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.

Research progress of non-coding RNAs regulation on intramuscular adipocytes in domestic animals.

Intramuscular fat (IMF) is the main determinant of the economic value of domestic animal meat, and has a vital impact on the sensory quality characteristics, while the content of IMF is mainly determined by the size and number of intramuscular adipocytes. In recent years, due to the development of sequencing technology and omics technology, a large number of non-coding RNAs have been identified in intramuscular adipocytes. Non-coding RNAs are a kind of RNA regulatory factors with biological functions but without translation function, which mainly include microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). These non-coding RNAs regulate the key genes of intramuscular adipocyte growth and development at post-transcriptional level through a variety of regulatory mechanisms, and affect the number and size of intramuscular adipocytes, thus affecting the content of IMF. Here, the review summarizes the candidate non-coding RNAs (miRNAs, lncRNAs, circRNAs) and genes involved in the regulation of intramuscular adipocytes, the related regulation mechanism and signaling pathways, in order to provide reference for further clarifying the molecular regulation mechanism of non-coding RNAs on intramuscular adipocytes in domestic animals.

MYH1F promotes the proliferation and differentiation of chicken skeletal muscle satellite cells into myotubes.

In diploid organisms, interactions between alleles determine phenotypic variation. In previous experiments, only MYH1F was found to show both ASE (spatiotemporal allele-specific expression) and TRD (allelic transmission ratio distortion) characteristics in the pectoral muscle by comparing the genome-wide allele lists of hybrid populations (F1) of meat- and egg- type chickens. In addition, MYH1F is a member of the MYH gene family, which plays an important role in skeletal muscle and non-muscle cells of animals, but the specific expression and function of this gene in chickens are still unknown. Therefore, qRT-PCR was used to detect the expression of MYH1F in different tissues of chicken. Proliferation and differentiation of chicken skeletal muscle satellite cells (SMSCs) have been detected by transfection of MYH1F-specific small interfering RNA (siRNA). The results showed that the expression of MYH1F in chicken skeletal muscle was higher than that in other tissues. Combined with CCK-8 assay, EdU assay, immunofluorescence, and Western blot Assay, it was found that MYH1F knockdown could significantly suppress the proliferation of chicken SMSCs and depress the differentiation and fusion of the cells. These results suggest that MYH1F plays a critical role in myogenesis in poultry, which is of great significance for exploring the regulatory mechanisms of muscle development and improving animal productivity.

Whole transcriptome analysis reveals the key genes and noncoding RNAs related to follicular atresia in broilers.

Broodiness, a maternal behavior, is accompanied by the atresia of follicles and the serious degradation of poultry reproductive performance. The comparison of follicles between brooding and laying hens is usually an ideal model for exploring the regulation mechanism of follicle atresia. In this study, we selected three brooding hens and three laying hens to collect their follicles for whole transcriptome sequencing. The results demonstrated different expression patterns between the follicles of brooding hens and laying hens. In the top 10 differentially expressed genes with the highest expression, MMP10 was relatively low expressed in the follicles of brooding hens, but other nine genes were relatively highly expressed, including LRR1, RACK1, SPECC1L, ABHD2, COL6A3, RPS17, ATRN, BIRC6, PGAM1 and SPECC1L. While miR-21-3p, miR-146a-5p, miR-142-5p and miR-1b-3p were highly expressed in the follicles of brooding hen, miR-106-5p, miR-451, miR-183, miR-7, miR-2188-5p and miR-182-5p were lowly expressed in brooding hen. In addition, we identified 124 lncRNAs specifically expressed in the follicles of brooding hens and 147 lncRNAs specifically expressed in the follicles of laying hens. Our results may provide a theoretical basis for further exploration of the molecular mechanism of broodiness in broilers.

Transcriptome analysis of breast muscle and liver in full-sibling hybrid broilers at different ages.

In China, the production mode of hybrid broilers with meat-type chicken as male parent and egg-type chicken as female parent is common, but few studies pay attention to the economic characteristics of hybrid broilers. In this experiment, we constructed a full-sib F1 population (n = 57) from male Recursive White broiler and female Lohmann Pink layer. Total 6, 6 and 7 hybrid broilers at days 1, 28 and 56 were selected randomly to collect breast muscle and liver tissues, respectively. After performing strand-specific RNA-Seq on these samples, we obtained 252.12 Gb sequencing data. Principal component analysis presented that the effects of different factors on gene expression were as below: tissue difference > age difference > sex difference. The ten genes with the highest expression in breast muscle were GAPDH, ACTA1, ATP2B3, COII, ATP6, COX3, COX1, MYL1, TNNI2 and ENSGALG00000042024. Through the analysis of differentially expressed transcripts (DETs) between different ages, we found that the number of DETs decreased progressively with the prolongation of ages in breast muscle. The same results were also observed in liver. GO enrichment analysis of DETs demonstrated that total 11 BP terms closely related to growth and development of breast muscle were annotated, such as cardiac muscle contract, muscle contract, cell division and so on. KEGG annotation presented that total 5 pathways related to growth and development were determined in breast muscle, including Cell cycle, Insulin signaling pathway, FoxO signaling pathway, Focal adhesion and Adrenergic signaling in cardiomyocytes. Our results may provide theoretical foundation for hybrid broiler production.

miR-24-3p Dominates the Proliferation and Differentiation of Chicken Intramuscular Preadipocytes by Blocking ANXA6 Expression.

Intramuscular fat (IMF) is one of the crucial factors determining meat quality. IMF deposition depends on the hyperplasia and hypertrophy of intramuscular preadipocytes, in which genes and noncoding RNAs play an important regulatory role. According to previous transcriptome analysis, ANXA6 and miR-24-3p were identified as involved in lipid metabolism in breast muscle. In this study, we further investigated their function in the proliferation and differentiation of chicken intramuscular preadipocytes. The results indicated that overexpression of ANXA6 inhibited proliferation and promoted differentiation of intramuscular preadipocytes, while knockdown of ANXA6 promoted cell proliferation and inhibited adipogenic differentiation. miR-24-3p was proved to directly bind to the 3' untranslated region (3'UTR) of ANXA6 by dual-luciferase reporter assay. The regulatory effect of miR-24-3p on the proliferation and differentiation of intramuscular preadipocytes was opposite to that of ANXA6. Besides, the overexpression vector of ANXA6 eliminated the impact of miR-24-3p mimics on intramuscular preadipocytes. In brief, we revealed that miR-24-3p promoted proliferation but inhibited differentiation of intramuscular preadipocytes by blocking ANXA6 expression, thus dominating IMF deposition in broilers. These findings may provide a novel target for improving chicken meat quality.

miR-122-5p regulates proliferation and apoptosis of chicken granulosa cells of hierarchal follicles by targeting MAPK3.

Chicken follicles plays a crucial role in the reproductive performance, especially in laying period. Recently, miR-122-5p has been found to be differentially expressed in the ovaries of rats with polycystic ovary syndrome and normal rats, indicating the potential role of miR-122-5p in the development of granulosa cells (GCs). In present study, we found that miR-122-5p was highly expressed in chicken atrophic ovaries. Herein, we investigated its function on GC proliferation and apoptosis of chicken in vitro. We found that overexpression of miR-122-5p significantly inhibited proliferation and promoted apoptosis of GCs, whereas the opposite effects were detected in miR-122-5p knockdown GCs. Meanwhile, mitogen-activated protein kinase 3 (MAPK3) was confirmed as a new target gene of miR-122-5p by bioinformatics software prediction and the dual-luciferase reporter assay verification. Furthermore, after knockdown of MAPK3, the function of MAPK3 for GC proliferation and apoptosis was opposite to that of miR-122-5p. Collectively, our results indicated that miR-122-5p impeded chicken GC proliferation and promoted apoptosis through the post-transcriptional downregulation of MAPK3.

Effects of Lactobacillus plantarum on growth traits, slaughter performance, serum markers and intestinal bacterial community of Daheng broilers.

Probiotics are expected to be an ideal alternative for antibiotics in the poultry industry. This study aimed to investigate the effect of Lactobacillus plantarum on growth traits, slaughter performance, serum markers and intestinal bacterial community of Daheng broilers. A total of 2400 healthy one-day-old Daheng broilers were randomly divided into 5 groups with 6 replicates per group and 40 individuals per replicate. Birds in control group were fed a basal diet, and others were fed basal diets supplemented with 105 , 106 , 107 and 108  CFU/kg Lactobacillus plantarum, respectively. It turned out that adding Lactobacillus plantarum to diet could significantly improve the serum immune performance of broilers (p < 0.05), enhance the antioxidant capacity to a certain extent (p > 0.05), but had no significant effect on growth traits and slaughter performance. Moreover, Lactobacillus plantarum could improve the diversity of intestinal bacterial community, but with the increase of addition concentration, the diversity would gradually decrease. In conclusion, Lactobacillus plantarum can be used as feed additive in broiler production, but whether it is more effective than antibiotics needs further investigation.

Transcriptomics analysis of Daheng broilers reveals that PLIN2 regulates chicken preadipocyte proliferation, differentiation and apoptosis.

BACKGROUND: Intramuscular fat content, an important meat quality trait, strongly affects flavor, juiciness, and tenderness. Sex hormones regulate lipid metabolism, and female hormones stimulate fat deposition, thereby making the female chickens always fatter than males. In this study, the effect of sex on IMF deposition was screened following transcriptomics in chickens. METHODS AND RESULTS: Results confirmed significantly higher IMF content of 150-day female chickens as compared to the male chickens. The female chickens manifested higher serum TG, LDL-C, and VLDL, and significantly lower HDL-C contents than male chickens. Moreover, differential expression of genes involved in lipid metabolism were obtained in the muscle and liver between female and male chicken, which could partly interpret the possible reasons for the sex-mediated differences of IMF content. Cellular results revealed that inhibition of PLIN2 significantly inhibited chicken preadipocyte proliferation and induces apoptosis of preadipocytes, as well as promoted adipocyte differentiation. CONCLUSIONS: According to our results, PLIN2 may be considered as a molecular marker for poultry meat quality and applying this gene in early breed selection.