Chick Oviduct Research Articles

Intravaginally CpG-ODN and Salmonella enteritidis on TLR21, cytokines, and AvBD10 gene expressions in the reproductive tract of native chicken.

Salmonella enterica subsp. enterica serovar Enteritidis (SE) is a global concern for the poultry industry due to its association with foodborne illnesses. The transmission occurs through the transovarial route which initiates from colonization in oviducts and ascending to ovaries. Though there are studies on cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) and the increase of innate immune response, there is limited research on the intravaginal treatment using CpG-ODN. Previous studies have shown that stimulating CpG-ODN can induce the production of antimicrobial peptide avian beta-defensins (AvBDs) in vaginal cell cultures, there is limited information on the use of intravaginal treatment to induce the innate immune system, particularly in the Kampung Unggul Balitbangtan (KUB-1) chickens (Gallus gallus domesticus). This study investigates the impact of intravaginal CpG-ODN stimulation on the innate immune response in KUB-1 chicken ovaries and oviducts when challenged to SE. A total of 39 KUB-1 chickens were divided into four groups namely T1 (treated with CpG-ODN, n=12), T2 (SE group, n=12), T3 (CpG-ODN and SE, n=12), and Control (without CpG-ODN and SE, n=3). Chickens were observed from day 1 to 4 post-intravaginal (PI) inoculation. The results suggest that intravaginal CpG-ODN treatment modulates AvBD10 production through toll-like receptor (TLR)21, with interleukin (IL)1B and IL10 playing reciprocal roles, providing insights into the potential of this treatment to prevent transovarial Salmonellosis in poultry. The novelty of this study adds valuable insights to the current body of knowledge.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken oviduct tract

Utilizing publicly available RNA-seq data to screen for ideal reference genes is more efficient and accurate than traditional methods. Previous studies have identified optimal reference genes in various chicken tissues, but none have specifically focused on the oviduct (including the infundibulum, magnum, isthmus, uterus, and vagina), which is crucial for egg production. Identifying stable reference genes in the oviduct is essential for improving research on gene expression levels. This study investigated genes with consistent expression patterns in the chicken oviduct, encompassing both individual oviduct tract tissues and the entire oviduct, by utilizing multiple RNA-seq datasets. The screening results revealed the discovery of 100 novel reference genes in each segment of oviduct tissues, primarily associated with cell cycle regulation and RNA binding. Moreover, the majority of housekeeping genes (HKGs) showed inconsistent expression levels across distinct samples, suggesting their lack of stability under varying conditions. The stability of the newly identified reference genes was assessed in comparison to previously validated stable reference genes in chicken oviduct and commonly utilized HKGs, employing traditional reference gene screening methods. HERPUD2, CSDE1, VPS35, PBRM1, LSM14A, and YWHAB were identified to be suitable novel reference gene for different parts of the oviduct. HERPUD2 and YWHAB were reliable for gene expression normalization throughout the oviduct tract. Furthermore, overexpression and interference assays in DF1 cells showed LSM14A and YWHAB play a crucial role in cell proliferation, highlighting the importance of these newly reference genes for further research. Overall, this study has expanded the options for reference genes in RT-qPCR experiments in different segments of the chicken oviduct and the entire oviduct.

Sustainable production of multimeric and functional recombinant human adiponectin using genome-edited chickens

BackgroundAdiponectin (ADPN) plays a critical role in endocrine and cardiovascular functions, but traditional production methods, such as Escherichia coli and mammalian systems, have faced challenges in generating sufficiently active middle molecular weight (MMW) and high molecular weight (HMW) forms of recombinant human ADPN (hADPN). In our previous study, we proposed genome-edited chickens as an efficient platform for producing multimeric hADPN. However, the consistency of multimeric hADPN expression in this system across generations had not been further investigated.ResultsIn this study, subsequent generations of ovalbumin (OVA) ADPN knock-in chickens showed stable multimeric hADPN production, yielding ~ 26% HMW ADPN (0.59 mg/mL) per hen. Comparative analysis revealed that egg white (EW)-derived hADPN predominantly consisted of hexameric and HMW forms, similar to serum-derived hADPN. In contrast, hADPN obtained from human embryonic kidney (HEK) 293 and High-Five (Hi-5) cells also exhibited the presence of trimers, indicating variability across different production systems. Furthermore, transcriptional expression analysis of ADPN multimerization-associated endoplasmic reticulum chaperone genes (Ero1-Lα, DsbA-L, ERP44, and PDI) indicated upregulation in the oviduct magnum of ADPN KI hens, suggesting the chicken oviduct magnum as the optimal site for HMW ADPN production. Lastly, the functional analysis demonstrated that EW-derived hADPN significantly reduced lipid droplets and downregulated lipid accumulation-related genes (LOX-1, AT1R, FAS, and FABP4) in human umbilical vein endothelial cells (HUVECs).ConclusionIn summary, stable and functional multimeric hADPN can be produced in genome-edited chickens even after generations. This highlights the potential of using chicken bioreactor for producing various high-value proteins.

The H9N2 avian influenza virus increases APEC adhesion to oviduct epithelia by viral NS1 protein-mediated activation of the TGF-β pathway.

H9N2 avian influenza is a low-pathogenic avian influenza circulating in poultry and wild birds worldwide and frequently contributes to chicken salpingitis that is caused by avian pathogenic Escherichia coli (APEC), leading to huge economic losses and risks for food safety. Currently, how the H9N2 virus contributes to APEC infection and facilitates salpingitis remains elusive. In this study, in vitro chicken oviduct epithelial cell (COEC) model and in vivo studies were performed to investigate the role of H9N2 viruses on secondary APEC infection, and we identified that H9N2 virus enhances APEC infection both in vitro and in vivo. To understand the mechanisms behind this phenomenon, adhesive molecules on the cell surface facilitating APEC adhesion were checked, and we found that H9N2 virus could upregulate the expression of fibronectin, which promotes APEC adhesion onto COECs. We further investigated how fibronectin expression is regulated by H9N2 virus infection and revealed that transforming growth factor beta (TGF-β) signaling pathway is activated by the NS1 protein of the virus, thus regulating the expression of adhesive molecules. These new findings revealed the role of H9N2 virus in salpingitis co-infected with APEC and discovered the molecular mechanisms by which the H9N2 virus facilitates APEC infection, offering new insights to the etiology of salpingitis with viral-bacterial co-infections.IMPORTANCEH9N2 avian influenza virus (AIV) widely infects poultry and is sporadically reported in human infections. The infection in birds frequently causes secondary bacterial infections, resulting in severe symptoms like pneumonia and salpingitis. Currently, the mechanism that influenza A virus contributes to secondary bacterial infection remains elusive. Here we discovered that H9N2 virus infection promotes APEC infection and further explored the underlying molecular mechanisms. We found that fibronectin protein on the cell surface is vital for APEC adhesion and also showed that H9N2 viral protein NS1 increased the expression of fibronectin by activating the TGF-β signaling pathway. Our findings offer new information on how AIV infection promotes APEC secondary infection, providing potential targets for mitigating severe APEC infections induced by H9N2 avian influenza, and also give new insights on the mechanisms on how viruses promote secondary bacterial infections in animal and human diseases.

Morphological examination of the isthmus of the oviduct in the cross Hysex white chicken

The reproductive function of birds and the hatchability of eggs are one of the most important and determining factors in raising poultry, and depend on a number of not only genetic, physiological, environmental, but also morphological factors. The main goal of this article is to identify the features of the macro- and microscopic structure of the isthmus of the oviduct in a Highsex white cross chicken. The object of the study is the oviduct of a Highsex white cross chicken at the age of 180 days. For histological examination, the material was fixed in a 10% aqueous solution of neutral formaldehyde, followed by embedding in paraffin. The research materials were subjected to statistical processing using the STATISTICA 6.1 program. As a result of our research, we noted that the isthmus of the oviduct is located between the albumen section and the bird's uterus and has a small diameter, representing a narrow strip of tissue (translucent zone) with the presence of tubular glands. The mucous membrane of the isthmus is lined with multirow ciliated epithelium. Between the primary folds there are deep crypts, which with their base are embedded deep into the lamina propria; they contain many branched glands that open into ducts onto the surface of the mucous membrane. In the subepithelial layer of the lamina propria, fibers of loose connective tissue are located, tightly surrounding the branched tubular glands. The mucous membrane of the isthmus occupies 59.74% of the total thickness of the isthmus. Muscular membrane – 29.16%. While the serous membrane accounts for 11.1% of the total thickness of the isthmus wall. The mucous membrane of the isthmus, due to the active secretory function of the glands, is 2 times the thickness of the muscular layer.

Vitamin D3 Receptors and Metabolic Enzymes in Hen Reproductive Tissues

In recent years, vitamin D3 has been revealed as an important regulator of reproductive processes in humans and livestock; however, its role in the female reproductive system of poultry is poorly known. The aim of this study was to examine vitamin D3 receptor (VDR and PDIA3) and metabolic enzyme (1α-hydroxylase and 24-hydroxylase) mRNA transcript and protein abundances, and protein localization within the hen ovary, oviductal shell gland, pituitary, liver, and kidney. We demonstrated, for the first time, the patterns of the relative mRNA and protein abundances of examined molecules in the ovary, dependent on follicle development and the layer of follicle wall, as well as in other examined organs. Immunohistochemically, PDIA3, 1α-hydroxylase, and 24-hydroxylase are localized in follicular theca and granulosa layers, luminal epithelium and tubular glands of the shell gland, pituitary, liver, and kidney. These results indicate that reproductive tissues have both receptors, VDR, primarily involved in genomic action, and PDIA3, probably participating in the rapid, non-genomic effect of vitamin D3. The finding of 1α-hydroxylase and 24-hydroxylase expression indicates that the reproductive system of chickens has the potential for vitamin D3 synthesis and inactivation, and may suggest that locally produced vitamin D3 can be considered as a significant factor in the orchestration of ovarian and shell gland function in hens. These results provide a new insight into the potential mechanisms of vitamin D3 action and metabolism in the chicken ovary and oviduct.

Protein profiles in the transfected oviductal secreting cells of laying hen (Gallus gallus domesticus)

Due to the intensive development of novel biopharming applications, there is a need for the in vitro verification models prior to in vivo testing. Laying hen has been already applied as an animal bioreactor to produce the therapeutical enzyme in a rare disease called lysosomal acid lipase deficiency. In this study, we aimed to verify how the proteome of the transfected oviduct epithelial cells would be affected by genetic nonviral modification with the human exogene. The study was based on a previously developed method to cultivate chicken oviduct epithelial cells (COEC). The typical characteristics of the COEC epithelial cells were retained across the experiments. The mean efficiency of nucleofection ranged from 2.6 to 19.7% depending on the cells' isolation and location in the oviduct (upper, infundibulum site, or magnum). The PCR confirmed the incorporation of human interferon alpha2a (hIFNα2a) exogene into the nucleofected COEC but, the production of hIFNα2a protein did not exceed the detection level in this study. The ovalbumin protein was detected in the nontransfected and transfected COEC, which confirmed the normal secreting functions of the cells subject to modification. Proteomic analysis revealed an increase in abundance of the cell adhesion molecules and collagen molecules after introducing gene under ovalbumin promoter. According to the bioinformatic analyses there was a limited negative impact of transfection on cells, and the normal biochemical pathways were not severely disordered. In conclusion, the observations provide new knowledge about the proteomic profile of the manipulated COEC with regard to the retained normal functionality of the cells, which can be informative for avian biopharma research.

Effect of tamoxifen treatment on catalase (CAT) and superoxide dismutase (SOD) expression and localization in the hen oviduct

The imbalance between free reactive oxygen species (ROS) generation and removal (e.g., by antioxidative enzymes) leads to the damage of important biomolecules and cells. Earlier studies in hens showed that treatment with tamoxifen (TMX; estrogen receptor inhibitor) modulates oxidative stress and causes the reproductive system regression realized by cell apoptosis. The aim of the present study was, therefore, to examine the expression and immunolocalization of the key enzymatic antioxidants, i.e. catalase (CAT) and superoxide dismutase (SOD), in the chicken oviduct following TMX treatment. Laying hens were treated daily with TMX until a pause in egg-laying occurred and then euthanized on day 8 of the experiment. Quantitative real-time PCR and western blot analyses showed the presence of CAT and SOD transcripts and proteins, respectively, in all oviductal segments, i.e., the infundibulum, magnum, isthmus, shell gland and vagina. In control hens (laying), the mRNA expression of CAT was the highest in the shell gland, lower in the isthmus and the lowest in other oviductal parts, whereas protein expression was the highest in the magnum, lower in the isthmus and the lowest in other segments. The SOD transcript and protein abundances only were lower in the magnum than in other segments. Immunoreactive CAT and SOD products were localized in all layers of the oviductal wall, but the intensity of staining depended on the cell type. TMX treatment affected CAT and SOD expression and the effect of TMX depended on gene, protein, cell type and oviductal part. Generally, CAT expression was elevated, while SOD expression was decreased under TMX treatment. These results point to the importance of CAT and SOD in the maintenance of proper oviduct health and function. Changes in ROS scavenging enzymes after estrogen receptor blockage indicate the significance of estrogen in the regulation of oxidative status in the avian oviduct.

Electroporation-based Easi-CRISPR yields biallelic insertions of EGFP-HiBiT cassette in immortalized chicken oviduct epithelial cells

Laying hens are an excellent experimental oviduct model for studying reproduction biology. Because chicken oviduct epithelial cells (cOECs) have a crucial role in synthesizing and secreting ovalbumin, laying hens have been regarded an ideal bioreactor for producing pharmaceuticals in egg white through transgene or gene editing of the ovalbumin (OVA) gene. However, related studies in cOECs are largely limited because of the lack of immortalized model cells. In addition, the editing efficiency of conventional CRISPR-HDR knock-in in chicken cells is suboptimal (ranging from 1 to 10%) and remains elevated. Here, primary cOECs were isolated from young laying hens, then infected with a retrovirus vector of human telomerase reverse transcriptase (hTERT), and immortalized cOECs were established. Subsequently, an electroporation-based Easi-CRISPR(Efficient additions with ssDNA inserts-CRISPR) method was adopted to integrate an EGFP-HiBiT cassette into the chicken OVA locus (immediately upstream of the stop codon). The immortalized cOECs reflected the self-renewal capability and phenotype of oviduct epithelial cells. This is because these cells not only maintained stable proliferation and normal karyotype and had no potential for malignant transformation, but also expressed oviduct markers and an epithelial marker and had a morphology similar to that of primary cOECs. EGFP expression was detected in the edited cells through microscopy, flow cytometry, and HiBiT/Western blotting. The EGFP-HiBiT knock-in efficiency reached 27.9% after a single round of electroporation, which was determined through genotyping and DNA sequencing. Two single cell clones contained biallelic insertions of EGFP-HiBiT donor cassettes. In conclusion, our established immortalized cOECs could act as an in vitro cell model for gene editing in chicken, and this electroporation-based Easi-CRISPR strategy will contribute to the generation of avian bioreactors and other gene-edited (GE) birds.

Functional molecular characterization and the assessment of the transmission route of multidrug-resistant Helicobacter pullorum isolates from free-range and broiler chickens

BackgroundSome of the life-threatening, food-borne, and zoonotic infections are transmitted through poultry birds. Inappropriate and irrational use of antimicrobials in the livestock industry has resulted in an increased incidence of multi-drug resistant bacteria of epidemic potentials. Materials and methodsThe adhesion and invasion properties of 11 free-range and broiler chicken derived Helicobacterpullorum isolates were evaluated. To examine the biofilm formation of H. pullorum isolates, crystal violet assay was performed. A quantitative assay of invasion-associated genes was carried out after infecting HepG2 cells with two different representative (broiler and free-range chicken) H. pullorum isolates, using RT-PCR assay. Furthermore, we investigated the prevalence of H. pullorum, Campylobacter jejuni and Salmonella spp. in chicken caeca and oviducts to determine the possibility of trans-ovarian transmission. ResultsAll H. pullorum isolates adhered to HepG2 cells significantly but a notable difference towards their invasion potential was observed between free-range and broiler chicken isolates wherein broiler isolates were found to be more invasive compared to free-range isolates. Furthermore, cdtB, flhA and flaB genes of H. pullorum were upregulated post infection of HepG2 cells, in broiler chicken isolates compared to free-range chicken isolates. Moreover, all isolates of H. pullorum were found to form biofilm on the liquid-air interface of the glass coverslips and sidewalls of the wells with similar propensities. Despite presence of H. pullorum and C. jejuni in high concentrations in the caecum, they were completely absent in oviduct samples, thus ruling out the possibility of vertical transmission of these bacterial species. In contrast, Salmonella spp. was found to be present in a significant proportion in the oviduct samples of egg-laying hens suggesting its vertical transmission. ConclusionsOur findings suggest that H. pullorum, an emerging multi-drug resistant (MDR) pathogen could be transmitted from poultry sources to humans. In addition to this, its strong functional similarity with C. jejuni provides a firm basis for H. pullorum to be an emerging food-associated, MDR pathogenic bacterium that could pose risk to public health.

Effects of Salmonella Enteritidis infection on TLRs gene expression and microbial diversity in cecum of laying hens

Salmonella Enteritidis (SE) is an important foodborne pathogen primarily causing human disease through contaminated food and water. In the current study, to assess the effect of Salmonella Enteritidis infection on the immune system and the microbial diversity of cecum and oviduct in chickens, twelve 24-week-old SE-negative White Leghorn layers were randomly selected and divided into 2 groups. Chickens in the challenge group were orally inoculated with SE, and chickens in the control group received an equal amount of sterilized Phosphate Buffered Saline solution. Serum and tissue samples (cecum, oviduct, ovary, liver, spleen, and pancreas) were collected at 7 days and 14 days post-infection (dpi). Quantitative PCR was used to detect the expression of Toll-like receptors (TLRs) in the cecum, oviduct and ovary. To understand the influence of SE infection on the microbial profile of the cecum and oviduct, microbial community composition of the cecal contents and oviducal contents were analyzed through 16S rRNA sequencing. Results showed that SE infection caused damage to the digestive organs, reproductive organs, and immune organs in laying hens. The expression of TLR1a, TLR1b, TLR2, TLR4, TLR5, TLR7 and TLR15 in the cecum were induced, and the content of IFN-γ, TNF-α, IL-2 and IL-18 in serum increased after SE infection. The composition of the microbial community significantly changed in cecal content, the dominant phylum of Firmicutes increased, and Bacteroidetes decreased significantly. In the oviduct, the microbial diversity became complicated, the dominant bacteria Faecalibacterium was significantly increased, and Bacteroides was significantly decreased. This study investigated the effects of SE infection in laying hens, including host innate immunity, the expression of TLRs, and changes in the composition of microbes in the cecum and reproductive tract. Our results may provide a scientific basis for the Salmonella Enteritidis control in chicken, the maintenance of oviduct function, and the guarantee of clean egg production.

Histological study of different oviduct parts of Gallus domesticus

This study included the histological structure of the oviduct of white chicken, The wall of this duct consisted of four layers, the mucosa made up from pseudostratified columnar epithelium, the epithelium is continuous layer of columnar cells with alternating basal cells which stand on the loose connective tissue form the lamina propria, the mucosa forms leaf shaped longitudinal folds contained glands which lined with cuboidal cells , the second layer was the submucosa which was loose connective tissue, The muscularis was the third layer contained smooth muscle with an inner circular and outer longitudinal layers. loose connective tissue formed the serosa

The conserved L1089 in the S2 subunit of avian infectious bronchitis virus determines viral kidney tropism by disrupting virus-cell fusion

The virulence of avian gamma-coronavirus infectious bronchitis viruses (IBV) for the kidney has led to high mortality in dominant-genotype isolations, but the key sites of viral protein that determine kidney tropism are still not fully clear. In this study, the amino acid sequences of the S2 subunit of IBVs with opposing adaptivity to chicken embryonic kidney cells (CEKs) were aligned to identify putative sites associated with differences in viral adaptability. The S2 gene and the putative sites of the non-adapted CN strain were introduced into the CEKs-adapted SczyC30 strain to rescue seven mutants. Analysis of growth characteristics showed that the replacement of the entire S2 subunit and the L1089I substitution in the S2 subunit entirely abolished the proliferation of recombinant IBV in CEKs as well as in primary chicken oviduct epithelial cells. Pathogenicity assays also support the decisive role of this L1089 for viral nephrotropism, and this non-nephrotropic L1089I substitution significantly attenuates pathogenicity. Analysis of the putative cause of proliferation inhibition in CEKs suggests that the L1089I substitution affects neither virus attachment nor endocytosis, but instead fails to form double-membrane vesicles to initiate the viral replication and translation. Position 1089 of the IBV S2 subunit is conservative and predicted to lie in heptad repeat 2 domains. It is therefore reasonable to conclude that the L1089I substitution alters the nephrotropism of parent strain by affecting virus-cell fusion. These findings provide crucial insights into the adaptive mechanisms of IBV and have applications in the development of vaccines and drugs against IB.

Sequential verification of exogenous protein production in OVA gene-targeted chicken bioreactors

The chicken has potential as an efficient bioreactor system because of its outstanding protein production capacity and low cost. The CRISPR/Cas9-mediated gene-editing system enables production of highly marketable exogenous proteins in transgenic chicken bioreactors. However, because it takes approximately 18 mo to evaluate the recombinant protein productivity of the bioreactor due to the generation interval from G0 founders to G1 egg-laying hens, to verification of the exogenous protein at the early stage is difficult. Here we propose a system for sequential validation of exogenous protein production in chicken bioreactors as in hatching female chicks as well as in egg-laying hens. We generated chicken OVALBUMIN (OVA) EGFP knock-in (KI) chicken (OVA EGFP KI) by CRISPR/Cas9-mediated nonhomologous end joining at the chicken OVA gene locus. Subsequently, the estrogen analog, diethylstilbestrol (DES), was subcutaneously implanted in the abdominal region of 1-wk-old OVA EGFP KI female chicks to artificially increase OVALBUMIN expression. The oviducts of DES-treated OVA EGFP KI female chicks expressed OVA and EGFP at the 3-wk-old stage (10 d after DES treatment). We evaluated the expression of EGFP protein in the oviduct, along with the physical properties of eggs and egg white from OVA EGFP KI hens. The rapid identification and isolation of exogenous protein can be confirmed at a very early stage and high-yield production is possible by targeting the chicken oviduct.

Alterations in connexin 43 gene and protein expression in the chicken oviduct following tamoxifen treatment

Connexins (Cxs) are a group of gap junction proteins involved in the direct exchange of small molecules between neighboring cells. Information concerning the expression and regulation of Cxs in the chicken oviduct is lacking, but likely has potential implications for functioning of the oviduct and the quality of the egg laid by commercially used hens. The present study was designed to examine whether selected Cxs are present in the chicken oviduct and, if so, whether expression of the most abundant Cx changes following tamoxifen (TMX; estrogen receptor modulator) treatment. Hy-Line Brown laying hens were injected (s.c.) daily with a vehicle (n = 6) or with TMX (n = 6), at a dose of 6 mg/kg of body weight for 7 days until complete cessation of egg laying by TMX-treated hens. All oviductal segments (infundibulum, magnum, isthmus, shell gland, and vagina) were collected from hens on day 8 of the experiment. First, the gene expression of GJA1 (i.e. Cx43 protein), GJA4 (Cx39), GJB1 (Cx32), and GJD2 (Cx36) was investigated by real-time PCR in tissues of control birds. The results demonstrated gene- and oviductal segment-dependent expression of GJB1, GJD2, GJA4, and GJA1 mRNA. Since the GJA1 transcript was the most abundant in all oviductal parts, subsequently, the Cx43 expression and localization were examined in the oviduct of all hens. The relative expression of GJA1 mRNA in control hens was highest in the infundibulum and vagina and lowest in the magnum. The pattern of Cx43 protein abundance evaluated by Western blot was similar to that of mRNA. Treatment of hens with TMX decreased the GJA1 mRNA levels in the magnum and isthmus, and Cx43 protein abundances were reduced in the isthmus and vagina. Immunofluorescence demonstrated cell- and segment-dependent localization of Cx43 protein in the oviductal wall; the most intense immunoreactivity was observed in the muscle cells of the shell gland and vagina. In TMX-treated hens, the immunoreactivity for Cx43 in all oviductal segments was slightly reduced and had a different signal pattern compared with control chickens. These results suggest that Cx43 likely takes part in the regulation of oviduct functioning, especially in the coordination of muscle contraction required for egg transport and oviposition. In addition, the results suggest a contribution of estrogen in the regulation of Cx43 expression and/or fates in the chicken oviduct. New insights into the expression and regulation of Cxs in the hen oviduct, indicating their potential involvement in the mechanisms of egg formation and transport that may affect poultry production, were obtained in this study.

Chemerin is secreted by the chicken oviduct, accumulates in egg albumen and could promote embryo development

Understanding of the distribution of chemerin and its receptors, Chemokine-like Receptor 1 (CMKLR1), G Protein-coupled Receptor 1 (GPR1) and Chemokine (C–C motif) receptor-like 2 (CCRL2), in the egg and the embryonic annexes is currently lacking, and their role during embryogenesis remains unknown. By immunoblot using monoclonal anti-chicken antibodies and Enzyme Linked Immunosorbent Assays (ELISA), we found that chemerin is expressed 10 times higher in albumen eggs than in blood plasma, and it is also abundant in the perivitelline membrane but undetectable in yolk. Chicken chemerin can inhibit bacterial growth. By Reverse Transcription—quantitative Polymerisation Chain Reaction (RT-qPCR), western-blot, and immunofluorescence, we show that chemerin is locally produced by the oviduct magnum that participates in albumen formation. Using cultures of magnum explants, we demonstrate that progesterone (P4) and oestradiol (E2) treatment increases chemerin secretion into cultured media and expression in magnum. Chemerin and its three receptors are present in amniotic and Chorio Allantoic Membranes (CAM). Only CMKLR1 expression decreased from embryonic day (ED) 7 to ED11 and remained low until ED18. Chemerin concentrations strongly increased in amniotic fluid at D14 when egg albumen crossed the amniotic membrane. In ovo injections of neutralising chemerin and CMKLR1 antibodies (0.01, 0.1 and 1 µg) increased embryo mortality, which occurred mainly at ED12-13, in a dose-dependent manner. Chemerin treatment increased primary CAM viability. Finally, chemerin and CMKLR1 inhibition within the CAM led to a decrease in blood vessel development and associated angiogenic gene expression. Our results show an important function of the chemerin system during embryo development in chickens, suggesting the potential use of this adipokine as a predictive marker for egg fertility or hatchability.

Development and in vitro evaluation of recombinant chicken promoters to efficiently drive transgene expression in chicken oviduct cells

Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in the chicken oviduct. Although a reconstructed chicken ovalbumin promoter that links an ovalbumin promoter and estrogen-responsive enhancer element (ERE) is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. We newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. Putative enhancer fragments of the genes, such as ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), were placed at the 5`-flanking region of the 2.8-kb ovalbumin promoter. Basal promoter fragments of the genes, namely, pTF, lysozyme (pLYZ), and ovomucoid (pOVM), were placed at the 3`-flanking region of the 1.6-kb ovalbumin ERE. The recombinant promoters cloned into each reporter vector were evaluated using a dual luciferase assay in human and chicken somatic cells, and LMH/2A cells treated with 0-1,000 nM estrogen, and cultured primary chicken oviduct cells. The recombinant promoters with linking ovalbumin and TF, OVOA, pOVM, and pLYZ regulatory regions had 2.1- to 19.5-fold (P < 0.05) higher luciferase activity than the reconstructed ovalbumin promoter in chicken oviduct cells. Therefore, recombinant promoters may be used to efficiently drive transgene expression in transgenic chickens.

Microbiota continuum along the chicken oviduct and its association with host genetics and egg formation

The microbiota of female reproductive tract have attracted considerable attention in recent years due to their effects on host fitness. However, the microbiota throughout the chicken oviduct and its symbiotic relationships with the host have not been well characterized. Here, we characterized the microbial composition of six segments of the reproductive tract, including the infundibulum, magnum, isthmus, uterus, vagina and cloaca, in pedigreed laying hens with phenotypes of egg quality and quantity. We found that the microbial diversity gradually increased along the reproductive tract from the infundibulum to the cloaca, and the microbial communities were distinct among the cloaca, vagina and four other oviductal segments. The magnum exhibited the lowest diversity, given that the lysozyme and other antimicrobial proteins are secreted at this location. The results of correlation estimated showed that the relationship between host genetic kinship and microbial distance was negligible. Additionally, the genetically related pairwise individuals did not exhibit a more similar microbial community than unrelated pairs. Although the egg might be directly contaminated with potential pathogenic bacteria during egg formation and oviposition, some microorganisms provide long-term benefits to the host. Among these, we observed that increased abundance of vaginal Staphylococcus and Ralstonia was significantly associated with darker eggshells. Meanwhile, vaginal Romboutsia could be used as a predictor for egg number. These findings provide insight into the nature of the chicken reproductive tract microbiota and highlight the effect of oviductal bacteria on the process of egg formation.

Prevention of diseases of growing of replacement young chickens kept in cages

Acute and chronic diseases of the extremities (arthrosis, arthritis, necrosis of the thighbone, dislocation, injury, etc.) of chickens, especially of roosters, with their subsequent raising and keeping in cages, cause significant economic damage to poultry farms. The article presents the results of scientific and production experience consisting in using non-steroidal anti-inflammatory medications “Paracetam-AVZ” and “Ketoquin 10%” for the prevention of limb diseases in the terms of raising chickens of cross Hisex Brown in the cages. The studied medications contributed to an increase in the live weight in the experimental group: females – by 3.8% (P <0.05), males – by 6.6% (P <0.05), homogeneity in the live weight of females – by 2.3%, males – by 15.6% compared with the results in control group. The safety of males taking into account mortality and culling in the experimental group exceeded this parameter in control group by 6.3%. At the same time, the number of rejected males due to limb disease in the experimental group was 2.2 times lower than in the control group. Evaluation of the development of reproductive organs showed that in males the difference in testes mass in favor of the experimental group was 16.7% (P <0.01), the length of the oviduct in chickens was 20.8% (P <0.01). Thus, the use of the studied medications had a positive effect on the development of the reproductive organs of hens and roosters in the experimental group, which accelerated their sexual maturation and increased the intensity of egg-laying.

Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia.

Objective:The avian influenza virus (AIV) subtype H9N2 circulating in Indonesia has raised increasing concern about its impact on poultry and its public health risks. In this study, the H9N2 virus from chicken poultry farms in Java was isolated and characterized molecularly.Materials and Methods:Thirty-three pooled samples of chicken brain, cloacal swab, trachea, and oviduct were taken from multiple chickens infected with AIV in five regions of Java, Indonesia. The samples were isolated from specific pathogenic-free embryonated eggs that were 9 days old. Reverse transcription polymerase chain reaction and sequencing were used to identify H9N2 viruses.Results:This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of hemagglutinin genes revealed a 96.9%–98.8% similarity to the H9N2 AIV isolated from Vietnam in 2014 (A/muscovy duck/Vietnam/LBM719/2014). According to the phylogenetic analysis, all recent H9N2 viruses were members of the lineage Y280 and clade h9.4.2.5. Nine of the H9N2 isolates studied showed PSKSSR↓GLF motifs at the cleavage site, while four had PSKSSR↓GLF. Notably, all contemporary viruses have leucine (L) at position 216 in the receptor-binding region, indicating that the virus can interact with a human-like receptor.Conclusion:This study described the features of recent H9N2 viruses spreading in Java’s poultry industry. Additionally, H9N2 infection prevention and management must be implemented to avoid the occurrence of virus mutations in the Indonesian poultry industry.