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Ikuomola et al
INOSR Experimental Sciences 8(1): 74 83, 2022.
©INOSR PUBLICATIONS
International Network Organization for Scientific Research
ISSN: 2705 1692 Evaluation of the Effect of Secnidazole on Sperm Motility, Morphology, Viability and Total Sperm Count of Wistar Rats
1,2E.O. Ikuomola, 1O.S Akinsonmisoye, 1R.O. Owolabi and 3M. B. Okon
1Department of Physiological Sciences, Faculty of Basic Medical Sciences, ObafemiAwolowo University,Ile Ife,OsunState,Nigeria;
2Department of Physiological Sciences, Kampala International University, Western Campus, Uganda.
3DepartmentofBiochemistry,KampalaInternationalUniversity,WesternCampus,Uganda.
ABSTRACT
The effect of secnidazole on sperm motility, morphology, viability and total sperm count of Wistar rats were evaluated. Forty adult male Wistar rats weighing 150 200g were used for this study. The animals were obtained from the Animal House, College of Health Science, Obafemi Awolowo University, Ile Ife, Nigeria. The rats were housed in the Animal House, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile Ife, Nigeria. The rats were kept in cages under normal environmental conditions and given free access to standard pellet diet and water. They were allowed to acclimatize to the laboratory environment for two weeks before the commencement of the experiment. Thirty five male rats were grouped into 7 groups containing 5rats each. Group A1 (n=5) were given distilled water (2ml/kg) which was the vehicle used to dissolve the drug. A2 (n=5) were given distilled water and allowed to recover for another 8 weeks Group B (n = 5) received 14.3mg/kg of secnidazole (low dosage) for 8 weeks only. Group C (n = 5) Received 28.6mg/kg of secnidazole (medium dosage) for 8 weeks only. Group D (n = 5) Received 57.2mg/kg of secnidazole (high dosage) for 8 weeks only. Group E (n = 5) Received 14.3mg/kg of secnidazole (low dosage) for 8 weeks once daily and allowed to recover for another 8 weeks. Group F (n = 5) Received 28.6mg/kg of secnidazole (medium dosage) for 8 weeks once daily and allowed to recover for another 8 weeks Group G (n=5) Received 57.2mg/kg of secnidazole (high dosage) for 8 weeks once daily and allowed to recover for another 8 weeks. All administrations were done orally with the aid of an oral cannula. The results indicated that there was a significant reduction in the sperm motility of the drug treated rats when compared with the control (p = 0.0001, F=73.18) and this was observed to be dose dependent. However, this effect was reversed in the recovery group. The reduction persisted even after recovery as there was still significant reduction in the sperm motility between the recovery group and the control. There was no significant difference in the sperm morphology of the rats between the drug treated and the control (p=0.0798, F=2.7088) and after recovery (p=0.7891, F=0.3508). There was no significant difference in the sperm viability of the rats between the drug treated and the control (p= 0.4277, F= 0.9779) and after recovery (p=0.9173, F=0.1667). There was a significant reduction in the sperm count of drug treated rats when compared with the control (p= 0.0001, F= 34.06) and this was observed to be dose dependent. However, this effect was reversed in the recovery group as there was no significant difference in sperm count between recovery groups and the control (p= 0.0597, F= 3.0342). In conclusion, there was a significant reduction in percentage sperm motility in the treated rats when compared with the control. The reduction was dose dependent, which may be partly due to reduction in serum FSH level as observed in this study. Also, there was a significant reduction in sperm count in the treated rats when compared with the control. The effect was dose dependent. This may be partly due to reduction in serum FSH level as observed in this study. Furthermore, there was no significant difference in the sperm viability between the treated rats and the control and after recovery. This may be due to the fact that sperm viability is the least affected among the sperm characteristics (sperm count, motility, morphology and sperm viability) after the administration of secnidazole drug. Finally, there was no significant difference in abnormal sperm morphology in the drug treated rats when compared with the control and after recovery. This may be due to the fact that secnidazole does not cause mutagenic effect in the development of sperm cells, also may be due to the facts that secnidazole does not induce its toxicity through permeation of the blood epididymis barrier.
Keywords: Secnidazole, sperm motility, morphology, viability, total sperm count and Wistar rats.
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Ikuomola et al
INOSR Experimental Sciences 8(1): 74 83, 2022.
INTRODUCTION
Male reproductive toxicity is also associated with derangement in the structures and functions of the male reproductive system such as sperm parameters, abnormal hormonal secretion and abnormal testicular and epididymal functions. It also induces altered variables indicative of oxidative stress like glutathione (GSH) depletion. Infertility has become an ominous problem in Africa on the average, about 10% of all couples face difficulty in startingafamilyandthiscreatesafeeling ofgreat personalfailure[1] Many factors both extrinsic and environmental factors including the increased use of antibiotics and anti effective drugs have been implicated as potential causes of male infertility [2]. Studies have shown that antimicrobial combination therapy such as metronidazole, quinolones, tetracycline, ketoconazole, fluconazole and other imidazole group of antibiotic drugs are among the most prescribed classes of drugs in medicine. There is a high possibility that some of the couples presenting with history of infertility or inability to conceive may be due to these groups of drugs [3] Nitro imidazole, an antimicrobial drug is among the most clinically prescribed classes of drugs for couples presenting with infertility in the clinics. However, these classes of drug have been shown to possess mutagenic activities in bacterial assay and reproductive toxicity including inhibition of spermatogenesis in rats [4, 3] Studies have also shown that some nitro imidazole class of drugs such as metronidazole and ketoconazole may have being responsible for some reproductive toxicity which causes inhibition of spermatogenesis in rats earlier reported by [5] Secnidazole is one of the representatives of nitro imidazole group of drugs. It is highly effective and differs from other compounds in this group by a prolonged serum half life of about 17 29 hours [6] The prescription of secnidazole have increased due to its
pharmacological advantages in treatment of adults and children with intestinal amoebiasis, urinary tract infection (UTI), pelvic inflammatory disease (PID) and vaginal infectionsmostly of mild or moderate severity [6] Secnidazole is an anti biotic with distinct pharmacological properties that are different from other imidazole group of drugs, giving it a relative pharmacological advantage [7] The frequency of prescription of secnidazole has increased tremendously in recent years due to its pharmacological advantages [6; 7] The tolerability profile of secnidazole does not differ markedly from other 5 nitroimidazoles. The most commonly reported adverse events in clinical trials involved the gastrointestinal tract (nausea, vomiting, glossitis, anorexia, epigastric pain and a metallic taste) and occurred in 2 to 10% of patients. Headache and dizziness were experienced by about 2% of patients. Secnidazole was reportedtobewelltoleratedinadultsand children, and no adverse event required therapeutic intervention or treatment withdrawal [8]Secnidazole, from its pharmacological properties has a mutagenic effect in bacterial assay. This mutagenic effect has been proved to reduce a nitro group and attacks the nucleic acid of the micro organism. Furthermore, it inhibits further DNA synthesis and causes degradation of already existing DNA [9] The effect of secnidazole on reproductive system was studied because reports from previous studieshave shown increasing number of people using secnidazole in clinical settings especially among the young ones andthoseinthereproductiveage bracket. Also, the pharmacological properties of secnidazole have been predicted to be reprotoxic.Secnidzole isoneofthenitro imidazole drugs mostly prescribed in the clinical settings because of it increase in patient’s compliance and most of the infections that it is used to treat are infections of the reproductive system suchasurinarytraitinfection(UTI),Pelvic
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Ikuomola et al
INOSR Experimental Sciences 8(1): 74 83, 2022.
inflammatory disease (PID), Vaginal infections.
Objective
The specific objective of the study is to assess the effect of secnidazole on sperm
Animals
parameters like sperm motility, morphology, viability and total sperm countofWistarrats
METHODOLGY
FortyadultmaleWistarratsweighing150 200g were used for this study. The animals were obtained from the Animal House,CollegeofHealthScience,Obafemi Awolowo University, Ile Ife, Nigeria. The rats were housed in the Animal House, Faculty of Basic Medical Sciences, Obafemi Awolowo University, Ile Ife, Nigeria.Therats werekeptincagesunder normal environmental conditions and given free access to standard pellet diet and water. They were allowed to acclimatize to the laboratory environment
for two weeks before the commencement oftheexperiment.
Drugs
Secnidazole was procured by May & Baker Company, Sangto Otta, Ogun State, Nigeria.
Ethical Clearance
Ethical clearance for this study was obtained from the Health Research Ethics Committee (HREC) of the Institute of Public Health, College of Health Sciences, ObafemiAwolowoUniversity,Ile Ife,Osun State Nigeria. (HREC Assigned Number: IPHOAU/12/1624).
Experimental Design
GROUPS NUMBER OF OF RATS TREATMENTS
GROUP A1 A2 5 5
2mlsofdistilled Water(control)
NUMBER OF DAYS
8Weekstreated 8WeeksRecovery
GROUP B 5 14.3mgsecnidazole (lowdose) 8Weeks
GROUP C 5 28.6mgsecnidazole (medium dose) 8Weeks
GROUP D 5 57.2mgsecnidazole (highdose) 8Weeks
GROUP E 5 14.3mgsecnidazole (lowdose)+Recovery
GROUP F 5 28.6mgsecnidazole (medium dose)+ Recovery
GROUP G 5 57.2mgsecnidazole (highdose)+Recovery
Secnidazole Drug Administration
Rats in group 1 received 2 ml/kg of distilled water only, group 2 rats received 14.3 mg/kg of secnidazole (low dosage) for 8 weeks; group 3 rats received 28.6 mg/kg of secnidazole (normal dosage) for 8 weeks; group 4 rats received 57.2 mg/kg secnidazole (high dosage) for 8 weeks; Rats in group 5, 6 and 7 received 14.3 mg/kg, 28.6 mg/kg, 57.2 mg/kg of
8Weekstreated+8 WeeksRecovery
8Weekstreated+8 WeeksRecovery
8Weekstreated+8 WeeksRecovery
secnidazole respectively for 8 weeks and they were allowed to recover from treatmentforanother8weeks.
Stock Solution Preparation
A stock solution of 500mg was prepared in20mlsofdistilledwaterandfromthisa dosage of 2mls/kg was administered. All administration was done orally with the aidofanoralcannula.
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Ikuomola et al
INOSR Experimental Sciences 8(1): 74 83, 2022.
Animal Groupings and Administration
Thirty five male rats were grouped into 7 groupscontaining5ratseach.
Group A1 (n=5) were given distilled water (2ml/kg) which was the vehicle usedtodissolvethedrug.
A2 (n=5) were given distilled water and allowedtorecoverforanother8weeks
Group B (n = 5) received 14.3mg/kg of secnidazole (low dosage) for 8 weeks only [7].
Group C (n = 5) Received 28.6mg/kg of secnidazole (medium dosage) for 8 weeks only[7]
Group D (n = 5) Received 57.2mg/kg of secnidazole (high dosage) for 8 weeks only[7].
Group E (n = 5) Received 14.3mg/kg of secnidazole(lowdosage)for8weeksonce dailyandallowedto recoverforanother8 weeks[7]
Group F (n = 5) Received 28.6mg/kg of secnidazole (medium dosage) for 8 weeks once daily and allowed to recover for another8weeks[7].
Group G (n = 5) Received 57.2mg/kg of secnidazole (high dosage) for 8 weeks once daily and allowed to recover for another 8 weeks [7]. All administrations were done orally with the aid of an oral cannula.
Mode of Sacrifice and Organ Collection
After eight weeks of administration of secnidazole, the rats were sacrificed by cervical dislocation. Their blood was collected through cardiac puncture. The testis and epididymis were harvested and weighed using digital weighing scale. The right testis of each rat was immediately fixed in Bouin’s fluid for histological processing. The semen for sperm parameters was obtained from the caudal epididymis and their sperm parameters were assessed under the microscope. The same sacrificial procedure was repeated forratsintherecoverygroups.
Sperm Characteristics Analysis
After the separation of the epididymis from the testis, the caudal part was minced in pre warmed normal saline (37°C and 1.8mls) in a clean sterile petri dish. This was left for 2 4 mins before thecommencementofspermassay.
Sperm Motility
From the sperm suspension in the petri dish and with the aid of a pipette, one drop of sperm suspension was placed on a glass slide for analysis. About 200 motilespermsinfourdifferentfieldswere analyzed at a magnification of x40 [10; 11]
Sperm Viability
Viability study (percentage of live/death spermatozoa) was done using eosin/nigrosin stain according to the method of [11]. From the sperm suspension in the petri dish and with the aid of a pipette, one drop of sperm suspension was placed on a glass slide and two drops of the stain were added. The motile (live) sperm cells were unstained, while the non motile (dead) sperm absorbed the stain. The stained and the unstained sperm cells were counted using ×40 objectives of the microscope and an average for each was takenfromwhichpercentage viabilitywas calculated.
Sperm Morphology
From the sperm suspension in the petri dish and with the aid of a pipette, one drop of sperm suspension was placed on a microscopic slide, two drops of Walls and Ewas stain was placed on it and air dried. The slides were examined under the microscope using ×100 objectives under oil immersion. The normal sperm cells were counted and the percentage wascalculated[10].
Sperm Count
From the sperm suspension in the petri dish and with the aid of a pipette, one drop of sperm suspension was placed on a hemocytometer and immediately covered with cover slip. The sperms were counted using a hemocytometer. The numbers of sperm in five squares (four corners and the center) in the center grid of both sides were counted based on the dilution factor and averaged following previousmethods[11]
Statistical analysis
Data were analyzed by using one way analysis of variance (ANOVA) followed by Students Neuman Keuls (SNK) and Turkey test for multiple comparisons. Results
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Ikuomola et al
INOSR Experimental Sciences 8(1): 74 83, 2022.
were expressed as mean ± S.E.M., p < 0.05 was taken as accepted level of significant
Effect of
Senidazole
on sperm motility
difference.
RESULTS
There was a significant reduction in the sperm motility of the drug treated rats when compared with the control (p = 0.0001, F = 73.18) and this was observed to be dose dependent. However, this effectwasreversedintherecoverygroup.
The reduction persisted even after recovery as there was still significant reduction in the sperm motility between the recovery group and the control (p= 0.0436,F=3.4000).(Fig1)
Figure 1: Effect of Secnidazole on sperm motility
GraphshowingtheeffectofSecnidazoleonspermmotility
ResultsarepresentedasMean SEM,n=5 *=significantlydifferentfromgroupA(control)(p<0.05) α=significantlydifferentfromgroupB(lowdose)(p<0.05) β=significantlydifferentfromgroupC(Mediumdose)(p<0.05)
Effect of Secnidazole on sperm morphology
There was no significant difference in the sperm morphology of the rats between the drug treated and the control (p= 0.0798, F= 2.7088) and after recovery (p=0.7891,F=0.3508).(Fig2)
Effect of Secnidazole on sperm viability
There was no significant difference in the sperm viability of the rats between the drug treated and the control (p= 0.4277, F= 0.9779) and after recovery (p=0.9173, F=0.1667) (Fig3)
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Ikuomola et al
INOSR Experimental Sciences 8(1): 74 83, 2022.
Effect of Secnidazole on total sperm count
34.06) and this was observed to be dose dependent. However, this effect was reversed in the recovery group as there was no significant difference in sperm count between recovery groups and the control(p=0.0597,F=3.0342).(Fig4)
GROUPA1-CONTROL GROUPB-LOWDOSE GROUPCMEDIUMDOSE GROUPDHIGHDOSE GROUPA2-CONTROL GROUPE-LD+RECOVERY GROUPF-MD+RECOVERY GROUPG-HD+RECOVERY
GraphshowingtheeffectofSecnidazoleonspermmorphology ResultsarepresentedasMean SEM,n=5(p<0.05)
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GROUPA1-CONTROL GROUPB-LOWDOSE GROUPC-MEDIUMDOSE GROUPD-HIGHDOSE GROUPA2-C0NTROL GROUPE-LD+RECOVERY GROUPF-MD+RECOVERY GROUPG-HD+RECOVERY
Fig: 3: Effect of Secnidazole on sperm viability GraphshowingtheeffectofSecnidazoleonspermviability ResultsarepresentedasMean SEM,n=5(p<0.05)
Figure 4: Effect of Secnidazole on total sperm count
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Ikuomola et al
INOSR Experimental Sciences 8(1): 74 83, 2022.
GraphshowingtheeffectofSecnidazoleontotalspermcount
ResultsarepresentedasMean SEM,n=5
*=significantlydifferentfromgroupA(control)(p<0.05)
α=significantlydifferentfromgroupB(lowdose)(p<0.05)
DISCUSSION
This study evaluated the effect of secnidazole on sperm motility, morphology, viability and total sperm count of Wistar rats. There was a significant reduction in percentage sperm motility in the treated rats when compared with the control. The reduction was dose dependent, which may be partly due to reduction in serum FSH level as observed in this study. [12] Also reported a reduction in sperm motility in the groups treated with either 200 or 400mg/kg of metronidazole for 4 weeks in male Wistar rats. However after 8 weeks of drug free recovery period, the sperm motility significantly recovers when compared with the control. This may be due to significant reverse in the serumFSH level in the recovery groups as observed in this study. Even after recovery, the rats that received high dose of secnidazole significantly reduced below the recovery control group. This showed secnidazole toxic effect, and may requiremoreprolongtimeforthe animals to recover fully. [13] Also reported tinidazole (a member of nitro imidazole group)significantlyreducespermmotility inmaleWistarratsfor4weeks.
Furthermore, there was a significant reduction in sperm count in the treated rats when compared with the control. The effect was dose dependent. This may be partlyduetoreductioninserumFSHlevel as observed in this study. [12] Also reported a reduction in sperm count in the groups treated with either 200 or 400mg/kg of metronidazole for 4 weeks in male Wistar rats. However, after 8 weeks of drug free recovery period, the sperm count significantly recovered when compared with the control. This may be due to significant reversal in the serum
This study concluded that there was a significant reduction in percentage sperm motility in the treated rats when compared with the control. The reduction
FSH level in the recovery groups as observed in this study. [13] also reported that tinidazole (a member of nitro imidazole group) significantly reduced sperm count in male Wistar rats for 4 weeks.
Furthermore, there was no significant difference in the sperm viability between the treated rats and the control and after recovery. This may be due to the fact that sperm viability is the least affected among the sperm characteristics (sperm count, motility, morphology and sperm viability) after the administration of secnidazole drug. [14] reported that sperm viability is the least affected among sperm parameters after the administration of xenobiotic. This may partly explain why there was no significant change in sperm viability after treatment in this study. However, there was significant reduction in sperm count and sperm motility respectively. In addition, there was no significant differenceinabnormalspermmorphology in the drug treated rats when compared with the control and after recovery. This may be due to the fact that secnidazole does not cause mutagenic effect in the development of sperm cells, also may be due to the facts that secnidazole does not induce its toxicity through permeation of the blood epididymis barrier [15,16]. It has been reported that tinidazole (600mg/kg/d) and ornidazole (400mg/kg/d) does not cause abnormal sperm morphology in respect to hook shaped head, mid piece and tail, after 4 and 8 weeks administration [13]. This may partly explain why secnidazole did not affect sperm morphology in this study.
CONCLUSION
was dose dependent, which may be partly due to reduction in serum FSH level as observed in this study. Also, there was a significant reduction in sperm count in
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the treated rats when compared with the control. The effect was dose dependent. This may be partly due to reduction in serumFSHlevelasobservedinthisstudy. Furthermore, there was no significant difference in the sperm viability between the treated rats and the control and after recovery. This may be due to the fact that sperm viability is the least affected among the sperm characteristics (sperm count, motility, morphology and sperm viability) after the administration of
secnidazole drug. Finally, there was no significant difference in abnormal sperm morphology in the drug treated rats when compared with the control and after recovery. This may be due to the fact that secnidazole does not cause mutagenic effect in the development of sperm cells, also may be due to the facts that secnidazole does not induce its toxicity through permeation of the blood epididymisbarrier.
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