Summary
This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.
Abstract
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.
Introduction
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'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' 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' toxicity to embryo development of other oviparous animals.
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Protocol
1. Preparation before exposure
- Select fertilized quail eggs born on the same day for the exposure test.
- Select quail eggs with similar weights. Each fertilized quail egg is about 10-12 g.
- Fully clean all fertilized quail eggs from external feces and other debris.
- 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 temperature). Sterilize the incubator with 75% ethanol.
- Open the eggs with the blunt end of a dental drill, leaving the eggshell at the tip for further use. Before transferring the fertilized eggs, the contents of the eggs are poured out. This is to keep the moisture of the eggshell. The opening diameter of the egg was about 3 cm.
NOTE: To reduce the damage to the quail embryo, use a dental drill to open the blunt end of the egg and make the crack as smooth as possible. - After sterilization, place the fertilized quail eggs in a 38 °C incubator with 60% humidity for 24-48 h. Ensure that the blunt end of the quail egg faces up.
- During the incubation of fertilized quail eggs, sterilize the tools needed in the subsequent experiments in a sterilization pot. These tools include plastic wrap, a beaker, sterile water, pipette tips, surgical straight scissors, tweezers, and a spoon.
NOTE: Use a film with a temperature tolerance high enough to avoid problems with the high-temperature sterilization.
2. Hatching the quail egg without a shell
- Transfer the pre-hatched fertilized quail eggs from the incubator to a clean bench and lay them flat on the container to stabilize them for about 1-2 min.
- Use scissors (12.5 cm surgical straight scissor) to poke a small hole (diameter 3 mm) in the central axis of the pre-hatched fertilized quail eggs and to cut 1-2 cm small opening. Carefully transfer the egg white and yolk of the fertilized quail eggs to the cut eggshell.
NOTE: When cutting a small opening with scissors, avoid touching of the yolk of quail eggs. - Add the control solution (without MPs) and the exposed solution of different masses (0.1, 0.2, and 0.3 mg) of microplastics with three particle sizes (100, 200, and 500 nm) to the egg contents by pipette. At the same time, add 1 drop of penicillin and 1 drop of streptomycin with a 1 mL syringe.
- Cover the opening of the eggshell with the sterilized film (step 1.6).
- According to step 2.1-2.4, treat all the fertilized quail eggs.
- Place the transferred quail embryos into the 38 °C incubator with 60% humidity for the necessary period. In this experiment, use an egg rotation angle of ±30°. Turn the eggs once an hour.
NOTE: The transfer should be as fast as possible, which requires more practice at the early stage.
3. Sample collection
- After seven days of culture, remove well-developed embryos observed by the naked eye from the yolk and wash with phosphate buffered solution (PBS).
- Dry the surplus solution outside the cleaned embryo with absorbent paper and weigh in a clean Petri dish.
- Open the whole chest cavity, separate the liver and the heart from the viscera with needle-nose pliers, and place in 1.5 mL centrifuge tubes immediately after clearing.
- Quickly record the weight on an electronic balance and calculate the hepatosomatic index (HIS = liver weight / body weight x 100). Measure the length of the sternum and body.
- Based on the above indicators, evaluate the impact of MPs on embryonic development.
NOTE: Embryo quality here refers to the quality of yolk removal.
4. Data analysis
- Report the experimental data in the form of mean ± standard error (SEM).
- Use single-factor analysis of variance to compare the means of multiple groups of samples. The significant difference value was α = 0.05.
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Representative Results
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' 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 contents into the hemispheric eggshells and put them into the incubator. Then we recorded the development of embryos in the middle period of incubation for three days. As shown in Figure 2, A-A2 is the control group, and B-B2 is one treatment group. From the perspective of macroscopic embryo development, the embryos developed normally without the adverse effects of microplastics.
Table 1 and Table 2 are the mean ± SEM of wet weight, body length, and sternum length of quail embryo after one-week exposure. The tables show that the wet weight and body length change significantly in different exposure groups. The weight and body length of the groups treated with 0.1 mg, 0.3 mg, 100 nm, and 500 nm MPs decreased slightly. The body weight and body length of 0.2 mg of 200 nm microplastic treated groups increased slightly (P < 0.05).
Hepatosomatic index (HIS) shows the proportion of liver in the quail embryo, which is an important sign to judge the degree of liver development. Moreover, HSI plays an important role in the pathogenesis of liver cell membrane injury and inflammatory infiltration. As shown in Figure 3 and Figure 4, compared with the control group, the proportion of liver in the whole treatment group increased significantly after exposure to microplastics. However, there was no significant difference between the 0.2 mg and 0.3 mg of 100 nm MPs treatment groups and the control group.
Figure 1: Hatching quail eggs without shell. Please click here to view a larger version of this figure.
Figure 2: The embryo development of quail on the 6th, 7th, and 8th day in the middle stage of hatching without eggshell. The green arrow points to the eyes; the blue arrow points to the limbs. Please click here to view a larger version of this figure.
Figure 3: Hepatosomatic index of quail embryos after exposure to MPs (nm) for 7 days. Significant differences between control and treatment groups are indicated by * P < 0.05. Please click here to view a larger version of this figure.
Figure 4: Hepatosomatic index of quail embryos after exposure to MPs (µm) for 7 days. Significant differences between control and treatment groups are indicated by * P < 0.05. Please click here to view a larger version of this figure.
| MPs treatment | Weight (g) | Length (cm) | Sternum length |
| Control | 2.509±0.324 | 5.425±0.477 | 1.025±0.094 |
| 100 nm | 1.812±0.155* | 4.632±0.315* | 0.950±0.152 |
| 200 nm | 2.272±0.368 | 5.297±0.268 | 1.025±0.076 |
| 500 nm | 1.785±0.127* | 4.892±0.154* | 1.017±0.082 |
Table 1: Wet weight, body length and sternum length of quail embryos after exposure to MPs (nm) for 7 days
| Treatment | Weight (g) | Length (cm) | Sternum length |
| Control | 2.161±0.166 | 5.23±0.26 | 1.10±0.04 |
| 0.1 mg | 1.960±0.338* | 4.82±0.75* | 1.04±0.04 |
| 0.2 mg | 2.410±0.366* | 5.25±0.26 | 1.07±0.10 |
| 0.3 mg | 1.901±0.759 | 4.95±0.15* | 1.02±0.09 |
Table 2: Wet weight, body length, and sternum length of quail embryos after exposure to MPs (µm) for 7 days. Compared with the control group, * indicates P < 0.05, ** indicates P < 0.01.
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Discussion
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 of the experiment. The research of embryotoxicity can only occur in the early and middle stages of embryo development. Second, the study of MPs on quail embryo development only occurs at the basic morphological analysis level. Thus, the conclusions are relatively simple and defects may exist. At the same time, the requirements for experimental conditions and operation are relatively high in the process of this experiment. Therefore, some noteworthy points are listed as follows:
It is very important to disinfect and sterilize fertilized quail eggs in the preparatory work due to the harmful pathogenic microorganisms on the surface of fertilized quail eggs. If disinfected, the microbes may intrude into the fertilized quail eggs during incubation, resulting in the quail embryos' death. Even if the transfer is successful, the death rate will be higher. Therefore, a good job should be done in disinfection and sterilization to reduce the experimental mortality.
When birds hatch eggs, they often change the position of the eggs and keep the air circulation to maintain a constant temperature for the eggs and the correct position for the fetus. This experiment used film to seal the eggshell. If the angle of egg rotation is too large, then the egg white will flow out. If it is too small, then adhesion between the embryo film and the eggshell film might occur, resulting in dead embryos. Therefore, set the rotation angle according to the actual situation.
During transfer of quail embryos, the pre-fertilized quail eggs are placed horizontally and then cut in the middle of the eggshell. In this way, a small part of egg white easily flows out, which destroys the normal proportion and distribution of the thick and thin egg white. This makes the yolk, which should have been on the top, lean to one side, causing the embryo to die. Therefore, take care to make all the egg white flow into the new hemispheric eggshell to ensure the normal proportion and distribution during transfer.
After the successful transfer, the experimenter must be careful not to drop the liquid directly. The liquid should rely on the eggshell wall to make it flow slowly during addition of pollutants and antibiotics.
In addition to the four points mentioned above, strictly control the incubation conditions. Coordinate the balance of temperature, humidity, and ventilation. Keep the incubation laboratory quiet and dark to achieve the best incubation environment.
In conclusion, this experiment provides a basic protocol for studying the effects of environmental pollutants on the development of quail embryos. There are also other types of indicators in the study of embryonic growth and development, including vascular development, oxidative stress, and cell damage. The above experiment is only a simple macroscopic evaluation of embryonic development from the morphological aspect. Finally, the improved research idea and protocol in the future could provide a new method for the toxicological study of embryo growth and development.
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Disclosures
The authors have nothing to disclose. All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work of this paper.
Acknowledgments
This work was supported by Key Research and Development projects in Xinjiang Uygur Autonomous Region (2017B03014, 2017B03014-1, 2017B03014-2, 2017B03014-3).
Materials
| Name | Company | Catalog Number | Comments |
| Multi sample tissue grinder | Shanghai Jingxin Industrial Development Co., Ltd. | Tissuelyser-24 | Grind large-sized plastics into small-sized ones at low temperature |
| Electronic balance | OHAUS corporation | PR Series Precision | Used for weighing |
| Fertilized quail eggs | Guangzhou Cangmu Agricultural Development Co., Ltd. | Quail eggs for hatching without shell | |
| Fluorescent polypropylene particles | Foshan Juliang Optical Material Co., Ltd. | Types of plastics selected for the experiment | |
| Incubator | Shandong, Bangda Incubation Equipment Co., Ltd. | 264 pc | Provide a place for embryo growth and development |
| Nanometer-scale polystyrene microspheres | Xi’an Ruixi Biological Technology Co., Ltd. | 100 nm, 200 nm, 500 nm | Types of plastics selected for the experiment |
| Steel ruler | Deli Group | 20 cm | Used to measure length |
| Vertical heating pressure steam sterilizer | Shanghai Shenan Medical Instrument Factory | LDZM-80KCS-II | Sterilize the experimental articles |
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