This work was supported by Key Research and Development projects in Xinjiang Uygur Autonomous Region (2017B03014, 2017B03014-1, 2017B03014-2, 2017B03014-3).

1. Preparation before exposure
<ol>
	<li>Select fertilized quail eggs born on the same day for the exposure test.</li>
	<li>Select quail eggs with similar weights. Each fertilized quail egg is about 10-12 g.</li>
	<li>Fully clean all fertilized quail eggs from external feces and other debris.</li>
	<li>Sterilize each pre-hatched fertilized quail egg and the eggs to be used (Choose eggs with similar shell shape, especially the tip of the egg) with an antibiotic solution (penicillin and streptomycin, 1:1000, room tempera...

<ol>
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M., Wilson, M. 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G., Kelly, C., Dawson, K. A., Simpson, J. 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Y., Neto, F. F., Freitas, P. F., Ribeiro, C. A. O., Ortolani-Machado, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadmium+effects+on+early+development+of+chick+embryos.">Cadmium effects on early development of chick embryos.</a> Environmental Toxicology and Pharmacology. 34 (2), 548-555 (2012).</li><li>Li, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+interferes+embryonic+development+through+over-stimulating+serotonergic+system+in+chicken+embryo.">Caffeine interferes embryonic development through over-stimulating serotonergic system in chicken embryo.</a> Food and Chemical Toxicology. 50 (6), 1848-1853 (2012).</li><li>Lokman, N. A., Elder, A. S. F., Ricciardelli, C., Oehler, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+Chorioallantoic+Membrane+(CAM)+Assay+as+an+In+Vivo+Model+to+Study+the+Effect+of+Newly+Identified+Molecules+on+Ovarian+Cancer+Invasion+and+Metastasis.">Chick Chorioallantoic Membrane (CAM) Assay as an In Vivo Model to Study the Effect of Newly Identified Molecules on Ovarian Cancer Invasion and Metastasis.</a> International Journal of Molecular Sciences. 13 (8), 9959-9970 (2012).</li><li>Burns, E. E., Boxall, A. B. 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This paper provides an effective experimental scheme to evaluate quail embryo development by detecting the basic development indexes. However, there are still some limitations to this experiment. 
First, the mortality of quail embryos in the later stage of hatching is higher because of the shell-less hatching. There are artificially uncontrollable factors such as the destruction of normal protein ratio in the experimental process. We limited the exposure time of embryos to ensure the accuracy ...

The production of plastic waste was about 6300 Mt in 2015, one-tenth of which was recycled, and the rest was burned or buried underground. It is estimated that about 12,000 Mt of plastic waste would be buried underground by 20501. With the international community&#39;s attention to plastic waste, Thompson first proposed the concept of microplastics in 20042. Microplastics (MPs) refer to small particle plastics with a particle diameter less than 5 mm. At present, researchers have detected the ubiquitous presence of MPs in the coastline of various continents, the Atlantic Islands, inland lakes, the Arctic, and deep-sea habitats3,4,5,6,7. Therefore, more researchers have begun to study the environmental hazards of MPs.
Organisms could ingest MPs in the environment. MPs were found in the digestive tract of 233 marine organisms worldwide (including 100% turtle species, 36% seal species, 59% whale species, 59% seabird species, 92 kinds of sea fish, and 6 kinds of invertebrates)8. Moreover, MPs may block the organisms&#39; digestive system, accumulate, and migrate in their bobies9. It has been found that MPs can be transferred via the food chain, and their intake differs with the changes of habitat, growth stage, feeding habits, and food sources10. Some researchers reported the existence of MPs in the droppings of seabirds11, which means that seabirds act as the carrier of MPs. In addition, ingestion of MPs can affect health of some organisms. For example, MPs can be entangled in the gastrointestinal tract, thus increasing the mortality of cetaceans12.
MPs alone have toxic effects on organisms as well as joint toxic effects on organisms with other pollutants. Ingestion of environmental-related concentrations of plastic debris may disturb the endocrine system function of adult fish13. The size of microplastics is one of the important factors that affect their uptake and accumulation by organisms14,15. The small-size plastics, especially the nanosize plastics, are prone to interaction with cells and organisms with high toxicity16,17,18,19. Although the harmful effects of nano-particle size microplastics on organisms exceed the current research level, the detection and quantification of microplastics with sizes less than several micrometers, especially the submicron/nano-plastics in the environment, is still a great challenge. In addition, nano-plastics also have some effects on embryos. Polystyrene can damage the development of sea urchin embryos by regulating protein and gene profiles20.
To explore the potential impact of MPs on organisms, we conducted this study. Due to the similarity between bird embryos and human embryos, they are usually used in developmental biology research21 including angiogenesis and antiangiogenesis, tissue engineering, biomaterial implant, and brain tumors22,23,24. Bird embryos have the advantages of low cost, a short culture cycle and easy operation25,26. Therefore, we chose quail embryos with a short growth cycle as the experimental animal in this study. Simultaneously, we can directly observe the morphological changes of quail embryos exposed to MPs during the embryonic development stage using an eggshell-free hatching technology. The experimental materials used were polypropylene (PP) and polystyrene (PS). Because PP and PS27 account for the largest proportion of polymer types obtained in sediments and water bodies worldwide, the most common polymer types extracted from captured marine organisms are ethylene and propylene28. This experimental protocol describes the whole process for visual evaluation of toxicological effects of MPs on quail embryos exposed to MPs. We can easily extend this method to examine other pollutants&#39; toxicity to embryo development of other oviparous animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Multi sample tissue grinder</td>
			<td>Shanghai Jingxin Industrial Development Co., Ltd.</td>
			<td>Tissuelyser-24</td>
			<td>Grind large-sized plastics into small-sized ones at low temperature</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>OHAUS corporation</td>
			<td>PR Series Precision</td>
			<td>Used for weighing</td>
		</tr>
		<tr>
			<td>Fertilized quail eggs</td>
			<td>Guangzhou Cangmu Agricultural Development Co., Ltd.</td>
			<td></td>
			<td>Quail eggs for hatching without shell</td>
		</tr>
		<tr>
			<td>Fluorescent polypropylene particles</td>
			<td>Foshan Juliang Optical Material Co., Ltd.</td>
			<td></td>
			<td>Types of plastics selected for the experiment</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Shandong, Bangda Incubation Equipment Co., Ltd.</td>
			<td>264 pc</td>
			<td>Provide a place for embryo growth and development</td>
		</tr>
		<tr>
			<td>Nanometer-scale polystyrene microspheres</td>
			<td>Xi&#8217;an Ruixi Biological Technology Co., Ltd.</td>
			<td>100 nm, 200 nm, 500 nm</td>
			<td>Types of plastics selected for the experiment</td>
		</tr>
		<tr>
			<td>Steel ruler</td>
			<td>Deli Group</td>
			<td>20 cm</td>
			<td>Used to measure&#160; length</td>
		</tr>
		<tr>
			<td>Vertical heating pressure steam sterilizer</td>
			<td>Shanghai Shenan Medical Instrument Factory</td>
			<td>LDZM-80KCS-II</td>
			<td>Sterilize the experimental articles</td>
		</tr>
	</tbody>
</table>

ecotoxicological effects of microplastics on bird embryo development by hatching without eggshell

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues and organs. Ecotoxicological effects of microplastics on the development of bird embryos are not known. The bird egg is a complete development and nutrition system, and the entire embryo development occurs in the eggshell. Therefore, a direct record of bird embryo development under the stress of pollutants such as microplastics is highly limited by the opaque eggshell in traditional hatching. In this study, the effects of microplastics on quail embryo development were visually monitored by hatching without an eggshell. The main steps include the cleaning and disinfection of fertilized eggs, the incubation before exposure, the short-term incubation after exposure, and the sample extraction. The results show that compared with the control group, the wet weight and body length of the microplastics-exposed group displayed a statistical difference and the liver proportion of the whole exposed group significantly increased. Additionally, we evaluated external factors that affect the incubation: temperature, humidity, egg rotation angle, and other conditions. This experimental method provides valuable information on the ecotoxicology of microplastics and a novel way to study the adverse effects of pollutants on the development of embryos.

For the analysis of experimental data, we compared wet weight, body length, sternum length and the change of hepatosomatic index between the control group and the 6 experimental groups, measuring and reflecting the quail embryos&#39; growth and development from a macro perspective. We detected six normal quail embryos in each group. Each embryo reached the required Hamburger and Hamilton (HH) stage.
In Figure 1, we transferred the pre-hatched fertilized quail egg ...

Watch this Scientific Journal Video about Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell at JoVE.com

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues ...

This experiment will enclose four parts.Preparation before exposure, hatching quail egg without shell, incubation process and a sample collection, And representative results.Please watch the following video for the specific experimental process.In the exposure test, the same batch of quail fertilized eggs were selected for the experiment.The eggs with similar size were selected to reduce the experimental error.At the same time all quail fertilize the eggs and eggs should be completely cleaned, because there will be feces and other substances on their shells.After that, the pre-hatched, the fertilized quail eggs, and the eggs to be used were disinfected with penicillin and a mycin When placing you need to pay attention to the top facing down and then the blunt end facing up.The egg's cleaning process isn't shown in the video.The next step is the process of grounding the eggshell.It's the most important step in preparation.The chanted egg shell is a cut in the centre egg size with a dental drill, but can't be broken.Trying to make the fracture surface of the incision smooth enough, then the incubator was disinfected with 75%Acinol solution.Quail fertilized egg placed in the incubator with a humidity of 60%and 38

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4.3K Views.  Chinese Academy of Sciences. This experiment will enclose four parts.Preparation before exposure, hatching quail egg without shell, incubation process and a sample collection, And representative results.Please watch the following video for the specific experimental process.In the exposure test, the same batch of quail fertilized eggs were selected for the experiment.The eggs with similar size were selected to reduce the experimental error.At the same time all quail fertilize the eggs and eggs should be completely cleaned...