Morphological Criteria for Staging Near‐Hatching Embryos of the Domesticated Mallard (Anas platyrhynchos) and Swan Goose (Anser cygnoides)

Mizia P.C., Rams-Pociecha I., Podmokła E., Piprek R.P.

The developing gonads constitute a valuable model for studying developmental mechanisms because the testes and ovaries, while originating from the same primordia, undergo two different patterns of development. So far, gonadal development among birds has been described in detail in chickens, but literature on the earliest stages of gonadogenesis is scarce. This study presents changes in the structure of the gonads in three species of breeding birds (chicken, duck, and pigeon), starting from the first signs of gonadal ridge formation, that is, the thickenings of the coelomic epithelium. It appears that both gonads show asymmetry from the very beginning of gonadal ridge formation in both genetic sexes. The left gonadal ridge is thicker than the right one, and it is invaded by a higher number of primordial germ cells. Undifferentiated gonads, both left and right, consist of the primitive cortex and the medulla. The primitive cortex develops from the thickened coelomic epithelium, while the primitive medulla - by the aggregation of mesenchymal cells. This study also describes the process of sex differentiation of the testes and ovaries, which is initiated at the same embryonic stage in all three studied species. The first sign of gonadal sex differentiation is the decrease in the number of cortical germ cells and a reduction in cortical thickness in the differentiating testes. This is followed by an increase in the number of germ cells in the medulla. The cortical asymmetry and difference in size between the left and right testes diminishes during later development. However, the differentiating left ovary shows an increase in the number of cortical germ cells and cortical thickness. No regression is seen in the right ovary, although its development is slower. The right ovarian cortex undergoes testis-specific reduction, while the medulla undergoes ovary-specific development. The process of gonadogenesis is similar in the three studied species, with only slight differences in gonadal structure.

Ran M., Ouyang Q., Li X., Hu S., Hu B., Hu J., Dong D., Li L., He H., Liu H., Wang J.

Abstract Background The development of asymmetric chick gonads involves separate developmental programs in the left and right gonads. In contrast to the left ovary developing into a fully functional reproductive organ, the right ovary undergoes gradual degeneration. However, the molecular mechanisms underlying the the degeneration of the right ovary remain incompletely understood. In the present study, we investigated the histomorphological and transcriptomic changes in the right ovary of ducks and geese during the the embryonic stage up to post-hatching day 1. Result Hematoxylin–eosin stainings revealed that the right ovary developed until embryonic day 20 in ducks (DE20) or embryonic day 22 in geese (GE22), after which it started to regress. Further RNA-seq analyses revealed that both the differentially expressed genes (DEGs) in ducks and geese right ovary developmental stage were significantly enriched in cell adhesion-related pathway (ECM-receptor interaction, Focal adhesion pathway) and Cellular senescence pathway. Then during the degeneration stage, the DEGs were primarily enriched in pathways associated with inflammation, including Herpes simplex virus 1 infection, Influenza A, and Toll-like receptor signaling pathway. Moreover, duck-specific DEGs showed enrichment in Steroid hormone biosynthesis, Base excision repair, and the Wnt signaling pathway, while geese-specifically DEGs were found to be enriched in apoptosis and inflammation-related pathways, such as Ferroptosis, Necroptosis, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway. These findings suggest that the degeneration process of the right ovary in ducks occurs at a slower pace compared to that in geese. Additionally, the observation of the left ovary of the geese varying degeneration rates in the right ovary after hatching indicated that the development of the left ovary may be influenced by the degeneration of the right ovary. Conclusion The data presented in this study provide valuable insights into the dynamic changes in histological structure and transcriptome during the degeneration of the right ovary in ducks and geese. In addition, through the analysis of shared characteristics in the degeneration process of the right ovary in both ducks and geese, we have uncovered the patterns of degradation and elucidated the molecular mechanisms involved in the regression of the right ovary in poultry. Furthermore, we have also made initial discoveries regarding the relationship between the degeneration of the right ovary and the development of the left ovary.

Bao Q., Wang L., Hu X., Yuan C., Zhang Y., Chang G., Chen G.

The liver is the main site of fat synthesis and plays an important role in the study of fat deposition in poultry. In this study, we investigated the developmental changes of duckling livers and isolated primary duck hepatocytes. Firstly, we observed morphological changes in duckling livers from the embryonic period to the first week after hatching. Liver weight increased with age. Hematoxylin-eosin and Oil Red O staining analyses showed that hepatic lipids increased gradually during the embryonic period and declined post-hatching. Liver samples were collected from 21-day-old duck embryos for hepatocyte isolation. The hepatocytes showed limited self-renewal and proliferative ability and were maintained in culture for up to 7 days. Typical parenchymal morphology, with a characteristic polygonal shape, appeared after two days of culture. Periodic acid-Schiff (PAS) staining analysis confirmed the characteristics of duck embryo hepatocytes. PCR analysis showed that these cells from duck embryos expressed the liver cell markers ALB and CD36. Immunohistochemical staining and immunofluorescence analysis also confirmed ALB and CK18 expression. Our findings provide a novel insight regarding in vitro cell culture and the characteristics of hepatocytes from avian species, which could enable further studies concerning specific research on duck lipid metabolism.

Guo B.B., Dai Z.C., Ren Y.H., Zhu H.X., Shao X.B., Sun A.D., Shi Z.D.

Egg turning during incubation plays important roles in achieving high hatching performance and gosling quality. The objective of this study was to improve embryonic and muscular developments so to achieve better gosling quality by wider egg turning angles during incubation, and to unravel the associated regulatory molecular mechanisms. In each of three consecutive incubations, 1,728 goose eggs were divided into 3 groups that were set in the same type of commercial incubators with turning angles adjusted differently to 50°, 60°, and 70°, respectively. On average of the 3 tests, incubation with wider 70° turning angle reduced the post-18-day embryo mortality, promoted embryonic growth and development, improved the hatchability and gosling quality. On embryonic day of 29, gene mRNA expression levels of the hypothalamic growth hormone-releasing hormone (GHRH), pituitary growth hormone (GH), and liver insulin-like growth factor 1 (IGF-1) were higher in the 70° turning group than in the 50° or 60° groups. Wider angle turning also increased mRNA expression levels of the muscle development regulatory genes such as MYF5, MyoD, Myogenin (MyoG), and MRF4. Changes in expression of the above genes, together with the upregulation of the Pax3 and Pax7 genes in leg muscles, well explained the enhancement of the muscular growth and development when eggs were incubated by wider turning angles. These results also extended our understanding of the impacts and mechanisms of egg turning during incubation on hatching performance and gosling quality.

Ducatez S., Field D.J.

The altricial-precocial spectrum describes patterns of variation in avian developmental mode that greatly influence avian life histories. Appraising a given species’ position on this spectrum is therefore fundamental to understanding patterns of avian life history evolution. However, evaluating avian developmental mode remains a relatively subjective task reliant on untested assumptions, including the notion that developmental strategies are distributed along a single dimension of statistical variation. Here, we present a quantitative multivariate framework that objectively discriminates among meaningfully different modes of avian development. We gathered information on seven hatchling and post-hatching traits for up to 4000 extant bird species, and find that most traits related to developmental mode show high phylogenetic signal and little intraclade variation, allowing unknown values to be reliably interpolated. Principal component analyses (PCAs) of these traits illustrate that most variation in hatchling state can be quantified along one dimension of trait space. However, our PCAs also reveal an important second dimension explaining variation in post-hatching behavior, enabling factors related to hatchling state and post-hatching behavior to be disentangled. In order to facilitate future macroevolutionary studies of variation in avian developmental strategies, as well as explorations of covariation between developmental mode and other aspects of avian biology, we present PC scores for 9993 extant avian species.

Cooney C.R., Sheard C., Clark A.D., Healy S.D., Liker A., Street S.E., Troisi C.A., Thomas G.H., Székely T., Hemmings N., Wright A.E.

The duration of the developmental period represents a fundamental axis of life-history variation, yet broad insights regarding the drivers of this diversity are currently lacking. Here, we test mechanistic and ecological explanations for the evolution of developmental duration using embryological data and information on incubation and fledging for 3096 avian species. Developmental phases associated primarily with growth are the longest and most variable, consistent with a role for allometric constraint in determining the duration of development. In addition, developmental durations retain a strong imprint of deep evolutionary history and body size differences among species explain less variation than previously thought. Finally, we reveal ecological correlates of developmental durations, including variables associated with the relative safety of the developmental environment and pressures of breeding phenology. Overall, our results provide broad-scale insight into the relative importance of mechanistic, ecological and evolutionary constraints in shaping the diversification of this key life-history trait. Developmental duration is a key life-history trait. Cooney et al. compile data on 3096 bird species to quantify the degree to which phylogenetic history, body size and ecological variables like predation risk or breeding phenology influence variation in developmental duration.

Li S., Bai S., Qin X., Zhang J., Irwin D.M., Zhang S., Wang Z.

Embryos from aquatic birds are the primary models for the study of flipper development. While some staging of early embryogenesis in duck have been studied, characterization of the stages of the entire embryonic development period in water birds has not been described. This study aimed to establish a comparison of complete morphological development staging for ducks (Anas platyrhynchos) and geese (Anser cygnoides) with the embryonic staging system by Hamburger and Hamilton (HH) for the chicken (Gallus gallus). Our results show that morphological development in the chicken, duck, and goose are similar in the early stages. The major differences occurred after stage 27 of embryonic development, where the beak shape in ducks and geese was wider and longer than in chickens. In addition, the second and third interdigital webs of the hind limb of the chicken were found to be degraded from stage 31, and eventually vanished at stage 35; however, they were retained in ducks and geese. Rapid physical development occurred in the mid-to-late stages in ducks and geese. To our best knowledge, this is the first description of complete embryonic development for the duck and goose. Establishment of an embryonic staging system for duck and goose provides new models for the study of waterfowl development.

Stern C.D.

Łukaszewicz E., Lasoń M., Rosenberger J., Kowalczyk A., Bakst M.

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.

Fu Y., Chen Z., Li C., Liu G.

Until recently, there was no cell line that could produce continuously high-titer duck hepatitis virus type 1 (DHV-1). In this study, a duck embryo fibroblast (DEF) cell line was established, and the susceptibility of this cell line to DHV-1 was determined. The primary culture of DEF cells was from a duck embryo that was partially digested with trypsin. Digested tissue pieces were cultured at 37°C in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. The cultured DEF cells, which had the morphology of fibroblast, proliferated to 100% confluence four days later. An immortalized DEF cell line, named DEF-TA, was established and subcultured to passage 33, and the susceptibility of that cell line to DHV-1 was determined. In the DHV-1 susceptibility tests, cytopathic effects and the propagation of virus were observed in DEF-TA cells after DHV-1 infection. This continuous DHV-1-susceptible DEF cell line may serve as a valuable cell line for studies of cell-virus interactions and the pathogenesis of DHV-1 and may be useful for the development of an inactivated vaccine.

Ainsworth S.J., Stanley R.L., Evans D.J.

Developmental biology research has used various avian species as model organisms for studying morphogenesis, with the chick embryo being used by the majority of groups. The focus on the chick embryo led Hamburger and Hamilton to develop their definitive staging series nearly 60 years ago and this series is still the mainstay of all laboratories working with avian embryos. The focus on the chick embryo has somewhat overshadowed the importance of another avian embryo that has proved to be equally powerful, the Japanese quail. Since the late 1960s, chimeras have been produced using chick and quail embryos and this technique has revolutionized the approach taken to the investigation of the cellular and molecular interactions that occur during development. Reviews of the literature demonstrate that many research groups are using the quail embryo in a number of established and new ways, and this species has become a primary animal model in developmental biology. Some staging of quail has been performed but this has been incomplete and variations in descriptions, stages and incubation timings mean that comparisons with the chick are not always easily made. There appears to be general agreement that, at the early stages of embryogenesis, there is little developmental difference between chick and quail embryos, although the basis for this has not been established experimentally. The accelerated ontogeny of quail embryos at mid to late stages of development means that registration with the chick is lost. We have therefore developed a definitive developmental stage series for Japanese quail so that differences are fully characterized, misconceptions or assumptions are avoided, and the results of comparative studies are not distorted.

Brugmann S.A., Powder K.E., Young N.M., Goodnough L.H., Hahn S.M., James A.W., Helms J.A., Lovett M.

Maxwell E.E.

Ossification sequences are poorly known for most amniotes, and yet they represent an important source of morphogenetic, phylogenetic, and life history information. Here, the author describes the ossification sequences of three ducks, the Common Eider Somateria mollissima dresseri, the Pekin Duck Anas platyrhynchos, and the Muscovy Duck Cairina moschata. Sequence differences exist both within and among these species, but are generally minor. The Common Eider has the most ossified skeleton prior to hatching, contrary to what is expected in a subarctic migrant species. This may be attributed to a tradeoff between growth rate and locomotory performance. Growth rate is higher in hatchlings with more cartilaginous skeletons, but this may compromise locomotion. No major ossification sequence differences were observed in the craniofacial skeleton when compared with Galliformes, which suggests that the influence of adult morphology on ossification sequence might be relatively minor in many taxa. Galliformes and Anseriformes, while both highly ossified at hatching, differ in the location of their late-stage ossification centers. In Anseriformes, these are most often located in the appendicular skeleton, whereas in Galliformes they are in the thoracic region and form the ventilatory apparatus.

Xu R.F., Wu W., Xu H.

In the present study, the process of feather follicle formation in the Zi goose, a Chinese indigenous breed, was investigated during various stages of embryonic development by using a modified histological processing method. The results showed that the feather placodes evolved initially at embryonic day (E) 12 on the spinal feather tract, emerging as symmetrical structures. Sequentially, the buds elongated from E14 to E16 with anterior-posterior and proximal-distal asymmetries, and invaginated to form the primary feather follicles, which were identified to develop the contour feathers or remiges. The remarkable observation at this stage was the formation of the feather follicle wall, which was understood to be the result of the epidermis surrounding the base and further invaginating into the dermis. With the differentiation of the barbule plates, the various types of feathers were determined. We proved that the secondary feather follicles simply had radially symmetrical barb ridges, with much smaller diameters than the primary follicles, and that they developed only downy feathers. The primary and secondary follicles evolved independently of each other and formed ranks in a linear fashion. Moreover, quantitative measurements of the densities of both follicles confirmed that the density of the primary follicles sharply reached the maximum at E18, and then decreased gradually. Coincidentally, the secondary follicles started to increase from the age of E18, and up to E26 the density of the secondary follicles exceeded that of the primary follicles. Each of the primary feather follicles was richly encircled with muscles, which pointed to a quadrangularly arranged network in the dermis. The present work lays the foundation for further study of the cellular and molecular mechanisms of feather follicle morphogenesis in geese.

Bernáth S., Farsang A., Kovács A., Nagy E., Dobos-Kovács M.

Goose embryos were infected with goose haemorrhagic polyomavirus (GHPV) onto the chorioallantoic membrane (CAM) in order to examine the effect of GHPV on the embryos and to obtain data on whether embryos could develop into infected, virus-shedding goslings, as well as to present an accurate biological method for virus titration. The reported method of infection could offer a possibility to express the virus titre as the median embryo infective dose (EID(50)). As a special pathological feature of the disease, extensive cerebral haemorrhages were observed, which protruded the skullcap in many cases. Some embryos infected with 10(1.25) or 10(0.25) EID(50)/0.2 ml were able to hatch; however, they were in poor physical condition and died by post-hatching day 4 showing haemorrhagic nephritis and enteritis of geese. Virus shedding was revealed by polymerase chain reaction. The ability of some of the infected goose embryos to hatch may indicate the potency of GHPV to spread vertically, although this needs further study for confirmation.