Avian Embryos Research Articles

Developmental toxicity of short-chain chlorinated paraffins on early-stage chicken embryos in a shell-less (ex-ovo) incubation system

Short-chain chlorinated paraffins (SCCPs) are listed as a category of globally controlled persistent organic pollutants (POPs) by the Stockholm Convention in 2017. However, SCCP toxicity, particularly their developmental toxicity in avian embryos, has not been well studied. In this study, we observed the early development of chicken embryos (Gallus gallus domesticus) by applying a shell-less (ex-ovo) incubation system developed in our previous studies. After exposing embryos at Hamburger Hamilton stage (HHS) 1 to SCCPs (control, 0.1% DMSO; SCCPs-L, 200 ng/g; SCCPs-M, 2000 ng/g; SCCPs-H, 20,000 ng/g), we observed the development of embryos from the 3rd to 9th incubation day. Exposure to SCCPs-M and -H induced a significant reduction in survival, with an LD50 of 3100 ng/g on the 9th incubation day. Significant dose-dependent decreases in body length were observed from days 4–9. We also found that SCCPs-H decreased the blood vessel length and branch number on the 4th incubation day. Additionally, SCCPs-H significantly reduced the heart rate on the 4th and 5th incubation days. These findings suggest that SCCPs may have potential of developmental and cardiovascular toxicity during the early stages of chicken embryos. Quantitative PCR of the mRNA of genes related to embryonic development showed that SLC16A10 (a triiodothyronine transporter) level decreased in the SCCPs-H group, showing a significant positive correlation with the body length of embryos. THRA level, a thyroid hormone receptor, was significantly decreased in the SCCPs-H group, whereas that of DIO3 level, a deiodinase was significantly increased. These results suggest that SCCPs exposure induces developmental delays via the thyroxine signaling pathway. Analysis of thyroid hormones (THs) in blood plasma also indicated a significant reduction in thyroxine (T4) levels in the SCCPs-H group on the 9th incubation day of embryos. In conclusion, SCCPs induce developmental toxicity by disrupting thyroid functions at the early-life stage of chicken embryos.

The Effect of Maternal Hormones on the Avian Offspring: A Review

Maternal hormones have a significant impact on the development of avian embryos, controlling vital physiological functions such as immune system development, metabolism control, and productive traits. This review will examine the different types of maternal hormones, synthesis, regulation, and effects on avian physiology and productivity, as well as the implications for embryonic development. Additionally, it appears that how they might affect hatchability and chick quality. Studies have shown that maternal hormones transfer to eggs and subsequently affect the offspring's development, greatly influencing the behavior and phenotype of avian offspring overall. Understanding these mechanisms can help improve our knowledge of developmental biology, parental investment, and evolutionary processes that influence avian traits. Different species of birds may experience different effects when exposed to maternal hormones. For example injecting testosterone into bird eggs can increase the rate at which certain species hatch, such as quail, other species may see a decrease in hatchling weight. This emphasizes the complexity of hormonal influences on bird development and the necessity of more research. In particular, it is still unclear how yolk steroids affect birds' immune systems; early research indicates that they may chickens' ability to produce antibodies. Undertaking further research is necessary to completely understand the implications of avian immunity to fill in these gaps in knowledge across species and developmental stages.

Ethynylestradiol feminizes gene expression partly in testis developing as ovotestis and disrupts asymmetric müllerian duct development by eliminating asymmetric gene expression in japanese quail embryos.

In avian embryos, xenoestrogens induce abnormalities in reproductive organs, particularly the testes and Müllerian ducts (MDs). However, the molecular mechanisms remain poorly understood. We investigated the effects of ethynylestradiol (EE2) exposure on gene expression associated with reproductive organ development in Japanese quail embryos. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the left testis containing ovary-like tissues following EE2 exposure highly expressed the genes for steroidogenic enzymes (P450scc, P45017α, lyase, and 3β-HSD) and estrogen receptor-β, compared to the right testis. No asymmetry was found in these gene expression without EE2. EE2 induced hypertrophy in female MDs and suppressed atrophy in male MDs on both sides. RNA sequencing analysis of female MDs showed 1,366 differentially expressed genes between developing left MD and atrophied right MD in the absence of EE2, and these genes were enriched in Gene Ontology terms related to organogenesis, including cell proliferation, migration and differentiation, and angiogenesis. However, EE2 reduced asymmetrically expressed genes to 21. RT-qPCR analysis indicated that genes promoting cell cycle progression and oncogenesis were more highly expressed in the left MD than in the right MD, but EE2 eliminated such asymmetric gene expression by increasing levels on the right side. EE2-exposed males showed overexpression of these genes in both MDs. This study reveals part of the molecular basis of xenoestrogen-induced abnormalities in avian reproductive organs, where EE2 may partly feminize gene expression in the left testis, developing as the ovotestis, and induce bilateral MD malformation by canceling asymmetric gene expression underlying MD development.

Abstract 6907: Micro-implantation of patient tumor samples into the avian embryo, a fast and reliable alternative in vivo technology for preclinical studies on melanoma and follicular lymphoma

Abstract Despite recent efforts to develop reliable and predictive patient-derived cancer models, drug attrition rates in oncology remain high. Moreover, conventional models such as patient-derived xenograft (PDX) mouse models face major cost and time limitations, not supporting real-time clinical decision making. To overcome such limitation, ONCOFACTORY developed a proprietary PDX platform enabling the engraftment of small size tumor biopsy samples within targeted tissue of the avian embryo at early stage of development, to conduct preclinical proof of efficacy studies and provide PDX clinical trial solution helping with patient’s selection prior to clinical phase II/III. On one hand, we performed prospective co-clinical trial aiming to evaluate the utility of the AVI-PDXTM model in predicting the response to standard therapy, consisting of BRAF and MEK inhibitors, for patient with metastatic melanoma harboring BRAF mutations. Skin biopsy samples were implanted into specific tissue driving tumor formation under the skin of avian embryos. On the other hand, we conducted a retrospective cohort study with 20 follicular lymphoma (FL) patient samples divided into “partial” and “complete” responders to the standard immuno-chemotherapy RCHOP regimen to establish FL AVI-PDXTM, and examine sensitivity to RCHOP in ovo. Twenty-four hours post-implantation, avian melanoma and FL replicas were respectively exposed to BRAFi/MEKi or RCHOP and their vehicles, over 24h. Then, embryos were harvested, and tumor response was examined by measuring tumor volume using light sheet microscopy. Alongside, in independent series of avian embryos implanted with FL samples, tumors were harvested for single-cell transcriptomic analyses to explore mechanism of response to RCHOP. For melanoma, we provide the proof of concept that the AVI-PDXTM reliably predict sensitivity or resistance to BRAF and MEK inhibitors within days, matching patient clinical outcome observed in clinic. For FL, we showed that the FL-AVI-PDXTM efficiently captures clinical response to RCHOP in a heterogenous cohort of patients, enabling to discriminate partial and complete responder within short-time frame. Next, we identified a robust genetic signature reflecting exposure of tumoral cells to RCHOP and showed that drug-mediated targeting of one of these genes with a chemotherapy resulted in strong potentiation of RCHOP. Thus, we provide evidence of the high predictive power of the AVI-PDXTM models allowing fast and reproducible generation of tumors replicas from small size patient tumor samples. This places the AVI-PDXTM as a valuable tool for clinical research to rationalize patient inclusion in clinical trials but also for the design of personalized medicine assays. Citation Format: Clelia Costechareyre, Loraine Jarrosson, Marjorie Lacourrege, Romain Teinturier, Julie Caramel, Manon Zala, Stéphane Dalle, Pierre Sujobert, Céline Delloye-Bourgeois, Frédéric Berget, Valérie Castellani. Micro-implantation of patient tumor samples into the avian embryo, a fast and reliable alternative in vivo technology for preclinical studies on melanoma and follicular lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6907.

Abstract 1459: The immuno-AVI-PDX, am innovative patient-derived xenograft model for preclinical immuno-oncology studies

Abstract Over the last decade, the success of immunotherapy in treating numerous solid and hematological tumors is unprecedent. However, the poor preclinical predictive power of current model systems explains in part the failures during late-stages clinical drug development for the numerous potential immunotherapies. Thus, there is a crucial need for innovative technologies offering fast and reliable evaluation of immunotherapies on patient-derived models. Using its proprietary process of micro-implantation of patient tumor cells within selected organs of series of avian embryos, Oncofactory developed two humanized avian xenograft models, namely IMMUNO-AVI-patient derived xenograft (iAVI-PDXTM) and IMMUNO-AVI-cell line derived xenograft (iAVI-cellDXTM). These models, consisting in the co-implantation of human peripheral-blood mononuclear cells (PBMC) and human cancer cells were validated through a sequence of experimental steps. First, we found that transplantation of PBMCs into avian tissues exhibited good survival and low allogenicity against the avian embryonic organism. Second, in embryos engrafted with tumoral cells, we established that intravenously injected PBMCs successfully populated the tumor. Third, to reduce the dilution of immune cells in the embryonic tissues and to better standardize the number of implanted cells in between embryos, we optimized the process by direct co-implantation of both PBMCs and cancer cells. We analyzed the immune cell contingent sorted from dissected tumors and found that all major immune cells populations, including T and B lymphocytes, monocytes as well as NK cells were preserved post-grafting, when compared to pre-grafted PBMCs. The AVI-cellDXTM and AVI-PDXTM models initially developed by Oncofactory allows fast and reproducible tumor growth with statistical evaluation of molecule efficacy already at 24 hours post-injection. To assess whether our humanized models retain these advantages, we analyzed the outcome of anti-PD1 antibody (Pembrolizumab) intravenous injection in series of avian embryos co-engrafted with human PBMCs and PDL1-expressing breast and colorectal cancer cell lines. Twenty-four hours post vehicle or Pembrolizumab exposure, we analyzed the tumor volumes in grafted embryos using light sheet microscopy. In all three cancer models, we found a statistically significant reduction of the volume upon pembrolizumab, compared with vehicle. Using a panel of molecular markers in flow cytometry, we found that the Pembrolizumab efficiently targeted T lymphocytes in the tumors. Finally, we validated the efficiency of Pembrolizumab treatment in models of co-implantation of human PBMCs with melanoma and colorectal patient samples. Thus, humanized iAVI-cellDXTM or -PDXTM technology bring powerful and fast alternative to mouse models, to strongly accelerate preclinical developments in immuno-oncology. Citation Format: Marjorie Lacourrege, Clélia Costechareyre, Loraine Jarrosson, Romain Teinturier, Frédéric Berget, Céline Delloye-Bourgeois, Valérie Castellani. The immuno-AVI-PDX, am innovative patient-derived xenograft model for preclinical immuno-oncology studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1459.

ECR Spotlight – Alexandra Cones

ECR Spotlight is a series of interviews with early-career authors from a selection of papers published in Journal of Experimental Biology and aims to promote not only the diversity of early-career researchers (ECRs) working in experimental biology but also the huge variety of animals and physiological systems that are essential for the ‘comparative’ approach. Alexandra Cones is an author on ‘ The incubation environment does not explain significant variation in heart rate plasticity among avian embryos’, published in JEB. Alexandra conducted the research described in this article while a PhD student in David F. Westneat's lab at the University of Kentucky in Lexington, KY, USA. She is now a DFG postdoctoral fellow in the lab of Dr Niels Dingemanse at LMU München, Germany, investigating environmental effects on the expression and covariation of plastic traits.

The incubation environment does not explain significant variation in heart rate plasticity among avian embryos.

The conditions an organism experiences during development can modify how they plastically respond to short-term changes in their environment later in life. This can be adaptive because the optimal average trait value and the optimal plastic change in trait value in response to the environment may differ across different environments. For example, early developmental temperatures can adaptively modify how reptiles, fish and invertebrates metabolically respond to temperature. However, whether individuals within populations respond differently (a prerequisite to adaptive evolution), and whether this occurs in birds, which are only ectothermic for part of their life cycle, is not known. We experimentally tested these possibilities by artificially incubating the embryos of Pekin ducks (Anas platyrhynchos domesticus) at constant or variable temperatures. We measured their consequent heart rate reaction norms to short-term changes in egg temperature and tracked their growth. Contrary to expectations, the early thermal environment did not modify heart rate reaction norms, but regardless, these reaction norms differed among individuals. Embryos with higher average heart rates were smaller upon hatching, but heart rate reaction norms did not predict subsequent growth. Our data also suggests that the thermal environment may affect both the variance in heart rate reaction norms and their covariance with growth. Thus, individual avian embryos can vary in their plasticity to temperature, and in contrast to fully ectothermic taxa, the early thermal environment does not explain this variance. Because among-individual variation is one precondition to adaptive evolution, the factors that do contribute to such variability may be important.

In a century from agitated cells to human organoids

Reaching back more than a century, suspension cultures have provided major insights into processes of histogenesis; e.g., cell communication, distinction of self/nonself, cell sorting and cell adhesion. Besides studies on lower animals, the vertebrate retina served as excellent reaggregate model to analyze 3D reconstruction of a complex neural laminar tissue. Methodologically, keeping cells under suspension is essential to achieve tissue organisation in vitro; thereby, the environmental conditions direct the emergent histotypic particulars. Recent progress in regenerative medicine is based to a large extent on human induced pluripotent stem cells (hiPSCs), which are cultured under suspension. Following their genetically directed differentiation into various histologic 3D structures, organoids provide excellent multipurpose in vitro assay models, as well as tissues for repair transplantations. Historically, a nearly fully laminated retinal spheroid from avian embryos was achieved already in 1984, foreshadowing the potential of culturing stem cells under suspension for tissue reconstruction purposes.

Maternally derived avian corticosterone affects offspring genome-wide DNA methylation in a passerine species.

Avian embryos develop in an egg composition which reflects both maternal condition and the recent environment of their mother. In birds, yolk corticosterone (CORT) influences development by impacting pre- and postnatal growth, as well as nestling stress responses and development. One possible mechanism through which maternal CORT may affect offspring development is via changes to offspring DNA methylation. We sought to investigate this, for the first time in birds, by quantifying the impact of manipulations to maternal CORT on offspring DNA methylation. We non-invasively manipulated plasma CORT concentrations of egg-laying female zebra finches (Taeniopygia castanotis) with an acute dose of CORT administered around the time of ovulation and collected their eggs. We then assessed DNA methylation in the resulting embryonic tissue and in their associated vitelline membrane blood vessels, during early development (5 days after lay), using two established methods - liquid chromatography-mass spectrometry (LC-MS) and methylation-sensitive amplification fragment length polymorphism (MS-AFLP). LC-MS analysis showed that global DNA methylation was lower in embryos from CORT-treated mothers, compared to control embryos. In contrast, blood vessel DNA from eggs from CORT-treated mothers showed global methylation increases, compared to control samples. There was a higher proportion of global DNA methylation in the embryonic DNA of second clutches, compared to first clutches. Locus-specific analyses using MS-AFLP did not reveal a treatment effect. Our results indicate that an acute elevation of maternal CORT around ovulation impacts DNA methylation patterns in their offspring. This could provide a mechanistic understanding of how a mother's experience can affect her offspring's phenotype.

Functional precision oncology for follicular lymphoma with patient-derived xenograft in avian embryos.

Functional precision oncology for follicular lymphoma with patient-derived xenograft in avian embryos.

The effect of ambient temperature on bird embryonic development: a comparison between uniparental incubating silver‐throated tits and biparental incubating black‐throated tits

The temperature experienced by avian embryos during development has important impacts on their growth and post‐hatching phenotypes. Ambient temperature can directly affect avian nest temperature and indirectly affect it through its impact on parental incubation behaviours. Because the nests of uniparental incubators are usually left unattended more frequently than the nests of biparental incubators whose nests can be attended by another bird when one bird leaves the nest, we predict that the effect of ambient temperature on nest temperature and thus on embryonic development (specifically, incubation period length and hatching success) and post‐hatching phenotype (i.e. potential carry‐over effect on nestling body mass and condition) should be greater in uniparental incubators than in biparental incubators. To test this prediction, we studied two congeneric species, the biparental incubating black‐throated tits Aegithalos concinnus and the uniparental incubating silver‐throated tits A. glaucogularis. We found that although the embryos of the two species both developed faster (shorter incubation period length) when ambient temperature was higher, the slope was significantly greater for silver‐throated tits than for black‐throated tits, consistent with our prediction of a greater effect of ambient temperature on embryonic development in silver‐throated tits. However, the result was not due to a greater effect of ambient temperature on nest temperature, because nest temperatures of the two species had positive relationships with ambient temperature in a similar way. Therefore, it implies a greater response of silver‐throated tit embryos to temperature change. In addition, ambient temperature during the incubation stage did not affect either hatching success or nestling body mass and condition in both species. Although our predictions were not fully supported, our findings highlight the different responses of embryonic development to environmental changes between a uniparental incubator and a biparental incubator, and suggest further research to explore the mechanisms.

How the Brain Develops from the Epiblast: The Node Is Not an Organizer.

Studies using early-stage avian embryos have substantially impacted developmental biology, through the availability of simple culture methods and easiness in tissue manipulation. However, the regulations underlying brain and head development, a central issue of developmental biology, have not been investigated systematically. Yoshihi et al. (2022a) devised a technique to randomly label the epiblast cells with a green fluorescent protein before their development into the brain tissue. This technique was combined with grafting a node or node-derived anterior mesendoderm labeled with a cherry-colored fluorescent protein. Then cellular events were live-recorded over 18hours during the brain and head development. The live imaging-based analyses identified previously undescribed mechanisms central to brain development: all anterior epiblast cells have a potential to develop into the brain tissues and their gathering onto a proximal anterior mesendoderm forms a brain primordium whereas the remaining cells develop into the covering head ectoderm. The analyses also ruled out the direct participation of the node's activity in the brain development. Yoshihi et al. (2022a) also demonstrate how the enigmatic data from classical models can be reinterpreted in the new model.This chapter was adapted from Yoshihi K, Iida H, Teramoto M, Ishii Y, Kato K, Kondoh H. (2022b). Epiblast cells gather onto the anterior mesendoderm and initiate brain development without the direct involvement of the node in avian embryos: Insights from broad-field live imaging. Front Cell Dev Biol. 10:1019845. doi: 10.3389/fcell.2022.1019845.

Nearly (?) sterile avian egg in a passerine bird.

During early ontogeny, microbiome affects development of the gastrointestinal tract, immunity, and survival in vertebrates. Bird eggs are thought to be (1) initially sterile (sterile egg hypothesis) and (2) colonized after oviposition through horizontal trans-shell migration, or (3) initially seeded with bacteria by vertical transfer from mother oviduct. To date, however, little empirical data illuminate the contribution of these mechanisms to gut microbiota formation in avian embryos. We investigated microbiome of the egg content (day 0; E0-egg), embryonic gut at day 13 (E13) and female faeces in a free-living passerine, the great tit (Parus major), using a methodologically advanced procedure combining 16S rRNA gene sequencing and microbe-specific qPCR assays. Our metabarcoding revealed that the avian egg is (nearly) sterile, but acquires a slightly richer microbiome during the embryonic development. Of the three potentially pathogenic bacteria targeted by qPCR, only Dietzia was found in E0-egg (yet also in negative controls), E13 gut and female samples, which might indicate possible vertical transfer. Unlike in poultry, we have shown that major bacterial colonization of the gut in passerines does not occur before hatching. We emphasize that protocols that carefully check for environmental contamination are critical in studies with low-bacterial biomass samples.

Single-cell profiling coupled with lineage analysis reveals vagal and sacral neural crest contributions to the developing enteric nervous system.

During development, much of the enteric nervous system (ENS) arises from the vagal neural crest that emerges from the caudal hindbrain and colonizes the entire gastrointestinal tract. However, a second ENS contribution comes from the sacral neural crest that arises in the caudal neural tube and populates the post-umbilical gut. By coupling single-cell transcriptomics with axial-level-specific lineage tracing in avian embryos, we compared the contributions of embryonic vagal and sacral neural crest cells to the chick ENS and the associated peripheral ganglia (Nerve of Remak and pelvic plexuses). At embryonic day (E) 10, the two neural crest populations form overlapping subsets of neuronal and glia cell types. Surprisingly, the post-umbilical vagal neural crest much more closely resembles the sacral neural crest than the pre-umbilical vagal neural crest. However, some differences in cluster types were noted between vagal and sacral derived cells. Notably, RNA trajectory analysis suggests that the vagal neural crest maintains a neuronal/glial progenitor pool, whereas this cluster is depleted in the E10 sacral neural crest which instead has numerous enteric glia. The present findings reveal sacral neural crest contributions to the hindgut and associated peripheral ganglia and highlight the potential influence of the local environment and/or developmental timing in differentiation of neural crest-derived cells in the developing ENS.

Cardiovascular physiology of embryonic neotropic cormorants (Phalacrocorax brasilianus)

Cardiovascular maturation in avian species has primarily been studied in precocial species of birds, with few studies conducted on altricial species, which make up the majority of avian species. In the precocial species of birds studied to date, cardiovascular regulation is derived primarily from an adrenergic receptor stimulation that is present from approximately 50% to 60% of incubation until hatching. Conversely, the cholinergic modulation of heart rate differs in its timing of activation, as it is reported to be present in some studies at 60% of incubation to as late as after hatching in others. This has led to the speculation that, although adrenergic stimulation is critical to cardiovascular homeostasis, cholinergic stimulation prior to hatching in birds is species-specific and therefore is not critical for cardiovascular homeostasis in embryonic birds. In this work, we conducted a series of studies on an altricial species, the neotropic cormorant (Phalacrocorax brasilianus), to gain novel data regarding cardiovascular development in a largely unstudied group of birds. We investigated cholinergic and adrenergic receptor mediated control of both arterial blood pressure and heart rate. We predicted that, given the state of this altricial species at hatching, both cholinergic and adrenergic tone on the cardiovascular system would be functional in the embryo. Our findings indicate that cholinergic tone was present at 90% of incubation. However, there was a pronounced adrenergic tone on the cardiovascular system that was relatively greater than that reported in the other studies of avian embryos. Therefore, our findings support our prediction regarding the function of cholinergic tone and adrenergic tone prior to hatching.

No effects of the antiandrogens cyproterone acetate (CPA), flutamide and p,p'-DDE on early sexual differentiation but CPA-induced retardation of embryonic development in the domestic fowl (Gallus gallus domesticus).

Because a wide range of environmental contaminants are known to cause endocrine disorders in humans and animals, in vivo tests are needed to identify such endocrine disrupting chemicals (EDCs) and to assess their biological effects. Despite the lack of a standardized guideline, the avian embryo has been shown to be a promising model system which responds sensitively to EDCs. After previous studies on the effects of estrogenic, antiestrogenic and androgenic substances, the present work focuses on the effects of in ovo exposure to p,p'-DDE, flutamide and cyproterone acetate (CPA) as antiandrogenic model compounds regarding gonadal sex differentiation and embryonic development of the domestic fowl (Gallus gallus domesticus). The substances were injected into the yolk of fertilized eggs on embryonic day one. On embryonic day 19 sex genotype and phenotype were determined, followed by gross morphological and histological examination of the gonads. Treatment with flutamide (0.5, 5, 50 µg/g egg), p,p'-DDE (0.5, 5, 50 µg/g egg) or CPA (0.2, 2, 20 µg/g egg) did not affect male or female gonad development, assessed by gonad surface area and cortex thickness in both sexes and by the percentage of seminiferous tubules in males as endpoints. This leads to the conclusion that antiandrogens do not affect sexual differentiation during embryonic development of G. gallus domesticus, reflecting that gonads are not target organs for androgens in birds. In ovo exposure to 2 and 20 µg CPA/g egg, however, resulted in significantly smaller embryos as displayed by shortened lengths of skull, ulna and tarsometatarsus. Although gonadal endpoints were not affected by antiandrogens, the embryo of G. gallus domesticus is shown to be a suitable test system for the identification of substance-related mortality and developmental delays.

Thermal conditioning of quail embryos has transgenerational and reversible long-term effects

BackgroundIn the current context of global warming, thermal manipulation of avian embryos has received increasing attention as a strategy to promote heat tolerance in avian species by simply increasing the egg incubation temperature. However, because of their likely epigenetic origin, thermal manipulation effects may last more than one generation with consequences for the poultry industry. In this work, a multigenerational and transgenerational analysis of thermal manipulation during embryogenesis was performed to uncover the long-term effects of such procedure.ResultsThermal manipulation repeated during 4 generations had an effect on hatchability, body weight, and weight of eggs laid in Japanese quails, with some effects increasing in importance over generations. Moreover, the effects on body weight and egg weight could be transmitted transgenerationally, suggesting non-genetic inheritance mechanisms. This hypothesis is reinforced by the observed reversion of the effect on growth after five unexposed generations. Interestingly, a beneficial effect of thermal manipulation on heat tolerance was observed a few days after hatching, but this effect was not transgenerational.ConclusionsOur multigenerational study showed that thermal conditioning of quail embryos has a beneficial effect on post-hatch heat tolerance hampered by transgenerational but reversible defects on growth. Assuming that no genetic variability underlies these changes, this study provides the first demonstration of epigenetic inheritance of traits induced by environmental temperature modification associated with long-term impacts in an avian species.

Analysis of the Development of B-Cell Precursors in Day Three Incubated Chicken Embryos

Background: In mammals, birds, and amphibians, the B lineage of lymphoid cells first arise during embryogenesis and are distinguished by their capacity to produce immunoglobulin. For the purpose of researching the development of the B-cell repertoire and the development of self-tolerance, these early B-cell precursors are of utmost importance. The genetic and/or microenvironmental variables that control the beginning of immunoglobulin synthesis in embryonic haemopoietic cells are, however, poorly understood. Purpose: The ontogeny of B-cell precursors in chicken embryos from day three of incubation was examined in this work. Research methods: The terms "ontogeny, B-cell, precursors, chicken embryos, incubation" were used in a thorough literature search in the PubMed, NCBI, and Google Scholar databases. After all articles were picked based on the inclusion and exclusion criteria, 38 papers that satisfied the criteria for inclusion were collected. Result: The study's findings show that clglarge basophilic hemopoietic stem cells and cIg+ small lymphoid B-cell precursors are two types of migrant cells that appear to enter the embryonic bursa of Fabricius. Hence, B lymphopoiesis does not only take place in the bursa of Fabricius in the avian embryo. Although the yolk sac and the hemopoietic tissues around the dorsal aorta are strong candidates, the identity of the extra-bursal location remains unknown. Conclusion: Hence, general haemopoietic organs may serve as the initial site of B lymphopoiesis in both birds and mammals. Only later in the course of avian development do the bursal follicles become accessible and take over.

Developmental toxicity in early chicken embryos on exposure to an organophosphorus flame retardant, tris(2-chloroisopropyl) phosphate

Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage avian embryos are vulnerable to the toxic effects of chemicals. However, studies on the specific effects of TCIPP on avian embryonic development are limited. We aimed to investigate the toxicity of TCIPP in early chicken embryos using a previously developed shell-less incubation system. Fertilized chicken (Gallus gallus domesticus) eggs (n = 220) were exposed to 50 or 500 nmol TCIPP/(g egg) or dimethyl sulfoxide (DMSO) as a vehicle control on Day 0 of incubation. Development of 198 embryos was monitored from Days 3–9 of incubation, and 22 embryos on Day 4 and 74 embryos on Day 9 were dissected. Messenger RNA expression levels for several genes were measured in embryos on Day 4. Both TCIPP-exposed groups showed a significant reduction in survival rate. Imaging analyses revealed significant decreases in body length, head and bill length, eye diameter, and forelimb and hindlimb length in both TCIPP-treated groups. TCIPP exposure significantly impaired the development of extraembryonic blood vessels and the production of red blood cells. A TCIPP-dose-dependent decreasing trend in heart rate was observed on Days 4–7. The somitic angle increased significantly on Days 4–6, and embryos with curved somites showed cleavage in the back and gaps between somites, resulting in asymmetrical somite formation. A significant correlation was found between the somitic angle and FGF8 expression levels, suggesting that TCIPP exposure affects somite formation through an altered FGF-signaling pathway. Embryos with somitic deformities in TCIPP-exposed groups had significantly reduced survival rates, indicating that abnormal segment formation directly increased mortality. Finally, eye weight and ocular luminosity values were significantly reduced, suggesting that TCIPP may also affect eye development. Overall, these findings highlight severe toxic effects of TCIPP on avian embryonic development, including in vascularization, cardiac function, and somite and ocular development.

Heat-induced maternal effects shape avian eggshell traits and embryo development and phenotype at high incubation temperatures.

Phenotypic plasticity is an important avenue by which organisms may persist in the face of rapid environmental change. Environmental cues experienced by the mother can also influence the phenotype of offspring, a form of plasticity called maternal effects. Maternal effects can adaptively prepare offspring for the environmental conditions they will likely experience; however, their ability to buffer offspring against environmental stressors as embryos is understudied. Using captive zebra finches, we performed a maternal-offspring environmental match-mismatch experiment utilizing a 2 × 2 × 2 factorial design. Mothers were exposed to a mild heat conditioning (38°C) or control (22°C) treatment as juveniles, an acute high heat (42°C) or control (22°C) treatment as adults, then paired for breeding. The eggs produced by those females were incubated at a hyperthermic (38.5°C) or optimal temperature (37.2°C). We found that when mothers were exposed to a mild heat conditioning as juveniles, their embryos exhibited reduced water loss, longer development times, and produced hatchlings with heavier pectoralis muscles when incubated at high incubation temperatures, compared to embryos from control mothers. Mothers exposed to both the mild heat conditioning as juveniles and a high heat stressor as adults produced eggs with a higher density of shell pores and embryos with lower heart rates during development. However, there was a cost when there was a mismatch between maternal and embryo environment. Embryos from these conditioned and heat-stressed mothers had reduced survival at control incubation temperatures, indicating the importance of offspring environment when interpreting potential adaptive effects.