Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
533
A STUDY OF TOXIC EFFECT OF SODIUM BENZOATE, VIT. C
ALONE AND THEIR COMBINATION ON REPRODUCTIVE
FUNCTIONS OF ADULT MALE RABBITS
Shahid M., Nawras A. Alwan and Eman Aboud Al-Masoudi
Department of Physiology, Pharmacology and Chemistry. College of Veterinary
Medicine,University Basrah, Basrah,Iraq.
Key words: Sodium benzoate, Vitamin C, Testosterone.
Corresponding Author: [email protected]
ABSTRACT
The present study was designed to determine the adverse effect of sodium benzoate
(SB), vitamin C (Vit.C) alone and of their combination on reproductive functions in
rabbits. Thirty-six adult male rabbits were divided randomly into six equal groups. Group
1 (Control group) received orally distal water, group 2: Vit.C (200mg/kg BW/day)
received orally, group 3: received orally SB (60 mg/kg BW/day), group 4: received orally
SB (120 mg/kg BW/day). Group 5: received orally SB+Vit.C (60+200 mg/ kg BW/day)
and group 6: received orally SB+Vit.C (120+200 mg/ kg BW/day). The result revealed a
significant decrease in serum testosterone concentrations in all treated groups as
compared with Vit.C and control group. Significant differences were observed in sperm
concentration, viability, abnormality and significant decrease in sperm motility in groups
treated with combination of SB and Vit.C as compared with control. A significant
improvement in sperm concentration, sperm motility, viability and abnormality in G2
(Vit.C) group compared with all-treated groups. Histopathological changes in testicular
tissues in groups treated with SB+Vit.C showed marked vacuolation and degeneration of
seminiferous tubules, with arresting of spermatogenesis.
INTRODUCTION
Sodium benzoate is a salt of benzoic acid has chemical formula C7H5NaO2 it is
white in colour, odorless crystalline and found as Powder or grain. This compound is
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
534
easily dissolved in water and hardly dissolved in ethanol. Its molecular weight is 114.11
Its solubility is enhanced by increasing water temperature. Its harmlessness was
confirmed first in 1909 by the department of agriculture in America, during three separate
investigations (1& 2).
Sodium benzoate is regarded as preservative, food additive, and it has the E number
E211, It is bacteriostatic and fungi static under acidic conditions. This compound has
been reported to cause detrimental adverse effect in the body, when is indirectly
consumed by the human population as food additives (3). In the United States, benzoic
acid and sodium benzoate are on the Food and Drug Administration (FDA) list of
substances that are generally recognized as safe (GRAS). Both may be used as
antimicrobial agents, flavor in agents and as adjuvant with a current maximum level of
0.1% in food. The FDA did not determine whether significantly different conditions of
use would be GRAS.
Ascorbic acid (Vit.C) is considered as a potent antioxidant, which eliminates
reactive oxygen and nitrogen. Vit.C helps some of our most important body systems. It
helps the immune system to fight the foreign invaders and carcinogenic cells. Vit.C also
supports the cardiovascular system by facilitating fat metabolism and protecting tissues
from free radical damage, it assists the nervous system by converting certain amino acids
into neurotransmitters. In addition to its antioxidant functions, As it is a water –soluble
antioxidant, vitamin C is a unique position to "Scavenge" aqueous peroxyl radicals before
these destructive substances have a chance to damage the lipids. It works along with
vitamin E, a fat-soluble antioxidant and the enzyme glutathione peroxides to stop free
radical chain reactions (4, 5 and 6) .
MATERIALS AND METHODS
Animals and housing: Thirty sixty adult male rabbits were used in this study. All rabbits
were weighed about (205.00±19.00). They were kept in animal house under constant
environmental condition for 2 weeks to acclimatiz before that beginning of the
experiment. Food and drinking water were provided ad libitum throughout the
experiment.
Experimental Design: adult male rabbits were divided randomly into six equal groups as
follows:
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
535
Group 1 (control): six adult male rabbits orally administered distilled water (4
ml/animal) by gavage daily for 30 days.
Group 2: six adult male rabbits were orally administered Vit.C ٢00 mg/kg BW) (6)
dissolved in 4 ml distilled water by gavage daily for 30 days.
Group 3: six adult male rabbits were orally administered sodium benzoate (60 mg/Kg
BW) (7) dissolved in 4 ml distilled water by gavage daily for 30 days.
Group 4: six adult male rabbits were orally administered sodium benzoate (120 mg/Kg
BW) (7) dissolved in 4 ml distilled water by gavage daily for 30 days.
Group 5: six adult male rabbits were orally administered sodium benzoate (60 mg/Kg
BW) combination with Vit.C (٢00 mg/kg BW) dissolved in 4 ml distilled water by gavage
daily for 30 days.
Group 6: six adult male rabbits were orally administered sodium benzoate (120 mg/Kg
BW) combination with Vit.C (٢00 mg/kg BW) dissolved in 4 ml distilled water by gavage
daily for 30 days.
Epididymal sperm concentration:
The sperms were counted according to the method of Robb et al. (8) by using
Neubauer hemocytometer chamber which uses for RBC and WBC count.
Procedure
1- The epididymis were put in a Petri dish contained 5 ml of 0.9 % normal saline.
2- The epididymis was cut into 6 – 10 pieces by using sharp scalple.
3- The suspension resulted from the previous step was filtered by a clean piece of
gauze into a test tube.
4- One drop from the filtrate was dropped on the Neubauer chamber which covered
previously with cover slid.
5- The sperms found on the five squares that use for counting the RBCS by using the
objective lens (40 X).
6- The sperms were calculated in one mm3 as follows
Sperms/cm = n* X 10000 *n = number of sperm in 5 squires.
Sperm motility percentage:
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
536
The individual motility of epididymal sperms was measured depending upon the
graduation basis that was suggested by Chemineau et al. (9) as follows:
1- Drop of diluted epididymal sperm was dropped into a warm, clean slide at 37°C and
covered with cover slide. 2- Sperm were examined under microscope using 40X
power.
Depending upon the progressive, forward sperm's movement , the strength and
speed of their motion are converted into the percentage.
Table (1): The percentage sperm's movement and the strength and speed of their motion (9):
Type of motion Degree Percentage
Sperms move rapidly and straightly. 5 90 – 100
Sperms are rapid, move fast and some of them move
in a circle.
4 75 – 85
Sperms go on a straight slope, without a shivering
motion.
3 45 – 65
Simple irregular shivering motion, some of them move. 2 20 – 40
Movement is very slow, sperms shiver with swinging
tail.
1 10
No movement. 0 0
Sperm abnormality:
The percentage of abnormal spermatozoa were counted in the same slide that used
for measurement of the viability of epididymal sperm using account 200 sperms under a
light microscope using 100X power (10).
1. Diluted semen was dropped into a warm, clean slide.
2. Warm eosin – nigrosine stain was dropped on the semen and mixed together
carefully by use of a glass rod. A smear was done by using a clean slide which was
put angularly on the semen slide and was dragged horizontally.
3. The slide was left to dry.
4. The slide was examined under the light microscope using 40X power. Live
sperms appeared in white color and the dead sperms appeared in red color.
Live sperms appeared in white color and the dead sperms appeared in red color.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
537
Biochemical Measurements: the biochemical measurement was done on the serum after
separation by using special enzymatic kits as follow:
Testosterone ELISA Kit: The quantitative determination of total testosterone
concentration in serum or plasma by a microplate enzyme immunoassay (11).
RESULTS
Effect of SB, Vit.C alone and of their combination on serum testosterone concentration of
the treated adult male rabbits showed in table (2) after 30 days of treatment, the results
appeared that there is significant decrease (P<0.05) of sperm concentration, viability of
sperm and sperm motility in all groups except group 2 while the more significant decrease
was in the G5 and G6 as compared with control group and other groups. While the
abnormality of the sperm appeared to be increased in all groups but the most significantly
(p<0.05) increase in G5 and G6 (those with combination of SB. and Vit.C) as compared
with other groups. Regarding the level of serum testosterone the results revealed that,
there was significant decrease (p<0.05) in G5 and G6 as compared with other groups.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
538
Table (2): Effect of SB, Vit.C alone and their combination on testosterone hormone
and epididymal sperm characteristics in adult male rabbits:
Values expressed in capital letters mean significant differences at (p< 0.05) levels (M±SD).
Histological examination:
The figure (1) of testes of normal control group (1) showed normal seminiferous
tubules with normal spermatogenesis. In figure (2) testes of group 2 showed normal
architecture of seminiferous tubules. While in figure (3) the testes of group 3 (60 mg SB)
showed moderate vacuolation (V) of seminiferous tubules with complete suppression of
spermatogenesis. Figure (4) histological section of testes in group 4 rabbits (120 mg SB)
parameters
Groups
(n=6)
Sperm
Concentration
(×106/ ml)
Sperm
motility
(%)
Viability
(%)
Abnormality
(%)
Testosterone
ng/ml
G1
84.00±4.42 B 88.66±2.0A 89.83±2.40 A 11.66±1.63 f 9.31±0.34 A
G2
97.83±1.94 A 81.00±2.52 B 83.00±2.82 B 15.33±1.36E 8.51±0.46B
G3
63.50±3.39 C 63.16±2.92c 61.16±1.16C 25.50±2.66 D 8.06±0.43 B
G4
54.50±3.27 D 59.33 ±2.16 D 58.33±1.36 D 29.33±1.96 c 7.22±0.15C
G5
30.33±4.27 E 28.83±2.78 E 27.50±2.42 E 42.83±1.94 B 6.49±0.48 D
G6
28.66±2.58 E 21.66±2.16 F 19.83±1.60 F 56.56±3.16 A 4.88±0.64 E
LSD
6.61 7.16 2.83 3.66 0.733
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
539
showed severe vacuolation of seminiferous tubules with complete suppression of
spermatogenesis, while in figure (5) the testes of group 5 (60mg SB+Vit.C) showed
marked vacuolation of seminiferous tubules with arresting of spermatogenesis lastly in the
histological section of testes in group 6 (120mg SB +Vit.C) showed moderate vacuolation
of spermatogenesis with suppression of the spermatogenesis.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
540
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
541
DISCUSSION
Effect of SB and Vit.C alone and their combination on epididymal sperm
characteristics and testosterone hormone, after 30 day of treatment, the result showed that
significant decrease in all groups expect groups 1 and 2 while the more significant
decrease in group 5 and 6 as compared the control group and the other groups. These
results may be due to the adverse effect of combination of SB with Vit.C, these
combination form benzene, benzene can be form when benzoate is decarboxylated in the
presence of ascorbic acid and transition metals such as Cu +2 and Fe +3 and can be
accelerated by light and heat (12). Dieter et al. (13) showed that benzene has further toxic
effects after its metabolism in the liver by the cytochrome p450 E1 enzyme system.
These result are in agreement with 14. Ashamnu et al. (14) who found that Vit.C
treatment of lead acetate intoxicated male mice resulted in significant improvement in
sperm count and sperm abnormality compared with lead acetate group but still
significantly lower than those of control. Similarly showed that swimming stress causes
significant reduction in sperm count, sperm motility and sperm viability while
administration of Vit.C in doses of 200 mg\kg can causes elevation of above parameter in
a dose dependent manner compared with stress group but still significantly lower
compared with control (15).
The reduction of sperm count and elevation in sperm abnormalities in animal
stranded with pesticides (causes stress oxidation) so the reduction sperm count may be
due to the interaction of ROS with sperm cell membrane the improvement of those
parameter in Vit.C treated group be occurred due to the antioxidant effect of Vit.C which
and neutralize the free radicals that generated from the oxidation stress. The histological
section of testes in present study revealed histopathological changes in testes in groups
treated with combination SB+Vit.C which showed marked vacuolation of seminiferous
tubules (V), with arresting of spermatogenesis. These results agreed with previous studies
have shown that administration of SB could altered the histological architecture of testis
which appeared disruption of spermatogenic process and seminiferous tubules (16; 17).
Kehinde et al. (17) reported that the histological section of testes of group given SB
and Vit.C appeared alter in sperm quality and function, the histological section of testes in
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
542
group treated with SB showed distorted spermatogenic cells and seminiferous tubules,
deleterious basement membrane with lumen vacuolation while the group treated with
combination of SB and Vit.C appeared disrupted of basement membrane, hyperplasia of
Sertoli cells, deleterious lumen, degeneration of interstilium and disruption of
spermatogenic cells while in group treated with Vit.C only showed normal testicular
tissues with hyperplasia of Sertoli cells.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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