Search

Abstract Real-time visualisation of chick embryo development occurring inside an opaque eggshell is a major goal of developmental biology. This goal was partially achieved when 3-day-old embryos recovered from fertilised shelled eggs, preincubated in a conventional incubator, hatched after the egg contents had been transferred into shell-less embryo culture systems constructed of transparent plastic film placed in a plastic cup. However, the hatchable shell-less embryo culture systems described thus far cannot be used to visualise the dynamic morphological changes associated with the emergence of major organ systems during the first 3 days of embryo development, as the blastoderm area needs to be covered with an opaque membrane. Here, we present the development of a chick embryo culture system that enables omnidirectional real-time visualisation of the developing embryo, beginning from the newly laid blastoderm stage embryo until hatching.

ABSTRACT: This study aimed to determine the impact of the age of breeder hens and egg storage on egg quality, embryonic development, hatching events and chick quality in FUNAAB-alpha chickens. The study involved the use of 500 hatching eggs each collected from 32-wk and 60-wk-old of FUNAAB-alpha broiler breeder hens at the Animal Breeding and Genetic Unit of the Federal University of Agriculture, Abeokuta, Nigeria and subjected to 5 storage periods (0, 3, 7, 11, and 15 d). The quality traits of the eggs were recorded and incubated using the conventional protocol. Data were collected on the internal and external egg characteristics, embryonic development, hatching events, and chick quality. The data collected were laid out in 2 by 5 factorial design. The results showed that eggs from 32-wk-old breeder hens had higher albumen height and Haugh unit (HU) value than those from 60-wk-old breeders. The albumen height and HU decreased progressively with storage length in the 2 age groups. Extended storage duration linearly increased (P < 0.01) egg weight loss and decreased (P < 0.01) yolk height. The eggs from both breeder ages had increasing blastodermal diameters at oviposition up until d 11 of storage but decreased on d 15 of storage in eggs from 32 wk breeders. Eggs of 32-wk-old FUNAAB-alpha breeder hens had larger diameters at oviposition compared with 60-wk-old breeders. The chicks from 60-wk breeder had late internal pipping (469.06 h), early external pipping (474.46 h) and a shorter time lag between both pips (9.00 h) compared to chicks from 32 wk breeder. The highest fertility was recorded in eggs stored for 3 d (80.7% and 79.6%), while the lowest fertility was in eggs stored for 15 d (53.4% and 47.7%) in both 32-wk and 60-wk-old breeders, respectively. Chicks from young breeder hens stored for 3 d had better quality scores (100%) compared to 0, 7, 11, and 15-d storage duration and in 60-wk-old breeders across all storage duration. It was concluded that both egg storage duration and age of breeder affected egg quality, hatching events and hatchling quality of FUNAAB-alpha chickens and the interaction effects of both factors was recorded for some of these traits. However, extended storage beyond 7 d had a larger negative impact on egg quality and hatchability of eggs from an old breeder (60 wk) than on eggs of a young breeder (32 wk).

In vitro fertilization has been widely used to produce offspring in several mammalian species. We previously successfully produced Japanese quail chicks using intracytoplasmic sperm injection (ICSI), whereas in vitro insemination was not successful. This may be due to the difficulties associated with mimicking the sperm-egg fusion process and subsequent events in physiological polyspermic fertilization in vitro. In the present study, we observed egg development after in vitro insemination and investigated the inactivation of metaphase-promoting factor (MPF) and cytostatic factor (CSF), which are downstream of the Ca2+ signaling pathway in the egg, due to fertilizing sperm. We found a sperm number-dependent increase in hole formation caused by sperm penetration of the perivitelline membrane, the extracellular coat surrounding the egg. Egg development was observed following in vitro insemination; however, the developmental rate and stages after 24-h culture were inferior to those of ICSI eggs, even when insemination was performed with a high number of sperm (2 × 104). We also noted the downregulation of inositol 1,4,5-trisphosphate receptor-1, ryanodine receptor-3, cyclin B1, and c-MOS, which are important regulatory components of MPF and CSF in the egg, which was dependent on the number of sperm used for insemination. However, the decreases observed in these components did not reach the levels observed in the ICSI eggs. Collectively, the present results suggest that a sperm number higher than 2 × 104 is required for the progression of the Ca2+ signaling pathway, which initiates subsequent egg development in Japanese quail.

Abstract Background Cool temperature egg storage prior to incubation is a common practice in the broiler industry; however, prolonged egg storage causes increased embryonic mortality and decreased hatchability and growth in surviving chicks. Exposing eggs to short periods of incubation during egg storage (SPIDES) reduces the adverse consequences of prolonged storage. SPIDES increases blastodermal cell viability by reducing apoptosis, though the counteracting mechanisms are unclear. To define the impact of prolonged storage and SPIDES, transcriptome analysis compared gene expression from blastoderms isolated from eggs exposed to the following treatments: control (CR, stored at 17 °C for 4 days), prolonged storage (NSR, stored at 17 °C for 21 days), SPIDES (SR, stored at 17 °C for 21 days with SPIDES), and incubated control (C2, stored at 17 °C for 4 days followed by incubation to HH (Hamburger–Hamilton) stage 2, used as the ideal standard development) (n = 3/group). Data analysis was performed using the CLC Genomics Workbench platform. Functional annotation was performed using DAVID and QIAGEN Ingenuity Pathway Analysis. Results In total, 4726 DEGs (differentially expressed genes) were identified across all experimental group comparisons (q 20, |fold change| > 1.5). DEGs common across experimental comparisons were involved in cellular homeostasis and cytoskeletal protein binding. The NSR group exhibited activation of ubiquitination, apoptotic, and cell senescence processes. The SR group showed activation of cell viability, division, and metabolic processes. Through comparison analysis, cellular respiration, tRNA charging, cell cycle control, and HMBG1 signaling pathways were significantly impacted by treatment and potential regulatory roles for ribosomal protein L23a (RPL23A) and MYC proto-oncogene, BHLH transcription factor (MYC) were identified. Conclusions Prolonged egg storage (NSR) resulted in enriched cell stress and death pathways; while SPIDES (SR) resulted in enriched basic cell and anti-apoptotic pathways. New insights into DNA repair mechanisms, RNA processing, shifts in metabolism, and chromatin dynamics in relation to egg storage treatment were obtained through this study. Although egg storage protocols have been examined through targeted gene expression approaches, this study provided a global view of the extensive molecular networks affected by prolonged storage and SPIDES and helped to identify potential upstream regulators for future experiments to optimize egg storage parameters.

In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the ‘timer’ genes caudal, Dichaete, and odd-paired, whose expression dynamics correlate with the mode of segmentation. In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small ‘tail’ region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. We used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. We propose that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.

Summary: The aim of the study was to examine the effects of age of broiler breeders and storage duration on quality traits and blastoderm of eggs obtained from Marshall broiler breeders. A total of 320 eggs were collected from 2 flocks, 43 wk and 65 wk of age. Eggs were sampled immediately after collection and subjected to a storage duration of 0 (control), 4, 7, and 10 D at 16°C and 80% RH. Extended storage of eggs until 10 D from 65-wk-old breeders resulted in significant deterioration of egg quality traits. Egg weights decreased, whereas egg weight loss increased with storage duration for both breeder ages. Egg traits such as yolk diameter increased with interaction effects, whereas yolk heights declined. However, yolk weight and yolk percentage were observed to increase with age of breeders during egg storage. With a storage of 10 D, albumen diameter, albumen weight, and albumen percentage were higher with 43-wk-old breeders. Albumen pH rapidly turned alkaline from 4 D of storage period for both breeder ages and increased until about 9.85 at 10 D of storage. Haugh units declined with age and storage effects and were higher for eggs from 43-wk-old breeders. Blastoderm diameter was also affected as it decreased with storage for 65-wk-old breeders especially at day 7 of eggs storage. The results suggest that breeder's age and storage duration significantly influenced important egg quality traits and blastoderm expansion of Marshall broiler breeder eggs especially from older breeders.

ABSTRACT: At the time of oviposition, the chicken embryo is in its blastodermal stage. The blastoderm displays the unique ability to undergo developmental arrest at low temperatures in a process called “embryonic diapause.” In the wild, diapause occurs in freshly laid eggs until the last egg of the clutch has been laid, providing an evolutionary advantage to hens that can synchronously hatch their eggs. The poultry industry utilizes the diapause phenomenon to store eggs before incubation, thereby mitigating their logistic problems. The embryos can only be stored at particular embryonic stages—termed “diapause developmental window” (DW)—if they are to continue to develop normally thereafter. Both cellular and molecular mechanisms define the limits of this DW which broadly comply with onset of blastulation to early gastrulation. Storage conditions affect the cellular and molecular characteristics of the embryo during this window and their ability to successfully resume development (SRD). At storage temperatures of ~12°C to 18°C, embryos can undergo diapause for a short period (up to 7 days (d)) without affecting SRD. However, following longer period of diapause (up to 28 d), embryo stored at ~12°C, but not at ~18°C, can resume development normally. Moreover, eggs can be heated before or during the storage period which will lead to their commencing in development; however, unlike the non-heated embryos, the storage temperature for heated embryos, which are more advance in developing, is not clear. Thus, based on SRD, this review brings evidence supporting the notion that a lower storage temperature is beneficial for early-stage blastoderms whereas a higher storage temperature is favorable for later-stage/gastrulating embryos. Our understanding of the molecular mechanisms underlying the relationship between storage temperature and development stage within the DW is rather limited. However, it is expected to become relevant in light of the effect of selective breeding of modern avian birds on the advancement of embryonic development stage. Thus, this review discusses parameters that are regulated during the DW and affect SRD, and presents the need to adopt new storage techniques. The pre-managerial decision of required duration of storage with manipulation of storage temperature in the currently used storage techniques may improve SRD characteristics.

Longer egg storage times (>7 d) are common in broiler parent and grandparent hatcheries to obtain the requested flock size. However, prolonged storage is known to decrease hatchability. The aim of this study was to investigate the effects of turning and short period of incubation during egg storage (SPIDES) for 14 d on the stage of blastoderm development, embryonic mortality, and hatchability of eggs from young and old grandparent flocks. Hatching eggs were obtained from Ross female line grandparent flocks aged 29 wk (young) and 58 wk (old). Eggs were stored at 15°C, and turned 90° 0 or 4 times daily during storage. On day 5 after egg collection, the eggs were either held in the storage room (control) or subjected to SPIDES treatment. The development of the blastoderm in sample eggs was determined immediately after collection on a farm and again after the SPIDES treatment. Each of the 8 subtreatments was tested on 6 replicate trays of 150 eggs (900 eggs per subtreatment) with 7,200 hatching eggs set in a single-stage setter and hatcher for the trial. The stage of blastoderm development was advanced by the old flock, by SPIDES, and by turning 4 times daily during egg storage (P ≤ 0.05). There was a significant interaction effect of flock age × turning during storage on embryonic development, which suggested that turning advanced the stage of blastoderm development only in eggs from the old flock (P ≤ 0.05). Eggs from the young flock had a better hatchability than eggs from the old flock (P ≤ 0.05). Hatchability was increased by turning 4 times/day during the storage period compared with no turning because of a decrease in the percentage of late embryonic mortality (P ≤ 0.05). SPIDES decreased early and late embryonic mortality as well as the percentage of second-grade chicks (P ≤ 0.05), which increased the hatchability of fertile eggs at both flock ages (P ≤ 0.05). The results of this study showed that a combination of turning eggs 4 times daily along with one SPIDES treatment during 14 d of storage resulted in the highest hatchability in both young and old broiler grandparent flocks.

ABSTRACT In order to find out the main factors influenced the hatchability and improve the hatchability of the windowed chicken eggs at stage X, several experiments were made on the basis of the former patent of eggshell windowing methods on equatorial plane, such as cutting and sealing techniques, air cell recovering, laying position immediately after sealing, as well as the injection volumes into the subgerminal cavity of the blastoderm. The result showed that:1) the best sealing material combination was straw powder (SP) and instant glue (IG); 2) there was a highly positive correlation between air cell rate and hatchability; 3) the highest hatchability increased to 71.6% when the eggs were windowed and sealed with IG dropped firstly and then SP sprinkled, finally lay down with the blunt end upward immediately after being sealed; 4) the hatchability was significantly reduced as injection volume (DMEM) was increased (p

The early stages of development of the chick embryo, leading to primitive streak formation (the start of gastrulation), have received renewed attention recently, especially for studies of the mechanisms of large-scale cell movements and those that position the primitive streak in the radial blastodisc. Over the long history of chick embryology, the terminology used to define different regions has been changing, making it difficult to relate studies to each other. To resolve this objectively requires precise definitions of the regions based on anatomical and functional criteria, along with a systematic molecular map that can be compared directly to the functional anatomy. Here, we undertake these tasks. We describe the characteristic cell morphologies (using scanning electron microscopy and immunocytochemistry for cell polarity markers) in different regions and at successive stages. RNAseq was performed for 12 regions of the blastodisc, from which a set of putative regional markers was selected. These were studied in detail by in situ hybridization. Together this provides a comprehensive resource allowing the community to define the regions unambiguously and objectively. In addition to helping with future experimental design and interpretation, this resource will also be useful for evolutionary comparisons between different vertebrate species.

It has been widely accepted that prenatal exposure to ionizing radiation (IR) can affect embryonic and fetal development in mammals, depending on dose and gestational age of the exposure, however, the precise machinery underlying the IR-induced disturbance of embryonic development is still remained elusive. In this study, we examined the effects of gamma-ray irradiation on blastula embryos of medaka and found transient delay of brain development even when they hatched normally with low dose irradiation (2 and 5 Gy). In contrast, irradiation of higher dose of gamma-rays (10 Gy) killed the embryos with malformations before hatching. We then conducted targeted irradiation of blastoderm with a collimated carbon-ion microbeam. When a part (about 4, 10 and 25%) of blastoderm cells were injured by lethal dose (50 Gy) of carbon-ion microbeam irradiation, loss of about 10% or less of blastoderm cells induced only the transient delay of brain development and the embryos hatched normally, whereas embryos with about 25% of their blastoderm cells were irradiated stopped development at neurula stage and died. These findings strongly suggest that the developmental disturbance in the IR irradiated embryos is determined by the proportion of severely injured cells in the blastoderm.

A novel strategy has been developed to generate muscular dystrophy chickens by means of germline chimeras. Donor embryos were obtained from the New Hampshire chicken; NH-413 strain which have genes responsible for Fukuyama type muscular dystrophy (Saito et al., 2005). Donor cells were isolated from the center of area pellucida of the blastoderms. Recipient embryos were obtained from White Leghorn chicken; Line-M. The generated chimeric chickens had the donor derived brown plumage in the down in some extent, suggesting that the cells containing muscular dystrophy were introduced into the chimeras. These chimeric chickens have been raised until sexual maturity. The chimeric chickens were back-crossed to donor strain; the NH-413 strain. The phenotype of some of the offspring was very similar to that of the donor strain. The offspring showed some characters typical to the muscular dystrophy.It was suggested that the donor derived NH-413 strain offspring was generated. The established system should be one of the powerful strategies for breeding and regeneration of the muscular dystrophy chickens.

Primordial germ cells (PGCs) are the progenitor cells of gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. PGCs enter the circulation soon after the formation of blood vessels in incubating eggs and eventually settle in the gonadal primordium. We have now examined exposure of chicken embryos to soft (low-energy) x-rays as a means of depleting endogenous PGCs and thereby improving the efficiency of chimera production. The blastoderm of White Leghorn eggs was exposed to soft x-rays for 0, 20, 40 or 60s before incubation. The irradiated embryos manifested delayed development at 60h of incubation. They also showed reduced numbers of circulating PGCs at stages 14 and 15 and of gonadal PGCs at stage 30. The hatchability of irradiated embryos was lower than that of nonirradiated controls. Irradiation for 20s was found to provide the best outcome taking into consideration both the restriction of PGC proliferation and hatchability. Dispersed blastoderm cells of quail (black plumage) embryos were introduced into the blastoderm of chicken embryos irradiated for 20 s or of nonirradiated embryos. The number of donor-derived PGCs was higher in the irradiated embryos than in the nonirradiated controls at stage 30. These results suggest that soft x-irradiation of chicken embryos is a feasible approach to depletion of endogenous germ cells and consequent improvement in the efficiency of incorporation of donor PGCs.

The present study was conducted to improve the rate and stage of blastoderm and embryo development of quail oocytes by intracytoplasmic sperm injection (ICSI) with quail phospholipase Cζ (PLCζ) cRNA. Blastoderm development of quail oocytes cultured for 24 or 72h were staged under a stereomicroscope. The blastoderms were stained with 4,6-diamidino-2-phenylindole (DAPI) for cell division progress. A quail PLCζ cDNA clone was isolated from sperm by RT-PCR. The 1978bp nucleotide sequence contained an ORF encoding 637 amino acids of protein. The deduced quail PLCζ protein consists of EF-hand domain, X and Y catalytic domain and C2 domain, but lacked a plextrin-homology (PH) domain at the N-terminus. RT-PCR analysis revealed that PLCζ mRNA is expressed only in the testis. ICSI into a quail oocyte without PLCζ cRNA induced blastodermal development in 16% (4/25) of the oocytes which reached stages III-VI 24h after culture. In contrast, ICSI together with PLCζ cRNA (in 3nl at 60μg/ml) into a quail oocyte induced blastoderm development more than 2 fold (36.8%, 7/19) with embryos reaching stages VI-VII. Furthermore, 72h after culture ICSI into a quail oocyte without PLCζ cRNA induced embryo development in 15.4% (2/13) of the oocytes and reached stages V-VII. In contrast, ICSI together with PLCζ cRNA (in 3nl at 60μg/ml) into a quail oocyte induced 3 fold more embryo development (50%, 9/18) with embryos between stages VI and over X. The microinjection of PLCζ cRNA significantly improved the conventional ICSI method by enhancing the proportion and the stages of embryo development. Accordingly, the present PLCζ cRNA microinjection method in birds may contribute to assist in the production of transgenic birds and protection of endangered species of birds.

Over the past 3 decades, different strategies were launched to obtain transgenic animals and chicken in particular. Among various technical approaches, the direct modification of the embryo with DNA vectors or viruses is one of the ways of choice which led to the generation of transgenic chicken. In parallel, cellular approaches were also actively developed and were mainly based either on the use of embryonic stem cells (ESC) or of primordial germ cells (PGC). Both cellular types present advantages and disadvantages. ESC can be easily amplified in vitro and genetically modified but until now failed to contribute efficienctly to the germ line. In contrast, PGC colonize the germ line but present a limited proliferative potential and are hardly modified in vitro. The exact developmental and molecular relationships between these two cell types are not well documented. Finally, recent development and demonstration of the lentiviruses efficiency revivifies the interest for transgenic chicken as a tool for developmental studies as well as potential biotechnological and agronomic purposes.

In order to improve transfection efficiency of the stage X blastoderm of chickens by in vivo electroporation, the effect of the size of the upper and lower electrodes was examined. The plasmid DNA (pbAEGFP) was injected into the stage X blastoderm followed by applying electric pulses vertically to the blastoderm layer. The manipulated embryos were incubated using an embryo culture system, and the GFP gene expression was then examined. By decreasing the size of the upper electrode or by increasing the size of the lower electrode, the effect of the electric pulses was clearly enhanced. Although the introduced GFP gene was efficiently expressed in the embryonic and extra-embryonic tissues, the expression in the embryos was transient. No integration of the GFP gene into the chromosomes of germline cells of chickens was detected.

In order to introduce exogenous DNA into early chicken embryos by transfecting the stage X (EG&K) blastoderm, in vivo electroporation was applied to it. A pair of novel electrodes was devised for applying electric pulses vertically to the blastoderm layer. Fertilised eggs at stage X were broken and the blastoderm was placed on top of the yolk. One of the electrodes was placed on the blastoderm layer through the yolk membrane, and the other electrode was placed on the bottom of the yolk. DNA (GFP gene) was injected into the blastoderm, and electric square pulses were applied 5 times at 5-100V for loading periods of 50 msec per pulse at one-second intervals. The manipulated embryos were transferred into host eggshells and incubated for 4 days. The viability of the manipulated embryos was 67.0% (59/88) on day 3 of incubation. The expression pattern of GFP gene was mostly mosaic in the embryos, and the percentages of embryos that expressed the GFP gene were 97.7% (86/88), 93.2% (82/88) and 75.0% (66/88) on days 1, 2 and 3 of incubation. A high rate of GFP gene expression was observed in the embryonic and extra-embryonic tissues (54.2%, 32/59) and also in the extra-embryonic tissues only (27.1%, 16/59) on day 3 of incubation. The expression of the GFP gene throughout the embryonic tissues was also observed. These results suggest that the system for applying electric pulses vertically to the blastoderm layer is effective in introducing exogenous DNA into early chicken embryos.

Considerable interest exists in the development of transgenic poultry as a means of improving commercial poultry stocks and for applications in the pharmaceutical industry. The development of transgenic poultry requires stable insertion of genes into the germline. Because of the unique aspects of reproduction in birds, new methods of embryo manipulation had to be developed that include the ex ovo culture of embryos and the formation of germline chimeras using blastodermal cells and primordial germ cells. Currently, transgenic poultry can be produced using retroviral vectors and through the microinjection of DNA into the germinal disk of the newly fertilized egg. However, other approaches such as the development of embryonic stem cells, the culture of primordial germ cells, and sperm-mediated transfection are being explored for their potential to produce transgenic poultry.