Artificial sweeteners have been on the rise over the past 50 years, boasting of increased sweetness, reduced caloric intake as opposed to sugar, and cheap production. Aspartame, an artificial sweetener, is widely found in desserts, soft drinks, multivitamins, cereals, and even medications millions of Americans ingest yearly (Rencuzogullari et al., 2004). When compared to natural sugar, aspartame is 200 times sweeter with zero calorie value designed to combat diabetes (Marinovich et al., 2013); however, previous work suggests that there are positive associations of aspartame intake with type 2 diabetes (Imamura et al., 2015; Sanyaolu et al., 2018).  Although aspartame may not pose a direct threat, aspartic acid, phenylalanine, and methanol - metabolic products of aspartame - all harm the body (Czarnecka et al., 2021). The increased phenylalanine level competes with neutral amino acids in neutral acid transporter binding. As a result, this causes chemical/oxidative stress, increases norepinephrine and epinephrine synthesis, and results in the upregulation of the sympathetic nervous system (Choudhary, Sundareswaran, & Sheela Devi, 2016). Given these findings, one would expect aspartame to increase the heart rate after consumption, which might harm the embryo’s growth and development (Branum et al., 2013). 

Previous work has been done on rats and mice to understand better Aspartame’s effect on heart rate and development. Choudhary et al. studied aspartame in albino rats and noticed a significant increase in heart rate and CK/CK-MB marker enzymes. They propose that the metabolites released from Aspartame may be why cardiac function was impaired (Choudhary et al. 2016). Another study was similarly done on neonatal mice and observed a significant increase in heart weight and cardiac myocyte diameter (Gudadhe et al. 2013). Aspartame has also been tested on chicken embryos and has been shown to result in structure malformation and growth retardation as the concentration of Aspartame is increased (Kormsing et al. 2020). The problem with this study is that it only observes the effects after 3 days of incubation – not longer-term effects. In addition, it covers higher concentrations of around 10-30mg/ml which resulted in 8% or more mortality. Another study on chicken embryos identified growth retardation and eye development abnormalities with aspartame-treated embryos; however, the substance used was not pure Aspartame (Al-Rashdi and Al-Qudsi, 2020).

Given the previous findings, there is a need to study the longer-term impact of lower concentrated aspartame on embryonic development, as it may lead to premature birth and other complications for newborns (Englund-Ögge et al., 2012). In this study, our primary objective was to determine how aspartame affects embryonic weight and heart rate using chicken embryos. Chicken embryos are ideal models due to their similar four-chambered human heart anatomy, short development time, and low cost. This experiment explicitly observed the effects of aspartame on chicken embryonic heart rate and embryo weight gain during the first week of development to better understand what longer-term effects aspartame may have regarding these developmental characteristics. Based on these previous findings, we predict increased concentrations of aspartame will increase the heart rate, decrease the embryonic weight, and expect hindered embryonic development due to aspartame. Embryonic weight loss is predicted because a previous study observed embryonic weight hindrance due to aspartame metabolite-induced oxidative stress that restricted nutrients to the fetus (Shalaby, Ibrahim, & Aboregela, 2019). 

Concentration Calculations:

We examined the effect of different concentrations of aspartame on heart rate and embryonic weight. We had a total of four replicate trials and selected 3, 6, and 9mg/ml concentrations of aspartame because they are within a safe range of aspartame concentrations that haven’t been deeply investigated or known to cause greater than 50% mortality (Choudhary et al., 2016). We made these concentrations using DI water because Naganobu et al. found that DI water proved more effective as a drug diluent than saline for injections (Naganobu, Hasebe, Uchiyama, Hagio, & Ogawa, 2000). 

Injections:

We cleaned fifteen unincubated eggs with 70% ethanol to avoid contamination from the drug injections. Each egg was placed on an egg candler to identify the egg yolk location, and a small piece of medical tape was placed at the end of the egg where the injection into the egg yolk was to take place. 0.1mL of a specified concentration of aspartame was injected into the egg yolk using a 28G 1cc insulin needle. Medical tape was then used to cover the hole. DI water was selected as a positive control because it has been known to not cause a response of behavioral change due to its isotonic and nontoxic nature (Naganobu et al., 2000). All eggs were placed in Brinsea Mini Advance humidified incubators at 37C and were harvested for observation after 1 week of incubation.  This was done for 4 weeks totaling 66 eggs, with each week being a replicate trial.

Observations: 

The embryo was removed using a plastic spoon and fine forceps were used to assist this procedure while removing as much yolk and albumin found on the embryo as possible. The embryo was then placed on a Bioptech Delta T open dish heater set to 41C with approximately 1.5mL warmed CMRL media 1066 to help maintain the warmth and heartbeat of the embryo and prevent it from drying out. A heat lamp was also placed near to ensure proper warmth was provided to the embryo. The heart rate was observed using a dissecting microscope and counting the number of beats over a 30 second period - video recording using a smartphone was also performed to verify heart beat count. Embryos were then dried with kim tech wipes to standardize the weight and weighed using a digital scale (Accuris Precision Balance) to the nearest 0.0001. This was done for all eggs in the incubator for 4 weeks resulting in a total of 66 eggs observed. 

Statistical Analysis: 

Data was collected on the average heart rate, embryo weight, and egg weight loss before and after incubation for all treatments and control (DI). An ANOVA test was performed to determine significant differences. An R linear regression analysis was also performed on the egg weight loss data. 

A total of 66 eggs were used during the experiment over a span of 5 weeks. 17 eggs received the 3 mg/mL concentration, 16 eggs received the 6 mg/mL concentration, 17 eggs received the 9 mg/mL concentration and 12 eggs received the DI water. However, some eggs were infertile or had died during the process which decreased the usable data points.

As seen in Figure 1, the 3 mg/mL concentration had the highest average heart rate, while the 6 mg/mL concentration had the lowest average heart rate for aspartame concentration treatment. The DI control indicated the lowest average heart rate in comparison to all aspartame concentration treatments.  Furthermore, Figure 2 depicts that the 6 mg/mL concentration had the highest embryonic weight while the 9 mg/mL concentration had the lowest embryonic weight. The DI and 9 mg/mL concentrations had similar average embryonic weights. Figure 3 depicts that 9 mg/mL had the highest change in egg weight, while the 3 mg/mL concentration had the smallest change in egg weights.

Statistical analysis was performed using an ANOVA single factor data analysis. There was no significant difference between heart rate and the treatment groups F(3,43)=0.259,p=0.854. Again, there was no significant difference between embryo weight and the treatment groups F(3,52)=0.063,p=0.979. Similarly, there was no significant difference found between the difference in egg weight loss and treatment groups F(3,60)=1.628,p=0.192. An R linear regression analysis of the egg weight data showed that the 9 mg/mL treatment had higher weight loss than all other groups (t=2.02, p=0.04); however, no other significant differences were observed.

In this study, we examined the impact of 3, 6, and 9 mg/mL concentrations of aspartame on the heart rate and growth development of chicken embryos and found no significant difference between these treatments with the treatment of DI water injection. This is contrary to what we hypothesized because previous studies report that administration of aspartame leads to oxidative stress and autonomic nervous system activation, which leads to increased heart rate (Choudhary et al 2016). A study which assessed the effects of various concentrations of aspartame on Daphnia magna heart rate also concluded that aspartame metabolites have excitatory effects on the nervous system (Schleidt et al 2009). Kormsing et al. discovered significant heart rate results at higher concentrations; therefore, we hypothesize that there was a negligible effect on heart rate in our study possibly because the concentrations used may be lower than required to provide a significant heart rate change(Kormsing et al. 2020). 

This is also contrary to our hypothesis that embryonic weight would decrease which is backed by previous research. A study on the aspartame effect on mice fetuses showed a significant decrease in whole body length and weight (Al-Qudsi and Al-Hasan 2019). A further study concluded that the methanol metabolite produced by the breakdown of aspartame could damage the cell membrane upon aspartame administration which would explain the decrease in body weight (Rogers et al 1985). Additional research has stated that methanol and L-phenylalanine metabolites are capable of inducing oxidative stress within the microvilli of the placenta, therefore decreasing the transport of nutrients to the fetus and ultimately hindering embryonic weight. (Shalaby et al 2019). 

We found a significant difference between the egg weight loss with the 9 mg/ml aspartame treatment with other treatments. However, no other significant differences were identified. This was not a surprising finding as we hypothesized that the egg weight would decrease as aspartame concentration increased; however, it was surprising that the control had an even greater egg weight loss. The hypothesis was formed based on prior research, which suggests that metabolites of aspartame, methanol, and phenylalanine specifically decrease placental weight, decreasing the nutrient supply to the embryo (Shalaby et al 2019). Like our findings, a prior research study found no significant difference between egg weights of aspartame injections and control eggs; however, a slight decrease in egg weight was observed for a few of the aspartame injection eggs compared to the control (Al-Rashdi and Al-Qudsi 2020). We, therefore, hypothesize that as aspartame concentration increases in the egg, more metabolites will be produced, hindering the nutrient flow for embryo development. As a result, more contents will remain liquid and evaporate through the porous eggshell. In addition, higher concentrations of aspartame would increase the concentration of methanol produced, leaving the egg via evaporation. 

Aspartame is an ingredient that is found in over 5000 commercially available products, and its low caloric value makes it a viable option for artificial sweetener. Due to its popularity, there has been great interest in studying the effects of aspartame on physiology and its effects on embryonic development.  In our experiment, we attempted to analyze the effects of aspartame on chicken embryonic development and heart rate. Our findings suggest that when administered in lower doses, aspartame does not elicit statistically significant changes in embryonic weight and development. However, there are statistically significant differences in overall egg weight following a week of injections. In conclusion, while Aspartame does indicate some sort of effect on the chicken embryos, it may not pose a significant threat when administered in lower doses.