Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases in the world. It has been more than twenty years since the first report in China. The objective of this paper is to analyse the prevalent history and current status through a perspective of epidemiology, aiding to the prevention of PRRS virus (PRRSV). Both genotype 1 and genotype 2 PRRSV are prevalent in China. For genotype 2, majority of epidemic strains in the fields were clustered into four lineages (lineage 8, lineage 5, lineage 3 and lineage 1), among which lineage 8 (highly pathogenic PRRSV-like) and lineage 1 (NADC30-like) are dominant viruses. The genome homology between BJ-4 and VR2332 is 99.6%, which suggested that BJ-4 was probably imported from North America region. For genotype 1, all the isolated strains in China belong to subtype 1 and were detected in more than 9 provinces/regions. The Chinese isolated strains have highly conserved amino acid mutation sites on the GP5 and N protein compared with Lelystad virus.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases in the world. It has been more than twenty years since the first report in China. The objective of this paper is to analyse the prevalent history and current status through a perspective of epidemiology, aiding to the prevention of PRRS virus (PRRSV). Both genotype 1 and genotype 2 PRRSV are prevalent in China. For genotype 2, majority of epidemic strains in the fields were clustered into four lineages (lineage 8, lineage 5, lineage 3 and lineage 1), among which lineage 8 (highly pathogenic PRRSV-like) and lineage 1 (NADC30-like) are dominant viruses. The genome homology between BJ-4 and VR2332 is 99.6%, which suggested that BJ-4 was probably imported from North America region. For genotype 1, all the isolated strains in China belong to subtype 1 and were detected in more than 9 provinces/regions. The Chinese isolated strains have highly conserved amino acid mutation sites on the GP5 and N protein compared with Lelystad virus.

Due to the increasing severity of bacterial drug resistance, the demand for novel antimicrobial agents has been growing in recent years. The multi-target hybrid which combines two kinds of drugs by a certain way provides the possibility for expanding the antibacterial spectrum, enhancing the activity against drug-resistant bacteria and reducing the frequency of bacterial resistance, and has become a new strategy to the research and development of novel antimicrobial agents. Meanwhile, oxazolidinones and quinolones with their unique mechanism of action and the complementary antibacterial spectrum attract the attention of researchers. Oxazolidinone-quinolone hybrids incorporating oxazolidinone and quinolone pharmacophores have been synthesized and shown to be active against a variety of susceptible and resistant Gram-positive and Gram-negative bacteria. In the present paper, the design, synthesis and antimicrobial activities of oxazolidinone-quinolone hybrids, and the research progress of cadazolid, a oxazolidinone-quinolone hybrid which is currently at the stage of clinical trials, are reviewed.

Due to the increasing severity of bacterial drug resistance, the demand for novel antimicrobial agents has been growing in recent years. The multi-target hybrid which combines two kinds of drugs by a certain way provides the possibility for expanding the antibacterial spectrum, enhancing the activity against drug-resistant bacteria and reducing the frequency of bacterial resistance, and has become a new strategy to the research and development of novel antimicrobial agents. Meanwhile, oxazolidinones and quinolones with their unique mechanism of action and the complementary antibacterial spectrum attract the attention of researchers. Oxazolidinone-quinolone hybrids incorporating oxazolidinone and quinolone pharmacophores have been synthesized and shown to be active against a variety of susceptible and resistant Gram-positive and Gram-negative bacteria. In the present paper, the design, synthesis and antimicrobial activities of oxazolidinone-quinolone hybrids, and the research progress of cadazolid, a oxazolidinone-quinolone hybrid which is currently at the stage of clinical trials, are reviewed.

Seasonal reproduction is a common adaptive strategy that allows for breeding at times when it is most advantageous for survival and growth of offspring, a major reason for seasonal reproduction is a striking change in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the negative feedback effects of estradiol. In recent years, the neural and neuroendocrine circuitry responsible for sheep seasonal reproduction has been primarily studied, which concerned with the melatonin action and changes of the relevant neuron activities in hypothalamus. In this review, we first described the afferent signals, neural circuitry for seasonal reproductive transitions in sheep, and then compared these mechanisms with those discovered from studies in hamsters. These results can provide reference for the study of neural mechanisms controlling seasonal reproduction in sheep.

Seasonal reproduction is a common adaptive strategy that allows for breeding at times when it is most advantageous for survival and growth of offspring, a major reason for seasonal reproduction is a striking change in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the negative feedback effects of estradiol. In recent years, the neural and neuroendocrine circuitry responsible for sheep seasonal reproduction has been primarily studied, which concerned with the melatonin action and changes of the relevant neuron activities in hypothalamus. In this review, we first described the afferent signals, neural circuitry for seasonal reproductive transitions in sheep, and then compared these mechanisms with those discovered from studies in hamsters. These results can provide reference for the study of neural mechanisms controlling seasonal reproduction in sheep.

In order to test the cross performance of Berkshire and Licha black pigs and pick out the valuable cross combination, seven cross combinations were designed in this study, namely Berkshire×Luchuan (BLu, 2 224 heads), Berkshire×Licha black pig (BL, 18 555 heads), Berkshire×Yushan black pig (BY, 660 heads), Berkshire×Weizhu (BW, 366 heads), Licha black pig×Luchuan pig (LL, 2 689 heads), Licha black pig×Yushan black pig (LY, 706 heads) and Duroc×Licha black pig (DL, 1 026 heads), and 16 traits containing growth, fattening, body size and reproductive performance were used to compare pros and cons of the 7 cross combinations. The performance test results showed that DL's overall performance was the best. All of its recorded traits were excellent except litter size and hip width, which were at the ordinary level. BL's overall performance was in the second place:except litter size, feed conversion ratio in fattening period and backfat thickness at 100 kg were not as good as DL. Most recorded traits of the remaining 5 cross combinations were ordinary or bad, so they were not suitable to be commercialized. In order to further understand the productive performances of DL and BL, DL and BL were slaughtered to test their meat production performances. Except marbling score and intramuscular fat content, all carcass and meat quality traits of BL were better than their counterparts of DL. In particular, the thoracic vertebrae number of BL was 1.33 more than that of DL (P=0.003). For the hog, besides of the production performance, body appearances were also very important, especially the coat color. All BL pigs were black, while coat color segregation existed in DL. Most of DL pigs were black, but some of them were covered with some other coat colors. In general, our combining ability test results indicate that the overall performances of BL and DL are better than that of the rest 5 cross combinations, the market prospect of BL is better than that of DL.

In order to test the cross performance of Berkshire and Licha black pigs and pick out the valuable cross combination, seven cross combinations were designed in this study, namely Berkshire×Luchuan (BLu, 2 224 heads), Berkshire×Licha black pig (BL, 18 555 heads), Berkshire×Yushan black pig (BY, 660 heads), Berkshire×Weizhu (BW, 366 heads), Licha black pig×Luchuan pig (LL, 2 689 heads), Licha black pig×Yushan black pig (LY, 706 heads) and Duroc×Licha black pig (DL, 1 026 heads), and 16 traits containing growth, fattening, body size and reproductive performance were used to compare pros and cons of the 7 cross combinations. The performance test results showed that DL's overall performance was the best. All of its recorded traits were excellent except litter size and hip width, which were at the ordinary level. BL's overall performance was in the second place:except litter size, feed conversion ratio in fattening period and backfat thickness at 100 kg were not as good as DL. Most recorded traits of the remaining 5 cross combinations were ordinary or bad, so they were not suitable to be commercialized. In order to further understand the productive performances of DL and BL, DL and BL were slaughtered to test their meat production performances. Except marbling score and intramuscular fat content, all carcass and meat quality traits of BL were better than their counterparts of DL. In particular, the thoracic vertebrae number of BL was 1.33 more than that of DL (P=0.003). For the hog, besides of the production performance, body appearances were also very important, especially the coat color. All BL pigs were black, while coat color segregation existed in DL. Most of DL pigs were black, but some of them were covered with some other coat colors. In general, our combining ability test results indicate that the overall performances of BL and DL are better than that of the rest 5 cross combinations, the market prospect of BL is better than that of DL.

The aims of this study were to screen differentially expressed genes of skin tissue at different positions(dorsal and leg) of chicken, and investigate the molecular mechanism of phenotypic differences. Commercial broiler chickens (63 days old) were selected as materials, Agilent cDNA microarray analyses were conducted to determine gene expression profiles of dorsal and leg skin tissues with significant differences in skin thickness, hair follicle density and diameter. A systematic identification of candidate genes and signaling pathways associated with skin follicle initiation and development were conducted, and qRT-PCR assays were used to validate the microarray hybridization results. A total of 676 genes were screened (|FC| ≥ 2,P<0.05), compared with the leg skin, among them, 223 were up-regulated expression genes and 453 were down-regulated expression genes in dorsal skin. Real-time PCR results were consistent with microarray results in the expression trend of partial differentially expressed genes (DEGs). GO analysis showed that the DEGs were involved in the regulation of cell proliferation and apoptosis, Wnt signaling pathway, neuronal differentiation, regulation of cell-cell adhesion, cell fate commitment and other biological processes. KEGG pathway analysis revealed that, in addition to partly known signaling pathways associated with skin follicle development(Wnt and Hedgehog signaling pathways), ECM-receptor interaction, Focal adhesion, Cell adhesion molecules (CAMs), Adherens junction signaling pathways were also enriched. Protein interaction network analysis showed that the DEGs genes interacted with each other to form a regulatory network that may affect the skin follicle traits by affecting the growth and development of the skin follicle and the periodic variation. ECM-receptor interaction, Focal adhesion, transporter pathways and DKK1, WNT3A, SHH, FGF1 and IGF1 genes may be important regulatory pathways and candidate genes involved in the differences of skin follicle traits in different positions of the chicken skin.

The aims of this study were to screen differentially expressed genes of skin tissue at different positions(dorsal and leg) of chicken, and investigate the molecular mechanism of phenotypic differences. Commercial broiler chickens (63 days old) were selected as materials, Agilent cDNA microarray analyses were conducted to determine gene expression profiles of dorsal and leg skin tissues with significant differences in skin thickness, hair follicle density and diameter. A systematic identification of candidate genes and signaling pathways associated with skin follicle initiation and development were conducted, and qRT-PCR assays were used to validate the microarray hybridization results. A total of 676 genes were screened (|FC| ≥ 2,P<0.05), compared with the leg skin, among them, 223 were up-regulated expression genes and 453 were down-regulated expression genes in dorsal skin. Real-time PCR results were consistent with microarray results in the expression trend of partial differentially expressed genes (DEGs). GO analysis showed that the DEGs were involved in the regulation of cell proliferation and apoptosis, Wnt signaling pathway, neuronal differentiation, regulation of cell-cell adhesion, cell fate commitment and other biological processes. KEGG pathway analysis revealed that, in addition to partly known signaling pathways associated with skin follicle development(Wnt and Hedgehog signaling pathways), ECM-receptor interaction, Focal adhesion, Cell adhesion molecules (CAMs), Adherens junction signaling pathways were also enriched. Protein interaction network analysis showed that the DEGs genes interacted with each other to form a regulatory network that may affect the skin follicle traits by affecting the growth and development of the skin follicle and the periodic variation. ECM-receptor interaction, Focal adhesion, transporter pathways and DKK1, WNT3A, SHH, FGF1 and IGF1 genes may be important regulatory pathways and candidate genes involved in the differences of skin follicle traits in different positions of the chicken skin.

The purpose of this study was to explore the regulation mechanism of MSTN gene on the proliferation and differentiation of sheep myoblasts and further reveal the biological function of MSTN. The expression of endogenous MSTN gene of sheep myoblasts was interfered by recombinant adenovirus-mediated shRNA. CCK-8 assay was used to detect the proliferation of myoblasts, and flow cytometry analysis was used to detect cell cycle of myoblasts. The qRT-PCR was used to measure the mRNA expression of p21 in myoblasts. Immunocytochemistry was used to detect myotubes formation and the cell fusion rate was counted, qRT-PCR was used to measure the mRNA expression of the MyoD,MyoG,Myf5, Myf6 and HACD1 genes at 48, 72, 96 h after myoblast induced to differentiation. The results showed that the proliferation of myoblasts was inhibited after downregulating MSTN expression, the cell cycle was arrested at G0/G1 phase, and the expression of cell cycle inhibitor p21 increased. In the study on myoblast differentiation, we found the expression of 4 myogenic regulatory factors after inducing for 48 h were decreased, and the expression of MyoG gene decreased significantly(P<0.05);the myoblast fusion rate of interference group was significantly higher than that of negative control group(P<0.01) after inducing for 72 h, the expression of Myf5 and Myf6 genes decreased significantly (P<0.01), the expression of MyoD gene didn't increase significantly (P>0.05), the expression of MyoG gene increased significantly(P<0.01);the expression of Myf5(P<0.05), MyoD(P>0.05) and MyoG(P<0.01) decreased after inducing for 96 h, and the expression of Myf6 gene didn't increase significantly(P>0.05). In addition, the expression of HACD1 gene was significantly higher in the interference group than that in the negative control group (P<0.05) after inducing for 72 h, and no significant changes were detected after inducing for 48 or 96 h. The results show that the MSTN gene interfered could inhibit the proliferation while promote differentiation of myoblasts, and HACD1 gene is related to myoblast fusion.

The purpose of this study was to explore the regulation mechanism of MSTN gene on the proliferation and differentiation of sheep myoblasts and further reveal the biological function of MSTN. The expression of endogenous MSTN gene of sheep myoblasts was interfered by recombinant adenovirus-mediated shRNA. CCK-8 assay was used to detect the proliferation of myoblasts, and flow cytometry analysis was used to detect cell cycle of myoblasts. The qRT-PCR was used to measure the mRNA expression of p21 in myoblasts. Immunocytochemistry was used to detect myotubes formation and the cell fusion rate was counted, qRT-PCR was used to measure the mRNA expression of the MyoD,MyoG,Myf5, Myf6 and HACD1 genes at 48, 72, 96 h after myoblast induced to differentiation. The results showed that the proliferation of myoblasts was inhibited after downregulating MSTN expression, the cell cycle was arrested at G0/G1 phase, and the expression of cell cycle inhibitor p21 increased. In the study on myoblast differentiation, we found the expression of 4 myogenic regulatory factors after inducing for 48 h were decreased, and the expression of MyoG gene decreased significantly(P<0.05);the myoblast fusion rate of interference group was significantly higher than that of negative control group(P<0.01) after inducing for 72 h, the expression of Myf5 and Myf6 genes decreased significantly (P<0.01), the expression of MyoD gene didn't increase significantly (P>0.05), the expression of MyoG gene increased significantly(P<0.01);the expression of Myf5(P<0.05), MyoD(P>0.05) and MyoG(P<0.01) decreased after inducing for 96 h, and the expression of Myf6 gene didn't increase significantly(P>0.05). In addition, the expression of HACD1 gene was significantly higher in the interference group than that in the negative control group (P<0.05) after inducing for 72 h, and no significant changes were detected after inducing for 48 or 96 h. The results show that the MSTN gene interfered could inhibit the proliferation while promote differentiation of myoblasts, and HACD1 gene is related to myoblast fusion.

The research aimed to study the promoter activity area and to explore the regulation mechanism of some related transcription factors of goat DCT gene, which would help to find the theoretical reference for expression regulation of goat DCT. 5' flanking region and the first exon sequences were analyzed by bioinformatics and blasted with mouse and human DCT gene promoter sequences. Based on the results of bioinformatics analysis and online promoter prediction, five 5'-terminal and six 3'-terminal deleted fragment promoter reporter gene vectors were constructed with the rapid amplification kit. Six mutation reporter gene vectors for SOX10, MITF and OTX2 transcription factor binding sites were built using P8 fragment sequences as the template. These vectors were transfected into A375 cells by transient transfection and their promoter activities were detected with the dual-luciferase detection reagent. The results showed that 11 deleted fragment promoter reporter gene vectors with different length were successfully constructed. The luciferase activity of P3 vector for -990-+232 bp was significantly higher than the others (P<0.01). And P8 vector for -881——154 bp based on P3 fragment had the highest luciferase activity among the serial 3'-terminal deletion vectors (P<0.01). The mutation vectors of SOX10 binding site showed extremely significantly lower luciferase activity compared with the original vector (P<0.01), while the MITF and OTX2 binding sites mutation vectors both showed extremely significantly higher luciferase activity compared with the original vectors (P<0.01). The core promoter region of goat DCT gene is located at -881——154 bp. Transcription factor SOX10 up-regulate the expression of DCT gene, while the regulating role of transcription factor MITF and OTX2 should be thoroughly studied.

The research aimed to study the promoter activity area and to explore the regulation mechanism of some related transcription factors of goat DCT gene, which would help to find the theoretical reference for expression regulation of goat DCT. 5' flanking region and the first exon sequences were analyzed by bioinformatics and blasted with mouse and human DCT gene promoter sequences. Based on the results of bioinformatics analysis and online promoter prediction, five 5'-terminal and six 3'-terminal deleted fragment promoter reporter gene vectors were constructed with the rapid amplification kit. Six mutation reporter gene vectors for SOX10, MITF and OTX2 transcription factor binding sites were built using P8 fragment sequences as the template. These vectors were transfected into A375 cells by transient transfection and their promoter activities were detected with the dual-luciferase detection reagent. The results showed that 11 deleted fragment promoter reporter gene vectors with different length were successfully constructed. The luciferase activity of P3 vector for -990-+232 bp was significantly higher than the others (P<0.01). And P8 vector for -881——154 bp based on P3 fragment had the highest luciferase activity among the serial 3'-terminal deletion vectors (P<0.01). The mutation vectors of SOX10 binding site showed extremely significantly lower luciferase activity compared with the original vector (P<0.01), while the MITF and OTX2 binding sites mutation vectors both showed extremely significantly higher luciferase activity compared with the original vectors (P<0.01). The core promoter region of goat DCT gene is located at -881——154 bp. Transcription factor SOX10 up-regulate the expression of DCT gene, while the regulating role of transcription factor MITF and OTX2 should be thoroughly studied.

The aim of this study was to disclose the information of genetic structure and maternal origins of Tibetan goat populations based on the Cytb gene diversity. Mitochondrial Cytb gene of 157 individuals from 8 Tibetan goat populations in Tibet plateau areas were amplified and sequenced. The genetic diversity and genetic differentiation indexes were analyzed, and phylogenetic tree was constructed. The results showed that the length of Cytb was 1 140 bp, and 33 variation sites were detected. Thirty haplotypes were defined, and haplotype diversity (Hd) and nucleotide diversity (Pi) were 0.736 and 0.001 8, respectively, which suggested that there was rich genetic diversity in Tibetan goat populations in Tibet. There were significant genetic differentiation among the 78.6% populations (P<0.05) and extremely significant variation among populations within group (F_SC=0.321,P<0.001). Phylogenetic tree revealed 4 haplogroups (A-D), suggesting that Tibetan goats had 4 maternal origins. Network of haplotypes showed that Tibetan goats didn't cluster into a cluster from different Tibet plateau areas. There was at least one population expansion event occurred during the domestic history of Tibetan goats. These results demonstrate that Tibetan goats in Tibet have rich genetic diversity and 4 maternal origins. Although apparent genetic differentiation has been occurred among the populations, there is no strong phylogeographic structure. And the genetic structure difference may become weak. These results will provide a scientific basis for protecting and utilizing the genetic resources of Tibetan goats.

The aim of this study was to disclose the information of genetic structure and maternal origins of Tibetan goat populations based on the Cytb gene diversity. Mitochondrial Cytb gene of 157 individuals from 8 Tibetan goat populations in Tibet plateau areas were amplified and sequenced. The genetic diversity and genetic differentiation indexes were analyzed, and phylogenetic tree was constructed. The results showed that the length of Cytb was 1 140 bp, and 33 variation sites were detected. Thirty haplotypes were defined, and haplotype diversity (Hd) and nucleotide diversity (Pi) were 0.736 and 0.001 8, respectively, which suggested that there was rich genetic diversity in Tibetan goat populations in Tibet. There were significant genetic differentiation among the 78.6% populations (P<0.05) and extremely significant variation among populations within group (F_SC=0.321,P<0.001). Phylogenetic tree revealed 4 haplogroups (A-D), suggesting that Tibetan goats had 4 maternal origins. Network of haplotypes showed that Tibetan goats didn't cluster into a cluster from different Tibet plateau areas. There was at least one population expansion event occurred during the domestic history of Tibetan goats. These results demonstrate that Tibetan goats in Tibet have rich genetic diversity and 4 maternal origins. Although apparent genetic differentiation has been occurred among the populations, there is no strong phylogeographic structure. And the genetic structure difference may become weak. These results will provide a scientific basis for protecting and utilizing the genetic resources of Tibetan goats.

This study aimed to express Helicobacter Pylori nonheme iron-containing Ferritin protein in Escherichia coli, and obtain the Ferritin nano-particles with natural nanostructure, for the application in disease and antigen presentation study by surface modification. To improve the expression of Ferritin, Ferritin gene was synthesized according to the Escherichia coli biased codon and cloned to the pET-32a vector. The recombinant protein was purified by affinity chromatography using His-trap Ni²⁺ column, and refold by gradient dialysis. The refolding protein was detected by transmission electron microscope to identify the nano-structure of the self-assembling protein. The results showed that the prokaryotic expression vector pET-32a-Ferritin was successfully constructed, and the recombinant His-Ferritin protein was expressed after induced by IPTG. The recombinant Ferritin protein was mainly expressed in the form of inclusion body. By use of His-trap Ni²⁺ affinity chromatography column, we obtained the recombinant Ferritin protein with 96% purity. The purified protein was in gradient dialysis to 2 mol·L^-1 urea buffer with a desalting column to obtain the refold protein. The refold protein had a nanostructure with the diameter of 12-20 nm under the transmission electron microscope, which was similar to the natural nanoparticles. In this study, the prokaryotic expression system of Ferritin was successfully established and the recombinant protein with nanostructure was prepared by induction expression, affinity chromatography purification and refolding. This study lays the foundation for the application of Ferritin in biomedical field.

This study aimed to express Helicobacter Pylori nonheme iron-containing Ferritin protein in Escherichia coli, and obtain the Ferritin nano-particles with natural nanostructure, for the application in disease and antigen presentation study by surface modification. To improve the expression of Ferritin, Ferritin gene was synthesized according to the Escherichia coli biased codon and cloned to the pET-32a vector. The recombinant protein was purified by affinity chromatography using His-trap Ni²⁺ column, and refold by gradient dialysis. The refolding protein was detected by transmission electron microscope to identify the nano-structure of the self-assembling protein. The results showed that the prokaryotic expression vector pET-32a-Ferritin was successfully constructed, and the recombinant His-Ferritin protein was expressed after induced by IPTG. The recombinant Ferritin protein was mainly expressed in the form of inclusion body. By use of His-trap Ni²⁺ affinity chromatography column, we obtained the recombinant Ferritin protein with 96% purity. The purified protein was in gradient dialysis to 2 mol·L^-1 urea buffer with a desalting column to obtain the refold protein. The refold protein had a nanostructure with the diameter of 12-20 nm under the transmission electron microscope, which was similar to the natural nanoparticles. In this study, the prokaryotic expression system of Ferritin was successfully established and the recombinant protein with nanostructure was prepared by induction expression, affinity chromatography purification and refolding. This study lays the foundation for the application of Ferritin in biomedical field.

To investigate the function of SOX5 in mouse skins and the regulation of hair color formation, the differential expression of SOX5 in skins of different coat color mouse skin was examined. In this study, C57BL/6 strains of black, brown and gray mice were selected each of the 3 for 12 days after birth, randomly. Quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemistry methods were used in this study to analyze the expression of SOX5; we also constructed the eukaryotic expression vector of SOX5 and transfected the mouse melanocytes to detect the expression level of the pigmentation-related genes, and measured the melanin content. The results showed that:1) SOX5 could be expressed in mouse skin with different hair color. The mRNA and protein expression levels of SOX5 in black and gray mouse skin were higher than that in brown mouse skin with very significantly difference (P<0.01). And the main expression site of SOX5 was hair follicle outer root sheath. 2) Compared with empty vector group(Vector-GFP), the mRNA and protein expression levels of MITF-M,TYR, TYRP1,TYRP2,PMEL and OA1 were increased with significant difference (P<0.05) and very significantly difference (P<0.01) in SOX5 transfected group; the content of melanin was significantly increased with very significantly difference (P<0.01) in SOX5 transfected group. 3) MITF-M had a negative feedback on SOX5 by overexpression of MITF-M.These suggest that SOX5 affect the formation of pigments and then involve in the formation of coat color by regulating MITF-M.

To investigate the function of SOX5 in mouse skins and the regulation of hair color formation, the differential expression of SOX5 in skins of different coat color mouse skin was examined. In this study, C57BL/6 strains of black, brown and gray mice were selected each of the 3 for 12 days after birth, randomly. Quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemistry methods were used in this study to analyze the expression of SOX5; we also constructed the eukaryotic expression vector of SOX5 and transfected the mouse melanocytes to detect the expression level of the pigmentation-related genes, and measured the melanin content. The results showed that:1) SOX5 could be expressed in mouse skin with different hair color. The mRNA and protein expression levels of SOX5 in black and gray mouse skin were higher than that in brown mouse skin with very significantly difference (P<0.01). And the main expression site of SOX5 was hair follicle outer root sheath. 2) Compared with empty vector group(Vector-GFP), the mRNA and protein expression levels of MITF-M,TYR, TYRP1,TYRP2,PMEL and OA1 were increased with significant difference (P<0.05) and very significantly difference (P<0.01) in SOX5 transfected group; the content of melanin was significantly increased with very significantly difference (P<0.01) in SOX5 transfected group. 3) MITF-M had a negative feedback on SOX5 by overexpression of MITF-M.These suggest that SOX5 affect the formation of pigments and then involve in the formation of coat color by regulating MITF-M.

The objective of this study was to investigate the regulatory mechanism of the early development of hybrid embryos of the yak (Bos grunniens) in order to provide theoretical basis for improving efficiency of in vitro production (IVP). Total RNA were extracted from embryos that derived from yak oocytes in vitro fertilized with Jersey cattle at 5 stages of 2-cell, 4-cell, 8-cell, morula and blastocyst and then amplified via the Smart-Seq2 method to construct RNA libraries and sequenced using RNA-seq. A total of 47 791 850-63 332 216 Clean Reads for each in vitro stage after filtering the Raw Reads of HiSeq^TM2500 sequence were obtained, of which, 80.00% to 91.13% Reads were covered in the reference genome. Number of expressed genes were greatest at 4-cell stage (15 400) and lowest at blastocyst stage (9 604). When|log₂ ratio| ≥ 1 and Q-value<0.05 were set as thresholds for identifying differentially expressed genes (DEGs), a total of 3 690, 6 332, 8 965 and 10 298 DEGs were identified for 2-cell-4 cell, 4-cell-8-cell, 8-cell-morula and morula-blastocyst, respectively. The GO distributions of the DEGs for 4 stages were classified into 3 categories:biological processes (BP), cellular components (CC), molecular functions (MF) with a total of 67 subcategories.KEGG enrichment analysis of DEGs showed that the major enrichment pathways were Spliceosome, Neuroactive ligand-receptor interaction, Cytokine-cytokine receptor interactions, Ubiquitin mediated proteolysis, RNA transport etc. In conclusion, this was the report to investigate the mechanism that the early development of hybrid in vitro-derived yak embryos using high-throughput sequencing. A number of DEGs and pathways were discovered, which provided valuable information to improve in vitro production technique for yak crossbred embryos.

The objective of this study was to investigate the regulatory mechanism of the early development of hybrid embryos of the yak (Bos grunniens) in order to provide theoretical basis for improving efficiency of in vitro production (IVP). Total RNA were extracted from embryos that derived from yak oocytes in vitro fertilized with Jersey cattle at 5 stages of 2-cell, 4-cell, 8-cell, morula and blastocyst and then amplified via the Smart-Seq2 method to construct RNA libraries and sequenced using RNA-seq. A total of 47 791 850-63 332 216 Clean Reads for each in vitro stage after filtering the Raw Reads of HiSeq^TM2500 sequence were obtained, of which, 80.00% to 91.13% Reads were covered in the reference genome. Number of expressed genes were greatest at 4-cell stage (15 400) and lowest at blastocyst stage (9 604). When|log₂ ratio| ≥ 1 and Q-value<0.05 were set as thresholds for identifying differentially expressed genes (DEGs), a total of 3 690, 6 332, 8 965 and 10 298 DEGs were identified for 2-cell-4 cell, 4-cell-8-cell, 8-cell-morula and morula-blastocyst, respectively. The GO distributions of the DEGs for 4 stages were classified into 3 categories:biological processes (BP), cellular components (CC), molecular functions (MF) with a total of 67 subcategories.KEGG enrichment analysis of DEGs showed that the major enrichment pathways were Spliceosome, Neuroactive ligand-receptor interaction, Cytokine-cytokine receptor interactions, Ubiquitin mediated proteolysis, RNA transport etc. In conclusion, this was the report to investigate the mechanism that the early development of hybrid in vitro-derived yak embryos using high-throughput sequencing. A number of DEGs and pathways were discovered, which provided valuable information to improve in vitro production technique for yak crossbred embryos.

The study was conducted to investigate the effect of heat stress on lipid metabolism and the underlying molecular mechanisms in liver of broiler chickens. A total of 96 AA broilers at the age of 28 with similar body weight were randomly allocated into 4 groups with 4 replicates of 6 chicks. The birds in two experimental groups were administered with 24 or 72 h heat stress with the temperature of (33±1)℃. The birds in two control groups were raised under normal temperature of (23±1)℃. After 24 or 72 hours high temperature exposure, 8 chicks from each group were sacrificed, respectively. The samples of liver and plasma were collected and stored in -80℃ for further determination. The growth performance was calculated. The levels of plasma glucose (GLU), uric acid (UA), total cholesterol (TCHO), total bile acid (TBA), triglyceride (TG) in plasma were measured by spectrophotometer. The relative mRNA levels of SREBP-1c, FATP-1, LXR, APOB, CYP7A1, FXR and AMPKα1 in liver were detected by RT-PCR method. The results showed that:1) Heat stress significantly reduced body weight gain and feed intake of broiler chickens (P<0.05). 2) The content of glucose and total bile acid in plasma were significantly increased after 24 hours high stress (P<0.05), and the content of glucose, triglyceride and total cholesterol in plasma were significantly increased after 72 hours heat stress (P<0.05). 3) The mRNA levels of FATP-1, SREBP-1c, APOB, LXR genes were significantly increased after 72 hours heat stress treatment(P<0.05). Heat exposure of 72 hours tended to increase genes expression of liver FXR and CYP7A1 (0.05<P<0.1). The mRNA level of AMPKα1 gene was significantly increased after 72 hours heat stress(P<0.05).In conclusion, heat stress significantly reduced the growth performance of broiler chickens. Short-term heat stress showed little or no effect on liver lipid metabolism, however long-term heat exposure significantly changed liver lipid metabolism at the molecular levels. The similar alteration profiles of the genes expression of AMPKα1 and genes related to lipid metabolism indicated that AMPK signal pathway might play important roles in the liver lipid metabolism of broiler chickens under heat stress.

The study was conducted to investigate the effect of heat stress on lipid metabolism and the underlying molecular mechanisms in liver of broiler chickens. A total of 96 AA broilers at the age of 28 with similar body weight were randomly allocated into 4 groups with 4 replicates of 6 chicks. The birds in two experimental groups were administered with 24 or 72 h heat stress with the temperature of (33±1)℃. The birds in two control groups were raised under normal temperature of (23±1)℃. After 24 or 72 hours high temperature exposure, 8 chicks from each group were sacrificed, respectively. The samples of liver and plasma were collected and stored in -80℃ for further determination. The growth performance was calculated. The levels of plasma glucose (GLU), uric acid (UA), total cholesterol (TCHO), total bile acid (TBA), triglyceride (TG) in plasma were measured by spectrophotometer. The relative mRNA levels of SREBP-1c, FATP-1, LXR, APOB, CYP7A1, FXR and AMPKα1 in liver were detected by RT-PCR method. The results showed that:1) Heat stress significantly reduced body weight gain and feed intake of broiler chickens (P<0.05). 2) The content of glucose and total bile acid in plasma were significantly increased after 24 hours high stress (P<0.05), and the content of glucose, triglyceride and total cholesterol in plasma were significantly increased after 72 hours heat stress (P<0.05). 3) The mRNA levels of FATP-1, SREBP-1c, APOB, LXR genes were significantly increased after 72 hours heat stress treatment(P<0.05). Heat exposure of 72 hours tended to increase genes expression of liver FXR and CYP7A1 (0.05<P<0.1). The mRNA level of AMPKα1 gene was significantly increased after 72 hours heat stress(P<0.05).In conclusion, heat stress significantly reduced the growth performance of broiler chickens. Short-term heat stress showed little or no effect on liver lipid metabolism, however long-term heat exposure significantly changed liver lipid metabolism at the molecular levels. The similar alteration profiles of the genes expression of AMPKα1 and genes related to lipid metabolism indicated that AMPK signal pathway might play important roles in the liver lipid metabolism of broiler chickens under heat stress.

This study was conducted to estimate the net macromineral requirements for maintenance and growth of Dorper×Hu crossbred F₁ ewe lambs from 20 to 35 kg. Thirty-five ewe lambs were used (the initial body weight (BW) of (19.2±0.36) kg). Seven lambs were randomly chosen and slaughtered at 20 kg BW as the BL group for measuring initial body composition. Another 7 lambs were also randomly chosen and offered a pelleted mixed diet for ad libitum intake and slaughtered at 28 kg BW as the IM group. The remaining lambs (n=21) were allocated randomly to 3 groups (ad libitum intake group, 70% of the ad libitum intake group, 40% of the ad libitum intake group) with 7 lambs in each group. All lambs were slaughtered when the lambs in ad libitum intake group reached approximately 35 kg BW. The contents of macrominerals of empty bodies of the lambs (head+feet, hide, internal organs + blood and carcass) were weighed, ground, mixed, and subsampled for chemical analyses. The results showed that, in the growth phase from 20 to 35 kg BW, the daily net macromineral requirements for maintenance were 22.21 mg Ca, 11.65 mg P, 3.41 mg Na, 6.92 mg K and 1.23 mg Mg per kilogram empty BW (EBW) for ewes, the daily net macromineral requirements for growth were 11.93-11.68 g Ca, 6.12-5.71 g P, 1.34-1.24 g Na, 1.58-1.63 g K and 0.41-0.36 g Mg per kilogram EBW gain (EBWG) for ewes. These results for the nutritional requirements of macrominerals may help to formulate more balanced diets for Dorper×Hu ewe lambs in the growth phase from 20 to 35 kg BW.

This study was conducted to estimate the net macromineral requirements for maintenance and growth of Dorper×Hu crossbred F₁ ewe lambs from 20 to 35 kg. Thirty-five ewe lambs were used (the initial body weight (BW) of (19.2±0.36) kg). Seven lambs were randomly chosen and slaughtered at 20 kg BW as the BL group for measuring initial body composition. Another 7 lambs were also randomly chosen and offered a pelleted mixed diet for ad libitum intake and slaughtered at 28 kg BW as the IM group. The remaining lambs (n=21) were allocated randomly to 3 groups (ad libitum intake group, 70% of the ad libitum intake group, 40% of the ad libitum intake group) with 7 lambs in each group. All lambs were slaughtered when the lambs in ad libitum intake group reached approximately 35 kg BW. The contents of macrominerals of empty bodies of the lambs (head+feet, hide, internal organs + blood and carcass) were weighed, ground, mixed, and subsampled for chemical analyses. The results showed that, in the growth phase from 20 to 35 kg BW, the daily net macromineral requirements for maintenance were 22.21 mg Ca, 11.65 mg P, 3.41 mg Na, 6.92 mg K and 1.23 mg Mg per kilogram empty BW (EBW) for ewes, the daily net macromineral requirements for growth were 11.93-11.68 g Ca, 6.12-5.71 g P, 1.34-1.24 g Na, 1.58-1.63 g K and 0.41-0.36 g Mg per kilogram EBW gain (EBWG) for ewes. These results for the nutritional requirements of macrominerals may help to formulate more balanced diets for Dorper×Hu ewe lambs in the growth phase from 20 to 35 kg BW.

The study was conducted to evaluate the effects of dietary with different calcium and phosphorus levels on growth performance, nutrient digestibility and metabolism of weaned lambs in Shaanbei White Cashmere goat, which would provide scientific basis for formulating standard of calcium and phosphorus requirement of weaned lamb of Shaanbei White Cashmere goat. Thirty-six weaned lambs of Shaanbei White Cashmere goat with((16.48±2.37)kg) were randomly selected and assigned to 4 groups, and each group had 3 repeats with 3 goats. The goats were fed with 4 different diets:Ⅰgroup(0.60% Ca, 0.45% P),Ⅱgroup(0.60% Ca, 0.65% P),Ⅲ group(0.80% Ca, 0.45% P) and Ⅳ group(0.80% Ca, 0.65% P) in a 2×2 factorial arrangement. The experiment lasted for 65 d. On the 30th day of the trial, one goat from each repeat was selected and fed individually in cage to carry out the digestion and metabolism experiments. The results showed that:1) Calcium and phosphorus supplementation in the basal diet had no significantly influence on DMI (P>0.05), but increased ADG remarkably (P<0.05) with the increasing of dietary Ca and P levels. 2) Dietary Ca levels had significantly influence on apparent digestibility and metabolic rate of gross energy (P<0.05). 3) Calcium and phosphorus supplementation in the basal diet had significantly influence on intake Ca or P, excretion Ca or P and deposited Ca or P (P<0.05), respectively. The relationships between excretion Ca or P and intake Ca or P were showed in the following regression equations, for Ca:Y=0.662X+0.929 (R²=0.993) and for P:Y=0.776X+0.506 (R²=0.996), the endogenous loss of Ca and P of weaned lamb of Shaanbei White Cahmere goats were 0.929 and 0.506 g·d^-1, respectively. The relationships between deposited Ca or P and ADG were showed in the following regression equations, for Ca:Y=47.737X-21.622 (R²=0.649) and for P:Y=53.770X+18.973 (R²=0.448). These results indicated that the relationships between deposited Ca or P and ADG of weaned lambs in Shaanbei White Cashmere goat were linear. Under the conditions of the study, the optimal dietary supplementation levels of calcium and phosphorus are 0.80% and 0.45% for weaned lambs of Shaanbei White Cashmere goat, respectively.

The study was conducted to evaluate the effects of dietary with different calcium and phosphorus levels on growth performance, nutrient digestibility and metabolism of weaned lambs in Shaanbei White Cashmere goat, which would provide scientific basis for formulating standard of calcium and phosphorus requirement of weaned lamb of Shaanbei White Cashmere goat. Thirty-six weaned lambs of Shaanbei White Cashmere goat with((16.48±2.37)kg) were randomly selected and assigned to 4 groups, and each group had 3 repeats with 3 goats. The goats were fed with 4 different diets:Ⅰgroup(0.60% Ca, 0.45% P),Ⅱgroup(0.60% Ca, 0.65% P),Ⅲ group(0.80% Ca, 0.45% P) and Ⅳ group(0.80% Ca, 0.65% P) in a 2×2 factorial arrangement. The experiment lasted for 65 d. On the 30th day of the trial, one goat from each repeat was selected and fed individually in cage to carry out the digestion and metabolism experiments. The results showed that:1) Calcium and phosphorus supplementation in the basal diet had no significantly influence on DMI (P>0.05), but increased ADG remarkably (P<0.05) with the increasing of dietary Ca and P levels. 2) Dietary Ca levels had significantly influence on apparent digestibility and metabolic rate of gross energy (P<0.05). 3) Calcium and phosphorus supplementation in the basal diet had significantly influence on intake Ca or P, excretion Ca or P and deposited Ca or P (P<0.05), respectively. The relationships between excretion Ca or P and intake Ca or P were showed in the following regression equations, for Ca:Y=0.662X+0.929 (R²=0.993) and for P:Y=0.776X+0.506 (R²=0.996), the endogenous loss of Ca and P of weaned lamb of Shaanbei White Cahmere goats were 0.929 and 0.506 g·d^-1, respectively. The relationships between deposited Ca or P and ADG were showed in the following regression equations, for Ca:Y=47.737X-21.622 (R²=0.649) and for P:Y=53.770X+18.973 (R²=0.448). These results indicated that the relationships between deposited Ca or P and ADG of weaned lambs in Shaanbei White Cashmere goat were linear. Under the conditions of the study, the optimal dietary supplementation levels of calcium and phosphorus are 0.80% and 0.45% for weaned lambs of Shaanbei White Cashmere goat, respectively.

The experiment was conducted to investigate the effects of different amino acid levels in low crude protein dites on age of puberty, serum metabolites and hormone concentration of replacement gilts based on ideal dietary amino acid pattern. 96 Landrace×Large White gilts, with initial body weight of (69.01±3.43) kg and similar body conditions, were allocated to 4 groups with 4 replicates per group. The gilts were fed respectively with 4 low-protein diets with different digestible lysine(DLys) levels of 0.61%(group 1), 0.66%(group 2), 0.71%(group 3), 0.76%(group 4) for the early phase(70 to 100 kg) and 0.55%(group 1), 0.60%(group 2), 0.65%(group 3), 0.70%(group 4) for the late phase(100 to 125 kg), and based on the same digestible amino acid pattern by adding synthetic amino acids. The results showed that:Serum circulating lysine concentrations was higher with increasing DLys (P<0.05), but serum triglyeride, glucose concentrations were similar among groups(P>0.05). The concentrations of serum insulin(INS), insulin growth factor-I (IGF-I), luteinizing hormone(LH) and estradiol(E₂) increased, then decreased with increasing DLys levels, and reached a maximum value in group 3(P>0.05), the concentration of serum INS in group 3 was significantly higher than those in group 1(P<0.05). Back fat thickness and ratio of estrus of gilts increased, then decreased with increasing DLys levels(P>0.05), the age of puberty of gilts in group 3 was significantly lower than those in group 4(P<0.05). The results indicated, based on ideal dietary amino acid pattern, the daily DLys intake were 17.82 and 17.29 g·d^-1 for 70-100 kg and 100-125 kg (DLys level in the low-protein diets should be 0.71% and 0.65%), respectively,which could increase the concentration of serum metabolites hormones INS, IGF-I, and elevate LH, E₂ secretion. Meanwhile, reduced the age of puberty and improved the ratio of estrus in gilts.

The experiment was conducted to investigate the effects of different amino acid levels in low crude protein dites on age of puberty, serum metabolites and hormone concentration of replacement gilts based on ideal dietary amino acid pattern. 96 Landrace×Large White gilts, with initial body weight of (69.01±3.43) kg and similar body conditions, were allocated to 4 groups with 4 replicates per group. The gilts were fed respectively with 4 low-protein diets with different digestible lysine(DLys) levels of 0.61%(group 1), 0.66%(group 2), 0.71%(group 3), 0.76%(group 4) for the early phase(70 to 100 kg) and 0.55%(group 1), 0.60%(group 2), 0.65%(group 3), 0.70%(group 4) for the late phase(100 to 125 kg), and based on the same digestible amino acid pattern by adding synthetic amino acids. The results showed that:Serum circulating lysine concentrations was higher with increasing DLys (P<0.05), but serum triglyeride, glucose concentrations were similar among groups(P>0.05). The concentrations of serum insulin(INS), insulin growth factor-I (IGF-I), luteinizing hormone(LH) and estradiol(E₂) increased, then decreased with increasing DLys levels, and reached a maximum value in group 3(P>0.05), the concentration of serum INS in group 3 was significantly higher than those in group 1(P<0.05). Back fat thickness and ratio of estrus of gilts increased, then decreased with increasing DLys levels(P>0.05), the age of puberty of gilts in group 3 was significantly lower than those in group 4(P<0.05). The results indicated, based on ideal dietary amino acid pattern, the daily DLys intake were 17.82 and 17.29 g·d^-1 for 70-100 kg and 100-125 kg (DLys level in the low-protein diets should be 0.71% and 0.65%), respectively,which could increase the concentration of serum metabolites hormones INS, IGF-I, and elevate LH, E₂ secretion. Meanwhile, reduced the age of puberty and improved the ratio of estrus in gilts.

Viperin is an antiviral protein which could inhibit the replication of a wide range of viruses. The aim of this study was to explore the anti-CSFV activity of porcine Viperin protein. CSFV was inoculated in the cell line of PK-Vi over-expressing Viperin, viral load was detected by real-time qRT-PCR and virus titration. Knockdown of Viperin expression in PK-Vi by siRNA (siVi) was performed and CSFV replication was detected. Viral load in cell culture supernatants and cell lysates were examined to analyze the influence of Viperin on virus release. The co-localization and interaction of Viperin with CSFV E2 protein was determined by confocal laser scanning microscopy test and co-immunoprecipitation (Co-IP) assay. The genome copy numbers and viral titers of CSFV in PK-Vi was significantly decreased by 68.75%, 83.61%, 77.27% and 68.75%, 87.5%, 80.39% at 24, 48 and 72 hpi (P<0.05), comparing with control cells (PK-C1 expressing EGFP). Knockdown of Viperin expression retrieved the replication of CSFV, which was significantly higher than those in PK-Vi and siNC transfected PK-Vi (P<0.05) although it was still lower than those of PK-C1 and siRNA treated PK-C1 groups. Viperin expression had no effect on virus release from PK-15 cells. Confocal laser scanning microscopy test showed Viperin protein co-localized with E2 protein in CSFV infected cells and plasmid transfected 293T cells. Co-IP assay indicated that Viperin could interact with E2 protein. Porcine Viperin protein effectively inhibited CSFV replication in vitro, potentially via interaction of Viperin with CSFV proteins. The results provide foundation for further studies of the interaction of CSFV infection with host immune response.

Viperin is an antiviral protein which could inhibit the replication of a wide range of viruses. The aim of this study was to explore the anti-CSFV activity of porcine Viperin protein. CSFV was inoculated in the cell line of PK-Vi over-expressing Viperin, viral load was detected by real-time qRT-PCR and virus titration. Knockdown of Viperin expression in PK-Vi by siRNA (siVi) was performed and CSFV replication was detected. Viral load in cell culture supernatants and cell lysates were examined to analyze the influence of Viperin on virus release. The co-localization and interaction of Viperin with CSFV E2 protein was determined by confocal laser scanning microscopy test and co-immunoprecipitation (Co-IP) assay. The genome copy numbers and viral titers of CSFV in PK-Vi was significantly decreased by 68.75%, 83.61%, 77.27% and 68.75%, 87.5%, 80.39% at 24, 48 and 72 hpi (P<0.05), comparing with control cells (PK-C1 expressing EGFP). Knockdown of Viperin expression retrieved the replication of CSFV, which was significantly higher than those in PK-Vi and siNC transfected PK-Vi (P<0.05) although it was still lower than those of PK-C1 and siRNA treated PK-C1 groups. Viperin expression had no effect on virus release from PK-15 cells. Confocal laser scanning microscopy test showed Viperin protein co-localized with E2 protein in CSFV infected cells and plasmid transfected 293T cells. Co-IP assay indicated that Viperin could interact with E2 protein. Porcine Viperin protein effectively inhibited CSFV replication in vitro, potentially via interaction of Viperin with CSFV proteins. The results provide foundation for further studies of the interaction of CSFV infection with host immune response.

The purpose of this study was to investigate the biological characteristics of 4 H5N6 subtype avian influenza virus isolates, and their pathogenicity to chickens. In this study, the clinical samples between 2015-2016 year in Guangdong were used to isolate virus, and the isolates were identified, the genes of isolates were cloned, sequenced and analyzed, then 4 weeks old SPF chickens were infected by the way of intranasal and eyedrop inoculation. Our results showed that 4 strains of H5N6 avian influenza virus were isolated, their HA gene belonged to Clade 2.3.4.4, and the isolates contained multiple basic amino acids at the cleavage site, which were characteristic of highly pathogenic AIV. The inoculation results showed that all chickens of the attack group died in 5 d, and the chickens of cohabitation group died in 9 d; the infected chickens could shed virus continuously. And the avian influenza virus could be detected in the heart, liver, spleen, lung, kidney, brain in high titer and caused extensive pathological damage. The 4 strains of H5N6 subtype avian influenza virus isolates showed high pathogenicity and horizontal transmission ability to chickens, suggesting the need to strengthen the prevention and control of H5N6 subtype avian influenza.

The purpose of this study was to investigate the biological characteristics of 4 H5N6 subtype avian influenza virus isolates, and their pathogenicity to chickens. In this study, the clinical samples between 2015-2016 year in Guangdong were used to isolate virus, and the isolates were identified, the genes of isolates were cloned, sequenced and analyzed, then 4 weeks old SPF chickens were infected by the way of intranasal and eyedrop inoculation. Our results showed that 4 strains of H5N6 avian influenza virus were isolated, their HA gene belonged to Clade 2.3.4.4, and the isolates contained multiple basic amino acids at the cleavage site, which were characteristic of highly pathogenic AIV. The inoculation results showed that all chickens of the attack group died in 5 d, and the chickens of cohabitation group died in 9 d; the infected chickens could shed virus continuously. And the avian influenza virus could be detected in the heart, liver, spleen, lung, kidney, brain in high titer and caused extensive pathological damage. The 4 strains of H5N6 subtype avian influenza virus isolates showed high pathogenicity and horizontal transmission ability to chickens, suggesting the need to strengthen the prevention and control of H5N6 subtype avian influenza.

This experiment was conducted to explore the proliferation function of T, B lymphocytes and expression of Cyclin D1 and p27 in peripheral blood of SPF chicks infected with reticuloendotheliosis virus (REV). Eighty 1-day-old SPF chicks were randomly divided into REV infection group and control group. The T, B lymphocytes proliferation function and expression of Cyclin D1 at mRNA and protein level, transcription of p27 in peripheral blood were detected by CCK-8, RT-qPCR and Western Blot. The results showed that the proliferation ability of T and B lymphocytes in peripheral blood of SPF chicks infected with REV early was significantly lower than that of the control chicks, and the transcription of p27 mRNA was significantly higher than that of the control chicks. The expression of Cyclin D1 mRNA and protein content in the late stage of infection were significantly higher than that of the control chicks (P<0.05), but the transcription of p27 mRNA were significantly lower than those of control chicks (P<0.05). The above results showed that the decrease of T, B lymphocytes proliferative function and p27 mRNA transcription, and increase of Cyclin D1 at mRNA and protein level in blood of SPF chicks were closely related to the decrease in immune function of chicks infected with REV.

This experiment was conducted to explore the proliferation function of T, B lymphocytes and expression of Cyclin D1 and p27 in peripheral blood of SPF chicks infected with reticuloendotheliosis virus (REV). Eighty 1-day-old SPF chicks were randomly divided into REV infection group and control group. The T, B lymphocytes proliferation function and expression of Cyclin D1 at mRNA and protein level, transcription of p27 in peripheral blood were detected by CCK-8, RT-qPCR and Western Blot. The results showed that the proliferation ability of T and B lymphocytes in peripheral blood of SPF chicks infected with REV early was significantly lower than that of the control chicks, and the transcription of p27 mRNA was significantly higher than that of the control chicks. The expression of Cyclin D1 mRNA and protein content in the late stage of infection were significantly higher than that of the control chicks (P<0.05), but the transcription of p27 mRNA were significantly lower than those of control chicks (P<0.05). The above results showed that the decrease of T, B lymphocytes proliferative function and p27 mRNA transcription, and increase of Cyclin D1 at mRNA and protein level in blood of SPF chicks were closely related to the decrease in immune function of chicks infected with REV.

This experiment was conducted to explore the dynamic distribution of type 3 duck hepatitis A virus (DHAV-3) and the relationship between the induction of histopathological, antiviral cytokines and pro-inflammatory cytokines in 7-day-old ducklings. One week old ducklings were infected with DHAV-3. At 1, 3, 6, 9, 12, 24, 48, 72 and 96 h after infection, 11 tissue samples of blood, liver, spleen, pancreas, kidney, lung, brain, thymus, bursa of Fabricius, Harderian glands and duodenum were collected randomly from three ducklings, and then detected. The virus load was detected by TaqMan real-time one-step RT-PCR and the pathological damage changes of liver was observed by optical microscope, the transcription of anti-viral cytokines (IFN-α, IFN-β and IFN-γ) and pro-inflammatory cytokines (IL-1β, IL-2 and IL-6) in liver were detected by real-time RT-PCR at different time points. The links between these changes were analyzed. The results showed that the virus can be detected in all tissues of 1-week-old ducklings in 1 h after infection. The highest load of virus in tissues was at 24-48 h after infection, except that the highest of blood and pancreas were at 12 h and 96 h after infection respectively. The three organs with the highest viral load were liver, spleen and kidney (10^11.15, 10^10.37 and 10^10.30 copies·g^-1). Liver main histopathological changes were vacuoles at 3-12 h, and 24-48 h for the necrotic changes. The transcription of 6 cytokines in the liver reached the highest level at 24-48 h (except for IL-1β at 12 h) after infection. After 48 h of infection, the transcription level of the cytokines decreased with the decrease of the virus content in the liver and was positively correlated with the severity of liver disease. DHAV-3 has extensive tissue tropism in ducklings. Liver, spleen and kidney are the main target organ of virus attack. The cytokines in the liver are highly likely to play an important role in inhibiting viral proliferation and repairing tissue damage.

This experiment was conducted to explore the dynamic distribution of type 3 duck hepatitis A virus (DHAV-3) and the relationship between the induction of histopathological, antiviral cytokines and pro-inflammatory cytokines in 7-day-old ducklings. One week old ducklings were infected with DHAV-3. At 1, 3, 6, 9, 12, 24, 48, 72 and 96 h after infection, 11 tissue samples of blood, liver, spleen, pancreas, kidney, lung, brain, thymus, bursa of Fabricius, Harderian glands and duodenum were collected randomly from three ducklings, and then detected. The virus load was detected by TaqMan real-time one-step RT-PCR and the pathological damage changes of liver was observed by optical microscope, the transcription of anti-viral cytokines (IFN-α, IFN-β and IFN-γ) and pro-inflammatory cytokines (IL-1β, IL-2 and IL-6) in liver were detected by real-time RT-PCR at different time points. The links between these changes were analyzed. The results showed that the virus can be detected in all tissues of 1-week-old ducklings in 1 h after infection. The highest load of virus in tissues was at 24-48 h after infection, except that the highest of blood and pancreas were at 12 h and 96 h after infection respectively. The three organs with the highest viral load were liver, spleen and kidney (10^11.15, 10^10.37 and 10^10.30 copies·g^-1). Liver main histopathological changes were vacuoles at 3-12 h, and 24-48 h for the necrotic changes. The transcription of 6 cytokines in the liver reached the highest level at 24-48 h (except for IL-1β at 12 h) after infection. After 48 h of infection, the transcription level of the cytokines decreased with the decrease of the virus content in the liver and was positively correlated with the severity of liver disease. DHAV-3 has extensive tissue tropism in ducklings. Liver, spleen and kidney are the main target organ of virus attack. The cytokines in the liver are highly likely to play an important role in inhibiting viral proliferation and repairing tissue damage.

This study was aimed to analyze the sequences of collagen-binding protein A (CbpA) of Trueperella pyogenes Chongqing isolate from goat (CbpA-CQ) and identify the two potential heparin binding domains of NRB and B1. The sequences of CbpA-CQ gene and its coding product were analyzed by bioinformatics software. NRB and B1 genes were amplified by PCR and cloned into prokaryotic expression vector (pGEX-4T-1). The fusion proteins (GST-NRB and GST-B1) were induced in Escherichia coli strain DE3 and purified using Gluthathione-Sepharose 4 B beads. The adhesion of fusion proteins (GST-NRB and GST-B1) to HeLa cells and the adhesion inhibition of heparin were detected by Western blot. The results showed that the CbpA-CQ had 7 collagen-binding B domains covering 55% in the whole sequence of CbpA, and the domains had homology with those of bacteria, such as T. pyogenes, Staphylococcus. CbpA-CQ and those of other T. pyogenes strains were clustered into a clade in the phylogenetic tree. Fusion proteins (GST-NRB and GST-B1) were purified by affinity purification from supernatant of induced bacteria lysate. The 2 proteins adhered to HeLa cells in a dose dependent manner, but the adhesion was inhibited by heparin in a dose dependent manner. The results suggest that although CbpA-CQ has large variability, it has common sequence characteristics compared with the published CbpA sequences. Besides, the NRB and B1 regions of CbpA-CQ are heparin binding domains.

This study was aimed to analyze the sequences of collagen-binding protein A (CbpA) of Trueperella pyogenes Chongqing isolate from goat (CbpA-CQ) and identify the two potential heparin binding domains of NRB and B1. The sequences of CbpA-CQ gene and its coding product were analyzed by bioinformatics software. NRB and B1 genes were amplified by PCR and cloned into prokaryotic expression vector (pGEX-4T-1). The fusion proteins (GST-NRB and GST-B1) were induced in Escherichia coli strain DE3 and purified using Gluthathione-Sepharose 4 B beads. The adhesion of fusion proteins (GST-NRB and GST-B1) to HeLa cells and the adhesion inhibition of heparin were detected by Western blot. The results showed that the CbpA-CQ had 7 collagen-binding B domains covering 55% in the whole sequence of CbpA, and the domains had homology with those of bacteria, such as T. pyogenes, Staphylococcus. CbpA-CQ and those of other T. pyogenes strains were clustered into a clade in the phylogenetic tree. Fusion proteins (GST-NRB and GST-B1) were purified by affinity purification from supernatant of induced bacteria lysate. The 2 proteins adhered to HeLa cells in a dose dependent manner, but the adhesion was inhibited by heparin in a dose dependent manner. The results suggest that although CbpA-CQ has large variability, it has common sequence characteristics compared with the published CbpA sequences. Besides, the NRB and B1 regions of CbpA-CQ are heparin binding domains.

In order to know the antimicrobial resistance, virulence genes and the epidemiological characteristics of Staphylococcus aureus (SA) isolated from cows with mastitis and endometritis. The drug resistance profile for 155 clinical S. aureus isolates were determined using disk diffusion method, and methicillin-resistant Staphylococcus aureus (MRSA) strains were also identified. Antimicrobial genes, virulence genes, SCCmec genotyping and MLST genotyping were detected and analyzed by using PCR method. The results showed that a total of 22 MRSA strains have been detected among 155 SA isolates, with the detection rate of 14.2%. The antimicrobial resistant rates of most MRSA were significantly higher than that of the MSSA strains; and the detection rates of different virulence genes were different between MRSA and MSSA. The SCCmecⅠwas identified as a predominant genotype of SCCmec in Xinjiang region. Fourteen ST genotypes were detected, namely ST188, ST584, ST9, ST805, ST2373, ST968, ST2139, ST1, ST2700, ST903, ST2454, ST2990, ST63 and STX, respectively. ST1 and ST9 had higher detection rates than others. ST9 was higher among MSSA strains and ST1 was higher among MRSA strains. This study showed that SA strains were of multiple resistance with diverse distribution of virulence genes. While ST9 was the predominant genotype among MSSA strains and ST1-SCCmecⅠwas the predominant genotypes among MRSA strains in Xinjiang region.

In order to know the antimicrobial resistance, virulence genes and the epidemiological characteristics of Staphylococcus aureus (SA) isolated from cows with mastitis and endometritis. The drug resistance profile for 155 clinical S. aureus isolates were determined using disk diffusion method, and methicillin-resistant Staphylococcus aureus (MRSA) strains were also identified. Antimicrobial genes, virulence genes, SCCmec genotyping and MLST genotyping were detected and analyzed by using PCR method. The results showed that a total of 22 MRSA strains have been detected among 155 SA isolates, with the detection rate of 14.2%. The antimicrobial resistant rates of most MRSA were significantly higher than that of the MSSA strains; and the detection rates of different virulence genes were different between MRSA and MSSA. The SCCmecⅠwas identified as a predominant genotype of SCCmec in Xinjiang region. Fourteen ST genotypes were detected, namely ST188, ST584, ST9, ST805, ST2373, ST968, ST2139, ST1, ST2700, ST903, ST2454, ST2990, ST63 and STX, respectively. ST1 and ST9 had higher detection rates than others. ST9 was higher among MSSA strains and ST1 was higher among MRSA strains. This study showed that SA strains were of multiple resistance with diverse distribution of virulence genes. While ST9 was the predominant genotype among MSSA strains and ST1-SCCmecⅠwas the predominant genotypes among MRSA strains in Xinjiang region.

The aim of this study was to investigate the effects of macrophage migration inhibitory (MIF) on metabolism of Free-Fatty-Acid in broiler chicken cardiocytes in hypoxia. Firstly, chicken embryonic cardiomyocytes were isolated by trypsin and type Ⅱ collagenase, and cultured in DMEM, then identified by α-actinin immunocytochemistry. Secondly, hypoxia model of chicken cardiocyte induced by cobalt chloride was established, which was identified by the hypoxia sign protein, HIF-1α. Thirdly, the MIF blocker ISO-1 was used to test the relationship between MIF and p-AMPK. The expression levels of FAT/CD₃₆, p-ACC and CPT-1A in cardiomyocytes were determined by western blot during hypoxia and normoxia. Results were as follows:The chicken cardiocytes, above 90% purity, showed spherical and polygon shape in vivo, and they beat spontaneously about 80-130 times per minute. HIF-1α expression reached the highest at 24 h with the 600 μmol·L^-1 CoCl₂. The p-AMPK levels decreased significantly (P<0.01) along with the MIF which was blocked by 100 μmol·L^-1 ISO-1. The levels of MIF, p-AMPK, FAT/CD₃₆, p-ACC and CPT-1A were all increased significantly (P<0.01) in the hypoxia group compared with the normal one, while the levels of the target proteins were all decreased significantly (P<0.01) in the inhibitor group compared with the hypoxia one. MIF, a myocardial autocrine factor, plays an important protective role in the fatty acid adapted metabolism of chicken cardiomyocytes during hypoxia by activating the AMPK signal pathway.

The aim of this study was to investigate the effects of macrophage migration inhibitory (MIF) on metabolism of Free-Fatty-Acid in broiler chicken cardiocytes in hypoxia. Firstly, chicken embryonic cardiomyocytes were isolated by trypsin and type Ⅱ collagenase, and cultured in DMEM, then identified by α-actinin immunocytochemistry. Secondly, hypoxia model of chicken cardiocyte induced by cobalt chloride was established, which was identified by the hypoxia sign protein, HIF-1α. Thirdly, the MIF blocker ISO-1 was used to test the relationship between MIF and p-AMPK. The expression levels of FAT/CD₃₆, p-ACC and CPT-1A in cardiomyocytes were determined by western blot during hypoxia and normoxia. Results were as follows:The chicken cardiocytes, above 90% purity, showed spherical and polygon shape in vivo, and they beat spontaneously about 80-130 times per minute. HIF-1α expression reached the highest at 24 h with the 600 μmol·L^-1 CoCl₂. The p-AMPK levels decreased significantly (P<0.01) along with the MIF which was blocked by 100 μmol·L^-1 ISO-1. The levels of MIF, p-AMPK, FAT/CD₃₆, p-ACC and CPT-1A were all increased significantly (P<0.01) in the hypoxia group compared with the normal one, while the levels of the target proteins were all decreased significantly (P<0.01) in the inhibitor group compared with the hypoxia one. MIF, a myocardial autocrine factor, plays an important protective role in the fatty acid adapted metabolism of chicken cardiomyocytes during hypoxia by activating the AMPK signal pathway.

This study aims to investigate the effects of feeding Hericium erinaceus polysaccharide (HEP) to weaned piglets on ileal morphology and permeability under oxidative stress. Forty-five healthy long-white weaned piglets with similar weight were randomly divided into five groups (every group had 9 replications). Control group and model group were fed with basic diet only, meanwhile the other three test groups were fed with basic diet added with 0.2%, 0.4% or 0.6% HEP. On the 21st day, piglets in model group and three test groups were injected with 100 μg·kg^-1·BW lipopolysaccharide, meanwhile, piglets in control group were injected with equal dose sterilized normal saline. Blood samples were collected to detect antioxidant functions and intestinal mucosal integrity. The piglets were slaughtered and the ileums were collected to observe the morphological changes by HE staining; the expression of tight junction proteins of ileum were detected by qPCR methods and Western blot methods. The results showed that, compared with the model group, HEP could significantly improve the decrease of serum antioxidant indexes caused by LPS (P<0.05), significantly increase glutamine (GLN) content (P<0.05) and reduce diamine oxidase (DAO) activity (P<0.05); and significantly recovery the decrease of tight junction protein expression in ileum epithelial cells induced by LPS stress (P<0.05). Supplementation of HEP can effectively improve the permeability of ileal morphology under oxidative stress.

This study aims to investigate the effects of feeding Hericium erinaceus polysaccharide (HEP) to weaned piglets on ileal morphology and permeability under oxidative stress. Forty-five healthy long-white weaned piglets with similar weight were randomly divided into five groups (every group had 9 replications). Control group and model group were fed with basic diet only, meanwhile the other three test groups were fed with basic diet added with 0.2%, 0.4% or 0.6% HEP. On the 21st day, piglets in model group and three test groups were injected with 100 μg·kg^-1·BW lipopolysaccharide, meanwhile, piglets in control group were injected with equal dose sterilized normal saline. Blood samples were collected to detect antioxidant functions and intestinal mucosal integrity. The piglets were slaughtered and the ileums were collected to observe the morphological changes by HE staining; the expression of tight junction proteins of ileum were detected by qPCR methods and Western blot methods. The results showed that, compared with the model group, HEP could significantly improve the decrease of serum antioxidant indexes caused by LPS (P<0.05), significantly increase glutamine (GLN) content (P<0.05) and reduce diamine oxidase (DAO) activity (P<0.05); and significantly recovery the decrease of tight junction protein expression in ileum epithelial cells induced by LPS stress (P<0.05). Supplementation of HEP can effectively improve the permeability of ileal morphology under oxidative stress.

To provide the theoretical basis for exploring the regulatory mechanism of cystathionine beta synthase(CBS) gene expression in layer bone, the 5' regulatory region and core promoter characteristics were investigated. The tibias were collected from laying hen at 68 weeks old. The genomic DNA was extracted with phenol-chloroform method. The primers for amplifying 5' regulatory region of CBS gene were designed according to the template sequence downloaded from UCSC database. The characteristic of 5' regulatory region and prediction of transcription factors binding sites were analyzed by bioinformatics methods. The constructed dual-luciferase recombinant vectors were transfected into DF-1 cells for detecting luciferase activtity, and the core promoter of CBS gene was identified. The results showed that the potential core promoter, classical CpG island and multiple transcription factor Sp1 binding sites were located in 5' regulatory region of CBS gene. All the fragments had promoter activity, and there were significant difference in promoter activity among different fragments. The fragment F4 (-155-+131 bp) with the highest transcription activity was identified as core promoter region. The identification of core promoter and prediction of transcription factor binding sites of CBS gene provide the basis for studying the expression regulatory mechanism of CBS gene in bone.

To provide the theoretical basis for exploring the regulatory mechanism of cystathionine beta synthase(CBS) gene expression in layer bone, the 5' regulatory region and core promoter characteristics were investigated. The tibias were collected from laying hen at 68 weeks old. The genomic DNA was extracted with phenol-chloroform method. The primers for amplifying 5' regulatory region of CBS gene were designed according to the template sequence downloaded from UCSC database. The characteristic of 5' regulatory region and prediction of transcription factors binding sites were analyzed by bioinformatics methods. The constructed dual-luciferase recombinant vectors were transfected into DF-1 cells for detecting luciferase activtity, and the core promoter of CBS gene was identified. The results showed that the potential core promoter, classical CpG island and multiple transcription factor Sp1 binding sites were located in 5' regulatory region of CBS gene. All the fragments had promoter activity, and there were significant difference in promoter activity among different fragments. The fragment F4 (-155-+131 bp) with the highest transcription activity was identified as core promoter region. The identification of core promoter and prediction of transcription factor binding sites of CBS gene provide the basis for studying the expression regulatory mechanism of CBS gene in bone.

It is noteworthy that more extensive expansion of the DNase Ⅱ-like protein family (estimated 125 genes) in the genome of the parasitic nematode Trichinella spiralis (T. spiralis). However, only plancitoxin-1 possesses one predicted active site of DNase Ⅱ (HKD motif) in its C-terminus domain in 125 DNase Ⅱ-like protein family. To explore the molecular characteristics of plancitoxin-1 gene in T. spiralis, plancitoxin-1 gene was cloned by RT-PCR from total RNA of T. spiralis larvae, and analyzed with bioinformatics software. The results showed that the sequences of plancitoxin-1 cloned from T. spiralis were shorter (210 bp less) than the XM_003370715.1 sequence in GenBank, and named after plancitoxin-1-like. The gene encoded a protein of 294 amino acids. Intriguingly, there were two HKD motifs in the N-and C-termini in the cloned sequences. plancitoxin-1-like is a stable protein with a predicted molecular mass (Mr) of 33.19 kDa and theoretical pI of 9.17. By sequence alignment, plancitoxin-1-like protein revealed 35% sequence identity to previously reported DNase Ⅱ family members from other species. A total of 4 N-linked glycosylation sites, 3 O-linked glycosylation sites, 31 phosphorylation site 17 linear B cell piptope, 9 T cell binding sites, and 3 disulfide bond were predicted in plancitoxin-1-like. Analysis of secondary structure revealed that α-helix, extended strand, and random coil were 14.29% (42), 28.23% (83), and 55.10% (162), respectively. In this study, the plancitoxin-1-like gene was cloned and analyzed. The results laid the foundation for the further study on the role of development infection in T. spiralis.

It is noteworthy that more extensive expansion of the DNase Ⅱ-like protein family (estimated 125 genes) in the genome of the parasitic nematode Trichinella spiralis (T. spiralis). However, only plancitoxin-1 possesses one predicted active site of DNase Ⅱ (HKD motif) in its C-terminus domain in 125 DNase Ⅱ-like protein family. To explore the molecular characteristics of plancitoxin-1 gene in T. spiralis, plancitoxin-1 gene was cloned by RT-PCR from total RNA of T. spiralis larvae, and analyzed with bioinformatics software. The results showed that the sequences of plancitoxin-1 cloned from T. spiralis were shorter (210 bp less) than the XM_003370715.1 sequence in GenBank, and named after plancitoxin-1-like. The gene encoded a protein of 294 amino acids. Intriguingly, there were two HKD motifs in the N-and C-termini in the cloned sequences. plancitoxin-1-like is a stable protein with a predicted molecular mass (Mr) of 33.19 kDa and theoretical pI of 9.17. By sequence alignment, plancitoxin-1-like protein revealed 35% sequence identity to previously reported DNase Ⅱ family members from other species. A total of 4 N-linked glycosylation sites, 3 O-linked glycosylation sites, 31 phosphorylation site 17 linear B cell piptope, 9 T cell binding sites, and 3 disulfide bond were predicted in plancitoxin-1-like. Analysis of secondary structure revealed that α-helix, extended strand, and random coil were 14.29% (42), 28.23% (83), and 55.10% (162), respectively. In this study, the plancitoxin-1-like gene was cloned and analyzed. The results laid the foundation for the further study on the role of development infection in T. spiralis.