Clomipramine affects sexual behavior and reproductive functions in male rats.
Abstract: Aim of the study

The present study investigated the influence of Clomipramine (Clmp) on male rat's sexual behavior, histology of testes and serum testosterone concentration.

Material and Methods

The study was conducted on male Sprague-Dawley rats in four groups of 12 rats each. Group I served as control and the therapeutic dose of 27mg/kg was given to Group II, III and IV as half therapeutic dose (VaTD), therapeutic dose (TD) & double the therapeutic dose (1/2 TD) of Clmp respectively. Sexual behavior of male rats was studied on 0, 15th, 30th & 60th days by challenging them with female rats in estrous phase. After 30 days treatment, blood samples were collected from half of the animals in each group and sacrificed, remaining animals on 60th day. Testes were collected and processed for histopathological examination. Serum separated and stored at -20[degrees]C for testosterone estimation.

Results and Conlusions

Our study indicated that Clmp produced dose & time dependent sexual dysfunction. It also showed that 1/2 TD of Clmp impaired the sexual behavior without decreasing the testosterone levels, TD & 2TD of Clmp produced sexual dysfunction and also decreased the serum testosterone levels and testicular cells count. Reduction in percentage of rats mounted, intromitted and ejaculated was observed dose and time dependently in all the doses of Clmp tested. All the latencies like mount latency, intromission latency, ejaculation latency and post ejaculatory pause were increased with all the doses of Clmp. Significant fall in testosterone was observed in TD and 2TD at the end of 30 days and 60 days. Reduction in testicular cell count was observed in 1/2 TD, TD, 2TD after 30 days and further decline was observed at the end of 60 days treatment. However this was significant in TD and 2TD treatment groups.

Keywords: Clomipramine, Male rat sexual dysfunction, reproductive functions, Testosterone, Testes.
Article Type: Report
Subject: Sexual disorders (Health aspects)
Testosterone (Health aspects)
Sex (Health aspects)
Authors: Shekar, D. Sheshadri
Satyanarayana, S.
Kumar, K. Eswar
Vivek, B.
Velmurugan, C.
Kumar, B.S. Ashok
Pub Date: 07/01/2010
Publication: Name: International Journal of Health Science Publisher: Renaissance Medical Publishing Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 Renaissance Medical Publishing ISSN: 1791-4299
Issue: Date: July-Sept, 2010 Source Volume: 3 Source Issue: 3
Topic: Canadian Subject Form: Sexual behaviour
Accession Number: 263251122
Full Text: INTRODUCTION

Obsessive compulsive disorder (OCD) is a syndrome characterized by obsessions and or compulsions that together last at least an hour a day and is sufficiently bothersome that they interfere with ones normal, social or occupational functioning. Obsessions are experienced internally and subjectively by the patient as thoughts, impulses or images. According to standard definitions in the diagnostic and statistical manual of mental disorders (1) obsessions are intrusive, inappropriate, and cause marked anxiety and distress. Common obsessions are contamination, aggression, religion, safety / harm, need for exactness or symmetry, somatic (body) fears etc. Compulsions on the other hand are repetitive behaviors, or purposeful mental acts, that are sometimes observed by family members or clinicians. The common compulsions are checking, cleaning / washing, counting, repeating, ordering / arranging, hoarding / collecting etc. Interest in OCD sky rocketed once Clmp was recognized throughout the world to be an effective treatment in the mid 1980s. Originally thought to be rare condition, recent epidemiological studies suggest that OCD exists in the adult population in about 1 out of every 50 adults, and in 1 out of every 200 children.

It has only been recognized widely and on worldwide basis for the treatment of OCD in mid 1980s. Although some similarities exists between the treatment of OCD with SSRIs and the treatment of depression with SSRIs, there are also some important differences, in general & doses for SSRI in OCD are greater than the doses for the treatment of depression. Also onset of therapeutic effects may be more delayed in OCD than it is in depression. (2)

Clmp is the imipramine analogue of chlorpromazine. Due to its action against anxiety disorders and panic attacks, it is the only drug with 2 entries in the essential drugs list of the WHO. Regarding the compulsive disorders it is now the "gold standard" of therapy against which other drugs are measured. (3-7) Chronic use of Clmp leads to sexual dysfunction in humans (8-9) and in male neonatal rats after the attainment of adulthood. (10) Compared to other tricyclic antidepressants, it has greater effect on dopamine blockade and serotonin reuptake inhibition. (11) These implicate orgasmic dysfunction mediated through [5-HT.sub.2] receptors. (12) Moreover peripheral antimuscarinic 13 and alpha adrenergic blockade (14), effects have been implicated in the Clmp induced sexual dysfunction. The usual therapeutic dose of Clmp ranged from 75-300 mg/day in humans (15, 16), however not much information is available regarding its influence on serum testosterone levels and on the histology of the testes. Initially the present investigation was carried out to find out the dose dependent influence of Clmp on the sexual behavior of male rats, serum testosterone level and on the histology of the testes. The doses selected were 14 TD (Therapeutic Dose), TD, 2 TD considering 300 mg as the maximum human therapeutic dose per day and it was extrapolated to rats based on the body surface area. (17) The doses used were 13.5 mg/kg (1/2 TD), 27 mg/kg (TD) and 54 mg/kg (2 TD) of Clmp and were administered orally. The studies were carried out for 60 days, sexual behavior was studied on 0, 15th, 30th & 60th day and estimation of serum testosterone and histology of testes were carried out at the end of 30 days, and 60 days.

MATERIALS AND METHODS Animal preparation

A total of 4 months old 48 Male and 48 female Sprague Dawley albino rats were procured from central animal house NIMHANS, Bangalore. All animals were housed in a group of four males and females separately in plexi glass cages (62x40x21cm) in an acclimatized colony room maintained on 12/12 h light/dark cycle and temperature at 21[degrees]C [+ or -] 2[degrees]C. Male rats were weighed around 300-400 gm each, while the females 250-350 gm each. They were fed on commercial pellet feed and water ad libitum. Prior approval was obtained from Institutional Animal Ethical Committee for conducting the studies.

Drugs and chemicals

Clomipramine HCl (Psychotropic India Ltd), Diethyl ether (Nice chemicals), Eosin (Nice chemicals), Estradiol benzoate (Sigma Aldrich), Progesterone (Glenmark), Testosterone kit (DPC), Haematoxylin (Nice chemicals), Sesame oil (N. Ravindra Company).

Assessing male rat sexual competency:

Male rat sexual behavior

The rats were mated with ovariectomized females (18), sexually activated with exogenous 12[micro]g/rat Estradiol benzoate 56 hrs before and Progesterone 500[micro]g/rat s.c, 4-6 hrs before copulatory test. (19) Ovariectomization was done to re-use the female rats to study the sexual behavior of male rats. Copulatory behavior of male rat is characterized by a series of mounts with or without vaginal intromission from the rear of the female approximately once in every 30 to 120 sec, that eventually culminates in ejaculation. The female responds to each mount with a lordosis response; a dorsoflexion of the spine and deflexion of the tail to one side allowing vaginal access to the male. Typically the male achieves vaginal penetration on 5080% of the mounts. Intromission patterns can be distinguished behaviorally from mounts with penetration by the presence of deep thrust and springing dismount. (20) A mount was defined as the male's pelvis intentionally coming into contact with the female's haunch with accompanying hip movements from the male. An intromission was identified when the male mounted and achieved insertion of the penis into the female as marked by deeper than normal thrust usually followed by abrupt movement away from the female, urgent front leg movements and grooming.

Ejaculation was marked by a more profound thrust than that of regular intromission and was followed not by movement away from the female, but by sudden limpness and immobility until female moves away. After ejaculation and immobility, the male is engaged in the long period of grooming (5-10 min) post ejaculation pause. (21)

An ejaculation occurs after 6-15 intromissions and is followed by a period of 5-10 minutes post ejaculatory interval during which the male is refractory to further copulatory activity. The number of intromissions and latency to ejaculate at first decreases and then subsequently increases with increasing number of ejaculations. (20) The ejaculatory activities of male rat were confirmed by observing the vaginal smear of female for the presence of male sex cells.

In summary, sexual behavior can be analyzed broadly into two major components: libido and potency. These measures are confounded by the erectile processes (i.e., potency) necessary for successful execution of copulatory patterns. (20) The parameters recorded were Mount latency, Intromission latency, Ejaculation latency, Post ejaculatory pause, Intromission frequency and Mounting frequency.

Using these measures, the following parameters were computed % mounted, % intromitted, % ejaculated, copulatory efficiency (number intromissions / number of mounts + number intromission) and inter copulatory interval. (21) Experiment was conducted in a dark and silent room. Since the urine trails left by one rat may have marked effects on the behavior of successor, cleaning of the mating area was done after each trial. They were observed in the cage for 30min under dim red light. When there was no mount latency, intromission, ejaculation and post ejaculation pause their total latent period was taken as 1800 seconds. Tests were terminated immediately after the first post-ejaculatory intromission/mount.

Experimental design

Group I: This group comprised 12 male rats & served as the control group.

Group II: This group comprised 12 male rats. These male rats were treated with 13.5 mg/kg (1/2 TD) of Clmp hydrochloride per oral for 60 days daily.

Group III: This group comprised 12 male rats. These male rats were treated with 27 mg/kg (TD) of Clmp hydrochloride per oral for 60 days daily.

Group IV: This group comprised 12 male rats. These male rats were treated with 54 mg/kg (2TD) of Clmp hydrochloride per oral for 60 days daily.

All the male rats were mated with ovariectomized females that are activated with 12 ug/rat s.c estradiol benzoate 56 hrs before and 500 [micro]g/rat s.c, Progesterone 5 hrs before copulatory test on 0, 15th, 30th & 60th day for sexual dysfunction analysis.

Estimation of testosterone

The Immulite 600032K DPC USA automated immunoassay system is a continuous random access instrument that performs automated chemiluminescent immuno assays.

Collection of blood sample

Half of the animals in each group were sacrificed on 30th day and remaining on 60th day for blood sample collection and histopathological examination of testes. Blood was collected before sacrifice through cardiac puncture using a 16 no needle under mild ether anesthesia and allowed to settle for 10 min time. After centrifugation serum was separated and stored at 20[degrees]c for subsequent hormonal estimation. Testes were also collected and processed for the histopathological studies.

Histopathological studies of testes

The testis was fixed in 10% formalin and processed as follows, testes of each rat were fixed in Bouin's fluid passed through ascending series of ethanol and then through xylene, and embedded in paraffin wax. Tissues were sectioned at the thickness of 5 [micro]m stained with haematoxylin and eosin. The evaluation of cell population was based on the calculation made for each cell type per cross section of the seminiferous tubule. Sertoli cells, spermatogonia, primary spermatocytes, secondary spermatocytes and round spermatids were count ed under 100* magnification using ocular and stage micrometer from twenty randomly selected seminiferous tubules from each slides. The group counts of these cell types were designated as crude counts and these crude counts were corrected by using Abercrombie formula. (22)

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Statistical analysis

Data were expressed as mean [+ or -] SEM. Statistical comparisons were made using Mann-Whitney U-test for the sexual behavior studies and Student's t-test for hormonal and testicular cell counts. (23) P< 0.05 was accepted as indicating significance.

RESULTS

Influence on sexual behavior

There was no change in the control group in the parameters studied during the period of experiment. Clmp treatment produced dose dependent effect in reducing sexual function of male rats. The lowest dose tried 1/2 TD reduced ejaculation from 15th day which further reduced to 50% at the end of the 60 days treatment.

The TD reduced the number of rats intromitted and ejaculated from the 15th day and 0 day respectively which further reduced drastically to 50% and absence of ejaculation was observed at the end of the 60 days treatment. The 2 TD dose reduced the number of rats intromitted and ejaculated from the 0 day itself and further suppressed all the parameters including mounting from the 30th day.

All the latencies observed like mount latency, intromission latency, ejaculation latency and post ejaculation pause were increased with all the doses of Clmp tried in the studies. The increase in ejaculation latency in the Clmp treated group was 3-4 fold when compared to the control supporting its usefulness in the treatment of premature ejaculation. (24, 25) In the 1/2 TD, further increase in the ejaculation latency was observed on 60th day and failure of ejaculation was observed with therapeutic dose on 60th day. Absence of ejaculation was observed on 30th day itself in all the animals treated with 2 TD Clmp. A gradual increase in the mount frequency was also observed in all the Clmp treated groups followed by a fall. In contrast, to that numbers of intromissions were decreased. The suppression of intromission ratio suggested an impaired penile erection. This was also reflected by gradual decline in copulatory efficiency on all the days observed. Clmp treatment prolonged the intercouplatory interval on all the days observed.

Overall Clmp has altered all the sexual behavior parameters in the male rats significantly on all the days observed when compared to the control. The results were given in table no 1.

Influence on serum testosterone levels

After 30 days of treatment no significant change was observed between the control group and 1/2 TD group, whereas TD significantly reduced the serum testosterone level and with 2TD, testosterone levels was further reduced to 1.7 ng/ml.

At the end of 60 days treatment no significant change was observed between the control and 1/2 TD, whereas TD group testosterone levels were further reduced significantly when compared to the control. Similarly in 2TD treatment significant fall was observed. The results were presented in table no 2.

Influence on the histology of testes

Histopathological findings after 30 days treatment show no significant changes between control and 1/2 TD. But significant changes were observed with TD & 2TD. The TD treated animals were presented with degeneration of tubules to mild degree of vacuolation with or without spermatogenesis, degeneration of sertoli cells indicated by aggregation at one end of tubules. Spermatogonia present with fragmented nuclei. Hypoplasia like action was seen in seminiferous tubules. There were some tubules where spermatocytes production was arrested along milder degree of vacuolation. Inter tubular spaces and venous congestions were increased in the treatment group when compared with control.

The 2TD were presented with the tubules lost, the basement membrane and some seminiferous tubules with thickened basement membrane had less number of epithelial cells in fact much of the epithelium lining tubules are vacuolated. A remnant of spermatogenesis was evident. Interstitial connective tissue and Leydig cells were not showing any function. Adecrease in all testicular elements was observed. The results are given Table--3

At the end of 60 days treatment, the groups treated with 1/2 TD were presented with seminiferous tubules separated by interstitial connective tissue. No significant changes were observed when compared to the control.

In case of TD, seminiferous tubules were presented with high degree of degenerative changes like elongation of tubules, loss of architecture or basement membrane. A decrease in the sertoli cell count and absence of the primary stages of the Spermatogenesis were noted.

In 2TD, the degree of degeneration of epithelium lining in the tubules was more severe. There were areas of no connective tissue. Interstitial space increased leading to diffused network of tubules of testis. Sertoli cells observed in some of the tubules were vacuolated preformed spermatids.

DISCUSSION

Rats are one of the most widely used animals in reproductive studies and copulation is the most natural and frequently examined behavior of the laboratory tests of sexual function. (26, 27) It has been proposed that there are obvious homologies between human copulatory behavior and that of rat including the mechanism of penile erection. (28) Hence rats were used for the present study.

The decrease in number of rats ejaculated with Clmp treatment at 1/2 TD TD and 2TD was dose dependent. The percentage ejaculated decreased with 1/2 TD dose without total failure of ejaculation. The failure of ejaculation was seen on 60th day in the case of TD and by 30th day in 2TD. The ejaculation failure was confirmed by observing the vaginal smear of female rats after the copulatory act of animals at the above intervals where no sperm cells found in the vaginal fluid after mating.

The study also demonstrated that chronic Clmp treatment impairs sexual competence in male rats at all the 3 doses tested namely 1/2 TD, TD, and 2TD tested (table. 1). The number of rats mounted and intromitted was reduced with Clmp treatment which was indicative of impaired libido (table 1). Clmp caused prolongation in latencies of mounting, intromission, and ejaculation in the animals observed. An inverse relationship exists between latencies of time parameters and sexual arousability / motivation. (29) Further Clmp treatment decreased the copulatory efficiency suggesting the disruption of sexual vigor. The results of earlier studies support our data. (30) A decrease in copulatory efficiency could be due to erectile dysfunction.

Clmp hypothesized to act therapeutically by blocking the reuptake pumps for 5-HT and NE and to a lesser extent, dopamine. Clmp act at several other types of receptors, including histamine, cholinergic muscarinic and [alpha]-1 adrenergic receptors, as well as at sodium channels in the heart and brain. These actions are largely responsible for unwanted side effects such as orthostatic hypotension, dizziness, constipation, confusion, blurred vision, dry mouth, weight gain, sedation, cardiac arrhythmias and seizures in overdose. The adrenergic and cholinergic blockade may also be responsible for symptoms of sexual dysfunction, since both are involved in erectile function. (3, 31)

Pharmacological evidence demonstrated also that the elevation of the cerebral levels of 5- HT affects the secretion of FSH and luteinizing hormone, for inhibiting the liberation of gonadotrophins releasing factor in the hypothalamus, with subsequent effects on the process of spermatogenesis and steroidogenesis in adult rats. (32-35) Sexual behavior is impaired by many 5-HT agonists and agents that increase 5-HT availability and is facilitated by serotonergic lesions and its receptor antagonists. (36-38) The 5-HT may be released in serotonergic nerve terminal fields including the median preoptic areas and promote sexual refractoriness during the post ejaculatory interval. The treatment with Clmp enhances 5-HT due to its reuptake inhibitory activity which might be responsible for the decreased sexual function.

Testosterone

The normal level of testosterone was found to vary between 5 to 9 ng/ml of serum in control rats. The Clmp with 13.5 mg/kg did not alter the normal levels of testosterone at the end of 30 days and 60 days treatment. However it was found to reduce sexual function. The TD and 2TD of Clmp reduced testosterone levels at the end of 30days and 60days. The effect was higher with higher dose of Clmp indicating the dose dependent effect. The decrease in sexual function correlated with the testosterone levels at TD & 2TD dose level.

Peripherally testosterone activates (nitric oxide synthase) NO synthase in the corpora cavernous of the penis to create erection. (39) NO synthase inhibitor prevents the above agents from inducing yawning and sexual behavior and they have an anti-sexual effect in the rat. It has been demonstrated by in vitro and in vivo studies in both humans and animals that SSRIs act as potent NOS inhibitors and lower NO centrally and peripherally. (40)

Histopathology of testes

The development of gonads (testes) is under the influence of gonadotropic hormones secreted by anterior pituitary gland. The follicle stimulating hormone (FSH) and luteinizing hormones are responsible for gonadal development and for spermatogenesis. The testes contain three different types of cells namely Leydig cells responsible for production of testosterone, spermatogonia responsible for spermatogenesis (sperm cells) and Sertoli cells responsible for secretion of androgen binding protein, inhibin, sulfated glycoprotein-2 and transferrin. The FSH is responsible for the development of spermatogonia to secondary spermatocytes through different stages namely spermatogonia, spermatids, sertoli cells and secondary spermatocytes. (41) After puberty the Leydig cells start producing testosterone which was found to be responsible for the development of round spermatids to spermatozoon through elongated spermatids. The cross section of seminiferous tubules represent the cellular development hence cross sectional examination was conducted. The damage was significant with 2TD. The results correlates with decrease in testosterone level (table 2) with doses of Clmp tried. The decrease in the early process of spermatogenesis may be because of decrease in FSH which was not determined in our studies (Fig 1- 7).

CONCLUSION

In conclusion the study has demonstrated that Clmp in the doses tested produced significant sexual dysfunction in male rats. Clmp treatment even at sub therapeutic dose itself produced sexual dysfunction in male rats without altering the testosterone levels. The TD and 2TD produced dose dependent changes in the sexual behavior, decline in testosterone levels, and damage to the testes. It is important to point out that work is in progress to find out a suitable antidote to antagonize the neuronal, cellular, and hormonal actions of Clmp.

ACKNOWLEDGEMENT

Authors are thankful to Dr. Mahenderbalar for technical assistance & helpful discussions in histological studies. Authors also like to thank Mr. K.V. Naveen Kiran, Chairman, Sri K.V College of Pharmacy for providing necessary facilities to carry out the work.

Conflict of interest: None declared.

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D. Sheshadri Shekar (1), S. Satyanarayana (2), K. Eswar Kumar (2), B.Vivek (1), C. Velmurugan (1), B.S. Ashok Kumar (3)

(1) Department of Pharmacology, Sri K.V College of Pharmacy, Chickballapur, Karnataka, India

(2) Department of Pharmacy, College of Pharmaceutical Sciences, Visakhapatanam, Andhra Pradesh, India

(3) Department of Pharmacognosy, Sri K.V College of Pharmacy, Chickballapur, Karnataka, India

Corresponding author: Sheshadri Shekar Head of the Department of Pharmacology, Sri K.V College of Pharmacy, Chickballapur, Karnataka, India. Mobile No-+91-9900510200, e-mail: sheshadrishekar@gmail.com
Table 1--Effect of chronic oral administration of different doses
of Clomipramine (Clmp on sexual behavior parameters of male rats
(data given as mean [+ or -] SEM)

Parameter          Control
Studied

Days               0                          15

% mounted          100                        100

% intromitted      100                        100

% ejaculated       100                        100

Mount latency in   5.37 [+ or -] 0.84         8.5 [+ or -] 1.29
sec

Intromission       23.5 [+ or -] 4.9          20.65 [+ or -] 4.8
latency in sec

Ejaculation        360 [+ or -] 18.4          375.5 [+ or -] 9.4
latency in sec

No of              15.625 [+ or -] 0.3        15.625 [+ or -] 0.3
intromissions

No of mounts       2.5 [+ or -] 0.32          2.25 [+ or -] 0.36

Post ejaculation   262.5 [+ or -] 10.9        290 [+ or -] 30.9
Pause in sec

Copulatory         0.85 [+ or -] 0.01         0.86 [+ or -] 0.2
efficiency

Inter copulatory   23.13 [+ or -] 1.43        24.15 [+ or -] 0.5
interval in sec

Parameter          Control
Studied

Days               30                         60

% mounted          100                        100

% intromitted      100                        100

% ejaculated       100                        100

Mount latency in   8.87 [+ or -] 1.02         7.5 [+ or -] 0.8
sec

Intromission       18.62 [+ or -] 1.4         20.5 [+ or -] 1.8
latency in sec

Ejaculation        372.5 [+ or -] 8.8         375 [+ or -] 6.45
latency in sec

No of              15.37 [+ or -] 0.2         15.5 [+ or -] 0.2
intromissions

No of mounts       2.62 [+ or -] 0.4          3.5 [+ or -] 1.08

Post ejaculation   225 [+ or -] 15.0          270 [+ or -] 19.14
Pause in sec

Copulatory         0.85 [+ or -] 0.03         0.82 [+ or -] 0.04
efficiency

Inter copulatory   24.29 [+ or -] 0.8         24.2 [+ or -] 0.7
interval in sec

Parameter          Clmp 13.5 mg/kg
Studied

Days               0                          15

% mounted          100                        100

% intromitted      100                        100

% ejaculated       100                        75

Mount latency in   12.87 [+ or -] 1.63 **     80. [+ or -] 9.8 **
sec

Intromission       156.25 [+ or -] 14.3 **    253.8 [+ or -] 17.42 **
latency in sec

Ejaculation        1012.5 [+ or -] 47.3 **    1172.5 [+ or -] 154.9 **
latency in sec

No of              21.37 [+ or -] 0.77 **     20.62 [+ or -] 2.8
intromissions

No of mounts       3.875 [+ or -] 0.4         6.62 [+ or -] 0.49 **

Post ejaculation   395 [+ or -] 27.9 **       808.75 [+ or -] 216.51 **
Pause in sec

Copulatory         0.83 [+ or -] 0.019        0.69 [+ or -] 0.07 **
efficiency

Inter copulatory   47.67 [+ or -] 2.64 **     158.5 [+ or -] 106 *
interval in sec

Parameter          Clmp 13.5 mg/kg
Studied

Days               30                         60

% mounted          100                        100

% intromitted      100                        100

% ejaculated       75                         50

Mount latency in   93.7 [+ or -] 13.7 **      263.5 [+ or -] 19.38 *
sec

Intromission       271.3 [+ or -] 14.3 **     378.5 [+ or -] 42.73 *
latency in sec

Ejaculation        1187.5 [+ or -] 137.58 **  1362.5 [+ or -] 252.80 *
latency in sec

No of              18.6 [+ or -] 3.51         15.5 [+ or -] 4.3
intromissions

No of mounts       6.75 [+ or -] 0.95 **      11 [+ or -] 3.4 *

Post ejaculation   821.25 [+ or -] 213.6 **   1150 [+ or -] 375.28 *
Pause in sec

Copulatory         0.66 [+ or -] 0.06         0.61 [+ or -] 0.1
efficiency

Inter copulatory   168.14 [+ or -] 79.5 **    165.62 [+ or -] 96.9 *
interval in sec

Parameter          Clmp 27 mg/kg
Studied

Days               0                          15

% mounted          100                        100

% intromitted      100                        75

% ejaculated       50                         33.35

Mount latency in   40.75 [+ or -] 3.4 **      143.12 [+ or -] 8.81 **
sec

Intromission       200 23.6 **                668.12 [+ or -] 247.01
latency in sec

Ejaculation        1380 [+ or -] 162.5 **     1360 [+ or -] 168.48 **
latency in sec

No of              17.37 [+ or -] 2.3         14 [+ or -] 3.6
intromissions

No of mounts       8.75 [+ or -] 0.41 **      10.87 [+ or -] 1.85 **

Post ejaculation   1147.5 [+ or -] 246.13 **  13075 [+ or -] 240.33 *'
Pause in sec

Copulatory         0.63 [+ or -] 0.04 **      0.51 [+ or -] 0.11 **
efficiency

Inter copulatory   110.8 [+ or -] 39.9 **     67.8 [+ or -] 26.8
interval in sec

Parameter          Clmp 27 mg/kg
Studied

Days               30                         60

% mounted          100                        100

% intromitted      75                         50

% ejaculated       8.3                        0

Mount latency in   1627.14 [+ or -] 13.18 **  341.25 [+ or -] 64.3 *
sec

Intromission       672.14 [+ or -] 246.05     1050 [+ or -] 433.01 *
latency in sec

Ejaculation        1710 [+ or -] 90 **        1800 [+ or -] 0 *
latency in sec

No of              13.75 [+ or -] 3.4         7.25 [+ or -] 4.19 *
intromissions

No of mounts       10 [+ or -] 3.65 *         6.75 [+ or -] 0.47 *

Post ejaculation   1475 [+ or -] 212.76 **    1800 [+ or -] 0 *
Pause in sec

Copulatory         0.56 [+ or -] 0.12 *       0.33 [+ or -] 0.19 *
efficiency

Inter copulatory   77.55 [+ or -] 20.40       62.12 [+ or -] 35.91
interval in sec

Parameter          Clmp 54 mg/kg
Studied

Days               0                          15

% mounted          100                        75

% intromitted      91.66                      58.33

% ejaculated       33.33                      8.3

Mount latency in   43.125 [+ or -] 9.3 **     133.7 [+ or -] 517 **
sec

Intromission       473.75 [+ or -] 189.51 **  860 [+ or -] 257.21 **
latency in sec

Ejaculation        1435 [+ or -] 180.9 **     1710 [+ or -] 90 **
latency in sec

No of              17.75 [+ or -] 2.6 **      7.625 [+ or -] 2.9 *
intromissions

No of mounts       9.875 [+ or -] 0.97 **     5.125 [+ or -] 1.6

Post ejaculation   1310 [+ or -] 239.11 **    1637.5 [+ or -] 162.5 **
Pause in sec

Copulatory         0.60 [+ or -] 0.08 **      0.28 [+ or -] 0.08 **
efficiency

Inter copulatory   62.87 [+ or -] 14.29 *     110.07 [+ or -] 39.85
interval in sec

Parameter          Clmp 54 mg/kg
Studied

Days               30                         60

% mounted          0                          0

% intromitted      0                          0

% ejaculated       0                          0

Mount latency in   1800 [+ or -] 0.00 **      1800 [+ or -] 0 *
sec

Intromission       1800 [+ or -] 0 **         1800 [+ or -] 0 *
latency in sec

Ejaculation        1800 [+ or -] 0 **         1800 [+ or -] 0 *
latency in sec

No of              0 **                       0 *
intromissions

No of mounts       0 **                       0 *

Post ejaculation   1800 [+ or -] 0 **         1800 [+ or -] 0 *
Pause in sec

Copulatory         0 **                       0 *
efficiency

Inter copulatory   0 **                       0 *
interval in sec

Significant at P<0.05 *, 0.01 ** compared to control
(Mann Whitney 'U' test)

Table 2. Influence of Clmp (30 days & 60 days) on serum
testosterone levels in male rats (data given as mean
[+ or -] SEM n= 6)

                   Testosterone            Testosterone
Treatment          (30 days) (ng/ml)       (60 days) (ng/ml)

Control 1ml/kg     6.55 [+ or -] 0.66      5.075 [+ or -] 0.2
Clmp 13.5 mg/kg    7.025 [+ or -] 0.60     5.825 [+ or -] 0.76
Clmp 27 mg/kg      3.5 [+ or -] 0.5 *      2.307 [+ or -] 0.16 **
Clmp 54 mg/kg      1.7 [+ or -] 0.24 **    1.48 [+ or -] 0.14 **

Significant at P <0.05 *, 0.01 ** compared to control (Students t-test)

Table 3. Influence of Clmp (30 days) treatment)
(data given as mean  [+ or -]  SEM n = 6)

Treatment (30 days)   Sertoli Cells            Spermatogonia

Control 1ml/kg        4.33 [+ or -] 0.33       53.5 [+ or -] 1.94
Clmp 13.5 mg/kg       2.5 [+ or -] 0.2         46.50 [+ or -] 1.68 *
Clmp 27 mg/kg         2.33 [+ or -] 0.21       37.33 [+ or -] 2.0 *
Clmp 54 mg/kg         2.25 [+ or -] 0.25       20.66 [+ or -] 2.0 **

Treatment (30 days)   Spermatids               Spermatocytes

Control 1ml/kg        123.66 [+ or -] 3.11     61.66 [+ or -] 3.19
Clmp 13.5 mg/kg       110.16 [+ or -] 3.7      64.16 [+ or -] 1.51
Clmp 27 mg/kg         62.66 [+ or -] 6.9 *     40.16 [+ or -] 1.30 *
Clmp 54 mg/kg         44.33 [+ or -] 2.9 **    26.16 [+ or -] 1.99 **

Significant at P <0.05 *, 0.01 ** compared to control
(Students t-test)

Table 4. Influence of Clmp (60 days) treatment on the histology of
testes (data given as mean [+ or -] SEM n= 6).

Treatment (60 days)   Sertoli Cells            Spermatogonia

Control 1ml/kg        4.16 [+ or -] 0.3        49.50 [+ or -] 0.50
Clmp 13.5 mg/kg       2.25 [+ or -] 0.25       48.16 [+ or -] 0.70
Clmp 27 mg/kg         2.5 [+ or -] 0.28        34.16 [+ or -] 2.15
Clmp 54 mg/kg         1.75 [+ or -] 0.25 *     15.83 [+ or -] 0.94 *

Treatment (60 days)   Spermatids               Spermatocytes

Control 1ml/kg        105.83 [+ or -] 4.12     66.66 [+ or -] 2.14
Clmp 13.5 mg/kg       94.16 [+ or -] 2.9       61.66 [+ or -] 2.2
Clmp 27 mg/kg         37.66 [+ or -] 2.4 *     30.00 [+ or -] 1.2
Clmp 54 mg/kg         21.66 [+ or -] 0.71 **   17.66 [+ or -] 0.88 *

Significant at P <0.05 *, 0.01 ** compared to control (Students t-test)
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