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.     

Effects of temperature on embryonic development of the veiled chameleon, Chamaeleo calyptratus

Temperature dependence of development of the chameleon, Chamaeleo calyptratus, was assessed from observations on eggs incubated at 25, 28 and 30 degrees C. Overall, differentiation, growth in mass, and growth of the yolk sac and chorioallantois were the slowest at 25 degrees C but did not differ between 28 and 30 degrees C. The relative area of the yolk sac (YS), chorioallantoic membrane (CAM), and their precursor, the area opaca vasculosa (AV) was used to characterize developmental phases. During Phase 1, only the AV was present; development was characterized by differentiation with little increase in the size of the embryo. During Phase 2, the vascularized YS and CAM grew from about 10 to 100% coverage of the surface of the shell during a period of about two weeks. Differentiation and growth of the embryo were accordingly rapid. During Phase 3, the YS and CAM were fixed in size and the remainder of development was relatively slow. Characterization of embryonic development with respect to the relative area of the AV-YS-CAM highlighted the functional linkage between development and the systems that provide nutrients to embryos.     

The effects of simulated microgravity on avian embryonic development.

Based on the few reports available, microgravity (MG) can have adverse effects on the early development of vascularised extra-embryonic membranes in avian eggs. Whether gravity or oxygen availability is the stimulus for development of the blood vessels in the chorioallantoic membranes (CAM) remains unclear. Under gravity the blastoderm forms on top of the yolk sac, closest to the oxygen rich region beneath the shell membranes, and from there the CAM buds from an abdominal extension subsequently to form a close contact with shell membranes. Then as the embryo develops it spreads beneath the eggshell surface to maximise the surface area of the CAM vascular bed available for O2 uptake. To investigate how simulated MG influences development of the CAM and embryo we conducted experiments on chicken embryos during incubation in a 3D-clinostat (control or continuous MG treatment at 5 rpm). Further, to determine if CAM angiogenesis is directed towards regions of high O2 tension or gravity we investigated the effects of wax treatment (50% shell surface area) on development in MG. We found that clinostat MG caused embryonic failure between day 0-5 by preventing normal development of CAM-shell membrane complex. Thereafter acute MG promoted increases in CAM mass, but did not affect embryo mass. Preliminary findings suggest that combined acute MG and wax treatment did not significantly affect embryonic growth in either MG or control groups, but retarded CAM growth in control embryos only. Finally, we will present evidence to show that acute and prolonged exposure to MG does not prevent normal growth and hatching, but might have more subtle effects on hatchling physiology, including reduced heart mass.     

Embryonic growth and mobilization of energy and material during incubation in the checkered keelback snake, Xenochrophis piscator

We collected 20 checkered keelback snakes (Xenochrophis piscator) to study embryonic growth and mobilization of energy and material during incubation. Females laid eggs between late May and late June. The eggs were incubated at 27 degrees C (+/-0.3). One egg from each clutch was dissected at five-day intervals starting at oviposition. The mean incubation length at 27 degrees C was 48.9 days. We identified three phases of embryonic growth or yolk depletion in X. piscator. Phase 1, between oviposition and Day 20, was one of minimal transfer of energy and material from yolk to embryo. Phase 2, between Day 20 and Day 39-40, was characterized by increasingly rapid embryonic growth or yolk depletion. Phase 3, between Day 39-40 and hatching, was characterized by reduced embryonic growth or yolk depletion. Approximately 71% of dry mass, 53% of non-polar lipids and 66% of energy were transferred from the egg contents to the hatchling during incubation. Our data confirm that oviposition is not timed to coincide with the onset of rapid embryonic growth in oviparous squamate reptiles. The greater conversion efficiencies of energy and material from egg to hatchling in snakes can be attributed to their lower energetic costs of embryonic development and greater residual yolk sizes.     

Embryonic growth and mobilization of energy and material in oviposited eggs of the red-necked keelback snake, Rhabdophis tigrinus lateralis

We used the red-necked keelback (Rhabdophis tigrinus lateralis) as a model animal to study embryonic growth and mobilization of energy and material in oviposited snake eggs. Females (N=12) laid eggs between late May and early June. Eggs were incubated at 30 (+/-0.3) degrees C. One egg from each clutch was dissected at five-day intervals starting at oviposition. Incubation length averaged 27.9 days. Three phases of embryonic growth or yolk depletion could be detected in this study. The first phase, between oviposition and Day 10, was one of minimal transfer of energy and material from yolk to embryo. The second phase, between Day 10 and Day 22-23, was characterized by increasingly rapid embryonic growth and yolk depletion. The third phase, between Day 22-23 and hatching, was characterized by a gradual reduction in embryonic growth and yolk depletion. Approximately 73.6% of dry mass, 50.0% of non-polar lipids and 57.8% of energy were transferred from egg to embryo during incubation. Embryos withdrew mineral from the eggshell mainly during the last quarter of incubation. Our data show that oviposition does not coincide with the onset of rapid embryonic growth in oviparous species of squamate reptiles that are positioned midway within the oviparity-viviparity continuum, and that the greater conversion efficiencies of energy and material from egg to hatchling in snakes can be mainly attributed to their lower energetic costs of embryonic development and greater residual yolk sizes.     

Is water uptake by reptilian eggs regulated by physiological processes of embryos or a passive hydraulic response to developmental environments?

Moisture availability is critical for successful embryonic development in many organisms. In most oviparous reptiles, for example, water exchange between eggs and the surrounding environment can have substantial fitness consequences, but regulation of this process is unclear. Here, we evaluate whether water uptake by eggs of the lizard Anolis sagrei is regulated by the presence of a live embryo or is a passive hydraulic response to substrate moisture conditions. Many eggs laid in our captive colony were infertile or contained embryos that died during early stages of development, yet these 'dead' eggs continued to gain mass similar to that of 'live' eggs at least during the first half of incubation. Our results suggest that water uptake by eggs is largely a passive hydraulic process during the first half of incubation, but active regulation by embryos may be necessary during latter stages. Maternal effects (e.g., deposition of salts into yolk) could influence this passive process during early incubation.     

Effects of oxygen concentration during incubation and broiler breeder age on embryonic heat production,chicken development,and 7-day performance

Older breeder flocks produce eggs with a relatively larger yolk and thereby a higher nutrient availability than young breeder flocks. To optimise nutrient utilisation and embryonic development throughout incubation and posthatch period, embryos originating from older breeder flocks may require a higher oxygen availability. The current study investigated effects of broiler breeder flock age and incubational oxygen concentration on embryonic metabolism and chicken development until 7-day posthatch. Similar sized eggs of a young (28–32 week) or old (55–59 week) Cobb 500 breeder flock were incubated at one of three oxygen concentrations (17%, 21% or 25%) from day 7 of incubation until 6 h after emergence from the eggshell. Posthatch, chickens were reared until 7 days of age. Egg composition at the start of incubation, heat production during incubation, and embryo or chicken development at embryonic day (ED)14 and ED18 of incubation, 6 h after hatch and day 7 posthatch were evaluated. An interaction was found between breeder age and oxygen concentration for yolk-free body mass (YFBM) at ED18. A higher oxygen concentration increased YFBM in the old breeder flock, whereas no difference was found between 21 and 25% oxygen in the young breeder flock. Yolk size was larger in the old compared to the young flock from ED0 until 6 h after hatch. Breeder flock age did not affect YFBM at ED14 and 6 h after hatch nor daily embryonic heat production, but there were some effects on relative organ weights. Chickens of the old compared to the young breeder flock showed a higher weight gain at day 7, but at a similar feed conversion ratio (FCR). A higher oxygen concentration during incubation stimulated embryonic development, especially between 17% and 21% of oxygen, in both flock ages_. Although this growth advantage disappeared at 7 days posthatch, a low oxygen concentration during incubation resulted in a higher FCR at 7 days posthatch. Results indicated that breeder flock age seemed to influence body development, with an advantage for the older breeder flock during the posthatch period. Oxygen concentrations during incubation affected body development during incubation and FCR in the first 7 days posthatch. Although an interaction was found between breeder flock age and oxygen concentration at ED18 of incubation, there was no strong evidence that nutrient availability at the start of incubation (represented by breeder flock ages) affected embryo and chicken development at a higher oxygen concentration.     

Thyroid Hormone Deposition in Avian Eggs and Effects on Embryonic Development

Studies to date indicate that thyroid hormones are present inthe eggs of chickens and quail and that those hormones are primarilyin the yolk. Quail hens deposit thyroid hormones into eggs inproportion to their own thyroid status, but appear to show someregulation of this process. Indirect studies suggest that thyroidhormones are transferred into oocytes bound to lipoproteinsand transthyretin, both of which are taken up by receptor-mediatedprocesses. Thyroid hormones bound to yolk lipoproteins may enterembryos with yolk that is taken up by non-specific endocytosisor they may be transported into the embryo by specific carriers.To date most of these ideas about thyroid hormone transportinto egg yolk and from egg yolk into embryos have not been investigateddirectly. In quail, very high T₄ content of eggs is associatedwith accelerated differentiation and growth of embryonic pelviccartilage, a thyroid hormone-responsive tissue. We evaluatethese effects on embryonic tissues and the changes in yolk hormonecontent during incubation in relation to the timing of thyroiddevelopment and studies of the capability for tissue responsesto thyroid hormones during early embryonic life     

Magnetic resonance imaging study of the structure of the yolk in the developing avian egg

Magnetic resonance imaging (MRI) techniques were used to study the morphology of the latebra and concentric rings seen in the yolk of White Leghorn eggs during development of the avian embryo. Previous studies of the macroscopic structure of avian yolk have revealed the latebra, a vase-shaped structure beneath the blastoderm composed of white yolk. The bulbous portion in the center of the yolk is termed the body of the latebra. The thinner portion extending toward the blastoderm is referred to as the neck of the latebra. As the neck of the latebra approaches the blastoderm, it flares out to become the nucleus of Pander. The remainder of the yolk often features alternating concentric layers of white and yellow yolk. These layers, which appear as rings in sections, are thought to represent the daily accumulation of yolk during oogenesis. In this study eggs were imaged with a single slice spin echo sequence using MRI parameters that maximized the visualization of the latebra and concentric rings in the egg yolk. Some experiments were conducted for 2 to 3 day periods with eggs kept in the bore of the magnet using a small incubator that was constructed using a temperature-controlled water pump. The concentric rings of the yolk and the body of the latebra flatten and become more elliptical during development. The neck of the latebra becomes shorter and disappears around the 7th day of incubation. The body of the latebra starts to become incorporated into the embryo at about the 7th day of incubation and usually disappears by the 13th day. The concentric rings are no longer visible as distinct entities at this time. Histochemical procedures carried out as a result of MRI findings indicate that the latebra is an iron-rich structure.     

Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier     

Endogenous β-d-galactoside binding lectin during the expansion of the yolk sac in the developing chick embryo

Summary A lectin with an affinity for -d-galactoside-containing saccharides is present in the developing yolk sac from the chick embryo at stages from 2 to 7 days of incubation. This activity is present in the area vitellina (less differentiated) and the area vasculosa (more differentiated). In both areas, lectin activity increases significantly during the spreading of the yolk sac up to 5 days of incubation. At all of the stages studied lectin activity was significantly higher in the area vasculosa, as compared to the area vitellina.Lectins were purified by affinity chromatography and examined by SDS-PAGE. Under reducing conditions two components are evident. A more prominent band of subunit molecular weight of 14,200±100 for the area vitellina and 13,700±300 for the area vasculosa and a second band with molecular weight of about 68,000±700 and 68,000±1,200 for the area vitellina and area vasculosa respectively, were observed. The -d-galactoside-binding lectin appears to be similar if not identical to that of the early chick blastoderm.     

乌骨鸡胚胎发育过程中卵内蛋白质脂肪的变化研究

对乌骨鸡胚胎发育过程中卵内各组成成分(蛋清、蛋黄、胚胎)在不同胚龄(即入孵的第0、3、6、9、12、15、18d和出壳)时蛋白质、脂肪含量变化进行测定与分析．结果表明：在不同胚龄时,蛋清中蛋白质含量显著高于蛋黄,蛋黄中脂肪含量始终高于蛋清;而胚胎中的蛋白质、脂肪逐渐增加。另外,胚胎发育过程中胚胎蛋白质的主要来源是蛋黄中的蛋白质,说明蛋黄是禽类胚胎发育过程中营养需要的主要来源。     

Yolk androgens and embryo sex: maternal effects or confounding factors?

Maternal effects occur when offspring phenotype is affected by environmental factors experienced by the mother and, in egg-laying species, are often mediated via egg resources. There is currently great interest among behavioural ecologists in maternally allocated yolk androgens, especially their relationship with offspring sex and development. Such studies need embryonic tissue for sexing, however, requiring eggs to be incubated (usually for 3 days). Therefore, there are concerns about whether the androgen concentrations assayed reflect those allocated by the mother. In addition, studies showing sex biases in maternal allocation of androgens could be confounded if male and female embryos uptake or metabolise androgens at different rates. We ran a series of experiments using zebra finch (Taeniopygia guttata) eggs to address these potential confounding factors. First we showed, using eggs naturally incubated for up to 5 days, that eggs containing embryos had lower yolk androgen concentrations than eggs that had failed to form embryos. We then tested various hypotheses for this difference using controlled incubation treatments. Our results suggested that (a) embryo development causes the yolk to become progressively more diluted with albumin; and (b) between 3 and 5 days of incubation embryos start uptaking or metabolising androgens. Crucially, we found no decline in yolk androgen concentration at 3 days incubation, and no evidence for sex-specific rates of uptake or metabolism of androgens. This strongly suggests that yolk androgen levels up to 3 days incubation do reflect those allocated by the mother, and that studies of sex biased maternal allocation of yolk androgens are not confounded by sex differences in embryo development.     

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in the chick embryo area vasculosa

Vascular endothelial growth factor is an angiogenic factor in vivo and in vitro that plays a crucial role in the control of blood vessel development and in pathological angiogenesis. The vascularized extraembryonic membranes of the chick embryo include the area vasculosa and the chorioallantoic membrane. In this study, we investigated the expression of vascular endothelial growth factor and of its receptor-2, specifically expressed by the endothelial cells, in the chick area vasculosa at days 6, 10 and 14 of incubation. Our results indicate that, in all the three developmental stages examined, vascular endothelial growth factor is clearly expressed in the endodermal cells immediately adjacent to the mesodermal endothelial cells which, in turn, expressed vascular endothelial growth factor receptor-2. These observations suggest that during the development of the vascular system, endodermal cells, expressing vascular endothelial growth factor, initiate angiogenesis by stimulating directly mesodermal cells, which express vascular endothelial growth factor receptor-2. Moreover, our data demonstrate that vascular endothelial growth factor receptor-2 expression is also maintained by endothelial cells in the later stages of development, until day 14 of incubation. In accord with other literature data, this suggests that vascular endothelial growth factor is required not only for proliferation, but also for the survival of endothelial cells.     

No sex difference in yolk steroid concentrations of avian eggs at laying

Yolk steroids of maternal origin have been proposed to influence genetic sex determination in birds, based on sex differences in yolk steroid concentrations of peafowl eggs incubated for 10 days. More recent reports dispute this proposal, as yolk steroids in eggs incubated for 3 days do not show such sex differences. To date, research examining this phenomenon has only analysed incubated eggs, although sex in avian species is determined before incubation begins. This may be a serious methodological flaw because incubation probably affects yolk steroid concentrations. Therefore, we investigated sex differences in yolk steroid concentrations of unincubated avian eggs. We withdrew yolk for steroid analysis from fresh, unincubated Japanese quail (Coturnix japonica) eggs by biopsy, and then incubated those eggs for 10 days, after which we harvested the embryonic material for genetic sexing and the incubated yolk for further steroid analysis. We found no sex differences in fresh Japanese quail eggs; however, sex differences were apparent in yolk steroids by day 10 of incubation, when female eggs had significantly more oestrogen in relation to androgen than male eggs. Concentrations of all yolk androgens decreased dramatically between laying and day 10 of incubation, whereas oestradiol (E2) concentrations increased marginally. Thus, yolk concentrations of androgens and E2 do not appear critical for avian sex determination.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Toxic effects of phencyclidine on developing chick embryo brain

We studied the effects of Phencyclidine (PCP, Angel Dust) on the developing chick embryo brain. In Group-1, the eggs were injected with PCP on the 7th day of incubation and the embryo brains were studied on the 10th day. In Group-2, eggs were injected twice; first on the 7th day and then on the 10th day of incubation. Group-2 brains were then studied on the 16th day of incubation. PCP significantly depressed the development of embryo brains. Cerebral hemisphere weight, total protein and total DNA were significantly lower on day 10 of incubation in Group-1. Similar results were observed in Group-2. Concomitantly, the concentration of brain serotonin at day 10 was also significantly reduced when PCP was injected into the eggs on the 7th day of incubation. Since serotonin has been reported to influence development of the chick embryo brain, the present finding of the effect of PCP on brain development might be a secondary phenomenon. The possible implications of the effects of PCP on human brain development are also discussed.     

Periodic cooling of bird eggs reduces embryonic growth efficiency

For many bird embryos, periodic cooling occurs when the incubating adult leaves the nest to forage, but the effects of periodic cooling on embryo growth, yolk use, and metabolism are poorly known. To address this question, we conducted incubation experiments on eggs of zebra finches (Taeniopygia guttata) that were frequently cooled and then rewarmed or were allowed to develop at a constant temperature. After 12 d of incubation, embryo mass and yolk reserves were less in eggs that experienced periodic cooling than in controls incubated constantly at 37.5 degrees Celsius. Embryos that regularly cooled to 20 degrees Celsius had higher mass-specific metabolic rates than embryos incubated constantly at 37.5 degrees Celsius. Periodic cooling delayed development and increased metabolic costs, reducing the efficiency with which egg nutrients were converted into embryo tissue. Avian embryos can tolerate periodic cooling, possibly by adjusting their physiology to variable thermal conditions, but at a cost to growth efficiency as well as rate of development. This reduction in embryo growth efficiency adds a new dimension to the fitness consequences of variation in adult nest attentiveness.     

The embryo pancreas is a source of increased yolk amylase in the fertilized eggs of domestic fowls.

The amylases were studied in the yolk of fertilized eggs and in the pancreases of the embryos of domestic fowls. The amylase activity in the yolk increased markedly from 13 days of incubation until hatching, but the activity decreased when the embryos were taken out of the eggs. The isoamylases in the yolk and in the pancreas of the embryo were identical electrophoretically. The amylase occurs mainly in the pancreas of the embryo. We think that the increase in amylase activity in the yolk of fertilized eggs during incubation depends upon the accumulation of pancreatic amylase synthesized by the developing embryo in the egg.     

The Developing Chicken Yolk Sac Acquires Nutrient Transport Competence by an Orchestrated Differentiation Process of Its Endodermal Epithelial Cells

During chicken yolk sac (YS) growth, mesodermal cells in the area vasculosa follow the migrating endodermal epithelial cell (EEC) layer in the area vitellina. Ultimately, these cells form the vascularized YS that functions in nutrient transfer to the embryo. How and when EECs, with their apical aspect directly contacting the oocytic yolk, acquire the ability to take up yolk macromolecules during the vitellina-to-vasculosa transition has not been investigated. In addressing these questions, we found that with progressive vascularization, the expression level in EECs of the nutrient receptor triad, LRP2-cubilin-amnionless, changes significantly. The receptor complex, competent for uptake of yolk proteins, is produced by EECs in the area vasculosa but not in the area vitellina. Yolk components endocytosed by LRP2-cubilin-amnionless, preformed and newly formed lipid droplets, and yolk-derived very low density lipoprotein, shown to be efficiently endocytosed and lysosomally processed by EECs, probably provide substrates for resynthesis and secretion of nutrients, such as lipoproteins. In fact, as directly demonstrated by pulse-chase experiments, EECs in the vascularized, but not in the avascular, region efficiently produce and secrete lipoproteins containing apolipoprotein A-I (apoA-I), apoB, and/or apoA-V. In contrast, perilipin 2, a lipid droplet-stabilizing protein, is produced exclusively by the EECs of the area vitellina. These data suggest a differentiation process that orchestrates the vascularization of the developing YS with the induction of yolk uptake and lipoprotein secretion by EECs to ensure embryo nutrition.