Integrated transcriptomics and metabolomics study of embryonic breast muscle of Jiaji ducks.

Because number of matured muscle fibers in poultry does not increase after birth, the meat yield is mainly determined during embryogenesis. We previously indicated breast muscle grew rapidly from 18th day after hatching (E18) to E27, and almost stopped from E27 to E34 of Jiaji ducks, while the mechanism is unclear. This study utilized RNA-seq to explore the related genes of muscle development and their relationship with small molecule metabolites at E18, E27 and E34 of Jiaji ducks. Several thousand differentially expressed genes (DEGs) were detected among E18, E27 and E34. DEGs expression profiles included 8 trend maps, among which trend 1 was opposite to and trend 6 was consistent with breast muscle development trend of Jiaji ducks. Through joint analysis between trend 1 of DEGs and trend 1 of differential metabolites (DEMs), protein digestion and absorption pathway stood out. The decrease of COL8A2 gene expression will lead to the decrease of arginine content, which will inhibit the development of breast muscle in embryonic Jiaji duck. Similarly, joint analysis between trend 6 of DEGs and trend 6 of DEMs indicated the increase of GAMT gene expression will cause the increase of proline content, and then promote the development of breast muscle of Jiaji duck in embryonic period. These results will be helpful for further understanding the mechanism of muscle yields of Jiaji ducks.

Differential expression profile and in-silico functional analysis of long noncoding RNA and mRNA in duck embryo fibroblasts infected with duck plague virus.

BACKGROUND: Duck plague virus (DPV), belonging to herpesviruses, is a linear double-stranded DNA virus. There are many reports about the outbreak of the duck plague in a variety of countries, which caused huge economic losses. Recently, increasing reports revealed that multiple long non-coding RNAs (lncRNAs) can possess great potential in the regulation of host antiviral immune response. Furthermore, it remains to be determined which specific molecular mechanisms are responsible for the DPV-host interaction in host immunity. Here, lncRNAs and mRNAs in DPV infected duck embryonic fibroblast (DEF) cells were identified by high-throughput RNA-sequencing (RNA-seq). And we predicted target genes of differentially expressed genes (DEGs) and formed a complex regulatory network depending on in-silico analysis and prediction. RESULT: RNA-seq analysis results showed that 2921 lncRNAs were found at 30 h post-infection (hpi). In our study, 218 DE lncRNAs and 2840 DE mRNAs were obtained in DEF after DPV infection. Among these DEGs and target genes, some have been authenticated as immune-related molecules, such as a Macrophage mannose receptor (MR), Anas platyrhynchos toll-like receptor 2 (TLR2), leukocyte differentiation antigen, interleukin family, and their related regulatory factors. Furthermore, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, we found that the target genes may have important effects on biological development, biosynthesis, signal transduction, cell biological regulation, and cell process. Also, we obtained, the potential targeting relationship existing in DEF cells between host lncRNAs and DPV-encoded miRNAs by software. CONCLUSIONS: This study revealed not only expression changes, but also the possible biological regulatory relationship of lncRNAs and mRNAs in DPV infected DEF cells. Together, these data and analyses provide additional insight into the role of lncRNAs and mRNAs in the host's immune response to DPV infection.

Optimizing environmental safety and cell-killing potential of oncolytic Newcastle Disease virus with modifications of the V, F and HN genes.

Newcastle Disease Virus (NDV) is an avian RNA virus, which was shown to be effective and safe for use in oncolytic viral therapy for several tumour malignancies. The presence of a multi basic cleavage site (MBCS) in the fusion protein improved its oncolytic efficacy in vitro and in vivo. However, NDV with a MBCS can be virulent in poultry. We aimed to develop an NDV with a MBCS but with reduced virulence for poultry while remaining effective in killing human tumour cells. To this end, the open reading frame of the V protein, an avian specific type I interferon antagonist, was disrupted by introducing multiple mutations. NDV with a mutated V gene was attenuated in avian cells and chicken and duck eggs. Although this virus still killed tumour cells, the efficacy was reduced compared to the virulent NDV. Introduction of various mutations in the fusion (F) and hemagglutinin-neuraminidase (HN) genes slightly improved this efficacy. Taken together, these data demonstrated that NDV with a MBCS but with abrogation of the V protein ORF and mutations in the F and HN genes can be safe for evaluation in oncolytic viral therapy.

Mycotoxin Fumonisin B1 Interferes Sphingolipid Metabolisms and Neural Tube Closure during Early Embryogenesis in Brown Tsaiya Ducks.

Fumonisin B1 (FB1) is among the most common contaminants produced by Fusarium spp. fungus from corns and animal feeds. Although FB1 has been known to cause physical or functional defects of embryos in humans and several animal species such as Syrian hamsters, rabbits, and rodents, little is known about the precise toxicity to the embryos and the underlying mechanisms have not been fully addressed. The present study aimed to investigate its developmental toxicity and potential mechanisms of action on sphingolipid metabolism in Brown Tsaiya Ducks (BTDs) embryos. We examined the effect of various FB1 dosages (0, 10, 20 and 40 µg/embryo) on BTD embryogenesis 72 h post-incubation. The sphingomyelin content of duck embryos decreased (p < 0.05) in the highest FB1-treated group (40 µg). Failure of neural tube closure was observed in treated embryos and the expression levels of a neurulation-related gene, sonic hedgehog (Shh) was abnormally decreased. The sphingolipid metabolism-related genes including N-acylsphingosine amidohydrolase 1 (ASAH1), and ceramide synthase 6 (CERS6) expressions were altered in the treated embryos compared to those in the control embryos. Apparently, FB1 have interfered sphingolipid metabolisms by inhibiting the functions of ceramide synthase and folate transporters. In conclusion, FB1-caused developmental retardation and abnormalities, such as neural tube defects in Brown Tsaiya Duck embryos, as well as are partly mediated by the disruption of sphingolipid metabolisms.

Effect of Maternal Marginal Zinc Deficiency on Development, Redox Status, and Gene Expression Related to Oxidation and Apoptosis in an Avian Embryo Model.

Maternal severe zinc (Zn) deficiency resulted in growth retardation and high mortality during embryonic development in human. Therefore, this study is aimed at evaluating the effect of maternal marginal Zn deficiency on the development and redox status to avoid severe Zn deficiency using an avian model. A total of 324 laying duck breeders at 214 days old were randomly allotted into 3 dietary Zn levels with 6 replicates of 18 ducks per replicate. The birds were fed experimental diets including 3 dietary supplemental Zn levels of 0 mg/kg (maternal Zn-deficient group, 29.2 mg Zn/kg diet), 60 mg/kg (maternal Zn-adequate group), and 120 mg/kg (maternal Zn-high group) for 6 weeks. Dietary Zn levels had on effect on egg production and fertility (P > 0.05), whereas dietary Zn deficiency decreased breeder plasma Zn concentration and erythrocytic alkaline phosphatase activity at week 6 and inhibited erythrocytic 5'-nucleotidase (5'-NT) activity at weeks 2, 4, and 6 (P < 0.05), indicating that marginal Zn-deficient status occurred after Zn depletion. Maternal marginal Zn deficiency increased embryonic mortality and contents of superoxide anion radical, MDA, and PPC and reduced MT content and CuZnSOD activity in duck embryonic livers on E29. The MDA content was positively correlated with embryonic mortality. Maternal marginal Zn deficiency increased BCL2-associated X protein and Caspase-9 mRNA expressions as well as decreased B-cell lymphoma-2 and MT1 mRNA and signal AKT1 and ERK1 protein expressions (P < 0.05). Breeder plasma Zn concentration and erythrocytic 5'-NT activities at week 6 were positively correlated with GSH-Px activity and GPx, MT1, and BCL2 mRNA expressions in embryonic livers on E29. In conclusion, erythrocytic 5'-NT activity could be more rapid and reliable to monitor marginal Zn-deficient status. Marginal Zn deficiency impaired hatchability and antioxidant defense system and then induced oxidative damage and apoptosis in the embryonic liver, contributing to the greater loss of duck embryonic death.

Duck Tembusu Virus Infection Promotes the Expression of Duck Interferon-Induced Protein 35 to Counteract RIG-I Antiviral Signaling in Duck Embryo Fibroblasts.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused a substantial drop in egg production and severe neurological disorders in domestic waterfowl. Several studies have revealed that viral proteins encoded by DTMUV antagonize host IFN-mediated antiviral responses to facilitate virus replication. However, the role of host gene expression regulated by DTMUV in innate immune evasion remains largely unknown. Here, we utilized a stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics analysis of DTMUV-infected duck embryo fibroblasts (DEFs) to comprehensively investigate host proteins involved in DTMUV replication and innate immune response. A total of 250 differentially expressed proteins were identified from 2697 quantified cellular proteins, among which duck interferon-induced protein 35 (duIFI35) was dramatically up-regulated due to DTMUV infection in DEFs. Next, we demonstrated that duIFI35 expression promoted DTMUV replication and impaired Sendai virus-induced IFN-ß production. Moreover, duIFI35 was able to impede duck RIG-I (duRIG-I)-induced IFN-ß promoter activity, rather than IFN-ß transcription mediated by MDA5, MAVS, TBK1, IKKϵ, and IRF7. Importantly, we found that because of the specific interaction with duIFI35, the capacity of duRIG-I to recognize double-stranded RNA was significantly impaired, resulting in the decline of duRIG-I-induced IFN-ß production. Taken together, our data revealed that duIFI35 expression stimulated by DTMUV infection disrupted duRIG-I-mediated host antiviral response, elucidating a distinct function of duIFI35 from human IFI35, by which DTMUV escapes host innate immune response, and providing information for the design of antiviral drug.

Decorin regulates myostatin and enhances proliferation and differentiation of embryonic myoblasts in Leizhou black duck.

Skeletal muscle is one of the most important economic traits in the poultry industry whose development goes through several processes influenced by several candidate genes. This study explored the regulatory role of DCN on MSTN and the influence of these genes on the proliferation and differentiation of embryonic myoblasts in Leizhou black ducks. Embryonic myoblasts were transfected with over-expressing DCN, Si-DCN, and empty vector and cultured for 24 h, 48 h, and 72 h of proliferation and the comparative expression of DCN and MSTN were measured. The results showed that cells transfected with the over-expression DCN had a significantly (P < 0.05) higher expression of DCN mRNA than the normal group and the expression of MSTN mRNA showed a downward trend during the proliferation of myoblasts. DCN mRNA expression was lower in cells transfected with Si-DCN than the normal group in all stages of proliferation. While the expression of MSTN in the Si-DCN transfected group was higher than the normal group with a significant (P < 0.05) difference at the 72 h stage. DCN mRNA increased at the early stage of differentiation but decreased (P > 0.05) from the 6th day to the 8th day of differentiation. The level of MSTN increased gradually during the differentiation process of myoblasts until it decreased significantly on the 8th day. These results show that DCN enhances the proliferation and differentiation of Leizhou black duck myoblasts and suppresses MSTN activity.

DPV UL41 gene encoding protein induces host shutoff activity and affects viral replication.

The virion host shutoff (VHS) protein, encoded by the UL41 gene of herpes simplex virus (HSV), specifically degrades mRNA and induces host shutoff. VHS and its homologs are highly conserved in the Alphaherpesvirinae subfamily. However, the role of the duck plague virus (DPV) UL41 gene is unclear. In this study, we found that the DPV UL41 gene-encoded protein (pUL41) degrades RNA polymerase (pol) II-transcribed translatable RNA and induces protein synthesis shutoff. DPV pUL41 was dispensable for viral replication, but the UL41-deleted mutant virus exhibited a significant viral growth defect and plaque size reduction in Duck embryo fibroblast (DEF) cells. Furthermore, DPV pUL41 regulated viral mRNA accumulation to affect viral DNA replication, release and cell-to-cell spread.

Identification of key genes and pathways associated with duck (Anas platyrhynchos) embryonic skin development using weighted gene co-expression network analysis.

Skin and feather follicle morphogenesis are important processes for duck development; however, the mechanisms underlying morphogenesis at the embryonic stage remain unclear. To improve the understanding of these processes, we used transcriptome and weighted gene co-expression network analyses to identify the critical genes and pathways involved in duck skin development. Five modules were found to be the most related to five key stages in skin development that span from embryonic day 8 (E8) to postnatal day 7 (D7). Using STEM software, 6519 genes from five modules were clustered into 10 profiles to reveal key genes. Above all, we obtained several key module genes including WNT3A, NOTCH1, SHH, BMP2, NOG, SMAD3, and TGFß2. Furthermore, we revealed that several pathways play critical roles throughout the skin development process, including the Wnt pathway and cytoskeletal rearrangement-related pathways, whereas others are involved in specific stages of skin development, such as the Notch, Hedgehog, and TGF-beta signaling pathways. Overall, this study identified the pathways and genes that play critical roles in skin development, which may provide a basis for high-quality down-type meat duck breeding.

Differently Expression Analysis and Function Prediction of Long Non-coding RNAs in Duck Embryo Fibroblast Cells Infected by Duck Tembusu Virus.

Duck Tembusu virus (DTMUV), the causative agent of egg-drop syndrome, has caused substantial economic losses to duck industry. DTMUV infection leads to profound changes of host cells, including transcriptome and proteome. However, the lncRNA expression profile and the biological function of lncRNA have not been revealed. Therefore, DTMUV was used to inoculate duck embryo fibroblast cells (DEFs) for high-throughput RNA-sequencing (RNA-Seq). The results showed that 34 and 339 differently expressed lncRNAs were, respectively, identified at 12 and 24 h post-infection (hpi). To analyze their biological functions, target genes in cis were searched and the regulatory network was formed. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the target genes were strongly associated with immune system, signaling molecular and interaction, endocrine system, and signal transduction. The differently expressed lncRNAs were selected and verified by quantitative real-time polymerase chain reaction (RT-qPCR). Our study, for the first time, analyzed a comprehensive lncRNA expression profile in DEFs following DTMUV infection. The analysis provided a view on the important roles of lncRNAs in gene regulation and DTMUV infection.

Antioxidant activities and protective effects of duck embryo peptides against H2O2-induced oxidative damage in HepG2 cells.

Previous work showed that peptides from duck eggs incubated for 15 D presented high total antioxidant activities. Here, this work explore the antioxidant activities of different segments, ZT1 (≤3 KD), ZT2 (≤10 KD), and ZT3 (≤30 KD), derived from duck embryo peptides and their protective effects against H2O2-induced oxidative damage in HepG2 cells. Peptides present no cytotoxicity to HepG2 cells. Moreover, ZT1 exhibits a higher ability to scavenge several radicals as well as stronger inhibition of H2O2-induced oxidative stress than ZT2 and ZT3. The activities of catalase and glutathione peroxidase as well as total superoxide dismutase increase in a concentration-dependent manner. Peptides are isolated from ZT1 and then subjected to LC-MS/MS to identify their sequences, followed by functional annotation, bioinformatics prediction, and hot-spot motif recognition. As a result, 413 potential functional peptides are identified, with some compounds exhibiting more than 1 function. This work will help for exploring bioactive substances in duck embryo eggs and enhance the utilization value of duck or other poultry eggs.

Effect of carbon dioxide during the early stage of duck egg incubation on hatching characteristics and duckling performance.

Gaseous exchange is very important for embryonic development during the incubation of bird eggs. The present study was carried out to investigate the effect of carbon dioxide (CO2) pumping during the first 10 days of Pekin duck egg incubation on hatching characteristics, embryonic growth, hormonal concentrations and the post-hatch weight of the ducks. Two different ventilation conditions were used in this study. In one condition (V), the incubator was ventilated standard. In the other condition (NV), CO2 was gradually pumped into a non-ventilated incubator to reach and maintain a 1% concentration through the first 10 days of incubation, after which the incubator was ventilated for the rest of the incubation period. The CO2 percentage in the V incubator was constant at approximately 0.18%; by contrast, in the NV incubator, the concentration was increased gradually from 0.17% to 1%, where it was maintained through the 10th day of incubation. Throughout the incubation period, the egg weight loss and the embryonic mortality percentages were significantly lower in NV than in V. The hatchability of fertile eggs and the weights of ducklings at hatch were significantly higher in NV than in V. The embryonic T3, T4 and corticosterone levels in NV were significantly higher than those in V. Additionally, the haematological parameters (haemoglobin, packed cell volume and red blood cells) of the embryos were significantly higher in NV than in V. Furthermore, a total of 1198 ducklings for the V and NV conditions, all one day old, were individually weighed and then transferred into randomly assigned floor pens with 3 replicates for each ventilation condition. The feed conversion ratio of the ducks was significantly lower in NV than in V, while the reverse was true of body weight at different ages. Therefore, we can conclude that the NV condition with circulation of CO2 for the first 10 days of incubation is preferable to the V condition.

Anti-angiogenic activity of Gracilaria coronopifolia J.G. Agardh extract by lowering the levels of trace metals (iron, zinc and copper) in duck chorioallantoic membrane and in vitro activation of AMP-kinase.

AMP-activated protein kinase (AMPK) is an intracellular energy sensor important in metabolic regulation, cell growth, and survival. However, the specific role of AMPK signaling pathway in the inhibition of angiogenesis remains unclear. The study highlights the activity on AMP activated protein kinase signaling pathways of a marine algae, Gracilaria coronopifolia, and its effects on angiogenesis. It was found that the most potent extract, GCD, inhibited angiogenesis significantly in the duck chorioallantoic membrane assay and also activated the enzyme AMP-kinase, in vitro. The dichloromethane extract was found most active in inhibiting angiogenesis in the duck chorioallantoic membrane (IC50 = 1.21 µg/mL) followed by GCH (IC50 = 3.08 µg/mL) (p = 0.479) and GCM (IC50 = 8.93 µg/mL) (p = 0.042). Benferroni post hoc analysis revealed that there was no significant difference between the percent inhibitions of GCH and GCM extracts (p = 0.479). Consequently, angiogenic inhibition caused lowering of iron, zinc, and copper levels in the duck CAM. Thin layer chromatography and gas chromatography-mass spectrometry revealed the components of each extracts. Notably, this is the first report on the kinase activity of a red algae G. coronopifolia extracts and a colorimetric-based quantification of angiogenesis based on metal content of CAM. Our data also suggest a novel therapeutic approach for inhibiting angiogenesis through the AMPK pathway.

Comparison of whole embryonic development in the duck (Anas platyrhynchos) and goose (Anser cygnoides) with the chicken (Gallus gallus).

Embryos from aquatic birds are the primary models for the study of flipper development. While some staging of early embryogenesis in duck have been studied, characterization of the stages of the entire embryonic development period in water birds has not been described. This study aimed to establish a comparison of complete morphological development staging for ducks (Anas platyrhynchos) and geese (Anser cygnoides) with the embryonic staging system by Hamburger and Hamilton (HH) for the chicken (Gallus gallus). Our results show that morphological development in the chicken, duck, and goose are similar in the early stages. The major differences occurred after stage 27 of embryonic development, where the beak shape in ducks and geese was wider and longer than in chickens. In addition, the second and third interdigital webs of the hind limb of the chicken were found to be degraded from stage 31, and eventually vanished at stage 35; however, they were retained in ducks and geese. Rapid physical development occurred in the mid-to-late stages in ducks and geese. To our best knowledge, this is the first description of complete embryonic development for the duck and goose. Establishment of an embryonic staging system for duck and goose provides new models for the study of waterfowl development.

Effect of thermal manipulation during embryogenesis on the promoter methylation and expression of myogenesis-related genes in duck skeletal muscle.

Avian embryos are an ideal system to investigate the effect of incubation temperature on embryonic development, but the characteristics and mechanisms of temperature effects on poultry embryonic myogenesis are unclear. In this study, we investigated the effect of increasing the incubation temperature by 1 °C on the expression of nine myogenesis-related genes in ducks and then explored the correlation between the alteration of promoter methylation and the expression of two of the nine genes under thermal manipulation (TM). The qRT-PCR results showed that TM during embryonic days (ED) 1-10 promoted (P < 0.05) the expression of genes in breast muscle (PAX3, PAX7, MYOG, MCK, SIX1, TNNC1) and leg muscle (MYOD, MYOG, MYF5, MCK, AKIRIN2, TNNC1). TM during ED10-20 promoted the expression of PAX3, MYF5 and MCK and inhibited AKIRIN2 expression in breast muscle (P < 0.05); however, it inhibited the expression of PAX3, PAX7, MYOD, MYOG, MYF5, SIX1, AKIRIN2 and TNNC1 and promoted MCK expression in leg muscle (P < 0.05). TM during ED20-27 inhibited the expression of genes in breast muscle (PAX7) and leg muscle (MYOD, MYOG, MYF5, TNNC1) and promoted MCK expression in breast and leg muscle (P < 0.05). Furthermore, with the Sequenom MassARRAY platform, it was observed that the average methylation level of AKIRIN2 (ED10) and TNNC1 (ED20) in leg muscle decreased (P < 0.05) after TM. Notably, we found significant (P < 0.05) inverse correlations between the methylation and mRNA levels of AKIRIN2 under TM during ED1-10 (r = - 0.969) and ED10-20 (r = - 0.805). Taken together, TM during ED1-10 was more favorable for improving duck myogenesis-related gene expression than TM during ED10-20 and ED20-27. TM during duck embryogenesis seemed to have a greater effect on the development of leg muscle than breast muscle and might alter AKIRIN2 expression by changing its promoter methylation status. These findings may be helpful to understand temperature effects on the muscle development of avian embryos and to explore the role of epigenetic regulation during this process.

EDACO, a derivative of myricetin, inhibits the differentiation of Gaoyou duck embryonic osteoclasts in vitro.

1. This study determined the effects of (E)-3-(2-(4-(3-(2,4-dimethoxyphenyl)acryloyl)phenoxy)ethoxy)-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (EDACO) on the differentiation of Gaoyou duck embryonic osteoclasts cultured in vitro. 2. Bone marrow mononuclear cells (BM-MNC) were collected from 23-d-old Gaoyou duck embryos and induced by macrophage colony-stimulating factor and receptor activator of nuclear factor κB ligand in the presence of EDACO at different concentrations (i.e. 10, 20, 40, 80 and 160 µM). Tartrate-resistant acid phosphatase (TRAP) staining and resorption ability determination were conducted. 3. Results suggested that EDACO suppressed the shaping of positive multinucleated cells and the number of TRAP-positive cells in the 20, 40, 80 and 160 µM EDACO groups was significantly decreased (P < 0.05 or P < 0.01). Besides, the absorption activity of differentiated duck embryonic osteoclasts was significantly inhibited (P < 0.05) in both 80 and 160 µM EDACO groups. 4. Overall, EDACO can inhibit the differentiation of BM-MNC into mature osteoclasts in duck embryos.1.

Embryonic morphology observation and HOXC8 gene expression in crest cushions of Chinese Crested duck.

The Chinese Crested duck (Fengtou duck) reappeared in China recently. Along with white feathers and a black bill and feet, the Fengtou duck has a high feather crest. This breed can be used for ornamental purposes or as a model organism; however, little is known about the genetic basis and development of its distinct morphological features. In this study, we observed the skull and feather crest of Fengtou duck in the embryonic stages. As a result, the protuberances of the head integument could be clearly observed at embryonic stage E9, and small perforations in the skull were first visible at E13 and were clearer at E15. Besides, intracranial fat in a small number of individuals was found starting at E15, and a small number of osteophytes was found at E18. In addition, hematoxylin-eosin (HE) and Oil Red-O staining of the crest cushion and intracranial tissue revealed fat tissue accumulation. Previous studies demonstrated that homeobox c8 (HOXC8) played a critical role in chicken crest formation. Here, we cloned the HOXC8 from Fengtou duck and determined that its transcript was highly expressed in the crest cushion; moreover, HOXC8 was detected in this tissue with a molecular weight of 38 kDa in the Fengtou duck. In conclusion, embryos of Fengtou duck have different small protuberances and perforations in the skull, including accumulation of intracranial tissue and osteophytes in some cases. Furthermore, HOXC8 may regulate the formation of the crest. These findings provide novel insight into the ontogenesis of the crest cushion in crested ducks and a basis for future studies on their evolutionary origins.

Embryonic development of parakeratinized epithelium of the tongue in the domestic duck (Anas platyrhynchos f. domestica): LM, SEM, and TEM observations.

The parakeratinized epithelium is a common and widespread type of keratinized epithelium in the oral cavity in adult birds. In contrast to orthokeratinized epithelium, which mostly covers mechanical papillae and the lingual nail, parakeratinized epithelium covers almost the entire dorsal surface of the tongue in birds. The characteristic feature of parakeratinized epithelium is the presence of nuclei in the keratinized layer. The present study aimed to investigate for the first time the micro- and ultrastructural changes of parakeratinized epithelium during embryonic development and to assess the readiness of the epithelium to serve protective functions during food transport to the esophagus. Three developmental stages were distinguished: embryonic, transformation, and pre-hatching stages. The embryonic stage lasts from the 9th to the 14th day of incubation and the epithelium is composed of undifferentiated epithelial cells. The transformation stage lasts from the 15th to the 22nd day of incubation and the epithelium undergoes transformation into stratified epithelium consisting of basal, intermediate, and superficial layers. The characteristic feature of this stage is formation of the periderm with osmophilic granules. The pre-hatching stage starts on the 23rd day, and the epithelium with a fully developed keratinized layer resembles that of the epithelium in adult animals. No periderm was observed on the epithelial surface. It was confirmed that at the time of hatching the parakeratinized epithelium is fully differentiated and ready to fulfill its function during food transport. The presence of periderm is a common feature characteristic for para- and orthokeratinized epithelium in the oral cavity of birds. However, the formation of the keratinized/cornified layer is different for these two types of keratinized epithelia.

3-(4-methoxyl)-1-(2-(4-coumarin)prop)-2-en-1-one inhibits the differentiation of Gaoyou duck embryonic osteoclasts in vitro.

This study determined the influence of 3-(4-methoxyl)-1-(2-(4-coumarin)prop)-2-en-1-one (MCPEO) on the differentiation of Gaoyou duck embryo osteoclasts cultured in vitro. Bone marrow mononuclear cells (BM-MNCs) were harvested from 23-day-old Gaoyou duck embryos and induced by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in the presence of MCPEO at different concentrations (i.e., 1, 5, 10, 20, and 40 µM). Cell viability measurement, tartrate-resistant acid phosphatase (TRAP) staining, resorption activity assay, and co-staining with Tetramethylrhodamine (TRITC)-conjugated phalloidin and Hoechst 33,258 were conducted. Results indicated that MCPEO influenced the cell viability of the M-CSF + RANKL-induced BM-MNCs in a concentration-dependent manner, reduced the formation of positive multinucleated cells, and restrained the resorption capability of osteoclasts. Microfilament and nuclear staining indicated that MCPEO restricted the differentiation of BM-MNCs into large multinucleated osteoclasts. In short, MCPEO can inhibit the differentiation of BM-MNCs into mature osteoclasts in duck embryos. Therefore, MCPEO is a promising agent for the treatment of poultry osteoporosis.