1. Antioxidants Boost Male Fertility: The role of
Reactive Oxygen Species (ROS) in modulating
sperm viability & fertility
Weily Lang, Dr. Preethi Radhakrishnan
Department of Natural Sciences
LaGuardia Community College

2. Relationship between Reactive Oxygen
Species and the male reproductive
system
 The body produces large amounts of Reactive Oxygen
species (ROS) when subjected to:
• Environmental Stressors (Smoking, Drugs, Pollution)
• Systemic Pathologies (Cancer, Diabetes, Systemic
infections)
 This oxidative stress can be potentially harmful to male
reproductive tissue, especially sperm
 Sperm are very prone to lipid peroxidation under high
stress (Agarwal & Esteves, 2011)

3. Relationship between Reactive Oxygen
Species and the male reproductive
system
Agarwal & Esteves, 2011

4. Reactive Oxygen Species (ROS)
 What are ROS
- free radicals produced as byproducts of REDOX reactions
- produced during cellular stress and immune insult (Bonilla 2006)
 How is ROS produced?
ATP (energy) production in the
mitochondria
results in surplus of oxygen
molecules
some O2 utilized, some converted
into ROS radicals by mitochondrial
enzyme, Complex IV

5. Antioxidants
 What are Antioxidants?
• Molecules which inhibit oxidation
• Scavenges free radicals
• Capable of reducing Reactive Oxygen Species
(ROS)
• Examples are:
• Melatonin
• Lipoic Acid

6. Antioxidants used in our experiments:
Bonilla et al., 2002 & 2006, found that dietary supplementation of
Melatonin and Lipoic acid caused significant lengthening of life-span.
Melatonin Lipoic acid
- is a lipid soluble hormone that easily - are sulfur-containing
accesses cell membrane compounds
- reduces DNA destruction by - quench ROS by donating
quenching free radicals (Bonilla, E. electrons to ROS (Bonilla, E.
2006). 2006).

7. Interaction between Paraquat and ROS
 What is paraquat (PQ)?
- Paraquat (PQ) is a nitrogen-containing herbicide
- It is highly toxic to living organisms leading to poisoning and death.
 What is the association between paraquat (PQ) and ROS?
- Organisms respond to PQ by producing a type of free radical called
superoxide anions that are then converted into large quantities of ROS
(Bonilla 2006).
- Therefore, we used PQ in our experiments at known doses (10mM) to
artificially induce the production of ROS (Bonilla 2006).

8. Fertility
 What is fertility and how is it important?
- Fertility is a measure of number of offspring produced by an
individual.
- The more offspring that are conceived the higher the chance of
passing on genes to the next generation
 Sexual Selection:
Pre-Copulatory sexual selection:
- Copula duration: The duration of intercourse
where sperm is injected in female.
- Sperm Viability: The measure of alive
sperm capable of fertilization.
-Post-Copulatory sexual selection:
- No. of offspring produced
 Fertility is measured by the number of offspring hatched

9. Relationship between ROS & Fertility
ROS may directly impact fertility:
 During an infection (systemic, STD)
 Environmental stressors (smoking, radiation)
 Pesticides (Paraquat)
Drosophila sperm
ROS’s effect on Sperm (Lipid Peroxidation):
● Sperm membranes are high in polyunsaturated fats - making
their membranes prone to ROS attacks via lipid peroxidation.
● Sperm also contain many mitochondria, which are a prime
source of free radical production, again making sperm likely
targets of excess ROS.

10. Relationship between ROS & Fertility
Agarwal & Esteves, 2011

11. How did we assay live/dead sperm in
males?
 Sperm Viability Kit (L-7011, Molecular Probes)
• Seminal Vesicles were dissected and sperm counted
• SYBR 14 – is a membrane-permeable nucleic acid stain (emission max.
516 nm)
• PROPIDIUM IODIDE - a dead-cell stain (emission max. 617 nm). Both
solutions stain sperm heads only
• Ninety-six percent of the cells seen in light microscopy were found in the
fluorescence image.
• Overall, 5% (678 out of 13 654 sperm cells) of the cells were doubly-stained
(green in centre, red at ends), and were included as dead cells.
• We counted live and dead sperm were counted manually, in each frame of
the slide
• Slides were counted blind of treatment

12. Drosophila Sperm – Sperm Viability
Assay
Live sperm Dead sperm

13. So how can antioxidants help?
 Feeding Antioxidants:
Antioxidants quench and react with ROS to reduce the oxidation effect on
the cell membrane and DNA. This protects sperm from damage.
 Antioxidant enhanced diets, Melatonin (M), Lipoic Acid (L):
Data from these groups showed a significantly higher percentage of viable
sperm and higher progeny.
 Control (EtOH):
Fewer viable sperm & fewer progeny than from antioxidant-fed males.

14. Methodology
 Collect virgins
 Starve males for 24 hours / separate females into different
vials
 Feed males antioxidant-enhanced and control (Ethanol)
diets
 Paraquat assault (24 hours on filter paper)
 Sperm viability assay (Propidium Iodide & SYBR Green)
 Mating (males from treatments paired with females) –
observations made
 Copula duration (start of mating and end of mating)
 Fertility (count progeny) – Flips 1, 2 and 3.

15. Methodology
COLLECTED VIRGINS
STARVED MALES FOR 24 HOURS ISOLATED FEMALES
FEED DIETS WITH
TREATMENTS
MELATONIN LIPOIC ACID ETHANOL
(Antioxidant) (Antioxidant) (Control)
MATING
PARAQUAT ASSAULT (induce ROS) & Copula Duration
SPERM VIABILITY FERTILITY (Progeny Counts)

16. Predictions
 Antioxidant-enhanced feeding treatments of melatonin
and lipoic acid would positively affect male fertility.
 There will be a significant decrease in the male fertility
within the control group (Ethanol Fed) due to the negative
effects of paraquat on fertility.
 If we find a positive correlation between antioxidants and
fertility, we plan to focus directly on the effects of these
two antioxidants on sperm viability in the future.

17. Results

18. Results: Mating
mating %
Percentage
Probability

19. Results: Sperm Viability
Proportion of Live Sperm

20. Results: Fertility
No. of offspring

21. Conclusion
 Mating probability and Copula Duration
• We found significant differences in treatments in the
number of flies that mated (p = 0.002)
• We found that antioxidant fed flies had higher mating
percentages than ethanol fed controls
• However, we found no significant differences in copula
duration between treatments (p = 0.567)

22. Conclusion
 Sperm viability
• Males fed Melatonin and Lipoic acid had a significantly higher
proportion of live sperm (74% and 56%) than the Ethanol
treatment (32%) (p = 0.0020)
• This indicated that both antioxidants Melatonin and Lipoic Acid,
might protect sperm from the lipid peroxidation activity of
Paraquat

23. Conclusion
 Fertility
• Males fed Melatonin and Lipoic acid sired significantly
more offspring (mean = 187 and 148) than the Ethanol fed
controls (mean = 82) (p = 0.0325)
• This shows that antioxidant-fed males, passed more
viable sperm to females than the Ethanol treated flies,
thereby siring more offspring.

24. References
1. Bonilla, E., et al., 2006. Paraquat –induced Oxidative stress in D. melanogaster: Effects
of Melatonin, Glutathione, Serotonin, Minocycline, Lipoic Acid and Ascorbic Acid.
Neurochem Res, 31:1425-1432
2. Bonilla, E, et al., 2002. Extension of life-span and stress resistance of D.melanogaster
by long-term supplementation with melatonin. Experimental Gerontology, 37:629-638.
3. Dowling, D, Simmons, L.W. 2009. Reactive oxygen species as universal constraints in
life-history evolution. Proc. R. Soc. B. 276: 1737-1745.
4. Aitken, J., Roman, D, S., Antioxidant Systems and Oxidative stress in the testes. 2008.
Molecular mechanisims in Spermatogenesis.
5. Radhakrishnan, P., Fedorka, K.MF. 2011. Influence of female age, sperm senescence
and multiple mating on sperm viability in female D. melanogaster. 57: 778-83
6. Radhakrishnan, P., Taylor, P.W. 2007. Seminal fluids mediate sexual inhibition and short
copula duration in mated female Quuensland fruit flies. 53: 741-745.

25. Acknowledgements
 Jon Rodriguez
 Dr. Hendrick Delcham
 NIH-Bridges R25 PAR-11-285
 Dr. Thomas Onorato
 Cheryl Pinzone, University of Georgia