This study comprehensively investigated the effects of different dietary energy levels-low energy (LE, 10.62 MJ/kg), medium energy (ME, 11.87 MJ/kg), and high energy (HE, 13.06 MJ/kg)-on growth performance, meat quality, muscle metabolite profiles, and cecal microbiota in a local Chinese chicken breed (Guangyuan gray chicken). Growth and carcass traits, including interpubic width and liver weight, responded positively to increasing dietary energy. Compared to the ME control, the HE diet significantly increased drip loss, whereas the LE diet reduced both intramuscular fat and protein content (P <0.05). Untargeted metabolomic analysis identified 188 differentially abundant muscle metabolites across the dietary groups, revealing distinct metabolic profiles. The biosynthesis of the amino acids pathway was significantly affected by different dietary energy levels. Targeted metabolomics further demonstrated the differences in muscle amino acid composition: essential, sweet-taste, and umami-taste amino acids increased progressively with dietary energy, with the HE group achieving the most favorable profile for flavor and nutrition. Cecal microbiota analysis indicated that dietary energy levels significantly altered microbial community structure. Specific bacterial taxa, including Spirochaetota and Bacteroides, were positively correlated with muscle amino acid concentrations. Our findings demonstrate that dietary energy level modulated the cecal microbiota, which in turn influenced muscle amino acid deposition and overall meat quality. This study provides a comparative multi-omics perspective for optimizing dietary energy strategies in local chicken production to meet varying market demands for meat quality and nutritional value. IMPORTANCE This comparative study established a crucial link between dietary energy intake and the final quality of poultry meat through a multi-omics lens. We demonstrate that low, medium, and high dietary energy levels elicit distinct phenotypic, metabolic, and microbial profiles in a local chicken breed. The findings provide a scientific framework for selecting dietary energy strategies tailored to specific market demands: HE for premium quality segments, LE for lean or cost-driven production, and ME for efficient conventional production. We identified specific correlations between the cecal bacteria (Spirochaetota, Bacteroides) and muscle metabolites (key amino acids). Thus, this work offered the scientific data to optimize meat quality through targeted feeding interventions.

In broiler production, achieving tissue-specific deposition of intramuscular fat (IMF) and abdominal fat (AF) remains challenging because the regulatory mechanisms are not fully understood. In this study, we focused on the liver, a shared hub of lipid metabolism for IMF and AF, and integrated targeted lipidomics and proteomics to characterize coordinated liver-serum-adipose lipid alterations and identify key hepatic regulatory proteins. The results showed that both IMF and AF deposition were primarily characterized by increased lipids from the triacylglycerol (TAG) subclass, and hepatic lipids responding to adipose tissue growth were predominantly from the TAG and diacylglycerol subclasses. In contrast, in the liver-serum-IMF alterations, serum lipid changes were mainly assigned to the glycerophospholipid class, whereas those in the liver-serum-AF alterations were primarily associated with the TAG subclass. In addition, serum glucose and 45 hepatic proteins were identified as potential regulators underlying the tissue-specific deposition of IMF and AF, including CA13, RCJMB04_17b10, SDE2, and the secreted protein C8G. These findings provide new insights into the coordinated regulation of body fat distribution by hepatic lipids and proteins, and offer a theoretical basis for molecular marker-assisted breeding of high-quality broilers with high IMF and low AF deposition.

Muscle skeletal embryonic nuclear protein 1 (MUSTN1) is a microprotein expressed and secreted by skeletal muscle satellite cells (SMSCs) and has long been implicated in muscle regeneration, yet its molecular mechanism remains unclear. This study demonstrates that MUSTN1 is transcriptionally regulated by MyoD1 and exerts its regenerative effects by inhibiting ferroptosis. Transcriptome analysis revealed that the overexpression of MUSTN1 leads to the enrichment of genes associated with ferroptosis. Mechanistically, MUSTN1 directly binds to ACO1 (IRP1), enhancing its interaction with the TFRC 3' untranslated region (UTR) of TFRC, thereby promoting TFRC expression and inhibiting SLC39A14, which ultimately alleviates iron accumulation and lipid peroxidation. Functional experiments confirmed that MUSTN1 mitigates dexamethasone-induced atrophy by enhancing myotube area, proliferation, and mitochondrial membrane potential. Additionally, MUSTN1 is secreted via exosomes, and treatment with exosomes containing MUSTN1 significantly promotes in vitro cell proliferation and differentiation while regulating cellular ferroptosis. In summary, our study reveals MUSTN1 as a MyoD1-driven, exosome-transmissible regulatory factor that inhibits ferroptosis by activating the ACO1-TFRC axis, providing mechanistic insights into muscle regeneration and potential therapeutic strategies for muscle atrophy-related diseases.

Intramuscular fat (IMF) improves meat quality, but the mechanisms underlying its deposition remain unclear. The lipid and flavor profiles of chicken breast muscles with high (H-IMF) and low (L-IMF) fat content were analyzed using targeted lipidomics and electronic nose technology. In H-IMF meat, triglycerides and phospholipids, particularly TAG52:0(16:0) and PC(18:2/18:2), were more abundant than L-IMF, while monoacylglycerols, primarily MAG(18:1) and MAG(18:2), were reduced. H-IMF meat exhibited improved water-holding capacity, tenderness, and flavor. The enhanced aroma profile was primarily attributed to aldehydes, ketones, alkanes, nitrides, and aromatic compounds. However, differences in aroma were lost after frozen storage, with H-IMF meat showing a more pronounced decline over prolonged storage. PE(18:0/20:5), PG(16:0/18:0), and CE(16:1) were significantly positively correlated with key aroma compounds, while MAG(18:2) and MAG(20:3) showed negative correlations. These findings offer novel insights into IMF deposition mechanisms, providing guidance for cold storage strategies tailored to meats with varying quality characteristics.

Managing fat accumulation is a critical goal in the poultry industry, with the liver being the primary site for lipid metabolism in chickens. This study used non-targeted metabolomics to investigate dynamic changes in metabolite composition in chicken liver across five physiological stages. A total of 1121 metabolites were identified, with 749 and 372 detected in positive- and negative-ion modes, respectively. The regulation of hepatic lipolysis involves numerous metabolic pathways, making it a complex process. We performed trend analysis of lipid, carbohydrate and amino acid-related metabolites. Age exerted major effects on hepatic metabolism, significantly enriching pathways including alpha-linolenic acid metabolism, linoleic acid metabolism, steroid hormone biosynthesis, 2-oxocarboxylic acid metabolism and nicotinate and nicotinamide metabolism. Transcriptomic analysis identified 12 lipid-metabolism-related genes, showing a tendency of continuously increase. Potential functional genes influencing lipid metabolism-related pathways were identified through a comprehensive analysis of differential lipid-related metabolites and genes, including fatty acid desaturase 2 (FADS2), acetyl-CoA acetyltransferase 2 (ACAT2), apovitellenin 1 (APOV1), vitellogenin 1 (VTG1), membrane-bound O-acyltransferase domain-containing protein 2 (MBOAT2) and ELOVL fatty acid elongase 1 (ELOVL1). These findings could help improve understanding of hepatic metabolism during different physiological stages and identify valuable biomarkers for specific metabolite accumulation.

High-fat diets (HFDs) are commonly used to modulate growth and meat quality in poultry; however, the duration- and muscle-specific effects remain unclear. In this study, Guangyuan Grey chickens were randomly assigned at day 150 into control, short-term HFD (SHFD), and long-term HFD (LHFD) groups (9 pens/treatment; 10 birds/pen). The HFD was formulated by increasing soybean oil from 1 % to 7 %, replacing corn and wheat bran. The pectoralis major (PEM) and soleus (SOL) muscles were evaluated for color, pH (15 min/24 h), drip/cooking loss, and shear force. Fatty acids were analyzed by GC-MS, volatiles by electronic nose, and untargeted metabolomics by LC-MS. LHFD resulted in decreased redness (a*), increased drip/cooking loss, and shear force (P < 0.05) with higher palmitic/stearic acids and aldehydes/sulfur volatiles. SHFD caused an increase in monounsaturated fatty acids and modestly enhanced aroma-related alcohols/ketones without significantly impairment in tenderness or water-holding capacity. Metabolomics indicated that amino acid metabolism and lipid-stress shifted in PEM and oxidative-stress was remodeled in SOL, implicating linoleic/unsaturated-fatty-acid pathways. These findings show that HFD effects are muscle-type and duration-dependent, with SHFD enhances flavor while LHFD leads to quality deterioration. The results provide a basis for optimizing diet fat level and duration strategies to improve poultry meat quality.

Understanding the biochemical basis of chicken meat flavor is important for improving meat quality. An integrated metabolomic, lipidomic, and transcriptomic approach was applied to compare breast (BM) and leg meat (LM) of 120-day-old Tianfu broilers. Compared with BM, LM showed higher pH values, a 43.05% lower drip loss, and markedly higher intramuscular fat (IMF) content (6.217% vs. 1.954%), whereas BM contained more inosine monophosphate and exhibited greater shear force. GC-MS and lipidomics identified 147 volatile compounds and 1972 lipid species, respectively, with glycerophospholipids, particularly phosphatidylethanolamine and phosphatidylcholine, closely associated with flavor differences between muscles. Multi-omics factor analysis indicated circ_015424 as a key molecular feature correlated with lipid metabolism and flavor-related pathways. Functional assays further showed that circ_015424 enhanced lipid droplet accumulation in intramuscular adipocytes via the miR-196-1-3p/MOGAT1 axis. These results clarify lipid-related mechanisms underlying muscle-specific flavor formation in chicken.

Tibial dyschondroplasia (TD) is a chronic cartilage disorder commonly found in fast-growing broiler chickens, characterized by impaired tibial development and subsequent growth retardation, which collectively compromise poultry health and production efficiency. Circular RNAs (circRNAs), a class of non-coding RNAs with covalently closed loop structures, have recently garnered increasing attention in biological research. Growing evidence suggests that circRNAs are involved in the fine-tuned regulation of TD in broiler through competitive endogenous RNA (ceRNA) networks. However, the role of circRNA in TD pathogenesis via protein-binding mechanisms remains unclear. In our previous study, we identified circCOG6 (circ_0002951), a circular RNA derived from the COG6 gene, as significantly upregulated in a Thiram-induced TD model, indicating its potential critical role in TD onset and progression. In vitro functional assays revealed that overexpression of circCOG6 suppresses proliferation and differentiation of TD chondrocytes and promotes apoptosis. Mechanistically, through AGO2-RIP, RNA pull-down, and RIP experiments, we demonstrated that circCOG6 interacts with activin receptor type-1-like (ACVRL1) to inhibit chondrocyte proliferation and differentiation, promote apoptosis, and synergistically enhance BMP/Smad signaling activation, thereby contributing to TD pathogenesis. Furthermore, in vivo studies showed that intra-articular injection of adeno-associated virus carrying shRNA targeting circCOG6 (AAV-sh-circCOG6) alleviates TD lesions in a broiler chicken. In conclusion, this study is the first to elucidate a circRNA-mediated regulatory mechanism in broiler TD via RBP-binding protein interaction, thereby enriching the TD regulatory network and offering a potential therapeutic target for its treatment.

Postnatal skeletal muscle growth in poultry relies on the precise orchestration of skeletal muscle satellite cell (SMSC) fate, yet the regulatory non-coding RNA networks governing this process remain partially veiled. Integrating small RNA sequencing with developmental expression profiling, we identified miR-6553-3p as a key differentially expressed regulator. Functional assays showed that miR-6553-3p markedly reduced SMSC proliferation while promoting myogenic differentiation. Specifically, miR-6553-3p significantly suppressed PAX7 expression at both the mRNA and protein levels, while simultaneously upregulating the mRNA and protein levels of MYOG and MYH1. Mechanistically, dual-luciferase assays confirmed that miR-6553-3p directly targeted Fas Apoptotic Inhibitory Molecule (FAIM). As an indispensable regulator of cellular homeostasis and cell fate decisions, FAIM is known to modulate key signaling cascades such as PI3K/AKT. In this study, we showed that FAIM normally sustained the proliferative pool of SMSCs; however, its repression by miR-6553-3p effectively attenuated AKT phosphorylation, linking this miRNA specifically to the PI3K/AKT pathway. By characterizing the miR-6553-3p-FAIM-AKT axis, our findings provide a molecular basis for fine-tuning muscle development and enhancing production traits during the critical early post-hatch window in the poultry industry.

High concentrations of reactive oxygen species (ROS) induce oxidative harm in laying chickens, leading to a drop in egg production efficiency. Curcumin alleviates oxidative stress-induced dysfunction by eliminating excess ROS. To investigate the underlying molecular mechanisms, we simulated oxidative stress and antioxidant processes in primary chicken granulosa cells. The results demonstrated that oxidative stress induced proliferation dysfunction in chicken granulosa cells, while curcumin treatment restored normal cell proliferation. Transcriptome sequencing identified a set of key genes. Among them, MHCY14, WFIKKN1, STARD10, and POU3F3 were significantly upregulated under oxidative stress and significantly downregulated during the antioxidant process. In contrast, TMEM45L, GH, C10orf71, ESPN, TRIM7.1, HEPHL1, SPRY3, SRD5A2, and VTCN1L exhibited the opposite expression pattern. Subsequent pathway analysis revealed that curcumin significantly inhibited the protein expression of AKT1, leading to a corresponding reduction in p-AKT1 levels. This was accompanied by a significant decrease in Raf1 phosphorylation and a significant increase in ERK1/2 phosphorylation. In conclusion, oxidative stress induced proliferation dysfunction in chicken granulosa cells, while curcumin alleviated this oxidative damage through the AKT-Raf1-ERK1/2 signaling pathway. These results deepen our insight into oxidative stress regulation mechanisms and may offer important theoretical guidance for optimizing antioxidant selection in poultry egg production.

For commercial laying hens, the continuous high-intensity ovulation process leads to a significant accumulation of reactive oxygen species (ROS) in the granulosa cells, inducing oxidative stress, which accelerates ovarian aging and shortens the peak laying period. The molecular mechanisms underlying this process remain poorly understood. Therefore, we modeled the processes of oxidative stress and antioxidant in chicken granulosa cells. Small RNA sequencing revealed that miR-15c-3p expression was elevated by oxidative stress induction and attenuated by antioxidant curcumin. Functional validation with miR-15c-3p mimic and inhibitor confirmed the role of miR-15c-3p in exacerbating oxidative stress and resultant suppression of lipid droplet storage and progesterone secretion in chicken granulosa cells by targeting insulin-like growth factor 2 binding protein 3 (IGF2BP3). These regulatory effects were mediated through the sequential downstream signaling cascade of AKT-Raf1-ERK1/2. In conclusion, IGF2BP3 curbed by miR-15c-3p restores disrupted lipid storage and progesterone secretion in chicken granulosa cells under oxidative stress through AKT-Raf1-ERK1/2 signaling pathway. These findings offer new insights into the molecular mechanisms by which oxidative stress damages reproductive capacity and a theoretical basis for mitigating oxidative stress in laying hens through genetic improvement.

In this study, we established and characterized a continuous cell line derived from the gill tissue of turbot ( Scophthalmus maximus) and assessed its applicability in studying the gill's function as a mucosal immune barrier. This cell line, designated SMG, has been successfully subcultured for up to 70 passages. The SMG cells exhibited robust growth in DMEM medium supplemented with 20 % FBS at 24 degrees C. Karyotype analysis revealed that the modal chromosome number of SMG cells was 44. Amplification of the 18S rRNA confirmed the turbot origin of the SMG cells. The cell line was transfected with a pEGFP-N1 plasmid and FAM labeled siRNA, resulting in high transfection efficiency and successful expression of the transfection reporter gene and FAM fluorescence signals 48 h post-transfection. Edwardsiella piscicida replicated in SMG cells at low concentrations without altering the morphology of the cells in the early stages of infection. These findings indicate the potential utilization of SMG cells in studying gene function and antimicrobial responses. This research provides a foundation for future studies on the immune barrier of gill mucosa in marine species.

Probiotics benefit the health and production performance of chickens, but their impact on egg and eggshell quality, particularly in the later stage, remains unclear. Here, 1-day-old Tianfu green shell-laying hens were fed either non-probiotics feed (n = 180) or feed supplemented with 100 mg / kg probiotics (n = 180). 16S rDNA sequencing indicated that dietary probiotics decreased the distribution of uterine p_Firmicutes, g_Fusobacterium, and s_Fusobacterium_unclassified, while increased p_Proteobacteria, g_Ralstonia, and s_Ralstonia_unclassified. PICRUSt2 and Bug Base analysis revealed enrichment in fatty acid metabolism, thiamine metabolism, vitamin B6 metabolism pathways and increased relative abundance of Proteobacteria, Firmicutes, Bacteroidetes. With LDA > 4.5, 35 and 25 marker bacterial taxa were identified in the uterus and cecum, respectively. Probiotics significantly increased uterine villi length and width, and the expressions of ATP2B2,SLC26A9,TF,OC-17,OCX-32, and OVAL in the uterus at the early and peak laying stage. Meantime, probiotics improved egg quality, pore density of eggshell barrier layer, and levels of Ca2+, Na+, and Mg2+, whereas dropped levels of P3-, S2- and K+ in eggshell. In serum, Ca2+, K+, Na+ had a response to dietary probiotics at different laying stages, except Cl-. Furthermore, the changes of these phenotypes are closely related to the microbial structure of the uterus and cecum. Overall, the data suggest that dietary probiotics improved uterine and cecal microbiota, optimized egg quality, eggshell quality, uterus development, and regulated mineralization gene expression and ion content in serum and eggshell, thereby improving productivity of laying hens. These results provide reference for the application of probiotics in the laying industry.

BACKGROUND: In poultry, the nutritional status of the body significantly impacts sexual maturation. The hypothalamus, a key neuroendocrine regulator of energy metabolism and sexual maturation, integrates peripheral nutritional signals to modulate reproductive function. However, the precise molecular mechanisms governing this integration remain elusive. This study aimed to investigate the influence of energetic status on hypothalamic function during sexual maturation in chickens. METHODS: In this study, chickens were supplied diets with different energy level. The hypothalamus of chickens at age of first egg and non-laying chickens at same age from different energy level groups were collected. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to determine the proteomics and metabolomics of hypothalamus. RESULTS: Dietary energy levels during the rearing period may modulate hypothalamic function of chicken by altering energy metabolism and amino acid metabolism, as evidenced by the differential expression of hexokinase 1 (HK1), dihydrolipoamide S-acetyltransferase (DLAT), malate dehydrogenase 1 (MDH1), and glutamate, valine, leucine. Furthermore, dietary energy levels alter hypothalamic signaling pathways, with significant differences observed in the proteins, including glutamate receptor 1 (GluR1), cholecystokinin (CCK), proopiomelanocortin (POMC), sphingosine-1-phosphate receptor 1 (S1PR1), relaxin-3 (RFLB) across different energy groups. Notably, GABA and its receptors in the hypothalamus may mediate the regulation of energy metabolism signals on the reproductive center. CONCLUSION: This study reports that the dietary energy level may affect the function of hypothalamus by altering energy metabolism, amino acid metabolism, and signal transduction. This study provides deeper insights into the impact of energy metabolism on sexual maturation, with implications for both animal husbandry and human medicine.

Lentinula edodes C91-3 is an edible mushroom with demonstrated medicinal activity against various types of cancer in vitro and in vivo.The gene for Latcripin-5 (LP-5) was obtained from Lentinula edodes strain C 91-3 and inserted into a pET32a (+) vector, which was then, expressed in the Rosetta gami (DE3) strain.The purified LP-5 protein was analyzed using SDS-PAGE and western blot.The optimal solubilization parameters were found to be 0.6 mM IPGT for 6 h incubation at 37℃.The solubilized protein was refolded using a refolding buffer and then dialyzed and concentrated using a dialysis buffer and PEG 20000, respectively.Phyre2 bioinformatics tool was used for protein modelling.The concentrated LP-5 protein was tested for its effect on the viability and cytotoxicity of various cancerous and non-cancerous cell lines using a cell counting kit-8 (CCK-8) assay.The LP-5 protein had the lowest IC50 value against the liver cancer line HepG2, at 58.15 μg/ml.Dose-and time-dependent morphological changes, such as cell shrinkage, blebbing formation, and cell fragmentation, were observed in treated cells.Apoptosis markers were evaluated using qPCR, flow cytometry, and western blot, and it was found that LP-5 protein increased the expression of Bax, Caspass-3, -8, -9, Cytochrome-C, and PARP, while decreasing the expression of Bcl2.Cell cycle arrest was also analyzed through qPCR, flow cytometry.Western blot results showed upregulated p21 and p27while CDK2, CDK4, CDK6, Cycline D1, and Cycline E1 were downregulated.These results suggest that LP-5 protein has potential as an anticancer agent against liver cancer cells.

Tibial dyschondroplasia (TD) is a prevalent skeletal disorder in the modern broiler poultry industry. MicroRNAs (miRNAs) regulate various biological processes. Our previous studies suggest miR-1737 plays a role in cartilage development, but its mechanism in TD remains unclear. This study found that miR-1737 promotes chondrocyte proliferation and differentiation in TD chickens (P < 0.05), accelerating disease progression. Bioinformatics analysis predicted transforming growth factor β-activated kinase 1 (TAK1) as a target gene of miR-1737, which was confirmed through luciferase assays, qRT-PCR, and rescue experiments. TAK1 was shown to inhibit chondrocyte proliferation and differentiation (P < 0.05). Additionally, miR-1737 markedly upregulated the mRNA expression levels of Smad 1/5/8, key components of the BMP-Smad signaling pathway, and enhanced their phosphorylation, whereas TAK1 exhibited opposing effects. In conclusion, miR-1737 promotes TD chondrocyte proliferation and differentiation in vitro by targeting TAK1 and activating the BMP-Smad signaling pathway. These findings enhance our understanding of TD and suggest new molecular targets for its prevention and treatment.

OBJECTIVE:Tibial dyschondroplasia (TD) is a metabolic disorder of cartilage that impairs the development of the tibial growth plate in rapidly growing poultry. This study aimed to identify key genes and clarify the molecular mechanisms involved in TD in broiler chickens. The study evaluated the potential effect of vitamin D3 (VD3) in alleviating TD symptoms, focusing particularly on the role of Bone morphogenetic protein 8A (BMP8A) and its interaction with transforming growth factor-β1 (TGF-β1). METHODS:Ninety-four broiler chicks were allocated into three groups: healthy control, thiram-induced TD, and thiram-induced with VD3 supplementation. RNA sequencing was performed to identify differentially expressed genes (DEGs) among the groups. Target genes underwent additional validated using molecular biology techniques, such as gene expression analysis and in vitro functional assays on chondrocytes. RESULTS:VD3 effectively mitigated chondrocyte damage induced by thiram. RNA-seq revealed 625 DEGs enriched in pathways such as the TGF-β signaling pathway. Four co-DEGs (BMP8A, COL10A1, SDC3, and SCIN) were closely associated with collagen metabolism and reorganization. Functional assays, such as CCK8, EdU and IHC showed that BMP8A reduced collagen accumulation induced by elevated TGF-β1 levels, promoted the release of collagen types I, II, and X, and facilitated chondrocyte proliferation and differentiation while reducing apoptosis. CONCLUSION:BMP8A plays a protective role in TD by the regulation of collagen balance and the maintenance of chondrocyte function, especially in the presence of high TGF-β1 levels. VD3 supplementation effectively reduces TD-related damage. The interaction between BMP8A and TGF-β1 may provide a novel therapeutic target for the prevention and treatment of TD in poultry.

The northern snakehead (Channa argus) is an important economic fish species that holds a significant position in Chinese aquaculture due to its high nutritional value, fast growth, and anti-hypoxia capacity. There is a natural albino-mutant (AM) C. argus strain. Albinism, a stable genetic trait characterized by loss of body pigmentation, provides a unique opportunity to study the molecular mechanisms of vertebrate coloration. This study investigates the molecular mechanisms underlying albinism in C. argus through skin transcriptomic analyses of wild type (WT) and AM individuals. Morphological and histological analysis revealed no significant phenotypic difference between WT and AM in early development, whereas adult AM exhibited a severe reduction in melanocytes. Through RNA sequencing of skin tissues from WT-adult, AM-adult, and AM-15dpf, a total of 10,891 differentially expressed genes (DEGs) were identified. Through DEG enrichment analyses, we identified a series of enriched pathways and genes related to albinism, including melanogenesis (i.e., tyr, tyrp1b, kitb, kitlga, pmela, pmelb, mitfa, and mitfb) and xanthophore formation (i.e., pax3a, pax3b, pax7a, and sox10). In conclusion, this study not only enriches the research on fish pigmentation, but also has potential significance for the aquaculture and breeding of C. argus.

The comb, as a secondary sexual characteristic in chickens, serves as an important indicator of sexual maturity and potential reproductive. This study explored the relationship between comb types and production performance in Tianfu G01 chickens. Single-comb hens exhibited significantly lower laying rates compared to rose-comb hens during specific weeks (24-26, 27-28, 30, 32-34, and 38-43) but had higher average egg weights at 22, 23, 33, and 36-43 weeks. There were no significant differences in initial egg weight or 300-day body weight between the comb types. However, rose-comb hens showed higher 300-day egg production and green-shell egg rates, while single-comb hens had better egg weight and survival rates. The uterine morphology of single-comb hens was superior, with longer and wider uterine villi. Blood analysis revealed higher calcitonin and parathyroid hormone levels in single-comb hens, while rose-comb hens had higher blood calcium and phosphorus levels. In roosters, single-comb had higher germ cell counts, thicker seminiferous epithelium, and better semen quality, leading to improved fertilization rates and hatchability. These findings suggest that comb type influences both reproductive and production traits in Tianfu G01 chickens, with rose-comb favoring egg production and single-comb favoring egg weight and survival.