The bone morphogenetic protein (BMP) gene family comprises a group of multifunctional cytokines that play important roles in limb development, bone formation, fat deposition, and reproductive traits of vertebrates. However, no systematic and comprehensive investigations of the various traits of the whole family members have been conducted, particularly in chickens. Here, we performed genome-wide screening and identified 14 BMP genes, which were classified into the BMP2/4, BMP5/6/7/8A, growth differentiation factor (GDF) 2/BMP10, GDF5/6/7, and GDF11/BMP3/15 subfamilies. Genetic variation pattern analysis showed that BMP genes were responsible for the artificial selection of commercial broilers and layers, with BMP2, BMP6, and GDF7 likely contributing significantly to the formation of both specialized meat- and egg-type lines, whereas BMP7 likely contributed more to the formation of meat-type lines. Genetic association analysis showed that single nucleotide polymorphisms (SNPs) in the BMP7 intron region were associated with body weight, breast muscle weight, leg weight, abdominal fat weights and contents of total cholesterol (T-CHO), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in serum. Additionally, gain- and loss-of-function assays demonstrated that BMP7 promoted the proliferation, myogenic differentiation, and lipid droplet accumulation in myoblasts; enhanced lipid synthesis in hepatocytes; promoted the proliferation and inhibited adipogenic differentiation of intramuscular preadipocytes; and induced the proliferation and adipogenic differentiation of abdominal preadipocytes. These results provide novel insights into the role of BMP genes in chicken growth, reproductive regulation, and lipid deposition and could be used to develop genetic markers for breeding selection in chickens.

Accumulating evidence has indicated that microRNAs (miRNAs) participate in chicken skeletal muscle development by post-transcriptionally regulating myogenesis-related gene expression. Our previous study showed that miR-34c-5p inhibited proliferation and myogenic differentiation of chicken primary myoblasts (CPMs), but its molecular mechanisms remain unclear. Here, miR-34c-5p was overexpressed in CPMs for transcriptome sequencing. The 159 differentially expressed genes (DEGs) were identified and were mainly involved in myogenesis-related processes and signaling pathways, including cytoskeleton regulation, PPAR, and cardiac muscle contraction. Intersection of DEGs and predicted targets of miR-34c-5p yielded 15 candidate genes. Of these, miR-34c-5p could inhibit the mRNA expression of MYH7B and TGM4 genes by directly interact with their 3 ' untranslated regions as determined by dual-luciferase reporter systems Gain- and loss-of-function assays demonstrated that TGM4 gene could promote CPMs proliferation. These findings elucidate the regulatory network of miR-34c-5p underlying myogenesis and provide potential molecular marker for genetic improvement of meat production in chicken.

MicroRNAs (miRNAs) have been increasingly involved in mammalian lipid metabolism. However, their regulatory roles and molecular mechanisms in abdominal fat deposition in chicken remain largely unexplored. In this study, based on the previous miRNA transcriptome data during chicken abdominal preadipocytes' adipogenic differentiation, we explored the biological functions and regulatory mechanisms of a differentially expressed miRNA, gga-let-7c-3p, in adipogenesis. Gain- and loss-of-function assays elucidated that gga-let-7c-3p significantly decreased viability, proliferation, cell cycle progression, intracellular lipid droplet accumulation and triglyceride content, as well as the mRNA expression of proliferation- and lipid metabolism-related genes in chicken abdominal preadipocytes. Dual-luciferase reporter assay confirmed that gga-let-7c-3p could directly interact with the 3'UTR of the transcription factor-peroxisome proliferator activated the receptor delta (PPARD) gene and thus inhibited its post-transcriptional expression. The PPARD gene significantly decreased viability, proliferation, and cell cycle progression, while it increased intracellular lipid droplet accumulation and triglyceride content of chicken abdominal preadipocytes, paralleling with the mRNA expression of proliferation- and lipid metabolism-related genes. Collectively, gga-let-7c-3p could inhibit the proliferation and adipogenic differentiation of chicken abdominal preadipocytes, at least by targeting the PPARD gene. These findings reveal the regulatory mechanisms of the gga-let-7c-3p/PPARD axis in chicken abdominal adipogenesis, and could provide potential molecular markers for lean line broiler breeding.

Reproductive senescence in laying hens, characterized by a progressive decline in egg production, represents a major challenge for the poultry industry. Although microRNAs (miRNAs) are recognized as important regulators of aging, their specific roles and mechanisms in ovarian aging of hens remain largely unclear. This study was designed to comprehensively analyze miRNA expression patterns during ovarian aging in laying hens. The objectives of this study were to identify key functional miRNAs and to elucidate their molecular regulatory mechanisms. Specifically, this study evaluated ovarian senescence in hens at 350, 500 and 700 days of age, observing a decline in egg production, increased follicular atresia, and p53 upregulation. miRNA sequencing analysis identified 44 differentially expressed miRNAs (DEMs), among which gga-let-7i exhibited the highest abundance and showed progressive upregulation during aging. Functional assays revealed that gga-let-7i induces cell cycle arrest and promotes cellular senescence in ovarian follicle granulosa cells (GCs). Mechanistically, collagen type I alpha 2 chain (COL1A2) was confirmed as a direct target of gga-let-7i, and it has been demonstrated that gga-let-7i accelerates senescence by inhibiting the COL1A2/PI3K/AKT/MDM2 pathway, resulting in p53 accumulation and the downstream cellular senescence signaling pathways activation. These results uncover a novel gga-let-7i/COL1A2 regulatory axis involved in ovarian aging and suggest potential targets for extending reproductive longevity in laying hens.

Circular RNAs (circRNAs) exert crucial functions in mammalian reproduction; however, their regulation underlying chicken reproduction remains unclear. Here, time-course transcriptome profiles of circRNAs were constructed in chicken ovarian tissues across the entire egg-laying cycle (15 W, 20 W, 30 W, 70 W, 90 W). A total of 6551 circRNAs were identified, and most were derived from exon-exon backsplicing with 400-600 nt. Of these, 1556 circRNAs harbour translation potential in both IRES- and m6A modification-dependent manners. The 1894 differentially expressed circRNAs (DE-circRNAs) exhibited stage-specific expression patterns, and were clustered into 6 significantly co-expressed subclusters and enriched in multiple signaling pathways involving follicular development. Weighted gene co-expression network analysis detected 1016 hub circRNAs, of which 136 circRNAs were co-expressed DE-circRNAs and considered as key candidates regulating chicken ovarian development. These key circRNAs may post-transcriptionally regulate folliculogenesis-related gene via competitive endogenous RNA mechanism. This study reveals potential regulatory roles of circRNAs in chicken ovarian development and egg-laying performance.

N6-methyladenosine (m6A) is the most common and abundant internal chemical modification in eukaryotic mRNA and long non-coding RNA (lncRNA). This dynamic and reversible epitranscriptional modification is mediated by a collaborative action of methyltransferases (writers), demethylases (erasers), and recognition proteins (readers) and represents an important epigenetic regulatory mechanism at the post-transcriptional level. LncRNAs are a class of RNA molecules that are longer than 200 nucleotides and lack the potential to encode proteins. They can participate in the fine regulation of gene expression at the transcriptional, post-transcriptional and epigenetic levels through various mechanisms such as cis-regulation and trans-regulation. In recent years, the interactive regulation of m6A modification and lncRNA has become a research hotspot. The precise regulatory network jointly constructed by these two elements plays a core role in the formation and plasticity regulation of complex traits in animals. This article systematically elaborates on the regulatory roles and research progress of m6A modification-related lncRNAs in important economic traits such as muscle development, fat deposition, and immune response in poultry. In response to the current issues of insufficient functional validation and insufficient mechanism elucidation, it proposes that future research should integrate multi-omics combined analysis, temporal and spatial dynamic analysis, cross-species comparative studies, and gene editing methods to systematically clarify their intrinsic regulatory mechanisms. This will open up a new path for molecular design breeding of poultry for disease resistance, high yield, high quality, and high efficiency, and is of great significance for promoting genetic improvement of poultry and the sustainable development of the industry.

BACKGROUND:Long noncoding RNAs (lncRNAs) participate in various critical regulatory steps during myogenesis. LncRNAs can encode small peptides, which can regulate gene expression through multiple mechanisms, thereby participating in key biological processes. RESULTS:The lncRNAs were screened between proliferating and differentiating myoblast in chicken through RNA-seq and Ribo-seq. As a result, 178 DE-lncRNAs were identified in RNA-seq, and three of them were identified as differentially translated lncRNAs by Ribo-seq. Among them, lncMPD, which showed coding potential, was highly expressed in proliferating myoblast. It encoded a small peptide containing 74 amino acids, which was named MPD-74aa. MPD-74aa was validated via WB and mass spectrometry. We subsequently confirmed that MPD-74aa promotes myoblast proliferation and inhibits its differentiation. Co-IP revealed that MPD-74aa interacts with the protein CDK1. Moreover, MPD-74aa positively regulated the expression of CDK1. CONCLUSION:This study confirms that the lncMPD plays a crucial regulatory role in the chicken myogenesis by encoding the small peptide MPD-74aa. Mechanistically, MPD-74aa exerts its regulatory function through interaction with CDK1, a key protein marker of cell proliferation. These findings provide new insight into the molecular mechanisms about the coding capacity of lncRNA regulating chicken muscle development.

Declining egg production in hens is often accompanied by ovarian dysfunction. Emerging evidence suggests a link between intestinal abnormalities and reproductive endocrine disorders, yet whether gut dysbiosis contributes to this process via ferroptosis and mitochondria-associated pathways remains unclear. Here, we show that low-producing hens exhibit intestinal villus atrophy and downregulation of tight junction proteins in both ileum and ovary, accompanied by elevated inflammatory cytokines, impaired antioxidant defense, and aberrant expression of ferroptosis-related genes, indicating a coordinated disruption of intestinal–ovarian homeostasis in inflammation and iron metabolism. 16S rRNA sequencing revealed a markedly increased abundance of Lactobacillus species in high-producing hens, correlating positively with sex hormone levels and laying performance, whereas these bacteria were depleted in low-producing hens. Dietary supplementation with Lactobacillus paracasei R8 (L. paracasei R8) improved laying performance, alleviated intestinal and ovarian injury, and attenuated ferroptosis-associated alterations. In LPS-induced intestinal epithelial and ovarian cell models, L. paracasei R8 improved barrier function, increased mitochondrial membrane potential, reduced lipid peroxidation and intracellular Fe2⁺ accumulation, exerting effects comparable to the ferroptosis inhibitor Fer-1. Co-treatment with the mitochondrial inhibitor CsA partially abrogated these effects, implicating mitochondrial regulation in the mechanism of action. Taken together, these findings identify an intestinal–ovarian injury pattern in low-producing hens associated with gut microbiota imbalance and iron-dependent cell death, and suggest that L. paracasei R8 may serve as a potential probiotic intervention to improve laying performance by alleviating gut–ovary-related pathological alterations through modulation of mitochondrial function and ferroptosis-related pathways.

Sexual dimorphism is a defining vertebrate feature, yet its sex-specific molecular architecture remains poorly understood. Here we established a sex-balanced, uniformly reared chicken cohort to map this landscape, integrating individual whole-genome sequencing with 7,969 bulk and 779,380 single nucleus transcriptomes across 32 tissues from 280 birds. We identified 495,098 independent expression quantitative trait loci for 20,194 genes, including 10,937 loci modulated by cell-type composition. Notably, 340 genes were regulated by 449 loci in a sex-dependent manner, significantly enrichment in endocrine tissues like adipose and the adrenal gland. Furthermore, we fine-mapped 1,219 structural variants, demonstrating their unique roles to tissue- and sex-specific expression beyond SNPs. Ultimately, we showed the utility of these regulatory effects in elucidating the molecular basis of metabolism and complex traits in both chickens and humans. This comprehensive atlas of regulatory effects provides profound insights into the genomic and molecular basis of sexual dimorphism in vertebrates.

Frizzled 4 (FZD4), a member of the frizzled family, is involved in various cancers and neurological disorders because of its abnormal expression. However, its regulatory role in skeletal development and bone formation, especially in livestock and poultry, remains poorly characterized. RNA sequencing (RNA-seq) analysis of bone tissue samples collected during physiological bone remodeling in chickens identified FZD4 as a differentially expressed gene. To investigate the function of FZD4 in the osteogenic differentiation of chicken bone marrow mesenchymal stem cells (BMSCs), we assessed the expression of osteogenic marker genes and canonical Wnt signaling pathway factors at the mRNA and protein levels. Additionally, we evaluated differentiation and mineralization potential via alkaline phosphatase (ALP) and alizarin red S (ARS) staining. The results demonstrated that FZD4 expression was significantly upregulated during early differentiation. Furthermore, FZD4 overexpression increased the expression of early osteogenic markers, whereas FZD4 knockdown suppressed this expression. Although FZD4 acts as a receptor for the canonical Wnt pathway, its role in promoting osteogenesis in chickens through this pathway remains unclear. Importantly, FZD4 overexpression upregulated key canonical Wnt signaling factors at both the mRNA and protein levels, whereas FZD4 knockdown had the opposite effect. These findings indicate that FZD4 participates in chicken bone formation by regulating osteogenic marker expression, likely through activation of the canonical Wnt signaling pathway. This study elucidates the specific regulatory role of FZD4 in avian skeletal development, providing novel insights and theoretical support for understanding bone development and skeletal health in poultry and livestock.

Ovarian aging in laying hens contributes significantly to the decline in egg production performance during the late laying period, and is accompanied by extensive remodeling of miRNA-mRNA interaction networks, which are critical post-transcriptional regulators during this process. In this study, we investigated the role of miR-30e-3p, which was previously identified as significantly upregulated during ovarian aging in laying hens, in granulosa cell (GC) senescence. To identify its downstream effectors, we performed mRNA transcriptome sequencing and screened for downregulated genes. Among potential targets, the histone methyltransferase complex regulatory subunit dpy-30 (DPY30) was validated as a direct target of miR-30e-3p by luciferase reporter and expression analyses. Functional assays demonstrated that miR-30e-3p significantly suppressed GC proliferation and induced cell cycle arrest, whereas DPY30 exhibited opposite effects. Furthermore, inhibition of miR-30e-3p or overexpression of DPY30 attenuated D-galactose (D-gal) -induced GC senescence. Mechanistically, miR-30e-3p promoted cellular senescence at least in part through downregulation of DPY30. Collectively, our findings indicated that miR-30e-3p promotes GC senescence by targeting DPY30 in chicken ovarian follicles, and suggest that both miR-30e-3p and DPY30 may serve as potential biomarkers for improving reproductive longevity and mitigating ovarian aging-related disorders in poultry.

Skeletal muscle development is regulated by transcriptional and post-translational mechanisms. While ankyrin repeat proteins participate in post-translational modifications, their role in myogenesis remains unclear. This study identifies ankyrin repeat domain-containing protein 9 (ANKRD9) as a novel negative regulator of chicken skeletal muscle development. ANKRD9 showed dynamic expression during postnatal muscle growth and downregulated cell cycle and DNA replication-related genes. Functionally, ANKRD9 overexpression inhibited myoblast proliferation and differentiation, while its knockdown enhanced these processes. In vivo, siRNA-mediated knockdown of ANKRD9 markedly increased muscle mass and myofiber diameter in chicks. Mechanistically, ANKRD9 bound directly to inosine monophosphate dehydrogenase 2 (IMPDH2), a rate-limiting enzyme in purine synthesis, and promoted its ubiquitin-mediated degradation without affecting mRNA levels. Crucially, rescue experiments confirmed that restoring IMPDH2 expression effectively reversed the inhibitory effects of ANKRD9 on myoblast proliferation and differentiation. Thus, this study unveils a novel regulatory axis in which ANKRD9 negatively regulates skeletal myogenesis by mediating the ubiquitination of IMPDH2. This discovery not only provides new insights into the post-translational regulatory network governing muscle development but also offers a potential target for genetic improvement of meat yield in poultry.

The reproductive performance of hens is predominantly determined by the growth and development of their follicles. Follicular development in chickens represents a highly complex and multi-regulatory biological process, wherein granulosa cells (GCs) play a crucial role in follicle selection. Recent studies have emphasized the critical role of circular RNAs (circRNAs) in regulating follicular growth and development in animals. In our previous study, employing circRNA RNA-seq, we identified differential expression of circRALGPS2 between healthy and atretic follicles in chickens. In the current investigation, we observed a marked increase in autophagy and apoptosis levels within atretic follicles. Notably, circRALGPS2 was shown to promote follicular atresia by enhancing autophagy and apoptosis in GCs. Moreover, we demonstrated that circRALGPS2 functions as a molecular sponge for miR-200a-3p, revealing that miR-200a-3p and circRALGPS2 exert antagonistic effects on GC autophagy and apoptosis. Mechanistically, circRALGPS2 targets miR-200a-3p, which subsequently modulates the expression of transforming growth factor β2 (TGFβ2), thereby activating the canonical TGFβ/SMAD signaling pathway. Collectively, our findings demonstrate that circRALGPS2 promotes apoptosis and autophagy in chicken GCs through activation of the TGFβ2/SMAD pathway via miR-200a-3p sequestration. These results provide valuable insights for advancing chicken genetic breeding technologies through marker-assisted selection.

Tyrosine is known to influence melanin generation; however, its involvement in melanin production in chicken feathers is unknown. We evaluated the feather color of H-line chickens fed diets containing different concentrations of tyrosine (0, 0.4, 0.6, 0.8, and 1.0%). The results indicated that a diet containing 1.0% tyrosine fed for 40 days significantly increased melanin deposition in the feathers (p < 0.05). Following this observation, we collected feather follicle tissue from chickens fed either 0% or 1.0% tyrosine at the 40-day time point for transcriptome sequencing. RNA-seq analysis identified a total of 314 DEGs, comprising 116 upregulated and 198 downregulated genes. KEGG analysis of feather follicle tissue revealed that 7 DEGs (EDNRB2, WNT3, POMC, INS, FLT3, CACNA2D3, and CACNA1I) mapped to melanin-related pathways, including the melanogenesis, MAPK signaling and Wnt signaling pathways. We also identified specific protein interactions within the melanin pathway, including EDNRB2–MLPH and WNT3–FGF16 interactions. Notably, the expression level of the EDNRB2 gene reached its peak at 10 weeks within the 0–12 week growth period in H-line chickens. In primary chicken melanocytes, EDNRB2 expression was quantified following tyrosine supplementation and was found to be markedly elevated at a concentration of 10−6 mol/L, significantly higher than the control and other treatment groups (p < 0.05). Overall, our findings suggest the significant involvement of the tyrosine-induced EDNRB2 regulatory network in melanin levels in sub-Columbian plumage. Taken together, these findings increase our understanding of the molecular mechanisms that regulate tyrosine-mediated melanin deposition in chicken plumage.

Chickens are economically important animals commonly utilized as model organisms in immunological research, but their immune cell heterogeneity and the subtle relationship between chicken immune cells and inter-breed immune variation remain poorly characterized. To fully understand the heterogeneity of immune cells and immune variation between breeds, single-cell RNA sequencing (scRNA-seq) technology was used to analyze the peripheral blood mononuclear cell (PBMC) of Hy-Line white (HW) and Hy-Line brown (HB) laying hens. This analysis revealed differences in immune cell heterogeneity and gene expression profiles among cell subpopulations between the two breeds. A total of nine immune cell populations were identified using known marker genes. Due to species differences between chickens and mammals such as humans and mice, which rendered some of the markers inapplicable, we identified new marker genes in the classification of cellular subpopulations that applied to chicken PBMC. Cell composition and expression abundance of immune genes underlie differences in immune traits between breeds. HW had a high intrinsic immune cell count, with a significant difference in the cellular proportion of B cells. In the B cell subpopulation, this was specifically demonstrated by the fact that B atypical memory and Plasma occupied a higher cellular proportion in HW. In contrast, the number of adaptive immune cells was higher in HB, with a significant difference in the proportion of conventional dendritic cell (cDC), specifically represented by type 1 conventional dendritic cell (cDC1) occupying a higher proportion of cells in HB and type 2 conventional dendritic cell (cDC2) occupies a higher proportion of cells in HW. In summary, HW can quickly respond to foreign pathogens and has stronger humoral immunity, while HB has advantages in antiviral response and cross-presentation. This study enhances our understanding of chicken immune heterogeneity and immune traits, facilitating the identification and functional analysis of chicken immune cells.

Induced molt is an effective measure to reduce the introduction cost, cope with the continuous rise of feed cost, and realize the prolonged rearing of laying hens as well. Vitamins are beneficial to the antioxidant capacity and reproductive performance of laying hens, however, studies on vitamin metabolism during fasting are rarely reported. We analyzed the association between cecal metabolome and liver transcriptome of laying hens during molt. The results showed that 3009 differences genes (FDR < 0.05), among which there were 62 differential genes related to vitamin metabolism. Eight core genes (ALDH1A1, CYP1A1, CYP1A4, AOX2P, AOX1, CYP3A7, BCAT1, CYP26B1) were obtained by protein network interaction (PPI). These genes were mainly enriched in Metabolic pathways, Retinol metabolism, Folate biosynthesis, One carbon pool by Folate, and Chemical carcinogenesis. After association analysis between these genes and cecal metabolites, a total of 176 differential metabolites were obtained. Among them, the metabolites with higher connectivity were Bifemelane, L-valine, Butyryl Fentanyl-D5 and Rimcazole. During fasting, vitamin A and vitamin E stored in the liver of laying hens are released in large quantities with the oxidative decomposition of lipids in the liver during fasting, which accelerates the metabolism of vitamin A in the liver. Folate and biotin may participate in the physiological remodeling of laying hens through epigenetic regulation. In addition, through association analysis, we constructed a data platform for vitamin metabolism-related pathway genes and cecal metabolites, laying a foundation for future research. However, whether the relationship between gene expression in the liver and metabolites in the cecum is bidirectional or unidirectional is still unclear and needs to be further studied.

RNA N6-methyladenosine (m6A) is one of the most common and widespread reversible epigenetic modifications of mRNAs, and m6A has been shown to play a positive role in regulating follicular development. However, the role of RNA m6A methylation in chicken ovaries and egg production has not been fully studied. In this study, we comprehensively analyzed the m6A transcriptome profiles of high- and low-yield Gushi chickens at 43 weeks of age (43 w). We found that m6A modification differed between the two groups. The m6A peak was positively correlated with the gene expression level, indicating that m6A may play an important role in regulating chicken egg production. In total, 9008 and 15,415 m6A peaks were separately identified in the two groups, including 2241 differential m6A peaks. In addition, seven candidate genes related to egg laying that were significantly enriched in the KEGG pathway related to ovary development and egg laying were identified. In summary, we constructed the first m6A modification map of ovarian tissue of Gushi chickens, and the differences in egg laying in 43 w Gushi chickens may originate from the effect of RNA methylation on the expression of egg-related genes. These findings provide new insights into the regulatory mechanisms of m6A methylation during egg production in Gushi chickens.

Skeletal muscle satellite cells are stem cells characterized by their multipotency and capacity for in vitro proliferation. However, primary skeletal muscle satellite cells demonstrate limited proliferative capacity in vitro, which impedes their investigation in poultry skeletal muscle research. Cell immortalization techniques have emerged as valuable tools to address this limitation and facilitate the study of skeletal muscle satellite cell functions. This study achieved the immortalization of chicken skeletal muscle satellite cells through the transduction of primary cells with TERT (telomerase reverse transcriptase) amplified from chicken (chTERT) using a lentiviral vector via telomerase activity reconstitution. While the cells successfully overcame replicative senescence, complete immortalization was not achieved. Initial functional characterization revealed that the proliferative properties and cell cycle characteristics of the immortalized chicken skeletal muscle satellite cell lines (ICMS) closely resembled those of chicken primary muscle satellite cells (CPMSCs). Serum dependency analysis and soft agar assays confirmed that ICMS did not undergo malignant transformation. Furthermore, induced differentiation experiments demonstrated preserved differentiation capacity in ICMS. The established cell lines provide a fundamental framework for developing immortalized poultry cell lines and offer a cellular model for investigating poultry skeletal muscle-related functional genes.

Fasting is beneficial to alleviate fatty liver, lose weight and improve reproductive function. However, previous studies have shown that, during fasting, disorders of bile acid metabolism were strongly associated with intestinal inflammation. The physiological and biochemical parameters and gene expression of multiple tissues of chickens at every critical time node were measured by ELISA and qPCR. In addition, association analysis was performed based on liver transcriptome sequencing and cecum metabolome data. At the cellular level, the regulatory effects of cecal metabolites on host bile acid metabolism were verified. During fasting, hepatic FXR-SHP-CYP7A1 and ileum-hepatic FXR-SHP-FGF15/19-FGFR4-CYP7A1 negative feedback pathways were activated to inhibit hepatic bile acid synthesis. The ileum FXR-SHP-ASBT pathways are activated, hindered the ileal bile reflux. At the same time, it promotes the secretion of bile acids and cholesterol in the liver, accelerates the utilization of H2O and CO2, to maintain liver homeostasis during fasting. In addition, enhanced gallbladder contraction and increased hunger were observed in laying hens during fasting. At the cellular level, the correlation between CYP7A1 and L-valine was verified, revealing that cecal metabolites of laying hens was enabled to regulate host bile acid metabolism. This study explored the metabolic patterns of bile acids during fasting and identified the main reasons for the accumulation of bile acids in the cecum, which provides a basis for fasting research and offers a reference for the formulation of fasting protocols.

The growth and development of chicken skeletal muscle directly affects chicken meat production, which is very important for broiler industry. Matrix metallopeptidase 2 (MMP2) exists in skeletal muscle. However, the underlying regulating of MMP2 remain unknown. In this study, MMP2 promoted cell proliferation and inhibited cell differentiation after overexpression in chicken primary myoblasts cells (CPMs). When MMP2 was knocked down, it inhibited CPMs proliferation and promoted cell differentiation. Subsequently, RNA sequencing (RNA-seq) and bioinformatics analysis were performed on overexpressing MMP2. We identified 265 up-regulated genes and 229 down-regulated genes. Based on the fragments per kilobase million (FPKM) ≥ 10, the retained data were analyzed by Pearson correlation analysis. MMP2 was positively correlated with carboxypeptidase M (CPM), MSTRG.14120 and aldehyde dehydrogenase 1 family member A3 (ALDH1A3), and the correlation coefficient was the highest (0.998). MMP2 was negatively correlated with hes family bHLH transcription factor 1 (HES1), and the correlation coefficient was the highest (0.998). Go term was enriched in cellular components or biogenesis, cellular processes, and cell aggregation. KEGG was significantly enriched to the cancer pathway. qRT-PCR analysis validated the transcriptomic results of RNA-seq. In conclusion, these results provided new insights into the molecular mechanisms by which MMP2 affected the proliferation and differentiation of chicken myoblasts.