J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 90-98. https://doi.org/10.47277/JIRB/8(4)/90  
L-Arginine, Quercetin and Their Combination on  
Testicular Function and Sexual Behaviors in Rats  
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Johnson Olawumi Feyisike *, Oyewopo Adeoye Oyetunji , Adeleke Opeyemi Samson , Akingbade  
Olabanji3  
1Department of Anatomy, University of Ilorin, Ilorin, Nigeria  
Department of Anatomy, Osun state University, Osogbo, Nigeria  
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Department of Anatomy, Ekiti State University, Ado Ekiti, Nigeria  
Received: 14/09/2020  
Accepted: 19/11/2020  
Published: 20/12/2020  
Abstract  
L-arginine is the substrate for nitric oxide (NO) synthesis in biologic system. NO, being a free radical may induce some cytotoxic cascades  
related to impaired spermatogenesis. This study was undertaken to determine the effects of L-arginine, quercetin and their combination on  
the histology of the testes, oxidative stress biomarkers, hormones, sperm parameters and sexual behaviors in rats. 35 adult male rats divided  
into seven groups were used. A (Control), B (Sildenafil treated, 1.4mg/kg/day), C (L-arginine treated, 30 mg/kg/day), D (L-arginine treated,  
1
00 mg/kg/day), E (Quercetin treated, 10 mg/kg /day), F (L-arginine, 30 mg/kg/day and Quercetin, 10 mg/kg/day) co-administrated, G (L-  
arginine, 100 mg/kg/day and Quercetin, 10 mg/kg/day) co-administrated. Animals were sacrificed after 8 weeks and the histology of the  
testes, oxidative stress biomarkers, hormonal assay, sperm parameters and some sexual behaviors were done. The results obtained from the  
study showed that L-arginine has a relatively adverse effect on spermatogenesis which was improved by co-administration with Quercetin.  
The findings from this study suggests that synergism between L arginine and Quercetin can improve spermatogenesis as well as sexual  
behaviors in rats.  
Keywords: Antioxidants, Arginine, Nitric oxide, Quercetin, Spermatogenesis  
Introduction  
Quercetin, a dietary flavonoid ubiquitously present in a broad  
range of foods has received considerable attention because of its  
overwhelming presence in foods (8). Quercetin has a wide range  
of reported biologic effects, including antioxidant,  
antihypertensive, antimicrobial and antiprotozoan activities (9-  
Infertility has become a global concern affecting up to 186  
million people worldwide (1) and male factors put up to about  
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0%-55% of all cases, which are mainly related to decreased  
spermatogenesis (2). Infertility is the inability to achieve  
successful pregnancy after 12 months of consistent unprotected  
sex. It is estimated that approximately 15% of reproductive-age  
couples suffer from infertility (3). Male infertility refers to the  
incapability of a man to impregnate a woman after 12 months of  
regular and unprotected sexual intercourse. Factors including  
drug treatment, chemotherapy, toxins, air pollutions and  
inadequate vitamins intake have harmful effects on  
spermatogenesis and normal sperm output (4).  
To help improve male sexual performance, men consume L  
arginine, a semi essential amino acid as dietary supplement. L-  
arginine is one of the most commonly used food supplements for  
the management of erectile dysfunction. It is an amino acid used  
in the body to manufacture nitric oxide (5). It has various  
metabolic and immunologic effects and has been considered to be  
conditionally essential. L-Arginine is a notable precursor to nitric  
oxide (NO), a key component of endothelial-derived relaxing  
factor, an endogenous messenger molecule connected to diverse  
12). Most studies conducted on it had focused on the antioxidant  
properties of quercetin, its effects on several enzyme systems and  
effects on biological pathways involved in carcinogenesis,  
inflammation and cardiovascular diseases (13-16) as it is believed  
to prevent lipid peroxidation. It is also attracting intense scientific  
interest for its unique anti-aging and immune boosting activities  
(
17) and several recent studies show that organisms exposed to  
high levels of quercetin live longer, healthier lives and have good  
pregnancy outcome (17-21).  
Investigations in infertility has confirmed that excessive  
production of reactive oxygen and nitrogen species (ROS and  
RNS), in other words, oxidative stress, is connected to the  
aetiology of infertility, especially male infertility (22, 23). All of  
the reports have linked ROS/RNS, generated both exogenously  
and endogenously, with some aspects of male infertility, such as  
decreased sperm motility, abnormal morphology and reduced  
spermegg penetration. There are conflicting arguments on the  
role or effects of nitric oxide, the product of L-arginine on male  
fertility (24-31). Despite the important physiological benefits, NO  
as a free radical can induce cytotoxic cascades in the biological  
endothelium-dependent  
physiological  
effects  
in  
the  
cardiovascular system (6). Nitric oxide (NO) is produced from L-  
arginine by the activity of NO synthase (NOS), an enzyme that  
occurs in three isoforms (7).  
Corresponding author: Johnson Olawumi Feyisike, Department of Anatomy, University of Ilorin, Ilorin, Nigeria. Email:  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 90-98. https://doi.org/10.47277/JIRB/8(4)/90  
system. Quercetin is a naturally occurring flavonoid with  
antioxidant properties (8). It has been found to prevent testicular  
toxicities (32).  
Glutathione peroxidase was assayed according to the method  
of (38). Nitric oxide is an unstable molecule with a short half-life  
that reacts with molecular oxygen and accumulates in serum or  
plasma as nitrate (NO3−) and nitrite (NO−) ions; thus, the  
quantification of NOx in biological samples provides essential  
information about NO production. NO level was assayed by  
assessing the nitrites content (i.e. the stable end product of NO  
according to the method of (39)  
This study would provide useful insights into the interactions  
between compounds used in the management of erectile  
dysfunction and testicular function and the role of quercetin on  
these interactions. The aim of the study therefore was to evaluate  
the effect of L-arginine and quercetin oral administration and  
their combination on histology of the testes, oxidative stress  
biomarkers, hormones (FSH, LH & TT), sperm parameters and  
sexual behaviors.  
Measurement of serum LH, FSH and testosterone levels  
The serum levels of LH, FSH and testosterone were analyzed  
using an enzyme-linked immuno-absorbent assay (ELISA) kit  
(
Shanghaicrystat Day Biotech Co. LTD, Shanghai, China for LH  
Materials and methods  
Animal materials  
& FSH and IBL International GMBH Flughafenstrasse Hamburg,  
Germany for testosterone) according to the manufacturer’s  
guidelines.  
35 adult male and 5 female Wistar rats (180g-200g) were  
procured and used for the experiment. The rats were randomly  
assigned into seven (7) groups with 5 male rats in each group.  
Thereafter, the rats were allowed to acclimatize to the animal  
house condition for two weeks with free access to feed and water  
and the grouping was maintained throughout the period of the  
experiment. Ethical approval for the study was obtained from the  
University of Ilorin Ethical Committee on the Use of Animals for  
Experiments. The studies were conducted in accordance with the  
laws and regulations guiding the use of animals. The seven (7)  
groups were as follows: A (Negative Control - Normal healthy  
animals), B (Sildenafil citrate treated rats, 1.4mg/kg/day) positive  
control, C (L-arginine treated rats, 30 mg/kg/day), D (L-arginine  
treated rats, 100 mg/kg/day), E (Quercetin treated rats, 10 mg/kg  
Sperm parameters  
The left epididymis of each rat was dissected and put in 1 mL  
of pre-warmed Ham’s F10, then minced into small sections and  
incubated for a few minutes (37 °C, 5% CO2) to allow the  
spermatozoa to swim out of the epididymal tubules.  
Sperm parameters (count, motility, viability, and  
morphology) were studied in all groups. An aliquot of sperm  
suspension was diluted 1: 20 with Ham’s F10 medium and  
transferred into a Neubar’s hemocytometer under a coverslip to  
estimate the sperm count. Spermatozoa were counted under a  
light microscope at ×400 (Olympus Light Microscope; Olympus  
Corp., Tokyo, Japan) and expressed as million per ml of  
suspension. A drop of sperm sample was placed on clean and 37  
°C pre-warmed slides, and a minimum of 200 spermatozoa  
motility was accessed under a light microscope. Motility was  
shown as the percentage of motile sperm according to the World  
Health Organization manual criteria. Sperm viability was  
analyzed by eosin-nigrosin staining in 500 spermatozoa. A drop  
of stained sperm suspension was put on a clean slide, and a thin  
smear was made and allowed for drying. This slide was analyzed  
under a light microscope at ×1000 and spermatozoa with white  
and pink heads were considered as alive and dead, respectively  
/
day), F (L-arginine, 30 mg/kg/day and Quercetin, 10 mg/kg/day)  
co-administrated rats, G (L-arginine , 100 mg/kg/day and  
Quercetin , 10 mg/kg/day) co-administrated rats.  
L-Arginine and quercetin preparation  
The pure L-arginine and Quercetin were purchased in powder  
form (Sigma Chemical). The powder forms were dissolved in  
distilled water (33) and administered to rats as suspension in  
distilled water by oral gavage for 8 weeks.  
(
40).  
Sildenafil citrate (Viagra) preparation  
Viagra was purchased from Pfizer pharmaceuticals. Each 100  
mg tablet of sildenafil citrate was dissolved in 100 ml of distilled  
water so each 1ml contained 1mg of the drug and administered  
Sexual behaviors  
For behaviorsal studies, female rats were introduced in the  
cages of the male rats 3 weeks after the start of administration and  
using a camcorder the following parameters were monitored:  
Mounting frequency, intromission frequency and sexual  
motivation (41).  
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.4 mg/kg/day (34) to rats in group B by oral gavage for the 8  
weeks of experiments.  
Animal sacrifice, Sample collection and Histology  
Animals were euthanized with ketamine (100 mg/kg  
bodyweight) at the end of the period of treatment. Blood was  
collected through cardiac puncture. For histological studies, the  
testis was excised and fixed in Bouin’s fluid and processed using  
Haematoxylin and Eosin (H&E)(35) staining. Blood samples  
were centrifuged at 3500rpm for 10 min and then sera were stored  
at -80ºC until analysis.  
Data Analysis  
Data obtained was analyzed using GraphPad prism 5  
software. All results were expressed as mean± standard error of  
mean (SEM), and one way ANOVA analysis, followed by  
Tukey’s post hoc test was performed. P<0.05 was considered  
statistically significant.  
Oxidative stress biomarkers  
Results  
Histology  
Superoxide dismutase (SOD) was assayed according to the  
method of (36). Reduced glutathione (GSH) level was determined  
according to the method of (37).  
Figure 1 shows the representative histological sections of the  
testes of rats after the administration of L-arginine, Quercetin and  
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