Prime editing system is a novel CRISPR/Cas9-derived genome-editing technology, which enables all 12 base-to-base conversions, precise small insertions and deletions without requiring double-strand breaks or exogenous donor DNA templates. This paper gives a detailed review of the basic research in recent years from the development process of gene editing, the composition and characteristics of the prime editing system, the optimization of the prime editing system, the current application of the prime editing system in animals and plants, the design of the prime editing system and off-target effects. The information presented may facilitate interested researchers to understand the prime editing system and further promoting the application of prime editing system in basic research and breeding of animals and plants.

The problem of bacterial resistance become a common challenge in the world, which leads to a decline in the effect of antimicrobial drugs and a rise in the incidence and mortality of bacterial diseases. The increase of treatment difficulty, expensive therapeutic cost and continuous decrease of animal productivity have caused serious economic losses to animal husbandry. Therefore, it is particularly necessary to find new schemes to fight resistant bacteria. Nanotechnology rose in modern times and has been widely used in many fields, such as biomedicine. It has a significant effect in fighting resistant bacteria, which can reduce bacterial resistance through various mechanisms such as destroy bacterial cell membrane, inhibiting efflux pump, produce reactive oxygen species (ROS) and degrading biofilm. This article will briefly summarize nanotechnology, about its development history, strategies to fight resistant bacteria and mechanisms to fight bacterial resistance, in order to provide some reference for veterinary drug researchers.

The study aimed to explore the polymorphisms of FABPs, SLC13A5 and NR1H4 genes in Ningxiang pigs and their association with intramuscular fat (IMF) content. In the experiment, the polymorphisms of FABPs, SLC13A5 and NR1H4 genes in 172 Ningxiang barrows were detected using PCR-RFLP and Sanger sequencing, and the association between them and IMF content was explored. The results showed that the Hinf Ⅰ digestion site of H-FABP gene was polymorphic in Ningxiang pigs, and the IMF content of individuals with Hh genotype was the highest, which was significantly different from that of individuals with HH and hh genotypes (P<0.01). Only DD and Dd genotypes were detected in H-FABP using Hae Ⅲ enzyme, and the IMF content of individuals with Dd genotype was significantly higher than that of individuals with DD genotype (P<0.01). There were 3 haplotypes and 5 haplotype combinations in the analysis of the Hinf Ⅰ and Hae Ⅲ sites of H-FABP gene, and the pigs with haplotype combination HD/hd had the best IMF content. The results of H-FABP Msp I digestion and A-FABP sequencing showed that there was no polymorphism at the detected sites in Ningxiang pigs. No significant difference was observed in IMF content among different genotypes at the polymorphism site of SLC13A5 (g.50705299T/C) and NR1H4 (g.83607915G/A) gene (P>0.05). In conclusion, the polymorphisms of H-FABP Hinf Ⅰ and Hae Ⅲ sites were significantly associated with IMF content in Ningxiang pigs, and the pigs with haplotype combination HD/hd had the highest IMF content. The above results could provide reference for the early molecular breeding of IMF content trait in Ningxiang pigs.

The aim of this study was to investigate the haplotype characteristics of the mitochondrial D-loop region in chickens and its correlation with growth rate, and to analyze its genetic origin. Six different types of broiler breeds (synthetic lines) were selected to determine their production performance. The full length of the mitochondrial D-loop region of 314 individuals from the 6 groups were sequenced to analyze their haplotype characteristics, and clustered with different jungle fowl subspecies to analyze their maternal origin. The results showed that a total of 37 variant loci were detected in the whole sequences of D-loop regions of the 6 populations, constituting 40 haplotypes, and were divided into 4 haplotype groups A, B, C and E. Among them, AA broilers, Ross 308, Qinyan partridge chicken and Yuhe No.1 broilers were all mainly E haplotypes, accounting for 85.92%, 50.00%, 100.00% and 70.21%, respectively. Yuanfeng partridge chicken No.2 was mainly B haplotype (66.67%) and Gangfeng yao black partridge chicken was mainly C haplotypes (40.30%). While the proportions of E haplotype in the two chicken populations were relatively low, with 15.00% and 22.39%, respectively. Correlation analysis showed that birth weight was positively correlated with the proportion of haplotype E (P<0.01), and negatively correlated with the proportion of haplotype A, B and C. There was a significantly negative correlation between the ratio of E haplotype and the age of the average body weight of males and females at about 1.8 kg (P<0.05). And there was a very significantly negative correlation between the ratio of E haplotype and the feed conversion ratio of average body weight of males and females at about 1.8 kg (P<0.01). Cluster analysis showed that all individuals with A and B haplotypes clustered with gallus gallus spadiceus, all individuals with E haplotypes clustered with gallus gallus murgha, all individuals with C haplotypes clustered with gallus gallus murghi, gallus gallus spadiceus, gallus gallus gallus and gallus gallus bankiva. The results indicated that there is a significant correlation between mitochondrial haplotype and the growth rate of broilers, E haplotype had a strong positive correlation with broiler growth rate. The local chicken breeds in China had multiple maternal origins, mainly originated from Gallus gallus spadiceus.

The aim of this study was to estimate genetic parameters of body weight (BW) at different ages in Holstein heifers using models considering different source of information. A data set including 32 338 BW records of 0-12 months old on 7 122 female Holstein cattle measured from 2019 to 2020 was collected. Variance components and parameters were estimated using linear mixed model with pedigree relationship matrix (LM_A) and genotype-pedigree joint relationship matrix (LM_H). For calf birth weight (CBW), animal models with maternal genetic effects were applied. The single-trait animal models with or without a CBW as a covariate were used for the estimation of heritability of monthly body weight from month 2 to 12. Genetic correlations between CAW and monthly BW were estimated using bivariate animal models. The results showed that models with LM_H had better goodness of fit based on Akaike information criterion (AIC), although similar parameters for CBW were obtained through LM_A and LM_H methods. For CBW, direct heritabilities estimated by LM_A and LM_H were 0.30 and 0.32, respectively, and maternal heritabilities were 0.08 and 0.09, respectively. The correlation coefficients between individual direct genetic effect and maternal genetic effect was antagonistic for LM_A (-0.65) and LM_H (-0.64). Monthly BW traits had moderate to high heritabilities. Estimates from LM_A and LM_H with model including CBW as a covariate ranged from 0.15 to 0.55 and 0.28 to 0.49, respectively, and heritabilities from LM_A and LM_H with model without CBW ranged from 0.16 to 0.54 and 0.28 to 0.51, respectively. BW at age of month 2 and 5 were highly genetically correlated with CBW (correlation coefficient was higher than 0.6). After 5 months old, genetic correlations between CBW and BW at different months were decreased with increasing time spans. Compared with LM_A, the LM_H method was more stable, with a smaller AIC value (that is, a larger goodness of fit) and a smaller standard error of the genetic parameters. In conclusion, using the LM_H method to estimate target traits can obtain more accurate and stable genetic parameters. This study provides a theoretical basis for the establishment of a genomic selection system for growth traits in Chinese Holstein cattle.

In order to identify genetic markers related to reproduction and milk production traits of Chinese Holstein cattle, the relationships between single nucleotide polymorphisms (SNPs) of MET gene and reproduction and milk production traits was explored. Candidate SNPs of MET were screened through direct sequencing of pooled DNA samples in 70 healthy unrelated Chinese Holstein bulls, and the genotyping of partial SNPs in 1 160 healthy Holstein lactating cattle was performed using kompetitive allele-specific PCR (KASP) method, diplotype information based on linkage disequilibrium analysis was obtained. The linear models were employed to conduct the association analysis between SNPs and their diplotypes and EBVs of 5 reproductive traits and 6 milk production traits. The bioinformatics analysis and the association analysis were conducted on SNPs located in functional areas. In this study, a total of 19 SNPs were detected in MET, indicating rich genetic variations existing within this gene in the population. Among them, 7 SNPs was genotyped, and the association analysis revealed that these SNPs were significantly (P<0.05) or highly significantly (P<0.01) associated with multiple traits. The individuals with GG genotype of g.51737889T>C located in upstream had the lowest EBVs of the age at the first calving (AFC), the interval from the first to last insemination in heifer (IFL_H), the interval from the first to last insemination in cows (IFL_C), and somatic cell score (SCS), which suggested the good reproduction and milk production performance of animals carrying this genotype. The individuals with GG genotype of g.51736640A>C located in upstream had the lowest EBVs of the age at the first calving, the interval from the first to last insemination in heifers, the interval from the first to last insemination in cows, and somatic cell score; however, they had the lowest EBVs of protein percentage (PP), and fat percentage (FP), which suggested animals carrying this genotype had the good performance in reproductive, but with relatively poor performance in milk production. The individuals with the TC genotype at g.51660569G>A in 6^th exon had the lowest EBVs of the interval from the first to last insemination in heifers and the interval from the first to last insemination in cows, which suggested the good reproduction performance of animals carrying this genotype. Linkage disequilibrium analysis revealed that 7 SNPs linked to form 2 haplotype blocks. The association analysis between diplotypes and target traits showed that the individuals with H1H3 diplotype at BLOCK1 had good reproduction performance but medium milk production performance, the individuals with H4H4 diplotype at BLOCK2 had good reproduction and milk production performance, and these two were dominant hiplotypes. In addition, H1H3 diplotype included the TC genotype at g.51660569G>A, H4H4 diplotype was combination of the GG genotype at g.51737889T>C and g.51736640A>C, the results of diplotype association analysis were consistent with that of SNP. The results of motif analysis showed that G allele at g.51737889T>C and g.51736640A>C enriched in transcription factors related to gene activation, cell proliferation and differentiation. The individuals with GG genotype at both SNPs had the highest gene expression, and the individuals with corresponding H4H4 diplotype had higher gene expression, which further verified the results of association analysis. In conclusion, the polymorphism map of MET gene was derived in Chinese Holstein cattle, and the genetic association between MET gene and reproductive and productive traits was identified through association analysis, and verified by bioinformatics prediction and gene expression analysis. The results provided useful genetic markers for genetic selection of Chinese Holstein cattle for higher production as well as higher efficiency.

The purpose of the experiment was to primary culture and study the in vitro differentiation potential of Simmental bovine pancreatic mesenchymal stem cells (PMSCs), and to provide new seed cells for cell therapy and tissue engineering. Pancreatic tissue was aseptically isolated from 3-month-old Simmental cattle embryos, and PMSCs were isolated by collagenase digestion and tissue block adherence, respectively, and then primary cultured. The growth curves of 3rd, 9th and 15th passage cells were drawn and the population doubling time and clone formation ability were determined. Stem cell surface markers (CD29, CD44, CD73, CD90, CD34 and CD45) were detected by immunofluorescence, and stem cell surface markers (CD29, CD44, CD73, CD90, CD106, CD166, CD34 and CD45) were detected by RT-PCR. Its gene stability was detected by karyotype analysis, the multi-directional differentiation potential was detected by inducing adipogenic, osteogenic, chondrogenic and hepatocyte-like cells. The results showed that PMSCs could be successfully isolated by both methods. After the cells adhered, they were all long spindle-shaped, swirling growth, and the growth trend was typical S-shaped. The doubling time of the cell population in the 9th passage was significantly lower than that in the 15th passage and higher than that in the 3rd passage (P<0.05); The clone formation rate of the 9th passage of PMSCs was significantly lower than that of the 3rd passage and significantly higher than that of the 15th passage (P<0.05); Immunofluorescence results showed that CD29, CD44, CD73 and CD90 specifically expressed in PMSCs, and RT-PCR results showed that CD29, CD44, CD73, CD90, CD106 and CD166 specifically expressed in PMSCs, but the hematopoietic cell surface markers CD34 and CD45 did not express, which were consistent with international corresponding to the surface markers of MSCs designated by the Tissue Stem Cell Committee of the Society for Cell Therapy; Karyotype analysis showed that PMSCs were normal diploid (2n=60, XY), and there was no mutation in the chromosomal genome; Specific staining and RT-PCR results showed that PMSCs obtained from Simmental cattle could differentiate into adipocytes, osteocytes, chondrocytes and hepatocyte-like cells. This experiment confirmed that both methods could successfully establish the in vitro separation and culture system of Simmental cattle PMSCs. PMSCs showed good proliferation activity, similar biological properties and multiple differentiation potentials with MSCs. The results could provide new seed cells for tissue engineering.

This study aimed to compare the changes in rumen tissues morphology and transcriptome profile of the yak at different ages to explore the key genes and signaling pathways affecting the rumen development, and lay a foundation for further in-depth exploration of the rumen development mechanism of the yak. In this study, the histomorphological and transcriptomic analysis of the healthy yak's rumen at 5 age groups was performed, including non-ruminant stage (1 day old and 20 days old), transitional stage (60 days old) and ruminant stage (15 months old and 3 years old), 3 samples per group. The body and rumen weights of the samples were measured. The rumen tissue samples were collected and carried out histomorphology observation, the muscle layer thickness, papillae height and width were measured and counted. Total RNA was extracted from rumen tissue for sequencing, and transcriptome analysis was performed with 1-day-old as control. The differentially expressed genes were selected for RT-qPCR, the results of RT-qPCR was compared with the expression trend of transcriptome data. The results of histomorphological analysis showed that the rumen muscle layer thickness and papillae morphology of yaks from 1 day to 3 years old developed and differentiated to a great extent at the histomorphological level. The rumen weight and index increased significantly at 15 months and 3 years old (P<0.05). From 20 days old, the muscle layer thickness increased significantly with the increase of age (P<0.05). After 20 days old, the height and width of rumen papillae increased significantly with the increase of age (P<0.05). In the 20 days old group, transcriptome results showed that the pathways related to VFA metabolism were enriched in cholesterol synthesis, butyrate metabolism and PPAR signaling pathway; The pathways related to immunity were enriched in the immune response biological process and Th17 cell differentiation pathway; The pathways related to the metabolism of rumen epithelial cells xenobiotics was enriched in the metabolic pathway of cytochrome P450 exogenous substance. In the 60 days old group, the pathways related to VFA metabolism were enriched in the fatty acid metabolism, propionic acid metabolism pathway and pyruvate metabolism pathway; The pathways related to VFA absorption was enriched in mineral absorption. The 15 months old and 3-year-old groups enriched in ECM receptor interaction pathways that maintain the integrity and barrier function of rumen epithelium. HMGCS2, SLC26A3, PPARG, PPARD and CCL5 genes were selected from the above pathways, which related to the nutrient absorption, metabolism and immunity of rumen. The expression pattern of differentially expressed genes detected by RT-qPCR was consistent with the results of the transcriptome sequencing. The above results indicate that the rumen morphology of the yak develops early, and 20 days old is an important critical point for ruminal development; The development of ruminal muscle layer thickness was earlier than that of ruminal papillae, muscular thickness increased rapidly from 20 days old; and the rumen papillae developed rapidly after 20 days old; The rumen started VFA metabolism at 20 days old, which promotes the rapid and healthy development of rumen.

The purpose of this study was to elucidate the dynamic metabolism of parous yaks in the late perinatal period and explore the mechanism of nutritional metabolism affecting reproductive hormones by metabolomics. Eight parous female yaks with similar age ((7.13±0.78)years), body condition score (2.69±0.35) and parity (1.75±0.43) were randomly selected. Serum was collected every 7 d from the date of the calving day to the 28th days after calving, in total 5 time points. The serum samples at 5 time points were detected by liquid chromatography mass spectrometry, and the key metabolites were screened by bioinformatics analysis to construct the dynamic metabolic network mechanism of parous yaks in the late perinatal period. The qualitative analysis results of 2 841 and 1 326 metabolites were obtained in the positive and negative ion modes, respectively. A total of 117 differential metabolites were screened by calculating the fold change of metabolite expression at each time point, VIP value and P value of T-test at each two time points. According to bioinformatics analysis, 3, 7 and 8 key metabolites related to reproductive performance, lipid metabolism and amino acid metabolism were obtained. The mechanism of constructing dynamic metabolic network based on key metabolites found that the initial efficiency of glucose metabolism in the late perinatal period was low and increased in the late perinatal period, while the efficiency of lipid metabolism and amino acid metabolism was high at the beginning and decreased in the late, it was speculated that the negative energy balance of parous female yaks occurred. The efficiency of reproductive hormone synthesis and secretion remained at a low level in the late perinatal period. According to the detection of dynamic metabolic spectrum and the construction of network mechanism of parous female yaks, it is found that there is a negative energy balance after calving, and the synthesis and secretion of estradiol are blocked due to excessive fat mobilization, which may be the main reason for the failure of postpartum reproductive function of parous female yaks to recover in time, but the specific mechanism needs to be further verified.

This aim of the study was to analyze the transcriptome and metabolome data by WGCNA technology, and explore the regulatory mechanism of donkey meat tenderness. Healthy female Guangling donkeys aged 24-36 months (average body weight 236.10 kg) were used as experimental animals. The shear force and intermuscular fat content of donkey meat were used as phenotypic data, phenotypic data was detected in 3 replicates per sample. In this study, based on the transcriptome and metabolome sequencing data of 14 longissimus dorsi muscle samples with significant shear force and intramuscular fat content differences in previous studies, WGCNA technology was used to screen genes and metabolites related to donkey meat tenderness, and combined transcriptome and metabolome analysis was performed to analyze tenderness related genes and metabolites. The results showed that 3 key gene modules Greenyellow, Darkgrey, Darkgreen and 2 key metabolite modules Brown and Yellow were obtained by using the WGCNA technology through|r| ≥ 0.5 and P ≤ 0.05. GO enrichment of key gene modules revealed that the genes in the modules were mainly enriched in GO functions such as glycerophospholipid biosynthesis, lipid oxidation, fatty acid β-oxidation, cellular macromolecular catabolism, muscle organ development, and calcium ion binding. KEGG functional enrichment analysis showed that most of genes and metabolites in the key modules were enriched in arginine and proline metabolism, Wnt signaling pathway, protein digestion and absorption, fatty acid metabolism, TCA cycle, glucagon signaling pathway, glycerophospholipid metabolism, purine metabolism, β-alanine metabolism and other pathways. Combined analysis indicated that alanine, aspartate and glutamate metabolism, arginine and proline metabolism, β-alanine metabolism and PPAR signaling pathway could regulate donkey meat tenderness. Alanine, aspartate and glutamate metabolism, arginine and proline metabolism, β-alanine metabolism and PPAR signaling pathway screened by WGCNA and combined KEGG co-enrichment analysis might play an important role in regulating donkey meat tenderness; while GAD1, PPAT, NIT2, AGMAT, CARNS1, ACOXL and adenylate succinic acid, L-proline, L-glutamic acid, creatine, homocarnosine, carnosine, pantothenic acid, (9S)-Hydroxy octadecadienoic acid might be the candidate genes and metabolites affecting donkey meat tenderness. This experiment results can provide a theoretical basis for the molecular regulation and improved breeding in meat tenderness of Guangling donkey in the future.

The study aimed to construct PLC-γ1 eukaryotic overexpression vector and preparing its polyclonal antibody, and provide basic data for exploring the effect of sheep PLC-γ1 gene on oocyte activation and early embryonic development. In this study, the Puc57-P2A-Flag-PLC-γ1 strain preserved in the laboratory was used as a template to design primers and introduce Hind III and Age I restriction sites. PCR was used to amplify and purify the P2A-Flag-PLC-γ1 gene. It was connected into pcDNA3.1-EGFP vector for transformation, and the plasmid was extracted by bacterial liquid PCR, double restriction digestion and sequencing identification. Then HEK-293T cells were transfected with the constructed recombinant plasmid for 24 h, and were divided into the control group, no-load group and PLC-γ1 overexpression group. The cell fluorescence was observed under a microscope, and the expression of PLC-γ1 in the transfected cells was detected by real-time quantitative PCR (qPCR) and Western blot (WB). PLC-γ1 protein was purified by Anti-Flag immunomagnetic beads; Polyclonal antibodies were prepared from 10-month-old New Zealand white rabbits (healthy condition, about 50 kg, n=2). The titer of polyclonal antibodies was determined by indirect ELISA, and its specificity was identified by WB. The oocytes were parthenogenetically activated by microinjection of recombinant plasmids and ionomycin (Ion) combined with 6-DMAP. The expression of PLC-γ1 was detected by qPCR and WB at different stages (2 cell stage, 4 cell stage, 8 cell stage and the morula stage) after cleavage in early embryo cells of sheep. The results showed that eukaryotic overexpression vector was successfully constructed. After transfection 24 h, the expression level of PLC-γ1 in overexpression group was significantly increased compared with blank control and negative control (P<0.01). WB results showed that the PLC-γ1 protein was successfully obtained and purified, for 149 ku. The PLC-γ1 polyclonal antibody titer was 1:12 800, and it could react with PLC-γ1 protein. The recombinant plasmid was injected into the cytoplasm of sheep MⅡ oocytes, and the results of qPCR and WB showed that PLC-γ1 was expressed in all stages of early embryo development of sheep. In conclusion, in this study, PLC-γ1 eukaryotic overexpression vector was successfully constructed and polyclonal antibody was prepared. The recombinant plasmid was injected into oocytes by the microinjection technique and realized its expression. It proved that PLC-γ1 was expressed in all stages of early embryo development of sheep and has the potential as a new oocyte activator. It not only provides a scientific basis for exploring the effects of PLC-γ1 gene on the activation of sheep oocytes and early embryo development, but also lays a foundation for further improving the reproductive performance of sheep.

The aim of this study was to explore the effect of altered O-GlcNAc modification on in vitro maturation (IVM) of bovine oocytes. Bovine in vitro-matured(IVM) oocytes were used to detect the distribution of OGT, OGA and O-GlcNAc proteins. Then cumulus-oocyte complexes were matured in IVM medium supplemented with 4 mmol·L^-1 OGT inhibitor BADGP or 100 μmol·L^-1 OGA inhibitor PUGNAc, respectively. The none supplementation group was used as control. The first polar body extrusion rate and subsequent embryonic development in vitro were detected, the O-GlcNAc level as well as the mRNA and protein expression levels of OGT, OGA, GFAT and TXNIP were detected by RT-qPCR and Western blot. Results showed that OGT and O-GlcNAc proteins were colocalized in the cytoplasm and nucleus of oocytes, while OGA and O-GlcNAc proteins were colocalized in the cytoplasm and primarily enriched at the cortex. Compared with control group, the first polar body extrusion rate ((52.8±5.1)% & (60.9±1.9)% vs. (70.8±5.4)%) and the blastocyst formation rate ((9.6±4.9)% & (10.0±5.8)% vs. (21.5±4.3)%) significantly reduced in BADGP and PUGNAc groups (P<0.05). BADGP treatment significantly reduced the expression of OGT, the level of O-GlcNAc modification in oocytes was down-regulated, and the expression of OGA was decreased; In the PUGNAc-treated group, the expression of OGA was significantly down-regulated, the modification level of O-GlcNAc was increased, and the protein expression of OGT was significantly decreased. Inhibition of OGT significantly up-regulated the expression of GFAT mRNA, a key rate-limiting enzyme in the hexosamine biosynthesis pathway, whereas inhibition of OGA significantly down-regulated the expression of GFAT mRNA. In addition, the altered level of O-GlcNAc modification significantly up-regulated the expression of TXNIP, a key factor in glucose regulation. The results indicated that changes in the level of O-GlcNAc modification would reduce the maturation and developmental ability of bovine oocytes in vitro, and oocytes responded to the fluctuation of O-GlcNAc modification levels by feedback-regulating the expressions of OGT, OGA, GFAT and TXNIP.

This study aimed to establish a method that can assess the chromosomal quality and production performance of early bovine embryos, and provide technical support for the industrial application of bovine in vitro embryos. In this study, 27 in vivo blastocyst, 21 in vitro blastocyst, 6 in vitro 2-cell embryos and 5 in vitro 8-cell embryos were selected, and the embryonic cell samples with different cell numbers of in vivo embryos and in vitro embryos were obtained by embryo splitting technique. Among that, in vivo embryos were divided into two groups, one was in vivo trophectoderm cells (tec) group (splitting some of the tec from the in vivo blastocyst), the other was the half in vivo embryo group (splitting half of the embryo from the in vivo blastocyst). The remaining groups were in vitro tec group (splitting some of the tec from the in vitro blastocysts), in vitro 2-cell embryo group (splitting one blastomere from the in vitro 2-cell embryo), and in vitro 8-cell embryo group (splitting one blastomere from the in vitro 8-cell embryo). After embryo splitting, whole-genome amplification (WGA) was conducted, and the samples with successful amplification (DNA amounts greater than 1 000 ng) were subjected to SNP chip detection. Data with a call rate greater than 90% were evaluated for production performance, those below 90% were subjected to chromosomal fragment deletion analysis and data filling. The results showed that:1) After splitting part of the trophectoderm cells, the developmental rates of bovine in vivo embryos (remaining portion of in vivo blastocysts after splitting some of the tec) and bovine in vitro embryos (remaining portion of in vitro blastocysts after splitting some of the tec) were (94.4±5.6)% and (90.5±6.6)%. However, the culture development rate of half of the in vivo embryo (the remaining part of the embryo after splitting half of the embryo from the in vivo blastocyst) was significantly reduced to (22.2±14.7)%. 2) The success rate of DNA amplification in the half in vivo embryo group and the in vitro 2-cell group were 100%, while the success rates of DNA amplification in the in vivo TE group, the in vitro TE group and the in vitro 8-cell embryo group were (94.4±5.6)%, (76.2±9.5)% and (60.0±24.5)%, respectively. Compared with the half in vivo embryo group, the amount of DNA after WGA was significantly lower in both of in vivo TE group and in vitro TE group (P<0.05), and the amount of DNA after WGA was significantly lower in the in vitro TE group than that in the in vivo TE group (P<0.05). 3) Compared to the half vivo embryo group (91.2±1.6)%, the chip call rates were significantly lower in the in vivo TE group (76.7±15.2)%, in vitro TE group (74.3±9.6)%, in vitro 2-cell group (76.1±6.9)%, and in vitro 8-cell group (61.2±19.0)% (P<0.05), and the call rates were all lower than 90%. The in vitro 8-cell group had the lowest chip call rate compared to the other groups. 4) If at least 7 consecutive SNPs deletions were considered as the chromosome fragment deletion selection criteria, a total of 188 and 388 chromosome deletion fragments were obtained in the in vitro TE group and in vivo TE group, respectively. Gene selection identified that a total of 46 genes were included in the deletion fragments of the in vitro TE group and 48 genes were included in the deletion fragments of the in vivo TE group. GO and KEGG enrichment analysis showed that the differentially deleted genes in the in vitro TE group were significantly enriched to cytoskeleton and cell differentiation, whereas the genes enriched in the in vivo TE group were mainly related to cellular material secretion and transport. 5) The filling accuracy (R²) of 52 334 SNPs filling sites out of 64 958 SNPs filling sites was between 0.99 and 1, and the mean value of R² for all filling sites was 91%, indicating a high filling accuracy. Breeding value estimation showed that the production performance of in vitro TE group was lower than that of in vivo TE group. Together, using of embryo splitting, WGA and SNP chips can enable to assess the chromosome quality and production performance of early preimplantation embryos with little impact on the quality of embryo development;In addition, the abnormal expression of genes such as cytoskeleton during in vitro development may contribute to the poor quality of in vitro embryos.

This study was designed to investigate the effects of Enterococcus faecium and Bacillus subtilis addition alone and mixed addition on growth performance, carcass traits and immune function of squabs, in order to explore the regulatory mechanism of probiotics on squabs growth. Two hundred and forty squabs at 12 days of age with similar body weight were selected and randomly divided into 4 groups, including the control group, Enterococcus faecium group, Bacillus subtilis group and mixed group, with 6 replicates per group and 10 squabs per replicate. The trial lasted for 16 days. At the end of the experiment, two squabs from each replicate (a total of 48 pigeons) were randomly selected. Serum immune indices, muscle quality, liver antioxidant indices and growth-related genes expression levels were determined. The results showed that:1) Compared with the control group, the average daily gain of squabs in the Enterococcus faecium group was significantly induced and was significantly higher than that of the other groups(P<0.05), while the Bacillus subtilis group significantly decreased the average daily gain of squabs(P<0.05); 2) There were no significant differences in the slaughter rate, semi-clear rate, full-clear rate, pectoral muscle rate, leg muscle rate or abdominal fat rate of squabs in each treatment group(P>0.05); Compared with control group, the brightness of breast muscle in Enterococcus faecium group and mixed group were significantly increased(P<0.05), and the redness of breast muscle in mixed group were significantly increased(P<0.05); 3) Compared with the control group, both Enterococcus faecium group and mixed group could significantly increase the contents of IgG in serum of squabs(P<0.05), and thymus index and bursa of fabricius index were significantly increased in mixed group(P<0.05). Serum GLB in Enterococcus faecium group was significantly higher than that in the control group, but there were no significant effect on the other serum biochemical indexes among all groups(P>0.05); 4) Compared with control group, the activity of superoxide dismutase (SOD) in Enterococcus faecium group was significantly higher (P<0.05), the glutathione peroxidase (GSH-Px) activity in mixed group was significantly higher (P<0.05); 5) The mRNA expression of GH and IGF-1 in liver of three experimental groups were significantly higher than those in the control group (P<0.05), and the mRNA expression of GHR in liver of Bacillus subtilis group as significantly higher than that in the control group (P<0.05). In conclusion, Enterococcus faecium can significantly increased the average daily gain of squabs, while Bacillus subtilis had the oppsite effect; Enterococcus faecium can improve the meat quality, enhance the immune capacity and antioxidant capacity, thus promote the growth of squabs.

This experiment was conducted to investigate the effects of different levels of lysolecithin (LPL) on growth performance, slaughter performance, meat quality, digestive enzyme activities and nutrient digestibility of Liangfenghua chickens fed with two different energy levels diets. A total of 1 080 healthy 1-day-old Liangfenghua chicken with similar body weight were randomly divided into 6 treatment groups:1) Control group:fed with basal diet; 2) Control+500LPL group:supplement 500 mg·kg^-1 of LPL to the basal diet; 3) Energy-reduced diet group:fed with low energy diet (metabolizable energy decreased by 251 kJ·kg^-1 at basal diet level); 4) Energy-reduced+500LPL group:supplement 500 mg·kg^-1 of LPL to the low energy diet; 5) Energy-reduced + 750LPL group:supplement 750 mg·kg^-1of LPL to the low energy diet; 6) Energy-reduced+1000LPL group:supplement 1 000 mg·kg^-1of LPL to the low energy diet, with 6 replicates in each group and 30 chickens in each replicate, respectively. The experimental period was 52 days, which was divided into starter phase (1~21 days) and grower phase (22~52 days). At the end of the experiment, chickens were slaughtered and sampled to determine certain indexes. The results showed that the F/G of Liangfenghua chicken in the energy-reduced diet group was higher than that in the control group, and after added 750 mg·kg^-1 LPL, the F/G of Liangfenghua chicken was almost the same as that in the control group (P>0.05). Compared with the energy-reduced diet group, LPL addition to the energy-reduced diet groups could improve the breast muscle rate and thigh muscle rate of Liangfenghua chicken on the 52nd day (P>0.05). Compared with the control group, added 500 mg·kg^-1 of LPL to the basal diet significantly improved the digestibility of dry matter and crude protein in the diet of Liangfenghua chickens at 21 d (P<0.05) and the digestibility of ether extract at 52 days (P<0.05). The addition of 750 and 1000 mg·kg^-1LPL to the energy-reduced diet significantly increased the villus height of the ileum and jejunum (P<0.05) and duodenal lipase activity (P<0.05) at 52 d of Liangfenghua chicken compared with the energy-reduced diet group. Compared with the energy-reduced diet group, added 750 mg·kg^-1 LPL to the energy-reduced diet significantly improved the apparent ileal digestibility of 11 amino acids such as Thr, Val, Ala in the diet of Liangfenghua chicken (P<0.05). Compared with the energy-reduced diet group, added 750 and 1 000 mg·kg^-1 LPL to the energy-reduced diet increased the gene expression of apolipoprotein A1 and apolipoprotein A4 (P>0.05). In conclusion, under the certain condition, LPL supplementation to the energy-reduced diets of Liangfenghua chicken can improve the slaughter performance of Liangfenghua chickens, and have a positive effect on its intestinal morphology, nutrient digestibility and liver lipid metabolism, and the addition amount of 750 mg·kg^-1is the best.

The goal of this experiment was to determine the apparent metabolizable energy (AME) and true metabolizable energy (TME) of chicken from various corn and sorghum sources using the free feeding method (FF) and tube feeding method (TF), and to compare the effects of the two evaluation methods on chicken available energy. The experiment was conducted in 3 phases. A total of 108 healthy mature Hyland brown shell roosters were chosen, with 96 roosters divided into FF and TF groups based on the principle of uniform body weight. Each method consisted of 12 dietary treatments, with four chickens treated with one diet, with a replicate of two chickens designed in the FF method group and a replicate of one chicken designed in the TF method group. The 12 dietary treatments included 6 sources of corn diet, corn-soybean meal basal diet, and 5 sources of sorghum diet. All excreta were collected to determine the AME and TME of diets and ingredients. Another 5 roosters weighing similar to the test group were selected to determine the endogenous loss. The results were showed as follows:1) The AME values range of 6 kinds of corn determinated by the FF method was 15.82~16.23 MJ·kg^-1 DM (P<0.05, CV=0.98%), and the TME values range was 15.95~16.36 MJ·kg^-1 DM (P<0.05, CV=0.99%). The AME values of 5 sorghums ranged from 13.43~15.37 MJ·kg^-1 DM (P<0.05, CV=5.16%), and the TME values ranged from 13.59~15.48 MJ·kg^-1 DM (P<0.05, CV=5.10%). 2) The AME values range of 6 kinds of corn assessed by the TF method was 14.35~15.01 MJ·kg^-1 DM (P<0.05, CV=1.66%), The TME values range was 16.00~16.64 MJ·kg^-1 DM (P<0.05, CV=1.45%). The AME values range of 5 sorghums was 12.51~14.87 MJ·kg^-1 DM (P<0.05, CV=6.74%), and the TME values ranged from 14.08 to 16.45 MJ·kg^-1 DM (P<0.05, CV=6.04%). 3) The AME values of the 6 corn measured by the FF method were 9.42% higher than the values measured by the TF method (P<0.05), but there was no significant difference in TME value determinated by FF on TF method. The AME values of the 5 sorghum measured by the FF method were 5.65% higher than the values measured by the TF method (P<0.05), while the TME values determined by the TF method were 4.82% higher than that measured by FF (P<0.05). It can be concluded that there are significant differences in the available energy values of chickens from different sources of corn and sorghum, and methodology will influence the determination of the available energy value of corn and sorghum fed to chickens.

To investigate the effect of bone marrow-derived mast cells (BMMCs) on the cytokines responses of FMDV-VLPs via mannose receptors, the recombinant plasmid pCMV-HA-HBcAg-VP1-VP4 was constructed and transfected into CHO-K1 cells to prepare the foot-and-mouth disease virus-like particles (FMDV-VLPs). The BMMCs treated with the mannose receptor inhibitor Mannan were loaded with FMDV-VLPs, and the BMMCs loaded with VLPs and BMMCs were used as controls. Quantitative cytokine chip was used to detect the content of cytokines secreted by BMMCs in different treatment groups. The results showed that compared with the control group, the levels of IL-1α, IL-2, IL-4, IL-15, IL-17A, IL-21, TNF-α, IFN-γ, CCL-17 and CCL21 of VLP group were all significantly up-regulated (P<0.05) and IL-10 showed no difference, while the levels of IL-1α, IL-2, IL-4, IL-15, IL-17A, TNF-α, IFN-γ, CCL-17 and L-selectin of the iMR-VLP group significantly down-regulated (P<0.05). Of note, the expression of IL-9, IL-10, CCL19 and CCL21 were further up-regulated in the iMR-VLP group. In conclusion, FMDV-VLPs can promote the secretion of a series of cytokines in BMMCs, and the ability to secrete cytokines of BMMCs is inhibited to a certain extent when MR was inhibited. The results indicate that FMDV-VLPs may enhance the secretion of Th1 cytokines through the mannose receptor of BMMCs, effectively inhibit the secretion of IL-10 that can cause immunosuppression, and reduce the formation of CCL19 and CCL21 that mediate the recycling of T cells. The results provide a theoretical basis and new ideas for the development of a new foot-and-mouth disease vaccine based on the roles of mannose receptors expressed on BMMCs.

US3 protein kinase is a critical virulence factor of pseudorabies virus (PRV). It is involved in many biological process, including cytoskeletal rearrangement, viral replication and transmission, antagonist apoptosis and interferon induced anti-viral response. The identification of US3 substrates is directly connected to understanding US3 functions and mechanisms. Here, we confirmed a host protein annexin A2 (ANXA2) can interact with US3 by co-immunoprecipitation and pull-down assay. Knock down the expression of ANXA2 can significantly affect the PRV proliferation on PK-15 cells, which was verified through quantitative PCR, viral titer and immunoblot analysis. Furthermore, we found that ANXA2 could inhibit apoptosis of PK-15 cells indued by PRV infection and apoptotic stimulator, suggesting that ANXA2 negatively regulates apoptosis. This study expands the host protein members that interact with US3 and deepens our understanding of the biological functions of ANXA2.

In this study, we screened the BHK-21 cells which were sensitive to pseudorabies (PRV) and analyzed their growth and virus production characteristics. The conditions of cell culture and virus production in the reactor were optimized, so we finally established the PRV propagation process of BHK-21 suspension cells in bioreactor. The optimal culture and virus propagation conditions of BHK-21 suspension cells in 1.2 L bioreactor based on cell growth dynamics and TCID₅₀ virus titer, were optimized by response surface methodology and single factor optimization method, and further batch culture was carried out in 5 L bioreactor. The results showed that one of adherent cells named BHK-21-02 and one of suspension cells named BHK-21-XF02 with high PRV sensitivity were screened. BHK-21-XF02 suspension cells could achieve good growth and virus propagation in serum-low medium containing 3% serum and serum-free medium. Using response surface methodology, the optimum cell culture conditions of 1.2 L reactor were as follows:inoculation density was 1.20×10⁶ cells·mL^-1, stirring speed was 120 r·min^-1 and DO was 40%, the cell density could reach (7.61±0.18)×10⁶ cells·mL^-1 and the cell viability was (96.93±1.18)% after batch culture in 5 L reactor for 72 hours. Using single factor method, the optimal conditions of virus propagation in 1.2 L reactor were as follows:MOI was 0.001, temperature was 37℃, cell density for virus inoculation was 2.0×10⁶cells·mL^-1 and stirring speed was 80 r·min^-1. Under conditions, the virus titer reached (7.13±0.11) lgTCID₅₀·mL^-1 at 48 hours in 5 L reactor batch culture. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of PRV vaccines.

The purpose of this study was to investigate the effect of African swine fever virus (ASFV) on porcine red blood cells (RBCs) and its effect on the porcine peripheral blood mononuclear cells (PBMs). Flow cytometry was used to detect the ability of ASFV to infect porcine primary alveolar macrophages (PAMs) and RBCs, and the percentage of ASFV-induced apoptosis of RBCs was detected. Whether induction of RBCs apoptosis affects the phagocytic ability of PBMs. The results showed that ASFV could not invade erythrocytes, but induced apoptosis of RBCs in a time- and dose-dependent manner. Apoptosis of RBCs at 1, 3, 5 and 7 days after inoculation with varying MOI (0.1,1,3) ASFV were as follows:at the MOI of 0.1, the data were 1.27%, 3.23%, 7.39% and 8.56%, respectively; at the MOI of 1, the data were 1.54%, 3.73%, 8.46% and 10.74%, respectively; at the MOI of 3, the data were 2.65%, 5.01%, 12.44% and 18.61%, respectively. At the same time, apoptotic RBCs can increase the number of yellow-green fluorescent microspheres phagocytosed by PBMs. In conclusion, ASFV cannot invade porcine RBCs, but ASFV induces RBCs apoptosis and promotes the phagocytic ability of PBMs in a time- and dose-dependent manner.

To investigate the molecular characteristics of ALV-K isolates from local breed chickens in Guangdong, 8 042 anticoagulant samples were collected from core flocks of healthy appearance in seven large-scale breeding poultry farms (A-G) in Guangdong that are undergoing eradication of exogenous ALV during 2020-2021. Among them, 357 positive samples were identified by DF-1 cell culture and p27 antigen ELISA test. PCR identification and env gene sequencing were performed on 150 positive samples with S/P values of 0.2 to 0.6 by ELISA, and 32 ALV-K strains were isolated. Sixteen isolates were further selected for whole-genome sequencing analysis. The results showed that the total genome length of the 16 ALV-K isolates ranged from 7 481 to 7 496 bp, and the genome conformed to the typical retrovirus structure of 5'-LTR-UTR-gag-pol-env-UTR-LTR-3', without known oncogenes. The gp85 gene of the isolates and ALV-K reference strains were located in the same evolutionary branch and had the highest similarity (>94.0%). The pol and gp37 genes were relatively conserved, with the similarity more than 94.0% to other ALV subgroups. The LTR showed highest similarity with endogenous ALV and most ALV-K reference strains (91.4%-99.5%), and the LTR U3 region had the same transcriptional regulatory elements as the endogenous ALV LTR. Additionally, 31.3% (5/16) of isolates were observed to have a 12 bp deletion at nucleotides 373-384 of the gag gene. Further, the full-length cDNA clones of the deletion strain (rGD20 JM10) and the complementary strain (rGD20 JM10 A12) were respectively constructed by PCR segmental amplification and homologous recombination based on the deletion strain GD20 JM10, and the infectious clones were transfected into DF-1 cells. The ELISA and IFA results showed the two viruses were successfully rescued, and the in vitro replication curves showed no significant difference between the two viruses. The epidemic ALV-K strains from seven breeding poultry farms in Guangdong investigated in this study all carried endogenous LTR, so it is particularly important to use a more sensitive detection technology when carrying out exogenous ALV eradication. Additionally, the growth curves showed that such gag gene with the 12 bp deletion had no evident effect on the virus replication in vitro.

In our study, five Clostridium perfringens isolates were obtained from diarrhea Guanzhong diary goats and named as 21-D-1 to 21-D-5. Toxin genes detection indicated the isolates all belonged to C. perfringens type D positive for etx and cpa, while cpe associated with foodborne diseases was detected in strain 21-D-5. After whole genome sequencing, we found the genome size, GC content and genes number were stable among the C. perfringens type D genomes. SNPs analysis showed there were few SNPs (<25) between 21-D-1 and 21-D-2, and 21-D-3 and 21-D-4, which revealed they might belonged to two C. perfringens strains, respectively. Fifteen toxin gens were detected among the C. perfringens type D isolates, sequence and genetic environment analysis of etx indicated sequence of etx were highly conserved and the genetic environments were similar among the genomes. In addition, cpe was detected downstream etx in the genome of C. perfringens strain 21-D-5. Besides, oxazolidinones-resistant gene optrA and the other eight antimicrobial resistant genes were also detected among the isolates, and erm(A), optrA and fexA were observed to have the the possibility of co-spread. This is the first study in China, which whole genome sequence and analyze the genomes of C. perfringens type D strains. The results of our study may provide reference for prevention and treatment of C. perfringens diseases, and further researches on C. perfringens genomes.

In this study, we report the analysis of the genes involved in the immune interaction between Echinococcus granulosus protoscoleces and macrophage RAW264.7, which provides a theoretical basis for further elucidating the mechanism of immune regulation and parasitic adaptation of the protoscoleces. The protoscolices and macrophage RAW264.7 cells were co-cultured for 24 hours, then the protoscolices were collected, total RNA was extracted and cDNA library was constructed. The results showed that there were 435 genes significantly differentially expressed in the protoscolices after co-cultured with macrophages RAW264.7 for 24 h compared with 0 h, which included 227 up-regulated genes, such as HSP70, HSP10, EG95 precursor, AgB, FABP and vesicular transporter SC22B; And 208 down-regulated genes, such as EF-hand, Cathepsin, transmembrane protein 144, endosterol receptor and MAPK7. GO analysis showed that the differentially expressed genes (DEGs) were mainly involved in heme transport, iron coordinated entity transport, extracellular Matrix, Ribonuclease MRP complex, serine type endopeptidase inhibitor activity and Galactosidases activity. KEGG analysis showed that the DEGs were mainly related to spliceosome, endocytosis, endoplasmic reticulum protein processing, phagosomes, MAPK signaling and calcium signaling. When macrophages RAW264.7 were co-cultured with protoscolex for 24 hours, compared with PBS control group, the expression of 3 745 genes in RAW264.7 cells was significantly changed, of which 1 159 genes were up-regulated, 2 586 genes down-regulated. The results of GO analysis showed that the DEGs were mainly concentrated in metabolic processes, intracellular components, intracellular, Organelles and membrane-structured Organelles. The analysis of KEGG showed that the DEGs were mainly involved in metabolic pathway, ribosome pathway, spliceosome pathway, RNA transport and ubiquitin-mediated protein hydrolysis. At the same time, some DEGs were selected randomly and verified by qRT-PCR. The results showed that the expression trend was consistent with that of RNA-seq results, which further indicates that the transcriptome data is credible. In conclusion, when the protoscoleces are subjected to immunologic pressure from host macrophages, they may cause gene differential expression, the immunoregulatory molecules such as AgB, FABP1 and Kunitz-type Serine protease inhibitors in protoscoleces were up-regulated, which might be involved in host immunoregulation, moreover, it is beneficial to parasitism and immune escape for E.granulosus.

The present study was carried out to investigate the impact of circRNA ciRS-7 on the propagation of Cryptosporidium parvum in HCT-8 cells via targeting miR-219a-5p. Using C. parvum-infecting HCT-8 cells as the model, we detected the expression of the autophagy associated-proteins of LC3B-II and p62 in HCT-8 cells by using Western bolt assays. The targeting relationship between ciRS-7 and miR-219a-5p were detected and verified by using qRT-PCR and dual luciferase reporter assays, and the infection burden of C. parvum (the mRNA level of C. parvum hsp70 gene) in HCT-8 cells was determined by using qRT-PCR. C. parvum infection induced significantly up-regulation of the protein levels of LC3B-II and p62 in HCT-8 cells at 8 h, 12 h, 24 h, 36 h and 48 h post-infection. Overexpression of ciRS-7 markedly inhibited the protein level of LC3B-II but promoted the protein level of p62 in C. parvum-infecting cells, whereas interfering the expression of ciRS-7 obtained the opposite results. C. parvum infection induced significant down-regulation of miR-219a-5p in HCT-8 cells, and ciRS-7 could target to regulate the expression of miR-219a-5p. miR-219a-5p mimics significantly increased the protein level of LC3B-II but decreased the protein level of p62 in C. parvum-infecting cells, while miR-219a-5p inhibitor had the opposite effects, and the inhibition effect on the protein level of LC3B-II and the promoted effect on the protein level of p62 by overexpression of ciRS-7 in C. parvum-infecting cells were reversed by miR-219a-5p mimics. miR-219a-5p mimics significantly inhibited the mRNA level of C. parvum hsp70 in infected cells, while miR-219a-5p inhibitor had the opposite effect. The upregulated effect of overexpression of ciRS-7 on the mRNA level of C. parvum hsp70 gene in infected cells was attenuated by miR-219a-5p mimics. The results revealed that ciRS-7 promoted the propagation of C. parvum in HCT-8 cells by targeting miR-219a-5p to inhibit C. parvum-induced cell autophagy.

The aim of this experiment was to study the effects of chicken transfer growth factor-β1 (TGF-β1) on the adhesion of Escherichia coli and Salmonella pullorum to DF1 cells. The expression of chicken TGF-β1 was detected by ELISA after DF1 cells were infected with Newcastle disease virus (NDV), then the overexpression and inhibitory expression vectors of chicken TGF-β1 were constructed by referring to the chicken TGF-β1 sequence in GenBank. The transfection efficiency of the recombinant TGF-β1 expression vector was observed under fluorescence microscope 48 h after transfection of DF1 cells. The mRNA level changes of chicken TGF-β1 in DF1 cells were detected by fluorescence quantitative PCR, and the changes of chicken TGF-β1 extracellar protein expression level were detected by ELISA. The effects of chicken TGF-β1 on the adhesion of pathogenic Escherichia coli and Salmonella pullorum to DF1 cells were detected by adhesion assay. The results showed that the extracellular expression level of TGF-β1 in DF1 cells infected with NDV was significantly higher than that in uninfected cells (P<0.05). Enzyme digestion and sequencing confirmed that the recombinant vectors with TGF-β1 inhibitory expression and overexpression were successfully constructed. The expression levels of TGF-β1 mRNA and extracellular protein in transfected chicken TGF-β1 recombinant overexpressed vector cells were significantly higher than those of untransfected cells (P<0.01), and the adhesion rates of pathogenic Escherichia coli and Salmonella pullorum to cells were significantly higher than those of untransfected cells (P<0.01). The expression levels of TGF-β1 mRNA and extracellular protein in transfected chicken TGF-β1 recombinant inhibitory vector cells were significantly lower than those of untransfected cells (P<0.01), and the adhesion rates of pathogenic Escherichia coli and Salmonella pullorum to cells were significantly lower than those of untransfected cells (P<0.01). In conclusion, the expression level of chicken TGF-β1 increased after NDV infection of DF1 cells, and chicken TGF-β1 can promote the adhesion of pathogenic Escherichia coli and Salmonella pullorum to DF1 cells, which provides experimental basis for further study on the role of chicken TGF-β1 in the bacterial disease secondary to poultry virus infection.

This study aims to systematically analyze the changes of hosts' T cells, NK cells, expression of Tim-3 and cytokines post infection with Plasmodium berghei (P. berghei) in mice. A total of 64 female C57BL/6 mice were selected and randomly divided into 8 groups, with 8 in each group. Mice spleen and peripheral blood immune cells were obtained on 0, 4, 7, 9, 11, 13, 16 and 19 days post infection, and flow cytometry was used to detect the changes of main immune cell subsets and the expression of immune checkpoint molecule Tim-3. Mice sera were collected to detect the changes in cytokine levels. Results showed that the proportions of CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells and NK cells in mice spleen were gradually reduced post infection with P. berghei (P<0.01), and this was accompanied by an increase in the expression of Tim-3 on the surface of these three types of cells (P<0.01). In mice peripheral blood, the proportion of CD3⁺CD4⁺ T cells decreased first and then increased, proportion of CD3⁺CD8⁺ T cells increased first and then decreased (P<0.05). The proportion of circulatory CD3^-NK1.1⁺ cells also decreased first and then increased, but at the end of infection time, its proportion was still lower than that of the uninfected group. The expression level of Tim-3 in peripheral blood of CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells and CD3^-NK1.1⁺ cells all increased significantly (P<0.05). Post infection with P. berghei, the secretion of pro-inflammatory cytokine IL-2 in mice serum was significantly higher than that in the uninfected group (P<0.05); The secretion of pro-inflammatory cytokine IFN-γ, TNF-α and IL-6 in mice sera all increased first and then decreased, while the immunosuppressive cytokine IL-10 showed a gradual increase trend, and increased sharply in the later stage of infection (P<0.001). The results of this study showed that post infection with Plasmodium, the specific immune response of mice played a certain killing effect, but due to the overexpression of immune checkpoint molecule (Tim-3 etc.) and some immunosuppressive cytokines (IL-10 etc.), which helps the malaria parasite to escape from the host's immune killing process. This work proposes the importance of studying Plasmodium infection from the perspective of host immunosuppression.

The aim of this study was to investigate the biological function of the 43K OMP gene of Fusobacterium necrophorum. In vitro growth rate, drug sensitivity, biofilm formation ability, bacterial adhesion ability, bacterial toxicity to cells and pathogenicity biological properties to Balb/c female mice between the wild type strain (A25 strain) and 43K OMP gene defective strain (A25Δ43K OMP strain) were detected to investigate the potential biological function of 43K OMP gene in the pathogenesis of Fusobacterium necrophorum. The results showed that when 43K OMP gene was mutated, there was no significant difference in growth rate or bacterial toxicity to cells of F. necrophorum (P>0.05); Resistance to claricid, kanamycin, neomycin, amikacin, netilmicin, furantoin, polymyxin B, rifampicin and ciprofloxacin were diminished; Biofilm formation ability was significantly enhanced after 72 and 96 h of incubation (P<0.05); After co-incubation with BEND cells for 1 h, the bacterial adhesion ability was significantly reduced (P<0.05); The pathogenicity to Balb/c female mice was significantly weakened (P<0.05). Therefore, 43K OMP gene is mainly related to the bacterial adhesion ability, biofilm formation ability and drug resistance of F. necrophorum.

The purpose of this study was to explore the epidemiological characteristics of ESBL-producing resistant bacteria in the dairy farming environments. A total of 160 samples were collected from 6 farms in Inner Mongolia and Ningxia from 2020 to 2021, including milk, milk area skin swabs and environmental sources. β-lactamase rapid test strip, with the isolation and identification, drug sensitivity, and genome sequencing of ESBL-producing resistant bacteria, were conducted respectively. The positive rate of β-lactamase in all farms was 75%-100% in 24 milk samples. A total of 6 ESBL-producing resistant strains were isolated from 153 samples in 5 farms, and these ESBL-producing resistant strains had high resistance rates to amoxicillin (100%), cefotaxime (91.0%) and ceftazidime (78.2%). And 14 ESBL-producing Escherichia coli strains sequenced were found to carry multiple drug resistance gene types, with bla_CTX-M (100%) carrying rate being the highest. At the same time, the sixth special farm with "long-term positive lactamase test" was found that ESBL-producing Acinetobacter baumannii was the main strain of β-lactamase positive in the milk of the farm, and the resistant strain carried bla_CTX-M, bla_TEM and bla_OXA β-lactamase resistance genes. This study identified the epidemiological characteristics of ESBL-producing resistant bacteria in farm environments in Inner Mongolia and Ningxia, and the causes of lactamase positivity in the milk of one farm in Inner Mongolia were successfully traced back.

This study aimed to investigate whether selenium deficiency induced apoptosis of chicken thymocytes by activating toll-like receptor signaling pathway. In vivo experiment, 200 healthy one-day-old broilers were randomly divided into control group (C group) and selenium deficiency group (L group) by replicating selenium deficiency model, the control group was fed a normal diet with 0.2 mg·kg^-1 selenium, and the se-deficient group was fed a se-deficient diet with 0.004 mg·kg^-1 selenium. At 15, 25, 35, 45, and 55 days of age, 15 chicks were selected from each group, the morphological and ultrastructural changes of thymus tissue were observed. TLR4 signaling pathway and apoptosis-related factors were detected in thymus tissue. The selenium deficiency model of MDCC-MSB1(MSB1) cells in vitro was established, and six groups were established on this basis:Control (C) group, Selenium deficiency (L) group, selenium deficiency+transfection empty plasmid (L+pCMV-HA-N) group, selenium deficiency+siRNA negative control (L+ NCsiRNA) group, selenium deficiency + overexpression of TLR4 (L+ pCMV-HA-TLR4) group, selenium deficiency +siChTLR4 (L+ siChTLR4) group. After 5 days of low selenium treatment, cell viability, apoptosis, TLR4 signal transduction pathway and expression of apoptosis-related factors were detected. Results were as follows:1) Compared with the C group, the number of lymphocytes in the medulla of the cortex in the L group was reduced, the cells were disordered, the medulla of the cortex was hyperemic, the nucleus was fragmented, and extensive focal necrosis was observed. The thymus tissue of chickens in L group showed fissure, reduced volume, cell fragmentation, and nuclear chromatin edge aggregation, mitochondrial swelling, cristae fracture; 2) Compared with C group at 15 days of age, mRNA and protein expressions of TLR4, MyD88, NF-κB, Caspase-3, Caspase-9 and Bax in the thymus of L group were up-regulated at 15 to 55 days of age, the expression levels of Bcl-2 mRNA and protein showed opposite trends; 3) Compared with C group, the viability of MSB1 cells in L group was significantly decreased, the number of apoptosis was significantly increased, the mRNA and protein expressions of TLR4, MyD88, NF-κB, Caspase-3, Caspase-9 and Bax were significantly increased, and the mRNA and protein expression levels of Bcl-2 were significantly decreased. Compared with the L group, the cell viability was significantly enhanced, the number of apoptosis was significantly decreased, and the mRNA and protein expression of related factors were significantly decreased in the L+siChTLR4 group, while the cell viability was significantly decreased, the number of apoptosis was significantly increased, and the mRNA and protein expression of related factors were significantly increased in the L+ pCMV-HA-TLR4 group. In conclusion, selenium deficiency mediates the apoptosis of chicken thymocyte through the TLR4/MyD88/NF-κB signaling pathway, and further induces thymus injury in chickens.

The purpose of this study was to investigate the inhibitory effects and potential mechanism of aspirin eugenol ester (AEE) on lipopolysaccharide (LPS)-induced inflammatory response in mouse macrophage (RAW264.7 cells). Inflammation in RAW264.7 cells was induced by LPS. Cells were divided into control group, LPS group, aspirin group (Asp, 150 μmol·L^-1), eugenol group (Eug, 150 μmol·L^-1), AEE low-(75 μmol·L^-1), medium-(150 μmol·L^-1) and high-dose (300 μmol·L^-1) groups. CCK-8 assay was used to detect the cytotoxicity of AEE in RAW264.7 cells. The levels of IL-1β, IL-6, IL-8 and TNF-α in each group were detected by ELISA. The mRNA expression levels of COX-2, COX-1, PLA2, CYP450 and 5-LOX, the key enzymes in arachidonic acid(AA) metabolism pathway, were detected by RT-PCR. Meanwhile, the interaction of Asp, Eug and AEE with key enzymes related to arachidonic acid metabolism pathway was studied by molecular docking. The results were showed as follows:1) CCK-8 results indicated that AEE with the concentration of 0 to 350 μmol·L^-1 was non-toxic to cells; 2) The results of ELISA showed that, compared with the control group, the expression levels of IL-1β, IL-6, IL-8 and TNF-α in the LPS group were extremely significantly increased (P<0.01); After treated with different doses of AEE extremely significantly reduced the expression levels of IL-1β, IL-8, and TNF-α (P<0.01); Compared with the Asp and Eug groups, no significant difference was observed for the equimolar AEE in inflammatory factors of IL-1β, IL-6, IL-8 (P>0.05), indicating the similar anti-inflammatory effects of AEE with Asp and Eug; And no significant difference of the levels of IL-1β, IL-6 and IL-8 among different doses of AEE was observed(P>0.05); 3) RT-PCR results showed that, compared with the control group, the expression of key enzymes in the AA metabolic pathway in the LPS group was extremely significantly increased (P<0.01); After treated with different doses of AEE significantly reduced the mRNA expression of key enzymes in the AA metabolic pathway (P<0.05); Compared with the Asp and Eug groups, the equimolar amount of AEE significantly reduced the expression of PLA2 (P<0.05); There was no significant difference of the expression levels of COX-1, COX-2, 5-LOX and CYP450 was observed among different doses of AEE (P>0.05); 4) The results of molecular docking showed that the binding energy of AEE to the target proteins was lower than -20.9 kJ, indicating the binding of AEE to target proteins was stable and high quality. Meanwhile, AEE could form hydrogen bonds with PLA2, COX-2, COX-1, CYP450 and 5-LOX. In summary, AEE could inhibit the inflammatory response of RAW264.7 cells induced by LPS, and the anti-inflammatory effects of AEE were similar to the precursor compounds Asp and Eug, which might be associated with the regulation of AA metabolic pathway.

circRNAs have important regulatory roles in enteric disease development of host infected by pathogenic bacteria. Clostridium perfringens type C (C. perfringens type C) is one of primary bacteria leading to a series of diarrhea and inflammatory diseases in piglets, which caused serious economic losses over the industry. However, the comprehensive and systematic understanding of circRNAs regulating piglet diarrhea caused by C. perfringens type C has not been reported. To illustrate the dynamics of piglet after C. perfringens infection, this study has investigated the expression profiles of ileum circRNAs of 7-day-old piglets challenged by C. perfringens type C through RNA sequencing, screened the differentially expressed circRNAs, and performed the GO and KEGG functional enriched functions. The targets of circRNAs were predicted by miRanda software, then constructing the circRNA/miRNA/mRNA interaction network and finally conducting the qPCR verification. A total of 3 162 circRNAs were identified with quantitative 694 differentially expressed circRNAs in treatment group(TI) and control group(CI) through sequencing, while 404 of which were up-regulated and 290 were down-regulated. Function analysis revealed that these dysregulated circRNAs were mostly enriched in several KEGG pathways including cell cycle, TGF-beta signaling pathway, Lysine degradation, Wnt signaling pathway, T cell receptor signaling pathway, MAPK signaling pathway and so on, regulating piglets resistance response to C. perfringens infection. Furthermore, the interactive network of circRNA-miRNA-mRNA were built in the C. perfringens type C-infected piglet ileum tissues, and the circRNA-associated ceRNA networks composed of 8 circRNAs-5 miRNAs-12 mRNAs was predicted to have important association with CAED. This study may provide valuable information for understanding the regulatory functions of circRNAs of piglet defensed to C. perfringens type C.

The aim of this study was to investigate the method of in vitro isolation and culture of bovine alveolar epithelial cells (BAECs) and the regulatory mechanism of Wnt5a on the autophagy of BAECs infected with Mycobacterium tuberculosis bacille Calmette-Guérin (BCG). BAECs were isolated by enzyme combined digestion method and mechanical scraping method, and purified by differential adhesion method. The expression of epithelial cell markers CK14 and CK5 were detected by immunofluorescence staining. BAECs were infected with BCG and the expression of Wnt5a was inhibited by Box-5. The expression of autophagy-related proteins and nonclassical Wnt signaling pathway related proteins were detected by Western blot and immunofluorescence staining. The results showed that BAECs were successfully isolated with high purity by enzyme combined digestion and mechanical scraping. Isolated cells were identified as positive by CK14 and CK5. BCG infection with BAECs promoted the expression of Wnt5a and increased autophagy. Box-5 pretreatment down-regulated the expression of BCG-induced autophagy-related proteins LC3II, P62, Atg7 and Atg5, and inhibited the expression of nonclassical Wnt/Ca²⁺ signaling pathway related proteins Wnt5a, CaMKII and NFAT. In conclusion, the isolation and culture method of BAECs was successfully established. BCG infection promoted the Wnt5a expression and autophagy of BAECs, and the inhibition of Wnt5a expression down-regulated the BCG-induced autophagy in BAECs. Moreover, Wnt5a regulates the autophagy induced by BCG in BAECs through the nonclassical Wnt/Ca²⁺ signaling pathway.

We aimed to investigate the effects of oleanolic acid (OLA) on oxidative damage of muscle and abnormal subchondral bone reconstruction in rats with osteoarthritis (OA). Eighteen Sprague-Dawley rats were selected and randomly divided into three groups:the control group (CON group), the model group (OA group) and the oleanolic acid administration group (OLA group).The OA and OLA groups were established using anterior cruciate ligament dissection combined with partial meniscectomy (ACLT+PMMx) to establish a rat knee OA model, and the CON group underwent sham surgery. Rats in the OLA group were given 50 mg·(kg·d)^-1 OLA by gavage daily, and rats in the CON and OA groups were given equal volumes of sterile saline containing 20 mL·L^-1 Tween 80. After four consecutive weeks of dosing, knee, quadriceps and serum samples were taken. The rats were tested for joint pain using knee extension vocalization test and heat sensitivity test. Enzyme-linked immunosorbent assay to detect the levels of citrate synthase (CS), myosin heavy chain (MHC), interleukin 1 (IL-1) and interleukin 6 (IL-6) in rat muscle and the levels of osteocalcin (OCN) and C-terminal telopeptide of type I collagen (CTX-Ⅰ) in rat serum. Western blot was used to detect changes in Nrf2/NQO1/HO-1 pathway protein expression in muscle tissue. Micro-CT was used for subchondral bone scanning and quantitative analysis of bone tissue microstructure. The results showed that compared with the OA group, OLA could reduce the OA pain score (P<0.05), activate the muscle Nrf2/NQO1/HO-1 pathway, promote the expression of muscle CS and MHC, and down-regulate the levels of IL-1 and IL-6 (P<0.05). In addition, percent bone volume (BV/TV), bone mineral density (BMD) and trabecular thickness (Tb.Th) were significantly increased (P<0.05) and expression of bone metabolic markers OCN and CTX-Ⅰ were significantly decreased (P<0.05) in subchondral bone of rats after OLA supplementation. The results suggest that OLA can inhibit the joint pain response in OA rats, inhibit muscle dysfunction in OA rats by regulating the Nrf2/NQO1/HO-1 pathway, improve the biomechanical properties and microstructural changes of subchondral bone in early OA, thereby delaying the occurrence of abnormal bone remodeling of subchondral bone in OA rats.

Based on the total material quality and polysaccharide content, the effects of cultivated and wild Artemisia rupestris L. crude polysaccharides (CARCP/WARCP) as adjuvant of inactivated foot-and-mouth disease vaccine (FMDV) on antibody level and T cell subsets in mice were compared to explore the activity and safety of CARCP/WARCP as adjuvant of vaccine. ICR mice were intramuscularly immunized with CARCP/WARCP combined with FMDV. Serum FMDV-specific antibody levels, T-cell subsets in spleen, serum IgE level, clinical signs, local reactions at the injection site and body weight after immunization were monitored. The results showed that when the content of total substance were the same, CARCP1/WARCP1 highly significantly improved FMDV-specific IgG andIgG_2aresponse, the percentages of T cell CD3⁺CD4⁺, CD4⁺CD44⁺, CD8⁺CD44⁺CD62L⁺(P<0.01). And significantly improved IgG₁, IgG_2ato IgG₁ratio, the percentages of CD3⁺CD8⁺, CD8⁺CD44⁺and CD8⁺CD44⁺CD62L^-(P<0.05), the adjuvant activities of CARCP1 were significantly higher than that of WARCP1 except the indexes of 28 d IgG and IgG₁(P<0.05). When the content of polysaccharides were the same, CARCP2/WARCP2 highly significantly increased FMDV-specific 28 d IgG, IgG_2ato IgG₁ ratio (P<0.01),and significantly improved 21 d IgG, 28 d IgG_2a,andpercentages of CD4⁺CD44⁺(P<0.05), there was no significant difference between CARCP2 and WARCP2 (P>0.05). CARCP/WARCP did not cause clinical symptom, adverse reactions such as granuloma and swelling at the injection site, no significant difference was observed in body weight after CARCP/WARCP immunization (P>0.05). IgE antibody was not detected among all groups (P>0.05). These results indicating that CARCP/WARCP has certain safety. In conclusion, when the content of total substance were the same, CARCP/WARCP could enhance the humoral and cellular immune responses of FMDV immunized mice, and the adjuvant activity of CARCP was better than that of WARCP; When the polysaccharide content is consistent, CARCP/WARCP as FMDV adjuvant has equivalent immune enhancement effect and is a safe adjuvant candidate.

This study aimed to explore the protective mechanism of baicalein against porcine deltacoronavirus (PDCoV) infection. The targets of baicalein were obtained through Pharmamapper, Pubchem, STITCH, TCMSP and Swiss Targer Prediction databases, and the targets of PDCoV infection were obtained according to the proteomics data from our previous study. The targets of baicalein-PDCoV interaction were obtained and analyzed by STRING database and Cytoscape 3.8.2 software to construct a network diagram of "baicalein-PDCoV-targets". The CytoNCA was used to analyze network topology and core network construction. Metascape database was used for GO and KEGG analysis of core network genes. The expression levels of genes in the predicted signaling pathways were detected in vitro. A total of 268 potential targets of baicalein were screened out. There were 75 potential targets of baicalein-PDCoV infection. GO enrichment results showed that baicalein was mainly involved in the formations of membrane raft, spindle and mitochondrial membrane, cell cycle and MAPK signaling pathways. A total of 277 signaling pathways (P<0.01) were screened out by KEGG enrichment. The PI3K-Akt, Ras and MAPK signaling pathways were the main pathways that involved in the protective effects of baicalein against PDCoV infection. The results showed that compared with the cellular control groups, the mRNA expressions of PI3K, AKT and NF-κB significantly increased in the PDCoV infection group. Compared with the PDCoV group, treatment of baicalein significantly decreased the mRNA expressions of PI3K, AKT and NF-κB (P<0.05). The effect of baicalein on PDCoV infection has the characteristics of multi-targets and multi-pathways, through the intervention of AKT1, HSP90AA1, SRC, EGFR, CASP3, MAPK, STAT3 and other core genes in regulating PI3K-Akt signaling pathway, Ras signaling pathway and MAPK signaling pathway, apoptosis, and virus infection. These results suggested that baicalein could be a potential therapeutic drug against PDCoV infection for further study.

To explore the possible binding sites of Hfq on GcvB, in this study, the Hfq binding preference U-rich motif in GcvB was screened. The mutant and truncated strains of the GcvB U-rich motif were constructed by the λ-Red homologous recombinase system, and the GcvB terminator stem extension strain was constructed at the same time. P22 phage transduction technology was used to construct corresponding hfq gene deletion strains and oppA::lacZ gene fusion strains. The gcvB gene transcription expression level of the recombinant strain was detected by qRT-PCR, and the oppA gene protein expression level was detected by the β-galactosidase test. The results showed that the mutation of U₅ motif did not cause obvious changes in the transcription and expression level of gcvB gene, but when the U₈ motif was truncated to U₅ and U₄, it was reduced by 40.5% and 37.5%, respectively. After the GcvB terminator stem of the U₄ truncated strain was extended, it was discovered that the transcription and expression level of gcvB gene was restored,but the ability to Hfq negatively regulating oppA mRNA was weakened after co-transcription. The above results show that the U₅ motif has no obvious relationship with Hfq maintaining the stability of GcvB,but the U₈ motif is important for Hfq to assist GcvB with regulating oppA mRNA negatively after co-transcription and maintain GcvB stability. In summary, we speculate that the U₈ motif is the binding site of Hfq on GcvB.

To understand the prevalence and variation of the H3N2 subtype avian influenza virus (AIV) in poultry in Zhejiang province, RT-PCR technology was utilized to detect a total of 923 samples from Zhejiang province in 2021, the isolated AIVs were subjected to molecular characteristics and genetic evolution analysis. The results showed that the positive rate of AIV in Zhejiang province in 2021 was 7.69% (71/923), 2 chicken-derived AIVs and 1 duck-derived H3N2 subtype AIVs were isolated, and their HA and NA genes homology were 93.4%-100% and 94.0%-99.9%, respectively. The origins of the internal gene fragments of the isolated AIVs were complex, and they were closely related to subtypes such as H1N2, H1N4, and H10N7. The genetic evolution analysis showed that H3N2 subtype AIV was mainly prevalent in East China, and the duck was its main host. The HA and NA genes of the three H3N2 subtype isolates belonged to avian-derived evolutionary branches. Amino acid analysis revealed that the cleavage site of HA protein was PEKQTR↓GLF, which was in line with the characteristics of the low pathogenic AIV. The 226Q and 228G of HA protein as well as the 627E of PB2 protein, which was related to receptor binding and mammalian adaptability respectively, were consistent with those of AIV, suggesting that the cross-species transmission to mammals appeared unlikely. However, amino acid 66 was mutated to S in the PB1 protein might increase the pathogenicity in mammals and need to be further investigated. In summary, the H3N2 subtype AIVs isolated in this study were consistent with the characteristics of low pathogenic avian influenza virus, and the origins of the gene fragments were complex. The potential of the isolates for cross-species transmission to humans was low, but whether they affect the pathogenicity of the host remains to be further explored in the near future.