Craniofacial studies in chicken embryos confirm the pathogenicity of human FZD2 variants associated with Robinow syndrome.

Robinow syndrome is a rare disease caused by variants of seven WNT pathway genes. Craniofacial features include widening of the nasal bridge and jaw hypoplasia. We used the chicken embryo to test whether two missense human FZD2 variants (1301G>T, p.Gly434Val; 425C>T, p.Pro142Lys) were sufficient to change frontonasal mass development. In vivo, the overexpression of retroviruses with wild-type or variant human FZD2 inhibited upper beak ossification. In primary cultures, wild-type and variant human FZD2 significantly inhibited chondrogenesis, with the 425C>T variant significantly decreasing activity of a SOX9 luciferase reporter compared to that for the wild type or 1301G>T. Both variants also increased nuclear shuttling of ß-catenin (CTNNB1) and increased the expression of TWIST1, which are inhibitory to chondrogenesis. In canonical WNT luciferase assays using frontonasal mass cells, the variants had dominant-negative effects on wild-type FZD2. In non-canonical assays, the 425C>T variant failed to activate the reporter above control levels and was unresponsive to exogenous WNT5A. This is the first single amino acid change to selectively alter ligand binding in a FZD receptor. Therefore, FZD2 missense variants are pathogenic and could lead to the altered craniofacial morphogenesis seen in Robinow syndrome.

Distinct molecular profile of the chick organizer as a stem zone during axial elongation.

The vertebrate organizer plays a crucial role in building the main (antero-posterior) axis of the embryo: it neuralizes the surrounding ectoderm, and is the site of emigration for cells making axial and paraxial mesendoderm during elongation. The chick organizer becomes a stem zone at the onset of elongation; it stops recruiting cells from the neighbouring ectoderm and generates all its derivatives from the small number of resident cells it contains at the end of gastrulation stages. Nothing is known about the molecular identity of this stem zone. Here, we specifically labelled long-term resident cells of the organizer and compared their RNA-seq profile to that of the neighbouring cell populations. Screening by reverse transcription-polymerase chain reaction and in situ hybridization identified four genes (WIF1, PTGDS, ThPO and UCKL1) that are upregulated only in the organizer region when it becomes a stem zone and remain expressed there during axial elongation. In experiments specifically labelling the resident cells of the mature organizer, we show that only these cells express these genes. These findings molecularly define the organizer as a stem zone and offer a key to understanding how this zone is set up, the molecular control of its cells' behaviour and the evolution of axial growth zones.

Fowl adenovirus serotype 4 (FAdV-4) infection induces inflammatory responses in chicken embryonic cardiac fibroblasts via PI3K/Akt and IκBα/NF-κB signaling pathways.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of the acute infectious disease hepatitis-hydropericardium syndrome (HHS). Previous studies have focused on the mechanisms of FAdV-4 caused liver injury, while studies revealing potential mechanisms of inflammatory injury in FAdV-4-infected chicken cardiac cells remain scare. Here we found that FAdV-4 successfully infected chicken embryonic cardiac fibroblasts (CECF) cells in vitro and significantly upregulated production of inflammatory cytokines including IL-1ß, IL-6, IL-8, and TNF-α, suggesting induction of a strong inflammatory response. Mechanistically, FAdV-4 infection increased expression of phosphorylated Akt in a time-dependent manner, while phosphorylation of Akt and production of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α were greatly reduced in FAdV-4-infected CECF cells after treatment with LY294002, a potent inhibitor of PI3K, indicating that the inflammatory response induced by FAdV-4 infection is mediated by the PI3K/Akt signaling pathway. Furthermore, FAdV-4 infection increased expression of phosphorylated IκBα, a recognized indicator of NF-κB activation, and treatment with the BAY11-7082, a selective IκBα phosphorylation and NF-κB inhibitor, significantly reduced IκBα phosphorylation and inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α) production in FAdV-4-infected CECF cells, suggesting a critical role of IκBα/NF-κB signaling in FAdV-4-induced inflammatory responses in CECF cells. Taken together, our results suggest that FAdV-4 infection induces inflammatory responses through activation of PI3K/Akt and IκBα/NF-κB signaling pathways in CECF cells. These results reveal potential mechanisms of inflammatory damage in chicken cardiac cells caused by FAdV-4 infection, which sheds new insight into clarification of the pathogenic mechanism of FAdV-4 infection and development of new strategies for HHS prevention and control.

Early Development of the Thalamo-Pallial Stage of the Tectofugal Visual Pathway in the Chicken (Gallus gallus).

The tectofugal pathway is a highly conserved visual pathway in all amniotes. In birds and mammals, retinorecipient neurons located in the midbrain roof (optic tectum/superior colliculus) are the source of ascending projections to thalamic relays (nucleus rotundus/caudal pulvinar), which in turn project to specific pallial regions (visual dorsal ventricular ridge [vDVR]/temporal cortex) organized according to a columnar recurrent arrangement of interlaminar circuits. Whether or to which extent these striking hodological correspondences arise from comparable developmental processes is at present an open question, mainly due to the scarcity of data about the ontogeny of the avian tectofugal system. Most of the previous developmental studies of this system in birds have focused on the establishment of the retino-tecto-thalamic connectivity, overlooking the development of the thalamo-pallial-intrapallial circuit. In this work, we studied the latter in chicken embryos by means of immunohistochemical assays and precise ex vivo crystalline injections of biocytin and DiI. We found that the layered organization of the vDVR as well as the system of homotopic reciprocal connections between vDVR layers were present as early as E8. A highly organized thalamo-vDVR projection was also present at this stage. Our immunohistochemical assays suggest that both systems of projections emerge simultaneously even earlier. Combined with previous findings, these results reveal that, in striking contrast with mammals, the peripheral and central stages of the avian tectofugal pathway develop along different timelines, with a tecto-thalamo-intrapallial organization arising before and possibly independently of the retino-isthmo-tectal circuit.

A new standardization for the use of chicken embryo: selection of target from the phage display library and infection.

The filamentous bacteriophage M13KO7 (M13) is the most used in phage display (PD) technology and, like other phages, has been applied in several areas of medicine, agriculture, and in the food industry. One of the advantages is that they can modulate the immune response in the presence of pathogenic microorganisms, such as bacteria and viruses. This study evaluated the use of phage M13 in the chicken embryos model. We inoculated 13-day-old chicken embryos with Salmonella Pullorum (SP) and then evaluated survival for the presence of phage M13 or E. coli ER2738 (ECR) infected with M13. We found that the ECR bacterium inhibits SP multiplication in 0.32 (M13-infected ECR) or 0.44 log UFC/mL (M13-uninfected ECR) and that the ECR-free phage M13 from the PD library can be used in chicken embryo models. This work provides the use of the chicken embryo as a model to study systemic infection and can be employed as an analysis tool for various peptides that M13 can express from PD selection. KEY POINTS: • SP-infected chicken embryo can be a helpful model of systemic infection for different tests. • Phage M13 does not lead to embryonic mortality or cause serious injury to embryos. • Phage M13 from the PD library can be used in chicken embryo model tests.

Chick sexing based on the blood analysis using Raman spectroscopy.

Efforts are underway to develop technology for automatically determining the sex of chick embryos, aimed at establishing a stable and efficient poultry farming system while also addressing animal welfare concerns. This study investigated the possibility of chick sexing through blood analysis using Raman spectroscopy. Raman spectra were obtained from whole blood and its constituents, such as red blood cells (RBCs) and blood plasma, collected from chicks aged 1-2 days, using a 785-nm excitation wavelength. Principal component analysis (PCA) revealed statistically significant sex-dependent spectral variations in whole blood and RBCs, whereas blood plasma showed less clear dependency. These spectral differences between male and female chicks were attributed to differences in the proportion of spectral components from oxygenated (oxy-) and deoxygenated (deoxy-) RBCs, with males exhibiting a slightly stronger contribution of oxy-RBCs compared to females. This reflects the higher oxygen affinity of hemoglobin (Hb) in males compared to females. A model for discriminating chick sex was built using the ratios of certain Raman band characteristics of oxy-RBCs and deoxy-RBCs, achieving a sensitivity of 100%. This spectroscopic method holds promise for developing technology to discriminate the sex of early chicken embryos in ovo by detecting differences in oxygen saturation of RBCs based on sex.

Atypical Course of the Habenulo-Interpeduncular Tract in Chick Embryos.

Classical studies of the avian diencephalon hardly mention the habenulo-interpeduncular tract (a.k.a. retroflex tract), although both the habenula (HB) (its origin) and the interpeduncular nuclear complex (its target) are present. Retroflex tract fibers were described at early embryonic stages but seem absent in the adult in routine stains. However, this tract is a salient diencephalic landmark in all other vertebrate lineages. It typically emerges out of the caudal HB, courses dorsoventrally across thalamic alar and basal plates just in front of the thalamo-pretectal boundary, and then sharply bends 90° caudalwards at paramedian basal plate levels (this is the "retroflexion"), to approach longitudinally via paramedian pretectum and midbrain the rostralmost hindbrain, specifically the prepontine median interpeduncular complex across isthmus and rhombomere 1. We systematize this habenulo-interpeduncular course into four parts named subhabenular, retrothalamic, tegmental, and interpeduncular. We reexamined the chicken habenulo-interpeduncular fibers at stages HH30 and HH35 (6.5- and 9-day incubation) by mapping them specifically with immunoreaction for BEN protein, a well-known marker. We found that only a small fraction of the stained retroflex tract fibers approaches the basal plate by coursing along the standard dorsoventral pathway in front of the thalamo-pretectal boundary. Many other habenular fibers instead diverge into atypical dispersed courses across the thalamic cell mass (implying alteration of the first subhabenular part of the standard course) before reaching the basal plate; this dispersion explains their invisibility. A significant number of such transthalamic habenular fibers cross orthogonally the zona limitans (ZLI) (the rostral thalamic boundary) and invade the caudal alar prethalamus. Here, they immediately descend dorsoventrally, just rostrally to the ZLI, until reaching the prethalamic basal plate, where they bend (retroflex) caudalwards, entering the thalamic basal paramedian area. These atypical fibers gradually fasciculate with the other groups of habenular efferent fibers in their final longitudinal approach to the hindbrain interpeduncular complex. We conclude that the poor visibility of this tract in birds is due to its dispersion into a diversity of atypical alternative routes, though all components eventually reach the interpeduncular complex. This case merits further analysis of the diverse permissive versus nonpermissive guidance mechanisms called into action, which partially correlate distinctly with successive diencephalic, mesencephalic, and hindbrain neuromeric fields and their boundaries.

Zinc oxide nanoparticles induces cell death and consequently leading to incomplete neural tube closure through oxidative stress during embryogenesis.

The implementation of Zinc oxide nanoparticles (ZnO NPs) raises concerns regarding their potential toxic effects on human health. Although more and more researches have confirmed the toxic effects of ZnO NPs, limited attention has been given to their impact on the early embryonic nervous system. This study aimed to explore the impact of exposure to ZnO NPs on early neurogenesis and explore its underlying mechanisms. We conducted experiments here to confirm the hypothesis that exposure to ZnO NPs causes neural tube defects in early embryonic development. We first used mouse and chicken embryos to confirm that ZnO NPs and the Zn2+ they release are able to penetrate the placental barrier, influence fetal growth and result in incomplete neural tube closure. Using SH-SY5Y cells, we determined that ZnO NPs-induced incomplete neural tube closure was caused by activation of various cell death modes, including ferroptosis, apoptosis and autophagy. Moreover, dissolved Zn2+ played a role in triggering widespread cell death. ZnO NPs were accumulated within mitochondria after entering cells, damaging mitochondrial function and resulting in the over production of reactive oxygen species, ultimately inducing cellular oxidative stress. The N-acetylcysteine (NAC) exhibits significant efficacy in mitigating cellular oxidative stress, thereby alleviating the cytotoxicity and neurotoxicity brought about by ZnO NPs. These findings indicated that the exposure of ZnO NPs in early embryonic development can induce cell death through oxidative stress, resulting in a reduced number of cells involved in early neural tube closure and ultimately resulting in incomplete neural tube closure during embryo development. The findings of this study could raise public awareness regarding the potential risks associated with the exposure and use of ZnO NPs in early pregnancy.

Lhx4 surpasses its paralog Lhx3 in promoting the differentiation of spinal V2a interneurons.

Paralog factors are considered to ensure the robustness of biological processes by providing redundant activity in cells where they are co-expressed. However, the specific contribution of each factor is frequently underestimated. In the developing spinal cord, multiple families of transcription factors successively contribute to differentiate an initially homogenous population of neural progenitors into a myriad of neuronal subsets with distinct molecular, morphological, and functional characteristics. The LIM-homeodomain transcription factors Lhx3, Lhx4, Isl1 and Isl2 promote the segregation and differentiation of spinal motor neurons and V2 interneurons. Based on their high sequence identity and their similar distribution, the Lhx3 and Lhx4 paralogs are considered to contribute similarly to these processes. However, the specific contribution of Lhx4 has never been studied. Here, we provide evidence that Lhx3 and Lhx4 are present in the same cell populations during spinal cord development. Similarly to Lhx3, Lhx4 can form multiproteic complexes with Isl1 or Isl2 and the nuclear LIM interactor NLI. Lhx4 can stimulate a V2-specific enhancer more efficiently than Lhx3 and surpasses Lhx3 in promoting the differentiation of V2a interneurons in chicken embryo electroporation experiments. Finally, Lhx4 inactivation in mice results in alterations of differentiation of the V2a subpopulation, but not of motor neuron production, suggesting that Lhx4 plays unique roles in V2a differentiation that are not compensated by the presence of Lhx3. Thus, Lhx4 could be the major LIM-HD factor involved in V2a interneuron differentiation during spinal cord development and should be considered for in vitro differentiation of spinal neuronal populations.

In Vivo Xenograft Model to Study Tumor Dormancy, Tumor Cell Dissemination and Metastasis.

Metastasis is a complex, multistep process. To study the molecular steps of the metastatic cascade, it is important to use an in vivo system that recapitulates the complex tumor microenvironment. The chicken embryo chorioallantoic membrane (CAM) is an in vivo system suitable for the implantation of xenograft tumor models. It allows the study of different aspects of the metastatic process, including the dormancy-awakening transition. The main advantages of this system are its high reproducibility, cost-effectiveness, and versatility. Here, by using two dormancy tumor models, one of head and neck squamous cell carcinoma and one of breast cancer, we described a detailed protocol for the use of the CAM model in metastasis assays and for the study of tumor growth and dormancy.

Lumen Pressure Modulation in Chicken Embryos.

Pressure exerted by fluid contained within a lumen plays a crucial role in the growth, morphogenesis, and patterning of epithelial organs. Accurate modulation of lumen pressure in the developing embryo requires sensitive and robust methods that can detect and vary pressure in the range of tens to hundreds of Pascals (Pa). Here we describe a simple, cost-effective protocol for setting up a pressure modulation apparatus combining a high-sensitivity pressure sensor and a water column whose height can be finely tuned. We demonstrate lumen pressure control using the developing brain of early chicken embryos.

The Role of Ovalbumin in Manganese Homeostasis during Chick Embryogenesis: An EPR Spectroscopic Study.

Ovalbumin (OVA), a protein vital for chick embryo nutrition, hydration, and antimicrobial protection, together with other egg-white proteins, migrates to the amniotic fluid and is orally absorbed by the embryo during embryogenesis. Recently, it has been shown that for optimal eggshell quality, the hen diet can be supplemented with manganese. Although essential for embryonic development, manganese in excess causes neurotoxicity. This study investigates whether OVA may be involved in the regulation of manganese levels. The binding of Mn(II) to OVA was investigated using electron paramagnetic resonance (EPR) spectroscopy. The results show that OVA binds a maximum of two Mn(II) ions, one with slightly weaker affinity, even in a 10-fold excess, suggesting it may have a protective role from Mn(II) overload. It seems that the binding of Mn(II), or the presence of excess Mn(II), does not affect OVA's tertiary structure, as evidenced from fluorescence and UV/vis measurements. Comparative analysis with bovine and human serum albumins revealed that they exhibit higher affinities for Mn(II) than OVA, most likely due to their essentially different physiological roles. These findings suggest that OVA does not play a role in the transport and storage of manganese; however, it may be involved in embryo protection from manganese-induced toxicity.

The developmental mechanics of divergent buckling patterns in the chick gut.

Tissue buckling is an increasingly appreciated mode of morphogenesis in the embryo, but it is often unclear how geometric and material parameters are molecularly determined in native developmental contexts to generate diverse functional patterns. Here, we study the link between differential mechanical properties and the morphogenesis of distinct anteroposterior compartments in the intestinal tract-the esophagus, small intestine, and large intestine. These regions originate from a simple, common tube but adopt unique forms. Using measured data from the developing chick gut coupled with a minimal theory and simulations of differential growth, we investigate divergent lumen morphologies along the entire early gut and demonstrate that spatiotemporal geometries, moduli, and growth rates control the segment-specific patterns of mucosal buckling. Primary buckling into wrinkles, folds, and creases along the gut, as well as secondary buckling phenomena, including period-doubling in the foregut and multiscale creasing-wrinkling in the hindgut, are captured and well explained by mechanical models. This study advances our existing knowledge of how identity leads to form in these regions, laying the foundation for future work uncovering the relationship between molecules and mechanics in gut morphological regionalization.

Emergence of large-scale cell death through ferroptotic trigger waves.

Large-scale cell death is commonly observed during organismal development and in human pathologies1-5. These cell death events extend over great distances to eliminate large populations of cells, raising the question of how cell death can be coordinated in space and time. One mechanism that enables long-range signal transmission is trigger waves6, but how this mechanism might be used for death events in cell populations remains unclear. Here we demonstrate that ferroptosis, an iron- and lipid-peroxidation-dependent form of cell death, can propagate across human cells over long distances (≥5 mm) at constant speeds (around 5.5 µm min-1) through trigger waves of reactive oxygen species (ROS). Chemical and genetic perturbations indicate a primary role of ROS feedback loops (Fenton reaction, NADPH oxidase signalling and glutathione synthesis) in controlling the progression of ferroptotic trigger waves. We show that introducing ferroptotic stress through suppression of cystine uptake activates these ROS feedback loops, converting cellular redox systems from being monostable to being bistable and thereby priming cell populations to become bistable media over which ROS propagate. Furthermore, we demonstrate that ferroptosis and its propagation accompany the massive, yet spatially restricted, cell death events during muscle remodelling of the embryonic avian limb, substantiating its use as a tissue-sculpting strategy during embryogenesis. Our findings highlight the role of ferroptosis in coordinating global cell death events, providing a paradigm for investigating large-scale cell death in embryonic development and human pathologies.

Lens placode modulates extracellular matrix formation during early eye development.

The role extracellular matrix (ECM) in multiple events of morphogenesis has been well described, little is known about its specific role in early eye development. One of the first morphogenic events in lens development is placodal thickening, which converts the presumptive lens ectoderm from cuboidal to pseudostratified epithelium. This process occurs in the anterior pre-placodal ectoderm when the optic vesicle approaches the cephalic ectoderm and is regulated by transcription factor Pax6 and secreted BMP4. Since cells and ECM have a dynamic relationship of interdependence and modulation, we hypothesized that the ECM evolves with cell shape changes during lens placode formation. This study investigates changes in optic ECM including both protein distribution deposition, extracellular gelatinase activity and gene expression patterns during early optic development using chicken and mouse models. In particular, the expression of Timp2, a metalloprotease inhibitor, corresponds with a decrease in gelatinase activity within the optic ECM. Furthermore, we demonstrate that optic ECM remodeling depends on BMP signaling in the placode. Together, our findings suggest that the lens placode plays an active role in remodeling the optic ECM during early eye development.

Effect of aripiprazole on neural tube development in early chick embryos.

INTRODUCTION: Aripiprazole (ARI) is a recently developed antipsychotic medication that belongs to the second generation of antipsychotics. The literature has contradictory information regarding ARI, which has been classified as pregnant use category C by the FDA. METHODS: 125 pathogen-free fertilized eggs were incubated for 28 h and divided into five groups of 25 eggs each (including the control group), and 18 eggs with intact integrity were selected from each group. After the experimental groups were divided, ARI was administered subblastodermally with a Hamilton micro-injector at 4 different doses (1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg). At the 48th hour of incubation, all eggs were hatched and embryos were removed from the embryonic membranes. And then morphologic (position of the neural tube (open or closed), crown-rump length, number of somites, embryological development status), histopathologic (apoptosis (caspase 3), cell proliferation (PCNA), in situ recognition of DNA breaks (tunnel)), genetic (BRE gene expression) analyzes were performed. RESULTS: According to the results of the morphological analysis, when the frequency of neural tube patency was evaluated among the experimental groups, a statistically significant difference was determined between the control group and all groups (p < 0.001). In addition, the mean crown-rump length and somite number of the embryos decreased in a dose-dependent manner compared to the control group. It was determined that mRNA levels of the BRE gene decreased in embryos exposed to ARI compared to the control group (p < 0.001). CONCLUSION: Morphologically, histopathologically, and genetically, aripiprazole exposure delayed neurogenesis and development in early chick embryos. These findings suggest its use in pregnant women may be teratogenic. We note that these results are preliminary for pregnant women, but they should be expanded and studied with additional and other samples.

Effect of post-gastrulation exposure to acrylamide on chick embryonic development.

The critical developmental stages of the embryo are strongly influenced by the dietary composition of the mother. Acrylamide is a food contaminant that can form in carbohydrate-rich foods that are heat-treated. The aim of this study was to investigate the toxicity of a relatively low dose of acrylamide on the development of the neural tube in the early stage chick embryos. Specific pathogen-free fertilized eggs (n = 100) were treated with acrylamide (0.1, 0.5, 2.5, 12.5 mg/kg) between 28-30th hours of incubation and dissected at 48th hours. In addition to morphological and histopathological examinations, proliferating cell nuclear antigen (PCNA) and caspase 3 were analyzed immunohistochemically. The brain and reproductive expression gene (BRE) was analyzed by RT-PCR. Acrylamide exposure had a negative effect on neural tube status even at a very low dose (0.1 mg/kg) (p < 0.05). Doses of 0.5 mg/kg and above caused a delay in neural tube development (p < 0.05). Crown-rump length and somite count decreased dose-dependently, while this decrease was not significant in the very low dose group (p > 0.05), which was most pronounced at doses of 2.5 and 12.5 mg/kg (p < 0.001). Acrylamide exposure dose-dependently decreased PCNA and increased caspase 3, with this change being significant at doses of 0.5 mg/kg and above (p < 0.001). BRE was downregulated at all acrylamide exposures except in the very low dose group (0.1 mg/kg). In conclusion, we find that acrylamide exposure (at 0.5 mg/kg and above) in post-gastrulation delays neural tube closure in chicken embryos by suppressing proliferation and apoptosis induction and downregulating BRE gene expression.

3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing.

BACKGROUND: Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. RESULTS: We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. CONCLUSIONS: We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

Protective effects of ferulic acid on embryonic development by improving antioxidant function in broilers embryo of thermal manipulation.

This study aimed to elucidate the effects of broiler embryos soaked in ferulic acid (FA) solution on alleviating the negative impact of thermal manipulation (TM) on chicken embryo development and to provide a theoretical and experimental basis for applying TM and FA in the poultry feeding industry. A total of 120 broiler fertilized eggs were randomly divided into three groups: control group, TM group, and comprehensive group (TM + FA), with 40 eggs in each group. The TM group and the comprehensive group from the 7th embryonic age to the 16th embryonic age received TM for ten days, treated with a temperature of 39.5 °C and relative humidity of 65% for 18 h a day. In the comprehensive group, broiler embryos were immersed in FA solution at a concentration of 80 mg/L for 6 min at 16:00 every day from the 6th to the 8th embryo age. They were incubated continuously after being soaked until the chicks hatched. The results showed that the rates of dead embryos and weak chicks in the TM group were significantly higher than those in the control group and comprehensive group. Chick body temperatures of the TM group and comprehensive group were significantly lower than those of the control group. The heart weights of the TM group and comprehensive group were significantly lower than those of the control group, and the leg weights of the TM group were significantly decreased compared with those of the control group and comprehensive group. The SOD activity of serum in the comprehensive group was significantly higher than that in the control group and TM group, while the CAT activity of serum in the comprehensive group and control group was significantly higher than that in the TM group; however, there was no difference between the comprehensive group and control group. The activities of SOD and CAT in the liver were significantly higher than those of the TM group; however, the MDA content of the liver in the comprehensive group and control group was significantly lower than that of the TM group. The gene expression of Nrf2 and SOD in the comprehensive group and TM group was significantly higher than that in the control group; however, there was no significant difference between the comprehensive group and TM group. Soaking broiler embryonic eggs in an FA solution can improve the antioxidant capacity of the liver by upregulating Nrf2-Keap1 signal pathway-related gene expression. FA can effectively alleviate the side effects of TM on chicken embryos and does not impact the effects of TM.

Protocol for observing lipid droplet dynamics in chicken cone cells.

Chicken cone cells are an excellent model for studying the regulation of lipid droplet dynamics. Here, we present a protocol for studying cone cell lipid droplets from in vivo and ex vitro cultured retinas of chicken embryos. We describe steps for dissecting chicken retinas, electroporating retinas, culturing retinas ex vivo and in vitro, and staining lipid droplets with neutral lipid dye. This protocol is also applicable to investigating other organelles in retinas. For complete details on the use and execution of this protocol, please refer to Pan et al.1.