Article

A Novel Shell-less Culture System for Chick Embryos Using a Plastic Film as Culture Vessels

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

The development of shell-less culture methods for bird embryos with high hatchability would be useful for the efficient generation of transgenic chickens, embryo manipulations, tissue engineering, and basic studies in regenerative medicine. To date, studies of culture methods for bird embryos include the whole embryo culture using narrow windowed eggshells, surrogate eggshells, and an artificial vessel using a gas-permeable membrane. However, there are no reports achieving high hatchability of >50% using completely artificial vessels. To establish a simple method for culturing chick embryos with high hatchability, we examined various culture conditions, including methods for calcium supplementation and oxygen aeration. In the embryo cultures where the embryos were transferred to the culture vessel after 55-56 h incubation, more than 90% of embryos survived until day 17 when a polymethylpentene film was used as a culture vessel with calcium lactate and distilled water supplementations. The aeration of pure oxygen to the surviving embryos from day 17 yielded a hatchability of 57.1% (8 out of 14). Thus, we successfully achieved a high hatchability with this method in chicken embryo culture using an artificial vessel.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... The development of a chick shell-less culture (cSLC; Tahara and Obara, 2014;Tahara et al., 2021) system has enabled uninterrupted omnidirectional visual observation of the development of chick embryos through transparent polymethylpentene film. This novel research tool is potentially valuable in multiple research areas, such as embryo manipulation (Boulland et al., 2010), bioimaging (Funahashi et al., 2014), and basic regenerative medicine research (Chiba et al., 2010). ...
... This novel research tool is potentially valuable in multiple research areas, such as embryo manipulation (Boulland et al., 2010), bioimaging (Funahashi et al., 2014), and basic regenerative medicine research (Chiba et al., 2010). One of the main factors contributing to the successful hatching of normal chicks in a cSLC is thought to be oxygen gas injection (OGI) into the cSLC starting on day 17 of embryo development (D17) (Tahara and Obara, 2014;Tahara et al., 2021). Here, the duration of embryo incubation in the cSLC is expressed in days, using the notation Di, where i=3, ... 19. ...
... The embryo culture vessel used in the cSLC was identical to that in our previous study (Obara et al., 2022) with slight modifications: a 2-cm-diameter hole was made in the side of the plastic cup, and a 2-mm-diameter tube (Atom Multipurpose Tube; Atom Medical, Tokyo, Japan) was inserted through this hole to supply oxygen (Tahara and Obara, 2014). Embryo Culture Embryos were cultured in a cSLC using an incubator (P-008; Showa Furanki, Saitama, Japan), as reported previously (Tahara and Obara, 2014). ...
Article
The effects of oxygen gas injection starting on day 17 of incubation (D17) in a chick shell-less culture system (cSLC) on the subsequent embryo development were examined on day 19 of incubation (D19). On D19 of cSLC, the plasma phosphorus and total cholesterol concentrations of the embryos were significantly higher (P<0.05), while the plasma calcium concentrations were significantly lower (P<0.05) than those in the intact control (IC) group. However, no significant differences in embryo viability and other major blood component levels were observed among the experimental groups (P>0.05). The percutaneous oxygen saturation was lower in D17-cSLC embryos before oxygen gas supplementation than in the IC (P<0.05) embryos. Severe renal tubular degeneration of the metanephros was observed in D19-cSLC embryos despite oxygen gas injection starting from D17. These results indicate that D19-cSLC embryos are hypoxia even after injecting oxygen gas starting on D17.
... Ex ovo culture is a system in which the original eggshell is removed, and the embryos are transferred to the new culture milieus, including a surrogate eggshell, Petri dishes, and artificial eggshell-like vessels (Ono, 2000;Borwompinyo et al., 2005;Liu et al., 2012;Tahara and Obara, 2014). By removing the eggshells, the ex ovo culture allows us to manipulate developing embryos at certain stages for surgical methods (Cloney and Franz-Odendaal, 2015). ...
... Significantly, the embryonic age is the key factor for manipulating ex ovo cultured embryos. To increase viability during culture, transferring the embryos to the new culture vessels after stage HH15-16 (Hamburger and Hamilton, 1951) is recommended (Tahara and Obara, 2014). Those embryos developing in a polymethylpentene film and supplemented with calcium lactate and distilled water resulted in a 90% survival rate (Tahara and Obara, 2014). ...
... To increase viability during culture, transferring the embryos to the new culture vessels after stage HH15-16 (Hamburger and Hamilton, 1951) is recommended (Tahara and Obara, 2014). Those embryos developing in a polymethylpentene film and supplemented with calcium lactate and distilled water resulted in a 90% survival rate (Tahara and Obara, 2014). Tahara and Obara (2014) also showed that the embryos transferred to the culture vessel after stage HH16 showed viability on day 8 of incubation. ...
Full-text available
Article
The avian embryos growing outside the natural eggshell (ex ovo) were observed since the early 19th century, and since then chick embryonic structures have revealed reaching an in-depth view of external and internal anatomy, enabling us to understand conserved vertebrate development. However, the internal environment within an eggshell (in ovo) would still be the ideal place to perform various experiments to understand the nature of avian development and to apply other biotechnology techniques. With the advent of genetic manipulation and cell culture techniques, avian embryonic parts were dissected for explant culture to eventually generate expandable cell lines (in vitro cell culture). The expansion of embryonic cells allowed us to unravel the transcriptional network for understanding pluripotency and differentiation mechanism in the embryos and in combination with stem cell technology facilitated the applications of avian culture to the next levels in transgenesis and wildlife conservation. In this review, we provide a panoramic view of the relationship among different cultivation platforms from in ovo studies to ex ovo as well as in vitro culture of cell lines with recent advances in the stem cell fields.
... Thus, much effort has been made to develop shell-less incubation systems, in which the egg contents are placed in a transparent plastic container that allows the observation of embryo development from all directions (Perry, 1988). Tahara and Obara (2014) have recently established a novel shell-less incubation system for chicken embryos using an artificial vessel. Using this simple, inexpensive culture method, they obtained more than 50% hatchability of chick embryos (Tahara and Obara, 2014). ...
... Tahara and Obara (2014) have recently established a novel shell-less incubation system for chicken embryos using an artificial vessel. Using this simple, inexpensive culture method, they obtained more than 50% hatchability of chick embryos (Tahara and Obara, 2014). This system that enables the in situ observation of embryos may be useful for assessing when and where TCEP affects the phenotype of avian embryos during development. ...
... To successively observe developing chicken embryos, incubation vessels, each comprising a 430-ml polystyrene plastic cup, were prepared according to the protocol reported by Tahara and Obara (2014). One hole of approximately 1.5 cm in diameter was made in the cup at a distance of 4.0 cm from the bottom to allow air exchange. ...
Article
Tris(2-chloroethyl) phosphate (TCEP) is an organophosphate flame retardant that used in textiles, industrial materials, and furniture to delay the spread of fire after ignition. TCEP has been detected in the tissues and eggs of fish and birds. However, there are no studies regarding the effects of TCEP on avian embryos. In the present study, we investigated the developmental toxicity of TCEP exposure on chicken embryos in a shell-less incubation system, which enables in situ observation. Chicken embryos were treated with graded doses of TCEP (50, 250, and 500 nmol/g egg) on incubation day 0. The survival rate, morphological biometrics, heart rate, and length and branch number of extraembryonic blood vessels were measured on incubation days 3-9. Survival rates were reduced from incubation day 3 and were significantly decreased until day 9. Body length, head + bill length and eye diameter were significantly reduced by TCEP exposure. Regarding skeletal effects, spine length was decreased in a dose-dependent manner on day 9. Body weight on day 9 significantly reduced in all TCEP treatment groups. These results suggest that TCEP exposure to >50 nmol/g egg retards development in chicken embryos. TCEP exposure to 500 nmol/g egg significantly increased heart weight to body weight ratio in the embryos. More than 250 nmol/g egg of TCEP significantly reduced the heart rate of embryos in the early developmental stage. The formation of extraembryonic blood vessels and the number of erythrocytes were significantly reduced even with 50 nmol/g egg of TCEP. These findings suggest that TCEP exposure specifically affects the cardiovascular system in chicken embryos, which leads to developmental delay. The results of this study also demonstrate that the shell-less incubation system can be used to continuously monitor the effects of chemicals on developing avian embryos.
... Therefore, a study of a shell-less culture system that hatches embryos completely in artificial vessels *2 was conducted and Kamihira et al. (1998) reported on their success in hatching quail embryos in a culture vessel that used polytetrafluoroethylene (PTFE) membranes. Following this, Tahara succeeded in hatching chick embryos in completely artificial culture vessels that used food wraps made of polymethylpentene and reported the detailed methods along with consideration for optimum conditions (Tahara and Obara, 2014). ...
... Using a cling film made of polymethylpentene as a culture vessel, adding calcium lactate as a calcium source to the embryo, and then pre-culturing the embryo for 55 hours (Stage s 16; Hamburger and Hamilton, 1951) before transferring the embryo to the culture vessel, continuing the culture, and directly supplying pure oxygen into the culture vessel from day 17, embryos can be successfully hatched (Tahara and Obara, 2014). As this method uses a transparent film as the culture vessel, it not only facilitates the observation of embryonic development but also provides easier access to the embryo than conventional methods. ...
... These studies have also been introduced in high school biology textbooks in Japan (Hotta et al., 2010). In 2012, Tahara succeeded in hatching chicks for the first time from shell-less culture using cling films at the Chiba Prefectural Oihama High School s and reported the experimental procedures in detail with further consideration of optimal conditions (Tahara and Obara, 2014;Tahara, 2016). ...
Article
Ex ovo culture of avian embryos can be applied not only to embryology but also to various fields of basic research such as embryo manipulation, toxicology, and regenerative medicine. The windowing method, which facilitates various manipulations and observations by opening a hole in one part of the eggshell, and culture systems using surrogate eggshells, are widely used. Despite this, biology lessons in high schools cover shell-less culture systems, which involve the development of avian embryos in artificial vessels, such as rice bowls, without using surrogate eggshells. However, as embryo development stops at its early stages in this method, it is not possible to continuously observe the development of the embryo. This led to attempts to develop an embryo culture method using a complete artificial culture vessel that does not use surrogate eggshells, and Kamihira et al. (1998) succeeded in hatching quail embryos in an artificial culture vessel using polytetrafluoroethylene membranes. In addition, Tahara succeeded in hatching chick embryos in artificial culture vessels that used cling film made of polymethylpentene and reported their detailed methodology (Tahara and Obara, 2014). These technologies are being applied not only to school education but also to various fields of research.
... 7,19,21-25 Current set-ups have now evolved to use gas-permeable artificial vessels made of materials such as polyethylene, polymethylpentene, polystyrene, polyurethane, and polytetrafluoroethylene, which are transparent, flexible, inexpensive, and provide better access to the embryos and its circulatory system. 16,[26][27][28][29] has further improved on existing set-up by devising a water circulation system to maintain the optimal temperature of chicken embryo culture. Table 1 summarizes the more commonly used and TA B L E 1 Ex ovo systems for avian embryo species ...
... Fertilized eggs were incubated for 55 to 56 hours before transferring into the bioengineered eggshell as recommended by Tahara and Obara. 28 Egg transfer into a different culture vessel is optimal on day 3 17,28,46 to minimize damage to the vitelline membrane covering the yolk and rupturing of blood vessels. The strength of the vitelline membrane decreases as it undergoes structural changes after being in contact with developing embryo for 10 hours. ...
... Fertilized eggs were incubated for 55 to 56 hours before transferring into the bioengineered eggshell as recommended by Tahara and Obara. 28 Egg transfer into a different culture vessel is optimal on day 3 17,28,46 to minimize damage to the vitelline membrane covering the yolk and rupturing of blood vessels. The strength of the vitelline membrane decreases as it undergoes structural changes after being in contact with developing embryo for 10 hours. ...
Full-text available
Article
The chicken embryo is widely used as an experimental model in the areas of regenerative medicine, tumor biology, and angiogenesis. Eggshell opacity and rigidity present restricted three‐dimensional (3D) viewing and accessibility to the embryo and its circulatory network despite egg windowing. The ability to engineer an eggshell, which eliminates the opacity yet provides 3D access for manipulation is beneficial and would potentially make it enticing for the use of chicken embryo as a cheaper alternative vertebrate model. Here, we present the feasibility of fabricating a morphologically analogous transparent chicken eggshell made of polydimethylsiloxane using a biaxial rotation computer‐controlled bioreactor to achieve uniform thickness. By culturing chicken embryo in the bioengineered eggshell, we demonstrated success in meeting developmental milestones and its practicality as an experimental platform by visualizing both embryo and vasculature development using common laboratory imaging tools. Although the viability of the embryo in the bioengineered eggshell was lower than in the normal egg, this was attributed to the absence of calcium source. The bioengineered eggshell, in its initial stage of development, provides a platform to be used to investigate beyond gross observations of the embryo and vascular network in 3D optical clarity and its imaging capabilities has the potential to be extended to other imaging modalities. We have developed a transparent ex ovo eggshell, biomimicking the elliptical geometry to address the lack of 3D optical clarity contributed by the eggshell opacity. This retains the natural shape that may support the development of the chicken embryo and allows easy access to the embryo, its circulatory system and respective organ development in far reaching applications.
... To show this, we use of a shell less culture system. To construct this hell-less culture system, we have used of the method in [9], however type of incubating, temperature and rotation were different. Similar to [9], a 450 ml polystylene plastic cup was applied as the pod for the culture vessel. ...
... To construct this hell-less culture system, we have used of the method in [9], however type of incubating, temperature and rotation were different. Similar to [9], a 450 ml polystylene plastic cup was applied as the pod for the culture vessel. A 1-1.5 cm diameter hole was made in the side of the cup approximately 2 cm from the bottom, and the hole was plugged with a cotton pledget as Some of fertilized chicken eggs were not incubated before transferring to the culture vessels. ...
... where a is the acceleration of system. On the other hand, it has been shown that this acceleration has a direct relation with temperature [9][10][11][12]: ...
Preprint
Recently, some authors have presented a short review of theoretical and experimental evidences of the formation of dissipative structures as the matrix of life in liquid water and shown their spontaneous quantum origin. We generalize this consideration and show that DNA can produce some hexagonal and pentagonal super-molecules in liquid water. We examine the model in shell less culture system and show that DNAs of stem cells of chick embryo produce some special structures in liquid water. We obtain the entropy for producing these DNA-like structures in water around a chick embryo in terms of temperature, rotating velocity and intensity of waves. Comparing this entropy with experimental data, we observe that our model conrms observations.
... And finally, some authors have suggested a shell-less culture system for chick embryos by using a plastic film as culture vessels. They have examined the conditions required for embryonic hatching by comparing factors such as the addition of calcium lactate and the presence or absence of an oxygen supply [5]. Motivated by these researches, we consider the effect of temperature and rotating velocity of vessels on the growth of cells in shell-less culture. ...
... Culture Vessels Our culture vessels are the same used in [5], however type of incubating, temperature and rotation were different. Similar to [5], a 450 ml polystylene plastic cup was applied as the pod for the culture vessel. ...
... Culture Vessels Our culture vessels are the same used in [5], however type of incubating, temperature and rotation were different. Similar to [5], a 450 ml polystylene plastic cup was applied as the pod for the culture vessel. A 1-1.5 cm diameter hole was made in the side of the cup approximately 2 cm from the bottom, and the hole was plugged with a cotton pledget as a filter. ...
Full-text available
Preprint
Recently, some authors have developed a shell-less culture system for chick embryos by using aplastic lm as culture vessels. We will show that velocity of rotation of vessels in incubator andtemperature have direct relations with growth of chick embryos. We also obtain a mathematicalrelation between velocity, temperature and rate of growth of chick embryo.
... Then, we break them and pour them in a tube or vessel of a shell-less culture system. In this system, similar to [19], we apply a 450 ml polystyrene plastic cup as the pod for the culture vessel. We also make a 1-1.5 cm diameter hole in the side of the cup approximately 2 cm from the bottom and plug the hole with a cotton pledget as a filter. ...
... We form a polymethylpentene film into a concave shape, carefully avoiding wrinkles and installed as an artificial culture vessel in the pod. Finally, we place a polystyrene plastic cover on top of the culture vessel [19]. In one of the vessels, we put normal embryo, and in another, we try to connect two embryos from their hearts. ...
Article
To recover chick embryos damaged the brain, two methods are presented. In both of them, somatic cells of an embryo introduced into an egg cell and an embryo have emerged. In one method, injured a part of the brain in the head of an embryo is replaced with a healthy part of the brain. In the second method, the heart of brain embryo dead is transplanted with the embryo heart. In this mechanism, new blood cells are emerged in the bone marrow and transmit information of transplantation to subventricular zone (SVZ) of the brain through the circulatory system. Then, SVZ produces new neural stem cells by a subsequent dividing into neurons. These neurons produce new neural circuits within the brain and recover the injured brain. To examine the model, two hearts of two embryos are connected, and their effects on neural circuits are observed.
... Our culture vessels for ex ovo method are the same used in [7]; however, type of incubating, temperature and rotation were different. Similar to [7], a 450-ml polystylene plastic cup was applied as the pod for the culture vessel. ...
... Our culture vessels for ex ovo method are the same used in [7]; however, type of incubating, temperature and rotation were different. Similar to [7], a 450-ml polystylene plastic cup was applied as the pod for the culture vessel. A 1-1.5-cm-diameter hole was made in the side of the cup approximately 2 cm from the bottom, and the hole was plugged with a cotton pledget as a filter. ...
Full-text available
Article
Recently, some authors have considered thermodynamics of DNA and obtained its entropy. In parallel, they have shown that phonons which are agents of temperature and entropy have direct effects on the electrons. Motivated by these researches, we obtain the relation between entropy and electronic properties of DNA like its radiated waves. We examine the model by considering the relation between entropy and the exchanged waves between DNAs of chick embryo in ovo and ex ovo models. To this aim, we put DNAs of chick embryo in ex ovo model and DNAs of chick embryo in ovo model in an inductor. We connect one end of this inductor to a generator of current and connect another end to an scope. We send a current to this inductor by a generator, produce a magnetic field and measure it’ changes after passing system of in ovo–ex ovo by an cope. By analyzing differences between input signals and currents with output signals and currents, we consider evolutions of entropy. Also, by considering changes in entropy, we determine the gender of chick embryo.
... Ex ovo or shell-less culture is a technique for the culture of chicken embryos outside of shell membrane using synthetic environments including artificial vessels, petri dishes, and cubeshaped egg shell like structures (Liu et al., 2013;Ono, 2000;Tahara and Obara, 2014). This A c c e p t e d M a n u s c r i p t 9 method could be helpful for embryo manipulations, developmental studies and biomedical research (Kyogoku et al., 2008). ...
... Whole embryo cultures can also be performed with the transfer of early embryos to the gas-permeable artificial vessels with water circulation systems (Yalcin et al., 2010). In these systems preparation of gas-permeable culture vessels from materials such as polymethylpentene film, polystyrene plastic and polyurethane membranes proved highly effective in successfully culturing embryos (Kamihira et al., 1998;Yalcin et al., 2010;Tahara and Obara, 2014) as water circulation systems are suitable for keeping the ex ovo chicken embryo culture at an optimal temperature (Yalcin et al., 2010). Therefore, this flexible, open, and inexpensive system by exclude the need for specific materials or it has made embryo manipulation easier (Dorrell et al., 2012;Saranya et al., 2016;Schmitz et al., 2016). ...
Full-text available
Article
1. The unique accessibility of the avian embryo have made them an ideal model for the study of development and genome editing. Chicken whole embryo culture has provided important insights into toxicity tests, gene manipulation, clarifying gene functions, cell transplantation, and cell tracking. 2. A simple technique for chicken manipulation is eggshell windowing, without or with seal, the latter having demonstrated some improvement in hatching rates. 3. Likewise, a surrogate eggshell system provides an accessible model for manipulation during chicken and quail development, with a higher hatchability compared to the simple windowing method. 4. The development of the chicken ex ovo culture systems in a synthetic environment as an efficient technique for imaging and microsurgery applications has enabled the study of important events of live chicken embryos at a specific time point. 5. This short review illustrates recent applications of well-designed whole embryo culture systems as a robust model for research into numerous biological mechanism, drug discovery, gene manipulating and production of functional proteins.
... This surrogate eggshell culture system from the blastoderm stage through to hatching has now extended to the blue-breasted quail (Coturnix chinensis) and bobwhite quail (Colinus virginianus), with hatching rates of about 25 and 40% respectively Kato et al. 2013). Artificial vessels such as those made from gas-permeable membranes can be used as substitutes for system III surrogate eggshells (Kamihira et al. 1998;Tahara and Obara 2014). In the shell-less culture system, calcium supplementation throughout development, as well as oxygen supplementation for the last 4 days until hatching, are critically important for hatching. ...
... In the shell-less culture system, calcium supplementation throughout development, as well as oxygen supplementation for the last 4 days until hatching, are critically important for hatching. A recently modified shell-less culture method for chicken embryos using a food wrap (polymethylpentene film) that corresponds to Perry's system III yielded a hatchability of 57.1% (Tahara and Obara 2014). Ex-vivo culture enables more easy access and precise manipulation of the developing avian embryos. ...
Chapter
Primordial germ cells (PGCs) generate new individuals through differentiation, maturation and fertilization. This means that the manipulation of PGCs is directly linked to the manipulation of individuals, making PGCs attractive target cells in the animal biotechnology field. A unique biological property of avian PGCs is that they circulate temporarily in the vasculature during early development, and this allows us to access and manipulate avian germ lines. Following the development of a technique for transplantation, PGCs have become central to avian biotechnology, in contrast to the use of embryo manipulation and subsequent transfer to foster mothers, as in mammalian biotechnology. Today, avian PGC transplantation combined with recent advanced manipulation techniques, including cell purification, cryopreservation, depletion, and long-term culture in vitro, have enabled the establishment of genetically modified poultry lines and ex-situ conservation of poultry genetic resources. This chapter introduces the principles, history, and procedures of producing avian germline chimeras by transplantation of PGCs, and the current status of avian germline modification as well as germplasm cryopreservation. Other fundamental avian reproductive technologies are described, including artificial insemination and embryo culture, and perspectives of industrial applications in agriculture and pharmacy are considered, including poultry productivity improvement, egg modification, disease resistance impairment and poultry gene “pharming” as well as gene banking.
... In these culture systems, the embryo remains on top of the intact yolk, and all the egg contents with the albumen are transferred to another container for incubation. The containers varied from a turkey or chicken surrogate eggshell (Nirasawa et al., 1992;Borwompinyo et al., 2005), a Petri dish (Auerbach et al., 1974), or cling wrap hammocks (Tufan et al., 2004;Yalcin et al., 2010;Schomann et al., 2013;Tahara and Obara, 2014) for culture. Some systems also include sequential culture in multiple containers involving a plastic cup (0-24 h), quail eggshell (24-76 h), and then in a chicken eggshell (until hatching) with a supply of chicken thin albumen for optimal growth (Ono et al., 1994). ...
Full-text available
Article
The avian egg is a closed system that protects the growing embryo from external factors but prevents direct observation of embryo development. Various culture systems exist in the literature to study the development of the embryo for short periods of incubation (from 12 h up to a maximum of 60 h of egg incubation). A common flaw to these culture techniques is the inability to culture the unincubated avian blastoderm with intact tissue tensions on its native yolk. The goal of this work is to create a unique novel egg-in-cube system that can be used for long-term quail embryo culture initiated from its unincubated blastoderm stage. The egg-in-cube acts as an artificial transparent eggshell system that holds the growing embryo, making it amenable to microscopy. With the egg-in-cube system, quail embryos can be grown up to 9 days from the unincubated blastoderm (incubated in air, 20.9% O2), which improves to 15 days on switching to a hyperoxic environment of 60% O2. Using transgenic fluorescent quail embryos in the egg-in-cube system, cell movements in the unincubated blastoderm are imaged dynamically using inverted confocal microscopy, which has been challenging to achieve with other culture systems. Apart from these observations, several other imaging applications of the system are described in this work using transgenic fluorescent quail embryos with upright confocal or epifluorescence microscopy. To demonstrate the usefulness of the egg-in-cube system in perturbation experiments, the quail neural tube is electroporated with fluorescent mRNA “in cubo”, followed by the incubation of the electroporated embryo and microscopy of the electroporated region with the embryo in the cube. The egg-in-cube culture system in combination with the “in cubo” electroporation and dynamic imaging capabilities described here will enable researchers to investigate several fundamental questions in early embryogenesis with the avian (quail) embryo on its native yolk.
... The development of avian embryos or oviparous embryos depends on the nutrients in egg yolk and calcium primarily utilized from the shell, especially during the later stage of development [2,3]. The nutrient intake by developing embryos from yolk, eggshells, or other parts attracted the attention of evolutionists as the ontogenic development has influenced the phenotypic characteristics [3], medical researchers [4], and developmental study researchers [5][6][7]. ...
Full-text available
Article
Growth and production of any animal husbandry including poultry depends on early development, growth and conversion efficiency apart from feeding, environmental and other factors. Early and later embryonic development forms a significant parameter in poultry production, during which human or technological intervention is practically impossible. Similarly embryonic development is governed by very many internal factors among which, egg calcium utilization bags prime importance. An experiment was set up to investigate the egg calcium utilization of different poultry breeds at its embryonic development. Four breeds of locally available chicken (Gallus gallus domesticus), Broiler, Local (Nadan), Vanaraja and Gramapriya breeds were selected and embryonic egg utilization was assessed by calcium estimation of egg shell before and after hatching and found out that Vanaraja breed utilizes maximum (0.20gm) and significantly (P < 0.01) higher quantity egg shell calcium for embryonic development followed by Local breed (0.16gm) and least used variety was Broiler breed (0.09gm).
... The minimum hatchability of this technique was 52.9%, which is higher than that reported (less than 37%) in most of the previous studies using a surrogate eggshell system [6,11,13,15,18,22,[40][41][42][43]. Although some improvements have been reported in the culture of chicken embryos [44][45][46][47], culture methods using surrogate eggshells or artificial vessels suffer from several disadvantages, such as high economic costs, long operating time, inconsistency between the morphology of the original eggs and the surrogate shells, and the risk of injury to or contamination in the embryo during the process of transferring egg content [48]. In addition, it is important to note that the rates of hatchability achieved in this study is following single-, double-and triple-injection and it is clear that over-manipulation could be more detrimental to embryo survival than a single manipulation. ...
Full-text available
Article
Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo’s bloodstream at Hamburger-Hamilton (HH) stages 14–15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14–15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.
... 85 Studies that require embryos to survive past day 18 of incubation demand the addition of these nutrients. 86 Despite these practical issues, the ex ovo CAM model has also a number of advantages. First of all, this approach results in a larger CAM area for experimental procedures compared to the in ovo method. ...
Article
Tissue engineering aims to structurally and functionally regenerate damaged tissues which requires the formation of new blood vessels that supply oxygen and nutrients by the process of angiogenesis. Stem cells are a promising tool in regenerative medicine due to their combined differentiation and paracrine angiogenic capacities. The study of their proangiogenic properties and associated potential for tissue regeneration requires complex in vivo models comprising all steps of the angiogenic process. The highly vascularized extraembryonic chorioallantoic membrane (CAM) of fertilized chicken eggs offers a simple, easy accessible and cheap angiogenic screening tool compared to other animal models. Although the CAM assay was initially primarily performed for evaluation of tumour growth and metastasis, stem cell studies using this model are increasing. In this review, a detailed summary of angiogenic observations of different mesenchymal, cardiac and endothelial stem cell types and derivatives in the CAM model is presented. Moreover, we focus on the variation in experimental set-up, including the benefits and limitations of in ovo and ex ovo protocols, diverse biological and synthetic scaffolds, imaging techniques and outcome measures of neovascularization. Finally, advantages and disadvantages of the CAM assay as a model for angiogenesis in tissue engineering in comparison with alternative in vivo animal models are described.
... Their eggshell was wiped and cracked and the whole egg contents were transferred to the culture vessel without pre-incubating period. The culture vessels were maintained at 38°C and rotated with 120° clockwise twice a day [8]. After 54 h, in most of vessels, chick embryo is emerged (See figure 1). ...
Full-text available
Article
BACKGROUND: Recently, some new methods for sexing of chicken eggs by fluorescence and Raman spectroscopy through the shell membrane have been proposed. On the other hand, in another investigation, a new virus medical imaging has been suggested. AIM: In this research, summing over these considerations, a new technique for sexing of chicken eggs by virus spectroscopy through the shell membrane is proposed. METHODS: (Please, add text …) RESULTS: (Please, add text …) CONCLUSION: It is shown that viruses outside the shell of egg can communicate with materials inside it and determine the gender of chick embryo and its evolutions.
... We put two types of chick embryos near each other, which one is emerged interior of ovo and another is produced outside ovo (EX-ovo model. See [12]). We radiate a magnetic field around 7 tesla to both of cells in and ex ovo. ...
Article
In this research, we calculate Tsallis δ-entropy and microstates of a rotating DNA by using concepts of hexagonal and pentagonal black branes. We argue that DNA is a massive brane-like object with more than 7 meter long which contracts in an small place. This leads to the emergence of a Rindler horizon and a curved space–time around a DNA. In this system, rotation of DNA leads to the radiation of some special waves. These waves lead to the motion of electrons in a metal wire and emergence of a current. This current has a direct relation with number of microstates of DNA and is different for females and males. By considering evolutions of this current, we determine some properties like entropy, number of microstates and gender.
... So far various artificial systems have been developed by other researchers, mainly based on plastic films or dishes [20]. Nevertheless, embryos relocated to artificial vessels face their own challenges aswell, including but not limited to, the material should preferentially be gas-permeable, such as polytetrafluoroethylene (PTFE), and the survival rate and health of the embryos suffers from the lack of physiologically relevant microenvironment factors such as sufficient calcium supply [18,19]. Surrogate eggs have proven to be a plausible improvement provided that the physiological relevant development environment and barrier can be maintained by the surrogate shells [18]. ...
... Ca consumption of laying hen was 4000 mg and deposit to the egg 3100 mg, faeces 500 mg and urine 400 mg (Leeson dan Summers, 2005). Ca of egg is needed for embryo development (Uni et al., 2012, Tahara andObara, 2014;Scanes, 2015). ...
Article
The aim of this research was to evaluate calcium deposition in egg using eggshell flour as a limestone substitute in feed. Two hundreds laying hen of Isa Brown strain of 25 weeks were used in this study. Treatments were diet with 7.5% limestone as control (T0), 2.5% limestone is substituted with eggshell flour(T1), 5% limestone is substituted with eggshell flour (T2) and limestone is substituted with eggshell flour (T3). A completly randomized design were used to allocated the treatments with 5 replications of each. Each experimental unit consists of 10 laying hens. Parameters measured were egg weight, yolk weight, albumen weight, eggshell weight, calcium of egg (yolk, albumen and eggshell), length, weight and Ca of tibia bone. The results showed that substitution of limestone with eggshell flour had significantly effect (P<0.05) on eggshell weight, Ca deposition on yolk, albumen, and Ca of tibia bone but non significantly effect on egg weight, weight and percentage of yolk, weight and percentage of albumen, percentage and Ca of eggshell, length and weight of tibia bone. In conclusion, calcium deposition in yolk was the highest in the use of 7.5% eggshell flour to substitute limestone but obtained the lowest Ca of bone , while calcium deposition in albumen was the highest in the use of 2.5% eggshell flour.
Article
Chicken (Gallus gallus) is a major protein source and an important model organism in the avian species. Although genome editing has enabled genetic modifications of various organisms and has a significant impact on academia and industry, its application to chickens has been comparatively delayed owing to difficulties in handling their one-cell fertilized eggs. Thus, researchers have attempted to produce genome-edited chickens using primordial germ cells (PGCs), which are precursor cells of sperm or eggs. Currently, genome-edited chickens can be produced with the development of avian biotechnologies, PGCs culture methods, and germline chimerism systems, in particular. In this review, we describe the current status of genome editing in chickens, including avian biotechnologies, with a primary focus on the achievements of Japanese researchers. In addition, we discuss the remaining issues and make suggestions for future research.
Article
This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation. In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.
Full-text available
Preprint
Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. We established a new method of eggshell windowing for embryo manipulation at different developmental stages. Compared to the other routes of viral vector administration, the embryo's bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.
Article
Developing chick embryos are a classical research tool in developmental biology. The whole embryo culture technique can be applied to various fields, such as embryo manipulation, toxicology, tumorigenesis, and basic research in regenerative medicine. When used for the generation of transgenic chickens, a high hatchability of genetically engineered embryos is essential to support normal embryonic development during culture. In this study, calcium carbonate, which is the main component of eggshells, was added as a calcium source in shell-less chick embryo cultures using a transparent plastic film as a culture vessel. In the absence of a calcium source in the shell-less culture system, embryogenesis ceased during culture, resulting in failed embryonic hatching. We found that the direct addition of calcium carbonate to the chorioallantoic membrane of the developing embryo was effective for the hatching of cultured chick embryos. The amount, timing, and location of calcium carbonate addition were investigated to maximize the hatchability of cultured embryos. Starting from the time of calcium carbonate supplementation, >40% hatchability was obtained with the optimal condition. This established method of shell-less chick embryo culture provides a useful tool in basic and applied fields of chick embryo manipulation.
Article
1. Birds’ newly oviposited blastoderms can survive several weeks in a dormant state during low-temperature storage. Previous studies demonstrated that there is a critical temperature range from 19 to 27℃ for chicken embryos. Within this range, the embryo will diapause in a dormant state; once the temperature rises above this range, the blastoderm will break dormancy. 2. Clarifying the mechanism that initiates duck embryo developmental recovery from blastoderm dormancy will be helpful to change temperature control to improve hatching in poultry production. It was hypothesised that there might be some temperature-sensitive genes involved in initiating duck embryo developmental recovery from blastoderm dormancy. 3. To test this hypothesis, the transcriptome of the newly oviposited duck blastoderm and duck embryo (incubated for 48 hours) were sequenced to screen for differentially expressed genes with functions that had been predicted by bioinformatics. 4. The results showed that there were 2416 differentially expressed genes between the two groups, 53 of which were involved in temperature-sensitive pathways. The protein-protein interaction network combined these 53 temperature-sensitive genes and another group of 65 genes, which enriched the development pathway. These results suggested that temperature-sensitive genes may be involved in growth and development related pathways. Keywords: Duck, blastoderm, incubation temperature, temperature-sensitive genes, RNA-seq
Article
Calcium carbonate in avian eggshell is used as a calcium source for bone formation during embryonic development. We studied ostrich eggshells as a representative avian egg and found that the calcium reserve assembly (CRA) region, eventually dissolving for bone formation, consists of nano-granules comprising amorphous calcium carbonate (ACC) by careful microscopic observation. ACC is unstable phase of calcium carbonate with high solubility and is stabilized by additives. As such a stabilizer, we found small molecules from eggshell internal region. Particularly, the ACC was recreated by the formation of calcium carbonate in the presence of d-myo-inositol 4,5-bisphosphate (Ins(4,5)P2), isolated from ostrich eggshell for the first time in this study. Further mass spectrometric imaging technique revealed that Ins(4,5)P2 is localized at the CRA region, suggesting that this compound could play a major role that makes it easier to dissolve and internalize eggshell calcium during embryogenesis.
Full-text available
Article
1. The PCO2 and PO2 levels of oxygenated and deoxygenated embryonic blood were measured in normal and shell-less cultured embryos. 2. Embryos in shell-less culture were both hypoxic and hypocapnic, suggesting that wetting the respiratory membranes and the reduction in their functional area is distorting their normal function. 3. Despite this hypocapnia, there was evidence for an increase in base excess during incubation. This suggests that plasma bicarbonate is derived from renal sources rather than by resorbing the eggshell calcium carbonate, which is, of course, absent from shell-less cultures.
Full-text available
Article
We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (∼5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs.
Full-text available
Article
The chick embryo is a classical model to study embryonic development. However, most researchers have not studied the effect of embryonic manipulation on chick hatchability. The objective of this study was to determine the effect of egg orientation and type of sealing film on the hatchability of cultured embryos. Windows were made in the small end of recipient surrogate chicken eggshells, and donor embryos were placed into the recipient eggshell for the first 3 d of incubation. Survival over the first 3 d was maximized (P < 0.05) when windowed eggs sealed with Saran Wrap were positioned with the window-end down compared with window-end up. Three-day-old cultured embryos were transferred into recipient turkey eggshells, sealed with cling film, and cultured until hatch. Water weight loss of the surrogate eggshell cultures regardless of cling film type was not significantly different from control intact eggs. The embryos cultured in turkey eggshells and sealed with Handi Wrap exhibited higher hatchability (75% +/- 10.2%) than cultures sealed with Saran Wrap (45.2% +/- 13.8%). Hatchability of control intact eggs (86.4% +/- 5.3%) was not significantly (P > 0.05) different from the hatchability of eggs sealed with Handi Wrap, which suggested that Handi Wrap was an excellent sealant for chick embryos cultured after 3 d of incubation.
Full-text available
Article
We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-* toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens.
Article
Quail are divided phylogenetically into two groups, Old World quail and New World quail. Old World quail, such as the Japanese quail (Coturnix japonica), belong to the Phasianidae and distributed in the Palaearctic region (Europe, North Africa, and Asia), whereas New World quail, such as the bobwhite quail (Colinus virginianus), belong to the Odontophoridae and are restricted to North and South America. Both the bobwhite quail and the Japanese quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been limited compared with those on the Japanese quail. We have therefore now developed an ex vivo culture protocol for bobwhite quail embryos from the blastoderm stage through hatching. Of the various culture conditions examined in the present study, a good hatching rate (39%) was obtained when the embryos were cultured ex vivo in a two-step procedure. Unincubated embryos (with egg yolk) were first cultured inside the shell of a Japanese quail egg (11.5 to 13.0 g whole egg weight) together with chicken thin albumen for 63 to 65 h and were then transferred to the shell of a small-sized chicken egg (38 g whole egg weight) until hatching. This ex vivo culture system should provide to be widely applicable to the maintenance and generation of manipulated birds for basic and applied studies on the bobwhite quail.
Article
1. Embryos of the domestic fowl (72 h old) have been explanted into shell‐less cultures or ‘surrogate’ eggshells, in order to investigate the possibility of rearing these embryos to hatching.2. Rocking embryo cultures during the first half of incubation enhanced embryo growth.3. Embryos explanted into ‘surrogate’ eggshells of either other individuals or other species have been successfully ‘hatched’.4. A normal chorioallantois is formed in these surrogate eggshells. This enables a functional albumen sac to form and eggshell resorption to be achieved.5. Embryos grown in ‘surrogate’ eggshells are slightly smaller than controls but otherwise normal.6. The technique provides opportunities for genetic engineering experiments.
Article
An experiment was carried out to investigate whether thick albumen is essential for the normal development of the chick embryo. Fertilized ova recovered from the oviducts of hens were cultured in vitro and transferred to recipient eggshells with (method A) or without (method B) replacement of the thick albumen by thin albumen. Embryos from freshly laid eggs were transferred to recipient eggshells with (method C) or without (method E) replacement of the thick albumen by thin albumen or with replacement of the thick albumen by thin albumen diluted with solution of salts (method D). Embryos were then incubated until hatching. The rates of hatching of the cultured embryos were 34.4%, 16.2%, 50.0%, 6.9-26.7%, and 47.5% for methods A, B, C, D, and E, respectively. Thus the rate of hatching of cultured fertilized ova was increased by replacement of the thick albumen by thin albumen at the blastoderm stage. Chicks obtained by method A reached maturity and produced viable offspring, and this technique provides an improved method for the culture of fertilized ova to hatching.
Article
The embryonic lifespan of the chick is 22 days. Development in the first day takes place in the oviduct, and in the remaining 21 days in the shelled egg. There have been few attempts to culture oviductal embryos, though methods covering the first few days of development in ovo are well established and a method for the final 18 days of development through hatching has recently been devised. I have now succeeded in culturing the fertilized ovum of the chick (Gallus domesticus) for the total embryonic period by growing it in a series of separate culture systems. This is the first report of a complete in vitro method for a homoiothermic animal. The technique opens the way to the investigation of developmental events in birds that require access to the embryo at the single-cell stage, and in particular to the genetic manipulation of the fertilized ovum.
Article
We report here a method to produce quail hatchlings by culture in vitro from the single-cell stage. The culture is composed of three steps. In the first step, the fertilized ovum surrounded by thick albumen obtained from the magnum is cultured for 24 hr at 41.5 degrees C in a tightly sealed 20-ml plastic cup with chicken thin albumen added to the equator level of the ovum (System Q1). In the second step, a quail egg shell, cut horizontally and emptied, is used as a bed shell. After the thick albumen is removed, the embryo with egg yolk is transferred to the bed shell and thin albumen from chicken eggs is added to fill the shell. Then, the embryo is cultured for an additional 52 hr at 37.5 degrees C while being rocked at an angle of 90 degrees at 30-min intervals (System Q2). The embryo is transferred again to a chicken bed shell and cultured at 37.5 degrees C with rocking at a 30-degree angle (System Q3). Just before hatching, the rocking of embryos is stopped. The procedure yielded a hatchability of 25%. For transgenesis, a plasmid construct containing a beta-actin-lacZ hybrid gene (pMiwZ) is microinjected into the ovum at the single-cell stage, which is cultured in vitro for 85-90 hr using Systems Q1 and Q2 consecutively. Seven out of 17 surviving embryos exhibited lacZ gene expression in embryonic tissues as detected by histochemistry. The procedure described here should be highly applicable for the production of transgenic birds.
Article
The establishment of avian embryonic culture is important both for the analysis of the developmental process and the establishment of transgenic chickens that produce useful biological materials in eggs. However, the hatchability of cultured embryos has been approximately 50%. We identified that the low rate of hatchability of cultured embryos was caused by limited oxygen and calcium availability. In quail embryo culture using chicken eggshell as a culture vessel, viability in the middle stage of culture was improved and 30% of embryos were hatched by oxygen enrichment. Furthermore, hatchability increased to 80% by supplementation with calcium lactate in addition to oxygen aeration. In the present study, a fully artificial vessel for quail embryo culture was designed using a gas-permeable Teflon membrane. By the addition of fine eggshell powder and calcium lactate, quail embryos grew and developed normally, and 43% of embryos hatched. Although the hatchability was lower than that of cultures using a surrogate eggshell, we achieved in hatching an avian embryo using a fully artificial vessel.
Article
The chick stage X blastoderm is routinely accessed through a small window in a freshly laid egg. However, windowing severely compromises embryo survival with hatch rates as low as a few percent. We previously reported a simple modification to the standard method that reduced introduction of air into the sealed egg and improved the hatchability to 32%. Here, we describe an even simpler and more rapid method for sealing a windowed egg using hot glue or paraffin in which the hatch rate increased to an average of 63% of the unwindowed control eggs. The primary reason for low hatchability can be attributed to air trapped within the egg during windowing and/or leakage during incubation, as shown by increased lethality by artificially introducing air into windowed and sealed eggs. Although the hatch rate was considerably improved, air can still enter the egg during incubation and is likely to account for less than 100% hatchability of the sealed eggs. The success of this new windowing method will facilitate high throughput for the production of transgenic birds and find use in developmental biology, toxicity testing, and avian disease research.
Article
Transgenic birds were expected to be an excellent transgenic bioreactor for the production of recombinant pharmaceutical proteins. However, the only successful transgenic bioreactors have been based on mammals. We have developed two key techniques for obtaining transgenic birds. For bird embryo culture, we identified that the low rate of hatchability of cultured embryos is caused by limited oxygen and calcium availability. In quail embryo culture using a chicken eggshell as a culture vessel, hatchability increased to 80% by the supplement of calcium lactate in addition to oxygen aeration. A fully artificial vessel for quail embryo culture using a gas-permeable Teflon membrane was also designed. Although the hatchability was lower than that of cultures using a surrogate eggshell, we succeeded in hatching of bird embryos using a fully artificial vessel. For transgene introduction, a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G) was injected to laid embryos at the blastodermal stage, and the embryos were hatched in vitro to generate G0 birds. The viral vector sequence was detected in the tissues of all G0 birds. The germ-line transmission efficiency was more than 80%. Plural copies of the transgene were inserted into the genome of G1 transgenic progeny.
Article
The blue-breasted quail (Coturnix chinensis), the smallest species in the order Galliforms, is a candidate model animal for avian developmental engineering because it is precocious and prolific. This species requires 17 days to hatch and 8 to 9 weeks to mature to an adult body weight of about 50 g, whereas the Japanese quail (Coturnix japonica) requires 16 days to hatch and 6 to 8 weeks to mature to an adult body weight of 100 to 150 g. The early embryo is the most challenging embryonic stage in terms of culture and manipulation for avian biotechnology. We have evaluated various conditions for the culture of blue-breasted quail embryos from the blastoderm stage to hatching. A hatchability rate of 26% (10/39) is among the best of the various culture conditions examined in the present study and the embryo culture system should facilitate advances in avian biotechnology.