Effect of feed restriction on the intestinal microbial community structure of growing ducks.

In poultry, feed restriction is common feeding management to limit poultry nutrients intake so that poultry only intake the essential energy, meeting the basic need of growth and development. Our study investigated whether feeding restriction affects the diversity of the intestinal microbiota of growing breeding ducks. In this research, the 60-120-day-old ducks were raised in restricted and free-feeding groups. After slaughtering, the carcass traits and the cecal contents were collected for 16S rRNA sequencing analysis. After feeding restriction, the growth rate of ducks was limited, the weight and rate of abdominal fat decreased, and the rate of chest and leg muscles increased. In addition, feeding restriction can also change the diversity of intestinal microorganisms in breeding ducks, such as the increase of Firmicutes abundance and the decrease of Bacteroidetes abundance. After analyzing of correlation, significant correlations between gut microbiota and carcass phenotypes were found. The results indicated that gut microbiota might be involved in the life activities associated with phenotypic changes. This study proved the effect of feeding methods on the intestinal microbiota of ducks, providing a theoretical basis of the microbial angle for raising ducks in a feeding-restricted period.

Silencing Pax3 by shRNA inhibits the proliferation and differentiation of duck (Anas platyrhynchos) myoblasts.

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 < 0.05). Silencing Pax3 by shRNA1 significantly reduced the proliferation and differentiation of duck myoblasts (P < 0.05) due to downregulated expression of myogenic regulator factors. These trends could be rescued by adding FA; and Pax7, a paralog gene of Pax3, was involved in those processes. Overall, Pax3 had a positive function in duck myoblast proliferation and differentiation by modulating the expression of myogenic regulation factors, and shRNA targeting of Pax3 might be a new approach for understanding the function of Pax3 in the development of diverse tissues.

In ovo feeding of IGF-1 to ducks influences neonatal skeletal muscle hypertrophy and muscle mass growth upon satellite cell activation.

To investigate reasons for the muscle increase observed when eggs are treated by IGF-1 and whether or not satellite cell activation is specific to different types of myofibers, duck eggs were administrated with IGF-1. After injection, during the neonatal stages, the duck breast muscle and leg muscle were isolated for analysis. The muscle weight, muscle fiber diameter (MFD), cross-sectional area (CSA), the number of myofibers per unit area (MFN) and frequency of satellite cell activation and mitosis at the embryo stage of 27 days (27E) and the postnatal stage of 2 days after hatching (P2D) were determined. In addition, expression of two important myogenic transcription factors MyoD and Myf5 were detected and compared in the two types of muscle tissues. Results indicated that IGF-1 administration increased the duck body weight, MFD, CSA, MFN, and quantity of activated satellite cells and mitotic nuclei in the two types of muscle tissues. The MyoD and Myf5 expressed at a higher level in IGF-1-treated muscle. IGF-1 stimulated muscle weight growth more in the leg muscle than in the breast muscle. These results indicate that in ovo feeding of IGF-1 can stimulate duck growth and, especially, lead to increased muscle hypertrophy. These increases appear to be mainly dependent on the activation of satellite cells, some of which proliferate and fuse to the myofiber, enabling increased muscle mass. IGF-1 can indirectly affect satellite cells by regulating the expression of two important myogenic transcription factors, MyoD and Myf5, which help activate satellite cells.

Cloning and expression of stearoyl-CoA desaturase 1 (SCD-1) in the liver of the Sichuan white goose and landes goose responding to overfeeding.

The EST sequence of goose (Anser cygnoides) Stearoyl-CoA desaturase 1(SCD-1) was obtained from a subtractive cDNA library. To further investigate the role of SCD-1 in lipid metabolism in geese, 5'-RACE and 3'-RACE were carried out in this study to obtain the complete cDNA sequence of goose SCD-1, which contained a 29-bp 5' UTR, a 1074-bp open reading frame (ORF) encoding 357 amino acids, and a 125-bp 3' UTR. The expression of SCD-1 was measured in several tissues, and the effects of overfeeding on the expression of SCD-1 were studied. The results of real time RT-PCR demonstrated that, compared to the brain, goose SCD-1 mRNA was more abundant in the liver. Overfeeding markedly increased the mRNA expression of SCD-1 in the liver of Sichuan White and Landes geese, and gene expression was markedly higher in the Sichuan White goose than in the landes goose. The mRNA abundance of SCD-1 in the liver had significant positive correlations with triacylglycerol (TG) content in liver lipids and in the levels of plasma insulin and very low-density lipoproteins (VLDL) levels in Sichuan white geese. However, the mRNA abundance of SCD-1 in the livers of Landes geese had only significant positive correlations with the TG content in liver lipids. In conclusion, SCD-1 is not only critical for hepatic steatosis in geese but is also important for the difference in lipid deposition in the livers of the two breeds.

Molecular evolutionary analysis of the duck MYOD gene family and its differential expression pattern in breast muscle development.

1. The objective of the research was to investigate the molecular evolutionary relationships between the duck myogenic determination factors (MYOD) gene family members and their roles in muscle development. 2. The four members of the duck MYOD gene family were cloned using RT-PCR, and their relative mRNA expression during duck muscle development was measured using qRT-PCR. 3. The results showed that MyoD and Myf5 clustered together, as did MyoG and MRF4 based on their complete amino acid sequence and the basic helix-loop-helix domain. Results of the evolutionary level analysis were consistent with that of the differential expression patterns during duck breast muscle development. As determined by qRT-PCR, MyoD and Myf5 were highly expressed in 22-day embryos, while MyoG and MRF4 expression was high in 14-day embryos. 4. We conclude that the entire MYOD gene family in the duck originated from a common ancestral gene and evolved after two duplication events. The roles of the MYOD gene family members in duck muscle development are similar to those in mammals.

Isolation, culture and differentiation of duck (Anas platyrhynchos) preadipocytes.

In the present study, we isolated preadipocytes from the adipose tissue of Peking duck and subsequently cultured them in vitro. Cell counting kit-8 assay was employed to establish the growth curve of duck primary preadipocytes. Meanwhile, after the cells reaching full confluency, they were induced to differentiate into mature adipocytes by the addition of a cocktail containing dexamethasone, insulin, 3-isobutyl-1-methylxanthine, and oleic acid for 8 days. Successful differentiation was demonstrated by the development of lipid droplets and the expression of key marker genes including peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (CEBP/α) and adipocyte fatty acid-binding protein (FABP4). Our results showed that duck primary preadipocytes began to adhere 12 h after seeding as short spindle shapes or litter triangles, which grew quickly 3 days post attachment and maintained stable after day 7. After 8 days the preadipocytes were induced to differentiate into mature adipocytes, which were stained red by oil red O. Additionally, it showed that during preadipocyte differentiation PPARγ mRNA was highly expressed at day 3, while CEBP/α and FABP4 mRNA peaked at day 5 and 8, respectively. These results indicate that we have successfully isolated and cultured Peking duck preadipocytes and successfully induced them to differentiate into mature adipocytes. This work could lay a foundation for further research into waterfowl adipogenesis.

Developmental expression and alternative splicing of the duck myostatin gene.

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 TGFß-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.