Nutritional supplementation of breeding hens may promote embryonic development through the growth hormone-insulin like growth factor axis.

The late stage of embryo development is a crucial period of metabolic changes, with rapid organ development requiring a substantial supply of nutrients. During this phase, maternal nutritional levels play a vital role in the growth, development, and metabolism of the offspring. In this study, we added 2 doses of ß-carotene (ßc) (120 mg/kg and 240 mg/kg) to the daily diet of Hailan Brown laying hens to investigate the impact of maternal nutritional enrichment on embryo development. Maternal nutrition supplementation significantly increased the expression of chicken embryo liver index, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF) in serum. At the same time, the expression of GH/growth hormone receptor (GHR), IGF-1 mRNA, and Proliferating Cell Nuclear Antigen (PCNA) protein in the liver was upregulated, indicating that maternal nutrition intervention may promote chicken embryo liver development through the GH-IGF-1 axis. Transcriptome sequencing results showed that differential genes in liver after maternal nutritional supplementation with ß-carotene were enriched in pathways related to cell proliferation and metabolism. Consequently, we postulated that maternal ß-carotene supplementation might operate via the GH-IGF-1 axis to regulate the expression of genes involved in growth and development, thereby promoting liver development. These results contribute to formulating more effective poultry feeding strategies to promote offspring growth and development.

A Comparative Analysis of the Cytotoxic and Vascular Activity Effects of Western Diamondback Rattlesnake (Crotalus atrox) and Eastern Diamondback Rattlesnake (Crotalus adamanteus) Venoms Using a Chick Embryo Model.

Crotalus snakebites induce various toxicological effects, encompassing neurological, myotoxic, and cytotoxic symptoms, with potentially fatal outcomes. Investigating venom toxicity is essential for public health, and developing new tools allows for these effects to be studied more comprehensively. The research goals include the elucidation of the physiological consequences of venom exposure and the assessment of toxicity using animal models. Chicken embryos serve as valuable models for assessing venom toxicity through the chick embryotoxicity screening test (CHEST) and the chick chorioallantoic membrane (CAM) assay, particularly useful for evaluating vascular impacts. C. adamanteus venom application resulted in higher embryotoxicity and morphological abnormalities, such as Siamese twins. The CAM assay demonstrated the hemorrhagic effects of venom, varying with venom type and concentration. The irritant potential of both venom types was classified as slight or moderate depending on their concentration. Additionally, acetylcholinesterase (AChE) activity was performed to receive information about organ toxicity. The results show that both venoms induced changes in the whole embryo, heart, and liver weights, but the C. adamanteus venom was identified as more toxic. Specific venom concentrations affected AChE activity in embryonic tissues. These findings underscore the embryotoxic and vasoactive properties of Crotalus venoms, providing valuable insights into their mechanisms of toxicity and potential applications in biomedicine.

Lignin-derivable alternatives to bisphenol A with potentially undetectable estrogenic activity and minimal developmental toxicity.

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.

Retinoic Acid-Mediated Control of Energy Metabolism Is Essential for Lung Branching Morphogenesis.

Lung branching morphogenesis relies on intricate epithelial-mesenchymal interactions and signaling networks. Still, the interplay between signaling and energy metabolism in shaping embryonic lung development remains unexplored. Retinoic acid (RA) signaling influences lung proximal-distal patterning and branching morphogenesis, but its role as a metabolic modulator is unknown. Hence, this study investigates how RA signaling affects the metabolic profile of lung branching. We performed ex vivo lung explant culture of embryonic chicken lungs treated with DMSO, 1 µM RA, or 10 µM BMS493. Extracellular metabolite consumption/production was evaluated by using 1H-NMR spectroscopy. Mitochondrial respiration and biogenesis were also analyzed. Proliferation was assessed using an EdU-based assay. The expression of crucial metabolic/signaling components was examined through Western blot, qPCR, and in situ hybridization. RA signaling stimulation redirects glucose towards pyruvate and succinate production rather than to alanine or lactate. Inhibition of RA signaling reduces lung branching, resulting in a cystic-like phenotype while promoting mitochondrial function. Here, RA signaling emerges as a regulator of tissue proliferation and lactate dehydrogenase expression. Furthermore, RA governs fatty acid metabolism through an AMPK-dependent mechanism. These findings underscore RA's pivotal role in shaping lung metabolism during branching morphogenesis, contributing to our understanding of lung development and cystic-related lung disorders.

Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine.

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Determination of the embryotoxic effects of propofol injected into eggs on the cerebellum and spinal cord using histologic methods: an animal study.

Background/aim: This study aims to determine the possible embryotoxic effects of propofol on the cerebellum and spinal cord using fertile chicken eggs. Materials and methods: A total of 430 fertile eggs were divided into 5 groups: control, saline, 2.5 mg.kg-1, 12.5 mg.kg-1, and 37.5 mg.kg-1 propofol. Injections were made immediately before incubation via the air chamber. On the 15th, 18th, and 21st day of incubation, 6 embryos from each group were evaluated. Serial paraffin sections taken from the cerebellum and spinal cord were stained with hematoxylin-eosin, Kluver-Barrera, toluidine blue, and periodic acid-Schiff's reaction. The outer granular layer and total cortex thickness were measured, and the linear density of the Purkinje cells was determined. The ratios of the substantia grisea surface area to the total surface area of the spinal cord were calculated. The transverse and longitudinal diameters of the canalis centralis were also assessed. Results: No structural malformation was observed in any embryos examined macroscopically. No significant difference was observed between the groups in terms of development and histologic organization of the cerebellum and spinal cord. However, on the 15th, 18th, and 21st day, the outer granular layer (p < 0.001 for all days) and the total cortex thickness (p < 0.01, p < 0.001, and p < 0.001, respectively) decreased significantly in different propofol dose groups in varying degrees in the cerebellum. Similarly, in the spinal cord, there were significant changes in the ratios of the substantia grisea surface area to the total surface area (p < 0.01 and p < 0.001, respectively). Conclusion: It was concluded that the in-ovo-administered propofol given immediately before incubation has adverse effects on the developing cerebellum and spinal cord. Therefore, it is important for anesthesiologists always to remain vigilant when treating female patients of childbearing age.

Persistent metabolic toxicities following developmental exposure to hexafluoropropylene oxide trimer acid (HFPO-TA): Roles of peroxisome proliferator activated receptor gamma.

BACKGROUND: Hexafluoropropylene oxide trimer acid (HFPO-TA), a perfluorooctanoic acid (PFOA) substitute, exhibited strong affinity and capability to activate peroxisome proliferator activated receptor gamma (PPARγ), a lipid metabolism regulator, suggesting potential to induce metabolic toxicities. METHODS: Fertile chicken eggs were exposed to 0, 0.5, 1 or 2 mg/kg (egg weight) HFPO-TA and incubated until hatch. Serum from 0- and 3- month-old chickens were subjected to liquid chromatography ultra-high resolution mass spectrometry for HFPO-TA concentration, while liver, pancreas and adipose tissue samples were collected for histopathological assessments. In ovo PPARγ reporter and silencing system were established with lentivirus microinjection. qRT-PCR and immunohistochemistry were utilized to evaluate the expression levels of PPARγ downstream genes. RESULTS: In 3-month-old animals developmentally exposed to HFPO-TA, adipose tissue hyperplasia, hepatic steatosis, pancreas islet hypertrophy and elevated serum free fatty acid / insulin levels were observed. Results of reporter assay and qRT-PCR indicated HFPO-TA-mediated PPARγ transactivation in chicken embryo. Silencing of PPARγ alleviated HFPO-TA-induced changes, while PPARγ agonist rosiglitazone mimicked HFPO-TA-induced effects. qRT-PCR and immunohistochemistry revealed that FASN and GPD1 were upregulated following developmental exposure to HFPO-TA in 3-month-old animals. CONCLUSIONS: Developmental exposure to HFPO-TA induced persistent metabolic toxicities in chickens, in which PPARγ played a central role.

The effect of methionine and folic acid administered in ovo on the blood biochemical parameters of chickens (Gallus gallus domesticus).

Methionine is one of the most frequently supplemented amino acids in raising of poultry. However, an overdose of methionine can cause hyperhomocysteinemia. Folic acid, taking part in the process of homocysteine remethylation, is a factor affecting the reduction of the concentration of this amino acid. The study was carried out in 2 stages. The experiment of step I was to investigate the effect of methionine and/or folic acid administration in ovo in the early stage of embryogenesis (E4), and the experiment of the second stage - in the late stage of embryogenesis (E17) on the following biochemical parameters of chicken blood: glucose concentration in whole blood and concentration of homocysteine and uric acid in plasma of domestic chickens (Gallus gallus domesticus). Our results confirm that methionine supplementation may increase the concentration of uric acid and homocysteine. Moreover, we demonstrated that folic acid administered during embryogenesis decreased homocysteine concentration, also in groups simultaneously supplemented with methionine, especially in the initial stage of postnatal life of the bird.

Dysregulated VEGF/VEGFR-2 Signaling and Plexogenic Lesions in the Embryonic Lungs of Chickens Predisposed to Pulmonary Arterial Hypertension.

Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.

Effects of Resveratrol on In Vivo Ovarian Cancer Cells Implanted on the Chorioallantoic Membrane (CAM) of a Chicken Embryo Model.

Ovarian cancer poses a significant threat to patients in its advanced stages, often with limited treatment options available. In such cases, palliative management becomes the primary approach to maintaining a reasonable quality of life. Therefore, the administration of any medication that can benefit patients without a curative option holds potential. Resveratrol, a natural compound known for its in vitro anticancer activities, has generated contrasting results in vivo and human studies. In this study, we aimed to assess the anticancer effects of resveratrol on ovarian cancer cells grown on the chorioallantoic membrane (CAM) of chicken embryos. Two ovarian cancer cell lines, OVCAR-8 and SKOV-3, were cultured in collagen scaffolds for four days before being implanted on the CAM of chicken embryos on day 7. Different doses of resveratrol were applied to the CAM every two days for six days. Subsequently, CAM tissues were excised, fixed, and subjected to histological analysis. Some CAM tumours were extracted to analyse proteins through Western blotting. Our findings indicate that specific doses of resveratrol significantly reduce angiogenic activities, pNF-κB levels, and SLUG protein levels by using immunohistochemistry. These results suggest that resveratrol may have the potential to impact the behaviour of ovarian cancer CAM tumours, thereby warranting further consideration as a complementary treatment option for women with incurable ovarian cancer.

Overview of chicken embryo genes related to sex differentiation.

Sex determination in chickens at an early embryonic stage has been a longstanding challenge in poultry production due to the unique ZZ:ZW sex chromosome system and various influencing factors. This review has summarized the genes related to the sex differentiation of chicken early embryos (mainly Dmrt1, Sox9, Amh, Cyp19a1, Foxl2, Tle4z1, Jun, Hintw, Ube2i, Spin1z, Hmgcs1, Foxd1, Tox3, Ddx4, cHemgn and Serpinb11 in this article), and has found that these contributions enhance our understanding of the genetic basis of sex determination in chickens, while identifying potential gene targets for future research. This knowledge may inform and guide the development of sex screening technologies for hatching eggs and support advancements in gene-editing approaches for chicken embryos. Moreover, these insights offer hope for enhancing animal welfare and promoting conservation efforts in poultry production.

Effectiveness of Rhenium(I)-diselenoether Low Doses in a Triple-negative Breast Cancer Chicken Embryo Model.

BACKGROUND/AIM: Rhenium(I)-diselenoether (Re-diSe) is a promising anticancer agent composed of one rhenium and two selenium atoms. Its effectiveness was established in inhibiting cancer cells while maintaining low toxicity toward normal cells at a 5 µM dose for 120 hours in MDA-MB-231 cells. In MDA-MB-231 breast tumor-bearing mice, anti-tumor and anti-metastatic effects were observed at a 10 mg/kg dose. However, contradictory results were observed in the 4T1 breast cancer model, where a dose of 60 mg/kg had a pro-tumor effect. To address these discrepancies, the efficacy of Re-diSe at the effective 10 mg/kg dose was validated in a transplanted MDA-MB-231 breast tumor model using the chicken chorioallantoic membrane assay. MATERIALS AND METHODS: MDA-MB-231 cancer cells were xenografted onto the chicken chorioallantoic membrane (CAM), and daily drug administration was carried out for nine days at doses of 0.1, 1, and 10 mg/kg. At the study's conclusion, a standard histological analysis was conducted. RESULTS: The low dose of 0.1 mg/kg showed a significant reduction in tumor weights compared to controls. The 1 mg/kg dose resulted in an increased inflammation score but did not induce a significant difference in tumor weights compared to the 0.1 mg/kg dose. Notably, at the 10 mg/kg dose, six out of 11 treated embryos displayed no visible signs of tumors. These tumors exhibited extensive tumor necrosis and significant infiltration by inflammatory cells. CONCLUSION: In this particular model, the anticancer efficacy of Re-diSe was achieved at the low dose of 0.1 mg/kg. The higher dose of 10 mg/kg, while eliminating visible tumors, might have immune-mediated effects, as indicated by substantial tumor necrosis and infiltration by inflammatory cells. Overall, this study successfully demonstrated the effectiveness of Re-diSe as an anticancer agent.

Evaluation of chicken chorioallantoic membrane model for tumor imaging and drug development: Promising findings.

BACKGROUND: The chicken chorioallantoic membrane (CAM) model is a potential alternative to the mouse model based on the 3R principles. However, its value for determination of the in vivo behaviors of radiolabeled peptides through positron emission tomography (PET) imaging needed investigation. Herein, the chicken CAM tumor models were established, and their feasibility was evaluated for evaluating the imaging properties of radiolabeled peptides using a 68 Ga-labeled HER2 affibody. METHODS: Two human breast cancer cell lines were inoculated into chicken CAM and mice, respectively. The tumor-targeting potential and pharmacokinetic profile of a 68 Ga-labeled affibody, 68 Ga-MZHER, in both tumor models were also determined. RESULTS: The tumor-formation time in chicken CAM model was shorter than that of mouse model. The uptake values of human epithelial growth factor receptor-2 (HER2)-positive Bcap37 tumors in chicken CAM and mouse models were 5.36 ± 0.26% ID/g and 5.26 ± 0.43% ID/g at 30 min postinjection of 68 Ga-MZHER, respectively. At the same time points, the uptake values of HER2-negative MDA-MB-231 tumors in the chicken CAM models and mouse models were 1.57 ± 0.15% ID/g and 1.67 ± 0.25% ID/g, respectively. Ex vivo biodistribution confirmed that more radioactivity accumulated in Bcap37 tumors than in MDA-MD-231 tumors in both CAM and mouse models. CONCLUSION: In this study, the CAM tumor model was successfully prepared. The chicken CAM model is a novel tool for quickly determining the in vivo properties of radiolabeled peptides targeting biomarkers. It may be beneficial for early monitoring of the therapeutic effect of a new drug through PET imaging with specific peptides.

Inflammatory responses and barrier disruption in the trachea of chicks following Mycoplasma gallisepticum infection: a focus on the TNF-α-NF-κB/MLCK pathway.

Mycoplasma gallisepticum (MG) can induce persistent inflammatory damage to the tracheal mucosa of poultry and cause chronic respiratory diseases in chickens. To further investigate the mechanism of MG-induced injury to the tracheal mucosa, we used chick embryo tracheal organ culture (TOC) as a model to study the invasion and reproduction of MG, the effect of MG on tracheal morphology, and the potential factors that promote MG tissue invasion. The results showed that MG infection significantly damaged the tracheal epithelial structure and weakened tracheal epithelial barrier function; MG also increased the occurrence of bacterial displacement, with a significant (p < 0.05) increase in the bacterial load of the infected TOCs at 5 and 7 days post-infection. In addition, MG significantly (p < 0.05) increased the expression levels of inflammatory cytokines, such as TNF-α, interleukin-1ß (IL-1ß), and IL-6, and activated the NF-κB signalling pathway, leading to increased nuclear translocation of NF-κB p65. Simultaneously, the map kinase pathway (MAPK) was activated. This activation might be associated with increased myosin light chain (MLC) phosphorylation, which could lead to actin-myosin contraction and disruption of tight junction (TJ) protein function, potentially compromising epithelial barrier integrity and further catalysing MG migration into tissues. Overall, our results contribute to a better understanding of the interaction between MG and the host, provide insight into the mechanisms of damage to the tracheal mucosa induced by MG infection, and provide new insights into the possible pathways involved in Mycoplasma gallisepticum infection in vivo.

Comamonas testosteroni as the cause of mortality in embryonated chicken eggs of breeding broiler hens in Costa Rica.

Mortality of chicken embryos and first-week chickens was reported in a commercial incubator company in Costa Rica. Six 1-day-old Cobb chickens and twenty-four embryonated chicken eggs were examined in the Laboratory of Avian Pathology and the Laboratory of Bacteriology of the National University of Costa Rica. Twelve dead-in-shell embryos showed maceration and were immersed in a putrid, turbid, slightly thick brown liquid. Additionally, the other 12 embryonated eggs had milky yellow-orange content. The livers of those embryos had congestion, haemorrhages and multifocal cream foci of necrosis. Granulocytic infiltration was observed in the bursa of Fabricius, myocardium, liver, lung and kidney. Livers and egg yolks from six embryonated chickens and all 1-day-old chickens were aseptically collected and cultured. In addition, tissues from six better conserved embryos and all 1-day-old chickens were fixed in buffered formalin and embedded in paraffin. Biochemical and molecular tests identified Comamonas testosteroni as the cause of the early, middle and late embryo mortality. As all the eggshells from the sampled embryonated eggs were dirty with soiled a fecal matter, contamination after manipulating the eggs was considered the source of infection. C. testosteroni is an environmental microorganism that has rarely been reported to cause human disease. To our knowledge, this is the first report of C. testosteroni causing mortality in a hatchery. Cleaning and disinfection using ozone were implemented in the hatchery to eliminate the embryo mortality associated with C. testosteroni.

Glycerol monolaurate improves intestinal morphology and antioxidant status by suppressing inflammatory responses and nuclear factor kappa-B signaling in lipopolysaccharide-exposed chicken embryos.

Medium-chain fatty acids and their derivatives are natural ingredients that support immunological functions in animals. The effects of glycerol monolaurate (GML) on intestinal innate immunity and associated molecular mechanisms were investigated using a chicken embryo model. Sixty-four Arbor Acres broiler embryos were randomly allocated into four groups. On embryonic day 17.5, the broiler embryos were administered with 9 mg of GML, which was followed by a 12-h incubation period and a 12-h challenge with 32 µg of lipopolysaccharide (LPS). On embryonic day 18.5, the jejunum and ileum were harvested. Results indicated that GML reversed the LPS-induced decline in villus height and upregulated the expression of mucin 2 (P < 0.05). GML decreased LPS-induced malondialdehyde production and boosted antioxidant enzyme activity (P < 0.05). GML alleviated LPS-stimulated intestinal secretion of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) (P < 0.05). GML also normalized LPS-induced changes in the gene expression of Toll-like receptor 4, nuclear factor kappa-B p65 (NF-κB p65), cyclooxygenase-2, NOD-like receptor protein 3, IL-18, zonula occludens 1, and occludin (P < 0.05). GML enhanced as well the expression of AMP-activated protein kinase α1 and claudin 1 (P < 0.05). In conclusion, GML improved intestinal morphology and antioxidant status by alleviating inflammatory responses and modulating NF-κB signaling in LPS-challenged broiler embryos.

Regulation of otic neurosensory specification by Notch and Wnt signalling: insights from RNA-seq screenings in the embryonic chicken inner ear.

The Notch and Wnt signalling pathways play key roles in the formation of inner ear sensory organs, but little is known about their transcriptional effectors and targets in this context. Here, we perturbed Notch and Wnt activities in the embryonic chicken otic vesicle using pharmacological treatment or in ovo electroporation of plasmid DNA, and used RNA-Seq to analyse the resulting changes in gene expression. Compared to pharmacological treatments, in ovo electroporation changed the expression of fewer genes, a likely consequence of the variability and mosaicism of transfection. The pharmacological inhibition of Notch activity induced a rapid change in the expression of known effectors of this pathway and genes associated with neurogenesis, consistent with a switch towards an otic neurosensory fate. The Wnt datasets contained many genes associated with a neurosensory biological function, confirming the importance of this pathway for neurosensory specification in the otocyst. Finally, the results of a preliminary gain-of-function screening of selected transcription factors and Wnt signalling components suggest that the endogenous programs of otic neurosensory specification are very robust, and in general unaffected by the overexpression of a single factor. Altogether this work provides new insights into the effectors and candidate targets of the Notch and Wnt pathways in the early developing inner ear and could serve as a useful reference for future functional genomics experiments in the embryonic avian inner ear.

No effects of the antiandrogens cyproterone acetate (CPA), flutamide and p,p'-DDE on early sexual differentiation but CPA-induced retardation of embryonic development in the domestic fowl (Gallus gallus domesticus).

Because a wide range of environmental contaminants are known to cause endocrine disorders in humans and animals, in vivo tests are needed to identify such endocrine disrupting chemicals (EDCs) and to assess their biological effects. Despite the lack of a standardized guideline, the avian embryo has been shown to be a promising model system which responds sensitively to EDCs. After previous studies on the effects of estrogenic, antiestrogenic and androgenic substances, the present work focuses on the effects of in ovo exposure to p,p'-DDE, flutamide and cyproterone acetate (CPA) as antiandrogenic model compounds regarding gonadal sex differentiation and embryonic development of the domestic fowl (Gallus gallus domesticus). The substances were injected into the yolk of fertilized eggs on embryonic day one. On embryonic day 19 sex genotype and phenotype were determined, followed by gross morphological and histological examination of the gonads. Treatment with flutamide (0.5, 5, 50 µg/g egg), p,p'-DDE (0.5, 5, 50 µg/g egg) or CPA (0.2, 2, 20 µg/g egg) did not affect male or female gonad development, assessed by gonad surface area and cortex thickness in both sexes and by the percentage of seminiferous tubules in males as endpoints. This leads to the conclusion that antiandrogens do not affect sexual differentiation during embryonic development of G. gallus domesticus, reflecting that gonads are not target organs for androgens in birds. In ovo exposure to 2 and 20 µg CPA/g egg, however, resulted in significantly smaller embryos as displayed by shortened lengths of skull, ulna and tarsometatarsus. Although gonadal endpoints were not affected by antiandrogens, the embryo of G. gallus domesticus is shown to be a suitable test system for the identification of substance-related mortality and developmental delays.

Liver proteomics analysis reveals the differentiation of lipid mechanism and antioxidant enzyme activity during chicken embryonic development.

Chicken embryo development is a dynamic process. However, no detailed information is available about the protein abundance changes associated with the lipid mechanism and antioxidant enzyme activity during the egg embryo development. Thus, in the present study, an TMT-based proteomic approach was used to quantify protein abundance changes at different stages of chicken embryonic development. A total of 289 significantly differentially abundant hepatic proteins were quantified, of which 180 were upregulated and 109 were downregulated in the comparison of Day 20 with Day 12 in chicken embryos. Pathway analysis showed that metabolic pathways were the most highly enriched pathways, followed by arachidonic acid metabolism and steroid biosynthesis. Integration of proteomic-based studies profiling of three incubation stages revealed that the two compare groups (Day 12 vs Day 20 and Day 16 vs Day 20) shared some key differentially abundant proteins (DAPs), including LBFABP, FABP5, CYP4V2, PDCD4, LAL, APOA1, APOA4, SAA, FABP2, ACBSG2, FABP2, CYP51A1, and FBXO9. The STRING database and GO analysis results showed that there was close connectivity between APOA4, LBFABP, SERPINC1, APOA1, FGB, FGA, ANGPTL3 and these proteins were involved in the oxidation-reduction process, lipid transport, iron ion, heme, and lipid binding. Importantly, APOA4, FABP2, and CYP51A1 might be key factors to control fat deposition and antioxidant enzyme activity during chicken embryonic development. These findings will facilitate a better understanding of antioxidant and lipid mechanisms in chicken embryo and these DAPs can be further investigated as candidate markers to predict lipid deposition and the activity of antioxidant enzymes.