L-Arginine, Quercetin and Their Combination on Testicular Function and Sexual Behaviors in Rats

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

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

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,

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

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

sperm–egg 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

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:

olawumi_2005@yahoo.co.uk

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).

.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