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Background: Developing chicken embryo culture techniques provides methods for manipulating chicken embryos. In this study, the cell-based approach investigated human Wharton’s jelly stem cells (HWJSCs) proliferation and migration into internal embryonic chick tissue. Methods: Wharton’s jelly cells were cultured on an acellular dermal matrix (ADM) and seeded on the chorioallantoic membrane (CAM) of a 4-day-old chicken embryo. Using an RT-PCR assay with the COLA-I human gene, molecular analysis was performed on chicken liver tissue after hatching to confirm the presence of these cells. Results: The chicken liver tissue from Wharton’s jelly stem cells/ADM sample tested positive for the human COLA-I gene. The COLA-I gene sequence analysis shows this sequence belongs to the COLA-I gene in humans. The cells cultured on CAM can migrate and proliferate in the liver of a chicken embryo. Conclusion: These findings suggest that human cells cultured on the CAM can appear in the liver of chickens. However, further investigations are necessary to confirm this finding. [ABSTRACT FROM AUTHOR]

BackgroundOrganic phosphate flame retardants are emerging environmental pollutants. While there have been multiple toxicities reported following organic phosphate flame retardants exposure, few studies focus on their potential developmental toxicities. It is necessary to elucidate these developmental toxicological effects and underlying mechanisms to improve risk assessments and better protect sensitive populations. ObjectiveTo evaluate potential developmental toxicities in early chicken embryos following exposure to triphenyl phosphate (TPhP) or cresyl diphenyl phosphate (CDP), to reveal TPhP and CDP’s capabilities to activate peroxisome proliferator-activated receptor γ (PPARγ) in vivo in an established chicken embryo gene reporter system, and to investigate the roles of PPARγ in TPhP/CDP-induced developmental toxicities with lentivirus-mediated in vivo gene silencing. MethodsFirstly, diverse doses of TPhP and CDP were injected into the air sacs of fertilized eggs to assess the development of chicken embryos after 6 d of incubation, and an optimal dose was chosen for subsequent experiments. Subsequently, the report gene system was employed to evaluate the intraembryonic activation of PPARγ by TPhP and CDP. Eventually, PPARγ was silenced using lentivirus, and the embryos were co-treated with TPhP and CDP to further disclose the roles of PPARγ in the observed developmental toxicity. ResultsFollowing developmental exposure to TPhP or CDP, significantly lower chicken embryo weights (normalized with egg weights) were observed in the 6 d embryos (10, 30 mg·kg−1 TPhP and 3, 10, 30 mg·kg−1 CDP), indicating that both chemicals have general developmental toxicities and CDP is more potent. Additionally, exposure to CDP also resulted in remarkably increased sagittal brain area (normalized to embryo weights) and decreased sagittal eye area (normalized to embryo weights) (P CDP > TPhP. The lentivirus microinjection successfully achieved in vivo silencing of PPARγ in developing chicken embryos, and the estimated silencing efficacy was approximately 55% according to the real-time quantitative polymerase chain reaction (qRT-PCR) results. The in vivo silencing of PPARγ effectively alleviated TPhP or CDP-induced decrease of embryo weights (P

Paracetamol (N-Acetylaminophenol) is the most widely used over-the-counter drug in the world. Although considered safe for pregnant mothers, there is a concern regarding the teratogenic effect of paracetamol. This study aims to observe the teratogenic effect of paracetamol developing embryos. Using chicken embryos, the embryos were incubated for 48 hours and then injected with paracetamol in 3 concentrations, namely 10 ppm, 15 ppm, and 20 ppm. The control group and treatment group consisted of 3 replicates of fertile embryos. Then, the embryos were incubated again for 48 hours in the incubator. Data analysis was carried out qualitatively by observing any developmental alterations. Results showed that paracetamol caused enlargement of the head and heart edema. Exposure to paracetamol at concentrations of 15 ppm and 20 ppm affects the morphology of chicken embryos, especially the formation of the head and disrupts the process of angiogenesis and normal heart formation, causing bleeding and edema at higher concentrations.

Introduction: The chicken egg, with its in ovo compartments, is a widely used and popular animal model in experimental studies. This study aimed to quantify the volumes of the yolk/yolk sac, amniotic fluid, and chicken embryo in ovo using non-invasive ultra-high-field magnetic resonance imaging (UHF-MRI). Materials and methods: In total, 64 chicken eggs were examined using a 7 T UHF-MRI scanner, acquiring T2-weighted anatomical images of the entire egg from developmental day 1 to 16 (D1-D16). Four eggs were scanned each developmental day, and the volumes of the yolk/yolk sac, amniotic fluid, and embryo were quantitatively assessed. Results: UHF-MRI facilitated the in ovo quantitative assessment of the yolk/yolk sac starting from D1 and the embryo from D5 onward. The yolk/yolk sac volume increased from D1 to D6 before progressively decreasing until D14. The amniotic cavity could be detected on D6, with its fluid volume increasing steadily until D14. The embryo's volume increased consistently throughout the developmental period, reaching its peak at D16. Discussion: UHF-MRI allows in vivo assessment of embryonic development, providing non-invasive, longitudinal insights into the volumes of the yolk/yolk sac, amniotic fluid, and chicken embryo. The investigation method described in this study may provide a standardized model for biomedical research in the developing chicken embryo, supporting various experimental applications. [ABSTRACT FROM AUTHOR]

The work was aimed at studying the role of the second messenger NAADP (Nicotinic Acid Adenine Dinucleotide Phosphate), which causes the release of Ca2+ ions from lysosomes and endosomes, in the regulation of heart contractions. Isolated hearts of the grape snail Helix pomatia and a chicken embryo were used as models. Using the membrane-permeable acetooxymethyl ester NAADP-AM, the authors showed that the nanomolar concentrations of NAADP increase the frequency and amplitude of spontaneous cardiac contractions in H. pomatia. The NAADP antagonist, NED19, dose-dependently reduces their frequency, completely blocking spontaneous contractions of the mollusk heart at a concentration of 5 μM without affecting the amplitude of contractions. The V-type H+-ATPase inhibitor bafilomycin A1 suppresses the accumulation of calcium ions in lysosomes and acidic endosomes. It has been shown that bafilomycin A1 causes attenuation of spontaneous contractions and cardiac arrest in H. pomatia. Serotonin increases the amplitude of contractions of the H. pomatia heart without affecting heart rate. NED19 at saturating concentrations (10 μM) reduces the frequency of contractions when the snail heart is activated by serotonin but only partially reduces their amplitude. The experiments with the isolated chick embryo heart showed that NED19 reduces the heart rate without affecting the amplitude of contractions. Based on the obtained data, a hypothesis was put forward that the release of calcium ions from lysosomes and endosomes under the influence of endogenous NAADP ensures the maintenance of spontaneous contractions of the heart. [ABSTRACT FROM AUTHOR]

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Genetic modification in vivo could provide direct functions of genes that could potentially contribute to diverse areas of research including genetics, developmental biology, and physiology. It has been reported that genes of interest could be introduced via recombinant adenovirus type 5 (Ad5) in poultry. Successful gene delivery to mammal fetuses in utero promises substantial progress in clinical and developmental biology, but it is limited because of difficulties in injecting specific sites and invasiveness. On the other hand, developing avian embryos are easily accessible by making a window on the eggshell. Therefore, the objective of this study is to determine permissive embryonic stages for gene transfer into specific avian tissue/organs by injection of Ad5 containing the green fluorescent protein (GFP) gene into blood vessels. At 2 d of post-injection, a strong GFP signal was predominantly identified in the heart of chicken embryos injected at Hamilton–Hamburger (HH) 14, 15, 16 and17 stages with the percentages (44%, 53%, 25%, and 14%, respectively) of GFP positive embryos. In quail embryos, the injection at the HH 15 resulted in heart-specific expression of GFP. Western blot analysis revealed that GFP was exclusively expressed in the avian hearts. These results suggest that the GFP gene is specifically delivered to the avian embryonic hearts when Ad5 is injected through the blood vessel at HH 14–17. This adenoviral transduction of genes of interest in avian embryonic hearts can provide new models for understanding functions of genetic factors on embryonic heart development and unravel genetic etiology of congenital heart diseases. [ABSTRACT FROM AUTHOR]

Genetic modification in vivo could provide direct functions of genes that could potentially contribute to diverse areas of research including genetics, developmental biology, and physiology. It has been reported that genes of interest could be introduced via recombinant adenovirus type 5 (Ad5) in poultry. Successful gene delivery to mammal fetuses in utero promises substantial progress in clinical and developmental biology, but it is limited because of difficulties in injecting specific sites and invasiveness. On the other hand, developing avian embryos are easily accessible by making a window on the eggshell. Therefore, the objective of this study is to determine permissive embryonic stages for gene transfer into specific avian tissue/organs by injection of Ad5 containing the green fluorescent protein (GFP) gene into blood vessels. At 2 d of post-injection, a strong GFP signal was predominantly identified in the heart of chicken embryos injected at Hamilton-Hamburger (HH) 14, 15, 16 and17 stages with the percentages (44%, 53%, 25%, and 14%, respectively) of GFP positive embryos. In quail embryos, the injection at the HH 15 resulted in heart-specific expression of GFP. Western blot analysis revealed that GFP was exclusively expressed in the avian hearts. These results suggest that the GFP gene is specifically delivered to the avian embryonic hearts when Ad5 is injected through the blood vessel at HH 14-17. This adenoviral transduction of genes of interest in avian embryonic hearts can provide new models for understanding functions of genetic factors on embryonic heart development and unravel genetic etiology of congenital heart diseases. [ABSTRACT FROM AUTHOR]

Gut peristalsis is a wave-like movement of a local contraction along the gut, and plays important roles in nutrient digestion and absorption. When peristaltic waves emerge in embryonic guts, randomly distributed origins of peristaltic waves (OPWs) become progressively confined to specific sites. We have investigated how this random-to-organized positioning is achieved using the caecum as a model in chicken embryos. While prominent OPWs, recognized as active (spontaneous) contractions, are located at endpoints of the intact caecum, other regions are also found to possess latent rhythm unveiled by fragmentation of a caecum into pieces, showing that the latent rhythm is normally suppressed in the intact gut. Analyses with caecum fragments demonstrate that the latent rhythm is spatially patterned in an early gut, to which negative impact by primitive passing waves contributes; the more passing waves a region experiences to undergo forced/passive contractions, the slower latent rhythm this region acquires. This patterned latent rhythm underlies the final positioning of OPWs at later stages, where a site with faster latent rhythm dominates neighboring slower rhythm, surviving as a “winner” by macroscopic lateral inhibition. Thus, the random-to-organized patterning of OPWs proceeds by self-organization within the caecum, in which two distinct mechanisms, at least, are employed; suppressive actions by primitive waves followed by macroscopic lateral inhibition.

Significant technological progress in the field of wireless devices that were primarily intended for military purposes, has resulted in their common manipulation by the general population. Wi-Fi, mobile phones, and other modern devices offer many advantages to their users. On the other hand, their excessive usage creates an environmental burden, also known as electrosmog. The objective of our current study was the observation of the Wi-Fi radiation effect on the histo-logical structure of the organs in the 9-day-old chicken embryo. On day 9 of incubation, the embryological material was routinely processed for preparation of hematoxylin-eosin, picrosirius red and periodic acid Schiff stained histological sections. Radiation with a frequency of 2.4 GHz and average power density of 300 µW.m−2 applied during the entire development up to the 9th embryonic day did not fundamentally affect general organogenesis. However, in the parenchyma of organs such as the liver, spleen, lungs, kidneys, and gonads, as well as in the developing mesenchyme, obvious vascular congestion of the blood vessels of different caliber was observed. Also, an increase in collagen and glycosaminoglycans production in the cartilaginous matrix and perichondrium of the future bone skeleton as well as developing connective tissue was noted. Although these morphological changes were just subtle, they point to the Wi-Fi radiation’s ability to influence the histogenesis of the individual.

Abstract The aim of this study was to investigate the mechanism of iron homeostasis and the ferroptosis pathway for yolk sac atrophy during late embryogenesis. To study the mechanism of yolk sac atrophy, 100 eggs were used. Further, 500 eggs were randomly divided into five treatments and in ovo feeding with different iron sources, such as FeSO4, ferrous glycinate (Fe‐Gly), or deferoxamine (DFO), to study the effects of free iron content on hatching quality and embryonic development. The results showed that total iron content of yolk decreased, but yolk sac increased from embryonic(E)13 to E19 (p

Due to the growing safety and environmental concerns associated with biocides, phenolic-soy branched chain fatty acids (phenolic-soy BCFAs) are synthesized as new bio-based antimicrobial agents. Safety evaluation is essential before the wide adoption of these new antimicrobial products. This study was initiated to evaluate the safety of four phenolic-soy BCFAs (with phenol, thymol, carvacrol, or creosote branches). Methyl-branched iso-oleic acid, phenol, and creosote were included in the study as controls. In silico toxicity simulation tools predicted that the phenolic BCFAs had much higher toxicities to aquatic organisms than free phenolics did, while the opposite was predicted for rats. The developmental toxicity of four phenolic-soy BCFAs was assessed using an in vivo chicken embryonic assay. Results showed that creosote-soy BCFA had much lower mortality rates than creosote at the same dosages. Additionally, creosotesoy BCFA and methyl-branched iso-oleic acid induced minimal estrogenic activity in the concentration range of 10 nM - 1 μM. Carvacrol-soy BCFA treatments significantly increased (p < 0.05) oxidative stress levels with higher thiobarbituric acid reactive substances in the livers of chicken embryos. Altogether, the phenolic-soy BCFAs, especially creosote-soy BCFA, reported in this study are potentially promising and safer bio-based antimicrobial products. [ABSTRACT FROM AUTHOR]

Drug use during pregnancy is an important issue that must be investigated due to its adverse effects on maternal and foetal health. This study aimed to determine the embryotoxic and teratogenic effects of in‐ovo administered metamizole (dipyrone), which can be used when needed during pregnancy and has potent analgesic, antipyretic, anti‐inflammatory, and long bone (tibia and femur) effects. This study used 240 fertile eggs from Atak S breed chickens, divided into eight equal groups: control, vehicle control, and 15.62, 31.25, 62.5, 125, 250 and 500 mg/kg metamizole. The eggs were hatched on the 21st day of incubation, and the chicks' body weights and mortality rates were determined. The right and left femur and tibia bones were resected from the chicks. Anatomical reference points were determined after removing the soft tissues of the bones, and necessary morphometric measures were taken from these points with a 0.01 mm precision using digital callipers. The 100% lethal dose (LD100) was identified in the highest examined dose (500 mg/kg) in the Chicken Embryotoxicity Screening Test (CHEST)‐I stage. The CHEST‐II stage determined the 50% lethal dose (LD50). High‐dose metamizole affected skeletal development, significantly decreasing tibia and femur lengths and corpus thicknesses and increasing mortality. [ABSTRACT FROM AUTHOR]

The experiment aimed to investigate the effects of injecting lactic acid bacteria into the embryo on the hatching performance and intestinal mucosa structure of chicks. A total of 150 SPF eggs were incubated until 18 days of embryonic age and then randomly divided into two groups, with each group having three replicates, each consisting of 25 eggs. The control group was injected with 0.2 mL of sterilized saline into the amniotic cavity, while the embryonic egg lactic acid bacteria injection group was injected with 0.2 mL containing 1.21×108 CFU/mL of live Lactobacillus salivarius XP132. The experiment spanned from incubation to hatching at 21 days, with a rearing period of 35 days. The results indicated that the injection of lactic acid bacteria into the fertilized eggs significantly increased the body weight of the chick (P<0.05). Compared to the control group, the embryonic egg lactic acid bacteria injection group showed a significant increase in the thymus index at 7 and 28 days of age, the spleen and bursa of Fabricius indices at 14 days of age, and the thymus and bursa of Fabricius indices at 21 days of age (P<0.05). Compared to the control group, the villus height (VH) of the duodenum in the embryonic egg lactic acid bacteria injection group was significantly increased (P<0.05), and the villus to crypt ratio (V/C) was extremely increased (P<0.01). The crypt depth (CD) of the duodenum at 7 days of age was extremely reduced (P<0.01), and the CD of the duodenum at 21 and 35 days of age was significantly reduced (P<0.05). At 7 and 21 days of age, the VH and V/C of the jejunum were significantly increased (P<0.05), while the CD was significantly decreased (P<0.05). At 21 days of age, the VH of the ileum was significantly increased (P<0.05), and the V/C was extremely increased (P<0.01). At 35 days of age, the VH of the ileum was significantly increased (P<0.05). Due to the absence of antibiotic use during the rearing process, some chicks in the control group experienced mild bacterial infections. Electron microscopy observation revealed certain damages to the microvilli and mitochondrial structures of the duodenal epithelial cells. However, the application of lactic acid bacteria prevented bacterial infections in the chicks at 7, 21, and 35 days of age, and improved the damage to the epithelial cell microvilli, mitochondrial structures, and tight junction proteins. The study shows that the injection of lactic acid bacteria into fertilized eggs can increase the body weight of SPF chicks and has a certain improving effect on their intestinal mucosal structure. [ABSTRACT FROM AUTHOR]

IntroductionThe chicken egg, with its in ovo compartments, is a widely used and popular animal model in experimental studies. This study aimed to quantify the volumes of the yolk/yolk sac, amniotic fluid, and chicken embryo in ovo using non-invasive ultra-high-field magnetic resonance imaging (UHF-MRI).Materials and methodsIn total, 64 chicken eggs were examined using a 7 T UHF-MRI scanner, acquiring T2-weighted anatomical images of the entire egg from developmental day 1 to 16 (D1-D16). Four eggs were scanned each developmental day, and the volumes of the yolk/yolk sac, amniotic fluid, and embryo were quantitatively assessed.ResultsUHF-MRI facilitated the in ovo quantitative assessment of the yolk/yolk sac starting from D1 and the embryo from D5 onward. The yolk/yolk sac volume increased from D1 to D6 before progressively decreasing until D14. The amniotic cavity could be detected on D6, with its fluid volume increasing steadily until D14. The embryo’s volume increased consistently throughout the developmental period, reaching its peak at D16.DiscussionUHF-MRI allows in vivo assessment of embryonic development, providing non-invasive, longitudinal insights into the volumes of the yolk/yolk sac, amniotic fluid, and chicken embryo. The investigation method described in this study may provide a standardized model for biomedical research in the developing chicken embryo, supporting various experimental applications.

Simple Summary: Crotalus snake envenomation poses a serious challenge due to its diverse toxicological effects, including neurological, myotoxic, and cytotoxic symptoms, often leading to death. The aim of the study was to elucidate the physiological effects of exposure to Crotalus atrox and Crotalus adamanteus venoms and to assess toxicity using chicken embryo models. Currently, there is not a lot of research demonstrating the physiological effects of venom, including its potential impact on embryos, in accordance with the 3R rules. The applied research model consisted of the chick embryotoxicity screening test (CHEST) and the chick chorioallantoic membrane (CAM) test, which allowed for (i) a demonstration of the greater toxicity of C. adamanteus venom and (ii) an observation of the embryotoxic effect and vasoactive nature of the tested venom species. Additionally, (iii) morphological abnormalities (such as Siamese twins) emerged, and (iv) changes in the activity of acetylcholinesterase (AChE) were identified (resulting solely from its presence in the examined tissues due to the lack of this component in exogenously applied venom). These results provide crucial insights into the mechanisms of Crotalus venom toxicity and their potential biomedical applications. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: Angiogenesis, defined as the formation of new blood vessels from pre-existing ones, plays a vital role in both physiological and pathological conditions. Understanding agents that can influence angiogenesis is crucial in managing diseases where angiogenesis is dysregulated. This study focuses on sevoflurane, a commonly used inhalation anesthetic, whose effects on angiogenesis and embryonic development are not well understood. Our objective was to assess the impact of sevoflurane exposure on angiogenesis using the ex-ovo chick chorioallantoic membrane model. Methods: In this model, fertilized chicken eggs were exposed to sevoflurane at concentrations of 2% and 4%, for varying durations of 1, 2, and 4 hours. The embryos were then divided into control and experimental groups for quantitative angiogenesis assessment using Image J software, followed by statistical analysis with one-way analysis of variance. Results: The results indicate that sevoflurane exposure has a dose-dependent positive effect on angiogenesis, with significant increases in vascular density observed in embryos exposed to both concentrations compared to the control group. Additionally, the length of exposure was found to further enhance these angiogenic effects. Conclusion: Despite the dose and duration-dependent impact of sevoflurane on angiogenesis, the existing literature presents mixed findings, highlighting the need for additional research to elucidate sevoflurane’s role in angiogenesis. This is particularly important for understanding its implications in various medical conditions, such as cancer, wound healing, and fetal development. Future investigations into sevoflurane’s effects on placental angiogenesis could also provide valuable insights into its potential consequences on intrauterine growth. [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.

ABSTRACT: 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 β-carotene (βc) (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 β-carotene were enriched in pathways related to cell proliferation and metabolism. Consequently, we postulated that maternal β-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.

ABSTRACT: 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.

Using in ovo injection method, specific nutrients and antioxidants can be provided in precise doses at certain times for maximum absorption by the embryo. The objective of this study was to examine the effect of in ovo injection of flaxseed oil on the quality and antioxidant properties as well as fatty acid profile of breast meat of day-old broilers. 20 one-day-old fertilized eggs were randomly divided into two groups of 10; one control and one FSO (flaxseed oil) which received 100 μL of flaxseed oil by injection into the allantoic sac. The amounts of fat, vitamin E, total phenol, carotenoid, TBARS (thiobarbituric acid reactive substances) value, color, texture and fatty acid profile of the chicken breast meat were measured. In ovo injection of flaxseed oil significantly increased the fat content. The mean amounts of vitamin E in control and FSO were 0.54 and 0.71 mg/100g, respectively (P

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

The article is devoted to the problem of increasing the resistance of chicks from hatching to the age of 3 weeks to infectious diseases by technological methods. Dosed low-temperature exposure to the embryo during the sensitive period of early embryogenesis was proposed as an impact factor; influenza vaccine virus was used as a test virus for experimental infection of embryos. It was found that in the embryos after cooling, the titer of the virus was significantly lower by 1.5-12.0 times (p

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. [ABSTRACT FROM AUTHOR]

Using in ovo injection method, specific nutrients and antioxidants can be provided in precise doses at certain times for maximum absorption by the embryo. The objective of this study was to examine the effect of in ovo injection of flaxseed oil on the quality and antioxidant properties as well as fatty acid profile of breast meat of day-old broilers. 20 one-day-old fertilized eggs were randomly divided into two groups of 10; one control and one FSO (flaxseed oil) which received 100 μL of flaxseed oil by injection into the allantoic sac. The amounts of fat, vitamin E, total phenol, carotenoid, TBARS (thiobarbituric acid reactive substances) value, color, texture and fatty acid profile of the chicken breast meat were measured. In ovo injection of flaxseed oil significantly increased the fat content. The mean amounts of vitamin E in control and FSO were 0.54 and 0.71 mg/100g, respectively (P<0.05). Carotenoid content, redness and yellowness of the broilers breast meat in FSO increased in comparison to control, while TBARS value and lightness index decreased insignificantly. FSO showed lower saturated fatty acids and higher unsaturated, monounsaturated, polyunsaturated, unsaturated/saturated and n-3 fatty acids, but these differences were not significant. Based on the results of this study, in ovo injection of flaxseed oil improved oxidative stability of day-old broiler breast meat through increasing vitamin E content. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The relevance of identifying pathological processes in the context of embryonic development is increasingly gaining attention in terms of professionalized prenatal care. To analyze local effects of prenatally administered drugs during embryonic development, the model organism of the chicken embryo can be used in a first exploratory approach. For the examination of local dexamethasone administration—as an exemplary drug—common bead implantation protocols have been adapted to serve as an in vivo technique for local drug testing during embryonic skin regeneration. For this, acrylic beads were soaked in a dexamethasone solution and implanted into skin incisional wounds of 4-day-old chicken embryos. After further incubation, the effects of the applied substance on the process of embryonic skin regeneration were analyzed using histological and molecular biological techniques. This data descriptor contains a detailed microsurgical protocol, a representative video demonstration, and exemplary results of local glucocorticoid-induced changes during embryonic wound healing. To conclude, this method allows for the analysis of the local effects of a particular substance on a cellular level and can be extended to serve as an in vivo technique for numerous other drugs to be tested on embryonic tissue. Dataset:  https://doi.org/10.3390/biomedicines10123125. Dataset License: CC-BY [ABSTRACT FROM AUTHOR]

Currently, the effect of prenatal ultrasound on foetal development is intensively discussed and the guidelines for prenatal diagnostics have been changed. However, data supporting these concerns are scarce. Therefore, we used an established in ovo model of the chicken embryo to investigate cell proliferation and apoptosis within the retina. A total of 21 chicken eggs were fenestrated on Day 5 and allocated to either the control (n = 8) or exposition group (n = 13). The exposition group was treated with pulsed‐wave Doppler ultrasound (PWD) for 10 min while controls remained without treatment. After subsequent incubation (6–48 h), the eggs were sacrificed, and chicken embryos were examined morphologically (HE‐staining) and immunohistochemically. Counting of apoptotic and proliferating cells per retina was performed using antibodies specific for phospho‐histone‐H3 and active caspase‐3 in combination with a biotin‐labelled secondary antibody and peroxidase conjugated avidin–biotin complex for chromogenic detection. Due to a rather low number of specimens at each time point after ultrasound exposition, we neglected the effects of incubation time and focused on treatment effects. This approach revealed that the median number of proliferating cells is reduced after 10 min of exposure to PWD (569 vs. 766), while the number of apoptotic cells is fairly comparable between groups (5 vs. 6). Our data contribute to a better understanding of prenatal US on foetal development by suggesting that PWD could have an impact on the number of proliferating cells in the developing chicken retina and therefore justify further investigations. [ABSTRACT FROM AUTHOR]

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 mg/g egg), p,p'-DDE (0.5, 5, 50 mg/g egg) or CPA (0.2, 2, 20 mg/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 mg 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. [ABSTRACT FROM AUTHOR]

ABSTRACT: Deoxynivalenol (DON) has a strong toxic effect on the gastrointestinal mucosa of poultry. In this study, we evaluated chicken embryo development and glandular stomach damage to clarify the immunotoxic effects of DON injected through the allantoic cavity of chicken embryos. The glandular stomach index, routine blood indices, plasma inflammatory factors, pathological changes in the glandular stomach, and transcriptome results were analyzed in the hatching chicks. The results showed that DON was supertoxic to chicken embryos, causing edema, shedding, and bleeding of the mucosa of the glandular stomach, which triggered inflammatory reactions. As the toxin concentration increased, the immune system was successively activated and inhibited, and regulation was carried out by the differential regulation of the mitogen-activated protein kinase (MAPK) signal pathway. These results suggested that the immunotoxic effect of DON on the glandular stomach of chicken embryos was closely related to the regulation of the MAPK signaling pathway.

Simple Summary: Chicken embryos are currently being increasingly used in various areas of research but are frequently not covered by animal protection legislation. In the food industry, it is often even common practice to kill male embryos because they are of no economic use. In both cases, there is a lack of knowledge about the sentience of these chicken embryos, especially their ability to perceive pain. The purpose of this exploratory study was to investigate whether—and if so, on which developmental day—a chicken embryo shows a behavioral change or a specific response to a noxious stimulus, both of which would be indicative of functioning nociception. Two complementary approaches were used for the evaluation: human observation and computer-assisted evaluation using a markerless pose estimation software (DeepLabCut). Through a combination of both approaches, it became apparent that developmental day 15 was the earliest stage at which a response to the applied stimulus was detectable. This result thus represents a contribution to the future improvement of animal welfare as it suggests that from developmental day 15 a chicken embryo in the egg has the capacity to show a nocifensive reaction. Many potentially noxious interventions are performed on chicken embryos in research and in the poultry industry. It is therefore essential and in the interest of animal welfare to be able to precisely define the point at which a chicken embryo is capable of nociception in ovo. The present part III of a comprehensive study examined the movements of developing chicken embryos with the aim of identifying behavioral responses to a noxious stimulus. For this purpose, a noxious mechanical stimulus and a control stimulus were applied in a randomized order. The recorded movements of the embryos were evaluated using the markerless pose estimation software DeepLabCut and manual observations. After the application of the mechanical stimulus, a significant increase in beak movement was identified in 15- to 18-day-old embryos. In younger embryos, no behavioral changes related to the noxious stimulus were observed. The presented results indicate that noxious mechanical stimuli at the beak base evoke a nocifensive reaction in chicken embryos starting at embryonic day 15. [ABSTRACT FROM AUTHOR]

Avian ceca play an important role in liquid absorption, cellulose digestion, and defensive mechanism. This study aims to demonstrate histological and histochemical characteristics of developing chicken cecum. For this purpose, 10 embryos on the 18th day of incubation, 10 chicks on hatching day and 10 chicks on the seventh day post‐hatching were used. The histological sections prepared from proximal, middle, and distal parts of cecum were stained with Crossmon's triple stain, periodic‐acid Schiff, Alcian blue (pH 2.5), Masson‐Fontana's argentaffin silver stain and Gordon and Sweets's silver stain. Alpha‐naphthyl acetate esterase (ANAE) and acid phosphatase (ACP‐ase) were also demonstrated in the sections. In the proximal part, although the villi were rudimentary on the 18th day of incubation, well‐developed villi were seen at seventh day post‐hatching. In middle and distal parts, while it was seen that rudimentary folds appeared on the 18th day of incubation, mucosal folds were prominent and short villi were formed on the hatching day and seventh day post‐hatching. Goblet cells and enteroendocrine cells were detected from the 18th day of incubation. The lymphoid follicles supported with reticular fibres were seen on the seventh day post‐hatching in proximal cecum wall. While ACP‐ase (+) lymphocytes were rarely observed, more ANAE (+) lymphocytes were in lymphoid follicles. As a result, development of cecum in chickens has been demonstrated by histological techniques in this study. [ABSTRACT FROM AUTHOR]

Simple Summary: Chicken embryos are frequently not protected by animal welfare laws. However, they are used in various research areas, and male embryos are commonly killed in food production as an alternative to culling day-old chicks. Increasing knowledge regarding the onset of nociception and pain perception in chicken embryos is fundamental for animal welfare protection. The aim of this exploratory study was to further narrow down the period when chicken embryos acquire the capacity for nociception. Therefore, changes in blood pressure and heart rate after the introduction of a noxious stimulus were assessed during the embryonic development of chickens. Embryos from 16 days of incubation onward showed cardiovascular changes after a noxious mechanical stimulus was introduced at the base of the beak, indicating a nociceptive response. Although it is assumed that chicken embryos acquire the capacity for nociception while developing in the egg, an exact time point has not yet been specified. The present research was an exploratory study aiming to determine when the capacity of nociception emerges during embryonic development in chickens. Changes in blood pressure and heart rate (HR) in response to a noxious mechanical stimulus at the base of the beak versus a light touch on the beak were examined in chicken embryos between embryonic days (EDs) 7 and 18. Mean arterial pressure (MAP) was the most sensitive parameter for assessing cardiovascular responses. Significant changes in MAP in response to a noxious stimulus were detected in embryos at ED16 to ED18, whereas significant changes in HR were observed at ED17 and ED18. Infiltration anesthesia with the local anesthetic lidocaine significantly reduced the response of MAP on ED18, so the measured cardiovascular changes may be interpreted as nociceptive responses. [ABSTRACT FROM AUTHOR]

There has been increased interest in phytochemical antioxidants to prevent protein damage and aggregate formation in cataract treatment. In this study, the protective effect of different doses of Rb1 (GRb1), one of the ginsenosides of Panax Ginseng, in the experimental cataract model formed in chick embryos was investigated. Five different experimental groups were formed with 100 SPF fertilized eggs: Control (0.9% NaCl to physiological saline), hydrocortisone hemisuccinate sodium (HC), low dose (HC + L-GRb1 (1 mg/kg)), medium dose (HC+). M-GRb1 (2.5 mg/kg)), and high dose (HC + H-GRb1 (5 mg/kg)). All solutions were given to air sack at 15 days of incubation. On the 17th day, the bulbous oculi of the chick embryos were dissected. Cataract formations of the lenses, glutathione (GSH), malondialdehyde (MDA), total antioxidant (TAS), total oxidant (TOS) levels, Caspase-3 H-score, and TUNEL index were determined. In addition, crystalline alpha A (CRYAA) gene expression was evaluated. Cataracts were observed in the control, HC, HC + L-GRb1, HC + M-GRb1, and HC + H-GRb1 groups with a frequency of 0%, 100%, 75%, 56.25%, and 100%, respectively. There were statistically significant differences between the control and HC groups in terms of TAS, TOS, MDA, GSH, Caspase-3 H-score, and TUNEL index (p <.05). When the therapeutic effect of the GRb1 groups was evaluated, the HC group showed significant differences with the HC + L-GRb1 and HC + M-GRb1 groups in almost all parameters (p <.05), while there was no statistical difference with the HC + H-GRb1 group (p >.05). In addition, gene expression levels differed between the groups, although not statistically significant (p >.05). 1 mg/kg and 2.5 mg/kg GRb1 applications show therapeutic properties on the HC-induced cataract model. This effect is more pronounced at 2.5 mg/kg. [ABSTRACT FROM AUTHOR]

The identification of a chicken's sex is a massive task in the poultry industry. To solve the problems of traditional artificial observation in determining sex, such as time-consuming and laborious, a sex identification method of chicken embryos based on blood vessel images and deep learning was preliminarily investigated. In this study, we designed an image acquisition platform to capture clear blood vessel images with a black background. 19,748 images of 3024 Jingfen No. 6 breeding eggs were collected from days 3 to 5 of incubation in Beijing Huadu Yukou Poultry Industry. Sixteen thousand seven hundred sixty-one images were filtered via color sexing in 1-day-old chicks and constructed the dataset of this study. A sex identification model was proposed based on an improved YOLOv7 deep learning algorithm. An attention mechanism CBAM was introduced for YOLOv7 to improve the accuracy of sex identification of chicken eggs; the BiFPN feature fusion was used in the neck network of YOLOv7 to fuse the low-level and high-level features efficiently; and α-CIOU was used as the bounding box loss function to accelerate regression prediction and improve the positioning accuracy of the bounding box of the model. Results showed that the mean average precision (mAP) of 88.79% was achieved by modeling with the blood vessel data on day 4 of incubation of chicken eggs, with the male and female reaching 87.91% and 89.67%. Compared with the original YOLOv7 network, the mAP of the improved model was increased by 3.46%. The comparison of target detection model results showed that the mAP of our method was 32.49%, 17.17%, and 5.96% higher than that of SSD, Faster R-CNN, and YOLOv5, respectively. The average image processing time was 0.023 s. Our study indicates that using blood vessel images and deep learning has great potential applications in the sex identification of chicken embryos. [ABSTRACT FROM AUTHOR]

Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage avian embryos are vulnerable to the toxic effects of chemicals. However, studies on the specific effects of TCIPP on avian embryonic development are limited. We aimed to investigate the toxicity of TCIPP in early chicken embryos using a previously developed shell-less incubation system. Fertilized chicken (Gallus gallus domesticus) eggs (n = 220) were exposed to 50 or 500 nmol TCIPP/(g egg) or dimethyl sulfoxide (DMSO) as a vehicle control on Day 0 of incubation. Development of 198 embryos was monitored from Days 3–9 of incubation, and 22 embryos on Day 4 and 74 embryos on Day 9 were dissected. Messenger RNA expression levels for several genes were measured in embryos on Day 4. Both TCIPP-exposed groups showed a significant reduction in survival rate. Imaging analyses revealed significant decreases in body length, head and bill length, eye diameter, and forelimb and hindlimb length in both TCIPP-treated groups. TCIPP exposure significantly impaired the development of extraembryonic blood vessels and the production of red blood cells. A TCIPP-dose-dependent decreasing trend in heart rate was observed on Days 4–7. The somitic angle increased significantly on Days 4–6, and embryos with curved somites showed cleavage in the back and gaps between somites, resulting in asymmetrical somite formation. A significant correlation was found between the somitic angle and FGF8 expression levels, suggesting that TCIPP exposure affects somite formation through an altered FGF-signaling pathway. Embryos with somitic deformities in TCIPP-exposed groups had significantly reduced survival rates, indicating that abnormal segment formation directly increased mortality. Finally, eye weight and ocular luminosity values were significantly reduced, suggesting that TCIPP may also affect eye development. Overall, these findings highlight severe toxic effects of TCIPP on avian embryonic development, including in vascularization, cardiac function, and somite and ocular development.

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.

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.