Injection of duck recombinant follistatin fusion protein into duck muscle tissues stimulates satellite cell proliferation and muscle fiber hypertrophy

Follistatin (FST) can inhibit the expression of myostatin, which is a predominant inhibitor of muscle development. The potential application of myostatin-based technology has been prompted in different ways in agriculture. We previously constructed an expression vector of duck FST and isolated the FST fusion protein. After the protein was purified and refolded, it was added to the medium of duck myoblasts cultured in vitro. The results show that the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide value of the myoblasts in the duck FST treatment group is higher than that in the control group, which indicates that the duck FST fusion protein exhibits the biological activities that can accelerate myoblast proliferation. To further investigate the roles of duck FST on muscle development, we injected the protein into the duck muscle tissues in vivo. The results show that both the duck muscle fiber cross-sectional area and the satellite cell activation frequency are influenced more in the FST treatment group than they are in the control group. In addition to these phenomena, expression of MyoD and Myf5 were increased, and the expression of myostatin was decreased. Together, these results suggest the potential for using duck FST fusion protein to inhibit myostatin activity and subsequently to enhance muscle growth in vivo. The mechanism by which FST regulates muscle development in the duck is similar to that in mammals and fishes.     

PCR-mtDNA技术鉴别检测不同动物肌肉组织和饲料中鸭源性成分

以鸭肌肉组织DNA为模板,利用PCR-mtDNA技术成功克隆出了鸭mtDNA COIII基因(GenBank Accession No.DQ655706).对所克隆的序列分析表明.其序列包括鸭细胞色素C氧化酶III(COIII)基因全序列784 bp,通过同源性分析可知,动物的线粒体DNA COIII基因是相对保守的,利用此特性设计PCR-mtDNA方法鉴别检测鸭源性成分的特异性引物;以各种动物肌肉组织及饲料DNA为模板进行PCR扩增、经反复验证筛选出只能扩增出鸭DNA的目的片段,而不能扩增出其他动物DNA片段的特异性强、稳定性好的引物P3、P4;利用此引物PCR扩增鸭DNA的特异性片段为226 bp,对PCR产物进行测序分析可知与已克隆的鸭mtDNA COIII基因同源性达到100%,证明了所筛选引物的准确性.通过对不同含量的DNA模板溶液进行PCR扩增的方法,对筛选出的特异性引物P3、P4进行灵敏度试验,结果分析表明灵敏度约为0.001%,证明该PCR方法具有特异性强、灵敏度高的特点,完全可作为鉴别不同动物肌肉组织和饲料中鸭源性成分的方法.     

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.     

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.     

Development of smooth and skeletal muscle cells in the iris of the domestic duck,chick and quail

Summary Embryonic development of the avian iris muscle was studied by light and electron microscopy in order to clarify the origin of the iridial skeletal muscle cells. In normal development of the domestic duck, chick, and quail, the muscle bundles appearing in the iris at stage 35 consisted solely of smooth muscle cells. Undifferentiated cells appeared at stage 36, and finally skeletal muscle cells were observed at stage 37. This sequence suggests that stromal mesenchymal cells migrate into the muscle bundles to become skeletal muscle cells.Tissue culture of whole indes removed from duck embryos at stages 30 through 34 produced skeletal muscle cells while culture of isolated iridial epithelia removed at stages 31 and 32 did not. Removal of the midbrain region of duck embryos at stage 10 frequently produced severe disorganization of the eye concomitant with craniofacial deformities typical of a neural crest mesenchymal defect. These severely disorganized eyes were devoid of iridial skeletal muscle cells. These results also suggest mesenchymal origin of iridial skeletal muscle cells.     

Analysis of the sternotrachealis muscle fibers in some Anseriformes: histochemistry and sex differences

Histochemical characteristics and sizes of the fibers of the sternotrachealis (ST) muscle have been investigated in some Anseriformes (mallard, Pekin duck, Muscovy duck, and goose) of both sexes. A sexual dimorphism has been shown in the muscle of the species examined. In the mallard and Pekin duck, the male ST muscle shows type IIIA fibers in addition to the type I, IIA, and IIB fibers observed also in the female. In the Muscovy duck, the male muscle has only type I and IIA fibers, whereas the female muscle presents type I fibers and both types IIA and IIB fibers. Moreover, the mean frequencies for each fiber type were significantly different between males and females. In the goose, both male and female muscles present only type I and IIA fibers. In all the species examined, the mean areas of each fiber type are significantly different between male and female, being always larger in the male muscles. The anatomical sexual dimorphism observed in the ST muscle is discussed in relation to function.     

The role of target size in neuronal survival.

A loss of about half of the trochlear motor neurons occurs during the course of normal development in duck and quail embryos. The role of the size of the target muscle in controlling the number of surviving motor neurons was examined by making motor neurons innervate targets either larger or smaller in size than their normal target. In one experiment the smaller trochlear motor neuron pool of the quail embryo was forced to innervate the larger superior oblique muscle of the duck embryo. This was accomplished by grafting the midbrain of a quail embryo in the place of the midbrain of a duck embryo. Results indicated that no additional quail trochlear motor neurons were rescued in spite of a considerable increase in target size. In another experiment the larger trochlear motor neuron pool of the duck embryo was made to innervate the smaller superior oblique muscle of the quail embryo. This resulted in loss of some additional neurons; however, the number of surviving motor neurons was not proportionate to the reduction in target size. These experiments failed to provide support for the hypothesis that the size of the target muscle controls the number of surviving motor neurons. Although contact with target is necessary for survival of neurons, factors other than the number or size of target cells are involved in the control of motor neuron numbers during development.     

The role of target size in neuronal survival

A loss of about half of the trochlear motor neurons occurs during the course of normal development in duck and quail embryos. The role of the size of the target muscle in controlling the number of surviving motor neurons was examined by making motor neurons innervate targets either larger or smaller in size than their normal target. In one experiment the smaller trochlear motor neuron pool of the quail embryo was forced to innervate the larger superior oblique muscle of the duck embryo. This was accomplished by grafting the midbrain of a quail embryo in the place of the midbrain of a duck embyro. Results indicated that no additional quail trochlear motor neurons were rescued in spite of a considerable increase in target size. In another experiment the larger trochlear motor neuron pool of the duck embryo was made to innervate the smaller superior oblique muscle of the quail embryo. This resulted in loss of some additional neurons; however, the number of surviving motor neurons was not proportionate to the reduction in target size. These experiments failed to provide support for the hypothesis that the size of the target muscle controls the number of surviving motor neurons. Although contact with target is necessary for survival of neurons, factors other than the number or size of target cells are involved in the control of motor neuron numbers during development. © 1992 John Wiley & Sons, Inc.     

鸭前胰岛素原基因多态性及其与屠体性状的关联分析

以384只北京鸭 (Z2系、Z4系、Z2×Z4杂交系)和樱桃谷鸭为材料, 利用PCR-SSCP结合测序技术, 对前胰岛素原基因外显子2与部分内含子的多态性进行了研究, 并分析对屠体性状的遗传效应。结果发现存在2个单核苷酸突变位点, 即在第179位和第195位分别发生了T→C和C→T的突变。适合性χ2检验结果表明, 北京鸭各品系和樱桃谷鸭均处于Hardy-Weinberg平衡状态(P>0.05)。最小二乘分析SNPs与屠体性状的关系表明, 在北京鸭3个品系中, 基因型 BB 在胴体重、全净膛重和胸肌重上极显著(P<0.01)高于基因型AA和AB, 在腿肌重和皮脂重上极显著(P<0.01)高于基因型AB; 基因型AA在皮脂率和全净膛重上极显著(P<0.01)和显著(P<0.05)高于基因型AB。而对于樱桃谷鸭, 只有AB型在皮脂重和腹脂重上显著(P<0.05)高于基因型AA。研究结果表明, 鸭前胰岛素原基因多态性与鸭的部分屠体性状存在显著相关性, 且B等位基因有利于增加鸭的胴体重和胸肌重。     

One novel SNP of growth hormone gene and its associations with growth and carcass traits in ducks

In this study, the growth hormone (GH) gene was studied as a candidate gene for growth and carcass traits of three duck populations (Cherry Valley duck, Muscovy duck and Jingjiang duck). Three pairs of primers were designed to detect single nucleotide polymorphisms of introns 2, 3 and 4 of the GH gene by polymerase chain reaction-restriction fragment length polymorphism and sequencing methods. Only the products amplified from intron 2 displayed polymorphism. The results showed one novel polymorphism: a variation in intron 2 of GH gene (C172T, JN408701 and JN408702). It was associated with some growth and carcass traits in three duck populations including birth weight, 8-week weight, carcass weight, breast muscle weight, leg muscle weight, eviscerated weight, lean meat rate, dressing percentage, etc. And the TT and CT genotypes were associated with superior growth and carcass traits in carcass weight, dressing percentage and percentage of eviscerated weight. Therefore, the variation in intron 2 of GH may be a molecular marker for superior growth and carcass traits in above duck populations.     

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.     

Identification and characterization of genes related to the development of breast muscles in Pekin duck

Pekin Duck is world-famous for its fast growth, but its breast muscle development is later and breast muscle content is lower compared with other muscular ducks. Therefore, it is very important to discover the genetic mechanism between breast muscle development and relative gene expression in Pekin duck. In current study, the genes which have relationships with breast muscle development were identified by suppression subtractive hybridization. A total of 403 positive clones were sequenced and 257 unigenes were obtained. The expression of 23 genes were analyzed in the breast muscle of 2-, 4-, 6-, 8- week old Pekin ducks. The results showed that unknown clone A233, C83 and C99 showed descending tendency as age increased; KBTBD10, HSPA8, MYL1, ZFP622, MARCH4, Nexilin, FABP4 and MUSTN1 had high expression levels at 6 weeks old; WAC, NT5C3, HSP90AA1, MRPL33, KLF6, TSNAX, CDC42EP3, HSPA4, TRAK1, NR2F2, HAUS1 and IGF1 had high expression levels at 8 weeks and showed ascending tendency as age increased. Expression of these 23 genes were also analyzed in breast muscle, leg muscle, heart, kidney, liver, muscular stomach and sebum cutaneum in 4-8-week old Pekin duck and results showed that most of these genes had high expression in breast muscle, leg muscle and heart.     

epsilon-crystallin from duck eye lens comparison of its quaternary structure and stability with other lactate dehydrogenases and complex formation with alpha-crystallin.

Taxon-specific epsilon-crystallin (epsilonC) from duck eye lens is identical to duck heart muscle lactate dehydrogenase. It forms a dimer of dimers with a dissociation constant of 2.2 x 10-7 M, far beyond the value observed for other vertebrate lactate dehydrogenases. Comparing the characteristics of wild-type epsilon-crystallin with those of three mutants, G115N, G119F and 115N/119F, representing the only significant peripheral sequence variations between duck epsilonC and chicken or pig heart muscle lactate dehydrogenase, no significant conformational differences are detectable. Regarding the catalytic properties, the Michaelis constant of the double mutant 115N/119F for pyruvate is found to be decreased; for wild-type enzyme, the effect is overcompensated by the high expression level of epsilonC in the eye lens. As taken from spectral analysis of the guanidine-induced and temperature-induced denaturation transitions, epsilonC in its dimeric state is relatively unstable, whereas the native tetramer exhibits the high intrinsic stability characteristic of common vertebrate heart and muscle lactate dehydrogenases. The denaturation mechanism of epsilonC is complex and only partially reversible. In the case of thermal unfolding, the predominant side reaction competing with the reconstitution of the native state is the kinetic partitioning between proper folding and aggregation. alpha-Crystallin, the major molecular chaperone in the eye lens, inhibits the aggregation of epsilonC by trapping the misfolded protein.     

Characterization of in vitro cultured myoblasts isolated from duck (<Emphasis Type="Italic">Anas platyrhynchos</Emphasis>) embryo

Myoblasts isolated from duck embryonic muscle were purified and in vitro cultured. External characteristics were observed by using the immunofluorescence technique, and growth curve of duck embryonic myoblasts was established after measuring with the MTT method. Moreover, mRNA expression of three marker genes, the Desmin, the muscle creatine kinase (Mck) and the troponin C (Tnnc), which could reflect the development status of myofibers, were detected each 24 h for cultured cells by using the qPCR technique. Results showed that the in vitro cultured duck myoblasts went through a series of developmental stages, including the proliferation of myoblasts, the differentiation of multi-nuclei myotubes, and the formation of myofiber. The cultured duck embryonic myoblasts entered into a logarithmic stage approximately on the fourth day after seeding. Accompanying with its progressive growth before entering into the logarithmic phase, the myoblasts also showed some differentiation phenomena, reflected by a low expression level of Desmin and high expression level of the Mck and Tnnc genes. During the rapid growth of the logarithmic phase, there was a high expression of the Desmin gene, and a low expression level of the Mck gene and the Tnnc gene in the cultured myoblasts. The expression profiles of the three marker genes for muscle development could be used for distinguishing the different developmental stages of in vitro cultured myoblasts at the molecular level, which would be more accurate and more feasible than observing the external characteristics of the cultured cells.