Identification of Differentially Expressed Genes in Breast Muscle and Skin Fat of Postnatal Pekin Duck

Lean-type Pekin duck is a commercial breed that has been obtained through long-term selection. Investigation of the differentially expressed genes in breast muscle and skin fat at different developmental stages will contribute to a comprehensive understanding of the potential mechanisms underlying the lean-type Pekin duck phenotype. In the present study, RNA-seq was performed on breast muscle and skin fat at 2-, 4- and 6-weeks of age. More than 89% of the annotated duck genes were covered by our RNA-seq dataset. Thousands of differentially expressed genes, including many important genes involved in the regulation of muscle development and fat deposition, were detected through comparison of the expression levels in the muscle and skin fat of the same time point, or the same tissue at different time points. KEGG pathway analysis showed that the differentially expressed genes clustered significantly in many muscle development and fat deposition related pathways such as MAPK signaling pathway, PPAR signaling pathway, Calcium signaling pathway, Fat digestion and absorption, and TGF-beta signaling pathway. The results presented here could provide a basis for further investigation of the mechanisms involved in muscle development and fat deposition in Pekin duck.     

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 13^th 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.     

Does overfeeding enhance genotype effects on energy metabolism and lipid deposition in breast muscle of ducks?

We evaluated the effects of genotype (Muscovy, Pekin and their crossbreed hinny and mule ducks) and feeding levels (overfeeding between 12 and 14 weeks of age vs ad libitum feeding) on energy metabolism and lipid deposition in breast muscle of ducks. Samples of breast muscle (Pectoralis major) were collected at 14 weeks of age from 8 birds per group. Overfeeding induced an accumulation of lipids in breast muscle (1.5- to 1.7-fold, depending on genotype) mainly induced by triglyceride deposition. It also induced a considerable increase in the amounts (expressed as g/100 g of tissue) of saturated and mono-unsaturated fatty acids (SFA, MUFA), while the amounts of poly-unsaturated fatty acids (PUFA) remained unchanged in hinny and Muscovy ducks or slightly increased in Pekin and mule ducks. In breast muscle, overfeeding decreased the activity of the main enzymes involved in lipogenesis from glucose (glucose-6-phosphate dehydrogenase, G6PDH, malic enzyme, ME, acetyl CoA carboxylase, ACX). Lipoprotein lipase (LPL) activity in Pectoralis major muscle was also significantly decreased (-21%). The ability of muscle tissues to catabolize long-chain fatty acids, as assessed by beta-hydroxyacyl CoA dehydrogenase (HAD) activity, was increased in Pectoralis major muscle, as was cytochrome-c oxidase (COX) activity. Hybrid and Pekin ducks exhibited higher levels of ACX and LPL activity in Pectoralis major muscle than Muscovy ducks, suggesting a greater ability to synthesise lipids in situ, and to take up circulating lipids. Total lipid content in breast muscle of hybrid and Pekin ducks was higher than in that of Muscovy ducks. In hybrid and Pekin ducks, lipid composition of breast muscle was characterized by higher amounts of triglycerides, SFA and MUFA than in Muscovy ducks. Finally, oxidative metabolism was greater in Pectoralis major muscles of hybrid and Pekin ducks than in Muscovy ducks, suggesting an adaptative strategy of muscle energy metabolism according to lipid level.     

Effects of heat stress on antioxidant defense system,inflammatory injury,and heat shock proteins of Muscovy and Pekin ducks: evidence for differential thermal sensitivities

Rising temperatures are severely affecting the mortality, laying performance, and meat quality of duck. Our aim was to investigate the effect of acute heat stress on the expression of heat shock proteins (HSPs: HSP90, 70, 60, 40, and 10) and inflammatory factors (nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)) and antioxidant enzyme activity (superoxide dismutase (SOD), malondialdehybe (MDA), catalase (CAT), total antioxidant capacity (T-AOC)) in livers of ducks and to compare the thermal tolerance of Pekin and Muscovy ducks exposed to acute heat stress. Ducks were exposed to heat at 39 ± 0.5 °C for 1 h and then returned to 20 °C for 1 h followed by a 3-h recovery period. The liver and other tissues were collected from each individual for analysis. The mRNA levels of HSPs (70, 60, and 40) increased in both species, except for HSP10, which was upregulated in Muscovy ducks and had no difference in Pekin ducks after heat stress. Simultaneously, the mRNA level of HSP90 decreased in the stress group in both species. Morphological analysis indicated that heat stress induced tissue injury in both species, and the liver of Pekin ducks was severely damaged. The activities of several antioxidant enzymes increased in Muscovy duck liver, but decreased in Pekin duck. The mRNA levels of inflammatory factors were increased after heat stress in both duck species. These results suggested that heat stress could influence HSPs, inflammatory factors expression, and the activities of antioxidant enzymes. Moreover, the differential response to heat stress indicated that the Muscovy duck has a better thermal tolerance than does the Pekin duck.     

Genome-wide association study reveals novel loci associated with fat-deposition and meat-quality traits in Pekin ducks

Meat-quality traits play an essential role in meat poultry production. To determine the genetic mechanisms of meat quality in Pekin ducks, we performed a large-scale GWAS to identify quantitative trait loci affecting meat quality in Pekin ducks. We measured 10 traits in 542 Pekin ducks and genotyped each duck using genotyping-by-sequencing. The genetic parameters (genomic heritability, genetic correlation) for 10 meat-quality related traits were evaluated. Based on the large genotype–phenotype dataset, we performed GWASs for all of these traits. A total of 33 significant QTL (P < 3.03 × 10⁻⁵) across 13 chromosomes were identified by loci-based analysis. Some newly identified candidate genes were discovered for fat-deposition and meat-quality traits, including PAG1 for body weight and eviscerated weight, INTU and NUP35 for abdominal fat weight and ratio, NUP3 and ARHGDIB for skin fat weight and ratio, GOLGA5 for breast muscle toughness and breast tenderness, and CTDSPL and PKP1 for breast muscle thickness. The current study is the first systematic report regarding duck meat quality.     

Genome-wide association studies demonstrate that TASP1 contributes to increased muscle fiber diameter

Muscle fiber diameter is an economically important trait because it affects meat yield and quality. However, the genetic basis underlying muscle fiber diameter has not been determined. In this study, we collected THREE muscular histological phenotypes in 479 ducks from an F₂ segregating population generated by mallard × Pekin duck crosses. We performed genome-wide association studies (GWAS) and identified a quantitative trait locus (QTL) significantly associated with muscle fiber diameter on chromosome 3. Then, we discovered the selection signatures using the fixation index among 40 mallards and 30 Pekin ducks in this QTL region. Furthermore, we characterized the recombination event in this QTL region and identified a 6-kb block located on TASP1 that was significantly associated with muscle fiber diameter. Finally, five SNPs were screened as potential causative mutations within the 6-kb block. In conclusion, we demonstrated that TASP1 contributes to an increase in muscle fiber diameter, which helps to characterize muscle development and contributes to the genetic improvement of meat yield and quality in livestock.Subject terms: Genome-wide association studies, Gene expression     

Quantitative real-time PCR primer design, cDNA amplification and sequence analysis from 22 genes mainly associated with lipid metabolism in Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) ducks

Few genomic tools are available in ducks. To produce some new resources, we have designed Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) duck-specific primers for 22 genes involved mainly in lipid metabolism, and to a lesser extent in carbohydrate metabolism and other functions. Primers were designed according to duck sequences when available and otherwise from the corresponding conserved regions in chicken and human sequences. These primers allowed quantitative RT-PCR amplification of RNA from Pekin and Muscovy ducks. Amplified cDNA products from both species were sequenced and were found to be very similar to chicken sequences (about 94%). This work provides additional genomic resources and polymorphism information for some genes in duck species and represents a first step towards gene expression analyses in Pekin and Muscovy ducks.     

A technique for liver biopsy performed in Pekin ducks using anesthesia with Telazol.

Infection of Pekin ducks with duck hepatitis B virus is a useful model for studying the hepadenoviruses, of which human hepatitis B virus is the prototype. The utility of this model has been limited, however, by the difficulties associated with anesthetizing and obtaining liver biopsies from ducks. We developed a technique using Telazol (13 mg/kg) to anesthetize ducks before surgical biopsy of the liver in ducks infected with duck hepatitis B virus. Eight Pekin ducks infected with duck hepatitis B virus underwent serial biopsies at 4- to 5-week intervals. There was one perioperative death in 34 surgical procedures with no evidence on intra-abdominal sepsis or wound complications. Telazol can be used safely and humanely to anesthetized ducks without the need for general endotracheal anesthesia.     

Is ability to hepatic steatosis influenced by age at the beginning of the overfeeding period in Muscovy and Pekin ducks?

The aim of this study was to analyse the effects of species (Muscovy and Pekin ducks) and age at the beginning of the overfeeding period on fatty liver production, carcass composition and lipid and moisture content of the liver and breast muscle. We reared four groups of 40 ducks per species for the study, starting at 2-week intervals in order to have four different ages together at the beginning of the overfeeding period (10, 12, 14 and 16 weeks). At the end of the overfeeding period, all ducks were slaughtered. Our results confirmed the high levels of difference in carcass composition and lipid content in the plasma, liver and breast muscle between Muscovy and Pekin ducks at all ages. Pekin ducks were not able to develop a high degree of hepatic steatosis, but had increased lipid storage in peripheral adipose and muscle tissues than Muscovy ducks. However, the fatty liver weight of Pekin ducks increased with age, with lipid deposition in the liver and peripheral tissues. The ability of Muscovy ducks to produce fatty livers remained unchanged with age in line, with lipid deposition in the liver and peripheral tissues. The sites of lipid deposition thus depend on species and not on the physiological maturity of ducks.     

Developmental origins of species-specific muscle pattern

Vertebrate jaw muscle anatomy is conspicuously diverse but developmental processes that generate such variation remain relatively obscure. To identify mechanisms that produce species-specific jaw muscle pattern we conducted transplant experiments using Japanese quail and White Pekin duck, which exhibit considerably different jaw morphologies in association with their particular modes of feeding. Previous work indicates that cranial muscle formation requires interactions with adjacent skeletal and muscular connective tissues, which arise from neural crest mesenchyme. We transplanted neural crest mesenchyme from quail to duck embryos, to test if quail donor-derived skeletal and muscular connective tissues could confer species-specific identity to duck host jaw muscles. Our results show that duck host jaw muscles acquire quail-like shape and attachment sites due to the presence of quail donor neural crest-derived skeletal and muscular connective tissues. Further, we find that these species-specific transformations are preceded by spatiotemporal changes in expression of genes within skeletal and muscular connective tissues including Sox9, Runx2, Scx, and Tcf4, but not by alterations to histogenic or molecular programs underlying muscle differentiation or specification. Thus, neural crest mesenchyme plays an essential role in generating species-specific jaw muscle pattern and in promoting structural and functional integration of the musculoskeletal system during evolution.     

Is the hepatic metabolism of glucose and linoleic acid influenced by species in overfed ducks?

There are genetic differences in the hepatic glucose and linoleic acid metabolisms between Muscovy and Pekin ducks ad libitum-fed. To understand the effect of overfeeding on the hepatic metabolisms in these two species of ducks, we compared the different pathways of glucose and linoleic acid reaching the liver of Muscovy (Cairina moschata) (n = 6) and Pekin (Anas platyrhynchos) (n = 6) ducks overfed for 1 week and sacrificed 2–4 h after their last meal by using the ex vivo method of liver slices incubated for 16 h with [U-¹⁴C]-glucose, [1-¹⁴C]-linoleic acid and [³⁵S]-methionine added to the survival medium. The glucose was the main precursor of triacylglycerol synthesis in the liver of these two species and its hepatic metabolism was similar between species. The hepatic uptake of linoleic acid was 1.7-fold higher (P = 0.020) in the Muscovy duck than in the Pekin duck leading to a 1.9-fold higher (P = 0.017) esterification of this fatty acid in the liver of the Muscovy duck than in that of the Pekin duck. Finally, both species after 1 week of overfeeding exhibited the same capacity to secrete VLDL remaining insufficient to avoid hepatic steatosis.     

Duck Enteritis Virus Glycoprotein D and B DNA Vaccines Induce Immune Responses and Immunoprotection in Pekin Ducks

DNA vaccine is a promising strategy for protection against virus infection. However, little is known on the efficacy of vaccination with two plasmids for expressing the glycoprotein D (gD) and glycoprotein B (gB) of duck enteritis virus (DEV) in inducing immune response and immunoprotection against virulent virus infection in Pekin ducks. In this study, two eukaryotic expressing plasmids of pcDNA3.1-gB and pcDNA3.1-gD were constructed. Following transfection, the gB and gD expressions in DF1 cells were detected. Groups of ducks were vaccinated with pcDNA3.1-gB and/or pcDNA3.1-gD, and boosted with the same vaccine on day 14 post primary vaccination. We found that intramuscular vaccinations with pcDNA3.1-gB and/or pcDNA3.1-gD, but not control plasmid, stimulated a high frequency of CD4⁺ and CD8⁺ T cells in Pekin ducks, particularly with both plasmids. Similarly, vaccination with these plasmids, particularly with both plasmids, promoted higher levels of neutralization antibodies against DEV in Pekin ducks. More importantly, vaccination with both plasmids significantly reduced the virulent DEV-induced mortality in Pekin ducks. Our data indicated that vaccination with plasmids for expressing both gB and gD induced potent cellular and humoral immunity against DEV in Pekin ducks. Therefore, this vaccination strategy may be used for the prevention of DEV infection in Pekin ducks.     

Genes involved in the establishment of hepatic steatosis in Muscovy,Pekin and mule ducks

Our main objectives were to determine the genes involved in the establishment of hepatic steatosis in three genotypes of palmipeds. To respond to this question, we have compared Muscovy ducks, Pekin ducks and their crossbreed the mule duck fed ad libitum or overfed. We have shown a hepatic overexpression of fatty acid synthase (FAS) and di-acyl glycerol acyl transferase 2 (DGAT2) in overfed individuals, where DGAT2 seemed to be more regulated. This increase in lipogenesis genes is associated with a decrease of lipoprotein formation in Muscovy and mule ducks, especially apolipoprotein B (ApoB) and Microsomal Triglyceride Transfer Protein (MTTP), leading to lipid accumulation in liver. In Pekin ducks, MTTP expression is upregulated suggesting a better hepatic lipids exportation. Regarding lipids re-uptake, fatty acid-binding protein 4 and very-low-density-lipoprotein receptor are overexpressed in liver of mule ducks at the end of the overfeeding period. This phenomenon puts light on a mechanism unknown until today. In fact, mule can incorporate more lipids in liver than the two other genotypes leading to an intensified hepatic steatosis. To conclude, our results confirmed the genotype variability to overfeeding. Furthermore, similar observations are already described in non-alcoholic fatty liver disease in human, and ask if ducks could be an animal model to study hepatic triglyceride accumulation.     

An SNP in the MyoD1 Gene Intron 2 Associated with Growth and Carcass Traits in Three Duck Populations

Myogenic differentiation 1 (MyoD1) genes belong to the MyoD gene family and play key roles in growth and muscle development. This study was designed to investigate the effects of variants in the MyoD1 gene on duck growth and carcass traits. Three duck populations (Cherry Valley, Jingjiang, and Muscovy) were sampled, their growth and carcass traits were measured, and they were genotyped using the PCR–RFLP method. The results showed one novel polymorphism, an alteration in intron 2 of the MyoD1 gene (A to T). It was associated with the traits of weight at 8?weeks, carcass weight, breast muscle weight, leg muscle weight, eviscerated percentage, percentage of leg muscle weight, dressing percentage, and lean meat percentage. This alteration in intron 2 of MyoD1 may be linked with potential major loci or genes affecting some growth and carcass traits.