The regulation of female mammalian follicular development and oocyte maturation involves the spatiotemporal regulation of those genes that play key roles in reproduction. miRNAs, as a class of small non-coding RNAs, regulate the expression of most of these genes. Over the last decade, a large number of miRNAs involved in mammalian follicular development and oocyte growth were found through high-throughput sequencing, knockout of key genes involved in miRNA generation, and functional inhibition or overexpression of miRNAs. The function of these miRNAs in mammalian follicular development and oocyte maturation were ultimately determined by studying the interaction with their target genes. The present review focuses on the expression and potential function of major miRNAs during follicular development and oocyte maturation in reproductive cells or extracellular. These results can provide a reference for further exploring the regulation mechanism of mammalian reproduction.

Hair follicles are important accessory structures on mammalian skin and are the most important organs for controlling hair growth. Development of hair follicle mainly includes hair follicle morphogenesis and hair follicle regeneration.The Wnt/β-catenin signaling pathway plays an important role in the development of hair follicle and has received extensive attention from scholars and research institutions. In this paper, regulating mechanisms and intranuclear activation of Wnt/β-catenin were reviewed. Regulating mechanisms of Wnt/β-catenin on hair follicle morphogenesis and hair follicle regeneration, regulatory roles of osteoblast inhibitory factor (Dkk) and a large number of nutrients on development of hair follicle in mammals were also summarized. The current review will provide references for the regulation of mammalian hair follicle development by Wnt/β-catenin signaling pathway.

The aim of this study was to analyze the effects of different factors on the reproductive performance of large populations of Large White and Landrace pigs. At the same time, the genetic parameters of reproductive traits of Large White and Landrace pigs were estimated. The breeding records of 65 546 litters of Large White sows and the breeding records of 9 552 litters of Landrace sows in Wen's from 2013 to 2018 were selected. The SAS software GLM process was used to analyze 8 reproductive traits, and analyze the effects of 3 factors, including the parity, the birth parity and the estrus condition on reproductive performance. At the same time, DMU software was used to estimate the heritability of these traits, genetic correlation and phenotype correlation. The results showed that:1) In the analysis of influencing factors:In the two breeds, the sow delivery parity had a significant effect on all reproductive traits (P<0.001); In Large White pig, the birth parity of sow had significant effect on the 5 reproductive traits(P<0.05), and the effects of estrus on all reproductive traits were not significant; In Landrace pig, the birth parity of sows had significant effect on the number of weak litters and the weight of litter(P<0.05), the estrus factor had significant effects on the 5 reproductive traits(P<0.05). 2) In the estimation of genetic parameters:In Large White pig, the heritability of the weight of litter was the highest, which was 0.281, the heritability of the total number of litters, the number of born alive, the number of healthy birth, the number of weak litters and the number of stillbirths were in the range of 0.117-0.179; In Landrace pig, the heritability of the total number of litters was the highest, which was 0.190, the heritability of the number of born alive, the number of healthy birth, the number of weak litters and the weight of the litter were in the range of 0.100-0.176; In Large White pig, the genetic correlation coefficients between the total number of litters and the number of born alive, the total number of litters and the number of healthy birth, the number of born alive and the number of healthy birth, the number of healthy birth and the weight of the litter were all above 0.738, the phenotype correlation coefficients were above 0.717; In Landrace pig, the genetic correlation coefficients between total number of litters and the number of born alive, the total number of litters and the number of healthy birth, the number of born alive and the number of healthy birth were higher than 0.895, the phenotype correlation coefficients were higher than 0.791. The results of this study provide experimental data and theoretical basis for improving the reproductive performance of sows and accelerating the genetic progress of reproductive traits.

The aim of this study was to explore the genome-wide copy number variations (CNVs) affecting porcine bone rate traits, and to study the action mode and mechanism of the genes covered by these CNVs. CNVcaller software which based on the depth of sequencing were used to detect the copy number variations of the F2 population of the Large White×Min pig, and TASSEL mixed-linear model (MLM) were used to perform genome association analysis on the bone rate traits. The genes covered by CNVs were analyzed by seeking databases. The expression of the 4 significant CNVs in leg cartilage were detected by RNA resequencing in 75-day-old Large White pigs. A total of 3 027 CNVs were detected in F2 population, including 1 251 deletions, 987 multiple copies, and 789 deletions and multiple copies. There were 4 copy number variations associated with bone rate at genomic level, all on chromosome 7. Two of the four variants did not overlap with any gene. The most significant CNV (CNV4) could cover the myeloid associated differention marker(MYADM) gene. The study of gene expression in 75-days-old pigs showed that only CNV2 was highly expressed in hind leg cartilage. It was speculated that CNV2 could affect bone rate by affecting the expression of internal genes, and CNV4 might play a regulatory role in embryonic development. In summary, four CNVs affecting bone rate traits were successfully identified in pigs. It was speculated that CNV2 affected bone rate by affecting the expression of internal genes, and CNV4 might play the regulation role in embryonic development and finally affect bone rate. This study laid a theoretical foundation for researching the regulation mechanism of bone rate traits in the future and provided a reference for the breeding of bone rate traits in pig.

The aim of this study was to edit the P53 gene, and analyze the expression of vital genes in its signaling pathway. Firstly, two designed sgRNAs were loaded into the PX459 V2.0 plasmid, and two single-stranded oligonucleotides (SSODN) carrying 241W, 242S, 266H mutations were ordered as donor DNA (one carrying 241W and 242S mutations, the other one carrying 266H mutation). The targeting vectors and SSODNs were co-transfected into PFF cells by electroporation, and monoclonal cells were collected. Then editing events in targeting region in isolated cells were detected, and expression of vital genes (MDM2, FAS, WIP1, BAX, P21 and DD1α) in P53 signaling pathway were detected and the proliferative capacity of the gene editing cells were identified. Among the isolated 107 monoclonal cells, each 4 clones were homozygous and heterozygous modification of R241 and R242, 1 clone was homozygous modification of R266, 63 clones contained homozygous deletions, 11 clones carried homozygous inversions, 11 clones contained homozygous insertions, 3 clones carried heterozygous deletions (or mixed clones) and 10 clones were wild-type. No cell with simultaneous modification of 3 sites was found. RT-PCR and Western blotting analysis showed that P53 expression was not detected in the cells with homozygous deletions. RT-PCR result indicated that the expression levels of MDM2, FAS, BAX and P21 were extremely significant decreased (P<0.01 or P<0.001) in cells carrying homozygous deletions and homozygous modification of R241 and R242. Western blotting result showed that P21 protein was undetectable, and MDM2 protein was obviously decreased in cells contained homozygous deletions. The CCK-8 test indicated that the proliferation capacity of the edited cells significantly (P<0.05) or extremely significantly (P<0.01 or P<0.001) increased compared with the wild-type cells. In conclusion, we successfully obtained PFF cells that simultaneously modified two sites in P53 gene related to human tumorigenesis. P53 gene editing obviously affected the expression levels of vital genes in its signaling pathway. The acquirement of cells with modification of two sites laid the solid foundation for further generating P53 point edited model in pigs.

The objective of this study was to explore the general characteristics of somatic cell count(SCC) and analyze the factors affecting SCC of Holstein in early lactation stage in Beijing area. From June to July 2018, daily SCC of 124 cows in a dairy farm was measured in first 7 days after calving. The factors influencing SCC in early lactation stage were analyzed by GLM procedure of SAS software, and the relationship between somatic cell score (SCS) during first 7 days after calving and test-day SCS in following months was analyzed by REG procedure. The results showed that the SCC in early lactation stage was higher than test day SCC (8-133 days after calving), and the average SCC from 0 to 7 days after calving was (860±1 287) 10³·mL^-1. Parity, days in milk, environmental temperature and humidity had significant impacts on SCS in early lactation stage (P<0.05). There was a significant regression relationship between SCS in early lactation stage and test day SCS in 39-68 days after calving(P<0.01), and the regression coefficient was 0.40. The average SCC of Holstein in early lactation stage(0-7 d) was higher than the average SCC in other stages. And the SCC of Holstein in the early lactation stage was sensitive to changes of different physiological states and environmental factors of cattle. This study laid the foundation for the genetic analysis of SCC in early lactation stage, and provided a reference for the management of mastitis in dairy farm according to SCC in the early lactation stage.

This research was conducted to identify and analyze piRNA in the testicular development of yaks of different ages. A total of 6 healthy male yaks (two in each group) in the fetal (4-5 months old), calves (1 year old) and juvenile (3 years old) stages were selected. Testis were separated for piRNA sequencing and two samples from yak at the same age were considered as biological duplications. The number, base preference, source and function of piRNA, as well as the chromosome distribution and expression levels of piRNA clusters in the samples were analyzed. The expression of PIWI gene family(PIWIL1-PIWIL4) in the testis of yak at 3 stages were detected by fluorescence quantitative PCR. The results showed that the number of piRNAs in testis of yaks in calves and juvenile was significantly more than that in fetal stage (P<0.01). piRNAs from yak testis showed a strong preference for uracil (U) at their 5' ends, and the most of the piRNA came from the other regions than the intergenic and gene regions. More than 60% piRNA clusters in fetal testes were at low abundance, while more than 70% clusters in testes of calves and juvenile yaks were at high abundance. The expressions of PIWIL1 and PIWIL4 in fetal testes were significantly different compared with those in the other two stages (P<0.05). GO function analysis result showed that the number of piRNA source genes ranked first in biological process, cell component and molecular function were metabolic process, cell and binding, respectively. The structure, function and source of piRNA in yak testes were specific. The number and expression abundance of piRNA in the post-embryonic stage (calves and juvenile) was significantly different from that in the fetal stage, which may be related to the expression difference of PIWIL1 and PIWIL4 between two stages.This research provides a theoretical basis for further studying the mechanism of piRNA regulating yak testicular development and improving yak production performance.

This study aimed to breed and identify XY female mice, and analyze their reproductive ability and the mechanism of sexual inversion, so as to provide an animal model for studying on gender determination during mouse development. The identification method of XY female mice was divided into two steps(phenotypic and genotypic identification). The first, phenotypic identification:B6.XY^TIR male was naturally mated with wild-type XX female mice, their offspring were analyzed. Male and female mice of 30-, 60- and 90-day-old were identified by external genital characteristics and anogenital distance, and the gender of fetuses of 17.5 days post coitum were identified by gonadal morphology and germ cell types. The second, genotype identification:In order to determine their sex chromosome as XX or XY, DNA of postnatal and fetal mice was extracted, primers were designed and Zfy gene (a unique gene on the Y chromosome) was amplified. Combined with phenotypic identification, B6.XY^TIR female mice were obtained. In this study, whole-tissue immunofluorescence staining was used to analyze the differentiation of gonads in fetal stage, and morphological observation was used to observe the internal reproductive system of postnatal mice, and the sequence of Zfy gene was preliminarily analyzed by sequencing. A type of XY sex reversed female mouse was obtained through phenotypic and genotypic identification. It was found by observation that the ovaries, fallopian tubes and uterine structures of the XY female mice were similar to those of wild-type XX female mice. Meanwhile, the preliminary study showed that the distribution of germ cells during gonad differentiation in XY female mice was similar to that in the wild-type XX female. Sequencing showed that only Zfy-1 gene, but not Zfy-2 gene, was amplified in B6.XY^TIR males and B6.XY^TIR females; XY females accounted for 47.47% of all 99 XY mice. The results showed that XY females had a complete female reproductive system, and sex reversed had already been formed during embryonic development. Zfy-2 might be missing on the Y chromosome of B6.XY^TIR female mice, which could be related to the occurrence of sexual inversion. The results provides a unique perspective for the further study of gonadal differentiation and gametogenesis.

The effect of different protein, nonfibrous carbohydrates(NFC) and cellulose nutritious ingredient on bacterial community structure in rumen microenvironment was studied. The aim was to explore the fluctuation of predominant bacteria in rumens, and to understand clearly the dependence of rumen bacteria on nutrients. It provided foundation for further study on scientific nutrition ratio for supplementary feeding under the traditional grazing and large scale breeding. Experiment was divided into 2 groups under traditional grazing and TMR fodder feeding, at each of which there were 10 Pengbo semi-fine wool ewes (2-year-old, 2 repetitions in each group, (23.77±2.34) kg of weight). The Kjeldahl, van soest and difference methods were used to determine the crude protein, cellulose and NFC content of forage grass mixed forage in the grazing group and forage in the feeding group. After half a year of feeding, the rumen fluid was extracted, the total DNA was extracted, and a recombinant standard plasmid was constructed to perform absolute fluorescence quantification(qRT-PCR) study on rumen bacteria. The results showed that:1) At the level of phylum, the preponderant population was Bacteroidetes, its abundance in grazing group((49.52±6.07)%) was 6.00% fewer than that in barn feeding group((55.52±12.18)%), but the difference was not significant (P>0.05). The abundance of Firmicutes in grazing group((43.28±4.59)%) was 6.60% higher than that in barn feeding group((36.68±9.78)%), the difference was significant(P<0.05). The abundance of Spirochaetes in grazing group((1.99±1.75)%) was 1.23% higher than that in barn feeding group((0.76±0.59)%), the difference was significant(P<0.05). 2) Analysis by PLS-DA and LEfSe revealed that the main bacteria that showed significant differences between the 2 groups were:the cellulose or its metabolites mainly depended on Pseudobutyrivibrio, Selenomonas, Coprococcus, Clostridium, Anaerovibrio, Shuttleworthia, CF231, Butyrivibrio, YRC22, Lachnospira, Moryella(in grazing group). The high-protein and NFC (in barn-feeding) or their metabolites mainly depended on Desulfobulbus, Atopobium, p-75-a5, Prevotella, Blautia, Ruminococcus and Streptococcus(in barn feeding group). The results indicated that the reasonable protein and energy levels in diet was not only beneficial to the proliferation of protein and starch-dependent bacteria, but also to the proliferation of main fiber-degrading bacteria (Ruminococcus) and synergistic fiber-degrading bacteria (Prevotella). Meanwhile, the abundance of cellulose decomposing bacteria (Firmicutes and Spirochaetes) had all declined obviously in barn feeding group.

This study was carried out to evaluate the effect of harvest time on the silage quality and the in vitro fermentation characteristics of silage maize. The KEYU1108 silage maize was harvested at the middle and later periods of the milk-ripe stage as well as the initial and middle periods of the waxy-ripe stage, respectively. After harvest, the silage was processed and the quality was investigated at 90 d of ensiling. Subsequently, the oven-dried silage was fermented for 72 h by an in vitro gas production technique. The results showed that:1) Before the ensiling, with the prolonging of harvest time of the silage maize, the whole-plant biomass (linear, P<0.000 1), the straw biomass (linear, P=0.019 0), the grain biomass (linear, P<0.000 1), the hundred-grain weight (linear, P<0.000 1; quadratic, P=0.006), the grain ratio (linear, P<0.000 1) and the ether extract concentration (linear, P=0.037) were increased, the straw ratio (linear, P<0.000 1) and the crude protein concentration (linear, P=0.028) were decreased, the concentrations of neutral detergent fiber (quadratic, P=0.029) and acid detergent fiber (quadratic, P=0.067) were firstly decreased and then increased, and the water-soluble carbohydrate concentration (quadratic, P=0.001) was firstly increased and then decreased; 2) In the process of the ensiling, with the prolonging of harvest time of the silage maize, the isovalerate molar proportion (linear, P=0.035) was decreased, the propionate molar proportion (quadratic, P=0.064) was firstly decreased and then increased, and the acetate molar proportion (quadratic, P=0.098) was firstly increased and then decreased; 3) In the process of in vitro fermentation after the ensiling, with the prolonging of harvest time of the silage maize, the potential maximum gas production (linear, P=0.015), total volatile fatty acids concentration (linear, P=0.017) and the dry matter disappearance (linear, P=0.099) were decreased, and the acetate molar proportion (linear, P=0.043) was increased. The results suggested that the prolonging of harvest time of the silage maize could increase the biomass, change the nutrient compositions, not affect the silage quality, but reduce the degradation characteristics in vitro after ensiling.

This study was conducted to explore the effect of interferon gene stimulating factor (STING) gene knockout on replication of pseudorabies virus (PRV). The STING gene of porcine pulmonary macrophage cell line 3D4/21 was knocked out by using CRISPR/Cas9 technique, the target gene knockout efficiency was detected by using T7 nuclease, and the activity of gene knockout cells was detected by using cell counting kit. The recombinant virus PRV-GFP expressing green fluorescent protein (GFP) was used to infect the knockout cells, and the fluorescence intensity of the infected cells was detected by flow cytometry. The expression of gB and TK genes in PRV and the transcription of IL-1β, IFN-β and ISG15 genes in infected cells were detected by quantitative RT-PCR, the expression level of PRV gE protein was detected by Western blot, and the viral titer of offspring was determined by viral titration. The results showed that all of three designed exon2 specific sgRNA of STING gene could cleave the target sequences of 3D4/21 cells, and the gene editing efficiency of sgRNA3 was the highest. SgRNA1-mediated STING gene knockout lines were cloned and three gene knockout cell lines were obtained. Gene knockout had no effect on cell activity. PRV-GFP positive cells cultured in parental cells accounted for 80.77%, and PRV-GFP positive cells cultured in STING knockout cells accounted for 95.55%. STING gene knockout could promote the expression of PRV gene. The virus titer of parent 3D4/21 cells was 10^6.2 TCID₅₀·0.1 mL^-1, and the virus titer of STING knockout cells was 10^8.3 TCID₅₀·0.1 mL^-1. The transcription of IL-1β, IFN-β and ISG15 in virus infected cells were significantly down-regulated. STING gene knockout can promote the replication of PRV in 3D4/21 cells, which may be related to the inhibition of IL-1β, IFN-β and ISG15 expression in infected cells.

This study was designed to identify the difference between no-hook Cysticercus pisiformis (C. pisiformis) and hook C. pisiformis. Scolex's ultrastructures of C. pisiformis were observed by scanning electron microscope; PCR amplification and sequences analysis of 18S DNA of C. pisiformis were conducted and evolutionary tree comparison was performed among C. pisiformis and other animals' cysticercus in this study. The results showed that different ultrastructures of no-hook and hook C. pisiformis were mainly observed on rostellum, but the ultrastructures of suck and somite were alike. Most of the C. pisiformis taken out from the vesicles were in relative rest state. The rostellum of no-hook C. pisiformis looked like several oblate pastries stacked up together. Its surface was flatter and had a bigger nodule in the center, in which the diameter was about 20 μm. There were several layers of gear like patterns around the rostellum,forming an overlapping gear like structure. The front end of rostellum of hook C. pisiformis was blunt, and was connected to the chicken claw like hamula through the tegument muscular columns. The hamula were about 120-150 μm long, and its surface was smooth, rich luster and possessed hard appearance. Using 18S-P1 and 18S-P2 primers, the same target gene bands were obtained by PCR in the target genes of no-hook and hook C. pisiformis, culture medium of competent cells and bacterial plasmid. The sequencing results showed that the difference of the 750 bp sequences amplified by 18S-P1 primers was more significant than that of the 666 bp sequences amplified by 18S-P2 primers. Compared with evolution of Cysticercus of other animals, the no-hook C. pisiformis was related to C. saginate, while the hook C. pisiformis was related to C. solium. In conclusion, although both no-hook and hook C. pisiformis belong to the same genus, no-hook C. pisiformis is a new C. pisifomis being discovered.

This experiment was conducted to investigate the expression level of ubiquitinated modified proteins in host cells infected with Brucella in early stage and screen out the key regulatory proteins that affect the process of immune response. By using Label-free and ubiquitination enrichment technology and high resolution LC-MS/MS combined quantitative proteomics research strategy, ubiquitination proteomics quantitative studies were carried out on Brucella 16M infected macrophages and uninfected macrophages after 11 hours (5 hours' infection, 6 hours' intracellular replication). Database retrieval analysis was performed on the proteins which were corresponding to the ubiquitination sites that differentially expressed by 16M infected macrophages and uninfected macrophages. The key proteins that can cause host immunosuppression after 16M infection of macrophages were screened out by bioinformatics method. In this study, 580 ubiquitination sites were identified on 349 proteins. The ubiquitination level of 259 sites on 167 proteins in the 16M infection group was up-regulated compared with the uninfected group, 321 sites on 212 proteins were down-regulated (difference multiple >1.5, P<0.05); There were 35 ubiquitination-modified differentially expressed proteins may be related to the host immune response after Brucella infection. Among them, we found 27 ubiquitinated down-regulated proteins such as Bcap31, Btk, Faf1 and Akap31; one up-regulated ubiquitinated protein, Ubqln1, may be the key protein causing immunosuppression after Brucella infection. In this study, ubiquitinated modified proteins differentially expressed in host cell immune response to Brucella 16M infection were screened and obtained. It was preliminarily revealed that Brucella can affect the ubiquitinated modification of related proteins in host immune signaling pathway, autophagy and apoptosis, so as to further study the regulation of perennial status after Brucella infection. The main ubiquitination modification to complete the molecular mechanism of immune escape provides a theoretical basis.

The objective of this study was to analyze the proportion of spleen regulatory T cells (Tregs) in chicks after Salmonella infection. In this study, Jing xing yellow chicken H lines were used as experimental groups, and 1-day-old chicks were randomly divided into control group (n=40) and infected group (n=80).Animals were kept in the same and sterile environment. At 7 days of age, the infected group was infected with Salmonella (Salmonella typhimurium CICC 21484) to establish an infection model. The control group was treated by saline instead of Salmonella. After 48 hours of infection, the expression levels of cecal tonsil immune response-related genes and serum inflammatory factors were measured in two groups. Then, two groups of chicks were randomly selected (control group, n=25; infected group, n=34) to detect CD4⁺ T cells and Tregs/CD4⁺T cells ratio of spleens. The results showed that there was no significant difference in the proportion of CD4⁺T cells in the spleens between control gruop and infected group (P>0.05). In infected group and control group, CD4⁺ CD25⁺/CD4⁺T cells (%) were 0.93±0.12 and 3.22±0.59, respectively (P< 0.01); CD4⁺ TGF-β⁺/CD4⁺T cells (%) were 0.55±0.07 and 1.42±0.25 (P<0.01), respectively; CD4⁺CD25⁺ TGF-β⁺/CD4⁺T cells (%) were 0.29±0.04 and 0.76±0.14, respectively (P<0.01), there were significant differences between the above data of the two groups. There was no significant difference in the proportion of CD4⁺T cells in the spleens of chicks after Salmonella infection. The ratio of Tregs/CD4⁺T cells were significantly lower than that in the control group (P<0.01). It is suggested that Tregs may be involved in the pathogenesis of Salmonella infection in chicks, which provides a theoretical basis for further study on the mechanism of chicks against Salmonella infection.

This experiment explored the mutual antagonism of ACE/AngⅡ/AT1R and ACE2/Ang1-7/MasR pathways in the renin angiotensin system (RAS) in rats nonalcoholic simple fatty liver (NAFL). Thirty male Sprague-Dawley rats were randomly divided into normal control group, model group and medication group. In addition to the normal control group, the other two groups were fed with high-fat diet, and each rat of medication group was given 50 mg·(kg·d)^-1 vitastatin. Blood was collected and all rats were killed after 6 weeks,liver samples were taken at the same time. The contents of TG, ALT and AST in serum of each group were determined. The activities of·OH, TNOS, SOD and T-AOC in liver tissue were determined. The release of AngⅡ, Ang1-7 and inflammatory factors in tissue homogenate were determined by ELISA. The levels of ACE, ACE2, AT1R and MasR in liver tissue were analyzed by Western blotting. HE staining was conducted to observe liver pathological changes. Results were as follows:In the model group,the liver index was significantly increased (P<0.05); Serum TG, AST and ALT levels were significantly increased (P<0.01); Liver pathology showed changes in steatosis; Oxidative stress and release of inflammatory factors in liver tissue increased significantly. The expression of ACE, AT1R protein and the amount of AngⅡ and Ang1-7 increased significantly (P<0.05); The protein expression of ACE2 and MasR decreased significantly (P<0.05), and the ratio of ACE/ACE2 increased (P<0.01). The medication group improved the damage of oxidative stress and inflammation, and improved liver damage by down-regulating the ACE/AngⅡ/AT1R pathway. High-fat feeding for 6 weeks can induce simple fatty liver in rats, and both the two pathways in the local RAS system are activated. Abnormal activation of the ACE/AngⅡ/AT1 pathway leads to oxidative stress and inflammatory response in the liver, while the ACE2/Ang1-7/MasR pathway has the opposite effect. It is suggested that endogenous ACE2 produced by the liver may play an important role in the prevention of NAFL by mediating the activation of the Ang1-7/MasR pathway in exogenous stimuli-induced liver injury.

The aim of this study was to investigate the effects of baeSR and acrB gene deletions on the expression of virulence genes associated with Salmonella typhimurium. The differentially expressed genes of ciprofloxacin-resistant strain (CR), acrB gene-deficient strain (CRΔacrB), baeSR and acrB gene double-deletion strain (CRΔbaeSRΔacrB) were screened by using RNA-Seq technique. GO enrichment and KEGG pathway enrichment analysis were carried out on the selected genes to further explore the main functions of differentially expressed genes and the biological processes involved, and 7 genes with significant differences were selected for verification by real-time PCR. The results showed that, a total of 1 320 differentially expressed genes (absolute value of Fold change>2, Q-value<0.005) were screened between CRΔacrB and CR, of which 426 were up-regulated and 894 were down-regulated. There were 1 377 differentially expressed genes (absolute value of Fold change ≥ 2, Q-value<0.005) were screened between CRΔbaeSRΔacrB and CR, of which 405 were up-regulated and 972 were down-regulated. The virulence-related genes screened by the two comparison groups were mainly enriched in pathways such as two-component system, flagellar component, Salmonella infection, and bacterial invasion of epithelial cells. The results of real-time PCR are basically consistent with those of RNA-Seq. This study preliminarily analyzed and explored the role of baeSR and acrB genes in the virulence of Salmonella typhimurium, laying a foundation for further study on the pathogenic mechanism of Salmonella typhimurium.

The present study aims to investigate the antimicrobial resistance,the distribution of efflux pump genes, and the relationship between the distribution of efflux pump genes and the antimicrobial resistance of bovine methicillin-resistant Staphylococcus aureus (MRSA). The antimicrobial resistance and frequency of efflux pump genes of MRSA were detected by the disk diffusion method and PCR, respectively. The results showed that all 79 MRSA isolates were resistant to penicillin, gentamicin and kanamycin, followed by ciprofloxacin (94.9%), levofloxacin (94.9%), clindamycin (87.3%) and tetracycline (79.7%). Moreover, all isolates were sensitive to nitrofurantoin, quinatudine/dafopretin, neostigmine/trimethoprim and linezolid. PCR results showed that all MRSA isolates carried the efflux pump genes norA, norB, norC, sepA, mepA and mdeA, follwed by qacA/B (24.1%). smr was not found in these isolates. Statistical analysis showed that there were significant correlations between the frequency of norA, norB, norC, sepA, mepA, mdeA and qacA/B and resistance phenotype of penicillin, gentamicin, kanamycin, ciprofloxacin, levofloxacin, clindamycin, rifampin and tetracycline (P<0.01), respectively. These results revealed high antimicrobial resistance to common antibiotics, which indicating that antimicrobial susceptibility testing should be performed prior to clinical administration for selecting suitable antimicrobial agents. Finally, the potential threat of high prevalence of efflux pump genes in MRSA should be of concern.

In order to investigate the changes in serum biochemical parameters during the course of acute laminitis in dairy cows, 12 healthy Chinese Holstein cows were used in this study. The experimental group was treated with 17 g·kg^-1 body weight of oligofructose which was dissolved in 20 mL·kg^-1 body weight of water, and the control group was given the same amount of water. Blood samples were collected at several fixed time points from 3 days before administration to 3 days after administration to detect blood glucose, insulin (INS), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (CHOL), leptin (LEP), adiponectin (ADP) and CD36. Results were as follows:After the administration, the cows developed symptoms such as diarrhea, lameness, increased hoof temperature, hyperthyroidism of the finger (toe), and slowing of rumen peristalsis. The blood glucose of the experimental group began to rise significantly at 6 h after administration and lasted untill 18 h, and the INS content decreased significantly at 6-12 and 36-48 h. CHOL showed a significant downward trend from 6 to 72 h after administration. TG and HDL-C showed a significant downward trend at 12 h. LDL-C began to decrease significantly at 36 h compared with the control group. LEP was increased significantly at 12, 18 and 36 h. Blood glucose and INS levels, as well as serum lipid levels, are significant and may be the diagnostic criteria for acute laminitis.

The aim of this study was to analyze the effects of 5-amino-imidazole-4-carboxamide nucleotide (AICAR) on autophagy and differentiation in osteoclasts. Bone marrow macrophages were derived from the tibia and femur of BALB/c mice and induced by 30 ng·mL^-1 M-CSF and 60 ng·mL^-1RANKL for 4 days. After 4 days differentiation, the cells were identified by tartrate resistant acid phophatase (TRAP) staining. The osteoclasts were treated with 0.5 mmol·mL^-1AICAR, AMPK activator for 4 h. Cell area, TRAP⁺ number, roundness and fluorescence intensity were analyzed by High Content Screening (HCS) and cell viability was detected by Cell Counting Kit-8 (CCK-8). Protein and mRNA expression levels of autophagy and differentiation-related proteins were detected by Western blot and qRT-PCR,respectively. After transfection of the EGFP-pmCherry-LC3 plasmid, the change of endogenous LC3 puncta was analyzed by confocal immunohistochemistry. Results from CCK-8 test indicated that the cell viability was not significantly affected by AICAR. The ratio, area and intensity of TRAP⁺cells were significantly lower than those of control group (P<0.05). The results of qRT-PCR showed that AICAR treatment significantly decreased the mRNA transcription level of c-Fos, TRAP (P<0.01) and p62 (P<0.05), and increased LC3, ATG5 mRNA transcription level (P<0.05). Data from Western blot showed that protein expression rate of p-AMPKα/AMPKα was significantly increased (P<0.01), and the protein expression rate of TRAP (P<0.05) were significantly dowm-regulated, and the protein expression rate of NFATc1, c-Fos, CTSK, p62 were extremely significantly down-regulated (P<0.01), while the protein expression level of ATG5, Beclin1, LC3-Ⅱ were extremely significantly increased (P<0.01). The results of EGFP-pmCherry-LC3 plasmid transfection showed that the yellow (EGFP+pmCherry) and red (pmCherry) puncta increased, while the green (EGFP) puncta decreased in osteoclasts after AICAR treatment. All the results suggest that AICAR could inhibit the differentiation of osteoclasts by inducing autophagy via AMPKα activation.

Rabbits were oral administrated with water extract of Artemisia argyi Levl. et Vant. (Artemisia argyi),subsequently the animals were injected with or without inactivated E. coli. After that,an indirect ELISA was used to detect the specific antibody induced by inactivated E. coli, and challenge test was conducted to assess the immune protection effect induced by inactivated E.coil. In addition,the innate immunity of rabbits against E. coli were measured by the methods of ELISA,blood routine,blood biochemistry,limulus amoebocyte lysate test with a chromogenic substrate,and RT-PCR. The results showed that the improved level of specific IgM, IgG (P<0.01), and the survival of rabbits challenged with E. coli were found in the animals administrated with Artemisia argyi for 7 days as compared to the control. More importantly, the supplement of Artemisia argyi alone clearly increased the total IgG, IgA (P<0.05),and the number of neutrophils(P<0.05), platelets(P<0.01) in blood, while along with the reduced viable cells of E. coli (P<0.05), LPS level (P<0.01),AST and ALT (P<0.05) were observed in blood of animals challenged with E. coli. Additionally,it was observed in the drug group that the mRNA expression of cytokines (TLR4, TNF-α, IL-1β, IRF7, IFN-β) which were correlated with the LPS-TLR4 signaling pathway and over-expressed after challenge with E. coli were significantly down-regulated (P<0.01 or P<0.05). Collectively,Artemisia argyi is an excellent immunopotentiator to increase rabbits resistance to E. coli, and has its potential clinical application value.

This experiment aimed to study the relationship between sperm motility and seminal plasma carnitine in Simmental bull. Twenty-six semen samples of Simmental bulls were collected, 12 of which were viewed as abnormal group, and 14 of which were viewed as normal group according to their sperm motility. Ultra-high Performance Liquid Chromatography-Quadrupole-Time-Of-Flight Mass Spectrometry (UPLC-Q-TOF MS) technique was used for non-target determination of samples, the Ultra-high Performance Liquid Chromatography-Quadrupole-Trap Mass Spectrometry (UPLC-Q-Trap MS) technology was used for broad-target lipid determination, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used for statistical analysis. The results of multivariate statistics showed that the metabolic profile of seminal plasma in the abnormal group changed obviously compared with the normal group. The carnitine with significant difference detected by the two methods was analyzed, in the abnormal group, the concentration of decanoyl-L-carnitine, L-palmitoylcarnitine, stearoylcarnitine, free carnitine, acetyl-carnitine, hydroxybutyryl-carnitine, 3-hydroxyoctanoyl carnitine, myristoyl-carnitine and decenoyl-carnitine were significantly higher than those in the normal group (P<0.05). These carnitine concentrations were negatively correlated with the sperm motility of Simmental bulls, which would provide new methods and ideas for studying the determination of bovine semen quality.

This study aims to localize the distribution of steroidogenic acute regulatory (StAR) protein in the brain of small mice, and to preliminarily investigate whether amylin affects the expression of StAR in the brain of small mice. C57BL/6 strain mice were purchased and fed under standard environment for one month. The offspring were fed for seven weeks. Nine male mice with similar weight and health status were selected as experimental animals, they were randomly divided into 3 groups:normal saline control group, amylin injection group and amylin + AC187 (amylin specific inhibitor) injection group. After 3 hours' continuous injection according to body weight (100 μg·kg^-1), brain tissue was dissected. The distribution of StAR in brain was studied by immunofluorescence staining, and the expression of StAR in different groups was compared by Western blot. The results showed that StAR immunopositive signals were mainly expressed in zone incerta (ZI), sporadically distributed in locus coeruleus (LC), ventromedial hypothalamic nucleus (VMH) and dorsal raphe nucleus (DR). Compared with the control group, intraperitoneal injection of amylin significantly reduced the expression of StAR in ZI (P<0.01); and amylin +AC187 significantly inhibited the effect of amylin on StAR expression in ZI (P<0.01). The results showed that intraperitoneal injection of amylin could significantly inhibit the expression of StAR in ZI. StAR is a rate-limiting enzyme that regulating steroid hormones, we speculate that amylin can not only regulate food intake and energy metabolism, but also affect the synthesis rate of steroids in the central nervous system by regulating the expression of StAR.