Developmental changes in free D-aspartic acid in the chicken embryo and in the neonatal rat

Free D-aspartic acid was measured in fertilized chicken eggs, chicken embryos, and neonatal rats. In each tissue examined a maximum value was found at a characteristic time of development. For the chicken embryo brain, the maximum was 9% D at 11 days of incubation; for the retina, 20% D at 13 days of incubation. In the neonatal rat, as in the chicken embryo, D-aspartic acid continued to increase in the retina after that in the brain and other tissues had begun to decline. The maximum, 29% D, was found 7 days after birth. Thus in two phylogenetically distant species, similar developmental patterns of D-aspartic acid change were observed. Some data on similarities between the D/L aspartic acid ratios of adult chicken and rat tissues are also reported. In addition, the total D-aspartic acid content of the egg, including the embryo, increased from 44 nmol at day 1 to 159 nmol at day 12, showing that release from a bound form or de novo synthesis is a continuing process during development.     

Laying, egg and hatchability characteristics in ostrich (Struthio camelus) at different age

The experimental material comprised 7 ostrich families (7 males and 14 females) of which five families were at the age of 7 and two at the age of 5 years. In the course of the entire reproductive season, the following parameters were analysed: length of the laying period, mean laying rate, number of eggs laid by one female, proportion ofhatching eggs, egg weight and shape, egg weight lost during incubation, egg fertilisation, percentage of dead embryos and unhatched chicks, hatchability from fertilised and set eggs. Seven year-old ostriches were characterised by shorter laying period (134 days) but, at the same time, by higher proportions of hatching eggs. This group was also characterised by high egg fertilisation (79.7%) as well as high hatchability indices at simultaneous highest embryo mortality during incubation (11.6 %). Five year-old ostriches exhibited a longer laying period (175 days) during which females laid more eggs (49 pcs.). In addition, this group was characterised by a smaller proportion of hatching eggs, better egg fertilisation indices (83.5%) and hatchability results. Moreover, the determined higher egg shape index indicates that the 5 year-old females laid eggs which were more spherical. Recapitulating, the obtained results indicate that, under Polish conditions, better indices of laying performance, egg fertilisation and hatchability were observed in the group of 5 year-old ostriches.     

Embryonic development of the domestic guinea fowl (Numida meleagris)

Inside the viable range 36–39°C, the development of the domestic guinea fowl embryo was studied during artificial incubation of about 5000 eggs. Equations giving internal and external pipping and hatching time as a function of incubation temperature were developed. Whatever temperature was used, internal and external pipping occurred at 89% and 95% of hatching time, respectively. The chronological development in ovo of the guinea fowl is illustrated by growth curves. Guinea fowl grew in ovo at the same rate as the chicken embryo. The embryonic mortality is significantly affected by incubation temperature. The thermal tolerance of the embryo follows a parabolic curve: temperatures below or above 37.2°C increase the rate of mortality. During the course of the incubation, the mortality frequency was mainly distributed around two peaks (each one third of the total), which occurred during the first days of incubation, that is, within the first 12% of the incubation time, and during the last days, that is, after 85% of the incubation time. At the optimal 37.2°C temperature, these two peaks occurred on days 1–3 and 23–27, respectively.     

Plasticity in incubation behavior and shading by king rails Rallus elegans in response to temperature

King rails experience a wide range of temperatures during the course of the breeding season throughout their rapidly contracting geographic range. Incubating parent birds are adapted to keep their eggs within a temperature range appropriate for embryo development, but king rail clutches are at risk of exceeding lethal temperatures in the latter half of the nesting season. We investigated whether behavioral plasticity during incubation enables parents to maintain clutch temperature within tolerable limits for embryo development. Video revealed that king rail parents interrupted incubation to stand above and shade their eggs. We tested the hypothesis that the onset of shading was a direct response to ambient temperature (adaptive plasticity). We monitored clutch temperature directly by experimentally adding into clutches a model egg embedded with a programmable iButton. We measured ambient temperature at the nest site simultaneously. Parents spent proportionately more time shading and less time incubating their eggs at higher ambient temperatures. Shading may primarily function in cooling the parent. The frequency and duration of shading bouts were significantly greater at higher ambient temperatures. Parents also took more frequent but shorter recesses in hotter conditions. Diurnal recesses exposed eggs to direct sunlight, and the highest clutch temperatures were recorded under these conditions. Complete hatching failure in at least one nest was attributable to high clutch temperature for an extended period. Because mean ambient temperature increases throughout the breeding season, we investigated seasonal patterns in onset of incubation and its effect on hatching rate. Later in the season, parents tended to initiate incubation earlier, and hatching asynchrony increased significantly. Together these results suggest that breeding king rails may be constrained in their ability to cope with sustained high temperatures should seasonal averages continue to rise as predicted.     

Plasticity of brood patch development and its influence on incubation periods in the yellow-eyed penguin Megadyptes antipodes: an experimental approach

In many bird species, eggs laid late in the laying period hatch after a shorter incubation period than early-laid eggs. However, the mechanisms that explain these seasonal declines in incubation periods among clutches remain poorly understood. In this study we investigated the plasticity of brood patch development during incubation in yellow-eyed penguins Megadyptes antipodes and established whether differences exist in brood patch formation among early, mean and late-breeding penguins. We also examined whether brood patch development was influenced by sex and age of birds. We then placed an artificial egg in nests a few days prior to egg laying to investigate whether the presence of an egg influences brood patch development and whether an advanced brood patch development at the time of egg laying causes declines in incubation periods. Initial brood patch width on the day the first egg was laid was dependent on sex and age, while the development of brood patch width after first egg laying was slower in early-laying birds than in mean- and late-laying birds. Initial brood patch temperature as well as temperature throughout incubation was largely dependent upon sex, whereby males had higher brood patch surface temperatures than females. Placement of an artificial egg in nests stimulated successfully brood patch development in manipulated birds, so that by the time they laid their own first egg, their brood patches were wider and had higher temperatures than those of control birds. Moreover, incubation periods of first eggs from manipulated nests were significantly shorter (43.5 days) than were those from control nests (47.3 days). Thus, variation in brood patch development and related differences in incubation temperature during early incubation could contribute to seasonal declines in incubation periods.     

Avian Egg Odour Encodes Information on Embryo Sex,Fertility and Development

Avian chemical communication is a rapidly emerging field, but has been hampered by a critical lack of information on volatile chemicals that communicate ecologically relevant information (semiochemicals). A possible, but as yet unexplored, function of olfaction and chemical communication in birds is in parent-embryo and embryo-embryo communication. Communication between parents and developing embryos may act to mediate parental behaviour, while communication between embryos can control the synchronicity of hatching. Embryonic vocalisations and vibrations have been implicated as a means of communication during the later stages of development but in the early stages, before embryos are capable of independent movement and vocalisation, this is not possible. Here we show that volatiles emitted from developing eggs of Japanese quail (Coturnix japonica) convey information on egg fertility, along with the sex and developmental status of the embryo. Specifically, egg volatiles changed over the course of incubation, differed between fertile and infertile eggs, and were predictive of embryo sex as early as day 1 of incubation. Egg odours therefore have the potential to facilitate parent-embryo and embryo-embryo interactions by allowing the assessment of key measures of embryonic development long before this is possible through other modalities. It also opens up the intriguing possibility that parents may be able to glean further relevant information from egg volatiles, such as the health, viability and heritage of embryos. By determining information conveyed by egg-derived volatiles, we hope to stimulate further investigation into the ecological role of egg odours.     

Changes of creatine concentration during development of chick embryo

Changes of creatine concentration during development of chick embryo. Acta physiol. pol., 1985, 36 (3): 208-215. Investigations were carried out on embryos of Star Cross chicken after 6, 8, 10, 12, 14, 16, 18 and 20 days of incubation. It was found that the concentration of total creatine increased from 0.18 to 0.67 mg/g of embryo weight. The increase of creatine concentration at the time of development of the embryo was proportional to the increase in nitrogen concentration, and the share of creatine nitrogen in the pool of total nitrogen was about 1% throughout the whole period of embryonal development and during the first two days after hatching. The amount of creatine in fresh egg and in the yolk sac of the newly hatched chicken was about 1.5 mg. It was estimated that chick embryos during their development synthesized, on the average, 18 mg (6 mMol) of creatine. The course of changes in creatine concentration in the developing chick embryo is very similar to the course of changes in the rate of heat production.     

Geographic variation in avian incubation periods and parental influences on embryonic temperature

Theory predicts shorter embryonic periods in species with greater embryo mortality risk and smaller body size. Field studies of 80 passerine species on three continents yielded data that largely conflicted with theory; incubation (embryonic) periods were longer rather than shorter in smaller species, and egg (embryo) mortality risk explained some variation within regions, but did not explain larger differences in incubation periods among geographic regions. Incubation behavior of parents seems to explain these discrepancies. Bird embryos are effectively ectothermic and depend on warmth provided by parents sitting on the eggs to attain proper temperatures for development. Parents of smaller species, plus tropical and southern hemisphere species, commonly exhibited lower nest attentiveness (percent of time spent on the nest incubating) than larger and northern hemisphere species. Lower nest attentiveness produced cooler minimum and average embryonic temperatures that were correlated with longer incubation periods independent of nest predation risk or body size. We experimentally tested this correlation by swapping eggs of species with cool incubation temperatures with eggs of species with warm incubation temperatures and similar egg mass. Incubation periods changed (shortened or lengthened) as expected and verified the importance of egg temperature on development rate. Slower development resulting from cooler temperatures may simply be a cost imposed on embryos by parents and may not enhance offspring quality. At the same time, incubation periods of transferred eggs did not match host species and reflect intrinsic differences among species that may result from nest predation and other selection pressures. Thus, geographic variation in embryonic development may reflect more complex interactions than previously recognized.     

Culture of naked quail (Coturnix coturnix japonica) ova in vitro for avian transgenesis: Culture from the single-cell stage to hatching with pH-adjusted chicken thick albumen

We first examined the pH change of the albumen of quail (Coturnix coturnix japonica) eggs before and after they were laid, as well as that of laid eggs. The pH rose rapidly after laying and continued to increase gradually in storage. Incubation at 37.5°C accelerated the increase in the pH of infertile eggs, while that of fertile eggs remained low during incubation. Referring to these results, we obtained a protocol for producing quail hatchlings by culture in vitro from naked ova. The naked ovum was filled with chicken (Gallus domesticus) thick albumen, the pH of which had been adjusted to 7.2–7.4. The ovum was cultured at 41.5°C in 20% CO₂ in air for the first 24 h. Then the embryo was moved to a surrogate quail egg shell that had been filled with non-pH-adjusted chicken thin albumen and cultured for a further 48 h in 100% air. The embryo was transferred again to a surrogate chicken egg shell and cultured under the same conditions until hatching. The culture yielded quail chicks with a hatchability of 19.4%. The method proposed here should be applicable to the production of transgenic birds.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Does solitary incubation enhance egg water uptake and offspring quality in a lizard that produces single-egg clutches?

Many organisms invariably produce one offspring per reproductive bout, but experimental tests of adaptive explanations for this reproductive pattern are rare. To address this issue, we studied a lizard (Anolis sagrei) that produces one egg at a time to test the hypothesis that solitary incubation (due to single-egg clutches) eliminates competition with adjacent eggs for moisture and thus enhances offspring quality via increased egg water uptake during development. Our findings suggest that solitary incubation does not affect rates of moisture uptake by eggs or offspring size. However, egg moisture uptake and offspring size were negatively affected when eggs were adjacent to an egg that died during development. Depending on rates of infertile eggs or embryo mortality in the field, single-egg clutches may improve developmental environments and enhance offspring fitness. These results highlight the importance of considering the role of plastic embryonic responses during development in explaining reproductive patterns.     

A new candling procedure for thick and opaque eggs and its application to avian conservation management

The ability to monitor developing avian embryos and their associated vascular system via candling enables the application of important reproductive management techniques. Egg candling facilitates the confirmation of egg viability throughout the incubation process and identification of a precise position on a vein for the safe extraction of blood. Blood samples may then be analysed to retrieve vital health and genetic information to assist in conservation management. However, the thick or opaque egg shell characteristics of some avian species prevents the observation of egg contents using traditional candling methods, thus limiting management options. This paper tests a novel method of preparing thick-shelled or opaque eggs so that traditional egg candling and blood extraction methods may be applied. Eggs from captive emu (Dromaius novaehollandiae, Latham 1790) and southern cassowary (Casuarius casuarius johnsonii, Linnaeus 1758) were obtained, and partial fenestration was performed on two areas of shell either before incubation or at ⅓ of incubation. Hatchability and weight loss were examined as a measure of effect of the fenestration process on the developing embryo. Clear observation of vascular development was successful in 97% of viable fenestrated eggs, without affecting hatchability or weight loss. Blood samples were taken from developing embryos and DNA was successfully extracted for proof of concept of this new technique. The ability to observe vascular development and monitor the developing embryo in thick and opaque eggs will significantly improve both in situ and ex situ population management options such as in ovo sexing in species of concern.     

Size and visibility of Baltic cod eggs with reference to size-selective and stage dependent predation mortality

Size of Baltic cod eggs from incubation experiments and from field samples was determined by microscopic analysis. Results from plankton samples were compared with corresponding size distributions of cod eggs found in herring stomachs. The influence of fixation on size of different developmental stages was studied. Live eggs from incubation experiments were also sized repeatedly throughout the developmental period with an optical plankton counter (OPC) based on light attenuance measurements as this was assumed to be more closely related to the visibility of the eggs for potential predators than egg diameter as obtained by microscopic analysis.
Preservation in formaldehyde solution caused a small reduction in egg diameter (2.2%) whereby no differences between the developmental stages were detected. Egg size decreased slightly during incubation (6.9%) while the OPC measurements revealed a substantial increase in light attenuance during egg development (42.2%). In the field, a general decrease in egg size with increasing depth was observed while no change between the developmental stages was detectable. The mean size of eggs ingested by herring was slightly lower than in the water column which was most pronounced for the late stages containing a well-developed embryo. The frequency of eggs in an advanced stage of development was considerably higher in the stomachs than in corresponding plankton samples. Therefore, it is suggested that the selection of further developed egg stages by predatory fish in the central Baltic Sea, i.e. herring and sprat, is due to an increase of visibility during egg development in relation to growth and pigmentation of the embryo. Thus it is likely that egg mortality due to predation is stage-dependent rather than strictly size-selective.     

The thermal energetics of an incubated chicken egg

I incubated chicken eggs using an artificial brood patch, which enabled me to measure the energy required to keep chicken eggs, 16–19 days old, warmer than the ambient air. To keep brood patch temperature (i.e. at the egg surface-brood patch interface) 9.4°C warmer than the ambient air, living chicken eggs require on average 541 mW, of which 403 mW is supplied by the parent, the remainder being supplied by the embryo. Killing the embryo reduces the power required to only 322 mW for the same elevation of the brood patch temperature, all of it supplied by the brood patch. Killing the embryo causes central egg temperature to decline by about 2.0°C. Contrary to widely held opinion, it is embryonic circulation, not embryonic metabolism that is the most important cause of elevated temperature of incubated eggs. The implications of this for incubation energetics are discussed.     

Developmental stage affects eggshell-breaking strength in two ground-nesting birds: the partridge (Alectoris rufa) and the quail (Coturnix japonica)

We examined the relationship between embryo development and egg hardness in two ground nesting bird species, the red-legged partridge (Alectoris rufa; n = 165 eggs) and the quail (Coturnix japonica; n = 148 eggs). For both species, we observed a strong effect of developmental stage on egg hardness. Eggs near hatching were significantly weaker than unincubated eggs (partridge: 18 and 23 N, respectively, and the quail 7 and 10 N, respectively). We additionally explored the effect of incubation on egg hardness in a control sample of non-fertilised quail eggs (i.e., without embryo development). The control eggs maintained in the incubator for the full incubation time (17 days) were significantly harder (7-9 N) than eggs containing fully developed chicks (5-7 N). Thus, the incubation conditions of high temperature and humidity alone seem not to have a significant effect on egg hardness, and support the important effect of calcium uptake.     

Egg Turning During Incubation has no Effect Upon the Growth of Embryos of Alligator mississippiensis

Alligator eggs are not turned during incubation, instead the embryo adheres to the top inside of the shell. Turning is alleged to shear off the embryo and kill it. Avian egg turning allegedly facilitates embryonic development by stimulating growth of the area vasculosa and minimizing the effects of unstirred yolk and albumen layers. From day 10 to day 45 of incubation, alligator eggs were experimentally turned, gently, through ± 60° in an hourly cycle. This turning regime killed only 6 out of 25 embryos. Compared with unturned controls, no significant effects were observed on the growth, production of extraembryonic fluids or utilization of albumen and yolk for those embryos that survived turning. The protein concentration of amniotic fluid at various stages of alligator development was examined in eggs incubated at 30 and 33°C. The fluid contained very little protein (max <8 mg) at any time: the protein concentration did not change consistently as development progressed. Differences in response to egg turning in birds and reptiles may be associated with the length of the incubation period, the protein content of the albumen and the mechanism of albumen utilization.     

Solubilization and Identification of Hen Eggshell Membrane Proteins During Different Times of Chicken Embryo Development Using the Proteomic Approach

A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca²⁺-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris–HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).     

Egg temperature and initial brood patch area determine hatching asynchrony in Magellanic penguin Spheniscus magellanicus

In birds, the adaptive significance of hatching asynchrony has been under debate for many years and the parental effects on hatching asynchrony have been largely assumed but not often tested. Some authors suggest that hatching asynchrony depends on the incubation onset and many factors have been shown to influence hatching asynchrony in different species. Our objective was to analyze the exact timing of the onset of incubation and if this affects hatching asynchrony; and, in addition, which other factors (brood patch development, incubation position, adult body condition, intra‐clutch egg dimorphism, laying date and year) affect hatching asynchrony in Magellanic penguins Spheniscus magellanicus. We first estimated the eggshell temperature at which embryo development starts, with a non‐destructive and novel method. We then recorded individual egg temperatures in 61 nests during incubation, and related them, and other breeding parameters, to hatching asynchrony. We also observed incubation positions in 307 nests. We found a significant positive relationship between hatching asynchrony and the temperature that the first‐laid egg experienced during egg laying and between hatching asynchrony and the initial brood patch area. We also found a negative relationship between hatching asynchrony and the difference in temperature between second and first‐laid eggs within a clutch, measured after the egg‐laying period was finished. We ruled out position of the eggs during incubation, adult body condition, egg volume, laying date, and study year as factors influencing hatching asynchrony. The egg temperature during laying and the difference in temperature between eggs of a clutch are determinants of hatching asynchrony in Magellanic penguins.     

Windowing chicken eggs for developmental studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model. The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation. These experiments have yielded important data on the development of the central and peripheral nervous systems. For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth. The embryo can be examined at successive developmental stages by re-opening the eggshell. While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times. In this method, the egg rests on its side and a small window is cut in the shell. After the experimental procedure, the shell is used to cover the egg for the duration of its development. Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period. This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.     

Nitrogen excretion during embryonic development of the green iguana, Iguana iguana (Reptilia; Squamata)

Development within the cleidoic egg of birds and reptiles presents the embryo with the problem of accumulation of wastes from nitrogen metabolism. Ammonia derived from protein catabolism is converted into the less toxic product urea or relatively insoluble uric acid. The pattern of nitrogen excretion of the green iguana, Iguana iguana, was determined during embryonic development using samples from allantoic fluid and from the whole homogenized egg, and in hatchlings and adults using samples of blood plasma. Urea was the major excretory product over the course of embryonic development. It was found in higher concentrations in the allantoic sac, suggesting that there is a mechanism present on the allantoic membrane enabling the concentration of urea. The newly hatched iguana still produced urea while adults produced uric acid. The time course of this shift in the type of nitrogen waste was not determined but the change is likely to be related to the water relations associated with the terrestrial habit of the adult. The green iguana produces parchment-shelled eggs that double in mass during incubation due to water absorption; the eggs also accumulate 0.02 mM of urea, representing 82% of the total measured nitrogenous residues that accumulate inside the allantois. The increase in egg mass and urea concentration became significant after 55 days of incubation then were unchanged until hatching.