Effect of Mycoplasma synoviae strains isolated from the respiratory and reproductive tracts of chicken on SPF chicken embryos

Mycoplasma synoviae (MS) infections in poultry are an important epidemiological and economic problem in poultry production all over the world. The differences between M. synoviae strains are related to the pathogenicity and the course of the disease. In recent years, the pathogenicity of M. synoviae strains has increased, and some of them are capable of causing serious infections. Both horizontal and vertical transmission routes play an important role in MS infection in flocks. The aim of the study was to determine the impact of infection with selected MS strains obtained from chickens showing a clinical form of MS infection on SPF chicken embryos. Ten strains of M. synoviae were used for this purpose. The strains were isolated from the respiratory tract and the oviduct of chickens with symptoms typical of infection with this pathogen. Genetic material isolated from liquid cultures of these strains was confirmed by molecular (PCR and LAMP) and microbiological methods. The selected M. synoviae strains belonged to six different genotypes. Significant differences in virulence between the strains were demonstrated. In nine infected groups of embryos, M. synoviae strains caused weight loss, and in seven groups they produced anatomopathological changes characteristic of mycoplasma infections. The most pathogenic for SPF chicken embryos turned out to be strains characterized as genotype F isolated from the chicken oviduct and strains of genotype C isolated from the respiratory tract. One strain of genotype H isolated from the respiratory tract showed no pathogenic effect on SPF chicken embryos. The study showed that infections with M. synoviae can have a significant impact on the production of chicken chicks in commercial hatcheries and the economy of the poultry industry.

TMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis

The location and regulation of fusion events within skeletal muscles during development remain unknown. Using the fusion marker myomaker (Mymk), named TMEM8C in chicken, as a readout of fusion, we identified a co-segregation of TMEM8C-positive cells and MYOG-positive cells in single-cell RNA-sequencing datasets of limbs from chicken embryos. We found that TMEM8C transcripts, MYOG transcripts and the fusion-competent MYOG-positive cells were preferentially regionalized in central regions of foetal muscles. We also identified a similar regionalization for the NOTCH ligand JAGGED2 along with an absence of NOTCH activity in TMEM8C+ fusion-competent myocytes. NOTCH function in myoblast fusion had not been addressed so far. We analysed the consequences of NOTCH inhibition for TMEM8C expression and myoblast fusion during foetal myogenesis in chicken embryos. NOTCH inhibition increased myoblast fusion and TMEM8C expression and released the HEYL transcriptional repressor from the TMEM8C regulatory regions. These results identify a regionalization of TMEM8C-dependent fusion and a molecular mechanism underlying the fusion-inhibiting effect of NOTCH in foetal myogenesis. The modulation of NOTCH activity in the fusion zone could regulate the flux of fusion events.

The Neonicotinoid Thiacloprid Interferes with the Development, Brain Antioxidants, and Neurochemistry of Chicken Embryos and Alters the Hatchling Behavior: Modulatory Potential of Phytochemicals

The present experiment was performed to investigate the toxic impact of thiacloprid (TH) on the brain of developing chicken embryos and also to measure its influence on the behavioral responses of hatchlings. The role of chicoric acid (CA) and rosmarinic acid (RA) in modulating the resulted effects was also investigated. The chicken eggs were in ovo inoculated with TH at different doses (0.1, 1, 10, and 100 ug/egg). TH increased the mortality and abnormality rates and altered the neurochemical parameters of exposed embryos dose-dependently. TH also decreased the brain level of monoamines and amino acid neurotransmitters and decreased the activities of acetylcholine esterase (AchE) and Na+/K+-ATPase. The brain activity of catalase (CAT) and superoxide dismutase (SOD) was diminished with downregulation of their mRNA expressions in the brain tissue. When TH was co-administered with CA and RA, the toxic impacts of the insecticide were markedly attenuated, and they showed a complementary effect when used in combination. Taken together, these findings suggested that TH is neurotoxic to chicken embryos and is possibly neurotoxic to embryos of other vertebrates. The findings also demonstrated the antioxidant and neuroprotective effects of CA and RA. Based on the present findings, the CA and RA can be used as invaluable ameliorative of TH-induced toxicity.

The Sexual Effect of Chicken Embryos on the Yolk Metabolites and Liver Lipid Metabolism

The metabolic processes of animals are usually affected by sex. Egg yolk is the major nutrient utilized for the growth and development of a chicken embryo. In this study, we explored the differences of yolk metabolites in male and female chicken embryos by LC–MS/MS. Furthermore, we investigated the mRNA expression of lipoprotein lipase (LPL) and fatty acid synthase (FAS) in chicken embryo liver with different sexes in different embryonic stages. The results showed that the nutrient metabolites in the yolk of female chickens were mainly related to lipid metabolism and amino acid metabolism in the early embryonic stage, and vitamin metabolism in the late embryonic stage. The male yolk metabolites were mainly associated with lipid metabolism and nucleic acid metabolism in the early developmental stage, and amino acids metabolism in the late embryonic stage. There was no significant difference in the expression of LPL or FAS in livers of male and female chicken embryos at different embryonic stages. Our results may lead to a better understanding of the sexual effect on yolk nutrient metabolism during chicken embryonic development.

Physiological Changes in Chicken Embryos Inoculated with Drugs and Viruses Highlight the Need for More Standardisation of This Animal Model

Several works have been developed using the Gallus gallus embryo as an experimental model to study the toxicity of drugs and infections. Studies that seek to standardise the evaluated parameters are needed to better understand and identify the viability of chicken embryos (CE) as an experimental model. Therefore, we sought to verify whether macroscopic, histopathological, blood count, metabolites and/or enzymes changes and oxidative stress in CE of different ages are peculiar to the model. To achieve this goal, in ovo assays were performed injecting a virus (Gammacoronavirus) and two drugs (filgrastim and dexamethasone) that cause known changes in adult chickens and other animals. Embryo response to virus and drug challenges may not occur as expected for adult chickens and even different species. While macroscopic and microscopic damage was visible in the case of viral infections, the white blood cell count and inflammation biomarkers did not change. Filgrastim (FG) testing did not result in the expected effects for CE. On the other hand, with dexamethasone (DX), changes in blood parameters and biomarkers were inherent to the model and depended on the stage of CE development. Our work reinforces the importance of standardization and correct use of the model so that the results of infection, toxicity and pharmacokinetics are recorded.

Influence of water concentration on the content of protein and nucleic acids in organs of chick embryos of different ages

The study was carried out in 10-, 14-15-, 18-20-day-old embryos and 1-2-day-old chickens of the Hy-Line breed. In chickens during embryogenesis, the peculiarities of the relationship between the main indicator of the ontogenetic growth of organs and tissues - the content of intracellular water by previously measured values: with the concentration of protein and nucleic acids in the organs of chicken embryos of different ages - were assessed. It was found that in the period of 10-19 days of embryogenesis, the growth of organs is accompanied by a significant decrease in water content to varying degrees: noticeably higher in the liver and in the cerebral hemispheres - up to 14 days; from the 19th day, these changes are less pronounced. The results of the studied indicator in the muscles indicate advanced embryonic development with a more pronounced decrease in the water content in the red oxidative muscles in comparison with white muscles, where the decrease in the indicator is less pronounced. Key words: chicken embryos, protein, nucleic acid, water content, pectoralis alba and gastrocnemius muscle.

Rational selection of hemagglutinin variants of H3N2 influenza viruses in preparation of live influenza vaccine strains to optimize growth characteristics

BACKGROUND: Up to date Russian live attenuated influenza vaccines are produced in developing chicken embryos. During passaging in embryos, the virus isolated from the human respiratory tract undergoes adaptation to the receptors in embryos. The population of the virus, at any passage in chicken embryos, is heterogeneous and contains variants of viruses with one or another set of adaptive substitutions. Before preparing the vaccine strain, the population of the epidemic virus is cloned and the genetic sequence of the hemagglutinin and neuraminidase clones is analyzed. The growth characteristics of the vaccine strain and its antigenic properties depend on the correct choice of the variant of the virus. AIM: The aim of the study was to select the variant of the H3N2 subtype virus for the preparation of a vaccine reassortant based on data on the composition of the population and an assessment of its growth properties. MATERIALS AND METHODS: Viruses were cloned in developing chicken embryos, sequencing of the hemagglutinin and neuraminidase genes of the clones was performed. On the basis of the clones selected based on the results of the analysis of the population, strains of a live influenza vaccine were obtained by the reassortment in the chicken embryos. The growth characteristics of the strains, the phenotype in eggs, and the antigenic properties by hemagglutination inhibition test were evaluated. RESULTS: The influenza virus A/Kansas/14/2017 recommended by WHO for the epidemic season 2019-2020 acquired a pair of D190N + N246T substitutions dominating in the population at the 7th passage in eggs. From the population of A/Kansas/14/2017-like strain A/Brisbane/34/2018, from the third passage in the eggs, it was possible to obtain a variant of the virus with substitutions G186V + S219Y in hemagglutinin. The growth characteristics of the strain based on A/Kansas/14/2017 (passage E7) were significantly inferior to the characteristics of the strain based on A/Brisbane/34/2018 (passage E3), in the absence of differences in antigenic properties. CONCLUSIONS: The variant of egg adaptation of hemagglutinin G186V in strains of clade 3c.3a is preferable for the preparation of live influenza vaccine strains; variant N246T is not optimal. When preparing strains, it is necessary to analyze the composition of the virus population by cloning and choose the most optimal option for preparing strains. The persistence of egg-adaptive substitutions in passaged variants of the virus is not always optimal for strains of live influenza vaccine, and therefore it is preferable to use the population as close as possible to the initial variant to start work on the strain.

Effect of Muscle Extract and Graphene Oxide on Muscle Structure of Chicken Embryos

The effects of CEME and it complex with GO injected in ovo on the growth and development of chicken embryo hindlimb muscle were investigated. First, the preliminary in vitro study on primary muscle precursor cell culture obtained from a nine-day-old chicken embryo was performed to assess toxicity (MTT assay) of CEME, GO (100 ppm) and it complex with different concentrations (1, 2, 5, and 10 wt.%). The effect on cell proliferation was investigated by BrdU assay. CEME at concentrations 1–5% increased cell proliferation, but not the complex with GO. In vitro cytotoxicity was highest in 10% and GO groups. Next, the main experiment with chicken embryos was performed with CEME, GO and it complex injected in ovo on day one of embryogenesis. On day 20 of embryogenesis survival, morphological development, histological structure of the muscle, and biochemical parameters of blood serum of the embryos were measured. No negative effect on mortality, body weight, or biochemistry of blood after use of CEME or GO-CEME complexes was observed. Interestingly, the slight toxicity of GO, observed in in vitro studies, was not observed in vivo. The use of CEME at the levels of 2% and 5% improved the structure of the lower limb muscle by increasing the number of cells, and the administration of 2% CEME increased the number of nuclei visible in the stained cross-section of the muscle. The complex GO-CEME did not further improve the muscle structure. The results indicate that CEME can be applied as an in ovo enhancer of muscle development in broilers.