CRISPR/Cas9 system is an immune mechanism that widely exists in bacteria and archaea. In recent years, it has been used as an efficient gene editing tool to investigate the function of coding or non-coding RNA. Non-coding RNA is a type of RNA that is not translated into proteins, and it plays an important role in biological processes such as animal growth, development and disease immunity through a variety of regulatory pathways. CRISPR/Cas9 technology can be used for targeting nucleotide sequence to get stable knockout mice or cell lines. When it is used in the research of non-coding RNA, it may interfere the expression of neighbor genes or host genes. Even though it has some shortages, this technology provides different ways for exploring the functional mechanism of non-coding RNA. In this review, we briefly summarize the history and functional principle of CRISPR/Cas system, and focus on its application in the functional research of miRNA, lncRNA and circRNA in animals in order to provide references for related research.

Beta-hydroxybutyric acid (BHBA) is one of the main components of ketones. BHBA is a metabolite of butyrate, which is produced by rumen microbial fermentation, in rumen epithelial cells. BHBA plays an important regulatory role in rumen epithelial cells proliferation and metabolism of ruminants. In recent years, most studies on BHBA have focused on liver ketogenesis, dairy cow ketosis and its effect on lactation. Little information is available in the literature regarding to the relationship and inherent mechanisms between BHBA and rumen epithelial cell growth in young ruminants. This review focuses on the formation and transport of BHBA in rumen epithelial cells, as well as the molecular mechanism of BHBA as a signal molecule regulating the metabolism and proliferation of rumen epithelial cells. This is of great significance to enrich the theory on regulation and development of rumen and the nutritional strategy for the rearing of young ruminants.

The purposes of this study were to reveal the genetic evolution of local chicken breeds at the genomic level and to discover genes with important germplasm characteristics. Restriction site-associated DNA sequencing (RAD-seq) technology was employed to identify single nucleotide polymorphism (SNP) markers in 19 indigenous chicken breeds. For each breed, thirty individuals(10 males and 20 females) were selected as experimental materials according to their family records. The genetic diversity and structure were analyzed by calculating the indicators of genetic statistics, which contained observed heterozygosity (Ho), nucleotide diversity (Pi), inbreeding coefficients (Fis), genetic differentiation coefficients (Fst) and gene flows (Nm). The selected genes were also identified through genome selective signals testing. The results showed that 400 562 SNP markers were identified in 19 indigenous chicken populations after data quality control. The PJ and WC populations had the highest genetic diversity, with Ho 0.246 8, 0.243 0, and Pi 0.278 1, 0.265 5, respectively. The DJ population had the lowest Ho and Pi, which were 0.156 0 and 0.157 2,respectively. The Fis in DX and BJ populations were higher than 0.160 0. The Fst among PJ and WC, HX, ZZ, WX, HX and WC, ZZ and CH were all lower than 0.100 0, correspondingly the Nm were all higher than 0.240 0. In terms of single population, the DJ population had higher Fst and lower Nm when compared to other chicken breeds. The phylogenetic tree, constructed using two foreign chicken breeds AK and RW as outgroups, divided the 19 indigenous chicken breeds into 5 clusters, which was consistent with their breeding history and geography distribution. In total, 26 selected areas and 31 candidate genes were identified through the Z transformation of heterozygosity (ZHp) signal testing. These selected genes were widely involved in the biological processes of immune system regulation, reproduction regulation, response to stress and metabolism. In conclusion, the genomic SNP markers have more advantage in revealing genetic diversity and structure of indigenous chicken breeds. The selection effects are mainly reflected in the shaping of local chicken breeds' resistance and disease resistance characteristics, gamete vitality and behavior.

The aim of this study was to analyze the tissue expression pattern and functional SNP of INSL3 gene in sheep. In this study, three each of male and female 1-year-old Small Tail Han sheep with healthy body condition and normal horn were used to detect tissue expression pattern of INSL3 by RT-PCR and real-time PCR. The gene resequencing data of 10 sheep breeds were used to explore the principal component analysis (PCA) and SNP of INSL3 gene region. The qPCR result showed that the expression level of INSL3 gene was the highest in ovary and testis, and INSL3 gene was also expressed in soft horn. The expression of INSL3 gene in testis was significantly higher than that in ovary (P<0.01), and the expression of INSL3 gene in soft horn of ram was significantly higher than that in ewe (P<0.05). PCA results showed that the INSL3 gene region was clustered according to the presence or absence of horn in a certain extent, which further indicated that this region might be related to horn. Functional site analysis revealed that 4 potential SNP sites were significantly different in distribution in horned and hornless populations. SNP1 was at the transcription factor binding site of KDM1A, SNP3 at the ESR1 binding site, and SNP6 at the terminator region. A missense mutation and a synonymous mutation were also found. This study shows that INSL3 gene may be related to the different horn size between male and female sheep as well as presence or absence of the horn, and multiple potential functional sites have been found. This study provides the basis for the future research on the functional mechanism of INSL3.

The aims of this study were to reveal the polymorphism and genetic regularity of the BMPR-IB gene in Hanper sheep population, and to explore the scientificity to use the A → G mutation (FecB mutation) at the 746th base of the BMPR-IB gene as a molecular marker for breeding sheep prolific breed. In this study, a total of 1 267 sheep individuals were selected, blood samples were collected from healthy breeding rams, breeding ewes, and lambs of the Hanper sheep breeding core population. PCR-RFLP method was used to determine the genotypes of BMPR-IB gene locus in sheep population and analyze the population genetics parameters. The statistics of the lambing records of a total of 980 breeding ewes were analyzed, and the effects of FecB mutation, parity and lambing season on litter size were analyzed. Genotype frequency of 167 healthy lambs from the offspring of the designed hybrid combination was counted. The results showed that BMPR-IB gene had BB, B+ and ++ genotypes in Hanper sheep population, the frequencies of the 3 genotypes were 1.97%, 73.40% and 24.63%, respectively, the allele frequencies of B and + were 38.67% and 61.33%, respectively. The litter size of breeding ewes with the BB, B+ and ++ genotypes were 2.69, 1.91 and 1.57, respectively, the average litter size of the current Hanper sheep population was 1.85; One copy of B allele was added to the individual of Hanper sheep by breeding, the litter size was expected to increase by 0.44. The ratio of B+ and ++ genotypes in the offspring of hybrid combination of B+×++ was 1.11:1, and the ratio of BB and B+ genotypes in the offspring of hybrid combination of B+×BB was 0.82:1, both conforming to Mendelian separation law. It was predicted that after 6 generations of breeding, litter size of Hanper sheep ewes could basically achieved 2 lamds. The results of this study provide a theoretical basis for the development of breeding and selection schemes in sheep breeding practice.

The aim of this study was to reveal the biological functions of miR-33a during the differentiation of ovine preadipocytes. In this study, preadipocytes from the back fat of 15-day-old male lambs were used as research material. All the experiments in this study had 3 replicates. Bioinformatics software was used to predict the target genes of miR-33a. The potential target genes were verified by double luciferase reporter system. In order to reveal the expression pattern of miR-33a and its targeting genes during the differentiation of ovine preadipocytes, qPCR and Western blotting were used to detect the expressions of miR-33a, Lipin1, and IRS2 and proteins. In order to elucidate the regulatory mechanism of miR-33a on its target genes, miR-33a was overexpressed or interfered using lentivirus-mediated method. The expressions of Lipin1, IRS2 and adipogenic marker genes in ovine preadipocytes were detected. The lipid droplet deposition ability was measured by Oil Red O staining. The binding sites of miR-33a with 3'-UTR of Lipin1 and IRS2 were found using bioinformatics prediction. miR-33a significantly down-regulated the relative fluorescence activity of Lipin1 and IRS2 wild-type double fluorescent plasmids (P<0.05). The expression levels of miR-33a and Lipin1, IRS2 exhibited opposite trends during ovine preadipocyte differentiation. The overexpression of miR-33a significantly down-regulated the expressions of Lipin1 (P<0.01), IRS2 (P<0.05) and their encoded proteins, and adipogenic marker genes. After the miR-33a was interfered, the expressions of these genes and proteins were significantly up-regulated. Overexpression of miR-33a reduced lipid droplet deposition, and interference of miR-33a promoted lipid droplet deposition. In conclusion, the expressions of miR-33a and Lipin1, IRS2 were negative correlated during ovine preadipocyte differentiation. miR-33a negatively regulates the differentiation of ovine preadipocytes and the lipid droplets deposition by targeting the 3'-UTR of Lipin1 and IRS2.

The aims of this study were to investigate the growth-related genes under selection in Angus cattle and provide a reference for identification of major genes for beef cattle growth. Blood samples of 72 Nanyang cows and 14 Black Angus cows were collected and used to isolate genomic DNA. The genome-wide SNP markers and genotypes of individuals were obtained by using SLAF-seq technology. Fst and π ratio values of each SNP were calculated for selecting the different genomic regions between the two breeds. Afterward, these identified regions were compared with the cattle growth QTLs in animal QTLdb. The overlapping regions were considered as candidate regions for further analysis. Genes within the overlapping regions were screened as candidate genes based on the gene annotation. Tissue expression status of candidate genes were checked in the "Expression Atlas" database. After filtering, 69 762 SNPs were remained for the further analysis. Using the 99% quantiles of Fst and π ratio values as thresholds, 33 genomic regions with high differences between the two breeds were screened. Among them, there were 16 genomic regions overlapped with the cattle growth QTLs. Within the 16 regions, 27 genes were located, among them, 4 genes (FXR1, ADAR, IGF1, MNF1) were related to bone/muscle development and growth regulation. FXR1 and MNF1 genes were highly expressed in the skeletal muscle. ADAR and IGF1 genes were highly expressed in the brain and liver, respectively. IGF1 gene can be considered as a major candidate gene for beef cattle growth, FXR1, ADAR and MNF1 genes are prioritized as potential candidate genes for further verification.

The objective of this study was to investigate the effect of RNF20 and it-mediated monoubiquitination of histone H2B at lysine120 (H2Bub) on brown adipocytes differentiation in mice. In this study, the expression of RNF20 and it-mediated H2Bub level in brown adipose tissue (BAT) from 1-day-old and 2-month-old male C57BL/6 mice were measured by Western blot (n=3). The brown preadipocytes were primarily isolated by collagenase digestion from 1-day-old mice. The brown preadipocytes and C3H10T1/2 cell lines were differentiated into mature adipocytes, respectively. Oil Red O staining was used to detect the adipogenic differentiation efficiency. The expression of RNF20 and it-mediated H2Bub level were detected in non-differentiated and differentiated cells (0 and 8 d) by Western blot. Furthermore, the siRNAs were applied to knockdown the expression of Rnf20 gene in C3H10T1/2 cells and the Oil Red O staining was used to detect adipogenic differentiation efficiency. qPCR and Western blot were used to detect the efficiency of interfering Rnf20 gene and it-mediated H2Bub level. The results showed that the expression of RNF20 and it-mediated H2Bub level were significantly higher in BAT from 2-month-old mice than those from 1-day-old mice. The expression of adipocyte differentiation marker proteins, PPARγ and CEBPα, and RNF20 and H2Bub level significantly increased in differentiated brown adipocytes and C3H10T1/2 cells. In addition, knockdown of Rnf20 gene in C3H10T1/2 cells significantly decreased the expression of RNF20 and it-mediated H2Bub level and reduced lipid droplets deposition in differentiated cells compared to those from negative control group. In summary, RNF20 was essential for mouse brown adipocyte differentiation. Knockdown of Rnf20 gene resulted in the decrease of H2Bub level and decreased the adipogenic differentiation efficiency of C3H10T1/2 cells. The study result adds more data to the epigenetic regulation of mouse brown adipocytes differentiation and provides new genetic material for further understanding the differentiation of animal brown adipocytes.

This study aimed to analyze the effects of age at first pregnancy (AFP) on milk yield and reproductive performance of cows. In this study, production data of 13 927 Holstein dairy cows from two large-scale dairy cow herds (A herd 8 091,B herd 5 836) in northern China were collected. The age at first pregnancy (AFP), milk production, post-partum first estrus time during the first 2 lactations, first 2 mating time and first conception time during the first 2 lactations were detected. The cows were divided into 8 groups according to the AFP(12-19-month-old).The milk yield and main reproductive performance during the first 2 lactation of cows in different groups were compared and analyzed. The results showed that:1) The AFP of Holstein heifers in two large-scale farms were mainly in 13- and 14-month-old (accounting for 70.1% of the total); 2) AFP could significantly affect the 305 d milk production of Holstein dairy cows in the first 2 lactations (P<0.05), the highest 305 d milk production for the first 2 lactation (15 102 kg vs 15 534 kg) was obtained on 14-month-old for AFP in A herd; 3) AFP could significantly affect the post-partum first estrus time and first conception time of Holstein dairy cows in the first 2 lactation (P<0.05), the optimal post-partum first estrus time and first conception time for the first lactation was obtained on 14-month-old for AFP; 4)The post-partum first mating time of cows with AFP at 16-month-old in A farm was significantly higher than that of cows with AFP at 17-month-old for the first lactation (P<0.05), there was no significant difference among the other groups (P ≥ 0.05), and the difference in mating time among the groups was less than 5 days; 5) Based on the analysis of the AFP records of the cattle in the farm (2 703 heads) and the culling cattle (660 heads) in A farm, it was found that the AFP of the cattle in the farm was 14.52-month-old, which is significantly higher than the AFP of the culling cattle (P<0.05). In the northern region, under the current production management level, 14-month-old may be the most suitable age at first pregnancy for Holstein heifers, which has certain reference value for the age at first pregnancy for Holstein heifers in large-scale dairy farms in China.

This study aimed to explore the effect of first cleavage time on the development potential and relative gene expression levels in parthenogenetic embryos of the pig. The oocytes were extracted from the ovary of a healthy sow for maturation in vitro, the pig parthenogenetic embryos were divided into two groups:early cleavage group(16-22 h) and late cleavage group (26-32 h), their cleavage rate and blastocyst rate were statistically compared. The expression levels of pluripotency-related genes Oct4, Sox2, Klf4 and other apoptosis-related genes Bcl-xl, Bax, Caspase-3 were detected and analyzed. The results showed that 50%-60% cleavage in parthenogenetically activated embryos of the pig occured at 16-22 h and less than 20% after 26 h. The embryos blastocyst development rate which completed the first cleavage before 18 h was 79%. Whereas, the embryos which completed the first cleavage after 42 h were not able to develop to the blastocyst stage. The blastocysts development rate in the early cleavage group of parthenogenetically activated pig embryos was significantly higher compared to the late cleavage group (P<0.05), the expression level of Oct4, Nanog, Sox2, and Klf4 genes in the early cleavage group were significantly higher than those in the late cleavage group (P<0.05). The expression level of Oct4, Sox2, Klf4 genes in the early cleavage group were extremely significantly higher than those in the late cleavage group (P<0.01); While the relative expression level of Bax and Caspase-3 genes in the early cleavage group were extremely significantly lower than those in the late cleavage group (P<0.01); However, the expression level of Bcl-xl as a protective factor was significantly up-regulated compared to the late cleavage embryos (26-32 h, P<0.05). The results indicate that the parthenogenetic activation potential of pigs with earlier first cleavage time is significantly higher than that of the late cleavage embryos. The expression of blastocyst pluripotency-related genes is up-regulated and the expression of apoptosis-related genes is down-regulated. The cleavage time as important parameters can be used to identify developmental potential of pig parthenogenetic activated embryos.

In order to clarify BMP15 gene expression characteristics in Rongchang pigs at different developmental stages in relation to their sexual maturity, as well as the relationship between the expression of the BMP15 gene and SMADs signaling pathways genes, ovarian tissues from 1 month, 3 months, and 5 months old Rongchang pigs were collected to analyze different BMP15 gene expression and cellular localization characteristics in ovarian tissues of the Rongchang pigs at different developmental stages using qRT-PCR and paraffin sectioning methods. BMP15 and SMADs signaling pathways related genes SMAD2, SMAD3, SMAD4, SMAD7, TGF-β1, TGF-β2, TGF-β3, TGF-β RⅠ and TGF-β RⅡ expression characteristics were also revealed using qRT-PCR and Western blot analysis by adding recombinant human BMP15 protein and TGF-β receptor inhibitor (LY215799 and LY2109761) to 5-month-old ovarian biopsy. The results showed that from the age of 1 month to 5 months, the mRNA expression of BMP15 gene in ovarian tissues were up-regulated (P<0.05)with the growth and development of Rongchang pigs. Immunofluorescence and paraffin sectioning experiments showed that BMP15 protein fluorescent signals existd in granulosa cells surrounding oocyte in the 5-month-old ovarian tissues. Within the ovarian tissues, from 3-5 months old, BMP15, SMAD4, TGF-β1 and TGF-β RⅡ genes mRNA expressions were raised and SMAD2, TGF-β2 and TGF-β RⅠ expressions reduced (P<0.05). By adding the re combinant human BMP15 protein, TGF-β RⅠ/ Ⅱ and TGF-β RⅠ receptor inhibitor in the age of 5 months old ovarian biopsy, the results showed that TGF-β RⅠ/ Ⅱ receptor inhibitor (LY2109761) significantly inhibited the expression of TGF-β RⅡ proteins and TGF-β RⅠ receptor inhibitor (LY2157299) could not inhibit the expression of TGF-β RⅡ proteins. In conclusion, BMP15 gene expression before sexual maturity in Rongchang pig ovarian tissues were raised and SMAD4, TGF-β1 and TGF-β RⅡ genes expression were also up-regulated. The study illuminated that BMP15 gene mediated SMAD4 signaling molecules and regulated the downstream genes expression through TGF-β R Ⅱ, which provided theoretical basis for the further study of Rongchang pig BMP15 gene roles in the follicles development.

This study was conducted to investigate the effect of different ages on intestinal micro-ecology of the giant pandas. Fresh feces of the giant pandas (Ailuropoda melanoleuca) under different ages[J₁ (sub-adult giant panda), J₂ (adult giant panda) and J₃ (old giant panda)] were collected. The bacterial composition in the feces was determined by 16S rRNA gene technology. The physical and chemical characteristics as well as activities of specific enzymes were analyzed. The correlation between the fecal microbiota and the fecal environmental factors of the giant pandas were analyzed by redundant analysis (RDA). The results showed that the relative abundance of the Proteobacteria increased with age at the phylum level. The relative abundance of Firmicutes decreased with age. The relative abundance of Streptococcus decreased with age at the genus level. The activity of amylase and cellulose were the highest in the feces of the J₂, the activity of amylase was the lowest in the feces of J₃, and the activity of cellulase was the lowest in the feces of J₁. The activity of protease was the highest in the feces of J₁, and the lowest in the feces of J₃. The relative abundance of Firmicutes and Bacteroidetes were positively correlated with amylase activity and reducing sugar content. The relative abundance of Proteobacteria was negatively correlated with reducing sugar content and amino acid content. The study showed that the fecal microbial of the giant pandas of different ages exhibited different characteristics. The relative abundance of dominant bacteria in the feces of the giant pandas was correlated with environmental factors such as fecal digestive enzymes. It is suggested that the diet and living environment of the sub-adult giant pandas should be improved, and probiotics can be added to the diet in order to improve gut health of the old giant pandas.

The aim of this study was to investigate the effects of dietary arginine levels on immune function in broilers and explore the mechanism of arginine anti-fowl adenovirus serotype 4 (FAdV-4). Three hundred one-day-old broilers(Ross 308) were randomly allocated to 5 groups with 5 replicates per group and 12 chickens per replicate. The experimental diets were set to 5 arginine levels of 0.96%, 1.20%, 1.44%, 1.68%, and 1.92%, respectively. The feeding trial lasted for 21 days. Four broilers were randomly selected from each replicate to determine serum immune index and immune relative organ index. Then, six broilers were randomly selected from each replicate and randomly divided into infected group and control group. The chickens in the infected group were individually infected with 0.2 mL of FAdV-4 NP strain (TCID₅₀=10^-6.23/0.01 mL) by intramuscular injection; the chickens of control group were individually injected with the same volume of sterile saline. Two days after infection, mortality rate and disease incidence were calculated. iNOS levels in livers were determined by ELISA. iNOS mRNA expression and viral load in livers were detected by qPCR. The results showed that:1) Three arginine levels of 1.44%, 1.68%, and 1.92% significantly increased spleen index (P<0.05) and arginine level of 1.68% significantly increased thymus index (P<0.05), while arginine level of 0.96% significantly reduced spleen index, thymus index and bursal index (P<0.05). 2) The arginine level of 1.68% significantly increased serum GLO, IgG levels and G/A value (P<0.05). Arginine level of 0.96% significantly decreased serum GLO and IgG levels (P<0.05). 3) The arginine level of 0.96% significantly decreased serum IL-1β, TNF-α, iNOS and NO levels (P<0.05), while the arginine levels of 1.68% and 1.92% significantly increased IL-1β, TNF-α, IFN-γ and iNOS (P<0.05). 4) After broilers infected with FAdV-4, the mortality rate in the 0.96% group was significantly higher than those of 1.20%, 1.44%, 1.68%, and 1.92% groups (P<0.05). 5) ELISA results showed that 1.44%, 1.68%, and 1.92% arginine levels could significantly increase iNOS levels in liver after infection. 6) The arginine levels of 1.44%, 1.68%, and 1.92% significantly increased the expression of iNOS mRNA in livers (P<0.05). After broiler were infected with FAdV-4, the level of iNOS mRNA in the livers were significantly decreased (P<0.05). High levels of arginine (1.44%, 1.68%, and 1.92%) increased the level of iNOS mRNA expression in the livers after broilers infected with FAdV-4 (P<0.05) and significantly reduced the viral load of the livers (P<0.05). It could be seen that deficiency of arginine in the diet could reduce the immune function and anti-FAdV-4 virus ability of the chicken. Increasing arginine levels in broiler diets not only improved immune organ index, serum immune factor levels, and iNOS mRNA expression levels in the livers, but also reduced viral load of livers, thereby improving immune function and antiviral capacity.

The study aimed to evaluate the effect of varying levels of defatted rice bran (DFRB) on carcass traits and meat quality of Suhuai pigs. Thirty-five healthy purebred Suhuai castrated boars with an initial body weight of (62.9±0.8) kg were randomly selected and allotted to 5 dietary groups (7 replicates for each group and 1 pig for each replicate). Pigs were fed by the Osborne Testing Stations System (OTSS). The control group (CTRL) was fed with basic diet without DFRB, and the experimental group Ⅰ to Ⅳ were fed the diet with 7%, 14%, 21%, 28% DFRB. Neutral detergent fiber (NDF) levels of 5 groups were 8.89%, 11.80%, 12.93%, 14.35% and 17.94%, respectively. Except the fiber content of the diet was different, the other nutrients were basically the same among 5 groups. The pre-feeding period was 10 days, and all pigs were fed basic diet; the formal period was 28 days, and pigs were fed basic diet and experimental diets, respectively. At the end of the experiment, all pigs were slaughtered to measure the carcass traits(carcass weight, dressing percentage, carcass straight length, carcass inclined length, loin eye area, average back fat thickness and skin thickness) of Suhuai pigs; meat samples were collected to measure the meat quality traits (drip loss, shear force, cooked meat percentage, intramuscular fat content, pH and meat color); and the longissimus dorsi muscle samples were collected for the expression analysis of phosphorylase kinase gamma 1(PHKG1), a major gene of drip loss in pigs. The results showed that:1) The substitution of DFRB had no effect on the carcass traits such as carcass weight, dressing percentage, carcass straight length, inclined length, loin eye area, average back fat thickness and skin thickness. 2) With the increase of DFRB level, the drip loss of Suhuai pork decreased first and then increased (quadratic, P<0.05), and the shear force of pork decreased linearly (P<0.05); the cooked meat percentage and pH_{24 h} intended to increase linearly with the increase of DFRB level (P=0.061, P=0.068); there was a tendency for a decrease in L_{24 h}^* as DFRB increased (linear, P=0.085). There was no significant difference in other meat quality traits among 5 groups. 3) The relative expression level of PHKG1 gene was not affected by the increase of DFRB level (quadratic, P=0.085). In conclusion, dietary DFRB fiber level had no effect on carcass traits of Suhuai fattening pigs. Adding proper level of DFRB to the diet could reduce the drip loss and shear force and improve the quality of Suhuai pork, but the mechanism behind it needs further study.

The aim of this study was to determine the effect of the replication level of Newcastle disease virus (NDV) on its oncolytic effect and to improve the research model for studying oncolytic mechanism of NDV. The human tumor cell line HCT116, A549 and the human non-tumor cell line HEK293T were infected with the NDV vaccine strain LaSota; the replication levels of viral genome in above mentioned cell lines were detected by NDV specific RT-qPCR; the expression levels of viral protein in above mentioned cell lines were assayed by Western blot; the effects of NDV infection on cell cycle and apoptosis of above mentioned cell lines were investigated using flow cytometry. The results revealed similar levels of viral replication but distinct oncolytic effects of NDV among these three cell lines; further flow cytometry showed that NDV infection induced G1 phase arrest in HEK293T and HCT116 but not in A549; in addition, NDV mainly induced early apoptosis in A549 but necrosis/late apoptosis in HCT116. The oncolytic effect of NDV does not depend on its replication levels, and NDV kills human tumor cells by cell type-specific mechanisms.

Pigs are regarded as a "mixing vessel" of influenza viruses, who could reassort with heterologous strains. They pose a potential threat to human health, due to the existence of the human-type (SA α-2,6-Gal) and the avian-type (SA α-2,3-Gal) receptors on the respiratory epithelial cells. Our previous study showed that two novel reassortants H3N2 swine influenza viruses(SIVs) with 2009 pandemic H1N1 internal genes were isolated in 2013 and 2014 from a large-scale pig farm of Nanning. To understand the genetic evolution of SIVs in the same pig farm, the consecutive surveillance was performed from 2018 to 2019. Two triple-reassortant H3N2 SIVs named as A/swine/Guangxi/JG13/2019 (JG13/2019) and A/swine/Guangxi/JG20/2019 (JG20/2019) were isolated once more in the pig farm mentioned above in 2019. The analysis of phylogenetic tree demonstrated that their reassortant form were similar to previous strains, A/swine/Guangxi/JGB4/2013 (JGB4/2013) and A/swine/Guangxi/JG1/2014 (JG1/2014), containing the surface genes HA and NA derived from the human-like H3N2 lineage, the internal genes NP, M, PA, PB1 and PB2 derived from the pdm09/H1N1 lineage and the NS gene derived from the classical swine H1N1 lineage. Besides, the homologies of HA and NA genes between the new isolates and previous isolates were 95.3%-97.4% and 93.9%-97.0%, respectively at the nucleotide level. The internal genes (NP, M, PA, PB1 and PB2) were 96.2%-98.1%, NS genes were 97.1%-97.6%. It was found that the protein HA still remained 190D, 226I and 228S which bind to human receptor. However, there were still two mutations at the position 223 (V→I) and 227 (P→S). Furthermore, there were two sites different from previous strains on PA protein and PB2 protein, which were R→K at the position 356 and I→T at the position 588. Whether these changes could affect the pathogenicity, replication and the ability of cross-species transmission needs to be further studied in future. After six years, H3N2 SIVs carrying internal gene fragments (PB2, PB1, PA, M and NP) of pdm/09 H1N1 and human-like surface genes (HA and NA) were still epidemic in pigs from the same farm. Although gene mutations have occurred in key functional domains, they still maintained the receptor binding characteristics to infect human. Therefore, the strength of surveillance for SIVs will provide the pre-alarm for the possible outbreak of human pandemic.

To study the epidemic situation of pseudorabies (PR) and genetic variation of epidemic strains of pseudorabies virus(PRV)in Guangxi from 2013 to 2018, 714 target tissues of suspected PR-infected pigs in various regions of Guangxi were collected and tested by PCR and virus isolation. gB, gE and TK genes of isolated strains were amplified, cloned, sequenced and analyzed by DNAStar and MEGA 6.0. The results indicated that the positive rate of suspected tissues was 24.5% (175/714), and 38 PRV strains were isolated. PRV infection of Pig is common in the tested herds. There co-existed classical strains and the variant strains, and the mutant strains are dominant. gB, gE and TK genes of different subtypes of isolated strains had the same amino acid variation characteristics and had closer relationship with domestic mutant strains. It is speculated that there were two different recombinant forms of PRV strains in Guangxi. The first was recombinant by vaccine strains (gB gene) and mutant strains (gE gene). The other one, classical strain (TK gene) was replaced by vaccine strains (TK gene). In recent years, PRV infection was common in the pig farms in Guangxi, and the epidemic strains were mainly mutant strains. And there may be two PRV recombinant forms of recombinant variant strains. Pig farms should pay more attention to practice PR targeted immunity plan.

The aim of this study was to clarify genomic characteristics, homology, and pathogenicity of Senecavirus A (strain FJLY). Strain SVA-FJLY isolated from a farm in Fujian Province was used as focus of study. Five pairs of primers were designed based on whole-genome sequence of SVV-001 (GenBank No. DQ641257.1) and whole-genome sequence of strain SVA-FJLY was obtained and analyzed by RT-PCR, sequencing, and splicing. Piglets aged 3-4 weeks old were also infected with this strain by intranasal spray. Results showed that genome of strain SVA-FJLY had a full length of 7 275 bp (excluding PolyA). It encoded a polyprotein, VP1 protein, and VP2 protein. Strain SVA-FJLY had high homology with reference strain and HeB01-2017 strain (MF967574.1), with highest homology reaching 98.5%, while it had low homology with SVA prototype strain SVV-001 (DQ641257.1), reaching 93.9%. Strain SVA-FJLY belonged to the same branch as several Senecavirus A strains isolated in China, and domestically had closest relationship with strain HeB01-2017, while regarding foreign strains it had closest relationship with Colombia-2016 (KX857728.1). Animal experiments showed that two of three animals in experimental group suffered from the disease, while all three animals in control group remained normal. All six piglets survived 10 days after challenge of experimental group. Whole-genome sequence of strain SVA-FJLY was obtained by genome sequencing. Genomic characteristics of strain SVA-FJLY and its homology with other strains were clarified. Pathogenicity of strain SVA-FJLY was also revealed. Moreover, a reference for reverse genetics and vaccine development for SVA was provided, and database with genome information on SVA was also enriched.

This study was conducted to investigate the role of outer membrane protein W (OmpW)of Gallibacterium anatis(G. anatis)in osmotic stress resistance. The survival rate and growth of wild type G. anatis RZ(WT) and its ompW deletion mutant ΔompW under different NaCl concentrations were determined. The expression of outer membrane proteins of WT and ΔompW under different NaCl concentrations were analyzed by SDS-PAGE and Western blot. Furthermore, transcription of OmpW mRNA was determined by real time PCR. In this study, we found that deletion of ompW result in reduced G. anatis growth in hypersaline culture conditions. Moreover, compared with survival rate of the WT strain, the survival rate of the ΔompW was reduced significantly in BHI with 3.0% NaCl(95.17% vs 83.50%, P<0.05). Compared with normal NaCl concentration (0.5%), transcription (1.19×) and expression of OmpW gene were up-regulated in cultures containing high NaCl concentrations. This study demonstrated that OmpW is involved in osmotic stress resistance of G. anatis.

There are two objectives of the study. First is to explore the relationship between HP-porcine reproductive and respiratory syndrome virus (HP-PRRSV) and pathological changes of pulmonary microthrombus, the other is to clarify the effect of endothelial nitric oxide synthase (eNOS) expression on thrombosis. Fifty 70-day-old healthy piglets were employed, control group consisted of four piglets and 46 piglets were infected with HP-PRRSV. Lungs were collected, HE technique was then applied to observe histopathological changes and to evaluate thrombus grade. Mild thrombus group, moderate thrombus group and severe thrombus group were assigned based on state of their thromboses according to the statistics, four piglets in each group were selected for follow-up study. Immunohistochemistry, Western blot, real-time PCR were employed in examining the distribution, expression and changes of HP-PRRSV and eNOS in the lungs of piglets. The results of histopathological studies showed that the alveolar space of the lung tissue was significantly widened, different degree of blood stasis and bleeding in blood capillary occurred, alveolar epithelial cells, macrophages peeled off in the alveolar cavity and amounts of microthrombus in the alveolar wall were also observed. HP-PRRSV was found mainly located in cytoplasm of alveolar macrophages and fewer in lymphocytes. The degree of thrombosis mounted associated with the increase of viral content and then lead to a higher mortality rate. In addition, eNOS was found mainly located in the cytoplasm of alveolar type Ⅱ cells, macrophages, vascular endothelial cells and bronchial smooth muscle cells. The expression of eNOS mRNA and protein in infected pig lungs was significantly lower compared with controls but there was no obvious difference between the three infected groups. In summary, HP-PRRSV develops the mortality of infected pigs by reducing the expression of eNOS and participated in the formation of microthrombosis.

The study aimed to investigate the effects of copper sulfate solution continuous induction on the antibiotic resistance ability and the expression of related resistance genes of methicillin-resistant Staphylococcus aureus(MRSA) isolates. The minimum inhibitory concentrations(MICs) of MRSA strains (MR-YB1224, MR-YS3 and MR-P318) to copper sulfate, β-lactam(including ampicillin, oxacillin and cefoxitin), fluoroquinolones(including ofloxacin), macrolides(including roxithromycin) and aminoglycosides(including streptomycin) were determined using the standard broth microdilution method. A quantitative real time-PCR was applied to monitor relative expression levels of anti-heavy metal genes (copA, arsB) and antibiotic resistance genes (mecA, norA, ermB, aac6'/aph2″). After continuous induction by copper sulfate solution for 7 days, the MIC values to copper sulfate solution increased for three MRSA strains; especially the MIC values to β-lactam antibiotics significantly increased after induction (P<0.05). The change of MIC values to the different antibiotics were as follows:oxacillin > ampicillin > cefoxitin > ofloxacin > streptomycin > roxithromycin. In addition, the expression of the anti-heavy metal genes and related antibiotic resistance genes for the three MRSA strains were significantly up-regulated (P<0.05). The results show that copper sulfate solution has a strong synergistic effect on the antibiotic resistance of MRSA strains.

The aim of this study was to investigate the effect of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene on milk fat synthesis and fatty acid composition in goat mammary gland epithelial cells by RNA interference. The complete CDS region of PTEN gene (GenBank accession number:MK158074.1) was amplified from the mammary gland tissues of Xinong Saanen dairy goat by RT-PCR. Sequence analysis and expression analysis of different lactation stages were performed. Then the siRNA targeting the PTEN gene was synthesized and screened by RT-qPCR to further detect PTEN interference on the transcription level of lipid synthesis-related genes and fatty acid composition. Results showed that:the CDS region of Capra hircus PTEN gene was cloned for the first time, with a total length of 1 212 bp. Compared with cattle (Bos Taurus), porcine (Sus scrofa) and human (Homo sapiens), the nucleic acid sequence homology of dairy goat PTEN was 99%, 98% and 97%,and the amino acid sequence homology was all reach up to 99%. The protein structure analysis showed that there was no transmembrane structure in PTEN protein. Subcellular localization analysis showed PTEN located in the cytoplasm. N terminal of PTEN protein has a phosphatase catalytic domain. The transcription of PTEN gene decreased dramatically by 51.5% in peak lactation compared with dry period. By transfecting the synthesized siRNA into mammary epithelial cells,the ideal siRNA was successfully screened of which the interference efficiency reached 94% (P<0.01). Compared with the control group,the mRNA transcriptions of SREBP1, FASN, ACACA and SCD1 were significantly up-regulated with PTEN interference (P<0.01 or P<0.05),and the mRNA transcriptions of LPL, FABP3, ACOX1, CPT1B, GPAM, DGAT2 and HSL were significantly down-regulated (P<0.01 or P<0.05). Fatty acid analysis showed that PTEN interference significantly up-regulated the desaturation index of C16:1 (P<0.05), but didn't affect C18:1. In summary, PTEN can regulate the transcription of lipid synthesis-related genes and fatty acid composition in mammary epithelial cells,which play an important role in milk fat metabolism in dairy goat.

The published clinical trial about treatment of weaned piglet stage with Chinese herbal medicine were systemically evaluated by meta-analysis method, in order to provide valuable reference for treatment of weaning piglet health care. The clinical randomised controlled trials (RCTs) about treatment were searched completely from databases of Science Direct, Web of Science, CNKI, Wanfang Database, VIP Database, etc, using the Review Manager 5.3 software recommended by the Cochrane Collaboration. A total of 27 RCTs and 1 870 weaned piglets were included, among them, 935 weaned piglets in treatment group treated with Chinese herbal medicine, 935 weaned piglets in control group treated with chlortetracycline, colistin sulfate, colistin sulfate + bacitracin zinc. Results showed that:the daily weight gain effect of Chinese medicine group was better than the overall antibiotic group[SMD=1.22, 95%CI(0.69-1.75), P<0.01], but there is no difference with the chlortetracycline group(P>0.05). The daily feed intake of Chinese medicine group was better than the overall antibiotic group[SMD=0.77, 95%CI (0.34-1.20), P<0.01], but equivalent to the chlortetracycline group (P=0.05).The difference in Feed weight ratio between the Chinese medicine group and the antibiotic group was not significant[SMD=-0.35, 95%CI(-0.78-0.09), P=0.12>0.05], However, it was significantly better than the colistin sulfate and chlortetracycline subgroup (P<0.01).The effect of Chinese medicine on diarrhea rate was significantly lower than antibiotics{SMD=-1.77, 95%CI[-2.52-(-1.01)], P<0.01}, However, the effect was comparable to that of the colistin sulfate + bacitracin group (P>0.05). The Chinese herbal medicine was added at the weaning stage of the piglets. The indexes of daily gain, daily feed intake and diarrhea rate were superior to that of the antibiotic group, that is, the traditional Chinese medicine has the potential of traditional Chinese medicine in the stage of weaning piglets.

The present study was conducted to investigate the effects of aqueous extract of Portulaca oleracea L. (AEP) on the structure and function of jejunum in mouse under the heat stress conditions. A total of 40 Kunming mice were randomly allocated to 4 groups:control group (C), heat stress group (HS), aqueous extract of Portulaca oleracea L. (AEP), and vitamin C group (Vc), heat stress (in groupHS, AEP &Vc) was conducted by exposing mice to (40±1)℃ for 0.5 h, and after 6 days of continuous heat stress, the mice were transferred to room temperature for treatment. Seven days after the administration, blood samples were collected from the orbital sinus and the jejunum of the mice was collected. The contents of D-xylose and glucose in the serum of mice were measured; the pathological changes of jejunum were observed by HE staining; qRT-PCR was used to detect the relative transcription levels of ZO-1, SGLT1 and GLUT2 mRNA in the jejunal mucosa. The results showed that compared with group C, the jejunal mucosal villi height of the HS group mice was significantly reduced (P<0.01); the level of D-xylose in the serum were significantly reduced (P<0.01); the relative transcription levels of ZO-1, SGLT1 and GLUT2 mRNA in the jejunal mucosa were significantly reduced (P<0.01); compared with the HS group mice, the jejunum villi of the AEP group mice were significantly increased (P<0.01); the levels of D-xylose and glucose in serum were significantly increased (P <0.01 or P<0.05); the relative transcription levels of ZO-1, SGLT1 and GLUT2 mRNA in the jejunal mucosa were significantly increased (P<0.05 or P<0.01). High temperature treatment can induce the jejunum mucosa to fall off and decrease the absorption function. After giving purslane extract treatment, the jejunum mucosa structure can be repaired by increasing the jejunum ZO-1 mRNA transcription, and absorption functioncan be improved by promoting the jejunum mucosa SGLT1 and GLUT2 mRNA transcription. AEP can repair the jejunum mucosa damage caused by heat stress to a certain level, and improve the intestinal absorption capacity of heat stress mice.

The aim of this study was to investigate the relationship between the content of swainsonine and the mRNA expression of catalytic enzyme gene related to the biosynthesis of swainsonine in the fermentation broth of Metarhizium anisopliae. Metarhizium anisopliae was inoculated and fermented in Czapek-Dox Medium, and then we determined the swainsonine content of fermentation broth by using Q Exactive high-resolution mass spectrometer and detected the expression of mRNA of catalytic enzyme genes, namely SwnA, SwnH₁, SwnH₂, SwnK, SwnN, SwnR, and SwnT by qRT-PCR method on the 1st, 3rd, 5th and 7th day of fermentation. The results showed that the regression equation calculated from the changes in the mass concentration and peak area of swainsonine was y=31 302.5x-45 910.5 (R²=0.996 7). The highest content of swainsonine in the fermentation broth on the 3rd day was 174.014 μg·mg^-1, the mRNA expression of SwnA, SwnH₁, SwnH₂, SwnK, SwnN, SwnR, and SwnT genes have great difference in different fermentation periods. Among them, the SwnR mRNA expression was consistent with the change of swainsonine content in the fermentation broth, and the SwnH2mRNA expression was opposite to the change of swainsonine content in the fermentation broth. The results indicated that the SwnR mRNA expression was closely related to synthesis of swainsonine in Metarhizium anisopliae, which would provide an important reference for exploring fermentation process and biosynthetic pathway of swainsonine in future.

Bovine kobuvirus (BKoV) is a potential diarrhea-causing pathogen in calves, and there is no real-time RT-PCR assay for detecting this virus until now. By designing primers targeted to 3D fragment of BKoV strains and optimizing reaction conditions and system, a TB Green real-time RT-PCR assay was successfully developed. The results demonstrated that the assay had a good linear relationship between 4.86×10⁸ and 4.86×10² copies·μL^-1 of virus concentration, the linear correlation coefficient was 0.999 5 and the amplification efficiency was 92.75%. This assay was only specific for detection of BKoV, no cross reaction was observed with other species of diarrhea-causing pathogens; the minimum detection limit was 4.86×10² copies·μL^-1; both the intra- and inter-group coefficients of variation were <2%. Comparing to other three reported RT-PCR assays, the assay had a significant high detection rate of BKoV for clinical feces samples from both diarrheal cows and yak. Total 131 yak diarrhea samples collected from Qinghai, Xizang and Sichuan region between May 2018 and May 2019, the BKoV detection rate was 31.30%. The real-time RT-PCR assay for detecting BKoV developed in this study has a good specificity and stability, which provide an effective method for detection and epidemiological investigation of BKoV.

In order to screen the target genes regulated by Salmonella typhimurium small RNA GcvB, this study predicted genes that can interact with GcvB R1, R2 and R3 regions by TargetRNA2 software based on the transcriptome sequencing results of Salmonella typhimurium gcvB knockout strain, annotated the KEGG database and GO enrichment analysis and validated the predicted genes using real-time PCR. The predicted results of Target RNA2 showed that the narY, yeaK, trpB and STM2732 genes with the highest hybridization energy were able to produce at least 7 consecutive base complementary to the small RNA GcvB. NarY, YeaK and TrpB were respectively related to anaerobic respiration, tRNA that recognizes proline, and synthesis of tryptophan. The results of real-time PCR showed that the transcript levels of narY, yeaK, trpB and STM2732 were up-regulated by 3.4, 9.8, 12.1 and 18.37 times, respectively, under gcvB gene knockout conditions. These results indicated that the narY, yeaK, trpB and STM2732 genes were negatively regulated by small RNA GcvB and may be directly negatively regulated. This study laid the foundation for further understanding of the interaction between small RNA GcvB and target genes, the regulation mechanism of small RNA, and the pathogenic mechanism of Salmonella.