Developmental morpho-analysis of the caecum in Japanese quail embryos (Coturnix coturnix japonica).

In the current study, we are focusing on the microanatomical structure of quail caecum during the prehatching time to try to understand the function and the role of each cell-built quail caecum reaching how caecum plays an essential role in immunity and absorption. The morpho-developmental features of the quail caecum were described in detail daily from the third incubation day (ID) till hatching time, investigating the gross morphology, microscopic, and ultrastructure using light and scanning electron microscope. The embryonic caecum appeared grossly as two lateral outpocketings with blinded ends, emerging laterally at the junction between the small and large intestine (the ileocaecal junction). The primordia of two caeca, represented by two lateral swellings from the hindgut on the fourth ID, continued growing till the day of hatching, where the caecal wall consisted of three apparent layers: mucosa, musculosa, and serosa. At the time of hatching, the quail caecum was still not fully mature and will continue growing posthatching. The findings in this study can be applied in further studies intended to understand the physiological mechanisms of the caecum during prehatching and posthatching periods. RESEARCH HIGHLIGHTS: Caecum is one of the hindgut derivatives that started as two lateral swellings. The caecal wall consisted of three layers; mucosa, musculosa, and serosa. The caecum plays an essential role in immunity maintenance. Caecum continues to grow posthatching as it is not fully mature at hatching time.

Adaptive patterning of vascular network during avian skin development: Mesenchymal plasticity and dermal vasculogenesis.

A vasculature network supplies blood to feather buds in the developing skin. Does the vasculature network during early skin development form by sequential sprouting from the central vasculature or does local vasculogenesis occur first that then connect with the central vascular tree? Using transgenic Japanese quail Tg(TIE1p.H2B-eYFP), we observe that vascular progenitor cells appear after feather primordia formation. The vasculature then radiates out from each bud and connects with primordial vessels from neighboring buds. Later they connect with the central vasculature. Epithelial-mesenchymal recombination shows local vasculature is patterned by the epithelium, which expresses FGF2 and VEGF. Perturbing noggin expression leads to abnormal vascularization. To study endothelial origin, we compare transcriptomes of TIE1p.H2B-eYFP+ cells collected from the skin and aorta. Endothelial cells from the skin more closely resemble skin dermal cells than those from the aorta. The results show developing chicken skin vasculature is assembled by (1) physiological vasculogenesis from the peripheral tissue, and (2) subsequently connects with the central vasculature. The work implies mesenchymal plasticity and convergent differentiation play significant roles in development, and such processes may be re-activated during adult regeneration. SUMMARY STATEMENT: We show the vasculature network in the chicken skin is assembled using existing feather buds as the template, and endothelia are derived from local bud dermis and central vasculature.

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 3ß-HSD) and estrogen receptor-ß, 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.

Signaling pathways and regulatory networks in quail skeletal muscle development: insights from whole transcriptome sequencing.

Quail, as an advantageous avian model organism due to its compact size and short reproductive cycle, holds substantial potential for enhancing our understanding of skeletal muscle development. The quantity of skeletal muscle represents a vital economic trait in poultry production. Unraveling the molecular mechanisms governing quail skeletal muscle development is of paramount importance for optimizing meat and egg yield through selective breeding programs. However, a comprehensive characterization of the regulatory dynamics and molecular control underpinning quail skeletal muscle development remains elusive. In this study, through the application of HE staining on quail leg muscle sections, coupled with preceding fluorescence quantification PCR of markers indicative of skeletal muscle differentiation, we have delineated embryonic day 9 (E9) and embryonic day 14 (E14) as the start and ending points, respectively, of quail skeletal muscle differentiation. Then, we employed whole transcriptome sequencing to investigate the temporal expression profiles of leg muscles in quail embryos at the initiation of differentiation (E9) and upon completion of differentiation (E14). Our analysis revealed the expression patterns of 12,012 genes, 625 lncRNAs, 14,457 circRNAs, and 969 miRNAs in quail skeletal muscle samples. Differential expression analysis between the E14 and E9 groups uncovered 3,479 differentially expressed mRNAs, 124 lncRNAs, 292 circRNAs, and 154 miRNAs. Furthermore, enrichment analysis highlighted the heightened activity of signaling pathways related to skeletal muscle metabolism and intermuscular fat formation, such as the ECM-receptor interaction, focal adhesion, and PPAR signaling pathway during E14 skeletal muscle development. Conversely, the E9 stage exhibited a prevalence of pathways associated with myoblast proliferation, exemplified by cell cycle processes. Additionally, we constructed regulatory networks encompassing lncRNA‒mRNA, miRNA‒mRNA, lncRNA‒miRNA-mRNA, and circRNA-miRNA‒mRNA interactions, thus shedding light on their putative roles within quail skeletal muscle. Collectively, our findings illuminate the gene and non-coding RNA expression characteristics during quail skeletal muscle development, serving as a foundation for future investigations into the regulatory mechanisms governing non-coding RNA and quail skeletal muscle development in poultry production.

Embryonic methionine triggers post-natal developmental programming in Japanese quail.

Embryonic development is one of the most sensitive and critical stages when maternal effects may influence the offspring's phenotype. In birds and other oviparous species, embryonic development is confined to the eggs, therefore females must deposit resources into the eggs to prepare the offspring for the prevailing post-natal conditions. However, the mechanisms of such phenotypic adjustments remain poorly understood. We simulated a maternal nutritional transfer by injecting 1 mg of L-methionine solution into Japanese quail eggs before the onset of incubation. The increase in early methionine concentration in eggs activated the insulin/insulin-like signalling and mechanistic target of rapamycin (IIS/mTOR) signalling pathways and affected post-natal developmental trajectories. Chicks from methionine-supplemented eggs had higher expression of liver IGF1 and mTOR genes at hatching but were similar in size, and the phenotypic effects of increased growth became apparent only a week later and remained up to three weeks. Circulating levels of insulin-like growth factor-1 (IGF-1) and expression of ribosomal protein serine 6 kinase 1 (RPS6K1), the mTOR downstream effector, were elevated only three weeks after hatching. These results show that specific nutritional cues may have phenotypic programming effects by sequentially activating specific nutrient-sensing pathways and achieving transgenerational phenotypic plasticity.

Expression atlas of avian neural crest proteins: Neurulation to migration.

Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.

Double-path parallel convolutional neural network for removing speckle noise in different types of OCT images.

Speckle noises widely exist in optical coherence tomography (OCT) images. We propose an improved double-path parallel convolutional neural network (called DPNet) to reduce speckles. We increase the network width to replace the network depth to extract deeper information from the original OCT images. In addition, we use dilated convolution and residual learning to increase the learning ability of our DPNet. We use 100 pairs of human retinal OCT images as the training dataset. Then we test the DPNet model for denoising speckles on four different types of OCT images, mainly including human retinal OCT images, skin OCT images, colon crypt OCT images, and quail embryo OCT images. We compare the DPNet model with the adaptive complex diffusion method, the curvelet shrinkage method, the shearlet-based total variation method, and the OCTNet method. We qualitatively and quantitatively evaluate these methods in terms of image smoothness, structural information protection, and edge clarity. Our experimental results prove the performance of the DPNet model, and it allows us to batch and quickly process different types of poor-quality OCT images without any parameter fine-tuning under a time-constrained situation.

Cyclin D1 gene expression is essential for cell cycle progression from the maternal-to-zygotic transition during blastoderm development in Japanese quail.

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.

The Trichohyalin-Like Protein Scaffoldin Is Expressed in the Multilayered Periderm during Development of Avian Beak and Egg Tooth.

Scaffoldin, an S100 fused-type protein (SFTP) with high amino acid sequence similarity to the mammalian hair follicle protein trichohyalin, has been identified in reptiles and birds, but its functions are not yet fully understood. Here, we investigated the expression pattern of scaffoldin and cornulin, a related SFTP, in the developing beaks of birds. We determined the mRNA levels of both SFTPs by reverse transcription polymerase chain reaction (RT-PCR) in the beak and other ectodermal tissues of chicken (Gallus gallus) and quail (Coturnix japonica) embryos. Immunohistochemical staining was performed to localize scaffoldin in tissues. Scaffoldin and cornulin were expressed in the beak and, at lower levels, in other embryonic tissues of both chickens and quails. Immunohistochemistry revealed scaffoldin in the peridermal compartment of the egg tooth, a transitory cornified protuberance (caruncle) on the upper beak which breaks the eggshell during hatching. Furthermore, scaffoldin marked a multilayered peridermal structure on the lower beak. The results of this study suggest that scaffoldin plays an evolutionarily conserved role in the development of the avian beak with a particular function in the morphogenesis of the egg tooth.

Morphometric evaluation of Japanese quail embryos and their extraembryonic vascular networks exposed to low-frequency magnetic field with two different intensities.

Animals developed or in an embryonic stage, are constantly subjected to magnetic pollution generated by electrical and electronic devices. Several researches have used the bird embryo as an experimental model to evaluate the action of magnetic field (MF) and electromagnetic field (EMF). This study proposed to perform a morphometric evaluation in the embryos and in the blood vascular network of the yolk sac membranes (YSM) of Japanese quail (Coturnix japonica) exposed to the 60 Hz MF with two different intensities (0.16 and 0.65 mT). A total of 30 eggs were used, 10 eggs were used for each assay. Each assay formed a group (control group, group submitted to the MF of 0.16 mT and 0.65 mT). The images of the skeletonized vascular network of YSM were evaluated by two methods of fractal dimension: box-counting dimension (Dbc) and information dimension (Dinf). The embryos were evaluated by body mass, percentage cephalic length and body area. The fractal dimensions revealed no difference among groups. There were no significant differences in relation to embryonic body mass among groups. However, the embryos exposed to 0.65 mT MF presented a smaller embryonic body development (body area and percentage cephalic length). In conclusion, 0.16 mT and 0.65 mT magnetic fields were not able to generate significant effects on vasculogenesis and angiogenesis. However, the embryos exposed to 6 h of magnetic field with 0.65 mT intensity and 60 Hz frequency showed a decrease in embryonic body development.

Three-dimensional alignment of microvasculature and cardiomyocytes in the developing ventricle.

While major coronary artery development and pathologies affecting them have been extensively studied, understanding the development and organization of the coronary microvasculature beyond the earliest developmental stages requires new tools. Without techniques to image the coronary microvasculature over the whole heart, it is likely we are underestimating the microvasculature's impact on normal development and diseases. We present a new imaging and analysis toolset to visualize the coronary microvasculature in intact embryonic hearts and quantify vessel organization. The fluorescent dyes DiI and DAPI were used to stain the coronary vasculature and cardiomyocyte nuclei in quail embryo hearts during rapid growth and morphogenesis of the left ventricular wall. Vessel and cardiomyocytes orientation were automatically extracted and quantified, and vessel density was calculated. The coronary microvasculature was found to follow the known helical organization of cardiomyocytes in the ventricular wall. Vessel density in the left ventricle did not change during and after compaction. This quantitative and automated approach will enable future cohort studies to understand the microvasculature's role in diseases such as hypertrophic cardiomyopathy where misalignment of cardiomyocytes has been observed in utero.

Influence of a 1000 Times Weakened Magnetic Field on Embryogenesis and Ontogenesis of the Japanese Quail in Several Generations.

The paper presents experimental data on the influence of a 1000-fold weakening of the Earth's magnetic field on the embryonic and postembryonic development of the Japanese quail in three generations. It has been shown that the weakening of the earth's magnetic field by a factor of 1000 affects the formation of blood vessels in Japanese quail embryos, in particular, causing a decrease in angiogenesis in seven-day-old embryos of both the first generation (F1) and the next two ones (F2 and F3). Pathological and anatomical studies of embryos of different ages in three generations have revealed various pathologies associated with vascular system disorders, as well as disorders in the development of the beak and eyes. In the ontogenesis of F3 quails, there is a decrease in the hatchability of chicks.

Visualizing mesoderm and neural crest cell dynamics during chick head morphogenesis.

Vertebrate head morphogenesis involves carefully-orchestrated tissue growth and cell movements of the mesoderm and neural crest to form the distinct craniofacial pattern. To better understand structural birth defects, it is important that we characterize the dynamics of these processes and learn how they rely on each other. Here we examine this question during chick head morphogenesis using time-lapse imaging, computational modeling, and experiments. We find that head mesodermal cells in culture move in random directions as individuals and move faster in the presence of neural crest cells. In vivo, mesodermal cells migrate in a directed manner and maintain neighbor relationships; neural crest cells travel through the mesoderm at a faster speed. The mesoderm grows with a non-uniform spatio-temporal profile determined by BrdU labeling during the period of faster and more-directed neural crest collective migration through this domain. We use computer simulations to probe the robustness of neural crest stream formation by varying the spatio-temporal growth profile of the mesoderm. We follow this with experimental manipulations that either stop mesoderm growth or prevent neural crest migration and observe changes in the non-manipulated cell population, implying a dynamic feedback between tissue growth and neural crest cell signaling to confer robustness to the system. Overall, we present a novel descriptive analysis of mesoderm and neural crest cell dynamics that reveals the coordination and co-dependence of these two cell populations during head morphogenesis.

Embryonic thermal manipulation has short and long-term effects on the development and the physiology of the Japanese quail.

In vertebrates, the embryonic environment is known to affect the development and the health of individuals. In broiler chickens, the thermal-manipulation (TM) of eggs during the incubation period was shown to improve heat tolerance at slaughter age (35 days of age) in association with several modifications at the molecular, metabolic and physiological levels. However, little is known about the Japanese quail (Coturnix japonica), a closely related avian species widely used as a laboratory animal model and farmed for its meat and eggs. Here we developed and characterized a TM procedure (39.5°C and 65% relative humidity, 12 h/d, from days 0 to 13 of incubation) in quails by analyzing its short and long-term effects on zootechnical, physiological and metabolic parameters. Heat-tolerance was tested by a heat challenge (36°C for 7h) at 35 days of age. TM significantly reduced the hatching rate of the animals and increased mortality during the first four weeks of life. At hatching, TM animals were heavier than controls, but lighter at 25 days of age for both sexes. Thirty-five days after hatching, TM decreased the surface temperature of the shank in females, suggesting a modulation of the blood flow to maintain the internal temperature. TM also increased blood partial pressure and oxygen saturation percentage at 35 days of age in females, suggesting a long-term modulation of the respiration physiology. Quails physiologically responded to the heat challenge, with a modification of several hematologic and metabolic parameters, including an increase in plasma corticosterone concentration. Several physiological parameters such as beak surface temperature and blood sodium concentration revealed that TM birds responded differently to the heat challenge compared to controls. Altogether, this first comprehensive characterization of TM in Japanese quail showed durable effects that may affect the response of TM quails to heat.

Uptake, Deposition, and Metabolism of Triphenyl Phosphate in Embryonated Eggs and Chicks of Japanese Quail (Coturnix japonica).

The toxicokinetics of triphenyl phosphate (TPHP) in vivo including the uptake, deposition, and biotransformation into the metabolite diphenyl phosphate (DPHP) is presently reported in embryonated eggs and chicks of Japanese quail. Quail were dosed with TPHP at 3 concentrations by air cell egg injection on embryonic day 0, followed by daily oral dosing after chicks hatched (5 d). Vehicle-only exposed controls were also used. In dosed eggs, only 33% of the TPHP remained 2 d after injection (no hepatic development); after 10 d (post-hepatogenesis), only 2% remained. The estimated TPHP half-lives in the eggs ranged from 1.1 to 1.8 d for the 3 dosed groups. In all exposed eggs and chicks, DPHP significantly increased with dose (0.001 < p < 0.044). It appears that DPHP is an important metabolite in quail, making up 41 to 74% of all metabolites formed in embryonated eggs. In chicks, at medium and high doses, DPHP concentrations significantly exceeded those of TPHP (p ≤ 0.007), making up 67 and 76% of the total burden, respectively. Our findings suggest that rapid TPHP metabolism occurred in chicks and embryonated quail eggs but that this may vary with the age of the embryonated egg and the stage of embryo development, which should be considered when evaluating concentrations of TPHP and DPHP measured in eggs of wild birds. Environ Toxicol Chem 2020;39:565-573. © 2019 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry © 2019 SETAC.

Cellular Invasion and Matrix Degradation, a Different Type of Matrix-Degrading Cells in the Cartilage of Catfish (Clarias gariepinus) and Japanese Quail Embryos (Coturnix coturnix japonica).

We previously studied the phenomena of the mesenchymal cell-dependent mode of cartilage growth in quail and catfish. Thus, we selected the two cartilage models in which mesenchymal cells participate in their growth. In such models, cartilage degradation occurred to facilitate cellular invasion. The studies do not explain the nature of the cartilage degrading cells. The current study aims to explore the nature of the cartilage-degrading cells using transmission electron microscopy (TEM) and immunohistochemistry. Samples of cartilage have been isolated from the air-breathing organ of catfish and the cartilage of the prospective occipital bone of quail embryos. Samples have been processed for TEM and immunohistochemistry. We found that two different cell types are involved in cartilage degradation; the macrophage in the cartilage of catfish and mesenchymal cells in the cartilage of the quail. Areas of cellular invasion in both catfish cartilage and quail embryo cartilage had an immunological affinity for MMP-9. In catfish, cartilage-degrading cells had identical morphological features of macrophages, whereas in quail embryos, cartilage-degrading cells were mesenchymal-like cells which had cell processes rich in vesicles and expressed CD117. Further study should consider the role of macrophage and mesenchymal cells during cartilage degradation. This could be valuable to be applied to remove the defective cartilage matrix formed in osteoarthritic patients to improve cartilage repair strategies.

Sequence alteration in the enhancer contributes to the heterochronic Sox9 expression in marsupial cranial neural crest.

Neonates of marsupial mammals are altricial at birth, because their gestation period is relatively short compared to placental mammals. Yet, as they need to travel to the teat from the birth canal, and suckle on the mother's milk, forelimbs and jaws develop significantly early. Previous studies in opossum (Monodelphis domestica), an experimental marsupial model, have revealed that cranial neural crest cells are generated significantly early compared to those in placental mammals, such as mouse, leading to an early development of jaw primordia. We have previously found that Sox9, an important neural crest-specifier gene, is expressed in the future cranial neural crest of the opossum embryonic ectoderm significantly earlier than that in mouse or quail embryos. As Sox9 is essential for neural crest formation in various vertebrates, it seems likely that the heterochronic expression of Sox9 is critical for the early cranial neural crest formation in the marsupial embryos. In this study, we show a marsupial-specific sequence in the Sox9 neural crest enhancer E3. We also reveal that the mouse E3 enhancer is activated in the cranial neural crest cells of quail embryos, that the E3 enhancer with marsupial-specific sequence is activated earlier in the Pax7-expressing neural border prior to the onset of endogenous Sox9 expression, and that a misexpression of cMyb, which is also a transcriptional activator of Pax7, in the neural border can ectopically activate the "marsupialized" enhancer. Thus, we suggest that the modification of the E3 enhancer sequence in the marsupial ancestor would have promoted the early expression of Sox9 in the neural border, facilitating the early formation of the cranial neural crest cells and the subsequent heterochronic development of the jaw primordia.

Spirulina platensis in Japanese quail feeding alters fatty acid profiles and improves egg quality: Benefits to consumers.

The aim of this study was to investigate whether microalgae in Japanese quail feed alters performance, fatty acid profiles in the eggs and egg quality. One hundred quails were distributed in four groups and five replicates of five birds per experimental group. The treatments consisted of four levels of Spirulina platensis microalgae (0%, 5%, 10%, and 15%) in the diets. We evaluated the productive performance and chemical-physical characteristics of eggs, the oxidant/antioxidant status in egg yolks, and the fatty acid profile in the diet and egg yolks. Microalgae in the diet did not influence egg production; however, it increased the yolk index as well as the color intensity of the yolk. Saturated and polyunsaturated fatty acid levels decreased in egg yolks, and monounsaturated fatty acid levels increased in the yolks. Lipid peroxidation levels were lower and total antioxidant capacity was higher in egg yolks of quails receiving microalgae in the diet. PRACTICAL APPLICATIONS: Microalgae in quail diets improves egg quality and provides benefits to consumer health, acting as an antioxidant and immune-stimulant. Microalgae in quail diets had positive effects on egg quality. This is because it reduced levels of saturated fatty acids that are undesirable, and increased monounsaturated fatty acid levels that are beneficial to the health of consumers. Finally, antioxidants increased in egg yolks, consequently reducing lipid peroxidation.

Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail.

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

Exposure of Avian Embryos to Cycling Incubation Temperatures Reduces Adult Bactericidal Ability.

In birds, the temperature at which eggs are incubated shapes many aspects of hatchling phenotype, but long-term effects are less studied. We studied the effect of incubation temperature and pattern on the subsequent development of innate immune function in Japanese quail (Coturnix japonica). We incubated quail eggs in one of three replicated treatments: control (37.5°C), low (36.0°C), and cyclical incubation. The cyclical treatment had the same average temperature as the low-temperature treatment (36.0°C) and an upper temperature that was the same as the control. When individuals were 5, 20, and 55 d of age (i.e., adults), we measured the ability of blood plasma to kill Escherichia coli. Throughout development there was a nonsignificant trend for immune function to be lower in the cycling treatment. In adulthood, however, individuals incubated at cycling temperatures had significantly lower immune function than control birds but did not differ from individuals incubated at constant low temperatures. Males and females responded similarly to the incubation treatment, but females developed a greater plasma bactericidal ability than males. We conclude that variation in innate immune function of adult birds is shaped by temperature fluctuations experienced during incubation.