Effect of Cimetidine on Semen Parameters, FSH and LH in Male Rats

ABSTRACT


INTRODUCTION
Cimetidine is a potent competitive H2 receptor blocker; it is extensively approved for peptic ulcers (1).Cimetidine is a prophylactic drug for colorectal cancer, in addition treatment of Zollinger -Ellison syndrome, heart burn, oesophagitis, upper gastrointestinal bleeding and paracetamol overdosage (2,3).Parker et al. (1984)  4 and Van Theil (1982)  5 reported the sexual dysfunction after cimetidine treatment.Franca et al., (2000)  6 and Winters et al. (1979)  7 recorded the antiandrogenic effects of cimetidine in Iraq, like other countries, cimetidine is wildly used usually in clinics and in hospitals, which approved in chronic diseases program without caution to their harmful effects for long-term treatment (8).Moreover, these medications may impair sperm maturation and epididymal function (9).The Food and Drug Administration (USA) has given the drug cimetidine approval to treat stomach acid secretion.It is used to treat hyper secretory disorders such Zollinger-Ellison syndrome, numerous endocrine adenomas, erosive gastroesophageal reflux, and peptic ulcer symptoms (10).The release of stomach acid brought on by food, histamine, pentagastrin , caffeine, and insulin is considerably reduced by cimetidine.It competes with DHT receptors in the pituitary gland, brain, and other organs that need DHT since it is an H2 receptor antagonist, in accessory glands, such as the prostate and seminal vesicle gland, DHT, a sterol that is produced when testosterone (T) is converted, performs the role of the socalled true male hormone.The term "weak nonsteroidal antiandrogen" is used to describe it (11).
Cimetidine has been shown to have significant immunoregulatory and anticancer effects, which furthered the therapeutic use of the compound (12, 13).Cimetidine's pharmacological activity is broad since it is extensively dispersed in many types of tissue Male patients on cimetidine have experienced side effects such impotence, reduced sperm count, and libido loss (14).Nevertheless, it has been noted that this medication has negative effects on males that are associated with its antiandrogenic activity, including impotence, gynecomastia, changes in the serum testosterone levels, and a drop in sperm concentration (14,15).Cimetidine lowers blood testosterone levels in animals (16,17), which is Corresponding Author: O. S. I. Al-janabi linked to Leydig cell death and decreased steroidogenesis (16).Loss of germ cells and Sertoli cell death have both been linked to this androgenic dysfunction (17)(18)(19)(20).The aim of this study is to find out the extent of the virrulence of cimetidine on the male reproductive system.

MATERIALS AND METHODS Animals
This study was conducted in the department of physiology and pharmacology University of Tikrit College of Veterinary Medicine.Male adult rats in good health were obtained from the Tikrit University College of Veterinary Medicine's animal house.In this investigation, 20 male rats were, age ranging from 8-10weeks and weight from (200 to 280) grams.These animals were maintained in an air-conditioned room with a temperature range of (20 -25) degrees and a 12-hour daily light cycle.The animals were kept in (46*28*13) cm.plastic cage enclosures.Food was provided in the form of freshly made ration pellets.Care was made to avoid any extra stress.Once every week, the cages were cleaned.The pets were housed for at least two weeks before being (adaptation period).

Ethical approve OR data collection permit
Trials, including laboratory rats, in animal house of the College were followed-up by the academic board of the Department of Physiology, Biochemistry, and Pharmacology at the University of Tikrit Veterinary Medicine College

Study design
The study was worked in the animal house of the Veterinary Medicine College\ Tikrit University during the period 38days from (20.Novmber.2022 to 28.Desmber.2022),20 male rats were randomly classified into two group 10 male rats to each group.

Control group (G1):
This group contain on 10 male rats receiving normal saline for 38 day.

Cimetidine group (2):
This group contain on 10 male rats receiving 100mg/kg.B.W. from cimetidine Preparation of cimetidine solution: -Cimetidine stock solution was made by dissolving 100% pure cimetidine product in distilled water.This solution was then administered to rats through stomach tube one time daily at a dose of 100 mg/kg B.W (20).Individual adjustments were made to the dosage of the provided solution based on body weight.

Sperm function test. ( sperm motility, sperm morphology and sperm count ):-Semen collection: -
Animals were slaughtered after the course of therapy, and an abdominal incision was made.The testis and epididymis are removed, cleaned from the accessory connective and adipose tissues, the analyses were achieved in diagnostic lab in animal house in collage of veterinary medicine/Tikrit university.

A. Sperm motility and immotilty: -
For analysis of sperm motion, collect samples from cauda epididymitis, (21) then making slicing by surgical blade to diffusion with a solution and making mix, then take drop of the sample and placed on the slide and then cover slide, to be examined under microscope on power( 10X and 40X).The percentage motile sperm, which is defined as the number of motile sperm/total number of sperm x 100.

B. Sperm morphology: -
The aberrant sperm morphology was assessed using Siegmund's approach from 1979.Two smears of each sample were created on microscope slides by first preparing a drop of sperm suspension, placing it over the edge of the slide, and adding a drop of Eosin-Nigrosin stain.Examining the smears for unusual morphology Abnormal sperm morphology was calculated as in the following equation: Percentage of morphologically abnormal sperms = (No. of morphologically abnormal sperms/Total sperms no.)×100.According to the sperm abnormalities, such as tapering head, tailless head, coiled tail, bifurcate tail, and broken tail, the morphologically abnormal sperms were calculated (20).

C. Sperm count: -
According to (Yuxin Ma, et al., 2011) 22 , sperm count was performed.Use a Haemocytometer (Neubauer Type).Sperms were counted in 25 tiny squares of the chamber that were divided into 5 l of a sperm suspension and covered by a cover slide in the Haemocytometer.The following formula was used to estimate the amount of sperm: Sperm concentration = Number of sperm X 1000.

Estimation of Sex Hormones (Testosterone, FSH and LH): -Blood collection: -
After the end of the experiment, animals were anesthetized by chloroform, blood samples were obtained via eye puncture and heart after anatomy from each anesthetized animal using disposable syringes.Samples were centrifuged at (2500 rpm) for 15 minutes, and then serum samples were stored in freezer at (-18ċ) untill use.

Statistical analysis:
The statistical analysis made use of the ANOVA Analysis of Variance.The significant differences were discovered at a significant level (P≤0.05) in accordance with Duncan's multiple ranges.( 23).Corresponding Author: O. S. I. Al-janabi

RESULTS
This study showed significantly decrease (p≤0.05) in sperm motility, normal sperm morphology and sperm count in group treated with cimetidine after 38 days from give drug compare with control group, but also showed increase (p≤0.05) in immotile sperm, abnormal sperm morphology.

Estimation of Sex Hormones
This study showed significant decrease (p≤ 0.01) in level of testosterone and FSH in group treated with cimetidine compared to control group, but also showed increase in LH level .Nonetheless, this study also revealed a gradual, substantially higher level of FSH and testosterone in the groups treated with OLE; this outcome is comparable to (34,35).

CONCLUSION
According to this study, administering Cimetidine has harmful on male reproductive system in rats, at a dose of 100mg/kg B.W for 38 days through its influence on sperm parameters( sperm motility, morphology and counts) and sex hormones ( testosterone, FSH and LH).

Acknowledgments:
The authors express their gratitude to the College of Veterinary Medicine at Tikrit University for all its assistance in achieving this work.

Conflicts of interests:
The authors declare that there is no conflict of interest in the publication of this paper.

Table 4 : -Effect of Cimetidine 100mg/kg B.W. on testosterone level in adult male rats.
, who noted that cimetidine therapy raises GnRH levels, which were linked to the involvement of LH negative feed back mechanism impairment in the response of LH to luteinizing releasing hormone, was the decrease in testosterone and FSH levels (LH-RH).Moreover, the disruption of the LH negative feedback system results in increased LH production, which alters LH testosterone secretion(31,32).According toLuiz et al. (2000)33 , using cimetidine causes the growth of the adrenal glands.