PROTECTIVE ROLE OF TRIBULUS TERRESTRIS ON ALUMINIUM CHLORIDE – INDUCED REPRODUCTIVE TOXICITY IN THE MALE LABORATORY MOUSE

PROTECTIVE ROLE OF TRIBULUS TERRESTRIS ON ALUMINIUM CHLORIDE - INDUCED REPRODUCTIVE TOXICITY IN THE MALE LABORATORY MOUSE

Praveen Kumar and Poonam Singh*

Department of Zoology, MMV, Banaras Hindu University, Varanasi, U.P., 221005, India

ABSTRACT:  The present study deals with the efficacy of Tribulus terrestris (TT) on aluminium chloride (AlCl₃) - induced alterations in the reproductive organs and fertility of the male laboratory mouse. Thirty adult male mice were divided into six groups of five each. Group I served as control while that of II, III and IV received various doses of AlCl₃ (25, 50, 100 mg/kgBW/day, respectively), for 30 days. The mice of groups V and VI were administered with TT (100mg/kgBW/day) only and AlCl₃ (100mg/kgBW/day) along with TT (100mg/kgBW/day) respectively, for the same duration. Only high dose of AlCl₃ (100mg/kgBW/day) - treated mice showed significant reductions in the body weight, sex organs relative weight, sperm count, motility, viability, epididymal sialic acid, seminal vesicular fructose, serum testosterone, antioxidant enzymes (SOD, Catalase, GPx), mating ability and fertility. However percentage of abnormal spermatozoa in the epididymis and level of testicular cholesterol and MDA were   significantly increased. Dose-dependent regressive histological changes in the seminiferous tubules and the Leydig cells were also noticed. Supplementation with TT in high dose of AlCl₃ - treated mice resulted in noticeable restorations in all the studied parameters as compared with only AlCl₃ - treated mice. The results thus indicated the protective role of TT against AlCl₃- induced adverse effects on the male reproductive organs and the fertility.

Aluminium chloride, Tribulus terrestris, Testosterone, Antioxidant enzymes, Fertility

INTRODUCTION: Herbal extracts are extensively used for the treatment of various diseases as these are natural and relatively safe with minimum or no side-effects. Therefore, intense research is growing for the analysis of plant products which are beneficial for human health. Recently, Abirami and Rajendran ¹ have reported the occurrence of more than 35,000 plant species, being used for medicinal purpose. Tribulus terrestris, a perennial ground spreading herb of family Zygophyllaceae, is widely distributed in many tropical and moderate areas of the world, including U.S. and Mexico, the Mediterranean region, and throughout Asia.

It is one of the well-known traditional medicinal plant with multiple beneficial effects such as liver tonic, aphrodisiac, analgesic, astringent, stomachic, anti-hypertensive, diuretic, lithon-triptic and urinary anti-infectives ². The fruit extract of TT contains saponins, glycosides, flavonoids, alkaloids, resins, tannin, sugars, steroid and essential oils ³. TT improves the sexual functions due to presence of protodioscin, a steroidal saponin ⁴. Protodioscin is suggested to be effective in treatment of low libido, erectile dysfunction and low seminological indices by elevating the level of testosterone, dihydrotestosterone, dehydroepiandro sterone and dehydroepiandrosterone sulphate ⁵.

The flavonoid, another active constituent of the TT, bears antioxidative properties ⁶. Bashir et al. ⁷ have reported the stimulating effects of TT on the germinative and endocrine function of the testis producing its precocious development in rat. Efficacy of TT has also been reported under various experimental conditions. It protects the testicular injuries in rat caused by cadmium ⁸ and cypermethrin ⁹. The extract of this plant also prevents the cadmium ⁶-induced hepatic injury and oxalate – induced renal injury ¹⁰ in rat.

Aluminium, a widely distributed metal in the environment, constitutes 8% of the earth’s crust ¹¹ and is extensively used in manufacture of cosmetics, cookware, utensils, food additives, toothpaste ¹² etc. It is also used in medicines and added to the drinking water for purification purposes ¹³.

It is released to the environment by natural process and from various anthropogenic sources. ATSDR ¹⁴ reports the distribution of aluminium mainly in bone, liver, brain, kidney and testis. It may impair the fertility in males directly by inhibiting the sperm or testicular function or indirectly by impairing the hypothalamic-pituitary-testicular axis resulting in poor semen quality. Aluminium ingestion in excessive amount leads to accumulation in target organs that damages the testicular tissue in human and animals both ¹⁵. High concentration of aluminium in human testis, seminal plasma, blood and urine is associated with impaired sperm quality and viability ¹⁶. Guo et al.^{17, 18} have reported aluminium accumulation in the testis leading to necrosis of spermatocytes / spermatids and significant decrease in fertility of the mice. AlCl₃-induced oxidative stress has also been reported as a contributing factor in testicular injuries ^{19, 20}.

Efficacy of several synthetic agents such as ascorbic acid ²⁰, Vit-E ²¹, a natural product of honey bee i.e. propolis¹⁵ and certain plant extracts such as of grape seeds ²², ginger ²³and onion ²⁴ has been reported in minimizing the AlCl₃- induced reproductive toxicity in the rat. As far known till now, there is no report indicating the protective efficacy of TT against AlCl₃-induced male reproductive impairments as majority of the populations are in continuous exposure to this metal through various sources. In this background, the present study has been designed to explore the effects of Tribulus terrestris on the aluminium chloride – induced reproductive toxicity in the adult male mice.

MATERIALS AND METHODS:

Plant and Extract preparation:

The fruits of Tribulus terrestris were collected from local market of Varanasi and identified morphologically by Department of Botany, BHU, Varanasi. Coarse powder of shade dried fruits of TT were extracted with 70% ethanol (1:10) in a Soxhlet apparatus and evaporated the extract in water bath at 60⁰C until a consistent solid material was formed. The extract was stored at 4⁰C in air tight bag and dissolved in distilled water for the treatment.

Animals:

Thirty healthy adult (12 weeks old) male mice weighing 20-30g were used in this study. They were maintained in an animal house as per the recommendation from Central Animal Ethical Committee of the University (CAECU) for the care and use of the laboratory animals. The mice were kept under 12h L/ 12h D cycle and ambient temperature. Standard pelleted food and tap water were provided ad libitum.

After recording the initial body weights all the animals were randomly divided into six groups of five each. Animals of group I served as control, administered with distilled water while that of groups II, III and IV were administered with aqueous solution of AlCl₃ at the doses of 25, 50 and 100mg/kg BW/day  respectively, for 30 consecutive days. The mice of groups V and VI were administered with TT (100mg/kgBW/day) only and AlCl₃ (100mg/kgBW/day) along with TT (100mg/kgBW/day), respectively, for the same duration. Twenty four hours after the last treatment, final body weights of the animals were recorded followed by sacrificing them by cervical dislocation. Testis, epididymis and seminal vesicle of both sides were dissected out, washed in normal saline and processed for the following studies:

Organs weight:

Wet weights of the testis, epididymis and the seminal vesicle of the left side were recorded to calculate the gonado-somatic index (GSI) by using the following formula:

Gonadal weight/total body weight ×100

Epididymal sperm assessment:

Cauda epididymides of five mice from each group were minced thoroughly in the physiological normal saline at 37⁰C and used for the assessment of motility, viability and count according to the WHO Laboratory Manual ²⁵. Morphology of the spermatozoa was assessed in the same slide as used for the sperm viability. Sperm abnormality was evaluated by the criteria of Wyrobeck and Bruce ²⁶ and Zaneveld and Polakoski ²⁷.

Histological Studies:

Bouin’s fixed testis and seminal vesicle were dehydrated in graded series of ethanol, cleared in xylene and embedded in paraffin wax. Sections of 5μm thickness were cut and stained with Periodic-Acid-Schiff (PAS) reagent followed by counterstaining with Harris Haematoxylin. The stained sections were observed under light microscope.

Biochemical Studies:

The concentrations of sialic acid in the epididymis and fructose in the seminal vesicle were estimated by using the methods of Aminoff ²⁸ and Linder and Mann ²⁹, respectively.

Antioxidant enzymes analysis:

Lipid peroxidation (LPO) was measured by thiobarbituric acid (TBA) method ³⁰ that determines aldehyde formed by degradation of hydroperoxide including malondialdehyde (MDA). The antioxidant enzymes e.g. superoxide dismutase, catalase and glutathione peroxidase, were assayed in the testis following the methods of Das et al.³¹, Aebi ³² and Paglia and Valentine ³³, respectively.

Cholesterol:

The level of cholesterol was estimated in the testis by using commercial diagnostic kit of ENZOPAK (Reckon Diagnostics Private Ltd.).

Hormonal analysis:  

Blood was collected by cardiac puncture immediately after sacrificing the animals. Serum was obtained by centrifuging the blood at 4500 rpm for 20 minutes in a cooling centrifuge and stored at -20^oC until utilized for the study. The level of serum testosterone was measured by using ELISA kit (LDN, Germany).

Mating ability and Fertility:

Five males from each group were caged individually with two virgin females for overnight and according to presence of vaginal plug and implantation sites in females, the mating ability and fertility of the males were assessed, respectively. The females caged with the treated males were sacrificed by cervical dislocation at day eighteenth of gestation and examined their uteri. The ovaries were removed to count the number of corpus luteum. To determine the total number of implantation sites, the dissected out uteri were placed in 10% ammonium sulfide solution, which stained the hemosiderin pigment of resorbed implanted sites blue black ³⁴. The numbers of live implants, as well as the pre- and the post-implantation losses were recorded. Pre-implantation loss was calculated by using the following formula:

Number of Corpus luteum – (number of resorbed implants + number of live implants + number of dead implants)

Post implantation loss was equal to the total number of resorbed and dead implants.

Statistical analysis:

Data were analyzed statistically by one way ANOVA followed by Newman- Keul’s test. Body weight and number of live implants as well as pre- and post-implantation losses were analyzed by using Student’s T test. Values were considered significant at P<0.05

RESULTS:

Body weight:

Low doses of AlCl₃  (25 and 50mg/kg BW/day) treatment did not induce any alteration in the final body weights of the mice, however a significant decrease was found in the final body weight of the mice treated with high dose of AlCl₃ (100mg/kgBW/day).The final body weights of  the mice administered with TT (100mg/kgBW/day) only and AlCl₃(100mg/kgBW/day) along with TT (100mg/kgBW/day), remained unaltered similar to that of the control (Table 1).

 TABLE 1: EFFECTS OF AlCl₃, TT AND AlCl₃ + TT ON THE BODY WEIGHT

Values are mean ± SEM of five animals

*Significant difference at P < 0.05

Organs weight:

Lowest dose of AlCl₃ (25mg/kgBW/day)   did not affect the weights the of testis, epididymis and seminal vesicle whereas a significant reduction was noticed in the weight of the epididymis, only in the mice treated with middle dose of AlCl₃ (50mg/kgBW/day). High dose (100mg/kgBW/day) of the treatment induced significant reductions in the weights of the testis, epididymis as well as the seminal vesicle, as compared with the control. Administration of TT (100mg/kgBW/day) alone was ineffective in inducing any alterations in the weights of these organs, however its administration at the same dose along with high dose of AlCl₃ treatment resulted in significant restorations in the organs weights as compared with high dose of only AlCl₃- treated animals, thus attained the values similar to that of the control (Table 2).

TABLE 2: RELATIVE WEIGHTS OF THE SEX ORGANS OF THE MICE TREATED WITH AlCl₃, TT AND AlCl₃ +TT

Values are mean ± SEM of five animals

Significant difference at P < 0.05 (a: I vs IV, b: IV vs VI)

Epididymal sperm assessment:       

Low dose of AlCl₃ (25mg/kg BW/day) neither affected the count nor the motility, viability and morphology of the spermatozoa obtained from the cauda epididymidis. However, significant reductions were noticed in the motility and viability of spermatozoa accompanied by a significant elevation in their percentage abnormality following administration of the middle dose of AlCl₃ (50mg/kg BW/day) treatment.

The sperm count remained unaffected at these doses of the treatment. By contrast, high dose of AlCl₃ (100mg/kg BW/day) treatment resulted in significant reductions in motility, viability as well as in the count of epididymal spermatozoa with significant elevation in the percentage of abnormal spermatozoa. Oral administration of TT (100mg/kgBW/day) only, did not alter any of the sperm parameters. Supplementation with TT (100mg/kg BW/day) in high dose of AlCl₃ (100mg/kgBW/day) - treated mice induced significant recovery in the sperm motility, viability, abnormality and count as compared to that of the high dose of  only AlCl₃ (100mg/kgBW/day)  – treated group, thus attained the control values (Table 3).

TABLE 3: EFFECTS OF AlCl₃, TT AND AlCl₃ + TT ON THE SPERM PARAMETERS

Values are mean ± SEM of five animals

Significant difference at P < 0.05 (a: I vs III and IV, b: IV vs VI)

Histology of testis:    

Histological examinations of the testes of the control, low dose of AlCl₃ (25mg/kgBW/day) and only the TT (100mg/kgBW/day)-treated mice exhibited normal histological features.   Administration of the middle dose of AlCl₃ (50mg/kgBW/day) caused mild degenerative changes in seminiferous tubules as indicated by thinning of the tunica propria, loosening of germ cells and mild regressive changes in the Leydig cells. By contrast testis of the high dose of AlCl₃ (100mg/kgBW/day)-treated mouse exhibited noticeable degenerative changes as indicated by shrinkage of the seminiferous tubules along with thinning of tunica propria, vacuolization, loosening and disorganization of the germ cells.

Lumina of the seminiferous tubules were either devoid of spermatozoa or contained PAS stained cell debris. Atrophy of the Leydig cells were also noticed in the testis of   such treated mice. However, complete recovery was noticed in the spermatogenic activity and the Leydig cells morphology following supplementation with TT (100mg/kgBW/day) in AlCl₃ (100mg/kgBW/day)-treated mice (Fig.1 A-D).

FIG. 1: (A-D) T.S. OF THE TESTIS OF  (A) CONTROL SHOWING NORMAL APPEARANCE OF SEMINIFEROUS TUBULES; (B) AlCl₃ (100 mg/kgBW/DAY)-TREATED MOUSE FOR 30 DAYS  SHOWING  SHRINKAGE OF THE SEMINIFEROUS TUBULES, DEPLETION AND DISORGANIZATION OF THE GERM CELLS, LUMINA DEVOID OF SPERMATOZOA AND LEYDIG CELLS ATROPHY; (C) TT (100 mg/kgBW/DAY)-TREATED MOUSE PRESENTING NORMAL APPEARANCE OF SEMINIFEROUS TUBULES; (D) AlCl₃ (100 mg/kgBW/day) AND TT(100 mg/kgBW/DAY)-TREATED MOUSE SHOWING THE COMPLETE RECOVERY IN SPERMATOGENESIS AND LEYDIG CELL.

Histology of seminal vesicle: The seminal vesicle of the low dose of AlCl₃ (25 mg/kgBW/day)-treated mouse exhibited normal histological features similar to that of the control. The organ of the middle dose of AlCl₃ (50mg/kgBW/day)-treated mouse showed slight increase in mucosal folding as compared with the control. However, the organ in the high dose of AlCl₃ (100mg/kgBW/day)-treated mouse presented noticeable regressive histological changes as indicated by an increase in the mucosal foldings with reduced amount of secretory material in the lumen as compared with that of the control. Supplementation with TT (100mg/kgBW/day) in  AlCl₃ (100mg/kgBW/day) treated- mouse resulted in  complete recovery  in the histology of the  seminal vesicle  as indicated by reduction in the mucosal foldings  and distension of the  lumen filled with large amount of secretion, comparable to that of the control (Fig. 2A-D).

FIG. 2: T.S. OF THE SEMINAL VESICLE OF (A)  CONTROL TO SHOW NORMAL HISTOLOGICAL FEATURES; (B) AlCl₃ (100 mg/kgBW/DAY)-TREATED MOUSE FOR 30 DAYS SHOWING MARKED REGRESSIVE CHANGES AS INDICATED BY AN INCREASE IN THE MUCOSAL FOLDINGS WITH REDUCED AMOUNT OF SECRETORY MATERIAL IN THE LUMEN; (C)TT (100 mg/kgBW/day)-TREATED MOUSE SHOWING NORMAL APPEARANCE; (D)AlCl₃ (100 mg/kgBW/day) AND TT (100 mg/kgBW/DAY)-TREATED MOUSE SHOWING  COMPLETE RECOVERY IN THE SEMINAL VESICLE  AS INDICATED BY REDUCED MUCOSAL FOLDINGS AND ENLARGED  LUMEN DISTENDED WITH THE LARGE AMOUNT OF SECRETION

Estimation of epididymal sialic acid:

Administration of AlCl₃ only at the high dose (100mg/kgBW/day) caused significant decrease in the level of sialic acid in epididymis as compared with the control. However it was restored following supplementation with TT (100mg/kgBW/day) in AlCl₃ (100mg/kgBW/day)-treated mice, thus attained the values of the control (Table 4).

 Estimation of seminal vesicular fructose:

Administration of AlCl₃ only at the high dose (100mg/kgBW/day) caused significant decrease in the level of seminal vesicular fructose as compared with the control. However supplementation with TT (100mg/kgBW/day) restored the level of this secretory product in the epididymis of the high dose of AlCl₃ (100mg/kgBW/day)-treated mice, similar to that of the control (Table 4).

Antioxidant enzymes:

The level of testicular MDA and the activities of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase were comparable in the control and only in the TT (100mg/kgBW/day)-treated mice. Administration of AlCl₃ only at the high dose (100mg/kgBW/day) caused significant increase in the level of MDA with significant decrease in the activities of testicular superoxide dismutase, catalase and GPx as compared with the control. However,  the altered levels of MDA, SOD, catalase and GPx were restored to the control values, following supplementation with TT (100mg/kgBW/day) in AlCl₃(100mg/kgBW/day) – treated mice (Table 5).

TABLE 4: EFFECTS OF AlCl₃, TT AND AlCl₃ +TT ON THE CONCENTRATIONS OF EPIDIDYMAL SIALIC ACID AND SEMINAL VESICULAR FRUCTOSE

Values are mean ± SEM of five animals

Significant difference at P < 0.05 (a: I vs IV, b: IV vs VI)

TABLE 5: EFFECTS OF AlCl₃, TT AND AlCl₃+TT ON THE ANTIOXIDANT MARKERS

Values are mean ± SEM of five animals

Significant difference at P < 0.05 (a: I vs IV, b: IV vs VI)

Cholesterol:

The level of cholesterol in the testis of AlCl₃ (100mg/kgBW/day)- treated mice was significantly increased as compared with the control. However, its level was significantly decreased in the testis of TT- supplemented AlCl₃- treated mice, hence attained the values similar to that of the control (Table 6).

Serum testosterone: The level of serum testosterone remained comparable in the control as well as in only TT (100mg/kgBW/day) -treated mice. Administration of only high dose of AlCl₃ (100mg/kgBW/day) caused significant decrease in the level of serum testosterone as compared with the control. However the reduced level of serum testosterone was restored to the control value following supplementation with TT (100mg/kgBW/day) in AlCl₃ (100mg/kgBW/day) – treated mice (Table 6).

TABLE 6: EFFECTS OF AlCl₃, TT AND AlCl₃+TT ON THE LEVELS OF TESTICULAR CHOLESTEROL AND SERUM TESTOSTERONE

Values are mean ± SEM of five animals

Significant difference at P < 0.05 (a: I vs IV, b: IV vs VI)

Mating ability and fertility test:

Mating ability of the males administered with high dose of AlCl₃ (100mg/kgBW) was reduced to approximately 20% as compared with the control. The number of pregnant females impregnated by AlCl₃ (100mg/kgBW/day)-treated males, was significantly reduced. The number of live blastocysts was also decreased with significant increase in the number of pre- and post- implantation losses in such females. By supplementation with TT (100mg/kgBW/day) in AlCl₃ (100mg/kgBW/day)-treated mice, the mating ability, fertility, the number of live implants as well as the pre- and post-implantation losses were restored to the control values (Tables7 and 8).

TABLE 7: EFFECTS OF AlCl₃, TT AND AlCl₃+TT ON THE MATING ABILITY AND FERTILITY

TABLE 8: EFFECTS OF AlCl₃, TT AND AlCl₃+TT ON LIVE BLASTOCYSTS AND PRE- AND POST- IMPLANTATION LOSS

Values are mean ± SEM of five animals

Significant difference at P< 0.05 (a: I vs IV, b: II vs VI)

DISCUSSION: The present study describes the protective efficacy of Tribulus terrestris against aluminium chloride - induced male reproductive toxicity. Oral administration of AlCl₃ at the dose of 100mg/kgBW/day caused significant decrease in the final body weight of the mice as compared with the control. This is consistent with the findings reported in mouse ³⁵, rat ²² and rabbit ³⁶. Reduction in the body weight following AlCl₃ treatment may be due to decrease in feed consumption and transient diarrhea ³⁷. Significant reductions in the relative weight of the testis and epididymis as noticed in the present study are also reported by previous authors ^{17, 18, 20}. Reduction in the weights of the testis and epididymis may be attributed to the depletion in the germ cells population ³⁸ and spermatogenic inhibition.

Restoration in the body weight and weights of the testis and epididymis following supplementation with TT (100mg/kgBW/day) in AlCl₃-treated mice may be attributed to the presence of an active phytochemical constituent, protodioscin, which is a steroidal saponin, bearing androgenic properties ⁴. Since androgens are anabolic in nature ³⁹, therefore, restorations in the body weight and the testicular weight in AlCl₃-treated mice cannot be ruled out due to existence of androgens present in the fruit extract of TT. Further spermatogenesis is an androgen-dependent process ⁴⁰, hence the presence of steroidal protodioscin in TT, possessing androgenic properties might have restored the AlCl₃-induced reduction in the germ cell population in the seminiferous tubules leading to the recovery in the weights of the testis and thus the epididymis.

In the present study, dose-dependent degenerative changes were observed in seminiferous tubules and Leydig cells resulting in the suppression of spermatogenic activity.  Testis of the high dose of AlCl₃ (100mg/kgBW/day)-treated mouse showed shrinkage of the seminiferous tubules, thinning of the tunica propria and necrotic changes presenting  lumina either devoid of spermatozoa or contained PAS stained cell debris.  Consistent findings have been reported in the mouse ^{35, 41}. A significant reduction noticed in the level of serum testosterone following treatment with 100mg/kg/bw/day of AlCl₃ is in agreement with that reported in the mouse ⁴². Appearance of degenerated Leydig cells and increased level of testicular cholesterol, a precursor of steroidogenesis, are reflected by reduced level of serum testosterone. Consistent finding has been reported by Chinoy et al .³⁵.

In our study it might be possible that the aluminium at the high dose would have crossed the blood-testis barrier ⁴³ causing the germ cell death by inducing significant reduction in the level of serum testosterone.   Since testosterone is essential for maintenance of spermatogenesis ⁴⁴, hence AlCl₃-induced reduction in the level of this hormone might have resulted in reduction in the epididymal sperm count which is the outcome of inhibited spermatogenic activity.

The inhibited spermatogenic activity with reduced sperm count noticed in our study is in accordance with the findings of Yousef et al.¹⁵ and Khattab et al.²². Significant reductions in the activities of antioxidative enzymes viz. SOD, catalase, GPx and an elevation in lipid peroxidation as reflected by an increase in MDA level, noticed in this study, are in accordance with the findings reported by previous authors ^{19, 20, 45}.  An increased oxidative stress caused by aluminium chloride treatment, as noticed in the present study, might have damaged the Leydig cells resulting in reduced level of serum testosterone which subsequently could have inhibited the spermatogenic activity in the testis and thus sperm count in the epididymis.

Restorations in the levels of serum testosterone, testicular MDA and the activities of antioxidative enzymes following supplementation with 100 mg/kgBW/day of TT in AlCl₃ (100 mg/kgBW/day) treated mice suggest the androgenic^4,46 and antioxidative ^47-49 properties, exhibited by protodioscin and flavonoids, respectively, present in its fruit extract, causing recovery in spermatogenic activity.

Restoration in the testicular oxidative stress following TT supplementation has also been reported in cadmium ⁸ and cypermethrin ⁹ intoxicated rats. It appears that the flavonoid, an antioxidative component of TT, might have prevented the free radical production   by enhancing the defense system during AlCl₃-induced testicular stress. This result is in line with the findings of previous authors ^{22-24, 43} who reported protective potential of flavonoids present in the grape seed, onion and ginger against aluminium-intoxicated testicular injury.  Significant reductions in the levels of epididymal sialic acid and seminal vesicular fructose noticed in the high dose (100mg/kg/BW/day) of AlCl₃-treated mice is consistent with that reported by Chinoy et al.³⁵

Sialic acid is an essential component for the maintenance of the structural integrity of epididymal membranes and maturation of spermatozoa in the epididymis ³⁵. Further degenerative changes noticed in the  seminal vesicle as indicated by an increase in the mucosal foldings with reduced amount of secretory material in the lumen after AlCl₃ treatment have also been reported by these authors. The decreased level of fructose in the seminal vesicle of such treated mice is reflected by reduced amount of secretory material present in the lumen.

Since the structural and functional integrity of the accessory sex glands in the males are androgen- dependent⁵⁰, therefore, significant decrease in the level of serum testosterone as noticed in our study is deflected by reduced levels of sialic acid in the epididymis and fructose in the seminal vesicle with its regressed histoarchitecture.   Significant restorations in the levels of these secretory products of the epididymis and the seminal vesicle following supplementation with TT in AlCl₃-treated mice indicate its androgenic property.

Significant reductions in the  motility and viability, with significant increase in the percentage of abnormal spermatozoa noticed in the epididymis of high dose of AlCl₃-treated mice is in accordance with the findings of others ^{15, 22, 23}. Previous reports have suggested the AlCl₃-induced decrease in sperm motility and viability may be due to reduction in the activity of mitochondrial protein, aconitase ^{22, 51} which is responsible for the isomerization of citrate to isocitrate in Kreb’s cycle, through the intermediate cis-aconitate.  Alteration in the activity of acotinase following aluminium accumulation thus leads to change the mitochondrial functions which may be reflected by the altered sperm motility and viability. Moreover, the observed decrease in the sperm motility may also be due to the reduction in the level of serum testosterone ^{17, 20}. An increased percentage of abnormal spermatozoa after treatment with AlCl₃ may be attributed to the free radical generation¹⁵.

Testosterone is an important hormone in stimulating the mating ability ¹⁵. High dose of AlCl₃-induced decrease in the level of testosterone have thus resulted in reduction in the mating ability leading to significant decrease in the fertility of the male mice.  Increase in the pre- and post- implantation losses with decrease in the number of live blastocysts in females impregnated with the high dose of AlCl₃-treated males is most probably attributed to the significant decline in the level of serum testosterone ^{15, 37} as well as the poor sperm quality ⁵².

Significant recovery noticed in mating ability and fertility of the TT supplemented AlCl₃-treated males and pre- and post-implantation losses in females impregnated with such males are probably attributed to the presence of steroidal protodioscin implicated in restoring the level of serum testosterone. Presence of flavonoids in TT might have improved the quality of spermatozoa by reducing the oxidative stress in AlCl₃ –treated mice resulting in marked reduction in pre- and post- implantation losses comparable to the controls.

CONCLUSION: The results of the present study showed the protective efficacy of the fruit extract of Tribulus terresrtis against aluminium chloride-induced reproductive toxicity in the male mice. Fruit extract of this plant thus may emerge as an effective herbal remedy in treatment of impaired fertility in the males, induced by certain ubiquitous metals.

ACKNOWLEDGEMENT: The first author is thankful to University Grant Commission, New Delhi and Banaras Hindu University, Varanasi, for providing financial assistance to carry out the present work.

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How to cite this article:

Kumar P and Singh P: Protective Role of Tribulus Terrestris on Aluminium Chloride - Induced Reproductive Toxicity in the Male Laboratory Mouse. Int J Pharm Sci Res 2015; 6(6): 2395-05.doi: 10.13040/IJPSR.0975-8232.6(6).2395-05.

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