Embryonic osmoregulation: consequences of high and low water loss during incubation of the chicken egg

The rates of water loss of domestic chicken eggs were varied during incubation to measure the osmoregulatory ability of the avian embryo. Egg water loss was increased by drilling holes in the eggshell over the airspace on day 13 (I = 21 days) and then placing these eggs in a low relative humidity (r.h.: 0-10%) incubator until hatch. Egg water loss was decreased by placing other eggs in a high-r.h. (85-90%) incubator on day 0. Eggs with low water loss (approximately 6% of initial fresh mass [IFM]) produced embryos and yolks that were not different in wet or dry mass when compared to control eggs that lost approximately 12% of IFM. However, 1-4 gm of excess albumen were left in low-water-loss eggs on day 21. Hatching success was 71% and 89% for low and control eggs, respectively. Low egg water loss did not appear to disturb embryonic growth. The allantoic fluid volume and millimolar allantoic Na+ and Cl- ions declined faster with high and slower with low rates of water loss. Thus, excess water was lost as a result of increased movement of water out of allantoic fluid, which was due to increased active transport of Na+ ions by the chorioallantoic membrane (CAM). Eggs with high water loss had elevated Cl- levels after day 17 in plasma and amniotic fluid, which indicated a period of osmotic stress after depletion of allantoic fluid between day 18 and hatch. The decrease in wet embryo mass measured in embryos from high-water-loss eggs was due principally to dehydration of skin. Embryonic skin may serve as an emergency water reservoir during osmotic stress. Dehydrated chicks produced from high-water-loss eggs were 6 gm less in wet mass at hatch compared to controls. However, these chicks regained the water deficit 7 days after hatch and grew at a rate not different from control chicks through 6 weeks of age. Total egg water loss of 12% of IFM results in highest hatching success. However, water losses between 6% and 20% of IFM do not appear to affect adversely the growth or water content of the chick. Water losses above 20% of IFM cause early depletion of allantoic fluid, prolong the period of osmotic stress, and result in subsequent dehydration of blood, amniotic fluid, and embryonic skin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Eggshell porosity and the water metabolism of the chick embryo

Chick embryos have been water stressed during the last week of incubation by either withdrawing 6 cm³ of allantoic fluid or by increasing shell porosity three to four fold. Despite the large increase in water loss from these eggs they were successfully incubated to produce hatchlings of apparently normal water content. The chicks produced by water deprivation were significantly smaller than controls and it appears that the growth of embryos is therefore modulated according to the metabolites available. If this interpretation is correct it suggests that the critical role of eggshell porosity in avian development has been over-emphasized.     

STUDIES ON THE pH AND Eh OF NORMAL AND INFLUENZA-INFECTED EGGS

1. pH values of allantoic fluids from non-infected eggs showed a rapid drop from the 12th to the 17th day of embryonic development. A definite age-pH relationship was shown to exist. 2. The allantoic fluid of eggs infected with influenza virus, contrasting with the non-infected eggs, revealed pH values which remained relatively stable about the neutral point at any age between 11 and 17 days. No relationship between pH and age could be established, in the infected series. 3. The Eh value of the fluid from non-infected eggs also exhibited a significant relationship to embryonic age. The majority of the determinations revealed a positive potential. 4. Similar determination on influenza-infected eggs gave results of a predominantly negative potential, indicating a marked state of reduction accompanying virus multiplication. As with the pH determinations, the Eh values of infected eggs were not a function of their age.     

The relationship of pores and mammillae on the inner surface of the eggshell of the alligator (Alligator mississippiensis)

The morphology of the eggshell of the alligator, Alligator mississippiensis, is similar to that of birds. In many avian species there is a positive linear correlation between the numbers of pores and mammillae on the inner surfaces of eggshells, indicating that the distribution and density of mammillae may determine the porosity of the shell. It is not known, however, if a relationship exists between pores and mammillae on the shell of the alligator. Using a scanning electron microscope, we counted pores and mammillae on the inner surfaces of pieces of shell from the middle of fertile and infertile eggs from wild and captive, pen-reared alligators. Data were analyzed by ANOVA, Duncan's multiple range tests and linear regression equations. Results demonstrate a positive linear correlation between the numbers of pores and mammillae on the shells of unincubated fertile and infertile eggs from wild and captive alligators; however, there is no correlation between pores and mammillae on shells of eggs that were incubated for 55 days. It is suggested that initially the porosity of the eggshell of the alligator is related to the density of mammillae on the inner surface of the shell and that erosion of the shell during incubation destroys the original relationship between pores and mammillae.     

Oxygen and CO2 Exchange and Acid-Base Regulation in the Avian Embryo

The avian embryo exchanges the oxygen and carbon dioxide withthe ambient air by diffusion. The respiratory organ is the chorioallantois,endowed with a rich circulation. Between ambient air and chorioallantoiccapillary blood are interposed the porous shell fibrous shellmembranes, and the chorioendothelium which compose the diffusionbarrier. The air cell is formed between the two shell membranesin the blunt end of the egg. The diffusion barrier is dividedinto an outer barrier (shell plus outer membrane) and an innerbarrier (inner membrane plus chorioendothelium and capillaryblood). The resistance to gas diffusion (the reciprocal of thediffusive conductance) in the outer barrier is almost fixedthroughout incubation while that in the inner barrier decreasesas the embryo develops. Because of the fixed outer barrier conductance,the embryo is obliged to take up oxygen under hypoxic conditionsagainst increasing metabolism with development and encountersa relative respiratory acidosis. In connection with the diffusivehypoventilation caused by the fixed outer barrier conductancethe respiratory factors of the allantoic circulation changeprogressively with development to moderate the restraint ofgas exchange through the shell. Blood oxygen capacity and hemoglobinincrease with development in association with an increase inerythrocyte count and hematocrit value. In addition, a progressiveleftward shift of the oxygen dissociation curve occurs. Theincreases in the allantoic blood flow and chorioallantoic capillaryvolume contribute to the increasing conductance of the innerbarrier. Furthermore regulation of acid base balance is inferredin the developing embryo.     

Adaptation of the Avian Egg to High Altitude

Theoretical predictions and experiments on eggs of domesticatedbirds indicate that the diffusion coefficient of gases is inverselyproportional to barometric pressure. Therefore potentially lethallosses of CO₂ and water vapor from eggs laid at high altitudemight result if the increased tendency of gases to diffuse atreduced barometric pressure were not counteracted in some manner.Limited data from two wild populations indicate that water lossfrom eggs is independent of altitude over a 3000 m elevationalgradient. Four different possibilities are discussed by whichcompensation for increased diffusion of water vapor might beachieved at high elevations 1) a reduction in eggshell conductance(GH₂O) 2) an increase in the initial water content of the eggs3) an increase in shell thickness, and 4) alteration of watervapor pressure in the nest microenvironment or incubation temperatureby variation in parental behavior. Mean GH₂O of eggs of twoprecocial and four altricial species breeding above 2800 m issignificantly reduced below values of related birds breedingat lower elevations, but no change in initial water contentor shell thickness has been observed in such eggs. Observationsof parental behavior in species breeding over wide elevationalgradients have not yet been made. Identification of the mechanismsby which eggshell structure is modified to achieve a reducedGH₂O the environmental cues used by females to determine theelevation of the breeding location and the rapidity with whichshell structuie can be modified awaits further research.     

The effect of calcium-regulating hormones on transport of calcium across the chorioallantoic membrane of the chicken embryo

The hormonal form of vitamin D3 (1,25(OH)2D3), parathyroid hormone (PTH), or appropriate vehicle were injected into the yolk sac of eggs of domestic fowl on days 16 and 17 of incubation. The chorioallantoic membrane (CAM) and overlying inner shell membrane were removed from eggs on day 18 and mounted in a Ussing-type apparatus. Transport of calcium was assessed by monitoring movements of radiolabeled calcium. Transport of calcium from the chorionic aspect of the CAM to the allantoic aspect increased considerably with time for all treatment groups except the one receiving PTH. "Back-flux" of calcium (movement of calcium from the allantoic aspect to the chorionic) was negligible for all treatment groups at all sampling periods. PTH treatment did not affect flux of calcium from allantois to chorion but reduced flux from chorion to allantois considerably. The underlying cause of this effect has not been identified. The hormonal form of vitamin D3 did not affect flux of calcium in either direction. These data raise the possibility that control of calcium transport by the CAM may not be the primary function of the vitamin D hormone.     

Gas Exchange of the Avian Egg Time, Structure, and Function

Data are presented for oxygen consumption water loss duringincubation water vapor conductance of the shell and pore numberof avian eggs and the way in which these values relate not onlyto egg mass but also to incubation time. It is proposed thatall these functions are proportional to the product of egg massand rate of development where the latter is defined as the inverseof incubation time. These interrelationships account at theend of incubation for similar O₂ and CO₂ tensions in the airspace of eggs utilization of calories (0.5 kcal g^–1) andwater loss (15 g g^–1)     

Variation in eggshell traits between geographically distant populations of pied flycatchers Ficedula hypoleuca

The expression and impact of maternal effects may vary greatly between populations and environments. However, little is known about large‐scale geographical patterns of variation in maternal deposition to eggs. In birds, as in other oviparous animals, the outermost maternal component of an egg is the shell, which protects the embryo, provides essential mineral resources and allows its interaction with the environment in the form of gas exchange. In this study, we explored variation of eggshell traits (mass, thickness, pore density and pigmentation) across 15 pied flycatcher populations at a large geographic scale. We found significant between‐population variation in all eggshell traits, except in pore density, suggesting spatial variation in their adaptive benefits or in the females’ physiological limitations during egg laying. Between‐ population variation in shell structure was not due to geographic location (latitude and longitude) or habitat type. However, eggshells were thicker in populations that experienced higher ambient temperature during egg laying. This could be a result of maternal resource allocation to the shell being constrained under low temperatures or of an adaptation to reduce egg water loss under high temperatures. We also found that eggshell colour intensity was positively associated with biliverdin pigment concentration, shell thickness and pore density. To conclude, our findings reveal large‐ scale between‐population variation of eggshell traits, although we found little environmental dependency in their expression. Our findings call for further studies that explore other environmental factors (e.g. calcium availability and pollution levels) and social factors like sexual selection intensity that may account for differences in shell structure between populations.     

Structure of fossil dinosaur eggshell from the Aix Basin,France

At least four types of dinosaur eggshell are distinguished among samples of fossil eggshell from Late Cretaceous deposits of southern France. Recognition is based on shell microstructure, porosity, and shell thickness. Estimated values of water vapour conductance for these dinosaur eggshells are much greather than predicted for avian eggs, suggesting that the eggs were incubated under conditions of high humidity, such as would occur underground.     

Effect of urea and uric acid on the hemolytic activity of Sendai virus

It was found that haemolytic activity of Fushimi strain of Sendai virus multiplied in allantoic cavity of chicken embryos is independent on its haemagglutinating titer and also on allantoic fluid urea and uric acid content. It was shown in experiments with embryonated eggs that these two compounds have no also influence on haemolytic activity induction in Sendai virus. Moreover, the results of an experiment in which allantoic fluid was replaced by Eagle's liquid suggest that most probably the other components present in allantoic fluid do not also influence the appearance of haemolytic activity of this virus.     

The effect of chlamydial infection on the initiation of premature labour: serial measurements of intrauterine prostaglandin E2 in amniotic fluid, allantoic fluid and utero-ovarian vein, using catheterised sheep experimentally infected with an ovine abortion strain of Chlamydia psittaci

The initiation of premature labour by an ovine abortion strain of Chlamydia psittaci was studied in relation to Prostaglandin E2 (PGE2), which plays a major role in parturition. The local intrauterine concentration of PGE2 was monitored after experimental infection with C. psittaci, during late gestation, using a catheterised sheep model. Indwelling catheters were implanted into the amniotic and allantoic cavities of six control sheep, and into six sheep infected with an ovine abortion of C. psittaci. The release of PGE2 into the utero-ovarian vein of these sheep was also monitored. Infection with C. psittaci was associated with a premature rise in PGE2 in the amniotic fluid between days 122 and 135 of gestation (P less than 0.01). A premature increase in PGE2 was defected between days 127 and 136 of gestation (P less than 0.05) in the allantoic fluid of sheep infected with C. psittaci, but not in the control uninfected sheep. Chlamydial infection significantly decreased the secretion of PGE2 into the utero-ovarian vein. The mean concentration of PGE2 in the utero-ovarian vein of infected sheep was 68.8 +/- 5.2% of the PGE2 concentration of control sheep between days 122 to 141 of gestation (P less than 0.01). The release of PGE2 into the utero-ovarian vein increased between days 126 to 136 of gestation in infected and control sheep (P less than 0.01). The results from the catheterised sheep model indicated that C. psittaci infection is associated with a local intrauterine release of PGE2. The magnitude of this release was similar to the PGE2 release in control sheep prior to parturition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the murine allantois by allantoic explants

The murine allantois will become the umbilical artery and vein of the chorioallantoic placenta. In previous studies, growth and differentiation of the allantois had been elucidated in whole embryos. In this study, the extent to which explanted allantoises grow and differentiate outside of the conceptus was investigated. The explant model was then used to elucidate cell and growth factor requirements in allantoic development. Early headfold-stage murine allantoises were explanted directly onto tissue culture plastic or suspended in test tubes. Explanted allantoises vascularized with distal-to-proximal polarity, they exhibited many of the same signaling factors used by the vitelline and cardiovascular systems, and they contained at least three cell types whose identity, gene expression profiles, topographical associations, and behavior resembled those of intact allantoises. DiI labeling further revealed that isolated allantoises grew and vascularized in the absence of significant cell mingling, thereby supporting a model of mesodermal differentiation in the allantois that is position- and possibly age-dependent. Manipulation of allantoic explants by varying growth media demonstrated that the allantoic endothelial cell lineage, like that of other embryonic vasculatures, is responsive to VEGF(164). Although VEGF(164) was required for both survival and proliferation of allantoic angioblasts, it was not sufficient to induce appropriate epithelialization of these cells. Rather, other VEGF isoforms and/or the outer sheath of mesothelium, whose maintenance did not appear to be dependent upon endothelium, may also play important roles. On the basis of these findings, we propose murine allantoic explants as a new tool for shedding light not only on allantoic development, but for elucidating universal mechanisms of blood vessel formation, including vascular supporting cells, either in the intact organism or in existing in vitro systems.     

A comparison of indices and measured values of eggshell thickness of different shell regions using museum eggs of 230 European bird species

The thickness of avian eggshells is used to assess shell quality in wild and domestic species, as an indicator of environmental pollution and as an adaptive explanation for shell maculation. Both direct measurements and calculated eggshell thickness indices (ETI) are used in such research, yet this is the first study to quantify, across a large spectrum of bird families (and thus egg shapes), the correlation between measured thicknesses and ETI. Furthermore, few studies have quantified thickness variation across the entire length of the shell, although this variation may influence both gas transfer and embryonic development. We measured the thickness of 942 eggshells of 230 European bird species from the Class II material at the Natural History Museum, Tring, UK, both in the conventional manner, at the equator through the blowhole and, uniquely, after a single longitudinal, cut at its equator at the blunt and pointed ends. Over half of the samples revealed shell defects, cautioning against the indiscriminate use of museum specimens. Strong positive associations were found between species‐specific means of shell thickness with each other and also with ETI, especially those derived from Schönwetter's ‘Handbuch der Oologie’ method, validating the interspecific comparative use of ETI. Thickness measurements and ETI factors are provided for all 230 species. Eggshells were usually thinner at the blunt end (the location of the air sac) than at the equator, but of equal thickness in passerine eggs. This difference was greatest in species producing elongate eggs and suggests that there is a functional significance of shell thickness variation among species that requires further investigation.     

Do pregnant lizards resorb or abort inviable eggs and embryos? Morphological evidence from an Australian skink,Pseudemoia pagenstecheri

Although pregnant viviparous squamates are sometimes claimed to be able to resorb inviable eggs and embryos from the uterus, definitive evidence for such resorption is not available. After placing pregnant female Pseudemoia pagenstecheri into conditions under which embryonic development is terminated, we periodically harvested the gravid oviducts and examined them histologically. Females contained abnormal and degenerating eggs and embryos that had died in various stages of development. Dead embryos had undergone extensive cytolysis, dissolution, and aseptic necrosis and vitelline masses showed signs of deterioration and passage down the oviduct. The uterine mucosa lay in direct contact with the vitelline material, with no intact shell membrane intervening between them. Yolk was sometimes displaced into the exocoelom and allantoic cavity due to rupture of the extraembryonic membranes. Histological examination revealed no evidence of the uptake of yolk by the uterine epithelium or its accumulation in the subepithelial connective tissue. In many specimens, the uterine epithelium showed minuscule, apical granules. The position, appearance, and staining properties of the granules suggests them to be secretory, a manifestation of placentotrophy. Our observations indicate that P. pagenstecheri females retain dead eggs and embryos for several weeks or longer, yet do not resorb them during that period. This lizard is the second placentotrophic skink species in which resorption has been suspected, but in which abortive eggs appear to be retained or extruded instead of being resorbed by the oviducts. Researchers should not assume that squamates can digest and resorb oviductal eggs without definitive morphological evidence.     

Why are birds’ eggs speckled?

Birds are unique in laying eggs with pigmented shells, but for most species (e.g. most passerines, which lay white eggs speckled with reddish spots of protoporphyrin) the pigments’ function is unknown. We studied a bird population at a geologically variable site, and considered a hitherto untested hypothesis: that protoporphyrin pigments might compensate for reduced eggshell‐thickness (caused partly by calcium deficiency), which is known to reduce eggshell‐strength and increase eggshell‐permeability. We found that pigment spots specifically demarcated thinner areas of shell, with darker spots marking yet thinner shell than paler spots. Variation in pigmentation was thus associated with variation in shell thickness both within and between clutches, so accounting for the eggshell's characteristic spot patterns. Geological variability at this site has resulted in a great range of calcium availability and, as predicted by the hypothesis, variation in calcium availability was found to affect between‐clutch variation in both eggshell‐mass (+) and pigmentation characteristics (?). We suggest a physiological mechanism and some important implications of these findings.     

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.     

Local exchange of oxytocin from the ovarian vein to ovarian arteries in sheep

Local transfer of 125I-labeled oxytocin from the ovarian vein to arteries supplying the ovary, the oviduct, and the tip of the uterine born has been investigated. In five sheep, 10 infusions of 125I-oxytocin over a period of 1 h were performed, and the concentration of labeled polypeptide in the peripheral plasma was compared to ovarian arterial plasma. During 2 consecutive infusions into each animal's ovarian vein, blood was collected simultaneously from the following sites: ovarian branch of the ovarian artery (OBOA), tubal branch of the ovarian artery (TBOA), uterine branch of the ovarian artery (UBOA), and from the jugular vein. In all experiments the concentration of 125I-oxytocin in ovarian arterial plasma was higher than in peripheral plasma. The ratio of ovarian artery/jugular vein for 125I-oxytocin was: OBOA 2.8, TBOA 1.8, UBOA 1.6. Based on a 4 ml/min blood flow through ovarian arteries supplying ovary, oviduct, and the tip of the uterine horn, the local transfer of the total amount of oxytocin infused was estimated to be about 1% (range: 0.1-4.4%). Analysis of variance did not reveal significant differences in the exchange ratios between OBOA, TBOA, and OBOA. However, the variances within these groups are significant, presumably because of anatomical variation in the degree of surface contact area between arteries and veins at the ovarian pedicle. It is concluded that polypeptides are locally recirculated to ovaries, oviduct, and the tip of the uterine horn in a higher concentration than is supplied by peripheral blood. This could provide a mechanism for local distribution and concentration of the ovarian peptides that regulate reproductive function.     

Differences in egg size, shell thickness, pore density, pore diameter and water vapour conductance between first and second eggs of Snares Penguins Eudyptes robustus and their influence on hatching asynchrony

Brood reduction in birds is frequently induced by hatching asynchrony. Crested penguins (genus Eudyptes) are obligate brood reducers, but in contrast to most other birds, first‐laid eggs are considerably smaller in size than second‐laid eggs; furthermore, first‐laid eggs hatch after their siblings. The mechanisms underlying this reversal in size and hatching order remain unclear. In this study, we tested whether the second‐laid eggs of Snares Penguins Eudyptes robustus have a higher eggshell porosity allowing them to maintain a higher metabolic rate throughout incubation and to hatch before their first‐laid siblings. We investigated differences in egg size, shell thickness, pore density, pore diameter and water vapour conductance between first and second eggs within clutches and examined the influence of these shell characteristics on hatching asynchrony. First‐laid eggs of Snares Penguins were approximately 78% of the size of the larger second eggs. Second‐laid eggs had considerably thicker shells and more pores per cm² than first eggs, whereas pore diameter did not differ between eggs. Water vapour conductance was greater in second‐ (16.8 mg/day/torr) than in first‐laid eggs (14.9 mg/day/torr). The difference in water vapour conductance between first‐ and second‐laid eggs within clutches was related to hatching patterns. In nests where second eggs hatched before first‐laid eggs, second eggs had a considerably greater water conductance than their sibling, whereas in nests where both eggs hatched on the same day, the difference in water conductance between eggs was very small, and in a few nests where small first eggs hatched before their larger sibling, they had a greater water conductance than their larger second‐laid nestmate. Surprisingly few studies have investigated differences in shell characteristics between eggs within clutches and associated effects on hatching asynchrony. This study has demonstrated that such differences exist between eggs within clutches and that they can influence hatching patterns.     

Development of calcium reabsorption by the allantoic epithelium in chick embryos grown in shell-less culture

The allantoic sac of the chick embryo functions as a primitive urinary bladder, storing and modifying the excretory fluid produced by the embryo. We have used chick embryos grown in shell-less culture to study the in situ handling of Ca2+ by the allantoic epithelium. Between Days 8 and 13 of incubation (38 degrees C, 5% CO2), the [Ca2+] of the allantoic sac fluid declines from about 1.5 mM to less than 0.3 mM, with most of this Ca2+ reabsorption occurring between Days 10 and 11. In 13-day-old embryos, the allantoic epithelium reabsorbs within 24 hr 85-92% of 45Ca2+ injected into the allantoic sac, while in 9-day-old embryos 45Ca2+ reabsorption is less than 40% by 24 hr. This is evidence for the developmental onset of a Ca2+ reabsorption process in the allantoic epithelium. The allantoic fluid Ca2+ is reabsorbed into the embryo's blood in which the serum [Ca2+] is about 1.5 mM. Also, electrical potential profiles reveal that the serosal (mesenchymal) side of the allantoic epithelium is 15-30 mV positive compared to the mucosal (luminal) side. Thus, by electrochemical criteria this reabsorption process appears to be active.