Germinal Discs Research Articles

Dietary yolk supplements and rate of yolk deposition do not influence sex ratios in Japanese Quail.

Birds appear to have the ability to bias offspring sex ratios in response to environmental and social conditions. The mechanisms responsible remain unknown, however one previous study indicated a link between rates of ovarian follicle growth and the sexes of resulting eggs. This could indicate that either follicles destined to retain male or female follicles grow at different rates, or the rate of ovarian follicle growth determines the sex chromosome that will be retained to influence the sex of the resulting offspring. We tested for evidence of both possibilities by staining yolk rings that are indicative of daily growth. First, we tested for a correlation between the number of yolk rings and the sexes of the resulting germinal discs collected from each egg, and second, we tested whether experimentally decreasing follicle growth rates with a dietary yolk supplement impacts the sexes of the resulting germinal discs. There was no significant correlation between the number of yolk rings and the sexes of the resulting embryos, and decreasing follicle growth rates did not impact sexes of resulting germinal discs. These results indicate that offspring sex is not related to the rate of ovarian follicle growth in quail.

Lab partners: oocytes, embryos and company. A personal view on aspects of oocyte maturation and the development of monozygotic twins.

The present review addresses the oocyte and the preimplantation embryo, and is intended to highlight the underlying principle of the "nature versus/and nurture" question. Given the diversity in mammalian oocyte maturation, this review will not be comprehensive but instead will focus on the porcine oocyte. Historically, oogenesis was seen as the development of a passive cell nursed and determined by its somatic compartment. Currently, the advanced analysis of the cross-talk between the maternal environment and the oocyte shows a more balanced relationship: Granulosa cells nurse the oocyte, whereas the latter secretes diffusible factors that regulate proliferation and differentiation of the granulosa cells. Signal molecules of the granulosa cells either prevent the precocious initiation of meiotic maturation or enable oocyte maturation following hormonal stimulation. A similar question emerges in research on monozygotic twins or multiples: In Greek and medieval times, twins were not seen as the result of the common course of nature but were classified as faults. This seems still valid today for the rare and until now mainly unknown genesis of facultative monozygotic twins in mammals. Monozygotic twins are unique subjects for studies of the conceptus-maternal dialogue, the intra-pair similarity and dissimilarity, and the elucidation of the interplay between nature and nurture. In the course of in vivo collections of preimplantation sheep embryos and experiments on embryo splitting and other microsurgical interventions we recorded observations on double blastocysts within a single zona pellucida, double inner cell masses in zona-enclosed blastocysts and double germinal discs in elongating embryos. On the basis of these observations we add some pieces to the puzzle of the post-zygotic genesis of monozygotic twins and on maternal influences on the developing conceptus.

Corticosterone and testosterone treatment influence expression of gene pathways linked to meiotic segregation in preovulatory follicles of the domestic hen.

Decades of work indicate that female birds can control their offspring sex ratios in response to environmental and social cues. In laying hens, hormones administered immediately prior to sex chromosome segregation can exert sex ratio skews, indicating that these hormones may act directly on the germinal disc to influence which sex chromosome is retained in the oocyte and which is discarded into an unfertilizable polar body. We aimed to uncover the gene pathways involved in this process by testing whether treatments with testosterone or corticosterone that were previously shown to influence sex ratios elicit changes in the expression of genes and/or gene pathways involved in the process of meiotic segregation. We injected laying hens with testosterone, corticosterone, or control oil 5h prior to ovulation and collected germinal discs from the F1 preovulatory follicle in each hen 1.5h after injection. We used RNA-sequencing (RNA-seq) followed by DESeq2 and gene set enrichment analyses to identify genes and gene pathways that were differentially expressed between germinal discs of control and hormone-treated hens. Corticosterone treatment triggered downregulation of 13 individual genes, as well as enrichment of gene sets related to meiotic spindle organization and chromosome segregation, and additional gene sets that function in ion transport. Testosterone treatment triggered upregulation of one gene, and enrichment of one gene set that functions in nuclear chromosome segregation. This work indicates that corticosterone can be a potent regulator of meiotic processes and provides potential gene targets on which corticosterone and/or testosterone may act to influence offspring sex ratios in birds.

Observations of turkey eggs stored up to 27 days and incubated for 8 days: embryo developmental stage and weight differences and the differentiation of fertilized from unfertilized germinal discs

For logistical reasons, egg storage prior to incubation is a growing practice in the commercial turkey industry. Yet the consequence of increasing egg storage over 7 d is a progressive increase in embryo mortality. The objective of this study was to provide the information necessary to differentiate an early dead embryo from an unfertilized egg after 8 days of incubation (DOI). Five groups of eggs each from inseminated and virgin hens were stored for progressively increasing periods of time (5-d or less, 6 to 10 d, 11 to15 d, 16 to 20 d, and 21 to 27 d) and incubated. At 8 DOI, eggs were examined and the stage of development (Hamburger and Hamilton, 1951) and embryo weights in normally developed eggs were determined. There was a significant negative correlation between the stage of development and embryo weight with increasing storage periods. All remaining eggs from the inseminated and virgin hens were broken-out and the appearance of the yolk and the fertilized and unfertilized germinal discs examined. The yolks of both hen groups with unfertilized ova maintained a homogeneous uniform yellow-orange color. In contrast, yolks of ova that had been fertilized, with or without early-dead embryos, and yolks from virgin hens that showed evidence of parthenogenetic development (3%) had a heterogeneous appearance. Using fluorescence microscopy, the heterogeneous appearance was due to sheets of aberrant cells and less frequently dispersed cells and folds of the perivitelline layer. It was concluded that clear egg breakouts need to be performed to more accurately assess the impact of egg storage on embryonic mortality. Furthermore, such breakouts should be performed with a high intensity light directed across the surface of the germinal disc to clearly differentiate the subtle differences between an early-dead embryo and an unfertilized germinal disc.

Health status and potential uptake of transgenic DNA by Japanese quail fed diets containing genetically modified plant ingredients over 10 generations

The hypothesis assumes that feed containing GMOs affects animal health and results in the transgene product accumulating in the body. Therefore, the objective of the study was to evaluate the impact of genetically modified (GM) ingredients used in poultry diets on aspects of bird health status and accumulation of transgenic DNA in eggs, breast muscle and internal organs.A total of 10 generations of Japanese quail were fed three types of diets: group A – containing GM soya (Roundup Ready) and non-GM maize, group B – containing GM maize (MON810) and non-GM soya, and group C – containing non-GM soya and maize.Bird performance traits were monitored throughout the trial. In 17-week-old animals of each generation, health examination took place on birds from each group including post-mortem necropsy and histological organ evaluation. For the purpose of transgenic DNA detection, samples of selected important tissues were taken. A molecular screening method of PCR amplification was used.The analysis of the sectional examination of birds used in the current experiment did not indicate the existence of the pathological changes caused by pathogens, nutritional factors or of environmental nature. The histopathological changes occurred in all three dietary groups and there were no statistically significant differences between the groups.There was no transgene amplification – neither CaMV35S promoter sequence nor nos terminator sequence, in the samples derived from breast muscle, selected tissues and germinal discs (eggs).According to the obtained results, it was concluded that there was no negative effect of the use of GM soya or maize with regard to bird health status or to the presence of transgenic DNA in the final consumable product.

Influence of storage on the position of the germinal disc in the fertilized unincubated chicken egg

The breeding of male layer chickens is currently considered to be highly uneconomical. In Germany alone, 40 to 50 million newly hatched male chickens were killed annually immediately after hatching. Therefore, it is necessary to find a method for sexing chickens early in the embryonic development, preferably before incubation. The genotypic sex of an egg can be determined using information found in the germinal disc, so knowledge of the exact position of the germinal disc is essential for further sexing, or for other actions such as the in ovo injection of agents. Previous studies have shown that the germinal disc is located somewhere on top of the yolk. However, no studies have yet been performed that investigate the influence of time spent in horizontal storage on the position of the germinal disc. Magnetic resonance imaging was chosen to determine this influence on the position of the germinal disc. It was found that eggs placed horizontally for long periods of time before scanning had significant changes in the positions of their germinal discs compared with those of eggs scanned minutes after positioning. The position of the germinal disc in eggs, minutes after horizontal positioning, deviated 14.7 ± 0.6 mm from the maximum vertical plane of the egg (zero position) in the z-direction; eggs scanned after 96 h of horizontal positioning showed a deviation of only 4.9 ± 1.6 mm. The x-axis also exhibited changes in the position of the germinal disc over time. Immediately after horizontal positioning, the eggs showed a deviation of 0.4 ± 0.4 mm in the x-direction, whereas the deviation after 96 h was 2.9 ± 0.5 mm. These results show that horizontal positioning of the egg hours before the measurement is necessary.

Molecular aspects of avian oogenesis and fertilisation

In this paper, we summarise studies which have been carried out on the metabolism of nucleic acids (maternal RNA, DNA, nucleolytic enzymes) in avian oocytes and embryos (Japanese quail, Coturnix coturnix japonica ) within the last 10 years in the Institute of Genetics and Animal Breeding of the Polish Academy of Sciences. The accumulation of maternal RNA in the quail oocyte during oogenesis is shown and discussed. Several individual transcripts were identified in RNA from the germinal disc and some also in extraembryonic RNA under the perivitelline membrane. The presence of the transcript encoding chick zona pellucida C protein (chZPC) points to the possibility of ZPC synthesis by the oocyte itself. The transcript encoding AA-NAT (arylalkylamine N-acetyltransferase, the penultimate enzyme in melatonin synthesis) was present in 2 forms (with and without an intron) and the ratio of the two forms changes during oogenesis. Melatonin and the two enzymes engaged in its synthesis (AA-NAT and HIOMT) have been found in the egg yolk; their transcripts and the transcripts of the melatonin receptors mel-1a,b and c are present in RNA from the germinal discs. This suggests a possible role for melatonin in early avian development. DNases I and II activity has been detected in the germinal disc and the cytoplasmic layer under the perivitelline membrane. We propose that they participate in degradation of supernumerary sperm entering avian oocytes during polyspermic fertilisation. A hypothesis to explain the selection of a single sperm participating in the formation of the zygotic nucleus is discussed; we propose that sperm entry into the centre of the germinal disc is the essential event underlying the selection mechanism.

Cell number and sex ratio in unfertilized chicken eggs (Gallus gallus domesticus)

In chickens and other birds, females have two different sex chromosomes (ZW), whereas males carry two homologous sex chromosomes (ZZ). The primary sex ratio can thus be determined by genetic analysis of the sex chromosome of the ovum before fertilization. Sex diagnosis is more reliable when there are more cells, i.e. sufficient DNA, for the analysis. In this study, eggs from virgin hens were incubated for 3 days and the number of cells in the germinal discs was counted. A median of 2.5 cells was counted with a range of two to 20 cells. We also counted cells in the germinal discs of unfertilized eggs of inseminated hens and recorded a median of three cells and a range of two to 40 cells. Sex diagnosis based on polymerase chain reaction (PCR) amplification of Z and W chromosomes specific fragments from the CHD1 gene in 30 incubated eggs obtained from 35-week-old virgin hens gave a ratio of 13 Z to 15 W chromosomes with two samples undetermined. The unfertilized eggs of three groups of chickens were subjected to sex diagnosis to supplement the sex ratio data of an incubation experiment (see companion paper). The high proportion of Z chromosomes diagnosed in all three groups by two independent gene products suggests a sex difference on developmental potential and/or a sex chromosome segregation biased toward males in unfertilized eggs especially at the beginning of reproduction.

DNase I and II present in avian oocytes: a possible involvement in sperm degradation at polyspermic fertilisation

During polyspermic fertilisation in birds numerous spermatozoa enter the eggs, in contrast to the situation in mammals where fertilisation is monospermic. However, in birds only one of the spermatozoa which have entered an egg participates in zygote nucleus formation, while the supernumerary spermatozoa degenerate at early embryogenesis. Our previous work has demonstrated the presence in preovulatory quail oocytes of DNase I and II activities able to digest naked lambdaDNA/HindIII substrate in vitro. In the present studies, the activities of both DNases in quail oocytes at different stages of oogenesis and in ovulated mouse oocytes were assayed in vitro using the same substrate. Degradation of quail spermatozoa by quail oocyte extracts was also checked. Digestion of the DNA substrate was evaluated by electrophoresis on agarose gels. The activities of DNase I and II in quail oocytes increased during oogenesis and were the highest in mature oocytes. The activities were present not only in germinal discs but also in a thin layer of cytoplasm adhering to the perivitelline layer surrounding the yolk. At all stages of oogenesis the activity of DNase II was much higher than that of DNase I. DNA contained in spermatozoa was also degraded by the quail oocyte extracts under conditions optimal for both DNases. In contrast to what is observed in quail oocytes, no DNase activities were detected in ovulated mouse eggs; this is logical as they would be useless or even harmful in monospermic fertilisation. The possible role of DNase activities in avian oocytes, in degradation of accessory spermatozoa during polyspermic fertilisation, is discussed.

Use of Nonsettable Eggs to Evaluate Turkey Hen Fertility

The use of nonsettable eggs (checked, cracked) to perform fresh egg breakouts to estimate true fertility and to assess the rate of sperm penetration of the perivitelline layer overlying the germinal disc was evaluated. Germinal discs and the perivitelline layer overlying the germinal disc were accessible for assessments. The stage of blastodermal development positively correlated (r = 0.65, P < 0.0001) with eggshell thickness. It was also determined that the perivitelline layer of nonsettable eggs could be isolated and stained to determine the presence or absence of sperm holes. True fertility of nonsettable eggs (checked and cracked only) and settable eggs was 90 and 95%, respectively. It was concluded that checked and cracked eggs could be used to estimate the true fertility of a flock.

Detection of deoxyribonuclease I and II activities in Japanese quail oocytes

Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the germinal disc of an oocyte and form pronuclei during fertilisation. However, only one of them unites with the female pronucleus to form a zygote nucleus; the supernumerary spermatozoal nuclei degenerate at the early cleavage stages. To establish a factor responsible for spermatozoal degeneration, the presence of DNase activity was studied in vitro in extracts of Japanese quail oocytes using lambda DNA/HindIII as a substrate. The experimental conditions were designed to reveal the presence of either DNase I or DNase II activities, separately. Degradation of the substrate DNA was evaluated by electrophoresis on agarose gels stained with ethidium bromide. High activities of DNase I and DNase II were found in the germinal discs of the largest vitellogenic oocytes. DNase I activity was estimated to be about 3 x 10(-3) Kunitz units and DNase II about 4 x 10(-2) Kunitz units per germinal disc. DNase I activity in an oocyte seems to increase during oogenesis since DNA degradation by the extracts from the germinal discs of the largest vitellogenic oocytes was much higher than by those from previtellogenic and small vitellogenic oocytes. The presence of high DNase I and II activities in the largest vitellogenic oocytes would point to their role in degradation of DNA from supernumerary spermatozoa entering the ovum during polyspermic fertilisation in birds. The enzymes could be a factor, or one of the factors, in the late block to polyspermy in the cytoplasm of avian eggs. It is suggested here that the DNase activities might also be responsible for poor efficiency in obtaining transgenic birds by microinjection of exogenous DNA into the fertilised chick ovum.

Immunization of chickens with quail and turkey perivitelline membrane proteins: production of antibodies and their effects on fertility

The cross-reactivity of antibodies developed against zona pellucida proteins and their subsequent deleterious effect on fertility in heterologous species is well documented. However, similar investigations have not been undertaken in avian species. In Experiment 1, White Leghorn hens were immunized with intact germinal discs (GD) of quail and chickens. Chicken GD proteins did not elicit an immune response in chicken hens, whereas quail GD proteins were highly immunogenic. Anti-quail GD antibodies did not bind with chicken inner perivitelline membrane (IPM) proteins as determined by immunoblot analysis. To examine the fertility of immunized hens, artificial insemination was performed at weekly intervals for 4 wk following the booster immunization. No significant differences were detected in fertility or hatchability of immunized hens when compared with unimmunized control hens. In Experiment 2, White Leghorn hens were immunized with intact turkey GD, solubilized turkey perivitelline membrane (PM) modified with dinitrophenol (DNP), and solubilized chicken IPM modified with DNP. High titers of antibodies were detected against the turkey GD and the DNP-modified turkey PM proteins. A weak immune response was observed in hens immunized with modified chicken IPM proteins. The fertility and hatchability of eggs laid by immunized hens, however, were not significantly different from those of unimmunized hens. Antibodies from immunized hens were further analyzed using an in vitro assay that assesses sperm penetration of intact IPM. Sperm penetration of intact IPM was inhibited to the same extent when IPM was preincubated with preimmune as well as anti-PM immunoglobulins. Collectively, these results suggested that the antibodies developed in these hens did not cross-react with the chicken IPM proteins involved in sperm-egg interaction and thus did not influence the fertility.

Gross appearance of the turkey blastoderm at oviposition

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.

Incorporation of uridine and leucine by the germinal discs of quail eggs kept cool or warmed periodically during storage

Nucleic acid and protein biosyntheses are characteristic of the fertilised germinal disc and continue during storage at 20 degrees C though at a reduced rate. There is no comparable activity in unfertilised eggs. 2. Warming the fertilised eggs for 30 min/d during storage increased the rate of incorporation of uridine and leucine into proteins and nucleic acids by a factor of three.

Comparative Weights and Body Lengths of Parthenogenetic and Normal Turkey Embryos

INTRODUCTIONDEVELOPMENT of the germinal discs of newly laid unfertilized turkey eggs is characterized by a pronounced time lag in onset of development. The developing blastoderm of a newly laid fertilized turkey egg starts to develop soon after eggs are placed in the incubator and can be detected before the candler by 18 to 24 hours of incubation (Olsen and Marsden, 1954a). In contrast, some of the developing blastoderms of unfertilized turkey eggs first become visible at 48–72 hours, while others may require as much as 144 hours of incubation before they can be detected by candling (Olsen and Marsden 1954b; Olsen, 1965a). This present investigation was conducted to establish comparative growth curves for parthenogenetic and normal embryos.MATERIAL AND METHODSParthenogenetic embryos employed developed in unfertilized eggs of young Beltsville Small White (BSW) and heterozygous bronze (HB) turkey hens. The HB hens were essentially Beltsville Small Whites having…