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.

Taxonomic insights and evolutionary history in East Asian terrestrial slugs of the genus Meghimatium.

East Asia, specifically the Japanese Archipelago, is a biodiversity hotspot of both vertebrates and invertebrates. Mollusks represent a burst of species diversity in this region due to the effects of biotic and abiotic factors on their morphological traits, such as shell shape and size. However, the evolutionary history of terrestrial slugs in East Asia remains unknown. In the present study, we investigated the molecular phylogeny of terrestrial slugs of the genus Meghimatium. This genus includes three described and eight undescribed species, and our study used all except for two. Based on phylogeny and the species delimitation tests, the genus Meghimatium was split into many putative species, suggesting higher species diversity than previously thought based on morphological and anatomical studies and that almost undescribed species may be inappropriate. Therefore, morphological traits, such as body size and colour, conventionally considered for classification may easily vary or be similar across geographic region. Moreover, the divergence time of this genus is almost concordant with the geographical time scale of the formation of the Japanese mainland. Our findings suggest that molecular phylogenetics helps classify Japanese Meghimatium slugs, but comprehensive taxonomic revisions using multi-locus analyses are needed.

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.

Effects of tris(2-chloroethyl) phosphate exposure on chicken embryos in a shell-less incubation system.

Tris(2-chloroethyl) phosphate (TCEP) is an organophosphate flame retardant that used in textiles, industrial materials, and furniture to delay the spread of fire after ignition. TCEP has been detected in the tissues and eggs of fish and birds. However, there are no studies regarding the effects of TCEP on avian embryos. In the present study, we investigated the developmental toxicity of TCEP exposure on chicken embryos in a shell-less incubation system, which enables in situ observation. Chicken embryos were treated with graded doses of TCEP (50, 250, and 500 nmol/g egg) on incubation day 0. The survival rate, morphological biometrics, heart rate, and length and branch number of extraembryonic blood vessels were measured on incubation days 3-9. Survival rates were reduced from incubation day 3 and were significantly decreased until day 9. Body length, head + bill length and eye diameter were significantly reduced by TCEP exposure. Regarding skeletal effects, spine length was decreased in a dose-dependent manner on day 9. Body weight on day 9 significantly reduced in all TCEP treatment groups. These results suggest that TCEP exposure to >50 nmol/g egg retards development in chicken embryos. TCEP exposure to 500 nmol/g egg significantly increased heart weight to body weight ratio in the embryos. More than 250 nmol/g egg of TCEP significantly reduced the heart rate of embryos in the early developmental stage. The formation of extraembryonic blood vessels and the number of erythrocytes were significantly reduced even with 50 nmol/g egg of TCEP. These findings suggest that TCEP exposure specifically affects the cardiovascular system in chicken embryos, which leads to developmental delay. The results of this study also demonstrate that the shell-less incubation system can be used to continuously monitor the effects of chemicals on developing avian embryos.

Intra-allantoic injection of calcium promotes hatching of chick embryos grown in shell-less culture.

The hatch rate of chick embryos cultured outside of the eggshell with 350 mg calcium l-lactate hydrate (CaL) and 3.5 mL water is fourfold greater in cultures in which the chorioallantoic membrane (CAM) surrounds the egg contents by incubation day 17.5 (E17.5) an event which occurs in ovo by E13. It was first investigated whether decreasing the volume of water added with 350 mg CaL would promote CAM expansion due to the smaller volume to enclose. When 350 mg CaL was present, the CAM did not surround the egg contents by E13. By E17.5, the CAM surrounded the egg contents in 53%-74% of cultures; however, CAM expansion was not significantly different when 0, 1, 2, or 3.5 mL water was present. The hatch rate with 2 or 3.5 mL water was greater than 50% but was not improved with less water. Second, it was investigated whether CaL or water inhibits CAM expansion. In the absence of CaL, the CAM surrounded the egg contents in up to two-thirds of cultures by E13, whether 2 mL water was present or not. Thus CaL, but not water, inhibits expansion of the CAM by E13, even though CaL promotes hatching. Finally, it was investigated whether injection of aqueous CaL into the allantoic fluid, in conjunction with not adding CaL to culture hammocks, would promote CAM expansion. Allantoic injection of CaL starting at E13 did not promote CAM expansion at E17.5 but resulted in hatch rates of approximately 30%. Allantoic injection is a novel route for supplementation of calcium in cultured chick embryos.

Supplemental calcium increases hatch rate but not hatchling mass of chick embryos in shell-less culture.

A method is described for culturing 64-70 h-old chicken embryos and egg contents outside of the eggshell through to hatching. Cultured egg contents were suspended in polymethylpentene kitchen wrap (F.O.R. wrap; Riken Fabro) supported in polyvinyl chloride tripods. Tripods were incubated in Plexiglas environmental chambers which were rocked automatically through an angle of ±20°. The concentration of CO2 was maintained at 2% throughout incubation, while that of O2 was increased from ambient to 50%, and relative humidity was decreased from 90%-92% to 83%-84% at incubation Day 9. Cultured embryos not supplemented with calcium did not hatch. The Hatch rate increased when supplemental calcium L-lactate hydrate was increased between 250 and 350 mg. A maximal hatch rate of 54.8% was achieved when cultures were supplemented with 350 mg of calcium L-lactate hydrate and 3.5 ml of sterile water. Adding 400 or 450 mg of calcium L-lactate hydrate did not increase the hatch rate further. The mass of cultured hatchlings (including the retracted yolk) and yolk-free carcass wet and dry mass and length of the right third toe were significantly less than the corresponding parameters observed in hatchlings in ovo. No statistically significant differences in hatchling mass, yolk-free carcass wet or dry mass, or length of the right third toe were noted among cultured hatchlings supplemented with 250-450 mg of calcium L-lactate hydrate. Failure to completely absorb albumen was the most common abnormality observed in cultures which failed to hatch. The present technique allows a unique approach to study the physiology of the developing chicken embryo.

Shell-less culture system for chick embryos from the blastoderm stage to hatching.

Since 2014, methods have been described to hatch chick embryos from shell-less culture after egg contents are first incubated within shells for 55-70 h. The present report describes for the first time a shell-less culture system for chick embryos from the blastoderm stage to hatching. For the first 69-70 h, egg contents suspended in polymethylpentene kitchen wrap (F.O.R. Wrap, Riken Fabro, Tokyo, Japan) supported in 6.35 or 6.67 cm inside diameter tripods and covered with a disc of immobilized Milli-Wrap, were rotated back and forth through 90° at 16 or 22 cycles per minute (CPM). Subsequently, the Milli-Wrap disc was removed and culture tripods were transferred to environmental chambers, which were rocked ±20° through incubation day 8.5 (E8.5). From E9, environmental chambers were maintained in the horizontal position through to hatching with controlled O2 and CO2 . To provide supplemental calcium, an aqueous solution containing 100 mg/mL of calcium l-lactate hydrate was injected through the plastic wrap into the albumen at E9 (2.5 mL) and at E13 (1.0 mL) or E15 (1.0 mL). After incubation for 69-70 h at 16 or 22 CPM, 80%-83% of previously unincubated egg contents yielded apparently normal embryos. Hatch rate of normal embryos resulting from turntable incubation at 16 or 22 CPM was approximately 43%. Of note, egg contents remained in the same culture tripod from blastoderm stage to hatching. This technique may find use as an educational tool and in basic investigations of early embryogenesis, teratogenesis, and gene transfer experiments.

Analysis of HDACi-Coupled Nanoparticles: Opportunities and Challenges.

Systemic administration of histone deacetylase inhibitors (HDACi), like valproic acid (VPA), is often associated with rapid drug metabolization and untargeted tissue distribution. This requires high-dose application that can lead to unintended side effects. Hence, drug carrier systems such as nanoparticles (NPs) are developed to circumvent these disadvantages by enhancing serum half-life as well as organ specificity.This chapter gives a summary of the biological characterization of HDACi-coupled NPs in vitro, including investigation of cellular uptake, biocompatibility, as well as intracellular drug release and activity. Suitable methods, opportunities, and challenges will be discussed to provide general guidelines for the analysis of HDACi drug carrier systems with a special focus on recently developed cellulose-based VPA-coupled NPs.

Effects of Calcium Lactate on the Development of Chicken Embryos in a Shell-less Culture System up to Day Seventeen of Incubation.

This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation. In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.

Effects of Oxygen Gas Injection on the Subsequent Development of Chick Embryos in a Shell-Less Culture System.

The effects of oxygen gas injection starting on day 17 of incubation (D17) in a chick shell-less culture system (cSLC) on the subsequent embryo development were examined on day 19 of incubation (D19). On D19 of cSLC, the plasma phosphorus and total cholesterol concentrations of the embryos were significantly higher (P<0.05), while the plasma calcium concentrations were significantly lower (P<0.05) than those in the intact control (IC) group. However, no significant differences in embryo viability and other major blood component levels were observed among the experimental groups (P>0.05). The percutaneous oxygen saturation was lower in D17-cSLC embryos before oxygen gas supplementation than in the IC (P<0.05) embryos. Severe renal tubular degeneration of the metanephros was observed in D19-cSLC embryos despite oxygen gas injection starting from D17. These results indicate that D19-cSLC embryos are hypoxia even after injecting oxygen gas starting on D17.

Effect of Organic or Inorganic Mineral Premix in the Diet on Laying Performance of Aged Laying Hens and Eggshell Quality.

In this study, we examined the effect of diets supplemented with organic and inorganic mineral premixes on the laying performance and eggshell quality of aged laying hens. A total of 600 68-week-old Hy-Line Brown laying hens were randomly assigned to 1 of 3 dietary treatments, repeated 5 times: Mash type basal diet, basal diet supplemented with an inorganic mineral premix (1.0 g/kg), and basal diet supplemented with an organic mineral premix (1.8 g/kg). The results showed that eggshell strength was higher (p < 0.01) in the inorganic mineral diet group than in the organic mineral and basal diet groups. Further, the levels of Fe and Mn in the liver were higher (p < 0.05) in the inorganic and organic mineral diet groups than in the basal diet group. The concentrations of Fe and Mg in the spleen were different (p < 0.05) among the treatment groups, with the highest levels reported in the organic mineral premix group. The concentrations of Cu, Zn, and Mn in the eggshell were different (p < 0.05) among the groups, with the highest levels reported in the inorganic and organic mineral premix diet groups. In conclusion, a diet containing organic mineral premix improved eggshell strength and had no detrimental effect on the laying performance of aged laying hens.

Ex Ovo Culture System for Avian Embryos and its Application.

Ex ovo culture of avian embryos can be applied not only to embryology but also to various fields of basic research such as embryo manipulation, toxicology, and regenerative medicine. The windowing method, which facilitates various manipulations and observations by opening a hole in one part of the eggshell, and culture systems using surrogate eggshells, are widely used. Despite this, biology lessons in high schools cover shell-less culture systems, which involve the development of avian embryos in artificial vessels, such as rice bowls, without using surrogate eggshells. However, as embryo development stops at its early stages in this method, it is not possible to continuously observe the development of the embryo. This led to attempts to develop an embryo culture method using a complete artificial culture vessel that does not use surrogate eggshells, and Kamihira et al. (1998) succeeded in hatching quail embryos in an artificial culture vessel using polytetrafluoroethylene membranes. In addition, Tahara succeeded in hatching chick embryos in artificial culture vessels that used cling film made of polymethylpentene and reported their detailed methodology (Tahara and Obara, 2014). These technologies are being applied not only to school education but also to various fields of research.

Calcium carbonate supplementation to chorioallantoic membranes improves hatchability in shell-less chick embryo culture.

Developing chick embryos are a classical research tool in developmental biology. The whole embryo culture technique can be applied to various fields, such as embryo manipulation, toxicology, tumorigenesis, and basic research in regenerative medicine. When used for the generation of transgenic chickens, a high hatchability of genetically engineered embryos is essential to support normal embryonic development during culture. In this study, calcium carbonate, which is the main component of eggshells, was added as a calcium source in shell-less chick embryo cultures using a transparent plastic film as a culture vessel. In the absence of a calcium source in the shell-less culture system, embryogenesis ceased during culture, resulting in failed embryonic hatching. We found that the direct addition of calcium carbonate to the chorioallantoic membrane of the developing embryo was effective for the hatching of cultured chick embryos. The amount, timing, and location of calcium carbonate addition were investigated to maximize the hatchability of cultured embryos. Starting from the time of calcium carbonate supplementation, >40% hatchability was obtained with the optimal condition. This established method of shell-less chick embryo culture provides a useful tool in basic and applied fields of chick embryo manipulation.

Effects of probiotic supplementation on performance traits, bone mineralization, cecal microbial composition, cytokines and corticosterone in laying hens.

Recent researches have showed that probiotics promote bone health in humans and rodents. The objective of this study was to determine if probiotics have the similar effects in laying hens. Ninety-six 60-week-old White Leghorn hens were assigned to four-hen cages based on their BW. The cages were randomly assigned to 1 of 4 treatments: a layer diet mixed with a commercial probiotic product (containing Enterococcus faecium, Pediococcus acidilactici, Bifidobacterium animalis and Lactobacillus reuteri) at 0, 0.5, 1.0 or 2.0 g/kg feed (Control, 0.5×, 1.0× and 2.0×) for 7 weeks. Cecal Bifidobacterium spp. counts were higher in all probiotic groups (P0.05). In addition, the plasma concentrations of cytokines (interleukin-1ß, interleukin-6, interleukin-10, interferon-γ and tumor necrosis factor-α) and corticosterone as well as the levels of heterophil to lymphocyte ratio were similar between the 2.0× group and the control group (P>0.05). In line with these findings, no differences of cecal tonsil mRNA expressions of interleukin-1ß, interleukin-6 and lipopolysaccharide-induced tumor necrosis factor-α factor were detected between these two groups (P>0.05). These results suggest that immune cytokines and corticosterone may not involve in the probiotic-induced improvement of eggshell quality and bone mineralization in laying hens. In conclusion, the dietary probiotic supplementation altered cecal microbiota composition, resulting in reduced shell-less egg production and improved bone mineralization in laying hens; and the dietary dose of the probiotic up to 2.0× did not cause negative stress reactions in laying hens.

Shell-Less Egg Syndrome (SES) Widespread in Western Canadian Layer Operations Is Linked to a Massachusetts (Mass) Type Infectious Bronchitis Virus (IBV) Isolate.

A disease with a sudden drop in egg production and shell-less eggs called, shell-less egg syndrome (SES) has been observed in Western Canada egg layer flocks since 2010. The etiology of this disease is not known. We hypothesize that SES is caused by an infectious bronchitis virus (IBV) strain since it is known that IBV replicates in the shell gland causing various eggshell abnormalities. In this study, we screened egg layer flocks, in the provinces of Alberta (AB) and Saskatchewan (SK), with and without a history of SES for the presence of IBV infection. During 2015⁻2016, a total of 27 egg layer flocks were screened in AB (n = 7) and SK (n = 20). Eighty-one percent of the screened flocks (n = 22) were positive for IBV infection. Thirty of these isolates were successfully characterized using molecular tools targeting the most variable spike (S) 1 gene. IBV isolates from this study clustered into three genotypes based on partial S1 gene variability. The majority of the IBV isolates (70%) were Massachusetts (Mass) type, and the rest were either Connecticut (Conn) type or an uncharacterized genotype with genetic characteristics of Mass and Conn types. Since the majority of the IBV isolates included within the Mass type, we used a Mass type IBV isolate to reproduce SES in specific pathogen free (SPF) white leghorn chickens in lay. Further studies are warranted to investigate whether other IBV isolates can cause SES, to clarify the pathogenesis of SES and to develop a vaccine in order to prevent SES as observed in Western Canadian layer flocks.

Quantitative Analysis of Human Cancer Cell Extravasation Using Intravital Imaging.

Metastasis, or the spread of cancer cells from a primary tumor to distant sites, is the leading cause of cancer-associated death. Metastasis is a complex multi-step process comprised of invasion, intravasation, survival in circulation, extravasation, and formation of metastatic colonies. Currently, in vitro assays are limited in their ability to investigate these intricate processes and do not faithfully reflect metastasis as it occurs in vivo. Traditional in vivo models of metastasis are limited by their ability to visualize the seemingly sporadic behavior of where and when cancer cells spread (Reymond et al., Nat Rev Cancer 13:858-870, 2013). The avian embryo model of metastasis is a powerful platform to study many of the critical steps in the metastatic cascade including the migration, extravasation, and invasion of human cancer cells in vivo (Sung et al., Nat Commun 6:7164, 2015; Leong et al., Cell Rep 8, 1558-1570, 2014; Kain et al., Dev Dyn 243:216-28, 2014; Leong et al., Nat Protoc 5:1406-17, 2010; Zijlstra et al., Cancer Cell 13:221-234, 2008; Palmer et al., J Vis Exp 51:2815, 2011). The chicken chorioallantoic membrane (CAM) is a readily accessible and well-vascularized tissue that surrounds the developing embryo. When the chicken embryo is grown in a shell-less, ex ovo environment, the nearly transparent CAM provides an ideal environment for high-resolution fluorescent microcopy approaches. In this model, the embryonic chicken vasculature and labeled cancer cells can be visualized simultaneously to investigate specific steps in the metastatic cascade including extravasation. When combined with the proper image analysis tools, the ex ovo chicken embryo model offers a cost-effective and high-throughput platform for the quantitative analysis of tumor cell metastasis in a physiologically relevant in vivo setting. Here we discuss detailed procedures to quantify cancer cell extravasation in the shell-less chicken embryo model with advanced fluorescence microscopy techniques.

A comparative analysis of chick culturing methods on skeletogenesis.

Chick embryos are desirable models for the study of developmental biology. Despite this, there are very few studies that examine the effect of different culturing methods on skeletogenesis, specifically, intramembranous and endochondral bones. This study presents a detailed description of these effects by comparing two different culturing methods: windowed (in the shell) eggs and ex-ovo or shell-less culturing to normal development. Using whole mount bone staining, we determined that there is no significant difference in the length of the ossified region of intramembranous and endochondral bones in control versus window cultured embryos. However, these bones are significantly underossified in shell-less embryos. Shell-less embryos also exhibit abnormalities in endochondral bones. Intramembranous bones, interestingly, are morphologically normal in shell-less embryos. This study provides the first detailed description of ossification in window (in-ovo) and shell-less (ex-ovo) cultured embryos compared with controls (in-ovo). Patterning of the skeleton is unaffected regardless of culturing method. We conclude that studies involving endochondral bones should not utilise shell-less culturing methods. This data has been lacking in the literature and will serve as an important resource for those using cultured chick embryos in the study of skeletogenesis.