DUSP8-attenuated ERK1/2 signaling mediates lipogenesis and steroidogenesis in chicken granulosa cells.

The lipogenesis and steroidogenesis of granulosa cells are crucial during follicular development, yet it remains unclear whether dual-specificity phosphatase 8 (DUSP8) is involved. In this study, the specific role of DUSP8 in lipogenesis and steroidogenesis was investigated through culturing chicken granulosa cells in vitro. The results revealed that the expression levels of adipogenic genes were elevated after DUSP8 overexpression and reduced after knockdown. The same was observed for lipid deposition in granulosa cells. Meanwhile, the steroidogenic gene expression and progesterone synthesis were promoted after DUSP8 overexpression and inhibited after knockdown. In addition, we also found that DUSP8 blocked the phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2). Based on the previous results that activated ERK1/2 signaling inhibited lipid deposition and progesterone synthesis in chicken granulosa cells, we demonstrated that DUSP8 promoted lipid deposition and progesterone synthesis through mediating the ERK1/2 signaling pathway. The results will improve our understanding of the molecular regulatory mechanisms regarding lipid metabolism and progesterone synthesis in chicken granulosa cells.

Physiological role of dietary energy in the sexual maturity: clues of body size, gonad development, and serum biochemical parameters of Chinese indigenous chicken.

Sexual maturity is a crucial factor in the formation and development of poultry reproductive capacity. The nutritional status has been confirmed to play an important role in the regulation of sexual maturity. To investigate the effect of dietary energy levels on sexual maturity in chicken, diets with 3 energy levels (group L: 2,573 kcal/kg, group C: 2,836 kcal/kg, group H: 3,122 kcal/kg) were implemented to feed Guangyuan Gray chickens. During this trial, body weight, body size, organ development, sexual maturity, reproductive performance and blood biochemical parameters were monitored. The earlier sexual maturity was observed in group H, as well as a heavier first egg weight, larger interpubic distance and higher total cholesterol (T-CHO) content at sexual maturity. The dietary energy levels had no significant effect on body weight at first egg and egg production at 300 d of age. Although dietary energy levels had a significant effect on body weight, comb length, tibia length and girth, abdominal fat weight, oviduct weight and length, T-CHO, triglyceride (TG) content and estradiol (E2) level during the rearing period. No significant difference of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) level among 3 groups was observed during the trial. The dietary energy levels had effects on mRNA expression of GnRH, estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2) in hypothalamus, gonadotropin inhibitory hormone receptor (GnIHR) in pituitary and luteinizing hormone receptor (LHR), ESR2 in ovary. The GnIHR/GnRHR ratio in pituitary was higher before sexual maturity and decreased at sexual maturity. The results of correlations analysis found that all the body size, carcass traits, serum biochemical parameters negatively correlated with age at first egg except for interpubic distance and serum blood glucose content. Collectively, dietary energy levels had effects on sexual maturity of chicken, which may be achieved by affecting body weight, gonad development, endocrine and the mRNA expression of genes related to hypothalamus-pituitary-gonad axis. These results further set our understanding of how dietary energy regulates sexual maturity.

A Comparison of the Meat Quality, Nutritional Composition, Carcass Traits, and Fiber Characteristics of Different Muscular Tissues between Aged Indigenous Chickens and Commercial Laying Hens.

The aim of this study is to assess the differences in the meat quality, nutritional composition, carcass traits, and myofiber characteristics between Hy-Line grey chickens (HLG, commercial breed) and Guangyuan grey chickens (GYG, indigenous breed). A total of 20 55-week-old chickens were selected for slaughter. The HLG exhibited a larger carcass weight, breast muscle weight, and abdominal fat weight (p < 0.05). The GYG exhibited a higher crude protein content, lower shear force, and smaller fiber size in the thigh muscles, whereas the HLG presented higher pH values and lower inosine-5'-monophosphate content in the breast muscles (p < 0.05). Darker meat based on higher redness and yellowness values was observed in the GYG instead of the HLG (p < 0.05). The research results also revealed parameter differences between different muscle types. Simultaneously, a correlation analysis showed significant correlations between the meat quality traits and myofiber characteristics (p < 0.05). In conclusion, aged indigenous chickens perform better in terms of tenderness and nutritional value in the thigh muscles, and may exhibit a better flavor in the breast muscles, but have a smaller breast muscle weight. Therefore, the current investigation provides a theoretical basis for the different needs of consumers and the processing of meat from old laying hens.

Comparative Analysis of Different Proteins and Metabolites in the Liver and Ovary of Local Breeds of Chicken and Commercial Chickens in the Later Laying Period.

The liver and ovary perform a vital role in egg production in hens. In the later laying period, the egg-laying capacity of female hens, particularly that of local breeds, declines significantly. Hence, it is essential to study the features and conditions of the ovary and liver during this period. In this research, we characterized the proteins and metabolites in the liver and ovary of 55-week-old Guangyuan gray chickens (Group G) and Hy-Line gray chickens (Group H) by using liquid chromatography chip/electrospray ionization quadruple time-of-flight/mass spectroscopy (LC-MS/MS). In total, 139 differentially expressed proteins (DEPs) and 186 differential metabolites (DMs) were identified in the liver, and 139 DEPs and 36 DMs were identified in the ovary. The upregulated DEPs and DMs in both the liver and ovary of Group G were primarily enriched in pathways involved in amino acid and carbohydrate metabolism. This suggests that energy metabolism was highly active in the Guangyuan gray chickens. In contrast, the upregulated DEPs and DMs in Group H were mainly enriched in pathways associated with lipid metabolism, which may explain the higher egg production and the higher fatty liver rate in Hy-Line gray hens in the later laying period. Additionally, it was found that the unique protein s-(hydroxymethyl) glutathione dehydrogenase (ADH4) in Group G was implicated in functions such as fatty acid degradation, glycolysis, and pyruvate metabolism, whereas the unique proteins, steroid sulfatase (STS), glucosylceramidase (LOC107050229), and phospholipase A2 Group XV (PLA2G15), in Group H were involved in the metabolism of steroid hormones and glycerol phosphate. In conclusion, variations in how carbohydrates, lipids, and amino acids are processed in the liver and ovary of local breeds of chicken and commercial hens towards the end of their laying period could explain the disparities in their egg production abilities.

Lipid Deposition and Progesterone Synthesis Are Increased by miR-181b-5p through RAP1B/ERK1/2 Pathway in Chicken Granulosa Cells.

Steroid hormones secreted by granulosa cells are essential for maintaining normal development of chicken follicles. Our previous sequencing data indicated that miR-181b-5p and RAS-related protein 1B (RAP1B) appeared to function in chicken granulosa cells, which was further explored in this study. The results suggested that miR-181b-5p facilitated the aggregation of lipid droplets and the synthesis of progesterone. In contrast, RAP1B astricted lipid deposition and progesterone secretion. Cotransfection of the RAP1B overexpression vector with miR-181b-5p mimic eliminated the promoting effect of miR-181b-5p. Dual-luciferase reporter assay confirmed that miR-181b-5p bound directly to the 3' untranslated region (3' UTR) of RAP1B. We also found that miR-181b-5p and RAP1B reduced and enhanced the phosphorylation levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2), respectively. The application of ERK1/2 activators and inhibitors demonstrated that ERK1/2 is a negative regulator of lipid deposition and progesterone synthesis. In conclusion, we revealed that miR-181b-5p accelerated lipid deposition and progesterone synthesis through the RAP1B/ERK1/2 pathway in chicken granulosa cells. miR-181b-5p and RAP1B may serve as new biomarkers in breeding to improve chicken reproductive performance and prevent ovary-related diseases.

Developmental Characteristics of Skeletal Muscle during the Embryonic Stage in Chinese Yellow Quail (Coturnix japonica).

The quail is an important research model, and the demand for quail meat has been increasing in recent years; therefore, it is worthwhile investigating the development of embryonic skeletal muscle and the expression patterns of regulatory genes. In this study, the expression of MyoD and Pax7 in the breast muscle (m. pectoralis major) and leg muscle (m. biceps femoris) of quail embryos on days 10 through 17 were determined using qRT-PCR. Paraffin sections of embryonic muscle were analyzed to characterize changes over time. Results showed that MyoD and Pax7 were expressed in both breast and leg muscles and played a significant role in embryonic muscle development. Compared to breast muscle, leg muscle grew faster and had greater weight and myofiber size. The findings suggested that embryonic day 12 (E12) may be a key point for muscle development. Correlation analysis showed that MyoD expression was significantly negatively correlated with muscle and embryo weight, whereas Pax7 gene expression had no significant correlation with these characteristics. These fundamental results provide a theoretical basis for understanding the characteristics and transition points of skeletal muscle development in quail embryos and an important reference for farmers raising quail from eggs.

The Implications in Meat Quality and Nutrition by Comparing the Metabolites of Pectoral Muscle between Adult Indigenous Chickens and Commercial Laying Hens.

Aged chickens are often a secondary dietary choice, owing to the poor organoleptic qualities of their meat. With regard to the meat quality of chickens, the metabolic profiles of pectoral muscle in Guangyuan grey chickens (group G) and Hy-Line grey hens (group H) aged 55 weeks were compared via ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A total of 74 metabolites were identified with differential changes in the ion model. Lipids and lipid-like molecules comprised the largest proportion among the different metabolites. The content of myristic acid and palmitic acid were found to be higher in the pectoral muscle of group G, while group H showed significantly higher levels of glycerophospholipid molecules, such as LPC(18:2/0:0), Pi(38:5), Pc(16:0/16:0), and Pe(16:1e/14-hdohe). KEGG pathway analysis indicated that the abundant metabolites in group G were mainly involved in energy metabolism and fatty acid biosynthesis and metabolism, whereas those of group H were mainly attributed to the metabolism of unsaturated fatty acids and amino acids. Overall, the differences in lipid and amino acid metabolism in pectoral muscle appear to be responsible for the difference in meat quality between indigenous chickens and commercial laying hens.

miR-136-5p/FZD4 axis is critical for Wnt signaling-mediated myogenesis and skeletal muscle regeneration.

Skeletal muscle can undergo a regenerative process in response to injury or disease to maintain muscle quality and function. Myogenesis depends on the proliferation and differentiation of myoblasts, and miRNAs can maintain the balance between them by precisely regulating many key factors in the myogenic network. Here, we found that miR-136-5p was significantly upregulated during the proliferation and differentiation of C2C12 cells. We demonstrate that miR-136-5p acts as a myogenic negative regulator during the development of mouse C2C12 myoblasts. In terms of mechanism, miR-136-5p inhibits the formation of ß-catenin/LEF/TCF DNA-binding factor transcriptional regulatory complex by targeting FZD4, a gating protein in the Wnt signaling pathway, thereby enhancing downstream myogenic factors and finally promoting myoblast proliferation and differentiation. In addition, in BaCl2 -induced muscle injury mouse model, miR-136-5p knockdown accelerated the regeneration of skeletal muscle after injury, and further led to the improvement of gastrocnemius muscle mass and muscle fiber diameter, while being suppressed by shFZD4 lentivirus infection. In summary, these results demonstrate the essential role of miR-136-5p/FZD4 axis in skeletal muscle regeneration. Given the conservation of miR-136-5p among species, miR-136-5p may be a new target for treating human skeletal muscle injury and improving the production of animal meat products.

Nutrition and Flavor Evaluation of Amino Acids in Guangyuan Grey Chicken of Different Ages, Genders and Meat Cuts.

The composition and content of amino acids in foodstuffs have a vital impact on the nutritional value and taste. With the aim of understanding the nutrition and flavor of Guangyuan grey chicken, the composition and content of amino acids in the pectoralis and thigh muscle of chickens at the age of 90 d, 120 d and 150 d were determine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an amino acid analyzer. A total of 17 amino acids were detected both in pectoralis and thigh muscle via the amino acid analyzer, of which the content of glutamate was the highest. Additionally, 21 deproteinized free amino acids were detected via LC-MS/MS. Among all samples, the content of glutamine in thigh muscle was the highest. The content of histidine in the pectoralis was the highest. In terms of the flavor amino acids (FAAs), the umami-taste and sweet-taste amino acids were higher in the thigh muscle of 120 d male chicken. From the perspective of protein nutrition, the essential amino acid was higher in pectoral muscle, and the composition was better. The results of the amino acid score showed that the content of leucine and valine were inadequate in Guangyuan grey chicken. Collectively, the content of amino acid in Guangyuan grey chicken was affected by age, gender and meat cut. This study confirms that meat of chicken in different ages, genders, and cuts presents different nutritional values and flavors owing to the variation of amino acids content.

Integration analysis of metabolome and transcriptome profiles revealed the age-dependent dynamic change in chicken meat.

To explore the chemical composition of chicken meat during different growth and development periods, the dynamic alterations of the metabolite composition were determined using LC-MS/MS-based metabolomics. Together, 573 metabolites were identified in chicken meat from five age stages. Generally, pentadecanoic acid, stearic acid, creatine, carnosine, IMP, L-histidine and L-isoleucine presented an upward trend with age, while anserine, DHA, L-aspartic acid, LPA 18:1 and LPI 18:1 decreased with age. The main pathways of chicken meat metabolism affected by age were fructose and mannose metabolism, arachidonic acid metabolism, steroid hormone biosynthesis, riboflavin metabolism, biosynthesis of unsaturated fatty acids, and linoleic acid metabolism. Using transcriptomic profiling data, we conducted Pearson correlation analysis between gene expression and metabolite profile data in each age comparison. Integration analysis of metabolome and transcriptome would be helpful to understand the biological processes underlying the development of meat quality and explore valuable biomarkers for specific metabolite accumulation.

Integrated analysis of microRNA and mRNA interactions in ovary of counter-season breeding and egg-ceased geese (Anser cygnoides).

Egg-ceasing is a phenomenon that occurs in most avian species and significantly reduces productivity. Although several factors are reported to regulate the reproduction progress, the underlying molecular mechanism of egg-ceasing remains obscure. Herein, we identified and explored the differentially expressed miRNAs and mRNAs involved in ovarian atrophy via high throughput sequencing. We identified a total of 901 mRNAs and 50 miRNAs that were differentially expressed in egg-laying and atrophic ovaries. Among them, numerous differentially expressed gene (DEG) transcripts and target genes for miRNAs were significantly enriched in Gene Ontology terms such as reproductive processes, cell proliferation, and apoptosis pathways. In addition, an interaction network was constructed by considering target relationships and correlation of the expression levels between ovary development-related genes, miRNAs and pathways. We discovered mRNA and miRNAs transcripts that are candidate regulators of ovary development in egg-ceased geese. Our findings expanded our understanding of the functional of miRNAs in ovarian atrophy and demonstrated that RNA-Seq is a powerful tool for examining the molecular mechanism in regulating egg-ceasing.

Comparative Analysis of Skeletal Muscle DNA Methylation and Transcriptome of the Chicken Embryo at Different Developmental Stages.

DNA methylation is a key epigenetic mechanism involved in embryonic muscle development and plays an important role in early muscle development. In this study, we sought to investigate the effects of genome-wide DNA methylation by combining the expression profiles of the chicken embryonic muscle. Genome-wide DNA methylation maps and transcriptomes of muscle tissues collected from different embryonic development points (E7, E11, E17, and D1) were used for whole-genome bisulfite sequencing (WGBS) and RNA sequencing, respectively. We found that the differentially methylated genes (DMGs) were significantly associated with muscle organ development, regulation of skeletal muscle satellite cell proliferation, and actin filament depolymerization. Furthermore, genes TBX1, MEF2D, SPEG, CFL2, and TWF2 were strongly correlated with the methylation-caused expression switch. Therefore, we chose the CFL2 gene to explore its function in skeletal muscle satellite cells, and the in vitro experiments showed that CFL2 acts as a negative regulator of chicken skeletal muscle satellite cell proliferation and can induce cell apoptosis. These results provide valuable data for future genome and epigenome studies of chicken skeletal muscle and may help reveal the molecular mechanisms of potential economic traits.

Whole-genome resequencing reveals loci with allelic transmission ratio distortion in F1 chicken population.

Allelic transmission ratio distortion (TRD) is the significant deviation from the expected ratio under Mendelian inheritance theory, which may be resulted from multiple disrupted biological processes, including germline selection, meiotic drive, gametic competition, imprint error, and embryo lethality. However, it is less known that whether or what extent the allelic TRD is present in farm animals. In this study, whole-genome resequencing technology was applied to reveal TRD loci in chicken by constructing a full-sib F1 hybrid population. Through the whole-genome resequencing data of two parents (30 ×) and 38 offspring (5 ×), we detected a total of 2850 TRD SNPs (p-adj < 0.05) located within 400 genes showing TRD, and all of them were unevenly distributed on macrochromosomes and microchromosomes. Our findings suggested that TRD in the chicken chromosome 16 might play an important role in chicken immunity and disease resistance and the MYH1F with significant TRD and allele-specific expression could play a key role in the fast muscle development. In addition, functional enrichment analyses revealed that many genes (e.g., TGFBR2, TGFBR3, NOTCH1, and NCOA1) with TRD were found in the significantly enriched biological process and InterPro terms in relation to embryonic lethality and germline selection. Our results suggested that TRD is considerably prevalent in the chicken genome and has functional implications.

Genome-wide analysis of spatiotemporal allele-specific expression in F1 hybrids of meat- and egg-type chickens.

In diploid organisms, each gene locus is composed of two parental alleles, which would interact with each other for determining the phenotypic variation. Better understanding of the allele-specific expression (ASE) in farm animals is much important to explore the genetic basis underlying economically important traits, which have been poorly understood yet. In this study, genome-wide analysis was applied to explore the spatiotemporal pattern of ASE in the F1 hybrids of chicken. First, meat- and egg-type chickens were selected for producing a full-sib F1 hybrid population (n = 57). Then, genome resequencing of two parents and 38 offspring were performed and liver and breast muscle samples (n = 38) were subjected to strand-specific RNA sequencing (ssRNA-seq) for ASE detection at 1, 28, and 56 days of age, respectively. The results accurately identified a total of 465 informative genes that could be distinguished with respect to their parental origins. There were 0.4% - 4.1% of informative genes showing ASE, and 57 of them were found across different tissues and time points. Besides, most ASE genes in chickens were tissue-specific, and no matter what the time-point pattern of one ASE gene, the same parental allele of this gene almost showed consistently higher or lower expression across all time points in the same type tissue. In conclusion, this study indicated that most of ASE genes were tissue-specific and time-dependent.

Genotype frequency distributions of 28 SNP markers in two commercial lines and five Chinese native chicken populations.

BACKGROUND: Modern breeding in the poultry industry mainly aims to produce high-performance poultry lines and breeds in two main directions of productivity, meat and eggs. To understand more about the productive potential of lowly selected Chinese native chicken populations, we selected 14 representative SNP markers strongly associated with growth traits or carcass traits and 14 SNP markers strongly associated with egg laying traits through previous reports. By using the MassArray technology, we detected the genotype frequency distributions of these 28 SNP markers in seven populations including four lowly selected as well as one moderately selected Sichuan native chicken populations, one commercial broiler line and one commercial layer line. RESULTS: Based on the genotype frequency distributions of these 28 SNP markers in 5 native chicken populations and 2 commercial lines, the results suggested that these Chinese indigenous chicken populations have a relatively close relationship with the commercial broiler line but a marked distinction from the commercial layer line. Two native chicken breeds, Shimian Caoke Chicken and Daheng Broilers, share similar genetic structure with the broiler line. CONCLUSIONS: Our observations may help us to better select and breed superior domestic chickens and provide new clues for further study of breeding programs in local chicken populations.

Transcriptome analysis reveals differentially expressed genes and pathways for oviduct development and defense in prelaying and laying hens.

PROBLEM: The oviduct plays an indispensable role in the formation of eggs, especially the magnum and uterus. The identification of oviduct development in different stages will help to target candidate genes and pathways in regulation of albumen and eggshell formation, as well as defense mechanism in oviduct and egg. METHODS: To identify the function differences and the molecular defense mechanism of the oviduct and egg, we performed transcriptome sequencing analysis of the magnum and uterus in 120-d-old and 300-d-old Lohmann layers, three birds in each group. RESULTS: With fold changes (log2 ratio) ≥ 2 and false discovery rate (FDR) < 0.01, RNA-Seq revealed 1,040 genes expressed differentially in the magnum and 595 genes in the uterus. By combining GO enrichment and KEGG pathway analysis, it served to show that gene activities of the magnum and uterus in prelaying chickens were more likely to concentrate on growth and development, and after egg-laying, they were mainly inclined to enhance the substances transmembrane transport and secretion activities. We further characterized 1579 new genes, while only 803 of them were functionally annotated. A complex mixture of proteins related to defense was measured in this study. A subset of avian ß-defensins (AvBDs) and ovodefensins (OvoDs), that is, AvBD12, AvBD11, AvBD10, OvoDA1, OvoDB1, OvoDA2, OvoDA3, and OvoDBß, was detected to express in the magnum of laying hens at high levels. CONCLUSION: Collectively, the identification and functional analysis of these differentially expressed genes (DEGs), as well as specific expression of avian defensins, may contribute to understand the development and defense mechanisms of oviduct and eggs.

Molecular Characterization, Expression and Functional Analysis of Chicken STING.

Innate immunity is an essential line of defense against pathogen invasion which is gained at birth, and the mechanism involved is mainly to identify pathogen-associated molecular patterns through pattern recognition receptors. STING (stimulator of interferon genes) is a signal junction molecule that hosts the perception of viral nucleic acids and produces type I interferon response, which plays a crucial role in innate immunity. However, relatively few studies have investigated the molecular characterization, tissue distribution, and potential function of STING in chickens. In this study, we cloned the full-length cDNA of chicken STING that is composed of 1341 bp. Sequence analyses revealed that STING contains a 1140-bp open-reading frame that probably encodes a 379-amino acid protein. Multiple sequence alignments showed that the similarity of the chicken STING gene to other birds is higher than that of mammals. Real-time polymerase chain reaction (PCR) assays revealed that STING is highly expressed in the spleen, thymus and bursa of fabricious in chickens. Furthermore, we observed that STING expression was significantly upregulated both in vitro and in vivo following infection with Newcastle disease virus (NDV). STING expression was also significantly upregulated in chicken embryo fibroblasts upon stimulation with poly(I:C) or poly(dA:dT). Taken together, these findings suggest that STING plays an important role in antiviral signaling pathways in chickens.

Molecular characterization of a novel ovodefensin gene in chickens.

Host defense peptides (HDPs) represent a large group of diverse small peptides that play important roles in host defense and disease resistance. In vertebrates, one of the main types of HDPs belong to defensins, which are less than 100 amino acid residues and characterized by a highly conserved motif of cysteine residues. Recently, a subfamily of defensins, namely ovodefensins (OvoDs), has been identified in birds and reptiles. However, both their family members and evolutionary relationships remain unclear. In the present study, we cloned and characterized a novel gene namely OvoDBß in chickens. Our results showed that the full length of chicken OvoDBß mRNA contains 344 bp nucleotides and encodes a 61-amino acid protein. We further revealed that the mRNA of OvoDBß is abundant in the oviduct of laying hens but absent in many other tissues. Additionally, sequences comparison and analyses suggested that OvoDBß is orthologous to the gene previously known as zebra finch OvoDB1, albeit it might exhibit specific structures. Furthermore, both OvoDBα and OvoDBß were existent in the genome of each bird, implying that two types of OvoDBs sharing same cysteine motif have already emerged before the species divergence. More importantly, recombinant OvoDBß mature peptide exerted antibacterial activity against Escherischia coli (CICC23657 strain) in vitro. These results collectively indicated that the putative sequence, namely chicken OvoDBß, is a function gene with potential antimicrobial property. Discovery and function characterization of novel HDP genes may help us develop novel antimicrobial agents in the future.