Developing Chick Embryo Research Articles

The developmental effects of isoflavone aglycone administration on early chick embryos

Soybeans contain the isoflavone aglycone, an endocrine disrupter. To determine the effects of small amounts of isoflavones on developmental processes, we administered 6.25, 62.5, or 625 μg isoflavone per egg to early stage (stage 10) developing chick embryos via the yolk just beneath the embryo. Eggs were kept at 37±0.5 °C and >80% relative humidity, with one rotation per hour for 48 hrs. The embryos were observed under a stereomicroscope for morphological abnormalities and number of somites. Relative to control eggs, there were no significant differences in the average number of somites in eggs administered isoflavone aglycone. Isoflavone, however, had a dose associated effect on abnormal embryogenesis. Embryos treated with isoflavone aglycone showed developmental arrest not reaching somitegenesis, dysmorphology of the neural tube, and shortening of entire embryos.

WITHDRAWN: An extremely low frequency-weak magnetic field can induce alterations in a biological system: A case study in chick embryo development

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). These articles are retracted at the request of the authors. The joint Editors-in-Chief agree with this decision.

Effects of N-cadherin on neuronal migration during chicken optic tectum development.

N-cadherin, a member of the cadherin family, plays an important role in neural development. In addition, N-cadherin has been reported to be crucial in neuronal migration, axonal outgrowth, and axonal path-finding. However, the mechanism underlying the effects of N-cadherin in neuronal migration is not entirely clear. In this study, we investigated the overexpression or knockdown of N-cadherin in the optic tectum during chicken embryo development, and then analyzed the effect of N-cadherin on neuronal migration. The results showed that compared with the control group, in the N-cadherin knockdown group, the neuronal migration of the optic tectum was significantly affected and could not arrive at destination. The stratum griseum central layer of the optic tectum mainly includes multipolar neurons, which could not be formed after the knockdown of N-cadherin, and more neurons form the bipolar or monopolar neurons compared with the control group. Compared with the control group, more cells stayed in the neuroepithelium layer. The axonal length in the optic tectum was significantly (P < 0.001) shorter in the N-cadherin knockdown group than in the control group. These results reveal that the knockdown of N-cadherin mainly affects the length of axons and formation of multipolar neurons in the development of the chicken optic tectum, which eventually results in the inhibition of neuronal migration.

Antifibrotic Activity and In Ovo Toxicity Study of Liver-Targeted Curcumin-Gold Nanoparticle.

Conjugation of curcumin and gold with green chemistry is an approach to improve the effectiveness of curcumin as anti-fibrosis. In this work, curcumin and gold were conjugated to deliver curcumin to the liver. Curcumin-gold nanoparticles (cAuNPs) were prepared by varying curcumin pH and concentration. The successful of cAuNPs formation were identified by using UV-visible and FTIR spectrophotometers. The particle size and morphology were analyzed using particle size analyzer and cryo-TEM respectively. In vitro antioxidant assay was performed to determine the curcumin activity after conjugation. Physical and chemical stabilities of cAuNPs were studied for one month at 5 °C, 25 °C, and 40 °C. Furthermore, the cAuNPs activity to modulate early marker of fibrosis was tested on NIH/3T3 cells. The optimum condition for cAuNPs synthesis was by using 1.5 mM curcumin at pH 9.3. As compared to free curcumin, cAuNPs showed higher antioxidant activity and maintained the nanosize after stored for one month. In line with the antioxidant activity, cAuNPs 0.25⁻1 μg/mL reduced the collagen production by NIH/3T3 cells. More importantly, cAuNPs did not demonstrate any effect on the development of chicken embryo. Taken together, the attachment of gold to curcumin in the form of cAuNPs is promising for curcumin targeting to treat hepatic fibrosis.

Non-invasive characterization of chick embryo body and cardiac movements using near infrared light

Embryonic movements, body and cardiac activity, are important physiological phenomena for chick embryo development. Currently, there is no complete non-invasive method for simultaneously quantifying chick embryonic body motility and cardiac rhythm during incubation. This study investigates the use of a near infrared sensor to simultaneously monitor embryonic body and cardiac movements. Light brown chicken eggs (ROSS 308) were incubated for chick embryos activity signals measurement from day 6 to 19. Signal features (peak frequencies and signal energy) of chick embryo periodical activity were extracted to quantify both body and cardiac movements using fast Fourier transform and numerical integration. Two types of body movement were found throughout the whole incubation period. During the early stage of incubation, the movement was periodic; with the pattern differing between embryos. In the mid to late stages of incubation, movements were irregular and had a lower frequency compared to the periodic motion. Heart rates throughout the incubation period varied from 3.8 to 4.8 Hz, while heart beat strength sharply increased during incubation, peaking at day 13 to 14, and then subsequently subsiding. These results indicate that near infrared sensing, combined with signal processing, has the potential to monitor embryo motility and cardiac rhythm that could be used in developmental physiology, cardiovascular medicine and precision poultry production systems.

The different roles of hcp1 and hcp2 of the type VI secretion system in Escherichia coli strain CE129.

Type VI secretion system (T6SS) is a secretory system found in Gram-negative bacteria. One of the main structures for T6SS is Hcp (hemolysin co-regulation protein) pipeline. To investigate the role of Hcp major sub-unit genes hcp1 and hcp2 , we deleted hcp1 and hcp2 genes for constructing the in-frame gene deletion mutants. The properties of biofilm formation and the adhesion to chicken embryo fibroblasts cells (DF1 cells) were reduced in the hcp2 mutant. The knockout of hcp1 and hcp2 genes reduced the ability of the avian pathogenic Escherichia coli (APEC) strain CE129 to infect developing chicken embryos. The expression of quorum sensing (QS)-associated genes luxS, lsrR, and pfs were down-regulated in the hcp1 mutant, and the expression of type 1 fimbriae gene fimA and the adhesion-related genes fimC and papC were decreased in the hcp2 mutant, as well as the expression of anti-serum survival factor genes ompA and iss were inhibited in both hcp1 and hcp2 mutants. These results described above from this study help to further elaborate the role of HCP in APEC.

The Impact of Prenatal Chronic Auditory Stimulation to the Development of Chicken Embryos’ Hippocampus: What Have Been Done and the Established Protocol

Auditory stimulation can differently modulate the development of auditory system and hippocampus. Using chicken embryos make it possible for researchers to focus on examining the direct impact of chronic auditoric stress to the development of fetal brain, especially to hippocampus without confounding factors from mother and placenta. We focus to review studies that examined the influence of prenatal chronic auditory stimulation exposure (both as environmental enrichment and as stressor) to the development of chicken embryos’ hippocampus. We wanted to gather information on what have been done and the what would be the established auditory stimulation protocol. We searched all in vivo studies in chicken that used prenatal chronic auditory stimulation to observed the changes in the developing hippocampus. The comparison was made in terms of characteristics of the studies, auditory stimulation regimen, parameters studied, results, conclusion and suggestion for further research. Eight studies fulfilled the selection criteria and were included in the analysis. Discussion: Researchers have been exploring the influence of auditory stimulation in form of music, species-specific sound and noise to the developing chicken embrios’ brainstem auditory nuclei, auditory imprinting area and hippocampus. They have used moderate intensity (65 dB) and high intensity (110 dB). The hippocampal parameters observed were morphology, molecular/biochemistry and behavioral/cognitive changes. Chicken embrios are representative for studying the impact of prenatal chronic auditory stimulation to the developing brain. Studies have convincingly shown the benefits of species-specific sound and music exposure and the negative effects of noise to the developing hippocampus. However, further research are still needed to address some issues. In doing so, researchers may replicate the established auditory stimulation protocol that have been consistently used in the studies reviewed.

Oxidative stress and NF-κB signaling are involved in LPS induced pulmonary dysplasia in chick embryos

ABSTRACTInflammation or dysbacteriosis-derived lipopolysaccharides (LPS) adversely influence the embryonic development of respiratory system. However, the precise pathological mechanisms still remain to be elucidated. In this study, we demonstrated that LPS exposure caused lung maldevelopment in chick embryos, including higher embryo mortality, increased thickness of alveolar gas exchange zone, and accumulation of PAS+ immature pulmonary cells, accompanied with reduced expression of alveolar epithelial cell markers and lamellar body count. Upon LPS exposure, pulmonary cell proliferation was significantly altered and cell apoptosis was inhibited as well, indicating a delayed progress of pulmonary development. LPS treatment also resulted in reduced CAV-1 expression and up-regulation of Collagen I, suggesting increased lung fibrosis, which was verified by Masson staining. Moreover, LPS induced enhanced Nrf2 expression in E18 lungs, and the increased reactive oxygen species (ROS) production was confirmed in MLE-12 cells in vitro. Antioxidant vitamin C restored the LPS induced down-regulation of ABCA3, SP-C and GATA-6 in MLE-12 cells. Furthermore, LPS induced activation of NF-κB signaling in MLE-12 cells, and the LPS-induced decrease in SP-C expression was partially abrogated by blocking NF-κB signaling with Bay-11–7082. Bay-11–7082 also inhibited LPS-induced increases of ROS and Nrf2 expression. Taken together, we have demonstrated that oxidative stress and NF-κB signaling are involved in LPS induced disruption of pulmonary cell development in chick embryos.

Adipokines expression profile in liver, adipose tissue and muscle during chicken embryo development

In broiler chickens, the intense genetic selection for rapid growth has resulted in an increase in growth rate and fat deposition. Adipose tissue is now recognized as an important endocrine organ that secretes a variety of factors including adipokines. However, the expression pattern of these adipokines is unclear in chicken embryo development. In the present study, we determined the expression profile of three novel adipokines, NAMPT, RARRES2 and ADIPOQ, and their cognate receptors in metabolic tissues (liver, muscles and adipose tissue) of chicken embryo/chicks from 15 days of incubation (E15) to hatching (D0). From E15 to hatching, embryos gradually gained weight and started to develop subcutaneous adipose tissue at E15. We conducted western blot and RT-qPCR tests and found that ADIPOQ expression increased over time and was positively correlated with adipose tissue weight. In addition, NAMPT expression increased only in muscles. By using a new homemade chicken RARRES2 specific antibody we showed that RARRES2 protein levels increased specifically at hatching in adipose tissue, liver and pectoralis major and this was associated with an increase in the weight of embryo. Taken together, these results support a potential involvement of adipokines in metabolic regulation during chicken embryo development.

Gut microbiota-derived endotoxin enhanced the incidence of cardia bifida during cardiogenesis.

Cytotoxicity and inflammation-associated toxic responses could be induced by bacterial lipopolysaccharides (LPS) in vitro and in vivo, respectively. However, the mechanism involved in LPS-induced cardiac malformation in prenatal fetus is still unknown. In this study, we demonstrated that LPS was induced in gut microbiota imbalance mice, and next, LPS exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Gene transfection and tissue transplantation trajectory indicated that LPS exposure restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. In vitro explant allograft of GFP-labeled anterior primitive streak demonstrated that LPS treatments could inhibit cell migration. A similar observation was also obtained from the cell migration assay of scratch wounds using primary culture of cardiomyocytes or H9c2 cells. In the embryos exposed to LPS, expressions of Nkx2.5 and GATA5 were disturbed. These genes are associated with cardiomyocyte differentiation when heart tube fusion occurs. Furthermore, pHIS3, C-caspase3 immunohistological staining indicated that cell proliferation decreased, cell apoptosis increased in the heart tube of chick embryo. Meanwhile, in vivo, pHIS3 immunohistological staining and Hochest/PI staining also draw the similar conclusions. The LPS exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. At last, we showed that LPS-induced cardia bifida could be partially rescued through the addition of antioxidants. Together, these results reveal that excess ROS generation is involved in the LPS-induced defects in heart tube during chick embryo development.

Cadmium-induced ultrastructural changes in primary target organs of developing chicken embryos (Gallus domesticus)

The aim of this study was to evaluate ultrastructural changes in kidney and liver tissue of chicken embryos exposed in ovo to cadmium. Embryonated eggs were injected on the 4th day of incubation with cadmium at the dose of 0, 2, 4 and 8 μg/egg (80 eggs/group). The samples of kidney and liver tissues were collected from embryos at the 14th and 18th day of incubation (E14 and E18) and at hatching day (D1). The tissue structure was evaluated by transmission electron microscopy (Tecnai G2 Spirit). The results indicate that hepatocytes responded to damage caused by toxic cadmium activity with a significant disturbance in the structure of mitochondria and a considerable expansion of the lysososmal system, while glomerular cells additionally reacted with an increased proliferation of peroxisomes. The range of changes observed on the subcellular level was dependent on the dose of cadmium, embryogenesis stage and cell type.

Evaluating the teratogenic effect of Eugenol in the development of the chick embryos

The aim of the present study is to investigate the effects of single Eugenol dose on chick embryo development. Fertilized treated eggs of Gallus gallus domesticus injected with a single volume of 0.1, 0.2, 0.3, 0.4 and 0.5 μl Eugenol to the egg air chamber and control egg group were incubated at 37ºC in the humidified incubator for 3, 6, 12 and 18 days. A visible damage effect caused by increasing of Eugenol doses on the vascular development with the cause of angiogenesis and brain acetychlorinesterase (AchE) activity suppression, incomplete embryonic development with the increasing of malformation and the mortality rate of the chick embryo. This work demonstrated that the treatment of the chick embryo in the early stages with Eugenol may be considered as teratogenic agents at the dose levels utilized in our laboratory conditions between days 3 to day 18 of incubation.

Effects of perfluorooctane sulfonate on genes controlling hepatic fatty acid metabolism in livers of chicken embryos

Per- and polyfluoroalkyl substances (PFAS) are synthetic surfactants with a wide variety of applications; however, due to their stability, they are particularly resistant to degradation and, as such, are classed as persistent organic pollutants. Perfluorooctane sulfonate (PFOS) is one such PFAS that is still detectable in a range of different environmental settings, despite its use now being regulated in numerous countries. Elevated levels of PFOS have been detected in various avian species, and the impact of this on avian health is of interest when determining acceptable levels of PFOS in the environment. Due to its similarities to naturally occurring fatty acids, PFOS has potential to disrupt a range of biological pathways, particularly those associated with lipid metabolism, and this has been shown in various species. In this study, we have investigated how in ovo exposure to environmentally relevant levels of PFOS affects expression of genes involved in lipid metabolism of developing chicken embryos. We have found a broad suppression of transcription of genes involved in fatty acid oxidation and PPAR-mediated transcription with more significant effects apparent at lower doses of PFOS. These results highlight the need for more research investigating the biological impacts of low levels of PFAS to properly inform environmental policy governing their regulation.

ESTIMATION OF CADMIUM FROM DRINKING WATER SOURCES AND ITS EFFECT ON CHICK EMBRYO DEVELOPMENT

The aim of the study was to screen Cd contamination from the River Mutha and to investigate its effects on chick embryo development. The study was correlated with the toxicity of fetal development in humans, as chick embryo provides an excellent model system for studying the development of higher vertebrates including humans. The estimated average concentration of Cd in water column of River Mutha was 0.0986 mg/l, exceeded the safe limit of drinking water (0.0003 mg/l). To study the effects of Cd on chick embryo development, analogous concentration observed in River water was introduced in fertilized chicken eggs. Aqueous solutions of Cd ranging from 0.05 to 1.00 mg/l were injected into fertilized chicken eggs at 72 hours of incubation and metal injected eggs were further incubated for 10 days to see the toxic effects if any. Control eggs were treated in the similar way with sterile distilled water. Results showed abnormal development of blood vessels and decrease in the embryo survival rate in the Cd treated eggs.

Compressed collagen and decellularized tissue \u2013 novel components in a pipeline approach for the study of cancer metastasis

BackgroundMetastasis is a complex process which is difficult to study and model. Experimental ingenuity is therefore essential when seeking to elucidate the biological mechanisms involved.Typically, in vitro models of metastasis have been overly simplistic, lacking the characteristic elements of the tumour microenvironment, whereas in vivo models are expensive, requiring specialist resources. Here we propose a pipeline approach for the study of cell migration and colonization, two critical steps in the metastatic cascade.MethodsWe used a range of extracellular matrix derived contexts to facilitate a progressive approach to the observation and quantification of cell behaviour in 2D, 3D and at border zones between dimensions. At the simplest level, cells were set onto collagen-coated plastic or encapsulated within a collagen matrix. To enhance this, a collagen compression technique provided a stiffened, denser substrate which could be used as a 2D surface or to encapsulate cells. Decellularized tissue from the chorioallantoic membrane of the developing chicken embryo was used to provide a more structured, biologically relevant extracellular matrix-based context in which cell behaviour could then be compared with its in vivo counterpart.ResultsCell behaviour could be observed and quantified within each context using standard laboratory techniques of microscopy and immunostaining, affording the opportunity for comparison and contrast of behaviour across the whole range of contexts. In particular, the temporal constraints of the in vivo CAM were removed when cells were cultured on the decellularized CAM, allowing for much longer-term cell colonization and cell-cell interaction.ConclusionsTogether the assays within this pipeline provide the opportunity for the study of cell behaviour in a replicable way across multiple environments. The assays can be set up and analysed using easily available resources and standard laboratory equipment. We believe this offers the potential for the detailed study of cell migration and colonization of tissue, essential steps in the metastatic cascade. Also, we propose that the pipeline could be used in the wider arena of cell culture in general with the increasingly more complex contexts allowing cell behaviours and interactions to be explored in a stepwise fashion in an integrated way.

Reduced embryonic blood flow impacts extracellular matrix deposition in the maturing aorta.

Perturbations to embryonic hemodynamics are known to adversely affect cardiovascular development. Vitelline vein ligation (VVL) is a model of reduced placental blood flow used to induce cardiac defects in early chick embryo development. The effect of these hemodynamic interventions on maturing elastic arteries is largely unknown. We hypothesize that hemodynamic changes impact maturation of the dorsal aorta (DA). We examined the effects of VVL on hemodynamic properties well into the maturation process and the corresponding changes in aortic dimensions, wall composition, and gene expression. In chick embryos, we found that DA blood velocity was reduced immediately postsurgery at Hamburger-Hamilton (HH) stage 18 and later at HH36, but not in the interim. Throughout this period, DA diameter adapted to maintain a constant shear stress. At HH36, we found that VVL DAs showed a substantial decrease in elastin and a modest increase in collagen protein content. In VVL DAs, up-regulation of elastic fiber-related genes followed the down-regulation of flow-dependent genes. Together, these suggest the existence of a compensatory mechanism in response to shear-induced delays in maturation. The DA's response to hemodynamic perturbations invokes coupled mechanisms for shear regulation and matrix maturation, potentially impacting the course of vascular development. Developmental Dynamics 247:914-923, 2018. © 2018 Wiley Periodicals, Inc.

Direct visualization of the Wntless-induced redistribution of WNT1 in developing chick embryos

Paracrine Wnt signals are critical regulators of cell proliferation, specification, and differentiation during embryogenesis. Consistent with the discovery that Wnt ligands are post-translationally modified with palmitoleate (a 16 carbon mono-unsaturated fatty acid), our studies show that the vast majority of bioavailable chick WNT1 (cWNT1) produced in stably transfected L cells is cell-associated. Thus, it seems unlikely that the WNT1 signal is propagated by diffusion alone. Unfortunately, the production and transport of vertebrate Wnt proteins has been exceedingly difficult to study as few antibodies are able to detect endogenous Wnt proteins and fixation is known to disrupt the architecture of cells and tissues. Furthermore, vertebrate Wnts have been extraordinarily refractory to tagging. To help overcome these obstacles, we have generated a number of tools that permit the detection of WNT1 in palmitoylation assays and the visualization of chick and zebrafish WNT1 in live cells and tissues. Consistent with previous studies in fixed cells, live imaging of cells and tissues with overexpressed cWNT1-moxGFP shows predominant localization of the protein to a reticulated network that is likely to be the endoplasmic reticulum. As PORCN and WLS are important upstream regulators of Wnt gradient formation, we also undertook the generation of mCherry-tagged variants of both proteins. While co-expression of PORCN-mCherry had no discernible effect on the localization of WNT1-moxGFP, co-expression of WLS-mCherry caused a marked redistribution of WNT1-moxGFP to the cell surface and cellular projections in cultured cells as well as in neural crest and surface ectoderm cells in developing chick embryos. Our studies further establish that the levels of WLS, and not PORCN, are rate limiting with respect to WNT1 trafficking.

STUDIES ON JOINT TOXIC EFFECTS OF A GLYPHOSATE HERBICIDE (FOZÁT 480) AND A HEAVY METAL (CADMIUM) ON CHICKEN EMBRYOS

The aim of the study was to determine the individual and combined toxic effects ofthe herbicide Fozát 480 (glyphosate [isopropylamine salt] 480g/l) and cadmiumsulphate (CdSO4) on the development of chicken embryos. On the first day ofincubation, chicken eggs were injected with 0.1 ml of cadmium sulphate solution(0.1%) and/or with 0.1 ml of Fozát 480 (2.0%). The chicken embryos wereexamined on the 19th day by measuring the rate of embryo mortality and bodyweight and by identification of different types of developmental anomalies andmacroscopic malformations. The body weight data were statistically evaluated byone-way ANOVA and Dunnett tests, while the embryonic mortality and thedevelopmental anomalies were analysed by Fisher test. Our teratogenicity studyrevealed, that the combined administration of cadmium sulphate and glyphosate(isopropylamine salt) containing herbicide formulation caused a significantreduction in the body weight of embryos and increased the rate of embryonicmortality. The joint toxic effect of cadmium sulphate and Fozát 480 is an additiveeffect compared to the individual toxicity of the test materials.

Role of innate immunity and oxidative stress in steroid-induced cataracts in developing chick embryos

Purpose: The exact etiopathogenesis of steroid-induced cataracts (SIC) is not known. Although oxidative stress is one of the most acceptable hypotheses, the exact molecular events in steroid-induced oxidative events in the lens need to be clarified. In addition, to the best of our knowledge, the innate immune system components in SIC formation have not been studied previously. The aim of the present study was to study the oxidative system and the innate immune system components in the cataractous lenses of a developing chick embryo SIC model.Materials and methods: The study included 20 specific pathogen-free (SPF) fertilized eggs divided into two groups, with one hydrocortisone (HC)-treated group (G1) and one non-HC-treated control group (G2). On the 15th day of incubation, the SPF eggs in the two groups were removed from the incubator; HC was injected into G1, and phosphate-buffered saline (PBS) was injected into G2 using insulin injectors into the chorioallantoic membrane. On day 17, all of the chick embryos were removed from the eggs, and all lens and liver tissues were dissected from the embryos. Cataract formation was evaluated in all lenses, and total antioxidant status (TAS), total oxidant status (TOS), reduced glutathione (GSH), oxidized glutathione (GSSG), interleukin-6 (IL-6), and interleukin-1β (IL-1β) levels were measured in all lens and liver tissues.Results: The lenses in the SIC group had lower levels of GSH, GSSG, TAS, and IL-6 (p = 0.016, 0.022, 0.000, 0.000, respectively), and higher levels of TOS (p = 0.000) than the control group. Furthermore, the liver tissues in the SIC group had decreased levels of TAS and IL-6, and increased levels of TOS compared to the control tissues (p = 0.000). Although the IL-1β levels in the lens and liver tissues in the HC-induced cataract group were lower than in the control group, these differences were not statistically significant. In addition, the GSH levels in the liver tissues did not statistically differ between the two groups despite the significant GSH difference in the lens tissues.Conclusions: Steroid therapy causes a decrease in GSH, GSSG, and TAS levels and an increase in TOS levels in lens tissues, which means there is increased oxidative stress and decreased antioxidant defence. Furthermore, lenses with SIC have lower IL-6 levels compared to non-cataractous lenses. The interaction between lenticular IL-6 and antioxidant defence needs to be further studied.

Development of Pyrosequencing-Based Assay for Analyzing the Origin of Genes in Preparing Reassortant LAIV Candidates

Live attenuated influenza vaccine (LAIV) is an effective measure to prevent influenza infection. The vaccine strain for LAIV is derived by reassortment of epidemic influenza strain with cold-adapted master donor virus (MDV) in developing chicken embryos. Molecular analysis of genomic composition of generated reassortants to select appropriate candidate for LAIV is a critical stage of vaccine strain development. Method of analysis permitting quick and precise detection of the genome composition of reassortants is required. The purpose of this study was to develop pyrosequencing-based assay using PyroMark Q24 for analysis of the origin of genes in preparing reassortant LAIV candidates for influenza A vaccine strains based on A/Leningrad/134/17/57 master donor virus and influenza B vaccine strains based on B/USSR/60/69 or B/Leningrad/14/77/55 master donor viruses, respectively. Objectives included development of primers set for genotyping of influenza A and B reassortants by pyrosequencing, establishing optimal dispensation order and assessment of effectiveness of protocol application for detecting the source of genes in virus samples. We have shown that a unified genome analysis protocol using PyroMark Q24 system is able to detect the source of internal genes in reassortant influenza strain by pyrosequencing. It allows analyzing the genome composition by a sensitive and specific innovative method in the first stages of vaccine strain development.