The effects of microplastics exposure on chicken embryonic development

1. Serial Dilutions: We prepared a control, and performed serial dilutions on a 109 particles/mL stock solution of spherical microplastics. 

1. Control - DI water solution - (n=23)

2. Low - 103 particles/mL - (n=20)

3. Medium - 105 particles/mL - (n=12)

4. High - 107 particles/mL - (n=24)

2. Injections: 

   1. Weigh eggs prior to injection

   2. Inject eggs in the yolk sac with 0.1 mL of solution, cover with lab tape

3. Measurements: Eggs were incubated for 7 days

   1. Candle eggs to check for development, and re-weigh

   2. Explant embryo and check for heartbeat

   3. Examine embryo under microscope to determine HH stage

   4. Photograph embryo and record head length, eyeball diameter

   5. Record embryo mass and embryo brain mass

4. Statistical analysis:

   1. Initial outliers were tested for and excluded from statistical analyses

   2. A chi-square test was performed for Viability

   3. ANOVA tests were were performed for Embryo Mass, Brain Mass, HH Stage, Head Length, and Eye diameter.

84 chicken eggs were used in our experiments to determine the extent to which increasing microplastics exposure affects chicken brain development and overall embryonic development. Preliminary analysis showed 2 outliers in HH stage of development, 1 outlier in head length, 1 outlier in eye diameter, and 1 outlier in embryo mass, all of which we excluded (n=79). We noted a positive trend in viability between the control group (n=23) to the low concentration group treated with 103 particles/mL (n=20) and medium concentration group treated with 105 particles/mL (n=12), with viability increasing with concentration (X2(3, N=79)=9.17, p=0.03; Figure 1). We observed the trend reverse, and viability was lowest in the high concentration group treated with 107 particles/mL (n=24). 

We did not observe any significant differences in brain mass between eggs in the control group and those treated with microplastics (F(3,26)=0.33, p=0.81; Figure 2A). there were no significant differences in embryo masses between eggs in the control group and those treated with microplastics (F(3,29)=0.042, p=0.99; Figure 2B). Neither were there any significant differences in the Hamburger Hamilton stages between eggs in the control group and those treated with microplastics (F(3,32)=0.4, p=0.75; Figure 3). 

There was also little to no indication to suggest a difference in eye diameter between treatment groups (F(3,28)=0.93, p=0.44; Figure 4B). However, there was some indication of a difference in head diameter between treatment groups (F(3,28)=1.9, p=0.16; Figure 4A). 

The aim of this study was to examine the impact of microplastics on chicken embyronic development, with a emphasis on brain mass. We hypothesized that a negative correlation would be found between the increasing concentration of microplastics and the brain and embryo mass of chickens after 7 days of incubation. We also examined the eye diameter, head length, and overall viability in each treatment group. There were no statistical differences in the masses and eye diameter and head length between treatment groups. Previous research has shown increased apoptosis caused by the introduction of microplastics, making our findings inconsistent with prior research (Nie et al. 2021). There was, however, a statistical difference in the viability between treatment groups. The high concentration of microplastics (107 particles/mL) had an overall viability that was much lower than the other treatment groups, leading to a probable correlation between increased microplastics concentrations and the ability for an embryo to survive This is consistent with the current research, with a previous study showing an overall viability of 50% in a high concentration treatment group (Wang et al. 2023). Overall, our findings have not indicated a statistical difference regarding the embryo mass or brain mass when introducing microplastics. However, a statistical difference has been found in the overall vialibility of the embryos, leading us to conclude that microplastics do influence embyronic development.

In future experimentation, we would repeat with our experiments with greater sample sizes, as a low sample size was our main limitations. Additionally, future experiments might involve microplastic injection in regions of the egg aside from the yolk such as the air sac or albumin, as yolk injection might have played a role in the low rate of development across all conditions.

The major limitations of our study are sample size and overall viability of the embryos, as we had an average viability much lower than the standard 90-95% (Biesek et al. 2023). Our low viability resulted in a very low effective sample size, despite starting with many eggs. There may have been something wrong with the eggs we were provided, which may have caused the unusual trends in viability across the control and microplastic conditions, or they may simply be a statistical anomaly. Future research with larger data sets and eggs sourced from a different farm would be needed in order to determine if this is the case.

Biesek, J., Wlaźlak, S., & Adamski, M. (2023). The biological value of hatching eggs of broiler chicken in the early-mid incubation period based on physicochemical and morphologic  features. Poultry science, 102(6), 102689. https://doi.org/10.1016/j.psj.2023.102689 

Nie, J. H., Shen, Y., Roshdy, M., Cheng, X., Wang, G., & Yang, X. (2021). Polystyrene nanoplastics exposure caused defective neural tube morphogenesis through caveolae-mediated endocytosis and faulty apoptosis. Nanotoxicology, 15(7), 885–904. https://doi.org/10.1080/17435390.2021.1930228 

Wang, M., Rücklin, M., Poelmann, R. E., de Mooij, C. L., Fokkema, M., Lamers, G. E. M., de Bakker, M. A. G., Chin, E., Bakos, L. J., Marone, F., Wisse, B. J., de Ruiter, M. C., Cheng, S.,  Nurhidayat, L., Vijver, M. G., & Richardson, M. K. (2023). Nanoplastics causes extensive congenital malformations during embryonic development by passively targeting neural crest cells. Environment International, 173, 107865. https://doi.org/10.1016/j.envint.2023.107865