Cells In Chickens Research Articles

Influence and Optimization of Diverse Culture Systems on Chicken Embryonic Stem Cell Culture

Background: The importance of embryonic stem cells (ESCs) in chickens is undeniable, as they can be applied across various fields, including animal modeling, developmental biology, cell fate research, drug screening, toxicity testing, and gene function studies. However, a widely applicable culture system for chicken ESCs has yet to be developed. Objectives: This study aimed to investigate the effects of different culture systems on the derivation and maintenance of chicken ESCs, with a focus on optimizing the selected culture conditions. Methods: To achieve this, we tested the effectiveness of various species-specific ESC media in the derivation and culture of chicken PGCs, while incorporating different small molecule compounds to optimize the process. The pluripotency and differentiation potential of the resulting ESC-like cells were also evaluated. Results: The combination of PD0325901, SB431542, and LIF (R2i+LIF system) was found to be effective in generating chicken ESC-like clones. Further experiments showed that enhancing the R2i+LIF system with cytokines such as SCF and FGF2 significantly extended the culture period and increased the passage number of chicken ESC-like cells. These ESC-like cells were characterized through positive alkaline phosphatase staining and the expression of pluripotency markers POUV, NANOG, and SOX2. Additionally, differentiation assays confirmed their ability to form the three germ layers. Conclusions: The newly developed culture system provides suitable conditions for the short-term culture of chicken ESCs. However, further optimization is required to establish a system that can sustain long-term maintenance.

Single-cell RNA sequencing reveals surface markers of primordial germ cells in chicken and zebra finch.

Primordial germ cells (PGCs) in avian species exhibit unique developmental features, including the ability to migrate through the bloodstream and colonize the gonads, allowing their isolation at various developmental stages. Several methods have been developed for the isolation of avian PGCs, including density gradient centrifugation, size-dependent separation, and magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) using a stage-specific embryonic antigen-1 (SSEA-1) antibody. However, these methods present limitations in terms of efficiency and applicability across development stages. In particular, the specificity of SSEA-1 decreases in later developmental stages. Furthermore, surface markers that can be utilized for isolating or utilizing PGCs are lacking for wild birds, including zebra finches, and endangered avian species. To address this, we used single-cell RNA sequencing (scRNA-seq) to uncover novel PGC-specific surface markers in chicken and zebra finch. We screened for genes that were primarily expressed in the PGC population within the gonadal cells. Analyses of gene expression patterns and levels based on scRNA-seq, coupled with validation by RT-PCR, identified NEGR1 and SLC34A2 as novel PGC-specific surface markers in chickens and ESYT3 in zebra finches. Notably, these newly identified genes exhibited sustained expression not only during later developmental stages but also in reproductive tissues.

Phage LysSA163-CBD mediated specific recognition coupled with ATP bioluminescence for the sensitive detection of viable Staphylococcus aureus in food matrices

BackgroundStaphylococcus aureus is a significant foodborne pathogen, commonly detected in milk and meat products. Conventional detection methods have limited sensitivity and specificity, which are time-consuming and susceptible to background interference from complex samples, and cannot effectively distinguish live and dead bacteria. ResultsHerein, we developed a novel adenosine triphosphate (ATP) bioluminescence method coupled with magnetic separation, which is based on phage-encoded endolysin LysSA163-CBD (CBD 163) for rapid and specific detection of viable Staphylococcus aureus. The expressed protein (CBD 163) was derived from the phage LSA2301 and was successfully expressed in Escherichia coli BL21 following an induction period of 4 h at 37 °C, with a molecular weight approximating 40 kDa. The optimal conditions for CBD-magnetic beads (cMBs) to capture S. aureus cells were determined to be 100 μL/mL cMBs at 25 °C for 30 min. The viable S. aureus cells were disrupted by the Cetyl trimethyl ammonium bromide (CTAB) to release intracellular ATP. Then, the ATP reacted with the firefly luciferase and D-Luciferin-based bioluminescence (BL) reagents solution to generate intensive BL signal. The CBD-magnetic separation-ATP bioluminescence (cMS-BL) assay was able to quickly detect viable S. aureus via ATP bioluminescence in 45 min, with a detection range from 5 × 103 to 5 × 107 CFU/mL and limit of detection (LOD) of 190 CFU/mL. Additionally, the cMS-BL method exhibited high specificity and anti-interference ability, which has been successfully applied to quantify S. aureus cells in crayfish-tail, chicken, and skim milk. Significance and noveltyThese results demonstrate the potential of CBD 163 as a versatile and robust biorecognition element for rapid and specific detection of viable S. aureus in food matrices. The proposed phage-derived bacteria-binding proteins-based protocol for BL detection shows various advantages, including high sensitivity, simple operation, and the capability to distinguish live bacteria, providing a strategy for designing high-quality biorecognition element toward foodborne pathogens.

Ultrastructural changes in the spermatogenic cells of domestic chicken (Gallus gallus domesticus) observed at different reproductive stages

Spermatogenesis is a complex process. It is the modification of progenitor spermatogonia into mature spermatozoa. The stages are similar in all-male vertebrates, as well as avian species. However, studies on spermatogenesis in birds are fewer compared to mammals. The current study investigated the ultrastructural changes in the spermatogenic cells of domestic chickens in different reproductive stages. Thirty (30) male birds, ten (10) in each of the three reproductive stages: pre-pubertal, pubertal, and adult were used in the study. Testicular tissues from all age groups were processed for transmission electron microscopy (TEM). TEM results showed spermatogonia and primary spermatocytes in the pre-pubertal testis, and the seminiferous tubule lumen was wide and empty. Also, the nuclei of spermatogonia at this stage did not contain condensed chromatin material at the center nor scattered at the periphery of the nuclear membrane. There were slight differences between the spermatogenic cells in the pubertal and adult age groups. The spermatogonia, primary and secondary spermatocytes, and round spermatids with scanty chromatin material were observed in both age groups. In the adult age group, round and elongated spermatids with condensed chromatin materials were observed besides the other spermatogenic cells. Also, the seminiferous tubule lumen was filled with sperm cells and cellular debris, unlike in the pre-pubertal and pubertal age groups where they were wide and empty. The presence of numerous oval mitochondria were observed in all age groups. This signifies the active process of spermatogenesis in pre-pubertal, pubertal, and adult male domestic chickens.

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 (FSHβ, LHβ, GnRHR, TSHβ, 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.

Alarming and calming: Dual functions of S100A9 on Mycoplasma gallisepticun infection in avian cells

Mycoplasma gallisepticum (MG) is the primary causative agent of chronic respiratory disease (CRD) in chickens, characterized by respiratory inflammation. S100A9 plays a pivotal role in modulating the inflammatory response to microbial pathogens. Our prior investigation revealed a significant upregulation of S100A9 in the lungs of chickens following MG infection. This study delves into the immunomodulatory effects of S100A9 during MG infection, demonstrating a notable increase in S100A9 levels in the lungs, immune organs, alveolar epithelial type II cells (AECII), and macrophage HD11 cells of MG-infected chicks and embryos. In MG-infected AECII cells, S100A9 overexpression significantly enhanced MG proliferation and adhesion, suppressed AVBD1, NFκB, pro-inflammatory factors (IL1β and TNFα), and chemokines, reduced apoptosis, and promoted cell proliferation, thereby facilitating MG infection. Conversely, inhibiting S100A9 produced opposing effects. In MG-infected HD11 cells, S100A9 impeded MG proliferation and adhesion, increased AVBD1, NFκB, pro-inflammatory factors, and chemokines, and induced cell apoptosis while inhibiting proliferation. Additional results demonstrated that S100A9 facilitates MG infection by modulating the TLR7/NFκB/JAK/STAT pathway in AECII/HD11 cells. In summary, S100A9 exhibits a dual role in activating/inhibiting the natural immune response through TLR7/NFκB/JAK/STAT pathway regulation. This dual role promotes MG infection in AECII cells while enabling MG to evade immune surveillance by HD11 cells, ultimately enhancing the overall infection process. These findings advance our understanding of host-pathogen interactions during MG infection and underscore S100A9's potential as a therapeutic target for CRD in chickens.

Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes deadly lymphomas in chickens. In chickens, up to 50% of all peripheral T cells are gamma delta (γδ) T cells. Until now, their role in MDV pathogenesis and tumor formation remains poorly understood. To investigate the role of γδ T cells in MDV pathogenesis, we infected recently generated γδ T cell knockout chickens with very virulent MDV. Strikingly, disease and tumor incidence were highly increased in the absence of γδ T cells, indicating that γδ T cells play an important role in the immune response against MDV. In the absence of γδ T cells, virus replication was drastically increased in the thymus and spleen, which are potential sites of T cell transformation. Taken together, our data provide the first evidence that γδ T cells play an important role in the pathogenesis and tumor formation of this highly oncogenic herpesvirus.IMPORTANCEGamma delta (γδ) T cells are the most abundant T cells in chickens, but their role in fighting pathogens remains poorly understood. Marek's disease virus (MDV) is an important veterinary pathogen, that causes one of the most frequent cancers in animals and is used as a model for virus-induced tumor formation. Our study revealed that γδ T cells play a crucial role in combating MDV, as disease and tumor incidence drastically increased in the absence of these cells. γδ T cells restricted virus replication in the key lymphoid organs, thereby decreasing the likelihood of causing tumors and disease. This study provides novel insights into the role of γδ T cells in the pathogenesis of this highly oncogenic virus.

Study on the effects of toosendanin against Eimeria tenella and its impact on the apoptosis of cecal cells in chicks

To investigate the therapeutic effect of toosendanin (TSN) against Eimeria tenella (E. tenella) in chicks. In this experiment, a chick model of artificially induced E. tenella infection was established. The anti-coccidial effect was investigated by treating different doses of TSN. A preliminary mechanism of action was conducted, using cecal cell apoptosis as a starting point. TSN at the concentration of 5 mg/kg BW showed the best effect against E. tenella with the ACI value of 164.35. In addition, TSN reduced pathological damage to cecal tissue, increased the secretion of glycogen and mucus in cecal mucosa, and enhanced the mucosal protective effect. It also elevated the levels of IFN-γ, IL-2, and IgG in serum, and raised the sIgA content in cecal tissue of infected chicks, thereby improving overall immune function. TSN was observed to promote the apoptosis of cecum tissue cells by TUNEL staining analysis. Immunohistochemistry analysis revealed that in TSN-treated groups, the expression of Caspase-3 and Bax was elevated, while the expression of Bcl-2 was reduced. TSN induced apoptosis in host cells by dose-dependently decreasing the Bcl-2/Bax ratio and upregulating Caspase-3 expression. In summary, TSN exhibited significant anticoccidial efficacy by facilitating apoptosis in host cecal cells, with the most pronounced effect observed at a dosage of 5 mg/kg body weight.

The development of chicken cerebellar cortex and the determination of AgNOR activity of the Purkinje cell nuclei

In this study, the embryonic and post-hatching development of the chicken cerebellar cortex, and the number, size and area of the silver-staining nucleolus-organizer regions (AgNORs) were determined in cerebellar Purkinje cells in layer-hen (Babcock-B380) embryos and chickens. The mean transverse diameter of the Purkinje cells and their nuclei, and the mean area of the Purkinje cell nucleus and AgNOR area increased during the entire experimental period. The mean ratio of the AgNOR area to the Purkinje cell nucleus area reached its highest value at the 15th day of incubation. There was no difference between the mean AgNOR numbers per nucleus. These findings provide reference data for future studies in this and related research topics, for example for establishing AgNOR parameters and we also hope that our data will be supplemented with other techniques such as PCNA and TUNEL in the near future.

Unraveling the chicken T cell repertoire with enhanced genome annotation.

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in β and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/β/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the β and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.

Response of genes effective in apoptosis in the liver and intestinal cells of broiler chickens fed silver nanoparticles coated on clinoptilolite under heat stress condition

Response of genes effective in apoptosis in the liver and intestinal cells of broiler chickens fed silver nanoparticles coated on clinoptilolite under heat stress condition

The exposure in ovo to glyphosate on the integrity of intestinal epithelial tight junctions of chicks

Glyphosate is an ingredient widely used in various commercial formulations, including Roundup®. This study focused on tight junctions and the expression of inflammatory genes in the small intestine of chicks. On the sixth day of embryonic development, the eggs were randomly assigned to three groups: the control group (CON, n = 60), the glyphosate group (GLYP, n = 60), which received 10 mg of active glyphosate/kg egg mass, and the Roundup®-based glyphosate group also received 10 mg of glyphosate. The results indicated that the chicks exposed to glyphosate or Roundup® exhibited signs of oxidative stress. Additionally, histopathological alterations in the small intestine tissues included villi fusion, complete fusion of some intestinal villi, a reduced number of goblet cells, and necrosis of some submucosal epithelial cells in chicks. Genes related to the small intestine (ZO-1, ZO-2, Claudin-1, Claudin-3, JAM2, and Occludin), as well as the levels of pro-inflammatory cytokines (IFNγ, IL-1β, and IL-6), exhibited significant changes in the groups exposed to glyphosate or Roundup® compared to the control group. In conclusion, the toxicity of pure glyphosate or Roundup® likely disrupts the small intestine of chicks by modulating the expression of genes associated with tight junctions in the small intestine.

Effects of L-Leu-L-Leu peptide on growth, proliferation, and apoptosis in broiler intestinal epithelial cells

Small peptides are nutrients and bioactive molecules that have dual regulatory effects on nutrition and physiology. They are of great significance for maintaining the intestinal health and production performance of broilers. We here cultured the primary small intestinal epithelial cells (IEC) of chicken in a medium containing L-Leu (Leu) and L-Leu-L-Leu (Leu-Leu) for 24 h. The untreated cells were considered as the control group. The growth, proliferation, and apoptosis of IEC were examined. By combining RNA-seq and label-free sequencing technology, candidate genes, proteins, and pathways related to the growth, proliferation, and apoptosis of IEC were screened. Immunofluorescence detection revealed that the purity of the isolated primary IEC was >90%. The Leu-Leu group significantly promoted IEC growth and proliferation and significantly inhibited IEC apoptosis, and the effect was better than those of the Leu and control groups. Using transcriptome sequencing, four candidate genes, CCL20, IL8L1, IL8, and IL6, were screened in the Leu group, and one candidate gene, IL8, was screened in the Leu-Leu group. Two candidate genes, IL6 and RGN, were screened in the Leu-Leu group compared with the Leu group. Non-quantitative proteomic marker sequencing results revealed that through the screening of candidate proteins and pathways, found one growth-related candidate protein PGM3 and three proliferation-related candidate proteins RPS17, RPS11, and RPL23, and two apoptosis-related candidate proteins GPX4 and PDPK1 were found in the Leu-Leu group compared with Leu group. In short, Leu-Leu could promote IEC growth and proliferation and inhibit IEC apoptosis. On combining transcriptome and proteome sequencing technologies, multiple immune- and energy-related regulatory signal pathways were found to be related to IEC growth, proliferation, and apoptosis. Three candidate genes of IL8, IL6, and RGN were identified, and five candidate proteins of RPS17, RPS11, RPL23, GPX4, and PDPK1 were involved in IEC growth, proliferation, and apoptosis. The results provide valuable data for preliminarily elucidating small peptide-mediated IEC regulation pathways, improving the small peptide nutrition theoretical system, and establishing small peptide nutrition regulation technology.

Characterization of Myosatellite Stem Cells from Black Bone Chicken and Effect of Different Animal Serums on Growth Performance

Isolation of muscle stem cells (myosatellite stem cells) of black bone chickens using enzymatic digestion, the pronase, and screening by low-speed centrifugation process combined with 40 μm cell strainer filtration showed that this method was able to separate chicken muscle stem cells efficiently. The results proved that the isolated cells were myosatellite stem cells expressing marker genes, c-met, Pax7, MyoD, and myogenin, by RT-PCR assay. Additionally, immunofluorescence staining with monoclonal antibodies confirmed the isolated cells were MSCs, with positive staining for canonical markers proteins: Pax7, Desmin, and actin. Evaluating the influence of fetal bovine serum (FBS), horse serum (HS), and chicken serum (CKS) supplements on MSC proliferation revealed that FBS emerged as the most effective despite its escalating cost and emerging need for alternatives. Notably, Chicken Serum (CKS) demonstrated potential as an alternative, closely following the cell proliferation rate achieved by FBS. Our findings affirm the efficacy of the devised isolation method and highlight the potential of using chicken serum in cell culture protocols. HIGHLIGHTS This research established a technique for isolating myosatellite stem cells from black bone chickens through enzymatic processes combined with gradient methods. Centrifugation aids in obtaining a purer isolation of these stem cells and allows for the evaluation of various animal serums’ effects. In cell cultures, the performance of chicken serum showed no significant difference compared to bovine serum. GRAPHICAL ABSTRACT

Renal Endocytic Regulation of Vitamin D Metabolism during Maturation and Aging in Laying Hens

Egg-laying hens undergo a specific and dramatic calcium metabolism to lay eggs with eggshells composed of calcium carbonate. Calcium metabolism is mainly regulated by vitamin D3. Although vitamin D3 metabolism is closely related to the deterioration of eggshell quality associated with aging and heat stress, the details of the mechanisms regulating vitamin D3 metabolism are not clear. In mammals, the vitamin D3 metabolite (25(OH)D3) produced in the liver binds to the vitamin binding protein (DBP), is subsequently taken up by renal proximal tubular cells via the endocytic receptors megalin (Meg) and cubilin (CUB), and is metabolized to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Therefore, the present study aimed to examine the expression and localization of Meg and CUB in the kidneys of immature chicks and mature and aged laying hens to prevent eggshell quality deterioration. As a result, we showed that as circulating 1,25(OH)2D3 concentrations increased from 156.0 ± 13.5 pg/mL to 815.5 ± 61.4 pg/mL with maturation in immature chicks, relative expression levels (arbitrary units; AU) of Meg and CUB mRNA in the kidneys of mature hens significantly increased 1.92- and 2.75-fold, respectively, compared to those in immature chicks. On the other hand, the Meg mRNA expression levels of mature hens did not change with age, while CUB mRNA expression levels (1.03 ± 0.11 AU) were significantly decreased compared to mature hens (2.75 ± 0.24 AU). Immunohistochemical observations showed that Meg and CUB proteins were localized to the apical membrane of renal proximal tubular epithelial cells in immature chicks, mature hens, and aged hens, and that DBP protein was observed as granular endosomes in the cytoplasm of proximal tubular cells from the apical membrane to the cell nucleus. Especially in mature hens, the endosomes were larger and more numerous than those in immature chicks. In contrast, in aged hens, DBP-containing endosomes were smaller and limited to the apical cytoplasm. These results indicate that with maturation, the expression of Meg and CUB is promoted in the renal proximal tubules of laying hens, facilitating the uptake of the 25(OH)D3-DBP complex and its conversion to 1,25(OH)2D3, and regulating calcium metabolism in eggshell formation. On the other hand, it is suggested that the age-related decrease in CUB expression suppresses the uptake of the 25(OH)D3-DBP complex in the kidney, resulting in a deterioration of eggshell quality.

Nano‑selenium alleviates the pyroptosis of cardiovascular endothelial cells in chicken induced by decabromodiphenyl ether through ERS-TXNIP-NLRP3 pathway

Decabromodiphenyl ether (BDE-209) is one of the most widely used flame retardants that can infect domestic and wildlife through contaminated feed. Nano‑selenium (Nano-Se) has the advantage of enhancing the anti-oxidation of cells. Nonetheless, it remains uncertain whether Nano-Se can alleviate vascular Endothelial cells damage caused by BDE-209 exposure in chickens. Therefore, we established a model with 60 1-day-old chickens, and administered BDE-209 intragastric at a ratio of 400 mg/kg bw/d, and mixed Nano-Se intervention at a ratio of 1 mg/kg in the feed. The results showed that BDE-209 could induce histopathological and ultrastructural changes. Additionally, exposure to BDE-209 led to cardiovascular endoplasmic reticulum stress (ERS), oxidative stress and thioredoxin-interacting protein (TXNIP)-pyrin domain-containing protein 3 (NLRP3) pathway activation, ultimately resulting in pyroptosis. Using the ERS inhibitor 4-PBA in Chicken arterial endothelial cells (PAECs) can significantly reverse these changes. The addition of Nano-Se can enhance the body's antioxidant capacity, inhibit the activation of NLRP3 inflammasome, and reduce cellular pyroptosis. These results suggest that Nano-Se can alleviate the pyroptosis of cardiovascular endothelial cells induced by BDE-209 through ERS-TXNIP-NLRP3 pathway. This study provides new insights into the toxicity of BDE-209 in the cardiovascular system and the therapeutic effects of Nano-Se.

Effect of RNA interference with HIF-1α on the growth of pulmonary artery endothelial cells in broiler chickens

Pulmonary artery remodeling is a characteristic feature of broiler ascites syndrome (BAS). Pulmonary artery endothelial cells (PAECs) regulated by HIF-1α play a critical role in pulmonary artery remodeling, but the underlying mechanisms of HIF-1α in BAS remain unclear. In this experiment, primary PAECs were cultured in vitro and were identified by coagulation factor VIII. After hypoxia and RNA interference, the mRNA and protein expression levels of HIF-1α and VEGF were determined by qPCR and Western blotting. The transcriptome profiles of PAECs were obtained by RNA sequencing. Our results showed that the positive rate of PAECs was more than 90%, hypoxia-induced promoted the proliferation and apoptosis of PAECs, and RNA interference significantly downregulated the expression of HIF-1α, inhibited the proliferation of PAECs, and promoted the apoptosis of PAECs. In addition, transcriptome sequencing analysis indicated that HIF-1α may regulate broiler ascites syndrome by mediating COL4A, vitronectin, vWF, ITGα8, and MKP-5 in the ECM, CAMs and MAPK pathways in PAECs. These studies lay the foundation for further exploration of the mechanisms of pulmonary artery remodeling, and HIF-1α may be a potentially effective gene for the prevention and treatment of BAS.

Fibroblast test cells of embryo of Super Java Chicken as an indicator to test toxicity and malignancy

Fibroblast is one component of connective tissue cells in polygonal or stellate with cytoplasmic processes or projections. In vitro, tissue culture is an excellent medium used in biomedical research. The goal of this study was to analyze Toxicity and Malignancy in Embryo of Super Java Chicken as Potential Candidates of Strong Poultry in Indonesia. This study used Preparation of fibroblast primary cell culture of Java super chicken embryos, Pathogenicity test using fibroblast cells from Super Java chicken, Toxicity test using fibroblast cells from Super Java Chicken. Primary fibroblast cell culture of Java super chicken embryos was prepared from 10-day-old brood chicken eggs free of pathogens. Cells were prepared in 96-well microplates with minimal essential medium (MEM) containing 10 % fetal calf serum (FCS) 100 IU/ug penicillin/streptomycinVirus isolates were diluted in stages from 10-2 to 10-5 and inoculated into Java Super Chicken Fibroblast cell culture. The result showed that the negative control of the samples had a faster proliferative power than the fibroblast cell culture of Java super chicken, which was treated with concentrations of 10-2; 10-3; 10-4; 10-5. Moreover, before being inoculated with the virus, the confluent fibroblast cells of Java super chicken looked oval and regular. The day after infection, syncytia (large multinucleated cells) began to form on a small scale and became more pronounced on the second and third post-infection days. CPE was found in the 10-2,10-3 and 10-4 virus dilutions, and CPE was not found in the 10-5 dilution.

Taurine-conjugated bile acids are the predominant form in hens and have potential impact on lipid metabolism in the liver

In mammals, bile acids composition varies with species, age, physiological conditions. However, the role of bile acids in the laying cycle of hens and their impact on lipid metabolism in chicken liver is not well understood. This study aimed to elucidate the role of bile acids in hen laying cycles, investigating their distribution across ages and assessing the impact of taurine-conjugated lithocholic acid (TLCA) on oleic acid-induced fatty liver cells in chickens. Results revealed an age-related increase in total bile acid concentration in hens blood, with taurine-conjugated bile acids prevailing. Among specific bile acids analysed, taurodeoxycholic acid (TDCA) decreased with age, while taurochenodeoxycholic acid (TCDCA) increased (n = 8). TLCA concentration also decreased gradually, while lycocholic acid (GCA) was elevated in 16-week-old hens (n = 8). Primary bile acids, cholic acid (CA), and chenodeoxycholic acid (CDCA) were higher at 16 weeks compared to egg production commencement (22 weeks) and 34 weeks (n = 8). Moreover, 5ß-Cholenic acid-7a-ol-3-one concentration was elevated in 74-week-old hens post-laying (n = 8). Investigating TLCA's impact on chicken liver lipid metabolism, TLCA significantly reduced lipid droplet accumulation, especially at 20 μM, while upregulating TLCA concentrations correlated with decreased SREBP1 protein expression responsible for triglyceride synthesis. In conclusion, taurine-conjugated bile acids potentially regulate lipid metabolism in hens, offering avenues for enhancing hens health and production through further mechanistic exploration and their potential integration into hens feed.

Loss of αβ but not γδ Tcells in chickens causes a severe phenotype.

The availability of genetically modified mice has facilitated the study of mammalian Tcells. No model has yet been developed to study these cells in chickens, an important livestock species with a high availability of γδ Tcells. To investigate the role of γδ and αβ Tcell populations in birds, we generated chickens lacking these Tcell populations. This was achieved by genomic deletion of the constant region of the Tcell receptor γ or β chain, leading to a complete loss of either γδ or αβ Tcells. Our results show that a deletion of αβ Tcells but not γδ Tcells resulted in a severe phenotype in KO chickens. The αβ Tcell KO chickens exhibited granulomas associated with inflammation of the spleen and the proventriculus. Immunophenotyping of αβ Tcell KO chickens revealed a significant increase in monocytes and expectedly the absence of CD4+ Tcells including FoxP3+ regulatory Tcells. Surprisingly there was no increase of γδ Tcells. In addition, we observed a significant decrease in immunoglobulins, B lymphocytes, and changes in the bursa morphology. Our data reveal the consequences of Tcell knockouts in chickens and provide new insights into their function in vertebrates.