IBBJ

Original Article
Summer 2018, Vol 4, No 3

Effects of Omega 3 on Testosterone Hormone Levels and

Quality of Spermatozoa in Obese Rattus Norvegicus
Wistar Albino Strain
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Nazwita Dewi Putri1*, Nur Indrawaty Lipoeto2, Mohamad Reza3

1. Faculty of Medicine Andalas University,West Sumatera, Indonesia.

2. Department of Nutrition, Faculty of Medicine, Andalas University, West Sumatera, Indonesia.
3. Department of Biology, Faculty of Medicine, Andalas University, West Sumatera, Indonesia.

Submitted 17 Aug 2018; Accepted 27 Sep 2018; Published 10 Oct 2018

Chronic obesity can lead to inflammation of the testes which can affect the production of testosterone and
spermatogenesis. As omega 3 is acting as an anti inflammatory, this study aimed to investigate the effects of
omega 3 on serum testosterone levels and quality of spermatozoa in obese rats. 25 male rats, 2-3 months old and
weighting 160-240 g were divided into 5 groups. The control group was given a standamrd diet. The treatment 1
group received high fat diet and cheese while the treatment 2, 3, and 4 groups were given high fat diet and cheese
for 6 weeks followed by omega 3 administration at doses of 28.8, 46.8, and 64.8 mg, respectively during the next
51 days . At the end of treatment period, serum testosterone levels were analyzed by ELISA, and sperm amount,
motility, viability, and morphology were observed under a digital microscope. Data were analyzed using one way
Anova test followed by Bonferroni post hoc test to analyze the differences of influence between groups. Results
showed that serum testosterone levels as well as the amount and viability of spermatozoa were not significantly
different between studied groups (P= 0.230, P= 0.071, and P= 0.241, respectively). The mobility of spermatozoa
(P= 0.039), and their morphology (P= 0.047) were significantly affected by omega 3 treatment. Omega 3 may
affect fertility by improving the motility and morphology of spermatozoa.

Keywords: Omega 3, testosterone, quality of spermatozoa, obesity

O besity is one of the most common nutritional

problems (1). Generally, obesity occurs due to
a much larger intake of energy within a certain time.
were obese (3). In addition, data from Basic Health
Research Indonesia 2013 reported that in Indonesia
the obesity rate increased from 15% in 2010 to 20%
The cause of obesity is complex and can be linked in 2013 for men, and for women the obesity rate
to behavioral, environmental, metabolic, and genetic increased from 26% to 35% (4).
factors (2). Obesity can affect the body's functional systems
The World Health Organization data (WHO) including the reproductive system by inhibiting
suggests that by 2014 more than 1.9 billion adults steroidogenesis (5). In addition, obesity induces
aged 18 years and older were overweight and more infiltration of immune cells and inflammation, with
than 600 million of these people were obese, 39% of large adipocytes producing more reactive oxygen
adults over the age of 18 were overweight and 13% species (ROS) (6). In obese men there will be a

*Correspondence: Faculty of Medicine Andalas University,West Sumatera, Indonesia.

E-mail: [email protected]
 Effects of Omega 3 on Testosterone and Spermatozoa

reduction in leydig cells and an increase in pro- in the head area. The presence of a transparent color
inflammatory markers in the testes. Chronic upon eosin staining means that the spermatozoa are
inflammatory conditions will negatively impact still alive and dead spermatozoa present a colored
steroidogenesis by leydig cells (7). head area. The surviving sperm has an acidic cover
Omega 3 is an essential fatty acid. Fatty acids can layer. In live spermatozoa, eosin solution cannot
effect on obesity by reducing inflammation through enter the sperm's body because they both are acidic.
the constraints of arachidonic acid formation on the But in dead sperm the outer layer is damaged and
phospholipid membranes that activate pro- alkaline, so it can absorb eosin (3). Viability was
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inflammatory cytokines. In addition, omega 3 may expressed as percentage. In normal morphological

also inhibit nuclear factor kappa beta (NF-kb), situation, the head is shaped like a sickle or hook,
which is a key factor for transcription of pro- the middle part is called short (middle piece), and
inflammatory cytokines (8). The aim of this study has a long tail (9, 10). The morphology of
was to investigate the effect of omega 3 spermatozoa is said to be abnormal when there are
administrations on testosterone levels, and the several abnormalities such as size abnormalities
quality of spermatozoa in obese rats. including large or small head, abnormal shape like
twin heads, neck disorders, the middle part
Materials and methods presenting a curved middle shape or being thin, and
Animals and study design abnormalities in the tail such as short, double tail,
This research was an experimental study with post rolled tail, no tail, and bent tail (11).
test only design. 25 adult male Rattus norvegicus Statistical analysis
starin wistar albino weighting 160-240 g and aged The data obtained were tested for normality by the
2-3 months old were divided into 5 groups. The shapiro-wilk test, and the data were processed by
control was given a standard diet, the treatment 1 parametric one-way Anova followed by post hoc
group was given high fat diet and cheese while the Bonferroni differences of influence test between
treatment 2, 3 and 4 groups were given high fat diet groups, with a significance level of P < 0.05.
and cheese for 6 weeks and then received omega 3
doses of 28.8, 46.8, and 64.8 mg, respectively for 51 Results
nd
days. On the 52 day, testosterone levels and Evaluation of testosterone levels
spermatozoa features were analyzed in all groups. Table 1 shows the levels of testosterone in
This study has obtained ethical approval from the different groups. The highest average testosterone
Research Ethics Committee at the Faculty of level was observed in the treatment group 4 while
Medicine, Andalas University the lowest level was observed in the treatment group
Hormonal analysis
Examination of rats serum testosterone levels was Table 1. Testosterone levels in controls and omega 3 treated
groups
performed using ELISA method. Results were
expressed as mean± SD. Testosterone (ng/ml)
Group
Characterization of spermatozoa N Mean± SD P
The amount, motility, viability, and morphology Control 5 13.33±6.88
of spermatozoa were determined under a digital Treatment 1 5 15.43±2.74 0.230
microscope. Spermatozoa viability shows the
Treatment 2 5 10.39±5.04
proportion of sperm that live normally after exiting
Treatment 3 5 14.84±9.06
the testes. Spermatozoa viability was obtained from
Treatment 4 5 23.21±14.36
the observation of the structure of the spermatozoa

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 Putri N d et al.

2. There was no significant difference in Table 2. Amount of spermatozoa in controls and omega 3
treated groups
testosterone levels between the control and
treatment groups after administration of omega 3 to Amount of spermatozoa
Group (milion/ml)
Rattus norvegicus strain wistar albino obese rats (P=
0.230). N Mean± SD P
Characterization of spermatozoa Control 5 44.30±14.33
Table 2 shows the amount of spermatozoa Treatment 1 5 26.80±9.19 0.071
determined for each tested group. The highest
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Treatment 2 5 41.60±6.41
average amount of spermatozoa was found in the
Treatment 3 5 34.90±7.48
control group while the lowest average was found in
Treatment 4 5 35.60±7.94
the treatment 1 group. There was no significant
difference in the amount of spermatozoa between
Table 3. Motility of spermatozoa in controls and omega 3
the control and treatment groups after administration treated groups
of omega 3 to Rattus norvegicus wistar albino obese
Spermatozoa motility (%)
animals (P= 0.071). 1Group Mean ±
Table 3 shows the percentage of motile sperma- N P
SD
tozoa in different groups. The highest sperm motility Control 5 61.28±10.31
percentage was observed in the control group while
Treatment 1 5 28.96±10.16 0.039
the lowest percentage was present in the treatment
Treatment 2 5 40.30±13.97
group 1. There was a significant difference in sperm
Treatment 3 5 51.09±19.58
motility between the control and treatment groups
after the administration of omega 3 to Rattus Treatment 4 5 49.55±20.42

norvegicus wistar albino obese strain (P= 0.039).

Based on the results of the post hoc bonferroni test control group while the lowest rate was observed in
in table 4 it can be concluded that there was a the treatment group 2. There was no significant
significant difference in sperm motility between the difference in spermatozoa viability between the
control group and treatment 1 (P= 0.036), while the control and treatment groups administered with
omega treated obese rat groups did not show a omega 3 (P= 0.241).
significant difference (P > 0.05). In table 6 the highest amount of normal
Sperm viability was determined as the ability of spermatozoa morphology was found in the treatment
live spermatozoa outside the body within a time group 4 while the lowest average was in the
range of 20-30 min after being expelled (3). In table treatment group 2. There was a significant
5 the highest spermatozoa viability was found in the difference in sperm morphology between the control

Table 4. Post hoc bonferroni multiple comparison test on spermatozoa motility

Significance level of spermatozoa motility (P)

Group
Control Treatment 1 Treatment 2 Treatment 3 Treatment 4
Control - 0.036 0.452 1.000 1.000

Treatment 1 0.036 - 1.000 0.356 0.489

Treatment 2 0.452 1.000 - 1.000 1.000

Treatment 3 1.000 0.356 1.000 - 1.000

Treatment 4 1.000 0.489 1.000 1.000 -

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 Effects of Omega 3 on Testosterone and Spermatozoa

and the groups administred with omega 3 (P = difference from the first to four quartiles (14). The
0.047). results of the present study are in contradiction with
Based on the results of the post hoc bonferroni test the results of Risso et al. (15) in which the
in table 7 it can be concluded that regarding sperm administration of fish oil supplements for 120 days
morphology there was no significant difference increased the concentration of testosterone in male
between groups after administration of omega 3. dogs.This difference can be attributed to the time of

Table 6. Morphology of spermatozoa in controls and omega

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Table 5. Viability of spermatozoa in controls and omega 3

treated groups 3 treated groups

Spermatozoa with normal

Spermatozoa viability (%)
Group morphology (%)
Group
N Mean ± SD P
N Mean ± SD P
Control 5 87.67±7.76
Control 5 73.99±13.95
Treatment 1 5 77.87±7.71 0.241
Treatment 1 5 60.12±8.34 0.047
Treatment 2 5 76.71±5.78 Treatment 2 5 57.79±19.4
Treatment 3 5 82.47±10.85 Treatment 3 5 64.2+8.81
Treatment 4 5 81.14±6.43 Treatment 4 5 81.16±10.57

Table 7. Post hoc bonferroni multiple comparison test on spermatozoa morphology

Significance level of spermatozoa morphology (P)

Group
Control Treatment 1 Treatment 2 Treatment 3 Treatment 4
Control - 1.000 0.606 1.000 1.000

Treatment 1 1.000 - 1.000 1.000 0.178

Treatment 2 0.606 1.000 - 1.000 0.095

Treatment 3 1.000 1.000 1.000 - 0.505

Treatment 4 1.000 0.178 0.095 0.505 -

Discussion administration of omega 3 which was relatively

The results of the present study showed that the unequal or shorter (ie 51 days later in our study), the
administration of omega 3 did not significantly composition of fish oil supplements (fish oil versus
influence the levels of the testosterone hormone of pure omega 3), as well as the type of animals (dogs
albino obese male rats. The mean testosterone levels versus obese rats). Increased adipose tissue in
were high, ranging from 10.39 ng/ml to 23.21 ng/ml, obesity cases results in hyperplasia and tissue
in comparison with the normal levels of total hypertrophy, while obesity and overweight over a
testosterone in white adult male rats which vary long period of time during prepubertal period
from 0.5 ng/ml to 5.4 ng/ml (12). In normal men the results in reduced amounts of leydig cells and an
testosterone value ranges from 300-1100 ng/dl (13). increased level of pro-inflammatory markers in the
Our data are concordant with those found by testes. This is related to chronic inflammation which
Alarcon et al. (14) where the intake of omega 3 was may have a negative impact on steroidegenesis by
performed in 4 quartiles with a 1 year duration of the leydig cells, meaning that chronic inflammation of
study. Relatively, free testosterone and total the testicular leydig cells results in impaired
testosterone levels did not show a significant production of testosterone (7).

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 Putri N d et al.

A study performed on Padang Indonesian Institute Mendeluk et al. (20) did not found any significant
of Health Science students revealed that there was difference in sperm concentration before and after
no significant relationship between obesity and consumption of omega 3, whereas Risso et al. (15)
testosterone levels (16). This supports the results of suggested that long-term fish oil supplementation
the present study where the average testosterone could increase sperm concentration in dogs. This
levels of the five studied groups did not differ discrepancy could be due to the relatively unequal
significantly. time of omega 3 administration, different fish oil
In addition to inflammation in obesity, there is supplements or different animal models studied.
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also resistance to the leptin hormone which can be The provision of high-fat food can increase the
described as endangering male fertility through a accumulation of energy in the body. High-fat
specific mechanism such as causing steroidegenesis feeding has been shown to cause increased levels of
barriers (5). glucose, insulin, and triglycerides and result in
As an anti-inflammatory, omega 3 or n-3 mitochondrial sperm cell disorders (21). The
polyunsaturated fatty acids (PUFA) is expected to increase of glucose levels may disrupt
suppress inflammation. Many clinical studies have spermatogenesis as the sperm needs energy for its
been conducted for decades to investigate the effects survival. This is evidenced by the presence of
of dietary fatty acids on the inflammatory response glucose transporters from the acrosome to the sperm
in obese people (8). tail. Therefore, if glucose cannot enter the sperm
Aside from being an anti-inflammatory, omega 3 cell, then the cell will lack energy and will be
also plays a role in the process of steroid hormones damaged (22). In addition, high-fat dietary feeding
namely testosterone biogenesis. In this process, performed by Fan et al. showed that obesity induced
omega 3 as a fatty acid can control the expression of in rats with high-fat foods for 10 weeks did not affect
the steroidogenic acute regulatory (STAR) gene sperm concentration, but caused sperm motility
which encodes for a cholesterol transporter that decrease (23). A study from Iran reported a
controls the transfer of cholesterol from the relationship between omega 3 fatty acids and sperm
cytoplasm into the mitochondria. This begins the quality and fertility. This study suggested that
conversion of cholesterol to produce testosterone infertile men have a lower concentration of omega 3
(17). fatty acids in their sperm in comparison with fertile
The mean amount of spermatozoa of the five men (8, 24). Besides being an anti-inflammatory,
groups was not significantly different. Therefore, omega 3 also acts as a sertoli cell fuel, and is also
the administration of omega 3 has not significantly used in membrane remodeling during germ cell
affected the amount of spermatozoa of albino obese development (15).
male rats. However, the average amount of The mean sperm motility of the five studied
spermatozoa in the present study was relatively low groups were significantly different. The average
and ranged from 26.8 to 44.3 million/ml. amount of sperm motility in the present study ranged
Comparatively, the normal range of spermatozoa in from 28.96% to 61.28%, implying that the increase
6 ,
wistar rats is 35.5-175 x 10 /ml (18), and the normal in omega 3 doses did not always improve sperm
amount of spermatozoa in humans is about 20 motility as witnessed by the mean spermatozoa
million spermatozoa/ml ejaculate (3). If the amount motility values observed in treatment 3 group which
of spermatozoa is reduced it can be said to be of no was higher than that of treatment 4 group.
quality. One of the factors causing the lack of Furthermore, the data analysis of bonferroni test
spermatozoa is damage to cells or tissues due to the revealed a significant difference in sperm motility
formation of free radicals (19). Similar to our study, between control and treatment 1 groups (P= 0.036)

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 Effects of Omega 3 on Testosterone and Spermatozoa

where the administration of high fat diet in the acid that is highly concentrated in the testicles and
treatment 1 group has decreased the sperm motility. influences the formation of acrosomes which are
This was also supported by Fan et al. who showed enzymes acting on ovum fertilization (29).
that obesity induced with high-fat foods for 10 The results of spermatozoa viability showed that
weeks in rats decreased sperm motility (23). The the mean spermatozoa viability of all studied groups
influence of omega 3 or fish oil supplements on was not significantly different.. It can be interpreted
sperm motility increase was also demonstrated in that the administration of omega 3 has not
other studies (12, 25). significantly affected the sperm viability of albino
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The motility of spermatozoa is influenced by obese male rats. The average viability of
several factors including the time of examination spermatozoa in this study was relatively high,
after ejaculation, the time between ejaculation, varying between 76.71% to 87.67%, in comparison
temperature, ionic composition, electromagnetic with normal standard sperm viability of > 58%. Our
radiation, ROS, viscosity, pH, osmotic pressure, data were in contradiction with those of Risso et al.
immunological aspects, the presence of stimulatory that used a long-term oil supplements among dogs
factors, and motility inhibition. Spermatozoa (15). The epididymis plays an important role in
damage caused by ROS can inhibit acrosomal spermatozoa viability as spermatozoa mature in the
reactions and tail damage which is very influential epididymis that provides food supplies, especially
on sperm motility (26). High ROS levels can damage glucose as a substrate for spermatozoa metabolism
the mitochondrial membrane causing loss of (30). If during spermatogenesis the hormonal
potential mitochondrial function which will interfere balance is disrupted, the viability of produced
with sperm motility because sperm motility energy spermatozoa will not be good. Ferramosca et al.
is supplied in the form of ATP synthesized by stated that obesity condition induced with a high-fat
mitochondria in the tail body (27). diet can cause mitochondrial spermatozoa damage
The administration of high-fat diets to rats caused (21). This is influenced by an increase in ROS in fat
a decrease in sperm motility. This was evidenced by tissue. An increase in ROS triggers the recruitment
Ferramosca's et al. who showed that sperm of macrophages in adipose tissue (6). Fatty acids and
mitochondrial damage resulted in impaired sperm especially DHA are needed for cell membrane
motility. This is due to the fact that sperm needs composition. DHA is highly concentrated in the
energy produced by mitochondria. Disruption of retina of the eye, cerebral cortex, testes, and sperm
energy formation in sperm leads to sperm motility (31).
decrease (21). The morphological data of spermatozoa showed
Under obesity, the fat cells will multiply. These fat that there was an effect of omega 3 administration
cells secrete the aromatase enzyme that converts on obese rat spermatozoa morphology. Based on the
testosterone to estradiol. Testosterone is results of the post hoc bonferroni multiple
transformed into estrogen according to weight gain. comparison test, no significant morphology
The more one gets weight gain, the lower the difference was observed among studied groups. The
testosterone level, and the higher the estrogen level mean sperm morphology in each study group was
(28). high with amounts ranging from 57.79% to
In addition, obesity predisposes to inflammation. 81.166%. The mean morphological value of rat
Omega 3 as an anti-inflammatory compound, spermatozoa in this study (57.79% to 81.16%) was
suppresses pro-inflammatory cytokines. Besides high compared to the normal 2010 WHO standard
omega 3 can increase sperm motility. of 40%.
Docosahexaenoic acid (DHA) is an omega-3 fatty The spermatozoa head functions to penetrate the

161 Int. Biol. Biomed. J. Summer 2018; Vol 4, No 3

 Putri N d et al.

ovum wall during fertilization, and also contains a References

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