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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.

The pioneering study of Eyal-Giladi and Kochav (EGK Eyal-Giladi and Kochav, 1976) on the early developmental stages-from fertilization, through oviposition, to the gastrulation process-set the standard for characterizing chicken embryos, and has been used in numerous studies over the years. During uterine development, the chicken embryo undergoes dramatic changes, extremely rapid cell cycles, massive cell death, and axial determination processes. However, once the egg is laid, the temperature drops and the embryo enters into a diapause-like state. This phenomenon is utilized to store fertile eggs prior to incubation. The ability to resume development to hatching, following storage, relies on several factors, including the number of living cells and the embryonic developmental stage. These factors are highly influenced by the storage conditions-mainly duration and temperature. Thus, to study the effects of storage conditions on embryonic viability, a comprehensive characterization of the starting point-shortly after oviposition-is needed. In this study, we characterized freshly laid broiler eggs from Ross 308 flocks for embryonic developmental stage, total cell count, and cell viability. Using the novel high-resolution episcopic microscopy (HREM) system, we show, for the first time, high-resolution 3D morphological models of blastoderms which allow for highly accurate embryonic staging. Staging was also done under a dissecting microscope thus allowing for a direct side-by-side comparison of the two methods. Analysis of freshly laid blastomeres showed that the total nucleus count increases with developmental stage from ∼60,000 at stage X EG&K to ∼130,000 at stage XIII EG&K, whereas the proportion of mitotic index and dying cells at oviposition are ∼2% and ∼5%, respectively. Moreover, staging embryos from young and old flocks revealed that the blastoderms of the old flocks are more developed. Specifically, the predominant embryonic stages were XI and XII EG&K in young and old flocks, respectively. Collectively, we characterized parameters that can serve to analyze the maladaptive effects of prolonged storage under various conditions on embryo survival.

Normal tables provide an objective step-wise description of the morphological development of an embryo. Such tables have been described for the chicken, turkey, quail, and duck embryos, but there is no such staging table for goose embryos. As the goose has one of the longest incubation periods of all the poultry species and embryo mortality during incubation is relatively high, a normal table of goose embryo development would be useful in assessing the morpho-genetic status of the goose embryo before and during incubation. In this study, embryos were isolated from commercial White Koluda goose eggs stored no longer than four days in a cool room (18°C) prior to incubation and after 4, 8, 12, and 16 h of incubation. Embryo staging was based on the normal tables described for the chicken by Eyal-Giladi and Kochav (EGK) and Hamburger and Hamilton (HH). Goose embryos from unincubated eggs were at Stage X and XI EGK and after 16 h of incubation the majority of embryos were between Stages 2 and 4 HH. Our results suggest that while the stage of development of the embryo in the unincubated goose egg is similar to that reported for the chicken, although the diameter of goose embryo is slighter larger. Following incubation, a goose embryo advances more slowly than a chicken embryo up to 16 h of incubation.

Cool temperature storage of eggs prior to incubation is a frequent practice by commercial broiler hatcheries. However, continued storage beyond 7 d leads to a progressive increase in the rate of early embryonic mortality. In this study, we examined the relative expression of 31 genes associated with fatty acid metabolism (8), apoptosis (7), and oxidative stress (16) pathways to better understand the basis of embryo mortality during egg storage. A total of 642 broiler eggs in 2 separate trials were subjected to the following egg treatments: stored 4 d (Control 1, C1); stored 21 d but subjected to short periods of incubation during egg storage (SPIDES); stored un-manipulated 21 d (NonSPIDES, NS); and stored 4 d then incubated for 10 h to advance the embryos to the same developmental stages as the SPIDES embryos (Control 2, C2). Hatchability trials (277 eggs) confirmed the efficacy of SPIDES compared to NS treatments in both trials. To determine relative expression of 31 selected genes, 365 blastoderms were isolated, staged, and flash frozen in batches of 5 to 10 blastoderms per vial (7 vials per egg treatment) prior to RNA extractions. Analysis of gene expression was performed using qRT-PCR and the results presented as relative expression normalized to C1. The relative expression of genes in which the SPIDES and C2 treatments were significantly up- or down-regulated in tandem indicated that the stage-specific expression of those genes was maintained by the SPIDES treatment. This study provides the relative gene expressions of blastodermal cells before and after prolonged egg storage as well as insight as to how SPIDES impacts blastodermal cell gene expression.

We examined the impact of egg storage on the hatchability of eggs from 2 lines (A and B) of commercial broiler breeders with known differences in fertility (line B slightly higher) and hatchability following egg storage (line A slightly lower). Eggs from both lines were stored in a 16°C room for 3 to 4, 10 to 12, and 17 d, the blastoderms were isolated, evaluated, and statistically compared. No significant interactions (line × duration of storage) were observed in the following traits: blastoderm diameter, total and percentage of viable blastodermal cells, stage of blastoderm development, and embryo weight and stage of development after 7 d of incubation. However, significant differences were observed when comparing line averages across storage treatments (diameter and total number of blastodermal cells) and storage treatment averages across lines (percentage of viable cells, and embryo weight and stages following incubation). To ascertain the basis for the observed differences in fertility, perivitelline sperm-hole numbers were determined in eggs within 72 h of lay (3 to 4 d storage group). Eggs from line B had a significantly higher number of eggs having greater numbers of sperm holes than eggs from line A. It is concluded that no single blastoderm trait or combination of traits can be definitively associated with the inter-line differences in hatchability following storage of eggs. Alternatively, differences in PVL sperm-hole counts between lines may be associated with the reported differences in fertility between the 2 lines.

It is well known that egg storage reduces embryo performance, but the fundamental reasons for reduced embryo quality remain unclear. The objective of this study was to investigate possible cellular and molecular mechanisms that might reduce embryo quality after egg storage. Broiler hatching eggs were obtained from the Ross 308 broiler strain, divided into 2 groups, and stored (4 and 14 d) under the same temperature and humidity conditions. Samples of the eggs were used to assess embryo quality by determining daily embryo weight (wet and dry) from 4 to 21 d of incubation. To understand possible cellular and molecular mechanisms that might affect embryo quality, blastoderms (unincubated embryos) were isolated from a sample of eggs from each storage group, dissociated into single cells, and subjected to flow cytometry analysis to differentiate between viable, apoptotic, and necrotic cell populations. Quantitative real-time PCR analysis was used to compare the expression of selected apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, Bcl-2-related ovarian killer gene, B-cell lymphoma 2 gene, and B-cell lymphoma xL gene) in blastoderms and embryos (6 d old after incubation). Data were analyzed by the MIXED model procedure of SAS (SAS Institute, Cary, NC), with significance set at P ≤ 0.05. After covariance analysis of initial egg weights, the results showed decreased daily embryo weights (wet and dry), an indication of decreased embryo quality that could affect hatch quality. In addition, a decrease in blastodermal cell viability was associated with an increased percentage of apoptotic cell deaths (P < 0.0001). Expression of pro-apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, and Bcl-2-related ovarian killer gene) were upregulated at the blastodermal level as the storage duration increased, but all genes were downregulated after 6 d of incubation. This suggests that an increase in egg storage duration could activate mechanisms of apoptotic cell death at the blastodermal level, which may be one of the molecular mechanisms that leads to reduced daily embryonic weight during incubation. Experimental controls capable of reducing the cellular and molecular mechanisms of egg storage should be used to increase embryo quality.

When eggs are stored beyond 7 d, hatchability and chick quality decrease. The cause of the negative effects of prolonged egg storage is not clear. The negative effects may be caused by a decrease in embryo viability due to an increase in cell death. The optimal time and curve of preincubation warming (the preincubation warming profile) may be different for eggs stored over short and long periods of time because embryo viability is dependent on egg storage time. The aim of this study was to investigate whether preincubation warming profiles affect embryonic development, hatchability, and chick quality when eggs are stored for a short or prolonged time. Two experiments were conducted. In both experiments, a 2x2 completely randomized design was used with 2 storage times (4 and 14 d at 17 degrees C in experiment I and 4 and 13 d at 19 degrees C in experiment II) and 2 preincubation warming profiles (within 4 or 24 h from storage temperature to 37.8 degrees C). In experiment I, results suggested that the effect of preincubation warming profile on hatchability was dependent on storage time. However, because a low number of eggs were used in this experiment, these differences were not significant. In experiment II, the interaction between storage time and preincubation warming profile was observed for embryonic mortality during the first 2 d of incubation and hatchability (P=0.006 and P=0.01, respectively). When storage time was 13 d, embryonic mortality during the first 2 d of incubation decreased by 4.4% and hatchability increased by 5.7% when the 24-h preincubation warming profile was used instead of the 4-h preincubation warming profile. However, no effect of preincubation warming profile was observed when storage time was 4 d. In both experiments, chick quality decreased when storage time increased but was not affected by preincubation warming profile. We concluded that a slow preincubation warming profile is beneficial for hatchability when storage time is prolonged but does not affect chick quality.

Our previous study demonstrated that elongated spermatids and sperm carrying the female-specific W-chromosome of the sex-reversed domestic fowl can activate the mouse oocyte, but whether they can fertilize the avian oocyte and lead to a developing zygote remains undetermined. A single sperm isolated from the semen and testis of normal rooster and from a testis of sex-reversed hen was microinjected into a quail oocyte and cultured for 20 to 24 h. Blastoderms were fixed, cleaved, nuclei stained by 4',6'-diamidino-2-phenylin-dole, and developmental stages were assessed. In the normal rooster group, ejaculated and testicular sperm induced blastodermal development in 22.6 and 20% of the quail oocytes, respectively. The developmental stages ranged from IV to VII. In the sex-reversal group, 20% of injected testicular sperm induced blastodermal development. The blastodermal stages varied from stage III to VI. Blastoderms after 4',6'-diamidino-2-phenylindole staining were assayed by PCR to identify the W chromosome of either chicken sperm or quail oocyte. The PCR assay results showed that 2 out of 9 developed blastoderms microinjected with sperm of sex-reversed hen were identified containing the female-specific W chromosome derived from sex-reversed hen. From these results, it is concluded that chicken sperm bearing the W chromosome possess fertilizing ability and can function to stimulate blastoderm development similar to that of normal chicken sperm carrying the Z chromosome.

Cryopreservation of avian germplasm is becoming better understood and more commonly practiced. However, one area that would be of great benefit for genome resource banking is the preservation of avian embryos. Little is know about the cryobiology of avian embryos, and they have never been successfully cryopreserved. However, it is likely that they share many of the challenges of other yolk-filled multicompartmental embryos. For example, the fish embryo has 1) a large overall size, resulting in a low surface-to-volume ratio, which retards water and cryoprotectant efflux/influx; 2) large-sized cells, such as the yolk, which could increase the likelihood of membrane disruption by intracellular ice formation; 3) compartments, such as the blastoderm and yolk, with differing permeability properties; and 4) susceptibility to chilling injury. Both the avian and fish systems share many physical and anatomical properties, and it is predicted that some of the same permeability barriers would exist in both as well. Although the systems are similar, some of the goals, and thus the practices, to protect the genome may be quite different. One of these major goals in avian developmental biology is to produce chicken:chicken transgenic animals, especially those with germ line transmission. Producing efficient germ line transmissions and being able to cryopreserve these transmissions would be extremely beneficial to both basic and agricultural science. This could be accomplished through the cryopreservation of embryonic gonadal tissue followed by grafting into a host. The gonadal/tail-graft system would provide an advantage for cryopreservation because it is small (in comparison with the whole embryo), has fairly uniform tissue, and contains the essential primordial germ line cells capable of recreating the genetic line of interest. Moreover, because the chicken is such a robust model for most other avian species, the cryopreservation of the gonadal/tail-graft may potentially open up similar treatments for other commercially important species.

Duck blastodermal cells isolated from Stage X embryos of Maya ducks were injected into subgerminal cavity of recipient Stage X chicken embryos treated with gamma-irradiation or untreated. Eleven somatic chimeras were obtained based on plumage color and were raised to sexual maturity. To test for germline chimerism, progeny tests were performed by mating the chimeras with Maya ducks. A total of 622 eggs was collected and incubated. Fertility rate and hatchability were 2.9% (18/622) and 1.0% (6/622), respectively. The six duck hatchlings were from Chimera 9801 and were considered to be derived from the germ cells developed from the donor Maya blastodermal cells, indicating that Chimera 9801 is a germline chimera.

The use of chicken blastodermal cells (CBC) in the production of transgenic chickens requires incorporation of the desired stable genetic modification into CBC. With greater proportions of stably transfected blastodermal cells in the embryo inoculum, the frequency of intermediate chimeric birds is greater. Magnetic-activated cell sorting (MACS) was evaluated as a method for enrichment of transfected CBC. This approach requires surface expression of a molecule that can be recognized by an antibody. Chicken blastodermal cells from fertilized Barred Plymouth Rock eggs were coelectroporated with pmiwZ and pMACS Kk and were sorted magnetically by expression of the mouse H-2Kk molecule on the surface of successfully transfected cells. The effectiveness of sorting was assessed using X-gal staining to detect lacZ expression from the pmiwZ plasmid. After 48 h of culture, lacZ-positive cells appeared to be enriched 1.4-fold in the MACS selected population. Cells from this enriched pool contributed to extra-embryonic and intra-embryonic tissues of 72-h White Leghorn recipient embryos with a marginal increase in levels of intra-embryonic contribution. Our demonstration that transfected, cultured, and magnetically sorted CBC maintain their ability to contribute to ectodermal and mesodermal lineages of intra-embryonic tissues illustrates the potential value of this technique for introducing genetic modifications into birds.

Chicken blastodermal cells (CBC) from Stage X embryos, which were isolated from newly laid, fertile, unincubated eggs, are pluripotent cells and can produce somatic and germline chimeras when injected into recipient stage X embryos. The CBC retain their pluripotential ability for up to 7 d in vitro. The molecular mechanisms that control proliferation and differentiation of CBC are largely unknown, although protein tyrosine kinases (PTK) are known to play important roles in these processes in similar cells. To understand better the molecular mechanisms of proliferation and differentiation in CBC, expression profiles of PTK and stem cell factor (SCF) were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific and degenerate oligonucleotide primers. Seventeen distinct PTK, including 14 receptor-type and 3 nonreceptor-type PTK and SCF were identified by RT-PCR. Expression of all of the genes was confirmed by northern blot analysis. The northern blot analysis showed that all probes hybridized with one or more transcripts at various expression levels. The expression of the 17 PTK and SCF genes in CBC suggests that they might play a role in signal transduction pathways that control the proliferation or differentiation in CBC.

Blastodermal cells isolated from newly laid, unincubated eggs are virtually uncommitted cells that exhibit many of the properties of pluripotential stem cells. They can be transferred from donor to recipient embryos and contribute to both somatic tissues and the germline. Blastodermal cells that have been maintained in culture for 7 d express the epitopes ECMA-7 and SSEA-1, which are also expressed by mouse embryonic stem cells. After culture for up to at least 7 d, blastodermal cells retain the ability to differentiate into somatic tissues and the germline both in vivo and in vitro. Proliferation in the absence of differentiation of blastodermal cells is stimulated by the presence of Leukemia Inhibitory Factor (LIF) and other ligands that interact with the gp130 receptor, and differentiation is stimulated by exposure to retinoic acid. Blastodermal cells also possess high levels of telomerase activity, which is shared by immortalized cells and cells within the germline. Blastodermal cells can be transfected and will express foreign genes both in vivo and in vitro. Transfected cells can be isolated by fluorescence activated cell sorting and can be cryopreserved without losing their ability to contribute to either somatic tissues or the germline. These properties of blastodermal cells make them ideal vectors for introducing genetic modifications to the germline.

The development of the turkey and chicken embryo from the first cleavage division through hypoblast formation is described. The early development of the chicken embryo has been categorized into 14 stages. A similar staging sequence for the turkey was not proposed until 1993, when we described the early development of the turkey embryo, which was divided into 11 stages. Comparatively, differences in the temporal and spatial development of the turkey and chicken blastoderm were evident. Of significance is the observation that at oviposition the turkey is in Stage VII and characterized by the first signs of area pellucida formation. In contrast, the chicken embryo at oviposition is in Stage X and area pellucida formation is completed. Similarly, the hypoblast, which is already apparent in the Stage X chicken embryo, does not appear in the turkey embryo until the egg is incubated. Furthermore, the anterior-posterior (head-tail) axis in the early embryo is achieved prior to oviposition in the chicken but after the onset of incubation in the turkey. It is apparent that the turkey embryo is less mature than the chicken embryo at oviposition. Whether this distinction is related to differences between the hatchability of turkey and chicken eggs is not yet known.

We describe a method of isolating blastodermal cells for characterization through flow cytometry. Particular attention was placed on cell viability and integrity issues faced by conventional protocols. The method allowed us to examine mechanisms behind cellular death. Our protocol was optimized by the spatial resolution of the ImageStream multispectral imaging flow cytometer. Overall, the technique provides both quantitative and qualitative information on blastodermal cells. The methodology was applied to the current biological problem in which prolonged (14 d) versus short-term (4 d) layer egg storage reduces embryo viability. Data were obtained between 3 egg storage treatments (unstored, 4 d, and 14 d); the data were analyzed by the PROC MIXED model procedure of SAS at P < or = 0.05 and least squares means separated by the PDIFF procedure of SAS. The results showed that egg storage increases the rate of cell death by both apoptosis and necrosis. Importantly, our study showed higher percentages of necrosis and late apoptosis in long-term versus short-term stored eggs. The percentage of live cells decreased significantly when eggs were stored for 14 d (71.42 + or - 3.36%) compared with eggs stored for 4 d (83.58 + or - 2.15%). The percentage of early apoptotic cells was not significantly different between the 2 treatments. The percentage of necrotic cells and late apoptotic-necrotic cells was higher in eggs stored for 14 d (16.75 + or - 1.73%; 7.36 + or - 1.53%) versus eggs stored for 4 d (3.56 + or - 1.64%; 2.31 + or - 1.52%), respectively. This could negatively affect embryo survival because of the potential effect that necrosis has on surrounding tissue integrity. The technique will be particularly relevant in studies requiring single cells from chicken blastoderms or as a basis to characterize genes that regulate apoptosis in avian species.

Twins and triplets of chick embryos were produced by microinjection treatment of cell suspension into the blastoderm. One pair of the twin embryos had an ability to develop normally and survived up to Day 21 of incubation, but did not hatch. The other twins and triplets died within 7 days.

The effects of injection and/or gamma-irradiation prior to injection on mortality, size of the gonads, and ultrastructure of primordial germ cell (PGC) were examined after 5 d of incubation. The mortality of embryos injected with donor cells was significantly higher than that of control and irradiated embryos. All irradiated embryos were alive, although their development was delayed compared to those not exposed to irradiation. The size of the gonads of embryos injected with donor cells were similar to those of control embryos, however, the size of the gonads in irradiated embryos was significantly smaller than those of control embryos. The number of PGC in the gonads was significantly decreased by irradiation. There was no notable effect of irradiation or injection on the nuclei and cytoplasmic organelles in PGC.

Modification of the chicken germline has been difficult, because it has been challenging to fractionate sufficient numbers of primordial germ cells for manipulation and implantation into developing embryos. A technique to enrich cell suspensions for primordial germ cells, using fluorescence-activated cell sorting (FACS), has recently been developed. The objective of the current study was to demonstrate that the FACS-enriched early embryonic gonocytes could fully participate in development of the germline. Therefore, cells were disassociated from stage 27 gonads, incubated with mouse anti-stage-specific embryonic antigen-1, which was detected with goat-antimouse IgM-fluorescein isothiocyanate, and the fluorescently labeled cells were sorted from the unlabeled cells using FACS. The isolated gonocyte population was injected into the blastoderm of unincubated stage X embryos, the germinal crescent of 3-d embryos, and into the circulation of stage 17 embryos that were pretreated with busulfan. Barred Plymouth Rock gonocytes were implanted exclusively into recipient White Leghorn embryos, and White Leghorn gonocytes were implanted exclusively into Barred Plymouth Rock recipient embryos. Embryos were cultured until hatch, and male putative chimeras were reared to sexual maturity. Germline chimerism was evaluated by observing feather color of the progeny. All injection methods resulted in germline chimeras demonstrating that FACS-sorted gonocytes can fully participate in development. Moreover, it was demonstrated that gonocytes isolated from stage 27 embryonic gonads can be introduced into embryos at an earlier stage of development, and the introduced gonocytes can fully participate in germline development.

Presently, avian genetic resources are best maintained as living collections of birds. Unfortunately, these stocks have been under constant pressure to be destroyed because of the decline in the number of Poultry Science Departments and pressures to cut costs at land grant institutions. Cryopreservation of semen is often suggested as a means to bank avian germplasm. However, this is only applicable for single-gene traits and does not allow for full reconstitution of the genetics of the original line. Over the last 15 yr, advances in the manipulation of the early chick embryo, manipulation of primordial germ cells (PGC), and the culture of embryonic stem cells (ESC) suggests that cryopreservation of blastodermal cells, ESC, or PGC might offer a means to preserve the entire genome of highly selected, specialized stocks of poultry. Freezing each of these cell types is possible with varying degrees of efficiency. Similarly, the effectiveness of generating germ line chimeras using blastodermal cells, ESC, or PGC also varies greatly. Other factors that must be considered include the choice of the recipient lines to develop the germ line chimeras and the number of individuals needed to reconstitute the line. Finally, the low efficiency rate of reconstitution and the high cost associated with current technologies makes these approaches prohibitive. Significant challenges remain to be overcome before the entire genome of poultry stocks can be routinely cryoperserved and reconstituted.

Chimeric chickens were evaluated as an intermediate for development of transgenic chickens. The transfer of Barred Plymouth Rock (BR) blastodermal cells into White Leghorn (WL) embryos results in BR-->WL chimeras, and some breeder males generate over 30% germline transmission of the BR genotype to offspring based on a feather-color trait. The objectives of the current study were to 1) identify the MHC (B haplotypes) in resident BR and WL lines, 2) establish that B antigens could be detected and quantified in red blood cells (RBC) of chimeras, 3) establish if there is a correlation in chimeras between percentage of RBC with donor B antigens and percentage germline transmission, and 4) evaluate if the MHC genotype influences chimera development. The RBC agglutination data indicated three B haplotypes were present in each line. The B*2-like, and B*19-like genes were unique to the WL line, and B*13-like and B-15-like genes were unique to the BR line, whereas a B*21-like gene was present in both lines. In adult BR-->WL chimeras, as well as 10- to 14 d-old WL-->WL chimeras, donor-type B antigens were detectable and quantifiable on RBC using flow cytometry. In BR-->WL chimeras, the percentage germline transmission was significantly correlated with the percentage of RBC with donor B antigen, as well as percentage of black feathers in the plumage. In a retrospective study using previously developed BR-->WL chimeras, the level of chimerism and germline transmission was higher in B*21/*21 type recipients, but this was not statistically significant in two prospective studies. It was concluded that MHC antigens on RBC can be used for identifying, quantifying, and selecting chicken chimeras developed by the transfer of blastodermal cells.

Blastoderm cells from chicken embryos of a donor breed (Green-legged Partridgelike; GP) were transferred to embryos of a recipient breed (White Leghorn; WL) to form chimeric progeny that, after inter se mating, permitted successful reconstitution of the donor breed. Among 23 chimeric chicks hatched from WL embryos injected with GP cells, 20 (87%) were raised until maturity, and progeny were tested by mating with GP birds to determine the ability of blastodermal cells to form germline chimeras. Six of the tested birds (30%) produced recipient-derived and donor-derived offspring, indicating that they were germline chimeras. The mean percentages of donor-derived germ cells in these birds were 21.1 (17.6 to 50.0%) and 16.9 (5.3 to 23.1%) in males and females, respectively. Among 477 chicks, resulting from mating the germline chimeric male with four germline chimeric females, 10 chicks (2.1%) exhibited a GP phenotype, indicating that the original donor stock had been reconstituted. Only one germline chimeric hen produced GP offspring, but the expected and calculated percentages of GP offspring were similar (2.99 and 2.08, respectively). Two methods of DNA analyses (RFLP and PCR amplification of polymorphic microsatellite loci) of chimeras and their offspring indicated that through mating of a relatively small number of chimeras it is possible to reconstitute a highly diverse population.

Two experiments were conducted to determine whether the presence of a blastoderm could affect albumen liquefaction of broiler hatching eggs prior to setting and during the initial stages of incubation. Eggs were stored for 0 to 3 d at 18 C and 75% RH. Albumen height and pH were measured after 0 or 2 h of incubation. In both experiments, albumen height was significantly lower in fertilized eggs compared with unfertilized eggs. The interaction of fertility with incubation time for albumen height in Experiment 1 was not significant (P ≤ 0.10). In Experiment 1, albumen pH was significantly higher for fertilized eggs compared with unfertilized eggs (P ≤ 0.01); the difference was similar but not significant in Experiment 2. The results suggest that the embryo contributes to albumen liquefaction and that ammonia production by the embryo may account for the effect.

In our first experiment, we studied the effect of injection method of blastoderm cells (BC) into the subgerminal cavity of White Leghorn embryos on hatchability of chicken chimeras. Freshly laid eggs were injected through a hole made in the equatorial plane of the eggshell (Method A). In Method B, eggs were stored pointed end down for 5 to 7 d prior to injection, and a hole was cut in the blunt end of the eggshell. An advantage of Method B was that the early embryonic mortality was reduced (P < or = 0.01) and resulted in higher hatchability (41.0%; 43/105) than Method A (9.8%; 14/143). In the second experiment, we studied chicken hatchabililty as influenced by windowing (no hole, Group 1; hole in the equatorial plane, Group 2; hole in the blunt end of egg, Groups 3 and 4) and egg turning (Groups 1 and 4) or not (Groups 2 and 3) during incubation. The hatchability percentages were as follows: 67.9 (Group 1) 0.0, (Group 2) 23.3, (Group 3), and 56.8 (Group 4). A statistically significant difference (P < or = 0.05) was noted between Group 1 or 4 and the other groups. We found no statistically significant differences in the weight changes (g) but did note certain differences in the egg weight loss (%) among different egg treatments. In the third experiment, we investigated the influence of origins of BC donors: Rhode Island Red (RIR), Barred Plymouth Rock (BPR), and Green-legged Partridgelike (GP) on hatchability of putative and somatic chimera chickens. The hatchability of chimeras was dependent on the adequate assortment of BC of the donor and ranged from 7.4% (RIR) to 56.1% (GP). In the case of BC injection of the GP breed, good hatchability was accompanied by very high percentage (86.9; 20/23) of somatic chimeras.

Stage X chick blastoderms following oviposition were accessed via a small window in the egg. Windowing, however, substantially reduces the hatchability of eggs containing early embryos. For example, only 32 of 389 (8.2%) eggs hatched after standard windowing with or without irradiation or injection. Ex ovo culture systems can overcome this problem but are labor intensive. A modification of a standard windowing technique has yielded an average hatch rate of 32% for 892 windowed eggs independent of incubator type, gamma-irradiation, or injection of the embryo. This was a fourfold increase over a standard windowing method. Similar hatch rates were observed using fertile eggs from five chicken lines [Barred Plymouth Rock (BR), Athens-Canadian (AC), Line 0, SPAFAS, and commercial White Leghorns (WL)]. The modification involves covering the egg shell membrane with PBS after grinding away the shell and before piercing the membrane. The window is then sealed by overlaying with fresh shell membrane and cementing it in place once it has dried. The method has been used successfully for the production of somatic and germline chimeras because donor BR blastodermal cells injected into Stage X, gamma-irradiated recipient embryos from WL or AC yielded a hatch of 33.7%, of which 42.3% were feather chimeras. Two of 11 cockerels tested were germline mosaics bearing at least 1% BR sperm. The modified windowing technique may be broadly applicable in emerging technologies in avian transgenesis and development.

Previous observations obtained from a culture of blastodermal cells on a mouse fibroblast feeder layer (STO) suggested that STO cells provide a factor or factors that facilitate development of avian primordial germ cells (PGC) from dispersed embryo cells. The purpose of the current study was to test the hypothesis that soluble factors produced by STO cells are responsible, at least in part, in supporting the development of PGC in culture and to examine the effect of stem cell factor (SCF), ciliary neurotrophic factor (CNTF), and basic fibroblast growth factor (bFGF) in the development of PGC in culture. Blastodermal cells on gelatin-coated plastic or on feeder layers of CV-1 cells yielded a small number of PGC. When blastodermal cells were cultured on STO cells, a marked increase in PGC was observed. The addition of STO cell-conditioned medium (STO-CM) to blastodermal cells cultured on gelatin-coated plastic and on feeder layers of CV-1 cells resulted in a significant increase in the number of PGC, indicating that soluble factors produced by STO cells can enhance the development of chicken PGC in culture. Supplementation of blastodermal cells with SCF (100 ng/mL) or CNTF (2 ng/mL) or with CNTF and SCF together resulted in a significant increase in the number of PGC after 48 h of culture on feeder layers of CV-1 cells. However, addition of bFGF (100 ng/mL) did not increase PGC. We concluded from these observations that the culture of blastodermal cells on feeder layers of STO and CV-1 cells can be used as a useful biological system in examining the regulatory factors that govern the ontogeny of the germ cell lineage in the avian embryo.

Using flow cytometry, the ploidy levels of parthenogenetic turkeys were quantified from blastodisc stage to adulthood. Eggs were collected from noninseminated hens of the Beltsville Small White flock, known for their high degree of parthenogenesis, and the blastodermal cells from developing embryos were compared with those of embryos produced by hens inseminated with semen from males of the same flock. Erythrocytes of parthenogens from Day 10 of incubation to 27 mo of age were also used for ploidy determination. Sperm and erythrocyte preparations from normal males of the above flock served as haploid and diploid standards, respectively. In parthenogenetically developing blastoderms, 40.3 +/- 14.5% of the cells were haploid and 48.9 +/- 11.9% diploid; blastoderms from fertilized eggs had no haploid cells. The haploid cell content of parthenogens declined from the blastodermal stage to adult life, with 1.9 +/- 2.3% at 10 to 20 d of embryonic development, 1.5 +/- 1.4% at 21 to 29 d of development, 1.4 +/- 2.6% at 4 wk posthatch, and 1.3 +/- 1.9% in adulthood, although changes between the 1st mo after hatch and adult stage were not significant. It is possible, therefore, that parthenogenetic embryos with a low proportion of haploid cells could be the ones that survive to Day 10 of development and beyond, whereas those with a higher proportion of haploid cells fail to develop. The semen volume of male parthenogens was significantly lower than that of normal males, although the concentration of spermatozoa and their fertilizing capacity did not vary significantly between groups, suggesting that the germ cells of these parthenogens are capable of normal meiosis and sperm maturation leading to a normal fertility.

The blastoderm (fertilized ovum) and unfertilized germinal disc (UGD) of fresh laid eggs and eggs stored prior to incubation exhibit subtle but definable morphological variations. Such variations may lead to difficulty when attempting to determine true flock fertility based on the appearance of the blastoderm/UGD. The objectives of this study were to define and categorize such morphological variations and to determine whether sperm influence the frequency distribution of the different categories. Eleven categories of blastoderms were defined based on the relative density and appearance of the area alba, area pellucida, area opaca, and the periblast. The majority of the blastoderms were included in the first four categories. Unfertilized germinal discs were divided into six categories and were best differentiated from the blastoderms by the presence of vacuoles around its central dense area. They were also discernible from blastoderms based on their overall denser appearance. Differences in the frequency distribution of some of the UGD categories between virgin and inseminated hens may be due to the effect that supernumary sperm may have on the organization of the UGD (no fertilization but supernumary sperm present) or blastoderm (fertilized but failed to develop). It is recommended that before starting true fertility determinations during fresh egg breakouts, one should study the appearance of the UGD from virgin hens and then the blastoderm from inseminated hens. One then will learn to appreciate the subtle differences in shape and density of the blastoderm/UGD structural components.

The origin of the germ cell lineage in vertebrates is a fundamental question that has preoccupied developmental biologists. Recent work on the origin of the avian germ line has extended and clarified our understanding of the temporal and spatial segregation of primordial germ cells (PGC) during prestreak stages of development. The germ cells first appear at Stage X (Eyal-Giladi and Kochav, 1976) in the ventral surface of the area pellucida in a scattered pattern among polyingressing cells. Subsequently, the PGC gradually translocate from the epiblast to the hypoblast. The entire process appears to be dependent upon the maintenance of an organized area pellucida. Little is known about the regulatory events governing germ cell emergence during this period; however, the culture of dispersed blastodermal cells on a mouse fibroblast feeder layer can compensate for a disorganized area pellucida and offers an in vitro system to examine the molecular basis of germ cell development. Such basic information is valuable for current approaches towards the production of transgenic poultry with targeted changes to the genome through the use of avian embryonic stem cells or primordial germ cells. Refinement of the culture of primordial germ cells or their precursors should allow academic and industrial research laboratories to answer significant biological questions and to improve the genetic potential of commercial poultry stocks. A better understanding of the biology of avian primordial germ cells during early embryo development can only enhance this process.

To execute a strategy for reconstituting genetic resources from cryopreserved blastodermal cells, experiments were conducted to optimize conditions for producing chimeric chickens from frozen-thawed blastodermal cells. Stage X blastodermal cells were collected from Barred Plymouth Rock embryos and dispersed. Cells were resuspended in 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, and distributed into plastic ampules. Cell suspensions were seeded to induce ice formation at -7 C, cooled from -7 to -35 C at 1 C/min and then ampules were plunged into liquid nitrogen. Thawing was done by plunging the ampules into warm water (37 C) for 3 min. After centrifugation, the supernatant was replaced with DMEM, and dead or broken cells were removed by density gradient centrifugation. Approximately 500 cells were injected into irradiated Stage X White Leghorn recipient embryos. Following incubation, several somatic chimeras were produced. The frequency of somatic chimerism when fresh (unfrozen) cells, or cells that were frozen and selected by density gradient centrifugation on Percoll or Nycoprep were injected into recipient embryos was 84, 79, and 85%, respectively. The percentage of donor-derived pigmentation in the down of these chimeric chickens was 79, 50, and 58%, respectively. Germline chimerism was determined by mating the chimeras that survived to sexual maturity to Barred Plymouth Rocks. Nine of 16 birds (56.2%) injected with fresh cells, 2 of 26 birds (7.7%) injected with cells that were frozen and selected by density gradient centrifugation on a Percoll gradient, and 3 of 26 birds (11.5%) injected with cells that were frozen and selected on a Nycoprep gradient showed germline transmission; the percentage of donor-derived progeny in these chimeras were 29.5, 5.2, and 6.8%, respectively. The Barred Plymouth Rock donor stock was “reconstituted” by inter se mating of germline male and female chimeras. These data demonstrate that the strategy described here for reconstituting genetic resources from cryopreserved blastodermal cells via chimeric intermediates can be performed successfully.

Germline chimeric chickens can be made by injecting dispersed cells from Stage X blastoderms into recipient embryos at an equivalent stage of development. Colonization of the chimera by donor-derived cells is facilitated when the recipient embryo is compromised by exposure to irradiation prior to injection of the donor cells. Donor cells can be genetically manipulated by lipofection-mediated gene transfer before they are introduced into the recipient. The genetic modification is expressed in the ectoderm, mesoderm, and endoderm of the chimera after incubation for 96 h. Donor cells can also be cultured as dispersed cells in a monolayer or as whole-embryo explants for at least 48 h before transfer into recipients and retain the ability to enter both somatic and germline tissues in the resulting chimera. A strategy is proposed for the production of transgenic chickens using lipofection-mediated gene transfer to blastoderm cells isolated from Stage X embryos, which are subsequently injected into compromised recipients to yield a germline chimera.

Twins and triplets of chick embryos were produced by microinjection treatment of cell suspension into the blastoderm. One pair of the twin embryos had an ability to develop normally and survived up to Day 21 of incubation, but did not hatch. The other twins and triplets died within 7 days.

We injected chick syncytial strain of reticuloendotheliosis virus (CS-REV) and wild type and recombinant avian leukosis virus (ALV) near the blastoderm of unincubated fertilized embryos and CS-REV intra-abdominally at day of hatch, and we progeny tested the surviving ALV viremic males and REV viremic males and females for transmitted viral genetic material. A number of positive progeny were identified and their deoxyribonucleic acid (DNA) analyzed for restriction enzyme fragments that hybridized with viral genetic material. Most of the progeny had simple restriction enzyme patterns unlike the viremic parents or congenitally infected progeny. This is suggestive evidence that retroviral genetic information has been inserted into the germ line of chickens.

A study of the in vitro growth of embryonic structures from dispersed blastoderm cells is reported. The specific type of blastoderm cells with the capability of growing on the coverslip developed into the discoidal embryonic structures resembling those of avian species. The glass surface was apparently an initiator of differentiation into at least three types of cells specifically distributed in the blastodisc. Groups of structures formed were evaluated at 6, 12, 24 and 36 hours to study the developmental pattern in vitro and to estimate the number of cells per structure. Microscopic examination of the area pellucida revealed that all three basic germ layers were established after 24 hours of incubation in vitro. The 36 hour stage was represented by bulky growth of mesodermal-like cells and changes in hypoblast layer where some of the cells degenerated and some were transformed to mesenchymal spindle-like cells.

The configuration of blastoderm-heart cell aggregates after cell sorting depended largely on the proportion of each cell type initially present in the culture. In heterotypic aggregates, blastoderm cells were always found to partially or completely surround heart cells with both cell types retaining their characteristic morphology. Specialized junctions (e.g., desmosomes) developed only between cells of the same type. These observations suggest that blastoderm cells are unable to recognize and form stable contacts with heart cells.