RNA-seq of breast in 3-week and 6-week age native Pekin and Cherry Valley Pekin ducks
ABSTRACT: Muscle development and lipid deposition are complex processes regulated by a coordinated expression profile of genes. Pekin ducks displayed a significant difference from Cherry Valley duck in muscle fiber development and IMF contents. Genetic comparisons between these two breeds would contribute to the exploration of mechanisms underlying the phenotypic differences. In the present study, breast tissues of Pekin (BD) and Cherry Valley (CD) ducks were used for RNA-seq in two different time points (3 and 6-week) to investigate the transcriptome basis. A total of 16705 genes from duck breast muscle were detected as positively expressed genes (RPKM>0.1) and 2273 genes were regarded as novel genes. Differentially expressed genes (DEGs) in BD3 VS CD3 were regarded as muscle development-related genes as CD3 grown faster than BD3 in muscle fibers. DEGs in BD6 VS CD6 were regarded as lipid-related genes as a much higher IMF contents in BD6 than CD6 while no differences observed in 3-week. DEGs obtained from BD3 VS BD6 and CD3 VS CD6 were also analyzed as relative genes here. In all, 9 genes (MAT1A, KAZALD1, SHROOM3, KLHL6, MYH13, ALDOA, TNNT2, PDLIM3 and PFKM) were validated as muscle development correlated candidate genes, and another 11 genes (DHCR24, CNTFR, ANGPTL4, BCL6, HIP1, TRIB1, ADORA1, C1QTNF2, CD36, PRKAG2 and ACSL1) were regarded as lipid correlated genes in duck. What’s more, functional analysis confirmed that the immune reaction, ECM and energy metabolism also correlated with muscle development and lipid deposition closely. The present study not only enlarged the duck genetic information pool but also provided a dozen of candidate genes related to muscle development and lipid deposition, which laid a firm foundation for the further studies on molecular mechanisms in duck. Overall design: RNA-deep sequencing of breast muscle in 3-week and 6-week age native Pekin duck and Cherry Valley Pekin duck under Hi-seq 2000
Project description:The meat quality of ducks is closely related to the intramuscular fat (IMF) content. This study explored the candidate regulatory genes of IMF formation and lipid deposition in Chaohu ducks. The IMF of breast muscle in 100 ducks was determined and statistically analysed by normal distribution test. Duck liver samples with high IMF (CH, n?=?3) and low IMF (CL, n?=?3) were selected for transcriptome analysis by RNA sequencing (RNA-Seq). The IMF was in accordance with normal distribution (T?=?0.001, P?=?0.999). The IMF from two tails of the normal distribution was significantly different with 2.9983%?±?0.3296% in the CH group and 1.1960%?±?0.1481% in the CL group (P?<?0.0001). RNA-Seq revealed 147 differentially expressed genes, including 78 up-regulated and 69 down-regulated genes in both groups. Validation by qRT-PCR was in agreement with RNA-Seq (R 2?=?0.838). Gene ontology analysis revealed that organophosphate catabolism, oxidation-reduction process, cellular lipid catabolism, lipid transport, lipid localisation, lipid biosynthesis and cellular lipid catabolism were involved in lipid metabolism. Meanwhile, Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that steroid hormone biosynthesis, ovarian steroidogenesis, alpha-linolenic acid metabolism, glycosylphosphatidylinositol anchor biosynthesis and linoleic acid metabolism were involved in lipid deposition, wherein the genes COMT, NT5E, PDE4D, PLA2G4F, A-FABP, ADRA2A, HSD17B2, PPP1R3C, PPP1R3B and NR0B2 were involved in lipid deposition. This study provided insights into the molecular mechanism for regulating lipid metabolism and identified candidate genes for selecting markers to control IMF formation in Chaohu ducks.
Project description:BACKGROUND:Since early 2015, mule duck and Cherry Valley duck flocks have been suffering from short beak and dwarfism syndrome. This widely spreading infectious disease is characterized by growth retardation, smaller beak and tarsus with high morbidity and low mortality rate. For better understanding, we identified and characterized virus isolates named AH and GD from diseased Cherry Valley duck and mule duck flocks and investigated the damage caused by novel parvovirus-related virus (NGPV) to tissues and organs, including kidney, brain, pancreas, liver, spleen, bursa of fabricius and myocardial tissues. RESULTS:AH and GD isolates shared high nucleotide identity with goose parvovirus (GPV). Alignment studies of AH and GD isolates showed 94.5-99.2% identity with novel parvovirus-related virus (NGPV), 98.7-91.5% identity with GPV and 79.9-83.7% with muscovy duck parvovirus (MDPV). Compared with other NGPV, classical GPV and MDPV sequences, a four 14-nucleotide-pair insertion in GD isolate was found in left open reading frame (ORF) (87-100 nt and 350-363 nt) and in right ORF (4847-4861 nt and 5122-5135 nt). However, in AH isolate, a five 14-nucleotide-pair deletions similar to other NGPV were found. The complete genome sequence comparison of eleven NGPV isolates from mule ducks and cherry valley ducks revealed no remarkable difference between them. Notably, the myocardium and bursa of fabricius of both disease and healthy animals are perfectly normal while other tissues have inflammatory cells exudation. CONCLUSIONS:The AH and GD strains are novel parvovirus-related virus that isolates from mule ducks or cherry valley ducks which DNA sequence has no remarkable difference. The histopathology of tissues and organs such as kidney, brain etc. revealed non-significant changes in experimental and control animals. Overall, this study has contributed better understanding of molecular biology of NGPV strains and will help to develop the candidate strain for vaccine preparation to get better protection against these viral infections.
Project description:Meat quality is closely related to adipose tissues in ducks, and adipogenesis is controlled by a complex network of transcription factors tightly acting at different stages of differentiation especially in ducks. The aim of this study was to establish the preadipocyte in vitro culture system and understand the biological characteristics of expansion of duck adipocyte tissue at the cellular and molecular level. We isolated pre-adipocytes from the subcutaneous fat of three breeds of duck and differentiated them into mature adipocytes using a mixture of insulin, rosiglitazone, dexamethasone, 3-isobutyl-1-methylxanthine, and oleic acid over 0,2, 4, 6, and 8 days. Successful differentiation was confirmed from the development of lipid droplets and their response to Oil Red O, and increasing numbers of lipid droplets were stained red over time. The expression of key marker genes, including peroxisome proliferator activated receptor γ (PPARγ), CCAAT/enhancer binding protein-α (C/EBPα), adipocyte fatty acid binding protein 4 (FABP4), and fatty acid synthetase (FAS), gradually increased during pre-adipocyte differentiation. Furthermore, it was verified by interference experiments that the knockdown of PPARγ directly reduced lipid production. Meanwhile we analyzed the role of unsaturated fatty acids in the production of poultry fat using different concentrations of oleic acid and found that lipid droplet deposition was highest when the concentration of oleic acid was 300 μM. We also compared the level of differentiated pre-adipocytes that were isolated from Jianchang ducks (fatty-meat duck), Cherry Valley ducks (lean-meat duck) and White-crested ducks (egg-producing duck). The proliferation and differentiation rate of pre-adipocytes derived from Jianchang ducks was higher than that of White-crested ducks. These results provide the foundation for further research into waterfowl adipogenesis.
Project description:Class II major histocompatibility complex (MHC-II) transactivator (CIITA) is a member of the pattern recognition receptor in cytoplasm, which is involved in host innate immune responses. In this study, the full-length cDNA of Cherry Valley duck CIITA (duCIITA) was cloned from the spleen of healthy Cherry Valley ducks for the first time. The CDs of duCIITA have 3648 bp and encode 1215 amino acids. The homology analysis of CIITAs amino acid sequence showed that the duCIITA has the highest identity with the Anas platyrhynchos (94.9%), followed by Gallus gallus and Meleagris gallopavo. Quantitative real-time PCR analysis indicated that duCIITA mRNA has a broad expression level in healthy Cherry Valley duck tissues. It was highly expressed in the lung and cerebellum, and lowly expressed in the rectum and esophagus. After the avian pathogenic Escherichia coli (APEC) O1K1 infection, the ducks exhibited the typical clinical symptoms, and a severe fibrinous exudate in the heart and liver surface was observed. Meanwhile, a significant up-regulation of duCIITA was detected in the infected liver. The inflammatory cytokines IL-1?, IL-6, and IL-8 have a significant up-regulation in the infected liver, spleen and brain. In addition, knockdown of the duCIITA reduces antibacterial activity and inflammatory cytokine production of the duck embryo fibroblast cells. Our research is the first study of the cloning, tissue distribution, and antibacterial immune responses of duCIITA, and these findings imply that duCIITA was an important receptor, which was involved in the early stage of the antibacterial innate immune response to APEC O1K1 infection of Cherry Valley duck.
Project description:Fat character is an important index in duck culture that linked to local flavor, feed cost and fat intake for costumers. Since the regulation networks in duck lipid metabolism had not been reported very clearly, we aimed to explore the potential miRNA-mRNA pairs and their regulatory roles in duck lipid metabolism. Here, Cherry-Valley ducks were selected and treated with/without 5% oil added in feed for 2 weeks, and then fat content determination was performed on. The data showed that the fat contents and the fatty acid ratios of C17:1 and C18:2 were up-regulated in livers of oil-added ducks, while the C12:0 ratio was down-regulated. Then 21 differential miRNAs, including 10 novel miRNAs, were obtain from the livers by sequencing, and 73 target genes involved in lipid metabolic processes of these miRNAs were found, which constituted 316 miRNA-mRNA pairs. Two miRNA-mRNA pairs including one novel miRNA and one known miRNA, N-miR-16020-FASN and gga-miR-144-ELOVL6, were selected to validate the miRNA-mRNA negative relation. And the results showed that N-mir-16020 and gga-miR-144 could respectively bind the 3'-UTRs of FASN and ELOVL6 to control their expressions. This study provides new sights and useful information for future research on regulation network in duck lipid metabolism.
Project description:The nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that is involved in host innate immunity and located in the cytoplasm. In the present study, the full-length cDNA of Cherry Valley duck NLRP3 (duNLRP3) (2,805 bp encode 935 amino acids) was firstly cloned from the spleen of healthy Cherry Valley ducks, and the phylogenetic tree indicated that the duNLRP3 has the closest relationship with Anas platyrhynchos in the bird branch. According to quantitative real-time PCR analysis, the duNLRP3 mRNA has a broad expression spectrum in healthy Cherry Valley duck tissues, and the highest expression is in the pancreas. There was significant up-regulation of duNLRP3 mRNA expression in the liver and down-regulation in the spleen after infection with avian pathogenic Escherichia coli (APEC) O1K1, especially at 3 days after the infection. Ducks hatched from NLRP3-lentiviral vector-injected eggs had significantly higher duNLRP3 mRNA expression in the liver, spleen, brain, and cecum, which are tissues usually with lower background expression. The mRNA expression levels of inflammatory cytokines IL-1β, IL-18, and TNF-α significantly increased after the APEC infection in those tissues. The bacterial content in the liver and spleen decreased significantly compared with the NC-lentiviral vector-injected ducks. In addition, in the duck embryo fibroblasts, both of the overexpression and knockdown of duNLRP3 can trigger the innate immune response during the E. coli infection. Specifically, overexpression induced antibacterial activation, and knockdown reduced the antibacterial activity of the host cells. The IL-1β, IL-18, and TNF-α mRNA expressions showed up-regulation or down-regulation. The results demonstrate that duNLRP3 has a certain antibacterial activity during E. coli infection. These findings also contribute to better understanding the importance of duNLRP3 in regulating the inflammatory response and the innate immune system of ducks.
Project description:BACKGROUND:Duck circovirus (DuCV) is a potential immunosuppressive virus that causes feather disorders in young ducks. In this study, DuCV obtained from various species of ducks was investigated by polymerase chain reaction (PCR) in southern and southwestern China (Guangdong, Guangxi and Yunnan provinces) from 2018 to 2019. RESULTS:A total of 848 bursa samples were collected from dead Mulard, Cherry Valley Pekin, Muscovy and Mallard ducks from duck farms. The positivity rate of DuCV in the total sample was approximately 36.91%. We found that the prevalence of DuCV in Yunnan (43.09%) was higher than those in Guangxi (34.38%) and Guangdong (34.4%). However, the positivity rates of DuCV in the four duck species were not significantly different (P?>?0.05). Nineteen randomly selected complete viral genomes were sequenced. The complete genomes of the DuCV were 1987 to 1995?nt in length, and were 81.7-99.3% homologous to the other 57 sequences in GenBank. Phylogenetic analyses based on the complete genomes of 76 DuCVs showed that the 19 novel DuCV sequences from Guangdong and Guangxi provinces mainly belonged to the DuCV-1 and DuCV-2 genetic groups, respectively. However, the two genotype groups coexisted in Yunnan Province. In addition, recombination analysis showed putative recombination sites in 3 strains in Yunnan that originated from strains Guangdong and Guangxi. Interestingly, the epidemiological investigation showed that Mulard ducks, Cherry Valley Pekin ducks and Muscovy ducks more than 4 weeks old were more susceptible to infection with the novel DuCV than ducks less than 4 weeks old. CONCLUSIONS:These data provide insight into the molecular epidemiology and genetic diversity of DuCVs circulating in southern and southwestern China for the first time.
Project description:Pectoral muscle (PM) comprises an important component of overall meat mass in ducks. However, PM has shown arrested or even reduced growth during late embryonic development, and the molecular mechanisms underlying PM growth during the late embryonic to neonatal period in ducks have not been addressed. In this study, we characterized potential candidate genes and signaling pathways related to PM development using RNA sequencing of PM samples selected at embryonic days (E) 21 and 27 and 5 days post-hatch (dph) in two duck breeds (Gaoyou and Jinding ducks). A total of 393 differentially expressed genes (DEGs) were identified, which showed higher or lower expression levels at E27 compared with E21 and 5 dph, reflecting the pattern of PM growth rates. Among these, 43 DEGs were common to all three time points in both duck breeds. These DEGs may thus be involved in regulating this developmental process. Specifically, KEGG pathway analysis of the 393 DEGs showed that genes involved with different metabolism pathways were highly expressed, while genes involved with cell cycle pathways showed lower expression levels at E27. These DEGs may thus be involved in the mechanisms responsible for the phenomenon of static or decreased breast muscle growth in duck breeds during the late embryonic period. These results increase the available genetic information for ducks and provide valuable resources for analyzing the mechanisms underlying the process of PM development.
Project description: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 ?-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.
Project description:Given the promising results of applying Bacillus subtilis (B.subtilis) as a probiotic in both humans and animals, the aim of this study was to systematically investigate the effects of B. subtilis on growth performance, immune response and disease resistance in Cherry Valley ducks. At 28 d post-hatch (dph), ducks fed a diet with B. subtilis weighed significantly more, had higher relative immune organ weights (e.g., bursa of Fabricius, thymus, and spleen), and exhibited greater villus heights, villus height to crypt depth ratios (duodenum and jejunum), and shallower crypt depths in the duodenum than controls fed a normal diet (p < 0.05). Moreover, the major pro-inflammatory factors and antiviral proteins, as measured in the thymus and the spleen, were higher at 28 dph in ducks fed probiotics than those of 14 dph. After 28 d of feeding, the ducks were challenged with Escherichia coli (E. coli) and novel duck reovirus (NDRV), and ducks fed B. subtilis achieved survival rates of 43.3 and 100%, respectively, which were significantly greater than the control group's 20 and 83.3%. Altogether, diets with B. subtilis can improve Cherry Valley ducks' growth performance, innate immune response, and resistance against E. coli and NDRV.