The excessive deposition of abdominal fat has become an important factor in restricting the production efficiency of chickens, so reducing abdominal fat deposition is important for improving growth rate. It has been proven that miRNAs play an important role in regulating many physiological processes of organisms. In this study, we constructed a model of adipogenesis by isolating preadipocytes (Ab-Pre) derived from abdominal adipose tissue and differentiated adipocytes (Ab-Ad) in vitro. Deep sequencing of miRNAs and mRNAs expressed in Ab-Pre and Ab-Ad groups was conducted to explore the effect of miRNAs and mRNAs on fat deposition. We identified 80 differentially expressed miRNAs (DEMs) candidates, 58 of which were up-regulated and 22 down-regulated. Furthermore, six miRNAs and six mRNAs were verified by qRT-PCR, and the results showed that the expression of the DEMs and differentially expressed genes (DEGs) in the two groups was consistent with our sequencing results. When target genes of miRNA were combined with mRNA transcriptome data, a total of 891 intersection genes were obtained, we predicted the signal pathways of cross genes enrichment to the MAPK signal pathway, insulin signal pathway, fatty acid metabolism, and ECM–receptor interaction. Meanwhile, we constructed miRNA and negatively correlated mRNA target networks, including 12 miRNA–mRNAs pairs, which showed a strong association with the abdominal adipocyte differentiation (miR-214−ACSBG2, NFKB2, CAMK2A, ACLY, CCND3, PLK3, ITGB2; miR-148a-5p−ROCK2; miR-10a-5p−ELOVL5; miR-146b-5p−LAMA4; miR-6615-5p−FLNB; miR-1774−COL6A1). Overall, these findings provide a background for further research on lipid metabolism. Thus, we can better understand the molecular genetic mechanism of chicken abdominal fat deposition.

The emopamil binding protein (EBP) is an important enzyme participating in the final steps of cholesterol biosynthesis in mammals. A predictive geneEBP-like, which encodes the protein with a high identity to human EBP, was found in chicken genome. No regulatory mechanisms and biological functions ofEBP-likehave been characterized in chickens. In the present study, the coding sequence ofEBP-likewas cloned, the phylogenetic trees of EBP/EBP-like were constructed and the genomic synteny ofEBP-likewas analyzed. The regulatory mechanism ofEBP-likewere explored within vivoandin vitroexperiments. The biological functions ofEBP-likein liver cholesterol biosynthetic were examined by using gain- or loss-of-function strategies. The results showed that chickenEBP-likegene was originated from a common ancestral with Japanese quailEBPgene, and was relatively conservative withEBPgene among different species. TheEBP-likegene was highly expressed in liver, its expression level was significantly increased in peak-laying stage, and was upregulated by estrogen. Inhibition of theEBP-likemRNA expression could restrain the expressions ofEBP-likedownstream genes (SC5D,DHCR24, andDHCR7) in the cholesterol synthetic pathway, therefore downregulate the liver intracellular T-CHO level. In conclusion, as substitute ofEBPgene in chickens,EBP-likeplays a vital role in the process of chicken liver cholesterol synthesis. This research provides a basis for revealing the molecular regulatory mechanism of cholesterol synthesis in birds, contributes insights into the improvement of the growth and development, laying performance and egg quality in poultry.

Background: Modern breeding strategies have resulted in significant differences in muscle mass between indigenous chicken and specialized broiler. However, the molecular regulatory mechanisms that underlie these differences remain elusive. The aim of this study was to identify key genes and regulatory mechanisms underlying differences in breast muscle development between indigenous chicken and specialized broiler. Results: Two time-series RNA-sequencing profiles of breast muscles were generated from commercial Arbor Acres (AA) broiler (fast-growing) and Chinese indigenous Lushi blue-shelled-egg (LS) chicken (slow-growing) at embryonic days 10, 14, and 18, and post-hatching day 1 and weeks 1, 3, and 5. Principal component analysis of the transcriptome profiles showed that the top four principal components accounted for more than 80% of the total variance in each breed. The developmental axes between the AA and LS chicken overlapped at the embryonic stages but gradually separated at the adult stages. Integrative investigation of differentially-expressed transcripts contained in the top four principal components identified 44 genes that formed a molecular network associated with differences in breast muscle mass between the two breeds. In addition, alternative splicing analysis revealed that genes with multiple isoforms always had one dominant transcript that exhibited a significantly higher expression level than the others. Among the 44 genes, the TNFRSF6B gene, a mediator of signal transduction pathways and cell proliferation, harbored two alternative splicing isoforms, TNFRSF6B-X1 and TNFRSF6B-X2 . TNFRSF6B-X1 was the dominant isoform in both breeds before the age of one week. A switching event of the dominant isoform occurred at one week of age, resulting in TNFRSF6B-X2 being the dominant isoform in AA broiler, whereas TNFRSF6B-X1 remained the dominant isoform in LS chicken. Gain-of-function assays demonstrated that both isoforms promoted the proliferation of chicken primary myoblasts, but only TNFRSF6B-X2 augmented the differentiation and intracellular protein content of chicken primary myoblasts. Conclusions: For the first time, we identified several key genes and dominant isoforms that may be responsible for differences in muscle mass between slow-growing indigenous chicken and fast-growing commercial broiler. These findings provide new insights into the regulatory mechanisms underlying breast muscle development in chicken.

MiRNAs have been found to be involved in the regulation of ovarian function as important post-transcriptional regulators, including regulators of follicular development, steroidogenesis, cell atresia, and even the development of ovarian cancer. In this study, we evaluated the regulatory role of gga-miR-449b-5p in follicular growth and steroid synthesis in ovarian granulosa cells (GCs) of laying hens through qRT-PCR, ELISAs, western blotting and dual-luciferase reporter assays, which have been described in our previous study. We demonstrated that gga-miR-449b-5p was widely expressed in granulosa and theca layers of the different-sized follicles, especially in the granulosa layer. The gga-miR-449b-5p had no significant effect on the proliferation of GCs, but could significantly regulate the expression of key steroidogenesis-related genes (StAR and CYP19A1) (p < 0.01) and the secretion of P4 and E2 (p < 0.01 and p < 0.05). Further research showed that gga-miR-449b-5p could target IGF2BP3 and downregulate the mRNA and protein expression of IGF2BP3 (p < 0.05). Therefore, this study suggests that gga-miR-449b-5p is a potent regulator of the synthesis of steroid hormones in GCs by targeting the expression of IGF2BP3 and may contribute to a better understanding of the role of functional miRNAs in laying hen ovarian development.

As an essential genetic and economic trait, chicken feather color has long been an important research topic. To further understand the mechanism of melanin deposition associated with coloration in chicken feathers, we selected feather follicle tissues from the neck and wings of chickens with differently colored feathers (yellow, sub-Columbian, and silver) for transcriptome analysis. We focused on genes that were expressed in both the wings and neck and were expressed with the same trends in breeds with two different plumage colors, specifically, SLC45A2, GPNMB, MLPH, TYR, KIT, WNT11, and FZD1. GO and KEGG enrichment analyses showed the DEGs were enriched in melanin-related pathways, such as tyrosine metabolic pathway and melanogenesis, and PPI analysis highlighted the genes SLC45A2 and GPNMB as associated with melanin deposition. Verification experiments in chicken melanocytes demonstrated that these two genes promote melanocyte melanin deposition. These data enrich our knowledge of the mechanisms that regulate chicken feather color.

The economic losses incurred due to reduced muscle pigmentation highlight the crucial role of melanin-based coloration in the meat of black-bone chickens. Melanogenesis in the breast muscle of black-bone chickens is currently poorly understood in terms of molecular mechanisms. This study employed whole-transcriptome sequencing to analyze black and white breast muscle samples from black-bone chickens, leading to the identification of 367 differentially expressed (DE) mRNAs, 48 DElncRNAs, 104 DEcircRNAs, and 112 DEmiRNAs involved in melanin deposition. Based on these findings, a competitive endogenous RNA (ceRNA) network was developed to better understand the complex mechanisms of melanin deposition. Furthermore, our analysis revealed key DEmRNAs (TYR, DCT, EDNRB, MLPH and OCA2) regulated by DEmiRNAs (gga-miR-140-5p, gga-miR-1682, gga-miR-3529, gga-miR-499-3p, novel-m0012-3p, gga-miR-200b-5p, gga-miR-203a, gga-miR-6651-5p, gga-miR-7455-3p, gga-miR-31-5p, miR-140-x, miR-455-x, novel-m0065-3p, gga-miR-29b-1-5p, miR-455-y, novel-m0085-3p, and gga-miR-196-1-3p). These DEmiRNAs competitively interacted with DElncRNAs including MSTRG.2609.2, MSTRG.4185.1, LOC112530666, LOC112533366, LOC771030, LOC107054724, LOC121107411, LOC100859072, LOC101750037, LOC121108550, LOC121109224, LOC121110876, and LOC101749016, as well as DEcircRNAs, such as novel_circ_000158, novel_circ_000623, novel_001518, and novel_circ_003596. The findings from this study provide insight into the mechanisms that regulate lncRNA, circRNA, miRNA, and mRNA expression in chicken melanin deposition.