Duck Skeletal Muscle Research Articles

Exploring Gene Expression and Alternative Splicing in Duck Embryonic Myoblasts via Full-Length Transcriptome Sequencing

The duck industry is vital for supplying high-quality protein, making research into the development of duck skeletal muscle critical for improving meat and egg production. In this study, we leveraged Oxford Nanopore Technologies (ONT) sequencing to perform full-length transcriptome sequencing of myoblasts harvested from the leg muscles of duck embryos at embryonic day 13 (E13), specifically examining both the proliferative (GM) and differentiation (DM) phases. Our analysis identified a total of 5797 novel transcripts along with 2332 long non-coding RNAs (lncRNAs), revealing substantial changes in gene expression linked to muscle development. We detected 3653 differentially expressed genes and 2246 instances of alternative splicing, with key genes involved in essential pathways, such as ECM–receptor interaction and Notch signaling, prominently featured. Additionally, we constructed a protein–protein interaction network that highlighted critical regulators—MYOM3, MYL2, MYL1, TNNI2, and ACTN2—associated with the processes of proliferation and differentiation in myoblasts. This extensive transcriptomic investigation not only sheds light on the intricate molecular mechanisms driving skeletal muscle development in ducks but also provides significant insights for future breeding strategies aimed at enhancing the efficiency of duck production. The results emphasize the efficacy of ONT sequencing in uncovering complex regulatory networks within avian species, ultimately contributing to progress in animal husbandry.

Fibronectin type III domain-containing protein 5 (FNDC5)-like immunoreactivity and mRNA abundance in domestic animal tissues

Irisin is a 112-amino acid peptide hormone that is cleaved from fibronectin type III domain-containing protein 5 (FNDC5), a type I transmembrane protein abundantly found in muscle tissue. Irisin is a putative mediator of the benefits of exercise, neuroprotection, bone growth, and cardiac health. However, few studies have focused on irisin in domestic animals. Further, whether processed irisin is detectable in domestic animal tissues remains uncertain. To address this, we determined FNDC5 mRNA and protein concentration in anatine (duck) and porcine (pig) skeletal muscle, and in equine (horse), swine, and anatine serum samples. RT-PCR analysis identified FNDC5 mRNA in all pig and duck skeletal muscle samples. An approximately 25 kDa band representing FNDC5 was detected in both pig and duck skeletal muscle. Fluorescence immunohistochemistry using a rabbit monoclonal FNDC5/irisin primary antibody and a goat polyclonal anti-rabbit secondary antibody localized FNDC5/irisin-like immunoreactivity in both the glandular and muscular regions of pig stomach. FNDC5/irisin-like immunoreactivity was also identified in horse, pig, and duck serum using a multispecies irisin ELISA. The average values of irisin-like immunoreactivity were 13.7 (duck), 15.4 (horse), and 7.0 (pig) ng/mL in samples tested. Our results support the presence of irisin precursor in several domestic animals. Processed irisin, however, was not detectable. Further studies are required to validate reliable tools to detect and quantify processed irisin in domestic animals.

Characterization and comparative transcriptomic analysis of skeletal muscle in female Pekin duck and Hanzhong Ma duck during different growth stages using RNA-seq

Duck is an economically important poultry, and there is currently a major focus on improving its meat quality through breeding. There are wide variations in the growth regulation mechanisms of different duck breeds, that fundamental research on skeletal muscle growth is essential for understanding the regulation of unknown genes. The study aimed to broaden the understanding the duck skeletal muscle development and thereby to improve the performance of domestic ducks. In this study, RNA-seq data from skeletal muscles (breast muscle and leg muscle) of Pekin duck and Hanzhong Ma duck sampled at d 17, 21, and 27 of embryo (E17d, E21d, and E27d), as well as at 6-mo-old following birth (M6), to investigate and compare the mRNA temporal expression profiles and associated pathways that regulate skeletal myogenesis of different duck breeds. There were 331 to 1,440 annotated differentially expressed genes (DEGs) in breast muscle and 380 to 1,790 annotated DEGs in leg muscle from different databases between 2 duck breeds. Gene ontology (GO) enrichment in skeletal muscles indicated that these DEGs were mainly involved in biosynthetic process, developmental process, regulation of protein metabolic process and regulation of gene expression. KEGG analysis in skeletal muscles showed that a total of 41 DEGs were mapped to 7 KEGG pathways, including ECM-receptor interaction, focal adhesion, carbon metabolism, regulation of actin cytoskeleton, calcium signaling pathway, biosynthesis of amino acids and PPAR signaling pathway. The differential expression of 8 selected DEGs was verified by qRT-PCR, and the results were consistent with RNA-seq data. The identified DEGs, such as SDC, SPP1, PAK1, MYL9, PGK1, NOS1, PHGDH, TNNT2, FN1, and AQP4, were specially highlighted, indicating their associations with muscle development in the Pekin duck and Hanzhong Ma duck. This study provides a basis for revealing the differences in skeletal muscle development between Pekin duck and Hanzhong Ma duck.

Transcriptome RNA Sequencing Reveals That Circular RNAs Are Abundantly Expressed in Embryonic Breast Muscle of Duck.

Circular RNAs are widespread in various species and have important roles in myogenesis. However, the circular RNAs involved in breast muscle development in ducks have not yet been studied. Here, to identify circular RNAs during duck skeletal muscle development, three pectorales from Shan Ma ducks at E13 and E19, which represent undifferentiated and differentiated myoblasts, respectively, were collected and subjected to RNA sequencing. A total of 16,622 circular RNAs were identified, of which approximately 80% were exonic circular RNAs and 260 were markedly differentially expressed between E19 and E13. The parental genes of the differentially expressed circular RNAs were significantly enriched in muscle-related biological processes. Moreover, we found that the overexpression of circGAS2-2 promoted cell cycle progression and increased the proliferation viability of duck primary myoblasts; conversely, knockdown of circGAS2-2 retarded the cell cycle and reduced the proliferation viability of myoblasts. Taken together, our results demonstrate that circular RNAs are widespread and variously expressed during the development of duck skeletal muscle and that circGAS2-2 is involved in the regulation of myogenesis.

Identification of genes related to growth traits from transcriptome profiles of duck breast muscle tissue

The growth and development of duck skeletal muscle is an important economic trait that is genetically regulated. The internal mechanism underlying the regulation of skeletal muscle growth and development in ducks remains unclear. The purpose of this study was to identify candidate genes related to the growth of duck skeletal muscle. RNA-sequencing technology was used to compare the transcriptome of duck breast muscles in an F2 population with the high breast muscle rate (HB) and the low breast muscle rate (LB). A total of 14,522 genes were confirmed to be expressed in the breast muscle, and 173 differentially expressed genes (DEGs) were identified between the HB and LB groups. Functional analysis showed that these DEGs were mainly involved in biological processes and pathways of fat metabolism and muscle growth, especially the FABP3 and MYL4 involved in the PPAR signaling pathway and cardiac muscle contraction pathway. These findings deepened our understanding of the molecular mechanisms involved in muscle growth in ducks and provided a theoretical basis for improving duck production and breeding of ducks.

Curcumin alleviates arsenic-induced injury in duck skeletal muscle via regulating the PINK1/Parkin pathway and protecting mitochondrial function

Arsenic is a well-known environmental pollutant due to its toxicity, which can do harm to animals and human. Curcumin is a polyphenolic compound derived from turmeric, commonly accepted to have antioxidant properties. However, whether curcumin can ameliorate the damage caused by arsenic trioxide (ATO) in duck skeletal muscle remains largely unknown. Therefore, the present study aims to investigate the potential molecular mechanism of curcumin against ATO-induced skeletal muscle injury. The results showed that treating with curcumin could attenuate body weight loss induced by ATO and reduced arsenic content accumulation in the skeletal muscle of duck. Curcumin was also able to alleviated the oxidative stress triggered by ATO, which was manifested by the increase of T-AOC and SOD, and MDA decrease. Moreover, we observed that curcumin could ease mitochondrial damage and vacuolate degeneration of nucleus. Our further investigation found that ATO disrupted normal mitochondrial fission/fusion (Drp1, OPA1, Mfn1/2) and restrained mitochondrial biogenesis (PGC-1α, Nrf1/2, TFAM), while curcumin could promote mitochondrial fusion and activated PGC-1α pathway. Furthermore, curcumin was found that it could not only reduce the mRNA and protein levels of mitophagy (PINK1, Parkin, LC3, p62) and pro-apoptotic genes (p53, Bax, Caspase-3, Cytc), but also increased the levels of anti-apoptotic genes (Bcl-2). In conclusion, curcumin was able to alleviate ATO-induced skeletal muscle damage by improving mitophagy and preserving mitochondrial function, which can serve as a novel strategy to take precautions against ATO toxicity.

Characterization and Comparative Transcriptomic Analysis of Skeletal Muscle in Pekin Duck at Different Growth Stages Using RNA-Seq.

Simple SummarySkeletal muscle is an important tissue and its development is strictly regulated by genes. In this study, in order to understand the muscle-related gene expression in Pekin duck, RNA-seq was performed to analyze and compare skeletal muscle at different growth stages. Alternative splicing, single nucleotide polymorphisms and insertion–deletions were detected, and 299 novel genes were discovered. MYL4, IGF2BP1, CSRP3, SPP1, KLHL31, LAMB2, LAMA2, ITGB1 and OPN played crucial roles in skeletal muscle development. Oxidative phosphorylation, ECM-receptor interaction, focal adhesion, carbon metabolism, and biosynthesis of amino acids participated in the regulation of skeletal muscle development in Pekin duck. This study provides an important reference for revealing the developmental mechanisms of pectoral and leg muscles in duck.Skeletal muscle, accounting for approximately 50% of body weight, is the largest and most important tissue. In this study, the gene expression profiles and pathways in skeletal muscle of Pekin duck were investigated and compared at embryonic day 17, 21, and 27 and postnatally at 6 months of age. An average of 49,555,936 reads in each sample was obtained from the transcriptome libraries. Over 70.0% of alternative splicing (AS) in each sample was mainly alternative 5′ first exon (transcription start site)—the first exon splicing (TSS) and alternative 3′ last exon (transcription terminal site)—the last exon splicing (TTS), indicating that TSS and TTS were the most common AS event in Pekin ducks, and these AS events were closely related to the regulation of muscle development at different growth stages. The results provided a valuable genomic resource for selective breeding and functional studies of genes. A total of 299 novel genes with ≥2 exons were obtained. There were 294 to 2806 differentially expressed genes (DEGs) in each pairwise comparison of Pekin duck. Notably, 90 DEGs in breast muscle and 9 DEGs in leg muscle were co-expressed at all developmental points. DEGs were validated by qPCR analysis, which confirmed the tendency of the expression. DEGs related to muscle development were involved in biological processes such as “endodermal cell differentiation”, “muscle cell cellular homeostasis”, “skeletal muscle tissue growth” and “skeletal muscle cell differentiation”, and were involved in pathways such as oxidative phosphorylation, ECM-receptor (extracellular matrix receptor) interaction, focal adhesion, carbon metabolism, and biosynthesis of amino acids. Some DEGs, including MYL4, IGF2BP1, CSRP3, SPP1 and KLHL31, as well as LAMB2, LAMA2, ITGB1 and OPN, played crucial roles in muscle growth and development. This study provides valuable information about the expression profile of mRNAs and pathways from duck skeletal muscle at different growth stages, and further functional study of these mRNAs and pathways could provide new ideas for studying the molecular networks of growth and development in duck skeletal muscle.

Skeletal Muscle Transcriptome Analysis of Hanzhong Ma Duck at Different Growth Stages Using RNA-Seq.

As one of the most important poultry worldwide, ducks (Anas platyrhynchos) are raised mainly for meat and egg products, and muscle development in ducks is important for meat production. Therefore, an investigation of gene expression in duck skeletal muscle would significantly contribute to our understanding of muscle development. In this study, twenty-four cDNA libraries were constructed from breast and leg muscles of Hanzhong Ma ducks at day 17, 21, 27 of the embryo and postnatal at 6-month-old. High-throughput sequencing and bioinformatics were used to determine the abundances and characteristics of transcripts. A total of 632,172,628 (average 52,681,052) and 637,213,938 (average 53,101,162) reads were obtained from the sequencing data of breast and leg muscles, respectively. Over 71.63% and 77.36% of the reads could be mapped to the Anas platyrhynchos genome. In the skeletal muscle of Hanzhong duck, intron variant (INTRON), synonymous variant (SYNONYMOUS_CODING), and prime 3′ UTR variant (UTR_3_PRIME) were the main single nucleotide polymorphisms (SNP) annotation information, and “INTRON”, “UTR_3_PRIME”, and downstream-gene variant (DOWNSTREAM) were the main insertion-deletion (InDel) annotation information. The predicted number of alternative splicing (AS) in all samples were mainly alternative 5′ first exon (transcription start site)-the first exon splicing (TSS) and alternative 3′ last exon (transcription terminal site)-the last exon splicing (TTS). Besides, there were 292 to 2801 annotated differentially expressed genes (DEGs) in breast muscle and 304 to 1950 annotated DEGs in leg muscle from different databases. It is worth noting that 75 DEGs in breast muscle and 49 DEGs in leg muscle were co-expressed at all developmental points of comparison, respectively. The RNA-Seq data were confirmed to be reliable by qPCR. The identified DEGs, such as CREBL2, RHEB, GDF6, SHISA2, MYLK2, ACTN3, RYR3, and STMN1, were specially highlighted, indicating their strong associations with muscle development in the Hanzhong Ma duck. KEGG pathway analysis suggested that regulation of actin cytoskeleton, oxidative phosphorylation, and focal adhesion were involved in the development of skeletal muscle. The findings from this study can contribute to future investigations of the growth and development mechanism in duck skeletal muscle.

Comparative Transcriptome Profiling of Skeletal Muscle from Black Muscovy Duck at Different Growth Stages Using RNA-seq.

In China, the production for duck meat is second only to that of chicken, and the demand for duck meat is also increasing. However, there is still unclear on the internal mechanism of regulating skeletal muscle growth and development in duck. This study aimed to identity candidate genes related to growth of duck skeletal muscle and explore the potential regulatory mechanism. RNA-seq technology was used to compare the transcriptome of skeletal muscles in black Muscovy ducks at different developmental stages (day 17, 21, 27, 31, and 34 of embryos and postnatal 6-month-olds). The SNPs and InDels of black Muscovy ducks at different growth stages were mainly in “INTRON”, “SYNONYMOUS_CODING”, “UTR_3_PRIME”, and “DOWNSTREAM”. The average number of AS in each sample was 37,267, mainly concentrated in TSS and TTS. Besides, a total of 19 to 5377 DEGs were detected in each pairwise comparison. Functional analysis showed that the DEGs were mainly involved in the processes of cell growth, muscle development, and cellular activities (junction, migration, assembly, differentiation, and proliferation). Many of DEGs were well known to be related to growth of skeletal muscle in black Muscovy duck, such as MyoG, FBXO1, MEF2A, and FoxN2. KEGG pathway analysis identified that the DEGs were significantly enriched in the pathways related to the focal adhesion, MAPK signaling pathway and regulation of the actin cytoskeleton. Some DEGs assigned to these pathways were potential candidate genes inducing the difference in muscle growth among the developmental stages, such as FAF1, RGS8, GRB10, SMYD3, and TNNI2. Our study identified several genes and pathways that may participate in the regulation of skeletal muscle growth in black Muscovy duck. These results should serve as an important resource revealing the molecular basis of muscle growth and development in duck.

Characterization of microRNAs during Embryonic Skeletal Muscle Development in the Shan Ma Duck.

Simple SummaryIt is of great commercial interest to elucidate the genetic mechanisms associated with skeletal muscle development in the duck. In this study, we performed high throughput microRNA (miRNA) sequencing to identify the candidate miRNAs during two developmental stages of duck embryonic breast muscle. We detected 1091 miRNAs and 109 of them were differentially expressed between embryonic day 13 (E13) and E19. We also predicted the target genes of the differentially expressed miRNAs and subsequently analyzed the enriched gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways, and finally constructed a protein–protein interaction (PPI) network with the target genes. Luciferase reporter assay showed that the growth-related genes, Fibroblast growth factor receptor like 1 (FGFRL1) and Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1), were target genes of miR-214-5p. These results can supplement the duck miRNA database and provide several candidate miRNAs for future studies on the regulation of embryonic skeletal muscle development.Poultry skeletal muscle provides high quality protein for humans. Study of the genetic mechanisms during duck skeletal muscle development contribute to future duck breeding and meat production. In the current study, three breast muscle samples from Shan Ma ducks at embryonic day 13 (E13) and E19 were collected, respectively. We detected microRNA (miRNA) expression using high throughput sequencing following bioinformatic analysis. qRT-PCR validated the reliability of sequencing results. We also identified target prediction results using the luciferase reporter assay. A total of 812 known miRNAs and 279 novel miRNAs were detected in six samples; as a result, 61 up-regulated and 48 down-regulated differentially expressed miRNAs were identified between E13 and E19 (|log2 fold change| ≥ 1 and p ≤ 0.05). Enrichment analysis showed that target genes of the differentially expressed miRNAs were enriched on many muscle development-related gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially mitogen-activated protein kinase (MAPK) signaling pathways. An interaction network was constructed using the target genes of the differentially expressed miRNAs. These results complement the current duck miRNA database and offer several miRNA candidates for future studies of skeletal muscle development in the duck.

Transcriptional regulatory region and DNA methylation analysis of TNNI1 gene promoters in Gaoyou duck skeletal muscle (Anas platyrhynchos domestica)

ABSTRACT 1. The slow skeletal muscle troponin I (TNNI1) gene has been found to be specifically expressed in slow muscle fibres and plays an important role in muscle development. The aim of this study was to determine the active control area of duck TNNI1 and identify the potential cis-regulatory elements in the promoter. 2. In this study, the TNNI1 promoter was first cloned by genome walking and the sequences were analysed using bioinformatics software. Firefly luciferase reporter gene vectors, driven by a series of constructs with progressive deletions, were used to identify the core transcriptional regulatory region of the duck TNNI1 gene. The methylation status of the CpG island in the TNNI1 promoter was detected in skeletal muscle on embryonic days 21 and 27, by bisulphite sequencing PCR (BSP). 3. The results showed two CpG islands presented in the promoter region, with one of the CpG islands located in the core transcriptional regulatory region (−2078/−885 bp). The total methylation levels of the 14 CpG sites were not altered between breast and leg muscles on embryonic days 21 and 27. However, four CpG sites (loci of positions 4, 11, 13, and 14) showed dramatically different methylation levels between breast and leg muscles at embryonic days 21 and 27. Analysis showed that multiple CpG sites had a significant correlation between the methylation levels of the CpG sites and mRNA expressions in skeletal muscle. Multiple transcription factor binding sites including Sp1, c-Myc, Oct-1 and NF-kB motifs were identified and might be responsible for transcriptional regulation of the TNNI1 gene. 4. These findings contribute to further understanding of the fundamental mechanism for transcriptional regulation of the TNNI1 gene in ducks.

Analysis of Anasplatyrhynchos genome resequencing data reveals genetic signatures of artificial selection.

Three artificially selected duck populations (AS), higher lean meat ratios (LTPD), higher fat ratios (FTPD) and higher quality meat (CMD), have been developed in China, providing excellent populations for investigation of artificial selection effects. However, the genetic signatures of artificial selection are unclear. In this study, we sequenced the genome sequences of these three artificially selected populations and their ancestral population (mallard, M). We then compared the genome sequences between AS and M and between LTPD and FTPD using integrated strategies such as anchoring scaffolds to pseudo-chromosomes, mutation detection, selective screening, GO analysis, qRT-PCR, and protein multiple sequences alignment to uncover genetic signatures of selection. We anchored duck scaffolds to pseudo-chromosomes and obtained 28 pseudo-chromosomes, accounting for 84% of duck genome in length. Totally 78 and 99 genes were found to be under selection between AS and M and between LTPD and FTPD. Genes under selection between AS and M mainly involved in pigmentation and heart rates, while genes under selection between LTPD and FTPD involved in muscle development and fat deposition. A heart rate regulator (HCN1), the strongest selected gene between AS and M, harbored a GC deletion in AS and displayed higher mRNA expression level in M than in AS. IGF2R, a regulator of skeletal muscle mass, was found to be under selection between FTPD and LTPD. We also found two nonsynonymous substitutions in IGF2R, which might lead to higher IGF2R mRNA expression level in FTPD than LTPD, indicating the two nonsynonymous substitutions might play a key role for the regulation of duck skeletal muscle mass. Taken together, these results of this study provide valuable insight for the genetic basis of duck artificial selection.

Effect of thermal manipulation during embryogenesis on the promoter methylation and expression of myogenesis-related genes in duck skeletal muscle

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.

Roles of miRNA‐1 and miRNA‐133 in the proliferation and differentiation of myoblasts in duck skeletal muscle

MicroRNA (miRNA)-1 and miRNA-133 are derived from the same bicistronic pairs with roles in skeletal muscle development. Many investigations have focused on the role of miRNA-1 and miRNA-133 in the regulation of skeletal muscle development in mammals and fish. However, the mechanisms of miRNA-1 and miRNA-133 underlying the differences in skeletal muscle development between different breeds are not well known. Our study found that the weights of body and breast at 42 days of age were greater in Cherry Valley ducks than in Putian ducks and the areas of breast muscle fibers increased with age; the areas of muscle fibers of Cherry Valley ducks were always greater than those of Putian ducks. Besides, quantitative reverse-transcriptase polymerase chain reaction analysis revealed that relatively high levels of miRNA-1 and miRNA-133 were detected in heart, breast, and leg muscles compared with the liver, spleen, lung, kidney, and the expression levels of miRNA-1 and miRNA-133 remained stable in the embryo stage, and in the growth period, the fluctuation in miRNA expression levels in Putian ducks was considerably higher than that in Cherry Valley ducks, especially from 7 to 28 days. However, in the late growth period, the expression of miRNA-1 and miRNA-133 of Cherry Valley duck was higher than that of Putian duck, which may indicate that miRNA-1 and miRNA-133 play a more important role during the growth period. To determine the function of miRNA-1 and miRNA-133 in skeletal muscle development, we found that the overexpression of miRNA-1, but not miRNA-133, promoted fusion of adjacent myoblasts. By contrast, a repressor of miRNA-1 promoted, whereas a miRNA-133 inhibitor inhibited, myoblast proliferation. Accordingly, the expression levels of myocyte enhancer factor 2D (MEF2D) and myogenic differentiation ( MYOD) were significantly increased by an miRNA-1 mimic and the miRNA-133 inhibitor. In addition, we found that the expression levels of miRNA-1 significantly affected the expression ofhistone deacetylase 4 ( HDAC4), and miRNA-133 affected serum response factor ( SRF) and transforming growth factor β receptor 1 ( TGFBR1) levels. However, dual-luciferase reporter assays revealed that only miRNA-1 directly inhibited pGL- HDAC4 luciferase reporter activity, whereas miRNA-133 did not affect pGL- SRF or pGL- TGFBR1 fluorescence activity. Taken together, these results suggest that miRNA-1 targets HDAC4 to promote the differentiation of duck myoblasts and miRNA-133 may affect SRF and TGFBR1 expression to promote proliferation, which indicates that miRNA-1 and miRNA-133 play different important roles in skeletal muscle development.

Expression profile of IGF-I-calcineurin-NFATc3-dependent pathway genes in skeletal muscle during early development between duck breeds differing in growth rates.

The insulin-like growth factor I (IGF-I)-calcineurin (CaN)-NFATc signaling pathways have been implicated in the regulation of myocyte hypertrophy and fiber-type specificity. In the present study, the expression of the CnAα, NFATc3, and IGF-I genes was quantified by RT-PCR for the first time in the breast muscle (BM) and leg muscle (LM) on days 13, 17, 21, 25, and 27 of embryonic development, as well as at 7days posthatching (PH), in Gaoyou and Jinding ducks, which differ in their muscle growth rates. Consistent expression patterns of CnAα, NFATc3, and IGF-I were found in the same anatomical location at different development stages in both duck breeds, showing significant differences in an age-specific fashion. However, the three genes were differentially expressed in the two different anatomical locations (BM and LM). CnAα, NFATc3, and IGF-I messenger RNA (mRNA) could be detected as early as embryonic day 13 (ED13), and the highest level appeared at this stage in both BM and LM. Significant positive relationships were observed in the expression of the studied genes in the BM and LM of both duck breeds. Also, the expression of these three genes showed a positive relationship with the percentage of type IIb fibers and a negative relationship with the percentage of type I fibers and type IIa fibers. Our data indicate differential expression and coordinated developmental regulation of the selected genes involved in the IGF-I-calcineurin-NFATc3 pathway in duck skeletal muscle during embryonic and early PH growth and development; these data also indicate that this signaling pathway might play a role in the regulation of myofiber type transition.

Gene expression patterns, and protein metabolic and histological analyses for muscle development in Peking duck

In this study, we aimed to use duck breast muscle and leg muscle, the 2 main productive muscle organs, as a model to elucidate the molecular mechanism controlling how the 2 muscles have different deposition capabilities, and to analyze the mechanisms facilitating duck muscle development posthatching. Peking duck breast muscle and leg muscle were collected 3, 7, and 16 wk posthatching. The morphology of the myofibers was observed by paraffin sectioning the muscles. The expression of genes involved in protein metabolism [mammalian target of rapamycin (mTOR), RPS6-p70-protein kinase (S6K), forkhead box O1 (FoxO1), muscle RING finger 1 (MuRF1), and atrogin-1 (MAFbx)] was detected using real-time quantitative PCR and Western blot assays, and the results indicated that breast muscle had a stronger capacity for both protein synthesis and protein degradation compared with leg muscle. Satellite cell frequency declined during muscle development in both tissues, and the expression of Pax3/7, satellite cell marker genes, was not significantly different between breast muscle and leg muscle. No notable apoptosis was observed in either tissue. The results of this study suggest that protein metabolism signaling is the main reason promoting duck skeletal muscle mass gain.

Identification and profiling of microRNAs in the embryonic breast muscle of pekin duck.

MicroRNAs (miRNAs) regulate gene expression by fully or partially binding to complementary sequences and play important roles in skeletal muscle development. However, the roles of miRNAs in embryonic breast muscle of duck are unclear. In this study, we analyzed the miRNAs profiling in embryonic breast muscle of Pekin duck at E13 (the 13th day of hatching), E19, and E27 by high-throughput sequencing. A total of 382 miRNAs including 359 preciously identified miRNAs 23 novel miRNA candidates were obtained. The nucleotide bias analysis of identified miRNAs showed that the miRNAs in Pekin duck was high conserved. The expression of identified miRNAs were significantly different between E13 and E19 as well as between E27 and E19. Fifteen identified miRNAs validated using stem-loop qRT-PCR can be divided into three groups: those with peak expression at E19, those with minimal expression at E19, and those with continuous increase from E11 to E27. Considering that E19 is the fastest growth stage of embryonic Pekin duck breast muscle, these three groups of miRNAs might be the potential promoters, the potential inhibitors, and the potential sustainer for breast muscle growth. Among the 23 novel miRNAs, novel-miRNA-8 and novel-miRNA-14 had maximal expression at some stages. The stem-loop qRT-PCR analysis of the two novel miRNAs and their two targets (MAP2K1 and PPARα) showed that the expression of novel-mir-8 and PPARα reached the lowest points at E19, while that of novel-mir-14 and MAP2K1 peaked at E19, suggesting novel-miRNA-8 and novel-miRNA-14 may be a potential inhibitor and a potential promoter for embryonic breast muscle development of duck. In summary, these results not only provided an overall insight into the miRNAs landscape in embryonic breast muscle of duck, but also a basis for the further investigation of the miRNAs roles in duck skeletal muscle development.

Effects of the regulation of follistatin mRNA expression by IGF-1 in duck (Anas platyrhynchos) skeletal muscle

The IGF-1 and TGF-β pathways have been shown to be involved in regulating muscle development. Many mediators that are associated with the regulation of muscle development have been found to participate in the cross-talk between these two pathways. To research the relationships between IGF-1 and the follistatin-mediated TGF-β pathways in duck skeletal muscle development, a series of studies were conducted. The results showed that follistatin had similar expression patterns to IGF-1 during duck embryonic muscle development. The in ovo feeding of IGF-1 to duck eggs was shown to increase follistatin expression in the duck skeletal muscle. Thus, IGF-1 may induce the mRNA expression of follistatin. These results suggest that follistatin may be a key regulator of multiple signaling cascades responding to the cross-talk between the IGF-1 and TGF-β pathways.

Apoptosis during postmortem conditioning and its relationship to duck meat quality

The aim of this work was to examine the relationship of skeletal muscle apoptosis and postmortem development of meat quality. Colour, cooking loss, myofibril fragmentation index (MFI) and shear force of duck breast and thigh meat postmortem were measured, and changes of positive nuclei were assessed with Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphophate nick end-labelling method (TUNEL). Correlation analysis revealed that apoptosis were positively correlated with colour (L∗, a∗ and b∗), cooking loss and MFI (P<0.05), while it is negatively correlated with shear force (P<0.05). Our results indicate the growing level of duck skeletal muscle cell apoptosis was associated with the postmortem development of meat quality traits such as meat colour, water holding capacity and tenderness.

Postmortem role of calpains in Pekin duck skeletal muscles

It is generally agreed that calpains are involved in postmortem proteolysis of skeletal muscle and improve meat tenderness. However, little information regarding the postmortem role of calpains in duck skeletal muscle is known. Therefore, the purpose of this study was to examine the role of calpains in Pekin duck postmortem breast muscles (BM) and leg and thigh muscles (LM) muscles at 5 °C. The postmortem pH was lower (P < 0.05) in BM than in LM. Western blots indicated that postmortem desmin degradation and the 30/32 kDa troponin-T degradation product accumulation were more rapid in BM than in LM. Casein zymograms showed that at-death µ-calpain activity was higher in BM than in LM. As time post mortem increased, µ-calpain was activated and autolyzed more rapidly and extensively in BM than in LM, but µ/m-calpain was activated at a relative slower rate compared with µ-calpain. Correlation results showed that µ-calpain activity, rather than µ/m-calpain activity, in BM samples was highly correlated with the abundance of desmin and the 30/32 kDa troponin-T degradation components across the postmortem period. However, no such correlations were found with LM µ- and µ/m-calpains. Therefore, our results suggest that BM µ-calpain with a faster and more extensive activation and autolysis would play a relatively dominant role in dictating degradation of desmin and troponin-T in postmortem duck muscle.