Host defense peptide is a multifunctional peptide produced by animals. It not only has broad-spectrum antimicrobial function, but also can be used as an innate immune regulator to enhance the body immunity and prevent the invasion of pathogenic microorganisms. It plays an significant role in ensuring animal health. The expression of host defense peptide is regulated by various nutrients in the diet. Some researchers suggested that promoting the expression of host endogenous defense peptide by nutritional regulation was beneficial to the improvement of animal health and production performance, and was one of the possible strategies to achieve antibiotic-free and healthy breeding in the future. In this review, the regulation of endogenous defense peptide expression by various nutrients is discussed comprehensively, which will provide a new theoretical reference for evaluating the effects of nutrients on host immunity and animal health and production.

This study was performed to investigate the expression of insulin-degrading enzyme (Ide) in different tissues of pig as well as its role in C2C12 myoblast proliferation and differentiation. RT-PCR and Western blotting were used to detect the expression of Ide in different tissues of 8-month old male mini-pigs. siRNA technology was used to knock down the expression of Ide in mouse C2C12 cell line to explore the role of Ide in myoblast proliferation,apoptosis and differentiation. Two groups containing Ide-siRNA treatment group and NC-siRNA (negative control) group were set up. There were 3 replicates in each group. The results showed that Ide was widely expressed in brain, quadriceps, biceps femoris, longest dorsal muscle, heart, liver, spleen, lung, kidney, and testis of pigs, among which its expression was relatively higher in quadriceps, kidney, and testis. After Ide-siRNA transfection into myoblasts for 48 hours, Ide expression was extremely significantly decreased(P<0.001). CCK-8 analysis showed that myoblast proliferation was promoted after Ide interference. Accordingly, the expressions of cell cycle-related genes were significantly increased. Meanwhile, the down-regulation of Ide by siRNA interference did not lead to myoblast apoptosis. Myoblasts induced to differentiation by 2% horse serum were transfected with Ide-siRNA, which resulted in the significant decrease in expression of differentiation marker genes Myog and Myhc at Day 2 and Day 5 compared with control group. This implied that the differentiation of myoblasts was inhibited after Ide interference. We further found that the expressions of Akt2 and phosphorylated AKT (P-AKT) were decreased at Day 5 of differentiation. In summary, Ide was widely expressed in different tissues of pigs, with higher expression in muscle, kidney and testis. Ide is involved in negatively regulating the proliferation of myoblasts, and interfering Ide can inhibit the differentiation of myoblasts through Akt2/P-Akt/Myog pathway.

The aim of this study was to explore the genetic stability of high-inbreeding Luchuan pig. Ear samples were collected from 20 inbreeding and 6 non-inbreeding Luchuan pigs and genomic re-sequencing was performed for each samples. Genetic diversity of inbreeding Luchuan pig population was analyzed by scanning the whole genome SNPs. The specific ROHs and associated genes existed in inbreeding Luchuan pig population were detected based on genomic homozygosity. The results showed that observed heterozygosity was 0.373 in inbreeding Luchuan pig population, which was less than expected heterozygosity (0.491), indicating that selection or inbreeding might happen in the population. The analysis of phylogenetic trees and population's genetic structure indicated that two inbred lines were generated during the inbreeding process of Luchuan pig and the number 230, 236, 239, 132, 130 and 126 individuals had closer relationship. In inbreeding population, the average of genomic homozygosity and ratio of genomic run of homozygosity were 59.64% and 0.12, respectively, which were extremely significantly higher than 53.18% and 0.05 in non-inbreeding Luchuan pig population (P<0.01), which indicated that the inbreeding Luchuan pig population had reached a high level of inbreeding. One thousand two hundred and fifty and 633 ROHs were found in inbreeding and non-inbreeding Luchuan pig populations, respectively. The results of overlapping analysis suggested that 224 pROHs were specifically existed in inbreeding population. Among them, homozygous mutations generated from inbreeding process might affect 1 322 genes, which involved in hormone biosynthetic process, pancreatic secretion, membrane lipid metabolic process, intestinal immune network for IgA production and Striated muscle cell differentiation. These results indicate that the inbreeding Luchuan pig population has lower genetic diversity, higher genomic homozygosity and reaches a high inbreeding level, which illustrate that the population has high genetic stability. Moreover, the homozygous mutations generated from inbreeding process might be associated with immune, growth and other production traits.

Both GBLUP and ssGBLUP methods involved in the inversion of genomic relationship matrices in genomic selection, and large-scale matrix inversion operations were time-consuming. The purpose of this study was to improve the efficiency of the inverse operation of large genomic relationship matrix. The genomic relationship matrix from real data and simulated data was constructed, and the Intel MKL matrix function was introduced, and the efficiency of matrix inversion was improved by reducing the number of iterations (method 1) and repeating the block (method 2), programming to implement algorithms and test computing time on desktop computers and servers. The results showed that the acceleration ratio of the direct inversion function with the MKL was 0.898 when calculating the genomic relationship matrix of 4 000×4 000 using method 1. The calculation speed of 16 000×16 000 was 1.006 times more than that of the inversion function in MKL; The calculation speed of method 2 in the 4 000×4 000 genomic relationship matrix was 1.084 times more than that of the MKL inversion function; For larger 128 000×128 000 matrix inversion operations, the acceleration ratio of this method was 1.805 times more than that of the inversion function in MKL. Compared with MKL direct inversion function, the improved two methods have higher efficiency.

The aim of this study was to clone the transcriptional sequence of chicken H+/myoinositol transporter (HMIT) gene, and to detect its expression in different tissues of chickens, and to explore the effect of exogenous insulin treatment on the relative expression of HMIT gene. In this study, AA broilers were used as experimental animals. The full-length coding region of chicken HMIT gene was cloned by RT-PCR technique, and the biological characteristics of its encoded protein were analyzed by bioinformatics technology. The expressions of HMIT gene in brain, liver, abdominal fat, leg muscle, heart, kidney, jejunum and ileum were detected by fluorescence quantitative PCR. The expressions of HMIT gene in kidney and brain were studied after insulin treatment for 120 and 240 min. The results showed that the chicken HMIT gene (1 694 bp, GenBank accession number: MN708211) was successfully cloned by using the chicken HMIT sequence(XM_001232939.5) predicted by NCBI as a template. The cloned full-length coding region of chicken HMIT was 1 380 bp, which was less than the predicted coding sequence(XM_001232939.5) of 561 bp, which contained a protein of 459 amino acids. Nucleotide and amino acid sequence alignment showed that chicken HMIT was highly homologous among species. The collinearity analysis showed that the region of chromosome 1 where HMIT was located was also highly conserved among species. The protein encoded by HMIT was an unstable hydrophilic protein, its secondary and tertiary structure were mainly composed of random coil and α-helix. Transmembrane structure analysis showed that there were 8 distinct transmembrane domains in chicken HMIT. The results of subcellular localization showed that chicken HMIT protein was distributed in plasma membrane (52.3%), endoplasmic reticulum (21.7%), vacuole (17.4%), mitochondria (4.3%) and Golgi apparatus (4.3%). The results of real-time fluorescence quantitative PCR showed that the expressions of HMIT gene were the highest in chicken brain and kidney, which was significantly higher than those in other tissues(P<0.05). At 240 min after insulin treatment, the expressions of HMIT in kidney and brain were significantly lower than that in PBS control group (P<0.05). In this study, the full-length coding region of chicken HMIT was cloned, the results of bioinformatics and tissue expression characteristics analysis laid a foundation for further exploration of the physiological function and regulatory mechanism of chicken HMIT gene.

The study was conducted to perform alternative splicing analysis on goat insulin receptor (INSR) gene, and to analyze its expression profiles in goat Longissimus dorsi muscle. The Longissimus dorsi muscle of Jianzhou Daer goat were collected from fetuses at 45, 60, and 105 days of gestation and lambs at the 3rd day after birth. Based on Sanger sequencing and high throughput sequencing data (accession number: SRR3567144), the alternative splicing patterns of the INSR gene and their expression difference in Longissimus dorsi muscle were studied. In addition, the characteristics of different alternative splicing variants of goat INSR gene were analyzed using online softwares. The results showed that 4 different INSR transcripts were identified and designated as INSR1, INSR2,INSR3 and INSR4, with CDS lengths of 4 110, 4 146, 4 113 and 4 149 bp, respectively, and the correspondingly amino acid residues were 1 369, 1 381, 1 370 and 1 382. The Exon 11 (36 bp) skipping and partial deletion of Exon 15 (3 bp) led to the main difference among 4 transcripts of INSR in goat. The two alternative splicing patterns were completely identical among INSR gene transcripts of bovine, pig, goat, human, sheep and mouse. These alternative splicing changed the serine phosphorylation sites and the second FN3 domain of goat INSR, and resulted in 3D structure change of the encoded protein in two regions. Meanwhile, there was no significant difference in the expression level of 4 splicing variants during 4 Longissimus dorsi muscle developmental periods of goats (P>0.05), and there was no significant difference among the expression level of 4 splicing variants within the same developmental period (P>0.05). In conclusion, the sequence and splicing model of INSR gene's splicing variants are highly conserved among mammals, and the results have provided the theoretical reference for further revealing the role of INSR gene in regulating animal muscle development.

This study was conducted to estimate the genetic parameters and genetic gains(2007—2017) of weight and average daily gain in different periods of 6 837 Chinese Simmental beef cattle born from 2000 to 2019, where these data were collected by National Center of Beef Cattle Genetic Evaluation. Heritabilities of traits were estimated using restricted maximum likelihood (REML) method in Chinese Simmental beef cattle. The estimated breeding value for each individual was calculated using ASREML software, and the genetic gain for each trait from 2007 to 2017 was also assessed. The results showed that the estimations of heritability for birth weight, 6-month weight, 12-month weight, 18-month weight, 24-month weight were 0.44, 0.42, 0.35, 0.38 and 0.46, respectively; The estimations of heritability of average daily gains of birth-to-six month, 6-12 month, 12-18 month, 18-24 month were 0.47, 0.32, 0.36 and 0.39, respectively in Chinese Simmental beef cattle. Average genetic gain per year from 2007 to 2017 for birth weight, 6-month weight, 12-month weight, 18-month weight, 24-month weight were 0.46, 2.65, 4.31, 4.12 and 2.44 kg, respectively; Average genetic gain per year for average daily gain of birth-to-six month, 6-12 month, 12-18 month, 18-24 month were 0.014, 0.016, 0.009 and 0.007 kg·d^-1, respectively. The systematic evaluation on genetic parameters for growth and development traits in Chinese Simmental beef cattle was conducted. Genetic gain for each trait was also assessed, and these results could provide theoretical basis for the future implementation of genetic improvement plan of beef cattle in China.

The aim of this study was to explore the differences in sperm storage tubules morphology, hormone concentration, and hormone receptor genes expression in hens with divergent sperm storage ability, and to further reveal the mechanism underlying sperm storage. A total of 158 White Leghorn laying hens and 28 rosters of 27 weeks of age were used in this study. After the artificial insemination with mixed semen for two consecutive days, the eggs were collected for each laying hen starting from the 3rd day for 21 days and incubated. According to the fertilization status of the eggs within 21 days after insemination, the individual fertilization rate was counted as the hen's sperm storage ability. The top 4 hens with the highest sperm storage ability and the 4 with the lowest sperm storage ability were selected and named high and low sperm storage ability groups, respectively. The concentrations of progesterone, estrogen, testosterone, and prolactin hormone in serum were detected. The uterine-vaginal junction area of the 8 laying hens was dissected and divided into two parts in the longitudinal direction. One was made into paraffin sections and HE stained for the morphology observation, and the other one was used to detect the expression of the corresponding hormone receptor genes using qRT-PCR assay. The results showed that there was no significant difference in mucosal area of uterine-vaginal junction, sperm storage tubules numbers and density of hens between high and low sperm storage ability groups (P>0.05). The average cross section area of sperm storage tubules of hens in high sperm storage ability group was higher than that of the low sperm storage ability group (P<0.05). The concentration of progesterone in serum of hens in high sperm storage ability group was higher than that of hens in low sperm storage ability group (P<0.05), but there was no significant difference in concentration of estrogen, testosterone or prolactin in serum between groups (P>0.05). Compared with the low sperm storage ability group, hens in the high sperm storage ability group had up-regulated expression of testosterone and prolactin receptor genes, and down-regulated expression of estrogen α/β receptor and progesterone receptor genes, but the differences were not significant (P>0.05). In conclusion, the cross section area of sperm storage tubules may be related to the sperm storage ability of laying hens; in addition, progesterone hormone may play an important role in sperm activation in the sperm storage tubules, which affects the hen's ability to continuous fertilization.

The objectives of this study were to explore the correlation between lncRNAs and the reproductive regulation for goat by analyzing the expression profile of lncRNAs in ovarian tissue of Chuanzhong Black goats with high and low fecundity, and to provide the base for further interpreting the molecule mechanism of lncRNA in reproduction of goats. A total of 10 healthy Chuanzhong female goats (3.5 to 4.5 years old) with more than 3 litters were divided into high fecundity group and low fecundity group. The litter size of female goats in high fecundity group (n=6) were more than 2 per litter, the litter size of female goats in low fecundity group (n=4) were only 1 per litter. Ovary tissue of high and low fecundity goats were collected to construct the RNA sequencing libraries by extracting total RNA after estrus synchronization. Total RNA sequencing was performed by HigSeq X ten platform and differentially expressed lncRNAs were screened. Then the cis-targeted genes of lncRNAs were predicted by exploring the positional relation and binding energy between lncRNAs and mRNAs, and the GO and KEGG pathway enrichment analysis were also performed. Finally, 6 randomly selected differentially expressed lncRNAs were verified by quantitative real-time PCR. The results showed that 168 differentially expressed lncRNAs were identified. Among them, 163 lncRNAs were significantly up-regulated in goat ovaries in high fecundity group and 5 lncRNAs were significantly up-regulated in goat ovaries in low fecundity group. The screened differentially expressed lncRNAs included ENSCHIG00000001479, ENSCHIG00000002617, and ENSCHIG00000002622, which might be candidate lncRNAs for regulating fecundity of goats. The target genes of candidate lncRNAs (MYC, CDH2, FGF9, PPARGC1A, etc) were mainly involved in Janus kinase/signal transducer and activator of transcription (Jak-STAT), cell adhesion molecules, adenosine monophosphate-activated protein kinase (AMPK) signaling pathways. The target gene MYC of ENSCHIG-00000001479 participated in Jak-STAT, transforming growth factor beta (TGF-β), MAPK pathways, and the target gene CDH2 of ENSCHIG00000002617 and ENSCHIG00000002622 participated in cell adhesion molecules pathway. The results of qRT-PCR were basically consistent with the sequencing results. It was inferred that those lncRNAs(such as ENSCHIG00000001479, ENSCHIG00000002617 and ENSCHIG00000002622) might play an important role in the regulation of ovarian development and ovulation of goats. It was considered that differentially expressed lncRNAs identified were associated with litter size of goats. In this study, RNA-Seq was used to obtain and analyze differentially expressed lncRNAs in high and low fecundity goats, and GO and KEGG functional analysis was performed. This provided more transcriptome data for further exploring the reproductive mechanisms of litter size of goats.

The study aimed to investigate the expression and function of absent, small, or homeotic 1-like (ASH1L) methyltransferase in healthy bovine cumulus cells. Location of ASH1L methyltransferase and the methylation patterns of histone H3 Lysine 36 (H3K36) were detected using immunofluorescence staining in healthy bovine cumulus cells. The quantitative PCR (qPCR) and Western blotting were used to screen the effective siRNA targeting Ash1L gene in siRNA-1, siRNA-2, siRNA-3 and control group. The expression level of apoptosis-related genes and polycomb repressive complex 2(PRC2) genes were estimated by qPCR after the Ash1L gene was interfered with siRNA in treatment and control groups. The results showed that ASH1L methyltransferase was located in the nucleus of bovine cumulus cells and distributed in a dotted pattern. The effective siRNA (siRNA-2) targeting the bovine Ash1L gene was successfully screened and its interfering efficiency reached 60%-70%. The monomethylation, dimethylation and trimethylation levels of H3K36 in cumulus cells transfected with siRNA-2 were significantly lower than those in the control group (P<0.05). Additionally, interfering Ash1L gene could significantly up-regulated the expressions of apoptosis-related genes Bax, Bcl-2 and caspase-3. However, the expression levels of apoptosis-related genes Bax and caspase-3 were higher than that of anti-apoptosis-related gene Bcl-2 (1.311 and 1.179 vs 1.146). At the same time, the mRNA expressions of PRC2 protein subunit EZH2 and Suz12 were significantly increased after interfering Ash1L(P<0.05). The expression and function of ASH1L methyltransferase in bovine cumulus cells were studied, ASH1L showed dot distribution in bovine cumulus cells, and the inhibition of Ash1L gene expression resulted in a significant decrease in H3K36me1/2/3 levels and an increase in the expressions of apoptosis-related genes and PRC2 protein related subunits EZH2 and Suz12 genes, which will provide technical and theoretical basis for further studying the effects of ASH1L on embryos development in livestock animals.

Lamb diarrhea is one of the common gastrointestinal diseases during the newborn and weaning stage that can result in lamb's growth retardation and even death. Fecal microbiota transplantation (FMT) has little been applied on the prevention and treatment of digestive tract diseases in young ruminants. In this study, FMT was used to treat diarrhea lambs with the comparison by using different antibiotics so as to explore the therapeutic effect of FMT on suckling lambs' diarrhea. Sixty suckling lambs (male and female in half) with significant clinical symptoms of diarrhea and 10 healthy lambs (male and female in half) at the age of (24±2) days and body weight of (6.12±1.01) kg were divided into 7 groups, namely, healthy control (HC), saline control (SC), fecal microbiota transplantation (FMT), gentamicin (GM), enrofloxacin (ENR), gentamicin+FMT (GM+FMT) and enrofloxacin+FMT (ENR+FMT) treatment, with 10 lambs each. The healing time (day), average daily gain (ADG), physio-biochemical indexes and the diarrhea related inflammatory factor markers were measured. The results showed: 1) ADG, RBC, HGB, the percentage of lymphocyte and the concentration of serum creatinine (CREA) in SC group were all significantly lower than those of HC group (P<0.05), whereas the MCHC and inflammatory factors in SC group were significantly higher than those of HC group (P<0.05). 2) Compared with SC group, after 5 methods of treatment, the FMT, ENR, ENR + FMT, and GM groups all significantly reduced the healing time (P<0.05) except for the GM + FMT group. Only after ENR treatment the ADG was significantly increased (P<0.05). 3) In comparison with SC group, FMT treatment significantly increased the blood hemoglobin content (HGB) and blood lymphocyte number (P<0.05). 4) After FMT, ENR and GM+FMT treatment the inflammatory factors measured were all significantly declined to the level as that in healthy lambs (P<0.05). It is indicated that FMT could treat the lamb's diarrhea effectively by shortening the healing time, improvement of physio-biochemical statues and the inhibition of inflammation in diarrheic lambs. FMT could be an alternative way of lambs' diarrhea treatment by antibiotics in practice.

The aim of this study was to investigate the effect of resveratrol on the transcription of inflammatory factors in heat stress (HS)-induced goat small intestinal epithelial cells(GIE cells). The fourth-generation well-growing GIE cells were used for the experiment and were divided into five groups: blank control group (37 ℃), heat stress group (42 ℃), and 42 ℃ heat stress adding resveratrol groups (5, 15, 30 μmol·L^-1); 6 hours' heat stress treatment was carried out after 6 hours' pretreatment with resveratrol supplementation. The resveratrol cytotoxicity was detected by CCK-8 method; the effect of resveratrol on the secretion of cytokines was measured by ELISA kits in HS-induced GIE cells; biochemical method was adopted to detect the effect of resveratrol on antioxidant properties of HS-induced GIE cells. Gene transcriptions of IL-1β, TNF-α, IL-8, HSP70 and NF-κB were detected by RT-PCR in HS-induced GIE cells, respectively. The results showed that resveratrol with different concentrations (5, 15, 30 μmol·L^-1) could increase GSH-Px, SOD and T-AOC activities in HS-induced GIE cells, and extremely significantly decrease MDA content of lipid peroxide (P<0.01); inhibit the secretion of IL-1β, IL-2, IFN-γ and TNF-α in GIE cells induced by heat stress, and extremely significantly reduce the gene transcription levels of IL-1β, TNF-α, IL-8, HSP70 and NF-κB in inflammatory model cells (P<0.01). These results demonstrated that resveratrol could regulate the expression of HSP70 gene and the production and expression of inflammatory factors in the inflammatory model of GIE cells induced by heat stress, promote the activity of antioxidant enzymes and have significant anti-inflammatory activities. Its anti-inflammatory effect might be related to the inhibition of NF-κB signaling pathway.

The aim of this study was to determine the effects of different dietary cobalt (Co) supplemental levels on lactation performance, nutrient digestibility and plasma biochemical parameters in lactating dairy cows. Thirty mid-lactation Holstein dairy cows were blocked and randomly divided into 5 groups which were fed with basal diet (control group) and experimental diets (4 experimental groups), and the experimental diets were supplemented with Co of 0.15, 0.30, 0.75 and 1.50 mg·kg^-1 DM as CoCO₃, respectively. The experiment was conducted for 56 d, including 14 d of adaptation and 42 d of trial period. Dairy feed intake and milk yield were recorded every day, milk samples were obtained for component determination, feed and fecal samples were collected for apparent nutrient digestibility determination, blood samples were taken for biochemical parameters determination. The results showed that there was no significant effect on dry matter daily intake (DMI) or milk contents at all Co supplemental levels compared with control group (P>0.05). However, the milk yield was increased by adding cobalt of 0.30 and 0.75 mg·kg^-1 DM (P<0.05). Acid detergent fiber (ADF) and crude protein (CP) digestibility increased significantly at 0.75 mg·kg^-1 DM group compared with control group (P<0.05). Co supplemental level of 1.50 mg·kg^-1 DM resulted in the decrease of plasma glucose and aspartate aminotransferase (AST) activity (P<0.05), while 0.75 mg·kg^-1 DM group decreased the activity of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) (P<0.05). Red blood cells (RBC), hemoglobin (HGB) concentration and hematocrit (HCT) were quadratically increased (P<0.05). The recommended level of dietary Co for mid-lactation Holstein dairy cows is between 0.30 to 0.75 mg·kg^-1 DM, the optimal level is 0.73 mg·kg^-1 DM in this study.

The present study was conducted to investigate the effects of dietary supplementation of N-carbamylglutamate (NCG) on growth performance, rumen fermentation, rumen microflora and methane emission of Holstein bulls. Forty-five healthy Holstein bulls (body weight (478±13.66) kg) were randomly assigned into 3 treatments with 15 replicates in each treatments, the bulls were supplemented with 0 (group Ⅰ, control), 15 (group Ⅱ) and 25 g·d^-1 (group Ⅲ) of NCG per head, respectively. A 7-day adaptation period and a 90-day experimental period were included in this study. The results showed that: 1) The feed-gain ratio (F/G) was reduced by 6.91% (P<0.01) and 7.91%(P<0.01) in bulls fed 15, 25 g·d^-1 NCG, respectively, compared with control; 2) The microbial protein concentration in rumen increased by 22.91% (P<0.01) and 15.17%(P<0.01) of bulls fed 15, 25 g·d^-1 NCG, respectively, compared with control; The concentrations of propionic acid in rumen increased linearly (P<0.01) while NCG supplementation increased, with the highest value for 15 g·d^-1 NCG group; 3) From the analysis of the relative abundance of rumen methanogenus at genus level, the relative abundances of Methanobrevibacter in rumen was the lowest when supplemented with 25 g·d^-1 NCG, which relative abundance was 80.05%;The relative abundance of Methanosphaera and Methanosaeta in rumen decreased linearly (P<0.01) while NCG supplementation increased; 4) Dietary supplemented with different levels of NCG all decreased the methane emission of Holstein bulls,the methane emission were decreased by 8.44% and 10.51% in bulls fed 15, 25 g·d^-1 NCG (P<0.01), respectively, compared with control. In conclusion, under the conditions of this experiment, supplemented wiht NCG can effectively regulate rumen fermentation of Holstein bulls, improve the efficiency of feed conversion ratio and reduce methane emissions. The recommended NCG supplementation level is 15 g·d^-1.

The study focused on isolation of the cattle origin bovine viral diarrhea virus (BVDV) from persistently infected dairy cattle in Ningxia and determination of the viral genomic nucleotide sequences, which would provide theoretical basis for the studying the genetic evolution of BVDV strains from various regions of China. Two consecutive anticoagulated blood samples were collected at a two weeks interval from 240 cattle on a demonstration farm of high-yielding dairy cows in Yinchuan, Ningxia Hui Autonomous Region. The samples were tested for the presence of BVDV viral antigen by BVDV antigen detection kit to screen cattle with persistent infection (PI). Lysis of blood lymphocytes was prepared from PI cattle and inoculated onto MDBK monolayers to isolate BVDV. The viral genomic sequence was obtained by cloning and sequencing and compared with referenced viral sequences to analyze the evolutionary relationships between viral strains. A total of two cattle persistently infected with BVDV were screened from cattle on the demonstration farm. A non-cytopathogenic strain of BVDV, NX2019/01, was isolated. A genomic nucleotide sequence of 12 107 nucleotide position (nt) with an 11 703 nt open reading frame (ORF) encoding 3 898 amino acids, was determined by sequencing. At the genomic level, NX2019/01 shared higher similarity ranged from 92.17% to 93.84% with domestic strains SD-15, ZM-95, XC and LN-1 that were segregated into the 1m sub-genotype, but large differences were observed in the E^rns, E1 and E2 genes. In the viruses from acutely infected cattle on the demonstration farm, nucleotide mutations in N terminus coding region of E2 protein led to variations of amino acid at position 9 or 67. Recombination analysis demonstrated similar recombination signals in a fragment between nt 179-288 in E2 gene of NX2019/01 strain with another fragment spanning nt 168 in E1 gene and nt 332 in E2 gene of ZM-95 strain. The signals indicated that NX2019/01 and ZM-95may originated from a recombination between the major parent SD-15 strain and the minor parent LN-1, sharing close relatedness with SD-15, LN-1 strains or highly similar viruses at earlier time during the evolutionary process. The strain of BVDV-1m sub-genotype originated from persistently infected cattle was isolated. The evolutionary relationship of viruses of BVDV-1m subtype was determined at the genomic level and genomic homologous recombination between strains within a same sub-genotype was first reported in this study, which may lay the foundation for further study about evolution analysis of BVDV in China.

Foot-and-mouth disease (FMD) caused by FMD virus (FMDV) is an acute, hot and highly contagious disease. FMDV causes a series of severe inflammatory response, and TLR3 pathway is one of the major cellular inflammatory. In order to study the effect of FMDV protein on TLR3 pathway, we screened the FMDV proteins affecting on TLR3 pathway by reporter assays. Then candidate protein effect on transcription of TLR3 pathway downstream genes was verified by Q-PCR assay. TLR3 signaling pathway proteins interacted with candidate protein was further verified by co-immunocoprecipitation. Finally, we detected phosphorylation level of TLR3 pathway downstream proteins in candidate protein-overexpressed 293-TLR3 cells by Western blot. The luciferase assays and Q-PCR results showed that FMDV 3D potentiated poly (I:C)-triggered TLR3 signaling pathway and transcription of TLR3 pathway downstream genes, respectively. In addition, co-immunocoprecipitation showed that FMDV 3D interacted with TLR3. Furthermore, FMDV 3D increased the phosphorylation level of TLR3 pathway downstream protein. In conclusion, FMDV 3D potentiated type Ⅰ interferon mediated by TLR3 pathway, which regulate innate immune response.

To study the effects of orfv infection on different miRNA expression profile in goat skin fibroblasts (GSF) cells, and understand the roles and regulatory mechanisms of miRNA during orfv infection, total RNA was isolated from GSF cells with or without orfv infection. Differential expressed miRNAs were obtained and analyzed with high-throughput sequencing technology after the miRNA library being constructed. The target genes of differential expressed miRNA were predicted and analyzed by GO and KEGG methods. Ten different miRNAs were randomly selected for RT-qPCR verification. Results indicated that there were total 678 differential expressed miRNAs (fold change≥1.5) between orfv infected and control group, 509 were up-regulated miRNAs, 169 were down-regulated miRNAs, and only 8.21% were conjunct miRNAs between infection group and control group according to the Venn diagram analysis of uniq_miRNA. GO and KEGG analysis showed that the differentially expressed miRNAs were mainly involved in cell biological processes such as lipid metabolism, receptor and cytokine signal transduction. RT-qPCR results of miRNA were consistent with high-throughput sequencing results. In a word, this study obtained a large number of different miRNAs encoded by GSF cells and it indicated that infection of GSF cells with orfv has a significant impact on the miRNA, which provide a reference for further revealing the infection and pathogenesis mechanisms of orfv at the host cells miRNA level.

Atypical porcine pestivirus (APPV) is a pathogen that causes congenital tremors in newborn piglets. To investigate the epidemical and phylogenetic characterization of APPV in China, 285 serum samples collected from 93 pig farms distributed among 22 provinces, during 2015-2017, were detected for APPV, and the sequences of E2 gene were further analyzed in this study. Meanwhile, the full-genomic sequencing and phylogenetic analysis on an APPV strain from a piglet with congenital tremors were also carried out. The results showed that APPV-positive rate of the tested samples was 25.26% (72/285, 95% CI=20.3% to 30.7%), with a prevalence of 38.71% (36/93, 95% CI=59.7% to 94.8%) at farm level, and the positive rate has increased year by year from 2015 to 2017. The nucleic acid homology of thirty-five APPV E2 gene sequences obtained from the positive samples is 83.4%-100.0% and the similarity of deduced amino acids was 90.5%-100.0%. Phylogenetic analysis of the E2 gene showed that they were highly diverse, and all listed strains could be divided into four subgroups. Most of the Chinese strains belong to subgroup 1, which are closely related to 000515 from the United States; and few sequenced strains are clustered into subgroup 2, together with European strains Bavaria S5/9 and NL1 Farm1, as well as ISDVDL2014016573 from the United States; six obtained sequences belong to subgroup 3; and all the strains in subgroup 4 are from Guangdong province; and the subgroups 3 and 4 are all composed of the Chinese strains. Whole-genomic sequence alignment shows that the identity of CHheb1701 with other referenced strains is 81.3% to 98.6%. The phylogenetic analysis indicates that all APPV strains could be divided into two main branches. The CHheb1701 is clustered into the branch containing all previous strains from the countries out of China and it is closed to Chinese virus GX04/2017 and the US strain 000515, which are clustered into the same subbranch. Meanwhile, another branch is composed of Chinese strains only from Guangdong province. The results above indicate that APPV is widely distributed in China, with wide variations, and the strains in China might be introduced from different resources. This study provides valuable information for further prevention and research of APPV.

Nucleic acid detection is the main method to diagnose the infection of classical swine fever virus (CSFV). To monitor performance and compare the measuring results of CSFV nucleic acid obtained by different laboratories, Certification and Accreditation of the People's Republic of China organized the national proficiency testing program “Diagnostic Techniques for Classical Swine Fever” (CNCA-19-A01). This program was undertaken by National/OIE Reference Laboratory for Classical Swine Fever (NRLCSF) which is affiliated to China Institute of Veterinary Drug Control. Positive and negative samples were prepared and numbered randomly. Four samples were sent to each participant and measured according to the participant's instruction. All participants should submit the qualitative results and the original reports to NRLCSF. If the results are not in line with expectation, the participant would have another chance to retest. If the result was still incorrect, the participant would be considered unqualified to detect CSFV nucleic acid. Ninety-seven institutions have participated this program. The accurate rate is 93.81% for the first test, and 98.97% for the first and second tests. This program shows that over 90% institutions have the capability for CSFV nucleic acid detection. These institutions could provide correct results and meet the requirements for CSF diagnosis and surveillance. For the unqualified institutions, the program could help them to investigate the reason for disagreement and implement their deficiencies.

Marek's disease, a rapid-onset neoplastic disease in poultry caused by the serotype 1 Marek's disease virus (MDV-1), has been historically regarded as an ideal model for the research on virally-induced cancers. Previous studies have demonstrated that the deletion of the miR-M4-5p, a viral analog of cellular oncogene miR-155 encoded by MDV-1, from the viral genome significantly reduces virus pathogenicity and oncogenicity, suggesting an important regulatory role responsible for MD tumorigenesis. To further reveal the regulatory mechanism mediated by miR-M4-5p in MD biology, we used CEF total cellular RNA as the template to produce a cDNA library utilizing the hybrid-PCR, for screening the host mRNA targets for miR-M4-5p. A total of 128 candidate host genes were obtained, of which there are 73 candidate binding sites were located in the 3'-UTRs and 23 of them contain the perfect matches of miRNA:mRNA. Further experiments of the dual luciferase reporter assay (DLRA) and the quantitative real-time PCR (qRT-PCR) analysis characterized three host genes, DPT, TMEM230 and DCLK1, as the in vivo targets for miR-M4-5p. Our work provides an important basis for further elucidating the regulatory mechanism of miR-M4-5p in MD oncogenesis.

Meq gene is commonly regarded as the main oncogene that plays critical role in the tumorigenesis of Marek's disease (MD). Evolutionarily, there is an obvious sequence difference between the meq genes of vaccine and pathogenic strains with differ virulence. In this study, we have designed the specific guide RNAs (gRNAs) targeting to the meq gene of the vaccine strain CVI988/Rispens, to make the plasmid pX459-gRNA. The CEF cells were transfected with pX459-gRNA plasmids and were infected with CVI988/Rispens virus to produce meq-gene mutated viral particles. Utilizing the CRISPR/Cas9-based gene editing technique, together with the viral plaque purification, PCR analysis, DNA sequencing and indirect immunofluorescence assay (IFA), we successfully generated a meq-deleted mutant named as CVI988Δmeq-C7. Our data provides an important basis for further screening of the monoclonal antibodies against MD vaccine MEQ protein and the development of differential diagnostic reagents.

This experiment was conducted to study immune responses induced by Brucella transcriptional regulatory factor HFQ. In this study, a pair of primers were designed according to hfq gene sequence of B. abortus S2308 from GenBank (BAB1_1134), and the heat inactivated S2308 was used as template to amplify hfq gene by PCR. And then the hfq gene was cloned into pET-32a vector and transformed to E. coli BL21 (DE3) and induced the expression of recombinant HFQ protein (rHFQ). The expressed proteins were analyzed by SDS-PAGE and Western blot. Expression levels of cytokines IFN-γ and IL-4 in murine macrophages (RAW 264.7) stimulated with pET-32a empty plasmid, rHFQ and current vaccine M5-90 were detected by ELISA. Mice were immunized with pET-32a empty plasmid, rHFQ and current vaccine M5-90, and IFN-γ, IL-4 in splenocytes and IgG antibody levels in serum of mice were detected by ELISA. The results showed that the full length of hfq gene was 237 bp, encoding 79 amino acids. rHFQ was approximately 25.8 ku by the SDS-PAGE. There was a single band after purification. Results of Western blot indicated that the rHFQ had good reactogenicity. When RAW 264.7 cells were stimulated with pET-32a, rHFQ and M5-90, the levels of IFN-γ and IL-4 in rHFQ group were similar to those in M5-90 group, significantly higher than those in PBS group and pET-32a empty plasmid control group. The levels of IFN-γ and IL-4 were increased over time. When mice were immunized with pET-32a, rHFQ and M5-90, the levels of IFN-γ and IL-4 in splenocytes and IgG antibody levels in serum in rHFQ group were similar to those in M5-90 group, significantly higher than those in PBS group and pET-32a empty plasmid control group. The HFQ protein of Brucella has good reactivity and can induce the body to produce high levels of cellular and humoral immunity. It is an ideal candidate antigen for the development of Brucella subunit vaccines.

This experiment was conducted to prepare the first batch of national standard of tylvalosin to determine the potency of tylvalosin in tylvalosin tartrate and its preparations. The refined tylvalosin tartrate was used as raw material. After dispersal of the raw materials, the quality inspection and uniformity evaluation were tested according to the requirements of quality standard and Chinese Veterinary Pharmacopoeia 2015 edition. Ultraviolet spectrophotometry was applied to determine the maximum absorption wavelength. Vacuum drying at 60 ℃ was applied to determine the loss on drying. HPLC was applied to determine the tylvalosin A. Based on the standard products of the original invention factory, the three dose tube dish method in antibiotic microbiological assay was used for collaborative calibration. The results showed that the weight loss was 2.0%, the uniformity evaluation was in accordance with the regulations, the maximum absorption wavelength was 289 nm, the potency value was 822 U·mg^-1. The prepared national standard of tylvalosin met all the relevant requirements. It is convenient for production enterprises and quality inspection departments to control the quality of tylvalosin tartrate and its preparations, and it is of great significance to improve and ensure the quality of veterinary drugs.

A new formulation of sarafloxacin/β-cyclodextrin (SAR/β-CD) inclusion complex microcapsules was prepared and characterized, and its solubilization ratio and entrapment efficiency were determined. The dissolution and pharmacokinetics of SAR/β-CD inclusion complex microcapsules were studied in vitro and in vivo. The inclusion complex microcapsules were prepared by the stirring method. The characterization was carried out by transmission electron microscope (TEM), scanning electron microscope (SEM) and powder X-ray diffraction (PXRD). The solubilization ratio and entrapment efficiency of SAR/β-CD inclusion complex microcapsules were determined by liquid chromatography, and the release of SAR/β-CD cyclodextrin inclusion complex microcapsules in phosphate buffer (pH6.8) was studied by dissolution experiment. Finally, the pharmacokinetics of inclusion complex microcapsules was evaluated in chicken with oral administration. Physical and chemical characterization showed that the drug entered the β-cyclodextrin cavity and SAR/β-CD inclusion complex microcapsules were successfully obtained. The average solubilization ratio and entrapment efficiency of the three batches of SAR/β-CD inclusion complex microcapsules were 25.3 times and 90.3%, respectively. The dissolution rate of SAR/β-CD inclusion complex microcapsules in phosphate buffer (pH6.8) was 95.6%, while that of common powder was only 72.2%. The solubility and dissolution of the preparation were significantly improved after the inclusion reaction. In pharmacokinetic experiments, the standard curve of pharmacokinetic determination of plasma concentration was y=1.563 2x-0.189 6, R²=0.999 3, which showed a good linear relationship in the range of 0.25 -10.00 μg·mL^-1. The precision RSD of the analytical method is less than 10%, and the accuracy of the analytical method is more than 90% and less than 110%. The recovery experiments showed that the recovery rates of low concentration (0.50 μg·mL^-1), medium concentration (1.00 μg·mL^-1) and high concentration (2.50 μg·mL^-1) were 90.55%±3.81%, 93.85%±3.14% and 98.19%±5.41%, respectively. The freeze-thaw experiment shows that the freeze-thaw stability (n=3) accords with the regulations of China Veterinary Pharmacopoeia (2015 edition). The pharmacokinetic tests showed that the AUC (mg·h^-1·L^-1), T_max(h) and C_max (μg·mL^-1) of SAR/β-CD inclusion complex microcapsules and sarafloxacin powder were 43.59±0.50, 2.18±0.09, 5.99±0.30 and 17.27±0.30, 0.98±0.07, 1.19±0.10, respectively. A new dosage form of SAR/β-cyclodextrin inclusion complex microcapsule was obtained, which can significantly improve the drug dissolution rate and bioavailability. It is of great significance to the popularization and application of quinolones.

The aim of this study was to screen the differentially expressed pathogenic genes in liver of Wenshang Barred chicken infected by subgroup J avian leukosis virus(ALV-J). ALV uninfected and infected Wenshang Barred chickens were pre-screened by virus isolation from blood at 42 and 300 days of age. The liver tissue samples from both ALV infected chickens which had the pathological changes and ALV uninfected chickens were identified accurately by PCR detection,then the liver tissues from 3 ALV-positive (G1 group)individuals and 3 ALV-negative (G2 group) individuals were sampled for further study. PCR ampilification products of 3 ALV-positive individuals were confirmed as subgroup J by sequencing and cluster analysis. The liver tissue samples from both 3 ALV-J positive individuals and 3 ALV-negative individuals were selected for screening the differentially expressed genes by RNA-Seq technology,then functional analysis was conducted. The partial differentially expressed genes were verified by qRT-PCR. The results showed that 42 differentially expressed genes were enriched in GO and KEGG databases, which included 18 up-regulated genes and 24 down-regulated genes. Moreover, expressions of 5 differentially expressed genes randomly selected were verified by qRT-PCR, which confirmed that mRNA expression levels were consistent with RNA-Seq sequencing results. GO analysis results indicated that the differentially expressed genes were mainly involved in cellular processes, metabolic processes,response to stimulus, immune system, and so on. KEGG analysis results showed that the differentially expressed genes were mainly enriched in cellular processes, signal transduction, disease, metabolism pathways. This study found many genes and critical signaling pathways which affected the pathogenicity of ALV-J by transcriptome analysis and would provide new ideas for understanding the pathogenic mechanism of ALV-J at molecular level.

The purpose of this study was to compare the differential expression of HIF-1α and IL-17 in the testicular tissues of different bovines, and to further explore the correlation between the regulatory functions of HIF-1α and IL-17 in bovine testes. The testes of adult yak, cattle-yak,and yellow cattle were used as the research objects. Western-blot and real-time quantitative PCR were used to detect the differential expression of HIF-1α and IL-17 proteins and genes in 3 types of bovine testes, respectively. The results showed that the expression of HIF-1α and IL-17 genes and proteins were significantly different among 3 bovine testicular tissues, and the expression in cattle-yak testes were extremely significantly higher than those in yak and yellow cattle (P<0.01); In addition, the expression levels of HIF-1α and IL-17 proteins in yak were significantly higher than those of yellow cattle (P<0.05,P<0.01), while the expression level of IL-17 gene was not significantly different between yak and yellow cattle (P>0.05). The differences in expression of HIF-1α and IL-17 genes and proteins in different bovine testicular tissues indicated species specificity; The significantly higher expression of HIF-1α and IL-17 in testis tissues of yak and cattle-yak suggested that they might have important regulatory effects on the adaptation to hypoxic environment.

This study aimed to isolate an epidemic strain of the canine adenovirus (CAV) from Beijing. Nasopharyngeal swabs were collected from 2 to 4 months dogs with cough and other respiratory symptoms in a pet hospital in Beijing. The sample showing positive with canine adenovirus after initial screening by colloidal gold and PCR, was propagated in MDCK cells after processing. Continuous subculture was carried out, and typical cytopathic lesions such as rounding, shedding, and grape stringing appeared. The sample was identified by morphological observation, PCR identification and animal regression test. The PCR amplification results indicated that the isolate was a canine adenovirus type II, and it was named as CAV-BJ02 (GenBank：MN744708) strain. Virus titers were determined according to the method of Reed and Muench, and TCID₅₀ of CAV-BJ02 isolated was 10^6.7·(100 μL)^-1 in MDCK cells. Canine adenovirus particles with a regular hexagonal icosahedron and a diameter of about 86 nm can be observed clearly under electron microscope. Phylogenetic analysis showed that the sequence of the CAV-BJ02 strain was consistent with that of CAV type Ⅱ. Animal regression test results showed that CAV-BJ02 strain can cause mild clinical symptoms such as fever, with oral and nasal secretions as the main routes for viral shedding, and the viral shedding period is about 5-6 days, which does not cause dogs to die. The results above provided insights into the prevalence of CAV in Beijing and laid a theoretical foundation for the diagnosis, prevention and treatment of canine adenovirus disease.

To investigate the epidemic status and characteristics of Maedi-Visna disease in Xinjiang, this study collected 2 647 samples of sheep sera from Aksu, Aletai, Bazhou, Changji, Hami, Shihezi, Yining, from 2017 to 2019. Enzyme-linked immunosorbent assay (ELISA) was performed to detect Maedi-Visna antibody. Statistical analysis of the positive rate of sheep Maedi-Visna antibody in different varieties, genders and regions. The detection rate of Aksu, Altay, Bazhou, Changji, Hami, Shihezi and Yining were 28.95% (121/418), 0.67% (5/751), 2.50% (2/80), 4.91% (8/163), 1.14% (3/264), 1.22% (1/82) and 17.10% (152/889). The positive rate of Aletai sheep, Hetian sheep, Kazakh sheep, Merino sheep, Suffolk sheep and Small Tail Han sheep, were 0.35% (1/288), 1.14% (3/264), 22.87% (231/1 010), 13.86% (42/303), 1.85% (13/702) and 2.50% (2/80), respectively. The positive detection rate of local variety and introduced variety were 0.79% (8/1015), 1.58% (15/947), respectively. The positive detection rate of female and male were 3.59 % (34/946) and 15.17 % (258/1 701), respectively. These results indicated that it is necessary to carry out early quarantine of Meadi-Visna disease, identify and eliminate the sick sheep timely, and improve the feeding and management conditions, which is of great significance to promote the healthy development of the sheep industry.

The aim of this study was to investigate the infection of feline coronavirus (FCoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in partial areas of China during the novel coronavirus outbreak. Nose swabs and rectal swabs were collected from cats presented with respiratory disease symptoms and gastrointestinal disease symptoms in pet hospitals of 14 cities in China. Total RNAs were extracted from the samples and then were transcribed to cDNA. The FCoV and SARS-CoV-2 were detected by the reported PCR methods. As a result, no SARS-CoV-2 case has been detected in 269 cats, while there were 35 cats infected with feline coronavirus. It was suggested that there was no case infected with SARS-CoV-2 in fourteen cities in China during novel coronavirus outbreak.

This experiment was conducted to isolate and characterize a lytic bacteriophage against Salmonella choleraesuis (S. choleraesuis) from the healthy pig gut. Chyme and mucosal samples from pig jejunum and colon were assayed for the presence of bacteriophage capable of forming plaques on S. choleraesuis 13122. Bacteriophage classification was confirmed by TEM observation and sequencing. The host range was determined by the spot test method and double-layer agar plate method. Bacteriostatic ability was evaluated by turbidity measurement. A bacteriophage named L6jm was isolated, which had a regular polyhedron head approximately 75 nm in diameter and a long noncontractile tail with 190 nm in length. As the sequencing result showed no integrase or bacterial genome was in the genome of L6jm, L6jm was defined as lytic bacteriophage. L6jm can infect S. choleraesuis 13312 and Escherichia coli (E. coli) 244. Also, L6jm lysed 9 E. coli strains including one enterotoxigenic E. coli (ETEC) strain. The optimal MOI of L6jm against S. choleraesuis 13122 and E. coli 244 were 0.01 and 0.001 respectively. The latent periods were both 15 min in two strains whereas the rise periods were 145 min and 125 min. L6jm was stable over a wide range of temperatures (30-60 ℃) and pH (3.0-11.0). L6jm could significantly (P<0.05) inhibit the growth of S. choleraesuis 13312, and inhibit ETEC 104 at MOI 100. The polyvalent bacteriophage L6jm infecting S. choleraesuis and E. coli as well as lysing ETEC104 has potential application value in preventing and controlling S. choleraesuis and E. coli infection.