Genome-wide variation study and inter-tissue communication analysis unveil regulatory mechanisms of egg-laying performance in chickens.

Egg-laying performance is of great economic importance in poultry, but the underlying genetic mechanisms are still elusive. In this work, we conduct a multi-omics and multi-tissue integrative study in hens with distinct egg production, to detect the hub candidate genes and construct hub molecular networks contributing to egg-laying phenotypic differences. We identifiy three hub candidate genes as egg-laying facilitators: TFPI2, which promotes the GnRH secretion in hypothalamic neuron cells; CAMK2D, which promotes the FSHß and LHß secretion in pituitary cells; and OSTN, which promotes granulosa cell proliferation and the synthesis of sex steroid hormones. We reveal key endocrine factors involving egg production by inter-tissue crosstalk analysis, and demonstrate that both a hepatokine, APOA4, and an adipokine, ANGPTL2, could increase egg production by inter-tissue communication with hypothalamic-pituitary-ovarian axis. Together, These results reveal the molecular mechanisms of multi-tissue coordinative regulation of chicken egg-laying performance and provide key insights to avian reproductive regulation.

The role of AdipoQ on proliferation, apoptosis, and hormone Secretion in chicken primary adenohypophysis cells.

Adiponectin (AdipoQ), an adipokine secreted by adipocytes, has been reported to exist widely in various cell types and tissues, including the adenohypophysis of chickens. However, the molecular mechanism by which AdipoQ regulates the function of chicken adenohypophysis remains elusive. In this study, we investigated the effects of AdipoQ on proliferation, apoptosis, secretion of related hormones (FSH, LH, TSH, GH, PRL and ACTH) and expression of related genes (FSHß, LHß, GnRHR, TSHß, GH, PRL and ACTH) in primary adenohypophysis cells of chickens by using real-time fluorescent quantitative PCR (RT-qPCR), cell counting kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) assays. Our results showed that AdipoQ promoted the proliferation of chicken primary adenohypophysis cells, up-regulated the mRNA expression of proliferation-related genes CDK1, PCNA, CCND1 and P21 (P < 0.05), as well as the increased protein expression of CDK1 and PCNA (P < 0.05). Furthermore, AdipoQ inhibited apoptosis of chicken primary adenohypophysis cells, resulting in down-regulation of pro-apoptotic genes Caspase3, Fas, and FasL mRNA expression, and decreased Caspase3 protein expression (P < 0.05). Moreover, there was an up-regulation of anti-apoptotic gene Bcl2 mRNA and protein expression (P < 0.05). Additionally, AdipoQ suppressed the secretion of FSH, LH, TSH, GH, PRL, and ACTH (P < 0.05), as well as the mRNA expression levels of related genes (P < 0.05). Treatment with AdipoRon (a synthetic substitute for AdipoQ) and co-treatment with RNA interference targeting AdipoQ receptors 1/2 (AdipoR1/2) had no effect on the secretion of FSH, LH, TSH, GH, PRL, and ACTH, as well as the mRNA expression levels of the related genes. This suggests that AdipoQ's regulation of hormone secretion and related gene expression is mediated by the AdipoR1/2 signaling axis. Importantly, we further demonstrated that the mechanism of AdipoQ on FSH, LH, TSH and GH secretion is realized through AMPK signaling pathway. In conclusion, we have revealed, for the first time the molecular mechanism by which AdipoQ regulates hormone secretion in chicken primary adenohypophysis cells.

Dynamic alternations of three-dimensional chromatin architecture contribute to phenotypic characteristics of breast muscle in chicken.

Breast muscle growth rate and intramuscular fat (IMF) content show apparent differences between fast-growing broilers and slow-growing indigenous chickens. However, the underlying genetic basis of these phenotypic characteristics remains elusive. In this study, we investigate the dynamic alterations of three-dimensional genome architecture and chromatin accessibility in breast muscle across four key developmental stages from embryo to starter chick in Arbor Acres (AA) broilers and Yufen (YF) indigenous chickens. The limited breed-specifically up-regulated genes (Bup-DEGs) are embedded in breed-specific A compartment, while a majority of the Bup-DEGs involving myogenesis and adipogenesis are regulated by the breed-specific TAD reprogramming. Chromatin loops allow distal accessible regions to interact with myogenic genes, and those loops share an extremely low similarity between chicken with different growth rate. Moreover, AA-specific loop interactions promote the expression of 40 Bup-DEGs, such as IGF1, which contributes to myofiber hypertrophy. YF-specific loop interactions or distal accessible regions lead to increased expression of 5 Bup-DEGs, including PIGO, PEMT, DHCR7, TMEM38B, and DHDH, which contribute to IMF deposition. These results help elucidate the regulation of breast muscle growth and IMF deposition in chickens.

Dietary restriction promote sperm remodeling in aged roosters based on transcriptome analysis.

BACKGROUND: The breeder rooster has played a pivotal role in poultry production by providing high-quality semen. Typically, fertility peaks between 30 and 40 weeks of age and then declines rapidly from 45 to 55 weeks of age. Research into improving fertility in aging roosters is essential to extend their productive life. While progress has been made, enhancing fertility in aging roosters remains a significant challenge. METHODS: To identify the genes related to promoting sperm remodeling in aged Houdan roosters, we combined changes in testis and semen quality with transcriptome sequencing (RNA-seq) to analyze the synchrony of semen quality and testis development. In this study, 350-day-old Houdan breeder roosters were selected for RNA-seq analysis in testis tissues from induced molting roosters (D group) and non-induced molting roosters (47DG group). All analyses of differentially expressed genes (DEGs) and functional enrichment were performed. Finally, we selected six DEGs to verify the accuracy of the sequencing by qPCR. RESULTS: Compared with the 47DG group, sperm motility (P < 0.05), sperm density (P < 0.01), and testis weight (P < 0.05) were significantly increased in roosters in the D group. Further RNA-seq analysis of the testis between the D group and 47DG group identified 61 DEGs, with 21 up-regulated and 40 down-regulated. Functional enrichment analysis showed that the DEGs were primarily enriched in the cytokine-cytokine receptor interaction, Wnt signaling pathway, MAPK signaling pathway, TGF-ß signaling pathway, and focal adhesion pathway. The qRT-PCR results showed that the expression trend of these genes was consistent with the sequencing results. WNT5A, FGFR3, AGTR2, TGFß2, ROMO1, and SLC26A7 may play a role in testis development and spermatogenesis. This study provides fundamental data to enhance the reproductive value of aging roosters.

The long noncoding RNA lncMPD2 inhibits myogenesis by targeting the miR-34a-5p/THBS1 axis.

Long noncoding RNAs (lncRNAs) participate in regulating skeletal muscle development. However, little is known about their role in regulating chicken myogenesis. In this study, we identified a novel lncRNA, lncMPD2, through transcriptome sequencing of chicken myoblasts at different developmental stages. Functionally, gain- and loss-of-function experiments showed that lncMPD2 inhibited myoblast proliferation and differentiation. Mechanistically, lncMPD2 directly bound to miR-34a-5p, and miR-34a-5p promoted myoblasts proliferation and differentiation and inhibited the mRNA and protein expression of its target gene THBS1. THBS1 inhibited myoblast proliferation and differentiation in vitro and delayed muscle regeneration in vivo. Furthermore, rescue experiments showed that lncMPD2 counteracted the inhibitory effects of miR-34a-5p on THBS1 and myogenesis-related gene mRNA and protein expression. In conclusion, lncMPD2 regulates the miR-34a-5p/THBS1 axis to inhibit the proliferation and differentiation of myoblasts and skeletal muscle regeneration. This study provides more insight into the molecular regulatory network of skeletal muscle development, identifying novel potential biomarkers for improving chicken quality and increasing chicken yield. In addition, this study provides a potential goal for breeding strategies that minimize muscle damage in chickens.

Biodegradation and bioaugmentation of tetracycline by Providencia stuartii TX2: Performance, degradation pathway, genetic background, key enzymes, and application risk assessment.

The antibiotic tetracycline (TC) is an emerging pollutant frequently detected in various environments. Biodegradation is a crucial approach for eliminating TC contamination. However, only a few efficient TC-degrading bacteria have been isolated, and the molecular mechanisms of TC degradation, as well as their application potential, remain poorly understood. This study isolated a novel TC-degrading bacterium, Providencia stuartii TX2, from the intestine of black soldier fly larvae. TX2 exhibited remarkable performance, degrading 72.17 % of 400 mg/L TC within 48 h. Genomic analysis of TX2 unveiled the presence of antibiotic resistance genes and TC degradation enzymes. Transcriptomic analysis highlighted the roles of proteins related to efflux pumps, enzymatic transformation, adversity resistance, and unknown functions. Three TC degradation pathways were proposed, with TC being transformed into 27 metabolites through epimerization, hydroxylation, oxygenation, ring opening, and de-grouping, reducing TC toxicity. Additionally, TX2 significantly enhanced TC biodegradation in four TC-contaminated environmental samples and reduced antibiotic resistance genes and mobile genetic elements in chicken manure. This research provides insights into the survival and biodegradation mechanisms of Providencia stuartii TX2 and evaluates its potential for environmental bioremediation.

Duck pan-genome reveals two transposon insertions caused bodyweight enlarging and white plumage phenotype formation during evolution.

Structural variations (SVs) are a major source of domestication and improvement traits. We present the first duck pan-genome constructed using five genome assemblies capturing ∼40.98 Mb new sequences. This pan-genome together with high-depth sequencing data (∼46.5×) identified 101,041 SVs, of which substantial proportions were derived from transposable element (TE) activity. Many TE-derived SVs anchoring in a gene body or regulatory region are linked to duck's domestication and improvement. By combining quantitative genetics with molecular experiments, we, for the first time, unraveled a 6945 bp Gypsy insertion as a functional mutation of the major gene IGF2BP1 associated with duck bodyweight. This Gypsy insertion, to our knowledge, explains the largest effect on bodyweight among avian species (27.61% of phenotypic variation). In addition, we also examined another 6634 bp Gypsy insertion in MITF intron, which triggers a novel transcript of MITF, thereby contributing to the development of white plumage. Our findings highlight the importance of using a pan-genome as a reference in genomics studies and illuminate the impact of transposons in trait formation and livestock breeding.

Fasting-Induced Molting Impacts the Intestinal Health by Altering the Gut Microbiota.

Fasting-induced molting (FIM) is a common method used to improve the laying performance of aged laying hens. Nevertheless, this approach may impose various stresses on chickens, such as disruptions in intestinal flora and inflammation issues within the intestines. However, the impact of an imbalance in intestinal flora on intestinal health during the FIM process remains elusive. Therefore, intestinal injury, the microbiome, and the metabolome were analyzed individually and integrated to elucidate the impact of the intestinal flora on intestinal health during the FIM process. The findings indicated that fasting resulted in a notable reduction in villus height and villus/crypt ratio, coupled with elevated levels of intestinal inflammation and permeability. During the fasting period, microbiota compositions changed. The abundance of Escherichia_Shigella increased, while the abundance of Ruminococcaceae_UCG-013 and Lactobacillus decreased. Escherichia_Shigella was positively correlated with Citrinin and Sterobilin, which lead to intestinal inflammation. Ruminococcaceae_UCG-013 and Lactobacillus exhibited positive correlations with Lanthionine and reduced Glutathione, thereby reducing intestinal inflammation. This study screened the intestinal probiotics, Ruminococcaceae UCG-013 and Lactobacillus, that influence gut health during the fasting period, providing an experimental basis for improving gut microbiota and reducing intestinal inflammation during the FIM process.

Long Noncoding RNA 6302 Regulates Chicken Preadipocyte Differentiation by Targeting SLC22A16.

The excessive deposition of abdominal adipocytes in chickens is detrimental to poultry production. However, the regulatory factors that affect abdominal adipogenesis in chickens are still poorly understood. SLC22A16 is differentially expressed in abdominal preadipocytes and 10-day differentiated adipocytes in chickens, but its role in regulating chicken adipogenesis has not been reported. In this study, the function of SLC22A16 in chicken abdominal preadipocytes was investigated. SLC22A16 is significantly upregulated during abdominal adipocyte differentiation. The overexpression of SLC2A16 upregulated the expression of adipogenic marker genes and proliferation-related genes, and promoted the proliferation of adipocytes and the accumulation of triglycerides. The knockdown of SLC22A16 downregulated the expression of adipogenic marker genes and proliferation-related genes, inhibited the proliferation of adipocytes, and impaired the accumulation of triglycerides in adipocytes. In addition, LNC6302 was differentially expressed in abdominal preadipocytes and mature adipocytes, and was significantly positively correlated with the expression of SLC22A16. Interference with LNC6302 inhibits the expression of adipogenic marker genes and proliferation-related genes. The data supported the notion that LNC6302 promotes the differentiation of chicken abdominal adipocytes by cis-regulating the expression of SLC22A16. This study identified the role of SLC22A16 in the differentiation and proliferation of chicken adipocytes, providing a potential target for improving abdominal adipogenesis in chickens.

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken.

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

Whole transcriptome profiling reveals a lncMDP1 that regulates myogenesis by adsorbing miR-301a-5p targeting CHAC1.

Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in different developmental stages of chicken primary myoblasts (CPMs) using RNA sequencing (RNA-seq) technology. The dual luciferase reporter system was performed using chicken embryonic fibroblast cells (DF-1), and functional studies quantitative real-time polymerase chain reaction (qPCR), cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry cycle, RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence, and western blotting assay. Our research demonstrated that miR-301a-5p had a targeted binding ability to lncMDP1 and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). The results revealed that lncMDP1 regulated the proliferation and differentiation of myoblasts via regulating the miR-301a-5p/CHAC1 axis, and CHAC1 promotes muscle regeneration. This study fulfilled the molecular regulatory network of skeletal muscle development and providing an important theoretical reference for the future improvement of chicken meat performance and meat quality.

Divergent Regulatory Roles of Transcriptional Variants of the Chicken LDB3 Gene in Muscle Shaping.

LIM domain binding 3 (LDB3) serves as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mammalian skeletal muscle development, but its regulatory role and molecular mechanism in avian muscle development are still unclear. In this study, we reanalyzed RNA sequencing data sets of 1415 samples from 21 chicken tissues published in the NCBI GEO database. First, three variants (LDB3-X, LDB3-XN1, and LDB3-XN2) generated by alternative splicing of the LDB3 gene were identified in chicken skeletal muscle, among which LDB3-XN1 and LDB3-XN2 are novel variants. LDB3-X and LDB3-XN1 are derived from exon skipping in chicken skeletal muscle at the E18-D7 stage and share three LIM domains, but LDB3-XN2 lacks a LIM domain. Our results preliminarily suggest that the formation of three variants of LDB3 is regulated by RBM20. The three splice isomers have divergent functions in skeletal muscle according to in vitro and in vivo assays. Finally, we identified the mechanism by which different variants play different roles through interactions with IGF2BP1 and MYHC, which promote the proliferation and differentiation of chicken myoblasts, in turn regulating chicken myogenesis. In conclusion, this study revealed the divergent roles of three LDB3 variants in chicken myogenesis and muscle remodeling and demonstrated their regulatory mechanism through protein-protein interactions.

Effects of immunosuppression-associated gga-miR-146a-5p on immune regulation in chicken macrophages by targeting the IRKA2 gene.

Stress-induced immunosuppression (SIIS) is one of the common problems in intensive poultry production, which brings enormous economic losses to the poultry industry. Accumulating evidence has shown that microRNAs (miRNAs) were important regulators of gene expression in the immune system. However, the miRNA-mediated molecular mechanisms underlying SIIS in chickens are still poorly understood. This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken SIIS. A stress-induced immunosuppression model was successfully established via daily injection of dexamethasone and analyzed miRNA expression in spleen. Seventy-four differentially expressed miRNAs (DEMs) was identified, and 229 target genes of the DEMs were predicted. Functional enrichment analysis the target genes revealed pathways related to immunity, such as MAPK signaling pathway and FoxO signaling pathway. The candidate miRNA, gga-miR-146a-5p, was found to be significantly downregulated in the Dex-induced chicken spleen, and we found that Dex stimulation significantly inhibited the expression of gga-miR-146a-5p in Chicken macrophages (HD11). Flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8) and other assays indicated that gga-miR-146a-5p can promote the proliferation and inhibit apoptosis of HD11 cells. A dual-luciferase reporter assay suggested that the Interleukin 1 receptor associated kinase 2 (IRAK2) gene, which encoded a transcriptional factor, was a direct target of gga-miR-146a-5p, gga-miR-146a-5p suppressed the post-transcriptional activity of IRAK2. These findings not only improve our understanding of the specific functions of miRNAs in avian stress but also provide potential targets for genetic improvement of stress resistance in poultry.

LncRNA lncMGR regulates skeletal muscle development and regeneration by recruiting CDK9 and sponging miRNAs.

Long non-coding RNAs (lncRNAs) play an essential role in vertebrate myogenesis and muscle diseases. However, the dynamic expression patterns, biological functions, and mechanisms of lncRNAs in skeletal muscle development and regeneration remain largely unknown. In this study, a novel lncRNA (named lncMGR) was differentially expressed during breast muscle development in fast- and slow-growing chickens. Functionally, lncMGR promoted myoblast differentiation, inhibited myoblast proliferation in vitro, and promoted myofiber hypertrophy and injury repair in vivo. Mechanistically, lncMGR increased the mRNA and protein expression of skeletal muscle myosin heavy chain 1 A (MYH1A) via both transcriptional and post-transcriptional regulation. Nuclear lncMGR recruited cyclin-dependent kinase 9 (CDK9) to the core transcriptional activation region of the MYH1A gene to activate MYH1A transcription. Cytoplasmic lncMGR served as a competitive endogenous RNA (ceRNA) to competitively absorb miR-2131-5p away from MYH1A and subsequently protected the MYH1A from miR-2131-5p-mediated degradation. Besides miR-2131-5p, cytoplasmic lncMGR could also sponge miR-143-3p to reconcile the antagonist between the miR-2131-5p/MYH1A-mediated inhibition effects and miR-143-3p-mediated promotion effects on myoblast proliferation, thereby inhibiting myoblast proliferation. Collectively, lncMGR could recruit CDK9 and sponge multiple miRNAs to regulate skeletal muscle development and regeneration, and could be a therapeutic target for muscle diseases.

Differential responses to avian pathogenic E. coli and the regulatory role of splenic miRNAs in APEC infection in Silkie chickens.

Avian pathogenic Escherichia coli (APEC) is a bacterial disease that harms the poultry industry worldwide, but its effect on Chinese Silkie has not been reported. Studies on whether there are differences in Silkie individual resistance to APEC and the regulatory role of spleen miRNAs lay the foundation for strategies against APEC. Therefore, 270 Silkie chickens were infected with the median lethal dose of an E. coli O1, O2, and O78 mixture. These chickens were divided into a susceptible group (Group S) and a recovery group (Group R) according to whether they survived 15 days postinfection (dpi). Moreover, 90 uninfected APEC Silkie served as controls (Group C). The splenic miRNA expression profile was examined to evaluate the role of miRNAs in the APEC infection response. Of the 270 Silkies infected with APEC, 144 were alive at 15 dpi. Cluster analysis and principal component analysis (PCA) of splenic miRNAs revealed that the four Group R replicates were clustered with the three Group C replicates and were far from the three Group S replicates. Differentially expressed (DE) miRNAs, especially gga-miR-146b-5p, play essential roles in immune and inflammatory responses to APEC. Functional enrichment analyses of DEmiRNAs suggested that suppression of immune system processes (biological processes) might contribute to susceptibility to APEC and that FoxO signaling pathways might be closely associated with the APEC infection response and postinfection repair. This study paves the way for screening anti-APEC Silkies and provides novel insights into the regulatory role of miRNAs in APEC infection.

Combined analysis of the microbiome, metabolome and transcriptome of silkie chickens in response to avian pathogenic E. coli (APEC).

Avian colibacillosis is a bacterial disease caused by avian pathogenic Escherichia coli (APEC) that results in great losses in the poultry industry every year. Individual Silkie chickens of the same breed that are given the same feed in the same feeding conditions have different levels of resistance or susceptibility to APEC. Differences in gut microbes, gut metabolites, and gene expression in the spleen of APEC-resistant and APEC-susceptible chickens were compared, and multiple omics associations were analyzed to explore the mechanism of resistance to APEC in Silkie chickens. Compared with those in the APEC-susceptible group, the APEC-resistant group showed significantly increased abundances of many gut microorganisms, including Bacillus, Thermoactinomyces, Arthrobacter, and Ureibacillus, which were positively correlated with norvaline, l-arginine, and valyl-glycine levels. Intestinal tryptophan, indole, and indole derivative-related differentially abundant metabolites played an active role in combatting APEC infection. In the spleen, "response to stimulus" was the most significantly enriched GO term, and "cytokine‒cytokine receptor interaction" was the most significantly enriched KEGG pathway. The arginine biosynthesis and PPAR signaling pathways were the KEGG pathways that were significantly enriched with differentially abundant metabolites and differentially expressed genes. This study provides new insight into the prevention and treatment of APEC infection in Silkie chickens and lays a foundation to study the mechanism of APEC infection in poultry.

Whole-transcriptome sequencing reveals a melanin-related ceRNA regulatory network in the breast muscle of Xichuan black-bone chicken.

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.

STC2 Inhibits Hepatic Lipid Synthesis and Correlates with Intramuscular Fatty Acid Composition, Body Weight and Carcass Traits in Chickens.

Stanniocalcin 2 (STC2) is a secreted glycoprotein involved in multiple biological processes. To systemically study the biological role of STC2 in chickens, phylogenetic tree analysis and conservation analysis were conducted. Association analysis between variations in the STC2 gene and the economic traits of Gushi-Anka F2 was conducted. The tissue expression patterns of STC2 expression in different chicken tissues and liver at different stages were detected. The biological role of STC2 in chicken liver was investigated through overexpression and interfering methods in the LMH cell line. Correlation analyses between STC2 expression and lipid components were conducted. (1) The phylogenetic tree displayed that chicken STC2 is most closely related with Japanese quail and most distantly related with Xenopus tropicalis. STC2 has the same identical conserved motifs as other species. (2) rs9949205 (T > C) found in STC2 intron was highly significantly correlated with chicken body weight at 0, 2, 4, 6, 8, 10 and 12 weeks (p < 0.01). Extremely significant correlations of rs9949205 with semi-evisceration weight (SEW), evisceration weight (EW), breast muscle weight (BMW), leg muscle weight (LMW), liver weight and abdominal fat weight (AFW) were revealed (p < 0.01). Significant associations between rs9949205 and abdominal fat percentage, liver weight rate, breast muscle weight rate and leg muscle weight rate were also found (p < 0.05). Individuals with TT or TC genotypes had significantly lower abdominal fat percentage and liver weight rate compared to those with the CC genotype, while their body weight and other carcass traits were higher. (3) STC2 showed a high expression level in chicken liver tissue, which significantly increased with the progression of age (p < 0.05). STC2 was observed to inhibit the content of lipid droplets, triglycerides (TG) and cholesterol (TC), as well the expression level of genes related to lipid metabolism in LMH cells. (4) Correlation analysis showed that the STC2 gene was significantly correlated with 176 lipids in the breast muscle (p < 0.05) and mainly enriched in omega-3 and omega-6 unsaturated fatty acids. In conclusion, the STC2 gene in chicken might potentially play a crucial role in chicken growth and development, as well as liver lipid metabolism and muscle lipid deposition. This study provides a scientific foundation for further investigation into the regulatory mechanism of the STC2 gene on lipid metabolism and deposition in chicken liver.

RRM2 promotes the proliferation of chicken myoblasts, inhibits their differentiation and muscle regeneration.

During myogenesis and regeneration, the proliferation and differentiation of myoblasts play key regulatory roles and may be regulated by many genes. In this study, we analyzed the transcriptomic data of chicken primary myoblasts at different periods of proliferation and differentiation with protein‒protein interaction network, and the results indicated that there was an interaction between cyclin-dependent kinase 1 (CDK1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Previous studies in mammals have a role for RRM2 in skeletal muscle development as well as cell growth, but the role of RRM2 in chicken is unclear. In this study, we investigated the effects of RRM2 on skeletal muscle development and regeneration in chickens in vitro and in vivo. The interaction between RRM2 and CDK1 was initially identified by co-immunoprecipitation and mass spectrometry. Through a dual luciferase reporter assay and quantitative real-time PCR, we identified the core promoter region of RRM2, which is regulated by the SP1 transcription factor. In this study, through cell counting kit-8 assays, 5-ethynyl-2'-deoxyuridine incorporation assays, flow cytometry, immunofluorescence staining, and Western blot analysis, we demonstrated that RRM2 promoted the proliferation and inhibited the differentiation of myoblasts. In vivo studies showed that RRM2 reduced the diameter of muscle fibers and slowed skeletal muscle regeneration. In conclusion, these data provide preliminary insights into the biological functions of RRM2 in chicken muscle development and skeletal muscle regeneration.

miR-19b-3p regulated by estrogen controls lipid synthesis through targeting MSMO1 and ELOVL5 in LMH cells.

miR-19b-3p is reported to undertake various biological role, while its function and action mechanism in chicken hepatic lipid metabolism is unclear. Conservation analysis and tissue expression pattern of miR-19b-3p and its target gene were evaluated, respectively. Dual luciferase reporter system and Western blot technologies were adopted to validate miR-19b-3p target gene. Overexpression and knockdown assays were done to explore the biological functions of miR-19b-3p and target gene in Leghorn Male Hepatoma cell line (LMH). Regulatory approaches of estrogen on miR-19b-3p and target gene expressions are analyzed through site-directed mutation combined with estrogen receptors antagonist treatment assays. The results showed that chicken miR-19b-3p mature sequences are highly conserved among Capra hircus, Columba livia, Rattus norvegicus, Mus musculus, Cricetulus griseus, Danio rerio, Danio novaehollandiae, Orycodylus porosus, Crocodylus porosus, Gadus morhua, and widely expressed in lung, ovary, spleen, duodenum, kidney, heart, liver, leg muscle, and pectoral muscle tissues. miR-19b-3p could significantly increase intracellular triglyceride (TG) content and decrease intracellular cholesterol (TC) content via targeting methylsterol monooxygenase 1 (MSMO1) and elongase of very long chain fatty acids 5 (ELOVL5), which are highly conserved among species, in both mRNA and protein levels. Estrogen could inhibit miR-19b-3p expression, but directly promoted MSMO1 transcription via estrogen receptor α (ERα) and indirectly regulated ELOVL5 expression at the transcription level. Meanwhile, estrogen could also upregulate MSMO1 and ELOVL5 expression through inhibiting miR-19b-3p expression at the post-transcription level. Taken together, these results highlight the role and regulatory mechanism of miR-19b-3p in hepatic lipid metabolism in chicken, and might produce useful comparative information for human obesity studies and biomedical research.