Search Results (77)

Long non-coding RNAs (lncRNAs) regulate granulosa cell function, but their stage-specific dynamics across the chicken follicle hierarchy remain unclear under matched endocrine conditions. We performed Ribo-Zero RNA sequencing on granulosa cells from small yellow follicles (SYF), F5, F2, and F1 follicles collected at the preovulatory luteinizing hormone (LH) surge. A multi-predictor pipeline (CPC2, CNCI, CPAT, PfamScan) identified 26,923 stringently filtered lncRNAs together with 15,838 mRNAs. Consecutive stage comparisons detected 2094, 1085, and 4318 differentially expressed genes and 671, 267, and 2762 differentially expressed lncRNAs in F5 vs. SYF, F2 vs. F5, and F1 vs. F2, respectively, with the most extensive remodeling at the F2-to-F1 transition. The F1 vs. SYF contrast captured the cumulative transcriptional difference across the hierarchy. Enrichment and temporal clustering showed that early hierarchical stages were characterized by proliferative, metabolic, and steroidogenic programs, whereas F1 granulosa cells were enriched for extracellular-matrix remodeling, MAPK signaling, and calcium ion binding. Weighted gene co-expression network analysis identified 10 stage-associated modules and highlighted candidate lncRNAs linked to lipid metabolism, angiogenesis, extracellular-matrix remodeling, and DNA repair, including G5825–MYLIP, G66587–VEGFA, and G60212–CKS1B. qPCR validation confirmed concordant expression trends for eight representative pairs. These results define a stage-resolved lncRNA–mRNA landscape across chicken follicle development and provide candidates for mechanistic studies of follicle maturation and periovulatory remodeling. Full article

As a representative form of extreme weather, typhoons inflict widespread and systemic damage, posing a severe threat to the livestock industry. The stress they induce, typhoon stress (TS), is an unavoidable and complex environmental challenge that severely disrupts the ovarian function of Wenchang chickens. In this preliminary study, we employed a two-group comparison design (n = 6 per group) integrating behavioral observations, serum biochemical assays, histopathological examinations, and molecular analyses (qPCR, 16S rDNA sequencing, and transcriptome sequencing) to explore the role of the microbiota–gut–brain–ovarian axis (MGBOA) in this process. The findings revealed that TS markedly reduced water intake and locomotor activity, while it elevated serum corticosterone (CORT) and oxidative stress markers. It also induced shifts in gut microbiota composition, including a decrease in Bacteroides and an increase in Escherichia–Shigella. Furthermore, TS compromises duodenal intestinal barrier integrity, as evidenced by downregulation of the tight junction proteins TJP1 and CLDN1, structural damage to intestinal villi, and a reduced villus-to-crypt ratio. In the hypothalamus, VIP mRNA expression was upregulated, while GHSR expression was downregulated; the expression of the tight junction protein CLDN5 was also reduced. In the ovary, reproductive potential was suppressed, manifested by a reduction in follicle number and downregulation of STAR expression. Ovarian transcriptome analysis highlighted enrichments in pathways associated with inflammation (e.g., Toll-like receptor signaling) and lipid metabolism (e.g., PPAR signaling). These results support the hypothesis that TS impairs egg production via the MGBOA, providing preliminary mechanistic insights into how environmental stressors might disrupt animal productivity through MGBOA-mediated pathways. Full article

The use of colored LED lights is a tool for controlling the development of lymphoid organs and the immune system in general. This study aims to analyze the effects of using simple and combined colored LED lights throughout a 6 week period (1–42 days of age). In this study, 336 one-day-old chicks were used, separated randomly into four groups with different sex and lighting systems, with each group being divided into four separate replicates (4 × 21 birds). The chicks in the WL-Male and WL-Female were exposed to white LED light (WL, 400–760 nm) for 6 weeks, while the chicks in the G-GxB-BL-Male and G-GxB-BL-Female were exposed to a combination of monochromatic lights as follows: green (560 nm) from 1 to 14 days of age, green and blue (480–560 nm) for 15–28 days of age, and blue lights (480 nm) for 29–42 days of age. The use of a mixture of green and blue LED lights (G-GxB-BL) resulted in a significant decrease in the average daily feed intake and feed conversion ratio compared to white light, without causing changes in the body weight of the chicks, average daily gain, mortality rate, and coefficient of variability. G-GxB-BL lights also improved the morphological development of the bursa of Fabricius (BF) compared to white light by significantly increasing the organ index and the lymphoid follicle area. At the same time, G-GxB-BL light compared to white light improved B lymphocytes proliferation in the BF by significantly increasing the lymphocyte density in lymphoid follicles, as well as the number of PCNA-positive cells. This light treatment had these results due to the activation of melatonin receptors, which led to a significant increase in Mel1a-positive cells and a significant decrease in the number of RORα-positive cells. These results demonstrate that G-GxB-BL lights improved the growth performance and immune response in the BF of broiler chickens. Full article

The sustainability of egg production in the poultry industry is frequently challenged by the progressive decline in ovarian function as laying chickens age. A primary driver of this reproductive transition is the functional deterioration of small white follicles (SWFs), which constitute the vital pre-hierarchical follicular reserve necessary for sustained egg production. However, the molecular mechanisms underlying age-associated SWF atresia remain poorly understood. In this study, we investigated the protective efficacy of apigenin (AP), a natural bioactive flavonoid, in mitigating follicular senescence by targeting calcium ion (Ca²⁺)-mediated endoplasmic reticulum stress (ERS) in D-galactose (D-gal)-induced SWFs and in naturally aged chickens. Our results revealed that AP treatment effectively rebalanced the D-gal-induced disruption of cell proliferation and survival. Molecular analysis of SWFs revealed that AP treatment promoted the coordinated restoration of transcriptional profiles of key Ca²⁺-handling genes, effectively counteracting the age-related disruption of ionic regulation. In addition, AP suppressed the aberrant upregulation of IP₃R and modulated the expression of other key Ca²⁺-regulatory genes, including CACNA1C, CACNA1D, CAMKII, MCU, and ATP2B1. This restoration of intracellular Ca²⁺ homeostasis was associated with attenuation of the ERS response, as evidenced by the decreased levels of GRP78 and CHOP, and the suppression of Caspase-3-mediated apoptotic signaling. The biological relevance of these findings was further validated in vivo using naturally aged chickens. Dietary supplementation with AP significantly enhanced pre-hierarchical follicle recruitment in aged laying chickens, and improved egg production and eggshell quality in aged laying chickens. Collectively, these findings indicate that AP can modulate ERS signaling in laying chickens by maintaining intracellular Ca²⁺ homeostasis, thereby enhancing laying performance. These results highlight AP as a promising nutritional intervention to enhance reproductive performance and extend productive longevity in poultry. Full article

Background: Broodiness is a major limiting factor for reproductive efficiency in indigenous avian breeds, a phenomenon underpinned physiologically by granulosa cell (GC) apoptosis and subsequent follicular atresia. While Serine Protease 23 (PRSS23) has been implicated in mammalian ovarian remodeling, its specific regulatory function in avian follicular dynamics remains elusive. Methods: Utilizing the Wuding chicken—an indigenous breed distinguished by robust environmental adaptability but compromised by high broodiness frequency—as a biological model, this study dissected the molecular mechanism of PRSS23-mediated follicular regression. We cloned the complete coding sequence of the Wuding chicken PRSS23 gene, characterized its spatiotemporal expression profile, and interrogated its function in primary GCs via gain- and loss-of-function assays. Results: RT-qPCR analysis revealed that PRSS23 is differentially expressed across the hypothalamic–pituitary–ovarian (HPO) axis, with ovarian expression being significantly upregulated during the broody period compared to the laying period. Mechanistically, PRSS23 overexpression significantly downregulated the expression of follicle-stimulating hormone receptor (FSHR) and key steroidogenic enzymes (STAR, CYP19A1, HSD3β1), thereby suppressing the expression of genes governing the biosynthesis potential of progesterone and estradiol. Concurrently, PRSS23 overexpression was associated with transcriptional repression of components of the PI3K/AKT/mTOR signaling cascade; this transcriptional regulation further induced cell cycle arrest at the G0/G1 phase, and activated the mitochondrial apoptotic pathway characterized by BAX upregulation and BCL2 downregulation. Conversely, siRNA-mediated knockdown of PRSS23 alleviated these inhibitory effects, promoting GC proliferation and survival. Conclusions: These findings establish PRSS23 as a pivotal pro-atretic factor in Wuding chickens, driving ovarian atrophy through the dual transcriptional-level inhibition of steroidogenesis and survival signaling pathways. This study identifies a potential molecular target for marker-assisted selection programs aimed at attenuating broodiness while preserving the superior meat quality traits of indigenous poultry. Full article

Ovarian follicular development determines the egg-laying performance in chickens. Besides hormonal signaling, epigenetic and post-transcriptional regulators, long non-coding RNAs (lncRNAs) also play a vital role in follicular development. We previously identified that RAR-related orphan receptor B-intronic transcript 1 (RORB-IT1), a novel lncRNA located in the intron of RORB, was differentially expressed in chicken pre-hierarchical and hierarchical follicular granulosa cells (Post-GCs). However, it remains unknown whether RORB-IT1 participates in regulating the development of chicken ovarian follicles. In this study, we further characterized the expression pattern of RORB-IT1 and explored its role in regulating the progesterone synthesis, proliferation and apoptosis of chicken Post-GCs. The results showed that RORB-IT1, with a full length of 383 bp, exhibits a uniform distribution in both the cytoplasm and nucleus of chicken Post-GCs. RORB-IT1 was specifically expressed in Post-GCs and upregulated by follicle-stimulating hormone (FSH), progesterone (P4) and estradiol (E2) in a dose-dependent manner. Functionally, RORB-IT1 promoted P4 synthesis and proliferation, while inhibiting the apoptosis of Post-GCs. Furthermore, we demonstrated that RORB-IT1 encoded a functional micropeptide exhibiting dual localization in both cytoplasmic and nuclear compartments. This micropeptide enhanced progesterone synthesis and proliferation, but paradoxically induced the apoptosis of Post-GCs when overexpressed independently. Collectively, this study uncovered the expression pattern and function of RORB-IT1 in chicken Post-GCs and provided a theoretical basis for improving the egg-laying performance in chickens. Full article

Objectives: Fibroblast growth factor 9 (FGF9), a crucial member of the FGF family, functions as an intercellular signaling molecule involved in angiogenesis, embryogenesis, and tissue repair. Our previous study demonstrated that FGF9 expression in chicken hierarchical granulosa cells (Post-GCs) is regulated by LSD1 Ser54 phosphorylation and that FGF9 promotes cell proliferation. This study aims to analyze the expression and regulation of the FGF9 gene in chicken ovarian follicles and its genetic effect on laying traits in hens. Methods: Chicken FGF9 mRNA expression patterns were examined by real-time quantitative PCR (RT-qPCR). Detection of single nucleotide polymorphisms (SNPs) was performed using PCR amplification and Sanger sequencing. Transcription activity was compared using dual-luciferase reporter assay. Results: Following follicle selection, chicken FGF9 expression significantly decreased in granulosa cells (p < 0.05) while it increased in theca cells (p < 0.05). Hormonal treatments revealed differential regulation; estradiol and FSH downregulated FGF9 in both pre-hierarchical and hierarchical granulosa cells (p < 0.05), whereas progesterone exhibited opposing effects, suppressing expression in pre-hierarchical granulosa cells (Pre-GCs) but stimulating its expression in Post-GCs (p < 0.05). In theca cells, estradiol consistently inhibited FGF9 expression (p < 0.05), while FSH only affected FGF9 expression in pre-hierarchical follicles. Six SNPs in the promoter region (g.−1965G>A, g.−2177G>A, g.−2289G>A, g.−3669A>G, g.−3770A>G, g.−3906G>A) were identified, five of which (g.−1965G>A, g.−2177G>A, g.−2289G>A, g.−3669A>G, g.−3906G>A) showed significant associations with egg production traits. Notably, alleles A (g.−2289), G (g.−3669), and A (g.−3906) enhanced the transcription activity of chicken FGF9 in Pre-GCs. Conclusions: These findings provide novel insights into the expression pattern and regulatory mechanisms of chicken FGF9 during follicular development and identify some genetic markers for egg-laying traits in chickens. Full article

Follicle selection is a pivotal process that determines which dominant prehierarchical follicle will enter the preovulatory hierarchy in the hen ovary and directly affects egg-laying productivity, in which granulosa cells (GCs) are characterized by active proliferation and significantly enhanced FSHR mRNA expression. Increasing evidence has shown that the PI3K/Akt signaling pathway and its important target and effector FOXO1, which promotes GC apoptosis, play crucial roles in ovarian follicular development in mammals. To investigate the molecular mechanism by which follicle-stimulating hormone (FSH)-mediated forkhead box O1 (FOXO1) participates in follicle selection, we treated granulosa cells from 6–8 mm prehierarchical follicles of chickens with FSH and leptomycin B (LMB). The results showed that under FSH and/or LMB treatment, the expression levels of FSHR, FOXO1, and its phosphorylated forms (p-FOXO1) at the predicted protein kinase B (PKB/Akt) phosphorylation sites Thr²⁴, Ser²⁴⁸, and Ser³¹¹ were differentially regulated. The subcellular localization of p-FOXO1 in hen ovarian GCs was determined by Western blotting and immunofluorescence staining (IF) analysis. And the expression of FOXO1 was significantly reduced, whereas the expression of p-FOXO1 corresponding to the PKB phosphorylation sites Ser²⁴⁸ and Ser³¹¹ was noticeably boosted in cultured GCs induced by FSH, accompanied by exclusion of FOXO1 from the nucleus to the cytoplasm. Subsequently, the effects of the PI3K/Akt signaling pathway on phosphorylation levels and nuclear exclusion of p-FOXO1 at the sites Ser²⁴⁸ and Ser³¹¹ were examined. The results indicate that the PI3K/Akt-dependent phosphorylation at these sites directly resulted in nuclear exclusion of FOXO1 in ovarian GCs, in which the Ser²⁴⁸ site is more essential than the Ser³¹¹ site. Subsequently, the FSH-induced acetylation of FOXO1 mediated by the cAMP/PKA pathway can enhance the phosphorylation level of FOXO1 at the Ser²⁴⁸ site. In summary, our findings demonstrate that FSH induces FOXO1 phosphorylation, nuclear exclusion, and functional inactivation by activating the PI3K/Akt signaling pathway. Moreover, during follicular development and selection, FOXO1 acts as a pivotal mediator linking the PI3K/Akt and P62/Keap1/Nrf2 signaling pathways to regulate granulosa cell proliferation and apoptosis, thereby exerting a central regulatory role. Full article

Corticosterone (CORT) is the primary avian stress hormone and regulates key physiological processes, but chronically elevated levels can be detrimental. This study simulated chronic stress by exogenously increasing CORT to assess effects on gait score, bone morphometry, immune indicators, and fluctuating asymmetry (FA) in broiler chickens. A total of 60 one-day-old male Ross 308 broiler chickens were allocated to six treatment groups (T0–T5; n = 10 per group). T0 received a placebo and served as the control group, while T1–T5 were gavaged daily with increasing corticosterone doses (1–5 mg/kg body weight). Treatments were administered from day 1 to day 42. CORT did not affect gait score on day 11 (p = 0.232) or day 42 (p = 0.112), but gait score was significantly correlated with body weight (day 11: p < 0.001; day 42: p = 0.004). Elevated CORT reduced bursa diameter (p < 0.001) and increased bursa follicle scores (p < 0.001), while heterophil-to-lymphocyte ratios remained unaffected (p = 0.349). Carcass and bone dimensions decreased consistently with higher doses (p < 0.001), and body weight correlated positively with all bone traits (p < 0.001). Length-to-width ratios increased with CORT dose (p < 0.001). Composite carcass FA showed dose-related variation in carcass asymmetry, with highest in T4, intermediate in T2, T3, and T5, and lowest in T0–T1 (p = 0.026). Trait-specific FA effects were detected for metatarsus length (p = 0.005) and wattle (p = 0.018), while bone FA remained unaffected (p = 0.272). Overall, exogenously increasing CORT impaired growth, suppressed immune function, and increased fluctuating asymmetry, indicating reduced developmental stability. Full article

This study investigates the regulatory role of circFBN1 in chicken follicular granulosa cells (GCs) and its underlying molecular mechanisms through the miR-206/E2F5 pathway. circFBN1 was found to significantly enhance GC proliferation and inhibit apoptosis, as evidenced by increased expression of proliferation-related genes (PCNA, CDK1, and CCND1) and decreased expression of apoptosis-related genes (Caspase-3). Additionally, circFBN1 overexpression promoted the secretion of estradiol (E2) and progesterone (P4) by upregulating steroidogenesis-related genes (StAR and CYP11A1). Mechanistic studies revealed that circFBN1 functions as a molecular sponge for miR-206, thereby alleviating its inhibitory effect on the target gene E2F5. Dual-luciferase reporter assays confirmed the specific binding between circFBN1 and miR-206. Overexpression of miR-206 had the opposite effects, inhibiting GC proliferation, inducing apoptosis, and reducing E2 and P4 secretion by downregulating StAR and CYP11A1. Furthermore, E2F5 was identified as a direct target of miR-206, and its knockdown significantly reduced GC proliferation, increased apoptosis, and decreased steroid hormone secretion. These findings elucidate the regulatory mechanisms of the circFBN1/miR-206/E2F5 axis in avian follicle development and provide potential molecular targets for improving poultry reproductive performance. Future research should focus on exploring the upstream regulators of this axis and its interactions with other signaling pathways. Full article

Oxidative stress-mediated dysfunction of granulosa cells (GCs) is recognized as a pivotal driver of prehierarchical follicular atresia in poultry, contributing substantially to the reduced egg production in aged laying hens. Here, we investigated the protective effects of the natural flavonol, fisetin, on aged chicken follicular GCs. A D-galactose (D-gal)-induced aging model of GCs was established to evaluate the protective role of fisetin against cellular senescence. Small yellow follicles (SYFs) from 580-day-old hens were cultured with fisetin for 72 h to verify its ameliorative effect on naturally aged follicles. Fisetin reduced the typical characteristic of senescence in D-gal-induced GCs, as reflected by decreased senescence-associated β-galactosidase (SA-β-gal) activity and increased expression of proliferation-related proteins, including cyclin D1 (CCND1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 1 (CDK1), and Cyclin B1. Furthermore, fisetin enhanced the activity of antioxidant enzymes by activating the Nrf2/HO-1 signaling pathways, while attenuating mitochondrial dysfunction and promoting ATP production in senescent GCs. Additionally, fisetin significantly promoted nuclear translocation of β-catenin, and suppressed the expression of senescence marker proteins p53 and p21, thereby alleviating cell cycle arrest in D-gal-induced senescent GCs. Simultaneous inhibition of Nrf2/HO-1 and β-catenin signaling also abolished the beneficial effects of fisetin on oxidative stress and cell proliferation in naturally senescent follicles. These findings indicate that fisetin prevents follicular atresia by suppressing GCs oxidative damage and improving cell cycle arrest via activating the Nrf2/HO-1 and Wnt/β-catenin signaling pathways. Full article

In poultry production, the laying rate is a critical economic trait, as high egg production significantly enhances profitability. Mitogen-activated protein kinase-activated protein kinase 3 (MK3) is a member of the mitogen-activated protein kinase (MAPK) family, which plays an important role in follicular development. Our previous RNA-seq analysis revealed that MK3 expression was significantly altered in the ovaries of laying hens exposed to normal versus light-deprivation conditions. Based on previous RNA-seq analysis of chicken ovaries, this study focused on the MK3 gene to explore its role in regulating apoptosis of follicular granulosa cells in laying hens. The results demonstrated that MK3 overexpression induced granulosa cell apoptosis by modulating the expression of key proliferation- and apoptosis-related genes, including FAS, Caspase3, BCL2, and C-myc. These findings were further validated using specific siRNA-mediated knockdown of MK3. Flow cytometry, CCK-8, and EdU assays consistently showed that MK3 facilitated apoptosis and inhibited granulosa cell proliferation. Additionally, dual-luciferase reporter assays revealed that the transcription factor WT1 bound to the MK3 promoter and enhanced its transcriptional activity. Mechanistically, MK3 regulated granulosa cell apoptosis through the TNF/P38 MAPK pathway. This conclusion was corroborated by treatment with the P38 inhibitor GS-444217 and specific siRNA targeting components of the pathway. In summary, MK3 promotes granulosa cell apoptosis in the follicles of laying hens, is transcriptionally regulated by WT1, and exerts its pro-apoptotic effects via the TNF/P38 MAPK pathway. Full article

Given the increasing global concern over antimicrobial resistance in poultry health, this study investigated the potential of Javanese cardamom essential oil (JCEO) and dried turmeric (DT) as alternative therapies for colibacillosis by evaluating their effects on the spleen organ. A total of 72 Cobb-strain day-old chickens were allocated into eight groups, which received different doses of JCEO (0.06 mL/kg BW and 0.1 mL/kg BW), DT (400 mg/kg feed) and ciprofloxacin. Infection was induced intraperitoneally (E. coli O78 strain, 10⁶ CFU/mL/chicken) at 28 days, while the treatments were administered orally from day 7 to week 5. Histopathological evaluation was graded on a 1–5 scale based on the five primary lesion criteria. The herb combination groups had the lowest severity grade, characterized by compact lymphoid follicles and reduced vasculitis. The JCEO single-dose group, both in vitro and in vivo, reduced bacterial growth and had a mild vasculitis score, indicating its antibacterial activity. In contrast, the single treatment of DT and the antibiotic groups showed moderate spleen lesion damage. These findings suggest that JCEO acts bactericidally, while DT provides an anti-inflammatory effect, and both combinations work synergistically. Full article

The evolution of the chilled processing technology has precipitated the emergence of ice-fresh poultry meat as a significant sales channel. The aesthetic appearance of chicken carcasses has become increasingly important in the context of poultry ice-fresh sales, in conjunction with the comprehensive implementation of China’s policies for poultry. Feather follicle development is a significant factor in determining the aesthetic appearance of the carcass. Recent studies have focused on the molecular mechanisms associated with feather follicle development. The WNT, EGF, FGF, SHH, and BMP signalling pathways have been identified as the regulatory mechanisms involved in the development of feather follicles in various segments of poultry skin. However, the BMP signalling pathway, acting as an inhibitor, has been demonstrated to impede the regulatory processes governing feather follicle development via these signalling pathways. This review summarises the structure and overview of feathers and feather follicles, the research progress of signalling pathways that affect the development of poultry feather follicles, the research progress of poultry follicle traits, and the research progress of feather follicle development biotechnology. The present review focuses on summarising the molecular mechanisms that affect feather follicle development, and on providing a summary of the application of biotechnology in this field. It also offers ideas and theoretical references for the molecular mechanism of poultry feather follicle development. Full article

The objective of the study was to investigate the effects of low-protein amino acid-balanced (LPAB) diets supplemented with Astragalus polysaccharides (APSs) on the production performance, antioxidants, immunity, and biochemical index of laying hens in an elevated-temperature environment. Fifty-two-week-old Hy-Line Brown chickens (n = 768) were randomly divided into four groups, with eight replicates of 24 hens each. The control group was kept at 24 °C with a basal diet (CON), while the treatment groups were exposed to 32 °C and given the following diets: basal (HB), LPAB (HL), and LPAB with 0.5% APSs (HLA). Under heat stress, APSs increased the egg production rate and number of small white follicles, improved the yolk color, and lowered the feed conversion ratio. LPAB diets increased follicle-stimulating hormone, antioxidant enzyme activities, and anti-inflammatory cytokine activity and up-regulated related genes, whereas they reduced stress-related hormones, malondialdehyde concentrations, and triglyceride concentrations and down-regulated related genes. The addition of APSs enhanced immunoglobulin concentrations and cholesterol recovery and altered the expression of related genes. The study found that the adverse effects of high temperatures are directly related to oxidative stress. LAPB diets and APSs relatively alleviate these adverse effects. Therefore, the importance of feeding strategies such as LPAB diets and APSs for laying hens under heat stress conditions has been identified. Full article