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Background: bones and muscles originated together from the mesoderm during embryogenesis, and they can influence each other through mechanical stimulations and chemical signals. The sclerostin (SOST) is secreted from mature osteocytes. Here, we used a bird model to illustrate the potential roles of SOST on duck myoblasts to verify the hypothesis that SOST might play functions in coordinating the development of bones and muscles. Methods and Results: Firstly, a recombinant adenovirus vector carrying duck SOST was constructed. Then, the adenovirus-mediated duck SOST was transfected into duck myoblasts. The results revealed by CCK-8 showed that the cell proliferation of myoblasts was inhibited after 12 h, 36 h, and 48 h treatment by transfection of SOST. The labeling rates of EdU positive cells in the Ad-duSOST group were significantly lower than the Ad-NC group (P

Background As a major economic trait in poultry, egg production efficiency attracts widespread interest in breeding and production. However, limited information is available about the underlying genetic architecture of egg production traits in ducks. In this paper, we analyzed six egg production-related traits in 352 F2 ducks derived from reciprocal crosses between mallard and Pekin ducks. Results Feed conversation ratio (FCR) was positively correlated with feed intake but negatively correlated with egg-related traits, including egg weight and egg production, both phenotypically and genetically. Estimates of pedigree-based heritability were higher than 0.2 for all traits investigated, except hip-width. Based on whole-genome sequencing data, we conducted genome-wide association studies to identify genomic regions associated with these traits. In total, 11 genomic regions were associated with FCR. No genomic regions were identified as significantly associated with hip-width, total feed intake, average daily feed intake, and total egg production. Analysis of selective sweeps between mallard and Pekin ducks confirmed three of these genomic regions on chromosomes 13, 3 and 6. Within these three regions, variants in candidate genes that were in linkage disequilibrium with the GWAS leader single nucleotide polymorphisms (SNPs) (Chr13:2,196,728, P = 7.05 × 10–14; Chr3:76,991,524, P = 1.06 × 10–12; Chr6:20,356,803, P = 1.14 × 10–10) were detected. Thus, we identified 31 potential candidate genes associated with FCR, among which the strongest candidates are those that are highly expressed in tissues involved in reproduction and nervous system functions of ducks: CNTN4, CRBR, GPR63, KLHL32, FHL5, TRNT1, MANEA, NDUFAF4, and SCD. Conclusions For the first time, we report the identification of genomic regions that are associated with FCR in ducks and our results illustrate the genomic changes that occurred during their domestication and are involved in egg production efficiency.

1. High stocking densities can lead to animal stress responses and lead to changes in bird behaviour, egg production and the fertility of laying birds. The oviduct plays a crucial role during the process of laying eggs. Therefore, it is essential to know how high stocking density affects oviduct function.2. In this study, a total of 2,115 differentially expressed genes (DEGs) were identified in duck oviduct tissues between different stocking density groups. These genes are mainly enriched in membrane components, calcium ion binding, cytokine-cytokine receptor interaction and focal adhesion. These pathways were closely related to the formation of eggs. This indicated that secretion and material transport functions of the oviduct are affected under high-density stocking. Further analysis showed that a total of 408 genes related to the transportation process were expressed in the oviduct, of which 96 genes were differentially expressed (LogFC≥1, P 0.05). Forty-two of these DEGs belonged to the solute carrier family. The data showed that the expression of 31 transcripts was different between the two density groups. Expression of

Egg internal quality traits are important traits related to egg production in poultry industry. To better understand the genetic architecture of egg internal quality traits in ducks, we performed genetic parameters estimates and a genome-wide association study (GWAS). The phenotypic values of egg weight, yolk color, albumin height (AH), yolk weight, and Haugh unit (HU) were collected individually from 352 F2 laying ducks produced by reciprocal crosses between mallards and Pekin ducks, and their genotypes were assayed by whole genome re-sequencing. The results showed that the AH and HU traits have a clear coefficient of variance, around 15% for both mallards and Pekin ducks. The pedigree-based genetic parameters estimates rane from 0.26 to 0.71 for all eight egg quality traits, while the highest heritability was 0.71 for egg weight. The GWAS showed that a clear signal was associated with AH and HU traits. The locus zoom analysis and conditional GWAS helped to narrow the candidate region to ~5.8-Mb spanning from 14.7 to 20.5 Mb on Chromosome 5, which harbored 111 candidate genes. MUC6 and LDLRAD3 were finally promised as the major candidate genes affecting albumen composition. Our data revealed the egg internal quality traits for the first time in ducks, which provides a theoretical basis and technological support for improving duck egg internal quality.

Avian egg coloration is shaped by natural selection, but its genetic basis remains unclear. Here, we used genome-wide association analysis and identity by descent to finely map green egg colour to a 179-kb region of Chr4 based on the resequencing of 352 ducks (Anas platyrhynchos) from a segregating population resulting from the mating of Pekin ducks (white-shelled eggs) and mallards (green-shelled eggs). We further narrowed the candidate region to a 30-kb interval by comparing genome divergence in seven indigenous duck populations. Among the genes located in the finely mapped region, only one transcript of the ABCG2 gene (XM_013093252.2) exhibited higher uterine expression in green-shelled individuals than in white-shelled individuals, as supported by transcriptome data from four populations. ABCG2 has been reported to encode a protein that functions as a membrane transporter for biliverdin. Sanger sequencing of the whole 30-kb candidate region (Chr4: 47.41-47.44 Mb) and a plasmid reporter assay helped to identify a single nucleotide polymorphism (Chr4: 47,418,074 G>A) located in a conserved predicted promoter region whose variation may alter ABCG2 transcription activity. We provide a useful molecular marker for duck breeding and contribute data to the research on ecological evolution based on egg colour patterns among birds.

Skin color is colorful for birds, which has been reported to be associated with multi-biological functions, such as crypsis, camouflage, social signaling and mate choice, but little is known about its underlying molecular mechanism. Studies on the major genes affecting the black skin color of ducks. For this purpose, Silver ammonia staining and RNA-seq analysis were carried out to identify the differences in tissue morphology and gene expressions between black and yellow skin ducks. The silver ammonia dyes slice results showed that in the development of black duck, the content of melanin in black skin gradually increased and then decreased, and the content of melanin in yellow and black skin was significantly different. Through transcriptome, a total of 102 and 84 differentially expressed genes (DEGs) were identified in beak skin and web skin, respectively. These DEGs were enriched in melanin biosynthesis and play a critical role in melanogenesis pathway. Co-expression analysis showed that EDNRB2 was the only gene associated with black skin color in DEGs, which was also consistent with qRT-PCR. The melanin synthesis pathway dominated by EDNRB2 up-regulated the amount of melanin synthesis, leading to the formation of black skin in ducks.

There are great differences in physiological and biological functions between animals of different sexes. However, whether there is a consensus between sexes in duck intestinal development and microorganisms is still unknown. The current study used Nonghua ducks to estimate the effect of sex on the intestine by evaluating differences in intestinal growth indexes and microorganisms. The intestines of male and female ducks were sampled at 2, 5, and 10 wk from the duodenum, jejunum, ileum, and cecum. Then, the intestinal length and weight were measured, the morphology was observed with HE staining, and the intestinal content was analyzed by 16S rRNA sequencing. The results showed that male ducks have shorter intestinal lengths with higher relative weights/relative lengths. The values of jejunal villus height (VH)/crypt depth (CD) of female ducks were significantly higher at 2 wk, whereas the jejunal VH/CD was significantly lower at 10 wk. There was obvious separation of microorganisms in each intestinal segment of ducks of different sexes at the 3 time periods. The dominant phyla at different stages were Firmicutea, Proteobacteria, Bacteroidetes, and Actinobacteria. The duodenal Chao index at the genus level of male ducks was significantly higher at 10 wk than that of female ducks. Significantly different genera were found only in the jejunum, and the abundances of Escherichia_Shigella, Pseudomonas, Clostridium_sensu_stricto_1, Sphingomonas, and Desulfovibrio in male ducks were higher than those in female ducks, whereas the abundance of Rothia was lower, and the abundance of viral infectious diseases, lipid metabolism, metabolism of terpenoids and polyketides, parasitic infectious diseases, xenobiotic biodegradation and metabolism, cardiovascular disease, and metabolism of other amino acids in male ducks were higher than that in female ducks, whereas gene folding, sorting and degradation pathways, and nucleotide metabolism were lower. This study provides a basic reference for the intestinal development and microbial symbiosis of ducks of different sexes.

1. When geese or ducks are overfed with a high-energy diet rich in carbohydrates, their liver increases in size by 5- to 10-fold in 2 weeks, which is accompanied by the occurrence of hepatic steatosis. As a result, this distinctive genetic characteristic of waterfowl has been taken advantage of to produce foie gras. 2. When overfed geese were fed a regular diet for a 20-d period of recovery, their liver was restored to the original state. Hence, the entire process is reversible, and cause no lasting cirrhosis or necrosis in the liver. This suggests that waterfowl have a mechanism to protect their liver from the harm caused by severe hepatic steatosis. 3. This paper reviews the formation, physiological changes to metabolic pathways and the protective mechanisms in geese and ducks with hepatic steatosis. Not only will understanding these mechanisms provide ideas for the waterfowl breeding selection for foie gras, it is conducive to improving production efficiency and quality of foie gras. This provides a scientific basis to ensure animal welfare and an approach to the prevention and treatment of fatty liver disease in human.

Rearing system is a critical nongenetic factor influencing meat quality of ducks. In this study, a total of 360 birds were randomly allocated into floor rearing system (FRS) and net rearing system (NRS) to compare their effects on intramuscular fat (IMF) deposition, fatty acid composition, and related gene expression in muscles of Nonghua ducks. Sawdust bedding and stainless mesh bed were equipped in FRS and NRS, respectively. At the eighth week (8w) and 13th week (13w), the breast and thigh muscles of ducks were collected to determine the profiles of lipids composition and the expressions of lipid metabolism-related genes. The IMF content was higher in 13w-FRS than 8w-FRS and 8w-NRS in breast muscle, whereas it was higher in 13w-NRS than other groups in thigh muscle (P < 0.05). C16:1, C20:5(n-3) of muscles were higher in 8w-NRS than 8w-FRS, whereas C18:1(n-9)c, C18:2(n-6)c, Ʃ monounsaturated fatty acid (MUFA), and ƩMUFA/Ʃsaturated fatty acid (SFA) ratio of muscles were higher in 13w-NRS than 8w-FRS and 8w-NRS (P < 0.05). C22:6(n-3), C20:4(n-6) of breast muscle and C20:3(n-6) of thigh muscle were higher in 13w-NRS than 13w-FRS (P < 0.05). Fatty acids variation was studied by principal component analysis, exhibiting extensive positive loadings on principal components. SREBP1, ACADL, and FABP3 were downregulated in breast muscle, whereas PPARα and ELOVL5 were upregulated in thigh muscle of NRS ducks at 13w. Principal components were extensively correlated with lipids composition parameters, and principal components of breast muscle 1 and principal components of thigh muscle 1 were correlated with SREBP1 and PPARα, respectively (P < 0.05). In conclusion, with increasing age, FRS enhanced IMF deposition in breast muscle, and the same promotion in thigh muscle was because of NRS. The variation of fatty acids in muscles was uniform, and the change of single fatty acid was unable to distinguish NRS and FRS. However, as NRS downregulated SREBP1, ACADL and FABP3 in breast muscle and upregulated PPARα and ELOVL5 in thigh muscle, NRS could improve nutrient value and meat quality by increasing ƩMUFA, ƩMUFA/ƩSFA ratio, and important PUFA levels. Therefore, NRS was more recommended than FRS for Nonghua ducks during week 8 to 13 posthatching.

1. Birds' newly oviposited blastoderms can survive several weeks in a dormant state during low-temperature storage. Previous studies demonstrated that there is a critical temperature range from 19 to 27°C for chicken embryos. Within this range, the embryo will diapause in a dormant state; once the temperature rises above this range, the blastoderm will break dormancy. 2. Clarifying the mechanism that initiates duck embryo developmental recovery from blastoderm dormancy will be helpful to change temperature control to improve hatching in poultry production. It was hypothesised that there might be some temperature-sensitive genes involved in initiating duck embryo developmental recovery from blastoderm dormancy. 3. To test this hypothesis, the transcriptome of the newly oviposited duck blastoderm and duck embryo (incubated for 48 hours) were sequenced to screen for differentially expressed genes with functions that had been predicted by bioinformatics. 4. The results showed that there were 2416 differentially expressed genes between the two groups, 53 of which were involved in temperature-sensitive pathways. The protein-protein interaction network combined these 53 temperature-sensitive genes and another group of 65 genes, which enriched the development pathway. These results suggested that temperature-sensitive genes may be involved in growth and development related pathways.

The Jianchang duck is mainly distributed in Southwest China, and has the characteristics of fast growth rate and strong abilities in lipid deposition in the liver. In order to investigate the effects of domestication process on formation of the unique characteristics of Jianchang duck, the whole genome of sixteen individuals and three pooling of Jianchang duck were re-sequenced, and genome data of 70 mallards and 83 domestic ducks from thirteen different places in China were obtained from NCBI. The population stratification and evolution analysis showed gene exchanges existed between the Jianchang and other domestic duck populations, as well as Jianchang ducks and mallards. Genomic comparison between mallards and Jianchang ducks showed genes, including CNTN1, CHRNA9, and SHANK2, which is involved in brain and nerve development, experienced strong positive selection in the process of Jianchang duck domestication. The genomic comparison between Jianchang and domestic duck populations showed that HSD17B12 and ESM1, which affect lipid metabolism, experienced strong positive selection during the domestication process. FST analysis among populations of Jianchang duck with different plumage colors indicated that MITF was related to the phenotype of a white feather, while MC1R was related to the phenotype of hemp feather. Our results provided a base for the domestication process of Jianchang duck and the genomic genes for unique traits.

The feather is an important epidermal appendage, plays an important role in the life activities of avian specie, and has important economic value. Revealing the molecular regulation mechanism of feather growth has a significant meaning in studying adaptive evolution, physiology, and mating of avian species and also provides a theoretical reference for poultry breeding. In this study, the genome-wide association analysis (GWAS) of 358 ducks was based on primary feather length phenotypic data (28-60 d), length growth rates (LGRs), and maturity scores (60 d) to explore the genetic basis affecting feather growth and maturation. The results showed that, among the primary feather 1 to 5 in ducks, the mean LGR of primary feather 2 was the fastest, with the longest length. The primary feathers in males grew and matured slightly faster than in females. The mean maturity scores of primary feather 10∼7 were higher than primary feather 1 to 3 in ducks. GWAS further showed 116 SNPs associated with feather length traits. In addition, 2 candidate regions (Chr1: 127,407,230-127,524,879 bp and Chr21: 182,061,707-183,616,298 bp) were associated with LGR, which contain total 13 candidate genes (The extremely significant SNPs were mainly located in 2 genes: Chr1: REPS2 and Chr21: PTPRT). Four candidate regions (Chr1: 29,113,036-28,675,018 bp, Chr2: 18,253,612-149,111,290 bp, Chr15: 6,489,774 to 12,138,221 bp and Chr21: 6,578,021-8,472,904 bp) were associated with feather maturity, which contain total 24 candidate genes (The extremely significant SNPs were mainly located in 4 genes: Chr1: IMMP2L, DOCK4 and DDX10, Chr2: LDLRAD4). In conclusion, sex factors influence feather growth and maturity, and the genetic basis of the growth /maturity trait between different feathers is similar. REPS2, PTPRT genes, and IMMP2L, DOCK4, DDX10, and LDLRAD4 are important candidate genes that influence feather growth and maturity, respectively.

Background Skin pigmentation is a broadly appearing phenomenon of most animals and humans in nature. Here we used a bird model to investigate why melanin spot deposits on the skin. Results Our result showed that growth age and the sunlight might induce melanin deposition in bird beak skin which was determined by genetic factors. GWAS helped us to identify two major loci affecting melanin deposition, located on chromosomes 13 and 25, respectively. The fine mapping works narrowed the candidate regions to 0.98 Mb and 1.0 Mb on chromosomes 13 and 25. The MITF and POU2F3 may be the causative genes and synergistically affect melanin deposition during duck beak skin. Furthermore, our data strongly demonstrated that the pathway of melanin metabolism contributes to melanin deposition on the skin. Conclusions We demonstrated that age and sunlight induce melanin deposition in bird beak skin, while heredity is fundamental. The MITF and POU2F3 likely played a synergistic effect on the regulation of melanin synthesis, and their mutations contribute to phenotypic differences in beak melanin deposition among individuals. It is pointed out that melanin deposition in the skin is related to the pathway of melanin metabolism, which provided insights into the molecular regulatory mechanisms and the genetic improvement of the melanin deposition in duck beak.

Eggs are essential food sources as they provide low cost and high nutritional content of animal protein. The preservation period is one of the apparent factors affecting egg quality. Previous studies based on traditional detection techniques demonstrated that storage period would significantly influence egg weight, eggshell weight, albumen height, haugh unit (HU) and albumen viscosity. Herein, we employed non-targeted metabolome technology to reveal the comprehensive changes in metabolite composition in duck eggs under the impacts of storage period.The results showed that the primary metabolites in the yolk of duck eggs are amino acids, carbohydrates and lipids. In contrast, the primary metabolites in the albumen are amino acids, benzene and indoles. We screened 43 and 16 different metabolites, respectively, in the albumen and yolk of duck eggs with different preservation periods. In addition, kyoto encyclopedia of genes and genomes (KEGG) enrichment was performed, and the results showed that various nutrients were degraded in the egg after preservation, thus affecting the quality of duck eggs. These nutrients included amino acids, fatty acids, nucleotides, sugars and vitamins; meanwhile, ammonia, biogenic amines and some flavor substances were produced, affecting the quality of the eggs.Ourfindings can contribute to a holistic understanding of metabolite composition changes in duck eggs during deterioration in storage. © 2022 Society of Chemical Industry.

1. The thymus and bursa of Fabricius are important immune organs in poultry as they play essential roles in sustaining the normal immune function to maintain health. The following trial investigated whether the stocking density affected gene expressions in immune organs. 2. Jinding ducklings were raised in either low or high density (4 or 8 birds/m2) conditions from four to 14 weeks of age, and were then slaughtered and tissues removed. Samples were subjected to high-throughput sequencing to sequence RNA extraction. After filtering calculations with R software, a total of 508 (thymus) and 1,356 (bursa of Fabricius) differentially expressed genes (DEGs) were identified, suggesting that stocking density has an effect on gene expression in duck immune organs. 3. Out of a total of 112 immune factor genes and 112 immune pattern receptor genes in ducks, four thymus and 18 bursa of Fabricius genes were differentially expressed in ducks, which indicated that the change of stocking density could affect the expression of immune genes in poultry.

Poultry products are an essential animal source of protein for humans. Many factors could destroy the balance of the poultry production chain and cause an overstock of products, which need to be stored in the frozen storage warehouse for a long time. The long-term frozen storage may affect the quality of meat products. In this study, the changes of small molecular substances were revealed in duck meat during long-term storage using non-targeted metabolomics. The results showed that compared with fresh meat, even if the meat is stored under frozen storage conditions, the number of differential metabolites of frozen storage meat continues to increase with the prolongation of storage time, indicating that the meat composition has changed significantly with the storage time increased. With the increase in storage time, the nitrogen-containing small molecular compounds in duck meat increased (carnosine and anserine, aspartic acid, and tyrosine, 1H-indole-3-acetamide, 2-Hydroxyphenethylamine, 2-Naphylamine, allocystathionine, and O-phosphoethanolamine), the nucleotides decomposition process strengthened (IMP and AMP, GMP and UMP), and the content of organic acid increased (5-hydroxy indole acetic acid, 5-hydroxypentanoic acid and phenylacetate, taurine) and carbohydrate (1-O-sinapoyl-beta-d-glucose, 4-O-beta-d-glucopyranosyl-d-mannose, and alpha-d-glucose). These small molecular substances can be used as biomarkers to detect long-term stored duck meat deterioration. KEGG enrichment analysis showed that protein catabolism, nucleotide catabolism, fat decomposition and oxidation, and carbohydrate decomposition were the main metabolic processes of meat deterioration during the long-term storage of duck meat. In addition, Non-target metabolome technology is a powerful tool to reveal the meat deterioration process during long-term storage systematically. This study provided a reference for optimizing domestic poultry meat storage methods and ensuring food safety.

The light intensity can affect the production performance of animals. The retina and pineal gland, closely linked, are directly photosensitive organs. This study evaluated the effect of light intensity on duck growth and investigated the effects of varying light intensities on retina and pineal gland transcriptome changes. The increase of light intensity will significantly decrease production performance, such as body weight, eviscerated weight, breast muscle weight, percentage of abdominal fat, etc. The RNA-seq revealed 967 and 201 differentially expressed genes (DEGs) in the retina and pineal gland under different light intensities, respectively. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) in the retina showed the DEGs were enriched in ECM-receptor interaction, Focal adhesion, Cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction, Melanogenesis, etc. Meanwhile, the DEGs in the pineal gland were mainly enriched in pathways associated with the mTOR signaling pathway, melanin production pathway, etc. Our results indicated that different light intensities might affect the function of the retina and pineal gland, including the melanin production of the retina and the secretion of melatonin in the pineal gland. Our study can provide a theoretical basis for the molecular mechanism of the effects of different light intensities on the retina and pineal gland.

As the largest single bone, avian sterna are very different from those of mammals in terms of morphology and functions. Moreover, years of artificial selection in poultry led to incomplete sternal ossification at slaughter age, which may cause diseases, sternal injury, and restriction to breast muscle growth. However, in living birds, studies have rarely described the ossification pattern and underlying mechanisms of the sterna. Here, we examined the pattern (timeline, ossification centers, ossification directions, weekly changes of different parts, quantified differences in ossification degree among sexes and parts) and developmental changes (histological structure, gene expression) of postnatal duck sternal ossification. Direct observation and alcian blue and alizarin red staining of whole sterna samples revealed that, duck sterna mainly ossified during 5 to 9 wk old with five ossification centers. These centers and their ossification directions were different from and more complex than the previously studied birds. The weekly changes of sterna and the quantitative analysis of ossification-related traits showed that ossifications in the three parts of duck sterna (sternum body, keel, posterolateral processes) were mutually independent in space and time, meanwhile, the male duck sterna were more late-maturing than the female. The results of hematoxylin-eosin, alcian blue, and toluidine blue stainings and the expression levels of COL2A1, COL10A1, COL1A2, and CTSK together supported that, duck sternal ossification was highly similar to typical endochondral ossification. Furthermore, continuously high expression of MMP13 and SPARC and their significant (P < 0.05) co-expression with COL2A1, COL10A1, COL1A2, and CTSK suggested the importance of MMP13 and SPARC in duck sternal ossification. Taken together, our results may be helpful for the understanding of avian sternal ossification and the improvement of the performance and welfare of poultry from a new perspective.

Background Birds have various plumage color patterns, and spot is a common phenotype. Herein, we conducted genome-wide association studies (GWAS) in a population of 225 ducks with different sized black spots to reveal the genetic basis of this phenomenon. Results First, we quantified the black spot phenotype within the duck population. The results showed that the uncolored area of the body surface first appeared on the ventral side. With increasing duck age, the area of the black spots was highly conserved across the whole body surface. The GWAS results identified a 198 kb (Chr4: 10,149,651 bp to 10,348,068 bp) genetic region that was significantly associated with the black spot phenotype. The conditional GWAS and linkage disequilibrium (LD) analysis further narrowed the ultimate candidate region to 167 kb (Chr4: 10,180,939 bp to 10,348,068 bp). A key gene regulating melanoblast migration and differentiation, EDNRB2 (Endothelin B receptor-like), was found in the candidate region and having significant mRNA expression level changes in embryonic duck skin tissue with different spot sizes. The significant SNPs (single nucleotide polymorphisms) associated with the EDNRB2 gene were annotated, and two mutations (Chr4: 10,180,939 T > C and Chr4: 10,190,671 A > T) were found to result in the loss of binding sites for two trans-factors, XBP1 and cMYB. The phenotypic effect of these two mutations suggested that they can regulate the size of black spots in a dose-dependent manner, and Chr4: 10,180,939 T > C was the major allele locus. Conclusions Our results revealed that EDNRB2 was the gene responsible for the variation in duck body surface spot size. Chr4: 10,180,939 T > C was the major allele that explained 49.5 % (dorsal side) and 32.9 % (ventral side) of the variation in duck body surface spot size, while 32.1 % (dorsal side) and 19.1 % (ventral side) of the variation could be explained by Chr4: 10,190,671 A > T. The trans-factor prediction also suggested that XBP1 and cMYB have the potential to interact with EDNRB2, providing new insights into the mechanism of action of these genes.

Muscles and bones are anatomically closely linked, and they can conduct communication by mechanical and chemical signals. However, the specific regulatory mechanism between the pectoral muscle and sternum in birds was largely unknown. The present study explored the potential relationship between them in ducks. The result of the sections showed that more nuclei in proliferate states were observed in the pectoral muscle fibers attached to the calcified sternum, than those attached to the un-calcified sternum. The RNA-seq identified 328 differentially expressed genes (DEGs) in the sternum between the calcified and un-calcified groups. Gene ontology (GO) showed that the DEGs were mainly enriched in pathways associated with calcification. In addition, DEGs in the muscles between the calcified and un-calcified sternum groups were mainly annotated to signal transduction receptor pathways. The expression patterns of genes encoding for secreted proteins, in bone (CXCL12, BMP7 and CTSK) and muscle (LGI1), were clustered with muscle development (MB) and bone calcification (KCNA1, OSTN, COL9A3, and DCN) related genes, respectively, indicating the regulatory relationships through a paracrine pathway existing between the sternum and pectoral muscles in ducks. Together, we demonstrated that the pectoral muscle development was affected by the sternal ossification states in ducks. The VEGFA, CXCL12, SPP1, NOG, and BMP7 were possibly the key genes to participate in the ossification of the duck sternum. We firstly listed evidence supporting the regulatory relationships through a paracrine pathway between the sternum and pectoral muscles in ducks, which provided scientific data for the study of the synergistic development of bone and skeletal muscle.

Duck hepatitis A virus 3 (DHAV-3) is a wild endemic virus, which seriously endangers the duck industry in China. The present study aims to elucidate the mechanism of duck resistance to DHAV-3 infection. Both resistant and susceptible ducks were challenged with DHAV-3 in this experiment. The histopathological features and serum biochemical indices (ALT and AST) were analyzed to estimate liver injury status at 6, 12, 15, and 24 h post-infection (hpi). The dynamic transcriptomes of liver were analyzed to explain the molecular regulation mechanism in ducks against DHAV-3. The result showed that the liver injury in susceptible ducks was more serious than that in the resistant ducks throughout the four time points. A total of 2,127 differentially expressed genes (DEGs) were identified by comparing the transcriptome of the two populations. The expression levels of genes involved in innate immune response increased rapidly in susceptible ducks from 12 hpi. Similarly, the expression of genes involved in cytokine regulation also increased at the same time points, while the expression levels of these genes in resistant ducks remained similar between the various time points. KEGG enrichment analysis of the DEGs revealed that the genes involved in cytokine regulation and apoptosis were highly expressed in susceptible ducks than that in resistant ducks, suggesting that excessive cytokine storm and apoptosis may partially explain the mechanism of liver injury caused by DHAV-3 infection. Besides, we found that the FUT9 gene may contribute to resistance towards DHAV-3 in resistant ducklings. These findings will provide insight into duck resistance and susceptibility to DHAV-3 infection in the early phases, facilitate the development of a strategy for DHAV-3 prevention and treatment, and enhance genetic resistance via genetic selection in animal breeding.

Background Rearing systems can affect livestock production directly, but whether they have effects on intestinal growth states and ceca microorganisms in ducks is largely unclear. The current study used Nonghua ducks to estimate the effects of rearing systems on the intestines by evaluating differences in intestinal growth indices and cecal microorganisms between ducks in the floor-rearing system (FRS) and net-rearing system (NRS). Results The values of relative weight (RW), relative length (RL) and RW/RL of the duodenum, jejunum, ileum and ceca in the FRS were significantly higher than those in the NRS during weeks 4, 8 and 13 (p p Conclusions The rearing system influences duck intestinal development and microorganisms. The FRS group had higher intestinal RL, RW and RW/RL and obviously separated ceca microorganisms compared to those of the NRS group. The differential metabolic pathways of cecal microorganisms decreased with increasing age, and the abundance of translation pathways was higher in the NRS group at week 13, while cofactor and vitamin metabolism were more abundant in the FRS group.

Background In animals, many factors affect the small intestinal function and cecal microorganisms, including body weight and genetic background. However, whether paternal weight impacts the small intestinal function and cecal microorganisms remains unknown to date. The current study used Nonghua sheldrake to estimate the effect of paternal weight on the intestine of the offspring by evaluating differences in small intestinal morphology, digestive enzyme activity, and cecal microorganisms between the offspring of male parents with high body weight (group H) and that of male parents with low body weight (group L). Results The results of the analysis of small intestinal morphology showed that the villus height of the jejunum of group H ducks was higher than that of group L ducks, and the difference was significant for ducks at 10 weeks of age. Moreover, the villus height/crypt depth of the duodenum in group H significantly exceeded that of group L at a duck age of 2 weeks. The amylase activity in the jejunum content of group H exceeded that of group L at 5 and 10 weeks of age. Furthermore, the proportion of the Firmicutes to Bacteroidetes was significantly higher in group H (duck age of 2 weeks). Among the genera with a relative abundance exceeding 1%, the relative abundances of genera Desulfovibrio, Megamonas, Alistipes, Faecalibacterium, and Streptococcus observed in group H were significantly different between group H and group L. Conclusions For the first time, this study identifies the effect of paternal weight on offspring small intestinal function and cecal microorganisms. Consequently, this lays a foundation for further research on the relationship between male parents and offspring intestinal function.

MiR-33a is found as a regulator of cell proliferation in many cancer cells. However, it remains unknown if and how miR-33a plays a role in myoblast proliferation. To investigate the effect of miR-33a on myoblast proliferation, miR-33a mimic or inhibitor was co-administered with or without insulin-like growth factor 1 (IGF1) to simulation myoblasts. Our study showed that up-regulation of miR-33a impaired myoblast proliferation, while down-regulation of miR-33a enhanced myoblast proliferation. Mechanistically, we examined that miR-33a can inhibit the transcription of IGF1, follistatin (FST) and cyclin D1 (CCND1) by targeting their 3′UTR region in both HEK293T cells and duck myoblasts. Moreover, up-regulation of miR-33a decreased and its down-regulation increased the mRNA expression of PI3K, Akt, mTOR and S6K. Importantly, the decreased PI3K, Akt, mTOR and S6K expression by miR-33a mimics was abrogated by co-administered with IGF1. Altogether, our results demonstrated that miR-33a may directly target IGF1, FST and CCND1 to inhibit myoblast proliferation via PI3K/Akt/mTOR signaling pathway. In conclusion, miR-33a is a potential negative regulator of myoblast proliferation and by modulating its expression could promote the early development of skeletal muscle.

Fatty acid composition contributes greatly to the nutritional value of meat, and breeds/strains are important factors affecting the composition of fatty acid. Recently, few studies have focused on the fatty acid composition in breast muscle of different duck breeds. Therefore, the objective of the present study was to compare the fatty acid composition and lipid metabolism-related genes expression in breast muscle of Jianchang duck (J), Cherry Verry duck (CV) and 3 crossbred strains (BH1, BH2 and MC♂ × (BGF2♂ × GF2♀)♀ (MBG)). Our results showed that the breast muscle of J had the highest contents of C22:1(n-9) but the lowest ratios of Ʃ-omega 6 (Ʃn-6)/Ʃ-omega 3 (Ʃn-3), Ʃ-mono-unsaturated fatty acid (ƩMUFA)/Ʃ-saturated fatty acid (ƩSFA) and Ʃ-polyunsaturated fatty acid (ƩPUFA)/ƩSFA. The ƩPUFA/ƩSFA ratio was higher in breast muscle of MBG than in that of BH2 and CV, and the contents of C22:1(n-9), ƩMUFA and ƩPUFA were higher in BH1 than in BH2 and CV. Furthermore, the mRNA levels of SCD1, FADS2, ELOVL2, and ELOVL5 were significantly higher in MBG (P0.05), while those of FASD1 and ACACA were significantly higher in BH1 than in BH2 and CV (P0.05). Principal component analysis showed that fatty acids variation exhibited extensive positive loading on principal components (PCs). Correlation analysis showed that PC1 and PC3 of BH1, as well as PC1 of MBG were correlated with the mRNA levels of ACACA and FABP3, respectively. Thus, it could be concluded that the breast muscles of MBG and BH1 have better fatty acid composition, which was closely related to the increased expression levels of SCD1, FADS2, ELOVL2, and ELOVL5 genes in MBG but FADS1 and ACACA in BH1. Moreover, these results also showed that crossbreeding could optimize the composition of fatty acid in breast muscle of ducks.

The cage rearing model of the modern poultry industry makes the bones of birds more vulnerable to deterioration. In this study, at 8 wk of age, a total of 60 birds were randomly allocated to 2 groups, including the floor rearing group (FRD) and cage rearing group (CRD), and their body weight was measured every 2 wk. At the age of 20 wk, the tibia, femur, and humerus were collected from each group (n = 12) to determine the bone quality parameters such as weight, size, bone mineral density (BMD), breaking strength, cortical thickness, and area, ash content, calcium (Ca) content, and phosphorus (P) content. Meanwhile, the serum metabolome composition of both groups was detected by untargeted metabolome technology. The results showed that there were no significant differences in body weight, bone weight, and size between the 2 groups (P > 0.05), but the humerus mineral density and the breaking strength, cortical bone thickness, cortical bone area percentage of tibia, femur, and humerus of CRD was significantly lower than those of FRD (P < 0.05), indicating that the cage rearing system caused the deterioration of bone quality. Based on nontarget metabolomics, 49 metabolites were correlated with bone quality parameters, and 10 key metabolites were strongly correlated, including erucic acid, citric acid, and ketoleucine. In addition, the KEGG analysis showed that the caged system mainly perturbed amino acid metabolism, lipid metabolism, and energy metabolism, which led to changes in related metabolite levels, produced ROS, and altering energy supply, thus leading to a deterioration of bone quality of cage rearing ducks. Therefore, our findings were helpful to further understand the potential mechanism of the deterioration of duck bone quality in cage rearing system, provided a theoretical basis for reducing the occurrence of poultry osteoporosis, and ensuring the healthy development of poultry breeding.

miR-365 is found to be involved in cancer cell proliferation and apoptosis. However, it remains unknown if and how miR-365 plays a role in myoblast proliferation. In the present study, we found that overexpression of miR-365 can inhibit duck myoblast proliferation. To uncover the mechanism by which miR-365 inhibits duck myoblast proliferation, we showed that miR-365 can down-regulate insulin-like growth factor-I (IGF-I) by directly targeting its 3′untranslated region (UTR). Moreover, enhanced miR-365 decreased the mRNA expression of PI3K, Akt, mTOR and S6K. Importantly, the enhanced PI3K, Akt, mTOR and S6K expression by miR-365 inhibitor (anti-miR-365) was abrogated by treatment with LY294002, a PI3K inhibitor. Together, our results indicated that miR-365 may target IGF-I to inhibit duck myoblast proliferation via PI3K/Akt pathway.

As potential substitutes for traditional free-range rearing system, floor-rearing system (FRS) and net-rearing system (NRS) are the current predominant dryland duck rearing systems. However, the influence of these two systems on production performance and duck health is poorly understood. In this study, a 2 × 3 factorial arrangement with two rearing systems (FRS and NRS) and three ages (4w, 8w and 13w) was conducted to study the effects of FRS and NRS on production, antioxidant capacity and immune status of Nonghua ducks. The production performance was mainly affected by the effect of rearing systems at 8w. Body weight, average daily gain, eviscerated weight and semi-eviscerated weight were higher in NRS ducks at 8w, but carcass yield at 8w and 13w was decreased (P 0.05), however, in NRS, slightly better antioxidant capacity was seen at 4w, while glutathione peroxidase (GSH-Px) activity was higher at 13w (P

The Akirin gene family normally contains two members that are essential to myoblast differentiation. Noticeably, the avian Akirin gene family comprises only one gene (Akirin2), However, it remains unknown whether avian Akirin gene family still has the function of Akirin1; moreover, it is still unclear whether and how Akirin2 plays a role in myoblast proliferation and differentiation. Interestingly, the unexpected functions of duck Akirin2 were revealed in the present study. The Real-time PCR results showed that between 12 and 48h during the process of duck myoblasts differentiation, the overexpression of Akirin2 did not significantly increase the expression of myogenic regulatory factors. Flow cytometry analysis revealed that the cell cycle transition was accelerated by Akirin2 overexpression. Moreover, the overexpression of Akirin2 did not influence the myotube formation. Strikingly, when duck myoblasts were cultured in the growth medium, the overexpression of Akirin2 significantly enhanced cell viability. Although the expression of cyclin-dependent proteins did not significantly increase after transfection, the expression of the mammalian targets of rapamycin (mTOR) and p70 S6 kinase (p70S6K) increased. Furthermore, the protein expression of phospho-p70S6K (Ser 417) also increased. However, when rapamycin and pEGFP-N1-Akirin2 plasmids were added together to the growth medium, the positive impact of Akirin2 on cell viability and the mRNA expression of mTOR and p70S6K were significantly blocked. Furthermore, the expression of phospho-mTOR (Ser 2448) and phospho-p70S6K (Ser 417) were also blocked. Taken together, these results could suggest that duck Akirin2 could promote myoblast proliferation via the activation of the mTOR/p70S6K signaling pathway.

Background Fatty acid composition contributes greatly to the quality and nutritional value of meat. However, the molecular regulatory mechanisms underlying fatty acid accumulation in poultry have not yet been cleared. The aims of this study were to characterize the dynamics of fatty acid accumulation in duck breast muscle and investigate its correlations with gene expression. Results Here, we analyzed the fatty acid profile and transcriptome of breast muscle derived from Pekin ducks and mallards at the ages of 2 weeks, 4 weeks, 6 weeks and 8 weeks. Twenty fatty acids were detected in duck breast muscle, with palmitic acid (C16:0, 16.6%~ 21.1%), stearic acid (C18:0, 9.8%~ 17.7%), oleic acid (C18:1n-9, 15.7%~ 33.8%), linoleic acid (C18:2n-6, 10.8%~ 18.9%) and arachidonic acid (C20:4n-6, 11.7%~ 28.9%) as the major fatty acids. Our results showed that fatty acid composition was similar between the two breeds before 6 weeks, but the compositions diverged greatly after this point, mainly due to the stronger capacity for C16:0 and C18:1n-9 deposition in Pekin ducks. By comparing the multistage transcriptomes of Pekin ducks and mallards, we identified 2025 differentially expressed genes (DEGs). Cluster analysis of these DEGs revealed that the genes involved in oxidative phosphorylation, fatty acid degradation and the PPAR signaling pathway were upregulated in mallard at 8 weeks. Moreover, correlation analysis of the DEGs and fatty acid composition traits suggested that the DEGs involved in lipogenesis, lipolysis and fatty acid β-oxidation may interact to influence the deposition of fatty acids in duck breast muscle. Conclusions We reported the temporal progression of fatty acid accumulation and the dynamics of the transcriptome in breast muscle of Pekin ducks and mallards. Our results provide insights into the transcriptome regulation of fatty acid accumulation in duck breast muscle, and will facilitate improvements of fatty acid composition in duck breeding.

Plasma cholinesterase (PCHE) activity is an important auxiliary test in human clinical medicine. It can distinguish liver diseases from non-liver diseases and help detect organophosphorus poisoning. Animal experiments have confirmed that PCHE activity is associated with obesity and hypertension and changes with physiological changes in an animal's body. The objective of this study was to locate the genetic loci responsible for PCHE activity variation in ducks. PCHE activity of Pekin duck × mallard F2 ducks at 3 and 8 weeks of age were analyzed, and genome-wide association studies were conducted. A region of about 1.5 Mb (21.8-23.3 Mb) on duck chromosome 9 was found to be associated with PCHE activity at both 3 and 8 weeks of age. The top SNP, g.22643979C>T in the butyrylcholinesterase (BCHE) gene, was most highly associated with PCHE activity at 3 weeks (-logP = 21.45) and 8 weeks (-logP = 27.60) of age. For the top SNP, the strong associations of CC and CT genotypes with low PCHE activity and the TT genotype with high PCHE activity indicates the dominant inheritance of low PCHE activity. Problems with block inheritance or linkage exist in this region. This study supports that BCHE is a functional gene for determining PCHE levels in ducks and that the genetic variations around this gene can cause phenotypic variations of PCHE activity.

Akirin1 is found to be involved in myoblast differentiation. However, the mechanism by which the Akirin1 gene regulates myoblast differentiation still remains unclear. In the present study, we found that ectopic expression of Akirin1 promoted myoblast differentiation by increasing the expression of myogenic regulatory factor (MRF) 4 (MRF4) and myocyte enhancer factor 2B (MEF2B) mRNA. Additionally, we showed that ectopic Akirin1 induced cell cycle arrest by up-regulating p21 mRNA. To further uncover the mechanism by which Akirin1 promotes myoblast differentiation, we showed that the enhanced Akirin1 increased the mRNA expression of P38α. Importantly, the enhanced MRF4 expression by Akirin1 can be abrogated by treatment of SB203580, a p38 inhibitor. Similarly, we found that enhanced MEF2B expression by Akirin1 can be abrogated by treatment with LY294002, a PI3K inhibitor. Together, our results indicate that Akirin1 promotes myoblast differentiation by acting on the p38 and PI3K pathways and subsequently inducing the expression of myoblast differentiation factors.

The blue-shelled egg not only plays a key role in helping birds to avoid predation as a result of crypsis and mimetism, but it also provides eggshell strength and filters solar radiation; moreover, it has an important economic trait for poultry. However, the source of biliverdin for blue-shelled egg remains unsolved in ducks. The current study detected the biliverdin content and localization of heme oxygenase 1 (HMOX1) in duck shell gland; moreover, RNA-seq analysis was performed in the shell gland of blue-shelled and white-shelled ducks. Results indicated that biliverdin is a primary pigment for blue-shelled egg in ducks, and the HMOX1 protein showed high expression in ciliated epithelial cells of shell gland between blue-shelled and white-shelled ducks. In the pathway of biliverdin synthesis, only 5-aminolevulinate synthase 1 expression level was significantly upregulated in blue-shelled ducks, and nuclear factor, erythroid 2 like 1 and period circadian clock 2 may be the essential elements in biliverdin synthesis of duck shell gland. Furthermore, some of the transporter genes, such as activator-Like and solute carrier family 13 member 5, may be involved in the formation of blue egg in duck. Results of the current study suggested that the biliverdin is most likely synthesized and secreted from epithelial cells of shell gland. In addition, ALAS1 may play a key role in the formation of blue egg in ducks.

Incubation temperature has an immediate and long-term influence on the embryonic development in birds. DNA methylation as an important environment-induced mechanism could serve as a potential link between embryos' phenotypic variability and temperature variation, which reprogrammed by DNA (cytosine-5)-methyltransferases (DNMTS) and Methyl-CpG binding domain proteins (MBPS) 3&5 (MBD3&5). Five genes in DNMTS and MBPS gene families were selected as target genes, given their important role in epigenetic modification. In this study, we aimed to test whether raising incubation temperature from 37.8°C to 38.8°C between embryonic days (ED) 1-10, ED10-20 and ED20-27 have effect on DNA methylation and whether DNMTS, MBPS play roles in thermal epigenetic regulation of early development in duck. Real-time quantitative PCR analysis showed that increased incubation temperature by 1°C has remarkably dynamic effect on gene expression levels of DNMTS and MBPS. Slight changes in incubation temperature significantly increased mRNA levels of target genes in breast muscle tissue during ED1-10, especially for DNMT1, DNMT3A and MBD5. In addition, higher temperature significantly increased enzyme activities of DNMT1 in leg muscle during ED10-20, liver tissue during ED1-10, ED20-27 and DNMT3A in leg muscle and breast muscle tissue during ED10-20. These results suggest that incubation temperature has an extended effect on gene expression levels and enzyme activities of DNMTS and MBPS, which provides evidence that incubation temperature may influence DNA methylation in duck during early developmental stages. Our data indicated that DNMTS and MBPS may involved in thermal epigenetice regulation of embryos during the early development in duck. The potential links between embryonic temperature and epigenetic modification need further investigation.

Avian embryos are an ideal system to investigate the effect of incubation temperature on embryonic development, but the characteristics and mechanisms of temperature effects on poultry embryonic myogenesis are unclear. In this study, we investigated the effect of increasing the incubation temperature by 1 °C on the expression of nine myogenesis-related genes in ducks and then explored the correlation between the alteration of promoter methylation and the expression of two of the nine genes under thermal manipulation (TM). The qRT-PCR results showed that TM during embryonic days (ED) 1-10 promoted (P 0.05) the expression of genes in breast muscle (PAX3, PAX7, MYOG, MCK, SIX1, TNNC1) and leg muscle (MYOD, MYOG, MYF5, MCK, AKIRIN2, TNNC1). TM during ED10-20 promoted the expression of PAX3, MYF5 and MCK and inhibited AKIRIN2 expression in breast muscle (P 0.05); however, it inhibited the expression of PAX3, PAX7, MYOD, MYOG, MYF5, SIX1, AKIRIN2 and TNNC1 and promoted MCK expression in leg muscle (P 0.05). TM during ED20-27 inhibited the expression of genes in breast muscle (PAX7) and leg muscle (MYOD, MYOG, MYF5, TNNC1) and promoted MCK expression in breast and leg muscle (P 0.05). Furthermore, with the Sequenom MassARRAY platform, it was observed that the average methylation level of AKIRIN2 (ED10) and TNNC1 (ED20) in leg muscle decreased (P 0.05) after TM. Notably, we found significant (P 0.05) inverse correlations between the methylation and mRNA levels of AKIRIN2 under TM during ED1-10 (r = - 0.969) and ED10-20 (r = - 0.805). Taken together, TM during ED1-10 was more favorable for improving duck myogenesis-related gene expression than TM during ED10-20 and ED20-27. TM during duck embryogenesis seemed to have a greater effect on the development of leg muscle than breast muscle and might alter AKIRIN2 expression by changing its promoter methylation status. These findings may be helpful to understand temperature effects on the muscle development of avian embryos and to explore the role of epigenetic regulation during this process.

Tyrosinase, encoded by the TYR gene, is the rate-limiting enzyme in the production of melanin pigment. In this study, plumage color separation was observed in Cherry Valley duck line D and F1 and F2 hybrid generations of Liancheng white ducks. Gene sequencing and bioinformatic analysis were applied to the 5'-regulatory region of TYR, to explore the connection between TYR sequence variation and duck plumage color. Four SNPs were found in the 5'-regulatory region. The SNPs were in tight linkage and formed three haplotypes. However, the genotype distribution in groups with different plumage color was not significantly different, and there were no changes in the transcription factor binding sites between the different genotypes. In conclusion, these SNP variations may not cause the differences in feather color observed in this test group.

As a central immune organ unique to birds, the bursa of Fabricius (BF) provides a proper microenvironment for B-cell development. The bursal B-cells undergo rapid proliferation and differentiation at the embryonic stages, but 95% of them undergo apoptosis after hatching. Few studies have focused on the cause of bursal B-cells apoptosis at the embryonic stages in birds. To explore the cause, we compared the transcriptional profiles of three characteristic embryonic stages in duck, including embryonic day 14 (ED14), 22 (ED22) and 1 day after hatching (D1). Our results showed that the apoptotic B-cells were first observed at ED22 while there were no apoptotic B-cells at ED14. By performing enrichment analysis for DEGs and qRT-PCR, our results demonstrated that both mitochondrial and Fas signaling pathways mediated bursal B-cell apoptosis during the duck embryonic development. Further, protein-protein interactions (PPIs) and KEGG enrichment analysis together showed that BMP4, FoxO1 and IGF-1 may regulate bursal B-cells apoptosis. In addition, the DEGs showed two stage-specific expression patterns. By analyzing the genes of two expression patterns, the results indicated that B-cell false differentiation may be one of the reasons of apoptosis in the duck embryonic BF. Overall, these data demonstrated that from ED14-ED22, apoptosis of bursal B-cells was mediated by mitochondrial and Fas signaling pathways and could be regulated by BMP4, FoxO1 and IGF-1 in duck. One of the primary causes of bursal B-cell apoptosis may be false differentiation in B-cells.

Poultry embryos are easily affected by environmental changings during incubation, thereinto, the temperature modification is the most important one, but the mechanism of temperature effects on bird eggs is not clear. By using RNA-seq, we have previously found that endoplasmic reticulum stress (ERS) may involve in regulating embryonic muscle development of duck under the influence of temperature alteration. To further clarify the role of ERS in the effect, in the present study, we detected the impact of increasing the incubation temperature by 1℃ during embryonic days 10-27 (E10-27) on the development of duck embryos, and investigated the changes in mRNA and protein expression of ERS marker genes and muscle-related genes under the thermal manipulation (TM). The results of relative weight comparison showed that only the relative weight of breast muscle was steadily decreased by TM from E10 to the first day after hatching (W0). Meanwhile, the real-time PCR and western-blot analysis revealed that raising the incubation temperature stimulated the expression of ERS marker genes in breast muscle at E20. The mRNA expressions of muscle hypertrophy and atrophy-related genes were also detected, and were not changed regularly, however, the protein expressions of hypertrophy-related genes were all decreased at both E20 and W0, and the protein expression of atrophy-related genes were up-regulated at E20. The protein expression of muscle proliferation-related genes were also decreased at E20. Additionally, these results were the same as that in the ERS positive control groups. Taken together, these results indicated that long-term TM during late embryonic period could block the development of duck breast muscle by inhibiting muscle hypertrophy and proliferation, and promoting muscle atrophy at a post-transcriptional level via the activation of ERS.

The growth and development of poultry embryos are easily affected by environmental factors, such as the incubation temperature and humidity. Metabolism, including lipid metabolism, during the embryonic stage is also important for the growth and development of poultry. Our study aimed to investigate the effects of incubation temperature on embryonic lipid metabolism in the liver of ducks. To fully evaluate the effects, thermal treatment was given between embryonic ages 11 and 24 days with a 1 °C higher incubation temperature than the control group, and lipid metabolism parameters in the liver and blood serum were analyzed both at embryonic stage day 20 and 2 weeks post-hatching. Our results showed no significant changes in the embryonic stage in total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in the blood serum (P>0.05). Additionally, the mRNA expression levels and enzyme activities of fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and elongase of very long chain fatty acids (ELOVL) did not show significant changes either in the embryonic stage or at hatching day 20 (P>0.05). However, there were significant changes in the gene expression and enzyme activities of TC, LDL-C and FAS at post-hatching stages (P≤0.05). These results may indicate that the thermal treatment has less influence on lipid metabolism in the embryonic stage but has a much stronger effect in the post-hatching stage.

The Pax3 gene has been proven to play a crucial role in determining myogenic progenitor cell fate during embryonic myogenesis; however, the molecular role of Pax3 in myoblast development during later stages of myogenesis is unknown. We hypothesized that Pax3 would function in myoblast proliferation and differentiation; therefore, we employed three short hairpin RNAs (shRNAs) (shRNA1, shRNA2, and shRNA3) that target Pax3 to characterize the function of Pax3 in duck myoblast development. The mRNA and protein expression levels of Pax3 in duck myoblasts were detected using real-time PCR and Western blotting. Cell proliferation was assessed using the MTT and BrdU assays, while cell differentiation was assayed using immunofluorescence labeling with a MyoG antibody. Additionally, folic acid (FA), which is a rescue tool, was added into the medium of duck myoblasts to indirectly examine the function of Pax3 on duck myoblast proliferation and differentiation. The results revealed that one of the shRNA vectors, shRNA1, could significantly and stably reduce the expression of Pax3 (P

Summary Follistatin (FST) acts as a positive regulator of muscle development by inhibiting the activities and expression of myostatin. The recombinant duck FST protein was injected into hatching eggs and was also added to the medium of duck myoblast to study its role on duck embryonic muscle development and gene expressions. Duck embryo weight increased 3.49% (p > 0.05) in FST treatment group as compared with control group, but minor effects were found on leg or breast muscle weights of ducklings at 2 days post-hatching (p > 0.05). Relative expression of Pax7 was upregulated in both leg and breast muscle tissues (p

Myocyte enhancer transcription factor 2D (MEF2D) is an important transcription factor for promoting the growth and development of muscle. CAG repeats have been found in the coding sequence (CDS) of avian MEF2D; however, their functions remain unknown and require further investigation. Here, we examined the characteristics and functional role of MEF2D CAG repeat in duck. The full-length CDS of duck MEF2D was cloned for the first time, and a novel CAG repeat was identified and located in exon 9. Sequence analysis indicated that the protein domains of duck MEF2D are highly conserved relative to other vertebrates, whereas MEF2D CAG repeats with variable repeat numbers are specific to avian species. Furthermore, sequencing has revealed polymorphisms in MEF2D CAG repeat at both DNA and mRNA levels. Four MEF2D CAG repeat genotypes and 10 MEF2D cDNA variants with different CAG repeat numbers were detected in two duck populations. A t-test showed that the expanded CAG repeat generated significantly longer transcription products (p 0.05). Association analysis demonstrated positive correlations between the expansion of the CAG repeat and five muscle-related traits. By using protein structure prediction, we suggested that the polymorphisms of the CAG repeat affect protein structures within protein domains. Taken together, these findings reveal that duck MEF2D CAG repeat is a potential functional element with polymorphisms and may cause differences in MEF2D function between duck and other vertebrate species.

The objective of the current research was to investigate the pattern of subcutaneous adipose tissue growth during Peking duck (Anas platyrhynchos) early development and to determine the reasons for regional differences. The morphological characteristics in 5 regions of subcutaneous tissue, including the neck area (NSF), chest area (CSF), lower abdomen area (ASF), back area (BSF), and leg area (LSF), were analyzed by comparing the morphology of the sections, adipocyte volume and number, and lipid content from wk 1 to 8. Moreover, the mRNA expression of several molecular marker genes, including 47-kDa tail interacting protein (TIP47), adipose differentiation-related protein (ADRP), and perilipin, were detected from wk 1 to 8 using quantitative real-time PCR. Our results revealed that the average cell number declined greatly as fattening proceeded (except in the NSF) and changed very little after wk 4 in all 5 regions. In contrast, the average cell volume and triglyceride content per cell increased gradually during early duck growth. The BSF and LSF lipid content had a different pattern of change than the other regions. The NSF, CSF, and ASF regions had the highest lipid content values at all stages, the BSF was intermediate, and the LSF was the lowest at all weeks except wk 3. The highest TIP47 expression level was found in the NSF from wk 1 to 2 and BSF at wk 1. The relative expression level of TIP47 was higher in the CSF than in the ASF and BSF at wk 4, and was higher in the NSF than in the ASF at wk 6. The highest levels of ADRP and perilipin were detected in the LSF. These results suggest that a combination of adipocyte hyperplasia and hypertrophy is mainly responsible for the development of duck adipose tissue before wk 4, after which adipose expansion is accomplished by adipocyte hypertrophy only. Adipocyte hyperplastic and hypertrophic capacity, fat storage capacity, and metabolic activity may be partial explanations for the regional differences during duck growth.

Myostatin (MSTN) plays a key role in the negative regulation of muscle growth and development during embryogenesis. The MSTN genes have different genetic characteristics in vertebrates: sole gene in mammals, gene duplication in fish, and alternative splicing in birds. To investigate the alternative splicing sites and developmental expression patterns of the duck MSTN genes, the mRNA and genome sequences were cloned, and the expression patterns were detected during breast muscle and leg muscle development by real-time PCR. In our study, four alternatively spliced forms of MSTN mRNA were found in the developing skeletal muscle of Peking duck, including two novel alternatively spliced transcripts, MSTN-c and MSTN-d. As a result of alternative splicing at the common GT-AG processing sites, MSTN-b and MSTN-c retained only the N-terminal TGFβ-propeptide superfamily domains. However, MSTN-d was not missing these domains, in contrast to MSTN-a. The real-time PCR results showed that there was no significant difference between breast muscle and leg muscle in MSTN-a mRNA expression, also in MSTN-b and MSTN-c. MSTN-a and MSTN-b have significant higher expressions than MSTN-c and MSTN-d, suggesting that they play the major role during embryo muscle development.

Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value

RNA-binding motif proteins 24 (Rbm24) and 38 (Rbm38) are known to regulate genes expression in a post-transcriptional way. However, it remains unclear about the similarities and differences between Rbm24 and Rbm38 in terms of their sequence characteristics and expression profiles during myoblast differentiation and skeletal muscle development. In this study, we found that the coding domain sequences (CDSs) of duck Rbm24 and Rbm38 consisted of 678 and 648 nucleotides, respectively. Both of them contain a conserved RNA-recognition motif (RRM). Phylogenetic analysis showed that duck Rbm24 and Rbm38 were clustered with other Aves, nevertheless, avian Rbm24 was clustered with mammalian and reptilian Rbm24; avian Rbm38 was clustered with amphibian and reptilian Rbm38. Real-time PCR results exhibited that during embryonic stage, Rbm24 and Rbm38 in leg and breast muscle increased to their peak (P

Changes in temperature will influence poultry embryonic muscle development. However, little is known about the changes in molecular processes impacted by incubation temperature in avians. In this study, we investigated the effects of increasing the incubation temperature by 1 °C from day 11–20 on the embryonic and posthatch skeletal muscle development of the Peking duck, and identified the differentially expressed genes using RNA-seq of leg muscle tissues. The results showed that altering the incubation temperature had immediate and long-lasting effects on phenotypic changes in the embryonic and post-hatching muscle development. It was shown that expression levels of total 1370 genes were altered in muscle tissues by the thermal treatments. The gene ontology (GO) analyses indicated that cellular processes including metabolism, cell cycle, catalytic activity, and enzyme regulatory activity may have involved in the muscle mass impacted by thermal manipulation. TGF-beta and insulin pathways as two classical muscle development related pathways may also involve in regulating muscle mass. These data may be helpful for understanding the physiological and biochemical processes of muscle development under environmental treatments in embryonic avians.