With the universal use of molecular identification, species evolution analysis and whole-genome breeding, accurate genomic-wide SNP genotyping has become the key to genome research in livestock and poultry. Genotyping array, whole genome resequencing (WGS), reduced-representation genome sequencing (RRGS) and target capture sequencing have been widely used in livestock and poultry genomic research. In this paper, the principle of genomic-wide SNP genotyping technology and its application in genome-wide association studies, selection signal analysis and background analysis of livestock and poultry genetic resources were summarized, in order to provide reference for livestock and poultry genome research and genetic breeding.

Maternal-fetal dialogue is a complex dialogue between mother and fetus during pregnancy, which is a necessary process for successful implantation and pregnancy and is regulated by many factors. Abnormal mother-fetus dialogue can lead to the failure of embryo implantation and pregnancy, and can affect the normal development of fetus during pregnancy. Therefore, mother-fetus dialogue during pregnancy is of great significance for improving the litter size and reproductive efficiency of pigs. The review summarized the factors influencing the mother-fetus dialogue from pregnancy recognition (before pregnancy), embryo implantation (pregnancy) to fetal development (after pregnancy) using sows as a model, including hormones, cytokines, adhesion factors, genes and proteins, etc, which can provide reference for pig maternal-fetal dialogue study and increasing litter size in pig industry.

Complex and diverse intestinal microbial communities play an important role in the host health. Butyrate-producing bacteria represent an important functional group in host intestinal microecology, and most of them are Gram-positive anaerobic bacteria. It is found that butyrate-producing bacteria are closely related to the gastrointestinal inflammation caused by microbial imbalance, so butyrate-producing bacteria became an industry research hotspot. Many studies have shown that butyrate-producing bacteria can confer a health benefit on the host by producing butyrate with indigestible carbohydrates to maintain intestinal bacterial balance, supply energy for intestinal epithelial cells and enhances the function of intestinal mucosal barrier. The biological classification distribution of butyrate-producing bacteria, the mechanism of digestive tract acid production, the regulation of intestinal inflammation and its application progress are systematically reviewed in this paper, in order to elucidate the mechanism of the interaction between butyrate-producing bacteria and host health, and future directions for butyrate producing bacteria are suggested as well.

The reproductive stress of ewes mainly includes active reproductive stress, passive reproductive stress, and fetal intrauterine stress. Appropriate reproductive stress is an important physiological basis for normal estrus, maintenance of pregnancy, initiation of parturition, and smooth lactation. However, long-term or excessive reproductive stress causes metabolic disorders, immune function damage, reproductive disorders, and fetus or lamb development retardation. This series of diseases and clinical manifestations that can cause maternal health damage has been defined as “ewe’s reproductive stress syndrome”. This paper defines ewe’s reproductive stress and ewe’s reproductive stress syndrome and discusses the comprehensive clinical application of the reproductive stress theory, which provided a theoretical basis for preventing and treating reproductive diseases (such as toxemia during pregnancy, postpartum anestrus, endometritis, etc.).

The purpose of this study was to investigate the selection signature differences of immune and lipid metabolic related genes between Wuzhishan pig and Duroc pig. The ear tissues were sampled from 30 individuals of 4 months old healthy Wuzhishan pig (10 boar and 20 sows) from Hainan National Conservation Farm of Wuzhishan Pig and performed whole genome sequencing (WGS), and the Duroc WGS data was download from the NCBI database (SRA: PRJNA378496). The single nucleotide polymorphisms (SNPs) of the WGS data of 59 samples were analyzed by bioinformatics methods. The SNPs were filtered, located in the genome, their structural characteristics, genotype frequency were analyzed, the functions of corresponding genes were annotated. The strongly selected regions of the Wuzhishan pig genome were screened using the XP-CLR method, and the functional differences of genes in related pathways of the two breeds was analyzed. The results showed that each Wuzhishan pig had 36 961 902 SNPs, and intron region had the largest number of 16 729 364 SNPs, taking account of 45.26%, and the coding region (start, end codons) had 2 073 SNPs. The selected signal regions screened by XP-CLR was focus on the immune response, metabolic, and neurological functions related pathways. In the immune response pathway, there was only one kind of mutation homozygous genotype in Wuzhishan pigs in the functional region SNPs of 9 genes (TGFBR2, IL26, IL15, BMPR2, TNFSF15, TNFSF4, TNFSF8, ACKR4, TNFRSF11B), while in Duroc, there are three kinds of genotypes (wild homozygous genotype, heterozygous, mutant homozygous genotype) in most individuals, among which the wild homozygous genotype had the highest proportion. In the lipid metabolism pathway, the genotype frequencies of key regulatory genes IRS2, PRKG1, and ADCY5 were similar to those of immune response genes. The proportion of mutant homozygous genotypes in Wuzhishan pigs was 100%, and the proportion of wild homozygous genotypes in Duroc pigs was 48%-93% (14/29-27/29). In this study, 9 candidate genes related to immune response and 3 candidate genes related to lipid metabolism were screened out in Wuzhishan pigs, and the candidate genes related to immune response, metabolic, and neurological functions showed stronger selection signature in Wuzhishan pig than that in Duroc pig, which provide a reference for revealing the molecular mechanism of the formation of characteristic traits of Wuzhishan pigs.

Pedigree is an important information source for animal breeding. This experiment was conducted to study the effect of high-density SNP markers on reconstructing genealogy in production populations and to fill the gap of using high-density SNP information to reconstruct multi-breed, large-scale realistic production pig populations. In this study, 1 471 great-grandparent purebred Duroc (n=986) and Landrace (n=485) pigs born from 2017 to 2021 in a pig farm in Sichuan province were genotyped by Illumina GeneSeek GGP Porcine 50K chip. Genomic relationship between the two populations was analysed and reconstructed using the common ancestor fragment method, and genomic relationship was used to reconstruct the genealogy of the two populations. At the same time, to measure the accuracy of the common ancestor fragment method,115 breeding pigs with individual chip genotype information and pedigree records were selected, and their pedigree records were ensured by strictly controlling the production operation process. The results showed that the identity by descent(IBD)-based method could use genomic information to simultaneously infer the distribution and proportion of common ancestral fragment among individual pairs in a multi-generational, breed-mixed true production population, and could more accurately differentiate kinship between individuals than identical by state(IBS), thereby determining inter-individual relatedness and further inferring lineage structure. A total of 702 kinship pairs were inferred in the validation population of 115 pigs, including all parent-offspring pairs (n=184), full sibling pairs (n=175), half sibling pairs (n=109) and grandparent and grandchild pairs (n=18) recorded in the pedigree. It was also possible to infer additional unrecorded 3rd (n=8) kinship and 4th (n=18) kinship between individuals than that with recorded genealogies. The reconstructed genealogy provides a clearer picture of kinship relationships between individuals within the family line than the common three-generation genealogy. In this study, the method of reconstructing the pedigree of a multi-variety population based on the analysis of high-density SNP markers by the IBD fragment method can quickly and easily judge the correctness of the pedigree of a multi-variety mixed population, and reconstruct the genealogy of individuals with missing genealogies, which provides a basis for breeding work such as selection and matching, calculation of breeding value and GWAS mining.

The study aimed to investigate the relationship between far upstream element binding protein 3 (FUBP3) or ubiquitin specific peptidase 43 (USP43) gene polymorphisms and loin muscle area traits in Beijing black pigs, so as to guide the breeding work at the molecular level. A total of 408 Beijing black pigs were selected to collect data on loin muscle area traits and meat samples for DNA extraction. A total of 43 pairs of primers were designed for PCR amplification and sequencing according to FUBP3 and USP43 genes sequence, and the sequencing results were analyzed by DNAstar software. Association analysis was performed on different genotypes of FUBP3 and USP43 genes with loin muscle area traits of Beijing black pigs, and gene expression differences of the two genes were analyzed by fluorescent quantitative PCR. There were 8 mutations in the promoter region of FUBP3 gene, among which rs701769847G>A was significantly associated with the 5-6 rib loin muscle area and the last rib loin muscle area(P<0.05), rs332131528C>G was only significantly associated with 5-6 rib loin muscle area (P<0.05), rs325550799T>C, rs339464012T>C and rs326069041T>C were only significantly associated with the last rib loin muscle area (P<0.05). Differential gene expression analysis showed that the mRNA level of FUBP3 gene in individuals with GG genotype at rs701769847G>A was significantly lower than those with AA genotype(P<0.05). rs335310752C>T in the promoter region and rs323463345G>A in the shear region of USP43 gene were both significantly associated with the last rib loin muscle area (P<0.05). Differential gene expression analysis showed that the mRNA levels of USP43 gene in rs323463345G>A were not significantly different between GG-type individuals and AA-type individuals. Two loci in the above two genes were significantly associated with 5-6 rib loin muscle area trait (P<0.05), and 6 loci were significantly associated with the last rib loin muscle area traits(P<0.05)， which can be used as candidate genes for loin muscle area trait variation in Beijing black pigs, and as potential molecular markers for loin muscle area traits.

The objective of this study was to screen differentially expressed mRNAs (DEMs) and lncRNAs (DELs) in skeletal muscle of Tibetan chicken and Daheng broiler in the embryonic stage and construct a lncRNA-miRNA-mRNA regulation network, so as to provide a basis for further investigation of the difference mechanisms in skeletal muscle development between the two breeds. Three embryos of Tibetan chicken and Daheng broiler that has been incubated for 18 days were randomly selected, respectively. The embryonic leg muscle tissues were collected for trans-criptome sequencing. The DEMs and DELs were screened and verified by real-time quantitative PCR (qRT-PCR). The DEMs and DELs were performed by GO and KEGG enrichment analysis. The miRNA that not only as a target for lncRNA but also targeting mRNA were selected to construct a lncRNA-miRNA-mRNA regulatory network. The results showed that a total of 106 DEMs were screened in the two breeds, of which 48 mRNAs were up-regulated and 58 mRNAs were down-regulated. These DEMs significantly enriched in GO terms including skeletal muscle cell differentiation and KEGG pathways including lipid metabolism. Twenty eight DELs were screened, of which 10 lncRNAs were up-regulated and 18 lncRNAs were down-regulated. KEGG analysis of the target genes of lncRNAs showed that many target genes significantly enriched in signaling pathways related to lipid metabolisms, such as steroid biosynthesis and fatty acid biosynthesis. The DEMs and DELs associated with skeletal muscle development were screened and lncRNA-miRNA-mRNA competitive regulation network was constructed. The result of qRT-PCR showed that the expression trend of selected DEMs and DELs was consistent with the sequencing result. The DEMs and DELs related to skeletal muscle development of Tibetan chicken and Daheng broiler were screened to construct the lncRNA-miRNA-mRNA regulatory network. The study provided a theoretical basis for revealing the variability of skeletal muscle growth and development in different chicken breeds.

This study aimed to analyze the expression profiles of long non-coding RNA (lncRNA) in sheep ovarian tissues at different reproductive cycles, understand the lncRNA expression and its regulatory mechanism, and provide a theoretical basis for sheep breeding research. In this study, Hu sheep were selected as the research object, and 3 ewes aged from 1.5 to 2.5 years old in luteal phase and follicular phase were selected, respectively. lncRNA in ovarian tissues were screened by high-throughput sequencing. Bioinformatics analysis was used to predict the target genes of differentially expressed lncRNAs. GO and KEGG enrichment analysis were used to identify pathways related to sheep reproduction. The results showed that a total of 1 379 differentially expressed lncRNAs were obtained in this study, of which 1 158 were up-regulated and 221 down-regulated. GO and KEGG enrichment analysis showed that the differentially expressed lncRNAs and their target genes mainly involved in ovarian follicle development, ovulation cycle process, calcium signaling pathway, oocyte meiosis, oxytocin signaling pathway, MAPK signaling pathway, thyroid hormone synthesis signaling pathway, and estrogen signaling pathway. Key lncRNAs might regulate reproduction by regulating genes involved in these signaling pathways and biological processes. Specifically, MAPK1, ADCY1, ADCY5, PPP3CA and CDC23, which are targeted by LNC_011239, LNC_012847, LNC_003902, LNC_003906, LNC_003907 and others, might play key regulatory roles. The results of qRT-PCR showed that the expression levels of 5 randomly selected lncRNAs were consistent with the sequencing results. In this study, RNA-Seq technology was used to screen lncRNAs in luteal and follicular ovarian tissues, and the differences were analyzed to provide the basis for revealing the molecular mechanism of sheep reproductive capacity.

Haplotypes have a stronger linkage disequilibrium (LD) relationship with quantitative trait loci (QTLs). Haplotypes tend to have higher application value in gene mapping and searching for causal mutations. In order to evaluate the role of haplotype markers in genome studies, a total of 1 478 individuals in Huaxi cattle with an average age of 24 months, including 1 333 bulls and 145 cows, were slaughtered from 2008 to 2021. The LD thresholds was set at r²>0.3 and the number of fixed-SNPs was 5 for haploblock construction with 770K chip data, GWAS using GCTA with mixed linear model based on SNP and haplotype (a total of 3 markers were used: SNP marker and two types of haplotype marker) were performed on Huaxi cattle to detect the significant SNPs, haploblocks and candidate genes associated with LW and DP traits. In addition, the GWAS results of the 3 markers were compared to evaluate the advantages and disadvantages of the 3 markers. The results showed that 16 significant SNPs and haploblocks were detected in the whole genome, mainly distributed on chromosome 1, 5, 6, 14, 16, 17 and 28. A total of 10 candidate genes, such as FAM184B,PPM1K,LCORL,RIMS2 were identified, which significantly associated with the slaughter traits. And 3 candidate genes identified by SNP markers could also be identified by haplotype method, most of the significant loci or regions identified by haplotype were located within the gene. Among the two haplotype construction methods, the LD-based construction method identified more candidate genes than the one based on the fixed-SNPs for GWAS. In this study, the results showed that haplotype-based GWAS can comprehensively consider the LD between SNPs and can better reveal the genetic structure of complex traits.

This study aimed to investigate the key miRNAs and target mRNAs affecting the process of milk fat metabolism in Chinese Holstein cows. In this study, 8 Ningxia Holstein cows with extreme differences in milk fat rate in their first lactation at mid to late lactation stage(150-220 d) were selected and divided into two groups according to differences in milk fat rate: high and low milk fat groups, with 4 replicates in each group. Milk samples from the 8 cows were collected to isolate mammary epithelial cells for transcriptome sequencing and bioinformatics analysis to screen out differentially expressed miRNA-mRNA, and their correlation with milk fat rate was analyzed. The miRNAs and mRNAs expression were verified by real-time fluorescence quantitative PCR. The results showed that 8 small RNA (sRNA) libraries were sequenced to obtain 11 976 914-16 235 680 clean reads, accounting for more than 97% of the total raw reads, with Q30 reaching more than 91%. The length of sRNA fragments were mainly distributed in the range of 21-24 nt. A total of 34 differentially expressed miRNAs (16 up-regulated and 18 down-regulated) were identified between the high and low milk fat groups. GO and KEGG enrichment analysis showed that the target genes of differentially expressed miRNAs were significantly enriched in functional items of intracellular signaling transduction, single organism processes, kinase binding and phosphotransferase activity, and significantly enriched in TNF signaling pathway, MAPK signaling pathway, Ras signaling pathway, Rap1 signaling pathway and oxytocin signaling pathway. A total of 16 miRNA-target gene pairs were obtained by comprehensive miRNA-mRNA analysis. miR-1343-3p and MTM1 were significantly negatively correlated with milk fat rate, and miR-370, miR-2285cb and SRRM2 were significantly positively correlated with milk fat rate. The expression trend of differentially expressed miRNAs and mRNAs verified by RT-qPCR were consistent with the result of transcriptome sequencing. These results explored the functional mechanisms of milk fat metabolism in Holstein cows in the Ningxia region at the miRNA and mRNA levels and identified miR-370-MTM1, miR-1343-3p-DIS3L2, miR-2285cb-XLOC-122799 and miR-2387-SRRM2 interaction pairs as possible key factors regulating milk fat metabolism in cows.

The purpose of this experiment was to detect the volatile compounds in donkey meat, explore the key differential flavor substances related to the tenderness of Guangling donkey, and analyze the function and relationship between key volatile compounds and genes related to tenderness. The 30 female Guangling donkeys with the same growth environment and rearing conditions and similar age were selected as the research objects. The shear force and intramuscular fat were measured and divided into high tenderness group (HT, n=4) and low tenderness group (LT, n=4) according to the content difference. The volatile components in longissimus dorsi muscle of Guangling donkey were detected by HS-SPME-GC-MS technology, the key flavor compounds of donkey meat were screened by odor activity value (OAV). In addition, multivariate statistical analysis was used to obtain variable importance in the projection (VIP) to screen the key difference flavor substances related to tenderness, and then Pearson coefficient and transcriptomics were used for joint analysis. The relationship between key flavor substances and differential genes in donkey meat tenderness were explored. A total of 41 volatile substances were identified in the longissimus dorsi of Guangling donkey, including alcohols, aldehydes, hydrocarbons, ketones, esters and 2 other substances. Thirteen key flavor compounds were screened by OAV, and it was found that the main volatile compounds and contributors affecting donkey meat were aldehydes. Through VIP and OAV values, 1-octen-3-ol, 1-octanol and lauryl aldehyde were screened out as the difference substances of tenderness and the key flavor substances that contributed to the flavor of donkey meat. Pearson coefficient was used to screen the differential genes related to key flavor substances and KEGG function analysis was performed. It was found that 1-octen-3-ol related genes were mainly enriched in fructose and mannose metabolism, glycolysis/gluconeogenesis, pancreatic Glucagon signaling pathway, etc.; 1-octanol related genes were mainly enriched in insulin signaling pathway, butyrate metabolism, type 2 diabetes, various adipocytokine signaling pathways, etc.; lauraldehyde related genes were mostly enriched in secondary metabolites biosynthesis, insulin signaling pathway, glycolysis/gluconeogenesis, pentose phosphate pathway, etc.. These pathways might be involved in the formation of key flavor compounds 1-octen-3-ol, 1-octanol and lauraldehyde in donkey meat. In this study, SPME-GC-MS technology was used to detect the flavor volatile substances in donkey meat and analyze the flavor differences of donkey meat with different tenderness, and 3 different tenderness substances and key flavor substances were screened out, including 1-octene-3-ol, 1-octanol and lauryl aldehyde. KEGG enrichment results showed that glycolysis/gluconeogenesis, glucagon signaling pathway, PPAR signaling pathway, fructose and mannose metabolism, and MAPK signaling pathway may be involved in the formation of 1-octene-3-ol, 1-octanol and lauryl aldehyde, which provides a new direction for molecular improvement and breeding of Guangling donkey meat tenderness and flavor.

Investigating caging stress in laying ducks has become extremely important since the environmental policies have changed and the duck industry has grown. This study aims to explore the plasma metabolite profiles of laying duck raised in floor-water rearing system (FWR) and cage-rearing system (CR) using nontargeted metabolomics. The metatarsal vein blood was taken from ten laying ducks from each experimental group for HPLC-HRMS. Differential metabolites were identified with Fold-change ratio≥2 or ≤1/2, VIP>1 and q value<0.05. A total of 30 differential metabolites were obtained based on the screening conditions, of which 14 were up-regulated (P<0.05) and 16 were down-regulated (P<0.05). Differential metabolites included 16 glycerophospholipids, 4 fatty acyls, 2 carboxylic acids and derivatives, as well as phenylpropionic acid, phenols, organic sulfuric acids and derivatives, sterol lipids, purine nucleosides, organooxygen compounds, steroids and steroid derivatives, steroids and steroid derivatives. Primary bile acid biosynthesis and glycerophospholipid metabolism (P<0.01), as well as the secondary bile acid biosynthesis and taurine and hypotaurine metabolism (P<0.05) were found to be enriched by the pathway enrichment analysis. The results showed substantial variations in plasma bile acids, taurine, and glycerophospholipids between FWR and CR. The insights gained from this study may be useful for the development and promotion of waterfowl husbandry by clarifying the mechanism of caging stress in laying duck industry.

The aim of this study was to investigate the effects of cytochrome P450arom (CYP19A1) on the secretion of endogenous 17β-estradiol (E₂), and its effects on autophagy of oocyte and subsequent embryonic development were also evaluated. During the in vitro culture of the yak cumulus-oocyte complexes (COCs), saline, E₂ (10^-7 mol·L^-1), aflatoxin B1 (AFB1) and bisphenol A (BPA) were respectively supplemented to M199 at same volume. The expression and location of CYP19A1 in COCs from different groups were detected by qRT-PCR, Western blot and immunofluorescence, as well as the autophagy related genes 5(ATG5), Bcl-2 homologous domain protein (BECLIN1) and microtubule associated protein light chain 3 (LC3). The levels of endogenous E₂ in mature medium of COCs from the groups treated with saline, AFB1 and BPA were measured by enzyme-linked immunosorbent assay (ELISA). Rates of mature oocyte, cleavage embryo and blastocyst were calculated and compared among different groups. The results showed that both gene and protein levels of CYP19A1 were enhanced significantly in the AFB1 group, along with the increase of the endogenous secretion of E₂, ATG, BECLIN1 and LC3 levels in mature COCs (P<0.05). The level of CYP19A1 were reduced significantly in the E₂ group, while the expression of autophagy related factors in mature COCs were enhanced (P<0.05). In BPA treatment group, the CYP19A1 and endogenous E₂ were significantly reduced (P<0.05), which leading to the lower expressions of ATG5, BECLIN1 and LC3 (P<0.05). Comparing to the control group, the rates of mature oocyte and cleavage embryo were enhanced by E₂ and AFB1 (P<0.05), but reduced in BPA group (P<0.05). There was no significant difference about blastocyst rates among 4 groups (P>0.05). It can be concluded that endogenous E₂ was positive regulated by CYP19A1 during COCs maturation, furthermore both endogenous and exogenous E₂ can induce autophagy and improved the early developmental capacities of yak oocyte. The results provide a theoretical basis for further exploring the molecular mechanism of reproductive hormones regulating the maturation of oocytes in mammalian.

The objectives of the present study were to investigate the effect of parity time on ruminal fermentation characteristics and methane (CH₄) production using the GreenFeed system, which examines relationships between methane (CH₄) output and animal body weight and parity time, and to use these relationships to develop prediction equations for CH₄ emission from Holstein cows at dry off in the normal physiological experimental under the housing condition. A total of 48 dry Holstein cows were selected and divided into 4 groups (12 cows in each group): first-, second-, third-, and fourth- and above-parity. The pre-trial period was 5 days and the formal trial period was 40 days. The results showed as follows: 1) The live weight and metabolic weight of third- and fourth- and above-parity groups were significantly higher than those of first- and second-parity groups (P<0.05). 2) Ammoniacal nitrogen (NH₃-N) of third-parity group was significantly higher than that of first-parity group (P<0.05), but no significant difference were found compared with the second-parity group and fourth- and above-parity groups (P>0.05); Compared with fourth- and above-parity group, microbial protein (MCP) of first-parity and second-parity groups were significantly increased (P<0.05), but there was no significant difference compared with the third-parity group (P>0.05); Compared with third-parity, valerate of first-parity group was significantly decreased (P<0.05), but there was not significantly different from the second-parity and fourth- and above-parity groups (P>0.05); The total volatile fatty acid (TVFA), pH, acetate, propionate, butyrat, isobutyrate, isovalerate, and acetate to propionate ratio were not significantly different among the treatment groups (P>0.05). 3) The average CH₄ emission of dry cows was 336 g·d^-1. The CH₄ emissions of dry cows within third-parity and fourth- and above-parity groups were significantly higher than those of first-parity group (P<0.05), and there was no significant difference compared with the group second-parity (P>0.05). 4) CH₄ emission was positively related to parity time and body weight (BW) of dry cows in the correlation coefficients of 0.47 or 0.61, respectively (P<0.01). Based on body weight to establish a prediction model CH₄(g·d^-1)=0.348×BW (kg)+64.018 (R²=0.46). It is concluded that CH₄ emission of Holstein cows at dry off can be predicted from BW. The dataset can also be used to validate a range of prediction equations for CH₄ production of cows under other physiologicalstates.

The aim of this study was to investigate the effects of dietary L-malic acid supplementation on intestinal health and inflammatory response of weaned piglets. A total of 192 healthy 28-day-old crossbred (Duroc×Landrace×Yorkshire) weaned piglets (initial body weight (9.12±0.52) kg) were randomly divided into 4 treatments, with 6 replicates per treatment and 8 piglets per replicate. The control group was fed a corn-soybean meal based diet, and the three treatment groups were fed the basal diet supplemented with 0.25%, 0.5% and 1% L-malic acid products (containing 20% L-malic acid and 80% carrier) for 28 days, respectively. Levels of circulating inflammatory factors were detected on days 14 and 28. Moreover, the inflammatory response, antioxidant capacity and jejunum morphology of piglets were measured after slaughter, and protein expression levels related to barrier function in jejunum were analyzed as well. The results showed as follows: 1) Compared with the control group, diet supplemented with 1% L-malate products significantly decreased the levels of pro-inflammatory factors tumor necrosis factor -α (TNF-α) and interleukin-6 (IL-6) in plasma of piglets on day 14 (P<0.01), and significantly increased the level of anti-inflammatory factor interleukin-10 (IL-10) (P<0.01). Plasma IL-6 level was significantly decreased (P<0.01) and plasma IL-10 level was significantly increased (P<0.01) on the 28th day of the experiment. This suggests that dietary supplementation with 1% L-malate product alleviates the inflammatory response of weaned piglets. 2) Dietary 1% L-malic acid product significantly decreased the levels of TNF-α, interferon-γ (IFN-γ) and IL-6 in jejunum mucosa (P<0.05 or P<0.01), and significantly increased the level of IL-10 (P<0.01). Meanwhile, L-malic acid product markedly increased the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and the level of total antioxidant capacity (T-AOC) in jejunum mucosa (P<0.05 or P<0.01), and also decreased the malondialdehyde (MDA) level significantly (P<0.05). 3) L-malic acid supplementation significantly increased the villus height of jejunum (P<0.01) and thus elevated the ratio of villus height to crypt depth (P<0.05). 4) Compared with the control group, L-malic acid supplementation tended to increase the protein expression level of Occludin (P=0.07) and decreased the protein expression level of Claudin-3 significantly (P<0.01). In conclusion, dietary supplementation of 1% L-malic acid product can improve the anti-inflammatory capacity of weaned piglets. Importantly, 1% L-malic acid supplementation can improve the anti-inflammatory and antioxidant capacity of jejunum, improve digestion and absorption function, and thus improve intestinal health, but the effect on intestinal barrier function needs further research.

This experiment was conducted to produce E2 protein of bovine viral diarrhea virus (BVDV) by using suspension cultured CHO cell expression system, and to identify the immunogenicity of purified E2 protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E2 was constructed based on the gene sequence of BVDV-1 NADL strain. The recombinant plasmid pcDNA3.1-BVDV-E2 was transfected into CHO cells in suspension culture, and the E2 protein was secreted and expressed in cells supernatant. The expression and purification of the E2 protein was determined by SDS-PAGE electrophoresis, and the reactivity was determined with anti-His antibody and BVDV positive serum by Western blot. Analysis of immunogenicity was determined after immunizing New Zealand white rabbits with E2 protein, and the antibodies of the E2 protein were tested by indirect ELISA and indirect immunofluorescence (IFA). The concentration of purified E2 protein was 1.228 mg·mL^-1 by BCA protein quantification kit. Western blot results showed that specific bands of the E2 protein could be detected with His antibody and BVDV positive serum. Serum antibody could be detected by indirect ELISA in the 7th day after prime immunization, and serum antibody was maintained at a high level until the 28th day after immunization. The antibody titer was up to 1:1 024 000. The result of IFA indicated that the expression of the E2 protein could be detected by the immunized rabbit serum in BVDV-infected MDBK cells. These results demonstrated that the purified E2 protein with good immunogenicity and specificity. In this study, BVDV E2 protein was produced using CHO suspension culture system successfully, which has a superior immunogenicity. This result will lay the foundation for the development of diagnosis method and novel subunit vaccine of BVDV.

Here, we report the development of an indirect ELISA antibodies detection method for African swine fever virus (ASFV). Two purified monoclonal antibodies (mAbs) against ASFV p30 and p54 protein were used as targets and a phage-displayed 12-mer peptide library was used to conduct four rounds of biopanning to screen peptide epitopes, then amino acids GGG was used as a linker to synthesize tandem-epitope peptide of ASFV p30 and p54 protein which was used as coating antigen. The optimum reaction conditions of indirect ELISA were determined by chessboard titration, and clinical serum samples were used to evaluate the specificity, sensitivity, stability and conformity of this method. The biopanning experiment indicated that ¹⁴⁶PAEPYTT¹⁵² was a core domain of the B cell linear epitope of p54 protein. The optimization results of ELISA reaction conditions showed that the tandem-epitope peptide coupled with ovalbumin (OVA) at N-terminal had low background of non-specific serum reaction. And the optimum reaction effect was obtained when the polypeptide antigen was coated with carbonate buffer in 2 μg·mL^-1, the serum was diluted 100-fold with blocking solution (1% gelatin solution), and the HRP-antibody was diluted 5 000 times with 0.05% PBST solution. The cut-off value was determined to be 0.339. Furthermore, the results of specificity, sensitivity and stability tests showed that there is no cross-reaction in positive serum samples of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV), the detection limit of ASFV positive sera is 1∶1 600, and the method had high repeatability. Finally, Total 320 swine serum samples were detected simultaneously by the present established method and commercial ASFV antibody detection kit. The results showed that the relative specificity and sensitivity of the two methods were 97.6% and 97.3%, respectively. And the coincidence rate was 97.5%. In conclusion, this method showed good specificity, sensitivity, repeatability and coincidence rate, that had the potential value of developing clinical diagnostic kit.

In order to establish a blocking ELISA method for detection of African swine fever virus (ASFV) antibody, the prokaryotic expressed ASFV p30 protein was used to immunize BALB/c mice for preparation of monoclonal antibody (MAb). A blocking ELISA method was developed after optimization of the reaction conditions with the recombinant p30 protein as a coating antigen and the horseradish peroxidase (HRP) conjugated MAb as detecting antibody. The receiver operating characteristic (ROC) curve calculated an optimal cut-off value of 16.63%. This method had no cross-reaction with positive serum samples of CSFV, FMDV-O/A, PRRSV, PEDV or SVA. The sensitivity test showed that a 1∶128 dilution of positive serum can be detected by this method. The coefficient of variation (CV) of the intra- and inter-assay was less than 10%. A total of 208 serum samples were detected in parallel by the method and the commercial ELISA kit, the Kappa value was 0.96, which demonstrated its high consistency. In conclusion, the established blocking ELISA method with high sensitivity and specificity can be applied to the detection of ASFV antibodies in serum, which provides technical support for the epidemiological investigation and epidemic monitoring of ASFV.

This study was designed to investigate the biological characteristics of a meningitis causing Pasteurella multocida and the biological basis for this bacterium causing brain infections. Serum bactericidal tests, intracellular survival tests, mouse infection tests, and nanopore sequencing were performed on a P. multocida strain SD001 which was isolated from the brain of a pig with neurological symptoms. Results were as follows: Compared to those of P. multocida isolates from the other organs of pigs, SD001 displayed a stronger anti-serum bactericidal capacity, and a stronger capacity of surviving in mouse macrophages. In mouse models, SD001 formed a higher bacterial load in both blood and brain than a pneumonic P. multocida strain at the same time points post challenge, and the challenge of SD001 led to inflammatory and other pathological damages in the experimental mice. These findings suggest that SD001 may cause bloodstream infections and meningitis animals. Results from nanopore sequencing revealed that the complete genome sequence of SD001 was 2.45 Mb in size, with an average GC content of 40.25%. The complete genome of SD001 encoded 2 281 putative proteins and 77 RNAs. It also contained two CRISPR sequences, five genomic islands, seven prophages, and 233 virulence factor encoding genes. Genotyping using the complete genome sequence showed that SD001 belonged to capsular: LPS: MLST type A: L6: ST10. SD001 infection could cause meningitis and its strong capacity of anti-serum bactericidal activity as well as inducing bloodstream infection may confer the bacterium capacity of causing brain infections. P. multocida infection should be considered for the diagnosis of brain infections in veterinary clinical settings.

This study was conducted to understand the pathogenicity and drug sensitivity of Klebsiella pneumoniae (Kp) isolated from pig lung tissue. In this study, the isolate was identified based on morphology observation, PCR detection, 16S rRNA sequencing and MLST multi-locus sequence analysis. The pathogenicity of the isolate was determined by virulence gene detection and mouse challenge test, and the drug sensitivity was analyzed by antibiotics and traditional Chinese medicine. The results showed that the isolate was Gram-negative bacilli, and the biochemical results were consistent with the biochemical characteristics of Kp. 16S rRNA gene sequence alignment analysis and phylogenetic tree construction identified the isolated strain as Kp, named KP-0728. MLST analysis showed that KP-0728 belonged to ST-35 type and converged with ST-875. KP-0728 contained four virulence genes, uge, wabG, fimH and kfu. When KP-0728 was infected with mice intraperitoneally, different degrees of pathological changes in various organs of the infected mice were induced, and high dose infection of KP-0728 can cause death in mice. KP-0728 carried four resistance genes bla-SHV, sul2, tetA and aadA1, and was resistant to ampicillin, imipenem, gentamicin, tetracycline and cotrimoxazole and sensitive to ceftriaxone, cefalexin, cefmetazole, azithromycin, ofloxacin, polymyxin B and teicoplanin. The results of in vitro antibacterial experiments of nine Chinese medicines showed that Poria cocos had the most significant antibacterial effect on KP-0728. The data of animal treatment test also showed that Poria cocos had inhibitory effect on KP-0728 in vivo. In conclusion, a porcine Kp type ST-35 was isolated in this study, carrying a variety of virulence genes and drug resistance genes. Artificial infection of Kp in mice can lead to pathological changes and the median lethal dose (LD₅₀) was 4.56×10⁶ CFU, and KP-0728 is sensitive to traditional Chinese medicine Poria cocos and many kinds of antibiotics. The results provide a reference for the prevention and treatment of Kp and vaccine research.

Avian vaccines have problems of low immunization efficiency, short immunization duration and insufficient cellular immune stimulation. New avian vaccine adjuvants need to be developed and used to solve these problems. As reported in our former studies, CpG ODN has high safety and good cellular and humoral immune stimulation functions. However, the expensive cost of CpG ODN prevents its promotion in avian vaccines. In order to reduce cost and improve efficacy, based on the potential synergistic relationship between TLR2 and TLR21, this study investigated the relationship between Rhodococcus ruber and CpG ODN to synergistically stimulate immunity by utilizing the avian influenza vaccine as a model vaccine. Through the HI test, cell proliferation test and detection of the expression of related cytokines, we confirmed that the compound adjuvant which induced Th1 immunity has stronger humoral immunity and cellular immunity, a long-last time protection, as well as systemic and local immunity enhancement. By examining the relative expression levels of the receptors, we confirmed that there is a synergistic relationship between CpG ODN and Rhodococcus ruber. In addition, in the compound adjuvant stimulation experiment of halving the dose of CpG ODN, we found that due to the existence of synergy, the compound adjuvant with half dose CpG ODN still had good immune stimulation effect. This study made a preliminary exploration for further reducing the production and use costs of CpG preparations, and laid a foundation for the efficient and cost-controllable promotion of CpG preparations in poultry animal breeding and even animal husbandry.

Apicomplexan Ap2 domain proteins (ApiAP2) may be transcription factors that control the development and sexual differentiation of parasites. This study aims to clone and express transcription factor ApiAP2 of Eimeria necatrix, and detect the native ApiAP2 protein and its localization in the parasites. The gene (EnApiAP2) was cloned from the total RNA of the third generation merozoites (MZ-3) of E. necatrix by RT-PCR, and inserted to pMD18-T vector by TA cloning. After sequencing analysis, EnApiAP2 cDNA was subcloned to pET-28a(+) vector to obtain a recombinant prokaryotic plasmid. After transformed into expression strain BL21 (DE3), the recombinant plasmid pET28a(+)-EnApiAP2 was induced to express by IPTG, which was purified and renatured. BALB/c mice were immunized with purified recombinant protein, and the polyclonal anti-EnApiAP2 antibodies were prepared, which were used to detect the native EnApiAP2 protein and its localization in the second generation merozoites (MZ-2) and MZ-3 of E. necatrix by Western blot and indirect immunofluorescence assay, respectively. Finally, the transcriptional level of EnApiAP2 in MZ-2 and MZ-3 was analyzed by qRT-PCR. The results showed that the target gene was 1 830 bp, coding 610 amino acids with a predicated molecular weight of 67.69 ku and one AP2 domain. The recombinant protein was about 74 ku and predominately expressed in inclusion body. Western blot analysis indicated that the recombinant protein could be specifically recognized by 6×HIS tag monoclonal antibodies, the convalescent serum of chicken infected with E. necatrix or E. tenella. Native EnApiAP2 protein was detected in MZ-3 and had a molecular weight of 85 ku. EnApiAP2 protein was located in the nucleus of MZ-3. The transcription level of EnApiAP2 in MZ-3 was significantly higher than that in MZ-2 (P < 0.01). In conclusion, EnApiAP2 gene was successfully cloned and expressed. EnApiAP2 protein located in the nucleus of MZ-2 and MZ-3. These results lay a foundation for further study on the transcription factor function of EnApiAP2 protein.

This study aimed to clarify the role of Enolase on Riemerella anatipestifer invading duck brain microvascular endothelial cells (DBMEC) and blood brain barrier. R. anatipestifer enolase gene deletion mutant ΔEnolase and the complemented strain cΔEnolase were constructed by homologous recombination and combined transfer assay using RA-LZ01 strain as the parental strain. The differences of adherence and invasion abilities of RA-LZ01, ΔEnolase and cΔEnolase to DBMEC were detected. At the same time, bacterial loads in blood and brain of ducklings infected with RA-LZ01, ΔEnolase and cΔEnolase were determined respectively.The results showed that, compared with RA-LZ01, the adhesion and invasion rates of ΔEnolase to DBMEC were significantly reduced, and cΔEnolase restored the adhesion and invasion abilities to DBMEC. Bacterial loads in blood had no significant difference among the animals infected with RA-LZ01, ΔEnolase or cΔEnolase. Compared with the ducklings infected with RA-LZ01 and cΔEnolase strains, the loads of bacteria in the brain of the ducklings infected with ΔEnolase strain was significantly decreased. These results indicated that Enolase was significantly correlated with the adhesion and invasion of R. anatipestifer into DBMEC and the invasion of ducklings’ brain. Hence, Enolase should be a virulence factor mediating R. anatipestifer crossing duck blood brain barrier.

This study was conducted to study the changes of transcriptome in Theileria annulata infected bovine monocytes (TaNM Ⅰ) before and after treatment with Bupavaquone (BW720) for laying a foundation for further study on the molecular mechanism of host cell transformation induced by T. annulata. Bovine TaNM Ⅰ cells infected by T. annulata were used as experimental materials, control group (DMSO) and experimental group (BW720) were set up. High-throughput sequencing was carried out by Illumina HiSeq4000 sequencing platform. The original reading segment (Raw reads) was compared with GenBank and Rfam database, and the clean value (Clean reads) was obtained by quality control. The differentially expressed genes in DMSO treatment group and BW720 treatment group were screened by Venn analysis software, and functional annotation (GO) and signal pathway (KEGG) analysis were performed. Ten genes were randomly selected and the expression of genes in cells under different treatment conditions was detected by fluorescence quantitative PCR (qPCR). After verifying the accuracy of the sequencing results, the differentially expressed genes were selected and their roles in apoptosis and proliferation of TaNM Ⅰ cells were analyzed. Results showed that 6 854 019 704 and 6 627 265 854 Raw reads were obtained in BW720 treatment group and DMSO treatment group, and 22 925 606 and 22 171 427 Clean read were obtained after filtration and quality control, respectively. A total of 4 054 genes were expressed in BW720 group and DMSO group, of which 2 146 genes were significantly up-regulated and 1 908 genes were significantly down-regulated. Three hundred and sixty-seven differentially expressed genes were further screened, of which 196 were significantly up-regulated and 171 were significantly down-regulated. At the same time, the expression trend verified by qPCR was consistent with that of transcriptome sequencing, indicating that the sequencing results were reliable. Then GO functional annotation was performed on the differentially expressed genes, and 20 biological processes, 15 cell components and 11 molecular functional items related to cell proliferation were screened. KEGG enrichment analysis showed that some signaling pathways related to T. annulata transformation cells were screened out in Top20 signaling pathway, such as cancer signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, etc. The progress analysis of these signaling pathways found that PI3KR3, FOXO1, IL23A, FZD3, AKT, MMP9 and other genes have also been reported in the study of transformation cells of T. annulata. This study shows that DAPK1,FZD3,FOXO3 and PI3KR3 may be the key candidate genes affecting the infinite proliferation of infected cells in TaNM Ⅰ cells. It lays a foundation for the follow-up study of the mechanism of infinite proliferation of TaNM Ⅰ cells.

Echinococcus multilocularis is a severe zoonotic parasite. To explore the mechanism of non-coding RNA in the polarization of mice liver Kupffer cells (KCs) induced by E. multilocularis infection, we herein infected the BALB/c mouse with E. multilocularis and isolated the KCs from the mice liver at 2 months post infection (Mpi). After RNA high-throughput sequencing, we analyzed and constructed the ceRNA regulatory network related to the M2 polarization of KCs induced by E. multilocularis infection. The qPCR and miRNA overexpression assays were used to verify the ceRNA regulatory network. The results showed that, after E. multilocularis infection 2 Mpi, the M1-type markers were significantly down-regulated, while the M2-type markers were significantly up-regulated. Among them, 5 circRNAs and 9 miRNAs were identified to be involved in regulating the expression of M2-type markers, such as IL10, IL13, and Arg1, and their expression patterns were consistent with the sequencing results and the circRNA-miRNA-mRNA regulatory relationship. Moreover, overexpression of miR-466c-5p led to the down-regulation of circ_0000372 and IL13, indicating that the circ_0000372-miR-466c-5p-IL13 axis plays vital role in the polarization of mouse liver macrophages induced by E. multilocular infection. This study provides an essential theoretical basis for further revealing the mechanism of E. multilocularis regulating the polarization of host macrophages.

The aim of this study was to investigate the function of MGF360-13L gene of African swine fever virus (ASFV) multigene family. The MGF360-13L gene was analyzed by bioinformatics software. The eukaryotic expressing plasmid pVAX1-MGF360-13L was constructed and transfected into 293T cells for expression. The MGF360-13L gene deleted strain ASFV-ΔMGF360-13L was constructed by homologous recombination. After infecting the gene deleted virus strain and parent virus strain (ASFV CN/GS/2018) in porcine alveolar macrophages (PAMs) with the same multiplicity of infection (MOI=0.01), the virus titer was calculated by erythrocyte adsorption test (HAD) and the in vitro growth curve was drawn; ASFV-ΔMGF360-13L and ASFV CN/GS/2018 were infected with porcine bone marrow macrophages (BMDM) with MOI=1. The expression levels of inflammatory factors IL-1β, IL-6 and TNF-α were determined by qPCR and ELISA. The results showed that, MGF360-13L gene was relatively conservative in ASFV gene type II. The coding protein belongs to non secretory type, without transmembrane region and with good hydrophilicity; The eukaryotic expression plasmid pVAX1-MGF360-13L was expressed by 293T cells; The MGF360-13L deleted strain ASFV-ΔMGF360-13L was successfully constructed. There was no significant difference between ASFV-ΔMGF360-13L and ASFV CN/GS/2018 in in vitro replication. After ASFV-ΔMGF360-13L infected BMDM, the levels of transcription and secretion of inflammatory cytokines IL-1β, IL-6 and TNF-α were significantly higher than those of ASFV CN/GS/2018. In this study, the MGF360-13L gene deleted strain of ASFV were successfully constructed, and it was confirmed as a non-essential gene for ASFV replication and have the function of inhibiting the expression of inflammatory factors. This study provides clues for further annotation of ASFV MGF360-13L gene function.

The study was conducted to explore the repair effect of 3, 6-Dibromo-beta-fluoro-N-(3-methoxyphenyl)-9H-carbazole-9-propanamine (P7C3-A20) on traumatic brain injury (TBI) of rat adrenal medullary pheochromoma differentiated cell line (PC12 cells). The cells were divided into control group (group A), model group (group B) and 0.03 μmol·L^-1 drug treatment group (Group C), 0.3 μmol·L^-1 drug treatment group (Group D), 3 μmol·L^-1 drug treatment group (Group E) and drug control group (Group F). TBI cell models were prepared by scribing straight lines 4 mm apart horizontally and vertically using a 10 μL gun tip. The cell viability was detected by CCK8 kit, the apoptosis and reactive oxygen (ROS) situation were observed by fluorescence microscope, and the relative mRNA expression of cysteine aspartate-specific protease-3 (Caspase-3), B-cell lymphoma/leukemia-2 (Bcl-2), Heme Oxygenase-1 (HO-1), NAD (P) H: quinone oxidoreductase 1 (NQO1) and glutamate-cysteine ligase catalytic subunit (GCLC) genes were detected by fluorescence quantitative PCR instrument. The results showed that the cell viability was extremely significantly decreased after TBI modeling (P<0.01). P7C3-A20 at concentrations of 0.03 μmol·L^-1 could significantly increase the cell viability after TBI (P<0.05). P7C3-A20 at a concentration of 0.3 μmol·L^-1 could extremely significantly increase the cell viability after TBI (P<0.01). After TBI modeling, the proportion of early and late apoptotic cells increased extremely significantly (P<0.01). P7C3-A20 at a concentration of 0.03 μmol·L^-1 could significantly reduce the proportion of early apoptotic cells after TBI (P<0.05), and extremely significantly reduce the proportion of late apoptotic cells after TBI (P<0.01), P7C3-A20 at concentrations of 0.3 and 3 μmol·L^-1 could extremely significantly reduce the proportion of early and late apoptotic cells after TBI (P<0.01). After TBI modeling, the proportion of reactive oxygen species increased extremely significantly (P<0.01). P7C3-A20 at concentrations of 0.03 and 3 μmol·L^-1 could significantly reduce the proportion of reactive oxygen species after TBI (P<0.05)， 0.3 μmol·L^-1 concentration of P7C3-A20 can significantly reduce the proportion of reactive oxygen species after TBI (P<0.01). P7C3-A20 at a concentration of 0.3 μmol·L^-1 can extremely significantly reduce the relative mRNA expression of Caspase-3 after TBI (P<0.01), and significantly increase the relative mRNA expression of Bcl-2, HO-1, NQO1 and GCLC genes expression level after TBI (P<0.05). P7C3-A20 has the repair effect of anti apoptosis and reducing oxidative stress on PC12 cells after traumatic brain injury.

The purpose of this experiment was to study the anti-allergic effect of hypoallergenic pet core material and preliminarily explore its possible mechanism. Five weeks old female BALB/c mice were used in the experimental. Ovalbumin (OVA) was administered to establish a food-borne allergy mouse model. Meanwhile, hypoallergenic pet core material was given to investigate the effect of this core material on the intestinal structure and allergic reaction of food-borne allergic mice. The anti-allergic mechanism was explored through the detection of immunoglobulin, cytokines, histamine, and immune organ index in mice. Compared with the control group, the average daily gain of food-induced allergic mice was significantly decreased (P<0.01); the villus height, crypt depth, and the ratio of which were significantly decreased (P<0.05 or P<0.01); diarrhea rate was up to 86% which was highly significantly increased (P<0.01), the rectal temperature was significantly decreased (P<0.01), and the allergic reaction score was significantly increased (P<0.01); the serum levels of IgE, IgG, His, and IL-4 were extremely significantly increased (P<0.01), the IL-10 levels were significantly decreased (P<0.01), and the number of degranulated mast cells was significantly increased (P<0.05); thymus and spleen index were significantly decreased (P<0.01); the above indexes of mice given hypoallergenic pet core material were significantly improved compared with the model mice. The hypoallergenic pet core material can inhibit the degranulation of mast cells, reduce the release of histamine, inhibit the occurrence of food induced allergic reaction in mice induced by OVA, reduce the symptoms of allergic reaction, increase the height of villi, improve the intestinal tissue structure of allergic mice and the absorption of nutrients of mice.

Sodium acetate at different concentrations (2, 4, 8 mmol·L^-1) was added to oleic acid-induced steatosis model of normal rat liver cells (BRL-3A). To investigate the regulation mechanism of steatosis cells model lipid metabolism and repair of cell damage, the following experiments were conducted: 1) BRL-3A cells were stimulated with oleic acid at different concentrations (0, 0.03, 0.06, 0.12, 0.24, 0.48 mmol·L^-1) for 24 h. The cell relative activity, total lipid droplet area, triglyceride (TG) content, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were detected to establish cell steatosis model. 2) BRL-3A cells were added with different concentrations of sodium acetate, and the apoptosis rate was detected by flow cytometry. 3)BRL-3A cells were incubated with different concentrations of sodium acetate and 0.12 mmol·L^-1 oleic acid. The experiment was divided into 4 groups, including oleic acid treatment group, 2 mmol·L^-1 sodium acetate + oleic acid treatment group, 4 mmol·L^-1 sodium acetate + oleic acid treatment group and 8 mmol·L^-1 sodium acetate + oleic acid treatment group. Lipid droplets and TG content, AST and ALT activities, AMPK signaling pathway proteins and key genes of lipid metabolism were detected. Results: 1) BRL-3A cells were treated with 0.12 mmol·L^-1 oleic acid for 24 h, and the steatosis model of BRL-3A cells was established successfully. 2)Different concentrations of sodium acetate had no effect on the apoptosis rate of BRL-3A cells. 3)Compared with oleic acid treatment group, the total lipid drop area, lipid drop number per mm², TG content, AST and ALT activities of cell steatosis model treated with 8 mmol·L^-1 sodium acetate were significantly decreased (P<0.05) or extremely significantly decreased (P<0.01). The expression level of P-AMPK was significantly (P<0.05) or extremely significantly increased (P<0.01). The mRNA expression levels of lipid anabolism-related genes ACC, FAS and SCD-1 all decreased to a certain extent; the mRNA expression levels of lipid catabolism-related genes CPT-1, CPT-2 and ACO all increased to a certain extent. This study showed that sodium acetate activates lipid catabolism through the AMPK pathway, attenuated the lipid accumulation in hepatocytes, and alleviated the oleic acid-induced damage of BRL-3A cell steatosis model.

This experiment aims to study the regulatory effects of Guiqiyimu compound preparation on rumen microbes and short-chain fatty acids in postpartum dairy cows. Nineteen healthy parturient Holstein cows were selected and randomly assigned to 2 groups with 10 cows in control group and 9 in experimental group. The dairy cows in experimental group were given 500 mL Guiqiyimu compound preparation everyday after delivery for 6 consecutive days, whereas the cows in the control group were given an equal volume of drinking water. The rumen fluid of each cow was collected before feeding in the morning at 7th day after delivery, 16S rRNA high-throughput sequencing and GC-MS/MS targeted metabolomics technology were used to determine the abundance of the rumen flora and the content of short-chain fatty acids in the two groups of dairy cows. The results of 16S rRNA sequencing exhibited that the abundances of the Proteobacteria, Escherichia-Shigella and Bacteroides were extremely significantly higher (P<0.01), Lactobacillus and Subdoligranulum were significantly higher (P<0.05) in experimental group than those of the control group. Correspondingly, the concentration of propionic acid (P<0.05) and isobutyric acid (P<0.01) in experimental group increased significantly. This study reveals the health care mechanism of Guiqiyimu formulae on postpartum dairy cows from the changes of rumen microbial flora and short-chain fatty acids.

The aim of this study was to investigate the effect of Thrombospondin-1 (THBS1) on canine mammary tumor cell CHMp, and to clarify its mechanism of action on the development of canine mammary tumors. THBS1 lentivirus-overexpressing cell lines and THBS1-silencing cell lines were constructed from canine mammary tumor cell CHMp. Then CCK-8 assay, scratch assay, Transwell assay, in situ fluorescence detection and flow cytometry were used to detect the effect of THBS1 on the proliferation, migration, invasion, apoptosis and cell cycle of CHMp cells. Finally, the effect of THBS1 on the expression of apoptosis-related factors (p53, Bcl-2, Bax) in CHMp cells was detected by qRT-PCR and Western blot, and the effect of THBS1 on the apoptosis pathway of CHMp cell was further verified. The results showed that overexpression of THBS1 can effectively enhance the proliferation, migration and invasion of canine mammary tumor cells CHMp, and reduce the number of apoptotic CHMp cells, while THBS1 silencing results in the opposite; Flow cytometry showed that THBS1 could affect the cell cycle distribution of CHMp cells; qRT-PCR and Western blot detection showed that after THBS1 overexpression, the expression levels of p53 and Bcl-2 were significantly increased, and the expression level of Bax was significantly decreased, and the results were the opposite after silencing THBS1. The results showed that abnormal expression of THBS1 could affect the proliferation, migration, invasion and cell cycle of canine mammary tumor cell CHMp. In addition, THBS1 can affect the related pathways of CHMp apoptosis factors by affecting the expression of apoptosis factors p53, Bcl-2 and Bax, and play an important role in canine mammary tumors, thereby affecting the occurrence and development of canine mammary tumors.

The purpose of this study was to investigate the effect of nano-selenium as a therapeutic drug on the expression and apoptosis of renal tissue ion transporters in dogs with acute renal failure induced by adenine. Twenty poodles weighing about 4 kg and aged about 1 year were randomly divided into blank control group (Control, fed with basal diet for 30 days) and acute renal failure model (Model, after basic health examination and adaptive feeding for two weeks, 15 days of basal diet + 15 days of feeding adenine, 75 mg·(kg·d)^-1), conventional infusion treatment group (Infusion, 15 days of feeding adenine, 75 mg·(kg·d)^-1+15 days glucose and sodium chloride injection 60 mL·(kg·d)^-1 and diuretic intravenous furosemide, 2-4 mg·kg^-1), nano-selenium treatment group (Nano-Se, 15 days of feeding adenine, 75 mg·(kg·d)^-1+15 days of feeding nano-selenium, 0.5 mg·(kg·d)^-1), nano-selenium prevention group (Prevention, 15 days of feeding nano-selenium 0.5 mg·(kg·d)^-1+15 days of feeding adenine, 75 mg·(kg·d)^-1), four dogs in each group. The expression levels of related protein genes were detected by blood biochemical analysis, urine routine analysis, immunohistochemistry of renal tissue paraffin sections, Western blot and RT-qPCR. The results show that nano-selenium treatment and prevention can effectively reduce the characteristic indicators of renal failure CRE and BUN, restore the urine specific gravity and urine pH of abnormal urine routine indicators of renal failure, and improve calcium and phosphorus metabolism in dogs with renal failure; compared with acute renal failure model, nano-selenium treatment effectively increased the mRNA and protein expression of CaSR and WNKs in renal tissue (P<0.05), and decreased the mRNA and protein expression of NKCC2 (P<0.05), thereby weakening the overcompensated reabsorption function in acute renal failure. The mRNA and protein expressions of pro-apoptotic Bax and Caspase3/9 in renal tissue of the renal failure model group were significantly down-regulated (P<0.05), which reduced the apoptosis effect of the kidney in acute renal failure.

The mechanism of high levels of copper on liver injury in rats was investigated in this research. One hundred and twenty SD rats were randomly divided into 5 groups, including control group, high level copper group I, high level copper group Ⅱ, high level copper group Ⅲ and high level copper group Ⅳ. The liver tissues were collected from the rats which were continuously intragastric administration of 0, 20, 40, 80 and 160 mg·kg^-1 copper for 63 days, respectively. The copper content in liver was determined by ICP-MS, and the pathological changes of liver were observed by pathological sections. The mitochondrial morphology and mitophagosomes were observed by transmission electron microscopy (TEM). The mRNA and protein expression levels of NLRP3, Caspase-1, GSDMD, IL-1β, IL-18, Pink1, Parkin, LCB3 and p62 in liver were detected by RT-qPCR and Western blot, respectively. The results showed that the Cu content in liver exhibited a significant dose-dependent effect with increased copper concentration, and long-term exposure to high levels of copper would cause significant structural damage and pathological changes in liver. With the increase of copper exposure level, the levels of mitophagy and pyroptosis decreased after initially increasing. Compared with the control group, the mRNA and protein expression levels of mitophagy-related genes and proteins PINK1 and LC3II/LC3I in high level copper group II and high level copper group III increased significantly (P<0.05). The mRNA and protein expressions of p62 in high level copper group Ⅱ were significantly decreased (P<0.05), and the mRNA and protein expressions of NLRP3, Caspase-1, GSDMD and IL-1β were significantly increased in high level copper group III (P<0.05), but down regulated in high level copper group IV. High levels of copper exposure could induce liver injury in rats by affecting mitophagy and pyroptosis.

Dystocia is a common clinical disease of dairy cows, and assisted calving is a common method to deal with it. The assisted calving can lead to the reduction of conception rate, the increase of frozen sperm use, the increase of empty pregnancy days and the delay of the first mating time. This study aims to analyze the effect of assisted calving on postpartum uterine microbiota of dairy cow, and to provide data reference for improving the conception rate in pasture. In a large dairy farm in Ningxia, a total of 7 samples of lavage fluid were collected from both unassisted and assisted calving cows at 45 days after delivery. Bacteria were isolated, cultured, and sequenced by 16S rRNA sequencing. A total of 10 bacterial genera and 12 kinds of bacteria were isolated, among which 5 genera of bacteria including Streptomyces, Streptococcus, Enterobacter, Staphylococcus and Aerococcus were isolated from the uterine washing fluid of non-assisted calving cows. Nine genera of bacteria such as Shigella, Acinetobacter, Pseudomonas, Corynebacterium, Bacillus, Aerococcus, Enterobacter, Staphylococcus and Streptococcus were isolated from the uterine washing fluid of assisted calving cows. Our results showed that the uterine microbiota of assisted calving and non-assisted calving cows were significantly different. Assisted calving resulted in increased diversity of uterine microflora in cows.

The purpose of this experiment was to investigate the effect of lycopene on the semen cryopreservation and the fresh semen quality, and to provide a basis for lycopene as a new semen diluent additive and dietary additive for breeding males.Fresh semen from 3 healthy Qinchuan bulls aged 3 to 5 years old was collected and cryopreserved with diluents containing lycopene of 0, 1, 2, 3 and 4 μmol·L^-1, respectively. The motility, acrosome integrity, membrane integrity, mitochondrial activity and antioxidant capacity of sperms were measured by computer-aided analyzer, hypo-osmotic swelling test, peanut lectin fluorescent labeling assay, rhodamine fluorescence assay, and so on. Qinchuan bulls were fed with 15 mg·kg^-1 BW lycopene, fresh semen quality and serum antioxidant indexes were detected before and after feeding.The results showed that the supplementation of the semen extender with lycopene produced a higher sperm motility, acrosome integrity, membrane integrity, mitochondrial activity, CAT and GSH-Px enzyme activities, resulted in a lower content of MDA in sperm than that of the control, the optimal concentration of lycopene was 2 μmol·L^-1 (P<0.05). After 15 mg·kg^-1 BW lycopene supplementation in diet, compared with before feeding, the sperm motility of Qinchuan bulls had a significant elevation (P<0.05), the sperm deformity rate significantly decreased (P<0.05), and the activities of antioxidant enzymes such as SOD, CAT and GSH-Px significantly elevated (P<0.05), with the content of MDA significantly decreased in serum (P<0.05). In conclusion, a certain concentration of lycopene had a good protective effect on sperm damage caused by semen cryopreservation and the reproductive performance reduction caused by stress in Qinchuan bull, which might be illustrated by the potential function of lycopene on reducing the oxidative damage of sperm during the freezing-thawing process and improving the antioxidant capacity of the animal body.

The purpose of this study was to explore the differential expression of small molecule metabolites in sika deer antler in different growth stages, and to lay a foundation for the study of the molecular mechanism of rapid growth and ossification of deer antler. Four years old male Northeast Sika deer in good condition were selected, and antler growing for 25, 45, 65, 100 and 130 d were collected as experimental samples, with 3 biological replicates in each period. Metabolomic analysis of the samples was performed by UHPLC-TOF-MS. The results of principal component analysis and hierarchical cluster analysis showed that the 5 growth periods could be divided into two stages: growth stage (25, 45 and 65 d) and ossification stage (100 and 130 d). A total of 171 significantly differentially expressed metabolites (VIP>1, P<0.05, |fold change|>2) were screened and identified, which were compared to 112 metabolites in the HMDB database, divided into 7 superclass. The contents of L-pyroglutamic acid, L-histidine, L-carnosine and acemic acid were the highest at 100 days, and 15-deoxy-δ12, 14-prostaglandin J2, prostaglandin H2 and prostaglandin I2 were significantly up-regulated at 130 days, while the expressions of other amino acids and organic acids were all up-regulated during the growth phase. The differential metabolites were significantly enriched in 13 KEGG metabolic pathways, such as aminoacyl-trNA biosynthesis and histidine metabolism. This study results provides data support for exploring the molecular mechanism of rapid growth and ossification of antler at the metabolome level.

The aim of this study was to investigate the differential expression characteristics of miRNA in bone marrow of chicks infected with Salmonella enterica serovar Pullorum (S. Pullorum), thus to provide a theoretical basis for an in-depth understanding of the pathogenic mechanism of S. Pullorum. Seven-day-old SPF chicks were randomly divided into two groups and orally administered Salmonella enterica serovar Pullorum and PBS, respectively. Bone marrow was collected 24 h after infection for miRNA sequencing, and differentially expressed miRNAs with differential multiplicity ≥2 and P value ≤0.05 were screened for target gene prediction, GO and KEGG enrichment analysis, while six miRNAs of which were randomly selected for qRT-PCR validation. An miRNA-mRNA target network related to the immune process was constructed. A total of 20 known differentially expressed miRNAs were obtained by miRNA sequencing, of which 11 were up-regulated and 9 were down-regulated. qRT-PCR results showed that the miRNA change trend was consistent with the sequencing results. GO analysis showed that the differentially expressed genes were mainly enriched in membrane transport, signal transduction, immune system, carbohydrate metabolism, sugar biosynthesis and metabolism, etc. The signaling pathways of KEGG were mainly enriched in Notch signaling pathway, Hedgehog signaling pathway, PPAR signaling pathway, AMPK signaling pathway, Hippo signaling pathway, etc. The miRNA-mRNA network interactions identified gga-miR-1466 and gga-miR-6643-5p as possible key miRNAs involved in immune process. This experiment resolved the bone marrow miRNA expression profiles of S. Pullorum-infected chicks, providing new insights into the complex molecular pathogenesis of the interaction between S. Pullorum and chickens, and offering new strategies for the prevention and control of S. Pullorum infection in chickens.