Genome-wide association study of 17 serum biochemical indicators in a chicken F2 resource population.

BACKGROUND: Serum biochemical indicators are often regarded as direct reflections of animal metabolism and health. The molecular mechanisms underlying serum biochemical indicators metabolism of chicken (Gallus Gallus) have not been elucidated. Herein, we performed a genome-wide association study (GWAS) to identify the variation associated with serum biochemical indicators. The aim of this research was to broaden the understanding of the serum biochemical indicators in chickens. RESULTS: A GWAS of serum biochemical indicators was carried out on 734 samples from an F2 Gushi× Anka chicken population. All chickens were genotyped by sequencing, 734 chickens and 321,314 variants were obtained after quality control. Based on these variants, a total of 236 single-nucleotide polymorphisms (SNPs) on 9 chicken chromosomes (GGAs) were identified to be significantly (-log10(P) > 5.72) associated with eight of seventeen serum biochemical indicators. Ten novel quantitative trait locis (QTLs) were identified for the 8 serum biochemical indicator traits of the F2 population. Literature mining revealed that the ALPL, BCHE, GGT2/GGT5 genes at loci GGA24, GGA9 and GGA15 might affect the alkaline phosphatase (AKP), cholinesterase (CHE) and γ-glutamyl transpeptidase (GGT) traits, respectively. CONCLUSION: The findings of the present study may contribute to a better understanding of the molecular mechanisms of chicken serum biochemical indicator regulation and provide a theoretical basis for chicken breeding programs.

miR-128-3p inhibits intramuscular adipocytes differentiation in chickens by downregulating FDPS.

BACKGROUND: Intramuscular fat (IMF) content is the major indicator for evaluating chicken meat quality due to its positive correlation with tenderness, juiciness, and flavor. An increasing number of studies are focusing on the functions of microRNAs (miRNAs) in intramuscular adipocyte differentiation. However, little is known about the association of miR-128-3p with intramuscular adipocyte differentiation. Our previous RNA-seq results indicated that miR-128-3p was differentially expressed at different periods in chicken intramuscular adipocytes, revealing a possible association with intramuscular adipogenesis. The purpose of this research was to investigate the biological functions and regulatory mechanism of miR-128-3p in chicken intramuscular adipogenesis. RESULTS: The results of a series of assays confirmed that miR-128-3p could promote the proliferation and inhibit the differentiation of intramuscular adipocytes. A total of 223 and 1,050 differentially expressed genes (DEGs) were identified in the mimic treatment group and inhibitor treatment group, respectively, compared with the control group. Functional enrichment analysis revealed that the DEGs were involved in lipid metabolism-related pathways, such as the MAPK and TGF-ß signaling pathways. Furthermore, target gene prediction analysis showed that miR-128-3p can target many of the DEGs, such as FDPS, GGT5, TMEM37, and ASL2. The luciferase assay results showed that miR-128-3p targeted the 3' UTR of FDPS. The results of subsequent functional assays demonstrated that miR-128-3p acted as an inhibitor of intramuscular adipocyte differentiation by targeting FDPS. CONCLUSION: miR-128-3p inhibits chicken intramuscular adipocyte differentiation by downregulating FDPS. Our findings provide a theoretical basis for the study of lipid metabolism and reveal a potential target for molecular breeding to improve meat quality.

The Chicken Pan-Genome Reveals Gene Content Variation and a Promoter Region Deletion in IGF2BP1 Affecting Body Size.

Domestication and breeding have reshaped the genomic architecture of chicken, but the retention and loss of genomic elements during these evolutionary processes remain unclear. We present the first chicken pan-genome constructed using 664 individuals, which identified an additional approximately 66.5-Mb sequences that are absent from the reference genome (GRCg6a). The constructed pan-genome encoded 20,491 predicated protein-coding genes, of which higher expression levels are observed in conserved genes relative to dispensable genes. Presence/absence variation (PAV) analyses demonstrated that gene PAV in chicken was shaped by selection, genetic drift, and hybridization. PAV-based genome-wide association studies identified numerous candidate mutations related to growth, carcass composition, meat quality, or physiological traits. Among them, a deletion in the promoter region of IGF2BP1 affecting chicken body size is reported, which is supported by functional studies and extra samples. This is the first time to report the causal variant of chicken body size quantitative trait locus located at chromosome 27 which was repeatedly reported. Therefore, the chicken pan-genome is a useful resource for biological discovery and breeding. It improves our understanding of chicken genome diversity and provides materials to unveil the evolution history of chicken domestication.

Global investigation of estrogen-responsive genes regulating lipid metabolism in the liver of laying hens.

BACKGROUND: Estrogen plays an essential role in female development and reproductive function. In chickens, estrogen is critical for lipid metabolism in the liver. The regulatory molecular network of estrogen in chicken liver is poorly understood. To identify estrogen-responsive genes and estrogen functional sites on a genome-wide scale, we determined expression profiles of mRNAs, lncRNAs, and miRNAs in estrogen-treated ((17ß-estradiol)) and control chicken livers using RNA-Sequencing (RNA-Seq) and studied the estrogen receptor α binding sites by ChIP-Sequencing (ChIP-Seq). RESULTS: We identified a total of 990 estrogen-responsive genes, including 962 protein-coding genes, 11 miRNAs, and 17 lncRNAs. Functional enrichment analyses showed that the estrogen-responsive genes were highly enriched in lipid metabolism and biological processes. Integrated analysis of the data of RNA-Seq and ChIP-Seq, identified 191 genes directly targeted by estrogen, including 185 protein-coding genes, 4 miRNAs, and 2 lncRNAs. In vivo and in vitro experiments showed that estrogen decreased the mRNA expression of PPARGC1B, which had been reported to be linked with lipid metabolism, by directly increasing the expression of miR-144-3p. CONCLUSIONS: These results increase our understanding of the functional network of estrogen in chicken liver and also reveal aspects of the molecular mechanism of estrogen-related lipid metabolism.

Gut microbiota profiles of commercial laying hens infected with tumorigenic viruses.

BACKGROUND: Studies have shown that some viral infections cause structural changes in the intestinal microflora, but little is known about the effects of tumorigenic viral infection on the intestinal microflora of chickens. RESULTS: A 29-week commercial layer flock positive for avian leukosis virus-J (ALV-J), Marek's disease virus (MDV) and avian reticuloendotheliosis virus (REV) was selected, and fresh fecal samples were collected and examined for the composition of the gut microflora by Illumina sequencing of the V3-V4 region of the 16S rRNA gene. The operational taxonomic units (OTUs) of the fecal microbiota differentiated the chickens infected with only ALV-J and those coinfected with ALV-J and MDV or REV from infection-negative chickens. The enrichment and diversity of cloacal microflora in chickens infected with ALV-J alone were slightly different from those in the infection-negative chickens. However, the diversity of cloacal microflora was significantly increased in chickens coinfected with both ALV-J and MDV or REV. CONCLUSIONS: The intestinal microbiota was more strongly disturbed in chickens after coinfection with ALV-J and MDV or REV than after infection with ALV-J alone, and there may be underlying mechanisms by which the capacity for the stabilization of the intestinal flora was impaired due to viral infection and tumorigenesis.

Transcriptom analysis revealed regulation of dexamethasone induced microRNAs in chicken thymus.

Stress-induced immunosuppression is one of the serious threats to the poultry industry, especially obvious for young chicken. However, the molecular mechanism of stress-induced immunosuppression has not been clear in chicken. Here, we established an immunosuppression model of 7-day-old chickens with injecting dexamethasone (Dex) to analyze the molecular regulation in the chicken thymus. The microRNAs (miRNAs) transcripts profiles of thymuses from the model and control group were identified by the Solexa sequencing technology. The results showed 121 significantly differently expressed (SDE) miRNAs, including 119 known and two novel miRNAs (novel-58 and novel-350). A total of 391 target genes of the SDE miRNAs were predicted and annotated. We verified the potential negative correlation between gga-miR-103-3p and TGM2 by quantitative real-time polymerase chain reaction (qRT-PCR), as well as between novel-350 and PCBD2, and the results were positive. Gene ontology (GO) enrichment analysis showed that there was 298 significant enrichment GO terms, in which 31 were related to immune or stress, such as lymphocyte apoptotic process and response to stress. KEGG pathway analysis suggested that the SDE miRNAs were involved in autophagy regulation, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, Jak-STAT signaling pathway, and so on (although not significantly enriched). In these immune signaling pathways, the SDE miRNAs (such as gga-miR-2954, gga-miR-146b-3p, gga-miR-106-3p, and gga-miR-214) and the predicted target genes (such as IL11Ra, CSF3R, IFNGR1, CNTF, and MAP2K2) might affect the thymus immune function of chicken. The above results would provide a basis for uncovering the molecular regulation mechanism of immunosuppression in poultry.

Transcriptome profile in bursa of Fabricius reveals potential mode for stress-influenced immune function in chicken stress model.

BACKGROUND: The molecular mechanisms underlying stress-influenced immune function of chicken (Gallus Gallus) are not clear. The stress models can be established effectively by feeding chickens corticosterone (CORT) hormone. The bursa of Fabricius is a unique central immune organ of birds. RNA-Seq technology was used to investigate differences in the expression profiles of immune-related genes and associated pathways in the bursa of Fabricius to clarify molecular mechanisms. The aim of this study was to broaden the understanding of the stress-influenced immune function in chickens. RESULTS: Differentially expressed genes (DEGs) in the bursa of Fabricius between experimental group (basal diet with added CORT 30 mg/kg; C_B group) and control group (basal diet; B_B group) were identified by using RNA-seq technology. In total, we found 1434 significant DEGs (SDEGs), which included 199 upregulated and 1235 downregulated genes in the C_B group compared with the B_B group. The immune system process GO term was the top significantly GO term, including MYD88, TLR4, IL15, VEGFA gene and so on. The cytokine-cytokine receptor interaction pathway and the Toll-like receptor signaling pathway were the key pathways affected by stress. The protein-protein interaction (PPI) analysis of the SDEGs showed that VEGFA, MyD88 and IL15 were hub genes and module analysis showed that MYD88, TLR4 and VEGFA play important roles in response to stress. CONCLUSION: This study showed that the VEGFA and ILs (such as IL15) via the cytokine-cytokine receptor interaction pathway, MYD88 and TLR4 via the Toll-like receptor signaling pathway may play important roles in the regulation of immune function under stress condition with CORT administration. The results of this study provide a reference for further studies of the molecular mechanisms of stress-influenced immune function.

Characterization of miRNA transcriptome profiles related to breast muscle development and intramuscular fat deposition in chickens.

Studies of the miRNA expression profiles associated with the postnatal late development of skeletal muscle and IMF deposition are lacking in chicken. Here, we evaluated the patterns of muscle fiber growth and IMF deposition in breast muscle in the Chinese domestic breed called Gushi chicken, where we constructed four small RNA libraries from breast muscle tissues at 6, 14, 22, and 30 weeks. A total of 388 known miRNAs and 31 novel miRNAs were identified based on four small RNA libraries. Comparative analysis identified 92 significant differentially expressed (SDE) miRNAs based on six combinations. KEGG pathway analysis for the SDE miRNAs showed that metabolic pathways such as glycolysis and biosynthesis of amino acids were significantly enriched before 22 weeks, and pathways such as biosynthesis of unsaturated fatty acids and fatty acid elongation were significantly enriched after 22 weeks. This trend was consistent with the patterns of breast muscle fiber growth and IMF deposition in Gushi chickens. We also constructed miRNA-mRNA interaction networks related to breast muscle development and IMF deposition. The results showed that miRNAs such as gga-miR-1a-3p, and gga-miR-133a-5p may play important roles in breast muscle development, and miRNAs such as gga-miR-103-3p, and gga-miR-138-2-3p may have key roles in IMF deposition. This study determined the dynamic miRNA transcriptome in breast muscle tissue for the first time in Gushi chickens. The results provide a valuable resource for investigating the post-transcriptional regulation mechanisms during postnatal late development of breast muscle and IMF deposition and for evaluating the muscular disease.

Sequencing and characterization of lncRNAs in the breast muscle of Gushi and Arbor Acres chickens.

Chicken muscle quality is one of the most important factors determining the economic value of poultry, and muscle development and growth are affected by genetics, environment, and nutrition. However, little is known about the molecular regulatory mechanisms of long non-coding RNAs (lncRNAs) in chicken skeletal muscle development. Our study aimed to better understand muscle development in chickens and thereby improve meat quality. In this study, Ribo-Zero RNA-Seq was used to investigate differences in the expression profiles of muscle development related genes and associated pathways between Gushi (GS) and Arbor Acres (AA) chickens. We identified two muscle tissue specific expression lncRNAs. In addition, the target genes of these lncRNAs were significantly enriched in certain biological processes and molecular functions, as demonstrated by Gene Ontology (GO) analysis, and these target genes participate in five signaling pathway, as revealed by an analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Taken together, these data suggest that different lncRNAs might be involved in regulating chicken muscle development and growth and provide new insight into the molecular mechanisms of lncRNAs.

MicroRNA-101-2-5p targets the ApoB gene in the liver of chicken (Gallus Gallus).

Apolipoprotein B (ApoB) is a major protein component of plasma lipoproteins. It is involved in many important biological processes such as lipid transportation, enzyme activity regulation, and receptor recognition. Extensive studies have shown that the expression of ApoB is regulated at multiple levels. However, the regulation of ApoB expression by microRNAs (miRNAs) still remains unknown. In the present study, identified are miRNAs that are predicted to interact with ApoB in chicken. The predicted relationship between the identified miRNAs and ApoB was verified through dual luciferase reporter assay in chicken DF1 cells, and the effect of miRNAs on ApoB expression was analyzed in chicken embryo hepatocytes stimulated by 17ß-estradiol. The results show that miR-101-2-5p was predicted to interact with ApoB. Dual luciferase reporter assay together with the miR-101-2-5p mimics study demostrate that ApoB is the target of miR-101-2-5p, which suppresses the expression of ApoB through binding with the 3'UTR of ApoB. Our experiments suggest that miR-101-2-5p might be involved in lipid metabolism through binding to the 3'UTR of ApoB in the liver of egg-laying chickens.

De novo assembly and characterization of the spleen transcriptome of common carp (Cyprinus carpio) using Illumina paired-end sequencing.

Common carp (Cyprinus carpio) is one of the most important aquacultured species of the family Cyprinidae, and breeding this species for disease resistance is becoming more and more important. However, at the genome or transcriptome levels, study of the immunogenetics of disease resistance in the common carp is lacking. In this study, 60,316,906 and 75,200,328 paired-end clean reads were obtained from two cDNA libraries of the common carp spleen by Illumina paired-end sequencing technology. Totally, 130,293 unique transcript fragments (unigenes) were assembled, with an average length of 1400.57 bp. Approximately 105,612 (81.06%) unigenes could be annotated according to their homology with matches in the Nr, Nt, Swiss-Prot, COG, GO, or KEGG databases, and they were found to represent 46,747 non-redundant genes. Comparative analysis showed that 59.82% of the unigenes have significant similarity to zebrafish Refseq proteins. Gene expression comparison revealed that 10,432 and 6889 annotated unigenes were, respectively, up- and down-regulated with at least twofold changes between two developmental stages of the common carp spleen. Gene ontology and KEGG analysis were performed to classify all unigenes into functional categories for understanding gene functions and regulation pathways. In addition, 46,847 simple sequence repeats (SSRs) were detected from 35,618 unigenes, and a large number of single nucleotide polymorphism (SNP) and insertion/deletion (INDEL) sites were identified in the spleen transcriptome of common carp. This study has characterized the spleen transcriptome of the common carp for the first time, providing a valuable resource for a better understanding of the common carp immune system and defense mechanisms. This knowledge will also facilitate future functional studies on common carp immunogenetics that may eventually be applied in breeding programs.

Identification and characterization of microRNAs in the spleen of common carp immune organ.

MicroRNAs (miRNAs) play an important role in the regulation of many fundamental biological processes in eukaryotes; however, miRNAs associated with immune functions in the common carp have not been reported. In this study, a small-RNA cDNA library was constructed from the spleen of the common carp. A total of 10,603,456 high-quality clean reads, representing 293,603 unique sequences, were obtained from the small-cDNA library using the Solexa sequencing. By the bioinformatic analysis, 194 conserved miRNAs and 12 novel miRNAs were identified in the carp spleen. The abundant miRNAs principally belong to 30 miRNA gene families such as let-7, mir-10, mir-15, mir-30, and so on. The conservation analysis showed that 23 families were present both in protostomes and deuterostomes, 46 families were conserved only in vertebrates, and 5 families (mir-430, mir-722, mir-724, mir-734, and mir-738) were identified only in fish species. Furthermore, GO enrichment analysis and KEGG pathway analysis suggested that miRNAs expressed in the spleen of common carp are involved in immune system development, lymphoid organ development, lymphocyte activation, immune response, B cell receptor signaling pathway, T cell receptor signaling pathway, Fc gamma R-mediated phagocytosis, Toll-like receptor signaling pathway, and so on. This study described the miRNA transcriptome in spleen tissue for the first time in the common carp. The results expand the number of known common carp miRNAs and provides a meaningful framework to understand the common carp immune system and defense mechanisms.

Dietary supplementation with pseudostellaria heterophylla polysaccharide enhanced immunity and changed mRNA expression of spleen in chicks.

In recent years, increasing interest has focused on natural components extracted from plants, among which plant polysaccharides as natural immunomodulators that can promote animal immunity. The present study was performed to investigate the effect of feed supplement Pseudostellaria Heterophylla Polysaccharide (PHP) on serum Immunoglobulins, T lymphocyte subpopulations, Cytokines and Lysozyme (LZM) activity in chicks. In addition, the influence of PHP on splenic gene expression was investigated by transcriptome sequencing. Four hundred 7-day-old Gushi cocks were randomly divided into four groups in a completely randomized design. The chicks were fed with a basal diet supplemented with 0 (CON-A), 100 (PHP-L), 200 (PHP-M) and 400 (PHP-H) mg/kg PHP. Blood and spleen samples were collected from 6 randomly selected chicks in each group at 14, 21, 28, and 35 days of age. The results showed that compared to the CON-A group, the PHP-M group exhibited significant increases in the levels of IgA, IgG, IgM, CD3, and LZM in the serum at 14, 21, 28, and 35 days (P < 0.05), and at 28 d, there was a significant quadratic relationship between the levels of dietary PHP and the levels of IgG, IgM, IFN-γ, IL-2, CD3, and LZM. Furthermore, a total of 470 differentially expressed genes (DEGs) were identified in spleen from PHP-M and CON-A at 28 d. These DEGs were significantly enriched in the Phagosome, Intestinal immune network for IgA production and Cytokine-cytokine receptor interaction pathways. The present investigation highlights the ameliorating effect of dietary PHP on immunological variables and spleen of chicks, the study suggests that PHP supplementation can enhance immunity and positively impact spleen mRNA expression in chicks.

Effects of immunosuppression-associated gga-miR-146a-5p on immune regulation in chicken macrophages by targeting the IRKA2 gene.

Stress-induced immunosuppression (SIIS) is one of the common problems in intensive poultry production, which brings enormous economic losses to the poultry industry. Accumulating evidence has shown that microRNAs (miRNAs) were important regulators of gene expression in the immune system. However, the miRNA-mediated molecular mechanisms underlying SIIS in chickens are still poorly understood. This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken SIIS. A stress-induced immunosuppression model was successfully established via daily injection of dexamethasone and analyzed miRNA expression in spleen. Seventy-four differentially expressed miRNAs (DEMs) was identified, and 229 target genes of the DEMs were predicted. Functional enrichment analysis the target genes revealed pathways related to immunity, such as MAPK signaling pathway and FoxO signaling pathway. The candidate miRNA, gga-miR-146a-5p, was found to be significantly downregulated in the Dex-induced chicken spleen, and we found that Dex stimulation significantly inhibited the expression of gga-miR-146a-5p in Chicken macrophages (HD11). Flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8) and other assays indicated that gga-miR-146a-5p can promote the proliferation and inhibit apoptosis of HD11 cells. A dual-luciferase reporter assay suggested that the Interleukin 1 receptor associated kinase 2 (IRAK2) gene, which encoded a transcriptional factor, was a direct target of gga-miR-146a-5p, gga-miR-146a-5p suppressed the post-transcriptional activity of IRAK2. These findings not only improve our understanding of the specific functions of miRNAs in avian stress but also provide potential targets for genetic improvement of stress resistance in poultry.

Prevalence of the optrA gene among Streptococcus suis isolates from diseased pigs and identification of a novel integrative conjugative element ICESsu988S.

Aim of this study was to investigate the prevalence and genetic environment of the oxazolidinone resistance gene optrA in Streptococcus suis (S. suis) isolates from diseased pigs in China. A total of 178 S. suis isolates were screened for the optrA gene by PCR. The phenotypes and genotypes of optrA-positive isolates were investigated by antimicrobial susceptibility testing, core genome Multilocus Sequence Typing (cgMLST), capsular serotypes determination and whole-genome sequencing (WGS). Fifty-one (28.7%) S. suis isolates were positive for optrA. Phylogenetic analysis indicated that the spread of the optrA among S. suis isolates was primarily due to horizontal transfer. Analysis of S. suis serotypes from diseased pigs revealed substantial diversity. The genetic environment of optrA was complex and diverse and could be divided into 12 different types. Interestingly, we identified a novel integrative and conjugative element ICESsu988S, carrying optrA and erm(T) genes. This is to the best of our knowledge the first report of the optrA and erm(T) co-located on an ICE in S. suis. Our results showed a high prevalence of optrA gene in S. suis isolates in China. Further research is needed to evaluate the importance of ICEs, as they horizontally propagate important clinical resistance genes.

Effect of Pseudostellaria heterophylla polysaccharide on the growth and liver metabolism of chicks.

In this study, the effects of Pseudostellaria heterophylla polysaccharide (PHP) on the growth, development, and liver metabolism of chicks were investigated by feeding chicks diets. Four hundred 7-d-old Gushi roosters were selected and randomly divided into four groups, labeled A, B, C, and D. Group A was fed the basal diet, and Groups B, C, and D were fed 100, 200, and 400 mg PHP per kilogram of basal diet, respectively. At 14, 21, 28 and 35 d of age, five chicks were randomly selected from each group to collect samples for index detection. The results showed that compared with Group A, there were significant reduction in average daily feed intake (ADFI) and feed-to-weight ratio (F/G) at 14, 21, and 28 d (P < 0.05), significant increase in average daily gain (ADG) at 21, 28 d (P < 0.05), significantly increased levels of total protein (TP), albumin (ALB), insulin (INS), thyroxine (T3), growth hormone (GH) at 14, 28 d (P < 0.05), significantly decreased levels of glucose (GLU), total cholesterol (TC), glucagon (GC), and triglyceride (TG) at 28 d in Group C (P < 0.05). There were significantly increased levels of TP, ALB at 14, 21 d (P < 0.05), significantly increased level of TP at 35 d (P < 0.05), significantly increased level of GH at 28 d (P < 0.05), significantly decreased levels of GLU, GC at 28 d (P < 0.05), significant reduction in F/G at 14, 21 d in Groups B and D (P < 0.05). Based on the above results, the livers from chicks in Groups A and C at 28 d were selected for transcriptome sequencing. The sequencing results showed that significantly differentially expressed genes (SDEGs) were enriched in growth and development, oxidative phosphorylation, the PPAR signaling pathway and the lipid metabolism pathway. All these results revealed that the addition of 200 mg/kg PHP in the diet promoted the growth and development, lipid metabolism and energy metabolism of chicks, inhibit inflammation and tumor development, and improve the function of the liver.

In order to explore the possibility of Pseudostellaria heterophylla polysaccharide (PHP) as green and healthy feed additive, we evaluated the effects of PHP on the growth, development and liver metabolism of chicks by feeding chicks diets in this study. The results revealed that the addition of 200 mg/kg PHP in the diet promoted the growth and development, lipid metabolism and energy metabolism in chicks and improved liver function. PHP may be a potential natural and safe feed additive applied in poultry production.

Effect of HSPA8 gene on the proliferation, apoptosis and immune function of HD11 cells.

HSPA8 (Heat shock 70 kDa protein 8) is a molecular chaperone involved in a variety of cellular processes. This gene may affect the proliferation, apoptosis and immune function of chicken macrophages, but the specific mechanism remains unclear. The purpose of this study was to explore the effect of the HSPA8 gene on the proliferation, apoptosis and immune function of chicken macrophages. In this study, a chicken HSPA8 overexpression plasmid, interference fragment and corresponding controls were transfected into HD11 cells, and then the expression of the HSPA8 gene, cell proliferation, cell cycle, apoptosis rate and immune function of each group were detected. The results showed that transfection of the HSPA8 overexpression plasmid significantly upregulated the level of HSPA8 expression in HD11 cells compared with the control; significantly promoted the proliferation of HD11 cells and the expression of PCNA, CCND1 and CCNB3; decreased the number of cells in the G1 phase and increased the number of cells in the S phase; decreased the rate of apoptosis and upregulated the expression of Bcl-2; and promoted the expression of the LPS-induced cytokines IL-1ß, IL-6 and TNF-α. Transfection of the HSPA8 interference fragment significantly downregulated the level of HSPA8 expression in HD11 cells; significantly inhibited the proliferation of HD11 cells and the expression of PCNA, CCND1 and CDK1; increased the number of cells in the G1 phase and decreased the number of cells in the S phase; increased the rate of apoptosis, downregulated the expression of Bcl-2 and upregulated the expression levels of Fas and FasL; and inhibited the expression of the LPS-induced cytokines IL-1ß and NF-κB. The results suggested that HSPA8 promotes the proliferation of and inhibits the apoptosis of HD11 cells and has a proinflammatory effect.

Transcriptomic analysis of mechanism underlying the effect of induced molting on semen quality and reproductive performance in aged Houdan roosters.

The reproductive performance of breeder roosters has significant economic importance in the poultry industry. Breeder roosters have severely reduced semen quality with age and will be at risk of culling in the following years. In order to extend the use of breeder roosters, we drew on the induced molting model of hens and selected 35 Houdan roosters aged 50 wk for induced molting. By comparing the body weight, testicular weight, semen quality, and reproductive performance before and after induced molting, we found that induced molting could restore the body weight and testicular weight to the levels before molting (P > 0.05). At the same time, it significantly improved sperm motility (P < 0.05) and also improved reproductive performance such as fertilization rate and hatching rate. To further reveal the mechanism underlying the effects of induced molting on semen quality and reproductive performance in aged Houdan roosters, we collected testes from 3 periods: 1 d before fasting (F0), 15 d after fasting (F15), and 32 d after recovery feeding (R32) for transcriptome sequencing analysis. A total of 5,671 genes were detected in F0, F15, and R32, and trend analysis of the 5,671 differential genes showed 2 significant trends (profile 5 and profile 2). KEGG enrichment analysis of the genes in the 2 profiles, revealed significantly enriched pathway regulation of actin cytoskeleton. In the regulation of actin cytoskeleton pathway, we found a protein kinase gene (SRC) and a senescence gene (ROCK2). SRC was highly expressed at F15, leading to the phosphorylation of key substrates, which in turn disrupted the Sertoli cell spermatid connection and the spermiogenesis process, resulting in no mature spermatozoa produced from F15, SRC expression was inhibited at R32, the expression level was reduced, and mature spermatozoa reappeared. The senescence gene ROCK2 was highly expressed at F15 compared to F0 and R32, which may have been responsible for inducing senescence atrophy in the testes.

Synergistic antibacterial activity of baicalin and EDTA in combination with colistin against colistin-resistant Salmonella.

The emergence and rapid spread of multidrug resistant (MDR) Gram-negative bacteria have posed a serious threat to global health and security. Because of the time-consuming, high cost and high risk of developing new antibiotics, a significant method is to use antibiotic adjuvants to revitalize the existing antibiotics. The purpose of the study is to research the traditional Chinese medicine baicalin with the function of inhibiting the efflux pump and EDTA whether their single or combination can increase the activity of colistin against colistin-resistant Salmonella in vitro and in vivo, and to explore its molecular mechanisms. In vitro antibacterial experiments, we have observed that baicalin and EDTA alone could enhance the antibacterial activity of colistin. At the same time, the combination of baicalin and EDTA also showed a stronger synergistic effect on colistin, reversing the colistin resistance of all Salmonella strains. Molecular docking and RT-PCR results showed that the combination of baicalin and EDTA not only affected the expression of mcr-1, but also was an effective inhibitor of MCR-1. In-depth synergistic mechanism analysis revealed that baicalin and EDTA enhanced colistin activity through multiple pathways, including accelerating the tricarboxylic acid cycle (TCA cycle), inhibiting the bacterial antioxidant system and lipopolysaccharide (LPS) modification, depriving multidrug efflux pump functions and attenuating bacterial virulence. In addition, the combinational therapy of colistin, baicalin and EDTA displayed an obvious reduction in bacterial loads cfus of liver and spleen compared with monotherapy and 2-drug combination therapy. In conclusion, our study indicates that the combination of baicalin and EDTA as a novel colistin adjuvant can provide a reliable basis for formulating the therapeutic regimen for colistin resistant bacterial infection.

Integrative analyses of the mRNA expression profile reveal the involvement of STC1 in chicken folliculogenesis.

Efficient ovarian follicle development, maturation, and ovulation are critical for egg production performance. Previous research has underscored the importance of messenger RNAs (mRNAs) in regulating development and folliculogenesis in chicken ovarians. However, the molecular mechanism is not fully understood, especially in the late period of the laying cycle. In the present study, ovarian tissues from 80-week-old Hy-Line Brown layers (three with high and three with low rates of egg laying) were collected for transcriptome sequencing. A total of 306 differentially expressed genes (DEGs) were identified in this study, at a false discovery rate (FDR)-corrected P-value < 0.05 and a log2|fold change| (log2|FC|) ≥1.5. Among these DEGs, stanniocalcin 1 (STC1) was mainly related to cellular processes, single-organism processes, biological regulation, metabolic processes, developmental processes, and reproductive processes. Then, we further investigated the regulation of STC1 during chicken follicle development and found that STC1 inhibited the proliferation and stimulated the apoptosis of follicular granulosa cells (GCs), and decreased the expression of progesterone (P4) and estradiol (E2). Collectively, these results suggest that STC1 plays an important role in chicken follicle development by decreasing GC proliferation and steroidogenesis and stimulating GC apoptosis. This study contributes to the understanding of the reproductive biology of laying hens in the late period of the laying cycle and further lays a foundation for the improvement of egg production in poultry breeding.

The egg production performance of chickens is an essential economic trait that differs significantly between high- and low-egg-laying breeds. In addition to external factors such as feeding, light, and environment, the periodic recruitment of pre-hierarchical follicles and the normal development of hierarchical follicles affect this difference. Thus, we used high-throughput sequencing technology to perform transcriptome analysis of ovarian tissues from 80-wk-old Hy-Line Brown layers with high- and low-egg-laying rates (HH and HL), and an association with the laying performance gene stanniocalcin 1 (STC1) was found. The proliferation and apoptosis of granulosa cells (GCs), as the basic functional cells of ovarian follicles, are highly correlated with the normal development and regression of follicles. Therefore, this study used ovarian follicular GCs cultured in vitro to study the effects of the STC1 gene on the proliferation, apoptosis, and secretion function of GCs and to explore its mechanism of action, laying a foundation for the study of the regulation of the STC1 gene on follicular development.