Chicken muscle antibody array reveals the regulations of LDHA on myoblast differentiation through energy metabolism.

Myoblast proliferation and differentiation are highly dynamic and regulated processes in skeletal muscle development. Given that proteins serve as the executors for the majority of biological processes, exploring key regulatory factors and mechanisms at the protein level offers substantial opportunities for understanding the skeletal muscle development. In this study, a total of 607 differentially expressed proteins between proliferation and differentiation in myoblasts were screened out using our chicken muscle antibody array. Biological function analysis revealed the importance of energy production processes and compound metabolic processes in myogenesis. Our antibody array specifically identified an upregulation of LDHA during differentiation, which was associated with the energy metabolism. Subsequent investigation demonstrated that LDHA promoted the glycolysis and TCA cycle, thereby enhancing myoblasts differentiation. Mechanistically, LDHA promotes the glycolysis and TCA cycle but inhibits the ETC oxidative phosphorylation through enhancing the NADH cycle, providing the intermediate metabolites that improve the myoblasts differentiation. Additionally, increased glycolytic ATP by LDHA induces Akt phosphorylation and activate the PI3K-Akt pathway, which might also contribute to the promotion of myoblasts differentiation. Our studies not only present a powerful tool for exploring myogenic regulatory factors in chicken muscle, but also identify a novel role for LDHA in modulating myoblast differentiation through its regulation of cellular NAD+ levels and subsequent downstream effects on mitochondrial function.

Identification of candidate genes associated with skin yellowness in yellow chickens.

The yellow color of the skin is an important economic trait for yellow chickens. Low and non-uniform skin yellowness would reduce economic efficiency. However, the regulatory mechanism of chicken skin yellowness has not been fully elucidated. In this study, we evaluated the skin yellowness of 819 chickens by colorimeter and digital camera, which are from the same batch and the same age of 2 pure lines with significant differences in skin yellowness. A total of 982 candidate differential expressed genes (DEGs) were detected in duodenal tissue by RNA-seq analysis for high and low yellowness chickens. Among the DEGs, we chose fatty acid translocase (CD36) gene and identified a single nucleotide polymorphism (SNP) upstream of the CD36 gene that was significantly associated with skin yellowness at multiple parts of the chicken, and its different genotypes had significant effects on the promoter activity of the CD36 gene. These findings will help to further elucidate the molecular mechanism of chicken skin yellowness and is helpful for improving chicken skin yellowness.

QPCTL Affects the Daily Weight Gain of the F2 Population and Regulates Myogenic Cell Proliferation and Differentiation in Chickens.

Molecular breeding can accelerate the process of animal breeding and improve the breeding efficiency. To date, many Indel molecular markers have been identified in livestock and poultry, but how Indels affect economic traits is not well understood. For molecular breeding, it is crucial to reveal the mechanism of action of Indels and to provide more accurate information. The purpose of this study was to investigate how the 52/224-bp multiallelic Indels of the chicken QPCTL promoter area affect the daily weight gain of chickens and the potential regulatory mechanism of the QPCTL gene. The analysis was conducted by association analysis, qPCR, dual-fluorescence assay and Western blotting. The results showed that Indels in the QPCTL promoter region were significantly associated with the daily weight gain in chickens and that QPCTL expression showed a decreasing trend in embryonic breast muscle tissues. Furthermore, QPCTL expression was significantly higher in breast muscle tissues of the AC genotype than in those of the AB and BB genotypes. Based on the transcriptional activity results, the pGL3-C vector produced more luciferase activity than pGL3-A and pGL3-B. In addition, overexpression of QPCTL promoted chicken primary myoblast (CPM) proliferation and inhibited differentiation. The results of this study suggest that Indels in the promoter region of the QPCTL gene may regulate the proliferation and differentiation of CPMs by affecting the expression of QPCTL, which ultimately affects the growth rate of chickens. These Indels have important value for the molecular breeding of chickens, and QPCTL can be used as a candidate gene to regulate and improve chicken growth and development.

Mining of chicken muscle growth genes and the function of important candidate gene RPL3L in muscle development.

The birth weight of chickens does not significantly affect the weight at slaughter, while the different growth rate after birth was one of the important reasons for the difference in slaughter weight. Also, the increase in chickens' postnatal skeletal muscle weight is the main cause of the slaughter weight gain, but which genes are involved in this biological process is still unclear. In this study, by integrating four transcriptome datasets containing chicken muscles at different developmental times or different chicken tissues in public databases, a total of nine candidate genes that may be related to postnatal muscle development in chickens were obtained, including RPL3L, FBP2, ASB4, ASB15, CKMT2, PGAM1, YIPF7, PFKM, and LDHA. One of these candidate genes is RPL3L, whose 42 bp insertion/deletion (indel) mutation significantly correlated with multiple carcass traits in the F2 resource population from Xinghua chickens crossing with White Recessive Rock (WRR) chickens, including live weight, carcass weight, half eviscerated weight, eviscerated weight, breast meat weight, wing weight, leg muscle shear force, and breast muscle shear force. Also, there was a very significant difference between different genotypes of the RPL3L 42 bp indel mutation in these trains. Further experiments showed that RPL3L was highly expressed in chicken skeletal muscle, and its overexpression could promote the proliferation and inhibit the differentiation of chicken myoblasts by regulating ASB4 and ASB15 expression. Our findings demonstrated that the RPL3L 42 bp indel may be one of the molecular markers of chicken weight-related traits.

Sox6 Differentially Regulates Inherited Myogenic Abilities and Muscle Fiber Types of Satellite Cells Derived from Fast- and Slow-Type Muscles.

Adult skeletal muscle is primarily divided into fast and slow-type muscles, which have distinct capacities for regeneration, metabolism and contractibility. Satellite cells plays an important role in adult skeletal muscle. However, the underlying mechanisms of satellite cell myogenesis are poorly understood. We previously found that Sox6 was highly expressed in adult fast-type muscle. Therefore, we aimed to validate the satellite cell myogenesis from different muscle fiber types and investigate the regulation of Sox6 on satellite cell myogenesis. First, we isolated satellite cells from fast- and slow-type muscles individually. We found that satellite cells derived from different muscle fiber types generated myotubes similar to their origin types. Further, we observed that cells derived from fast muscles had a higher efficiency to proliferate but lower potential to self-renew compared to the cells derived from slow muscles. Then we demonstrated that Sox6 facilitated the development of satellite cells-derived myotubes toward their inherent muscle fiber types. We revealed that higher expression of Nfix during the differentiation of fast-type muscle-derived myogenic cells inhibited the transcription of slow-type isoforms (MyH7B, Tnnc1) by binding to Sox6. On the other hand, Sox6 activated Mef2C to promote the slow fiber formation in slow-type muscle-derived myogenic cells with Nfix low expression, showing a different effect of Sox6 on the regulation of satellite cell development. Our findings demonstrated that satellite cells, the myogenic progenitor cells, tend to develop towards the fiber type similar to where they originated. The expression of Sox6 and Nfix partially explain the developmental differences of myogenic cells derived from fast- and slow-type muscles.

Integrative Analysis of Metabolomic and Transcriptomic Data Reveals the Antioxidant Potential of Dietary Lutein in Chickens.

Lutein can increase the body's skin color and has antioxidant potential. However, how it affects lipid metabolism and oxidative stress in chickens remains unknown. In this study, 74-day-old male chickens raised on feed supplemented with lutein had higher hip, back, breast, leg, shin and abdominal fat yellowness than the control group, and the livers of chickens in the lutein group had higher superoxide dismutase and glutathione peroxidase and lower malondialdehyde activities. To clarify the potential regulatory network regulated by lutein, we used RNA-seq and nontargeted metabolomics to detect changes in the male chicken liver and plasma, respectively. A total of 243 differentially expressed genes were significantly enriched in cytokine-cytokine receptor interaction signaling pathways, among others. A total of 237 significantly different metabolites were enriched in lysine biosynthesis and degradation and glycerophospholipid metabolism signaling pathways, among others. Finally, we comprehensively analyzed metabolome and transcriptome data and found that many differentially expressed genes and significantly different metabolites play crucial roles in lipid metabolism and oxidative stress. In summary, dietary lutein can improve male chicken skin yellowness and antioxidant indices and affect liver gene expression and plasma metabolites and may help improve the health of chickens.

Novel 61-bp Indel of RIN2 Is Associated With Fat and Hatching Weight Traits in Chickens.

The Ras and Rab interactor 2 (RIN2) gene, which encodes RAS and Rab interacting protein 2, can interact with GTP-bound Rab5 and participate in early endocytosis. This study found a 61-bp insertion/deletion (indel) in the RIN2 intron region, and 3 genotypes II, ID, and DD were observed. Genotype analysis of mutation sites was performed on 665 individuals from F2 population and 8 chicken breeds. It was found that the indel existed in each breed and that yellow feathered chickens were mainly of the DD genotype. Correlation analysis of growth and carcass traits in the F2 population of Xinghua and White Recessive Rock chickens showed that the 61-bp indel was significantly correlated with abdominal fat weight, abdominal fat rate, fat width, and hatching weight (P < 0.05). RIN2 mRNA was expressed in all the tested tissues, and its expression in abdominal fat was higher than that in other tissues. In addition, the expression of the RIN2 mRNA in the abdominal fat of the DD genotype was significantly higher than that of the II genotype (P < 0.05). The transcriptional activity results showed that the luciferase activity of the pGL3-DD vector was significantly higher than that of the pGL3-II vector (P < 0.01). Moreover, the results indicate that the polymorphisms in transcription factor binding sites (TFBSs) of 61-bp indel may affect the transcriptional activity of RIN2, and thus alter fat traits in chicken. The results of this study showed that the 61-bp indel was closely related to abdominal fat-related and hatching weight traits of chickens, which may have reference value for molecular marker-assisted selection of chickens.

Accelerate the Promotion of Mobile Payments during the COVID-19 Epidemic.

COVID-19 is caused by a novel SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). During the COVID-19 epidemic, when people are infected with the virus, they can transmit the virus onto paper or coin money through touch and droplets, potentially making any physical currency a carrier of the virus. Although there is no report confirming that people can become infected with viruses by cash circulation, relevant research on the survival of viruses on solid surfaces supports this hypothesis. Mobile payments can help individuals avoid coming in direct contact with any paper or coin money. Therefore, we strongly recommend the promotion of mobile payments during the COVID-19 epidemic.

A 31-bp indel in the 5' UTR region of GNB1L is significantly associated with chicken body weight and carcass traits.

BACKGROUND: G-protein subunit beta 1 like (GNB1L) encodes a G-protein beta-subunit-like polypeptide. Chicken GNB1L is upregulated in the breast muscle of high feed efficiency chickens, and its expression is 1.52-fold that in low feed efficiency chickens. However, no report has described the effects of GNB1L indels on the chicken carcass and growth traits. RESULTS: This study identified a 31-bp indel in the 5' untranslated region (UTR) of GNB1L and elucidated the effect of this gene mutation on the carcass and growth traits in chickens. The 31-bp indel showed a highly significant association with the body weight at 8 different stages and was significantly correlated with daily gains at 0 to 4 weeks and 4 to 8 weeks. Similarly, the mutation was significantly associated with small intestine length, breast width, breast depth and breast muscle weight. Moreover, DD and ID were superior genotypes for chicken growth and carcass traits. CONCLUSIONS: These results show that the 31-bp indel of GNB1L significantly affects chicken body weight and carcass traits and can serve as a candidate molecular marker for chicken genetics and breeding programs.

A Novel 65-bp Indel in the GOLGB1 Gene Is Associated with Chicken Growth and Carcass Traits.

Golgin subfamily B member 1 (GOLGB1) gene encodes the coat protein 1 vesicle inhibiting factor, giantin. Previous study showed that mutations of the GOLGB1 gene are associated with dozens of human developmental disorders and diseases. However, the biological function of GOLGB1 gene in chicken is still unclear. In this study, we detected a novel 65-bp insertion/deletion (indel) polymorphism in the chicken GOLGB1 intron 5. Association of this indel with chicken growth and carcass traits was analyzed in a yellow chicken population. Results showed that this 65-bp indel was significantly associated with chicken body weight (p < 0.05), highly significantly associated with neck weight, abdominal fat weight, abdominal fat percentage and the yellow index b of breast (p < 0.01). Analysis of genetic parameters indicated that "I" was the predominant allele. Except for the yellow index b of breast, II genotype individuals had the best growth characteristics, by comparison with the ID genotype and DD genotype individuals. Moreover, the mRNA expression of GOLGB1 was detected in the liver tissue of chicken with different GOLGB1 genotypes, where the DD genotype displayed high expression levels. These findings hinted that the 65-bp indel in GOLGB1 could be assigned to a molecular marker in chicken breeding and enhance production in the chicken industry.

Association Between the Methylation Statuses at CpG Sites in the Promoter Region of the SLCO1B3, RNA Expression and Color Change in Blue Eggshells in Lushi Chickens.

The formation mechanism underlying the blue eggshell characteristic has been discovered in birds, and SLCO1B3 is the key gene that regulates the blue eggshell color. Insertion of an endogenous retrovirus, EAV-HP, in the SLCO1B3 5' flanking region promotes SLCO1B3 expression in the chicken shell gland, and this expression causes bile salts to enter the shell gland, where biliverdin is secreted into the eggshell, forming a blue shell. However, at different laying stages of the same group of chickens, the color of the eggshell can vary widely, and the molecular mechanism underlying the eggshell color change remains unknown. Therefore, to reveal the molecular mechanism of the blue eggshell color variations, we analyzed the change in the eggshell color during the laying period. The results indicated that the eggshell color in Lushi chickens can be divided into three stages: 20-25 weeks for dark blue, 26-45 weeks for medium blue, and 46-60 weeks for light blue. We further investigated the expression and methylation levels of the SLCO1B3 gene at eight different weeks, finding that the relative expression of SLCO1B3 was significantly higher at 25 and 30 weeks than at other laying weeks. Furthermore, the overall methylation rate of the SLCO1B3 gene in Lushi chickens increased gradually with increasing weeks of egg production, as shown by bisulfite sequencing PCR. Pearson correlation analysis showed that methylation of the promoter region of SLCO1B3 was significantly negatively correlated with both SLCO1B3 expression in the shell gland tissue and eggshell color. In addition, we predicted that CpG5 and CpG8 may be key sites for regulating SLCO1B3 gene transcription. Our findings show that as the level of methylation increases, methylation of the CpG5 and CpG8 sites hinders the binding of transcription factors to the promoter, reducing SLCO1B3 expression during the late period and resulting in a lighter eggshell color.

Transcriptom analysis revealed regulation of dexamethasone induced microRNAs in chicken thymus.

Stress-induced immunosuppression is one of the serious threats to the poultry industry, especially obvious for young chicken. However, the molecular mechanism of stress-induced immunosuppression has not been clear in chicken. Here, we established an immunosuppression model of 7-day-old chickens with injecting dexamethasone (Dex) to analyze the molecular regulation in the chicken thymus. The microRNAs (miRNAs) transcripts profiles of thymuses from the model and control group were identified by the Solexa sequencing technology. The results showed 121 significantly differently expressed (SDE) miRNAs, including 119 known and two novel miRNAs (novel-58 and novel-350). A total of 391 target genes of the SDE miRNAs were predicted and annotated. We verified the potential negative correlation between gga-miR-103-3p and TGM2 by quantitative real-time polymerase chain reaction (qRT-PCR), as well as between novel-350 and PCBD2, and the results were positive. Gene ontology (GO) enrichment analysis showed that there was 298 significant enrichment GO terms, in which 31 were related to immune or stress, such as lymphocyte apoptotic process and response to stress. KEGG pathway analysis suggested that the SDE miRNAs were involved in autophagy regulation, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, Jak-STAT signaling pathway, and so on (although not significantly enriched). In these immune signaling pathways, the SDE miRNAs (such as gga-miR-2954, gga-miR-146b-3p, gga-miR-106-3p, and gga-miR-214) and the predicted target genes (such as IL11Ra, CSF3R, IFNGR1, CNTF, and MAP2K2) might affect the thymus immune function of chicken. The above results would provide a basis for uncovering the molecular regulation mechanism of immunosuppression in poultry.

Sequencing and characterization of lncRNAs in the breast muscle of Gushi and Arbor Acres chickens.

Chicken muscle quality is one of the most important factors determining the economic value of poultry, and muscle development and growth are affected by genetics, environment, and nutrition. However, little is known about the molecular regulatory mechanisms of long non-coding RNAs (lncRNAs) in chicken skeletal muscle development. Our study aimed to better understand muscle development in chickens and thereby improve meat quality. In this study, Ribo-Zero RNA-Seq was used to investigate differences in the expression profiles of muscle development related genes and associated pathways between Gushi (GS) and Arbor Acres (AA) chickens. We identified two muscle tissue specific expression lncRNAs. In addition, the target genes of these lncRNAs were significantly enriched in certain biological processes and molecular functions, as demonstrated by Gene Ontology (GO) analysis, and these target genes participate in five signaling pathway, as revealed by an analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Taken together, these data suggest that different lncRNAs might be involved in regulating chicken muscle development and growth and provide new insight into the molecular mechanisms of lncRNAs.

Identification and association of novel lncRNA pouMU1 gene mutations with chicken performance traits.

The biological functions of long noncoding RNAs (lncRNAs), which play an important role in regulating development and gene expression, may be affected by variations in lncRNA gene loci or associated genomic sequences. However, the functions of many lncRNAs remain unknown. To analyse correlations between mutations in pouMU1 with chicken growth and carcass traits, 860 chickens from a Gushi×Anka F2 resource population and 96 Lushi, Xichuan, Changshun and recessive white chickens were used to evaluate the genetic effect of the pouMU1 gene. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to analyse the relative expression levels of pouMU1 in nine different tissues and stages of development. pouMU1 expression was highest in pectoralis and leg muscles, whereas no expression was observed in the heart, liver and abdominal fat. Using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods, two novel sequence mutations (g.1198A>G and g.1238-1239del/insGA) were detected in the pouMU1 gene. SPSS software was used for statistical analysis in association studies. Based on the association data, the presence of both variants was significantly associated with leg muscle fibre width and leg muscle fibre roundness (P < 0.05) and highly associated with leg muscle fibre girth and body weight at 0 week of age (P < 0.01). These data suggest that pouMU1 might participate in regulating chicken muscle development and growth, and the findings offer new insight into the functions of sequence mutations in lncRNAs.