Bulk Actin Dynamics Drive Phase Segregation in Zebrafish Oocytes.

Segregation of maternal determinants within the oocyte constitutes the first step in embryo patterning. In zebrafish oocytes, extensive ooplasmic streaming leads to the segregation of ooplasm from yolk granules along the animal-vegetal axis of the oocyte. Here, we show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the oocyte. This wave functions in segregation by both pulling ooplasm animally and pushing yolk granules vegetally. Using biophysical experimentation and theory, we show that ooplasm pulling is mediated by bulk actin network flows exerting friction forces on the ooplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. Our study defines a novel role of cell-cycle-controlled bulk actin polymerization waves in oocyte polarization via ooplasmic segregation.

Mathematical modeling of the interaction between yolk utilization and fish growth in zebrafish, Danio rerio.

Optimal embryonic development plays a major role in the health of an individual beyond the developmental stage. Nutritional perturbation during development is associated with cardiovascular and metabolic disease later in life. With both nutritional uptake and overall growth being risk factors for eventual health, it is necessary to understand not only the behavior of the processes during development but also their interactions. In this study, we used differential equations, image analyses, curve fittings, parameter estimation and laboratory experiments to quantify the rate of yolk absorption and its effect on early development of a vertebrate model (Danio rerio). Findings from this study establish a nonlinear functional relationship between nutrient absorption and early fish growth. We found that the rate of change in fish length and yolk utilization is logistic, that is the yolk decays rapidly for a period of time before leveling out. An interesting finding from this study is that yolk utilization reaches its maximum at 84 h post-fertilization. We validated our mathematical models against experimental observations, making them powerful tools for replication and future simulations.

In ovo metabolism of estradiol to estrone sulfate in chicken eggs: Implications for how yolk estradiol influences embryonic development.

The steroid 17ß-estradiol (herein "estradiol") is a potent regulator of sexual differentiation that exerts wide-ranging effects on the developing brain and other tissues. The developing gonads are an important source of estradiol but most, if not all, vertebrate embryos are also exposed to maternally derived estradiol during development. In birds, this maternally derived estradiol is present in the egg at the time of oviposition but very little is known about how this source of estradiol influences development. A critical aspect of understanding yolk estradiol effects is deciphering how steroid metabolism may regulate embryonic exposure to yolk estradiol. In this study, we examine the metabolic fate of estradiol during the first five days of incubation in chicken (Gallus gallus) eggs. Using tritiated estradiol to trace the movement and metabolism of estradiol, we demonstrate that estradiol is metabolized to estrone, which is subsequently conjugated to estrone sulfate as the primary metabolite. Estrone sulfate then accumulates in the albumen by day five of incubation. Overall, these findings have important implications for how yolk estradiol may influence development and alter offspring phenotype. Mechanisms through which estradiol, as well as estrone sulfate, might elicit effects are discussed.

Are there synergistic or antagonistic effects of multiple maternally derived egg components on offspring phenotype?

Eggs are 'multivariate' in that they contain multiple maternally derived egg components (e.g. hormones, antibodies, mRNA, antioxidants) which are thought to influence offspring phenotype. However, most studies have focused on single egg components and on short-term effects. Here, we simultaneously manipulated two egg components, maternally derived antibodies (MAb) and yolk testosterone, to assess potential synergistic or antagonistic effects on zebra finch offspring phenotype from hatching to sexual maturity. We found no evidence for short- or long-term effects of either MAb or yolk testosterone alone, or their interaction, on hatching mass, size at fledging (tarsus length), body mass at sexual maturity (day 82), chick survival, humoral immune function or any measured female reproductive trait at sexual maturity. There was a positive effect of yolk testosterone, but not MAb, on offspring phytohaemagglutinin (PHA) response at 26 days of age but at 82 days of age, MAb, but not yolk testosterone, had a positive effect on PHA response. There was also a MAb×sex interaction on 30 day chick mass, and a positive effect of yolk testosterone on male courtship behaviour at sexual maturity. However, we found no evidence for synergy, i.e. where offspring treated with both MAb and yolk testosterone had higher trait values than offspring treated with either MAb or yolk testosterone alone for any measured trait. Similarly, evidence for antagonistic (compensatory) effects, where offspring treated with both MAb and yolk testosterone had intermediate trait values compared with offspring treated with either MAb or yolk testosterone alone, was equivocal.

Genetic parameters of the thick-to-thin albumen ratio and egg compositional traits in layer-type chickens.

1. Generating a robust egg albumen is one of the major factors contributing to interior egg quality owing to its nutritive value and superior appearance. However, the genetic factors regulating the proportion of thick albumen are poorly understood. 2. In this study, 1330 eggs were collected from 450 Rhode Island White layers, aged 40 weeks, to measure egg compositional traits for three successive days. The restricted maximum likelihood method was applied to estimate genetic parameters for the thick-to-thin albumen ratio and other egg compositional traits. A univariate animal model was fitted to calculate heritability for each trait. 3. The heritabilities of egg weight, yolk weight, albumen height, Haugh units, percentages of yolk, thick albumen, thin albumen and the thick-to-thin albumen ratio were 0.32, 0.34, 0.28, 0.47, 0.61, 0.39, 0.31, and 0.45, respectively. The percentage of thick albumen was negatively correlated genetically with all traits, and percentage of thin albumen was negatively correlated genetically with all traits except for Haugh units. The thick-to-thin albumen ratio was positively correlated genetically with egg weight, albumen height and Haugh units, with correlations ranging from 0.21 to 0.54. 4. The results indicated that the percentage of thick albumen and the thick-to-thin albumen ratio were found to be moderately to highly heritable, and selection for the thick-to-thin albumen ratio could be conducive to the improvement of egg albumen quality.

High post-thaw survival of ram sperm after partial freeze-drying.

BACKGROUND: Recrystallization damages occur when a frozen sample is held at high subzero temperatures and when the warming process is too slow. METHODS: In this work, ram semen diluted in two different concentrations of sugar solutions (Lyo A consisted of 0.4 M sorbitol and 0.25 M trehalose, and the second, Lyo B composed of 0.26 M sorbitol and 0.165 M trehalose) in egg yolk and Tris medium were compared after freezing 10 µL samples to: (1) - 10, - 25, and - 35 °C and thawing. (2) Freezing to - 10 and - 25 °C, holding for 1 h and then thawing, and (3) freezing to - 10 and - 25 °C and drying for 1 h at these temperatures at a vacuum of 80 mTorr, prior thawing. For drying, we used a new freeze-drying apparatus (Darya, FertileSafe, Israel) having a condensation temperature below - 110 °C and a vacuum pressure of 10-100 mTorr that is reached in less than 10s. RESULTS: Results showed that samples in Lyo B solution frozen at - 25 °C had significantly higher sperm motility in partially freeze-dried samples than frozen samples (46.6 ± 2.8% vs 1.2 ± 2.5%, P < 0.001). Moreover, partially dried samples in Lyo B showed higher motility than Lyo A at - 25 °C (46.6 ± 2.8% vs 35 ± 4%). Cryomicroscopy and low-temperature/low-pressure environmental scanning electronic microscope demonstrated that the amount of the ice crystals present in partially dried samples was lower than in the frozen samples. CONCLUSION: Holding the sperm at high subzero temperatures is necessary for the primary drying of cells during the freeze-drying process. Rapid freeze-drying can be achieved using this new device, which enables to reduce recrystallization damages.

Nervous system development in cephalopods: How egg yolk-richness modifies the topology of the mediolateral patterning system.

Cephalopods possess the most complex centralized nervous system among molluscs and the molecular determinants of its development have only begun to be explored. To better understand how evolved their brain and body axes, we studied Sepia officinalis embryos and investigated the expression patterns of neural regionalization genes involved in the mediolateral patterning of the neuroectoderm in model species. SoxB1 expression reveals that the embryonic neuroectoderm is made of several distinct territories that constitute a large part of the animal pole disc. Concentric nkx2.1, pax6/gsx, and pax3/7/msx/pax2/5/8 positive domains subdivide this neuroectoderm. Looking from dorsal to ventral sides, the sequence of these expressions is reminiscent of the mediolateral subdivision in model species, which provides good evidence for "mediolateral patterning" conservation in cephalopods. A specific feature of cephalopod development, however, includes an unconventional orientation to this mediolateral sequence: median markers (like nkx2.1) are unexpectedly expressed at the periphery of the cuttlefish embryo and lateral markers (like Pax3/7) are expressed centrally. As the egg is rich with yolk, the lips of the blastopore (that classically organizes the neural midline) remain unclosed at the lateral side of the animal pole until late stages of organogenesis, therefore reversing the whole embryo topology. These findings confirm - by means of molecular tools - the location of both ventral and dorsal poles in cephalopod embryos.

Does the oviparity-viviparity transition alter the partitioning of yolk in embryonic snakes?

BACKGROUND: The oviparity-viviparity transition is a major evolutionary event, likely altering the reproductive process of the organisms involved. Residual yolk, a portion of yolk remaining unutilized at hatching or birth as parental investment in care, has been investigated in many oviparous amniotes but remained largely unknown in viviparous species. Here, we used data from 20 (12 oviparous and 8 viviparous) species of snakes to see if the oviparity-viviparity transition alters the partitioning of yolk in embryonic snakes. We used ANCOVA to test whether offspring size, mass and components at hatching or birth differed between the sexes in each species. We used both ordinary least squares and phylogenetic generalized least squares regressions to test whether relationships between selected pairs of offspring components were significant. We used phylogenetic ANOVA to test whether offspring components differed between oviparous and viviparous species and, more specifically, the hypothesis that viviparous snakes invest more in the yolk as parental investment in embryogenesis to produce more well developed offspring that are larger in linear size. RESULTS: In none of the 20 species was sex a significant source of variation in any offspring component examined. Newborn viviparous snakes on average contained proportionally more water and, after accounting for body dry mass, had larger carcasses but smaller residual yolks than did newly hatched oviparous snakes. The rates at which carcass dry mass (CDM) and fat body dry mass (FDM) increased with residual yolk dry mass (YDM) did not differ between newborn oviparous and viviparous snakes. Neither CDM nor FDM differed between newborn oviparous and viviparous snakes after accounting for YDM. CONCLUSIONS: Our results are not consistent with the hypothesis that the partitioning of yolk between embryonic and post-embryonic stages differs between snakes that differ in parity mode, but instead show that the partitioning of yolk in embryonic snakes is species-specific or phylogenetically related. We conclude that the oviparity-viviparity transition does not alter yolk partitioning in embryonic snakes.

A Novel Pattern of Yolk Processing in Developing Snake Eggs (Colubridae: Lampropeltini) and its Functional and Evolutionary Implications.

Early amniotic vertebrates evolved large-yolked eggs that permitted production of well-developed, terrestrial hatchlings. This reproductive pattern required new mechanisms for cellularizing the yolk and mobilizing it for embryonic use. In birds, cells that line the yolk sac cavity phagocytose and digest the yolk material, a pattern that is commonly assumed to be universal among oviparous amniotes. However, recent evidence challenges the assumption that all squamate reptiles conform to the avian developmental pattern. In this paper, scanning electron microscopy and histology were used to study mechanisms of yolk processing in two colubrid snakes, the kingsnake Lampropeltis getula and the milksnake L. triangulum. Endodermal cells from the yolk sac splanchnopleure proliferate massively as they invade the yolk sac cavity, forming elaborate chains of interlinked cells. These cells grow in size as they phagocytose yolk material. Subsequently, vitelline capillaries invade the masses of yolk-laden cells and become coated with the endodermal cells, forming an elaborate meshwork of cell-coated strands. The close association of cells, yolk, and blood vessels allows yolk material to be cellularized, digested, and transported for embryonic use. The overall pattern is like that of the corn snake Pantherophis guttatus, but contrasts markedly with that of birds. Given recent evidence that this developmental pattern may also occur in certain lizards, we postulate that it is ancestral for squamates. Studies of lizards, crocodilians, and turtles are needed to clarify the evolutionary history of this pattern and its implications for the evolution of the amniotic (terrestrial) vertebrate egg.

Multicomponent T2 relaxation studies of the avian egg.

PURPOSE: To investigate the tissue-like multiexponential T2 signal decays in avian eggs. METHODS: Transverse relaxation studies of raw, soft-boiled and hard-boiled eggs were performed at 3 Tesla using a three-dimensional Carr-Purcell-Meiboom-Gill imaging sequence. Signal decays over a TE range of 11 to 354 ms were fitted assuming single- and multicomponent signal decays with up to three separately decaying components. Fat saturation was used to facilitate spectral assignment of observed decay components. RESULTS: Egg white, yolk and the centrally located latebra all demonstrate nonmonoexponential T2 decays. Specifically, egg white exhibits two-component decays with intermediate and long T2 times. Meanwhile, yolk and latebra are generally best characterized with triexponential decays, with short, intermediate and very long T2 decay times. Fat saturation revealed that the intermediate component of yolk could be attributed to lipids. Cooking of the egg profoundly altered the decay curves. CONCLUSION: Avian egg T2 decay curves cover a wide range of decay times. Observed T2 components in yolk and latebra as short as 10 ms, may prove valuable for testing clinical sequences designed to measure short T2 components, such as myelin-associated water in the brain. Thus we propose that the egg can be a versatile and widely available MR transverse relaxation phantom.

Maternal corticosterone deposition in avian yolk: Influence of laying order and group size in a joint-nesting, cooperatively breeding species.

Glucocorticoid hormones play a key role in day-to-day adjustments to fluctuating metabolic needs. These hormones also mediate physiological and behavioral responses to stressful events, allowing individuals to cope with stressors. Various environmental insults, such as a food shortages, predation attempts, and agonistic encounters often elevate plasma glucocorticoid levels in vertebrates. Because exposure to maternally-derived (via circulation or egg) glucocorticoids may be detrimental to the developing embryo, maternal stress can have negative carryover effects on offspring fitness. We examined corticosterone, the primary avian glucocorticoid, concentrations in egg yolk in a plural-breeding, joint-nesting species, the smooth-billed ani (Crotophaga ani), in which females compete among themselves to lay eggs in the final incubated clutch. We investigated whether yolk corticosterone levels varied with laying order and group size. Because egg-laying competition leads to physiological and social stress that is intensified with group size and laying order, we predicted that yolk corticosterone levels should increase from the early to the late egg-laying period and from single female to multi-female groups. In this two-year field study, we found that yolk corticosterone levels of late-laid eggs within the communal clutch were higher in multi-female groups than in single female groups. Results from this study suggest that laying females experience higher levels of stress in multi-female groups and that this maternal stress influences yolk corticosterone concentrations. This study identifies a novel cost of group-living in plural-breeding cooperatively breeding birds, namely an increase in yolk corticosterone levels with group size that may result in detrimental effects on offspring development.

Production of Interspecific Germline Chimeras via Embryo Replacement.

In avian species, primordial germ cells (PGCs) use the vascular system to reach their destination, the genital ridge. Because of this unique migratory route of avian germ cells, germline chimera production can be achieved via germ cell transfer into a blood vessel. This study was performed to establish an alternative germ cell-transfer system for producing germline chimeras by replacing an original host embryo with a donor embryo, while retaining the host extraembryonic tissue and yolk, before circulation. First, to test the migratory capacity of PGCs after embryo replacement, Korean Oge (KO) chick embryos were used to replace GFP transgenic chick embryos. Four days after replacement, GFP-positive cells were detected in the replaced KO embryonic gonads, and genomic DNA PCR analysis with the embryonic gonads demonstrated the presence of the GFP transgene. To produce an interspecific germline chimera, the original chick embryo proper was replaced with a quail embryo onto the chick yolk. To detect the gonadal PGCs in the 5.5-day-old embryonic gonads, immunohistochemistry was performed with monoclonal antibodies specific to either quail or chick PGCs, i.e., QCR1 and anti-stage-specific embryonic antigen-1 (SSEA-1), respectively. Both the QCR1-positive and SSEA-1-positive cells were detected in the gonads of replaced quail embryos. Forty percent of the PGC population in the quail embryos was occupied by chick extraembryonically derived PGCs. In conclusion, replacement of an embryo onto the host yolk before circulation can be applied to produce interspecies germline chimeras, and this germ cell-transfer technology is potentially applicable for reproduction of wild or endangered bird species.

Influence of mate preference and laying order on maternal allocation in a monogamous parrot species with extreme hatching asynchrony.

It is well established that in many avian species, prenatal maternal resource allocation varies both between and within clutches and may affect offspring fitness. Differential allocation of maternal resources, in terms of egg weight and yolk composition, may therefore allow the female to adjust brood reduction and to fine-tune reproductive investment in accordance with the expected fitness returns. The adaptive value of such maternal resource allocation is thought to be context-dependent as well as species-specific. We investigated the effects of female preference for her mate on the allocation of prenatal maternal resources in the budgerigar, Melopsittacus undulatus, a monogamous species of parrot that shows an extreme hatching asynchrony. We assessed mate preferences in a two-way preference test and allowed females two breeding rounds: one with the preferred and one with the non-preferred partner. We found no effect of preference on either latency to lay or clutch size, but females mated with the preferred partner laid eggs that contained significantly more yolk. Their eggs also contained significantly more androstenedione but not testosterone. Our results suggest that in this species, female preference may influence maternal resource allocation, and that the functional roles of each androgen in the yolk should be considered separately. In addition, we found a significant effect of laying order on egg and yolk weight as well as on yolk testosterone and androstenedione levels. These measures, however, did not change linearly with the laying order and render it unlikely that female budgerigars compensate for the extreme hatching asynchrony by adjusting within-clutch allocation of prenatal maternal resources.

Phosvitin phosphorus is involved in chicken embryo bone formation through dephosphorylation.

The aim of this study was to investigate the role of phosvitin in bone formation in chicken embryos. The yolk P content, P/N ratio and secondary structure of phosvitin, alkaline phosphatase (ALP) activity of the tibia, and body length were determined during incubation. A high correlation was found between the phosphate group content of phosvitin and both secondary structure and bone metabolism (ALP activity in the tibia, body length). The ALP activity and body length growth slightly lagged behind changes in the P/N ratio and the secondary structure of phosvitin. The phosphate content of phosvitin decreased, the γ-random coil and ß-turn gradually transformed into α-helixes, and the secondary structure of protein tended to become more orderly; these changes mainly occurred on d 13 to 16. Bone formation of the chicken embryos occurred primarily on d 14 to 18, whereas ALP activity and body length growth increased substantially. The results indicate that phosvitin P is involved in chicken embryo bone formation through dephosphorylation.

Hatching and residual yolk internalization in lizards: evolution, function and fate of the amnion.

Most egg-laying vertebrates hatch without depleting the entire yolk reserve. The residual yolk is internalized before emergence from the egg is completed and the yolk is subsequently metabolized during early neonatal life. Here we provide the first description of the mechanism of yolk internalization in non-avian reptiles. We describe the hatching of two lizard species (Physignathus lesueurii and Varanus rosenbergii) and provide a step-by-step account of sequence of events leading to yolk internalization and emergence from the egg. We also conducted incubation experiments to determine the cause of failed yolk internalization. Contraction of the ruptured amnion is the mechanism by which the residual yolk is internalized, which provides an explanation for the functional significance of amniotic contractions. Failures of internalization occur when the amount of residual yolk exceeds that which can be enclosed by the ruptured amnion. We conclude that, because of the connections formed between the amnion and both the allantois and chorion, the pipping and retraction of the amnion pulls the chorioallantoic membrane (CAM) off the surface of the eggshell, which impairs the capacity for gas exchange and forces the embryo to breach the eggshell to commence breathing. We further speculate that the loss of amniotic contractions in mammals may indicate an incompatibility of amnion-assisted yolk internalization with viviparity, an evolutionary process that could be tested by examining viviparous squamates.

Embryonic growth and antioxidant provision in avian eggs.

Avian embryos undergo extremely rapid development over a relatively short period of time, and so are likely to suffer high levels of oxidative damage unless this is mitigated by sufficient maternal allocation of appropriate antioxidants. At a species level, it is therefore predicted that antioxidants should be allocated to eggs according to the rate of embryonic growth, such that eggs containing embryos that grow faster are furnished with higher antioxidant levels, independent of egg size. We tested this prediction for three potentially important classes of dietary-derived yolk antioxidants: carotenoids, vitamin E and vitamin A. Across species, we found positive relationships between embryonic growth rate and total yolk levels of each of the three antioxidant classes. Moreover, there were consistent differences in antioxidant provision between pairs of species that share a common initial egg mass yet have differing rates of embryonic growth, such that the eggs of the faster-developing species have higher levels of carotenoids and vitamin E. These results may explain the marked interspecific variation in antioxidant provision and provide evidence for the role that these antioxidants play during embryonic development.

Sequestration of PDC-109 protein by specific antibodies and egg yolk cryoprotects bull spermatozoa.

PDC-109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time- and concentration-dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC-109 either by anti-PDC-109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY + Ab in minimizing cryoinjury to bull spermatozoa. PDC-109 protein was purified by applying two-step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion-exchange chromatography. After checking the Ab cross-reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris-glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY + TG (group I), Ab + TG (group II) or EY + Ab + TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC-PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein-sequestered-semen (zona-penetration assay) were evaluated. A significant (p < 0.05) improvement in post-thaw SQPs as well as in non-capacitated spermatozoa observed at pre-freeze and post-thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein-mediated cryoinjury. From this study, it can be concluded that sequestration of PDC-109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra-low temperatures.

Effects of dietary rosemary and oregano volatile oil mixture on quail performance, egg traits and egg oxidative stability.

1. This study was conducted to determine the effects of volatile oil mixture on quail laying performance, egg traits and egg malondialdehyde (MDA) concentration. 2. A total of 260 Pharaoh quails (Coturnix coturnix Pharaoh) aged 6 weeks were equally divided into 5 groups of 65 (4 replicates of 13 quails each). The mixture of diets was as follows: a control treatment with 0 mg volatile oil/kg of diet; (1) 200 mg/kg rosemary volatile oil; (2) 200 mg/kg oregano volatile oil; (3) 40 mg/kg rosemary volatile oil plus 160 mg/kg oregano volatile oil (ratio 20:80) and (4) 160 mg/kg rosemary volatile oil plus 40 mg/kg oregano volatile oil (ratio 80:20). The diets were prepared fresh for each treatment. The experimental period lasted 10 weeks. 3. At the end of the experiment, there were no significant differences amongst the groups in body weight, egg weight, egg mass, egg shape index, Haugh unit, egg shell thickness or egg shell-breaking strength. 4. Diets containing rosemary volatile oil increased the egg production significantly. Feed intake significantly increased in the groups containing volatile oil mixture (groups 4 and 5). The inclusion of rosemary volatile oil at 200 mg/kg improved feed efficiency. 5. Egg albumen and egg yolk index values showed significant increases in the group given diets containing rosemary volatile oil. Egg yolk colour became darker with the addition of rosemary and oregano volatile oil. The treatment group had lower egg yolk MDA concentration than the control group. 6. It is concluded that, alone or in combination, rosemary and oregano volatile oil can be used in quail diets without adverse effects on the measured parameters. Inclusion of rosemary and oregano volatile oil in quail diets enhanced the antioxidant status of eggs.

Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation.

The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during pre-hatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to early somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.

Strategic female reproductive investment in response to male attractiveness in birds.

Life-history theory predicts that individuals should adjust their reproductive effort according to the expected fitness returns on investment. Because sexually selected male traits should provide honest information about male genetic or phenotypic quality, females may invest more when paired with attractive males. However, there is substantial disagreement in the literature whether such differential allocation is a general pattern. Using a comparative meta-regression approach, we show that female birds generally invest more into reproduction when paired with attractive males, both in terms of egg size and number as well as food provisioning. However, whereas females of species with bi-parental care tend to primarily increase the number of eggs when paired with attractive males, females of species with female-only care produce larger, but not more, eggs. These patterns may reflect adaptive differences in female allocation strategies arising from variation in the signal content of sexually selected male traits between systems of parental care. In contrast to reproductive effort, female allocation of immune-stimulants, anti-oxidants and androgens to the egg yolk was not consistently increased when mated to attractive males, which probably reflects the context-dependent costs and benefits of those yolk compounds to females and offspring.