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The objective of this study was to determine the single and simultaneous toxic effects of chlorpyrifos containing insecticide formulation (Pyrinex 48 EC) and tebuconazole containing fungicide formulation (Mystic 250 EW) on the development of chicken embryos. Amount of 0.1 ml of 1% Pyrinex 48 EC and of 0.4% Mystic 250 EW was alone and concomitantly injected into the air chamber of eggs on the first day prior to incubation. The chicken embryos were examined on day 19 of incubation for the followings: number of embryonic deaths, body, liver and heart weight of the embryos, and type of developmental anomalies. The body, liver and heart weight data were evaluated statistically by One-Way ANOVA, Tukey and Dunnett tests, the embryonic mortality and the developmental abnormalities were analysed by Fisher's exact test. The combined administration of Pyrinex 48 EC and Mystic 250 EW pesticides on the chick embryo had shown to be embryotoxic to the chick. The rate of mortality and the incidence of developmental anomalies were increased due to the simultaneous application of them. The body and liver weight were significantly reduced. Our teratogenicity study revealed that the combined administration of both chlorpyrifos (Pyrinex 48 EC) and tebuconazole (Mystic 250 EW) pesticides on the chick embryo had shown to be embryotoxic to the chick. They had a slight addition effect on the rate of embryonic mortalities, however, the toxic interaction of both pesticides on developmental anomalies, liver and body weight was not proven. [ABSTRACT FROM AUTHOR]

Dysregulation of the immune system is associated with many pathologies, including cardiovascular diseases, diabetes, and cancer. To date, the most commonly used models in biomedical research are rodents, and despite the various advantages they offer, their use also raises numerous drawbacks. Recently, another in vivo model, the chicken embryo and its chorioallantoic membrane, has re-emerged for various applications. This model has many benefits compared to other classical models, as it is cost-effective, time-efficient, and easier to use. In this review, we explain how the chicken embryo can be used as a model for immune-based studies, as it gradually develops an embryonic immune system, yet which is functionally similar to humans’. We mainly aim to describe the avian immune system, highlighting the differences and similarities with the human immune system, including the repertoire of lymphoid tissues, immune cells, and other key features. We also describe the general in ovo immune ontogeny. In conclusion, we expect that this review will help future studies better tailor their use of the chicken embryo model for testing specific experimental hypotheses or performing preclinical testing.

A total of 220 birds of age ranging from 3 to 14 weeks old were collected from several backyards and different farms in Sharkia Governorate, Egypt, and surveyed for the presence of fowl cholera. Twenty Pasteurella multocida from chickens (15/145, 10%) and ducks (5/75, 6%) were bacteriologically isolated, and it was shown that the infection was significantly related to age and breed. Capsular typing, using multiplex polymerase chain reaction (PCR), demonstrated that all strains were type A (100%). Disk diffusion assay towards ten antimicrobials revealed high susceptibilities to amikacin, doxycycline, chloramphenicol, and neomycin with varying degrees. Doxycycline was effective at the lowest concentration (MIC 0.125–1 µg/ml). Multidrug resistance was detected with a percentage of 25%. Multidrug-resistant isolates (five isolates) were subjected to study their pathogenicity in embryonated chicken eggs (ECE). The results showed a variation in indices between different dilutions of the tested strains. The resulting pathogenicity indices showed significant differences (P < 0.05) according to the origin and dilution of the isolate. From the original inoculum to 10–4 dilutions, the mortality of inoculated embryos occurred within 1–2 days with pathological findings, including maceration and lesions on chorioallantoic membrane (CAM). From dilutions ranging from 10–5 to 10–9, no death occurred until 7 days post-inoculation, but a variation in the lesions on CAM was observed. In conclusion, P. multocida serogroup A could be intensely pathogenic for mature chickens thus causing considerable economic losses, and PCR provides a suitable technique for early and rapid diagnosis of fowl cholera. [ABSTRACT FROM AUTHOR]

Single and simultaneous toxic effects of glyphosate (Amega Up, 360 g L−1, 4%) and copper sulphate (0.01%) were studied in avian embryos treated either with injection directly into the air chamber or by immersion application for 30 min on day 0 of incubation. Alterations of the chicken embryos were evaluated during necropsy performed on day 19 of incubation, together with mortality, body weight and the type of developmental abnormalities. Based on the results, the injection application appeared to be more toxic than the immersion method, as it induced increased mortality and reduced the average body weight, and resulted in a higher incidence of congenital anomalies. Supposedly, a toxicodynamic interaction occurs between copper sulphate and glyphosate, which may reduce the vitality of embryos and thus decrease the number of offspring in wild birds. [ABSTRACT FROM AUTHOR]

Dysregulation of the immune system is associated with many pathologies, including cardiovascular diseases, diabetes, and cancer. To date, the most commonly used models in biomedical research are rodents, and despite the various advantages they offer, their use also raises numerous drawbacks. Recently, another in vivo model, the chicken embryo and its chorioallantoic membrane, has re-emerged for various applications. This model has many benefits compared to other classical models, as it is cost-effective, time-efficient, and easier to use. In this review, we explain how the chicken embryo can be used as a model for immune-based studies, as it gradually develops an embryonic immune system, yet which is functionally similar to humans'. We mainly aim to describe the avian immune system, highlighting the differences and similarities with the human immune system, including the repertoire of lymphoid tissues, immune cells, and other key features. We also describe the general in ovo immune ontogeny. In conclusion, we expect that this review will help future studies better tailor their use of the chicken embryo model for testing specific experimental hypotheses or performing preclinical testing. [ABSTRACT FROM AUTHOR]

• Excess 5-HT affects the serotonergic and dopaminergic systems during embryogenesis. • Effect of 5-HT on fetal neurodevelopment is via unique paths based on its dosage. • Multisystemic view is critical for examining SSRIs and birth defects, linked to ASD. • Chicken embryo is suitable for studying maternal 5-HT fluctuation on neurogenesis. The placenta is the primary source of serotonin (5-HT) for fetal development, programming fetal neural wiring in humans and other mammals. The fluctuation in maternal 5-HT affects fetal neurogenesis with life-long consequences, however, its mechanisms have not been well known. The chicken embryo, independent of maternal neurohormonal influence, may offer an ideal model for studying the mechanisms of prenatal 5-HT exposure altering postnatal physiological homeostasis and behavioral exhibition. To investigate the fine-tuning of 5-HT to the early embryonic neurodevelopment, 10 µg and 20 µg 5-HT were secretively injected to chicken embryos before incubation. 5-HT exposure mainly affected the neural development in the pons and midbrain, altered the serotoninergic and dopaminergic (DAergic) neuronal morphology, nucleus distribution, and their metabolisms and related gene expressions. The comprehensive effect of 5-HT exposure was not dosage-dependent but the working pathways differed, 10 µg 5-HT exposure reduced 5-HT turnover rate, increased 5-HT 1a receptor expression, and facilitated the ventral tegmental area neuronal development; while 20 µg 5-HT exposure increased the serotoninergic and DAergic neurotransmission and enhanced serotoninergic regulation to the hypothalamus. These findings indicate that the 5-HT exposure effect can be achieved via different paths by modifying the embryonic serotonergic (5-HTergic) and DAergic systems and altering fetal 5-HTergic influence on the thalamocortical circuit and hypothalamic–pituitary–adrenal axis. These results may offer a novel sight for understanding the function of 5-HT during neurodevelopment and raise the possibility for using selective 5-HT reuptake inhibitors to regulate emotional and mental wellness during early pregnancy and possible risks of complications for babies. [ABSTRACT FROM AUTHOR]

6:2 fluorotelomer carboxylic acid (6:2 FTCA) is a perfluorooctanoic acid (PFOA) substitute, which is supposedly less accumulative and toxic than PFOA. However, 6:2 FTCA is structurally similar to PFOA, and there had already been reports about its toxicities comparable to PFOA. The aim of the current study is to assess potential effects of developmental exposure to 6:2 FTCA on the development of kidney in chicken embryo and to investigate underlying mechanism. Fertile chicken eggs were exposed to 1.25 mg/kg, 2.5 mg/kg or 5 mg/kg doses of 6:2 FTCA, or 2 mg/kg PFOA, then incubated to hatch. Serum and kidney of hatchling chickens were collected. Blood urea nitrogen (BUN) and creatinine (Cre) levels were measured with commercially available kits. Morphology of kidney was assessed with histopathology. To further reveal molecular mechanism of observed endpoints, IGF signaling molecules were assessed in the kidney samples with qRT-PCR, results indicated that IGFBP3 is a potentially crucial molecule. Lentiviruses overexpressing or silencing IGFBP3 were designed and applied to enhance/suppress the expression of IGFBP3 in developing chicken embryo for further verification of its role in the observed effects. Disrupted nephron formation, in the manifestation of decreased glomeruli number/area and increased serum BUN/Cre levels, was observed in the animals developmentally exposed to 6:2 FTCA. Correspondingly, IGF signaling molecules (IGF1, IGF1R and IGFBP3) were affected by 6:2 FTCA exposure. Meanwhile, overexpression of IGFBP3 effectively alleviated such changes, while silencing of IGFBP3 mimicked observed effects. In conclusion, developmental exposure to 6:2 FTCA is associated with disrupted chicken embryo renal development, in which IGFBP3 seems to be a remarkable contributor, suggesting potential health risks for human and other species. Further risk assessments and mechanistic works are necessary. • 6:2 FTCA exposure resulted in developmental nephrotoxicity in chicken embryo. • IGF signaling molecules (IGF1, IGF1R and IGFBP3) were affected by 6:2 FTCA exposure. • IGFBP3 seems to be a major target of 6:2 FTCA-induced renal effects. [ABSTRACT FROM AUTHOR]

Lignin-derivable bisguaiacols/bissyringols are viable alternatives to commercial bisphenols; however, many bisguaiacols/bissyringols (e.g., bisguaiacol F [BGF]) have unsubstituted bridging carbons between the aromatic rings, making them more structurally similar to bisphenol F (BPF) than bisphenol A (BPA) – both of which are suspected endocrine disruptors. Herein, we investigated the estrogenic activity (EA) and developmental toxicity of dimethyl-substituted bridging carbon-based lignin-derivable bisphenols (bisguaiacol A [BGA] and bissyringol A [BSA]). Notably, BSA showed undetectable EA at seven test concentrations (from 10−12 M to 10−6 M) in the MCF-7 cell proliferation assay, whereas BPA had detectable EA at five concentrations (from 10−10 M to 10−6 M). In silico results indicated that BSA had the lowest binding affinity with estrogen receptors. Moreover, in vivo chicken embryonic assay results revealed that lignin-derivable monomers had minimal developmental toxicity vs. BPA at environmentally relevant test concentrations (8.7–116 μg/kg). Additionally, all lignin-derivable compounds showed significantly lower expression fold changes (from ∼1.81 to ∼4.41) in chicken fetal liver tests for an estrogen-response gene (apolipoprotein II) in comparison to BPA (fold change of ∼11.51), which was indicative of significantly reduced estrogenic response. Altogether, the methoxy substituents on lignin-derivable bisphenols appeared to be a positive factor in reducing the EA of BPA alternatives. [Display omitted] • Bissyringol A had undetectable estrogenic activity in MCF-7 cell assays. • Lignin-derivable monomers had reduced estrogenic responses vs. bisphenol A and F. • Developmental toxicity of lignin-derivable monomers was tested for the first time. • Lignin-derivable monomers had minimal developmental toxicity in chicken embryos. [ABSTRACT FROM AUTHOR]

Some studies have shown that the excessive metabolic heat production is the primary cause for dead chicken embryos during late embryonic development. Increasing heat shock protein (HSP) expression and adjusting metabolism are important ways to maintain body homeostasis under heat stress. This study was conducted to investigate the effects of in ovo injection (IOI) of vitamin C (VC) at embryonic age 11th day (E11) on HSP and metabolic genes expression. A total of 320 breeder eggs were randomly divided into normal saline and VC injection groups. We detected plasma VC content and rectal temperature at chick’s age 1st day, and the mRNA levels of HSP and metabolic genes in embryonic livers at E14, 16 and 18, analysed the promoter methylation levels of differentially expressed genes and predicted transcription factors at the promoter regions. The results showed that IOI of VC significantly increased plasma VC content and decreased rectal temperature (P < 0.05). In ovo injection of VC significantly increased heat shock protein 60 (HSP60) and pyruvate dehydrogenase kinase 4 (PDK4) genes expression at E16 and PDK4 and secreted frizzled related protein 1 (SFRP1) at E18 (P < 0.05). At E16, IOI of VC significantly decreased the methylation levels of total CpG sites and −336 CpG site in HSP60 promoter and −1137 CpG site in PDK4 promoter (P < 0.05). Potential binding sites for nuclear factor-1 were found around −389 and −336 CpG sites in HSP60 promoter and potential binding site for specificity protein 1 was found around −1137 CpG site in PDK4 promoter. Our results suggested that IOI of VC increased HSP60, PDK4 and SFRP1 genes expression at E16 and 18, which may be associated with the demethylation in gene promoters. Whether IOI of VC could improve hatchability needs to be further verified by setting uninjection group.

Understanding the complex processes of fetal wound healing and skin regeneration can help to improve fetal surgery. As part of the integumentary system, the skin protects the newborn organism against environmental factors and serves various functions. Glucocorticoids can enter the fetal circulatory system by either elevated maternal stress perception or through therapeutic administration and are known to affect adult skin composition and wound regeneration. In the present study, we aimed at investigating the effects of local glucocorticoid administration on the process of embryonic wound healing. We performed in-ovo bead implantation of dexamethasone beads into skin incisional wounds of avian embryos and observed the local effects of the glucocorticoid on the process of skin regeneration through histology, immunohistochemistry and in-situ hybridization, using vimentin, fibronectin, E-cadherin, Dermo-1 and phospho-Histone H3 as investigational markers. Local glucocorticoid administration decelerated the healing of the skin incisional wounds by impairing mesenchymal contraction and re-epithelialization resulting in morphological changes, such as increased epithelialization and disorganized matrix formation. The results contribute to a better understanding of scarless embryonic wound healing and how glucocorticoids might interfere with the underlying molecular processes, possibly indicating that glucocorticoid therapies in prenatal clinical practice should be carefully evaluated.

Salmonella spp. is recognized as an important zoonotic pathogen. The emergence of antimicrobial resistance in Salmonella enterica poses a great public health concern worldwide. While the knowledge on the incidence and the characterization of different S. enterica serovars causing chick embryo death remains obscure in China. In this study, we obtained 45 S. enterica isolates from 2,139 dead chick embryo samples collected from 28 breeding chicken hatcheries in Henan province. The antimicrobial susceptibility assay was performed by the broth microdilution method and the results showed that 31/45 (68.8%) isolates were multidrug-resistant (≥3 antimicrobial classes). Besides the highest resistance rate was observed in the aminoglycoside class, all the isolates were susceptible to chloramphenicol, azithromycin, and imipenem. Furthermore, genomic characterization revealed that S. Enteritidis (33.33%; 15/45) was a frequent serovar that harbored a higher number of virulence factors compared to other serovars. Importantly, genes encoding β-lactamases were identified in three serovars (Thompson, Enteritidis, and Kottbus), whereas plasmid-mediated quinolone resistance genes (qnrB4) were detected in certain isolates of S. Thompson and the two S. Kottbus isolates. All the examined isolates harbored the typical virulence factors from Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). Additionally, a correlation analysis between the antimicrobial resistance genes, phenotype, and plasmids was conducted among Salmonella isolates. It showed strong positive correlations (r < 0.6) between the different antimicrobial-resistant genes belonging to certain antimicrobial classes. Besides, IncF plasmid showed a strong negative correlation (r > −0.6) with IncHI2 and IncHI2A plasmids. Together, our study demonstrated antimicrobial-resistant S. enterica circulating in breeding chicken hatcheries in Henan province, highlighting the advanced approach, by using genomic characterization and statistical analysis, in conducting the routine monitoring of the emerging antimicrobial-resistant pathogens. Our findings also proposed that the day-old breeder chicks trading could be one of the potential pathways for the dissemination of multidrug-resistant S. enterica serovars.

ABSTRACT: Epigenetics is defined as the study of changes in gene function that are mitotically or meiotically heritable and do not lead to a change in DNA sequence. Epigenetic modifications are important mechanisms that fine tune the expression of genes in response to extracellular signals and environmental changes. In vertebrates, crucial epigenetic reprogramming events occur during early embryogenesis and germ cell development. Chicken embryo, which develops external to the mother's body, can be easily manipulated in vivo and in vitro, and hence, it is an excellent model for performing epigenetic studies. Environmental factors such as temperature can affect the development of an embryo into the phenotype of an adult. A better understanding of the environmental impact on embryo development can be achieved by analyzing the direct effects of epigenetic modifications as well as their molecular background and their intergenerational and transgenerational inheritance. In this overview, the current possibility of epigenetic changes during chicken embryonic development and their effects on long-term postembryonic development are discussed.

Salmonella spp. is recognized as an important zoonotic pathogen. The emergence of antimicrobial resistance in Salmonella enterica poses a great public health concern worldwide. While the knowledge on the incidence and the characterization of different S. enterica serovars causing chick embryo death remains obscure in China. In this study, we obtained 45 S. enterica isolates from 2,139 dead chick embryo samples collected from 28 breeding chicken hatcheries in Henan province. The antimicrobial susceptibility assay was performed by the broth microdilution method and the results showed that 31/45 (68.8%) isolates were multidrug-resistant (≥3 antimicrobial classes). Besides the highest resistance rate was observed in the aminoglycoside class, all the isolates were susceptible to chloramphenicol, azithromycin, and imipenem. Furthermore, genomic characterization revealed that S. Enteritidis (33.33%; 15/45) was a frequent serovar that harbored a higher number of virulence factors compared to other serovars. Importantly, genes encoding β-lactamases were identified in three serovars (Thompson, Enteritidis, and Kottbus), whereas plasmid-mediated quinolone resistance genes (qnrB4) were detected in certain isolates of S. Thompson and the two S. Kottbus isolates. All the examined isolates harbored the typical virulence factors from Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). Additionally, a correlation analysis between the antimicrobial resistance genes, phenotype, and plasmids was conducted among Salmonella isolates. It showed strong positive correlations (r < 0.6) between the different antimicrobial-resistant genes belonging to certain antimicrobial classes. Besides, IncF plasmid showed a strong negative correlation (r > −0.6) with IncHI2 and IncHI2A plasmids. Together, our study demonstrated antimicrobial-resistant S. enterica circulating in breeding chicken hatcheries in Henan province, highlighting the advanced approach, by using genomic characterization and statistical analysis, in conducting the routine monitoring of the emerging antimicrobial-resistant pathogens. Our findings also proposed that the day-old breeder chicks trading could be one of the potential pathways for the dissemination of multidrug-resistant S. enterica serovars. [ABSTRACT FROM AUTHOR]

During early embryogenesis in amniotic vertebrates, the gonads differentiate into either ovaries or testes. The first cell lineage to differentiate gives rise to the supporting cells: Sertoli cells in males and pre-granulosa cells in females. These key cell types direct the differentiation of the other cell types in the gonad, including steroidogenic cells. The gonadal surface epithelium and the interstitial cell populations are less well studied, and little is known about their sexual differentiation programs. Here, we show the requirement of the homeobox transcription factor gene TGIF1 for ovarian development in the chicken embryo. TGIF1 is expressed in the two principal ovarian somatic cell populations: the cortex and the pre-granulosa cells of the medulla. TGIF1 expression is associated with an ovarian phenotype in estrogen-mediated sex reversal experiments. Targeted misexpression and gene knockdown indicate that TGIF1 is required, but not sufficient, for proper ovarian cortex formation. In addition, TGIF1 is identified as the first known regulator of juxtacortical medulla development. These findings provide new insights into chicken ovarian differentiation and development, specifically cortical and juxtacortical medulla formation. [ABSTRACT FROM AUTHOR]

We investigated the antioxidant effects of vitamin E on a glucocorticoid (GC) induced model of cataracts in chick embryos. We used 70 fertilized eggs divided into seven groups as follows: phosphate-buffered saline (PBS) group, olive oil treatment (OO) group, hydrocortisone treatment (HC) group, olive oil and hydrocortisone treatment (OO + HC) group, 50 mg/kg vitamin E and hydrocortisone treatment (HC + VE (50)) group, 25 mg/kg vitamin E and hydrocortisone treatment (HC + VE (25)) group and 15 mg/kg vitamin E and hydrocortisone treatment (HC + VE (15)) group. On day 17, chick embryos were removed from the eggs and lens and liver tissues were excised. Cataract formation was evaluated and total antioxidant status (TAS), total oxidant status (TOS), malondialdehyde (MDA) and glutathione peroxidase (GPx) were measured in lens and liver tissues; MDA was measured only in liver. The lenses in the HC + VE (50) group exhibited significantly higher levels of GPx and TAS, and lower levels of TOS than for HC and OO + HC groups. The livers of the HC + VE (50) group exhibited significantly higher levels of GPx and lower levels of MDA than for the HC and OO + HC groups. The HC + VE (50) group lenses were evaluated as grade 1, because the nuclei were completely free of cataracts, likely due to the antioxidative effect of high dose VE. VE is an effective antioxidant agent that exhibits a dose-response effect, for ameliorating the negative effects of GCs. [ABSTRACT FROM AUTHOR]

Background: Lung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ultimately define the airway conducting system. Throughout this process, energy and structural macromolecules are necessary to sustain the high proliferative rates. The extensive knowledge of the molecular mechanisms underlying pulmonary development contrasts with the lack of data regarding the embryonic lung metabolic requirements. Here, we studied the metabolic profile associated with the early stages of chicken pulmonary branching. Methods: In this study, we used an ex vivo lung explant culture system and analyzed the consumption/production of extracellular metabolic intermediates associated with glucose catabolism (alanine, lactate, and acetate) by 1H-NMR spectroscopy in the culture medium. Then, we characterized the transcript levels of metabolite membrane transporters (glut1, glut3, glut8, mct1, mct3, mct4, and mct8) and glycolytic enzymes (hk1, hk2, pfk1, ldha, ldhb, pdha, and pdhb) by qPCR. ldha and ldhb mRNA spatial localization was determined by in situ hybridization. Proliferation was analyzed by directly assessing DNA synthesis using an EdU-based assay. Additionally, we performed western blot to analyze LDHA and LDHT protein levels. Finally, we used a Clark-Type Electrode to assess the lung explant's respiratory capacity. Results: Glucose consumption decreases, whereas alanine, lactate, and acetate production progressively increase as branching morphogenesis proceeds. mRNA analysis revealed variations in the expression levels of key enzymes and transporters from the glycolytic pathway. ldha and ldhb displayed a compartment-specific expression pattern that resembles proximal–distal markers. In addition, high proliferation levels were detected at active branching sites. LDH protein expression levels suggest that LDHB may account for the progressive rise in lactate. Concurrently, there is a stable oxygen consumption rate throughout branching morphogenesis. Conclusions: This report describes the temporal metabolic changes that accompany the early stages of chicken lung branching morphogenesis. Overall, the embryonic chicken lung seems to shift to a glycolytic lactate-based metabolism as pulmonary branching occurs. Moreover, this metabolic rewiring might play a crucial role during lung development. [ABSTRACT FROM AUTHOR]

Introduction: Yangambin, a lignan predominant in the leaves of Ocotea duckei Vattimo-Gil, has several biological activities, such as anti-convulsant, analgesic, anti-inflammatory and leishmanicidal. Objectives: The aim of this study was to evaluate the embryotoxicity of yangambin on the neurodevelopment of Gallus gallus domesticus embryos. Materials and Methods: 120 fertilized eggs were divided in three groups: G1 (PBS with 0.1 % Tween 80), G2 (50 µg/ml yangambin) and G3 (65 µg/ml yangambin) and each egg was inoculated with 100 µL of the respective solutions. The fertilized eggs were incubated at a temperature of 37.5°C, with a relative humidity of 65% to 75%, for 48 hr and then their embryos were histologically processed. Results: In staging, carried out according to Hamburger and Hamilton (1951), variations of stages were identified. In all groups, the morphological analysis revealed the closure of the anterior neuropore and absence of malformations in the optic vesicles and in the secondary encephalic vesicles. In the caudal region, a standard development of the neural tube was observed, with well-segmented somites and regression of the primitive line. The cross sections showed that the internal structure of the somite's, composed of dermatome, myotome and sclerotome, was preserved. The statistical analysis did not show significant differences between the groups regarding the morphometry of the cephalic and caudal regions of the neural tube. Conclusion: Yangambin did not show embryotoxic effects on the neurodevelopment of Gallus gallus domesticus embryos, under the tested conditions. [ABSTRACT FROM AUTHOR]

ABSTRAK. Penelitian ini bertujuan melihat perkembangan embrio ayam secara histologi pada waktu 1, 2, 3, 4, 5, dan 7 hari inkubasi. Sampel yang digunakan adalah telur fertil yang didapat dari teaching farm Fakultas Pertanian Jurusan Peternakan Universitas Syiah Kuala. Pengamatan secara makroskopis dilakukan pada setiap sampel embrio ayam, sementara pengamatan mikroskopis struktur embrio ayam dilakukan dengan pembuatan preparat histologi. Hasil penelitian ini menampilkan data perkembangan embrio ayam secara spesifik dari umur inkubasi hari ke-1 sampai hari ke-7. Secara makroskopik dan mikroskopis, gambaran umum perkembangan embrio ayam selaras dengan informasi tabel pertumbuhan embrio ayam. Secara histologis, gambaran perkembangan struktur embrio ayam baru dapat diamati dengan baik yaitu pada umur inkubasi hari ke-3 sampai hari ke-7. Dapat disimpulkan bahwa secara histologi perkembangan embrio ayam baru dapat diamati pada masa inkubasi hari ke-3. Penelitian ini juga dapat dijadikan sebagai bahan acuan dasar untuk penelitian lanjutan terutama terhadap organogenesis pada embrio ayam. (Histological evaluation on the development of chicken embryo during the incubation periodfrom 1st to 7th days) ABSTRACT. The aim of this study was to find out the development of chicken embryos at day 1st, 2nd, 3rd, 4th, 5th, and 7th of incubation by histological method. The samples used were the fertile eggs obtained from the teaching farm of the Faculty of Agriculture, Department of Animal Husbandry, Syiah Kuala University. Macroscopic observations were made on each embryos sample, while microscopic observations of the embryos structure were carried out by histological preparations procedure. The results of this study presented the specific data of chicken embryos development from day 1st to day 7th of incubation. The macroscopic and microscopic data showed the general description of chicken embryos development similar to the information on the chicken embryo growth table. The microscopic results showed the structure description of the chicken embryos can be well observed at day 3rd to the day 7th of incubation. It can be seen the development of chicken embryos can be observed by histology method on day 3rd of incubation. This research can also be used as a basic reference for further research, especially on organogenesis of chicken embryos.

Aim: The purpose of this study was to evaluate the antifungal activity and toxicological parameters of 8‐hydroxyquinoline derivatives PH151 and PH153 using alternative animal models, to understand their behaviour when subjected to in vivo experiments. Methods and results: We used Toll‐deficient Drosophila melanogaster to test the protective effect of compounds against Candida albicans infection. Toxicological parameters were investigated in chicken and zebrafish embryos. PH151 and PH153 showed low toxicity and the treated flies with these compounds had a significantly higher survival rate than untreated flies after 7 days of infection. The compounds did not cause interruption of chicken embryogenesis. Zebrafish embryos exposed to compounds showed dose‐dependent toxicity. Conclusions: The data supported the potential of PH151 and PH153 for the treatment of systemic candidiasis and demonstrated to be appropriate drug candidates for further studies using mammalian models. Significance and Impact of the Study: The increased incidence of Candida infections resistant to antifungals currently available requires acceleration of the discovery of new agents with properties of inhibiting this fungal pathogen. In this study, we have described the antifungal potential and toxicity of two 8‐hydroxyquinoline derivatives using in vivo alternative models, and the results confirm their potential to be developed as new drug candidates. [ABSTRACT FROM AUTHOR]

Current ischemic models strive to replicate ischemia-mediated injury. However, they face challenges such as inadequate reproducibility, difficulties in translating rodent findings to humans, and ethical, financial, and practical constraints that limit the accuracy of extensive research. This study introduces a novel approach to inducing persistent ischemia in 3-day-old chicken embryos using endothelin-1. The protocol targets the right vitelline arteries, validated with Doppler blood flow imaging and molecular biology experiments. This innovative approach facilitates the exploration of oxidative stress, inflammatory responses, cellular death, and potential drug screening suitability utilizing a 3-day-old chicken embryo. Key features • This model enables the evaluation and investigation of the pathology related to persistent ischemia • This model allows for the assessment of parameters like oxidative stress, inflammation, and cellular death • This model enables quantification of molecular changes at the nucleic acid and protein levels • This model allows for the efficient screening of drugs and their targets Graphical overview., Competing Interests: Competing interestsThe authors declare no competing interest., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Gut peristaltic movements transport ingested materials along the gut axis, which is critical for food digestion and nutrient absorption. While a large amount of studies have been devoted to analyzing the physiological functions of peristalsis in adults, little is known about how the peristaltic system is established during embryogenesis. In recent years, the chicken developing gut has emerged as an excellent model, in which specific sites along the gut axis can be genetically labeled enabling live imaging and optogenetic analyses. This review provides an overview of recent progress in optogenetic studies of gut peristalsis. Analyses with an improved channelrhodopsin-2 variant demonstrated that the peristalsis can artificially be generated in the developing gut. These studies unveiled novel functional coordination between different regions along the gut axis. In addition, imaging with GCaMP6s, a genetically encoded calcium indicator, enabled a fine mapping of developmental changes in the peristaltic patterns as Ca2+ signals. These advanced techniques will broaden our knowledge of how embryonic peristalsis is established at the cellular and molecular level, leading to the understanding of physiological and pathological processes in adult peristalsis., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

A study was conducted to understand the effects of 25-hydroxyvitamin D3 (25OHD) and 1,25-dihydroxyvitamin D3 (1,25OHD) administration on the expression of key genes related to osteogenesis, adipogenesis, myogenesis, and vitamin D3 metabolism in the chicken embryo. A total of 120 fertilized Cobb 500 eggs were used in the current study and were reared under standard incubation conditions. On embryonic day 3 (ED 3), PBS (C), PBS with 40ng 1,25OHD (1,25D-L), 200ng 1,25OHD (1,25D-H), 40ng 25OHD (25D-L), or 200ng 25OHD (25D-H) were injected into the dorsal vein of developing embryos. Whole embryos were harvested at 1, 3, and 6h post-injection for gene expression analyses (n=8). Gene expression for key osteogenesis markers (RUNX2: runt-related transcription factor 2; BMP2: bone morphogenetic protein 2; COL1A2: collagen type I alpha 2 chain; BGLAP: bone gamma-carboxyglutamate protein; SPP1: secreted phosphoprotein 1; and ALP: alkaline phosphatese), adipogenesis markers (PPAR-γ: peroxisome proliferator-activated receptor gamma; FASN: fatty acid synthase; and FABP4: fatty acid binding protein 4), myogenesis markers (MYOG: myogenin; MYOD1: myogenic differentiation 1; and MYF5: myogenic factor 5), and the enzyme responsible for vitamin D3 inactivation (CYP24A1: cytochrome P450 family 24 subfamily A member 1) were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data were normalized by the ΔΔCT method and analyzed using a one-way ANOVA. Results indicated that at 1h post-injection, no differences were found among treatments. At 3h, the early osteogenesis differentiation marker, ALP, was increased by 1,25D-H and 25D-H, and 25D-H also stimulated the expression of adipogenesis markers (FAPB4 and FASN). In contrast, the expression of myogenesis markers (MYOD1 and MYF5) was suppressed by 25OHD or 1,25OHD treatments, respectively. At 6h, a late osteogenic differentiation marker, SPP1, was increased by 25D-H. MYOD1 and MYF5 were continuously suppressed by 25OHD treatments or 1,25D-H. The evidence of vitamin D3 metabolite retention was assessed by measuring CYP24A1 expression. At 1h, there were no differences in CYP24A1 expression. At 3h, all treatments upregulated CYP24A1 expression relative to control (PBS) embryos. However, at 6h, only the 25D-H group retained higher CYP24A1 expression compared to the other treatments. In conclusion, the results suggested both 1,25OHD and 25OHD induced chicken embryo osteogenesis and adipogenesis, but inhibited myogenesis during early chicken embryo development. The higher dosage of 25OHD showed a possibility of a longer retention time in the embryos.

Tris(2-chloroethyl) phosphate (TCEP) is an organophosphate flame retardant that used in textiles, industrial materials, and furniture to delay the spread of fire after ignition. TCEP has been detected in the tissues and eggs of fish and birds. However, there are no studies regarding the effects of TCEP on avian embryos. In the present study, we investigated the developmental toxicity of TCEP exposure on chicken embryos in a shell-less incubation system, which enables in situ observation. Chicken embryos were treated with graded doses of TCEP (50, 250, and 500 nmol/g egg) on incubation day 0. The survival rate, morphological biometrics, heart rate, and length and branch number of extraembryonic blood vessels were measured on incubation days 3–9. Survival rates were reduced from incubation day 3 and were significantly decreased until day 9. Body length, head + bill length and eye diameter were significantly reduced by TCEP exposure. Regarding skeletal effects, spine length was decreased in a dose-dependent manner on day 9. Body weight on day 9 significantly reduced in all TCEP treatment groups. These results suggest that TCEP exposure to >50 nmol/g egg retards development in chicken embryos. TCEP exposure to 500 nmol/g egg significantly increased heart weight to body weight ratio in the embryos. More than 250 nmol/g egg of TCEP significantly reduced the heart rate of embryos in the early developmental stage. The formation of extraembryonic blood vessels and the number of erythrocytes were significantly reduced even with 50 nmol/g egg of TCEP. These findings suggest that TCEP exposure specifically affects the cardiovascular system in chicken embryos, which leads to developmental delay. The results of this study also demonstrate that the shell-less incubation system can be used to continuously monitor the effects of chemicals on developing avian embryos.

The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (β2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring β2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. β2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the β2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the β2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The β2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The β2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The β2GPI dimer proved to be largely more harmful than the β2GPI monomer. β2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.

The gonads of vertebrate embryos are unique among organs because they have a developmental choice; ovary or testis formation. Given the importance of proper gonad formation for sexual development and reproduction, considerable research has been conducted over the years to elucidate the genetic and cellular mechanisms of gonad formation and sexual differentiation. While the molecular trigger for gonadal sex differentiation into ovary of testis can vary among vertebrates, from egg temperature to sex-chromosome linked master genes, the downstream molecular pathways are largely conserved. The cell biology of gonadal formation and differentiation has long thought to also be conserved. However, recent discoveries point to divergent mechanisms of gonad formation, at least among birds and mammals. In this mini-review, we provide an overview of cell lineage allocation during gonadal sex differentiation in the mouse model, focusing on the key supporting and steroidogenic cells and drawing on recent insights provided by single cell RNA-sequencing. We compare this data with emerging information in the chicken model. We highlight surprising differences in cell lineage specification between species and identify gaps in our current understanding of the cell biology underlying gonadogenesis.

Particulate matter poses health risk to developing organisms. To investigate particulate matters with a diameter smaller than 2.5 um (PM2.5)-induced developmental cardiotoxicity, fertile chicken eggs were exposed to PM2.5 via air cell injection at doses of 0.05, 0.2, 0.5, 2, and 5 mg/egg kg. Morphological changes in the embryonic day four (ED4) and hatchling hearts were assessed with histological techniques. Heart rates of hatchling chickens were measured with electrocardiography. The protein expression levels of nuclear factor kappa-light-chain-enhancer of activated B cells p65 (NF-kb p65), inducible nitric oxide synthase (iNOS), and matrix metallopeptidase 9 (MMP9) were assessed with immunohistochemistry or western blotting in hatchling hearts. PM2.5 exposure elevated areas of heart in ED4 embryo, increased heart rate, and thickened right ventricular wall thickness in hatchling chickens. Immunohistochemistry revealed enhanced NF-kb p65 expression in hatchling hearts. Western blotting results indicated that both iNOS and MMP9 expression were enhanced by lower doses of PM2.5 exposure (0.2 and 0.5 mg/kg) but not 2 mg/kg. In summary, developmental exposure to PM2.5 induced developmental cardiotoxicity in chicken embryo and hatchling chickens, which is associated with NF-kb p65, iNOS, and MMP9.

Nutrients and xenobiotics cross the blood–placenta barrier, potentially depositing in the fetal brain. The prenatal exposure affects the neuroendocrine and microbial development. The mechanism underlying maternal risk factors reprograming the microbiota–gut–brain axis with long-term effects on psychosocial behaviors in offspring is not clear. In humans, it is not possible to assess the nutrient or xenobiotic deposition in the fetal brain and gastrointestinal system for ethical reasons. Moreover, the maternal–fetal microbe transfer during gestation, natural labor, and breast-feeding constitutes the initial gut microbiome in the progeny, which is inevitable in the most widely utilized rodent models. The social predisposition in precocial birds, including chickens, provides the possibility to test behavioral responses shortly after being hatched. Hence, chickens are advantageous in investigating the ontogenetic origin of behaviors. Chicken embryos are suitable for deposition assessment and mechanistic study due to the accessibility, self-contained development, uniform genetic background, robust microbiota, and easy in vivo experimental manipulation compared to humans and rodents. Therefore, chicken embryos can be used as an alternative to the rodent models in assessing the fetal exposure effect on neurogenesis and investigating the mechanism underlying the ontogenetic origin of neuropsychiatric disorders.

Objective(s): In veterinary medicine, zeolites are used as toxin binder in animal feed, ammonia purification of aquatic pools, animal smell and moisture control. This study investigated on the risk assessment of nano zeolite on development of embryonic chicken models, anomalies, tetratogenic and embryotoxic effects. Methods: Eggs (120 = n) were accidentally divided into 4 groups. In the experimental groups, 0.3 ml of the solution of nanozeolite (5,50,100 mg/L) injected into egg albumin, The eggs were then incubated for 19 days at 60% humidity and 37.5 ° C. At the end of incubation time, the fetus and organs (liver, heart, brain, spleen) weight and congenital anomalies were investigated. Results: The administration of nano-zeolite in chick embryos as a model for evaluating human embryonic damage showed teratogenic effects including deformity of legs and wings, liver and heart disformation at the doses of 50,100 mg/L. The embryo were smaller and significant morphological anomalous changes were observed. The comparison between the three experimental groups showed that the dose of 5 mg/L improved viability of chicken and showed increasing the dose of zeolite increases teratogenic effects and increased fetal mortality rate. Conclusions: The teratogenic effects of nanozeolits on chick embryo should be considered in the risk assessment of nanoparticles on human embryo and fetous.

Objective: To determine the effects of chicken embryo brain extract (BE) on transects sciatic nerve in male rats.Methods: Thirty adult male Sprague-Dawley rats weighing 200 to 250 g, were randomized into three groups treated with (1) sham surgery, (2) normal saline (NS), and (3) brain extract (BE). The BE was taken from incubating chick embryos at day 8. The sciatic nerve was exposed and sharply transected at the mid thigh level. Immediate epineurial repair was then performed. The BE treated animals were given 400 µl/kg of the chick embryo BE intraperitoneal, once daily, for 2 weeks. All animals were evaluated by sciatic functional index (SFI), electrophysiology, histology, and immunohistochemistry at days 28, 90 after surgery.Results: The mean SFI difference between BE and NS groups at days 28, 60 and 90 after surgery was statistically significant (p=0.086). The mean number of myelinated fibers in the BE group was significantly greater than that of the NS group on days 28 and 90 after surgery (p=0.034). At days 28 and 90 after surgery, the mean nerve conduction velocity (NCV) in the BE group was significantly faster than that of the NS group (p=0.041).Conclusion: These results indicate for the first time that chick embryo brain extract can enhance peripheral nerve regeneration in rat.

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression. [ABSTRACT FROM AUTHOR]

[Objectives] The aim of the study was to establish a stable technique for isolation, purification and culture of chicken type II alveolar epithelial cells, and to provide an in vitro research model for investigation of the effects of air pollutants and other environmental stress factors on chicken alveolar function. [Methods] Digestion of lung tissue from 16-day-old chicken embryos was conducted with type I collagenase. After differential centrifugation, and differential attachment, chicken immunoglobulin G (lgG) immunoadsorption purification of cells was performed, and immunofluorescence detection and alkaline phosphatase were used for cell identification. [Results] The cells were adhered for 18-24 h, and they were polygonal in shape and grew like islands. After 24-72 h, the cells had proliferated and metabolized vigorously, the nucleus was obvious, the cytoplasm was rich, and the cells gradually connected to form a monolayer. After 72 h, the intracellular particulate matter decreased and the cell morphology changed. lmmuno-fluorescence detection showed the expression of epithelial cell marker cytokeratin-19(CK19). Alkaline phosphatase staining showed diffuse red or red-brown particles in the cytoplasm. [Conclusions] This method is an effective technique for the isolation, purification and culture of chicken alveolar epithelial cells with high cell yield and purity, which can satisfy general research in vitro. [ABSTRACT FROM AUTHOR]

This study was carried out to determine the effect of sodium nitroprusside (SNP), a nitric oxide (NO) exogenous donor, and NG-nitro-L-arginine methyl ester (L-NAME), a NO inhibitor, on the development of chicken embryos by injected into the egg. A total of 210 hatching fertilized chicken eggs used into control, Sham control (SC), serum physiological (SP) and treatment groups containing 30 eggs. The air space of the remaining eggs was pierced at the beginning of the incubation, whereas the eggs in the control group were not subjected to any application (negative control). The SC group of the drilled eggs was immediately closed with paraffin. After the SP group was called a positive control, 50 and 200 mg/kg SNP respectively and L-NAME solutions at the same levels were injected into the hole opened to the air space into the experimental groups consisting of the remaining eggs. The eggs were opened on some days 7th and 14th of the embryonic period and the following parameters of each group were determined: Relative embryo weights, weights and lengths of the embryos (on days 7th and 14th), hatching weight, hatching length, hatching rate and embryonal deaths (on day 21st). The results showed that the relative embryo weight and embryo length on the days 7th and 14th of the hatching and the hatching weight on the day 21st of the hatching were not affected by injection of SNP and L-NAME (P>0.05). Hatching rate on the day 21st was highest in the LNAME50 group, whereas this rate was lowest in the SNP200 group (P<0.05). Hatching length increased in the LNAME200 group (P<0.05). It was observed that NO was functional in embryonic development of poultry, and the increase in its metabolism decreased in the hatching rate increasing embryonal death. [ABSTRACT FROM AUTHOR]

Aiming at to solve the problem of inoculation failure caused by the cracks and the needle impact during the injection of chicken embryo vaccine, a mechanical model of impact during eggshell injection was established and analysied. First, the relationship between the resultant force per unit length of the injection hole and the initial injection velocity, the mass of the injection system and the cone angle of the needle was derived from the impulse theorem and the law of conservation of momentum. In order to verify the correctness of the mechanical model, and to further explore the relationship between eggshell deformation, stress and strain with taper angle, initial injection speed and mass of the injection system during the injection of chicken embryos, ANSYS software was used to simulate the impact of the needle on the eggshell. The simulation results verified the accuracy of the mechanical model, and it was preliminarily determined that the range of the initial injection velocity was 0.5-0.8 m/s, and the range of the injection system mass was 0.5-1.2 kg. The needle cone angles are 10°and 15°. Second, in order to complete the chicken embryo injection test, a chicken embryo injection and micro-crack detection device was established. The device consists of the high-speed camera, injection system, buffer mechanism and crack detection device of the injection hole of chicken embryo. The high-speed camera can measure the initial speed of injection, and the injection system can realize the injection of chicken embryo, even can change the mass of the injection system arbitrarily, and select different specifications the needle can change the cone angle, and the buffer mechanism can effectively avoid the crack of chicken embryo due to vibration. The crack detection device is mainly composed of CCD industrial camera, egg support and cold light source, which can ensure the accurate identification of chicken embryo crack and the smooth progress of injection parameter optimization test. Furthermore, an orthogonal experiment was designed to extract the characteristics of the injection hole cracks with image processing technology, and calculate the injection hole crack effective area index and the crack effective contour index, judgment whether the injection of cracked egg embryos and intact egg embryos. Eggshell was a micro porous structure, and the permeability of the eggshell was inconsistent. The eggshell need to be irradiated with a cold light source during the image processing, and the collected source image has bright spots. Eggshell bright spots cannot be effectively removed, and effective crack features cannot be retained using traditional threshold segmentation algorithm. Therefore, an eggshell bright spot elimination algorithm was used to retain the crack features of the chicken embryo injection hole and remove the bright spots to the maximum extent. Finally, a three-factor two-level interaction analysis of variance method was used to determine the optimal injection parameter combination: the initial injection velocity was 0.6 m/s, the needle cone angle was 10°, and the injection system mass was 1.0 kg. 7-14 day-old chicken embryos were used to test under the optimized parameters, the injection success rate was 98.75%. [ABSTRACT FROM AUTHOR]

Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-offunction experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (Lzts1), the expression of which is also activated by HoxB4 and HoxB9. Gain- and loss-of-function experiments showed that Lzts1, which is expressed endogenously in the IZ, controls neuronal delamination. These data collectively indicate that HoxB genes have a generic function in the developing spinal cord, controlling the expression of Lzts1 and neuronal delamination. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance during development and disease, including muscle disease. Advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species, however, for many of these miRNAs specific roles have not yet been determined. LNA in situ hybridisation has revealed expression patterns of somite-enriched miRNAs, here we focus on characterising the functions of miR-128. We show that antagomiR-mediated knockdown (KD) of miR-128 in developing chick somites has a negative impact on skeletal myogenesis. Computational analysis identified the transcription factor EYA4 as a candidate target consistent with the observation that miR-128 and EYA4 display similar expression profiles. Luciferase assays confirmed that miR-128 interacts with the EYA4 3′UTR. In vivo experiments also suggest that EYA4 is regulated by miR-128. EYA4 is a member of the PAX-SIX-EYA-DACH (PSED) network of transcription factors. Therefore, we identified additional candidate miRNA binding sites in the 3′UTR of SIX1/4 , EYA1/2/3 and DACH1. Using the miRanda algorithm, we found sites for miR-128, as well as for other myogenic miRNAs, miR-1a, miR-206 and miR-133a, some of these were experimentally confirmed as functional miRNA target sites. Our results reveal that miR-128 is involved in regulating skeletal myogenesis by directly targeting EYA4 with indirect effects on other PSED members, including SIX4 and PAX3. Hence, the inhibitory effect on myogenesis observed after miR-128 knockdown was rescued by concomitant knockdown of PAX3. Moreover, we show that the PSED network of transcription factors is co-regulated by multiple muscle-enriched microRNAs. • miR-128 directly targets EYA4 in vitro and in vivo in developing somites. • Knockdown of miR-128 inhibits myogenesis indicated by loss of MYOD1 expression. • miR-128 knockdown deregulates EYA4 expression and other members of the PSED network, including PAX3. • The negative effect of miR-128 knockdown on myogenesis is restored by inhibition of PAX3. [ABSTRACT FROM AUTHOR]

Vitamin A mediates many important biological functions in humans and animals. Presence of vitamin A receptors on immune system cells emphasizes their role in immune functions. To assess the effects of in ovo inoculation of vitamin A on the immune system of chicken embryos, 18 days old embryonated eggs were inoculated with 3 different concentrations of retinoic acid (the active form of vitamin A) at 30, 90, and 270 μmol/egg via the amniotic sac. After 6, 18, and 24 h, the spleen and bursa of the embryos were collected for RNA extraction and real-time polymerase chain reaction. The results were dose dependant. After 24 h, inoculation with 270 μmol/egg downregulated relative expression of interferon IFN-α, IFN-β, IFN-γ, interleukin (IL)-1β, IL-2, CXCLi2, IL-12, and IL-13 compared to control in the spleen, indicating an anti-inflammatory effect at this concentration. In comparison, 90 μmol/egg induced greater expressions of the above genes at the same timepoint compared to the 270 μmol. The results of this study indicate that in ovo inoculation of vitamin A can modulate immune functions of the chicken embryo, which might be beneficial for induction of immune responses by in ovo vaccines. [ABSTRACT FROM AUTHOR]

Cell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18–HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail. [ABSTRACT FROM AUTHOR]

This work aimed to evaluate the antimicrobial effect of Ganoderma lucidum mycelial growth filtrates (MGF) on the phytopathogen Erysiphe diffusa and their potential effects on the embryonic development of Gallus gallus. The antimicrobial activity was evaluated on E. diffusa spores by the microdilution broth method. To evaluate embryotoxic and teratogenic effects, fertile eggs of G. gallus received injections of solutions containing the filtrates of G. lucidum through the air chamber. After three days of incubation, we opened the eggs and evaluated egg viability, embryo survival, malformation occurrence, embryonic staging and heart rate. Live embryos were prepared using whole mount technique and the morphological analysis was performed. We used the generalized linear model to fit embryotoxicity and teratogenicity data. We verified that G. lucidum MGF showed inhibitory activity in vitro against E. diffusa and the minimum inhibitory concentrations ranged from 5 to 10 mg/mL. We could also observe that the filtrates did not present embryotoxic or teratogenic effects on the early embryonic development of G. gallus, but induced significant differences in the embryonic mean heart rate and on the stage of embryonic development. [ABSTRACT FROM AUTHOR]

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In this study, we examined the effect of sodium fluoride (NaF) on oxidative stress in chicken embryonic gonads. Following exposure to varying concentrations of NaF for 6 h, mRNA expression and immunolocalisation of catalase (CAT), sodium dismutase (SOD1 and SOD2) and nuclear respiratory factors (Nrf1 and Nrf) were analysed in the gonads. In the ovary, a dose-dependent increase in mRNA expression of CAT , Nrf1 and Nrf2 following NaF exposure was found, while the intensity of immunolocalised CAT, SOD2 and Nrf1 was higher in NaF-treated groups. In the testis, no effect of NaF on CAT , SOD1 and Nrf1 mRNA levels was observed; however, NaF (3.5–14.2 mM) elevated Nrf2 mRNA expression. NaF, at a dose of 7.1 mM, increased the immunoreactivity of Nrf1 and SOD2. Further experiments evaluated the ovary and testes when incubated with NaF (7.1 mM), vitamin C (Vitamin C, 4 mM) or NaF + Vitamin C. mRNA expression of all four examined genes in the whole ovary and immunoreactivity of Nrf1 and CAT in the ovarian medulla increased in each experimental group. Similar effects were observed in the testis, where mRNA expression, as well as CAT and Nrf2 immunoreactivity, increased in Vitamin C and NaF + Vitamin C-treated groups. In summary, NaF exposure generated oxidative stress which is manifested by increased expression of free radical scavenging enzymes in chicken embryonic gonads. High doses of Vitamin C did not reverse this effect. • Short-term exposure of chicken gonads to NaF generates oxidative stress. • NaF increases expression of antioxidant enzymes (CAT and SOD) and Nrfs primarily in the ovary. • High doses of Vit C do not attenuate gonadal oxidative stress evoked by NaF exposure. [ABSTRACT FROM AUTHOR]

Objectives: Bisphenol A (BPA) is one of the most heavily produced chemicals in the world. BPA is involved in the production of many substances such as cosmetics, various foodstuffs, toys, personal care products, detergents and plastic bottles all that are frequently used in daily life. Depending on BPA exposure, sexual maturation and reproductive function, and bone and brain development are adversely affected. The aim of this study is to investigate the possible effects of BPA on the development of the nervous system and neural tube in 48‐hr chicken embryos. Methods: Thirty specific pathogen‐free (SPF) fertilized eggs were used in the study. SPF eggs were placed in the incubator and divided into three groups at 28 hr of incubation; control, BPA 1 and BPA 2 (10 eggs in each group). At this stage of incubation, two different doses of BPA were injected sub‐blastodermically with the Hamilton microinjector. At the end of 48 hr of incubation, all eggs were opened and embryos were dissected and separated from the embryonic membrane. All embryos were evaluated morphologically and histopathologically. Results: As the BPA dose increased, delays in the development of the nervous system and midline closure increased in the early period of chicken embryos. Depending on the dose, it was found that the embryo's crown‐rump length and somite number decreased (p <.05). Conclusion: It was determined that BPA application on early chicken embryos adversely affected neural tube development. It was also found to delay midline closure. [ABSTRACT FROM AUTHOR]

Intensive selection in modern lines of fast-growing chickens has caused several skeletal disorders. Therefore, current research is focused on methods to improve the bones of birds. A new potential solution is in ovo technology using nanoparticles with a high specificity for the bone tissue. Thus, the objective of the present study was to evaluate the effect of in ovo application of hydroxyapatite nanoparticles (HA-NP) in different concentrations (50, 100 and 500 μg/ml colloids) on chicken embryo development, with a particular focus on the oxidative status and bone characteristics of the embryo. The results showed that in ovo treatment with HA-NP did not negatively affect hatchability and body weight. However, bone weight was reduced in 500 μg/ml group. The concentrations of calcium, phosphorus and crude ash were not affected. The modulatory effect of HA-NP was observed on the basis of antioxidative markers – superoxide dismutase, total antioxidant status, malondialdehyde in serum and selected tissues. Glutathione concentration in serum suggested higher metabolic stress. Among bone turnover markers, the concentration of osteocalcin was found to be significantly affected by HA-NP injection. Thus, the in ovo application of HA-NP could modify the molecular responses at the stage of embryogenesis but these changes were not reflected in embryo growth and even slowed down bone development. Nevertheless, the question for the follow-up research is whether in ovo administration of HA-NP would affect the antioxidative status and bone turnover resulting in improved bone conditions and body gain in post hatch chickens. [ABSTRACT FROM AUTHOR]

Prenatal treatment with magnesium sulfate (MgSO4) has neuroprotective effects in very preterm infants but its use has been associated with an increased rate of patent ductus arteriosus (DA). MgSO4 is a vasodilator and thus may exert a direct relaxant effect in the DA. We aimed to investigate the vasoactive effects of MgSO4 in the DA using the chicken embryo as experimental model. DA rings from 15-d (E15), 17-d (E17) and 19-d (E19) chicken embryos (total incubation: 21-d) were mounted in a wire myograph for isometric tension recordings. Exposure of DA rings to 21% O2 induced a tonic contraction which was relaxed by MgSO4 (2.4 -- 7.2 mmol L--1) in a concentration-dependent manner (mean maximal relaxation E19: 51.4%, SE 6.3; EC50: 3.5 mmol L--1, SE 0.7). The relaxation evoked by MgSO4 was not significantly different between E15, E17 and E19 DA and was not affected by removal of the endothelium or by the presence of the nitric oxide synthase inhibitor L-NAME, the soluble guanylate cyclase inhibitor ODQ, or the cyclooxygenase inhibitor indomethacin. In contrast, when the DA rings were incubated in Ca2+-free solution, or in the presence of inhibitors of L-type Ca2+ channels (nifedipine), or largeconductance Ca2+-activated K+ (BKCa) channels (iberiotoxin), MgSO4-induced relaxation was impaired. Preincubation of the DA rings with MgSO4 concentrations ranging from 0 to 6.0 mmol L--1 did not significantly affect O2-induced contraction that was only impaired by a concentration of 7.2 mmol L--1. In conclusion, MgSO4 induced endothelium-independent relaxation of chicken DA and this relaxation appeared to be mediated through stimulation of BKCa channels and blockade of transmembrane flux of extracellular Ca2+. However, O2-induced DA contraction was only impaired by suprapharmacological concentrations of MgSO4 (> 6.0 mmol L--1). Therefore, our data suggest that the higher incidence of patent DA in preterm infants exposed to MgSO4 is unlikely to be due to a direct pharmacological effect of the drug on the DA. [ABSTRACT FROM AUTHOR]

Background: Müllerian ducts are paired embryonic tubes that give rise to the female reproductive tract in vertebrates. Many disorders of female reproduction can be attributed to anomalies of Müllerian duct development. However, the molecular genetics of Müllerian duct formation is poorly understood and most disorders of duct development have unknown etiology. In this study, we describe for the first time the transcriptional landscape of the embryonic Müllerian duct, using the chicken embryo as a model system. RNA sequencing was conducted at 1 day intervals during duct formation to identify developmentally-regulated genes, validated by in situ hybridization. Results: This analysis detected hundreds of genes specifically up-regulated during duct morphogenesis. Gene ontology and pathway analysis revealed enrichment for developmental pathways associated with cell adhesion, cell migration and proliferation, ERK and WNT signaling, and, interestingly, axonal guidance. The latter included factors linked to neuronal cell migration or axonal outgrowth, such as Ephrin B2, netrin receptor, SLIT1 and class A semaphorins. A number of transcriptional modules were identified that centred around key hub genes specifying matrix-associated signaling factors; SPOCK1, HTRA3 and ADGRD1. Several novel regulators of the WNT and TFG-β signaling pathway were identified in Müllerian ducts, including APCDD1 and DKK1, BMP3 and TGFBI. A number of novel transcription factors were also identified, including OSR1, FOXE1, PRICKLE1, TSHZ3 and SMARCA2. In addition, over 100 long non-coding RNAs (lncRNAs) were expressed during duct formation. Conclusions: This study provides a rich resource of new candidate genes for Müllerian duct development and its disorders. It also sheds light on the molecular pathways engaged during tubulogenesis, a fundamental process in embryonic development. [ABSTRACT FROM AUTHOR]

Particulate matter poses health risk to developing organisms. To investigate particulate matters with a diameter smaller than 2.5 um (PM2.5)-induced developmental cardiotoxicity, fertile chicken eggs were exposed to PM2.5 via air cell injection at doses of 0.05, 0.2, 0.5, 2, and 5 mg/egg kg. Morphological changes in the embryonic day four (ED4) and hatchling hearts were assessed with histological techniques. Heart rates of hatchling chickens were measured with electrocardiography. The protein expression levels of nuclear factor kappa-light-chain-enhancer of activated B cells p65 (NF-kb p65), inducible nitric oxide synthase (iNOS), and matrix metallopeptidase 9 (MMP9) were assessed with immunohistochemistry or western blotting in hatchling hearts. PM2.5 exposure elevated areas of heart in ED4 embryo, increased heart rate, and thickened right ventricular wall thickness in hatchling chickens. Immunohistochemistry revealed enhanced NF-kb p65 expression in hatchling hearts. Western blotting results indicated that both iNOS and MMP9 expression were enhanced by lower doses of PM2.5 exposure (0.2 and 0.5 mg/kg) but not 2 mg/kg. In summary, developmental exposure to PM2.5 induced developmental cardiotoxicity in chicken embryo and hatchling chickens, which is associated with NF-kb p65, iNOS, and MMP9. [ABSTRACT FROM AUTHOR]

This work was designed to investigate incidence of P. aeruginosa in dead in shell chicken embryos and molecular detection of virulence genes and study their pathogenicity effect on embryonated chicken eggs and chicks. Out of 100 pooled samples collected from liver, heart and yolk sac (as one mixed pool/sample) of dead in shell chicken embryos, a total of 24 (24%) P. aeruginosa isolates were isolated. On carrying PCR test for 24 P. aeruginosa isolates there were high distribution of (opr L) and ( lasI ) genes with 100% while distribution of other virulence genes was as follows: toxA (83.3%), lasR (75%), Rhl1 (70.8%). Pathogenicity of P. aeruginosa on embryonated chicken eggs was studied either by inoculation of P. aeruginosa strain via yolk sac route in a dose of 0.1 ml suspension 14x107. Which gave 100% embryonic mortalities after 3 days or by dipping group of 17 days -old embryonated chicken eggs in a dose of 6.1X 109 /ml which gave 90% embryonic mortality. Experimentally infected chicks showed 70% mortality. Histopathological changes in dead embryos from infected embryonated eggs and sacrificed, dead infected chicks revealed lymphocytic hepatitis with presence of gram negative bacteria. Lymphocytic nephritis with hemosiderosis found in renal parenchyma. Heart showed lymphocytic myocarditis. Lung showed alveolar emphysema, interstitial pneumonia and perivascular fibrosis. P. aeruginosa was re-isolated from internal organs of sacrificed hatched chicks and from liver and yolk sac of dead embryos. [ABSTRACT FROM AUTHOR]

In amniotes, unlike primary neurulation in the anterior body, secondary neurulation (SN) proceeds along with axial elongation by the mesenchymal-to-epithelial transition of SN precursors in the tail bud. It has been under debate whether the SN is generated by neuromesodermal common progenitor cells (NMPs) or neural restricted lineage. Our direct cell labeling and serial transplantations identify uni-fated (neural) precursors in the early tail bud. The uni-fated SN precursor territory is further divided into two subpopulations, neural-differentiating and self-renewing cells, which are regulated by high- and low levels of Sox2, respectively. Unexpectedly, uni-fated SN precursors change their fate at later stages to produce both SN and mesoderm. Thus, chicken embryos adopt a previously unappreciated prolonged phase with uni-fated SN stem cells in the early tail bud, which is absent or very limited in mouse embryos. [ABSTRACT FROM AUTHOR]

Acute exposure to hypoxic conditions is a frequent natural event during the development of bird eggs. However, little is known about the effect of such exposure on the ability of young embryos in which cardiovascular regulation is not yet developed to maintain a normal heart rate (HR). To address this question, we studied the effect of 10–20 min of exposure to moderate or severe acute hypoxia (10% or 5% O2, respectively) on the HR of day 4 (D4) chicken embryos. In ovo, video recording of the beating embryo heart inside the egg revealed that severe, but not moderate, hypoxia resulted in significant HR changes. The HR response to severe hypoxia consisted of two phases: the first phase, consisting of an initial decrease in HR, was followed by a phase of partial HR recovery. Upon the restoration of normoxia, after an overshoot period of a few minutes, the HR completely recovered to its basal level. In vitro (isolated heart preparation), the first phase of the HR response to severe hypoxia was strengthened (nearly complete heart silencing) compared to that in ovo, and the HR recovery phase was greatly attenuated. Furthermore, neither an overshoot period nor complete HR recovery after hypoxia was observed. Thus, the D4 chicken embryo heart can partially maintain its rhythm during hypoxia in ovo, but not in vitro. Some factors from the egg, such as catecholamines, are likely to be critical for avian embryo responding to hypoxic condition and survival. [ABSTRACT FROM AUTHOR]

The transcription factor (TF) SOX2 regulates various stem cells and tissue progenitors via functional interactions with cell type‐specific partner TFs that co‐bind to enhancer sequences. Neural progenitors are the major embryonic tissues where SOX2 assumes central regulatory roles. In order to characterize the partner TFs of SOX2 in neural progenitors, we investigated the regulation of the D1 enhancer of the Sox2 gene, which is activated in the embryonic neural tube (NT) and neural crest (NC), using chicken embryo electroporation. We identified essential TF binding sites for a SOX, and two ZIC TFs in the activation of the D1 enhancer. By comparison of dorso‐ventral and antero‐posterior patterns of D1 enhancer activation, and the effect of mutations on the enhancer activation patterns with TF expression patterns, we determined SOX2 and ZIC2 as the major D1 enhancer‐activating TFs. Binding of these TFs to the D1 enhancer sequence was confirmed by chromatin immunoprecipitation analysis. The combination of SOX2 and ZIC2 TFs activated the enhancer in both the NT and NC. These results indicate that SOX2 and ZIC2, which have been known to play major regulatory roles in neural progenitors, do functionally cooperate. In addition, the recently demonstrated SOX2 expression during the NC development is accounted for at least partly by the D1 enhancer activity. Deletion of the D1 enhancer sequence from the mouse genome, however, did not affect the mouse development, indicating functional redundancies of other enhancers. [ABSTRACT FROM AUTHOR]