Discover QTLs for the level of blood components in Shaoxing duck using GWAS and haplotype sharing analysis.

Blood composition is indicative of health-related traits such as immunity and metabolism. The use of molecular genetics to investigate alterations in these attributes in laying ducks is a novel approach. Our objective was to employ genome - wide association studies (GWAS) and haplotype - sharing analysis to identify genomic regions and potential genes associated with 11 blood components in Shaoxing ducks. Our findings revealed 35 SNPs and 1 SNP associated with low-density lipoprotein cholesterol (LDL) and globulin (GLB), respectively. We identified 36 putative candidate genes for the LDL trait in close proximity to major QTLs and key loci. Based on their biochemical and physiological properties, TRA2A, NPY, ARHGEF26, DHX36, and AADAC are the strongest putative candidate genes. Through linkage disequilibrium analysis and haplotype sharing analysis, we identified three haplotypes and one haplotype, respectively, that were significantly linked with LDL and GLB. These haplotypes could be selected as potential candidate haplotypes for molecular breeding of Shaoxing ducks. Additionally, we utilized a bootstrap test to verify the reliability of GWAS with small experimental samples. The test can be accessed at https://github.com/xuwenwu24/Bootstrap-test.

Cu exposure induces liver inflammation via regulating gut microbiota/LPS/liver TLR4 signaling axis.

Copper (Cu) serves as an essential cofactor in all organisms, yet excessive Cu exposure is widely recognized for its role in inducing liver inflammation. However, the precise mechanism by which Cu triggers liver inflammation in ducks, particularly in relation to the interplay in gut microbiota regulation, has remained elusive. In this investigation, we sought to elucidate the impact of Cu exposure on liver inflammation through gut-liver axis in ducks. Our findings revealed that Cu exposure markedly elevated liver AST and ALT levels and induced liver inflammation through upregulating pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and triggering the LPS/TLR4/NF-κB signaling pathway. Simultaneously, Cu exposure induced alterations in the composition of intestinal flora communities, notably increasing the relative abundance of Sphingobacterium, Campylobacter, Acinetobacter and reducing the relative abundance of Lactobacillus. Cu exposure significantly decreased the protein expression related to intestinal barrier (Occludin, Claudin-1 and ZO-1) and promoted the secretion of intestinal pro-inflammatory cytokines. Furthermore, correlation analysis was observed that intestinal microbiome and gut barrier induced by Cu were closely related to liver inflammation. Fecal microbiota transplantation (FMT) experiments further demonstrated the microbiota-depleted ducks transplanting fecal samples from Cu-exposed ducks disturbed the intestinal dysfunction, which lead to impaire liver function and activate the liver inflammation. Our study provided insights into the mechanism by which Cu exposure induced liver inflammation in ducks through the regulation of gut-liver axis. These results enhanced our comprehension of the potential mechanisms driving Cu-induced hepatotoxicity in avian species.

Two cis-regulatory SNPs upstream of ABCG2 synergistically cause the blue eggshell phenotype in the duck.

Avian eggshell color is an interesting genetic trait. Here, we report that the blue eggshell color of the domestic duck is caused by two cis-regulatory G to A transitions upstream of ABCG2, which encodes an efflux transporter. The juxtaposed blue eggshell allele A-A exhibited higher promoter activity and stronger nuclear protein binding capacity than the white eggshell allele G-G. Transcription factor analysis suggested differential binding capability of CTCF between blue eggshell and white eggshell alleles. Knockdown of CTCF expression significantly decreased the promoter activity of the blue eggshell but not the white eggshell allele. DNA methylation analysis revealed similar high methylation of the region upstream of the CTCF binding sites in both blue-eggshelled and white-eggshelled ducks. However, DNA methylation levels downstream of the binding sites were decreased and 35% lower in blue-eggshelled ducks than in white-eggshelled ducks. Consistent with the in vitro regulatory pattern of causative sites, ABCG2 exhibited higher expression in uteruses of blue-eggshelled ducks and also showed polarized distribution in their endometrial epithelial cells, distributing at the apical surface of endometrial epithelial cells and with orientation toward the uterine cavity, where the eggshell is pigmented. In conclusion, our results suggest that two cis-regulatory SNPs upstream of ABCG2 are the causative mutations for blue eggshells in ducks. The blue eggshell variant up-regulated ABCG2 expression through recruiting CTCF binding, which may function as a barrier element to shield the downstream region from high methylation levels present upstream. ABCG2 was identified as the only candidate causative gene for blue eggshells; it may function as an efflux transporter of biliverdin to the uterine cavity.

Genome-wide association studies and haplotype sharing analysis targeting the growth traits in Yandang partridge chickens.

The body size of a chicken is an economically important trait as it directly influences the benefits of the poultry industry, but the relevant genetic mechanisms have not yet been elucidated. In this study, we measured eight growth traits for 94 Yandang partridge chickens, then undertook genome-wide association studies (GWAS) for those traits in using a linear mixed model based on 10× whole genomic sequencing data to better understand the knowledge of the genetic architecture of growth traits. Ninety-four individuals and 7647883 SNPs remained after quality control and removal of the sex chromosomes, and these data were used to carry out a GWAS analysis. The result showed that only one significant single-nucleotide polymorphisms (SNP) locates at 14852873 bp on SSC13 surpassed the genome-wide significance level for Keel length (KL). Through linkage disequilibrium analysis and haplotype sharing analysis, we identified one haplotype underlying the SSC13 significantly associated with KL, which could be selected as a potential candidate haplotype that is used in molecular breeding of Yandang partridge chicken. On the other hand, we have learned from a method called bootstrap testing to verify the reliability of GWAS with small experimental samples, which users can access at https://github.com/xuwenwu24/Bootstrap-test.

Immunopromoter improves liver apoptosis and immune response in Shaoxing ducklings.

Antibiotics as feed additives, play a vital role in animal husbandry. However, overused antibiotics could cause endogenous infections in animals, and even endanger human health through the food chain. And immunopotentiators can make the low immune function improve and accelerate the induction of immune response. The aim of this study was to investigate the effects of five different immunopotentiators on the expression of liver apoptosis and immune factor related genes in Shaoxing ducklings (Anas Platyrhynchos). A total of 150 one-day-old Shaoxing ducklings were randomly divided into six groups including saline, chlorogenic acid, ß-D-glucan, astragalus flavone, CpG DNA and chicken IgG, which were injected subcutaneously into the neck, respectively. At 18 days old, the liver tissues were collected to detecte the mRNA and protein expression levels of inflammatory and apoptosis-related genes. The results showed that compared with the control group, the mRNA and protein levels of liver Bcl2 with chlorogenic acid, ß-D-glucan, astragalus flavone, CpG-DNA and chicken IgG were significantly decreased (p < 0.05), while the expression level of Caspase3 was up-regulated in some different degrees. In addition,The expression levels of liver iNOS and COX2 were significantly increased after the injection of five immunopotentiators (p < 0.05), and the mRNA levels of IFN-α, IFN-ß, IL-1ß, RIG-I, TLR3 and TLR7 genes were also significantly up-regulated compared with the control group (p < 0.05). In conclusion, chlorogenic acid, ß-D-glucan, astragalus flavone, CpG-DNA and chicken IgG can be used as immunopotentiators to regulate duck innate immunity. This study provides a new way to prevent important infectious diseases of ducks, and also provides a certain reference for the application of antibiotic substitutes in animal production.

Chromosome-level genome assembly of the Muscovy duck provides insight into fatty liver susceptibility.

The Muscovy duck (Cairina moschata) is an economically important poultry species, which is susceptible to fatty liver. Thus, the Muscovy duck may serve as an excellent candidate animal model of non-alcoholic fatty liver disease. However, the mechanisms underlying fatty liver development in this species are poorly understood. In this study, we report a chromosome-level genome assembly of the Muscovy duck, with a contig N50 of 11.8 Mb and scaffold N50 of 83.16 Mb. The susceptibility of Muscovy duck to fatty liver was mainly attributed to weak lipid catabolism capabilities (fatty acid ß-oxidation and lipolysis). Furthermore, conserved noncoding elements (CNEs) showing accelerated evolution contributed to fatty liver formation by down-regulating the expression of genes involved in hepatic lipid catabolism. We propose that the susceptibility of Muscovy duck to fatty liver is an evolutionary by-product. In conclusion, this study revealed the potential mechanisms underlying the susceptibility of Muscovy duck to fatty liver.

Chlorogenic Acid Alleviates LPS-Induced Inflammation and Oxidative Stress by Modulating CD36/AMPK/PGC-1α in RAW264.7 Macrophages.

Chlorogenic acid (CGA) is a bioactive substance with anti-inflammatory activities. Clusters of CD36 have been suggested to be widely involved in inflammatory damage. However, the mechanism of CGA protecting against LPS-induced inflammation involving the CD36 regulation is unclear. Here, we demonstrated that CGA protected against LPS-induced cell death and decreased the production of ROS. Moreover, the SOD, CAT, and GSH-Px activities were also upregulated in CGA-treated cells during LPS stimulation. CGA reduced COX-2 and iNOS expression and IL-1ß, IL-6, and TNF-α secretion in LPS-stimulated RAW264.7 macrophages. In addition, CGA treatment widely involved in immune-related signaling pathways, including NF-κB signaling, NOD-like receptor signaling, and IL-17 signaling using transcriptomic analysis and CD36 also markedly reduced during CGA pretreatment in LPS-induced RAW264.7 cells. Furthermore, the CD36 inhibitor SSO attenuated inflammation and oxidative stress by enabling activation of the AMPK/PGC-1α cascade. These results indicate that CGA might provide benefits for the regulation of inflammatory diseases by modulating CD36/AMPK/PGC-1α to alleviate oxidative stress.

Single-cell RNA sequencing revealed the liver heterogeneity between egg-laying duck and ceased-laying duck.

BACKGROUND: In the late phase of production, ducks untimely cease laying, leading to a lower feed conversion. Liver plays a vital role in the synthesis and transport of yolk materials during egg formation in birds. However, the molecular mechanism of liver in ceased-laying duck is far from clear, higher resolution and deeper analysis is needed. Sing-cell RNA-sequencing of 10 × Genomics platform can help to map the liver single cell gene expression atlas of Shaoxing duck and provide new insights into the liver between egg-laying and ceased-laying ducks. RESULTS: About 20,000 single cells were profiled and 22 clusters were identified. All the clusters were identified as 6 cell types. The dominant cell type is hepatocyte, accounted for about 60% of all the cells. Of note, the heterogeneity of cells between egg-laying duck and ceased-laying duck mainly occurred in hepatocytes. Cells of cluster 3 and 12 were the unique hepatocyte states of egg-laying ducks, while cells of cluster 0 and 15 were the unique hepatocyte states of ceased-laying ducks. The expression mode of yolk precursor transporters, lipid metabolizing enzymes and fibrinogens were different in hepatocytes between egg-laying duck and ceased-laying duck. APOV1, VTG2, VTG1, APOB, RBP, VTDB and SCD might be activated in egg-laying ducks, while APOA1, APOA4, APOC3, FGB and FGG might be activated in ceased-laying ducks. CONCLUSIONS: Our study further proofs that APOV1 and APOB play key roles in egg production, rather than APOA1 and APOA4. It is also the first to detect a correlation between the higher expression of APOC3, FGB, FGG and ceased-laying in duck.

Transcriptome analyses of potential regulators of pre- and post-ovulatory follicles in the pigeon (Columba livia).

To identify the dominant genes controlling follicular maturation, ovulation and regression for pigeon, we used RNA-seq to explore the gene expression profiles of pre- and post-ovulatory follicles of pigeon. We obtained total of 4.73million (96% of the raw data) high-quality clean reads, which could be aligned with 20282 genes. Gene expression profile analysis identified 1461 differentially expressed genes (DEGs) between the pre- (P4) and post-ovulatory follicles (P5). Of these, 843 genes were upregulated, and 618 genes were down-regulated. Furthermore, many DEGs were significantly enriched in some pathways closely related to follicle maturation, ovulation and regression, such as ECM-receptor interaction, vascular smooth muscle contraction, progesterone-mediated oocyte maturation, phagosome. Importantly, the DGEs in ECM-receptor interaction pathway included COL1A1 , COL1A2 , COL4A1 , COL4A2 , ITGA11 , ITGB3 and SDC3 , in the progesterone-mediated oocyte maturation pathway involved CDK1 , CDC25A , CCNB3 , CDC20 and Plk1 , and in the vascular smooth muscle contraction covered CALD1 , KCNMA1 , KCNMB1 , CACNA1 , ACTA2 , MYH10 , MYL3 , MYL6 , MYL9 , closely related to promoting follicular maturation and ovulation in pre-ovulatory follicles. Moreover, it seems that the lysosomal cathepsin family has a decisive role in the regression of early stage of post-ovulatory follicle. Taken together, these data enrich the research of molecular mechanisms of pigeon follicular activities at the transcriptional level and provide novel insight of breeding-related physiology for birds.

Molecular cloning, tissue distribution and function analysis of duck TLR7.

Toll-like receptors (TLRs) play an important role in detecting pathogen-associated molecular patterns (PAMPs). Among the TLRs, TLR7 is involved in the recognition of antiviral compounds and single-stranded RNA. This study was designed to explore the structure and function of TLR7 in duck (Anas platyrhynchos), a natural host for avian influenza virus. Firstly, the full-length cDNA of Shaoxing egg-laying duck TLR7 (duTLR7) was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). It consisted of 38 base pairs (bp) 5'-untranslated region (UTR), 187 bp 3'-UTR, and 3270 bp open reading frame that encodes a single protein of 1089 amino acid residues. DuTLR7 shares high identity with TLR7 genes from other vertebrates. In healthy ducks, duTLR7 transcripts were broadly expressed in different tissues, with higher expression levels in the liver, kidney, and thymus. The highest relative transcript level of duTLR7 could be induced with R848 stimulation. In addition, overexpression of duTLR7 by stimulating with poly(I:C) significantly promoted IFN-ß, NF-κB, IRF7, TRIF, Mx, STAT1 and STAT2 expressions. Taken together, these results suggest that TLR7 may play an important role in the innate immune response of ducks.