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Journal of Visualized Experiments : JoVE Jan 2021The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed...
The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.
Topics: Animals; Chickens; Egg Proteins; Electrophoresis, Polyacrylamide Gel; Female; Solubility; Vitelline Membrane
PubMed: 33586711
DOI: 10.3791/61739 -
Journal of Agricultural and Food... Feb 2021The weakening of chicken egg vitelline membrane (CEVM) is one of the most important factors influencing egg quality during high-temperature storage. Therefore, a...
The weakening of chicken egg vitelline membrane (CEVM) is one of the most important factors influencing egg quality during high-temperature storage. Therefore, a comparative N-glycoproteomic analysis of CEVM after 10 days of storage at 30 °C was performed to explore the roles of protein N-glycosylation in membrane deterioration. In total, 399 N-glycosites corresponding to 198 proteins were identified, of which 46 N-glycosites from 30 proteins were significantly altered. Gene ontology analysis revealed that these differentially N-glycosylated proteins (DGPs) were involved in antibacterial activity, glycosaminoglycan binding, lipid binding, and aminopeptidase activity. Removal of the N-glycans in Mucin-5B may result in a loss of CEVM's mechanical properties. The N-glycosites enriched in the apolipoprotein B β2 domain in CEVM were significantly changed, which may contribute to lipid composition modifications during storage. Moreover, N-glycosites in several metalloproteases were located within the functional domain or active site region, indicating that the decreased N-glycosylation levels may affect their structural stability, specific substrate binding, or enzyme activity. These findings provide novel insights into the roles of protein N-glycosylation during membrane weakening.
Topics: Animals; Chickens; Egg Proteins; Glycoproteins; Temperature; Vitelline Membrane
PubMed: 33566602
DOI: 10.1021/acs.jafc.0c07557 -
Scientific Reports Nov 2020In this study, we aimed to perform structural and proteomic analysis of the vitelline membrane (VM) of two species birds belonging to the family Turdidae: blackbird...
In this study, we aimed to perform structural and proteomic analysis of the vitelline membrane (VM) of two species birds belonging to the family Turdidae: blackbird (Turdus merula) and song thrush (Turdus philomelos). We performed structural analyses using scanning electron microscopy. The VM proteins were identified and compared to the best-known chicken VM proteins. According to our results, VM of both species has a typical three-layered structure: the outer layer, inner layer, and the continuous membrane between them. An unusual observation was the finding of "convexity" formed by the inner layer in blackbird. The role of these convex structures is not known, but they can be typical for the species and can be used in their identification. In addition, we identified two proteins in the VM of both species of birds, of which U3KEZ1 FICAL was not previously identified in any other bird species, and the U3JXV8 FICAL protein was confirmed only once in cockatiel parrot VM. The function of these proteins is not exactly known, but their structure shows similarities to the SERPIN proteins that are involved in microbiological defense, i.e., they are immune proteins. This study contributes to the current knowledge about the structure and composition of proteins of VM, especially because similar analyses have never been performed for Turdidae family. Knowledge of the structure and specific proteins of blackbird and song thrush VM can be beneficial in research on ecology and bird biology and also helpful in developing noninvasive and nongenetic identification methods.
Topics: Animals; Chickens; Egg Proteins; Egg Yolk; Female; Immune System; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Proteome; Songbirds; Species Specificity; Vitelline Membrane
PubMed: 33168893
DOI: 10.1038/s41598-020-76559-4 -
Veterinary Microbiology Dec 2020Since 2017, duck spleen necrosis caused by new variant duck orthoreovirus (N-DRV) infection had been observed in several provinces in China. This disease retards the...
Since 2017, duck spleen necrosis caused by new variant duck orthoreovirus (N-DRV) infection had been observed in several provinces in China. This disease retards the growth and development of ducks, thereby reducing feed return rate. N-DRV infection causes damage to duck spleen and other immune organs, leading to immunosuppression and susceptibility to other pathogens. In this study, we successfully constructed a breeding duck artificial infection model and found that N-DRV infection can cause pathologic changes, such as ovarian hemorrhage, follicle atrophy, and fallopian tube bleeding in breeding ducks, resulting in significantly reduced fertilization rate and egg hatching rate. Viral RNA was present in egg vitelline membrane, duck embryo, and duckling's spleen samples, as determined through quantitative polymerase chain reaction (qPCR). Autopsy revealed obvious pathologic changes in the spleen and other organs, although there were no obvious early clinical symptoms observed in ducklings. Sequence distance and phylogenetic analysis confirmed that N-DRV-SD19 re-isolated from the spleen samples of ducklings was consistent with the strain N-DRV-XT18 used for infecting breeding ducks. The findings in this study confirmed that N-DRV can be vertically transmitted through eggs, which provide an important reference for the disease prevention and control.
Topics: Animals; Ducks; Female; Infectious Disease Transmission, Vertical; Male; Orthoreovirus, Avian; Ovum; Phylogeny; Poultry Diseases; RNA, Viral; Reoviridae Infections; Sequence Analysis, DNA; Spleen
PubMed: 33059276
DOI: 10.1016/j.vetmic.2020.108861 -
Poultry Science Sep 2020A study was conducted to determine differences between Histomonas meleagridis-infected and control pullets based on disease signs, hen growth, and egg production and...
A study was conducted to determine differences between Histomonas meleagridis-infected and control pullets based on disease signs, hen growth, and egg production and quality. Ross 708SF females were weighed and then placed in pens on the day of hatch (92 chicks/pen). At 25 D, 4 pens were infected with H. meleagridis in the cloaca, whereas 4 pens were control. At 5, 10, and 20 D after inoculation, 5 birds per pen (2 birds per pen at 20 D) were subjectively scored for blackhead disease. Birds were feed restricted based on BW and/or egg production. Individual BW were collected at 3, 5, 13, 15, 20, and 64 wk. Egg production was recorded at 24-63 wk. Egg quality was measured at 30, 34, 39, 42, and 56 wk and included shell and vitelline membrane strength, shell thickness, egg weight, and Haugh units. Hatchability was measured at 27, 37, and 60 wk and fertility at 27 and 37 wk. Treatment effects were determined by JMP Pro 14 using GLM with means separated using the Student t test (P ≤ 0.05). Cecal lesions were apparent on 5, 10, and 20 D and liver lesions on 10 and 20 D for the infected birds. The control had no histomoniasis lesions. Flock uniformity differed on wk 13 and 20 (P = 0.04; 0.04). Infected birds weighed less at 64 wk (P = 0.002). The onset of lay was not delayed. Infected birds produced more eggs during 1 period (P = 0.02). The infected birds produced heavier eggs at 30 wk (P = 0.04), eggs with a stronger and thicker shell at 42 wk (P = 0.05, 0.03), and eggs with a stronger vitelline membrane at 56 wk (P = 0.049). Hatchability and fertility did not differ (P > 0.05). H. meleagridis was observed in the infected birds' cecal samples at trial termination. This study indicates early infection with H. meleagridis has limited effects on pullet egg production and quality.
Topics: Animals; Body Weight; Chickens; Female; Fertility; Oviposition; Poultry Diseases; Protozoan Infections, Animal; Trichomonadida
PubMed: 32867968
DOI: 10.1016/j.psj.2020.05.020 -
International Journal of Biological... Dec 2020The chicken egg vitelline membrane (CEVM) is an important structure for the transmembrane transport of egg yolk components, protection of the blastodisc, and separation...
The chicken egg vitelline membrane (CEVM) is an important structure for the transmembrane transport of egg yolk components, protection of the blastodisc, and separation of egg white and egg yolk. In this study, the N-glycoproteome of the CEVM was mapped and analyzed in depth. Total protein of the CEVM was digested, and the glycopeptides were enriched by a hydrophilic interaction liquid chromatography microcolumn and identified by nano liquid chromatography/tandem mass spectrometry. A total of 435 N-glycosylation sites on 208 N-glycoproteins were identified in CEVM. Gene Ontology enrichment analysis showed that CEVM N-glycoproteins are mainly involved in the regulation of proteinases/inhibitors and transmembrane transport of lipids. Mucin-5B is the primary N-glycoprotein in the CEVM. Comparison of the main N-glycoproteins between the CEVM and other egg parts revealed the tissue specificity of N-glycosylation of egg proteins. The results provide insights into protein N-glycosylation in the chicken egg, CEVM functions and underlying mechanisms.
Topics: Animals; Chickens; Chromatography, Liquid; Egg Proteins; Gene Ontology; Glycoproteins; Mucin-5B; Tandem Mass Spectrometry; Vitelline Membrane
PubMed: 32860793
DOI: 10.1016/j.ijbiomac.2020.08.193 -
Journal of Agricultural and Food... Sep 2020To explore the thermally induced alterations in chicken egg vitelline membrane (CEVM) protein abundances, a comparative proteomic analysis of CEVM after 10 days of...
To explore the thermally induced alterations in chicken egg vitelline membrane (CEVM) protein abundances, a comparative proteomic analysis of CEVM after 10 days of storage at 30 °C was performed. Altogether, 981 proteins were identified, of which 124 protein abundances were decreased and 79 were increased. Bioinformatic analysis suggested that the altered proteins were related to structure ( = 10), mechanical properties ( = 13), chaperone ( = 15), antibacterial ( = 12), and antioxidant ( = 3). Alterations in abundances of structural proteins, possibly resulting from the disintegration of these complexes, were observed in this study, suggesting a loss in fibrous structure. Several proteins involved in mechanical strength ( = 10), elasticity ( = 3), and chaperone were decreased in abundances, which indicated that deficits in these proteins might affect the CEVM mechanical properties. These findings will extend our understanding of CEVM deterioration during high-temperature storage from a proteomic perspective.
Topics: Animals; Chickens; Egg Proteins; Eggs; Food Storage; Hot Temperature; Proteomics; Vitelline Membrane
PubMed: 32809818
DOI: 10.1021/acs.jafc.0c03538 -
Parasitology Research Dec 2020This study of the fish blood fluke Aporocotyle simplex represents the first detailed transmission electron microscopical (TEM) investigation of the vitellarium of an...
Interrelationships of vitelline and muscle cells within the vitelline follicles of the blood fluke Aporocotyle simplex (Digenea, Aporocotylidae) and morphological evidence for the modification of vitelline material for eggshell formation.
This study of the fish blood fluke Aporocotyle simplex represents the first detailed transmission electron microscopical (TEM) investigation of the vitellarium of an aporocotylid digenean blood fluke. It revealed some unusual characteristics in the cytoarchitecture of the vitelline follicles and demonstrated modifications of the vitelline granules for eggshell formation. The vitelline follicles consist of vitellocytes at different developmental stages surrounded by sarcoplasmic processes of myocytes which occur throughout each follicle. Sites of intimate contact occur between the vitellocytes and the myocytes. Individual vitelline globules (0.1-0.2 μm in diameter) accumulate in quite small clusters of 10-20 and have a dense, heterogeneous matrix possessing central and peripheral regions with a greater density. Modifications of the vitelline globules take place within the clusters and are first apparent when the vitellocytes reach the lumen of the vitelline duct and vitelline reservoir. Globules within the clusters become confluent, and, when the vitellocytes reach the lumen of the oviduct and proximal ootype, these consolidated clusters contain a shapeless, loosely packed, dense material which is released from the vitellocytes by exocytosis. This investigation has provided morphological evidence for shell formation from modified vitelline globules in the form of a discontinuous, thin layer (~ 0.07 μm in thickness) of electron-dense shell material around the fertilized ovum and associated vitellocytes in the proximal ootype. The eggshell of intra-uterine eggs acquires an additional thin, heterogeneous outer layer, increasing its thickness to ~ 0.1 μm. The cytoarchitecture of the vitellarium, modifications of the vitelline globules within the clusters and the structure of the eggshell of A. simplex may prove to be of value in studies examining relationships between the three distinct lineages of aporocotylid digeneans.
Topics: Animals; Egg Shell; Female; Fishes; Microscopy, Electron, Transmission; Muscle Cells; Oogenesis; Ovarian Follicle; Ovum; Schistosomatidae; Trematode Infections; Vitelline Membrane
PubMed: 32808101
DOI: 10.1007/s00436-020-06849-3 -
Fish Physiology and Biochemistry Jun 2021Assessing female fish reproductive success requires a thorough evaluation of egg characteristics, including egg number, size, and variability as well as egg...
Assessing female fish reproductive success requires a thorough evaluation of egg characteristics, including egg number, size, and variability as well as egg developmental potential through the monitoring of embryo survival after fertilization. While embryonic success relies, at least in part, on paternal contribution, some parameters are strictly related to egg characteristics, one of the main ones being the viability of the egg when released into the water at spawning. It is however not necessarily possible, at least in salmonid fish that lay nontransparent eggs, to separate the different causes of egg/embryo failure. In this context, our aim was (i) to develop a simple and rapid system to capture images of rainbow trout eggs combined with computerized processing of these images to perform a fully automatic individual characterization of egg features including number and size (ii) to estimate unfertilized egg viability through the monitoring of the percentage of eggs that will not survive to water hydration. To evaluate the VisEgg system, unfertilized eggs (approximatively 400 eggs per batch) originating from 105 different females were hydrated in water. After 24 h, a picture of the eggs was obtained using a dedicated shooting system consisting of a light source and a digital single-lens reflex (SLR) camera. An image processing algorithm was developed to allow the automatic detection and separation of the eggs and to perform automatic measurements of egg number and individual egg size. The presence of white egg was used as an indirect measure of egg integrity, the "whitening" being the result of water entry into the egg through the vitelline membrane. These white eggs were therefore considered nonviable, as a result of their lack of physical integrity. Fertilization assays were performed in parallel using a subsample of the same egg batch. Embryonic development was monitored and hatching rate was calculated. A significant correlation between white egg percentage after hydration and hatching rate was observed (Spearman coefficient = -0.557, p < 0.001), in consistency with the fact that nonviable egg will not allow successful embryonic development. In contrast, the percentage of eggs that do not successfully hatch includes egg/embryo failures of different nature including reduced egg viability. Using the VisEgg, we were able to quantify the lack of viability of the eggs separately from the different other events that may occur during fertilization and incubation. the VisEgg is a convenient and reliable tool to obtain individual measures on trout eggs. It can be used to assess not only egg size and egg number but also unfertilized egg viability before fertilization.
Topics: Animals; Embryonic Development; Female; Oncorhynchus mykiss; Ovum; Phenotype
PubMed: 32607663
DOI: 10.1007/s10695-020-00844-2 -
Veterinary Microbiology May 2020In 2019, a novel goose astrovirus (GoAstV) epidemiological investigation on geese was conducted in Shandong province, China. During the investigation, a high prevalence...
In 2019, a novel goose astrovirus (GoAstV) epidemiological investigation on geese was conducted in Shandong province, China. During the investigation, a high prevalence of novel GoAstV was observed in symptom-free breeding geese flocks. Moreover, the novel GoAstV-specific RNA was detected in either breeder birds or their progenies. To verify the hypothesis that the novel GoAstV could be transmitted vertically, a total of 42 WuLong breeder geese, aged 335 days, were equally divided into three groups for experimental infection. The SDPY isolate of novel GoAstV (A/goose/Shandong/SDPY/2018, SDPY), preserved in our laboratory, was injected intramuscularly to subjects of group A while orally inoculated to those of group B. After the inoculation, novel GoAstV RNA was detected in vitelline membrane, embryos, and allantoic fluid of goose embryos in novel GoAstV infected groups. Moreover, the ORF2 gene of novel GoAstV from vitelline membrane, embryo, allantoic fluid as well as gosling shared almost 100 % nucleotide homology to a novel GoAstV virus isolated from the goose ovary which produced the egg, suggesting that the novel GoAstV can be vertically transmitted in the goose. Taken together, the findings provide evidence of possible vertical transmission of novel GoAstV from breeding goose to goslings.
Topics: Animals; Astroviridae; Astroviridae Infections; China; Female; Geese; Infectious Disease Transmission, Vertical; Male; Phylogeny; Poultry Diseases; RNA, Viral
PubMed: 32402337
DOI: 10.1016/j.vetmic.2020.108657