1. DETERMINING THE EFFECTS OF
ARTIFICIAL SWEETENERS AS
TERATOGENS ON CHICK EMBRYO CELL
MORPHOLOGY
Alexandria Emery
Katharine Meola
Katrina Nikitsina
Lorraine Salterelli

2. Introduction
 Teratogen: any factor that causes malformations of an
embryo
 There are many types of teratogens from the environment,
drugs, or infectious agents.
 Examples: Alcohol, nicotine, caffeine, mercury, cat feces,
pesticides, and chlamydia.
 Increasing populations means increasing pregnancies
along with high costs of healthcare illustrates the
importance of proper prenatal care

3. Artificial Sweeteners as Possible
Teratogens
 Why is this important?
 Epidemic levels of obesity cause an increase in use of artificial
sweeteners
 Women are especially prone to dieting and use, possibly more so when
weight gain is expected due to pregnancy
 People use this freely without fear of possible consequences
 Currently approved by the FDA, though controversy remains as amount
consumed is increased in diet
 Previous studies have shown risks to using substitutes including
increasing risks for cancer, or increasing cravings for sweets
 Effects: altering or halting development, ending with malformations or
death of the developing embryo, or tumor growth within the consumer.

4. Artificial Sweeteners
 Sucrose
 Sugar
 Disaccharide made from
glucose and fructose
 Used as a source of energy
and to increase sweetness of
foods
 Form of added sugar which
should be restricted to less
than 25% of energy intake2
 Sucralose
 Splenda
 0 calories
 Not readily broken down by
body: passes through
unmetabolized1
 No energy gain
 Widely believed to be the
safest alternative

5. Artificial Sweeteners
 Aspartame
 Equal
 Low calories
 Negligent energy gain
 Not used in cooking
 Made of amino acid Phenylalanine
 Denatures in high temperatures3
 Saccharin
 Sweet N' Low
 0 calories
 No energy gain
 Used for over 100 years
 Studied intensively
 Concerns arise from study
that found correlation of use
with bladder tumors in male
mice4

6. Why study the chick embryo as a model for the human
embryo?
 First studied by Aristotle
 Tissue differentiation lead to disproving
preformation, supporting epigenesis5
 Eggshell parts can be replaced by clear material to
allow a visual of the development
 Short developmental period
 Eggs may be frozen to halt development until ready
for use
 Cheap to maintain
 Similar development
Chick Embryo
Figure 1: Comparison of
embryos Chick and human
embryo similarities made

7. Hypotheses
1. Changes in the cell morphology will appear at the
macroscopic level and at the cellular level.
 Null: There will be no changes in cell morphology at either
macroscopic or cellular levels
2. The damage to embryos will be increased in those
that receive the higher concentrated sugar solutions
 Null: The damage observed will be equal in those that received
low and high concentrated solutions
Of the four experimental groups, we predict saccharin will have the greatest
negative effects on the embryo development.

8. Objectives
1. To determine if the sweeteners will have an effect at the
cellular level on the cell morphology.
2. To determine if there is a visible, larger morphological effect
on the organ systems.
3. To determine how different the effects of the different
sweeteners are.

9. Methods
Solutions were made by adding sugar to the Ringer’s
Solution

10. Methods
Eggs were injected with sugar solutions and
weighed before incubated for 7 days
Figure 2: Aspartame High 1 Egg After
Injection
Figure 3: Eggs weighed before incubation

11. Methods
Chick Eggs were:
1. Placed in freezer after 7 days of
incubation to ensure death
2. Weighed again
3. Cracked open to observe embryos
Embryos were:
1. Weighted and measured for length
2. Preserved in ethanol until observations
were completed
Figure 4: Eggs after being placed in
freezer

12. Methods
Homogenization
occurred and cells
were stained with
methylene blue and
eosin stains
Figure 5: Eosin staining

13. Results
Deformations and
mutations
Table 2: Physical deformations and mutations observed

14. Results
Figure 7: Untreated vs. Aspartame
High Concentration
Sucrose LowSucrose High
Figure 6: Embryos injected with Sucrose High
and Low Concentrations

15. Results
Control
Aspartame High Sucralose Low
Saccharin Low
Cell Staining:
Eosin
Figure 8: Eosin staining of cells from different groups

16. Results
Sucralose Low
Saccharin Low
Sucrose Low
Control
Cell Staining:
Methylene Blue
Figure 9: Methylene blue staining of cells
from different groups

17. Results
Initial weight after 7 days incubation change
Untreated 65.094 61.089 -4.005
Ringer’s solution 66.658 63.160 -3.498
Aspartame H 66.619 62.805 -3.814
Aspartame L 66.316 62.040 -4.276
Saccharin H 66.865 63.030 -3.835
Saccharin L 69.813 65.430 -4.382
Sucralose H 62.628 58.800 -3.828
Sucralose L 65.901 61.960 -3.941
Sucrose H 66.201 62.810 -3.391
Sucrose L 69.558 66.080 -3.478
Table 2: Average Weight of Embryos in Ovo
and their Change
0
1
2
3
4
5
6
∆EggMass
Solutions
Change in Egg Weight
Figure 10: Average Weight of Change in Egg Weight after
the Incubation Period. Error is shown in SD bars. [F(9) = 1.36,
P = 0.22]

18. Results
Figure 11: Average Body Weight of Chick Embryos. Error is shown in SD bars. [F(7) = 2.73, P =
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Untreated Aspartame
0.6M
Aspartame
2.2M
Saccharin 0.6M Saccharin 2.2M Sucrose 0.6M Sucrose 2.2M Sucrolose 0.6M Sucrolose 2.2M Ringer's 0.9%
Saline
EmbryoWeight
Solutions
Embryo
Weight

19. Result
s
Figure 12: The Significant Mean Difference of the Embryo Length
Tukey Test: Embryo
Weight
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Control Aspartame
Low
Aspartame
High
Sucrose High Sucralose
High
Sucralose
Low
Sucrose Low Ringers
Weight(g)

20. Results
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Untreated Aspartame
0.6M
Aspartame
2.2M
Saccharin
0.6M
Saccharin
2.2M
Sucrose 0.6M Sucrose 2.2M Sucrolose
0.6M
Sucrolose
2.2M
Ringer's 0.9%
Saline
EmbryoLength
Solutions
Figure 13: Average Body Length of Chick Embryos. Error is shown in SD bars. [F(7) = 3.04, P =
0.021]
Embryo Length

21. Result
s
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Control Aspartame Low Aspartame
High
Sucrose High Sucralose High Sucralose Low Sucrose Low Ringers
Length(cm)
Figure 14: The Significant Mean Difference of the Embryo
Tukey Test: Embryo
Length

22. Figure 15: Average Cell Size. Error is shown in SD bars. [F(5) = 10.54, P =
1.82 X 10-7 ]
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Untreated Aspartame
0.6M
Aspartame
2.2M
Saccharin
0.6M
Saccharin
2.2M
Sucrose 0.6M Sucrose 2.2M Sucrolose
0.6M
Sucrolose
2.2M
Ringer's 0.9%
Saline
CellSize
Solutions
Result
s
Cell Size

23. Results
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Sucrose High Sucrose Low Saccharin High Saccharin Low Sucralose Low Control Aspartame Low
CellDiameter(µ)
Figure 16: The Significant Mean Difference of the Cell
Tukey Test: Cell Size

24. Conclusion
Lowest Body Weight:
Sucralose High
Smallest Body Length:
Sucralose High
Irregular shaped cells:
Aspartame Low
Sucralose
Highest Body
Weight:
Saccharin High
Largest Body Length:
Saccharin High
Enlarged Cell Size:
Sucrose High
Sucrose Low

25. Conclusion
 A significant difference was found among the
groups for body weight, body length, and cell size.
 Aspartame: produced differences among all observations.
 Sucralose Low may be harmful because of its effects on body weight and length
 Saccharin High might affect body weight. Low concentrations may effect body height.
 Sucrose may cause low body weight and stunt growth. However, this was not
observed in an experiment7 that found an increase in body weight of chicks injected
with carbohydrates
 A follow-up of immunofluorescence stain would reveal if there were damages to the
cytoskeleton. This is time consuming and was not able to be performed, but might
provide information on how irregular shapes were produced

26. References
4. Calorie Control Council [Internet]. Saccharin. cited 2014 Mar 9. Available from:
http://www.caloriecontrol.org/sweeteners-and-lite/sugar-substitutes/saccharin
2. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol,
Protein, and Amino Acids (Macronutrients)[Internet]. 2005. National Academy of Sciences.
Institute of Medicine. Food and Nutrition Board. Available from:
http://www.nal.usda.gov/fnic/DRI//DRI_Energy/R1-26.pdf
6. Gilbert, S F 1997. Developmental Biology.http://9e.devbio.com/about.php.
5. Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates; 2000.
Comparative Embryology. Available from: http://www.ncbi.nlm.nih.gov/books/NBK9974/
1. International Food Information Council [Internet] 2009. Everything You Need to Know About
Sucralose. cited 2014 Mar 9. Available from:
http://www.foodinsight.org/Content/5519/Sucralose%20cons%20piece_web.pdf
3. International Food Information Council [Internet] 2011. Everything You Need to Know About
Aspartame. cited 2014 Mar 9. Available from:
http://www.foodinsight.org/Content/3848/FINAL_Aspartame%20Brochure_Web%20Version_11-
2011.pdf
7. Shafey, TM, Alodan, MA, Al-Ruqaie, IM, Abouheif, MA. 2012. In ovo feeding of carbohydrates and
incubated at a high incubation temperature on hatchability and glycogen status of chicks.
South African Journal of Animal Science. 42(3), 210-220.

27. Acknowledgements
 Dr. Rosch
 Dr. Stone
 Kutztown University