutritional composition, amino acid content, meat colour, pH, tenderness and meat production performance were detected after slaughtered at 8-month-old. The results showed that the crude protein and water content of meat in the grazing group were significantly higher than those of the confinement group (P<0.05). The dry matter of meat in the confinement group was significantly higher (P<0.05) and the crude fat was extremely significantly higher (P<0.01) than those of the grazing group. The meat contents of methionine, aspartic acid and serine were significantly higher (P<0.05) and the contents of threonine, valine, isoleucine, leucine, lysine, glutamic acid and histidine in confinement lambs were extremely significantly higher (P<0.01) than those in grazing lambs. The total amount of essential amino acids, non-essential amino acids and total amino acids in confinement lambs were also extremely significantly higher than those in grazing lambs (P<0.01). Compared with the confinement group, the yellowness (b^*) and brightness (L^*) values of longissimus dorsi were extremely significantly higher (P<0.01), the diameter of muscle fibre was extremely significantly smaller (P<0.01), the density of muscle fibre was extremely significantly larger (P<0.01) in the grazing group. The cooked meat rate was extremely significantly higher (P<0.01) and the loin-eye area was extremely significantly larger (P<0.01) in the confinement group than those of the grazing group. In conclusion, grazing lambs have a higher content of protein, lower content of fat, better characteristics of muscle fiber, and confinement lambs have a higher content of fat and amino acids, better meat color and meat production performance. Two feeding systems should be combined in production to balance feeding.

The purpose of this study was to investigate the role of miR-133c-3p in the process of cell apoptosis caused by porcine epidemic diarrhea virus (PEDV) infection, and to explore its mechanism. In this study, PEDV-infected MARC-145 cells were used as a model to detect the expression differences of 6 apoptosis-related microRNAs during PEDV infection. The expression levels of 6 apoptosis-related microRNAs were measured by RT-qPCR during PEDV infection. The apoptosis of PEDV-infected, miR-133c-3p transfected MARC-145 cells was detected by flow cytometry. The effect of miR-133c-3p on cell apoptosis was determined by flow cytometry. The target gene of miR-133c-3p was predicted by bioinformatics method. The binding of miR-133c-3p and the 3'UTR of target gene was determined by the luciferase reporter genet. The expression levels of BCL-w and PEDV protein were determined by Western blot when miR-133c-3p was over-expressed. The cell apoptosis rate was determined by flow cytometry when knock-down of BCL-w. The results showed that PEDV infection could induce apoptosis of MARC-145 cells, and the expression of microRNAs related to apoptosis, such as miR-133c-3p and miR-149-5p, were upregulated (P<0.05 or P<0.001), the expression of miR-138-3p was down-regulated (P<0.05). The apoptosis-related microRNAs miR-133c-3p and miR-149-5p were up-regulated (P<0.05 or P<0.001), and miR-138-3p was down-regulated (P<0.05). Among these, the expression of miR-133c-3p was upregulated almost 5 folds (P<0.01). The cell apoptosis rate was significantly increased after overexpression of miR-133c-3p (P<0.01) and the cell apoptosis rate was reduced after knock-down of miR-133c-3p (P<0.05). Bioinformatics methods were used to predict the binding sites between the miR-133c-3p and the 3'UTR of BCL2L2 gene. The result of luciferase reporter gene experiment showed that miR-133c-3p could bind to 3'UTR of BCL2L2 gene (P<0.01). The expression of BCL-w in cells was significantly down-regulated after over-expression of miR-133c-3p (P<0.01). PEDV could inhibit the expression level of BCL-w. Knock-down of BCL-w could induce cell apoptosis. PEDV infection could down-regulate the expression of BCL-w by up-regulating the expression level of miR-133c-3p, thereby promoting cell apoptosis.

The purpose of this study was to establish a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of bluetongue viruses (BTVs) prevalent in China. Specific primers were designed according to the highly conserved regions of the Seg-5 gene of BTV strains isolated in China. By optimizing the reaction condition, the RT-LAMP method was established, and the specificity and sensitivity of the method were evaluated. To verify the accuracy and reliability of the RT-LAMP method, 46 BTV strains belonging to 12 sero-types (BTV-1,-2, -3, -4, -5, -7, -9, -12, -15, -16, -21 and -24) were tested, further 120 BTV nucleic acid-positive blood samples collected from BTV-infected animals and 60 blood samples collected from sentinel animals in 2020 were tested through RT-LAMP and qRT-PCR simultaneously. The optimal reaction temperature of the RT-LAMP was 64℃, and the optimal reaction time was 45 min, the optimal concentration and ratio of the outer primer:inner primer:loop primer in the reaction mixture was 0.2 μmol· L^-1:0.6 μmol·L^-1:0.4 μmol·L^-1. This method can specifically detect the nucleic acids of BTV belonged to the 12 epidemic serotypes in China without any cross-reaction with the nucleic acids of epidemic hemorrhagic virus (EHDV), Chuzan disease virus (CHUV), Akabane disease virus (AKAV), foot-and-mouth disease virus (FMDV) and African horse sickness virus (AHSV). The lower detecting limit of the method was 4.5 copi-es·μL^-1 of BTV genomes. The detection results of 46 BTV strains belonged to 12 serotypes were positive. There was no significant difference (McNemar test P>0.05) between the detection results of RT-LAMP and qRT-PCR for 120 BTV nucleic acid positive blood samples with a coincidence rate of 95.0%. The detection results of 60 blood samples were completely consistent between RT-LAMP and qRT-PCR. These data demonstrated that the RT-LAMP method established in this study was accurate and reliable to detect the BTV strains isolated in China and the clinical blood samples collected from animals. The BTV RT-LAMP method established in this study was rapid, visual, strong specific and highly sensitive to detect samples, which provided technical means for the detection, diagnosis and epidemiological investigation of the BTVs prevailing in China.

Bordetella bronchiseptica and Klebsiella pneumoniae infections are extremely harmful to the breeding of rabbits. They often cause multiple respiratory diseases. To determine the pathogens that cause dyspnea and diarrhea in rabbits, suspected dead rabbits in the rabbit farm were subjected to necropsy. Subsequently, we collected tissues for the isolation and identification of pathogenic bacteria and drug sensitivity tests. The results showed that we obtained a gram-negative, blunt-round, small rod-shaped suspected pathogen and a gram-negative, oval, suspected pathogen; their 16S rRNA gene sequence amplified fragment sizes were 1 492 and 1 494 bp, respectively. The homology of B. bronchiseptica AU 12671 and K. pneumoniae strain F5feb.57 was 99.85% and 100.00%, respectively. The rabbits were identified as B. bronchiseptica and K. pneumoniae mixed infection. The drug susceptibility test found that they are all sensitive to gentamicin, ceftriaxone, and cefazolin, and they have varying degrees of resistance to other drugs. In summary, the bacteria we isolated are Bordetella bronchiseptica and Klebsiella pneumonia. This work provides a reference for the isolation and identification of B. bronchiseptica and K. pneumoniae coinfection and clinical scientific medications during rabbit breeding.

The study aimed to understand the effects of Radix paeoniae rubra and Cortex phellodendri preparation on the intestinal flora diversity of chickens infected with pathogenic Escherichia coli. Two hundred and forty 15-day-old chicks were randomly divided into 4 groups:blank control group (CON), infection group (BC), low-dose treatment group (TL) and high-dose treatment group (TH). Except for the control group, all the treatment groups (BC, TL, TH) were infected with pathogenic Escherichia coli. TL (4 mL·L^-1 sterilized water) and TH (12 mL·L^-1 sterilized water) groups were treated with different concentration of Radix paeoniae rubra and Cortex phellodendri preparation by drinking water after infection of 0.5 d. After 5 days of treatment, the morbidity and mortality of each group were calculated, and high-throughput sequencing technology was used to detect the diversity of bacterial community in the duodenum of chicks. The results showed that, compared with the BC group, the TH group significantly reduced the morbidity and mortality of chicks (P<0.05). Bacterial community analysis showed that, the preparation processing changed the intestinal bacterial community in the chicks. The dominant bacteria were Firmicutes and Enterococcus in CON group; Firmicutes, Proteobacteria, Enterococcus, Escherichia and Staphylococcus in BC group; Firmicutes, Lactobacillus, Enterococcus and Staphylococcus in TL group; Firmicutes and Lactobacillusin in TH group. The heatmap analysis showed that Lactobacillus was negatively correlated with morbidity and mortality, while Escherichia was positively correlated with morbidity and mortality (P<0.05). In summary, the infection of Escherichia coli and the preparation treatment of Radix paeoniae rubra and Cortex phellodendri changed the diversity and composition of intestinal bacterial community in chicks, and the changes of intestinal bacterial community may have an important impact on the incidence and disease resistance of diarrhea in chicks.

Staphylococcus pseudintermedius is the main pathogenic bacterium of canine pyoderma, which has multi-drug resistance and serious threat to the health of dog and human. In this study, 20 strains of Staphylococcus pseudintermedius were isolated from dogs with pyoderma, and the antibacterial activity and antibacterial mechanism of rhein against Staphylococcus pseudintermedius were revealed. In this study, the antibacterial activity of rhein against Staphylococcus pseudintermedius was determined by minimum inhibitory concentration, minimum bactericidal concentration, bacteriostatic curve and adhesion assay. The cell membrane integrity and permeability, ROS level of Staphylococcus pseudintermedius were detected after rhein treated. The morphology and ultrastructure of bacterial cells were observed by electron microscopy, so as to explore the antibacterial mechanism of rhein against Staphylococcus pseudintermedius. The results showed that the minimum inhibitory concentration of rhein against Staphylococcus pseudintermedius was 12.5 μg·mL^-1, and subinhibitory concentration could significantly inhibit the growth and adhesion. The permeability of bacterial cell membrane was changed and the activity of β-galactosidase was increased after treated with rhein (P<0.05). The results of ROS assay showed that the level of ROS increased by 3.9 times (1×MIC) and 6.4 times (2×MIC) after rhein treatment. Electron microscopy showed that the morphology was changed after rhein treatment, there was abundant secretions on the cell surface and it was shrunk, the cell wall was broken, electron density was reduced, and the contents was leaked. This study indicate that rhein can destroy cell membrane integrity and permeability, and induce bacterial cells to produce a lot of ROS to achieve antibacterial effect.

To reconstruct noncanonical pyroptosis in vitro, the coding sequences of Caspase-4, hGSDMD-FL and hGSDMD-p30 were amplified by PCR, and these PCR products were inserted into corresponding plasmids to get the following recombinant eukaryotic expression vectors:pCMV-MYC-Caspase-4, p3XFLAG-hGSDMD-FL. The expressions of both recombinant vectors in HEK293T cells were confirmed by Western blot. Whether the noncanonical pyroptosis model was successfully constructed was confirmed by testing the production of LDH, active N-terminal GSDMD-p30 and PI staining after transfection into HEK293T cells. The results showed that recombinant vectors including pCMV-MYC-Caspase-4, p3XFLAG-hGSDMD-FL, and pcDNA3.1-hGSDMD-p30-MYC were constructed and each protein was successfully expressed in HEK293T cells. After co-transfection, significant production of LDH was detected, Caspase-4 cleaved the full-length GSDMD into active N-terminal GSDMD-p30 domain and obvious PI staining was observed by fluorescence microscope, which indicated that the noncanonical pyroptosis was successfully reconstructed in vitro. Therefore, the noncanonical pyroptosis is reconstructed in vitro, which establishes foundation for further revealing the mechanism of noncanonical pyroptosis activation.

The aim of this study is to screen out strains that can efficiently degrade aflatoxin B₁ (AFB₁), and to investigate the detoxification effect of its detoxification active components. In this study, the target bacteria were primarily screened by coumarin as the sole carbon source, and rescreened based on the degradation ratio of AFB₁.The selected strain was identified by morphological observation, physiological and biochemical tests, and 16S rDNA sequence analysis. The detoxification active components produced by the bacteria were located by measuring the AFB₁ degradation rate of the different fermentation components. The cytotoxicity of AFB₁degradation products to LMH cells was analyzed by CCK-8 method. And the effects of heat treatment, ultraviolet radiation, strong acid, strong alkali and proteinase K treatment on the detoxification effects of active ingredients were also studied. The results showed that 24 bacteria strains could grow well in the primary screening medium. After detoxification re-screening, strain Y1-B1 could efficiently degrade AFB₁, and its degradation ratio of AFB₁ reached 73.2%. Through morphological observation, physiological and biochemical tests and 16S rDNA sequence analysis, this strain Y1-B1 was identified as Bacillus amyloliquefaciens. Studies on the detoxification activity of different fermentation components of the cultures of strain Y1-B1 showed different degradation rate of AFB₁, with the supernatant of cell culture exhibited the strongest detoxification activity, followed by bacterial lysates, suspension of bacteria, and supernatant of bacterial lysate. The results demonstrated that the detoxification active components of Bacillus amyloliquefaciens Y1-B1 was an extracellular product in the supernatant. The results of cytotoxicity showed that the toxi-city of AFB₁ degraded by the detoxification active components was significantly reduced compared with the toxicity of AFB₁ to LMH cells. The detoxification active components present different performances to different physical and chemical factors, and has strong resistance to high temperature and ultraviolet radiation, relatively poor resistance to strong acids and strong bases, and proteinase K only partially weakens the detoxification ability. This study will provide new microbial germplasm resources for solving the problem of aflatoxin pollution and also lay foundation for the development of subsequent microbial detoxification products.

The purpose of this study was to investigate the effect of dexmedetomidine on ketamine induced nerve injury in developing rats and its possible mechanism. Seven-day-old SD rats were randomly divided into control groups; ketamine group (20 mg·kg^-1 ketamine, i.p. once every 1.5 hours, 5 times); dexmedetomidine group (15 μg·kg^-1 dexmedetomidine, i.p); ketamine + dexmedetomidine group (30 min before ketamine injection, 15 μg·kg^-1 dexmedetomidine, i.p.). Ninety minutes after the last administration, the brain tissue was taken and fixed for Nissl staining; the contents of CAT, GSH, MDA, IL-1β and IL-18 in the hippocampus and cortical tissue were measured. The results of Nissl staining showed that pre-administration of dexmedetomidine could alleviate the loss of neurons in hippocampal CA1, CA3, and cortex caused by ketamine compared with the control group. Dexmedetomidine pretreatment could also significantly reduce (P<0.05) the levels of MDA, IL-1β and IL-18 in the hippocampus and cortex, and significantly increase (P<0.05) the content of CAT and GSH. The results of the study show that dexmedetomidine pretreatment can effectively reduce hippocampal and cortical MDA levels, increase CAT and GSH content, and inhibit the secretion of IL-1β and IL-18, and exert neuroprotection when ketamine-induced neurological damage in developmental rats.

Hou-hai (GV-1), Hou-san-li (ST-36), Da-chang-shu (BL-25) are commonly used acupoints for treating animal constipation. The present study aimed to clarify the beneficial effect of the three acupoints on rat constipation and the effect of acupuncture on gastrointestinal motility-related neurotransmitters, thus to know what degree and how the acupoints affecting the gastrointestinal system when relieving the constipation and which point being the best, also to differentiate the 3 acupoints for relieving constipation. Sixty SPF healthy rats were randomly divided into 6 groups, except for group A, rats in other 5 groups were given 3 mg·(kg·d)^-1 loperamide once daily to induce constipation. In day 6, other than daily loperamide, rats in group C were given 1.38 mg·(kg·d)^-1 suspension of mosapride for 6 days as western medicine control group. Rats in groups D, E, and F were dry needled at GV-1, ST-36, BL-25 respectively for 30 minutes for 6 days. And then 5-hydroxytryptamine (5-HT), somatostatin (SS), vasoactive intestinal peptide (VIP), substance P (SP), 5-HT₃R and 5-HT₄R mRNA were tested accordingly. Results showed that rats in model group B had typical constipation signs with the increasing of 5-HT and SS, while the contents of VIP and SP reduced, as well as the levels of 5-HT₃R mRNA and 5-HT₄R mRNA. The constipation symptoms for the rats in group C and the 3 acupoint groups were significantly relieved, the time period for the first stool was shortened, the water content in feces was increased, and the feces size was increased. Acupuncture at GV-1 reduced the content of SS in serum by 36.34% (P<0.01), the content of 5-HT was significantly reduced as well. Acupuncture at bilateral ST-36 down-regulated the serum 5-HT content by 23.93% (P<0.01), and up-regulated the relative expression of 5-HT₃R mRNA and 5-HT₄R mRNA in colon tissue (P<0.01). Acupuncture at bilateral BL-25, both VIP and SP increased by 27.48% and 22.75% respectively, while the 5-HT content was significantly reduced. All the 3 acupoints could increase the expression of colonic goblet cells. Dry needle at GV-1, ST-36, BL-25 would alleviate rat constipation, improve gastrointestinal motility and adjust the content of gastrointestinal neurotransmitters with some different pathways, and GV-1 being the best points on rat constipation induced by loperamide.

In recent years, the researches on the anti-inflammatory mechanisms of Traditional Chinese Medicine formula have been in-depth. Pulsatilla decoction served as a classic Traditional Chinese Medicine prescription for clearing away heat and toxic material, is usually employed to prevent and treat bacterial diarrhea. However, its anti-inflammatory mechanisms and target cells are still unclear. In this study, rat intestinal mucosa microvascular endothelial cells (RIMVECs) were used as the model cells, aiming to illustrate the regulation effects of Pulsatilla decoction on LPS-induced inflammation of RIMVECs. The levels of mRNA and protein expressions of key proteins TLR4, TRAF6 and ERK in the inflammatory signaling pathway of RIMVECs after LPS stimulation were detected by RT-PCR and Western blot. The release levels of IL-6, IL-8, IL-1β, and TNF-alpha were determined by ELISA. The results showed that Pulsatilla decoction could significantly reduce the mRNA levels and protein expressions of TLR4, TRAF6 and ERK induced by LPS, and also suppress the secretions of the downstream inflammatory factors induced by LPS. Pulsatilla decoction alleviates the inflammatory responses of LPS-induced RIMVECs by inhibiting TLR4-ERK1/2 signaling pathway and plays an anti-inflammatory role.

In order to explore the role of Fas death receptor in rat pheochromocytoma cell line (PC12) apoptosis induced by cadmium (Cd) and its regulation mechanism on the mitochondrial pathway, Fas gene silencing PC12 cells were treated with 10 μmol·L^-1 Cd for 12 hours. The activation of BH3 interacting domain death agonist (BID), cysteine aspartate-specific protease-9 (caspase-9), cysteine aspartate-specific protease-3 (caspase-3), poly ADP ribose polymerase (PARP), the expression of Bcl-2 associated X protein (Bax), B-cell lymphoma/leukemia-2 (Bcl-2), apoptosis inducing factor (AIF), endonuclease G (Endo G), and the distribution of cytochrome C (Cyt C) in cells were detected by Western blot. Nuclear translocation of AIF was detected by immunofluorescence staining. The results showed that Cd significantly increased the tBID/BID and Bax/Bcl-2 ratios, the release of Cyt C from mitochondria into the cytosol, the activation of caspase-9, caspase-3, PARP, and the expression of AIF, Endo G (P<0.01). Meanwhile, Cd induced the nuclear translocation of AIF. Fas silencing significantly inhibited Cd-induced increase of the tBID/BID and Bax/Bcl-2 ratios, the release of Cyt C from mitochondria into the cytosol, the activation of caspase-3, PARP, the expression of AIF, Endo G (P<0.01). Cd-activated caspase-9 was significantly inhibited by Fas silencing (P<0.05). Cd-induced nuclear translocation of AIF was abated by Fas silencing. The above results indicate that PC12 cells apoptosis induced by Cd via the mitochondrial pathway is mediated by Fas.

This study aimed to evaluate the levels of short chain fatty acids in dogs with chronic renal failure and healthy dogs, and explore the causes of changes in short chain fatty acids and its impact on renal function. Twenty-two dogs with mild chronic renal failure (M-CRF group), 29 dogs with severe chronic renal failure (S-CRF group) and 26 healthy dogs (HC group) were selected. The diversity of gut microbiota was analyzed by 16S rDNA sequencing technology and the concentration of short chain fatty acids in feces was detected by gas chromatography. The effects of gut microbiota and sodium butyrate on renal function of 5/6 nephrectomized dogs were observed by fecal bacteria transplantation and sodium butyrate supplementation. The results showed that:1) the observed species and Simpson index of gut microflora diversity in S-CRF group were lower than those in HC group (P<0.05). PCoA analysis showed that the gut microflora in S-CRF group was different from that both in M-CRF and HC group. 2) Lefse analysis showed that there were a large number of different flora between S-CRF group and HC group. Seven species (bacteroideae, Bacteroides, Pseudomonas, et al) were enriched in S-CRF group, while eleven species, such as Prevotellaceae, Clostridiaceae, Prevotella, Faecalibacterium and so on, were enriched in HC group. CCA analysis showed that the species richness in S-CRF group and HC group were positively correlated and negatively correlated with renal function indexes, respectively. 3) The fecal concentrations of acetic acid, propionic acid and butyric acid in S-CRF group were significantly lower than those in HC group and M-CRFgroup, and butyric acid concentration in M-CRF group was significantly lower than that in HC group too (P<0.05). Furthermore, butyric acid concentration was negatively correlated with serum cystatin C (Cys-C), creatinine (Cr) and urea nitrogen (BUN),and the correlation coefficients were -0.451, -0.583 and -0.514 (P<0.01), respectively. 4) Compared with chronic renal failure model group (5/6 Nx group), the serum Cr and BUN level of 5/6 nephrectomized dog fed sodium butyrate were significantly decreased after 8 weeks (P<0.05). Serum Cr and BUN level of 5/6 nephrectomized mice transplanted fecal bacteria from dogs with CRF were more higher than those in 5/6 Nx group after 8 weeks (P<0.05), while sodium butyrate could reverse these changes. In summary, chronic renal failure of dogs can lead to the decrease of gut microflora diversity, the change of flora structure and abundance, and the decrease of fecal short chain fatty acids concentration. It provides a new theoretical basis for the prevention and treatment of chronic renal failure in dogs.

In order to explore the effect of the Shenhu Baidu granules against porcine reproductive and respiratory syndrome virus (PRRSV), 20 piglets free of PRRSV were randomly divided into 4 groups. Group A were given medicine at 3 days before the infection and Group B were given medicine and infection at the same time. Group C and D were the infection and non-infection control, respectively. Medicine were mixed in the basal diet (10 g per pig per day) of A, B groups and supplied from the start time points to the end of the experiment. The rectal temperature of each piglet was recorded everyday. On the 14th day post infection (PI), one piglet in each group was randomly selected and euthanized to observe the lesions caused by viral infection. The viral loads in lungs were detected with RT-qPCR. And on day 1, 3, 5, 7, 14, 21, 28, and 35 PI, anticoagulant and non anticoagulant blood samples were collected for detection viral loads and antibodies, respectively. The rectal temperature of the piglets in Group A and B were raised up to 40℃ at 6th PI and returned to normal at the 11th and 12th days PI. While, high rectal temperature (more than 40℃) of the piglets in Group C lasted to the end of the experiment. The lung lesions in piglets from Group A and B were lighter and with less alveolar structure damage, inflammatory cell invasion and less exudate in the bronchus. However, the lesions in lung from Group C was serious. The alveolar structures were missing and the bronchus in the lung were filled with full of exudates. Serious inflammatory cell invasion were also observed around the bronchus. The viremia of the Group A were highly significantly lower than Group C at 3, 5, 7, and 14 days PI of PRRSV (P<0.01). And Group B was highly significantly lower than Group C at 3, 5 days PI (P<0.01), significantly lower at 7 and 14 days PI (P<0.05). Antibodies against PRRSV in Group A and B reached to the peak at 21 days PI, and the antibodies of the Group C reached to the peak at 28 days PI. In summary, the Shenhu Baidu granules significantly reduced the lesions caused by PRRSV and also the viral loads in bloods and lungs. The drug brought forward of the peak value of PRRSV-specific antibody and reduced the duration of viremia. This study proved that the Shenhu Baidu granules is effective in prevention and treating PRRSV infection.

Nebovirus (NeV) is an emerging diarrhea-causing pathogen in calves in China. Its VP1 protein contains receptor binding sites and neutralizing antigen epitopes, which are closely related to virus infection and immunity. The purpose of this study was to analyze the molecular characteristics of VP1 genotype 1.2 strains. In this study, diarrhea fecal samples of calves were collected from Ningxia and Henan in 2019 for detecting NeV by RT-PCR, and the complete major capsid proteins (VP1) and RNA-dependent RNA polymerase (RdRp) were amplified from tested positive samples. The results showed that the NeV detection rates in Ningxia and Henan were 11.32% and 8.62%, respectively. The complete VP1 and RdRp sequences of genotype 1.2 were successfully obtained from 4 positive samples, which shared 75.4%-97.8% amino acid identity with 73 complete VP1 sequences available in GenBank. Compared with all other 3 VP1 of genotype 1.2 in GenBank, the 4 strains in this study shared 1 identical amino acid variation in P2 domain and 2 identical amino acid variations in P1 domain. Compared with genotype 1.1, 1.3 and 1.4 strains from China, the 4 strains shared 9, 18 and 14 identical amino acid variations in P2 domain, respectively, and shared 2 identical amino acid variations in P1 domain and 1 identical amino acid variations in S domain, respectively. The 4 complete RdRp sequences in this study were NB-like genotype and shared 67.2%-94.8% nucleotide identity with all other 8 complete RdRp sequences in GenBank. This is firstly detection of VP1 1.2 genotype NeV strains in China, and 4 complete VP1 and RdRp sequences of genotype 1.2 strains were successfully obtained, contributing to better understanding molecular prevalence of NeVs.

This study aimed to investigate the infection of mammalian orthoreovirus (MRV) in Northwest Sichuan yaks and isolate MRV from positive samples. 72 fecal samples of diarrhea yaks from 15 pastures in northwest Sichuan and 15 serum samples of diarrhea yaks from 5 pastures were collected for detecting MRV by PT-PCR, and the positive samples were further serotyped by PCR assay. The results showed that the detection rates of MRV in diarrhea samples and serum samples were 20.83% (15/72) and 40% (6/15), respectively; and MRV2 was 60% (9/15) in MRV-positive diarrhea samples and 83.33% (5/6) in MRV-positive serum samples, respectively; other serotypes were not detected. One strain of MRV2 (TCID₅₀ is 4×10^-8.56·mL^-1) was isolated from a fecal sample, and its complete genome was successfully sequenced with 23 587 bp in length. In phylogenetic analysis of MRV genomes, the strain yak/AB14/2018 is closely related to China swine strains. Compared with all S1 sequences availble in GenBank, this strain shared 4 amino acids variations. In conclusion, MRV was detected from yaks, and one MRV2 strain was successfully isolated, contributing to further research the biological characteristics of MRV2.