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The avian embryo
... Chicken fetal liver was initially considered a non-haematopoietic organ [9], however, it appears to exhibit intra-and/or extra-vascular haematopoiesis [11,12,3]. Thus, correlations between chicken fetal liver and other hematopoietic structures and understanding of the hematopoietic process in more detail require further investigation. An integrated morphological study is important to better understand the temporal and spatial distribution of hematopoietic sites during avian development. ...
... Chicken fetal liver is not considered a relevant haematopoietic organ, as is fetal liver in mammals [3,11,12,13,14]; however, intra-or extra-vascular haematopoiesis occurs in this organ [3,11,12,15]. Haff [12] considered two important moments in haematopoiesis during liver development in chickens. ...
... Chicken fetal liver is not considered a relevant haematopoietic organ, as is fetal liver in mammals [3,11,12,13,14]; however, intra-or extra-vascular haematopoiesis occurs in this organ [3,11,12,15]. Haff [12] considered two important moments in haematopoiesis during liver development in chickens. The first corresponds to erythropoiesis within the capillaries between E7 and E9. ...
Haematopoiesis is the process of proliferation, self-renewal and differentiation of hematopoietic stem cells (HSC), which results in the formation of blood cells in both the embryonic and adult stages. The purpose of this paper is to evaluate the degree of dissemination of progenitor hematopoietic cell populations in the liver of Broiler chickens during the hatching period. Research has begun on a breeding farm of S.C. Avicola Crăieşti-Mureş, with the collecting and preparation of the biological material from 21-day embryo (n=10) and one day broiler chickens (n=10). Sample processing and primary preparation was completed in the Pathological Anatomy and Physiology Laboratories of Veterinary Medicine Faculty, Cluj-Napoca. Thus, liver and bone marrow samples were collected, adapting the avian necropsy technique to the small size and fragility of the investigated biological material. From the samples, cytological direct imprints were made on slides, which were then stained (Dia Quick Panoptic method) and microscopically (x100) examined. Haematopoiesis in birds has some notable differences from that of mammals. These are due to the presence of nucleated erythrocytes, heterophilic neutrophils, and cell type nuclear platelets. Unlike mammals, hepatic haematopoiesis in birds is predominantly performed on the granulocytic line and less on the erythrocytic one. Accumulation of progenitor granulocyte cells in chicken embryo liver can result from their migration towards the bone marrow where they meet appropriate conditions for granulopoiesis.
... In chicks, the foregut differentiates into several organs from the pharynx to the stomach, the midgut into the small intestine, and the hindgut into the large intestine, cloaca, and common gut-urogenital opening. The tailgut is known as the portion of the hindgut beyond the cloacal membranes [18], [34], [48], [46]. The cloacal membrane is derived from ectoderm and becomes perforated later in embryonic development to become the anus [3] (Fig. 1d). ...
... After disconnection from the cloaca, degeneration of the tailgut progresses from the most proximal end, leaving the cloaca with a foramen. This will be closed before the end of the fourth day of incubation [3], [33], [48], [53]. ...
... Spatially, the tailgut is typically found dorsomedial to the tailbud, the pluripotent mesenchymal cell mass that is the main player in secondary neurulation [48]. However, as the developmental morphology of both structures is complex, the relative spatial relationship according to embryonic stages cannot be described simply. ...
The tailgut is an ephemeral structure located in the caudal end of the gut during the development of the GI tract. It has been noted that incomplete degeneration of the tailgut is relevant to the pathogenesis of tailgut cysts and caudal agenesis. In this thesis, to elucidate the morphology and degeneration of the tailgut, the period of degeneration is examined. The TUNEL assay demonstrated apoptosis in the tailgut, supported by basal lamina degradation, and the putative mechanisms of apoptosis induction are suggested. The results of this study may help to elucidate the normal development of the human tailgut and the pathogenesis of tailgut cysts and caudal agenesis.
... Eggs of oviparous amniotes are characterized by an eggshell, plus a relatively large yolk (as compared to typical amphibians), and a series of fetal (extraembryonic) membranes. Descriptions are widely available in textbooks (e.g., Barresi & Gilbert, 2019;Raven & Johnson, 2003) and reviews (Romanoff, 1960;Romanoff & Romanoff, 1949). The shell protects against mechanical shock as well as dehydration. ...
... During oviparous development, the yolk material becomes overgrown and surrounded by a cellular yolk sac that processes it for embryonic use (Blackburn, 2020;Blackburn et al., 2020;Freeman & Vince, 1974;Starck, 1998Starck, , 2020Stewart, 2020;Stewart & Thompson, 2017). Another fetal membrane, the chorioallantois, provides for gas exchange across the eggshell (Birchard & Reiber, 1995;Romanoff, 1960;Starck, 1998). It also supplies calcium derived from the eggshell to the developing embryo, in birds as well as reptiles (M. ...
... J. Packard, 1994;Stewart, 2020;Stewart & Ecay, 2010). The amnion suspends the embryo in a pool of amniotic fluid that sequesters embryonic wastes and is thought to prevent adhesions (Mossman, 1987;Romanoff, 1960). The amnion is often said to protect against dehydration and mechanical shock (Balinsky, 1975;Miller & Harley, 2016). ...
We review morphological features of the amniote egg and embryos in a comparative phylogenetic framework, including all major clades of extant vertebrates. We discuss 40 characters that are relevant for an analysis of the evolutionary history of the vertebrate egg. Special attention is given to the morphology of the cellular yolk sac, the eggshell and extraembryonic membranes. Many features that are typically assigned to amniotes, such as a large yolk sac, delayed egg deposition and terrestrial reproduction have evolved independently and convergently in numerous clades of vertebrates. We use phylogenetic character mapping and ancestral character state reconstruction as tools to recognize sequence, order and patterns of morphological evolution and deduce a hypothesis of the evolutionary history of the amniote egg. Besides amnion and chorioallantois, amniotes ancestrally possess copulatory organs (secondarily reduced in most birds), internal fertilization, and delayed deposition of eggs that contain an embryo in the primitive streak or early somite stage. Except for the amnion, chorioallantois, and amniote type of eggshell, these features evolved convergently in almost all major clades of aquatic vertebrates possibly in response to selective factors such as egg predation, hostile environmental conditions for egg development, or to adjust hatching of young to favorable season. A functionally important feature of the amnion membrane is its myogenic contractility that moves the (early) embryo and prevents adhering of the growing embryo to extraembryonic materials. This function of the amnion membrane and the liquid filled amnion cavity may have evolved under the requirements of delayed deposition of eggs that contain developing embryos. The chorioallantois is a temporary embryonic exchange organ that supports embryonic development. A possible evolutionary scenario is that the amniote egg presents an exaptation that paved the evolutionary pathway for reproduction on land. As shown by numerous examples from anamniotes, reproduction on land has occurred multiple times among vertebrates – the amniote egg presenting one “solution” that enabled the conquest of land for reproduction. This article is protected by copyright. All rights reserved.
... Eggs of oviparous amniotes are characterized by an eggshell, plus a relatively large yolk (as compared to typical amphibians), and a series of fetal (extraembryonic) membranes. Descriptions are widely available in textbooks (e.g., Barresi & Gilbert, 2019;Raven & Johnson, 2003) and reviews (Romanoff, 1960;Romanoff & Romanoff, 1949). The shell protects against mechanical shock as well as dehydration. ...
... During oviparous development, the yolk material becomes overgrown and surrounded by a cellular yolk sac that processes it for embryonic use (Blackburn, 2020;Blackburn et al., 2020;Freeman & Vince, 1974;Starck, 1998Starck, , 2020Stewart, 2020;Stewart & Thompson, 2017). Another fetal membrane, the chorioallantois, provides for gas exchange across the eggshell (Birchard & Reiber, 1995;Romanoff, 1960;Starck, 1998). It also supplies calcium derived from the eggshell to the developing embryo, in birds as well as reptiles (M. ...
... J. Packard, 1994;Stewart, 2020;Stewart & Ecay, 2010). The amnion suspends the embryo in a pool of amniotic fluid that sequesters embryonic wastes and is thought to prevent adhesions (Mossman, 1987;Romanoff, 1960). The amnion is often said to protect against dehydration and mechanical shock (Balinsky, 1975;Miller & Harley, 2016). ...
Evolution of the terrestrial egg of amniotes (reptiles, birds, and mammals) is often considered to be one of the most significant events in vertebrate history. Presence of an eggshell, fetal membranes, and a sizeable yolk allowed this egg to develop on land and hatch out well‐developed, terrestrial offspring. For centuries, morphologically‐based studies have provided valuable information about the eggs of amniotes and the embryos that develop from them. This review explores the history of such investigations, as a contribution to this special issue of Journal of Morphology, titled Developmental Morphology and Evolution of Amniote Eggs and Embryos. Anatomically‐based investigations are surveyed from the ancient Greeks through the Scientific Revolution, followed by the 19th and early 20th centuries, with a focus on major findings of historical figures who have contributed significantly to our knowledge. Recent research on various aspects of amniote eggs is summarized, including gastrulation, egg shape and eggshell morphology, eggs of Mesozoic dinosaurs, sauropsid yolk sacs, squamate placentation, embryogenesis, and the phylotypic phase of embryonic development. As documented in this review, studies on amniote eggs and embryos have relied heavily on morphological approaches in order to answer functional and evolutionary questions.
... The surrounding mesenchyme develops at the same pace as the epithelium. The endodermal pouch then elongates (Romanoff, 1960). The bronchial tree, also elongated, and gives off evaginations proximal to the leading sac of expansion (Romanoff, 1960) . ...
... The endodermal pouch then elongates (Romanoff, 1960). The bronchial tree, also elongated, and gives off evaginations proximal to the leading sac of expansion (Romanoff, 1960) . ...
... In the chick, the pancreas arises from three evaginations from the gut wall, one dorsal and the other two are ventral in relation to the dorsal evagination. The pancreas was thought to be composed of cellular cords which later cavitated (Romanoff, 1960). ...
The development of the chicken stomach, particularly its asymmetrical and glandular morphogenesis is poorly understood. From a straight epithelial tube the gizzard endoderm evaginates dorsally to form the basic asymmetrical shape. It is suggested that evagination might be brought about by contractile elements in the dorsal bulging epithelium. The cell proliferation rate, examined by tritiated thymidine autoradiography, decreased in the bulging epithelium. Thus cell proliferation was not responsible for evagination morphogenesis. Expansion of the gizzard dorsally was achieved by differential localization of cell proliferation to the dorsal-most epithelium. There was also differential localized cell proliferation to the dorsal-most mesenchyme. The morphogenesis of the proventricular exocrine tubular glands was described using transmission electron microscopy and tritiated thymidine autoradiography. Development began by numerous evaginations in the rapidly dividing endoderm in which population pressure might have been developing. Evaginations were also brought about by cell shape changes which were thought to be caused by contractile elements. Localization of cell proliferation to the distal expanding tubule was the mechanism by which the glands expanded. The morphogensis of the gizzard tubular glands was also examined using light and electron microscopy. From columnar cells the epithelium became stratified in morphology and penetrated the underlying tunica propria. Deep in the stratified layer, microlumina formed between the cells which were delineated with junctional complexes. These were similar to the apical junctional complexes. The microlumina coalesced, expanded, and eventually opened up to the main lumen. A changing pattern of mitotic figures was noted. The pits developed by cell detachment and invasion by the underlying mesenchymal papilla. Epithelial cells from the transient stratified stage were examined in tissue culture. They mimicked their in vivo behaviour and managed to form small microlumina when plated on gizzard fibroblasts of the same age.
... Avian beaks are formed from multiple facial prominences, each of which has a neural crestderived mesenchymal core that is covered externally by an epithelial layer of ectoderm (Francis-West et al., 1998;Helms and Schneider, 2003;Richman and Lee, 2003). The upper beak comprises five prominences, including a fused frontonasal mass and paired maxillary and lateral nasal prominences (Romanoff, 1960;Wu et al., 2006). The lower beak is formed from two highly proliferating sites (one on each side) that are later fused into a single mandibular prominence (Romanoff, 1960;Wu et al., 2006). ...
... The upper beak comprises five prominences, including a fused frontonasal mass and paired maxillary and lateral nasal prominences (Romanoff, 1960;Wu et al., 2006). The lower beak is formed from two highly proliferating sites (one on each side) that are later fused into a single mandibular prominence (Romanoff, 1960;Wu et al., 2006). All prominences are coordinated with proportional sizes to produce a species-specific beak morphology (Abzhanov et al., 2004;Wu et al., 2004a;Wu et al., 2006). ...
... Beak morphogenesis consists of three major processes: the outgrowth of beak primordial mesenchyme (skeletal basis), the development of integument inside the oral cavity (oral mucosa) and the growth of the external keratinous sheath covering the snout (rhamphothecae). In chickens, beak growth starts by the end of embryonic day 5 (E5) and is marked by the initiation of the fused mandibular processes elongation (Romanoff, 1960). Later, the mesenchymal cell proliferation increases in the frontonasal mass, resulting in relatively faster growth of the upper beak than that of the lower beak (Romanoff, 1960). ...
Recent discoveries of exquisitely preserved nonavialan and avialan theropod dinosaurs have not only prompted studies of theropod tooth morphologies, but have also provided information about the origin and early evolution of avian beaks. Recent studies on beak morphologies and morpho-genesis in Darwin's finches have greatly improved our understanding of how avian beaks adapt to various ecological niches, but the question of how birds lost their teeth during the course of evolution has long been debated. Evolutionary developmental experiments performed on extant bird embryos bridge the gap between paleontological and neontological evidence, suggesting that the avian beak could have originated through heterochronic truncation of odontogenesis over evolutionary time. Here, we systematically review independently evolved regional and complete edentulism present in nonavialan and avialan theropod dinosaurs, and suggest that the tooth-reduction processes of different jaw bones are likely to be independently controlled. Through reviewing the recent advances of molecular regulations involved in tooth and avian beak morphogenic processes, we suggest that several molecules regulating the development of the avian beak also mediate the growth of keratinous rhamphothecae, and the divergence of odontogenic signalling pathways are likely to have accounted for both of these processes.
... A ganglion in the nerve of Remak consists of neuronal and satellite cells, which do not fully ensheathe the neurons, myelinated and unmyelinated nerve cell processes, fibroblasts, collagen fibrils, is densely vascularized and surrounded by a thick capsule of fibroblasts and collagen fibrils (Young, 1990). Romanoff (1960) regarded the nerve as a representative of the hypogastric plexus, and thus, believed it was sympathetic in origin. Yntema and Hammond (1952) claimed it is a derivative of sacral parasympathetic system and this was confirmed later (Le Douarin and Teillet, 1973). ...
... Early in development, the small intestine is a short tube running almost straight from the gizzard to the rectum. Subsequently, around the fifth day of incubation, two loops appear: the duodenal loop and the umbilical loop (Romanoff, 1960). After E6, these loops rotate around the longitudinal axis of the body and grow in length. ...
... The duodenal loop consists of an oral (descending limb) and an aboral segment (ascending limb) and extends from the gizzard-duodenal junction to the duodenal-ileal flexure; the latter marks the boundary between the duodenum and successive intestinal segments. By day 7 in ovo (E7), the pancreas fills the space encompassed by the duodenal loop (Romanoff, 1960). At E13, the duodenal loop folds on the right side of the gizzard (Romanoff, 1960). ...
The chemical and structural development of enteric plexuses was studied in the chick from embryonic day 4 to hatching. The enteric ganglia consist of neural crest-derived neuronal and glial cells, which can be distinguished morphologically and immunohistochemically in late developmental stages. At what stage different cell lineages originate from uncommitted precursors remains unknown. While the primordial neurons and glial cells could not be distinguished ultrastructurally from each other until E16, by means of polyclonal antibodies against GFAP and tubulin, distinct populations of neuronal and glial cells were identified from E5 in the duodenal myenteric ganglia. The enteric ganglia display a compact structure with exclusion of blood vessels and collagen, from the earliest stages investigated. A basement membrane, of unknown origin, is recognized ultrastructurally around ganglia and connecting strands from E12. The distribution of laminin, a basement membrane component, was examined by light and electron microscope immunohistochemistry; immunoreactivity was detected inside enteric ganglia, associated with glial cells, from E13 in the duodenum and from E11 in the rectum. The presence of laminin on the entire glial cell surface and probably in the cytoplasm suggests that glial cells are responsible for the production of the laminin-containing periganglionic basement membrane and that laminin is a marker of enteric glia in the chick. In addition, laminin immunolabelled the basement membranes of mesothelium, epithelium, muscle cells and endothelium. An important aspect of the functional maturation of enteric ganglia is the establishment of synapses. Synapse formation was studied by immunohistochemistry and electron microscopy. Synaptic vesicles were detected from E9, when immunoreactivity for the vesicle protein synaptophysin also appeared in ganglionic nerve endings. Because programmed cell death plays a major role in nerve tissue development, the presence of apoptotic cells in enteric ganglia, was studied using the TUNEL method and electron microscopy. The observations suggest that cell death contributes little, if at all, to the morphogenesis of the chick enteric plexuses. In other experiments, the gastrointestinal tract was excised from embryos of E5 to E8 and placed unsegmented in organ culture. The gut not only survived, but also continued to differentiate. The intramural migration of AChE positive ganglion cells into the hindgut, which is aganglionic at E5, proceeded and was completed in vitro, at the same rate as in controls. Separate populations of ganglion cells were identified by means of tubulin and GFAP immunoreactivity, but not ultrastructurally; basement membranes, visible at the ultrastructural level and as laminin immunoreactive structures developed in muscle cells, epithelium and ganglia. Synaptic contacts were established, small synaptic vesicles were visible and synaptophysin immunolabelling was also present. Submucosal ganglia contain fewer cells than myenteric ganglia in the duodenum and therefore, immunolabelling for all the antibodies was consistently less extensive or sometimes even absent. Immunoreactivities appeared in the myenteric ganglia, a day or two earlier than in the submucosal. Similar was the case in the rectum, where ganglia in both plexuses are of comparable size; laminin immunoreactivity, however, appeared earlier and was more extensive in the submucosal than in the myenteric ganglia.
... Birds are known to have an asymmetry in the female reproductive system. Previous research stated that this asymmetry occurs during sex differentiation of gonads when the right ovary regresses (Romanoff, 1960;Stahl and Carlon, 1973;Ebensperger et al., 1988;Van Krey, 1990;Ukeshima and Fujimoto, 1991;Smith, 2007;González-Morán, 2011;Intarapat and Stern, 2013;Jung et al., 2019). This concept was re-examined in the present study. ...
... Numerous publications repeat the false statement that gonadal asymmetry in birds appears after the sex differentiation of gonads, when the right ovary starts to regress and becomes smaller than the left ovary (Romanoff, 1960;Stahl and Carlon, 1973;Ebensperger et al., 1988;Van Krey, 1990;Ukeshima and Fujimoto, 1991;Smith, 2007;González-Morán, 2011;Intarapat and Stern, 2013;Jung et al., 2019). Carlon and Stahl (1985) did not find asymmetry of the gonadal ridges at earliest stages of their formation. ...
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.
... During the 21 days of embryonic development, the chick embryo utilizes essential nutrients from the egg compartment for tissue growth, extraembryonic tissue development and for its energy needs. The content of albumen is approximately 65-75% of the total content of eggs and consists of 12% protein and approximately 88% waterall of these two components are completely used by the embryo at the time of incubation (Romanoff, 1960). While yolk is composed of protein (15% of the total yolk), fat (33%), carbohydrates (less than 1%) as well as water (about 50%); yet, such contents bank significantly on the weight of the egg, strain (genetic) plus the age of hen (Vieira and Moran, 1999). ...
... In the final phase of incubation, nutrients, which are ingested with the amniotic fluid, arrive at the intestine of the embryo (Romanoff, 1960;Moran, 2007). Through this time, the intestine of the embryo experiences molecular, cellular and morphological changes, rapid proliferation and differentiation of enterocytes, enhanced capacity of absorption and nutrient uptake, and higher gene expression concerned in the process of digestion and absorption in the epithelial enterocytes (Geyra et al., 2001;. ...
Incubation period including pre-hatch few days are the critical time during which the embryo undergoes rapid
growth and development, and prepares for emergence and life outside the egg. This period is also important to chicken
survival during the first 1-3 days post-hatch when stored glycogen and residual yolk components aid in the transition to
feeding grains. In practical conditions, the time-consuming process in the hatching window additionally hinders newly
hatched chicks’ access to feed. Therefore, to supply nutrients timely and properly at the very early stage of life of the birds,
the concept of in ovo feeding as early nutritional intervention is coming up. This technique is promising to yield numerous
advantages, including reduced post-hatch morbidity and mortality, greater efficiency of nutrient utilization at an early
age, enhanced immune response to enteric antigens, reduced occurrence of developmental skeletal disorders, improved
muscle enlargement and breast meat yield. However, in ovo feeding method needs to be applied in order to supply the
embryo with additional nutrients prior to hatching, and those nutrients would likely be utilized by the chick throughout
the perinatal period. Further advancement of in ovo feeding system can lead to improved feed conversion efficiency, early
attainment of marketable body weight and overall performances of chicks. If the present limitations of in ovo feeding
technology are properly addressed, its wide acceptability and implementation are highly prospective in poultry.
... The movements of the embryo and the amnion could influence gas exchange by stirring the albumen and amniotic fluid, but the effects remain unclear. Muscles in the amnion cause rhythmic contractions that begin at D4, increase in frequency until D7-D9, and then decrease (Romanoff 1960). Body axis flexing is known to begin at D3 at a rate of 14.4 ± 2.4 movements per hour and increases to 125 ± 24 movements per hour at D5 (Wu et al. 2001) In this study, the greatest intensity of movement occurred in D5 embryos, which coincided with the lowest Po 2 readings (Table 2), so it is likely that they improved oxygenation. ...
... In the present case, the chicken embryos are effectively at rest and not exposed to experimental hypoxia, so the anaerobiosis results primarily from natural hypoxia that is evident in Po 2 transects. The values of whole-body lactate measured in this study align with evidence that lactate levels rise steeply in the albumen and yolk between D1 and D2 (Romanoff and Romanoff 1967). About half of the glucose use in the D1 embryo results in lactate production (Kučera et al. 1984). ...
Respiratory gas exchange in avian embryos progresses through three stages inside the egg. During the first 3–5 days of incubation, the chicken embryo has no specialised respiratory organs and is not reliant on blood circulation. At this stage, it obtains oxygen mainly by diffusion through the eggshell, albumen, amniotic fluid and embryonic tissues. In the second stage, gas exchange relies on diffusion through the shell in the gas phase and convection by blood circulation through the chorioallantoic membrane and body. Day 19 starts the third stage, the transition from chorioallantoic to pulmonary gas exchange, which is complete when the chick hatches on day 20. Metabolism is thought to be aerobic throughout incubation, although the early embryo is covered by fluids (albumen and amniotic fluid) which would greatly resist oxygen diffusion. This study uses fibre-optic sensors to measure oxygen partial pressure (Po2) near, and inside of, the embryo during days 3–5, and relates the data to total body lactate levels. The study shows that fluids surrounding the embryo greatly impede oxygen diffusion, with Po2 becoming severely hypoxic near the embryo, occasionally almost anoxic inside it. Meanwhile, lactate rises to high levels, and the stored lactate can be later oxidised by the embryo when the chorioallantois takes over and metabolism becomes entirely aerobic.
... The yolk sac membrane eventually develops elaborate folds and a microvillus structure (Yadgary et al. 2011). The yolk sac membrane plays an important role in the delivery of the required nutrients for embryo development (Romanoff 1960). The functional ability of the yolk sac membrane is dependent on numerous factors such as 1) morphological and structural changes (Romanoff 1960), 2) nutrient digestion, and transportation changes (Yadgary et al. 2013). ...
... The yolk sac membrane plays an important role in the delivery of the required nutrients for embryo development (Romanoff 1960). The functional ability of the yolk sac membrane is dependent on numerous factors such as 1) morphological and structural changes (Romanoff 1960), 2) nutrient digestion, and transportation changes (Yadgary et al. 2013). Nutrient transportation in the yolk sac membrane can be conducted via transporters for amino acids or receptor-mediated endocytosis of lipoproteins (Yadgary et al. 2013). ...
Optimal skeletal development prior to sexual maturity is pivotal in mitigating progressive structural bone and eggshell quality deterioration associated with egg production. Herein are investigations on the impact of omega-3 polyunsaturated fatty acids (n-3 PUFA) on skeletal development during embryonic and rearing
phases and subsequent effects on egg production, bone and eggshell quality in ISA brown and Shaver white hens. Hatching eggs were procured from breeders fed control or diets supplemented with n-3 PUFA sources: docosahexaenoic acid (DHA) or α-linolenic acid (ALA). During rearing, pullets from breeders fed control diet were fed control or supplemented diets, and pullets from supplemented diets either continued with respective n-3 PUFA diets or control diet. Pullets were transitioned to a common layer diet at 18 weeks of age (WOA) and monitored to 42 WOA. The use of n-3 PUFA in breeder feed increased embryonic utilization of DHA. The effects of n-3 PUFA on skeletal development were strain-dependent. Although feeding ALA did not affect bone quality, DHA fed to breeders supported tibia and femur structural (cortical) development in Shaver white hens. However, egg production, bone, and eggshell quality were not influenced by pre-lay exposure to n-3 PUFA. Although n-3 PUFA supported skeletal development pre-lay, there were no residual effects evident in 42 WOA ISA brown and Shaver white hens. Further studies should focus on the impact of various doses of n-3 PUFA, mainly DHA, and the subsequent effect on bone and eggshell quality over the entire lay cycle.
... The pattern of yolk processing in birds is substantially different from that of extant reptiles. Basic structural and functional features that are involved have been known in the domestic chicken since the late 19th through mid-20th century (Patten, 1920;Romanoff, 1960). ...
... Functions of the yolk sac endoderm of birds have been clarified through biochemical and molecular studies. Such studies have resolved a long-standing controversy (Freeman & Vince, 1974;Romanoff, 1960;Speake, Murray, & Noble, 1998) over whether the endodermal cells secrete enzymes into the yolk sac cavity and subsequently absorb the products of extracellular digestion. Strong evidence now exists that digestive enzymes and other factors contained within the ovulated egg (and within the yolk spheres) function in extracellular breakdown of nutrients, releasing catabolic products that are taken up by the endodermal cells (Yoshizaki et al., 2004). ...
Evolution of the terrestrial, amniotic egg of vertebrates required new mechanisms by which yolk material could be processed for embryonic use. Recent studies on each of the major extant reptile groups have revealed elaborate morphological specializations for yolk processing, features that differ dramatically from those of birds. In the avian pattern, liquid yolk is housed in a yolk sac whose endodermal lining absorbs and digests yolk material and sends resultant nutrients into the blood circulation. In snakes, lizards, turtles, and crocodilians, as documented herein, the yolk sac becomes invaded by endodermal cells that proliferate and phagocytose yolk material. Blood vessels then invade, and the endodermal cells become arranged around them, forming elongated "spaghetti-like" strands that fill the yolk sac cavity. This pattern provides an effective means by which yolk material is cellularized, digested, and transported by vitelline vessels to the developing embryo. Phylogenetically, the (non-avian) "reptilian" pattern was ancestral for sauropsids and was modified or replaced in ancestors to birds. This review postulates that evolution of the "avian" pattern involved increased reliance on extracellular digestion of yolk, allowing embryonic development to occur more rapidly than in typical reptiles. Comparative studies of yolk processing that draw on morphological, biochemical, molecular approaches are needed to explain how and why the "reptilian" pattern was replaced in birds or their archosaurian ancestors.
... Most of the Ross chicks (57.2%) were hatched 13 h before the hatch pull-out time, and only 3% chicks from Ross were hatched at terminal phase. In earlier studies, light provision has been found to affect the pull-out time of hatch (Romanoff 1960;Rozenboim et al. 2003). The scientific studies gave the reason of this early hatching partly due to increase in egg temperature that causes an elevation in yolk temperature resulting in an early hatch of broiler chicks and turkey poults (Romanoff 1960;Rozenboim et al. 2003). ...
... In earlier studies, light provision has been found to affect the pull-out time of hatch (Romanoff 1960;Rozenboim et al. 2003). The scientific studies gave the reason of this early hatching partly due to increase in egg temperature that causes an elevation in yolk temperature resulting in an early hatch of broiler chicks and turkey poults (Romanoff 1960;Rozenboim et al. 2003). However, with specific to strains variations, information is still lacking, and there is need to conduct studies to find how the embryos of different broiler strains respond to different light durations during incubation. ...
Present study was aimed to evaluate the hatching traits and subsequent performance of broilers strains under the intermittent and continuous light regime during incubation. In total, 2250 eggs from Hubbard classic, Cobb-500, and Ross-308 strains (750 eggs from each of same age breeders) were incubated under three different light durations. First treatment was the incubation totally under darkness where no light was able to penetrate in the assigned section of machine. In the second treatment, eggs were incubated at 12 h of lightness and 12 h of darkness. In the third treatment, the eggs received lightning of 24 h. Data were collected for hatching traits and hatch window, growth performance, welfare aspects, and meat quality. A two-way factorial analysis was performed using SAS software applying Duncan's multiple range test. The results showed that hatching traits were improved when Hubbard breeder eggs were provided with light period of 12 h. However, gait score was non-significantly different among the treatment. The meat quality was better in Hubbard broilers obtained after 12 h of intermittent light during incubation. Blood biochemistry was also improved in Hubbard broilers of 12 h of light duration. It was concluded that 12 h of light period during incubation is beneficial for getting better hatchability and subsequent performance of Hubbard broilers.
... Les fibres musculaires se mettent en place progressivement au cours de la vie embryonnaire (Romanoff 1960) et leur nombre final est fixé à la naissance. Les modifications post éclosion concernent la taille de la fibre musculaire (développement longitudinal et radial) et le nombre de noyaux par fibre. ...
... Comme chez les mammifères, il existe chez les oiseaux des organes lymphoïdes primaires (bourse de Fabricius et thymus) et secondaires (rate, diverticule de Meckels, glande de Harderian, plaques de Peyer et amygdale caecale). Ces tissus sont mis en place et colonisés par les cellules lymphoïdes au cours de la vie embryonnaire (Romanoff 1960). A la naissance, le système immunitaire est immature (Dibner et al 1998b). ...
Depuis une dizaine d’années, l’alimentation du poussin nouveau-né suscite un intérêt croissant car elle influence, à terme, les performances des poulets de chair. La résorption rapide du résidu vitellin représente une réserve relativement modeste de nutriments (1 jour) par rapport au développement intense du tube digestif et de ses fonctions d’une part et du muscle pectoral d’autre part pendant les deux ou trois premiers jours de la vie. Le délai d’alimentation dû aux conditions d’éclosion et de transport peut varier d’un individu à l’autre de 10 à 60 heures. Plusieurs travaux concluent que ce jeûne, à un moment critique du développement, retarde de manière irréversible la croissance et réduit sans doute les capacités de défense immunologique du poussin. De plus, les rares travaux portant sur la composition des régimes alimentaires pour très jeunes poussins suggèrent que les rations couramment distribuées sont peut être trop pauvres en protéines et trop riches en lipides. Il est, par ailleurs, nécessaire de freiner la croissance entre une et trois semaines d’âge pour limiter les problèmes locomoteurs et métaboliques ultérieurs. Les recherches actuelles s’orientent vers un ’pilotage’ plus précis de la nutrition des jeunes poulets de chair en stimulant le développement initial au cours de la première semaine, puis en le freinant, avant d’optimiser économiquement l’alimentation après l’âge de trois semaines. Dans un tel schéma général, les effets de stimulations initiales du développement des poussins par une alimentation plus précoce ou/et mieux adaptée sur la qualité de la viande et les performances globales de l’élevage restent à quantifier avec précision.
... All of nutrients available for embryonic development are stored in the albumen, yolk and eggshell (Romanoff, 1960;Speake, Noble, & Murray, 1998). The yolk sac is the main source of energy (via fatty acid oxidation) during embryonic development and the only source of lipids for embryo tissue growth (Speake et al., 1998). ...
... A reduction in embryonic metabolism during incubation may suggest that the embryo will not have enough cells to make effective use of the available O 2 to oxidize carbohydrates, fats or proteins to produce the needed energy for embryonic growth and organ development (Christensen, Wineland, Fasenko, & Donaldson, 2001). This may result in the lower embryo and hatchling weight after prolonged egg storage (Byerly, 1932;Christensen et al., 2001;Fasenko, 1996;Hamidu et al., 2011;Kooijman, 1986;Lilja & Olsson, 1987;Romanoff, 1960). In the same context, Uddin and Hamidu (2014) noted that embryos from long-term stored eggs not only lag behind in development, but their metabolism was also changed. ...
... All of nutrients available for embryonic development are stored in the albumen, yolk and eggshell (Romanoff, 1960;Speake, Noble, & Murray, 1998). The yolk sac is the main source of energy (via fatty acid oxidation) during embryonic development and the only source of lipids for embryo tissue growth (Speake et al., 1998). ...
... A reduction in embryonic metabolism during incubation may suggest that the embryo will not have enough cells to make effective use of the available O 2 to oxidize carbohydrates, fats or proteins to produce the needed energy for embryonic growth and organ development (Christensen, Wineland, Fasenko, & Donaldson, 2001). This may result in the lower embryo and hatchling weight after prolonged egg storage (Byerly, 1932;Christensen et al., 2001;Fasenko, 1996;Hamidu et al., 2011;Kooijman, 1986;Lilja & Olsson, 1987;Romanoff, 1960). In the same context, Uddin and Hamidu (2014) noted that embryos from long-term stored eggs not only lag behind in development, but their metabolism was also changed. ...
Prolonged hatching egg storage (>7 days) influences internal egg quality and embryo survival during both storage and subsequent incubation. Moreover, effects of storage of hatching eggs interact with the breeder age. The aim of this review was to investigate how this interaction between storage duration and breeder age affects egg, embryo, hatchling and chicken characteristics. Prolonged storage resulted in a reduction in egg quality in both young and old breeders. This reduction was more pronounced in young flocks than in older flocks. For example, albumen pH increased more after 8 days of storage in younger flocks than in older flocks. Additionally, the embryonic morphological stage appears to increase as well with storage duration, but this increase is again more pronounced in younger flocks than in older flocks. Short storage (<7 days) seems to increase hatchability of eggs from young breeders, probably as a result of albumen liquefaction with consequently better oxygen availability for the embryo. However, long storage (>7 days) resulted in a decline in hatchability, which was stronger in older breeders than in younger breeders. Prolonged storage duration resulted in lower chicken quality in both young and old breeders, but interaction between storage duration and breeder age on multiple chicken quality parameters is not clear. Based on this review, it can be concluded that (a) Short storage can improve hatchability of eggs from young breeders, but not from older breeders. (b) Negative impact of long storage appears to be lower with young breeders than with old breeders. (c) Adapted storage conditions related to the age of breeders might be an option to reduce negative effects of prolonged storage on hatching egg quality and chicken quality.
... Albumin contains very small amounts of IgM and IgA 14 . Although the vitelline membrane and eventually an extension of the chorion continue to separate the yolk and albumin, it is unlikely that these macromolecules will contaminate early yolk sac cells 15 . Large lymphoid cells with IgM or possibly IgA were found in the early yolk sac (3 days of incubation and onward). ...
Background: In mammals, birds, and amphibians, the B lineage of lymphoid cells first arise during embryogenesis and are distinguished by their capacity to produce immunoglobulin. For the purpose of researching the development of the B-cell repertoire and the development of self-tolerance, these early B-cell precursors are of utmost importance. The genetic and/or microenvironmental variables that control the beginning of immunoglobulin synthesis in embryonic haemopoietic cells are, however, poorly understood. Purpose: The ontogeny of B-cell precursors in chicken embryos from day three of incubation was examined in this work. Research methods: The terms "ontogeny, B-cell, precursors, chicken embryos, incubation" were used in a thorough literature search in the PubMed, NCBI, and Google Scholar databases. After all articles were picked based on the inclusion and exclusion criteria, 38 papers that satisfied the criteria for inclusion were collected. Result: The study's findings show that clglarge basophilic hemopoietic stem cells and cIg+ small lymphoid B-cell precursors are two types of migrant cells that appear to enter the embryonic bursa of Fabricius. Hence, B lymphopoiesis does not only take place in the bursa of Fabricius in the avian embryo. Although the yolk sac and the hemopoietic tissues around the dorsal aorta are strong candidates, the identity of the extra-bursal location remains unknown. Conclusion: Hence, general haemopoietic organs may serve as the initial site of B lymphopoiesis in both birds and mammals. Only later in the course of avian development do the bursal follicles become accessible and take over.
... The splenic primordium first appears as a mass of mesenchymal cells in the 48-hour embryo [73]. Sinusoids with erythrocytes appear in the mesenchyme at E5, granulopoiesis begins from E7 and erythropoiesis follows at E11. ...
Our ability to understand the function, pathology, and regeneration ability of an organ system is handicapped without knowledge of its normal structure. Electron microscopy and, later, immunocytochemistry made it possible to extend our knowledge about the structure of cells and the communication between them. These novel techniques helped to recognize the avian dendritic cells among others. Contemporary research on the structure of the lymphoid system contributed to recognition of the microenvironment and secretory functions of the primary lymphoid organs. Determining the histology of organs, using electron microscopy and immunocytochemistry of cells, will catalyze interactions between morphologists and immunologists to create novel ideas and hypotheses and will result in a more comprehensive understanding of avian immunology. This chapter is a comprehensive immune-morphological report on the primary and secondary lymphoid organs of birds.
... Small intestinal development can also be enhanced prior to hatch by nutritional stimulation, through in-ovo feeding (IOF). IOF is a method for supplying nutrients to the small intestine of chicken embryos by injecting their amniotic fluid with a formulated nutrient solution at E 17, prior to amniotic ingestion, which occurs up until DOH (Hamilton, 1952;Romanoff, 1960;Uni and Ferket, 2003). Various studies showed that IOF of specific nutrients enhanced peri-hatch intestinal development and functionality by expanding villi and crypt dimensions and increasing nutrient digestion and absorption capacities (Cheled-Shoval et al., 2011;Dai et al., 2020;Foye et al., 2007;Gao et al., 2017;Kadam et al., 2013;Tako et al., 2004;Wang et al., 2020). ...
Nutritional stimulation of the developing small intestine of chick embryos can be conducted by in-ovo feeding (IOF). We hypothesized that IOF of glutamine and leucine can enhance small intestine development by promoting proliferation and differentiation of multipotent small intestine epithelial cells. Broiler embryos (n = 128) were subject to IOF of glutamine (IOF-Gln), leucine (IOF-Leu), NaCl (IOF-NaCl) or no injection (control) at embryonic d 17 (E 17). Multipotent, progenitor and differentiated cells were located and quantified in the intestinal epithelium between E 17 and d 7 after hatch (D 7) in all treatment groups by immunofluorescence of SRY-box transcription factor 9 and proliferating cell nuclear antigen, in situ hybridization of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) and peptide transporter 1 (PepT1) and histochemical goblet cell staining. The effects of IOF treatments at E 19 (48 h post-IOF), in comparison to control embryos, were as follows: total cell counts increased by 40%, 33% and 19%, and multipotent cell counts increased by 52%, 50% and 38%, in IOF-Gln, IOF-Leu and IOF-NaCl embryos, respectively. Only IOF-Gln embryos exhibited a significant, 36% increase in progenitor cell counts. All IOF treatments shifted Lgr5+ stem cell localizations to villus bottoms. The differentiated, PepT1+ region of the villi was 1.9 and 1.3-fold longer in IOF-Gln and IOF-Leu embryos, respectively, while goblet cell densities decreased by 20% in IOF-Gln embryos. Post-hatch, crypt and villi epithelial cell counts were significantly higher IOF-Gln chicks, compared to control chicks (P < 0.05). We conclude IOF of glutamine stimulates small intestine maturation and functionality during the peri-hatch period by promoting multipotent cell proliferation and differentiation, resulting in enhanced compartmentalization of multipotent and differentiated cell niches and expansions of the absorptive surface area.
... This transition required the production of shelled (calcified) eggs that minimise water loss and could hold large amounts of yolk (Fig. 2). Hence, reptiles and birds have specialized regions of the oviduct that facilitate these functions [18][19][20][21][22][23]. While fertilisation takes place in the infundibular region, the development of shelled egg has led to significant structural differentiation of the oviduct. ...
The vertebrate female reproductive tract has undergone considerable diversification over evolution, having become physiologically adapted to different reproductive strategies. This review considers the female reproductive tract from the perspective of evolutionary developmental biology (evo-devo). Very little is known about how the evolution of this organ system has been driven at the molecular level. In most vertebrates, the female reproductive tract develops from paired embryonic tubes, the Müllerian ducts. We propose that formation of the Müllerian duct is a conserved process that has involved co-option of genes and molecular pathways involved in tubulogenesis in the adjacent mesonephric kidney and Wolffian duct. Downstream of this conservation, genetic regulatory divergence has occurred, generating diversity in duct structure. Plasticity of the Hox gene code and wnt signaling, in particular, may underlie morphological variation of the uterus in mammals, and evolution of the vagina. This developmental plasticity in Hox and Wnt activity may also apply to other vertebrates, generating the morphological diversity of female reproductive tracts evident today.
... Addition of a nutrient solution to the embryonic amniotic fluid would deliver essential nutrients into the embryo's intestine because pre-hatch birds naturally consume the amniotic fluids towards hatch (Romanoff, 1960;Kadam et al., 2013). Salahi et al. (2011) determined the best in ovo injection time may be 453 h of incubation. ...
... This finding may explain the result of Bai et al. (2016) who reported that Japanese quail embryo growth and development were relatively slow during ED10-ED16. Earlier studies on avian species reported that the elevated incubation temperature at a very early stage leads to acceleration in embryonic growth and development (Ricklefs 1987;Romanoff 1960). The continuous (24 h) long-term high incubation temperature resulted in reduced yolk consumption and regressed means of chicken embryo weights (Ozaydın and Celik 2014). ...
Embryonic thermal manipulation led to several modifications in molecular, physiological, and biochemical parameters which affect pre- and post-hatch growth performance. The current study aims to elucidate the onset and long-term effects of intermittent thermal manipulations (TM) during two-time windows, early/late, of embryogenesis in Japanese quail (Coturnix japonica) on embryonic development, hatchability, muscle histogenesis, and post-hatch growth performance. Four groups were created; quail eggs in the control group were incubated at 37.7 °C and relative humidity (RH) 55%. Three thermally treated groups were incubated intermittently at 41 °C and 65% RH intermittently (3 h/day): early embryogenesis group (EE) was thermally treated during embryonic days (ED) 6–8, late embryogenesis group (LE) was thermally treated during (ED12–ED14), and early and late embryogenesis group (EL) was thermally manipulated in both time windows. Relative embryo weights in EL and EE were significantly lighter than those in LE and Ctrl groups. The hatched chicks were reared under optimal managemental conditions (three replicates per treatment). Average daily feed intake was recorded, and feed conversion ratio (FCR) was calculated. Histological and quantitative analyses of muscle fibers were performed. The results revealed that TM led to significant hypertrophy of quail breast muscle in (EE). Intermittent short-term (3–6 h) thermal manipulation (39–40 °C) protocols during early embryogenesis (ED6–ED8) could be recommended to enhance muscle mass growth and breast muscle yield in the Japanese quail.
... In avian species, hypoxia during incubation inhibits embryonic development and even causes damage to the development of some organs, especially the brain Ophelders et al., 2016;Veenith et al., 2016). The avian brain is an organ that develops earlier at the embryonic stage and the development of the brain plays a key role in the development of the entire embryo (Romanoff, 1960). The oxygen consumed in chicken brain is not accurately studied, but in adults, the brain accounts for about 2% of body weight but consumes 20% of the total oxygen consumed by the body under normoxia (Schönfeld and Reiser, 2013;Raichle, 2015). ...
The Tibetan chickens (Gallus gallus; TBCs) are an indigenous breed found in the Qinghai-Tibet Plateau that are well-adapted to a hypoxic environment. As of now, energy metabolism of the TBCs embryonic brain has been little examined. This study investigated changes in energy metabolism in TBCs during hypoxia, and compared energy metabolism in TBCs and Dwarf Laying Chickens (DLCs), a lowland chicken breed, to explore underlying mechanisms of hypoxia adaptation. We found TBCs exhibited decreased oxygen consumption rates (OCR) and ATP levels as well as an increased extracellular acidification rate (ECAR) during hypoxia. Nevertheless, OCR/ECAR ratios indicated aerobic metabolism still dominated under hypoxia. Most important, our results revealed significant differences in TBCs brain cellular metabolism compared to DLCs under hypoxia. Compared to DLCs, TBCs had higher OCR and TCA cycle activities during hypoxia. Also, TBCs had more mitochondrial content, increased mitochondrial aspect ratio and MFN1, MFN2, and OPA1 proteins which have previously been reported to control mitochondrial fusion were expressed at higher levels in TBCs compared to DLCs, suggesting that TBCs may regulate energy metabolism by increasing the level of mitochondrial fusion. In summary, TBCs can reduce aerobic metabolism and increase glycolysis to enable adaptation to hypoxia. Regulation of mitochondrial fusion via MFN1, MFN2, and OPA1 potentially enhances the ability of TBCs to survive on the Qinghai-Tibet Plateau.
... During development, the bursa grows from a rounded to a more oval shape, and, in the direction of the bursa lumen, several longitudinal plicae are visible. It originates due to hypertrophy of the mesoderm surrounding the epithelial layer of the bursa [6]. ...
Hieronymus Fabricius ab Aquapendente (1533-1619) described the homonymous bursa in the "De Formatione Ovi et Pulli", published posthumously in 1621. He also included a figure in which the bursa was depicted. We here present the figure of the bursa of Fabricius, along with corrections of some mislabeling still presents in some anastatic copies. The bursa of Fabricius is universally known as the origin of B-lymphocytes; morphogenetical and physiological issues are also considered.
... On the day of lay, the embryo of a fertilized egg is 23 h old and its development is very rapid afterwards, if the egg is incubated. Indeed, embryonic development and the growth of some extraembryonic structures expand using PL as a substratum, which is thus rapidly degraded, and replaced by the extraembryonic yolk sac 19 . ...
The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success. © 2021, Journal of Visualized Experiments. All rights reserved.
... However, the earliest development of this cartilage is unclear. Previous authors had described it as the result of fusion of several earlier cartilages, with diverging accounts on the number of distal tarsal cartilages initially present (summarized in Romanoff, 1960). ...
The adult ankle of early reptiles had five distal tarsal (dt) bones, but in Dinosauria, these were reduced to only two: dt3 and dt4, articulated to metatarsals (mt) mt3 and mt4. Birds have a single distal tarsal ossification center that fuses to the proximal metatarsals to form a new adult skeletal structure: the composite tarsometatarsus. This ossification center develops within a single large embryonic cartilage, but it is unclear if this cartilage results from fusion of earlier cartilages. We studied embryos in species from four different bird orders, an alligatorid, and an iguanid. In all embryos, cartilages dt2, dt3, and dt4 are formed. In the alligatorid and the iguanid, dt2 failed to ossify: only dt3 and dt4 develop into adult bones. In birds, dt2, dt3, and dt4 fuse to form the large distal tarsal cartilage; the ossification center then develops above mt3, in cartilage presumably derived from dt3. During the entire dinosaur-bird transition, a dt2 embryonic cartilage was always formed, as inferred from the embryology of extant birds and crocodilians. We propose that in the evolution of the avian ankle, fusion of cartilages dt3 and dt2 allowed ossification from dt3 to progress into dt2, which began to contribute bone medially, while fusion of dt3 to dt4 enabled the evolutionary loss of the dt4 ossification center. As a result, a single ossification center expands into a plate-like unit covering the proximal ends of the metatarsals, that is key to the development of an integrated tarsometatarsus.
... In the case of the present study, the putrescine was injected inside the amniotic fluid at 17 days of incubation, and these solutions containing putrescine may have plated the animals for different periods of time until they started to swallow the amniotic fluid. Amniotic fluid prevents the embryo from dehydrating, protects the embryo from temperature fluctuations and mechanical shocks, prevents the embryo from adhering to other membranes, and also at the end of incubation provides buffer pH capacity (Randles & Romanoff, 1954;Romanoff, 1960). Hence, the doses of putrescine could disturb the amnion functions and the period of exposure of the embryo plated in the solutions may act as a toxic agent, increasing the mortality of more fragile animals. ...
The aim of this study was to evaluate the effects of increasing doses of putrescine injected in ovo on hatchability, intestinal morphology and pre-starter performance of broilers. For this purpose, 720 eggs from broiler breeders were separated into a negative control (no injection) and injection treatments with increasing doses of putrescine (0.05; 0.1; 0.15 and 0.2%), totalling five treatments of 144 eggs each. Eggs were distributed in a completely randomized design inside the setter and the injection of solutions occurred at 17 days of incubation. After hatch, 330 birds were housed in mixed lots following the original treatments, totalling 5 treatments of 6 replicates with 11 birds each. Six birds per treatment were weighed and euthanized by cervical dislocation to collect the liver, intestine and breast 24 hr after injection, at hatch and 24 hr after hatch. At 2 days of age, intestines were collected from 4 animals per treatment to analyse histomorphology. The effects of putrescine levels were evaluated by polynomial regression models, ANOVA and Tukey test at 5% probability. The hatchability decreased linearly in response to increased doses of putrescine. The percentage of residual yolk was lower in animals that received putrescine compared to the control. After injection, the percentage of breast increased linearly, and the percentage of intestine had a quadratic response to increased doses of putrescine. However, 24 hr after hatch, the percentage of intestine linearly decreased, and the percentage of liver linearly increased in response to increased doses of putrescine. Villus height increased quadratically, crypt depth decreased linearly, and goblet cells increased linearly in response to the putrescine dose. FI and BWG were not affected in the pre-starter phase; however, FCR increased in response to increased levels of putrescine. Due to putrescine effects on embryos, it is recommended that the doses injected in ovo not exceed 0.1%.
... In the avian pattern, the yolk sac is a hollow structure that is filled with non-cellularized yolk. Yolk material is absorbed by endodermal cells that line the yolk sac cavity, and products of yolk digestion are delivered to the underlying vitelline blood vessels (Lambson, 1970;Mobbs & McMillan, 1979, 1981Romanoff, 1960). ...
Recent studies have demonstrated a mechanism of embryonic yolk processing in lizards, snakes and turtles that differs markedly from that of birds. In the avian pattern, cells that line the inside of the yolk sac take up products of yolk digestion and deliver nutrients into the vitelline circulation. In contrast, in squamates and turtles, proliferating endodermal cells invade and fill the yolk sac cavity, forming elongated strands of yolk‐filled cells that surround small blood vessels. This arrangement provides a means by which yolk material becomes cellularized, digested, and transported for embryonic use. Ultrastructural observations on late‐stage Alligator mississippiensis eggs reveal elongated, vascular strands of endodermal cells within the yolk sac cavity. The strands of cells are intermixed with free yolk spheres and clumps of yolk‐filled endodermal cells, features that reflect early phases in the yolk‐processing pattern. These observations indicate that yolk processing in Alligator is more like the pattern of other reptiles than that of birds. Yolk processing in embryos of the American alligator is accomplished through formation of elongated, vascular strands of endodermal cells that fill the yolk sac cavity. This pattern is very different from that of birds, but shows similarities to those of other reptiles.
... Secondly, there is readily available information on the developmental physiology of broiler chicks. For example, it is now known that developing chick embryos consume the amniotic fluids prior to hatch [2]. Hence, the introduction of isotonic nutrient solutions to the amniotic fluid may deliver essential nutrients into the developing chick embryo. ...
... As cited by Bellville [12] and Romanoff [13], the early research works of Bogue [14] in 1932, showed that the heart rate of chicken embryos could in those days only be studied using an electrocardiogram (ECG). In this technique, at least three electrodes have to be inserted into the tissues surrounding the embryo. ...
The chicken embryo is a widely used experimental animal-model in many studies such as developmental biology and to study the physiological responses and adaptation to altered environments as well as for cancer and neurobiology research. Embryonic heart rate is an important physiological variable useful as an index reflecting the embryo's natural activity and is considered one of the most difficult parameters to measure. An acceptable measurement technique of embryonic heart rate should provide a reliable cardiac signal quality while maintaining adequate gas exchange through the eggshell along the incubation and embryonic developmental period. In this paper, we presented a detailed design and methodology for a non-invasive PPG-based prototype (Egg-PPG) for real-time and continuous monitoring of embryonic heart rate during incubation. An automatic embryonic cardiac wave detection algorithm, based on normalised spectral entropy, is described. The developed algorithm successfully estimated the embryonic heart rate with 98.7% accuracy. We believe that the developed overall system presented in this paper is showing a promising solution for non-invasion, real-time monitoring of embryonic cardiac signal, which can be used in both experimental studies (e.g., developmental embryology and cardiovascular research) and in industrial incubation applications.
... The development of the gut occurs throughout incubation (Romanoff, 1960), but the functional abilities of the gut only begins to develop about the time the amniotic fluid is orally consumed by the 18 th day old embryo. The weight of the intestine, as a proportion of embryonic weight, increases from approximately 1 % at 17 days of incubation to 3.5 % at hatch. ...
This experiment was on 350 uniform sized Cobb broiler hatching eggs (60 g) to assess the response of trace mineral supplementation (Zinc and copper) on growth performance and gastrointestinal tract development in broiler chicken. The fertile eggs were divided into groups with in ovo trace mineral solution containing zinc (80 µg) and copper (16 µg) and without in ovo administration. After hatching, the chicks were further divided into four groups: Group I served as control without in ovo and without post-hatch supplemented diet (WoINOVO-WoPHS), birds in Group II were without in ovo and with post-hatch supplemented diet (WoINOVO-WPHS) (100 % higher level of zinc 200 ppm, copper 30 ppm in diet), birds in Group III had in ovo (zinc, 80 µg; copper,16 µg) and without post-hatch supplemented diet (WINOVO-WoPHS) and birds in Group IV had in ovo and with post-hatch supplemented diet (WINOVO-WPHS). Data collected were subjected to completely randomized design. Hatchability, live weight gain, feed intake and feed conversion ratio at 0–3 wk were not affected (p > 0.05) by in ovo administration of the mineral. Post-hatch supplementation of zinc and copper without in ovo supplementation showed better feed conversion ratio at 3–5 wk of age. It could be recommended that for improved post-hatch performance, broiler chickens diets could be supplemented with inorganic zinc and copper.
Background
Tibetan chickens (Gallus gallus; TBCs), an indigenous breed distributed in the Qinghai-Tibet Plateau, are well adapted to the hypoxic environment. Currently, the molecular genetic basis of hypoxia adaptation in TBCs remains unclear. This study investigated hypoxia adaptation patterns of embryonic brain at different development stages by integrating analysis of the transcriptome with our previously published metabolome data in TBCs and Dwarf Laying Chickens (DLCs), a lowland chicken breed.
Results
During hypoxia, the results revealed that 1334, 578, and 417 differentially expressed genes (DEGs) (|log2 fold change|>1, p-value < 0.05) on days 8, 12, and 18 of development, respectively between TBCs and DLCs. Gene Ontology (GO) and pathway analyses revealed that DEGs are mainly related to metabolic pathways, vessel development, and immune response under hypoxia. This is consistent with our metabolome data that TBCs have higher energy metabolism than DLCs during hypoxia. Some vital DEGs between TBCs and DLCs, such as EPAS1, VEGFD, FBP1, FBLN5, LDHA, and IL-6 which are involved in the HIF pathway and hypoxia regulation.
Conclusion
These results suggest varied adaptation patterns between TBCs and DLCs under hypoxia. Our study provides a basis for uncovering the molecular regulation mechanism of hypoxia adaptation in TBCs and a potential application of hypoxia adaptation research for other animals living on the Qinghai-Tibet Plateau, and may even contribute to the study of brain diseases caused by hypoxia.
Even in mammals with the diaphragm, the lung and liver are likely to attach mutually without separation by any structure in embryos. The aim of this study was to examine whether or not the lung attaches to the liver in embryonic development of birds without diaphragm. First, we ensured the topographical relation between the lung and liver in 12 human embryos at 5 weeks. After the serosal mesothelium was established, the human lung sometimes (3 embryos) attached tightly to the liver without interruption by the developing diaphragm in the pleuroperitoneal fold. Second, we observed the lung-liver interface in chick and quail embryos. At 3-5 days' incubation (stages 20-27), the lung and liver were fused at bilateral narrow areas just above the muscular stomach. Therein, mesenchymal cells, possibly derived from the transverse septum, were intermingled between the lung and liver. The interface tended to be larger in the quail than the chick. At and until 7 days' incubation, the fusion of the lung and liver disappeared and, instead, a membrane connected them bilaterally. The right membrane extended caudally to attach to the mesonephros and caudal vena cava. At 12 days' incubation, bilateral thick folds, containing the abdominal air sac and pleuroperitoneal muscle (striated muscle), separated the dorsally located lung from the liver. Therefore, the lung-liver fusion occurred transiently in birds. Rather than the presence of the muscular diaphragm, whether the lung and liver were fused seemed to depend on a timing and sequence of development of the mesothelial coverings of these viscera.
Since 2014, methods have been described to hatch chick embryos from shell-less culture after egg contents are first incubated within shells for 55-70 h. The present report describes for the first time a shell-less culture system for chick embryos from the blastoderm stage to hatching. For the first 69-70 h, egg contents suspended in polymethylpentene kitchen wrap (F.O.R. Wrap, Riken Fabro, Tokyo, Japan) supported in 6.35 or 6.67 cm inside diameter tripods and covered with a disc of immobilized Milli-Wrap, were rotated back and forth through 90° at 16 or 22 cycles per minute (CPM). Subsequently, the Milli-Wrap disc was removed and culture tripods were transferred to environmental chambers, which were rocked ±20° through incubation day 8.5 (E8.5). From E9, environmental chambers were maintained in the horizontal position through to hatching with controlled O2 and CO2 . To provide supplemental calcium, an aqueous solution containing 100 mg/mL of calcium l-lactate hydrate was injected through the plastic wrap into the albumen at E9 (2.5 mL) and at E13 (1.0 mL) or E15 (1.0 mL). After incubation for 69-70 h at 16 or 22 CPM, 80%-83% of previously unincubated egg contents yielded apparently normal embryos. Hatch rate of normal embryos resulting from turntable incubation at 16 or 22 CPM was approximately 43%. Of note, egg contents remained in the same culture tripod from blastoderm stage to hatching. This technique may find use as an educational tool and in basic investigations of early embryogenesis, teratogenesis, and gene transfer experiments.
A method is described for culturing 64-70 h-old chicken embryos and egg contents outside of the eggshell through to hatching. Cultured egg contents were suspended in polymethylpentene kitchen wrap (F.O.R. wrap; Riken Fabro) supported in polyvinyl chloride tripods. Tripods were incubated in Plexiglas environmental chambers which were rocked automatically through an angle of ±20°. The concentration of CO2 was maintained at 2% throughout incubation, while that of O2 was increased from ambient to 50%, and relative humidity was decreased from 90%-92% to 83%-84% at incubation Day 9. Cultured embryos not supplemented with calcium did not hatch. The Hatch rate increased when supplemental calcium L-lactate hydrate was increased between 250 and 350 mg. A maximal hatch rate of 54.8% was achieved when cultures were supplemented with 350 mg of calcium L-lactate hydrate and 3.5 ml of sterile water. Adding 400 or 450 mg of calcium L-lactate hydrate did not increase the hatch rate further. The mass of cultured hatchlings (including the retracted yolk) and yolk-free carcass wet and dry mass and length of the right third toe were significantly less than the corresponding parameters observed in hatchlings in ovo. No statistically significant differences in hatchling mass, yolk-free carcass wet or dry mass, or length of the right third toe were noted among cultured hatchlings supplemented with 250-450 mg of calcium L-lactate hydrate. Failure to completely absorb albumen was the most common abnormality observed in cultures which failed to hatch. The present technique allows a unique approach to study the physiology of the developing chicken embryo.
In the thorax of higher vertebrates, ribs and intercostal muscles play a decisive role in stability and respiratory movements of the body wall. They are derivatives of the somites, the ribs originating in the sclerotome and the intercostal muscles originating in the myotome. During thorax development, ribs and intercostal muscles extend into the lateral plate mesoderm and eventually contact the sternum during ventral closure. Here, we give a detailed description of the morphogenesis of ribs and thoracic muscles in the chicken embryo (Gallus gallus). Using Alcian blue staining as well as Sox9 and Desmin whole‐mount immunohistochemistry, we monitor synchronously the development of rib cartilage and intercostal muscle anlagen. We show that the muscle anlagen precede the rib anlagen during ventrolateral extension, which is in line with the inductive role of the myotome in rib differentiation. Our studies furthermore reveal the temporary formation of a previously unknown eighth rib in the chicken embryonic thorax. In this study, we give a detailed description of chicken thorax development. By staining the cartilage lineage with Sox9 and Alcian blue, and the muscle lineage with Desmin, we show that the intercostal muscle anlagen grow in advance of the cartilage anlagen, the musculature thus paving the way for the developing ribs. We furthermore show the existence of a hitherto unknown temporal eighth rib anlage in almost half of the embryos.
Pesticides are major contaminants of our environment and pose a serious threat to non target organisms. The present experimental design aims to study the toxic effect of Flash on the embryo of chick treated at the post organogenesis stage of its developmental period. Fertilized eggs of pure breed (BV 300) of chick were immersed in low, moderate and high concentrations of the toxicant for one hour on day 7 of incubation and recovered on day 15 to observe skeletal development. The treated group's revealed malformations at all doses. The mortality rate was found to increase with significant decrease in wet body weight with increase in the dose. The pesticide caused overall reduction in ossification of skeleton; with significant malformations at only high dose, and in thoracic and caudal vertebrae. Present findings show that Flash is a potent teratogenic with reference to avian species.
Mammalian TRPC5 channels are predominantly expressed in the brain, where they increase intracellular Ca²⁺ and induce depolarization. Because they augment presynaptic vesicle release, cause persistent neural activity, and show constitutive activity, TRPC5s could play a functional role in late developmental brain events. We used immunohistochemistry to examine TRPC5 in the chick embryo brain between 8–20 days of incubation, and provide the first detailed description of their distribution in birds and in the whole brain of any animal species. Stained areas substantially increased between E8 and E16, and staining intensity in many areas peaked at E16, a time when chick brains first show organized patterns of whole-brain metabolic activation like what is seen consistently after hatching. Areas showing cell soma staining match areas showing Trpc5 mRNA or protein in adult rodents (cerebral cortex, hippocampus, amygdala, cerebellar Purkinje cells). Chick embryos show protein staining in the optic tectum, cerebellar nuclei, and several brainstem nuclei; equivalent areas in the Allen Institute mouse maps express Trpc5 mRNA. The strongest cell soma staining was found in a dorsal hypothalamic area (matching a group of parvicellular arginine vasotocin neurons and a pallial amygdalo-hypothalamic cell corridor) and the vagal motor complex. Purkinje cells showed strong dendritic staining at E20. Unexpectedly, we also describe neurite staining in the septum, several hypothalamic nuclei, and a para-median raphe area; the strongest neurite staining was in the median eminence. These novel localizations suggest new unexplored TRPC5 functions, and possible roles in late embryonic brain development.
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The remarkable reproductive success of avian species relies upon the integrated defensive features of the eggshell, acellular membranes, physicochemical characteristics and antimicrobial molecules. These egg structures are dramatically modified during embryonic development: solubilization of internal eggshell components, disintegration of acellular membranes that serve as substratum for the growth of extraembryonic structures, and assimilation/transfer of egg white and yolk antimicrobials to other compartments. The apparent degradation in basic egg defenses is counter balanced by the establishment of other protective and functional structures (namely the amniotic, the allantoic and the yolk sacs) that support several vital functions until hatching. Their cellular complexity, as well as the functional and molecular specificity of these structures, add an additional defensive layer. This chapter reviews the different levels of these interconnected egg defenses that assist the embryo throughout its development, beginning with the egg defenses from maternal origin, to the development of extra-embryonic protective structures.
We have studied the expression of the protein kinase activity of NCP98, the c-fps gene product, in several hemopoietic tissues of chickens as a function of the developmental stage of these organs. We found that in bone marrow, spleen, and bursa, maximum NCP98 kinase activity on a per-cell basis correlates with the peak of granulopoiesis in these organs. Furthermore, in a bovine serum albumin density gradient fractionation of bone marrow cells, granulocytic cells appeared to account for most of the NCP98 kinase activity. No correlation was found between the distribution of erythrocytic, lymphocytic, or thrombocytic cells and the distribution of the expression of NCP98 kinase activity. However, NCP98 protein and kinase activity were 10-fold higher in macrophages than in bone marrow. In addition, depletion by complement-mediated lysis of erythrocytic cells in bone marrow did not significantly reduce the total recovery of NCP98 kinase activity. These results argue for the specific expression of the c-fps gene product in granulocytic cells and macrophages.
This study aimed to assess the effects of breeder age on egg quality and amino acid and mineral transfer to the egg yolk and yolk sac of newly hatched chicks. Three ages (32, 42 and 52 weeks) of the same commercial flock of Hubbard breeders were studied. A total of 465 eggs were used for each age, with 60 being used for determining egg quality and amino acid and mineral content of yolk, and 405 for incubation period to obtain and evaluate the yolk sac of chicks. Breeders aged 52 weeks had heavier eggs and a higher percentage of yolk (p < 0.05), whereas 32‐week‐old breeders had higher eggshell percentage and thickness (p < 0.05). The percentage of protein deposited in egg yolk for 52‐week‐old breeders was higher than that for 32‐ and 42‐week‐old breeders (p < 0.05). Percentages of methionine, cysteine, met + cysteine, lysine, threonine, tryptophan, arginine and isoleucine in egg yolk for 32‐week‐old breeders were higher than that for 42‐ and 52‐week‐old breeders (p < 0.05). The transfer from breeder of phosphorus, potassium, calcium, magnesium, copper, iron, manganese and zinc to the yolk of eggs from 32‐week‐old breeders was greater than that for eggs from 42‐ and 52‐week‐old breeders (p < 0.05). Chicks from 32‐week‐old breeders had greater deposition of phosphorus and calcium in the yolk sac (p < 0.05). Breeder age did not affect the deposition of potassium, magnesium, copper, iron, manganese and zinc in the yolk sac of newly hatch chicks (p > 0.05). It can, however, be concluded that younger breeders deposit more amino acids and minerals in egg yolk, while embryos of older breeders seem to use the nutrients present in the yolk more efficiently during embryonic development.
The concept of the sugar code interprets the cellular glycophenotype as a rich source of information read by glycan‐lectin recognition in situ. This study's aim is the comprehensive characterization of galectin expression by immunohistochemistry during chicken nephrogenesis along with mapping binding sites by (ga)lectin histochemistry. Light and two‐color fluorescence microscopy were used. First, six plant/fungal lectins that are specific for galectin‐binding parts of N‐ and O‐glycans were applied. The spatiotemporally regulated distributions of these glycans in meso‐ and metanephros equip cells with potential binding partners for the galectins. Complete galectin profiling from HH Stage 20 (about 70–72 hr) onward revealed cell‐, galectin‐, and stage‐dependent expression patterns. Representatives of all three types of modular architecture of the galectin family are detectable, and overlaps of signal distribution in light and two‐color fluorescence microscopy illustrate a possibility for functional cooperation among them. Performing systematic galectin histochemistry facilitated comparisons between staining profiles of plant lectins and galectins. They revealed several cases for differences so that tissue lectins appear to be selective among the β‐galactosides. Notably, selectivity is also disclosed in intrafamily comparison. Thus, combining experimental series with plant and tissue lectins is a means to characterize target populations of glycans presented by cellular glycoconjugates for individual galectins. Our results document the presence and sophisticated level of elaboration among β‐galactosides and among the members of the family of galectins during organogenesis, using chicken galectins and kidney as model. Thus, they provide a clear guideline for functional assays using supramolecular tools, cells, and organ cultures.
A spectrophotometric procedure for standardizing red blood cell suspensions for use in viral hemagglutination tests is described. The procedure is based on highly reproducible cyanmethemoglobin absorbance readings at 540 nm on any suitable spectrophotometer. Target values for milligrams of cyanmethemoglobin per 100 ml are given for the red cell suspensions of all mammalian and avian species employed in viral hemagglutination tests. By use of these values and a cyanmethemoglobin standard curve for a particular photometer, approximate 4% erythrocyte suspensions can be diluted to any lesser concentration. Coefficients of variation for the various diluted suspensions range from 4 to 7%.
Amniote embryos are supported and nourished by a suite of tissues, the extraembryonic membranes, that provide vascular connections to the egg contents. Oviparous reptiles share a basic pattern of development inherited from a common ancestor; a vascular chorioallantoic membrane, functioning as a respiratory organ, contacts the eggshell and a vascular yolk sac membrane conveys nutrients to the embryo. Squamates (lizards, snakes) have evolved a novel variation in morphogenesis of the yolk sac that results in a unique structure, the yolk cleft/isolated yolk mass complex. This structure is a source of phylogenetic variation in architecture of the extraembryonic membranes among oviparous squamates. The yolk cleft/isolated yolk mass complex is retained in viviparous species and influences placental architecture. The aim of this paper is to review extraembryonic membrane development and morphology in oviparous and related viviparous squamates to explore patterns of variation. The survey includes all oviparous species for which data are available (11 species; 4 families). Comparisons with viviparous species encompass six independent origins of viviparity. The comparisons reveal that both phylogeny and reproductive mode influence variation in extraembryonic membrane development and that phylogenetic variation influences placental evolution. Models of the evolution of squamate placentation have relied primarily on comparisons between independently derived viviparous species. The inclusion of oviparous species in comparative analyses largely supports these models, yet exposes convergent patterns of evolution that become apparent when phylogenetic variation is recognized.
In the last decade, the chicken chorioallantoic membrane (CAM) assay has been re-discovered in cancer research to study the molecular mechanisms of anti-cancer drug effects. Literature about the CAM assay as an alternative in vivo cancer xenograft model according to the 3R principles has exploded in the last 3 years. Following a summary of the basic knowledge about the chicken embryo, we compare advantages and disadvantages with the classical mouse xenograft model, exemplify established and innovative imaging techniques that are used in the CAM model, and give examples of its successful utilization for studying major hallmarks of cancer such as angiogenesis, proliferation, invasion, and metastasis.
This chapter uses continuum theories for growth and contraction to simulate morphogenesis in embryos. First, the cellular activities underlying tissue-scale morphogenesis are discussed. Next, to illustrate basic concepts in epithelial morphogenesis, a linear theory for growing beams and plates is presented and used to solve illustrative examples involving some basic morphogenetic processes. The full nonlinear theory is then used to solve problems in embryonic development, including gastrulation, neurulation, and organogenesis. Examples of organogenesis include the development of the early heart and brain, the eyes, the gut, and the lung. A buckling analysis is used to simulate folding of the cerebral cortex. Finally, mechanical feedback and a theory for mesenchymal morphogenesis are discussed.
Effects of in-ovo intoxication of an insecticide formulation Nurelle D 505 EC (chlorpyrifos 50% EC + cypermethrin 5% EC) was evaluated in two generations of domestic chick. The investigation covered four groups of fertilized RIR eggs-three for experimental and one for control. Experimental groups of eggs received doses of 0.01, 0.05 and 0.1 µg/egg of combination insecticide (Ci) and the control received corn oil on day zero of incubation. The doses were selected based on our previous study. The eggs were incubated and upon hatching the F1 generation chicks were sorted as per their treatment, tagged and reared on standard diet. Upon sexual maturity the birds from the same groups were allowed pen mating. The fertilized eggs were collected and incubated for the next generation (F2). The eggs were regularly candled every two days till the 18th day of incubation to cull out the unfertile or dead embryos. The dead embryos and hatchlings were given a meticulous visual examination and the rate of morphological malformations was calculated. The study showed that chlorpyrifos and cypermethrin applied in a mixture caused significant increase in the rate of mortalities and decreased hatchability in chick of F1 and F2 generation. The morphological abnormalities caused by the treatment of the Ci were also observed in chicks of F1 & F2 generation. This is the first report of Ci induced structural anomalies in the second generation of chicks and we believe that the test system can evolve as an alternate model for testing two generation developmental toxicity.
The early stages of development of the chick embryo, leading to primitive streak formation (the start of gastrulation), have received renewed attention recently, especially for studies of the mechanisms of large-scale cell movements and those that position the primitive streak in the radial blastodisc. Over the long history of chick embryology, the terminology used to define different regions has been changing, making it difficult to relate studies to each other. To resolve this objectively requires precise definitions of the regions based on anatomical and functional criteria, along with a systematic molecular map that can be compared directly to the functional anatomy. Here, we undertake these tasks. We describe the characteristic cell morphologies (using scanning electron microscopy and immunocytochemistry for cell polarity markers) in different regions and at successive stages. RNAseq was performed for 12 regions of the blastodisc, from which a set of putative regional markers was selected. These were studied in detail by in situ hybridization. Together this provides a comprehensive resource allowing the community to define the regions unambiguously and objectively. In addition to helping with future experimental design and interpretation, this resource will also be useful for evolutionary comparisons between different vertebrate species.
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