Neroli Oil

The Scent of Growth: Can Neroli Oil Influence Cardiac Development and Size in the Embryonic Chicken?

Introduction

Essential oils are widely used across the globe for various applications, including aromatherapy, skincare, perfumery, cleaning, and medicinal purposes. Among them, neroli essential oil (EO) is highly used in perfumery due to its floral fragrance. While essential oils are generally considered safe when used at appropriate doses, they can be toxic at higher concentrations. For instance, rosemary oil has been shown to increase post-injection mortality rates in Japanese quail eggs at dosages of 3µL/egg, whereas a lower dose of 1µL/egg improved both hatchability and chick weight (Aberbour et al., 2023). Neroli oil contains several bioactive components, including limonene (27.5%), linalool (~20%), (E)-nerolidol (17.5%), alpha-terpineol (14%), alpha-terpinyl acetate (11.7%), and (E,E)-farnesol (8%). These compounds contribute to its antimicrobial, antifungal, antioxidant, and anti-inflammatory properties (Ammar et al., 2012; Khodabakhsh et al., 2015). Neroli EO also demonstrated antinociceptive effects, potentially reducing pain perception. Studies on the embryotoxicity of essential oils in animal models such as zebrafish and rat embryos have shown adverse effects, including reduced heart rate, decreased hatchability, and increased morphological abnormalities (Thitinarongwate et al., 2021). Limonene's toxicity in higher dosages is due to limonene hydroperoxide, which spontaneously forms from Limonene in aerobic environments (Chubukov et al., 2015.) Limonene hydroperoxide causes oxidative stress and forms free radicals. However, research specifically examining neroli EO's impact on embryonic development is extremely limited. To address this gap, our research investigates the effects of in ovo injection of neroli oil in chicken embryos. Specifically, we aim to determine how neroli EO influences overall growth and heart rate during embryonic development.

Material & Methods

Methods

Chemical: We will be using neroli EO (essential oil) as our chemical of study. We will have a high concentration group containing 16.67% neroli oil and a low concentration group containing 6.67% Neroli EO. To create the high and low concentration groups, we will use corn oil to dilute pure Neroli EO down to the respective concentration.

Control: Control eggs will receive an injection of only corn oil.

Procedures:

Day 0: To deliver the treatment, we will inject .05mL of stock solution into the eggs. The solutions will either be a high concentration of neroli oil (16.67%), low concentration of neroli oil (6.67%), or corn oil only.

Day 7: We will open the eggs and collect measurements. To measure heart function, we will cut a window in the egg shell and observe the embryo inside the egg. We will count the beats for 30 seconds and then multiply by 2 to get beats per minute. The embryo will remain in the shell for this measurement in order to keep warm and connected to all vessels. To assess overall growth, we will take pictures of the embryos. We will then upload the images into ImageJ to obtain measurements for head-to-tail length. We will also weigh each of the embryos and determine their Hamburger-Hamilton developmental stage.

Results

A total of 104 chicken eggs were injected with neroli EO to determine its effects on embryonic development in a dose-dependent manner, with a focus on heart rate. Of those eggs, 24 were unfertilized, which resulted in a total sample size of 80 eggs. The control group contained 30 eggs. 24 eggs received a low concentration of 6.67% neroli EO, and 26 eggs received a high concentration dose with 16.67% neroli EO.

Of the eggs that were fertilized, eggs treated with the high concentration of neroli EO had a statistically significant higher number of underdeveloped embryos than any other group (Figure 1; 𝜒2=9.32, df=2, p=0.01). The heart rate was similar across all groups (Figure 2C; F=0.82, df=2, p=0.45). Additionally, the embryo weight was also similar across groups (Figure 2D; F=0.76, df=2, p=0.47). Finally, the head-to-tail length also did not show a statistical difference (Figure 2B; F=1.9, df=3, p=0.16). Combining high and low concentration groups into a single treated group showed no significant differences between the heart rates (t-0.09, p=0.85), the embryo weights (t=0.69, p=0.5), and the head-to-tail lengths (t=1.6, p=0.12). Hamburger Hamilton stages were similar across groups (Figure 2A; F=1.6, df=2, p=0.21).

Conclusions

We aimed to identify if neroli EO significantly impacted the growth of embryos, predicting that the higher concentration treatment will amplify the number of developmental abnormalities, and the lower concentration will increase healthy embryo development. This outcome is useful in identifying the possible application of neroli EO in medical settings to reduce pain and anxiety in animals, as it does in humans. While the heart rate, weight, and head-to-tail length measurements were similar across all 3 groups, we found that the number of underdeveloped embryos was highest in the high concentration group, indicating that the high dosage may have blocked growth within the first few days of development. Additionally, there was a trend that the eggs injected with the control, corn oil, developed slower than the experimental groups, injected with neroli EO. This trend could indicate that the corn oil had a negative impact on early embryonic development. Potential causes of this could be the corn oil clogging forming blood vessels or preventing the cardiovascular system from forming within the first days of incubation.

While the study gave us valuable insight into the effects of neroli EO on embryogenesis, it was not without its limitations. The small sample size increased random variation, making it hard to determine if the results were significant or due to chance. This also reduced the number of concentrations that were able to be tested. Another limitation is that the method for recording the embryos’ heart rate varied slightly. For a majority of the eggs, the embryo was able to stay inside the shell, connected to all vessels. However, there were some embryos that had to be completely extracted in order to visualize their heart. In the future, it would be beneficial to investigate the individual components of neroli EO to determine which constituents are responsible for the recorded results and what the mechanism of action is. Including a wider variety of concentrations could also narrow down the safe range of neroli EO. Lastly, a study using multiple essential oils could lay the groundwork for creating a mixture that has the greatest positive impact on embryonic development.

References

Ammar, A. H., Bouajila, J., Lebrihi, A., Mathieu, F., Romdhane, M., & Zagrouba, F. (2012). Chemical composition and in vitro antimicrobial and antioxidant activities of Citrus aurantium l. flowers essential oil (Neroli oil). Pakistan journal of biological sciences : PJBS, 15(21), 1034–1040. https://doi.org/10.3923/pjbs.2012.1034.1040

Aberbour, A., Touazi, L., Benberkane, A., Aissanou, S., Sherasiya, A., Iguer-Ouada, M., Hornick, J. L., & Moula, N. (2023). The Effect of In Ovo Administration of Rosemary Essential Oil on Hatchability, Relative Hatching Weight, and Embryo Mortality Rate in Japanese Quail (Coturnix coturnix japonica). Animals : an open access journal from MDPI, 13(7), 1217. https://doi.org/10.3390/ani13071217

Chubukov, V., Mingardon, F., Schackwitz, W., Baidoo, E. E., Alonso-Gutierrez, J., Hu, Q., Lee, T. S., Keasling, J. D., & Mukhopadhyay, A. (2015). Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC. Applied and environmental microbiology, 81(14), 4690–4696. https://doi.org/10.1128/AEM.01102-15

Fouyet, S., Olivier, E., Leproux, P., Dutot, M., & Rat, P. (2022). Evaluation of Placental Toxicity of Five Essential Oils and Their Potential Endocrine-Disrupting Effects. Current issues in molecular biology, 44(7), 2794–2810. https://doi.org/10.3390/cimb44070192

Khodabakhsh, P., Shafaroodi, H., & Asgarpanah, J. (2015). Analgesic and anti-inflammatory activities of Citrus aurantium L. blossoms essential oil (neroli): involvement of the nitric oxide/cyclic-guanosine monophosphate pathway. J Nat Med, 69(3), 324–331. https://doi.org/10.1007/s11418-015-0896-6

Scandurra, C., Mezzalira, S., Cutillo, S., Zapparella, R., Statti, G., Maldonato, N. M., Locci, M., & Bochicchio, V. (2022). The Effectiveness of Neroli Essential Oil in Relieving Anxiety and Perceived Pain in Women during Labor: A Randomized Controlled Trial. Healthcare (Basel, Switzerland), 10(2), 366. https://doi.org/10.3390/healthcare10020366

Thitinarongwate, W., Mektrirat, R., Nimlamool, W., Khonsung, P., Pikulkaew, S., Okonogi, S., & Kunanusorn, P. (2021). Phytochemical and Safety Evaluations of Zingiber ottensii Valeton Essential Oil in Zebrafish Embryos and Rats. Toxics, 9(5). https://doi.org/10.3390/toxics9050102

Volpato, G. T., Francia-Farje, L. A. D., Damasceno, D. C., Oliveira, R. V., Hiruma-Lima, C. A., & Kempinas, W. G. (2015). Effect of essential oil from Citrus aurantium in maternal reproductive outcome and fetal anomaly frequency in rats. Anais da Academia Brasileira de Ciencias, 87(1), 407–415. https://doi.org/10.1590/0001-3765201520140354

Anslee Barrett

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Sarah Cait Osborn

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Young Bui

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